Nature Chemistry Volume 02 No 06 Pp425-510

editorial
Bringing science to the party

Although politics has been defined as the science of government, there is little science in government. Recent events in UK politics have highlighted the lack of scientifically literate elected representatives a situation that must change for the good of society.
The relationship between science and politics has been likened to a marriage1, with the inference being that, to develop, the partners must not become alike but must respect their differences and that the odd quarrel along the way is no big deal. Recently, however, science has taken the role of the meek, misunderstood spouse that has little influence over their all-powerful partner. Science must become stronger in this relationship; at present it does not have the respect it deserves from most politicians, and so its champions must become louder within the political arena if we are to address the grand challenges of the coming century. Two recent incidents in the UK suggest that scientists hold little political power, with the real crux of the matter being a lack of science-literate politicians. Although some prominent politicians have science backgrounds (Margaret Thatcher and Angela Merkel were chemists) out of the 650 (pre-2010 election) UK members of parliament (MPs), 27 held science degrees and 584 admitted to having no political interest in science and technology and taking into account upcoming retirements, its about to get worse2. This alarming finding calls into question whether the people responsible for making important policy decisions, either based on scientific research or about its funding, fully understand its importance or crucially the scientific method at its core. The scientific method relies on the search for and critical consideration of evidence on which to base explanations and decisions, but it is often trumped by political or economic considerations, or more worryingly, is just not understood. Although UK government policy may be more informed by evidence than in the past, the way in which hard scientific evidence is handled and debated often seems to result in two steps forward and one step back. Take for example the way in which a frightening number of UK MPs have reacted to a recent government report on homeopathy a practice currently funded publicly by the National Health Service. In a progressive move, a science and technology committee, made up of the UKs more science-minded MPs, was tasked to look at the supporting evidence for homeopathy to help re-evaluate government policy. The thorough, evidence-led report, found that homeopathy is not efficacious (that is, it does not work beyond the placebo effect) and that explanations for why homeopathy would work are scientifically implausible. The committee therefore recommended the withdrawal of funding. On publication of the report, MP David Tredinnick also an advocate of unscientific practices such as medical astrology and remote energetic healing spearheaded a movement to reject the findings. Even though the rigours of the scientific method are there to behold in this report which dismisses outright the value of homeopathy 70 MPs supported Tredinnicks campaign. Science has fought back in the form of science writer, Michael Brooks. He took the view that if our politicians dont listen to rational, reasoned arguments he would change things by trying to become one of them, standing against Tredinnick as a candidate in the recent 2010 election3. Although ultimately unsuccessful, this unusual move has gone some way to raising the profile of science. The case of homeopathy is just one extreme example of how politicians can harm the development of science by undermining and questioning its credibility. Further troubles undoubtedly lie ahead because so many of the challenges faced by society today rely on knowledge afforded through scientific research, in topics far more complicated and with greater ramifications than homeopathy: climate change, energy provision and genetic modification to name but a few. To deal with them ably, governments must become more science-literate. This doesnt require our politicians to be scientists (although this would help), rather they must have an appreciation of science. Foremost, however, they must learn how scientific research actually works to reach the best possible explanation for a given set of hypotheses. There are obviously no expectations that politicians must be experts in cuttingedge science any government must also have professional science advisers who present evidence around which policy can be moulded. Again, however, the UK government has shown how, even on the most scientific of subjects where expert testimony is of paramount importance, their policies can be chosen without full regard for the evidence with which they are provided, as in the case of Professor David Nutt, the governments former chief drug adviser. In the first of a series of spats over Nutt contradicting the governments hard line on illegal drugs, it reprimanded him for his efforts to show how the harm drugs can cause compares with other potentially harmful activities for example, he highlighted that horse riding was statistically riskier than using the drug ecstasy. Then, after ignoring his advice to not reclassify cannabis from class C to B, it later sacked him for publicly presenting evidence suggesting that LSD, ecstasy and cannabis were in fact less harmful than alcohol and tobacco again contradicting the governments stance. Although the issues involved in the classification of illegal drugs are not clear cut, and governments must balance many different factors, ignoring expert advice and attempting to silence its communication to the public rather than explaining their decisions in the face of it undermines science and its worth. Winston Churchill once said that courage is what it takes to stand up and speak; courage is also what it takes to sit down and listen. Science needs courage. It needs courage from scientists like David Nutt and Michael Brooks to stand up and speak out assertively to communicate its importance to politicians and to not back down when the evidence is clear. Scientists must question policies that ignore the evidence and must try to educate those who make them. In return, we need courage from governments and politicians. Governments must try to teach their members more about the basics and value of science so that policy decisions can be informed by a greater understanding of the technical issues. Politicians should seek advice from scientists and be open to learning more about what may seem like difficult topics. They must be willing to trust the scientific method and, in doing so, make tough decisions that not only consider the political ramifications, but take into account the underlying evidence.
References
1. Price, D. K. The Scientific Estate (Harvard Univ. Press, 1965). 2. http://go.nature.com/6UsL5q 3. http://www.scienceparty.org.uk/
nature chemistry | VOL 2 | JUNE 2010 | www.nature.com/naturechemistry
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2010 Macmillan Publishers Limited. All rights reserved
research highlights
Water-OXiDatiOn cataLysts
stable and able
Science 328, 342345 (2010)
Developing a cheap and effective wateroxidation catalyst (WOC) is one of the key breakthroughs needed to produce clean energy from artificial photosynthesis. Although heterogeneous WOCs have some advantages over homogeneous ones, the latter are easier to study and this increased understanding makes them easier to improve. Many homogeneous WOCs, however, have organic ligands that undergo oxidative degradation, or include expensive and rare metals, such as ruthenium.
reDuctiVe aLKyLatiOn
Now, Craig Hill and colleagues from Emory University in Atlanta have developed a homogeneous WOC with a cobalt oxide core, stabilized by polytungstate ligands. The catalyst is made from cheap and readily available starting materials in a fairly simple one-pot synthesis. Although Hill and co-workers made a range of similar cobaltbased polyoxometalate compounds, only one showed catalytic activity. This had a turnover frequency the number of molecules of water oxidized by a molecule of the catalyst of 5 s1, much higher than a similar heterogeneous cobalt phosphate catalyst. To show that their catalyst was stable and did not break down into aqueous cobalt ions which can themselves act as WOCs Hill and colleagues performed a range of experiments. As well as UVvisible and NMR spectroscopy showing no changes over a month, adding a ligand known to inhibit the catalytic activity of aqueous cobalt ions had no effect on the catalyst. Furthermore, computational studies showed that the highest occupied molecular orbitals of the catalyst were located on the cobalt core, with no contribution from the polytungstate, showing that the ligands are effectively inert.
eXhaust cataLysts
Perovskites prove potent

Science 327, 16241627 (2010)
easier by increasing the proportion of NO2 compared with NO. Current exhaust systems require the use of expensive platinum-based catalysts to do this, and these also have poor stability at the high operating temperatures. Now, Wei Li and colleagues from General Motors Global Research and Development in Michigan have developed perovskite oxide catalysts that do the job as well or better than the commercially available ones. The oxides were LaCoO3 and LaMnO3 with some of the lanthanum replaced with a small amount of strontium. This doping not only almost doubled the surface area of the solids, but also promoted the catalysis itself in LaCoO3, by increasing the number of weakly bonded oxygen atoms. A useful exhaust catalyst also must be able to oxidize carbon monoxide and unburnt hydrocarbons. Although the perovskite catalysts on their own were less effective at catalysing these reactions, combining them with palladium particles made them as effective as commercial platinum catalysts. The palladium additive also helped prevent the new catalysts from becoming poisoned by sulfur, a common problem in exhaust catalysts. The manganese-based catalyst was structurally stable enough to withstand the high temperatures as well as the reducing environments required to regenerate them.
DenDrimers
2010 AAAS
Diesel engines are more fuel efficient than petrol ones but, because they run at higher airto-fuel ratios, they give off more harmful NOx (NO and NO2). Removing NOx in this oxygenrich environment is challenging, but made
sticky situation
Angew. Chem. Int. Ed. 49, 30303033 (2010)
twice as nice
Angew. Chem. Int. Ed. 49, 30373040 (2010).
The synthesis of complex molecules relies on methods for the formation of carbon carbon bonds. Methods that allow more than one carboncarbon bond to be formed in a single step improve the efficiency of the process. The reaction of organometallic reagents with carbonyl compounds is one of the most popular methods of carboncarbon bond formation. Adding two different groups to one carbonyl group, however, usually requires a cycle of reductive addition, followed by oxidation and then a second addition. Now, Pei-Qiang Huang and co-workers from Xiamen University in China have developed a sequential addition of two different organometallic reagents to an amide or a lactam to produce a tertiary alkyl amine in a single pot. The carbonyl oxygen of the amide or lactam is first activated by formation of a triflate, which is a good leaving group. The iminium triflate thus formed is reacted with a Grignard reagent. The loss of the triflate group then results in a new iminium species that can react with a further organometallic reagent and, importantly, this can be a different one from the first to form the product. A wide variety of different organometallic reagents can be employed for the second addition, including further Grignard reagents, alkynyl lithium reagents and even lithium enolates. Huang and co-workers also demonstrate that for a reaction with a lactam that already bears another substituent, the second addition can be made in a diastereoselective fashion.
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Muscles contract because of the concerted molecular-level interactions between numerous actin and myosin proteins that work together to shorten muscle fibre. Myosin acts like an active ratchet, which binds to a filament of actin, pulls it in one direction, lets go, and then realigns itself before binding again. ATP drives the process and, in particular, it binds to myosin, which reduces the affinity of myosin for binding with actin, causing the two proteins to detach. Now Takuzo Aida, Kazushi Kinbara and colleagues at the Universities of Tokyo and Tohoku have shown that this process can be hindered or completely stopped by gluing the proteins together with a dendrimer that binds to both actin and myosin. The dendrimer is made up of ether branches and is terminated with nine guanidinium ions, which can bind to the oxyanions prevalent on protein surfaces. To show that it could bind to both proteins and arrest the motion of actin, Aida, Kinbara and colleagues attached numerous myosin proteins to a surface and observed the behaviour of fluorescently tagged actin molecules when added to the mix. When no dendrimer was present the actin filaments
research highlights
moved with a velocity of approximately 4.6 m s1 but when the dendrimer was added, their motion stopped completely. A lower-generation dendrimer with only three guanidinium-terminated branches does not act like a molecular glue, so it seems that the number of sites on the dendrimer that can interact with the proteins is key to its ability to bind two proteins simultaneously.
PermeaBLe memBranes
The AA dipeptide retained its crystallinity after oxygen permeation, suggesting that its guest-induced flexibility is reversible. This dynamic response was also guest-dependent: AA was more permeable to oxygen than to helium even though helium is a smaller species. This excellent selectivity suggests that dipeptide crystals hold promise for a variety of separation processes.
GLucOse cOnVersiOn
smart cookies
Peptides make the difference

Angew. Chem. Int. Ed. 49, 30343036 (2010)
a solid combination
Word goes chemistry, science goes cool and cookies go nuclear. A chemistry add-in for Word (http:/ / go.nature.com/KynNV9), created by Microsoft in collaboration with the Unilever Centre for Molecular Science Informatics at Cambridge University, aims to make it easier to get your chemistry into the widely used word processor. Lauren Wolf on Newscripts (http:/ /go.nature.com/xkBm6h) pointed out that you need either Word 2007 or 2010, so as she operates in the Stone Age (or is it Bronze Age?Im not sure) and have only Word 2003, she hasnt had a chance to try it yet. Wolf also picks up on the fact that many academics use Macs, for which there isnt a version available. Worry not, Mac users: Peter Murray-Rust, one of the collaborators, hopes that Chem4Words open-source nature will mean it should be available to the Mac Word and Open Office communities. We must confess that our office uses Stone Age versions of Word too, so we havent been able to test it out either. Is science cool? This question was asked by KJHaxton on Endless Possibilities (http:/ /go.nature.com/HBgcyt) after reading a newspaper article suggesting that it is (http:/ /go.nature.com/NXGfxO). The article quotes a Large Hadron Collider physicist, astronomers, numerous science writers and a comedian, but Haxton isnt entirely convinced. She welcomes the increase in science on TV and that the press are reporting more scientific experiments, because We need publicity for the science of the future, the science that the viewer or their children or grandchildren will help fashion. But Haxton concludes that the only measure is the number of young people who view it as a viable career choice. And finally, following on from last months atomic-emission-spectra scarves, we have atomic-orbital cookies. Windell Oskay at Evil Mad Scientist Laboratories (http:/ /www.evilmadscientist.com/article. php/atomiccookies) cut some melamine extrusion plates for a cookie press in the shape of the s, p, d and f orbitals we all know and love. One standard cookie recipe later and, hey presto, weve got atomic orbitals in a new and tasty form.
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Proc. Natl Acad. Sci. USA 107, 61646168 (2010)
The materials that come to mind when porous solids are mentioned are usually zeolites, silicates and carbon-based frameworks. Dipeptides, however, have recently been shown to form hydrogen-bonded microporous crystals, and have attracted interest for their gas sorption properties. Now, a team of Portuguese researchers led by Lus Gales at the Institute of Molecular and Cell Biology in Porto have observed that dipeptide single-crystals can act as permeable membranes able to distinguish between argon, nitrogen and oxygen a process of interest for air separation, but difficult to carry out because of the similarity in size between the species. The permeability of three dipeptides l-leucyl-l-serine (LS), l-valyl-l-isoleucine (VI), and l-alanyl-l-alanine (AA), which crystallize into different lattices with different porosities was found to be size-dependent. The crystals that displayed narrower pores combined lower gas absorption abilities and guest diffusivities, resulting in poorer permeabilities. Thus, LS, with nanochannels that are larger than argon, nitrogen and oxygen, is permeable to all three. VI, whose channel size is close to that of the gas molecules, was only permeable to oxygen and nitrogen. And although AAs porosity consists of discrete pockets rather than channels, it was found to be permeable to oxygen.
The conversion of glucose into fructose is widespread in the food industry for the production of the sweetener high-fructose corn syrup, and has also emerged in the field of renewable energy as a path to degrading biomass into fuel or other valuable chemicals. Enzymatic catalysts are typically used for this isomerization, but their lifetime is limited, they require pre-purified substrates, and only work under specific conditions (neutral pH and around 333 K). To remedy these limitations, research is now focusing on chemical catalysts. Basic catalysts have shown promising activity, but are not practical as glucose and fructose easily decompose in alkaline solutions. Now, Mark Davis and colleagues from the California Institute of Technology have prepared an efficient inorganic, heterogeneous catalyst by incorporating tin centres into a microporous zeolite with large pores (Beta). Much lower or no conversion was observed when tin was inserted into an ordered mesoporous silica or a mediumpore zeolite, respectively, showing that the catalysts activity greatly depends on the size of the pores. The tinBeta heterogeneous catalyst was efficient even under high glucose concentrations (up to 45%), reusable over three cycles, and remained active after subsequently undergoing a typical zeolite regeneration process (involving calcination at 813 K). Its activity in acidic aqueous solutions also makes the tin-containing zeolite promising for one-pot processes, as demonstrated with the degradation of starch into fructose, coupling hydrolysis and isomerization steps. The isomerization mechanism remains to be elucidated, but the researchers suggest that it occurs through the formation of a five-membered ring involving the tin centre, followed by an intramolecular hydrogen transfer.
The definitive versions of these Research Highlights first appeared on the Nature Chemistry website, along with other articles that will not appear in print. If citing these articles, please refer to the web version.
2010 Wiley
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MOLECULAR MACHINES
Springing into action

Ben L. Feringa
Controlling the movements of molecular systems through external stimuli is crucial for the construction of nanoscale mechanical machines. A spring-like compound has now been prepared a double helicate that retains its handedness under ion-triggered extension and contraction.
irtually every biological process, from intracellular transport to muscle contraction, is driven by a molecular machine. These biological machines carry out pretty much the same functions as the macroscopic ones, but they take very different forms. Whereas macroscopic machines typically operate under the constraints of friction, inertia and gravity, their biological counterparts operate in the nanoworld against the Brownian storms created by the random movements of molecules in solution1. The apparent ease with which biological machines operate fascinates and inspires scientists, while at the same time challenging us to learn how to construct compounds that can perform mechanical functions2,3. A variety of ingenious systems have been prepared, including molecular muscles, motors and shuttles46 but it remains notoriously difficult to control these movements and translate them from the nanoscale into a macroscopic motion. In particular, despite its ubiquity in biological systems, mimicking springlike function at the nanoscale has proven highly challenging. Writing in Nature Chemistry, Yoshio Furusho and co-workers now describe7 a molecular double helix that behaves in a similar manner to a macroscopic spring that is, undergoes contraction and extension while winding and unwinding in a unidirectional sense. Ion binding and release processes are key mechanisms in biological machines. The functioning of some muscle tissue, for example, relies on calcium ions binding. The interaction between the muscle components myosin and actin is governed by the calciumsensing complex troponin C, which activates or inhibits muscle contraction through calcium binding and release. Taking a leaf out of natures book, Furusho and co-workers have used ion-binding events to trigger the spring-like motion of a helicate a motif that is also omnipresent in nature, the best known being the DNA double helix. The researchers had previously constructed a double-stranded helicate8
consisting of two hexaphenol strands bridged by two boron atoms through the formation of two spiroborate (BO4) moieties, and accommodating a sodium cation in its central position. The sodium ion was coordinated to eight oxygen atoms the two central hydroxyl groups of each hexaphenol strand and the two closest oxygen atoms of each spiroborate moiety. Furusho and colleagues noticed, however, that the four central hydroxyl groups werent necessary to hold the complex together and replaced them with hydrogen atoms, thus preparing a new double helicate in which the central sodium cation is coordinated only to the spiroborate moieties (shown in Fig. 1). The inclusion and removal of the central sodium ion triggers the contraction and extension of the helix. As a sodium ion binds to the spiroborate moieties, it shields the electrostatic repulsion within the helixs core, causing its contraction along its long axis. When cryptands are added to the solution, they bind to the sodium ions, removing them from the doublehelicate complex. This unmasks the negative charges between the spiroborate moieties,
causing them to repel each other and resulting in the partial unwinding of the helix. A detailed analysis of the structure by X-ray crystallography and nuclear magnetic resonance reveals that the helix approximately doubled its length, from 6 to 13 (Fig. 1). In macroscopic and biological springs, the contraction and expansion of springs is typically accompanied with unidirectional twisting, but this has rarely been observed in synthetic molecular systems. Typically, on contraction or extension, synthetic helicates adopt a non-helical conformation, which leads to a racemization of the helicate and a twisting in both the rightand left-handed directions. The double helicate described here, however, retained its inherent chirality; circular dichroism studies show that extension by unwinding of the double-stranded helix proceeds clockwise, and contraction by winding proceeds anticlockwise. The spring-like motion can be repeated many times simply by adding sodium ions or cryptands (which equates to removing sodium ions) to the solution. The rate of the extension process is slower than that
N O O O O N O
Na
Figure 1 | Spring-like molecular motion. Inclusion and removal (through trapping by a cryptand) of a sodium ion triggers the contraction and extension of a double helicate. These events are accompanied by a unidirectional twisting.
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of the contraction one a feature that is attributed to the differences between the sodium binding processes involved (sodiumhelicate and sodiumcryptand). This elegant system shows how a clever design taking advantage of the unique features of helices can lead to a springlike mechanical motion at the nanoscale. The next step could be an autonomous spring-like motion, in which the metal ion would bind successively to different helical strands. Making the leap to translating the molecular systems operation to macroscopic movement 9, reminiscent of the actinmyosin system that achieves muscle contraction, will be a challenge. Any such system will necessitate binding an ensemble of molecular springs to a surface and achieving concerted action. One could also foresee associating the ion binding and release events to a catalytic function, or controlling it by light irradiation. This would set the stage for the construction of a truly molecular mechanical device. The molecular spring presented by Furusho and co-workers7 combines the beauty of molecular helicity with a useful function and is a significant step on the long and winding road towards molecular nanotechnological devices.
Ben L. Feringa is at the Centre for Systems Chemistry, Stratingh Institute for Chemistry and Zernike Insitute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. e-mail: b.l.feringa@rug.nl References
1. Astumian, R. D. Science 276, 917922 (1997). 2. Browne, W. R. & Feringa, B. L. Nature Nanotech. 1, 2535 (2006). 3. Euan, R., Kay, E. R., Leigh, D. A. & Zerbetto, F. Angew. Chem. Int. Ed. 46, 72191 (2006). 4. Huang, J. et al. Appl. Phys. Lett. 85, 53915393 (2003). 5. Collin, J-P., Dietrich-Buchecker, C., Gavina, P., Jimenez-Molero, M. C. & Sauvage, J-P. Acc. Chem. Res. 34, 477487 (2001). 6. Kinbara, K. & Aida, T. Chem. Rev. 105, 13771400 (2005). 7. Miwa, K., Furusho, Y. & Yashima, E. Nature Chem. 2, 444449 (2010). 8. Katagiri, H., Miyagawa, T., Furusho, Y. & Yashima, E. Angew. Chem. Int. Ed. 45, 17411744 (2006). 9. Percec, V., Rudick, J. G., Peterca, M. & Heiney, P. A. J. Am. Chem. Soc. 130, 75037508 (2008).
REACtION kINEtICS
Catalysis without a catalyst

Raoul kopelman
Can two identical reactors with the same concentrations, under identical physical conditions, have reaction rates that differ by a factor of a thousand? A study now shows that, although not true in uncrowded environments, a reactants starting point makes a large difference to reaction kinetics in identically crowded systems, such as cellular nuclei.
or a reaction to have superfast kinetics, compared with expectations from textbook equations, it helps if a higher power intervenes and microscopically arranges the reacting molecules to be close to each other closer than in a random distribution. Such non-classical kinetics do occur for some heterogeneous chemical reactions and may play a large role in biology. Writing in Nature Chemistry, Olivier Bnichou and colleagues use theory to investigate such geometrically controlled reactions1. They study reactions in a geometrically confined (topologically tortuous) reaction space in which the reactants have a spatially ordered distribution (Fig. 1). Such situations are of much interest at present because they may be typical of highly significant subcellular biochemical reactions of potential biomedical importance, such as gene transcription. Similar situations are also encountered in condensed-state physical and chemical reactions, such as exciton and electronhole recombination or trapping, which have relevance to photonics and solarenergy science. Bnichou and colleagues1 use theory that goes beyond what has been generally termed non-classical or fractal-like kinetics, but still use a random-walkbased approach (that is, diffusion-limited
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reaction kinetics) to obtain analytical expressions that allow straightforward computations. To understand such theory we must first introduce the classical concepts of chemical reaction kinetics, where an elementary bimolecular reaction, at time t, is described by R(t) = kA(t) B(t). Here, A(t) is the instantaneous concentration (or activity) of reactant A, and B(t) is that of reactant B. R(t) is the instantaneous reaction rate and k is a constant that doesnt change with time and depends on the transport coefficients of the reactant molecules, as well as on the so-called reaction cross-section, or reaction probability at collision. What this classical formula does not seem to depend on is the size and shape of the reaction vessel, or the locations of the molecules. What is assumed implicitly in this expression is that the chemistry is taking place within a large reaction vessel with a homogeneous and random distribution of molecules at all times, that is, with perfect stirring. Furthermore, it is implicit that the so-called exploration volume, V(t), which is the volume that a reactant visits in a given period of time, increases (at least) linearly with time. As soon as any of these idealized conditions is relaxed, the equation above has to be modified. For instance, without perfect stirring,
k becomes a time-dependent quantity, that is, k(t), which has a monotonically descending dependence on time2. To understand this better, lets describe the distinction between diffusion in onedimensional and three-dimensional (3D) exploration spaces with the following analogies: (1) the drunk in an alley always returns to the bar; (2) the drunk space pilot hardly ever manages to return to the bar planet. Implicit in the above analogies is that the drunk performs a diffusive (random) walk along the alley, and likewise, the pilot randomly changes the direction of flight, that is, performs a random walk in 3D space. Mathematics teaches us that, in one dimension, V(t) increases (asymptotically) as t 1/2 (even if the alley is infinitely long), whereas in three dimensions it increases linearly with t. Two equivalent ways of looking at that 3 are: in one dimension (even if infinitely long, and in infinite time) the probability of the drunk (random walker) returning to the bar (origin) is unity, and thus his escape probability is zero; however, the probability of the drunk space pilot returning to the bar planet is far from unity, for the drunk pilot manoeuvres in three dimensions, and thus his escape probability is finite. The above considerations distinguish
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could they potentially be used to control the kinetics? The general derivation uses a random walker in a fractal medium with a fractal dimension, and the dynamics characterized by the dimension of the walk3. The results differ significantly depending on the relative size of these dimensions. When the fractal dimension is larger resulting in noncompact exploration and an exploration volume that grows linearly in time it is the mean of the FPT that characterizes the kinetics, as in the simple case of regular 3D diffusion. The result is that the initial position of the reactants has little effect on the kinetics (except in recombination reactions, which are not characteristic of biological situations and are not addressed). For the opposite case of compact exploration however with exploration volume growing sublinearly in time the full distribution of the FPT is required and not just the mean; the result is that the kinetics depend strongly on the original distance between the initial starting position and the target. It is the real-estate principle of location, location, location that is key. Bnichou and colleagues use the term geometrically controlled kinetics to describe such situations and it is typical of certain important reactions inside biological cells and nuclei, as is shown schematically in Fig. 1. To illustrate the power of such geometrically localized reactions, they discuss the specific situations of transcription kinetics in cases of gene co-localization, showing that a 100 nm co-localization in the nucleus may speed up the transcription kinetics by three orders of magnitude. These results demonstrate the importance of correctly treating such chemical reactions, rather than using the classical reactionkinetics approach. One also wonders if evolution has in fact used the idea of geometrically controlling the reactants to speed things up.
Raoul Kopelman is in the Department of Chemistry, University of Michigan, 930 N. University, Ann Arbor, Michigan 48109-1055, USA. e-mail: kopelman@umich.edu References
1. Bnichou, O., Chevalier, C., Klafter, J., Meyer, B. & Voituriez, R. Nature Chem. 2, 472477 (2010). 2. Kopelman, R. Science 241, 16201626 (1988). 3. Ben-Avraham, D. & Havlin, S. Diffusion and Reactions in Fractals and Disordered Systems (Cambridge Univ. Press, 2000). 4. Kopelman, R. in Radiationless Processes in Molecules and Condensed Phases Vol. 15 (ed. Fong, F. K.) 297346 (Topics in Applied Physics, Springer-Verlag, 1976). 5. Parson, R. P. & Kopelman, R. Chem. Phys. Lett. 87, 528532 (1982). 6. Kopelman, R. J. Phys. Chem. 80, 21912195 (1976). 7. Monson, E. & Kopelman, R. Phys. Rev. Lett. 85, 666669 (2000).
S2
S1
Figure 1 | A random race through crowded space: here reactant 1 starts at site S1 and reactant 2 at S2. Which one will get to the target T first? Both travel equally fast, and randomly change direction equally often. As may be intuitively expected here, the closer reactant is much more likely to win the race because of the shorter random path. Had there been no obstacles, however, both reactants would be equally likely to win the race. The obstacles lead to apparent catalysis in the kinetics of reactant 2, and this demonstrates the concept of geometry controlled kinetics. To apply this picture to biology, consider the elliptical area to be a cell nucleus, the reactants to be transcription factors, and the target to be a gene. Has nature ordered all transcription factors to originate in close lying locations such as site 2, and not in farther ones like site 1, so as to speed up this basic reaction of life? Image reproduced from ref. 1.
between diffusion in compact exploration spaces with zero escape probability and a sublinear V(t), and in non-compact exploration spaces with finite escape probability and a linear V(t). As most fractal topologies (even if embedded in three dimensions) are compact, the resulting non-classical reaction kinetics, with sublinear exploration space, V(t), and time-dependent reaction coefficient, k(t), have been termed fractal-like reaction kinetics2. Although the above analogy considers one reactant exploring a volume, when we consider a distribution it turns out that fractal-like reaction kinetics are also accompanied by non-random reactant distributions in space, even if the initial distribution was random. Bnichou and co-workers look at how such a non-random distribution of reactants influences the kinetics of a reaction in confined, compact reaction spaces. Historically, the early demonstrations of non-classical reaction kinetics were those of photophysical reactions in confined and tortuous domains. Such systems include exciton trapping in disordered molecular crystals4,5, or biological photosynthetic units6, and photochemical reactions in confined domains with co-localization of
photoexcited molecules, that is, a nonrandom initial distribution owing to laser speckles (a laser optics phenomena that causes a non-random distribution of photons at the target)7. Specifically, the situation that Bnichou et al.1 study is that of a spatially confined reaction space with labyrinthine topology, and a non-random (quasi-intelligently designed) reactant distribution. Their contribution is to give a unified theoretical treatment, with universal results, using the first passage time (FPT) approach the FPT is the time it takes a diffusing molecule to reach a target with the implicit assumption that reaching the target means reacting with it. The approach taken by Bnichou and colleagues allows them to derive the full distribution of the FPT, rather than just the mean, and this is crucial for quantifying the reaction kinetics. Although the mean gives satisfactory results for noncompact exploration, it does not for the compact case. The main questions posed are: how does the FPT distribution depend on the volume of the confining domain and the initial position of the diffusing molecule, and are these factors important? If so,
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CRYStAL ENGINEERING
towards artificial enzymes

Joseph t. Hupp
Despite knowing that the active centres of many metalloprotein enzymes are iron porphyrin haem complexes, chemists find them difficult to imitate. Now, the assembly of haem-like centres into a crystalline, stable, nanoporous array shows promise for biomimetic catalysis.
any enzymes rely on iron porphyrin haem centres (Fig. 1a) to carry out important tasks in biology, in particular using their ability to bind small molecules such as O2 and water. For example, the metalloproteins haemoglobin and myoglobin, found in red blood cells and muscle tissue, respectively, use haem centres to reversibly bind O2 through their Fe(ii) ions and act as oxygen carriers. In contrast, in cytochrome b5 proteins, their role is to transport electrons through changes in the oxidation state of iron. Both of these abilities oxygen binding and redox changes are used by the cytochrome P450 family to catalyse oxygen-transfer reactions, ranging from the enantioselective epoxidation of olefins to the conversion of alkanes to alcohols (depending on the enzyme). What if synthetic iron porphyrins could be coaxed to catalyse valuable chemical transformations in much the same fashion as cytochromes, but without the complex protein machinery? Over the years this idea has motivated some terrific biomimetic chemistry 1,2 and, at the same time, revealed some practical difficulties. Most notably, iron porphyrins were found to easily form oxo-bridged dimers, which renders them catalytically inactive. Furthermore, there are challenges in positioning the nitrogen- or sulfur-based ligands needed for catalyst activation at one of the two available axial iron coordination sites while avoiding coordination at the second. Collman and co-workers showed several years ago that both problems, in principle, could be overcome through sophisticated functionalization of the porphyrins (for example by using organic picket fences to prevent dimerization, or by overarching straps to control ligation)2. Unfortunately, these derivatives are difficult to synthesize, making them impractical for routine oxidative catalysis, either in academic labs or in industry. Writing in Science, McKeown and coworkers3 have now proposed an interesting alternative solution: build crystalline nanoporous arrays. Crystallinity ensures that the haem-like active sites are precisely positioned. Regular spacing (nanoporosity)
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Figure 1 | Schematic representation of the nanoporous crystalline haem-like array. a, The structure of iron porphyrin. b, Structure of a non-aggregating iron phthalocyanine. c, 3D nanoporous crystalline arrays of iron phthalocyanine molecules connected by bipyridine linkers.
ensures that they are suitably isolated from each other, and at the same time provides channels to transport reactants and products to and from the sites. Furthermore, with practical applications in mind, McKeown and colleagues chose to use phthalocyanines as building blocks. These macrocycles are closely related to porphyrins (Fig. 1a,b) but are much easier to synthesize, and metallated
phthalocyanines behave catalytically much like their metallated porphyrin cousins. At first, phthalocyanines seem an odd choice of components to build nanoporous arrays. They are large planar molecules that easily stack, forming poorly soluble aggregates. In previous studies, however, McKeown and co-workers had addressed this problem and arranged zinc phthalocyanines
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into regular arrays first for phthalocyanines4 and subsequently for azaphthalocyanines5. This was achieved by introducing peripheral phenyl ethers eight of them per molecule as side groups that lie out of plane and prevent stacking. The arrays consist of stacked cubes, each filled with solvent molecules and delimited by phthalocyanines acting as faces. The corners of the cubes contain small openings about 4 in diameter forming small solvent-filled cavities that interconnect with the remarkably large solvent-filled voids defined by each cube 8 nm3 each. Why do the arrays form? They are examples of clathrates molecular crystals that trap a second type of molecule in this case, solvent. In these zinc phthalocyanine arrays, the clathrates were held together solely by van der Waals forces. This cube geometry, discovered serendipitously, obviously represented a minimum energy structure. McKeown and colleagues had shown that the solvent (mainly methanol) present initially could be exchanged for a variety of other solvents (including water) without loss of crystallinity. Nevertheless, the presence of solvent was essential, and removing it as opposed to exchanging it caused a loss of porosity, a loss of crystallinity, and therefore a loss of information about the nanoscale structure. Now, McKeown et al. have replaced Zn(ii) by Fe(ii) and neatly resolved the array stability problem by introducing molecular tie bars (linear ligands such as 4,4-bipyridine). The tie bars span the cavities between neighbouring cubes (separated by roughly 11 ) and coordinate to the iron centres of two adjacent faces, keeping them linked together (Fig. 1c). With these tie bars in place, the assemblies retain their structure on removal of solvent. This is directly characterized by single-crystal X-ray crystallographic structural measurements, and indirectly by the N2 adsorption measurements of the arrays microporosity and internal surface area. McKeown and co-workers point out that the tie bars could, in principle, also play the role of activating ligands in catalysis applications. The X-ray crystallographic measurements (carried out under a nitrogen atmosphere) show that the second iron axial coordination site is occupied not by a second tie bar, but instead by N2. It is therefore reasonable to assume that exposure to air would lead to coordination of O2. With the synthesis of these remarkable haem-like arrays demonstrated, the next step will clearly be to assess their catalytic competency. It will be interesting to see to what extent the arrays can mimic the activity of various types of cytochrome P450. Can the catalytic activity and reaction selectivity be modulated by changing the identity of the tie bars? Will the catalytic chemistry be limited to comparatively small substrates, given the small portals between cubes? Or will the portals prove to be sufficiently flexible to allow large molecules to reach reactive metal sites? Will the site-isolation inherent to the array structure lead to unusually high turnover numbers and exceptional catalyst longevity? And, will the solid-state nature of the catalytic arrays allow for recovery and reuse? Finally, will other kinds of catalytic chemistry be accessible based on available ruthenium and cobalt analogues3 of the ironcontaining nanoporous structures?
Joseph T. Hupp is in the Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA, and in the Materials Science Division, Argonne National Laboratory, Argonne, Illinois 60439, USA. e-mail: j-hupp@northwestern.edu References
1. Collman, J. P., Boulatov, R., Sunderland, C. J. & Fu, L. Chem. Rev. 104, 561588 (2004). 2. Groves, J. T. Proc. Natl Acad. Sci. USA 100, 35693574 (2003). 3. Bezzu, C. G., Helliwell, M., Warren, J. E., Allan, D. R. & McKeown, N. B. Science 327, 16271630 (2010). 4. McKeown, N. B. et al. Angew. Chem. Int. Ed. 44, 75467549 (2005). 5. Makhseed, S. et al. Chem. Eur. J. 14, 48104815 (2008).
DYNAMIC COVALENt CHEMIStRY
Catalysing dynamic libraries

Benjamin L. Miller
The composition of dynamic small-molecule libraries can be biased by the addition of a target compound such as a protein that binds selectively to one of the components in the mixture. The chemistry of the library must, however, be compatible with the target and it has now been shown that aniline-catalysed exchange of acylhydrazones fits the bill.
lthough laboratory applications of processes centred on the principle of Darwinian evolution are now commonplace for nucleic acid and peptide biopolymers, it is only recently that researchers have brought molecular evolution to bear on organic compounds. One example of this is dynamic combinatorial chemistry (or dynamic covalent chemistry, both abbreviated DCC), a conceptual framework whereby collections of molecules (dynamic combinatorial libraries, or DCLs) are generated under equilibrating conditions and allowed to undergo evolution as a function of some thermodynamic selection pressure1. In many experiments, the selection pressure is binding affinity for a small-molecule guest or a biopolymer host of biomedical relevance.
The exchange reactions used to provide mixture equilibration and evolution in DCC are in some ways the antithesis of modern synthetic chemistry. Ideally, they are completely reversible under certain conditions, but irreversible under others, and chemoselective (the reaction only occurs between specific functional groups) but otherwise not influenced by compound structure. If the goal of a DCC experiment is the selection of compounds that bind to a biopolymer target such as an oligonucleotide or protein, an even more stringent set of constraints must be considered, because the exchange reaction has to operate under conditions compatible with the biological target. As one might imagine, the list of transformations satisfying all of these criteria
is limited. This represents a problem for the field, as constraints on reaction diversity concomitantly limit the structural diversity accessible to DCLs. In arguably the earliest example of a biologically targeted DCL, Venton and colleagues showed that a biological catalyst (the enzyme thermolysin) could be employed to equilibrate peptide libraries targeting antibody binding 2. Much like chemical catalysis has transformed modern synthetic chemistry, we are now beginning to learn that non-biological catalysts can also extend the range of chemistry accessible to DCC, and make it applicable to problems in biomolecule-targeted library selection. Now, writing in Nature Chemistry, Greaney, Campopiano and co-workers show 3 that
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N H N Cl R2 NO2
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Figure 1 | An aniline-catalysed acylhydrazone dynamic cominatorial library and the influence of protein targets on its composition. Two different isoforms of GST lead to amplification of different members of the dynamic library, whereas bovine serum albumin has no effect.
a catalysed version of acylhydrazone formation enables formation of a DCL under biopolymer-friendly conditions, thereby providing a new strategy for the generation and screening of protein-targeted DCLs. Introduced in the context of DCC by the Sanders group4 and studied by them extensively, acylhydrazone formation has many of the characteristics of an ideal exchange reaction. The aldehyde and hydrazide components are easily obtainable, the reaction is readily reversible and chemoselective, and the exchange process can be halted by simply changing the pH of the solution. Unfortunately, the pH range at which acylhydrazone exchange occurs rapidly is inhospitable to most protein targets. Pointing the way towards a solution to this problem, Dawson and colleagues reported in 2006 that aniline could be employed as a convenient catalyst for the reaction, with 10 mM aniline providing as much as a 70-fold rate enhancement on the reaction of a 1 mM peptide substrate in a pH 5.7 buffer 5. This work paved the way for further studies by the Dawson group on the application of the reaction to the preparation of exchangeable chemical linkers for protein enrichment6, as well as to the library studies described here. Greaney and colleagues began their efforts by generating a small library of acylhydrazones from one aldehyde, chosen based on its structural resemblance to a known substrate of glutathione-S-transferase (GST), and 10 hydrazides. This library required five days to reach equilibrium in the absence of a catalyst. In contrast, incorporating aniline into the mixture accelerated the system dramatically, providing a fully equilibrated library in six hours in a pH 6.2 buffer. Control experiments (varying starting conditions) verified that the system
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had reached a true equilibrium. With these data in hand, it was demonstrated that anilinecatalysed library equilibration in the presence of two different GST enzymes (hGST P1-1, a human isoform of interest as a potential drug target in reducing drug resistance to chemotherapy, and SjGST, an isoform from the helminth worm) resulted in amplification of isoform-selective binders (Fig. 1). After halting equilibration by raising the pH to 8.0, HPLC analysis of the mixture showed that hGST P1-1 and SjGST had selected distinctly different library members. In contrast, the control protein bovine serum albumin did not alter the composition of the library in comparison with that obtained for the protein-free system. Moreover, incorporation of a glutathione moiety into the library enhanced the solubility of its members, and led to the selection of compounds with significant binding ability. Interestingly, a catalytically inactive SjGST mutant selected the same library member as its active counterpart, confirming that the catalytic activity of the enzyme was not critical to the selection process. Subsequent binding studies confirmed that the selected compounds were indeed the most potent members of the library. Although the libraries examined in this study are of modest size, and thus could have been screened using standard parallel techniques, the dual strengths of DCC are that it (1) enables researchers to rapidly generate and screen large numbers of chemical entities with a minimum of resources (or effort), and (2) as one moves from simple dimeric compounds to oligomers and macrocycles one can begin to identify surprising structures that would otherwise be difficult to access in the laboratory. We can anticipate that catalysed
acylhydrazone exchange will now find utility in both these areas. It is worth noting that even with a relatively small library it was possible, nonetheless, to identify compounds with significant selectivity for specific GST isoforms. The success of Greaney and co-workers in applying Dawsons simple organic catalyst to acylhydrazone equilibration for nonpeptide library evolution targeting GST will hopefully provide encouragement for researchers engaged in the search for catalysts for other reactions, thus expanding the chemical repertoire of DCC. Indeed, efforts are underway to develop catalysts suitable for amide formation7, although these are not yet in a form suitable for libraries targeting biomolecules. Jeremy Knowles famously used the title of a paper to state that enzyme catalysis was not different, just better8; we can look forward to an expanded range of chemical catalysts enabling DCC to fulfil its promise of being not just different but also better.
Benjamin L. Miller is at the University of Rochester Medical Center, Rochester, New York 14642, USA. e-mail: benjamin_miller@urmc.rochester.edu References
1. 2. 3. 4. 5. 6. 7. 8. Miller, B. L. (ed.) Dynamic Combinatorial Chemistry (Wiley, 2010). Swann, P. G. et al. Biopolymers 40, 617625 (1996). Bhat, V. T. et al. Nature Chem. 2, 490497 (2010). Furlan, R. L. E., Ng, Y. F., Otto, S. & Sanders, J. K. M. J. Am. Chem. Soc. 123, 88768877 (2001). Dirksen, A., Dirksen, S., Hackeng, T. M. & Dawson, P. E. J. Am. Chem. Soc. 128, 1560215603 (2006). Dirksen, A., Yegneswaran, S. & Dawson, P. E. Angew. Chem. Int. Ed. 49, 20232027 (2010). Stephenson, N. A., Zhu, J., Gellman, S. H. & Stahl, S. S. J. Am. Chem. Soc. 131, 1000310008 (2009). Knowles, J. R. Nature 350, 121124 (1991).
Published online: 16 May 2010
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tOPOLOGICAL CRYStAL CHEMIStRY
Polycatenation weaves a 3D web

Mechanical linking of small cage structures leads to a type of metalorganic framework with an architecture topologically distinct from those constructed so far.
Davide M. Proserpio
etalorganic frameworks (MOFs) and porous coordination polymers are promising materials for a variety of applications, including sorption, selection, catalysis, sensing or microelectronics, and the efforts of many research groups worldwide have produced numerous extended systems of ever growing structural complexity 1. A mathematical (topological) description of the structure of these complex systems is necessary to achieve a correct classification and thus help to clarify the fundamental relationship between structure and properties. Indeed, new topological configurations are continuously discovered, including mechanically linked arrays derived from the simplest two-ring link, the Hopf link (Fig. 1). Hopf links are well known to the chemistry community as they appear in catenanes compounds that consist of molecular rings held together by mechanical bonds2. A recent review 3 suggested the extension of catenation to create infinite objects in one, two or even three dimensions by the formation of several Hopf links. At the time, however, no real examples had actually been observed. It is only recently that a onedimensional (1D) [n]-catenane (Fig. 1a) has been observed in the realm of coordination polymers4, and no examples extending into two or three dimensions have appeared until now. Writing in this issue of Nature Chemistry, Can-Zhong Lu and co-workers5 describe the exciting discovery of a coordination material in which the catenation of adamantane-like molecular cages considered to be a zerodimensional (0D) building block extends in three directions to form a three-dimensional (3D) polycatenated architecture. They also show that two such identical extended structures interpenetrate one another in the final structure (Fig. 1b). Indeed, whereas interpenetration is frequently found in coordination networks, it is exceptional to observe structures based on polycatenated molecular basic motifs3,6. The unique polycatenated and interpenetrated array described by Lu and coworkers is obtained thanks to the templating effect of the polyoxometalate counter-anions employed in the synthesis. The very existence of the structure, however, suggests that the use of mechanically interlinked cages as
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0D 0D + 0D 3D pcu POLYCATENATION 3D + 3D 3D pcu twofold INTERPENETRATION
Cd(imidazolate)2 INTERPENETRATION 3D + 3D 3D dia twofold
Figure 1 | Interpenetrated and polycatenated arrays. a, The Hopf link (left) is the basic unit of inextricable entanglement. Multiple Hopf links (right) result in an [n]-catenane and an increase in dimensionality. b, Polycatenation of 0D cages results in a 3D octahedral array of cages. In the final structure, two of these octahedral arrays (shown in blue and red) are interpenetrated. c, Cd(imidazolate)2 is an example of the type of MOF more commonly observed in which there is only interpenetration, and precisely twofold dia. This MOF is formed between cadmium ions acting as nodes and the organic imidazolate ligands acting as bidentate linkers.
building blocks instead of the traditional use of metal ions as nodes coordinatively bound to polydentate organic linkers represents a new synthetic strategy to obtain interesting MOFs. A few words on the topology of the entanglements are necessary here. The Hopf link is the basic unit defining an inextricable entanglement the only way to separate the links is by breaking one of the rings.
Complexity arises when one considers the dimensionality of the starting building blocks versus that of the resulting final architecture. Polycatenation is defined by an increase in the dimensionality of the final architecture over the dimensionality of the building blocks. On the contrary, in interpenetration there is no change in dimensionality. This distinction is therefore truly topological rather than just semantic7 (Fig. 2).
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supramolecular interactions in particular hydrogen bonding as important building factors of solid-state architectures. Here, if we also consider secondary weak interactions, then the independent motifs become connected and a different entanglement arises. How can such complex topologies be detected? In the past, the crystallographer needed great experience in 3D visualization and the help of ball-and-stick models to explain these complicated arrangements. Thankfully, modern tailored software allows us to compute, detect and classify entanglements in periodic structures9,10. Such computer methods give us much better design capabilities for new extended architectures. The results of Lu and co-workers demonstrate that, provided it is theoretically possible, almost any even bizarre entanglement can be realized in nature. First, however, it is important to thoroughly explore the relationships between the intricate subarchitectures to identify possible pathways for their synthesis.
Davide M. Proserpio is in the Department of Structural Chemistry DCSSI, Universit degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy. e-mail: davide.proserpio@unimi.it References
1. Long, J. R. & Yaghi, O. M. Chem. Soc. Rev. 38, 12131214 (2009). 2. Fang, L. et al. Chem. Soc. Rev. 39, 1729 (2010). 3. Carlucci, L., Ciani, G. & Proserpio, D. M. Coord. Chem. Rev. 246, 247289 (2003). 4. Jin, C. M., Lu, H., Wu, L. Y. & Huang, J. Chem. Commun. 50395041 (2006). 5. Kuang, X. et al. Nature Chem. 2, 461465 (2010). 6. Blatov, V. A., Carlucci, L., Ciani, G. & Proserpio, D. M. CrystEngComm 6, 377395 (2004). 7. Francl, M. Nature Chem. 1, 334335 (2009). 8. OKeeffe, M., Peskov, M. A., Ramsden, S. J. & Yaghi, O. M. Acc. Chem. Res. 30, 17821789 (2008). 9. Blatov, V. A. IUCr CompComm Newsletter 7, 438 (2006). 10. www.topos.ssu.samara.ru/starting.html
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Figure 2 | Topologically distinct entanglements of hexagonal layers. Two different modes of polycatenation are shown, which both result in an increase in dimensionality, versus interpenetration in which the dimensionality remains the same.
By way of example, [n]-catenanes are the simplest example of polycatenation (0D + 0D 1D), whereas the structure of Cd(imidazolate)2 is an example of a twofold interpenetrated6 diamondoid (called dia) network (3D + 3D 3D) (see Figs 1 and 2). A further criterion for polycatenation is that, unlike interpenetration, each distinct building block is never interlaced with all the others in the array. So, in structures such as Cd(imidazolate)2, we can define a degree of interpenetration, because there is always a finite number of interpenetrated components. This is impossible for polycatenated arrays where there are an infinite number of entangled components, as shown in Fig. 2 for hexagonal layers3. Starting from simple rings, it is perfectly possible to imagine structures more complex than the [n]-catenane chains in which each
ring is linked to just two adjacent rings and obtain something two-dimensional (2D) akin to medieval chain-mail2,3. If the finite building block is a 3D cage it is possible to extend the catenation in three directions, as in the compound reported by Lu and co-workers. Here, each cage links to six other equivalent cages, giving rise to a type of octahedral six-coordination that is the basic node of the primitive cubic net (called pcu according to the modern nomenclature for nets)8 (Fig. 1). It is important to keep in mind that as is usual in solid-state chemistry such descriptions depend on the types of interaction that are being considered. If we take into account only the strongest interactions, such as covalent bonds, we describe the polycatenated and interpenetrated array as above. In general, topological descriptions may consider
MECHANOCHEMIStRY
Forcing a molecules hand

S. karthikeyan and Rint P. Sijbesma
Ultrasound can be used to control molecular processes as delicate as rotation around a single carboncarbon bond.
ug of War is a game that tests the strength of two teams pulling on a rope in opposite directions. If it is being played on a molecular scale, the game is known as polymer mechanochemistry, and the interest is not so much in the muscle power of the contestants as in the strength of the rope and its fate under stress. Writing in the Journal of the American Chemical
Society, Bielawski and co-workers1 have brought this increasingly popular game to a higher level of sophistication by showing that pulling on molecular ropes may be used to promote racemization of chiral molecules. In the past, chemists have focused almost exclusively on the use of light or heat to bring about chemical reactions. Promoting reactions with force is an alternative that is
much less popular, although the principle is well established, particularly when the transformation is simply a matter of breaking bonds in the main chain of a polymer mastication of rubber to reduce its molecular weight is an example of a bond breaking reaction that is widely used in industry. Recently there has been a flurry of reports that show how mechanical forces applied
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to polymer chains can be used for far more subtle manipulation of chemical bonds. Polymer mechanochemistry has become a burgeoning field that uses mechanical forces to bias reaction pathways2, to change the colour of materials3, to trigger multiple reactions in a single polymer chain4, and to selectively break weak coordinate bonds to activate dormant catalysts5. Recent theoretical work has helped to create a much better understanding of mechanical force as a unique stimulus for chemical reactions3,6. Chemists have begun to work in this area with renewed effort because of its potential applications in self-healing polymers, molecular strain gauges and controlled drug release, and because they recognize the possibility that it will provide greater understanding of the mechanochemical transduction mechanisms in biological systems. Bielawski and co-workers have now elegantly shown that mechanical force can be used to surmount the high energy barrier associated with rotation about the CC single bond in binaphthyl derivatives and thus convert one mirror image of this molecule to the other (Fig. 1). These mirrorimage molecules are called atropisomers stereoisomers resulting from the hindered rotation around a CC single bond. The barrier for CC bond rotation in binaphthyl derivatives is high (~30 kcal mol1), which makes their racemization at ambient temperatures extremely slow. In fact, even at 195 C, the half-life (t1/2) of the parent binaphthol molecule is as long as 4.5 hours (ref. 7). How did the team, led by Texas-based chemist Chris Bielawski, manage to isomerize these stable chiral molecules? One of the most efficient ways to exert pulling forces on a molecule is to use the intense flow fields around collapsing cavitation bubbles in sonicated solutions. For effective transfer of the mechanical force to the reactive unit, it is essential to functionalize it with polymer chains. Therefore, Bielawski and co-workers appended poly(methyl acrylate) chains with total molecular weights between 10 and 100 kDa to binaphthyl derivatives. When the polymeric (S)-binaphthyl derivative (with Mn = 98.7 kDa) was sonicated in acetonitrile solution, circular dichroism spectroscopy showed that more than 95% of the derivative had racemized after 24 hours. As a measure of the relative efficiency of the mechanical force, heating of the same (S)-binaphthylfunctionalized polymer was also investigated, but heating at 250 C for 72 hours gave no change in the intensity of the circular dichroism signal. The mechanical action of ultrasonication is always accompanied by thermal effects, and the heating effect of collapsing cavitation
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Figure 1 | Racemization of binaphthyl-based polymers with ultrasound. Starting with the (S)-configured binaphthyl polymer, ultrasonic irradiation leads to rapid racemization, which may occur through one of the planar transition structures shown.
bubbles can be strong. Therefore, control experiments that establish the contribution of heating are important. To this end, ultrasonication experiments were performed on binaphthyl derivatives without the attached polymer chains. In these experiments, no racemization was observed. Further convincing evidence for the mechanochemical origin of the racemization comes from the molecular-weight dependence of the racemization rate observed for binaphthyl derivatives attached to polymer chains with Mn varying from 10 to 100 kDa. The ultrasoundinduced isomerization shows a limiting molecular weight (between 25 and 50 kDa), below which no change was observed. The present work raises interesting questions concerning the pathway by which the stereoisomers are interconverted. The two stereoisomers are stable because the steric bulk of the substituents adjacent to the biaryl bond prevents free rotation. Isomerization of the binaphthyl may therefore proceed by passage of 2,8- and 2,8 substituents (the socalled anti route) or 2,2- and 8,8 substituents (the so-called syn route)7,8 (Fig. 1). Detailed theoretical studies on thermal racemization pathways of 1,1-binaphthyl analogues suggest that the most favourable pathway proceeds through the centrosymmetric anti transition state, but this is favoured over the syn route by only 4 kcal mol1. Does the mechanically facilitated reaction follow the preferred thermal reaction pathway, as the authors propose? Investigating this question may reveal unexpected complexities. It is, for instance, imaginable that the favoured pathway has several transition states and includes mechanically as well as thermally surmounted barriers. Given the fact that
applied mechanical forces change the potential energy surface in a direction-dependent manner, the question can be answered conclusively only with detailed calculations using methods specifically developed to study mechanochemical reactions2,6. The importance of the work lies in showing the path to selectivity to others who consider entering the fascinating field of mechanochemistry. Although polymer scission using ultrasound has been used for many decades, the use of mechanical forces to perform useful transformations and applications is still in its infancy. In the near future, efforts to use mechanochemistry productively will undoubtedly increase, and selectivity will be the focus of attention. Bielawski and co-workers have shown that the seemingly untamed force of ultrasound can be used to control a process as simple and fundamental as rotation about a CC single bond.
S. Karthikeyan and Rint P. Sijbesma are in the Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. e-mail: k.sivasubramanian@tue.nl; r.p.sijbesma@tue.nl References
1. 2. 3. 4. 5. 6. 7. 8. Wiggins, K. M. et al. J. Am. Chem. Soc. 132, 32563257 (2010). Hickenboth, C. R. et al. Nature 446, 423427 (2007). Davis, D. A. et al. Nature 459, 6872 (2009). Lenhardt, J. M., Black, A. L. & Craig, S. L. J. Am. Chem. Soc. 131, 1081810819 (2009). Piermattei, A., Karthikeyan, S. & Sijbesma, R. P. Nature Chem. 1, 133137 (2009). Ribas-Arino, J., Shiga, M. & Marx, D. Angew. Chem. Int. Ed. 48, 41904193 (2009). Meca, L., Reha, D. & Havlas, Z. J. Org. Chem. 68, 56775680 (2003). Kranz, M., Clark, T. & von Rague Schleyer, P. J. Org. Chem. 58, 33173325 (1993).
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PRESERVAtION PROCESSES
Protein under glass

Proteins are typically preserved in the form of dehydrated powders to avoid their degradation or the growth of microbes that can occur in solution. Such powders are typically obtained by freeze-drying, but some proteins can be damaged during the process. Now, using an organic solvent (decanol) as drying agent, David Needham and coworkers at Duke University in the USA have successfully dehydrated a protein (lysozyme) to form beads of controllable size through a simple glassification procedure (pictured; Biophys. J. 98, 10751084; 2010) In aqueous solutions, biological molecules are closely surrounded by hydration water molecules that are more difficult to remove than those of the bulk solution, and which keep the molecules apart. When small droplets of an aqueous lysozyme solution were structure. On re-hydration, the lysozyme recovered most of its activity. Using a packing model, the researchers determined the level of protein hydration, and therefore the separation distance between the lysozyme molecules, from the water activity measured in decanol (its concentration in the non-ideal mixture). This means that by adjusting the water activity in the drying solvent, they were able to control the final protein concentration, and thus the size of the resulting glassy beads. This drying method was also faster and cheaper than the freeze-drying process, showing great promise for biological applications.
ANNE PICHON The original version of this story first appeared on the Research Highlights section of the Nature Chemistry website.
added into a decanol solution, all the bulk and hydration water molecules dissolved into the organic solvent within minutes. This process was too fast for the protein to crystallize and instead it arranged into microbeads with a glassy, amorphous
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2010 The Biophysical socieTy
perspective
Published online: 16 may 2010|doi: 10.1038/nchem.654
robust dynamics
hexiang deng1, mark a. olson2, J. Fraser stoddart2* and omar m. yaghi1*
Although metalorganic frameworks are extensive in number and have found widespread applications, there remains a need to add complexity to their structures in a controlled manner. It is inevitable that frameworks capable of dynamics will be required. However, as in other extended structures, when they are flexible, they fail. We propose that mechanically interlocked molecules be inserted covalently into the rigid framework backbone such that they are mounted as integrated components, capable of dynamics, without compromising the fidelity of the entire system. We have coined the term robust dynamics to describe constructs where the repeated dynamics of one entity does not affect the integrity of any others linked to it. The implication of this concept for dynamic molecules, whose performance has the disadvantages of random motion, is to bring them to a standstill in three-dimensional extended structures and thus significantly enhance their order, and ultimately their coherence and performance.
titching molecular building blocks into extended frameworks using strong bonds reticular chemistry is one of the most widely investigated areas in chemistry today 1. A library of organic and inorganic building blocks has been used to build a large number of structures, named metalorganic frameworks2 (MOFs). Generally, the MOF construct is based on the principle of linking metal-oxide joints with organic struts as illustrated in Fig. 1. This process has been repeated over and over again in various different chemical contexts to afford extensive classes of porous MOFs with a diversity and multiplicity previously unknown in the realm of artificial materials. The rigidity and directionality of the joints and struts ensure the MOFs architectural stability and therefore permanent porosity: both are vitally important for their applications in catalysis, gas storage and separation3. It is these very same features, however, that rob MOFs of their dynamics and give rise to the question: how can we preserve the important characteristics and properties of MOFs, while accessing the dynamics that could provide the key to enhancing their functions? An obvious strategy is to make flexible frameworks from pliable struts4. Another strategy is to use multi-interpenetrating
frameworks, wherein one framework shifts with respect to the others, thereby closing or opening the pores5. Both of these strategies are severely limited because frameworks that flex back-and-forth fail when subjected to further repeated dynamics, and interpenetrating frameworks are highly dependent on the uncontrollable behaviour of guests that fill their pores. In fact, this problem is not unique to MOFs it is understood that structural failure is to be expected when the backbones of polymers and other extended structures are subjected to repeated dynamics. How then do we overcome the challenge of introducing dynamics into MOFs and, for that matter, other extended chemical structures, while retaining their robustness? To answer this question, we turn our attention to another field of endeavour that has been progressing at an equally rapid pace of late that of artificial molecular switches and machines6. In one of their most highly studied manifestations, they are composed (Fig. 2ad) of circular, and sometimes also linear, components that are linked together mechanically 7. Given the use of templation8 in their synthesis, they are capable of elaborate and repeated dynamics,
90
+
90 Zn4O
Six-way connection
Triphenylene unit
90
O-C-O Claws IRMOF-16 Organic strut Metal-oxide joint Framework backbone
Figure 1 | A rare view into the construction of metalorganic frameworks (MOFs). Herein the phenylene rings are bonded together by pivot joints to make the struts that link six-way tetra-zinc oxide clusters, likened to ball-joints, to form a MOF-5-type structure, previously named IRMOF-16. The carboxyl units act as claws to keep the zinc centres in invariant positions and disallow any major structural perturbations. These features, when combined with the perpendicular orientations of all the claws, provide a glimpse into the reason for the well-known architectural stability of this MOF construct. The phenylene rings (red and black), OCO claws (grey and blue), and zinc oxide joint (Zn4O, pink). Center for Reticular Chemistry, Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA, 2Center for the Chemistry of Integrated Systems, Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA. e-mail: stoddart@northwestern.edu; yaghi@chem.ucla.edu
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of MOFs will yield materials that are intrinsically robust and rigid, yet dynamic a property we term robust dynamics. The idea is that molecular switches and machines will be incorporated symmetrically into the struts of MOFs to graft dynamics onto their frameworks. In such materials, the repeated relative motions of the mechanically interlocked components will not affect the robustness of the framework backbone because their relative movements do not subject their constituent covalent bonds to undue stress and hence breakage: only non-covalent bonds are being ruptured and remade. In this way, the materials fidelity and longevity will be overwhelmingly enhanced. It is our opinion that a system capable of robust dynamics must inevitably encompass a rigid framework into which flexible units (Fig. 2ad) are inserted. The result of this thought process is illustrated in Fig. 2eh, using the well-known MOF-5-type structure10 and a bistable [2]catenane6,7. Here, we detail some of the underlying principles and thinking that now need to go into the blending of these two types of structural architecture, while emphasising the vast potential inherent in this union. The process of designing a MOF structure is not unlike how we design and construct macroscopic objects such as bridges and skyscrapers; we link together girders and junctions of various shapes according to a blueprint using fasteners and rivets. The way the construction is done on the ngstrm or nanometre scale is to employ the chemical architects blueprint, which is a net that is, a 3D array of points joined together by links, ideally related to each other by symmetry 11. There is virtually a limitless number of net topologies of widely varying connectivities: we select the simplest and most symmetrical as feasible targets for synthesis. One such net is the primitive cubic topology. It is composed of two-connectors (struts or girders) that link (riveting) the vertices (junctions) leading to six-way connectivity. In our MOF-5type structure, the intersecting points are joints with octahedral geometry. To make a MOF based on this topology, we take the phenylene ring of 1,4-benzenedicarboxylate as a two-way connector, and the zinc cluster as the six-coordinated unit and together they impose (Fig. 1) a primitive cubic structure on the MOF. Using related links, the same strategy can be applied to produce12 yet more MOFs based on the same net (isoreticular) with predetermined pore sizes and shapes. Ideally, the joints are rigid entities with well-defined geometries, which impart directionality and control over the resulting structure. By predetermining the geometry of the joint, one dictates the connectivity of the underlying net. The key to making rigid joints is to choose clusters that have an intrinsically 3D structure that is entirely composed of common-sized rings. In the case of these MOFs, the Zn4O(CO2)6 joints may be viewed as being made up of six 6-membered rings of Zn2OCO2 composition that are sharing edges and are perpendicular to adjacent rings while also positioned (Fig. 1) opposite to other such rings. Indeed, when one considers this arrangement and the fact that the rings act as OCO claws, which hold the zinc atoms in position, then it becomes apparent why this construction does not shear. Once reaction conditions are identified for forming a joint, a small number of high-symmetry nets can be targeted, employing struts of the appropriate shapes and symmetries. We note that the use of joints made up of one metal atom, where no common rings exist, leads to flexing motions of large amplitude that inevitably destroy the framework, and the variable connectivity of such metal atoms preclude making frameworks by design. By contrast, metal-cluster joints ensure an invariant connectivity and therefore allow the design of frameworks, whereas the presence of multiple six-membered rings within such joints imparts that crucial but slight flexing at those joints ultimately, leading to robust structures of high fidelity. With all these considerations in mind, the stage is now set for introducing bistable catenanes and rotaxanes as two-way
Struts
MOFs
Figure 2 | Illustrative examples of how elaborate units can be mounted onto the organic struts to introduce complexity and dynamics into MOFs. The struts (a to d) are linked by tetra-zinc oxide centres to produce MOFs (e to h) in which the metrics of the struts and their functionality can be varied to give highly ordered 3D systems with controlled complexity and of vast openness such as to allow access to, and dynamics at, the mounted units. The polyether oxygen (pink), carbon (black) and phenylene (red) form the crown ether receptor/template (a and e), which forms pseudorotaxanes with Paraquat dication (b and f), degenerate catenanes with a cyclophane (blue) containing two Paraquat units linked by phenylene rings interlocking the crown ether (c and g), and a cyclophane (blue) containing a dimethyl diazapyrenium unit (purple) in addition to a Paraquat unit linked by phenylene rings interlocking the crown ether (d and h).
yet so far, for the most part, in an incoherent manner in solution or in condensed phases9. The problem with their present design is that they lack the rigid backbone that could provide a platform for their strategic and precise placement in two- (2D) or three-dimensional (3D) space so that they can express their dynamics namely, the coherent switching between their mounted components. During a switching process, only weak non-covalent bonds get broken and reformed again in a wholly reversible and highly controllable fashion7,9. We therefore propose that coupling the dynamics of molecular switches and machines with the rigid structures
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connectors into exactly the same type of net that we just described for MOF-5-type structures. For mechanically interlocked molecules (MIMs), known13,14 as catenanes (Lat. Catena = chain) and rotaxanes (Lat. Rota = wheel, axis = axle) to behave as molecular switches and machines they must incorporate two characteristic features6,7,1517. One is that their components two mechanically interlocked rings (Fig. 2) in the case of a bistable [2]catenane, and a ring encircling a dumbbell in the case of a bistable [2]rotaxane must communicate with each other by means of intramolecular forces that can be modulated subsequently 6,7,1317 with chemicals (for example, pH change), electricity or light, that is, redox change. The other feature is that, as a result of some judiciously chosen constitutional dissymmetry, as well as the required orthogonality to stimuli associated with the two different recognitions sites the more interactive of which can be switched OFF and ON reversibly with complete fidelity in the presence of the weaker site. By means of this modulation, bistable MIMs can be raised from a ground to a metastable state, such that there are two translationally isomeric13 forms expressing their bistability 9. The first feature is associated intimately with the efficient synthesis of bistable MIMs by protocols that rely on templation8 that is, the use of molecular recognition processes involving a steadily increasing number of intermolecular forces, which become intramolecular on the formation of a mechanical bond7, a factor that guides the assembly of the components of the bistable MIMs. It is the very fact that the non-covalent bonding, which is introduced incrementally in a step-wise manner into these bistable MIMs each time a covalent bond is formed, lives on inside the molecules afterwards, which endows them with their unique properties. The situation is a true chicken-and-egg one: without the progressive build-up of non-covalent bonds during templation8, the outcome of a synthesis will be no better than statistical in nature that is, the yields will be miniscule and the product will contain little or no information. One attribute of their construction feeds off the other to the extent that, if reversibility is introduced into the covalent bond-forming steps, then proofreading and error checking will often lead18 to all but quantitative yields. In pursuit of systems expressing functions, there has been a drive to self-assemble them (for example, as thiols on gold) on surfaces19, or to place them at interfaces by self-organization for example, by LangmuirBlodgett transfer of monolayers20 of amphiphilic MIMs to create nanoelectromechanical systems21 or molecular electronic devices22, respectively. The incoherence that characterizes the operation of artificial molecular switches and machines only finds a practical expression when they are constituted as self-assembled monolayers on surfaces19 or in molecular switch tunnel junctions at interfaces20,22,23 one molecule thick between two electrodes. Not only do these self-assembly processes and self-organizational procedures all come with a downside in the shape of disorder, which introduces blemishes into devices, the artificial machinery is also often impeded in its function and usually becomes exhausted after tens, or, at the most, hundreds, of cycles. In other words, the distribution of orientation of the active molecules within these environments leads to their drastically reduced performance. So the inevitable question arises how can we remove these blemishes and impediments and, at the same time, improve on the performance of molecular switches and machines? The act of introducing MIMs into MOFs can be likened to the building of helicopters, jet planes and rockets. These particular flying machines are all constructed around robust fuselages to which engines are attached to the top, or on the sides, or at the back, or on the bottom. By appealing to a combination of robustness and motive power, we have all but overcome the hurdle of flight, which fascinated, yet evaded human beings for centuries. By the same token, we can envisage incorporating the artificial switches
perspective
a
Figure 3 | The 2D and 3D merger of MIMs and MOFs to bring about robust dynamics. A representation of a, 2D and b, 3D MOFs incorporating units (catenated light blue and red in a for degenerate rings of blue positions and blue/purple for the non-degenerate bistable rings in b) that typically have random motions as discrete molecules, but such motions are brought to a standstill when the units are mounted covalently to the rigid framework backbone (pink spheres and grey struts). This merger between MIMs and MOFs brings order to the appended units and endows them with the ability to carry out well-defined repeated dynamics without compromising the integrity of the entire system a concept termed robust dynamics.
and machines, as part of the two-way connectors held together by cluster joints during the template-directed synthesis8 of the enabled struts. We foresee the possibility of being able to locate arrays of molecular switches and machines symmetrically and efficiently inside the 3D structures of MOFs, while retaining their inherent robustness. By virtue of the highly ordered MOF structure, its ultrahigh porosity, and facile accessibility to all its internal sites, the dynamic components of the MIMs, mounted within the extended structure provided by the MOF, will, in principle, be completely addressable and, under the right set of circumstances, behave in a coherent and reproducible manner. In essence, the random motion that plagues untethered MIMs in solution and condensed phases is curbed (Fig. 3) in MOFs. Recently, the feasibility of mounting MIMs within MOFs has become evident from the successful introduction of a strut (Fig. 2a) into a MOF (Fig. 2e) and the formation of its [2]pseudorotaxane24-27 MOF (Fig. 2f). Although MOFs incorporating the degenerate28 and the non-degenerate29 [2]catenane (Fig. 2c,d and g,h) have not been synthesized as yet, a 2D MOF containing the strut in Fig. 2c, as illustrated in Fig. 3a, has already been reported30. In these extended structures (Fig. 2e,f), long organic struts (~2 nm) incorporating 34- and 36-membered polyether rings27 were used to build new MOFs (MOF-1001 and MOF-1002) whose structures are based on that of the MOF-5-type. The polyether chains are known to be highly dynamic in their free state, where they are folded in on themselves in the absence of guests, but are capable of readily unfolding to accommodate guests in their interior. Accordingly, this dynamic behaviour is also present in the new MOFs, in which the polyether units are found to be disordered in the crystals. There is, however, yet another reason for the macrocycles to be disordered in MOF-1001 and MOF-1002: the presence of planar chirality. As the method of their synthesis did not permit any control over the handedness of the macrocycles, enantiomeric forms of them are presumably distributed in three dimensions throughout the crystals. In the future, however, it is going to be possible
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perspective
Sorting domain
Coverage domain
Active domain
Figure 4 | The sorting, coverage and active domains of MOFs. The three porous domains that the new MOFs (Figs 2 and 3) combine when they recognize and bind incoming substrates (guests). These domains are principally characterized by use of the pore opening to sort guests by shape and size selection (sorting domain), the internal adsorption sites to compact guests (coverage domain), and the crown ether receptors designed to bind guests in a stereoelectronically selective manner (active domain).
to employ asymmetric catalysis31 to produce homochiral MOFs with receptors that incorporate planar chirality. Such dramatically new materials, in which the chiral receptor sites for the stereoselective docking of enantiomers are arranged precisely in space and are easily accessible to racemic analytes in a mobile phase, could revolutionize the production of chiral stationary phases for highperformance liquid chromatography for the efficient separation of enantiomeric compounds. Remarkably, when MOF-1001 (Fig. 2e) is exposed to Paraquat dications in acetonitrile solutions, its polyether loops unfold and bind to the Paraquat guests in a stereoelectronically specific manner. The process can be repeated many times with the full preservation of the MOF backbone structure and without leaving any imprint on it. We attribute this framework fidelity to the fact that the only segments of the MOF structure that are flexible are the polyether loops, and none of their dynamics require any alteration to the metrics of the framework. It is the ideal construct because the rigidity of the framework allows the permanent openness of the structure so that guests may move in and out without obstruction and the large interstices provide sufficient space for the polyether loops to fold and unfold, and to do so independently of the framework. Therefore, the key to achieving robust dynamics in extended systems is only present in the segment of the structure where dynamics is desirable leaving unperturbed the remainder of the structure. The stereoelectronically selective manner in which Paraquat dication is bound to the polyether loops in MOF-1001 introduces (Fig. 4) molecular recognition into porous crystals that so far have operated on either a shape/size selective or compacting capability. Further independence of the flexible units in MOFs is potentially achieved by employing mechanically interlocked components. In principle, the synthesis of such MOFs was shown30 to be feasible by the successful incorporation of degenerate, donor acceptor [2]catenanes28 into the 2D structure of MOF-1011. The layered nature of this MOF and the strong layerlayer interactions in the crystal preclude any dynamics involving the mechanically interlocked rings. Indeed, the construction of a 3D MOF structure, such as that of the MOF-5-type, would be necessary to realize the full potential of the interlocking rings dynamics (Figs 2h and 3b). Nevertheless, it is encouraging to observe that these mechanically interlocked components, which are prototypical molecular machines6, can be mounted successfully inside extended MOF systems. We believe that once the non-degenerate MOF-5-like structure is made, it will be just a matter of time before ultradense, 3D arrays
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of molecular memory based on switchable [2]catenanes make their way into state-of-the-art device settings23. Ultradense memory, however, is the tip of the iceberg, provided it can be addressed. In the fullness of time, coupling switching with the ever-increasing capabilities of carrying out recognition processes for example, microcontact printing on the surfaces and extending into the highly sophisticated interiors of these new switchable MOFs will become commonplace. In principle, MOFs with molecular machinery mounted appropriately within their extended structures would not be unlike airplanes carrying their strategically mounted propeller-driven or jet engines on robust wings. One can easily conceptualize a chemical world where chameleon-like MOF crystals can be induced under the influence of chemicals (pH change), electricity (redox change) and light to travel in solution from one environment to another. In essence, the concept of robust dynamics is not only a necessary requirement for the longevity of dynamic extended structures, but it is also a strategy for adding yet another layer of complexity to the present functioning capabilities of MOFs. The ability to build integrated systems that are capable of a complex set of functions is reminiscent of operations in the biological world. In a sense, robust dynamics is an example of how the concept of repeating dynamics, which is so prevalent in biology, can be transferred from the biological world to an artificial arena occupied by MOFs and MIMs and filled with integrated systems, without actually mimicking biology. Robust dynamics, therefore, is about concept transfer from the life sciences into the chemistry of materials. We can define32 concept transfer as adapting and applying the recognition processes, employed by living systems in achieving their forms and fulfilling their functions, to the construction of chemical systems with well-defined forms and prescribed uses. In the wake of this definition, we propose that concept transfer, rather than trying to mimic biology, is a more viable approach33 for making the next leap in the design and synthesis of useful materials. Moreover, the ordered, extended structures of MOFs and the flexibility with which they can be synthesized and functionalized with MIMs render the marriage between the two chemistries ideally suited for uncovering, testing and developing other concept transfer strategies.
references
1. Long, J. R. & Yaghi O. M. The pervasive chemistry of metal-organic frameworks. Chem. Soc. Rev. 38, 12131214 (2009). 2. Yaghi, O. M. et al. Reticular synthesis and the design of new materials. Nature 423, 705714 (2003).

3. Horike, S., Shimomura, S. & Kitagawa, S. Soft porous crystals. Nature Chem. 1, 695704 (2009). 4. Serre, C. et al. Role of solvent-host interactions that lead to very large swelling of hybrid frameworks. Science 315, 18281831 (2007). 5. Maji, T. K., Matsuda, R. & Kitagawa, S. A flexible interpenetrating coordination framework with a bimodal porous functionality. Nature Mater. 6, 142148 (2007). 6. Balzani, V., Credi, A. & Venturi, M. Molecular Devices and Machines Concepts and Perspectives for the Nanoworld (Wiley-VCH, 2008). 7. Stoddart, J. F. The chemistry of the mechanical bond. Chem. Soc. Rev. 38, 18021820 (2009). 8. Diederich, F. & Stang, P. J. (eds) Templated Organic Synthesis (Wiley-VCH, 1999). 9. Choi, J. W. et al. Ground-state equilibrium thermodynamics and switching kinetics of bistable [2]rotaxane switches in solution, polymer gels, and molecular electronic devices. Chem. Eur. J. 12, 261279 (2006). 10. Li, H., Eddaoudi, M., OKeeffe, O. & Yaghi, O. M. Design and synthesis of an exceptionally stable and highly porous metal-organic framework. Nature 402, 276279 (1999). 11. Ockwig, N., Friedrichs, O. D., OKeeffe, M. & Yaghi, O. M. Reticular chemistry: occurrence and taxonomy of nets, and grammar for the design of frameworks. Acc. Chem. Res. 38, 176182 (2005). 12. Eddaoudi, M. et al. Systematic design of pore size and functionality in metal-organic frameworks and application in methane storage. Science 295, 469472 (2002). 13. Schill, G. Catenanes, Rotaxanes and Knots (Academic Press, 1971). 14. Sauvage, J-P. & Dietrich-Buchecker, C. (eds) Molecular Catenanes, Rotaxanes and Knots: A Journey Through the World of Molecular Topology (Wiley-VCH, 1999). 15. Bissell, R. A., Cordova, E., Kaifer, A. E. & Stoddart, J. F. A chemically and electrochemically switchable molecular shuttle. Nature 369, 133137 (2004). 16. Livoreil, A., Dietrich-Buchecker, C. O. & Sauvage, J-P. Electrochemically triggered swinging of a [2]catenane. J. Am. Chem. Soc. 116, 93999400 (1994). 17. Kay, E. R., Leigh, D. A. & Zerbetto, F. Synthetic molecular motors and mechanical machines. Angew. Chem. Int. Ed. 46, 72191 (2007). 18. Chichak, K. S. et al. Molecular Borromean rings. Science 304, 13081312 (2004). 19. Klajn, R. et al. Metal nanoparticles functionalized with molecular and supramolecular switches. J. Am. Chem. Soc. 131, 42334235 (2009).
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20. Collier, C. P. et al. A [2]catenane-based solid-state electronically reconfigurable switch. Science 289, 11721175 (2000). 21. Juluri, B. K. et al. A mechanical actuator driven electrochemically by artificial molecular muscles. ACS Nano 3, 291300 (2009). 22. Luo, Y. et al. Two-dimensional molecular electronic circuits. ChemPhysChem 3, 519525 (2002). 23. Green, J. E. et al. A 160-kilobit molecular electronic memory patterned at 1011 bits per square centimetre. Nature 445, 414417 (2007). 24. Li, Q. et al. Docking in metal-organic frameworks. Science 325, 855859 (2009). 25. Kim, K. Entering the recognition domain. Nature Chem. 1, 603604 (2009). 26. Alavi, S. Selective guest docking in metal-organic framework materials. ChemPhysChem 11, 5557 (2010). 27. Zhao, Y-L. et al. Rigid strut-containing crown ethers and [2]catenanes for incorporation into metal-organic frameworks. Chem. Eur. J. 15, 1335613380 (2009). 28. Anelli, P-L. et al. Molecular meccano 1. [2]Rotaxane and a [2]catenane made to order. J. Am. Chem. Soc. 114, 193218 (1992). 29. Balzani, V. et al. Constructing molecular machinery. A chemically switchable [2]catenane. J. Am. Chem. Soc. 122, 35423543 (2000). 30. Li, Q. et al. A metal-organic framework replete with ordered donor-acceptor catenanes. Chem. Commun. 46, 380382 (2010). 31. Kandra, K., Koike, T., Endo, K. & Shibata, T. The first asymmetric Sonogashira coupling for enantioselective generation of planar chirality in paracyclophanes. Chem. Commun. 18701872 (2009). 32. Glink, P. T. & Stoddart, J. F. Concept transfer from the life sciences into materials science. Pure Appl. Chem. 70, 419424 (1998). 33. Stoddart, J. F. Thither supramolecular chemistry. Nature Chem. 1, 1415 (2009).
acknowledgements
We acknowledge the Department of Energy (BES-Separation Program), the Department of Defense (Defense Reduction Threat Agency), the Air Force Office of Scientific Research under their Multidisciplinary University Research Initiative (FA9550-07-1-0534), the Microelectronics Advanced Research Corporation and its Focus Center Research Program, the Center on Functional Engineered NanoArchitectonics, and the NSF-MRSEC Program through the Northwestern University Materials Research Science and Engineering Center for their continued support of this research.
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ARTICLES
PUBLISHED ONLINE: 2 MAY 2010 | DOI: 10.1038/NCHEM.649
Ion-triggered spring-like motion of a double helicate accompanied by anisotropic twisting

Kazuhiro Miwa, Yoshio Furusho* and Eiji Yashima*
Molecules that extend and contract under external stimuli are used to build molecular machines with nanomechanical functions. But although common in biological systems, such extension and contraction motions with helical molecules have rarely been accompanied by unidirectional twisting in synthetic systems. Here we show that sodium ions can trigger the reversible anisotropic twisting of an enantiomeric double-stranded helicate, without racemization. An optically active helicate consisting of two tetraphenol strands bridged by two spiroborate groups sandwiches a sodium ion. On removal of the central sodiumthrough addition of a cryptand [2.2.1] in solutionthe double helicate extends. Crystallographic and nuclear magnetic resonance studies reveal that the extended helicate is over twice as long as the initial molecule, and is twisted in the right-handed direction. Circular dichroism analysis suggests that the twisting doesnt affect the helicates handedness. This anisotropic extensioncontraction process is reversibly triggered by the successive addition and removal of sodium ions in solution.
he design and construction of molecular actuators that can undergo a reversible extensioncontraction motion at the molecular level triggered by chemical, electrochemical or photochemical stimulation has recently been the subject of considerable interest in the context of molecular machines116. The muscle-like extensioncontraction molecular motion has been achieved by a number of molecular devices, such as macrocycles5, rotaxanes68, oligomeric and polymeric helical systems916, and coordination solids10. In particular, helical molecules are of signicant interest as their inherently chiral structures suggest a seductive anisotropic twisting (unidirectional spring-like motion) during the extensioncontraction process. Several synthetic helical molecules and polymers exhibit such extensioncontraction motions triggered by ion binding5,9 and changes in pH12, solvent13 or temperature11,16; these helical systems have rarely undergone a unidirectional twisting motion, even though it is common in biological systems1719. Recently, Percec et al. reported one precedent using dendronized helical poly(phenylacetylene) molecules that exhibit a thermally induced anisotropic twisting16. In these biological and synthetic polymer systems, however, the resulting anisotropic twisting is controlled entirely by the homochirality of the biopolymers and by the introduction of stereocentres into the monomer units, respectively; therefore, one of the diastereomeric helices is favourably formed, leading to a unidirectional twisting motion. In contrast, the synthetic helical molecules readily racemize or take a non-helical conformation or transition state during the extensioncontraction process, which results in a bidirectional twisting motion. Helical molecules that undergo a spring-like motion accompanied by anisotropic twisting could generate a torque on microscopic objects and rotate them unidirectionally, when embedded in liquid crystals20. Ultimately, this could lead to a sophisticated unimolecular machine that can perform intelligent work. Over the course of our research on synthetic helical molecules based on oligo- and poly(m-phenylene) structures2123, we unexpectedly found that the reaction of a hexaphenol, H6L1 (1), with an equimolar amount of NaBH4 afforded a unique double-stranded helicate (DH1BNaB2.Na, Fig. 1), which consists of the two
hexaphenol strands bridged by two spiroborate groups accommodating a Na ion in the centre coordinated by eight oxygen atoms24. The structure of DH1BNaB2 predicts that the two hydroxyl groups on the two central benzene rings of each strand are not necessary for the formation of the helicate because they do not participate in the spiroborate bridges. With this in mind, we have designed and synthesized a new double-stranded helicate (DH2BNaB2) that contains a tetraphenol lacking the two central hydroxyl groups on the hexa(m-phenylene) backbone (H4L2, 2). Here we report an enantiomeric double helical molecule of DH2BNaB2 obtained by optical resolution of the racemic helices that undergoes Na ion-triggered, reversible extension contraction motion coupled with a twisting motion in one direction (right-handed).
Results and discussion

The ligand H4L2 (2) was allowed to react with an equimolar amount of NaBH4 in 1,2-dichloroethane-ethanol at 80 8C for 20 h, affording the boron complex [B2Na(L2)2]2.Na (DH2BNaB2.Na) in 22% yield (Fig. 1). Countercation exchange with N-benzyl-N,N,N-trimethylammonium bromide (BMAmm.Br2) gave DH2BNaB2.BMAmm, of which single crystals suitable for an X-ray study were grown from an acetonitrile solution. The X-ray crystallographic analysis unambiguously revealed that DH2BNaB2.BMAmm adopts a double helical structure with a pseudo-D2-symmetry, which is virtually isomorphic to that of the hexaphenol-based helicate DH1BNaB2 (Fig. 2 and Supplementary Fig. S1). The two tetraphenol strands are intertwined with each other through two spiroborate bridges, and a Na ion is embraced in the centre of the complex coordinated by four oxygen atoms of the spiroborate moieties. The two terminal benzene rings of each tetraphenol strand are twisted by approximately 2808. Interestingly, the two negatively charged spiroborate moieties are very close to each other, with a BB distance of 6.0 , as they are electrostatically attracted by the positively charged central Na ion. Electrospray ionization (ESI) mass measurements in the negative mode support the monoanionic nature of the boron helicate,
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan. * e-mail: furusho@apchem.nagoya-u.ac.jp; yashima@apchem.nagoya-u.ac.jp
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NATURE CHEMISTRY | VOL 2 | JUNE 2010 | www.nature.com/naturechemistry
2010 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY
a
t
DOI: 10.1038/NCHEM.649
ARTICLES
t tBu
Bu
tBu
tBu
Na+
Bu X
Y OH
tBu
OH
NaBH4 OH Cl(CH2)2Cl-EtOH 80 C, 20 h
O B O
O O
X Na X
X X
O B O
O O
OH
t
X Y
t
Bu
Bu
tBu t
1 H6L1: X = OH, Y = tBu 2 H4L2: X = Y = H
Bu
Bu
Bu
DH1BNaBNa+: [B2Na(H2L1)2]Na+ DH2BNaBNa+: [B2Na(L2)2]Na+
b
+ Precipitates (+)-DH2BNaB()-DMEph+ 93% d.e. DH2BNaBNa+ ()-DMEph+Br ()-DH2BNaB()-DMEph+ Filtrate 29% d.e. TBAmm+Br ()-DH2BNaBTBAmm+ 29% e.e. TBAmm+Br (+)-DH2BNaBTBAmm+ 93% e.e. Ph OH N
C12H25 Br
(()-DMEph+Br) C4H9 C4H9 + N C4H9 C4H9
Br
(TBAmm+Br )
Figure 1 | Synthesis and optical resolution. a, Synthesis of the boron helicates DH1BNaB2.Na and DH2BNaB2.Na. b, Optical resolution of DH2BNaB2.Na by a diastereomeric salt formation using ()-DMEph.Br2. The Na cation located outside the helicate moiety was exchanged with ()-DMEph in an acetonitrile solution to form a pair of diastereomeric salts, ()-DH2BNaB2.()-DMEph and ()-DH2BNaB2.()-DMEph. The former precipitated from the solution and the diastereomeric excess was determined to be 93%, whereas the latter was collected by evaporating the ltrate as a white solid with 29% d.e. Both diastereomeric salts were converted to a pair of enantiomeric salts through countercation exchange with achiral TBAmm.Br2 in acetonitrile without racemization.
()-DH2BNaB.BMAmm+ B
Ring current
~280 B Na Ring current B
()-DH1BNaB.H+ B B
Na
Figure 2 | Capped-stick representations of the crystal structures of the trinuclear boron helicates. a, Side (left) and top (right) views of the crystal structure of (+)-DH2BNaB2.BMAmm, which reveal the double-stranded helical structure of the helicate bridged by the spiroborates formed from the terminal biphenol units and the boron atoms. The t-Bu groups are located in close proximity to the benzene rings of the other strand, and their 1H NMR signals are therefore shifted upeld owing to an aromatic ring current effect. The two terminal benzene rings of each strand are twisted by 2808. b, Side (left) and top (right) views of the crystal structure of DH1BNaB2.H. Similar upeld shifts of the terminal t-Bu groups can be seen for each strand as the double-stranded helical structure of DH1BNaB2.H is virtually isomorphic to DH2BNaB2. All the hydrogen atoms, solvent molecules, and countercations are omitted for clarity.
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a
tBu
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Bu
Bu
tBu
t t
Bu
Bu
Bu
tBu
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DH2BNaB B DH2BB 1.4 1.3 1.2 1.1 (ppm)

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13.0 ()-DH2BB2(BMAmm+)2
Figure 3 | Synthesis and characterization of the dinuclear helicate. a, Schematic illustration of the removal of the central Na ion from DH2BNaB2.Na by cryptand [2.2.1]. b, 1H NMR spectra of H4L2 (2) (top), DH2BNaB2.Na (middle), and DH2 BB 2.(Na , [2.2.1])2 (bottom) in CD3CN. The 1H NMR signals of the t-Bu groups of DH2BB 22 did not show upeld shifts. c, The capped-stick representations of the crystal structure of (+)-DH2BB 22.(BMAmm)2 (side view (bottom) and top view (top)). All the t-Bu groups are pointing outwards and are therefore remote from benzene rings in this structure, which explains the lack of upeld shifts of the 1H NMR signals of the t-Bu groups as there is no ring current effect. The two terminal benzene rings of each strand are twisted by 1808, and the distance between the two boron atoms is 13.0 , indicating that the length of helicate DH2 BB 22 is extended by almost twofold. All the hydrogen atoms, solvent molecules, and countercations are omitted for clarity.
showing a strong signal due to the monovalent anion (DH2BNaB2) at m/z 1529.94 along with a minor signal due to the divalent anion (DH2BB22) at m/z 753.46 (Supplementary Fig. S2). The 1H nuclear magnetic resonance (NMR) spectrum of the complex in CD3CN also revealed the pseudo-D2-symmetric structure as determined by X-ray analysis in the solid state. The two t-Bu signals shifted upeld owing to the ring current effect of the benzene rings of the other strand, which is in good agreement with the crystal structure (Fig. 2). Furthermore, the 1H two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) experiments showed strong negative cross peaks between the phenol rings A and C (Supplementary Figs S3S7), which are attributed to the interstrand nuclear Overhauser effects (distance approximately 2.5 ). This indicates that the complex retains the double-stranded helical trinuclear structure in solution. The optical resolution of the helicate ((+)-DH2BNaB2.Na) was then carried out by diastereomeric salt formation through cation exchange with ()-N-dodecyl-N-methylephedrinium bromide (()-DMEph.Br2), which had been successfully used for the optical resolution of (+)-DH1BNaB2.Na (ref. 24; Fig. 1). On addition of a tenfold excess of ()-DMEph.Br2 to a solution of (+)-DH2BNaB2.Na in acetonitrile, ()-DH2BNaB2.()-DMEph was obtained as a white crystalline solid (the prexes () and ()
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for the helicate moieties denote the signs of the Cotton effect that is, the characteristic change in optical rotatory dispersion and/or circular dichroism (CD) in the vicinity of an absorption band of a substanceat 313 nm). Its diastereomeric excess (d.e.) was determined to be 93% on the basis of its 1H NMR spectra (Supplementary Figs S9 and S10). Evaporation of the mother liquor afforded the opposite diastereomer rich in ()-DH2BNaB2.()-DMEph with 29% d.e. The CD spectra of both diastereomers showed almost mirror image Cotton effects in their patterns in the range of 240330 nm except for the intensities because the CD signals due to the ()-DMEph moiety are negligible (Supplementary Fig. S8). The helix senses of the diastereomers were assigned to be right- and left-handed for ()-DH2BNaB2.()-DMEph and ()-DH2BNaB2. ()-DMEph, respectively, on the basis of their CD patterns relative to those of ()- and ()-DH1BNaB2.Na. The relationship between the handedness of the hexaphenol-based helicate and the CD patterns of these had been established in an X-ray single crytsallographic study (unpublished observations). The resulting diastereomeric double-stranded helicates were successfully converted to the corresponding enantiomers by exchanging the optically active ammonium cation ()-DMEph with achiral tetrabutylammonium bromide (TBAmm.Br2) (Supplementary
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13.0 [2.2.1] B Na+ B
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0 (+)-DH2BNaBTBAmm+ 200 + [2.2.1] + NaPF6 400
4 3 2 1 0 400 (105 M1 cm1)
200
250
300 350 Wavelength (nm)
Figure 4 | Anisotropic twisting motion of the helicate. a, Schematic representation of anisotropic twisting motion observed in a right-handed double helical helicate. b, CD and absorption spectra (CH3CN, 25 8C) of ()-DH2BNaB2.TBAmm (93% d.e.) (red). After the addition of 1.5 equivalents of cryptand [2.2.1] (blue), ()-DH2BNaB2 was quantitatively converted to the extended helicate ()-DH2 BB 22, which gave a completely different CD spectrum to that of ()-DH2BNaB2, reecting the difference in their helical structures (blue). The addition of 1.5 equivalents of NaPF6 reverted ()-DH2 BB 22 back to the contracted helicate, ()-DH2BNaB2, quantitatively and without racemization, as is apparent from the complete recovery of both CD and absorption spectra (dashed black).
Figs S11 and S12). Thus, the ()-DH2BNaB2.TBAmm bearing only the helical chirality, without any other chiral factors, was obtained. The Cotton effects and absorption spectra are perfectly identical to those of ()-DH2BNaB2.()-DMEph, indicating that the helicate is inert to changes in stereochemistry during the cation exchange process, and its enantiomeric excess (e.e.) (helical sense-excess) can be estimated to be 93% (Supplementary Fig. S8). The other diastereomer, ()-DH2BNaB2.()-DMEph with 29% d.e., was similarly converted to the corresponding enantiomer, ()-DH2BNaB2.TBAmm, of which the d.e. was determined to be 29% on the basis of its relative CD intensity to that of the opposite enantiomer (Supplementary Fig. S8). Furthermore, neither the racemization nor decomposition of the complex ()-DH2BNaB2.TBAmm took place on heating in acetonitrile at 60 8C for 9 days and at 80 8C for 24 h. The extended helicate ()-DH2BB22.TBAmm.Na , 221 (see below) was also stable and maintained its optical activity without racemization after heating at 60 8C for 9 days. In contrast, when 10 mol% of triuoroacetic acid was added to an acetonitrile solution of ()-DH2BNaB2.TBAmm, the Cotton effects completely disappeared within 1 h and there was a negligible change in the absorption spectra. This indicates that racemization took place, probably through an acid-catalysed BO bond cleavage and reformation of the spiroborate groups. We expected that the weakly bound central Na ion could be removed from the DH2BNaB2 helicate with the assistance of crown ethers or cryptands to give the divalent cationic helicate DH2BB22 (Fig. 3a). The rst attempt using 2 equivalents of 15-crown-5 ether failed, but cryptand [2.2.1], which is known to bind cations more strongly than its crown ether analogue, successfully trapped the Na ion. On the addition of 2 equivalents of cryptand [2.2.1] to a CD3CN solution of DH2BNaB2.Na, the 1H NMR signals assigned to Na , [2.2.1] (this denotes that the cryptand [2.2.1] includes a
Na ion) appeared and those of the DH2BNaB2.Na were drastically changed (Fig 3b and Supplementary Figs S19 and S20). In particular, the two t-Bu signals showed large downeld shifts of Dd 0.52 and 0.14 ppm, which indicates that the t-Bu groups are no longer under the ring current of the aromatic rings of the other strand. When a tenfold excess of BMAmm.Br2 was added to the solution, the countercation exchange readily proceeded to yield DH2BB22.(BMAmm)2 , of which single crystals were gradually formed from the solution. The X-ray single crystal analysis of DH2BB22.(BMAmm)2 conrmed that the Na ion is indeed removed from the centre of the DH2BNaB2 helicate by the cryptand and that the resultant boron helicate, DH2BB22.(BMAmm)2 , has a dianionic nature with two countercations, BMAmm, in the outer space (Fig. 3c and Supplementary Fig. S13). The crystal structure accounted for the absence of the ring current effect on the tBu groups in the 1H NMR spectrum and the absence of interstrand cross peaks in the rotating-frame nuclear Overhauser effect spectroscopy (ROESY) spectra (Supplementary Figs S16S18). The binding constant of DH2BB22 to the Na ion was determined to be 2.68 106 M21 in acetonitrile at 25 8C by the competitive binding titration experiment between DH2BNaB2 and dicylohexano-18-crown-6 ether (DC18C6) using 1H NMR spectroscopy (Supplementary Fig. S22). Of particular interest is that the length of the boron helicate DH2BB22 is signicantly extendedby almost twofold and with a BB distance of 13.0 when compared with DH2BNaB2 (6.0 ). This noticeably large extension is most likely attributable to the enhanced electrostatic repulsion between the two anionic spiroborate moieties due to the absence of the central Na ion. Close inspection of the crystallographic data of the DH2BNaB2 and DH2BB22 reveals that the torsion angles between the benzene rings of the spiroborate moieties are inverted during the extensioncontraction process, while
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maintaining their spirochirality (Supplementary Table S3). In accordance with the extension, the helicate unwinds with a twisting angle between the terminal benzene rings of each tetraphenol strand of 1808, demonstrating that the extension is coupled with the twisting motion in one direction, although the direction of twisting could not be dened because the DH2BNaB2 complex used was racemic. Adding a small excess of NaPF6 to the solution of DH2BB22 readily and quantitatively regenerated the trinuclear helicate, DH2BNaB2, as evidenced by its 1H NMR spectral change. Analogous trinuclear helicates embracing Li, K or NH4 ions in the centre were obtained by the addition of LiPF6 , KPF6 or NH4PF6 , respectively, instead of NaPF6. In order to realize the anisotropic right- or left-handed twisting motion, we next used the right-handed double helical DH2BNaB2.TBAmm. A unidirectional rotary motion has been achieved with an optically active helical alkene25 bearing two stereocentres that are essential to control the diastereomeric helical chirality, leading to the unidirectional behaviour of the molecular motor. The pure enantiomers of helical alkenes with no stereogenic centres will lose their optical activity owing to racemization, resulting in bidirectional rotating. The addition of a slight excess of [2.2.1] to a solution of the righthanded double helical ()-DH2BNaB2.TBAmm with 93% e.e. brought about complete removal of the central Na ion from the helicate, as conrmed by its 1H NMR spectrum (Supplementary Fig. S21). The CD spectra of the extended helicate ()-DH2 BB22 is quite different from that of the contracted helicate ()-DH2BNaB2, reecting the difference in their helical structures (Fig. 4). On further addition of NaPF6 , the extended helicate can quantitatively revert back to the contracted helicate, as demonstrated by the recovery of the initial absorption and CD spectra. This complete recovery of the CD spectrum unambiguously indicates the dynamic extension and contraction motions that proceed without any racemization or breaking and reformation process of the spiroborate groups. It should be noted that both helicates DH2BNaB2 and DH2 BB22 showed no spectral change within the temperature range of 70 to 10 8C (Supplementary Fig. S23), indicating that the spiroborate helicates DH2BNaB2 and DH2 BB22 most likely exist as single speciesthe contracted form with a sandwiched Na ion and the extended form, respectivelyirrespective of temperature. This extensioncontraction cycle can be repeated several times by the sequential addition of [2.2.1] and NaPF6 in an alternating manner. Similarly, Li and K ions also triggered an anisotropic extension/contraction motion, as supported by the CD and absorption spectral changes (Supplementary Fig. S24). In order to study the kinetics of the extension event of DH2BNaB2 induced by cryptand [2.2.1] and the contraction event of DH2 BB22 triggered by Na ions, stopped-ow CD measurements in acetonitrile at 22 8C were performed and the CD intensity changes at 240 nm were followed (Supplementary Fig. S25). Interestingly, the extension event of DH2BNaB2 induced by cryptand [2.2.1] was found to take place much more slowly than the contraction event of DH2 BB22 triggered by Na ions; the extension event took approximately 5 sec to nish. The data for the extension were tted to an equation based on pseudo-second order kinetics by the nonlinear least-squares curve-tting method to yield a rate constant kext of (5.38+0.05) 103 M21 s21. In contrast, the contraction event was too fast to follow under the present experimental conditions, indicating that it takes place within the dead time of the apparatus (7.8 msec), which would give a kcont much higher than 4.8 105 M21 s21. The slower rate of the extension process could be attributed to the steric hindrance generated by the cryptand [2.2.1]; the contraction process, in contrast, involves Na cations solvated by acetonitrile molecules. This suggests that the extension and contraction rates can be controlled by selecting Na ion trapping reagents (cryptand [2.2.1] in this case).
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The understanding gained in this study will serve as a starting point for future nanoscale mechanical device applications that incorporate molecules undergoing a specic double helical twisting motion although maintaining their one-handedness.
Methods
Synthesis of (+ )-DH2BNaB2.Na1. To a solution of 2 (100 mg, 134 mmol) in + 1,2-dichloroethane (18.0 ml) was added a solution of NaBH4 in ethanol (44.6 mM, 3.0 ml, 134 mmol) under an argon atmosphere. After stirring at 80 8C for 20 h, the mixture was cooled to ambient temperature to form a white precipitate, which was removed by ltration. The ltrate was evaporated to dryness, and 1,2-dichloroethane (2 ml) was added to the residual viscous oil to form a white precipitate. This was collected by ltration, washed with 1,2-dichloroethane (2 2 ml), and dried in vacuo to afford (+)-DH2BNaB2.Na as a white solid in 22% yield. Melting point . 300 8C; ESI-MS (CH3CN, negative): m/z 1,530 [MNa]2, 753 [M2Na]2. Analysis calculated for C104H108B2Na2O8: C, 80.40; H, 7.01; found, C, 80.40; H, 6.89. For 1H and 13C NMR data, see Supplementary Information. Synthesis of (+ )-DH2BNaB2.BMAmm1. To a solution of (+)-DH2BNaB2.Na + (10.0 mg, 6.4 mmol) in CH3CN (10 ml) was added a tenfold molar excess of benzyltrimethylammonium bromide (BMAmm.Br2, 14.7 mg, 64.0 mmol). The solution was allowed to stand at ambient temperature for a few days to form colourless crystals, which were collected by ltration to give (+)-DH2BNaB2.BMAmm in 42% yield. High resolution mass spectrometry (HRMS) (negative mode ESI, CH3CN/CHCl3 (1/1, v/v)): m/z calculated for [MBMAmm]2, 1,529.8156; found, 1,529.8134. For 1H NMR data, see Supplementary Information. Optical resolution of (+ )-DH2BNaB2.Na1. To a solution of (+)-DH2BNaB2.Na + (200 mg, 0.129 mmol) in CH3CN (168 ml) was added a tenfold molar excess of ()-N-dodecyl-N-methylephedrinium bromide (()-DMEph.Br2) (553 mg, 1.29 mmol). The mixture was stirred at room temperature for 36 h to form a white precipitate. After ltration, the collected white precipitate was washed with CH3CN (3 5 ml), and then dried in vacuo to give the diastereomeric salt, ()-DH2BNaB2.DMEph (93% d.e.) as a white solid in 38% yield (45.8 mg). The ltrate was evaporated in vacuo to give a mixture of the other diastereomeric salt, ()-DH2BNaB2.DMEph (29% d.e.) and an excess of ammonium salt ()-DMEph.Br2 (691 mg). ()-DH2BNaB2.DMEph: HRMS (negative mode ESI, CH3CN/CHCl3 (1/1, v/v)): m/z calculated for [MDMEph]2, 1,529.8156; found 1,529.8108. For 1H NMR data, see Supplementary Information. Conversion of (1)-DH2BNaB2.DMEph1 to (1)-DH2BNaB2.TBAmm1 (93% e.e.). To a solution of ()-DH2BNaB2.DMEph (93% d.e.) (10.0 mg, 5.3 mmol) in CH3CN (15 ml) was added a 100-fold molar excess of tetra-n-butylammonium bromide (TBAmm.Br2) (171 mg, 530 mmol). The mixture was stirred at room temperature for 24 h, and the solution was evaporated to dryness. The residue was titrated with CH3CN (2 ml), ltrated, washed with CH3CN (0.5 ml), and dried in vacuo to give the enantiomeric salt, ()-DH2BNaB2.TBAmm (93% e.e.), as a white solid in 51% yield (4.8 mg). HRMS (negative mode ESI, CH3CN/CHCl3 (1/1, v/v)): m/z calculated for [M2TBAmm]2, 1,529.8156; found 1,529.8107. For 1 H NMR data, see Supplementary Information. Conversion of (1)-DH2BNaB2.TBAmm1 to (1)-DH2BB22.TBAmm1.Na1 , 221. To a solution of ()-DH2BNaB2.TBAmm (93% e.e., 0.347 mg, 0.196 mmol) in CD3CN (0.7 ml) was added a solution of [2.2.1] in CD3CN (10 mM, 29.4 ml, 0.294 mmol) to yield ()-DH2 BB22.TBAmm.Na , 221 quantitatively. HRMS (negative mode ESI): m/z calculated for [MTBAmm Na]2, 753.4129; found 753.4125. For 1H NMR data, see Supplementary Information.
Received 5 October 2009; accepted 23 March 2010; published online 2 May 2010
References
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Acknowledgements
This work was supported in part by Grant-in-Aids for Scientic Research from the Japan Society for the Promotion of Science (JSPS) and for Scientic Research on Innovative Areas, Emergence in Chemistry (21111508) from the MEXT. We acknowledge Y. Kondo and K. Nagamori of JASCO for their help in the measurements of the stopped-ow CD spectra.
Author contributions
Y.F. and E.Y. designed and directed the project and K.M. performed the experiments. K.M., Y.F. and E.Y. analysed and discussed the results and co-wrote the manuscript.
Additional information
The authors declare no competing nancial interests. Supplementary information and chemical compound information accompany this paper at www.nature.com/ naturechemistry. Reprints and permission information is available online at http://npg.nature. com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to Y.F. and E.Y.
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Direct transformation of graphene to fullerene

Andrey Chuvilin1,2 *, Ute Kaiser1, Elena Bichoutskaia3, Nicholas A. Besley3 and Andrei N. Khlobystov3 *
Although fullerenes can be efciently generated from graphite in high yield, the route to the formation of these symmetrical and aesthetically pleasing carbon cages from a at graphene sheet remains a mystery. The most widely accepted mechanisms postulate that the graphene structure dissociates to very small clusters of carbon atoms such as C2 , which subsequently coalesce to form fullerene cages through a series of intermediates. In this Article, aberration-corrected transmission electron microscopy directly visualizes, in real time, a process of fullerene formation from a graphene sheet. Quantum chemical modelling explains four critical steps in a top-down mechanism of fullerene formation: (i) loss of carbon atoms at the edge of graphene, leading to (ii) the formation of pentagons, which (iii) triggers the curving of graphene into a bowl-shaped structure and which (iv) subsequently zips up its open edges to form a closed fullerene structure.
ver the past two decades, many different models have been proposed to explain the formation of fullerene from graphite. The generally accepted mechanisms can be categorized into four major groups according to the exact route leading to the fullerenes: the pentagon road13, the fullerene road4, ring coalescence5,6 and the shrinking hot giant model7,8. All of these can be classied as bottom-up mechanisms, because fullerene cages are considered to be formed from atomic carbon or small clusters of carbon atoms. Although there is a large body of experimental evidence supporting bottom-up mechanisms911, it is almost entirely based on mass spectrometry and its variants, which analyse only those species present in the gas phase. These experiments provide no direct structural information about the precursors of fullerenes and do not allow fullerene formation to be followed in situ. Any process of fullerene assembly on the surface of graphite, for example, would be overlooked by the traditional experimental methodology. An atomically thin single sheet of graphite, so-called graphene12, represents an ideal viewing platform for molecular structures using transmission electron microscopy (TEM), because it provides a robust and low-contrast support for molecules and other nanoscale species adsorbed on the surface. Under TEM observation while exposed to an 80-keV electron beam (e-beam), the edges of the graphene sheet appear to be continuously changing in shape (Fig. 1a; see also Supplementary Video). The high energy of the e-beam, when transferred to the carbon atoms of the graphene, can cause fragmentation of large sheets of graphene into smaller ake-like structures (Fig. 1c). The akes adsorbed on the graphene substrate can be visualized and their further transformations readily observed in TEM. The nal product of these transformations is often a perfect fullerene molecule (Fig. 1b). The sequence shown in Fig. 1ch presents a typical transformation route for an individual graphene ake, which changes its shape under the inuence of the e-beam, becoming increasingly round (Fig. 1ce). The contrast of its edges gradually increases (Fig. 1f ), indicating that the edges of the ake come progressively out of plane and rearrange into the spheroidal shape of a fullerene (Fig. 1g). The experimentally observed images can be related through TEM image simulation to models of a graphene ake (Fig. 2b ), curved graphene intermediates (Fig. 2d and e ), and
the resultant fullerene molecule (Fig. 2f ) adsorbed on the graphene substrate. Once fullerene formation is complete, the molecule appears to roll back and forth on the underlying graphene (Fig. 1h). This is possible, because the energy of the van der Waals interaction of a fullerene with the substrate is signicantly reduced compared to a at graphene ake13,14 due to the reduced surface area in contact with the underlying graphene sheet. Loss of carbon atoms at the edge of graphene is a key initial step in the graphene-to-fullerene transformation. Carbon atoms at the edge of a graphene ake are labile, because only two bonds connect them to the rest of the structure. Our density functional theory (DFT) calculations (for details see the Methods section) show that the loss of a carbon atom at the zigzag edge of a small graphene ake and the subsequent relaxation of the structure require 5.4 eV. As expected, it is approximately one-third less than the energy of carbon atom loss from the middle of the ake, which is estimated to be 7.47.6 eV (refs 1517). Indeed, recent TEM experiments have demonstrated that the edge atoms of graphene can be chipped away, one by one, by the e-beam18,19. Following the removal of one or several carbon atoms, the graphene edge undergoes structural reconstruction, normally leading to the most stable zigzag conguration18. The loss of carbon atoms at the edge and the subsequent reconstruction do not cause any signicant changes to the structure of a large graphene sheet18,19 (Supplementary Fig. S1). However, small fragments of graphene, as our extended observations demonstrate (Supplementary Fig. S2), undergo drastic structural transformations under the e-beam, leading to the formation of fullerene cages (Fig. 1ch). These observations provide direct evidence (unlike mass spectrometry) that a fullerene can be formed directly from graphene without the need for dissociation to small carbon clusters as is inherent to other mechanisms of fullerene formation1,2,20. The experimental TEM images provide compelling evidence for the graphene-to-fullerene transformation. However, the exact pathway of this process can be best explained through quantum chemical modelling of the key stages of the process (Fig. 2af ). The structures used for modelling represent one possible ake that is as close as possible to the experimental observations (Fig. 1ch). Induced by the high-energy e-beam, the initial loss of carbon atoms at the edge destabilizes the structure (Fig. 2b),
University of Ulm, Central Facility of Electron Microscopy, Electron Microscopy Group of Materials Science, Albert Einstein Allee 11, 89069 Ulm, Germany, IKERBASQUE, Basque Foundation for Science, E-48011, Bilbao, Spain, 3 School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK. * e-mail: a.chuvilin@nanogune.eu; andrei.khlobystov@nottingham.ac.uk
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b
1 nm
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Figure 1 | Experimental TEM images showing stages of fullerene formation directly from graphene. a, The black arrow indicates a double layer of graphene, which serves as the substrate. The white arrow indicates a strip of graphene (monolayer) adsorbed on this substrate. The dashed white line outlines a more extended island of graphene mono- or bi-layer, which has its edges slightly curved on the left side. b, The nal product of graphene wrapping: a fullerene molecule on the surface of graphene monolayer (carbon atoms appear as black dots). ch, Consecutive steps showing the gradual transformation of a small graphene ake (c) into fullerene (g,h). The graphene lattice is ltered out of images ch for clarity. (See Supplementary Video for a demonstration of the dynamics of the entire process.).
+0.201 eV/atom E(eV/atom) +0.3
+0.197 eV/atom
+0.178 eV/atom
0.261 eV/atom +0.059 eV/atom
(b)
+0.2 +0.1 Etched graphene
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Flat graphene with pentagons
(d)
Curved graphene with pentagons
(e)
Bowl-shaped structure
(a)
Graphene 0.1 0.2 0.3
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Fullerene
Figure 2 | Quantum chemical modelling of the four critical stages of fullerene formation from a small graphene ake. ad, Loss of carbon atoms at the edge (a b); formation of pentagons (b c); curving of the ake (c d); formation of new bonds, leading to zipping of the ake edges (d e). e, Top and side views of a bowl-shaped intermediate structure. Stabilization energies (in eV per carbon atom) of the intermediate structures and resultant fullerene C60 (f), relative to the at defect-free ake of graphene shown in a, are presented pictorially and graphically. b ,d f , Top views of the graphene ake (b ), curved graphene intermediates (d ,e ) and the fullerene C60 molecule (f ) adsorbed on the underlying graphene substrate and simulated TEM images corresponding to each structure, showing how they would appear in TEM experiments.
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because it increases the number of dangling bonds at the edge. The formation of pentagons at the edge (Fig. 2c) and subsequent curving of the ake are thermodynamically favourable processes, because they bring covalently decient carbon atoms close to one another (Fig. 2d), thus enabling them to form bonds. New carboncarbon bonds are then formed, leading to zipping of the ake edges and reducing the number of dangling bonds. This has a profound stabilizing effect on the structure (Fig. 2e). In a similar way, the bowlshaped structure (Fig. 2e) can evolve further by losing carbon atoms from its remaining open edge through e-beam etching, forming more pentagons and curling until the structure is sufciently small to close up into a cage (Fig. 2f ). Fullerene is the most stable conguration for a nite number of sp 2 carbon atoms because the molecular cage has no open edges (that is, etching is prohibited), and all the carbon atoms form three bonds. If the structure of the newly formed fullerene does not correspond to the most stable isomer, its structure can anneal by means of a series of StoneWales rearrangements1 that are facilitated by the e-beam in TEM or by an add-atom under the standard fullerene synthesis conditions21,22. A theoretical study exploring the possibility of the transformation of a graphene sheet into a fullerene conrms that the formation of defects at the edge of graphene is the crucial step in the process20. The structural defects considered in this earlier study are based on a series of rearrangements that give rise to pentagonal rings. However, the energy barrier for such rearrangements appeared to be extremely high, making this pathway plausible only at extremely high temperatures (3,500 K)20, signicantly higher than the temperatures used in fullerene production. Our calculations, however, show that the loss of the outermost carbon atoms in a graphene ake provides a viable route for fullerene formation under realistic experimental conditions. The initial size of the graphene ake is important, because it determines the size of the fullerene cage that can be formed. If the ake is too large, in the region of several hundreds of carbon atoms, there will be a signicant energetic penalty during the curving step (Fig. 2d) associated with the van der Waals interactions between the underlying graphene sheet and the ake. Its edges will continue to be etched until the ake reaches a size that enables the thermodynamically driven formation of fullerene described above. On the other hand, the transformation of very small akes (less than 60 carbon atoms) into fullerenes will be suppressed by excessive strain on CC bonds imposed by the high curvature of small fullerene cages and the violation of the isolated pentagon rule in fullerenes smaller than C60. Indeed, our experiments indicate that fullerene cages formed directly from graphene have a relatively narrow range of diameters averaging 1 nm, which corresponds to 60100 carbon atoms (Supplementary Fig. S2). This observation is in agreement with the consistent observation of a disproportionately high abundance of C60 and C70 fullerenes found in the different methods of fullerene production. Our in situ TEM experiments correlated with quantum chemical modelling demonstrate that a direct transformation of at graphene sheets to fullerene cages is possible. Etching of edge carbon atoms by the e-beam facilitates the formation of curved graphene fragments, which continue to be etched until it becomes possible for them to zip up into a fullerene. Previous studies have suggested that a piece of graphene of limited size may not be the most stable allotrope of carbon2325, particularly under e-beam radiation, so the latter stages of this thermodynamically driven process should occur with similar ease to the formation of C60 fullerene from carefully designed polyaromatic molecules26. Once the edges are sealed, no further carbon atoms can be lost, and the newly created fullerene remains intact under the e-beam. Could these TEM observations be relevant to real-life fullerene production methods, such as arc discharge or laser ablation of
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graphite? On the one hand, conditions inside the transmission electron microscope are quite different (for example, no direct heating, high vacuum, no buffer gas), but on the other hand, the e-beam supplies the energy required to break chemical bonds (just like a laser beam), and the underlying graphite substrate attached to a TEM sample holder absorbs the excess energy released on the formation of new bonds (just like a helium buffer-gas). Indeed, a mass spectrometry study27 has shown that C60 and C70 can form almost exclusively from graphite under 10-keV e-beam radiation in vacuumconditions very similar to those in TEMbut no mechanism explaining fullerene formation was suggested at the time. The top-down mechanism of fullerene formation proposed in our study does not exclude the bottom-up models, because different mechanisms may co-exist under the same experimental conditions (Supplementary Fig. S3). For example, in arc discharge, some fullerenes may form in the gas phase from C2 fragments and some may form on the surface of graphite electrodes from small akes of graphene. A laser ablation study28 has shown that the yield of fullerene is critically dependent on the orientation and quality of the graphitic surface, which conrms that the formation of fullerene directly on the graphite surface is certainly not unique for TEM experiments and may have relevance for preparative methods of fullerene production. We hope that our study will stimulate a reassessment of the current understanding of how fullerenes are formed.
Methods
Thin graphite akes were prepared from spectroscopically pure graphite by grinding in an agate mortar under a layer of ethanol. The dispersion was treated as prepared in an ultrasonic bath and deposited onto a holey carbon TEM grid. Stacks of graphene with thicknesses varying between one and several layers were observed. Images were acquired using a Titan 80-300 instrument (FEI) equipped with an imaging spherical aberration (Cs) corrector. We used an accelerating voltage of 80 kV and Cs optimized Scherzer conditions29 (Cs value, 20 mm; defocus, 23 nm), so the atoms were imaged dark. The exposure time was 1 s per frame, with an interval of 4 s between the frames in one sequence (Supplementary Video). Images of one sequence were aligned by cross-correlation and low-pass-ltered for noise reduction. The ltering did not affect the nal resolution because of signicant over-sampling of the original images. TEM image simulations were performed using MUSLI code30. Coherent aberrations corresponding to those in the experimental images were used. Parameters for the dumping envelope were as follows: focal distance, 1.5 mm (tabulated value for Titan 80-300); coefcient of chromatic aberration, 1.4 mm (measured experimentally); energy spread of the electron source, 0.2 eV (measured experimentally); stability of high tension, 1 1026 (tabulated value for Titan 80-300); stability of objective lens current, 3 1027 (tted by simulations); convergence semi-angle, 0.5 mrad (this parameter does not measurably inuence aberration-corrected imaging). Thermal vibrations were treated using the frozen phonons approach, with 100 phonon congurations averaged for every image at a corresponding DebyeWaller factor of 0.005 nm2. The sampling rate was 0.017 nm pixel21. Images were calculated at an electron dose of 1 106 e2 nm22 and further processed using the same routine as for the experimental images (see above). In theoretical calculations of the geometries and energies of the intermediates, the description of a small graphene ake, which initially contained 117 carbon atoms and was subsequently reduced to 86 and 84 atoms, was based on the DFT formalism using the Q-CHEM quantum chemistry package31 with the B3LYP exchangecorrelation functional32 and 6-31G* basis set. Dispersive van der Waals interactions between the graphene ake and the underlying graphene sheet contribute up to 30% reduction in its stabilization energy, depending on the size and curvature of the fragment. The energy of these non-covalent interactions was estimated using the empirical Girifalco potential, which has been successfully applied to describe the interactions between graphitic nanostructures13.
Received 5 November 2009; accepted 19 March 2010; published online 9 May 2010
References
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22. Ioffe, I. N. et al. Fusing pentagons in a fullerene cage by chlorination: IPR D2C76 rearranges into non-IPR C76Cl24. Angew. Chem. Int. Ed. 48, 59045907 (2009). 23. Ugarte, D. Curling and closure of graphitic networks under electron-beam irradiation. Nature 359, 707709 (1992). 24. Burden, A. P. & Hutchison, J. L. An investigation of the electron irradiation of graphite in a helium atmosphere using a modied electron microscope. Carbon 35, 567578 (1997). 25. Fuller, T. & Banhart, F. In situ observation of the formation and stability of single fullerene molecules under electron irradiation. Chem. Phys. Lett. 254, 372378 (1996). 26. Otero, G. et al. Fullerenes from aromatic precursors by surface-catalysed cyclodehydrogenation. Nature 454, 865868 (2008). 27. Bunshah, R. F. et al. Fullerene formation in sputtering and electron beam evaporation processes. J. Phys. Chem. 96, 68666869 (1992). 28. Xie, Z.-X. et al. Formation and coalescence of fullerene ions from direct laser vaporization. J. Chem. Soc. Faraday Trans. 91, 987990 (1995). 29. Scherzer, O. The theoretical resolution limit of the electron microscope. J. Appl. Phys. 20, 2029 (1949). 30. Chuvilin, A. & Kaiser, U. On the peculiarities of CBED pattern formation revealed by multislice simulation. Ultramicroscopy 104, 7382 (2005). 31. Shao, Y. et al. Advances in methods and algorithms in a modern quantum chemistry program package. Phys. Chem. Chem. Phys. 8, 31723191 (2006). 32. Becke, A. D. A new mixing of HartreeFock and local density-functional theories. J. Chem. Phys. 98, 13721377 (1993).
4. Heath, J. R. Synthesis of C60 from small carbon clusters: a model based on experiment and theory. ACS Symp. Ser. 481, 123 (1991). 5. Hunter, J. M., Fye, J. F., Roskamp, E. J. & Jarrold, M. F. Annealing carbon cluster ionsa mechanism for fullerene synthesis. J. Phys. Chem. 98, 18101818 (1992). 6. Rubin, Y., Kahr, M., Knobler, C. B., Diederich, F. & Wilkins, C. L. The higher oxides of carbon C8nO2n (n35): synthesis, characterization and X-ray crystal structure. Formation of cyclo[n]carbon ions C (n18, 24), C2 (n18, 24, 30), n n and higher carbon ions including C60 in laser desorption Fourier transform mass spectrometric experiments. J. Am. Chem. Soc. 113, 495500 (1991). 7. Irle, S., Zheng, G., Wang, Z. & Morokuma, K. The C60 formation puzzle solved: QM/MD simulations reveal the shrinking hot giant road of the dynamic fullerene self-assembly mechanism. J. Chem. Phys. B 110, 1453114545 (2006). 8. Huang, J. Y., Ding, F., Jiao, K. & Yakobson, B. I. Real time microscopy, kinetics and mechanism of giant fullerene evaporation. Phys. Rev. Lett. 99, 175503 (2007). 9. Yannoni, C. S., Bernier, P. P., Bethune, D. S., Meijer, G. & Salem, J. R. NMR determination of the bond lengths in C60. J. Am. Chem. Soc. 113, 31903192 (1991). 10. Hawkins, J. M., Meyer, A., Loren, S. & Nunlist, R. Statistical incorporation of carbon-13 13C2 units into C60 (buckminsterfullerene). J. Am. Chem. Soc. 113, 93949395 (1991). 11. Ebbesen, T. W., Tabuchi, J. & Tanigaki, K. The mechanistics of fullerene formation. Chem. Phys. Lett. 191, 336338 (1992). 12. Geim, A. K. & Novoselov, K. S. The rise of graphene. Nature Mater. 6, 183191 (2007). 13. Girifalco, L. A. & Hodak, M. Van der Waals binding energies in graphitic structures. Phys. Rev. B 65, 125404 (2002). 14. Ulbricht, H., Moos, G. & Hertel, T. Interaction of C60 with carbon nanotubes and graphite. Phys. Rev. Lett. 90, 095501 (2003). 15. El-Barbary, A. A., Telling, R. H., Ewels, C. P., Heggie, M. I. & Briddon, P. R. Structure and energetics of the vacancy in graphite. Phys. Rev. B 68, 144107 (2003). 16. Saito, M., Yamashita, K. & Oda, T. Magic numbers of graphene multivacancies. Jpn J. Appl. Phys. 46, L1185L1187 (2007). 17. Carlsson, J. M. & Schefer, M. Structural, electronic and chemical properties of nanoporous carbon. Phys. Rev. Lett. 96, 046806 (2006). 18. Girit, C. O. et al. Graphene at the edge: stability and dynamics. Science 323, 17051708 (2009). 19. Jia, X. et al. Controlled formation of sharp zigzag and armchair edges in graphitic nanoribbons. Science 323, 17011705 (2009). 20. Lozovik, Y. E. & Popov, A. M. Formation and growth of carbon nanostructures: fullerenes, nanoparticles, nanotubes and cones. Uspekhi Fizicheskikh Nauk 167, 751774 (1997). 21. Eggen, B. R. et al. Autocatalysis during fullerene growth. Science 272, 8790 (1996).
Acknowledgements
This work was supported by the Engineering and Physical Sciences Research Council (Career Acceleration Fellowship to E.B., grant no. EP/C545273/1 to A.N.K.), the European Science Foundation, the Royal Society, the DFG (German Research Foundation) and the State Baden-Wurttemberg within the SALVE (Sub Angstrom Low Voltage Electron Microscopy) project and by the DFG within Collaborative Research Centre (SFB) 569.
A.C. conceived, designed and carried out experiments. U.K. contributed to the development of the experimental methodology and the discussion of the results. E.B. and N.A.B. performed theoretical modelling and contributed equally to this work. A.N.K. proposed the mechanism and wrote the original manuscript. All authors discussed the results and commented on the manuscript.
The authors declare no competing nancial interests. Supplementary information accompanies this paper at www.nature.com/naturechemistry. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to A.C. and A.N.K.
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PUBLISHED ONLINE: 25 APRIL 2010 | DOI: 10.1038/NCHEM.623
Lattice-strain control of the activity in dealloyed coreshell fuel cell catalysts

Peter Strasser1,2 *, Shirlaine Koh2, Toyli Anniyev3,4, Jeff Greeley5, Karren More6, Chengfei Yu2, Zengcai Liu2, Sarp Kaya3,4, Dennis Nordlund4, Hirohito Ogasawara3,4, Michael F. Toney3,4 and Anders Nilsson3,4
Electrocatalysis will play a key role in future energy conversion and storage technologies, such as water electrolysers, fuel cells and metalair batteries. Molecular interactions between chemical reactants and the catalytic surface control the activity and efciency, and hence need to be optimized; however, generalized experimental strategies to do so are scarce. Here we show how lattice strain can be used experimentally to tune the catalytic activity of dealloyed bimetallic nanoparticles for the oxygen-reduction reaction, a key barrier to the application of fuel cells and metalair batteries. We demonstrate the coreshell structure of the catalyst and clarify the mechanistic origin of its activity. The platinum-rich shell exhibits compressive strain, which results in a shift of the electronic band structure of platinum and weakening chemisorption of oxygenated species. We combine synthesis, measurements and an understanding of strain from theory to generate a reactivitystrain relationship that provides guidelines for tuning electrocatalytic activity.
lectrocatalytic energy-conversion processes are expected to play a major role in the development of sustainable technologies that mitigate global warming and lower our dependence on fossil fuels. More specically, fuel cells that use polymer electrolyte membranes (electrochemical energy-conversion devices) are potentially useful as power sources in the transportation sector, one of the largest emitters of greenhouse gases and consumers of fossil fuels. Efcient and stable fuel-cell electrocatalysts, however, are largely unavailable13, and so fundamental progress in the design of these catalysts is needed. The ultimate goal in catalytic design is to have complete synthetic control of the material properties that determine the reactivity4,5. Catalysts that consist of two metals (bimetallic) allow greater reactivity and more exible design, and recent studies have focused on these catalysts614. There are three fundamental effects in bimetallic catalysis: ensemble, ligand and geometric. Ensemble effects arise when dissimilar surface atoms, individually or in small groups (ensembles), take on distinct mechanistic functionalities, as demonstrated for palladium atom pairs on gold for a gas-phase catalytic reaction7,15. Ligand effects are caused by the atomic vicinity of two dissimilar surface metal atoms that induces electronic charge transfer between the atoms, and thus affects their electronic band structure. Finally, geometric effects are differences in reactivity based on the atomic arrangement of surface atoms and may include compressed or expanded arrangements of surface atoms (surface strain)16. Ligand and geometric effects1,2,8,15,1720 and, in some cases, all three effects15, are generally simultaneously present and coimpact the observed catalytic reactivity. To date, however, no effective strategy to isolate and tune strain effects in electrocatalytic systems has been achieved. Considering the dimensions across which ensemble, geometric strain and ligand effects are effective, only geometric strain can impact surface reactivity over
more than a few atomic layers. Hence, a catalyst structure that consists of a few atomic monometallic layers, supported on a substrate with different lattice parameters (a coreshell structure), should isolate geometric strain effects. If the amount of strain in these structures can be controlled, we could use the rich effects of bimetallic catalysis to tune surface catalytic reactivity continuously. The preferential dissolution (removal) of the electrochemically more reactive component from a bimetallic alloy (precursor) that consists of a less reactive (here Pt) and more reactive metal (here Cu)2123 is commonly referred to as dealloying. We showed that dealloyed PtCu nanoparticles have uniquely high catalytic reactivity for the oxygen-reduction reaction (ORR) in fuel cell electrodes2428, which is of tremendous importance as it occurs at the cathode of virtually all fuel cells29,30, with pure Pt as the preferred catalyst. The ORR is sluggish, which is why signicant amounts of Pt metal are required in fuel cells, which makes them prohibitively expensive. Dealloyed Pt catalysts, however, meet and exceed the technological activity targets in realistic fuel cells24, as shown in Supplementary Fig. S1. Owing to their reactivity, dealloyed Pt catalysts can reduce the required amount of Pt by more than 80%. Despite such importance, the mechanistic origin of their enhanced reactivity remains poorly understood. To realize similar property improvements in related electrocatalytic systems, the principles that underlie their performance must be elucidated. Here, we demonstrate that the concept of strain tuning the catalytic properties, introduced above, provides such a unifying principle.
Results
We studied six different PtCu alloy nanoparticle precursors and their corresponding dealloyed counterparts. We used alloy precursors with various initial atomic Pt/Cu ratios and preparation, in
The Electrochemical Energy, Catalysis, and Materials Science Laboratory, Department of Chemistry, Chemical Engineering Division, Technical University Berlin, 10623 Berlin, Germany, 2 Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77204, USA, 3 Stanford Institute of Materials and Energy Sciences, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA, 4 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA, 5 Center for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois 60439, USA, 6 Materials Science & Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6064, USA. * e-mail: pstrasser@tu-berlin.de
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c 100
80 Intensity (c.p.s.) 60 40 20 Cu Pt
10 nm
10 nm 0 0.5 1 1.5 2 2.5 3 3.5 Distance (nm) 4 4.5
Figure 1 | Elemental maps and line proles of PtCu bimetallic nanoparticle precursors and dealloyed active catalysts. a,b, High-resolution EDS (HR-EDS) elemental maps of a Pt25Cu75 bimetallic nanoparticle alloy precursor (a) and of the active electrocatalyst after Cu dealloying from the precursor (b). Pt is given in blue, Cu in red and pink domains indicate well-alloyed PtCu domains. The precursor alloy was prepared at 800 8C and is supported on carbon black of high surface area. The dealloyed catalyst was obtained by voltammetric cycling of the precursor between 0.05 V and 1.2 V (with respect to a reversible hydrogen electrode) in 0.1 M HClO4 solution. PtCu alloy domains are discernible in the centres of the dealloyed particles with a Pt-enriched surface. EDS elemental maps were acquired using a probe-corrected STEM and were extracted from spectrum images acquired from an area that measured 40 nm 40 nm, with a beam size of 0.2 nm and a step (pixel) size of 0.2 nm. c, HR-EDS line prole across an individual 4 nm diameter dealloyed PtCu alloy active-catalyst particle. Data were acquired using a probe-corrected STEM with a probe diameter 0.2 nm and step size 0.2 nm. The thickness of the Pt-enriched particle shell was 0.6 nm, as shown by the dashed lines. c.p.s. cycles per second.
particular the annealing temperature. We focused on two sets of three Pt/Cu ratios (Pt25Cu75 , Pt50Cu50 and Pt75Cu25); one set was annealed at 800 8C and the other at 950 8C. To obtain the active dealloyed catalysts, the precursors were subjected to an identical electrochemical dealloying protocol in which Cu was removed preferentially from the precursor particles. We rst address the structure of the PtCu bimetallic precursors and the corresponding dealloyed catalysts. These form facecentred cubic disordered alloys31. Figure 1a,b shows energy-dispersive elemental colour-map overlays for Pt and Cu, acquired using a probe-corrected scanning transmission electron microscope (STEM), for the Pt25Cu75 800 8C alloy precursor and the dealloyed catalyst, respectively; the mean particle size prior to dealloying (Fig. 1a) was around 4.5 nm (see Supplementary Fig. S2e), but the dealloyed, catalytically active form (Fig. 1b) exhibited a decrease in the average particle size to 3.4 nm (Supplementary Fig. S2e). The corresponding Pt25Cu75 950 8C alloy precursor and dealloyed catalysts had average particle sizes of 6.0 nm and 5.1 nm, respectively. As Fig. 1b shows, for the dealloyed catalyst, Cu is conned to the centre of the majority of the dealloyed PtCu nanoparticles. The dealloyed particles exhibit a distinct Pt-enriched layer (blue in Fig. 1b) on the surfaces of the alloy PtCu cores (pink in Fig. 1b). A corresponding energy-dispersive spectroscopy (EDS) line prole taken across the diameter of typical ( 4 nm) dealloyed nanoparticles conrmed the presence of a 0.6 nm, Pt-enriched layer on the surface of the dealloyed particles (Fig. 1c). Hence, the Pt and Cu elemental maps and line proles shown in Fig. 1 provide evidence for the formation of a coreshell structure in the nanoparticles after dealloying, in which a Pt-enriched shell surrounds a PtCu core. Furthermore, aberration-corrected, high-angle annular dark-eld (HAADF) STEM images indicate a change in the Pt and Cu distributions within the nanoparticles, from a uniform PtCu alloy (Fig. 2a) to a morphology suggestive of a coreshell structure (Fig. 2b)32. Further evidence comes from X-ray photoelectron spectroscopy (XPS) data, which show a large enhancement in the surface concentration of Pt (Table 1). Based on the STEM, EDS and XPS data, we estimate the thickness of the Pt shell to be 0.61.0 nm, which corresponds to three or more Pt-rich layers, consistent with estimates from anomalous small-angle X-ray scattering of similar materials33. The propensity for Cu to dissolve means that dealloyed
2 nm
2 nm
Figure 2 | HAADF-STEM images of PtCu bimetallic nanoparticle precursors and dealloyed active catalysts. a,b, Sub-angstrom resolution images of individual ( 4 nm) PtCu nanoparticles that show a typical PtCu precursor alloy nanoparticle (a) and a typical PtCu dealloyed nanoparticle (b). The dealloyed nanoparticle exhibits an outer Pt-enriched shell (outer shell images brightly) and a PtCu alloy core (which images less brightly than the shell). Contrast variations in the HAADF-STEM images result from the atomic number (Z) difference between Pt and Cu (commonly referred to as Z-contrast imaging). The thickness of the Pt-enriched particle shell in (b) is 0.6 nm.
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Table 1 | Composition depth proles for Pt25Cu75.
264 8 84 Photoelectron energy (eV) 616 1,133 1,480 8,000 12 19 18 31 68 55 56 59
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Pt25Cu75 precursor (Pt at %) Pt25Cu75 dealloyed (Pt at %)
PtCu alloy core with lattice parameter acore. Using the AXRDderived nanoparticle compositions and lattice-parameter data3436, our coreshell model allowed the determination of ashell. The strain, s(Pt), in the particle shell relative to bulk Pt, is given by equation (1), where aPt is the bulk Pt lattice parameter. s(Pt) = ashell aPt 100 aPt (1)
Composition depth proles for Pt25Cu75 nanoparticles annealed at 950 8C before and after dealloying. Increasing photon energy correlates with increased probing depth. Pt at% platinum atomic per cent.
Pt50Cu50 and Pt75Cu25 nanoparticles possess a similarly Pt-enriched shell structure. From these combined, consistent results, we conclude that the geometry of the dealloyed Pt-enriched shell on the PtCu core leads to the high electrocatalytic activity. Specically, we hypothesize that lattice strain in the Pt shell controls the surface catalytic reactivity. We address our lattice-strain hypothesis using anomalous X-ray diffraction (AXRD), which permits independent measurements of the average lattice parameter and average composition of the scattering nanoparticles before and after dealloying. In AXRD, diffraction patterns are collected at a number of X-ray energies near the X-ray absorption edge of an element of interest (here Cu). Figure 3a shows (111) diffraction proles of a Pt25Cu75 precursor at several energies. The scattering power of Cu drops near the Cu absorption edge, and so the diffracted intensity of the alloy shows a characteristic decrease, as indicated by the arrow in Fig. 3a. In AXRD, peak positions yield average lattice parameters for the nanoparticles, and the relationship between scattering intensity and energy (Supplementary equation (S2)) provides the chemical composition of the scattering alloy phase. As illustrated in Fig. 3b, this model equation for diffraction intensity (black line) was tted to the integrated (111) peak intensities (red circles), and the compositions xPt and xCu were determined. Supplementary Fig. S3a provides a direct comparison of the chemical compositions, xPt , of the alloy precursors derived by AXRD and by inductively coupled plasma optical emission spectroscopy. Given the coreshell nature of the dealloyed particles, the lattice strain in the Pt shell is most relevant for surface catalysis. To estimate the lattice parameter in the particle shell, we approximated the structure of the dealloyed particles by a simple two-phase coreshell model, as shown schematically in Fig. 4a. We assumed a pure Pt shell with lattice parameter ashell that surrounded a
a
8,800 eV 8,890 eV 8,980 eV 8,990 eV (Cu edge) 9,060 eV 9,150 eV
Figure 4b shows ashell as a function of precursor composition and preparation temperature, and s(Pt) is shown in Supplementary Fig. S4. Foremost, the data show that for all catalysts the lattice parameter of the Pt shell, ashell , is smaller than that of pure Pt (dotted line, Fig. 4b) and so is strained compressively. With increasing Cu in the alloy precursor and with higher preparation temperature, ashell becomes smaller and the magnitude of s(Pt) larger. The observed synthesisstrain trends are understood within our coreshell model. The lattice mismatch between the Pt shell and the PtCu core causes a reduced PtPt interatomic distance in the shell. The richer in Cu the particle core, the smaller its lattice parameter, and hence the more strain induced in the shell. A similar argument holds for the increased strain in the high-temperature materials; for these, the bimetallic precursor phase is alloyed more uniformly with less residual, unalloyed Cu (ref. 25), which effectively makes the alloy phase richer in Cu. Both experimental and computational work suggest that lattice contraction induced by particle size (surface stress) in small Pt particles only becomes signicant below a particle diameter of 2.5 nm (refs 37,38) and can therefore be ruled out as source of strain for the dealloyed coreshell particles. To gain insight into how the strain of the dealloyed catalysts affects the catalytic surface reactivity, we studied the electronic band structure. The d-band model developed by Nrskov and co-workers has been successful in relating the adsorption properties of rate-limiting intermediates in catalytic processes to the electronic structure of the catalyst3941. For simple adsorbates, such as the ORR intermediates O and OH, this can be understood in a simple electron-interaction model in which the adsorbate valence p-level forms bonding and antibonding states with the metal d-band40,41. Population of any antibonding state leads to Pauli repulsion, and the bond strength is thereby weakened. A downward shift of the d-band pulls more of the antibonding states below the Fermi level, which results in increasing occupation and weaker adsorbate bonding.
b
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Intensity (a.u.)
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2.6
2.8
3.0
3.2 Q (A1)
3.4
3.6
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9,000
9,100
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Figure 3 | AXRD-based structural and phase-composition analysis of PtCu bimetallic nanoparticle precursors and dealloyed nanoparticle catalysts. a, AXRD intensity proles of a Pt25Cu75 alloy precursor as function of the scattering vector Q. Diffraction proles were taken as a function of the X-ray energy across the X-ray absorption edge of Cu at 8,990 eV. Scattering intensities of Bragg reections (shown are the (111) and (200) reections) decrease (arrow) as the Cu absorption edge energy is approached. The extent of intensity decrease correlates with the Cu content of the scattering phase and can be used to extract composition. b, Fit (black line) of the relation (Supplementary equation (S2)) between the square of the scattering amplitude |F|2 and incident X-ray energy to experimental values (red circles) to extract the molar fractions of Pt and Cu in the scattering phase. AXRD provided the lattice parameter of the scattering phase (structural information) as well as its actual composition (chemical information). a.u. arbitrary units.
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b
Unit cell parameter, ashell () 3.95 Bulk Pt 3.90
Pt shell 1 2
3.85
PtCu core
3.80
acore ashell
3.75
3.70 25 50 Precusor Cu content (%) 75
Figure 4 | A simple structural two-phase core-shell model for the dealloyed nanoparticles and evaluation of their lattice parameters. a, Scheme of a simple two-phase structural model for the dealloyed state of a bimetallic particle. Pure Pt layers surround an alloy particle core (ashell and acore represent the mean lattice parameters in shell and core, respectively). Deviations of ashell from the bulk unit-cell parameter of pure Pt (aPt) imply strain, s(Pt). Shell and core regions possess a face-centred cubic structure and are assumed to be structurally and compositionally uniform. Using a simple linear model (Supplementary equation (S3)), ashell can be estimated without further assumptions about the volume of the core or shell. As PtCu alloys exhibit unit-cell parameters that are smaller than those of pure Pt, the model predicts ashell , aPt; that is, compressive strain in the shell. Unlike the model, a real coreshell particle shows a gradient in lattice parameters, with strain in the surface layer (point 1) being less than in layers near the core (point 2). b, Determination of ashell for dealloyed PtCu bimetallic particles, plotted as a function of the alloy precursor Cu atomic composition at precursor annealing temperatures of 950 8C (blue) and 800 8C (red). Dealloyed catalysts derived from precursors richer in Cu or precursors annealed at higher temperatures display a smaller lattice parameter in the shell. All alloys show lattice parameters below that of pure bulk Pt (dotted line). Error bars refer to resulting uncertainty involved in obtaining ashell from experiments and relations (Supplementary equations (S2), (S3), (S17)).
We extended and applied these ideas to single-crystal Pt surfaces by preparing and characterizing bimetallic single-crystal model surfaces that consist of atomic layers of Pt with various thicknesses grown on a Cu(111) substrate. This model mimics the structural and electronic environment of Pt layers that surround a particle core with signicantly smaller lattice parameters, similar to the dealloyed PtCu catalysts42. Figure 5a shows the photoelectron spectra of the valence band of Pt in Pt(111) and for ve monolayers (5 ML) of Pt grown on Cu(111), measured at grazing electron emission to enhance the surface sensitivity. There is no detectable Cu signal in the 5 ML Pt spectrum, and it thereby represents pure Pt with no ligand effect from the underlying Cu substrate. Based on low-energy electron diffraction (LEED) probing of the lattice parameter during the growth of Pt on Cu(111), 5 ML Pt should give about 2.5+0.3% compressive strain, as shown in Supplementary Fig. S6. The spectrum of Pt on Cu(111) shows a much broader d-band compared to that of the Pt(111) surface, and the d-band centre is downshifted from 2.87 eV to 3.26 eV below the Fermi level. The broadening is related directly to the compressive strain, because the electronic state overlap between the metal atoms increases with shorter interatomic distances; furthermore, keeping the d-occupancy constant for a pure metallic system gives rise to a downward shift of the d-band centre43. Next, we used X-ray spectroscopy to monitor directly the position and atom-specic occupation of the oxygen 2p and Pt 5d antibonding states projected onto the oxygen atom40,41. Figure 5b shows the K-edge X-ray emission spectra (XES) and X-ray absorption spectra (XAS) of atomic oxygen adsorbed on thin lms of Pt on Cu(111) and Pt(111) to show the occupied and the unoccupied electronic states, respectively. For oxygen on Pt(111), we observed a broad occupied bonding state in the XES spectrum and an intense resonance related to the antibonding state in the XAS spectrum. For the two Pt lms on Cu(111), which correspond to strains of around 2.8+0.3% and 3.3+0.3% (ref. 42), we observed a decrease in the intensity of the antibonding resonance with increasing strain. Of primary interest is that, for the lm with maximum strain, the antibonding resonance vanishes in the XAS spectrum and is resolved in the XES spectrum, which directly indicates that the antibonding state is occupied fully with a peak around 1.5 eV below the Fermi level.
b
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6 4 2 0 Binding energy (eV)
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Figure 5 | Surface-science XAS and XES studies of single-crystal model systems that mimic dealloyed bimetallic coreshell structures. a, Photoelectron spectra of the valence-band region for Cu(111), Pt(111) and 5 ML of Pt on Cu(111) measured with a photon energy of 620 eV and with a 158 grazing electron-emission angle. The inelastic background has been subtracted. The spectra of the 5 ML Pt on Cu(111) is dominated completely by the emission from Pt because no sharp Cu d-band emission is seen at 2.3 eV from the underlying substrate (similar spectra are also found for 3 ML of Pt). b, In-plane polarized oxygen K-edge XAS and normal emission oxygen K-edge XES of 0.2 ML of oxygen chemisorbed on 3.6 ML and 2.6 ML lms of Pt on Cu(111) and on bare Pt(111). The XAS were normalized with respect to the high-energy region, where the cross-section is dominated by atomic effects related to the photoionization continuum, whereas the absolute intensity scaling between the XES and XAS spectra is arbitrary. The energy scale is with respect to the oxygen 1s binding energy, which represents the Fermi level38. The strain was estimated based on the Pt coverage and using the LEED data shown in Supplementary Fig. S6.
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0.12 0.10 0.08 ORR activity (eV) 0.06 0.04 0.02 0.00 0.02 4 3 2 1 Strain (ashell aPt )/aPt (%) Pt25Cu75 Pt50Cu50 Pt75Cu25 Pt 0 Pt75Cu25 Pt25Cu75 Pt50Cu50
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nanoparticles of sufciently high surface strain to access the true maximum of the volcano. When the strain passes a critical point, surface relaxation probably relieves further strain and thereby limits the accessibility of the high-strain side of the volcano. Figure 6 also reveals that the set of dealloyed nanoparticle catalysts prepared at the higher annealing temperature exhibits reduced activity at comparable lattice strain in the particle shells, presumably because of differences in the mean particle size. Hence, our strain-related conclusions generally refer to particles of comparable size.
Discussion
Dealloyed fuel-cell catalysts show unprecedented electrocatalytic activity for the electroreduction of oxygen, yet we did not have a fundamental understanding of the mechanistic origin of the catalytic enhancement. Here, we clarify the origin, on an atomic scale, of the exceptional electrocatalytic activity of dealloyed PtCu nanoparticles. We present microscopic and spectroscopic evidence for the formation of a PtCu alloy coreshell nanoparticle structure using STEM elemental maps (Fig. 1), XPS depth proling (Table 1) and anomalous small-angle X-ray scattering33. Given the thickness of the pure Pt shell and considering the limited range of ligand effects46, we conclude that compressive-strain effects rather than ligand effects are responsible for the exceptional reactivity of the particle surface. This is in contrast to other ORR electrocatalyst systems, such as Pt monolayer20 or Pt skin17 catalysts, in which strain and ligand effects are always convoluted. Using X-ray diffraction, we measured and quantied the presence of compressive lattice strain in the Pt shells of the dealloyed particles. To further corroborate the lattice-strain hypothesis in coreshell structures, we studied a surface-science coreshell model system. Our goal was to verify experimentally the predicted effects on the band structure for compressively strained Pt layers. In contrast to previous reports of correlations in reactivity-band structure19, in which band-broadening and band-centre shifts were adopted from computational predictions, we demonstrated experimentally a continuous change of the oxygen 2p and Pt 5d antibonding state from above to below the Fermi level as additional compressive strain was applied, which resulted in a weakening of the adsorbate bond. This represents the rst direct experimental conrmation of the computational prediction of band shifts of adsorbate-projected band structure. Finally, we correlated experimental synthesisstrainactivity data of dealloyed coreshell particles (Fig. 6). The resulting activitystrain relationships provide experimental evidence that the deviation of the Pt-shell lattice parameter from that of bulk Pt, that is the lattice strain in the shell, is the controlling factor in the catalytic enhancement of dealloyed Pt nanoparticles; in particular, these relationships are consistent with computational predictions that compressive strain enhances ORR activity. In conclusion, a coherent picture of the origin of the exceptional electrocatalytic reactivity for the ORR of dealloyed PtCu particles is now established. Strain forms in Pt-enriched surface layers (shells) that are supported on an alloy particle core with a smaller lattice parameter. The compression in the shell modies the d-band structure of the Pt atoms, and thereby weakens the adsorption energy of reactive intermediates compared to unstrained Pt and results in an increase in the catalytic reactivity, consistent with DFT-based predictions. A unique feature of the class of dealloyed catalysts is the experimental control over the extent of dealloying (shell thickness) and the alloy core composition (the upper limit for strain in the shell). The noble and the non-noble constituents can be adjusted in the alloy precursor, such that both expansive and compressive strain can be achieved to control the strengthening or weakening of surface bonds. This enables continuous tuning of catalytic reactivity. We demonstrate explicitly such strain-related tuning for the ORR, and this phenomenon will probably offer control over the activity of other important electrocatalytic reactions that require
Figure 6 | Experimental and predicted relationships between electrocatalytic ORR activity and lattice strain. The experimental ORR activity (in units of kT ln(js,alloy/js,Pt), T 298 K), based on relative surface area, of two families of dealloyed PtCu bimetallic coreshell nanoparticles plotted as a function of strain, s(Pt), in the particle shell (red and blue triangles denote dealloyed PtCu precursors prepared at annealing temperatures of 800 8C and 950 8C, respectively). The ORR activity is proportional to the logarithm of the ratio of the ORR current density of the dealloyed particles to that of pure Pt nanoparticles; this quantity is the effective difference in ORR activation energies (reaction driving force). The experimental curves are upper bounds of the strain in the surface layer. The dashed line is the DFT-predicted, volcano-shaped trend of the ORR activity for a Pt(111) single-crystal slab under isotropic strain. Moderate compressive lattice strain is predicted to enhance the rate of ORR catalysis. Absolute and relative values are provided in Supplementary Table S1. Vertical error bars refer to experimental uncertainty involved in measuring catalytic activities from electrochemical systems; horizontal errors bars refer to errors involved in obtaining ashell from experiments and modelling.
To quantify the relationship between surface strain, O and OH binding energies and the catalytic ORR reactivity, we carried out density functional theory (DFT) calculations to predict the changes in the PtO surface bond energy for a strained Pt(111) model surface. Our computations showed a linear relationship between lattice strain and the adsorbate bond energy, consistent with the experimental Xray spectroscopy data and previous computational analyses16,44. By combining the strain-bond energy relationships with a microkinetic model, originally developed by Hammer and Nrskov39, for the electroreduction of oxygen18,45, we derived a volcano relation between the predicted ORR rate and the strain (the dashed line in Fig. 6). The volcano shape implies that compressive strain rst enhances the overall ORR activity by reducing the binding energy of intermediate oxygenated adsorbates and, thereby, lowers the activation barriers for proton- and electron-transfer processes. Beyond a critical strain, however, the binding becomes too weak and the catalytic activity is predicted to decrease because of an increased activation barrier for either oxygen dissociation or the formation of a peroxyl (OOH) intermediate39. We also plotted (Fig. 6) the experimentally measured ORR electrocatalytic activities for our PtCu catalysts as a function of s(Pt) (electrochemical currents are reported in Supplementary Table S1). We did not, however, observe a decrease in the experimental activity values on the left side of the volcano curve, as predicted by theory; this is probably related to compressive-strain relaxation in the Pt shells. Pt atoms adjacent to the PtCu cores (Fig. 4a, point 2) adopt a lattice parameter closer to that of the cores32, but outer Pt shell atoms (Fig. 4a, point 1) relax towards the lattice constant of bulk Pt (ref. 42). Hence, the surface strain is less than that represented by ashell , which is an average strain in the Pt shell; if the surface strain was plotted in Fig. 6, we would expect a shift of all experimental data points to the right. Furthermore, it is difcult to prepare dealloyed
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References
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modication of the adsorption energy of reactive intermediates, such as the electrooxidation of small organic molecules, including ethanol, methanol and related species.
Methods
Synthesis. PtCu binary electrocatalyst precursors were synthesized using a liquid metalsalt precursor impregnation method followed by freeze-drying and thermal annealing. This method was applied previously to the synthesis of binary25,47 and ternary24 Pt alloys. The nanoparticle-catalyst precursors were prepared by adding appropriate stoichiometric amounts of a solid Cu precursor (Cu(NO3)2.2.5H2O, Sigma-Aldrich) to weighted amounts of commercial Pt electrocatalyst powder, of 30 weight per cent platinum nanoparticles supported on carbon with a high surface area (TEC10E30E from Tanaka Kikinzoku). Electrochemical measurements. The voltammetric response of the electrocatalysts was measured rst during the initial three cyclic voltammetry (CV) scans at 100 mV s21 to obtain the initial rapid Cu-dissolution proles. The catalysts were further pretreated using 200 CV scans between 0.05 V and 1.0 V at a scan rate of 500 mV s21, during which a large amount of Cu was lost from the alloy nanoparticles. Thereafter, the Pt electrochemical surface area (Pt-ECSA) was determined by cycling the treated catalysts at 100 mV s21 between 0.05 V to 1.2 V and integrating the Faradaic charge associated with stripping of underpotentially deposited hydrogen (Supplementary Fig. S1a). Pt-ECSA measurements using CO stripping resulted in comparable values of the surface area. Linear-sweep voltammetry (LSV) measurements were conducted under an oxygen atmosphere by sweeping the potential from 0.06 V anodically to the open-circuit potential ( 1.0 V) at a scan rate of 5 mV s21 (Supplementary Fig. S1b). The ORR activities of the dealloyed, activated catalysts were corrected for mass-transport limitation using equation (5) in Gasteiger et al.3. Specic activities for mass and surface area were established at 900 mV at room temperature (Supplementary Fig. S1c,d). Electron microscopy (STEM and EDS). EDS imaging was carried out in STEM mode at 200 kV with a Philips CM200FEG equipped with an EDAX detector/pulse processor and an Emispec Vision system (Supplementary Fig. S2). Pt-L and Cu-K elemental maps were extracted from the spectral data and PtCu (blue and red, respectively) colour map overlays were produced (Fig. 1a,b). High-angle annular dark-eld (HAADF) STEM images of individual PtCu nanoparticles were recorded at sub-angstrom resolution using a JEOL 2200FS Cs-corrected STEM (CEOS hexapole aberration-corrector) operated at 200 kV. Anomalous X-ray diffraction. Synchrotron-based XRD was used to characterize Pt-alloy electrocatalyst precursor powders as well as electrochemically treated activated catalyst lms using X-ray energies from 8,900 eV, through the Cu K-adsorption edge (8,979 eV) to 9,150 eV. Diffraction measurements were conducted at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 2-1. A detailed description of the analysis of the AXRD results is provided in the Supplementary Information. Computational methods. Computational analysis was carried out using DACAPO (ref. 48), a total-energy calculation code. All calculations were performed on a four-layer slab with a 2 2 unit cell. Full relaxation of the oxygen adsorbate and of the rst two metal layers was allowed. For strained Pt slabs, uniform expansion (or contraction) of the Pt(111) lattice was allowed in all three Cartesian directions, and no corrections to the interlayer Pt distance were included. This model provides a reasonable representation of the compression found in the Pt-base metal alloys that form the substrate of the Pt samples. X-ray photoelectron, X-ray emission and X-ray absorption spectroscopy. XPS, XES and XAS measurements were performed in an ultrahigh vacuum end-station with a base pressure better than 1010 torr at beamline 13-2 at SSRL, which contains an elliptically polarized undulator that allows control of the direction of the photon E vector about the propagation direction. An electron energy analyser (VG-Scienta SES-100 or R3000), mounted perpendicular to the incoming light, was used for the XPS measurements. This was also equipped with a partial-yield detector for X-ray absorption measurements. Samples were mounted on a rotatable sample rod at grazing angle ( 58 for the photon incidence angle) with respect to the incoming light. The independent rotation of the sample and the photon polarization (E vector) allowed for selection of arbitrary angles between the E vector and the sample surface and any choice of detection angle with respect to the sample surface. Composition of the catalysts was determined by measuring the ratio of Pt 4f to Cu 3p XPS intensities normalized to their respective subshell photoionization cross-sections49 for different photon energies. The kinetic energy of the photoelectron denes the inelastic mean free path and the probing depth of the analysis. We varied the photoelectron kinetic energy by changing the incident photon energy to obtain the composition at different probing depths (see Table 1). The estimated probing depths are 0.6 nm, 1 nm, 1.5 nm, 1.8 nm and 7 nm at photon energies of 250 eV, 620 eV, 1,130 eV, 1,480 eV and 8,000 eV, respectively. Further experimental details are provided in the Supplementary Information.
Received 27 October 2009; accepted 11 March 2010; published online 25 April 2010
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47. Mani, P., Srivastava, S. & Strasser, P. Dealloyed PtCu CoreShell Nanoparticle electrocatalysts for use in PEM fuel cell cathodes. J. Phys. Chem. C 112, 27702778 (2008). 48. Hammer, B., Hansen, L. B. & Nrskov, J. K. Improved adsorption energetics within density-functional theory using revised PerdewBurkeErnzerhof functionals. Phys. Rev. B 59, 74137421 (1999). 49. Yeh, J. J. & Lindau, I. Atomic Data and Nuclear Data Tables 32, 1155 (1985).
Acknowledgements
This project was supported by the Department of Energy, Ofce of Basic Energy Sciences, under the auspices of the Presidents Hydrogen Fuel Initiative. Acknowledgment is also made to the National Science Foundation (grant #729722) for partial support of this research. P.S. acknowledges support from the Cluster of Excellence in Catalysis (UNICAT) funded by the German National Science Foundation (Deutsche Forschungsgemeinschaft) and managed by the Technical University Berlin, Germany. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the US Department of Energy, Ofce of Basic Energy Sciences. Use of the Center for Nanoscale Materials was supported by the US Department of Energy, Ofce of Science, Ofce of Basic Energy Sciences, under contract No. DE-AC02-06CH11357. We acknowledge computer time at the Laboratory Computing Resource Center (LCRC) at Argonne National Laboratory, the National Energy Research Scientic Computing Center (NERSC) and the EMSL, a national scientic user facility sponsored by the Department of Energys Ofce of Biological and Environmental Research and located at Pacic Northwest National Laboratory. Microscopy research supported by ORNLs SHaRE User Program, which is sponsored by the Scientic User Facilities Division, Ofce of Basic Energy Sciences, US Department of Energy. The authors thank L. Pettersson for reading the manuscript.
P.S., M.F.T., J.G. and A.N. designed the research and co-wrote the paper, S.K., T.A., K.M., C.Y., Z.L., S.K., D.N. and H.O. performed the experiments and analysed the data, and J.G. performed the theoretical calculations.
The authors declare no competing nancial interests. Supplementary information accompanies this paper at www.nature.com/naturechemistry. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to P.S.
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Assembly of a metalorganic framework by sextuple intercatenation of discrete adamantane-like cages

Xiaofei Kuang1, Xiaoyuan Wu1, Rongmin Yu1, James P. Donahue2, Jinshun Huang1 and Can-Zhong Lu1 *
Metalorganic frameworks form a unique class of multifunctional hybrid materials and have myriad applications, including gas storage and catalysis. Their structure is usually achieved through the innite coordination of metal ions and multidentate organic ligands by means of strong covalent bonds. Threaded molecules such as catenanes and rotaxanes have largely been restricted to comprising components of two-dimensional interlocking rings or polygons. There are very few examples of the catenation of polyhedral cages. Although it has been postulated that the innite extended architecture can be obtained from the polycatenation of a discrete cage based on such threading, this has not been documented to date. Here we describe an innite three-dimensional metalorganic framework composed of catenated polyhedral cages, in which the framework is achieved by mechanical interlocking of all of the vertices of the cages. The three-dimensional polycatenated framework shows twofold self-interpenetration in its crystal packing. The penetration of polycatenanes creates nanosized voids into which the Keggin polyoxometalate anions are perfectly accommodated as counteranions.
nterpenetrated and interlocked assemblies such as catenanes and rotaxanes, in which components are held together by mechanical linkages rather than by chemical covalent bonds, can form prototypes for molecular machines1,2, molecular motors3 and molecular knots4, all of which have potential applications in information processing and storage5, molecular electronics6 and light-driven molecular machines7. Because of their ubiquity in biological systems, distinctive non-covalent mechanical interactions and unique dynamic behaviour8, catenanes and rotaxanes have been the subject of extensive interest in supramolecular chemistry911. In most cases, catenanes (such as organic species12,13, proteins14 and synthetic DNA assemblies15) have been predominantly limited in their use to components of two-dimensional molecular rings16. In 1999, Fujita and colleagues reported pioneering studies on the assembly of a 2[catenane] with two discrete, interlocking threedimensional metalorganic cages17. By 2008, only one further example, by Hardie and colleagues, had been reported18. However, both supramolecular architectures were conned to two interlocking three-dimensional discrete polyhedral cages. Recently, several groups have studied supramolecular assemblies with regular three-dimensional prismatic and polyhedral structures1925. In principle, each vertex of a three-dimensional polyhedral molecule is a potential linking point for the formation of a catenane. Thus, multiple catenations of polyhedral molecules may lead to the formation of three-dimensional innite polycatenanes. This mechanism has been recognized as a basis on which extended architectures might be constructed26. Despite the fact that both three-dimensional innite metalorganic frameworks and polycatenanes have been intensely investigated for nearly two decades, no example of a three-dimensional, innite polycatenane fabricated by the intercatenation of discrete polyhedral molecular components has been documented. The construction of polyhedral, threedimensional, periodic extended architectures from a discrete
1
polyhedral molecular motif by means of polycatenation has therefore remained an intriguing challenge for chemists. Here, we report the synthesis and structure of a new member of the polycatenanes, {[Ag2(trz)2][Ag24(trz)18]}[PW12O40]2 (1) (trz 1,2,4triazole), which, to the best of our knowledge, represents the rst example of a three-dimensional innite polycatenane that has been assembled directly from discrete polyhedral nanocages.
Results and discussion

Colourless or pale-coloured crystals of compound 1 were obtained from the reaction of the trilacunary Keggin [A-PW9O34]9 anion, Ag(O2CCH3) and 1,2,4-triazole in water under hydrothermal reaction conditions. The formula of the product was determined to be {Ag26(trz)20}[PW12O40]2 on the basis of the combined results of X-ray single-crystal structure analysis, thermogravimetric analysis (TGA) and elemental analysis. It is noteworthy that there is a structure transformation from the trivacant [A-PW9O34]9 species to the saturated Keggin [PW12O40]3 motif. The phase purity of the product was conrmed by powder X-ray diffraction, and its crystallinity was stable over a period of several months at ambient temperature in air. The asymmetric unit of compound 1 is composed of one-twelfth of a [PW12O40]3 polyanion, half each of two independent Ag cations, half of an independent triazole ligand, one-fourth of a second independent triazole ligand, and a third disordered Ag cation that is connected to the second triazole ligand. A third triazole anion could not be located, possibly due to a highly disordered and statistical arrangement of the third Ag cation to which it coordinates, which is a situation imposed by the very high symmetry of the total structure in the crystalline state. Despite the complications posed by disorder, crystals of compound 1 were strongly diffracting. The amount of the third Ag cation and the third triazole anion was established by elemental analysis to be one-twelfth of the
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China, 2 Department of Chemistry, Tulane University, 6400 Freret Street, New Orleans, Louisiana 70118, USA. * e-mail: czlu@fjirsm.ac.cn
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N N 3 HN N N N + 3Ag+ Ag N N N Ag N Ag N N
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DOI: 10.1038/NCHEM.618
3 Ag
Figure 1 | Schematic representation of the details of the adamantane-like {Ag24(trz)18}61 nanocage. a, Three Ag cations and three triazole ligands form trigonally symmetric Ag3(trz)3 fragments. b, The Ag3(trz)3 fragments are further coordinated with three Ag ions to form the extended trigonal [Ag3(Ag3(trz)3)]3 cationic unit. c, Four of these units assemble with six additional triazole ligands through AgN bonds, generating the adamantanelike {Ag24(trz)18}6 nanocage. Six triazoles are located at the vertices of the adamantane-like cage. Two of these act as threefold bridging ligands and the remaining four act as twofold bridging ligands. The twofold bridging ligands each also coordinate a further Ag ion. These additional Ag ions are disordered and are omitted for clarity. In the molecular triangle [Ag3(trz)3] fragment, the AgN bonds are 2.105(16) in length, and the NAgN angles are 172.0(9)8. The AgN bonds between the m-Ag ions and the triazole on the vertices of the nanocage as well as the molecular triangle [Ag3(trz)3] are 2.11(2) and 2.19(3) long, respectively. The NAgN angles are 171.1(9)8.
independent composition of the asymmetric unit. The average of the AgN bond distances (2.11(4) ) between the third Ag cation and the second triazole ligand is comparable to the other AgN bond lengths in the structure.
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The structure of compound 1 comprises a polyoxometalatecontaining metalorganic framework composed of catenated metalorganic cages, which can be described overall as two interpenetrating frameworks based on the primitive cubic geometry. Each framework is constructed from nodes consisting of a metal organic adamantane-like cage that is catenated by six others through its vertices. The existence of the adamantane-like nanocages in compound 1 was established unambiguously by X-ray single-crystal analysis. Each cage is composed of twenty-four Ag cations, twelve m3 1,2,4-triazole ligands and six m2 1,2,4-triazole ligands, and is formulated as {Ag24(trz)18}6. The several levels of structural complexity of compound 1 are best appreciated by reversing the description summarized in the previous paragraph and proceeding in a bottom-up fashion while referring to Figs 1 to 5. The structure of the adamantane-like nanocage can be understood from Fig. 1. Three Ag cations are rst linked by three triazole ligands in a pyrazole-like bridging mode to give a neutral trigonal [Ag3(trz)3] fragment with D3 local symmetry (Fig. 1a). The triazole ligands deviate slightly from the triangular plane dened by the three Ag ions. The non-bonding Ag...Ag distances are 3.5852(1) . A similar Ag3(trz)3 unit has been reported in the compound of [Ag5(trz)4]2[Ag2(Mo8O26)].4H2O, in which the trigonal units act as bidentate ligands27. In contrast, each Ag3(trz)3 unit in compound 1 behaves as a hypothetical tridentate ligand connecting to three Ag ions and leading to the formation of a [Ag3(Ag3(trz)3)]3 cationic unit (Fig. 1b). Subsequently, another six triazole ligands, which adopt the pyrazole-like coordination mode, and four [Ag3(Ag3(trz)3)]3 cationic units are connected to generate a perfectly enclosed adamantane-like metalorganic {Ag24(trz)18}6 nanocage (Fig. 1c). In the nanocage, each Ag ion is coordinated in nearlinear fashion by two triazole ligands. The nanocage shows Td symmetry, and its structure can be best visualized when superimposed over an octahedron (Fig. 1c). The m2 1,2,4-triazole ligands dene the six vertices of the octahedron, and the [Ag3(Ag3(trz)3)]3 cationic units and windows alternately occupy the eight faces of the octahedron. A geometrically related adamantane-like complex was rst reported by Saalfrank and colleagues in 198819. Since then, several similar three-dimensional cages have been reported2025. In these earlier examples, the frameworks were constructed from ten molecular species, that is, four tripodal organic ligands and six metal units, and represented as M6L4. The nanocage in compound 1 can also be represented as an M6L4 species if the neutral trigonal [Ag3(trz)3] fragment is considered to be a tridentate ligand (L) and the combination of the apical triazole ligand of the octahedral nanocage with its two bonding Ag ions (trzAg2) as an endcapped transition-metal building block (M) (Fig. 1). The nonbonding Ag...Ag distance in the hypothetical metal units is 4.1367(1) , which is signicantly longer than those within the triangular Ag3(trz)3 fragment (L). Although several geometrically related M6L4 adamantane-like coordination molecular nanocages have been reported previously, in which the L ligands are presynthesized or are known triangular tridentate ligands, the cage reported in this work shows new features. It is the rst example in which the trigonally symmetric building element (tridentate ligand) of a polyhedron is assembled in situ from simple starting components instead of from a pre-designed tridentate ligand. Second, it is an adamantane-like structure constructed from a considerably greater number of constituent parts than any previous example28. The most aesthetically pleasing structural feature of 1 is its unique polycatenation between discrete octahedral {Ag24(trz)18}6 nanocages. As illustrated in Fig. 2ac, each octahedral nanocage is catenated by six others through all its six vertices. Variously coloured adamantane-like nanocages are shown catenating a central, black-coloured nanocage (Fig. 2c) at each of the six vertices that dene the octahedron illustrated in Fig. 1c. It is noteworthy that the overall architecture is produced with mechanical linkages rather
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DOI: 10.1038/NCHEM.618
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c
Figure 2 | Schematic representation of the overall structure of the polycatenanes in 1 from a discrete {Ag24(trz)18}61 nanocage to three-dimensional innite polycatenation. a, Discrete {Ag24(trz)18}6 nanocage. b, Simplied structure of the {Ag24(trz)18}6 nanocage. In proceeding from a to b, the {Ag24(trz)18}6 nanocage is represented more simply by depicting each of the four threefold symmetric Ag3(trz)3 units as a vertex. c, View of a {Ag24(trz)18}6 nanocage (black) catenated by six others through its vertices. These six interlocking cages are shown in different colours, although they are crystallographically equivalent. d, Ball-and-cylinder model representation of c. Each nanocage is viewed as a six-connected node, with the catenation as linkage between nodes. The polycatenated nanocages (c) can be rendered as a ball-and-cylinder model in which the ball represents the octahedral nanocage and the cylinder represents the catenation as linkage. The nanocages in c are represented by correspondingly coloured balls in d, and the catenanes are represented by correspondingly coloured cylinders. e, Schematic view of the extended architecture of d. The NaCl-type a-Po topology represents the polycatenated three-dimensional innite extended framework in 1.
than by common coordination bonding interactions. The propagation of the sextuple polycatenation of each discrete adamantane-like cage generates an unprecedented three-dimensional extended polycatenated polyhedral architecture. The unique threedimensional structure of the discrete, robust and spacious {Ag24(trz)18}6 nanocage makes possible a continuous catenation between the nanocages in three perpendicular directions, thus forming the exquisitely beautiful three-dimensional polycatenated extended structure of compound 1. From a topological viewpoint, if each {Ag24(trz)18}6 nanocage is considered to be a six-connected node, with the catenation as linkage between nodes, the structure of 1 can be described as a NaCl-type aPo topological framework (Fig. 2d). To the best of our knowledge, the three-dimensional innite polycatenated structure of compound 1 represents the rst example of a zero- to three-dimensional polycatenated system, namely, one in which the catenation of individual molecules or ionic species (zero-dimensional) has directly produced a three-dimensional polycatenated assembly. Some of the detailed aspects of the interactions between adjacent {Ag24(trz)18}6 nanocages in the polycatenanes are pertinent to understanding its formation. A view of two interlocking {Ag24(trz)18)}6 nanocages is shown in Fig. 3. The interpenetrating corners are disposed orthogonally to one another and overlap by 4.0255(1) . It is particularly interesting that the two m3 triazoles in the interpenetrated corner and the one m2 triazole from the adjacent catenating cage are arranged in a face-to-face-to-face manner. The
separation between the centroids of the adjacent aromatic rings is 3.63 and the mean interplanar distance is 3.4864 , indicating weak pp interactions between the triazole ligands of the two interlocking cages29. Furthermore, weak argentophilic interactions between the interpenetrating corners are observed30, as illustrated by the proximity of pink and orange rods at the catenated vertex in Fig. 3. The Ag...Ag distances between the corners are 3.018(3) , which is shorter than the sum of their van der Waals radii. Thus, weak pp and argentophilic interactions likely have critical roles in initiating, propagating and stabilizing the polycatenated structure. Yet another remarkable feature of the structure of compound 1 is that two independent but identical NaCl-type a-Po topological polycatenated extended frameworks interpenetrate one other to form a highly ordered supramolecular aggregate (Fig. 4). The void space in the single polycatenated framework is so large that two identical three-dimensional polycatenated frameworks interpenetrate one another to achieve efcient packing (Supplementary Fig. S1). The window-to-window arrangement of the {Ag24(trz)18}6 nanocages between the interpenetrating frameworks creates nanosized pores in which [PW12O40]3 counteranions are located (Fig. 5). The arrangement of the [PW12O40]3 polyanions around a {Ag24(trz)18}6 nanocage is shown in Fig. 5a. A polyanion resides in each of the four windows of the nanocage. Weak coordination bonding interactions between the nanocage and the Keggin anion are observed in compound 1. The shortest Ag...O distances between the silver ions in the trigonal Ag3(trz)3 unit and the m2-O groups of the Keggin anions
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DOI: 10.1038/NCHEM.618
Figure 3 | Supramolecular architecture of adjacent interlocking {Ag24(trz)18}61 nanocages. X-ray crystallographic analysis shows that two identical nanocages have interlocked with one another through their vertices; each cage is shown in a different colour. The interpenetrating corners are disposed orthogonally to one another and overlap by 4.0255(1) . The two m3 triazoles in the interpenetrated corner and the one m2 triazole from the adjacent catenating cage are arranged in a face-to-face-to-face manner with the centroid of the aromatic rings, threaded by thin red string. The separation between the centroids of the adjacent aromatic rings is 3.63 , and the mean interplanar distance is 3.4864 , indicating weak pp interactions between the triazole ligands of the two interlocking cages. The Ag...Ag distances between the corners are 3.018(3) , indicating weak argentophilic interactions between the interpenetrating corners, as illustrated by the proximity of the pink and orange rods at the catenated vertex.
Figure 4 | Topological views of the twofold interpenetration of the polycatenation in 1. a, Two independent NaCl-type a-Po topological polycatenaned frameworks further interpenetrate one another, with the ball representing the cage and the cylinder representing the catenation. The two independent, equivalent and interpenetrating frameworks are distinguished here by different colours. b, Schematic view of the twofold interpenetrating, innite extended polycatenated framework.
Figure 5 | Arrangement of polyoxometalates and {Ag24(trz)18}61 nanocages. a, View showing four polyoxometalate species residing in four windows of the adamantane-like nanocage. b, View showing the supramolecular architecture of the interlocked cages from the catenated framework and the cage from the interpenetrating framework. The polyoxometalate anion is located in a nanosized pore created by the twofold interpenetration of the polycatenated framework in 1. c, X-ray single-crystal structure of {[Ag2(trz)2][Ag24(trz)18]}[PW12O40]2 , composed of two interpenetrating frameworks and polyoxometalate species. The two independent interpenetrating frameworks are distinguished by different colours, for clarity. Each framework is constructed from the polycatenation of a discrete adamantane-like cage, which is mechanically interlocked with six others through its six vertices.
are 2.6737 . If the weak Ag...O coordination interactions are considered, each [PW12O40]3 acts as an interpenetrating unit to connect two {Ag24(trz)18}6 nanocages from different polycatenated frameworks through six m2-O atoms (Fig. 5b). The entire supramolecular architecture of the interpenetrating polycatenated cationic framework and the Keggin polyoxometalate anions is shown in Fig. 5c. Furthermore, evidence of an anion template effect is obvious in compound 1. Without the presence of the Keggin polyoxometalate, the
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self-assembly of Ag cations and 1,2,4-triazole leads to the formation of a two-dimensional [Ag(trz)]1 network31. Therefore, the Keggin polyanions function not only as counteranions but also as templates to help direct the formation of the polycatenated framework in compound 1. In conclusion, the self-assembly of {[Ag2(trz)2][Ag24(trz)18]} [PW12O40]2 demonstrates that the construction of innite threedimensional polycatenated frameworks can be realized by the selfintercatenation of discrete polyhedral building blocks. The structure of compound 1 shows rst that an adamantane-like nanocage can be assembled from simple triazole ligands and does not require a
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15. Rybenkov, V. V., Ullsperger, C., Vologodskii, A.V. & Cozzarelli, N. R. Simplication of DNA topology below equilibrium values by type II topoisomerases. Science 277, 690693 (1997). 16. Fuller, A.-M. L., Leigh, D. A., Lusby, P. J., Slawin, A. M. Z. & Walker, D. B. Selecting topology and connectivity through metal-directed macrocyclization reactions: a square planar palladium [2]catenate and two noninterlocked isomers. J. Am. Chem. Soc. 127, 1261212619 (2005). 17. Fujita, M., Fujita, N., Ogura, K. & Yamaguchi, K. Spontaneous assembly of ten components into two interlocked, identical coordination cages. Nature 400, 5255 (1999). 18. Westcott, A., Fisher, J., Harding, L. P., Rizkallah, P. & Hardie, M. J. Self-assembly of a 3-D triply interlocked chiral [2]catenane. J. Am. Chem. Soc. 130, 29502951 (2008). 19. Saalfrank, R. W., Stark, A., Peters, K. & Schnering, H. G. The rst adamantoid alkaline earth metal chelate complex: synthesis, structure, and reactivity. Angew. Chem. Int. Ed. Engl. 27, 851853 (1988). 20. Beissel, T., Powers, R. E. & Raymond, K. N. Symmetry-based metal complex cluster formation. Angew. Chem. Int. Ed. Engl. 35, 10841086 (1996). 21. Fujita, M., Tominaga, M., Hori, A. & Therrien, B. Coordination assemblies from a Pd (II)-cornered square complex. Acc. Chem. Res. 38, 371380 (2005). 22. Leininger, S., Olenyuk, B. & Stang, P. J. Self-assembly of discrete cyclic nanostructures mediated by transition metals. Chem. Rev. 100, 853908 (2000). 23. Seidel, S. R. & Stang, P. J. High-symmetry coordination cage via self-assembly. Acc. Chem. Res. 35, 972983 (2002). 24. Cotton, F. A., Murillo, C. A. & Yu, R. Deliberate synthesis of the preselected enantiomer of an enantiorigid molecule with pure rotational symmetry T. Dalton Trans. 31613165 (2005). 25. Tranchemontagne, D. J., Ni, Z., OKeeffe, M. & Yaghi, O. M. Reticular chemistry of metalorganic polyhedra. Angew. Chem. Int. Ed. 47, 51365147 (2008). 26. Carlucci, L., Ciani, G. & Proserpio, D. M. Polycatenation, polythreading and polyknotting in coordination network chemistry. Coord. Chem. Rev. 246, 247289 (2003). 27. Zhai, Q.-G., Wu, X.-Y., Chen, S.-M., Zhao, Z.-G. & Lu, C.-Z. Construction of Ag/1,2,4-triazole/polyoxometalates hybrid family varying from diverse supramolecular assemblies to 3-D rod-packing framework. Inorg. Chem. 46, 50465058 (2007). 28. Fujita, M. et al. Self-assembly of ten molecules into nanometre-sized organic host frameworks. Nature 378, 469471 (1995). 29. Lu, J. et al. Octapi interactions: self-assembly of a Pd-based [2]catenane driven by eightfold p interactions. J. Am. Chem. Soc. 131, 1037210373 (2009). 30. Codina, A. et al. Do aurophilic interactions compete against hydrogen bonds? Experimental evidence and rationalization based on ab initio calculations. J. Am. Chem. Soc. 124, 67816786 (2002). 31. Zhang, J.-P., Lin, Y.-Y., Huang, X.-C. & Chen, X.-M. Copper (I) 1,2,4-triazolates and related complexes: studies of the solvothermal ligand reactions, network topologies, and photoluminescence properties. J. Am. Chem. Soc. 127, 54955506 (2005).
designed, pre-synthesized tridentate ligand, and second that polyhedral nanocages can interlock each other by means of mechanical linkages through all of their vertices to produce multiply catenated innite extended structures. The successful synthesis and structural characterization of compound 1 provides new insights for the design of complex types of metalorganic frameworks built from simple, readily available components.
Synthesis of {[Ag2(trz)2][Ag24(trz)18]}[PW12O40]2 (1). A mixture of Na9[A-PW9O34].7H2O (0.338 g, 0.13 mmol), CH3COOAg (0.108 g, 0.65 mmol) and 1,2,4-triazole (0.121 g, 1.75 mmol) was dissolved in 10 ml distilled water at room temperature. The pH value of the mixture was adjusted to 7.3 with 2.0 M NaOH, and the suspension was placed in a Teonw-lined autoclave and kept under autogenous pressure at 160 8C for 4 days. After slowly cooling to room temperature for another 4 days, highly pure and slightly green octahedral crystals were ltered and washed with distilled water (48% yield, based on tungsten). Crystallographic study of 1. Diffraction data for 1 were collected on a Saturn 70 charge-coupled device diffractometer equipped with confocal-monochromated Mo Ka radiation (l 0.71073 ) at room temperature. The CrystalClear program was used for absorption correction. The structure was solved by direct methods and rened on F 2 by full-matrix, least-squares methods using the SHELXL-97 program package. Crystal data are as follows: Ag26C40H40N60O80P2W24 , Mr 9,920.28, cubic, space group Pn3m, a b c 19.3329(5) , V 7,225.9(3) 3, Z 2, rcalcd 4.559 g cm23, m 22.564 mm21, F(000) 8,736, GOF 1.042. A total of 19,675 reections were collected, 1,205 of which were unique. R1 0.0791, wR2 0.2356 for 110 parameters and 1,163 reections. R1 0.0771, wR2 0.2306 for data with I . 2s(I). CCDC reference no. 745116. (See Supplementary Information for details.)
Methods
Received 11 September 2009; accepted 2 March 2010; published online 11 April 2010
References
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Acknowledgements
This work was supported by the 973 Key Program of the Ministry of Science and Technology of China (2006CB932904, 2007CB815304 and 2010CB933501), the National Natural Science Foundation of China (20873150, 50772113, 20821061 and 20973173), the Chinese Academy of Sciences (KJCX2-YW-M05, 319) and the National Science Foundation (CHE-0845829; J.P.D.).
X.K. and C.Z.L. conceived and designed the experiments. X.K. performed the experimental work. X.K., X.W., R.Y., J.H. and C.Z.L. analysed the X-ray structural data and interpreted the results. C.Z.L. was responsible for the overall design, direction and supervision of the project. X.K., R.Y., J.P.D. and C.Z.L. co-wrote the paper.
The authors declare no competing nancial interests. Supplementary information accompanies this paper at www.nature.com/naturechemistry. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to C.Z.L.
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Direct detection of CH/p interactions in proteins

Michael J. Plevin1,2,3 *, David L. Bryce4 * and Jerome Boisbouvier1,2,3 *
XH/p interactions make important contributions to biomolecular structure and function. These weakly polar interactions, involving p-system acceptor groups, are usually identied from the three-dimensional structures of proteins. Here, nuclear magnetic resonance spectroscopy has been used to directly detect methyl/p (Me/p) interactions in proteins at atomic resolution. Density functional theory calculations predict the existence of weak scalar (J) couplings between nuclei involved in Me/p interactions. Using an optimized isotope-labelling strategy, these J couplings have been detected in proteins using nuclear magnetic resonance spectroscopy. The resulting spectra provide direct experimental evidence of Me/p interactions in proteins and allow a simple and unambiguous assignment of donor and acceptor groups. The use of nuclear magnetic resonance spectroscopy is an elegant way to identify and experimentally characterize Me/p interactions in proteins without the need for arbitrary geometric descriptions or pre-existing three-dimensional structures.
he three-dimensional structures of biomacromolecules are dependent on an array of weak non-covalent interactions. As well as stabilizing secondary, tertiary and quaternary structure in proteins and nucleic acids, such interactions can also play essential roles in macromolecular function. The family of hydrogenbond-like interactions includes examples in which a delocalized system of sp 2-hybridized covalent bonds can act as an acceptor group1,2. These so-called XH/p interactions, in which X is an atom capable of forming a covalent bond with hydrogen and p refers to the delocalized p-electrons, have long been proposed to contribute to biomolecular structure and function1. XH/p interactions have been well studied in small-molecule systems2,3. Spectroscopic investigations of benzene and water4 or ammonium5 have revealed intermolecular hydrogen-bond-like interactions in which the p-electrons of benzene act as an acceptor group. XH/p interactions are best classied somewhere between conventional hydrogen bonds and weaker interactions dominated by dispersion. The hydrogen-bond-like nature of XH/p interactions is largely dependent on the polarity of the donor group. When X is nitrogen or oxygen, the interaction energy is dominated by an electrostatic term. However, when X is carbon, the interaction has a more dispersive character, and is thus less analogous to classical hydrogen bonds. XH/p interactions in simple model systems, such as between benzene and methane (CH/p), ammonia (NH/p) or water (OH/p), are predicted to have interaction energies between 1 and 4 kcal mol21 (ref. 6). CH/p interactions, in particular, are considered borderline cases under most denitions of the hydrogen bond. XH/p interaction energies are dependent on donoracceptor group geometry. Polydentate donor groups such as water, methane or ammonia preferentially form interactions in which the XH vector is pointing directly at the centre of the ring and is collinear with the ring normal6,7. Experimental or theoretical approaches applicable for studying non-covalent bonds in smaller molecules cease to be practical when studying larger systems containing multiple examples. In more complicated molecules such as proteins or nucleic acids, XH/p interactions are usually identied from a three-dimensional structural model. Cyclic aromatic moieties and other p-rich systems found in proteins, nucleic acids or other biomolecules can all act as acceptor groups in XH/p interactions. Surveys of the
protein three-dimensional structure database (PDB) have proposed that substantial numbers of XH/p interactions exist in nature810. A major advance in the study of classical hydrogen bonds came with the discovery that scalar (J) couplings exist between acceptor and donor nuclei and that they can be detected using highresolution nuclear magnetic resonance (NMR) spectroscopy experiments1113. This approach has allowed the unambiguous identication of donor and acceptor groups and the study of individual hydrogen bonds in proteins and nucleic acids. J couplings are detectable across many biologically relevant hydrogen bonds14, including weak CaHa...OC interactions between b-strands in proteins15. In this Article, direct experimental evidence of CH/p interactions between side-chain methyl groups and aromatic aminoacid residues in proteins is presented. The high concentration of aromatic and methyl groups in the hydrophobic cores of structured proteins and macromolecular interfaces suggests that methyl/p (Me/p) interactions play important structural or functional roles. A geometric analysis of a database of high-resolution protein structures shows a preference for methyl groups to be located above aromatic rings. Hybrid density functional theory (DFT) calculations predict the existence of weak J couplings between nuclei involved in Me/p interactions in proteins. With the aid of an optimized isotope-labelling strategy, these weak protoncarbon and carbon carbon couplings were used in two heteronuclear NMR experiments that directly identied the donor and acceptor groups involved in a Me/p interaction.
Results
Occurrence of Me/p interactions in proteins. Despite several general studies looking at XH/p interactions, the propensity for methyl groups to locate above aromatic rings in proteins has not yet been investigated. Three geometric parameters were used to identify putative Me/p interactions in a database of highresolution X-ray structures (Fig. 1a): d, the distance between the donor carbon atom and the centre of the acceptor ring; u, the angle between the ring normal and a vector connecting the methyl carbon atom and the centre of the ring; and w, the angle between the CH and ring centre-H vectors. The values used here to dene Me/p interactions (d , 4.3 ; u , 258;
CEA, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France, 2 CNRS, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France, Universite Joseph Fourier, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France, 4 Department of Chemistry and Centre for Catalysis Research and Innovation, University of Ottawa, Ottawa, K1N 6N5, Canada. * e-mail: michael.plevin@ibs.fr; david.bryce@uottawa.ca; jerome.boisbouvier@ibs.fr
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0.8 (deg) 0.4 10 0.0 2.0 4.0 6.0 Distance, d () 0 2.0 4.0 6.0 Distance, d () 0.8 c JCMeCaro (Hz) 0.8 30 20
Figure 1 | Analysis of Me/p interactions in a database of 183 three-dimensional protein structures (resolution, < 2.0 ). a, Schematic showing the three parameters (d, u and w ) used to describe Me/p interactions. b, Histogram of Me/p distances for six-carbon-ring systems (Phe, Tyr and Trp; for any Me/p pair satisfying u , 50.08). The frequency of distances was corrected for the increase in volume at larger values of d to give the density value plotted. c, Scatter plot of d versus u for six-carbon-ring systems. Turquoise points, w , 1208; black points, 1208 , w , 1808. The red line approximates the steric hindrance limit.
a JCMeCaro (Hz) 0.8 b JCMeCaro (Hz) d 0.4 H H d H
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Figure 2 | DFT analysis of hpJCMeCaro couplings in Me/p interactions in a model system consisting of toluene and ethane. ac, Plots showing the variation of hpJCMeCaro as a function of d (a), u (b) and w (c). The subject parameters in panels ac and the colour coding of the aromatic carbon atoms are drawn in d. The orientation of the ethane CC bond is dened by the C(ethane)C(ethane)H-centroid dihedral angle, which is 1808. Viewed from above, the CC bond bisects the bond between the green and red carbon atoms. In a, u and w were xed at 08 and 1808, respectively, and only d was varied. In b and c, starting values of d 3.66 , u 08 and w 1808 were used. Variation of u in b was achieved by a translation of the donor group such that the value of u increases from 08 as the ethane moiety moves towards the red and green carbon atoms. Variation of w (c) was achieved by rotation of the CH bond around the donor proton. See also Supplementary Fig. S1.
w . 1208) are similar to those from previous database studies of XH/p interactions and are generally less conservative than those describing a standard hydrogen bond810. It should be noted that these geometric cutoffs, although consistent with earlier studies, are somewhat arbitrary. A total of 1,014 putative Me/p interactions were identied from a database of 183 highresolution X-ray structures, giving an overall average of four putative interactions per 100 residues. When considering the multiple donor groups in isoleucine, leucine and valine residues and the sizes of aromatic side-chains, 3% of methyl groups and 15% of aromatic rings are involved in Me/p interactions matching the search parameters used (see Supplementary Tables S1 and S2). An analysis of the distribution of methylaromatic distances, d, shows a maximum density at 4.2 (Fig. 1b). A plot of u versus d shows a grouping of points towards the limit permitted before van der Waals clashes occur (Fig. 1c). Steric hindrance produces a distribution in which shorter methylring distances are associated with methyl groups located directly above the ring (that is, u , 108). However, it is noteworthy that the bottom-right quadrant of Fig. 1cthat is, values of d and u where no steric clashes occur is largely unoccupied. This observation suggests that methyl groups sitting more directly above an aromatic ring tend to be located at distances consistent with XH/p interactions.
DFT prediction of J couplings between nuclei involved in Me/p interactions. A series of DFT calculations was performed to assess whether J couplings exist between donor and acceptor nuclei involved in Me/p interactions. A small model system consisting
of toluene and ethane was used to explore the variation of hp JXMeCaro couplings, where X refers to the carbon or hydrogen of the donor methyl group, within the free parameters describing the geometry of the Me/p interaction. The size of the hpJCMeCaro coupling is unsurprisingly dependent on the distance between the ethane and aromatic moieties (Fig. 2a). At distances greater than 5.0 , the coupling becomes negligible. The location of the donor methyl group above the aromatic ring greatly affects the size of the coupling. With the methyl group directly above the ring (u 08), hpJCMeCaro is equal for all aromatic carbons (0.22 Hz for d 3.66 ; Fig. 2b). At larger values of u, the hpJCMeCaro couplings increase specically for the two carbon nuclei brought closer by the displacement of the donor carbon. Maximum couplings (0.7 Hz) are observed when the methyl group is directly above the ring carbons (u 20.78, d 3.9 ). The angle made by the methyl CH bond with the ring normal has less inuence on the magnitude of the coupling (Fig. 2c). When considering hpJHMeCaro couplings it is important to take account of the effect of rotation of the methyl donor group protons with respect to the ring. Rapid transitions between the three rotameric states of the methyl group will average the three individual hpJHMeCaro couplings at each site, resulting in a lower overall value of hpJHMeCaro. As with hpJCMeCaro couplings, the donoracceptor group distance affects the magnitude of the coupling (Supplementary Fig. S1). The effect of varying u or w on hpJHMeCaro is similar to that seen for hpJCMeCaro. In the majority of cases presented in Fig. 2, the isotropic hp JCMeCaro coupling constant is dominated by the Fermi-contact coupling mechanism. For hpJHMeCaro couplings, paramagnetic
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Figure 3 | Exploitation of weak hpJCMeCaro couplings in proteins by NMR spectroscopy. a, Aromatic region of a long-range two-dimensional HCC spectrum of U-[2H,13C,15N], Leu/Val-[13C1H3]proS-labelled ubiquitin. A one-dimensional 1H spectrum is shown above to demonstrate the efciency of the selective labelling scheme. One-dimensional traces taken from the 13C-dimension are shown. b,c, Selected images of the three-dimensional structure of ubiquitin (1ubq) showing L50/Y59 (b) and L67/F45 (c) Me/p interactions, with the magnitude of experimental couplings annotated on the gure. 1H and 13C assignments of ubiquitin are taken from BMRB entry 6427 (ref. 17).
and diamagnetic spinorbital terms are not negligible, but partially cancel to leave the Fermi-contact term dominant (Supplementary Fig. S2). Two proteins, the third domain of protein-G (GB3) and ubiquitin, for which high-precision solution and X-ray structures are available, were analysed for potential Me/p interactions. The threedimensional structure of ubiquitin16,17 revealed a potential Me/p interaction involving Y59 and L50 (d 3.6 ; u 5.68; w 140.68). The local structure shows the d2 methyl group of L50 sitting directly above the aromatic ring of Y59. In the three-dimensional structure of GB318,19 a possible Me/p interaction involving the g2 methyl group of V54 and the ve-membered ring of W43 (d 4.0 ; u 16.78; w 112.68) closely matches the search criteria. Both ubiquitin-L50 d2 and GB3-V54 g2 methyl groups have upeld-shifted proton resonance frequencies that are indicative of close proximity to an aromatic moiety.
L50
The magnitude of hpJXMeCaro couplings between nuclei involved in these two putative Me/p interactions was evaluated using DFT calculations performed at the B3LYP/6-311 G** level. In ubiquitin, hpJXMeCaro couplings for the L50/Y59 interaction vary, depending on the pair of nuclei considered. Both hpJCMeCaro and hpJHMeCaro couplings range between 0.1 and 0.2 Hz (Supplementary Fig. S3). The V54/W43 interaction in GB3 has smaller hpJHMeCaro couplings (00.1 Hz), and the maximum hpJCMeCaro coupling is 0.07 Hz (Supplementary Fig. S3). Direct detection of Me/p interactions by NMR spectroscopy. Long-range HCC and 1H,13C-heteronuclear multiple quantum coherence (HMQC) NMR experiments were designed to permit the transfer of magnetization between nuclei involved in Me/p interactions via hpJXMeCaro couplings (Supplementary Fig. S4). The examples of Me/p interactions identied in ubiquitin and GB3 involve proS methyl groups of Leu or Val. A recently reported isotope labelling scheme was used to prepare uniformly [2H,13C,15N]-labelled protein samples with proS-specic protonation (and [13C]-labelling) of Leu and Val methyl groups20 (Supplementary Fig. S5) to ensure maximal experimental sensitivity during the long transfer periods required to evolve hpJXMeCaro couplings. Long-range HCC spectra of U-[2H,13C,15N], Leu/Val13 1 [ C H3]proS-labelled ubiquitin showed crosspeaks originating from Me/p interactions (Fig. 3). At 20.2 ppm, the chemical shift of L50 d2-protons, two correlations are observed that resonate in the aromatic region of the carbon spectrum (Fig. 3a,b) with chemical shifts that have previously been assigned to Cd1/d2 and C11/12 nuclei of Y59 (ref. 17). These correlations support the existence of the L50/Y59 Me/p interaction identied in the three-dimensional structure of ubiquitin. Two-dimensional spectra of U-[2H,13C,15N], Leu/Val-[12C1H3]proS-labelled ubiquitin acquired with a long-range 1H,13C-HMQC experiment also yielded crosspeaks arising from the L50/Y59 interaction (Fig. 4). No correlations between the V54 g2 methyl group of GB3 and the aromatic carbon nuclei in W43 were observed, which is consistent with the small couplings predicted by DFT (Supplementary Figs S3,S6). However, this interaction was observed in long-range 1 13 H, C-HMQC spectra of U-[2H,13C,15N], Leu/Val-[12C1H3]proSlabelled GB3. In these data, correlations between V54 g2 methyl proton nuclei and C12, Cj2, Cd1, Cd2 and Ch2 aromatic carbon nuclei of W43 were detected (Supplementary Fig. S7). In HCC and HMQC spectra, crosspeaks were observed between nuclei not predicted to participate in standard Me/p interactions. For example, a correlation was observed in HCC spectra of ubiquitin between the L67 d2 methyl group and the Cd nuclei of F45
0.11 Hz Y59
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Figure 4 | Exploitation of weak hpJHMeCaro couplings in proteins by NMR spectroscopy. One-dimensional 13C trace (bottom panel) from the aromatic region of a long-range two-dimensional 1H,13C HMQC spectrum of U[2H,13C,15N], Leu/Val-[12C1H3]proS-labelled ubiquitin. Correlations between L50-Hd21/d22/d23 and Y59-Cd1/d2, C11/12 and Cj are observed (bottom panel) for the three-dimensional structure of the L50/Y59 Me/p interaction (top panel). 1H and 13C assignments of ubiquitin are taken from BMRB entry 6427 (ref. 17).
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(Fig. 3a,c). The three-dimensional structure of ubiquitin suggests a possible Me/p interaction involving F45 and L67, although the geometry (d 4.7 ; u 50.38; w 106.78) does not match the original search parameters. Likewise, HCC spectra of U-[2H,13C,15N], Leu/Val-[13C1H3]proS-labelled GB3 showed a correlation between the L5 d2 methyl group and the Cd1/d2 nuclei of F3021 (Supplementary Fig. S6). The same interaction was observed in a long-range HMQC spectrum of U-[2H,13C,15N], Leu/Val[12C1H3]proS-labelled GB3, as well as an additional correlation involving F30 Cg (Supplementary Fig. S7). These correlations match a putative Me/p interaction identied in the three-dimensional structure (d 4.0 ; u 16.78; w 112.68). The HMQC spectrum of GB3 also shows a correlation between the L5 d2 methyl group the and Cd1/d2 nuclei of Y33 (Supplementary Fig. S7). Again, the three-dimensional structure of GB3 reveals a putative Me/p interaction (d 4.6 ; u 60.28; w 129.88). These data demonstrate that the L5 d2 methyl group participates in Me/p interactions with two aromatic groups simultaneously. Comparison of theoretical and experimental values. Several crosspeaks were observed by NMR spectroscopy that correspond to methyl/aromatic interactions that do not match the geometries used in the original search criteria. In each case, DFT calculations revealed that these interactions have similar-sized couplings to classical Me/p interactions (Supplementary Fig. S8). The magnitude of hpJXMeCaro couplings can be directly quantied using the long-range HCC and HMQC experiments introduced here. The spectra presented show that it is possible to observe and quantify hpJCMeCaro couplings of 0.06 Hz in small proteins (corresponding to an experimental S/N 3) when a suitable isotope-labelling scheme has been used. However, owing to the more favourable relaxation properties of methyl protons, it is possible to detect hp JHMeCaro couplings as small as 0.03 Hz (corresponding to an experimental S/N 3) using a [1H,12C]-methyl-labelled sample and sensitive band-selective, optimized ip-angle, short transient (SOFAST)-HMQC experiments. Each experimental hpJXMeCaro coupling corresponds to interactions between different pairs of nuclei, the positions of which are averaged by ring ipping of the aromatic acceptor group and rapid rotation of the methyl donor group. In contrast, DFT calculations report hpJXMeCaro couplings for a single static conformation. To take into account the extremely short lifetime of interactions between each pair of atoms, the DFT calculated couplings have been averaged assuming a simple twoor three-step fast jump model for ring ipping and methyl rotation, respectively. For Me/p interactions in GB3, excellent agreement was observed between the experimental NMR values of both hpJCMeCaro and hpJHMeCaro couplings and the theoretical values predicted by DFT (root mean-squared deviation (rmsd) 0.06 Hz; Fig. 5). It should be noted that an ultrahigh-resolution, NMR-rened X-ray structure of GB3 (PDB code: 2oed)19 was available for the calculation of precise hpJXMeCaro couplings. The agreement between NMR and DFT data for Me/p interactions in ubiquitin is lower (rmsd 0.1 Hz). This disparity reects local structural uncertainty between the X-ray (1ubq16) and solution structures (1d3z17) used for DFT calculations. A detailed comparison of these two three-dimensional structures revealed small, but non-negligible, variations in the orientations of the donor and acceptor groups of both of the Me/p interactions described in ubiquitin. For example, the parameters of the L50/Y59 Me/p interaction calculated from the X-ray structure of ubiquitin (d 3.6 ; u 5.68; w 140.68) differ from the average across the NMR ensemble (d 3.17 ; u 8.578; w 134.08). These local structural differences affect the value of the couplings calculated by DFT. The rmsd between hpJXMeCaro couplings calculated from the NMR ensemble and those determined from the X-ray structure is 0.29 Hz (Supplementary Fig. S9).
JCMeCaro (DFT) JCMeCaro (exp)
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0.0 Aromatic C: Me/ pair: Protein: / 50/59 Ubiquitin 67/45 / 2 2 2 1 2 5/30 5/33 GB3 54/43
Figure 5 | Plot comparing calculated and observed hpJCMeCaro and hp JHMeCaro couplings. Experimental hpJCMeCaro couplings (black circles) were quantied from long-range two-dimensional HCC spectra of ubiquitin (L50/Y59 and L67/F45) and GB3 (L5/F30). Experimental hpJHMeCaro couplings (red circles) were quantied from long-range two-dimensional HMQC spectra of ubiquitin (L50/Y59) and GB3 (L5/F30 and V54/W43). Calculated hpJXMeCaro couplings (red and grey bars) for each interaction were determined using hybrid DFT protocols from the three-dimensional structures of ubiquitin (1ubq) and GB3 (2oed). Standard deviations (error bars) for each experimentally quantied coupling were extracted from the spectral noise.
A small number of couplings calculated to have magnitudes above the detection threshold were not observed. A 0.48-Hz hp JCMeCaro coupling is predicted for the L5/Y33 interaction in GB3, but no correlation was detected experimentally (Supplementary Figs S7,S8). Furthermore, the experimentally quantied couplings between the L5 d2 methyl group and the Cd2 nuclei of Y33 are lower than those predicted by DFT (rmsd 0.1 Hz). In the crystal structure of GB318, Y33 is located at an intermolecular interface and forms two potential crystal contacts. This is not the case for either W43 or F30. Analysis of the x1 rotameric state of Y33 in solution was not possible due to degeneracy of the b protons, which is likely due to side-chain exibility22. The same study reported a good agreement between the x1 angles of W43 and F30 calculated in solution and from the X-ray structure. Excluding couplings from the L5/Y33 interaction reduces the rmsd between experimental and DFT couplings for GB3 to 0.01 Hz. These observations suggest that the local structure of Y33 in solution may be different from that reported in the original 1.1- X-ray structure and a subsequent NMR-rened model19. The analysis presented here demonstrates that quantication of hp JXMeCaro couplings is particularly sensitive to small structural changes and that these coupling values could potentially be used to nely optimize side-chain positions in the hydrophobic cores of proteins.
Discussion XH/p interactions in biomolecules have received considerable

attention and their existence and role have been the subject of much debate1,23,24. From the rst three-dimensional structural studies of proteins and nucleic acids, there has been suspicion that interactions involving aromatic acceptor groups might constitute an important stabilizing force for biomolecular structure. However, in larger biomolecules there is only anecdotal, structurebased evidence of these interactions810. Despite the small size of hp JXMeCaro couplings, the spectra presented clearly demonstrate that it is possible to use NMR spectroscopy to study Me/p interactions in proteins at atomic resolution. The high sensitivity of
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the labelling and spectroscopic approaches also enabled the detection of crosspeaks corresponding to coupled nuclei that do not conform to a canonical Me/p description. High-level ab initio analysis of CH/p interactions between methane and benzene6,7,25 have shown that the interaction is strongest when a CH bond is pointing directly at the centre of the aromatic ring (that is, u 08 and w 1808). Changing the value of w such that the interaction becomes tridentate only slightly reduces the interaction energy. Lateral displacement of the donor group (that is, increasing u) also does not greatly affect the interaction energy. Only when the donoracceptor distance increases does the overall interaction energy diminish. Thus, although a classical XH/p geometry (u 08 and w 1808) is favoured, the location of the carbon determines the interaction energy rather than the location of the proton. These previously reported ab initio results seem to match well the NMR spectroscopy results presented here. That is, crosspeaks were detected in two-dimensional NMR data both for Me/p interactions with classical geometry (for example, L50/Y59 in ubiquitin) and for interactions that fall outside the classical XH/p cone (for example, L5/F30 in GB3). It is important to note that the detected J couplings do not directly report interaction energies. Although the magnitude of a coupling is proportional to the degree of overlap between electronic wavefunctions, the electron correlation indicated by J couplings can be attractive or repulsive. This said, the data from previously published ab initio studies6,7,25 and those presented here suggest that the cone commonly used to identify XH/p interactions810 does not offer a full description of interactions involving apolar donor groups. Studies of the electrostatic potential of aromatic groups in proteins show clear negative regions towards the centre of aromatic ring systems26. XH/p interactions involving polar donor groups seem to be well described by a cone centred on this negatively charged point. However, for XH/p interactions in which the donor group is apolar and where the interaction has a signicantly lower electrostatic component, it seems necessary to consider a much broader volume above and below the whole aromatic ring. Nishio and colleagues proposed a geometric denition for CH/p interactions that stated that the CH donor group must be located above the p-system and preferably above one of the sp 2-hybridized atoms27,28. This broader description, which does not focus on the most negatively charged region of the ring, seems to be largely consistent with the CH/p interactions for which hpJXMeCaro couplings could be experimentally detected. Me/p and XH/p interactions have been shown to be a common and functionally important feature in structured proteins. The data presented here represent the rst direct detection of individual XH/p interactions in proteins. Using NMR spectroscopy it has been possible to unambiguously identify donor and acceptor groups and to experimentally characterize these weak interactions, on an individual basis, and without a priori knowledge of the three-dimensional structure of the protein.
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ethane and toluene, and the positions of protons (including for calculations of protein model systems), were optimized at the same level of theory before the calculation of J couplings. hpJXMeCaro coupling values were calculated from 1ubq (ubiquitin) and 2oed (GB3). More detail is provided in the Supplementary Information. Sample preparation. Ubiquitin was expressed from cultures of E. coli BL21(DE3) transformed with a pET21b vector with ampicillin resistance32. Following cell lysis, soluble protein was passed over a Q-sepharose ion exchange resin. The ow-through was further puried by size-exclusion chromatography and fractions containing ubiquitin were identied by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). Samples of GB3 were prepared as previously reported19. Final NMR samples of ubiquitin (50 mM tris-HCl, pH 8.0) and GB3 (50 mM sodium phosphate, pH 5.5) of 2 mM protein were prepared in D2O and supplemented with sodium azide and protease inhibitors. Isotope labelling. All protein samples were prepared in standard M9 minimal media containing [15N]-labelled ammonium chloride (Cambridge Isotope Laboratories, Inc. (CIL)), [2H,13C]-d6-glucose (CIL) and 99.85% D2O (CIL). Specic labelling of leucine d2 and valine g2 methyl groups was achieved by supplementing the growth medium with 300 mg of acetolactate 1 h before induction. ProS-specic [1H,12C] or [1H,13C] labelling of Leu and Val methyl groups was achieved by supplementing the medium with either 2-[12C1H3]methyl-4-[12C2H3] acetolactate or 2[13C1H3]methyl-4-[12C2H3] acetolactate20. Both varieties of acetolactate were prepared in house. ProR/S nomenclature of Leu and Val methyl groups follows IUPAC/IUBMB/IUPAB guidelines33. NMR spectroscopy. NMR spectra were recorded on an 800 MHz Varian DirectDrive spectrometer equipped with pulsed-eld gradients and a cryogenically cooled triple-resonance probe head. Detailed information regarding the long-range HCC and HMQC experiments can be found in Supplementary Fig. S4. Twodimensional long-range HCC spectra were collected with 144 (t1) 1,200 (t2) complex points with acquisition times of 1.8 ms (t1) and 60 ms (t2). A total of 160 (ubiquitin) or 224 (GB3) scans were recorded per complex t1 increment with a recycle delay of 1.5 s. Two-dimensional long-range HMQC spectra were collected with 320 (t1) 2,600 (t2) complex points with acquisition times of 4.0 ms (t1) and 100 ms (t2). A total of 128 scans were recorded per complex t1 increment with a recycle delay of 1.0 s. Total experimental times were between 10 and 20 h. All NMR data were processed with NMRPipe34 and analysed using NMRDraw or AZARA (www.bio.cam.ac.uk/azara). Peak intensities and noise estimates were extracted using nmrDraw. Quantication of experimental hpJXMeCaro couplings. Experimental hpJCMeCaro and hpJHMeCaro couplings were determined from the ratio of signal intensity between corresponding peaks in transfer and reference spectra (see Supplementary Information for further explanation). DFT-calculated hpJXMeCaro couplings were obtained for all pairs of nuclei from the donor and acceptor groups involved in Me/p interactions. Under experimental conditions, aromatic Cd1/d2, C11/12 carbon (Phe and Tyr) and methyl protons were considered to have degenerate resonance frequencies due to ring ipping or methyl group rotation. To take into account the multiplicity of nuclei involved in each detected NMR magnetization transfer, DFT-calculated couplings for each pair of nuclei were suitably combined to give predicted values that were directly comparable to the experimental values obtained from NMR spectra.
References
1. Perutz, M. F. The role of aromatic rings as hydrogen-bond acceptors in molecular recognition. Phil. Trans. R. Soc. Lond. A 345, 105112 (1993). 2. Desiraju, G. R. & Steiner, T. The Weak Hydrogen Bond (Oxford Univ. Press, 1999). 3. Meyer, E. A., Castellano, R. K. & Diederich, F. Interactions with aromatic rings in chemical and biological recognition. Angew. Chem. Int. Ed. 42, 12101250 (2003). 4. Suzuki, S. et al. Benzene forms hydrogen bonds with water. Science 257, 942945 (1992). 5. Rodham, D. A. et al. Hydrogen bonding in the benzeneammonia dimer. Nature 362, 735737 (1993). 6. Tsuzuki, S., Honda, K., Uchimaru, T., Mikami, M. & Tanabe, K. Origin of the attraction and directionality of the NH/p interaction: comparison with OH/p and CH/p interactions. J. Am. Chem. Soc. 122, 1145011458 (2000). 7. Tsuzuki, S., Honda, K., Uchimaru, T., Mikami, M. & Tanabe, K. The magnitude of the CH/p interaction between benzene and some model hydrocarbons. J. Am. Chem. Soc. 122, 37463753 (2000). 8. Burley, S. K. & Petsko, G. A. Aminoaromatic interactions in proteins. FEBS Lett. 203, 139143 (1986). 9. Steiner, T. & Koellner, G. Hydrogen bonds with p-acceptors in proteins: frequencies and role in stabilizing local 3D structures. J. Mol. Biol. 305, 535557 (2001).
Methods
Database analysis of Me/p interactions. A database of high-resolution threedimensional protein structures (X-ray only; Supplementary Table S2) was prepared and rened against sequence redundancy (,25%), resolution (,2.0 ) and R-factor (,0.25) using PISCES29. Protons were added to each of 183 structures using the HGEN module of CCP4 (ref. 30). Putative Me/p interactions were identied using the following denition of an Me/p interaction: d , 4.3 ; u , 258; w . 1208 (ref. 10). Denitions of d, u and w are given in Fig. 1a. Distributions of methyl aromatic group distances (six-carbon rings only) were measured within a cone dened by d , 7.0 ; u , 508. The steric hindrance limit shown in Fig. 1c was estimated by a grid search using an idealized six-carbon ring (bond length 1.4 ) acceptor group. DFT calculations. All DFT calculations of indirect nuclear spinspin (J) coupling constants were carried out using Gaussian 03, revision C.02 (ref. 31). Three CPUs were used per job. Calculations of J couplings were performed using the hybrid B3LYP functional and the 6-311 G** basis set on all atoms. The geometries of
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26. Dougherty, D. A. Cationp interactions in chemistry and biology: a new view of benzene, Phe, Tyr and Trp. Science 271, 163168 (1996). 27. Umezawa, Y. & Nishio, M. CH/p interactions in the crystal structure of class I MHC antigens and their complexes with peptides. Bioorg. Med. Chem. 6, 25072515 (1998). 28. Umezawa, Y. & Nishio, M. CH/p interactions as demonstrated in the crystal structure of guanine-nucleotide binding proteins, Src homology-2 domains and human growth hormone in complex with their specic ligands. Bioorg. Med. Chem. 6, 493504 (1998). 29. Wang, G. & Dunbrack, R. L. Jr. PISCES: a protein sequence culling server. Bioinformatics 19, 15891591 (2003). 30. CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760763 (1994). 31. Frisch, M. J. et al. Gaussian 03, Revision C.02. (Gaussian, 2003). 32. Sass, J. et al. Purple membrane induced alignment of biological macromolecules in the magnetic eld. J. Am. Chem. Soc. 121, 20472055 (1999). 33. Markley, J. L. et al. Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy. J. Biomol. NMR 12, 123 (1998). 34. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277293 (1995).
10. Brandl, M., Weiss, M. S., Jabs, A., Suhnel, J. & Hilgenfeld, R. CH...pinteractions in proteins. J. Mol. Biol. 307, 357377 (2001). 11. Dingley, A. J. & Grzesiek, S. Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings J. Am. Chem. Soc. 120, 82938297 (1998). 12. Pervushin, K. et al. NMR scalar couplings across WatsonCrick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy. Proc. Natl Acad. Sci. USA 95, 1414714151 (1998). 13. Cordier, F. & Grzesiek, S. Direct observation of hydrogen bonds in proteins by interresidue 3hJNC scalar couplings. J. Am. Chem. Soc. 121, 16011602 (1999). 14. Grzesiek, S., Cordier, F., Jaravine, V. & Bareld, M. Insights into biomolecular hydrogen bonds from hydrogen bond scalar couplings. Prog. Nucl. Magn. Reson. Spectrosc. 45, 275300 (2004). 15. Cordier, F., Bareld, M. & Grzesiek, S. Direct observation of CaHa...OC hydrogen bonds in proteins by interresidue h3JCaC scalar couplings. J. Am. Chem. Soc. 125, 1575015751 (2003). 16. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin rened at 1.8 resolution. J. Mol. Biol. 194, 531544 (1987). 17. Cornilescu, G., Marquardt, J. L., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 68366837 (1998). 18. Derrick, J. P. & Wigley, D. B. The third IgG-binding domain from streptococcal protein G. An analysis by X-ray crystallography of the structure alone and in a complex with Fab. J. Mol. Biol. 243, 906918 (1994). 19. Ulmer, T. S., Ramirez, B. E., Delaglio, F. & Bax, A. Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy. J. Am. Chem. Soc. 125, 91799191 (2003). 20. Gans, P. et al. Stereospecic isotopic labeling of methyl groups for the NMR studies of high molecular weight proteins. Angew. Chem. Int. Ed. 49, 19581962 (2010). 21. Nadaud, P. S., Helmus, J. J. & Jaroniec, C.P. 13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy. Biomol. NMR Assign. 1, 117120 (2007). 22. Miclet, E., Boisbouvier, J. & Bax, A. Measurement of eight scalar and dipolar couplings for methinemethylene pairs in proteins and nucleic acids. J. Biomol. NMR 31, 201216 (2005). 23. Mitchell, J. B., Nandi, C. L., McDonald, I. K., Thornton, J. M. & Price, S. L. Amino/aromatic interactions in proteins: is the evidence stacked against hydrogen bonding? J. Mol. Biol. 239, 315331 (1994). 24. Weiss, M. S., Brandl, M., Suhnel, J., Pal, D. & Hilgenfeld, R. More hydrogen bonds for the (structural) biologist. Trends Biochem. Sci. 26, 521523 (2001). 25. Tsuzuki, S., Honda, K., Uchimaru, T., Mikami, M. & Fujii, A. Magnitude and directionality of the interaction energy of the aliphatic CH/p interaction: signicant difference from hydrogen bond. J. Phys. Chem. 110, 1016310168 (2006).
Acknowledgements
The authors wish to thank O. Hamlin and P. Gans for providing labelled acetolactate, B. Brutscher, J.-P. Simorre and D. Marion for a critical reading of the manuscript, I. Ayala for help in preparing protein samples, and the Partnership for Structural Biology for access to integrated structural biology platforms. The clone of GB3 was kindly provided by A. Bax and that of ubiquitin by S. Grzesiek. M.J.P. acknowledges funding from LAssociation pour la Recherche sur le Cancer and the EU (FP7-PEOPLE-IRG-2008), J.B. acknowledges funding from Agence Nationale de la Recherche, Human Frontiers Science Programme and Centre National de la Recherche Scientique, and D.L.B. acknowledges the Natural Sciences and Engineering Research Council of Canada and the High-Performance Virtual Computing Laboratory.
All authors conceived and devised the experiments, and co-wrote the manuscript. M.J.P. prepared samples. M.J.P. and J.B. recorded and analysed the NMR data. D.L.B. performed and analysed the DFT calculations.
The authors declare no competing nancial interests. Supplementary information accompanies this paper at www.nature.com/naturechemistry. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to M.J.P., D.L.B. and J.B.
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Geometry-controlled kinetics
O. Benichou1 *, C. Chevalier1, J. Klafter2, B. Meyer1 and R. Voituriez1
It has long been appreciated that the transport properties of molecules can control reaction kinetics. This effect can be characterized by the time it takes a diffusing molecule to reach a targetthe rst-passage time (FPT). Determining the FPT distribution in realistic conned geometries has until now, however, seemed intractable. Here, we calculate this FPT distribution analytically and show that transport processes as varied as regular diffusion, anomalous diffusion, and diffusion in disordered media and fractals, fall into the same universality classes. Beyond the theoretical aspect, this result changes our views on standard reaction kinetics and we introduce the concept of geometry-controlled kinetics. More precisely, we argue that geometryand in particular the initial distance between reactants in compact systemscan become a key parameter. These ndings could help explain the crucial role that the spatial organization of genes has in transcription kinetics, and more generally the impact of geometry on diffusion-limited reactions.
t is generally known that reaction kinetics can be inuenced by the transport properties of the reactants13. In fact, the transport step, before reactants meet and eventually react, can even be limiting in the case of conned systems such as cells or cell subdomains where a small number of reactants are involved49. In such systems, the rst step in estimating the kinetics of reactions consists of evaluating the properties of the rst encounter between reactants. Quantitatively, this amounts to calculating the distribution of the time it takes a diffusing molecule to reach a target sitethe rstpassage time (FPT) distribution. Although this quantity is well known in quasi-one-dimensional or unconned geometries (see ref. 10 for a review), determining the FPT distribution seems to be intractable in the realistic situation where the diffusing molecule is conned within a nite domain11. A rst estimate of the effect of geometrical parameters of connement on this search time is given by the mean of the FPT. This has recently been calculated, and a linear scaling with the volume has been demonstrated1215. However, as soon as several timescales are involved, the kinetics can not be determined by the mean of the FPT only, and the entire distribution is needed16,17. Gene transcription provides an extremely important example which we shall repeatedly invoke in what followsof reactions involving a small number of (or even single) reactants conned within a small (microsized) domain, and whose kinetics must be precisely regulated to fulll vital cell functions. Interest in the question of how geometrical parameters impact on the kinetics of such transcriptional reactions and how they could act as regulatory factors has recently increased, mainly because of new experimental tools that enable the observation of the real-time production of proteins at the single molecule level4. These techniques, which give access to the spatial organization of the genetic material, have revealed strong correlations between the spatial locations of successively activated genes, both for prokaryotes18 and eukaryotes19. Indeed, it has been found that successively activated genes are often colocalized, that is located in the very same regions. These observations raise the question of the importance of geometrical parameters in transcription kinetics, which has remained so far widely unanswered. In the broader context of chemical reactions in connement, we are interested here in several questions. (i) How does the FPT distribution depend on the volume of the conning domain? (ii) How
does it depend on the initial position of the diffusing molecule? (iii) Is this geometric dependence an important factor that could potentially control the kinetics? Note that the inuence of connement and crowding effects on biochemical reactions has already been studied (see refs 20, 21 for reviews) on the basis of a thermodynamical treatment of reaction kinetics. Although this approach is well suited to the case of a large number of reactants, it does not provide the dependence of the kinetics on the geometrical parameters mentioned above (such as the initial position of the reactants), which involve the individual nature of the reactants and their dynamical properties. In this work, we calculate analytically the distribution of the FPT at a target T for a diffusing particle released at a starting point S (see Fig. 1) and quantitatively answer questions iiii above. We highlight universal laws of the FPT distribution as functions of the volume N of the conning domain and of the distance between S and T (ST ; r), and show that two regimes emerge. More precisely, we nd that the key criterion is the compact versus non-compact nature of the diffusion process, to be dened mathematically below. In the non-compact case, which physically corresponds to a diffusing molecule that sparsely explores its environment and leaves unvisited regions (typically a molecule diffusing in a dilute solution), we show that the kinetics are widely independent of the starting point. In the contrasting compact case of a diffusing molecule that densely explores its environment (for example a molecule in a very crowded medium), the position of the starting point strongly inuences the search time of the target, which leads us to introduce the concept of geometry-controlled kinetics. In the context of gene transcription, this result implies that the kinetics of activation of a gene T by a transcription factor can be orders of magnitudes faster if the transcription factor is released from a site that is colocalized with (that is, in the vicinity of) T, as compared with the case where the transcription factor is released from a remote site (Fig. 1).
Results
We consider a Markovian random walker of position r(t), whose dynamics are characterized by the dimension of the walk dw , dened by the scaling of the mean squared displacement kr 2(t)l / t 2/dw . The walker is conned in a domain of N sites with reecting walls. Additionally, we assume that the medium is of
1 ` UPMC Univ Paris 06, CNRS-UMR 7600 Laboratoire de Physique Theorique de la Matiere Condensee, 4 Place Jussieu, F-75005 Paris, France, 2 School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel; and Institute for Advanced Studies (FRIAS), University of Freiburg, 79104, Freiburg, Germany. * e-mail: benichou@lptmc.jussieu.fr
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rescaled variable u takes the following general form in the large volume limit: GTS (u) = (1 PTS )d(u) + PTS c(u) (4)
S2
S1
Figure 1 | First-passage time distribution (FPT) and geometry-controlled kinetics. Is the initial position of the particle an important parameter of reaction kinetics? We show quantitatively that in the case of non-compact exploration (for example, for dilute solutions), the kinetics turns out to be widely independent of the starting point (S1 or S2), whereas in the compact exploration case (for example, for crowded environments), the position of the starting point strongly inuences the search time of the target, leading to geometry-controlled kinetics. This result in particular implies that the kinetics of activation of a gene T by a transcription factor can be orders of magnitudes faster if the transcription factor is released from a site S ; S2, which is colocalized with T, as compared to the case where the transcription factor is released from a remote site S ; S1.
where the Dirac d function corresponds to trajectories hitting the target without reaching the boundary within a time of order r dw , which is much smaller than kTlT . The geometrical factor 1 2 PTS can be interpreted as the weight of these trajectories. Similarly, the contribution PTSc(u) accounts for trajectories reaching the boundary before the target. Note that the dependence on the starting point lies entirely in the geometrical factor PTS , whereas the time dependence is contained in the scaling function c. The geometrical factor PTS and the rescaled variable u are explicitly determined in the Supplementary Information and their scaling with the volume N and the distance r are obtained under the standard scale-invariance assumption 1 of the unconned propagator Wij (n) / ndf /dw f (|ri 2 rj|/ n1/dw ) (ref. 22). Actually, the FPT distribution given by equation (4) falls into a few universality classes, dened according to a purely geometrical criterion as detailed below. In the case of non-compact exploration, dened here by dw , df (ref. 23), where the mean number of distinct sites visited by the walker in the absence of connement grows linearly with the number of steps, we nd: stat kTlT = HTT /WT / N kT l 1 P = TS / 1 a TS r kTlT u c(u) = e
df dw
fractal type, so that its characteristic linear size R scales as R / N 1/df , where df is the fractal dimension22. We are interested in the distribution P(TTS) of the time it takes a walker starting from the site S to reach for the rst time the target site T located at a distance r from S. We start from the backward equation satised by the probability P(TTS t) in discrete time t (ref. 10) P(TTS = t) =
j
(5)
wjS P(TTj = t 1)
(1)
obtained by partitioning over the rst step of the walk, where wij is the transition probability from site j to site i. It is shown in the Supplementary Information that this equation, after Laplace transform, leads to the following hierarchy satised by the moments kTn l Tj of the FPT: kTn l = TS 1 stat WT
n j k=1
n (1)k+1 k + (HjS
stat HjT )WT ]kTnk l Tj
(2)
[(HTT Here
HTS )Wjstat
1
Hji =
n=0
(Wji (n) Wjstat )
(3)
where Wji (n) denotes the propagator of the walk, that is, the probability to be at site j at step n starting from site i, and Wstat is the j probability of being at site j in the stationary state. At this stage, the hierarchy of equation (2) remains formal as it involves the unknown function Hji , and does not allow an explicit determination of the FPT distribution. However, this difculty can be circumvented by taking the large volume limit and by considering stat the rescaled time u = TTS /kTlT , where kTlT = S kTTS lWS is the mean FPT to the target site rT, averaged over the initial position. Note that here we implicitly assume kTlT (as well as its disorder average in the case of disordered systems to be discussed below) to be nite. Actually, a detailed analysis of equation (2) in the Supplementary Information shows that the distribution of the
where a is a lattice-dependent constant of order 1. Note that the linear dependence on N of the scaling variable kTlT is the same as found in ref. 13 for the mean FPT kTTSl, and in particular does not depend on the dimensions df and dw. This general result includes the special case of regular diffusion in three dimensions (for which dw 2 and df 3) that is given in ref. 24. Strikingly, the exponential form of C hold for any dimensions such that dw , df. Note that although in the limit of r . step size the FPT distribution is a mere exponential of weight, it departs signicantly from this distribution if the starting point is close to the target. In the case of compact exploration, dw . df (ref. 23), where the mean number of distinct sites visited by the walker in the absence of connement grows slower than linearly with the number of steps, further hypotheses on the unconned propagator are needed to estimate the relative importance of the terms involved in equation (2). Making use of the OShaughnessyProcaccia operator25 to evaluate the large volume behaviour of Hij in equation (2), we nd that the FPT distribution obeys the generic form of equation (4) with stat kTlT = HTT /WT / N dw /df 2 2df kTTS l r dw df P = TS / R dw (df + dw ) kTlT (6) a2 dw df k 1 ak 32n (2) Jn (ak ) 2(d 2 d 2 ) u G(n) c(u) = 2df dw w f e 2 d 2 G(2 n) J1n (ak ) dw f k=0 where u . 0, n df/dw and a0 , a1 , , . . . stand for the zeros of the Bessel function J2n. Strikingly, the scaling with N of the scaling variable u is no longer given by the mean FPT kTTSl, which clearly indicates that several timescales are involved in the problem. The interplay between these timescales leads to a non-trivial family of universal non-exponential scaling functions, parametrized by dw and df. The geometrical factor strongly depends on the sourcetarget
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distance r, and in particular is very small for r R, in contrast to the non-compact situation. We add that the marginal case dw df , which is compact according to the denition given in ref. 23, corresponds to an exponential scaling function c as given by equation (5) with logarithmic corrections in the scalings of kTlT and PTS with r and N (see Supplementary Information). Equations (5) and (6) fully dene universality classes of FPT distributions in connement. Additional comments are as follows. (i) Whereas the linear scaling with N of the rst moment is universal, the scaling of higher moments differ in compact and non-compact cases. In particular, this scaling implies that whereas the reduced variance of the FPT is always of order one in the non-compact case, in the compact case it reads (kT2 l 2 kTTSl2)/kTTSl2 / (R/r)dw df , so very TS large uctuations occur for r R. (ii) Remarkably, the FPT distribution is entirely determined as soon as the mean kTTSl of the FPT is known (as well as its average over the starting point kTlT )2630, even if the distribution is not exponential. (iii) For specic cases, this mean FPT can be calculated exactly, which provides a fully explicit expression of the FPT distribution (as used in Figs 2b, 3a,b). (iv) In all cases it can be calculated in the large volume limit using recent results13, leading to the scaling in the geometrical parameters given in equations (5) and (6). We note that our approach covers in particular the important case of subdiffusion31, which is characterized by a sublinear dependence of the mean squared displacement with time (that is, dw . 2). Subdiffusion is widespread in complex crowded environments such as biological cells32,33, and might physically originate from a few classes of models based on different underlying microscopic mechanisms34. Importantly, subdiffusive processes can be either compact or non-compact, which will prove below to be the relevant criterion in the context of reaction kinetics. The FPT distribution for one class of models for subdiffusion, which rely on spatial inhomogeneities22 as exemplied by diffusion in fractals, is directly given by equations (5) and (6). Another class of models stems from large trapping times, leading to the case of innite kTlT , which we have discarded so far. Whereas the quenched version of this type of model becomes quite involved in the case of broad distribution of trapping times over the disorder, the FPT distribution in the annealed casethe continuous time-random walk model (CTRW), which is a standard random walk with random waiting times, drawn from a distribution f(t) (refs 31,3537)is straightforwardly deduced from equations (5) and (6). In this case, the Laplace transform of the FPT distribution reads: PCTRW (s) = P( f (s)), where P(s) is the generating function (discrete Laplace transform) of the FPT distribution of the underlying discrete time-random walk, which is determined in equations (5) and (6). These analytical results are validated by Monte Carlo simulations and exact enumeration methods, applied to various models, which illustrate the universality classes dened above. These schematic models have been widely used to describe transport in disordered media16,22for example in the case of exciton trapping on percolation systems38 or anomalous diffusion in biological cells39,40as a rst step to account for geometrical obstruction and binding effects involved in real crowded environments20,21. (i) The non-compact and marginal cases (see Fig. 2) are exemplied by: regular diffusion on three-dimensional (3D) and two-dimensional (2D) cubic lattices; diffusion on a 3D percolation cluster above criticality; and diffusion in disordered systems such as the random barrier model (namely a symmetric random walk on a 3D cubic lattice with transition rates G drawn from the normalized distribution r(G) / G2a) and the random trap model (namely a symmetric random walk on a 3D cubic lattice with frozen waiting times ti at each site drawn from the normalized distribution r(t) / t 2(1a)). (ii) The compact case (see Fig. 3) is exemplied by diffusion on deterministic fractals such as a Sierpinski gasket and T-graph and on a critical percolation cluster, as dened in Supplementary Fig. S1. Figures 2 and 3 reveal an excellent quantitative agreement between the asymptotic analytical
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a
Rescaled FPT distribution () 1
DOI: 10.1038/NCHEM.622
0.1
0.01
2D and 3D simple lattices (5 pairs) 3D supercritical percolation (5 pairs) Random barrier model (4 pairs) Random trap model (4 pairs) Theory (Eq. 5)
2 3 Rescaled time ()
b
()
1 0.1 0.01 0 1 2 3 4 Rescaled time () 5
100 20 20, T(20,5,10), S(70,15,10) 60 40 20, T(4,4,4), S(56,36,16) 80 80 10, T(25,25,5), S(55,55,5) 200 150, T(4,4), S(2,4) 100 80, T(54,40), S(56,40) Theory (Eq. 5)
c
()
1 0.1 0.01 0 1 2 3 4 Rescaled time () 5

Random clusters and S/T pairs Theory (Eq. 5)
d
()
1 0.2 0.04
= 0.8, T(2,2,2), S(28,28,28) = 0.5, T(2,2,2), S(3,3,3) = 0.2, T(10,10,10), S(20,20,20) = 0.2, T(2,2,2), S(3,3,3) Theory (Eq. 5)
0.008 0 1 2 3 4 Rescaled time () 5
e
()
5 1 0.2 0.04 0.008 0 1 2 3 4 Rescaled time () 5

= 2, 100 20 10, T(2,2,2), S(98,18,8) = 1.5, 100 80 10, T(60,60,5), S(20,20,5) = 1.5, 100 80 60, T(3,3,3), S(4,4,4) = 1, 100 20 20, T(50,10,10), S(40,8,8) Theory (Eq. 5)
Figure 2 | Universal FPT distribution in the non compact and marginal cases. The simulated distribution GTS(u ) divided by the weight PTS is plotted against the universal theoretical prediction c(u ) (equation (5)). The collapse of various examples onto a single master curve shows the universality of the result. a, All non-compact and marginal cases (plotted independently in be) collapse onto a single exponential master curve. b, Regular diffusion on a 3D cubic lattice and 2D square lattice for various rectangular domains (of sizes L1 L2 L3 and L1 L2) and sourcetarget pairs S(x,y,z) and T(x,y,z) whose rectangular coordinates are indicated in the legend inset. Here u and PTS are calculated using exact results for kTTSl and kTlT given in ref. 24. ce, Examples of disordered systems. Here, kTTSl and kTlT are evaluated numerically. c, Diffusion on a 3D percolation cluster above criticality embedded in a 30 30 30 rectangular domain with link probability p 0.4. d, 3D random barrier model (see text) embedded in a 30 30 30 rectangular domain. e, 3D random trap model (see text) embedded in various rectangular domains.
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Rescaled FPT distribution () 10
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involving an immobile target B and searcher particles A (ref. 1). When only a small number of A are involved in the reaction, as is the case in a microdomain in a biological cell, this reaction has to be described at the single molecule level6 and can be quantied by the survival probability of a particle A, S(t) = 1 t =0 P(t ), t which gives the probability that A has not reacted with B until time t. The quantity S(t) depends on the initial position of the molecule A and is explicitly determined using equations (5) and (6), which indicate that such reactions in connement obey universal kinetic laws, depending on the non-compact versus compact nature of the underlying transport process. In the non-compact case, which corresponds qualitatively to trajectories leaving many sites unvisited, as in the case of a 3D medium dilute enough to lead to regular diffusion, for any r signicantly greater than a typical molecular length, the geometrical factor PTS is close to 1, and the dependence on the initial position is lost. We therefore recover a simple rst-order decay of the survival probability, which depends on the volume of the conning domain only, and not on the initial position of the reactant. In this case of noncompact exploration, the initial position is not an important parameter of the kinetics (see Fig. 4), except in specic cases involving return times, such as recombination reactions. On the contrary, in the compact case, which means that each visited site is on average oversampled, geometrical factors dominate. This is typically the case of a crowded medium described to a rst approximation as a fractal structure where the available space for diffusion is restricted. Here, the temporal evolution of S(t) strongly depends on the starting position. S(t) drops to small valuesindicating that the reaction occurred with high probabilityon a timescale that depends on the volume, but also critically on the starting position of the reactant. This timescale ranges according to equations (4) and (6) from rdw (for starting positions such that r R) to Rdw (for starting positions such that r R), which in practice can span several orders of magnitude (see Fig. 4). In these types of geometry-controlled reactions (not to be confused with fractal-like reactions2), spatial organization of reactants plays a crucial role, which can be quantied by our approach. We stress that the decisive criterion leading to geometry-controlled kinetics is not the subdiffusive versus diffusive nature of the transport process, but rather the compact versus non-compact nature. We expect this effect to impact a wide class of reactions involving either an inhomogeneous initial concentration of reactants, such as the speckled distribution experimentally realized in ref. 41, or a small number of particles, such as biochemical reactions in cell subdomains. Notably, in the context of gene colocalization, our results give access to the kinetics of elementary steps of activation by transcription factors. As an illustrative example, let us consider two genes A and B, which share a common transcription factor (for example the genes sog and zen of the Drosophila genome, which are both targeted by Dorsal42). Experimental results concerning subdiffusive motion of tracer particles in the nucleus32,43,44 on the one hand and observations of a fractal organization of the chromatin on the other hand4446, provide the estimates 2 dw 3 and df 2.4, and therefore suggest that both compact and noncompact exploration cases could occur. Relying on the analysis of the survival probability developed previously, we nd that in the case of compact exploration (with, for example, df 2.4, and dw 3 as in ref. 43) that the typical time needed for the transcription factor to reach gene B starting from a gene A colocalized with B (typically rAB rcoloc 100 nm, which is the size of a transcription factory19) can be (rremote/rcoloc)dw 103 times faster than for a remote gene A (typically rAB rremote 1 mm, which is the order of magnitude of a nucleus radius). This is in strong contrast with the case of noncompact exploration (with for example df 2.4 and dw 2)32 where the typical activation time of B starting from A has the same order of magnitude for A either colocalized with B or remote. This
475
1.5 1
Sierpinski gasket T-graph
1 0.2 Theory (Eq. 6) N = 123 N = 366 N = 1,095 0 1 2 Rescaled time () 3 4 0.4 0.6 0.8
0.1
10
1 ()
0.1
Theory (Eq. 6) N = 82 N = 244 N = 730 0 1 2 Rescaled time ( ) 3 4
c
10
()
0.1
Theory (Eq. 6) 20 20 20 domains 30 30 30 domains 40 40 40 domains 0 1 2 Rescaled time () 3 4
Figure 3 | Universal FPT distributions in the compact case. The simulated distribution GTS(u) divided by the weight PTS is plotted against the universal theoretical prediction c(u ) (equation (6)). The collapse for different system sizes N shows the universality of the results. a,b, Examples of deterministic fractals. Diffusion on a Sierpinski gasket (with a target at the apex site) (a) and on a T graph (with a target at the centre) (b). Here, exact expressions are used for calculating kTTSl and kTlT . The inset shows that the scaling function c weakly depends on the dimensions df and dw . c, Diffusion on a 3D critical percolation cluster (random fractal) embedded in rectangular domains of sizes (L1 L2 L3), as indicated in the inset. Here, kTTSl and kTlT are evaluated numerically, and average over pairs of points is performed, to fulll the scale-invariance hypothesis of the propagator (see text before equation (5)).
predictions and the numerical simulations, even for systems of small size. We emphasize that despite their very different nature, all these models fall into the above dened universality classes.
Discussion
We now discuss important implications of these results for reaction kinetics, using the ubiquitous example of a target search process
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a
1
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b
1
DOI: 10.1038/NCHEM.622
0.8 Survival probability Survival probability
0.8
0.6
0.6
0.4 r = 38 r = 20 r = 10 r=5 r=2 Theory 0.001 0.01 0.1 Rescaled time ()
0.4
0.2
0.2
r = 45 r = 23 r = 12 r=7 r=4 r=2 Theory 0.001 0.01 0.1 Rescaled time ()
0 0.0001
10
0 0.0001
10
Figure 4 | Reaction kinetics as quantied by the survival probability S(t), plotted for different sourcetarget distances r. a,b, The non-compact case (a) is exemplied by 3D regular diffusion and the compact case (b) by diffusion on a Sierpinski gasket (with a target at the apex). The theoretical prediction for S(t) is obtained from equations (5) and (6). Here u and PTS are calculated using exact results for kTTSl and kTlT. Quantitatively, the typical reaction time ttyp can be dened, for example, through the median S(ttyp) 1/2, indicated by the dotted line. In the non-compact case, ttyp weakly depends on the initial position of the reactant, which therefore is not an important parameter of the kinetics. On the contrary, in the compact case, ttyp runs over several orders of magnitude depending on the initial position, which, in turn, controls the kinetics.
suggests that gene colocalization is highly favourable for transcription kinetics, but only when it is geometrically controlled (that is, in the case of compact exploration), which makes the experimental characterization of the nature of transport in the nucleus a major issue. To conclude, we calculated analytically the FPT distribution of a diffusing particle to a target, and showed that transport processes as various as regular diffusion, anomalous diffusion, diffusion in disordered media and in fractals fall into the same universality classes. Our results put forward that geometry, and in particular the initial localization of reactants, can become a key parameter of reaction kinetics in connement. In particular, this regime of geometry-controlled kinetics could be relevant to transcription kinetics and could help understand the crucial role of spatial organization of genes.
Received 30 November 2009; accepted 9 March 2010; published online 18 April 2010
References
1. Rice, S. Diffusion-Limited Reactions (Elsevier, 1985). 2. Kopelman, R. Fractal reaction kinetics. Science 241, 16201626 (1988). 3. Shlesinger, M. F., Zaslavsky, G. M. & Klafter, J. Strange kinetics. Nature 363, 3137 (1993). 4. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 16001603 (2006). 5. Elf, J., Li, G.-W. & Xie, X. S. Probing transcription factor dynamics at the singlemolecule level in a living cell. Science 316, 11911194 (2007). 6. Schuss, Z., Singer, A. & Holcman, D. The narrow escape problem for diffusion in cellular microdomains. Proc. Natl Acad. Sci. USA 104, 1609816103 (2007). 7. Loverdo, C., Benichou, O., Moreau, M. & Voituriez, R. Enhanced reaction kinetics in biological cells. Nature Phys. 4, 134137 (2008). 8. Mirny, L. Biophysics: Cell commuters avoid delays. Nature Phys. 4, 9395 (2008). 9. Benichou, O., Loverdo, C., Moreau, M. & Voituriez, R. Optimizing intermittent reaction paths. Phys. Chem. Chem. Phys. 10, 70597072 (2008). 10. Redner, S. A Guide to First Passage Time Processes (Cambridge University Press, 2001). 11. Shlesinger, M. F. Mathematical physics: rst encounters. Nature 450, 4041 (2007). 12. Condamin, S., Benichou, O. & Moreau, M. First-passage times for random walks in bounded domains. Phys. Rev. Lett. 95, 260601 (2005). 13. Condamin, S., Benichou, O., Tejedor, V., Voituriez, R. & Klafter, J. First-passage times in complex scale-invariant media. Nature 450, 7780 (2007). 14. Benichou, O. & Voituriez, R. Narrow-escape time problem: Time needed for a particle to exit a conning domain through a small window. Phys. Rev. Lett. 100, 168105 (2008). 15. Benichou, O., Meyer, B., Tejedor, V. & Voituriez, R. Zero constant formula for rst-passage observables in bounded domains. Phys. Rev. Lett. 101, 130601 (2008).
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16. Bouchaud, J.-P. & Georges, A. Anomalous diffusion in disordered media: Statistical mechanisms, models and physical applications. Phys. Rep. 195, 127293 (1990). 17. Kurzynski, M., Palacz, K. & Chelminiak, P. Time course of reactions controlled and gated by intramolecular dynamics of proteins: Proc. Natl Acad. Sci. USA 95, 1168511690 (1998). 18. Kolesov, G., Wunderlich, Z., Laikova, O. N., Gelfand, M. S. & Mirny, L. A. How gene order is inuenced by the biophysics of transcription regulation. Proc. Natl Acad. Sci. USA 104, 1394813953 (2007). 19. Fraser, P. & Bickmore, W. Nuclear organization of the genome and the potential for gene regulation. Nature 447, 413417 (2007). 20. Zimmerman, S. B. & Minton, A. P. Macromolecular crowding: Biochemical, biophysical, and physiological consequences. Ann. Rev. Biophys. Biom. 22 (1993). 21. Zhou, H.-X., Rivas, G. & Minton, A. P. Macromolecular crowding and connement: Biochemical, biophysical, and potential physiological consequences. Ann. Rev. Biophys. 37, 375397 (2008). 22. Ben-Avraham, D. & Havlin, S. Diffusion and Reactions in Fractals and Disordered Systems (Cambridge Univ. Press, 2000). 23. de Gennes, P. G. Kinetics of diffusion-controlled processes in dense polymer systems. i. nonentangled regimes. J. Chem. Phys. 76, 33163321 (1982). 24. Condamin, S., Benichou, O. & Moreau, M. Random walks and brownian motion: a method of computation for rst-passage times and related quantities in conned geometries. Phys. Rev. E 75, 021111 (2007). 25. OShaughnessy, B. & Procaccia, I. Diffusion on fractals. Phys. Rev. A 32, 30733083 (1985). 26. Montroll, E. W. Random walks on lattices. iii. calculation of rst-passage times with application to exciton trapping on photosynthetic units. J. Math. Phys. 10, 753765 (1969). 27. Kozak, J. J. & Balakrishnan, V. Analytic expression for the mean time to absorption for a random walker on the sierpinski gasket. Phys. Rev. E 65, 021105 (2002). 28. Agliari, E. Exact mean rst-passage time on the t-graph. Phys. Rev. E 77, 011128 (2008). 29. Haynes, C. P. & Roberts, A. P. Global rst-passage times of fractal lattices. Phys. Rev. E 78, 041111 (2008). 30. Tejedor, V., Benichou, O. & Voituriez, R. Global mean rst-passage times of random walks on complex networks. Phys. Rev. E 80, 065104 (2009). 31. Metzler, R. & Klafter, J. The random walks guide to anomalous diffusion: a fractionnal dynamics approach. Phys. Rep. 339, 177 (2000). 32. Wachsmuth, M., Waldeck, W. & Langowski, J. Anomalous diffusion of uorescent probes inside living cell nuclei investigated by spatially-resolved uorescence correlation spectroscopy. J. Mol. Biol. 298, 677689 (2000). 33. Golding, E. & Cox, E. Physical nature of bacterial cytoplasm. Phys. Rev. Lett. 96, 981102 (2006). 34. Condamin, S., Tejedor, V., Voituriez, R., Benichou, O. & Klafter, J. Probing microscopic origins of conned subdiffusion by rst-passage observables. Proc. Natl Acad. Sci. USA 105, 56755680 (2008). 35. Montroll, E. W. & Weiss, G. H. Random walks on lattices. ii. J. Math. Phys. 6, 167181 (1965).
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44. Bancaud, A. et al. Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin. EMBO J. 28, 37853798 (2009). 45. Lebedev, D. V. et al. Fractal nature of chromatin organization in interphase chicken erythrocyte nuclei: Dna structure exhibits biphasic fractal properties. FEBS Lett. 579, 14651468 (2005). 46. Lebedev, D. et al. Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization. Crystallogr. Rep. 53, 110115 (2008).
36. Shlesinger, M. F. Asymptotic solutions of continuous-time random walks. J. Stat. Phys. 10, 421434 (1974). 37. Condamin, S., Benichou, O. & Klafter, J. First-passage time distributions for subdiffusion in conned geometry. Phys. Rev. Lett. 98, 250602 (2007). 38. Parson, R. P. & Kopelman, R. Percolative versus homogenous energy transport kinetics: time-resolved donor and acceptor uoresence of isotopic mixed naphthalene crystals. Chem. Phys. Lett. 87, 528532 (1982). 39. Saxton, M. J. A biological interpretation of transient anomalous subdiffusion. ii. reaction kinetics. Biophys. J. 94, 760771 (2008). 40. Malchus, N. & Weiss, M. Elucidating anomalous protein diffusion in living cells with uorescence correlation spectroscopyfacts and pitfalls. J. Fluoresc. (2009). 41. Monson, E. & Kopelman, R. Observation of laser speckle effects and nonclassical kinetics in an elementary chemical reaction. Phys. Rev. Lett. 85, 666669 (2000). 42. Markstein, M., Markstein, P., Markstein, V. & Levine, M. S. Genome-wide analysis of clustered dorsal binding sites identies putative target genes in the drosophila embryo. Proc. Natl Acad. Sci. USA 99, 763768 (2002). 43. Platani, M., Goldberg, I., Lamond, A. I. & Swedlow, J. R. Cajal body dynamics and association with chromatin are atp-dependent. Nat. Cell Biol. 4, 502508 (2002).
Acknowledgements
Support from ANR grants DYOPTRI and DYNAFT is acknowledged.
All authors contributed equally to this work.
The authors declare no competing nancial interests. Supplementary information accompanies this paper at www.nature.com/naturechemistry. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to O.B.
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Efcient stereo- and regioselective hydroxylation of alkanes catalysed by a bulky polyoxometalate

Keigo Kamata1,2, Kazuhiro Yonehara1, Yoshinao Nakagawa1, Kazuhiro Uehara1,2 and Noritaka Mizuno1,2 *
Direct functionalization of alkanes by oxidation of CH bonds to form alcohols under mild conditions is a challenge for synthetic chemistry. Most alkanes contain a large number of CH bonds that present difculties for selectivity, and the oxidants employed often result in overoxidation. Here we describe a divanadium-substituted phosphotungstate that catalyses the stereo- and regioselective hydroxylation of alkanes with hydrogen peroxide as the sole oxidant. Both cyclic and acyclic alkanes were oxidized to form alcohols with greater than 96% selectivity. The bulky polyoxometalate framework of the catalyst results in an unusual selectivity that can lead to the oxidation of secondary rather than the weaker tertiary CH bonds. The catalyst also avoids wasteful decomposition of the stoichiometric oxidant, which can result in the production of hydroxyl radicals and lead to non-selective oxidation and overoxidation of the desired products.
he selective transformation of inert CH bonds of alkanes into useful functional groups has attracted much attention because alkanes are less expensive and more readily available than the current petrochemical feedstocks13. The catalytic hydroxylation of alkanes under mild conditions remains a major challenge in industrial and synthetic chemistry47. Among various oxidants, molecular oxygen is used widely in homogeneous and heterogeneous industrial processes. However, the scope of homogeneous aerobic oxidation reactions is narrow because autoxidation reactions are only compatible with the production of terephthalic acid and cyclohexanone. Although hydrogen peroxide (H2O2) is an economically and environmentally desirable oxidant in comparison with peracids8, organic hydroperoxides9, N-oxides10,11 and iodosobenzene12, the combination of metal complexes with H2O2 often generates hydroxyl radicals. The disadvantages of H2O2-based systems are:
bonds (that is, adjacent to a heteroatom, a p-system and/or an electron-rich tertiary CH bond) are hydroxylated selectively in most metal-catalysed oxidation systems. Polyoxometalates (transition-metal oxygen anion clusters) were applied in various elds, such as structural chemistry, analytical chemistry, surface science, medicine, electrochemistry, photochemistry and catalysis2731. Our approach to the design of active catalysts for the regioselective hydroxylation of alkanes is to create strong electrophilic oxidants with high steric hindrance based on the concepts of: synthesis of a divanadium site for the cooperative activation of H2O2; control of the nature of the oxidant by changing the hetero atom; steric protection of the catalyst by using a rigid and bulky allinorganic polyoxometalate ligand3235. In this paper, we report the efcient, stereospecic and regioselective hydroxylation of alkanes with H2O2 catalysed by a divanadium-substituted phosphotungstate, [g-H2PV2W10O40]32 (1a). High selectivity for alcohols, efciency of H2O2 utilization and stereospecicity were observed. This system also showed specic regioselectivity for secondary alcohols in the oxidation of some cycloalkanes that have both secondary and tertiary CH bonds. This study provides the rst example of a synthetic catalyst that can achieve specic, regioselective, H2O2-based hydroxylation of secondary CH bonds, even in the presence of more reactive tertiary CH bonds.
selectivity for alcohols is low because of overoxidation; non-productive decomposition of H2O2 leads to low efciencies of H2O2 utilization; indiscriminate attack of hydroxyl radicals results in low stereospecicity and regioselectivity. Therefore, efcient H2O2-based catalytic oxidation systems were limited to a few examples of organocatalysts13 and transition-metal catalysts, such as iron1418, vanadium1921, osmium22 and manganese23,24, and the development of efcient, stereospecic and regioselective hydroxylation of various kinds of alkanes with H2O2 under mild conditions is in great demand. Natural enzymes (for example, methane monooxygenases and fatty-acid desaturases) use molecular recognition, such as size and/or shape selectivity and substrate orientation, to achieve high chemo-, regio- and stereoselectivities25. On the basis of these strategies, manganese- and iron-based biomimetic selective oxidations were studied2426. However, these systems need a directing carboxylic acid group in the substrate to achieve the high selectivity, and also susceptibility of the organic ligands to oxidative self-degradation has limited their usefulness. In addition, the activated CH
1
Results
Compound 1 was synthesized by the cation-exchange reaction of the caesium salt of deprotonated [g-PV2W10O40]52 with an alkylammonium and subsequent protonation with perchloric acid. An X-ray crystallographic structural analysis of anion 1a was carried out on the tetraethylammonium salt derivative. 1a had two vanadium atoms in the g-Keggin-type polyoxotungstate structure (Fig. 1a). The bond-valence sums of vanadium (5.20), tungsten (5.986.12) and phosphorous (5.17) indicated that the anion was composed of vanadium(V), tungsten(VI) and phosphorous(V) ions.
Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan, 2 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. * e-mail: tmizuno@mail.ecc.u-tokyo.ac.jp
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a
O101'
DOI: 10.1038/NCHEM.648
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b
OH C R2 V H O O H V
O101
O1' O2' O3' V101' O1 V101 O2
O3
R1
R3
H2O2 H2O Step 1 H2O
[-PV2W10O38(OH)2]3 1a H2O2 H R1 R3 R2 + H2O O O V H2O H O O O H C Step 3
[-PV2W10O38(O2)]3 1c Step 2
[-PV2W10O38(OH)(OOH)]3 1b
H2O
[(n-C4H9)4N]4[-HPV2W10O40] (1.3 mM), HClO4 (1.3 mM) 24% H218O2 (50 mM; 18O content: 90%) CH3CN/t-BuOH (0.67/1.33 ml) 333 K, 120 minutes
18
OH 80% yield 98% selectivity 18 O content: 90 1%
2 (9.7 M)
Figure 1 | Structure and reactivity of a divanadium-substituted phosphotungstate catalyst for the selective oxidation of alkanes. a, X-ray crystal structure of 1a (the tetraethylammonium counterion is omitted for clarity). Yellow, grey, green and red circles represent vanadium, tungsten, phosphorus and oxygen, respectively. b, Proposed mechanism for the hydroxylation of alkanes with H2O2 catalysed by 1a. c, Hydroxylation of cyclohexane catalysed by 1a using H218O2 gave cyclohexanol in 80% yield. The 18O content of the alcohol product (90+1%) remained constant throughout the reaction, which shows that all the oxygen atoms in the product originated from the H2O2 oxidant.
The bond-valence sum of atom O1 (1.34) was lower than those of the other oxygen atoms (1.712.08), which indicates that O1 and O1 are protonated (hydroxo ligands). The 31P NMR spectrum in CD3CN showed a single line at 213.8 ppm. The 51V NMR spectrum in CD3CN gave a single line at 2578 ppm, which shows two equivalent vanadium atoms. The 1H NMR spectrum in CD3CN gave a line at 7.0 ppm (2H per anion), which can be assigned to the bis(m-hydroxo) groups. All NMR data were consistent with the characterization of 1a in CD3CN as a single species of formula [g-H2PV2W10O40]32. The oxidation of various cyclic alkanes with H2O2 catalysed by 1a was investigated (Table 1). The efciency of H2O2 utilization was more than 80% in each case (Table 1, entries 16). The oxidation of cyclic alkanes 26 proceeded selectively to give the corresponding alcohols 812 (98% selectivity) without signicant formation of ketones, carboxylic acids and products of cleaved CC bonds (Table 1, entries 15). Under stoichiometric conditions (Table 1) the selectivity of the oxidation of cyclohexane 2 (50 mM) to give cyclohexanol 8 was 8798% (with a conversion range of 1.18.2%), a value larger than or comparable to those of the hydroxyl radical ( 50%) (ref. 36), Fe(III)-porphyrin/iodosobenzene (8394%) (ref. 36) and H2O2-based vanadium (5090%) (refs 1921), iron (081%) (refs 1418), manganese ( 50%) (ref. 23) and osmium (70%) (ref. 22) systems. Secondary alcohols react very poorly with the bis(m-hydroxo) divanadium site in [g-H2SiV2W10O40]42 because of the steric crowding between the polyoxometalate framework and substrates. Therefore, this high selectivity with 2 results from suppression of the oxidation of 8 by the steric hindrance of 1a. Adamantane (5) was hydroxylated preferentially at the electronrich tertiary CH bonds (Table 1, entry 4). The selectivity ratio of tertiary/secondary (38/28) CH activation normalized to the number of CH bonds was 18. This value is comparable to those of cytochrome P450 and haem catalysts (6 48) and much larger
than those of hydroxyl (2) and t-butoxy radical (10) systems36. Also, acyclic n-hexane (7) was hydroxylated to give the corresponding alcohols (13a13c) with 96% selectivity (Table 1, entry 6). This system was applied on a larger scale (scaled up 33-fold) to hydroxylations of cycloalkanes; 0.29 g of 8 (85% isolated yield based on H2O2), 0.37 g of cyclooctanol (9) (80%) and 0.47 g of cyclododecanol (10) (77%) were isolated with 99% purity according to 1H NMR spectroscopy (Table 1, entries 13). The turnover frequency and efciency of H2O2 utilization for the oxidation of 2 were 710 h21 and 90%, respectively, the highest values among those reported for H2O2-based catalytic systems so far: [Fe(TPA)(CH3CN)2](ClO4)2 , 17 h21, 43% (ref. 14) (TPA tris(2-pyridylmethyl)amine); [(N4Py)Fe(CH3CN)](ClO4)2 , 89 h21, 44% (ref. 15) (N4Py N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine); [Fe(CF3SO3)2((S,S,R)-mcpp)], 296 h21, 66% (ref. 18) ((S,S,R)mcpp N,N -dimethyl-N,N -bis[(R)-[4,5]-pineno-2-pyridylmethyl] [(1S,2S)-1,2-cyclohexanediamine]); [V(O)(Cl)(PBHA)2], 19 h21, 8% (ref. 19) (PBHA N-phenyl benzohydroxamate); [(VO)4(hptb)2(H2O)2(m-O)](ClO4)4 , 98 h21, 12% (ref. 20) (hptb N,N,N,N -tetrakis(2-benzimidazolylmethyl)-2-hydroxo-1,3-diamino propane); VOSO4/HNO3 , 9 h21, 11% (ref. 21). Although the turnover frequencies of [Fe(III)2OL2(NO3)2 (CH3OH)2](NO3)2 (785 h21) (ref. 17) (L 2,6-bis(N-methylbenzimidazol-2-yl)pyridine), [Mn2O3(TMTACN)](PF6)2/oxalic acid (1,500 h21) (ref. 23) (TMTACN 1,4,7-trimethyl-1,4,7-triazacyclononane) and [(n-C4H9)N][Os(N)Cl4]/FeCl3/CH3COOH (840 h21) (ref. 22) were higher than that of 1a, the selectivity to give 8 (1768%) was lower than that of 1a.
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Table 1 | H2O2 hydroxylation of alkanes catalysed by 1a.
Entry 1*
2
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Substrate
Time (h) 1
Yield (%) 92 (85)
Product (selectivity (%))

OH
H2O2 efciency (%) 94
8 (98)
2
3
84 (80)
9 (99)
OH
84
3
4
79 (77)
OH
80
10 (98)
4
5
98
OH
OH
OH
98
OH
11a (82)
11b (15)
11c (3)
5
6
80
12a (94)
OH OH 12b (6)
80
56
OH 13a (2) OH 13b (66)
OH
56
13c (26)
Reaction conditions: [(n-C4H9)4N]4[g-HPV2W10O40] (1.3 mM), HClO4 (1.3 mM), substrate (2.5 M), CH3CN/t-BuOH (0.67/1.33 ml), 30% aqueous H2O2 (50 mM), 333 K. Yield (%) products (mol)/initial H2O2 (mol) 100. H2O2 efciency (%) (alcohols (mol) 2 ketones (mol))/consumed H2O2 (mol) 100. The values in parentheses are isolated yields. *2 (4.7 M), cyclohexanone (2% selectivity). Cyclooctanone (2% selectivity). Cyclododecanone (2% selectivity). 5 (0.3 M). 7 (7.5 M), 342 K, 2-hexanone (4% selectivity).
The regioselectivity for the hydroxylation of cycloalkanes 1419, with both secondary and tertiary CH bonds, was investigated (Table 2). For all substrates, stereospecic hydroxylations were observed. The high stereospecicity is comparable to those (9299%) of stoichiometric organic oxidants, such as peruorodialkyldioxiranes, peruorodialkyloxaziridines and p-nitroperoxybenzoic acid, for which a concerted mechanism mediated by an electrophilic oxidant has been proposed3739. In systems catalysed by organometallic complexes that do not have bulky ligands and in stoichiometric systems with dioxirane complexes, the activated tertiary CH bonds of cycloalkanes with both secondary and tertiary CH bonds are hydroxylated selectively to give the corresponding tertiary alcohols (Table 3 and Supplementary Tables S1 and S2)4047. However, the unactivated secondary CH bonds were hydroxylated selectively with product ratios (28 alcohols)/(38 alcohols) that ranged from 78/22 to .99/,1 for the cycloalkanes listed in Table 2. Such specic selectivities are different from those of radical reactions and are observed for the reaction of platinum(II) complexes with alkanes via oxidative addition, in which steric factors play an important role in determining the unusual selectivity48. For the oxidation of trans-1,2-dimethylcyclohexane (14), selectivity for the secondary alcohols was 90% (Table 2, entry 1) and the value was higher than that (80%) of the sterically hindered metalloporphyrin Mn(II)(TPFPP)(ClO4)/m-chloroperoxybenzoic acid (m-CPBA) (TPFPP meso-tetrakis(pentauorophenyl)porphinato) system8 and much higher than those of H2O2-based oxidation systems, such as [(N4Py)Fe(CH3CN)](ClO4)2 (,1%) (ref. 15), [(n-C4H9)4N][Os(N)Cl4]/FeCl3/CH3COOH (69%) (ref. 22) and [Fe(TPA)(CH3CN)2](ClO4)2 (50%) (ref. 14). In addition, for the oxidation of 14 and trans-decalin (15), with two adjacent tertiary
480
CH groups, the selectivities for trans-3,4-dimethylcyclohexanol (20c) and trans-2-decalinol (21a) were 86% and 93%, respectively (Table 2, entries 1 and 2), and mixtures of various secondary alcohols and the corresponding ketones have been obtained for the iron- and osmium-based systems14,15,22. Such high regioselectivities for the secondary alcohols in the presence of the more electron-rich tertiary CH bonds have not been reported before. Also, monoalkyl-substituted cyclohexanes, such as methylcyclohexane (17), ethylcyclohexane (18) and t-butylcyclohexane (19), hydroxylated selectively to give the corresponding secondary alcohols with 81% selectivity (Table 2, entries 46). 5 is an exception to this pattern of oxidation of cyclic alkanes that have both secondary and tertiary CH bonds. The six-membered rings of both 5 and the cyclic alkanes 1419 have chair conformations, but the tertiary CH bonds of 5 are at the equatorial positions and the tertiary CH bonds of the cyclic alkanes 1419 are at the axial positions. Therefore, the steric hindrance around the tertiary CH bonds of 5 is much smaller than those of the cyclic alkanes 1419. Such a difference results in the low selectivity for secondary alcohol in the oxidation of 5. The competitive oxidation between 2 and cyclohexane-d12 shows an intermolecular kinetic isotope effect (the ratio of the hydrogen and deuterium reaction rate constants, kH/kD) of 3.2, larger than those (1 2) associated with radical chain autoxidations36. The addition of the radical scavenger 2,6-di-t-butyl-4-methylphenol (ve equivalents with respect to 1a) did not affect the reaction rate, selectivity and total yield for the oxidation of 5. All the data, including the alcohol/ketone ratio for cyclohexane oxidation, 38 for the oxidation of 5, regioselectivity, stereospecicity and /28 kinetic isotope effect show the formation of a non-free-radical, electrophilic, metal-based oxidant in 1a.
NATURE CHEMISTRY
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Product (selectivity (%))
OH HO OH (10) 20b (4) 20c (86)
Table 2 | Regioselective H2O2 hydroxylation of alkanes to give secondary alcohols catalysed by 1a.
Entry 1
14 20a
Substrate
Time (h) 1
Yield (%) 59
[28 alcohols]/ 8 [38 alcohols] 8 90/10
2*
51
OH (93)
.99/,1
H 15
H 21a
72
OH (22)
OH H (36) OH (40) H 22c
78/22
H 16
H 22a
H 22b
75
OH OH (19) (6) OH 23a 23b 23c (44) OH 23d (24)
81/19
17
64
OH (3) OH (7) (53) OH (25)
OH (4)
97/3
18 24a 24b 24c
OH 24d
24e
67
(63) OH (24)
.99/,1
19 25a
OH 25b
Hydrolysis reaction conditions: [(n-C4H9)4N]4[g-HPV2W10O40] (1.3 mM), HClO4 (1.3 mM), substrate (2.5 M), CH3CN/t-BuOH (0.67/1.33 ml), 30% aqueous H2O2 (50 mM), 333 K. Yield (%) products (mol)/initial H2O2 (mol) 100. *Trans-2-Decalone (7% selectivity). Cis-2-Decalone (1% selectivity). 3-Methylcyclohexanone (4% selectivity) and 4-methylcyclohexanone (2% selectivity). 3-Ethylcyclohexanone (6% selectivity) and 4-ethylcyclohexanone (2% selectivity). 3-Butylcyclohexanone (10% selectivity) and 4-butylcyclohexanone (3% selectivity).
The reactivity of 1a in dichloroethane with H2O2 (9095% aqueous solution) at 253 K was investigated. The cold-spray ionization mass spectrum of 1a in 1,2-dichloroethane shows that the most intense parent-ion peak (centred at m/z 3,583) has an isotopic distribution that agrees with the pattern calculated for [((n-C4H9)4N)4H2PV2W10O40] and a fragment peak (centred at m/z 3,565) with an isotopic distribution that agrees with the pattern calculated for [((n-C4H9)4N)4PV2W10O39] (Supplementary Fig. S3). On addition of H2O2 , a new peak centred at m/z (3,583 16) appeared and agrees with the pattern calculated for [((n-C4H9)4N)4HPV2W10O39(OOH)]. The 1H NMR spectrum of 1a in dichloroethane showed a signal at 5.48 ppm from the hydroxyl proton of the (VO-(m-OH)2-VO) core in 1a (Supplementary Fig. S1a). Two new 1H NMR signals appeared at 9.49 and 5.31 ppm with intensity ratios of 1:1 on the addition of H2O2 (Supplementary Fig. S1b). The intensity of the signal at 5.48 ppm decreased and the sum of the three signal intensities remained constant. The chemical shift of 9.49 ppm is close to those of [g-SiV2W10O38(OH)(OOH)]42 (9.45 ppm) and other hydroperoxide species such as H2O2 (8.74 ppm), t-butyl hydroperoxide (8.84 ppm) and cumen hydroperoxide (8.95 ppm) (refs 33,34). Therefore, the 1H NMR signals at 9.49 and 5.31 ppm can be assigned to the protons of the hydroperoxo and hydroxo
groups in [g-PV2W10O38(OH)(OOH)]32 (1b), respectively. One new 51V NMR signal appeared at 539 ppm on the addition of H2O2 , and the intensity of the signal of 1a at 574 ppm decreased, although the sum of the two signal intensities remained almost constant (Supplementary Fig. S2). The oxo hydroperoxo transformations produced 1530 ppm (from [HxMoO2(O2)2](2x) (0 x 1) to [MoO(O2)2OOH]222) and 32 ppm (from [g-SiV2W10O38(OH)2]42 to [g-SiV2W10O38(OH)(OOH)]42) downeld shifts in the 95Mo and 51 V NMR signals, respectively33,34,49. The downeld shift of 35 ppm in our experiments is in accordance with the oxo hydroperoxo transformation. Detection of 1b by 183W NMR spectroscopy was unsuccessful because the solubility of 1b in 1,2-dichloroethane is very low, below the detection limit of 183W nuclei. In CH3CN/ t-BuOH (volume/volume 1/2), a new signal at 630 ppm (1c) was observed in addition to the two signals of 1a and 1b (Supplementary Fig. S4). The effects of H2O2 and H2O on the formation of 1b and 1c were investigated (Supplementary Fig. S5). The ratio of [1b]/[1a] was proportional to [H2O2]/[H2O], which indicates the reversible formation of 1b. The ratio of [1c]/[1a] was proportional to [H2O2]/[H2O]2, which suggests the successive dehydration of 1b to form 1c. The two vanadium atoms in 1c are equivalent because only one 51V NMR signal was observed for 1c. In addition, 1c was formed by the dehydration of 1b. Therefore,
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Table 3 | Regioselectivity for the oxidation of 14.
OH + OH HO +
NATURE CHEMISTRY
DOI: 10.1038/NCHEM.648
O O + +
14
26
27
28
29
30
Entry 1 2 3 4 5 6 7},# 8},** 9 10}, 11 12 13 14
System 1a/H2O2 (this work) CH3ReO3/H2O2 [Fe(II)(TPA)(CH3CN)2](ClO4)2/H2O2 [(N4Py)Fe(II)(CH3CN)](ClO4)2/H2O2 [Mn(IV)2O3(TMTACN)2](PF6)2/peracetic acid [Mn(II)(BQEN)(CF3SO3)2]/peracetic acid Co(III)(TPFPP)(CF3SO3)/m-CPBA Mn(II)(TPFPP)(ClO4)/m-CPBA (n-Bu4N)[Os(VIII)(N)O3]/Fe(III)Cl3/Cl2PyO Fe(III)(TPFPP)Cl/PhI(OAc)2 Cr(VI)O2(OAc)2/H5IO6 Methyl(triuoromethyl)dioxirane/TFP Dimethyldioxirane Peruoro cis-2-n-butyl-3-n-propyloxaziridine
Yield (%) 59 20 23 2 7 48 51 45 66 27 18 89 45 67
Selectivity (%) 26 27 28 29 30 Others 10 4 86 .98* 50 50 .99 33 20 47 38 62 84 16 20 80 30 27 27 15 37 63 .99 90 6 4 .99 .99
[28 alcohols]/ 8 [38 alcohols] 8 90/10 0/.99 50/50 0/.99 67/33 62/38 16/84 80/20 69/31 63/37 0/.99 10/90 0/.99 0/.99
[cis-26]/ [trans-26]
E ox (%)
Ref. 40 14 15 41 42 43 8 10 45 46 38 47 39
0/.99 59 39 0/.99 23 58/42 2 9/91 10 0/.99 .48 0/.99 51 0/.99 45 ,5/.95 76 0/.99 27 0/.99 18 0/.99 98 0/.99 45 0/.99 67
The yield (%) for the regioselectivity is (26 27 28 29 30 others) (mol)/oxidant used (mol) 100; selectivity (%) product (mol)/total products (mol) 100; Eox (%) (26 27 28 29 2 30 2 others) (mol)/oxidant used (mol) 100. *7-Hydroxy-2-octanone (.99% selectivity). TPA tris(2-pyridylmethyl)amine, 27 28 (50% selectivity). N4Py N,N-bis(2-pyridylmethyl)-N-bis(2pyridyl)methylamine. TMTACN 1,4,7-trimethyl-1,4,7-triazacyclononane, 27 28 (20% selectivity) and 29 30 (47% selectivity). BQEN N,N dimethyl-N,N -bis(8-quinolyl)ethane-1,2-diamine, 27 28 29 30 (62% selectivity). }TPFPP meso-tetrakis(pentauorophenyl)porphinato. #27 28 (16% selectivity). **27 28 (80% selectivity). Cl2PyO 2,6-dichloropyridine N-oxide. 27 28 (63% selectivity). TFP 1,1,1-triuoropropanone.
the m-h 2:h 2-peroxo species [g-PV2W10O38(O2)]32 (1c) is a possible active species similar to that used in epoxidation catalysed by [g-H2SiV2W10O40]42 catalysed epoxidation (refs 33,34). Kinetic study of the 1a-catalysed hydroxylation revealed rst-order dependencies of the reaction rate on the concentrations of 1a (0.25 1.25 mM), 5 (35250 mM) and H2O2 (1580 mM), and an inversely second-order dependency on the concentration of water (190 530 mM). These kinetic results suggest that [g-PV2W10O38(O2)]32 (1c) is the active species for the hydroxylation and that the reaction of an alkane with 1c is the rate-determining step (Fig. 1b). The steric effect of the all-inorganic, rigid and bulky polyoxometalate framework in 1c leads to a specic regioselectivity for the hydroxylation of alkyl-substituted cycloalkanes. To investigate the origin of the oxygen atom incorporated into the alcohol, hydroxylation of 2 with H218O2 (24% solution, 90% enriched) catalysed by 1a was carried out. This gave 8 (98% selectivity) in 80% yield with an 18O content of 90+1% and the 18O content in the alcohol (18O-labelled alcohol/total alcohol ratio) did not change during the reaction (Fig. 1c), which shows that all the 18O atoms incorporated into the alcohol originated from H218O2.
Conclusions
The bis(m-hydroxo) divanadium-substituted phosphotungstate 1a reacted easily with H2O2 to produce a highly active, sterically hindered oxidant species. The strong electrophilic oxidants with high steric hindrance hydroxylated alkanes with high selectivity for the alcohols, complete stereospecicity and specic regioselectivity. Such unique properties of all-inorganic molecular polyoxometalates are promising as homogeneous and heterogeneous catalysts for stereo- and shape-selective H2O2-based hydroxylation of CH bonds.
(1.8 g, 5.6 mmol) was added with vigorous stirring. The precipitate was collected by ltration, washed with 400 ml of water and dried in vacuo. Recrystallization from acetone/ether gave analytically pure orange crystals of [(n-C4H9)4N]4 [g-HPV2W10O40].H2O, with a yield of 60%. 51V NMR (CD3CN), 581 ppm; 1 H NMR (CD3CN), 4.38 ppm (1H); 31P NMR (CD3CN), 14.1 ppm. Analytical calculation for [(C4H9)4N]4[HPV2W10O40].H2O was C, 21.4; H, 4.12; N, 1.56; P, 0.86; V, 2.83; W, 51.1; found was C, 21.3; H, 3.96; N, 1.61; P, 0.84; V, 2.92; W, 49.0. Infrared (KBr) 1,096, 1,062, 1,039, 1,001, 952, 870, 803, 752, 534, 489, 399, 358, 333, 282, 256 cm21. The tetraethylammonium (Et4N) salt of diprotonated derivative [(C2H5)4N]3[g-H2PV2W10O40] (Et4N)3.1a) was prepared by the reaction of [g-HPV2W10O40]42 with an acid followed by a cation-exchange reaction. Perchloric acid (0.1 mmol) and tetraethylammonium bromide (76 mg, 0.36 mmol) dissolved in CH3CN (0.5 ml) was added to the CH3CN solution (10 ml) of [(n-C4H9)4N]4 [g-HPV2W10O40].H2O (216 mg, 60 mmol). Diethyl ether (20 ml) was added to the clear yellow solution and the resulting precipitate collected by centrifugation. The crude product was dissolved in CH3CN (10 ml), followed by the addition of 1,4-dioxane (10 ml). Standing the solution in an open vessel for a few hours gave yellow crystals of [(C2H5)4N]3[H2PV2W10O40].3C4H8O2 , with a yield of 120 mg (63%). 51V NMR (CD3CN), 578 ppm; 1H NMR (CD3CN), 6.95 (2H, V-OH-V), 3.59 (24H, 1,4-dioxane), 3.19 (24H, cation), 1.23 ppm (36H, cation); 31P NMR (CD3CN), 13.8 ppm. Analytical calculation for [(C2H5)4N]3[H2PV2W10O40].3C4H8O2 was C, 13.2; H, 2.65; N, 1.29; P, 0.95; V, 3.17; W, 56.3; found was C, 13.6; H, 2.73; N, 1.37; P, 0.95; V, 3.15; W, 58.6. Infrared (KBr): 1,119 (1,4-dioxane), 1,094, 1,060, 1,040, 1,011sh, 972, 957, 871 (1,4-dioxane), 803, 716, 606, 585, 537, 490, 413, 397, 358, 333, 311, 279, 255 cm21. Crystal data (153 K): C24N3O40PV2W10; formular weight 2,941.62, orthorhombic, space group Pbcn (No. 60), a 15.5486(2) , b 21.1547(2) , c 23.1083(2) , V 7600.92(14) 3, Z 4, Dcalcd 2.571 g cm23, m (Mo Ka) 15.39 cm21, R1 0.0761 (I . 2s(I)), wR 2 0.2302 (all 10,413 data) and 194 parameters were used for renement. CCDC 746253 contains the supplementary crystallographic data, which can be obtained free of charge through www.ccdc. cam.ac.uk/data_request/cif. Typical procedure for the catalytic oxidation of alkanes. The catalytic reactions were carried out with a glass tube that contained a magnetic stir bar. The catalyst, solvent and substrate were charged in the reaction vessel. For 1a-catalysed reactions, diprotonated 1a was prepared in situ by the reaction of monoprotonated [(n-C4H9)4N]4[g-HPV2W10O40] with one equivalent of perchloric acid (70% aqueous solution) because of the low solubility of (Et4N)3.1a in the solvent. The formation of 1a was conrmed by 51V NMR spectroscopy (578 ppm). The reaction was initiated by the addition of 30% aqueous H2O2. The reaction solution was analysed periodically by gas chromatography, gas chromatographymass spectroscopy and NMR spectroscopy. Ce3/4 titration showed that no H2O2 remained after the reaction. All products are known and identied by comparison of their 1H and 13C NMR signals with the literature data.
Methods
Synthesis and characterization of divanadium-substituted phosphotungstate (1a). The caesium salt of deprotonated divanadium-substituted phosphotungstate Cs5[g-PV2W10O40] was synthesized according to published literature procedures50 and characterized by infrared spectroscopy. The tetra-n-butylammonium salt of the monoprotonated derivative [(n-C4H9)4N]4[g-HPV2W10O40] was prepared using a cation-exchange reaction. Sodium metavanadate (1.2 mmol) was dissolved in 120 ml of hot water. On cooling, the pH of the solution was adjusted to 2.0 with 3 M HCl. Cs5[g-PV2W10O40].6H2O (3.8 g, 1.1 mmol) was dissolved in the solution and the insoluble materials removed by ltration. Tetra-n-butylammonium bromide
482
Received 1 December 2009; accepted 22 March 2010; published online 2 May 2010
NATURE CHEMISTRY
References
DOI: 10.1038/NCHEM.648
ARTICLES
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Acknowledgements
We are grateful to K. Yamaguchi and K. Yonehara for discussions. This work was supported by the Core Research for Revolutional Science and Technology program of the Japan Science and Technology Agency, the Global COE Program Chemistry Innovation through Cooperation of Science and Engineering, the Development in a New Interdisciplinary Field Based on Nanotechnology and Materials Science Programs and a Grant-in-Aid for Scientic Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan.
K.K. and N.M. conceived and designed the experiments. K.K., K.Y. and Y.N. carried out the experiments. K.U. analysed the crystallographic data. K.K. and N.M. co-wrote the paper.
The authors declare no competing nancial interests. Supplementary information and chemical compound information accompany this paper at www.nature.com/ naturechemistry. Reprints and permission information is available online at http://npg.nature. com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to N.M.
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Using rst principles to predict bimetallic catalysts for the ammonia decomposition reaction
Danielle A. Hansgen, Dionisios G. Vlachos* and Jingguang G. Chen*
The facile decomposition of ammonia to produce hydrogen is critical to its use as a hydrogen storage medium in a hydrogen economy, and although ruthenium shows good activity for catalysing this process, its expense and scarcity are prohibitive to large-scale commercialization. The need to develop alternative catalysts has been addressed here, using microkinetic modelling combined with density functional studies to identify suitable monolayer bimetallic (surface or subsurface) catalysts based on nitrogen binding energies. The NiPtPt(111) surface, with one monolayer of Ni atoms residing on a Pt(111) substrate, was predicted to be a catalytically active surface. This was veried using temperature-programmed desorption and high-resolution electron energy loss spectroscopy experiments. The results reported here provide a framework for complex catalyst discovery. They also demonstrate the critical importance of combining theoretical and experimental approaches for identifying desirable monolayer bimetallic systems when the surface properties are not a linear function of the parent metals.
he ammonia decomposition reaction has recently been subject to an increasing level of attention due to the possibility of ammonia being used as a hydrogen storage medium in a possible hydrogen economy. Ammonia can be liqueed easily at a pressure of 8 atm at 293 K, leading to high energy densities. It is readily available because of its use in fertilizers, and is a CO-free source of hydrogen. Experimental studies of single-metal catalysts have shown that Ru is the most active decomposition catalyst13, but it is expensive and limited in supply; hence there is a need to develop either less expensive alternatives or catalysts with higher activity. Ammonia decomposition proceeds by means of dehydrogenation, NH3 NH2 NH N followed by recombination of N and H to form N2 and H2 , respectively. It has been shown that the heat of nitrogen chemisorption is a good descriptor for ammonia synthesis and decomposition4,5. The binding energy of the nitrogen atom to the surface must be strong enough for dehydrogenation of the NHx species to occur, but sufciently weak that the nitrogen recombines to desorb from the surface to complete the catalytic cycle. This trade-off leads to a volcano-type relationship between nitrogen binding energy and ammonia decomposition activity. Although Ru has the optimal heat of chemisorption among single metals, it is possible that bimetallic catalysts with higher activities might exist. Encouragingly for the ammonia synthesis reaction, a bimetallic catalyst (CoMo) has been found that is more active than Ru. However, because the activity for the CoMo catalyst decreases signicantly in the presence of ammonia, with concentrations as low as 5%, its use as a decomposition catalyst is restricted4,5. The CoMo bimetallic catalyst for the synthesis reaction was predicted through the concept of Periodic Table interpolation, in which the binding energy of a mixed metal (alloyed) surface is taken as a linear combination of the binding energies of the parent metals. A metal with a high nitrogen binding energy (Mo) and one with a low binding energy (Co) were chosen to give a surface with an intermediate binding energy. Although this specic alloy catalyst may be stable4, for many bimetallic alloys, one metal often segregates to the
H H H
surface either to minimize the surface energy of the alloyed system or due to adsorbate-induced reconguration611. This results in surface properties that are vastly different from the alloyed surface, and makes predictions from the periodic table interpolation method invalid. Monolayer bimetallic catalysts consist of a monolayer of an admetal in the top layers of a host metal1214. These surfaces can be used to represent the segregated surface of an alloy or can be used to model coreshell bimetallic nanoparticles. The admetal can be on the surface of the host metal, giving rise to the surface conguration, or below the surface layer, forming the subsurface conguration. These two congurations have been shown, both experimentally and through density functional theory (DFT) calculations, to have properties that differ drastically from one another and from the parent metals; these bimetallic surfaces are also very different from the corresponding alloyed system, where the topmost surface layer is intermixed with the two parent metals. Typically, the monolayer bimetallic surfaces have binding energies that are greater or less than both of the parent metals, depending on the conguration, and are not a function of the parent metal binding energies12,13. Owing to this nonlinear behaviour, there is currently no method to rationally design these novel catalysts. In this study, we present full microkinetic models for NH3 decomposition on various single-metal catalysts, including Co, Pt, Pd, Ni, Ru, Rh, Ir, Re and Mo. These models include adsorbate adsorbate interactions, which have been shown to have a signicant effect on calculated surface coverages, the rate-determining step and catalytic activity15. The results, combined with DFT data, are used to predict suitable monolayer bimetallic (surface or subsurface) systems, based on nitrogen binding energies, for the ammonia decomposition reaction. These bimetallic surfaces are then tested experimentally for their activity towards ammonia decomposition using temperature-programmed desorption (TPD) and highresolution electron energy loss spectroscopy (HREELS) to validate the model predictions. To the best of our knowledge, our approach represents the rst time that full microkinetic models and DFT predictions, together with experimental verication, have been combined to identify novel catalyst formulations and surface structures.
Center for Catalytic Science and Technology, Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716, USA. * e-mail: vlachos@udel.edu; jgchen@udel.edu
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Results
DOI: 10.1038/NCHEM.626
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Table 1 | Monometallic binding energies and interaction energies.
Metal Zero coverage N binding energy 110.1 107.4 117.3 122.3 125.4 125.4 141.6 152.0 154.3 Calculated N interaction parameter 242.0 231.7 232.8 242.6 239.5 234.0 236.9 228.6 227.0 Zero coverage H binding energy 67.0 63.3 62.1 64.4 64.8 64.8 66.6 71.6 69.8 Calculated H interaction parameter 21.6 21.4 22.7 0.1 20.5 21.6 21.4
Computational results. DFT calculations were performed to obtain the interaction energies for nitrogen and hydrogen adsorbates on the surface. The binding energies were determined at coverages of 1/9 to 1 monolayer (ML), which were then plotted as a function of coverage. Although not completely linear, approximating the data with a linear function is adequate for trends and screening studies as performed here. The binding energies extrapolated to the zero coverage limit (QA(0)) and the interaction parameters (IP), the slope of the line, for each metal studied are listed in Table 1. Plots of the binding energies and the linear ts can be found in the Supplementary Information. The zero coverage binding energy and the interaction parameter were used to approximate the binding energy at any surface coverage within the microkinetic models through the following equation: QA (uA ) = QA(0) + IP uA (1)
Pd Pt Ir Ni Rh Co Ru Re Mo
Binding energies extrapolated to the zero coverage limit and the calculated interaction parameters can be used to estimate the atomic binding energies at any surface coverage through equation (1). The negative sign indicates that the interactions are repulsive. All energies are in kcal mol21.
where uA is the coverage of adsorbate A. The interaction parameters for hydrogen are low, at approximately 21 kcal mol21 (ML hydrogen)21. This may be a result of the small size of the hydrogen atom, the low binding energy or a combination of both. Because the hydrogen interaction parameters are small, they have very little effect on the reaction mechanism and predicted ammonia conversions. The nitrogen interaction parameters, on the other hand, are much greater, ranging from 227.0 to 242.6 kcal mol21 (ML nitrogen)21 (depending on the metal), and have a signicant effect on conversion. For each metal, the microkinetic model was used to calculate the conversion at the reactor exit. The overall ammonia decomposition reaction was modelled with 12 elementary reaction steps, with no assumption of a rate-determining step. The elementary reaction steps are as follows: NH3 + NH 3 NH + 3 NH + H 2 NH + 2 NH + H NH + N + H N + N N2 + 2 H + H H2 + 2 NH 3 NH3 + NH + H 2 NH + 3 NH + H NH + 2 N + H NH + N2 + 2 N + N H2 + 2 H + H (R1) (R2) (R3) (R4) (R5) (R6) (R7) (R8) (R9) (R10) (R11) (R12)
100
Pt Pd
Rh Ir Ni Co
Ru
Re Mo
10 Conversion (%)
TOF (s1)
0.1
0.01 100
110
120
130
140
150
160
(kcal mol1)
where represents an adsorbed surface species. The coveragedependent atomic binding energies were used to calculate the molecular heats of chemisorption (QNHx) and the activation barriers of the elementary reactions using the bond-order conservation method16, resulting in activation barriers that were coveragedependent. Pre-exponentials for the elementary reaction steps were taken from a previous literature study in which the preexponentials were t to Ru experimental data with constraints on the overall entropic consistency15. Figure 1 shows the predicted conversion of ammonia versus the nitrogen heat of chemisorption (QN(0)) at a reactor temperature of 850 K. Among the single-metal catalysts studied, Ru was found to have the highest activity, consistent with experimental data13. The model results (circles) for the full microkinetic library are in good agreement with an extensive experimental study of 13 metals from
Figure 1 | Ammonia decomposition volcano curve. Ammonia conversion calculated from microkinetic modelling (circles, left axis) at 850 K for various transition-metal catalysts and experimental supported catalyst turnover frequencies (TOF) (triangles, right axis) from Ganley and colleagues1 at 850 K plotted against the nitrogen binding energies (QN(0)). The dotted line is calculated by assuming average interaction energies (see Table 1) and a correlation between nitrogen and hydrogen binding energies to aid in determining the volcano maximum. The nitrogen binding energy is found to be a good descriptor for this reaction. The peak of the volcano curve is at a nitrogen binding energy of 134 kcal mol21, and this value is used to identify bimetallic surfaces with desirable catalytic activity for the ammonia decomposition reaction.
Ganley and colleagues1 (triangles). Figure 1 reveals a volcano relationship, and shows that the heat of nitrogen chemisorption is a good descriptor to identify surfaces with desirable catalytic activity (other possible descriptors are listed in Supplementary Section 4). This conclusion is consistent with previous studies on ammonia synthesis and decomposition reactions4,5, although in the current study repulsive adsorbateadsorbate interactions were accounted for and no assumptions of the rate-determining step or surface coverages were made, as is frequently done in previous literature studies. Through the models, a maximum activity (peak of the volcano curve) is predicted to be at a nitrogen heat of chemisorption of 134 kcal mol21. To determine the kinetically signicant reaction steps, a sensitivity analysis was performed. The pre-exponentials of each
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Pt Pd 0.6 NH3 + * * Normalized sensitivity coefficient 0.5 0.4 0.3 0.2 0.1 0 100 110 120 130 140 150 160 NH2 + * * 2N* NH2 + H* * NH* + H* N2 + 2 * Ir Rh Ni Co Ru Re Mo
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Table 2 | Library of DFT binding energies and bond lengths of nitrogen atoms at a 1/9 ML coverage on various monolayer bimetallic surfaces.
Conguration Subsurface Metal (111) surface PtTiPt PtVPt PtCrPt PtMnPt PtFePt PtCoPt PtNiPt Pt Ni NiPtPt CoPtPt FePtPt MnPtPt CrPtPt VPtPt TiPtPt Nitrogen binding energy (kcal mol21) 70.7 81.0 76.3 77.6 78.4 83.4 87.5 102.1 113.8 130.7 126.5 134.1 207.2 188.3 188.1 176.1 Bond length d MN () 1.975 1.975 1.965 1.968 1.969 1.964 1.941 1.954 1.770 1.761 1.780 1.864 1.854 1.894 1.876 1.918
Single metal
Surface
(kcal mol1)
Figure 2 | Normalized sensitivity coefcients of the kinetically signicant elementary reaction steps for each monometallic metal. Pre-exponentials of the forward and reverse reaction pairs were perturbed simultaneously at a temperature of 850 K and the response of conversion was monitored. The reaction pairs that have the highest normalized sensitivity coefcient are kinetically signicant reaction steps. The sensitivity analysis shows that there are multiple kinetically signicant reaction steps and their sensitivity changes across the volcano curve. Only reaction pairs that have a normalized sensitivity coefcient above 0.01 for any metal are shown.
By adding a metal to the Pt(111) surface, in either the surface or subsurface conguration, the nitrogen binding energy is modied, creating a large range of binding energies on the bimetallic surfaces. The binding energies and bond lengths on the Pt(111) and Ni(111) surfaces are also included for comparison. The different colours in the structures indicate the different metals.
forward and reverse elementary reaction step pair (Ai) (that is, R1 and R2, R3 and R4, and so on) were perturbed simultaneously, thus ensuring thermodynamic consistency. The normalized sensitivity coefcient for each reaction pair (NSCi) was calculated using NSCi = D(ln X) D(ln Ai ) (2)
where X is the conversion at the end of the reactor. The largest (in absolute value) model responses among the reaction pairs indicate kinetically signicant reaction steps17,18 (see Supplementary Information). A sensitivity analysis performed on each of the surfaces shows the rate-determining step to be the removal of the second hydrogen (from the NH2; reaction R5) for surfaces with a nitrogen binding energy less than 125 kcal mol21. For surfaces with higher nitrogen binding energies, the removal of the rst and second hydrogens (R3 and R5) and nitrogen desorption (R9) are kinetically signicant, as shown in Fig. 2. At the peak of the volcano curve ( 134 kcal mol21), the removal of the second hydrogen is the most signicant elementary reaction step, although the removal of the rst hydrogen and nitrogen desorption are both kinetically signicant. Interestingly, the dominant surface coverage changes from the left to the right leg of the volcano curve (Supplementary Fig. S4). This analysis underscores the fact that the kinetically signicant step and dominant coverage may be changing along a volcano curve, the maximum in the volcano curve may be a result of multiple physical mechanisms, and the importance of performing a full microkinetic analysis rather than assuming a priori a rate-determining step and a dominant surface species. Because the nitrogen binding energy is a good activity descriptor, a DFT search was performed to identify catalyst surfaces that have a similar binding energy to the optimal value of 134 kcal mol21. Pt-based monolayer bimetallic surfaces were the focus of this study. These surfaces have been shown to form the surface (MPtPt) and subsurface (PtMPt) congurations, both
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experimentally and through DFT calculations11,12,19,20. Binding energies were calculated at the 1/9 ML coverage, which is a good approximation to the binding energies extrapolated to zero coverage. Table 2 shows the calculated nitrogen binding energies for several surface and subsurface congurations. Also included are the metalnitrogen bond lengths on each surface. The binding energies for the subsurface congurations are lower than both the parent metals due to a broadening of the d band, whereas the binding energies for the surface congurations are stronger than both the parent metals, due to a contraction of the d band21. For the subsurface congurations, the PtN bond lengths were similar to Pt(111), with only a slight lengthening of the bond. PtNiPt was the only subsurface conguration in which there was a shortening of the bond compared to the Pt(111) surface. For the surface congurations, the nitrogen surface bonds show much more variation, which is probably a result of the differences in the metals to which the nitrogen is bound. The nitrogen binding energies vary from 71 to 207 kcal mol21 by adding a second metal to the surface or subsurface layer of the Pt host. Based on the theoretical predictions in Fig. 1, the activity of these bimetallic surfaces should also follow a volcano relationship. The NiPtPt(111) bimetallic surface has a nitrogen binding energy of 130.7 kcal mol21, slightly lower than that of Ru, and is a potentially active catalyst (see the maximum in Fig. 1). The subsurface conguration, PtNiPt(111), and the parent metals, Pt(111) and Ni(111), are expected to have lower activities because of the weaker nitrogen binding energies (Table 2). The Ni/Pt bimetallic system was chosen as a test system to be studied experimentally in the current paper to assess the model predictions. Table 2 also identies the CoPtPt(111) and FePtPt(111) surfaces as additional promising systems. The activity of these additional surfaces will be evaluated in future studies. TPD studies of ammonia decomposition. We tested the DFT/ microkinetic model predictions using the NiPt bimetallic systems. The NiPtPt surface, together with PtNiPt, Pt(111) and a Ni(111) lm, were tested for their activity towards ammonia decomposition through TPD experiments. Depositing Ni at room temperature leads to the NiPtPt surface, whereas depositing at 600 K leads to the PtNiPt conguration22.
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0.4 0.3 0.2 0.1 0.0 200 300 400 N = 0.27 N = 0.29 N = 0.18 N = 0.14 N = 0.11 N = 0.07 N = 0.07
Depositing at least 5 ML of Ni achieves a surface with chemical properties similar to a Ni(111) surface23. Ammonia (3 L, equal to 1 1028 torr for 300 s, where L indicates Langmuir) was dosed onto each of the four surfaces at 350 K. Figure 3 shows the desorption spectra of the decomposition product, nitrogen, for each of the surfaces. The m/z 14 AMU was used to monitor N2 desorption (N atoms from N2 cracking) to eliminate the overlap between N2 and any background CO adsorbed to the surface (both seen at m/z 28 AMU). Detection of the peak at 626 K conrms that the NiPtPt surface is active towards decomposition. The absence of any peaks on the other three surfaces shows that these surfaces are not active towards decomposition at these dosing conditions. The results for Pt(111) are consistent with previous results, which showed that Pt(111) does not decompose ammonia under 400 K (ref. 24). The inactivity of the Pt(111), thick Ni(111) lm and the PtNiPt surfaces can be attributed to the low binding energy of nitrogen on these surfaces, conrming the predictions from the microkinetic models. TPD results following the desorption of hydrogen from the four surfaces as well as ammonia dosed at low temperatures are provided in the Supplementary Information. Because the NiPtPt surface showed decomposition activity, the surface was exposed to 3 L of ammonia at various temperatures from 150 to 425 K to determine the onset temperature for decomposition (Fig. 4). Nitrogen coverages on the NiPtPt surface were quantied by comparing the integrated nitrogen peak area to the area produced by a saturation coverage of CO (uCO 0.68) on the Pt(111) surface after taking into consideration the different sensitivity factors for N2 and CO, as described previously for other reactions on Ni/Pt(111) (ref. 25). The nitrogen coverage achieved at each dosing temperature is compared in the inset of Fig. 4. The coverage stays constant at 0.07 ML up to 300 K. At 325 K, the coverage begins to increase and continues to increase up to 375 K, indicating decomposition occurring during dosing at a phenomenally low temperature of 325 K. Above a dosing temperature of 375 K, the coverage stays constant because
N2 (14 AMU)
630 K
N2 (14 AMU)
400 K 375 K 350 K 340 K 325 K 300 K 250 K
Intensity (a.u.)
400
500 600 700 Temperature (K)
800
Figure 4 | TPD spectra of nitrogen desorption from a NiPtPt surface after dosing 3 L ammonia at the specied temperature. The nitrogen coverages (uN) resulting from ammonia decomposition are indicated. The inset shows the nitrogen coverage as a function of the dosing temperature. The saturation coverage of nitrogen on this surface is 0.3 ML, as indicated by the coverage plateau from the inset. The onset temperature for the sharp increase in the N2 peak area demonstrates that the NiPtPt surface is active towards the decomposition of ammonia at temperatures as low as 325 K.
626 K Ni(111) film
Ni-Pt-Pt
Pt-Ni-Pt
Pt(111)
400
500
600
700
800
Temperature (K)
Figure 3 | Ammonia decomposition on different NiPt surfaces. TPD results of nitrogen desorption from the decomposition of ammonia on Pt(111), PtNiPt, NiPtPt and a Ni(111) lm. 3 L of ammonia was dosed at 350 K, then the temperature was ramped at a heating rate of 3 K s21 and the desorption of nitrogen was monitored using a mass spectrometer. The NiPtPt surface is the only one that shows activity towards ammonia decomposition under these conditions, as indicated by the peak at 626 K. This conrms model predictions of the NiPtPt surface being active towards ammonia decomposition based on an optimal nitrogen binding energy and the other three surfaces being inactive due to a nitrogen binding energy that is too low.
the surface is saturated with nitrogen at a coverage of 0.3 ML. A longer exposure of 6 L at 400 K was also performed (not shown), resulting in identical desorption spectra, conrming that saturation coverage was achieved. The saturation value is consistent with the saturation coverage of 0.28 ML on the Ru(0001) surface26. Interestingly, the nitrogen desorption peak is constant at 630 K over all nitrogen coverages. This is surprising, because nitrogen desorption is a second-order reaction, and a peak shift towards lower temperatures at higher coverages is expected. The absence of this shift indicates that surface reconstruction may be occurring. Reconstruction of a Ni(111) surface to pseudo-Ni(100) has been reported in the presence of strong binding adsorbates such as N, C and S (ref. 27). Because the surface layer in Fig. 4 is Ni, a similar transition is likely to be occurring. In a study of ammonia decomposition on Ru(0001), an exposure of 3,500 L of ammonia was dosed at 500 K to achieve a nitrogen saturation coverage26. In comparison, a saturation coverage is achieved with 3 L at 375 K on the NiPtPt surface. The signicantly lower dosing temperature and ammonia exposure clearly indicate that the overall dehydrogenation barrier, which was shown through sensitivity analysis to be a kinetically signicant reaction step, is much lower for the bimetallic surface. The other kinetically signicant reaction step for ammonia decomposition was found to be the recombinative nitrogen desorption (Fig. 2). Nitrogen on Ru(0001) was shown to have a peak desorption temperature ranging from 770 K at low coverages to 680 K at a saturation coverage of 0.28 ML, using the same heating rate of 3 K s21 used in this study26. The lower desorption temperature on NiPtPt is comparable to the highly stepped Ru(109) and undercoordinated Ru(1121) and Ru(1010) surfaces, which have peak desorption temperatures at a saturation coverage of 600 K (at 2 K s21) (ref. 28), 610 K (ref. 29) and 620 K (ref. 30), respectively. Furthermore, the desorption peaks from NiPtPt are much narrower, with a temperature span of 560725 K, compared to 550900 K from the Ru(0001) surface. Our results indicate that not only is ammonia dehydrogenation on NiPtPt facile, but also nitrogen desorption is relatively fast. To support the DFT predictions and TPD experiments, HREELS was used to investigate the decomposition of ammonia on the
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NiPtPt surface. The HREELS results and the vibrational assignment of possible reaction intermediates are provided in the Supplementary Information. The most important observation from the HREELS results is that, furthermore to adsorbed NH3 , partially decomposed intermediates including NH2 and atomic nitrogen are produced on NiPtPt at 350 K, supporting the TPD detection of the N2 gas-phase product from the decomposition of ammonia on the NiPtPt surface.
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used. A 3 3 supercell of four layers and a vacuum region equivalent to seven metal layers were used to decouple consecutive slabs. All calculations were performed spinpolarized, with the bottom two layers frozen to represent the bulk structure and the top two metal layers allowed to relax. An electron smearing parameter of 0.2 eV was applied and the convergences were set to 1 1024 for the self-consistent electronic minimization loop and to 1 1023 for the ionic relaxation loop. DFT was used to calculate the binding energies of atomic species (N and H) on the single-metal and bimetallic surfaces using Q(adsorbate) = E(slab) + X E(adsorbate) E(slab+X adsorbates) X (3)
Discussion
Our experimental ndings support the proposed computational framework in which potential (complex) bimetallic catalysts can effectively be screened using a library of binding energies of bimetallic catalysts from DFT calculations guided from full, rst-principlesbased microkinetic models. The NiPtPt bimetallic surface was predicted to be very active for the ammonia decomposition reaction; this was conrmed by experimental measurements using TPD and HREELS. The TPD experiments indicate that a NiPtPt catalyst may be more active than Ru based on a lower nitrogen desorption temperature and a remarkably low dehydrogenation barrier, both of which were determined to be kinetically signicant reaction steps. For this reaction to take place under steady-state conditions, both NHx decomposition and nitrogen desorption must occur. Our results show that NHx decomposition occurs at temperatures as low as 325 K and the onset for nitrogen desorption is at 560 K. Therefore, steady-state decomposition activity should be seen at temperatures below 600 K. As the activity is highly dependent on the location of the Ni atoms, the stability of the surface is very important. Menning and Chen have shown that the relative thermodynamic stability of the surface and subsurface structures depends on the adsorbate coverage and the Pauling electronegativity of the adsorbate9. For oxygen, which has a similar Pauling electronegativity as nitrogen (3.44 for oxygen and 3.04 for nitrogen), the surface conguration was shown to be more stable than the subsurface conguration through DFT and was conrmed experimentally. Exposure to oxygen caused the subsurface Ni to reconstruct to the surface conguration (NiPtPt) under conditions of both low pressure (1 1027 torr O2) and atmospheric pressure9,31,32. A similar stability was predicted for the NiPtPt surface with nitrogen adsorbates9, and this surface is expected to be stable under reaction conditions. Previous studies within our group have shown a remarkable correlation between the activity on single-crystal surfaces, polycrystalline lms and supported bimetallic catalysts for reactions such as cyclohexene hydrogenation20,33,34. This, combined with strong agreement of the microkinetic models with the supported monometallic catalysts of Ganley and colleagues,1 gives reason to expect that the activity of the supported bimetallic catalysts for the ammonia decomposition reaction will also follow the predicted trends of the microkinetic models on the single-crystal surfaces. The ammonia decomposition reaction was used in the current study as a test system due to the relative simplicity of the molecule and the decomposition reaction pathways. This methodology for predicting active monolayer bimetallic surfaces should also be applicable to more complex chemistries, although care must be taken to choose bimetallic systems that are thermodynamically stable under reaction conditions.
where E(adsorbate) is the energy of the atom in vacuum, E(slab) is the energy of the bare slab without adsorbates, E(slabX adsorbate) is the energy of the slab with the adsorbates bound to the surface, and X is the number of adsorbates within the supercell. Atomic nitrogen and hydrogen binding energies were calculated at varying coverages, specically at 1/9, 2/9, 1/3, 2/3 and 1 ML. The adsorbate congurations used are shown in the Supplementary Information. For the microkinetic models, a xed-bed reactor with a length of 0.5 cm and a diameter of 0.32 cm was modelled. A ow rate of 70 s.c.c.m. and a catalytic surface area of 12,000 cm2 cm23 were used. Experimental methods. A two-level stainless steel ultrahigh vacuum (UHV) chamber, with a typical base pressure of 4 10210 torr, was used for the TPD experiments25. A heating rate of 3 K s21 was used for preparation methods and all TPD experiments. Anhydrous ammonia, of 99.99% purity, was used and the purity was checked in situ with the mass spectrometer before experiments each day. Ammonia was dosed in the background for all experiments. After dosing, the temperature was held constant for 10 min to allow the residual ammonia in the UHV background to be pumped away before ramping the temperature. The Pt(111) surface was cleaned by cycles of Ne bombardment at 600 K. The crystal was then heated to 890 K in 3 Langmuir (1 L 1 1026 torr s) oxygen, of 99.998% purity, to burn off any remaining surface carbon. The crystal was then heated to 1,100 K and held at this temperature for 5 min to anneal the crystal surface. This procedure was repeated until surface cleanliness was veried by Auger electron spectroscopy. The Ni/Pt(111) bimetallic surfaces were prepared for this study by depositing Ni through physical vapour deposition on the clean, freshly annealed Pt(111) surface. Three Ni-containing surfaces were created: the surface monolayer (NiPtPt), the subsurface monolayer (PtNiPt) and a thick Ni(111) lm that mimics a Ni(111) surface when over 5 ML are deposited on the Pt(111) surface23. Using preparation procedures described previously22,25, the PtNiPt surface was created by depositing Ni at 600 K until a Ni(849 eV)/Pt(241 eV) Auger ratio of 1.0 was achieved. For the NiPtPt surface, Ni was deposited for the same amount of time at 300 K, resulting in an Auger ratio of approximately 1.5. The thick Ni(111) lm was created by depositing Ni until an Auger ratio above 3.0 was achieved.
Received 31 December 2009; accepted 12 March 2010; published online 25 April 2010
References
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Methods
Theoretical predictions. DFT calculations were performed using the Vienna ab initio Simulation Package (VASP) version 4.6 (refs 35,36). A plane-wave basis set was used with an energy cutoff of 396 eV, together with ultrasoft Vanderbilt pseudopotentials37 and the PW-91 functional38. A 3 3 1 Monkhorst Pack k-point grid39 was used for all slab calculations. All calculations were performed on the close-packed surfaces: fcc(111) for Pd-, Pt-, Ir-, Ni-, Rh- and Pt-based monolayer bimetallic surfaces; hcp(0001) for Co, Ru and Re; and bcc(110) for Mo. Lattice constants that were previously optimized for the PW-91 functional were used40. For the Pt-based monolayer bimetallic surfaces, the Pt lattice constant of 4.01 was
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30. Dietrich, H., Jacobi, K. & Ertl, G. Vibrations, coverage, and lateral order of atomic nitrogen and formation of NH3 on Ru(10(1)0). J. Chem. Phys. 106, 93139319 (1997). 31. Menning, C. A. & Chen, J. G. Thermodynamics and kinetics of oxygen-induced segregation of 3d metals in Pt3dPt(111) and Pt3dPt(100) bimetallic structures. J. Chem. Phys. 128, 174709 (2008). 32. Menning, C. A. & Chen, J. G. Regenerating Pt3dPt model electrocatalysts through oxidationreduction cycles monitored at atmospheric pressure. J. Power Sources 195, 31403144 (2010). 33. Lu, S. L. et al. Low temperature hydrogenation of benzene and cyclohexene: a comparative study between gamma-Al2O3 supported PtCo and PtNi bimetallic catalysts. J. Catal. 259, 260268 (2008). 34. Humbert, M. P., Murillo, L. E. & Chen, J. G. Rational design of platinum-based bimetallic catalysts with enhanced hydrogenation activity. ChemPhysChem 9, 12621264 (2008). 35. Kresse, G. & Furthmuller, J. Efcient iterative schemes for ab initio total-energy calculations using a plane-wave basis set. Phys. Rev. B 54, 1116911186 (1996). 36. Kresse, G. & Furthmuller, J. Efciency of ab initio total energy calculations for metals and semiconductors using a plane-wave basis set. Comput. Mater. Sci. 6, 1550 (1996). 37. Vanderbilt, D. Soft self-consistent pseudopotentials in a generalized Eigenvalue formalism. Phys. Rev. B 41, 78927895 (1990). 38. Perdew, J. P. et al. Atoms, molecules, solids and surfacesapplications of the generalized gradient approximation for exchange and correlation. Phys. Rev. B 46, 66716687 (1992). 39. Monkhorst, H. J. & Pack, J. D. Special points for Brillouin-zone integrations. Phys. Rev. B 13, 51885192 (1976). 40. Pseudopotential Library. Center for Atomic-Scale Materials Design. https://wiki. fysik.dtu.dk/dacapo/Pseudopotential_Library. 41. Catlett, C. et al. TeraGrid: analysis of organization, system architecture, and middleware enabling new types of applications, in HPC and Grids in Action (Grandinetti, L. ed.) Advances in Parallel Computing series (IOS Press, 2007).
12. Chen, J. G., Menning, C. A. & Zellner, M. B. Monolayer bimetallic surfaces: experimental and theoretical studies of trends in electronic and chemical properties. Surf. Sci. Rep. 63, 201254 (2008). 13. Pallassana, V., Neurock, M., Hansen, L. B., Hammer, B. & Norskov, J. K. Theoretical analysis of hydrogen chemisorption on Pd(111), Re(0001) and PdML/Re(0001), Re-ML/Pd(111) pseudomorphic overlayers. Phys. Rev. B 60, 61466154 (1999). 14. Rodriguez, J. A. & Goodman, D. W. The nature of the metal metal bond in bimetallic surfaces. Science 257, 897903 (1992). 15. Mhadeshwar, A. B., Kitchin, J. R., Barteau, M. A. & Vlachos, D. G. The role of adsorbate2adsorbate interactions in the rate controlling step and the most abundant reaction intermediate of NH3 decomposition on Ru. Catal. Lett. 96, 1322 (2004). 16. Shustorovich, E. & Sellers, H. The UBI-QEP method: a practical theoretical approach to understanding chemistry on transition metal surfaces. Surf. Sci. Rep. 31, 5119 (1998). 17. Campbell, C. T. Finding the rate-determining step in a mechanismcomparing DeDonder relations with the degree of rate control. J. Catal. 204, 520524 (2001). 18. Campbell, C. T. Micro- and macro-kinetics: their relationship in heterogeneous catalysis. Top. Catal. 1, 353366 (1994). 19. Menning, C. A., Hwu, H. H. & Chen, J. G. Experimental and theoretical investigation of the stability of Pt3dPt(111) bimetallic surfaces under oxygen environment. J. Phys. Chem. B 110, 1547115477 (2006). 20. Humbert, M. P. & Chen, J. G. Correlating hydrogenation activity with binding energies of hydrogen and cyclohexene on M/Pt(111) (MFe, Co, Ni, Cu) bimetallic surfaces. J. Catal. 257, 297306 (2008). 21. Kitchin, J. R., Norskov, J. K., Barteau, M. A. & Chen, J. G. Role of strain and ligand effects in the modication of the electronic and chemical properties of bimetallic surfaces. Phys. Rev. Lett. 93, 156801 (2004). 22. Kitchin, J. R. et al. Elucidation of the active surface and origin of the weak metal hydrogen bond on Ni/Pt(111) bimetallic surfaces: a surface science and density functional theory study. Surf. Sci. 544, 295308 (2003). 23. Khan, N. A., Hwu, H. H. & Chen, J. G. Low-temperature hydrodesulfurization of thiophene on Ni/Pt(111) bimetallic surfaces with monolayer Ni coverage. J. Catal. 205, 259265 (2002). 24. Sun, Y. M., Sloan, D., Ihm, H. & White, J. M. Electron-induced surface chemistry: production and characterization of NH2 and NH species on Pt(111). J. Vac. Sci. Technol. A 14, 15161521 (1996). 25. Skoplyak, O., Barteau, M. A. & Chen, J. G. Reforming of oxygenates for H2 production: correlating reactivity of ethylene glycol and ethanol on Pt(111) and Ni/Pt(111) with surface d-band center. J. Phys. Chem. B 110, 16861694 (2006). 26. Dietrich, H., Jacobi, K. & Ertl, G. Coverage, lateral order and vibrations of atomic nitrogen on Ru(0001). J. Chem. Phys. 105, 89448950 (1996). 27. Gardin, D. E., Batteas, J. D., Vanhove, M. A. & Somorjai, G. A. Carbon, nitrogen, and sulfur on Ni(111)formation of complex structures and consequences for molecular decomposition. Surf. Sci. 296, 2535 (1993). 28. Kim, Y. K., Morgan, G. A. & Yates, J. T. Site-specic dissociation of N2 on the stepped Ru(109) surface. Surf. Sci. 598, 1421 (2005). 29. Dietrich, H., Jacobi, K. & Ertl, G. Decomposition of NH3 on Ru(11(2)1). Surf. Sci. 352, 138141 (1996).
Acknowledgements
This research was supported by the Ofce of Basic Energy Sciences, Department of Energy grants DE-FG02-06ER15795 and DE-FG02-00ER15104. The DFT calculations were performed using the TeraGrid resources provided by the University of Illinois National Center for Supercomputing Applications (NCSA)41.
D.A.H. and D.G.V. designed and developed the microkinetic models. D.A.H. and J.G.C. designed and developed the UHV experiments. D.A.H. performed and analysed all modelling and experimental work. All authors contributed to writing the paper.
The authors declare no competing nancial interests. Supplementary information accompanies this paper at www.nature.com/naturechemistry. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to D.G.V. and J.G.C.
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Nucleophilic catalysis of acylhydrazone equilibration for protein-directed dynamic covalent chemistry

Venugopal T. Bhat1, Anne M. Caniard1, Torsten Luksch2, Ruth Brenk2, Dominic J. Campopiano1 * and Michael F. Greaney1 *
Dynamic covalent chemistry uses reversible chemical reactions to set up an equilibrating network of molecules at thermodynamic equilibrium, which can adjust its composition in response to any agent capable of altering the free energy of the system. When the target is a biological macromolecule, such as a protein, the process corresponds to the protein directing the synthesis of its own best ligand. Here, we demonstrate that reversible acylhydrazone formation is an effective chemistry for biological dynamic combinatorial library formation. In the presence of aniline as a nucleophilic catalyst, dynamic combinatorial libraries equilibrate rapidly at pH 6.2, are fully reversible, and may be switched on or off by means of a change in pH. We have interfaced these hydrazone dynamic combinatorial libraries with two isozymes from the glutathione S-transferase class of enzyme, and observed divergent amplication effects, where each protein selects the best-tting hydrazone for the hydrophobic region of its active site.
ynamic covalent chemistry (DCC) uses reversible chemical reactions to set up equilibrating assemblies of molecules at thermodynamic equilibrium14. The resultant dynamic combinatorial library (DCL) is responsive to the addition of a template, which will selectively amplify the best binding compounds from the equilibrium distribution. The essence of the concept lies in the subsequent adjustment of the DCL equilibrium, which will express more of the best binding compounds at the expense of the poorer ones. A DCL is thus adaptive and capable of evolutionary behaviour, whereby individual components are either amplied or reduced in response to template-directed binding events. These concepts have been applied to diverse problems in biological and medicinal chemistry511, synthetic receptorligand interactions1216, self-replication1719, complex molecule synthesis2022 and materials science23,24. Taken together, they represent the best characterized examples to date of systems chemistry, which looks to synthesize complex molecular networks and study their properties and behaviour in macrocosm, rather than as a sum of their individual components25,26. We are interested in DCC systems that use a biological molecule, such as a protein, to template assemblies of small molecules at dynamic equilibrium27. Here, the DCC experiment provides a method for discovering, studying and ranking novel protein ligands, concepts fundamental to medicinal chemistry. In these terms, the DCC process bridges the gap between targeted chemical synthesis of drug candidates and their biological binding assay, meshing the two processes into a single step in which the structure of the biological target directs the assembly of its own best inhibitor in situ. A particular challenge for DCC in biological systems lies in the implementation of a suitable reversible reaction that can operate effectively under the physiological conditions required by the biotemplate. Lehn has dened two limiting cases for DCL construction:
adaptive and pre-equilibrated DCC28. The adaptive DCL represents the ideal scenario, where the DCL chemistry is fully compatible with the biological target and the ensuing binding events control the evolution of the DCL composition. Pre-equilibrated DCL refers to the cases where the reversible chemistry used to constitute the
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Figure 1 | Transimination reactions for DCC. a, Imine DCLs: reversible addition of amines to aldehydes gives unstable imines that cannot be isolated or analysed directly, necessitating an in situ reduction step. The resultant static library of amines may or may not share the binding prole of the imine precursors. b, Acyl hydrazone DCLs: reaction of aldehydes with hydrazides gives acylhydrazones that have good stability and are amenable to analysis. Equilibration requires acidic conditions that are incompatible with biological targetsa nucleophilic catalyst such as aniline may enable DCL formation at biocompatible pH.
EastChem, School of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JJ, UK, 2 College of Life Sciences, University of Dundee, James Black Centre, Dow Street, Dundee DD1 5EH, UK; These authors contributed equally to this work. * e-mail: Dominic.Campopiano@ed.ac.uk; Michael.Greaney@ed.ac.uk
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O R NHNH2 Aniline pH 6.2 Cl NO2 2 3 O NHNH2 N H 2e NHNH2 H N N O R
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Figure 2 | Aniline-catalysed acylhydrazone formation. a, Aldehyde equilibration with hydrazide to form an acylhydrazone. b, Hydrazide components of the ten-membered DCL. c, DCL established in the absence of aniline. Conditions: aldehyde (5 mM), hydrazides (20 mM each) in NH4OAc buffer (50 mM, pH 6.2) containing 15% DMSO. d, DCL established in the presence of aniline (10 mM).
DCL is not compatible with the biological target, meaning that the DCL and the target must be separated in some manner. This results in static libraries in which the molecular recognition events that control DCL composition are lost. Given the challenges associated with conducting fast, freely reversible chemistry under physiological conditions, it is not surprising that methods for true adaptive DCL generation are limited, with the majority of successful systems using sulfur-based transformations such as disulde bond formation or thiol conjugate addition2932. The development of new methods for adaptive DCLs is thus central to the application of DCC to biological systems, as the chemistry will dene the target scope and range of available DCL components. The reversible formation of CN imine-type linkages emerged early on as a DCL-forming reaction33. The ready availability of diverse carbonyl and amine building blocks, plus the extensive precedent of imine formation in biochemical systems, makes it an ideal candidate reaction. However, the inherent instability of imines in aqueous solution presents serious analytical and isolation problems in the DCC context. The solution to this in the eld of biological DCC has been to construct pseudo-adaptive DCLs where the imine linkage is reduced in situ to an amine with an external hydride source. The resulting library contains static amine components that can correspond to the imines in binding afnity,
although both false-positive and false-negative results are possible. In addition, the introduction of an in situ reduction step complicates the DCL equilibration and makes it difcult to distinguish between genuine thermodynamic selection of the best binders and selection of those compounds that are kinetically favoured. An advance on simple imine formation in DCC came from the Sanders group, who introduced acylhydrazones as reversible linkages34. The reaction has proven to be an excellent balance between facile reversibility and product stability; the acylhydrazone products formed are stable to analysis and isolation, and the reaction has very good equilibration properties, as is made evident by its application to a large number of elegant abiological DCC studies subsequently reported by the Sanders group3537. It has not, however, been generally possible to apply this reaction directly to adaptive biological DCC systems because of the acidic pH required for reversibility to occur in a reasonable timeframe (pH , 4)38,39. A single elegant study from Poulsen has shown that slow equilibration of acylhydrazones, taking one week at pH 7.2, can be accelerated in the presence of the enzyme carbonic anhydrase, enabling in situ identication of binders using mass spectrometry40. We were keen to apply this proven reaction to our DCC studies of enzymes, and reasoned that it could be harnessed as a powerful tool for biological investigation if a suitable catalyst
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Figure 3 | Structure of GST illustrating H- and G-sites. Grey, monomer 1; yellow, monomer 2; green, G-site; red, H-site.
could be found to accelerate the equilibration. Nucleophilic catalysis of semicarbazone formation using aniline derivatives was established in classic work from Jencks in the 1960s, and recently applied to hydrazone and oxime formation in peptide ligation systems by Dawson4144. We reasoned that an additive such as aniline could promote the equilibration of acylhydrazides and aldehydes at pH values closer to the physiological window required by biological targets in DCC (Fig. 1). We began by reacting aldehyde 1 (Fig. 2), related to the known glutathione S-transferase (GST) substrate chlorodinitrobenzene (CDNB, see below), with an excess of the ten aryl hydrazides 2a2j at room temperature. The hydrazides were chosen to randomly display aryl and heteroaryl groups and featured eight acyl and two sulfonyl hydrazides (2d and j). Equilibration at pH 6.2 was slow, and only two of the ten possible hydrazones could be observed by high-performance liquid chromatography (HPLC) after 1 h (Fig. 2c). Notably, there was a signicant amount of free aldehyde 1 present throughout the reaction, despite the presence of excess amounts of the ten different hydrazides. Equilibrium was not complete after 48 h, and required incubation for a further 5 days until the library composition reached a steady-state composition with signals for each of the ten hydrazones 3a3j being clearly identied. In contrast, repeating the experiment in the presence of excess aniline produced a far higher rate of equilibration. A distribution of acylhydrazones was observed after initial mixing and HPLC sampling, and complete equilibration of the ten components was observed after just 6 h (Fig. 2d). Aldehyde 1 could not be detected following initial mixing, indicating that it was continually being sequestered as an acylhydrazone component, reecting the faster exchange processes operating in the presence of aniline (see Supplementary Information for a study on the effect of varying aniline concentration on rates of hydrazone formation). We demonstrated the reversibility of the DCL by generating it from a different starting composition, hydrazone 3g plus the nine other hydrazides and aniline. An identical equilibrium distribution to Fig. 2 was observed, indicating true thermodynamic equilibrium. A second control experiment conrmed the reversibility of the DCL through the addition of excess hydrazide 2b to the preequilibrated DCL, which resulted in a large amplication of the corresponding acylhydrazone 3b (see Supplementary Information). Having established that aniline could act as an effective nucleophilic catalyst for hydrazone DCC formation at both a pH and timeframe reasonable for biomolecule stability, our next step was to introduce proteins to the DCL. Our target chosen for DCC interrogation was the GST enzyme superfamily45. The GSTs are responsible for cell detoxication, catalysing the conjugation of glutathione (GSH) to a wide variety of xenobiotic electrophiles, thereby
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protecting the cell from cytotoxic and oxidative stress. We have previously developed thiol conjugate addition DCLs directed towards GST inhibition, and successfully interfaced the enzyme with small molecules so that it controlled library evolution27. The GSTs are well suited to exploration using DCC methods, being well-characterized, robust proteins having nascent medicinal chemistry application46,47. There are relatively few ligands reported in the literature for GST bindinga plus point, as it would enable us to use DCC as a genuine discovery tool for new binding motifs, rather than as a proof-of-principle process for conrming the binding ability of known ligands. The cytoplasmic GSTs are inherent dimers with active sites composed of residues from both monomers, bifurcating between a highly conserved G-site, which binds the endogenous ligand GSH, and an H-site, which binds hydrophobic substrates for GSH conjugation (Fig. 3). This bisubstrate architecture is particularly appropriate for DCC interrogation, given that the method essentially uses a reversible linkage to couple two sets of fragment structures together48. Furthermore, within the GST superfamily, the large, heterogeneous H-sites are functionally evolved to accommodate many different hydrophobic substrates for conjugation, a classically difcult architecture to investigate using orthodox structure-based drug-design methods. We prepared two recombinant GST isozymes as targets, SjGST from the helminth worm Schistosoma japonicum, a drug target in tropical disease49, and hGST P1-1, a human isoform that has been targeted in the treatment of chemotherapy drug resistance50. An initial control experiment with SjGST established that the enzyme retained GSH conjugation activity in the presence of aniline (up to 20 mM). The acylhydrazone DCL prepared in Fig. 2 was then interfaced with the two protein targets and amplication was measured (Fig. 4). Both DCLs demonstrated strikingly clear amplication of hydrazone components; thiophene acylhydrazone 3g was selected by SjGST and t-butylphenyl hydrazone 3c by hGST P1-1. Synthesizing the DCL in the presence of bovine serum albumin (BSA, 1 equiv.) as a control experiment produced no measurable amplication of any component, indicating the GST enzymes as being responsible for component amplication. We further demonstrated that amplied components were bound in the target H-region of the active site of the enzyme by performing conjugation experiments with GSH. Conjugation of GSH to the aryl chloride group in hydrazones 3 by means of SNAr substitution is a slow reaction at pH 6.2, taking several days. In the presence of catalytic amounts of SjGST, however, rapid formation of the SNAr conjugation adduct for hydrazone 3g was observed at pH 6.2. The amplied hydrazone can thus act as a substrate for SjGST and binds in the targeted H-site. The amplied hydrazones were re-synthesized and assayed against both GSTs and found to be inhibitors of GSH conjugation of CDNB, but poor solubility prevented the determination of accurate IC50 values at the higher concentrations necessary to assay weak binding compounds. To solve this problem, and simultaneously increase the potency of our DCL components, we conjugated GSH to aldehyde 1 using an SNAr reaction. We anticipated that the highly soluble GSH tripeptide motif would act as an anchor at the G-site, enabling exploration of the H-site with assorted hydrazide fragments. This approach, in which a known enzymesubstrate interaction is used for inhibitor discovery, is well exemplied in classical medicinal chemistry drug design, GST inhibition51 and DCC methods. The IC50 value for SjGST inhibition of the anchored fragment 4 was measured in the CDNB conjugation assay as 280 mM. Initial DCC experiments using GS-conjugated aldehyde 4 and the same ten hydrazides used previously conrmed the utility of aniline as a nucleophilic catalyst (Fig. 5). Equilibration was complete in 6 h, compared to 4 days in the absence of aniline, and each of the ten acylhydrazones were clearly identied by liquid chromatographymass spectrometry (LC-MS) (see Supplementary Information). As before, clear amplications could be observed for both GST
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Figure 4 | GST-templated DCLs. a, DCL hydrazone composition in the absence of any target (blank). b, When the DCL is constituted in the presence of SjGST, the thiophene hydrazone 3g is clearly amplied. c, Changing the target protein to hGSTP1-1 produces a different distrubution, in which the t-butylphenyl derivative 3c is amplied. Targeted DCL conditions: GST (1 equiv.), aldehyde (5 mM), hydrazides (20 mM) and aniline (10 mM) in NH4OAc buffer (50 mM, pH 6.2) containing 15% DMSO for 16 h.
targets: in each case the same hydrazide fragment was selected as the best binder, thiophene (5g) for SjGST and t-butylphenyl (5c). Both components were amplied to over 300% of their concentrations in the blank DCL, at the expense of nearly all other competing hydrazones. Also of note, the anisyl sulfonylhydrazone 5j underwent 100% amplication using hGST P1-1 as the only other positively selected component. The most signicant reductions in equilibrium concentrations occurred for 5b, f and i (SjGST) and 5f, g and i (hGST P1-1). The GST-directed DCLs were synthesized with the protein present from the beginning of the experiment, that is, in the presence of aldehyde 4 and the ten hydrazides 2a2j. To verify that the amplication results were not due to a kinetic selection by means of target-accelerated synthesis, we added SjGST to the pre-equilibrated DCL. The same equilibrium distribution was achieved as is shown in Fig. 5, with hydrazone 5g strongly amplied, indicating that the amplied components are the result of genuine thermodynamic selection. Further controls involved a BSA control experiment, which was negative, and DCL synthesis in the presence of a large excess of the nonselective GST inhibitor ethacrynic acid. Component amplication was completely suppressed for both SjGST and hGST P1-1 DCLs, indicating that the GST active site is saturated by the ethacrynic acid and cannot inuence the DCL equilibrium composition. We completed our protein-directed DCL studies by preparing a catalytically inactive SjGST mutant. It was of interest to see whether a functionally disabled enzyme would exert the same control and selectivity on DCL composition as the wild-type enzyme. The conserved Tyr 7 active site residue is known to play a critical role in GSH conjugation for the Sj class of GSTs, stabilizing the GSH thiolate anion through H-bonding from the phenol group, with enzymes lacking this residue being catalytically inactive52. We prepared a Y7F mutant of SjGST, in which the crucial tyrosine
residue is replaced with phenylalanine. We observed essentially zero activity with this mutant in CDNB conjugation when compared with the wild-type SjGST. However, SjGST Y7F proved equally effective in controlling DCL composition, showing a clear preference for the same thiophene derivative 5g as was amplied by the wild-type SjGST (see Supplementary Information). Biological assay was then performed to establish whether the best binding compounds in the GST-directed DCLs were also the best inhibitors of the GST enzyme. To fully explore the isozyme-specic amplication effects of the two DCLs, we separately synthesized hydrazone conjugates 5a5j for study. We rst conrmed that the amplied ligands 5c and 5g bound to SjGST and hGST P1-1 by isothermal calorimetry (ITC) (see Supplementary Information). We then studied their inhibitory activity towards SjGST and hGST P1-1 using the CDNB conjugation assay. The IC50 values were slightly higher for all hydrazones against hGST P1-1 compared to SjGST (data ranging from 59 to 126 mM and 22 to 63 mM, respectively; see Supplementary Information). For each isozyme, the DCC amplied hydrazone was the most active; thiophene 5g had the lowest IC50 value (22 mM) among all the library members against SjGST, and t-butylphenyl 5c had the lowest value among the four conjugates tested against hGST P1-1 (57 mM). The DCL hydrazone selection process has successfully extended inhibitor structure in the GST H-site, increasing potencies by sixfold for hGST P1-1 (331 to 57 mM ) and by over tenfold for SjGST (279 to 22 mM) relative to the starting anchored aldehyde 4. Steady-state kinetic studies on the two amplied DCL components 5c (hGST P1-1) and 5g (SjGST) conrmed the expected competitive inhibition prole, with both compounds binding to the GST active sites. It was interesting to note slightly higher Ki values for both compounds when assayed against CDNB, a substrate for the H-site of the enzyme, relative to the endogenous G-site
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Figure 5 | GST-templated DCLs of GSH conjugates. a, Acyl hydrazone DCL based on GSH-conjugated aldehyde 4 (GS S-linked glutathione). b, DCL hydrazone composition in the absence of target (blank), in the presence of SjGST and in the presence of hGSTP1-1. DCL conditions: GST (1 equiv.), aldehyde (5 mM), hydrazides (20 mM) and aniline (10 mM) in NH4OAc buffer (50 mM, pH 6.2) containing 15% DMSO for 16 h. c, Changes in DCL component concentration for blank, SjGST and hGST P1-1 DCLs. The error bars represent the standard deviation over three experiments.
ligand GSH. The afnity of the two hydrazone conjugates towards both GST G-sites was relatively close (data ranging from 5.25 to 7.19 mM), as would be expected for two compounds sharing a common GSH-tagged nitrobenzene fragment. To obtain some molecular insight into the selectivity of our isozymes towards the two hydrazone inhibitors 5c and g, we carried out a molecular modelling study. We surveyed the available GST structures in the protein data bank (PDB) and retrieved those that contained a bound GSH-based ligand. The binding sites of these structures, together with the bound ligands, were aligned, and it became evident that the glutathione portions overlaid well,
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being bound in very similar conformations in the G-sites (Fig. 6a). In contrast, the conjugate parts of the various ligands showed great diversity in their conformations within the H-site, an unsurprising result given the respective functions of the G- and H-sites. Detailed analysis of the superimposed crystal structures identied the GSH conjugate of 1,2-epoxy-3-( p-nitrophenoxy)propane (EPNP) (6) bound to cGST M1-1 (PDB code 1c72)53 as the ligand that projected functionality into the H-site with the most similar geometry to the energy-minimized structure of hydrazone 5g (Fig. 6b). Analysis of the GSTEPNP complex shows the EPNP moiety orienting towards R107 and Q165 in the H-site of the enzyme
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reducing k EPNP by 59%, although Km showed only small cat changes53. Because the amino acids in the equivalent pocket of SjGST and hGST P1-1 are not highly conserved, these residues have such a great inuence on ligand binding that it is likely that these amino-acid exchanges across the isoforms are critical in determining ligand selectivity.
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H N O 5g
Conclusions
We have demonstrated that reversible synthesis of acylhydrazones can be compatible with protein targets by using aniline as a nucleophilic catalyst. The many advantages of this DCC tool (ready availability of easily customized building blocks, good kinetic and thermodynamic properties leading to ease of analysis, good biological compatibility forming amide-like linkages) may now be realized with biological targets. Most importantly, the acylhydrazone DCLs are truly adaptive, allowing amplication effects to be simply and directly related to structures present at equilibrium. The GST enzyme proved extremely effective as a DCL template, with two isozymes from the GST family smoothly integrating with the small molecule assemblies and strongly amplifying the best binding components. The selected hydrazones showed increased inhibitory activity of over one order of magnitude from the starting GSH-tagged benzaldehyde 4, validating the approach in the context of proteinligand discovery. Interestingly, a single, small DCL composed of only ten members displays isozyme selectivity according to which variant of the GST enzyme is used as the template. The study at hand has been deliberately conned to a small number of DCL components so as to thoroughly characterize equilibrium distributions and quantify amplications with the anilinecatalysed hydrazone method. In principle, much larger hydrazone DCLs may be accessed to thoroughly explore chemical space, both within the GST H-site and for other biological targets9,15. It may not be possible, or even desirable, to accurately characterize the equilibrium distribution of such complex DCLs, but this will not be necessary if one simply seeks to identify prominently amplied components from a ligand discovery perspective. To gain insight into isoform selectivity, we found that each amplied molecule could be effectively docked into its respective GST H-site, although the ne structural features of the SjGST versus hGST P1-1 H-site that discriminate between thiophene hydrazone 5g and t-butylphenyl hydrazone 5c are unclear at the present time. Structural determination of the complexes of various GST:GShydrazone conjugates will be needed for a deeper understanding of the factors that control H-site selectivity. Work in this
Y103 H162
Figure 6 | GST ligands. a, Superposition of a selection of GST ligands from the PDB. b, Conformation of the GST-bound EPNP ligand 6 as found in the crystal structure of cGST M1-1 (PDB code 1c72, green carbon atoms), relative to the energy-minimized structure of compound 5g (pink carbon atoms).
(Fig. 7c). The side chains of R107, F110, Q165, Q166 and F208 dene the pocket that connes the EPNP moiety. On this basis, we could generate a binding model for SjGST with thiophene hydrazone 5g and for hGST P1-1 with t-butyl hydrazone 5c (Fig. 7). The interactions in the generated binding modes for SjGST in complex with 5g (Fig. 7a) and for hGST P1-1 in complex with 5c (Fig. 7b) between the glutathione moiety and the proteins are identical to those reported in previous publications54,55. We predict that the hydrazone group of 5g forms hydrogen bonds to R103 and Q204 in SjGST, and equivalent interactions are observed for 5c in complex with hGST P1-1. Residue V161 in SjGST and I161 in hGST P1-1 make hydrophobic interactions in our models with the ligands 5c and 5g. As expected, the sub-pockets of both isoforms accommodating the hydrazones are rather hydrophobic, and complement the hydrophobic hydrazones amplied from the DCL. The thiophene hydrazone ts easily in the SjGST binding pocket, with only minor sidechain adjustments necessary (root mean square deviation (RMSD) 0.3 between model and crystal structure template), whereas the t-butylphenyl group would lead to a steric clash and would require some degree of induced t in order to bind. Induced t is also required to accommodate this ligand in the hGST P1-1 pocket, but in that case the binding mode could be stabilized by additional lipophilic interactions of the t-butyl group with Y103, H162 and I161. It is worth noting that Chern and colleagues have reported that mutations in that region of the H-site had a great impact on EPNP binding as a substrate, with mutation of cGST M1-1 Q165 to leucine (V161 in SjGST and I161 in hGST P1-1)
a
Q204 V161 V162 V106
b
N204 I161
c
N208 Q165
Q166
F110
R103 W38 W41 N54 Q67 K44
R100 W45 N58 Q51 Q64 K49 Q71
R107
K45
Figure 7 | Molecular modelling of amplied DCL components with the GST active site. a, Model of 5g bound to SjGST. b, Model of 5c bound to hGST P1-1. The binding pocket surfaces are shown in light blue and key amino acids as blue sticks. The ligands are represented in salmon pink, with atoms coloured by type. Hydrogen bonds of the conjugated ligand parts are shown as yellow dotted lines. c, The EPNPcGST M1-1 crystal structure (PDB code 1c72). The binding pocket surface is shown in raspberry pink and key amino acids as red sticks. The ligand is represented in green, with atoms coloured by type. Hydrogen bonds of the conjugated ligand parts are shown as yellow dotted lines.
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area, together with applications of acylhydrazone DCC to other biological targets, is the subject of our current research.
NATURE CHEMISTRY
DOI: 10.1038/NCHEM.658
Methods
Aniline catalysis of reversible hydrazone formation. The ten hydrazides 2aj (10 5 ml, 10 mM, DMSO), aldehyde 1 (2 ml, 10 mM, DMSO) and aniline (10 ml, 1 M, DMSO) were added to a mixture of DMSO (93 ml) and ammonium acetate buffer (845 ml, 50 mM, pH 6.2). The DCL was allowed to stand at room temperature with occasional shaking, and was monitored periodically by HPLC to establish the blank composition until the relative populations of the hydrazones became constant. The pH of all samples was raised to 8 by the addition of NaOH (15 ml, 1 M, aqueous). LC-MS veried that each of the expected hydrazones was present in the DCL (HPLC conditions: column, Luna 5 m C18(2), 30 mm 4.6 mm, and Luna 5 m C18(2), 50 mm 4.6 mm, in sequence; ow rate, 1 ml min21; wavelength, 254 nm; temperature, 23 8C; gradient, H2O/MeCN (0.01% TFA) from 95% to 80% over 6 min, then to 45% over 30 min, and eventually to 5% over 5 min) (Fig. 2d). The DCL was then re-synthesized in the absence of aniline, and the HPLC traces at different time intervals were compared (Fig. 2c). Templated DCL aldehyde 1. SjGST (111 ml, 180 mM, in potassium phosphate buffer 0.1 M, pH 6.8), the ten hydrazides 2aj (10 5 ml, 10 mM, DMSO), aldehyde 1 (2 ml, 10 mM, DMSO) and aniline (10 ml, 1 M, DMSO) were added to a mixture of DMSO (93 ml) and ammonium acetate buffer (734 ml, 50 mM, pH 6.2). The DCL was allowed to stand at room temperature, with occasional shaking, for 12 h. The pH of the sample was raised to 8 by the addition of NaOH (15 ml, 1 M, aqueous), and the protein was removed by ultraltration using a 10,000 MWCO lter (Vivaspin). HPLC analysis was performed and the traces were compared with the blank composition (HPLC conditions: column, Luna 5 m C18(2), 30 mm 4.6 mm, and Luna 5 m C18(2), 50 mm 4.6 mm, in sequence; ow rate, 1 ml min21; wavelength, 254 nm; temperature, 23 8C; gradient H2O/MeCN (0.01% TFA) from 95% to 80% over 6 min, then to 45% over 30 min, and eventually to 5% over 5 min). DCL composition was identical, regardless of whether the SjGST was present from the beginning or added after pre-equilibration, but equilibration took more than 24 h in the latter case. For the hGST P1-1 templated library, the ten hydrazides 2aj (10 5 ml, 10 mM, DMSO), aldehyde 1 (2 ml, 10 mM, DMSO), aniline (10 ml, 1 M, DMSO) and hGST P1-1 (100 ml, 200 mM, in potassium phosphate buffer 0.1 M, pH 6.8) were added to a mixture of DMSO (93 ml) and ammonium acetate buffer (734 ml, 50 mM, pH 6.2). After equilibration for 12 h, the DCL was analysed using HPLC. Control experiments were performed using the same equivalents of BSA in place of GST. Conjugate DCLs. To establish the blank DCL composition, the ten hydrazides 2aj (10 5 ml, 10 mM, DMSO), aldehyde 5 (5 ml, 10 mM, aqueous) and aniline (10 ml, 1 M, DMSO) were added to a mixture of DMSO (96 ml) and ammonium acetate buffer (839 ml, 50 mM, pH 6.2). The DCL was allowed to stand at room temperature, with occasional shaking, and was monitored periodically by HPLC to establish the blank composition until the relative populations of the hydrazones became constant. The pH of all the samples was increased to 8 by the addition of NaOH (15 ml, 1 M, aqueous). LC-MS veried that each of the expected hydrazones was present in the DCL (Fig. 5) (HPLC conditions: column, Luna 5 m C18(2), 50 mm 4.6 mm, and Luna 5 m C18(2), 250 mm 4.6 mm, in sequence; ow rate, 1 ml min21; wavelength, 254 nm; temperature, 23 8C; gradient H2O/MeCN (0.01% TFA) from 95% to 5% over 40 min). For re-synthesizing the DCL in the presence of the protein SjGST (278 ml, 180 mM, in potassium phosphate buffer 0.1 M, pH 6.8), the ten hydrazides 2aj (10 5 ml, 10 mM, DMSO), aldehyde 5 (5 ml, 10 mM, DMSO) and aniline (10 ml, 1 M, DMSO) were added to a mixture of DMSO (96 ml) and ammonium acetate buffer (561 ml, 50 mM, pH 6.2). The DCL templated by hGST P1-1 was synthesized by adding the ten hydrazides 2aj (10 5 ml, 10 mM, DMSO), aldehyde 5 (5 ml, 10 mM, DMSO), aniline (10 ml, 1 M, DMSO) and hGST P1-1 (250 ml, 200 mM, in potassium phosphate buffer 0.1 M, pH 6.8) to a mixture of DMSO (96 ml) and ammonium acetate buffer (589 ml, 50 mM, pH 6.2). The DCLs were allowed to stand at room temperature for 12 h, after which the pH was raised to 8 by the addition of NaOH (15 ml, 1 M). The protein was ltered off using a centrifuge lter of MWCO 10,000 followed by analysis of the ltrate by HPLC using conditions similar to those listed above. Molecular modelling. To establish ligand alignment, the superposition of GST ligands was carried out using Relibase 3.0.0 (ref. 56). A search was rst performed to nd binding sites that share a sequence identity between 40 and 100% with the target GST crystal structure 1m9a. The resulting 38 structures with bound ligand were superimposed by using binding site residues only. Finally, the ligands from the superimposed structures were extracted and visually analysed. To carry out a binding mode prediction with Moloc57, the SjGST crystal structure (PDB code 1m9a SjGST S-hexylGSH complex) and the hGST P1-1GSH complex crystal structure (PDB code 6gss)54 were used as starting conformations for binding mode generation. The glutathione groups of the synthesized ligands were mapped onto the glutathione groups of the ligands bound to the crystal structures. The hydrophobic hydrazone groups of the synthesized ligands were oriented towards the cavity, lying at the end of the S-hexyl site, as
496
observed for the EPNP ligand bound to cGSTM1-1 (PDB code 1c72). In the next step, the protein in complex with the modelled ligand was minimized, considering the ligand as fully exible. For the protein all residues were kept rigid, except for the amino acids that dene the pocket at the end of the S-hexyl site (R103, V106, V161, V162, Q204 for SjGST and R100, Y103, I161, H162, N204 for hGST P1-1).
Received 7 December 2009; accepted 30 March 2010; published online 16 May 2010
References
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28. Ramstrom, O. & Lehn, J.-M. In situ generation and screening of a dynamic combinatorial carbohydrate library against concanavalin A. ChemBioChem 1, 4148 (2000). 29. Otto, S., Furlan, R. L. E. & Sanders, J. K. M. Dynamic combinatorial libraries of macrocyclic disuldes in water. J. Am. Chem. Soc. 122, 1206312064 (2000). 30. Nicolaou, K. C. et al. Target-accelerated combinatorial synthesis and discovery of highly potent antibiotics effective against vancomycin-resistant bacteria. Angew. Chem. Int. Ed. 39, 38233828 (2000). 31. Milanesi, L., Hunter, C. A., Sedelnikova, S. E. & Waltho, J. P. Amplication of bifunctional ligands for calmodulin from a dynamic combinatorial library. Chem. Eur. J. 12, 10811087 (2006) 32. Shi, B. & Greaney, M. F. Reversible Michael addition of thiols as a new tool for dynamic combinatorial chemistry. Chem. Commun. 886888 (2005). 33. Huc, I. & Lehn, J.-M. Virtual combinatorial libraries: dynamic generation of molecular and supramolecular diversity by self-assembly. Proc. Natl Acad. Sci. USA 94, 21062110 (1997). 34. Cousins, G. R. L., Poulsen, S. A. & Sanders, J. K. M. Dynamic combinatorial libraries of pseudo-peptide hydrazone macrocycles. Chem. Commun. 15751576 (1999). 35. Furlan, R. L. E., Ng, Y. F., Otto, S. & Sanders, J. K. M. A new cyclic pseudopeptide receptor for Li from a dynamic combinatorial library. J. Am. Chem. Soc. 123, 88768877 (2001). 36. Roberts, S. L., Furlan, R. L. E., Cousins, G. R. L. & Sanders, J. K. M. Simultaneous selection, amplication and isolation of a pseudo-peptide receptor by an immobilised N-methyl ammonium ion template. Chem. Commun. 938939 (2002). 37. Liu, J. Y., West, K. R., Bondy, C. R. & Sanders, J. K. M. Dynamic combinatorial libraries of hydrazone-linked pseudo-peptides: dependence of diversity on building block structure and chirality. Org. Biomol. Chem 5, 778786 (2007). 38. Bunyapaiboonsri, T. et al. Dynamic deconvolution of a pre-equilibrated dynamic combinatorial library of acetylcholinesterase inhibitors. ChemBioChem 2, 438444 (2001). 39. Bunyapaiboonsri, T., Ramstrom, H., Ramstrom, O., Haiech, J. & Lehn, J.-M. Generation of bis-cationic heterocyclic inhibitors of Bacillus subtilis HPr kinase/phosphatase from a ditopic dynamic combinatorial library. J. Med. Chem. 46, 58035811 (2003). 40. Poulsen, S. A. Direct screening of a dynamic combinatorial library using mass spectrometry. J. Am. Soc. Mass Spectrom. 17, 10741080 (2006). 41. Cordes, E. H. & Jencks, W. P. Nucleophilic catalysis of semicarbazone formation by anilines. J. Am. Chem. Soc. 84, 826831 (1962). 42. Dirksen, A., Dirksen, S., Hackeng, T. M. & Dawson, P. E. Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J. Am. Chem. Soc. 128, 1560215603 (2006). 43. Dirksen, A. & Dawson, P. E. Rapid oxime and hydrazone ligations with aromatic aldehydes for biomolecular labeling. Bioconjugate Chem. 19, 25432548 (2008). 44. Rodriguez-Docampo, Z. & Otto, S. Orthogonal or simultaneous use of disulde and hydrazone exchange in dynamic covalent chemistry in aqueous solution. Chem. Commun. 53015303 (2008). 45. Hayes, J. D., Flanagan, J. U. & Jowsey, I. R. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 5188 (2005). 46. Mahajan, S. & Atkins, W. M. The chemistry and biology of inhibitors and pro-drugs targeted to glutathione S-transferases. Cell. Mol. Life Sci. 62, 12211233 (2005).
Acknowledgements
The authors would like to thank EastChem for the award of a studentship to V.T.B. and the Marie Curie Early Stage Training Network (Syn4chembio) and School of Chemistry at Edinburgh for awarding a studentship to A.M.C. R.B. is supported by an EC Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 223461. M.F.G. is an Engineering and Physical Sciences Research Council (EPSRC) Leadership Fellow. The authors thank A. Cooper (University of Glasgow) for ITC measurements and helpful discussions. N. Petitjean is thanked for the synthesis of hydrazoneGSH conjugates.
V.T.B., A.M.C., D.J.C. and M.F.G. conceived and designed the experiments, V.T.B. and A.M.C. performed the experiments, and T.L., R.B. and A.M.C. carried out molecular modelling. All authors discussed the results and co-wrote the manuscript.
The authors declare no competing nancial interests. Supplementary information and chemical compound information accompany this paper at www.nature.com/ naturechemistry. Reprints and permission information is available online at http://npg.nature. com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to D.J.C. and M.F.G.
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Electrochemistry through glass

Jeyavel Velmurugan, Dongping Zhan and Michael V. Mirkin*
In this Article we have used new approaches to investigate a well-known chemical process, the propagation of electrochemical signals through a thin glass membrane. This process, which has been extensively studied over the last century, is the basis of the response of a potentiometric glass pH sensor; however, no amperometric glass sensors have yet been reported because of its high ohmic resistance. Voltammetry at nanoelectrodes has revealed that water molecules can diffuse through nanometre-thick layers of dry glass and undergo oxidation/reduction at the buried platinum surface. After soaking for a few hours in an aqueous solution, voltammetric waves of other redox couples, such as Ru(NH3)631/21, could also be obtained at the glass-covered platinum nanoelectrodes. This behaviour suggests that the nanometre-thick insulating glass sheath surrounding the platinum core can be largely converted to hydrated gel, and electrochemical processes occur at the platinum/hydrogel interface. Potential applications range from use in nanometre-sized solid-state pH probes and determination of the water content in organic solvents to glass-modied voltammetric sensors and electrocatalysts.
n intriguing aspect of nanoelectrochemical experiments is the possibility of using a nanometre-sized electrode to observe processes and phenomena that are not accessible using macroscopic probes1. Examples include single molecular events2, unusual transport phenomena3 and electrical double-layer effects4. The subject of this articleelectrochemistry through glassmay sound like an oxymoron, because glass is commonly used as an insulating material in electrode fabrication. However, 100-nm-thick layers of glass have been found to be sufciently conductive for electrochemical measurements. The propagation of an electrical signal through glass membranes has been extensively studied since the beginning of the twentieth century because of its relevance to the potentiometric glass pH electrode5. Because the potential of the glass electrode is a linear function of solution pH, it is intuitive to assume that the voltage drop across the membrane is determined by proton transfer. However, numerous experiments using the 3H isotope and other methods have shown that protons do not cross the glass membrane6,7. The potentiometric response of the pH electrode originates in the ion-exchange equilibrium on the glass surface, and the diffusion of protons and water is essentially conned to a nanometre-thick surface layer of hydrated gel. This gel forms on both sides of the membrane when it is soaked for several hours in acidic aqueous solution8. This response mechanism was established for electrodes with micrometre-thick sensing membranes (typical thickness, 100 mm). The behaviour of nanometre-thick glass layers, however, is substantially different. We used nanometre-sized platinum electrodes to investigate the permeability of glass in aqueous and non-aqueous solutions. An electrode of this type with a conductive core radius of a 5 nm can be produced by pulling a platinum microwire into a borosilicate glass capillary with the help of a laser pipette puller. After pulling, the metal wire is completely sealed into the glass, and its nanometre-sized tip can be exposed by gentle polishing under video microscopic control9. The geometry of the polished nanoelectrodes was characterized by a combination of voltammetry, scanning electron microscopy (SEM) and scanning electrochemical microscopy (SECM). It was shown that the effective radius value (a) determined from steady-state voltammetry is close to the geometric radius of the conductive disk. The absence of detectable solution leakage through
the glass seal was also demonstrated 9. By selecting appropriate pulling parameters, the thickness of the glass at the tip could be varied between a few tens of nanometres and several micrometres. Pulled platinum probes encased in submicrometre-thick glass were used in the experiments described in this Article.
Results
Figure 1a shows cyclic voltammograms (CVs) obtained for two glass-covered platinum nanoelectrodes in 0.5 M H2SO4 solution. Curve 1, obtained for an electrode with a thicker glass sheath, exhibits very low background current and no cathodic or anodic waves within a wide potential window (+3 V). This response could be expected, because the conductive platinum core is completely buried in borosilicate glass, which is often used as an insulating material in the fabrication of electrodes. Curve 2 was obtained with the platinum electrode buried inside a signicantly thinner glass sheath (right image in Fig. 1b). This CV shows well-dened anodic and cathodic currents at electrode potentials of E 1.5 V and E 20.5 V versus a Ag/AgCl reference, respectively. These currents, which increase exponentially with applied potential and can reach relatively high values (nA), are clearly due to the electrochemical oxidation/reduction processes. The only redox species present in our system that can be reduced or oxidized within the above potential range are water and molecular oxygen. The current in curve 2 (Fig. 1a) was essentially unaffected by oxygen removal, thus suggesting that the anodic and cathodic processes are the oxidation and reduction of water. The difference between the onset potentials of the cathodic and anodic waves in curve 2 ( 2 V) is somewhat larger than the theoretical minimum voltage required for water electrolysis (1.23 V). To eliminate the possibility that water molecules diffuse to the platinum surface through microscopic cracks or pinholes in the glass sheath, we obtained voltammograms of hydrophilic (Ru(NH3)63; curve 1 in Fig. 1c) and relatively hydrophobic (for example, ferrocenemethanol, FcCH2OH; not shown) redox species at glass-covered platinum electrodes. The complete absence of a reduction wave observed with Ru(NH3)63 concentrations as high as 20 mM suggests that the platinum core is completely covered by glass. The sensitivity of this test to extremely small pinholes in
Department of Chemistry and Biochemistry, Queens CollegeCUNY, Flushing, New York 11367, USA; Present address: Department of Chemistry, Xiamen University, Xiamen 361005, China. * e-mail: mmirkin@qc.cuny.edu
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a
300 200 i (pA) 100 0 100 200 300 3,000 2 i (pA) 0 2 4 6 2,000 1 2,000 1
DOI: 10.1038/NCHEM.645
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b
2 1.23 m 152 nm
1,000
1,000
2,000
3,000 505 nm
E versus Ag/AgCl (mV)
2 1,000 0 1,000 2,000
Figure 1 | Characterization of glass-covered nanoelectrodes. a, Voltammograms obtained in 0.5 M H2SO4 solution at platinum nanoelectrodes with thicker (trace 1) and thinner (trace 2) insulating glass sheaths. b, SEM images of thick glass (left) and thin glass (right) electrodes. c, Voltammograms of 20 mM Ru(NH3)6Cl3 in 1 M KNO3 at two thin glass covered electrodes without (1) and with (2) a nanometre-sized pinhole. Potential sweep rate, n 500 mV s21.
a glass insulator can be seen from equation (1) for the diffusion limiting current to a disk-shaped electrode: id = 4FDac (1)
where F is the Faraday constant, D 6.5 1026 cm2 s21 is the diffusion coefcient of Ru(NH3)63 (ref. 9) and c* is its concentration in solution. Assuming that a 1-nm-radius platinum disk is exposed to the solution due to the presence of a pinhole, one
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Figure 2 | Hydrogen evolution by means of through-glass electrolysis of water. a, Dependence of current on time. The electrode potential was 2900 mV. The inset shows the initial portion of the curve (up to t 320 s, when the insulating sheath was broken) at higher current sensitivity. b, Voltammograms of 20 mM Ru(NH3)63 obtained before (1) and after (2) the hydrogen evolution transient shown in a.
obtains id 5 pA for c * 20 mM. Curve 2 in Fig. 1c is an example of such pinhole detection. The well-dened reduction wave of Ru(NH3)63 corresponds to a value of a as small as 0.7 nm. The actual defect size may be somewhat larger, because the measured current is affected by the diffusion of redox species through the pore in the glass lm. However, the smallest current measurable by our instrument (50 fA) is about two orders of magnitude lower than that in Fig. 1c, and therefore practically any microscopic defect in glass should be detectable. The currents in Fig. 1a cannot be attributed to diffusion of sodium in glass, because water has to physically cross the membrane to be reduced or oxidized at the platinum surface. To further prove this point we monitored the generation of hydrogen (Fig. 2) and oxygen (not shown) at sufciently high negative and positive potentials. Biasing a glass-covered nanoelectrode at 2900 mV in 0.5 M H2SO4 produced 300500 pA current of hydrogen reduction (Fig. 2a, inset). After 5 min, the pressure of the generated hydrogen became high enough to break the insulating sheath and expose the platinum surface to the solution, resulting in a dramatic increase in cathodic current. Figure 2b shows that the reduction of Ru(NH3)63, which did not occur at this electrode before the hydrogen evolution experiment (curve 1), produced a pronounced voltammetric wave after the rupture of the insulating glass (curve 2). Figure 3 shows the response of a glass-covered nanoelectrode to water dissolved in an aprotic organic solvent (1,2-dichloroethane, DCE). The voltammogram obtained with no water added to twice distilled DCE is essentially at and contains neither anodic (Fig. 3a) nor cathodic (Fig. 3b) waves. In contrast, several anodic and cathodic waves appear in the curves obtained when different water concentrations are added to the DCE (cH2 O ; from 1 to 130 mM, as shown by the colour code in Fig. 3a,b). The height of all waves increases with cH2 O . A more prominent anodic peak is observed at 3.3 V, which in fact represents two closely spaced peaks, as can be seen in the yellow curve. The dependence of this peak current (ip) on cH2 O is essentially linear (Fig. 3c). The behaviour of the glass-covered electrode changes dramatically after soaking in acidic solution for a few hours, which is known to result in glass swelling and the formation of a hydrogel surface layer. Figure 4a shows CVs of 0.5 M H2SO4 at a glasscovered nanoelectrode before (1) and after (2) it was kept overnight in 6 M HCl solution. In contrast to curve 1, curve 2 exhibits
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ten buffer solutions of different pH. The linear pH dependence (r 2 . 0.99) of the electrode potential over the range of pH from 1 to 10 exhibits a slightly sub-Nernstian slope of 52 mV pH21.
Discussion
Using nanometre-sized probes, we were able to observe oxidation/ reduction reactions at electrodes buried inside borosilicate glass, which should not have been possible according to conventional wisdom. Nanometre-thick layers of glass are sufciently permeable to observe the oxidation/reduction of water in either aqueous or organic media. From Fig. 1a it can be seen that the through-glass oxidation/reduction of water occurs with a total tip diameter of 150 nm (curve 2), but is not observed when the diameter is 1 mm (curve 1). The rupture of the insulating sheath by evolved hydrogen (Fig. 2) or oxygen unequivocally conrms the diffusion of water molecules through the lm, and a very sensitive voltammetric test (Fig. 1c) provides strong evidence against the possibility of microscopic pores or cracks in the glass layer. So far, no other electroactive specieseither hydrophilic or hydrophobic, ionic or neutralhave been found to cross the dry glass barrier. Voltammetry in organic solutions (Fig. 3) provides additional evidence for molecular diffusion through glass (as opposed to solution permeation through defects), because organic solution does not spontaneously enter nanometre-sized holes in hydrophilic glass12. Importantly, no waves appeared within the entire potential range from 23 to 3 V when no water was added to distilled DCE. The dependence of the peak current on cH2 O for the peak occurring at 3.3 V (Fig. 3c) is essentially linear for the entire range of concentrations from 1 to 130 mM (the latter corresponds to water-saturated DCE13). The linearity of the calibration curve and the detection limit of 0.5 mM attained without any optimization suggest that voltammetry at glass-covered nanoelectrodes may become an alternative to the well-known Karl Fisher titration technique14 for the determination of water in organic solvents. Obvious advantages of the nanoelectrochemical approach include an extremely small sample size and the fact that additional reagents need not be used. A hydrated gel layer can be formed by soaking a glass-covered nanoelectrode in acidic solution. Unlike conventional glass pH sensors, for which the thick ( 0.1-mm) membrane remains mostly dry when immersed in an aqueous solution, the 100-nmthick glass covering our electrodes seems to be largely (if not completely) converted to hydrated gel. The voltammograms of water oxidation/reduction before and after the formation of hydrated gel are completely different. The hydrogen and oxygen evolution currents in Figs 1 and 2 were produced by the diffusion of water molecules through the thin layer of dry glass. The currents corresponding to these processes can be recorded immediately after immersing a glass-covered nanoelectrode in aqueous solution. The response is stable and essentially time-independent on the timescale of several minutes, which is too short for the slow process of glass swelling. In contrast, hydrated gel is an aqueous environment, in which a monolayer of adsorbed water forms on the platinum surface. Accordingly, curve 2 in Fig. 4a exhibits characteristic voltammetric peaks corresponding to adsorption/desorption of hydrogen and oxygen, similar to those obtained at macroscopic platinum electrodes in acidic solutions (cf. the inset in Fig. 4a). Such peaks are not present in curve 1, obtained at a dry glass electrode. Ru(NH3)63/2 species, which were completely blocked by dry glass (curve 1 in Fig. 1c), yielded a pair of well-dened cathodic and anodic peaks at a hydrated glass electrode (Fig. 4b). An increased peak separation, DEp 95 mV, points to a signicant resistance of the hydrated glass layer that varies for different electrodes (as does the DEp value). The resistive potential drop apparently depends on the thickness of the hydrogel and the surface area of the platinum exposed to it. In Fig. 4b, the 35 mV increase in
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Figure 3 | Oxidation/reduction of water in DCE at glass-covered electrodes. a,b, Anodic (a) and cathodic (b) voltammograms obtained in DCE solutions containing different concentrations of water. n 500 mV s21. c, Dependence of the second anodic peak current in a on cH2O. The supporting electrolyte was 0.1 M tetrabutylammonium perchlorate.
well-dened hydrogen, double-layer and oxygen regions. This curve is quite similar to voltammograms of water oxidation/reduction obtained at macroscopic platinum electrodes (inset in Fig. 4a; ref. 10). The current was stable in time and reproducible; the two consecutive potential cycles shown in Fig. 4a produced essentially identical responses. After the acid treatment, a glass-coated electrode responds to redox species other than water. Figure 4b shows a voltammogram of Ru(NH3)63, which (unlike curve 1 in Fig. 1c obtained at a dry glass-coated electrode) contains both cathodic and anodic waves. The voltammograms of anionic IrCl632 (Fig. 4c) and neutral FcCH2OH (Fig. 4d) are not so well shaped. Figure 5a shows a voltammogram of copper electrodeposition in hydrated glass. The shape of the curve, with a characteristic cathodic peak appearing after the potential sweep reversal and a sharp anodic peak of copper stripping, is typical of metal nucleation/growth processes11. In a chronoamperometric experiment (Fig. 5b), the electrode potential was stepped to 2600 mV to deposit a larger amount of copper. The cathodic current was almost constant during the rst 8 s (the inset in Fig. 5b) and then increased sharply by a factor of .400 (the highest current of 10 nA in Fig. 5b corresponds to the overow of the potentiostat amplier). The growth of copper beyond the hydrogel limits and the formation of a micrometre-sized metal electrode were conrmed voltammetrically (not shown), and the deposited copper was observed by optical microscopy and SEM. The potentiometric response of the hydrated glass nanoelectrode to solution pH is shown in Fig. 6. After soaking a glass-covered platinum electrode overnight in 6 M HCl, its potential was measured in
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Figure 4 | Effect of acid pre-treatment of glass-encased nanoelectrodes on their voltammetric responses. ad, CVs obtained in 0.5 M H2SO4 solution before (1) and after (2) an electrode was soaked overnight in 6 M HCl (a), and CVs of 20 mM Ru(NH3)6Cl3 in 1 M KNO3 (b), 15 mM K3IrCl6 in 0.2 M KCl (c) and 2 mM FcCH2OH in 0.25 M KCl (d) at the glass-covered electrode after pre-treatment with 6 M HCl. n 500 (a) and 50 (bd) mV s21. The inset in a shows a CV obtained in 0.5 M H2SO4 with a macroscopic platinum working electrode (5.47 cm2) and saturated calomel electrode reference; n 200 mV s21 (ref. 10).
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Figure 5 | Electrodeposition of copper in hydrated glass. a,b, CV (a) and chronoamperogram (b) of copper deposition on the platinum nanowire buried in hydrated glass from 20 mM CuSO4 solution. In a, n 50 mV s21. In b, the electrode potential was stepped to 2600 mV versus Ag/AgCl. The inset shows the initial portion of the current transient at a higher current sensitivity.
peak separation (the peak separation expected for a reversible CV unaffected by the resistive potential drop is 59 mV) corresponds to the glass layer resistance of 100 MV. An unusual combination of the low (pA) current typical of nanoelectrodes and a peak-shaped CV that is normally obtained at macroscopic electrodes can be attributed to considerable viscosity (and, thus, low diffusion coefcients of Ru(NH3)63/2), which results in non-steady-state diffusion within a thin layer of hydrated gel. The time at which the diffusion in a thin layer approaches a steady state is of the order of d 2/D (ref. 15), where d is the layer thickness. Assuming a lm thickness of 100 nm and noting the experimental timescale in Fig. 4b of 1 s, the apparent D value is 1 10210 cm2 s21. The hydrated glass lm exhibits permselectivity; in comparison with cationic Ru(NH3)63, voltammograms of anionic IrCl632 (Fig. 4c) and the neutral, more hydrophobic FcCH2OH (Fig. 4d) exhibit larger peak separations and less-dened faradaic waves. Electrode surface modication by nanometre-thick hydrated glass may provide new opportunities for sensor preparation, protection of electrocatalysts from fouling and inhibitors, and other electrochemical applications. Another interesting possibilityelectrodeposition
of metals within the glass matrixis suggested by the nucleation and bulk deposition of copper in hydrated gel (Fig. 5). The pH response of nanometre-thick borosilicate glass is due to the formation of the hydrogel layer over a few hours of treatment with acidic solution. The high resistance of the hydrated gel and very slow diffusion within it are consistent with the development of a membrane potential across this layer. The strong dependence of the nanoelectrode potential on pH and excellent linearity of the calibration curve (Fig. 6) suggest that these electrodes can serve as all-solid-state pH nanoprobes. Electrodes containing a buffer solution (presently the most common type of pH sensors16) have many disadvantages, including storage problems, pressure and temperature dependence, mechanical instability and relatively large size and fabrication cost17. Numerous efforts to produce solid-state pH electrodes based on iridium oxide18, conductive polymer composites19 and other sensing strategies have met with limited success. The main advantages of a glass-coated platinum pH electrode its microscopic size and biocompatibilitymake it potentially useful for cell biology applications and other experiments in small volumes. The attainable tip size (tens of nanometres) is comparable
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References
1. Murray, R. W. Nanoelectrochemistry: metal nanoparticles, nanoelectrodes and nanopores. Chem. Rev. 108, 26882720 (2008). 2. Fan, F.-R. F. & Bard, A. J. Electrochemical detection of single molecules. Science 267, 871874 (1995). 3. Smith, C. P. & White, H. S. Theory of the voltammetric response of electrodes of submicron dimensions. Violation of electroneutrality in the presence of excess supporting electrolyte. Anal. Chem. 65, 33433353 (1993). 4. Sun, P. & Mirkin, M. V. Electrochemistry of individual molecules in zeptoliter volumes. J. Am. Chem. Soc. 130, 82418250 (2008). 5. Bach, H., Baucke, F. K. G. & Krause, D. (eds) Electrochemistry of Glasses and Glass Melts, Including Glass Electrodes (Springer, 2001). 6. Haugaard, G. The mechanism of the glass electrode. J. Phys. Chem. 45, 148157 (1941). 7. Schwabe, K. & Dahms, H. Permeability of the glass electrode to hydrogen ions with the aid of tritium tagging. Monatsber. Deutschen Akad. Wissen. 1, 279282 (1959). 8. Vetter, K. J. Electrochemical Kinetics: Theoretical and Experimental Aspects (Academic Press, 1967). 9. Sun, P. & Mirkin, M. V. Kinetics of electron transfer reactions at nanoelectrodes. Anal. Chem. 78, 65266534 (2006). 10. Attard, G. S. et al. Mesoporous platinum lms from lyotropic liquid crystalline phases. Science 278, 838840 (1997). 11. Fletcher, S. et al. The response of some nucleation/growth processes to triangular scans of potential. J. Electroanal. Chem. 159, 267285 (1983). 12. Shao, Y. & Mirkin, M. V. Fast kinetic measurements with nanometer-sized pipets. Transfer of potassium ion from water into dichloroethane facilitated by dibenzo-18-crown-6. J. Am. Chem. Soc. 119, 81038104 (1997). 13. Masterton, W. L. & Gendrano, M. C. Henrys Law studies of solutions of water in organic solvents. J. Phys. Chem. 70, 28952898 (1966). 14. Harris, D. C. Quantitative Chemical Analysis 6th edn, 397 (W. H. Freeman, 2002). 15. Hubbard, A. T. & Anson, F. C. The theory and practice of electrochemistry with thin layer cells, in Electroanalytical Chemistry (ed. Bard, A. J.) Vol. 4, 129214 (Marcel Dekker, 1970). 16. Vonau, W., Gabel, J. & Jahn, H. Potentiometric all solid-state pH glass sensors. Electrochim. Acta 50, 49814987 (2005). 17. Kreuer, K.-D. Solid potentiometric pH electrode. Sens. Actuat. B 1, 286292 (1990). 18. El-Giar, E. E.-D. M. & Wipf, D. O. Microparticle-based iridium oxide ultramicroelectrodes for pH sensing and imaging. J. Electroanal. Chem. 609, 147154 (2007). 19. Malkaj, P., Dalas, E., Vitoratos, E. & Sakkopoulos, S. pH electrodes constructed from polyaniline/zeolite and polypyrrole/zeolite conductive blends. J. Appl. Polym. Sci. 101, 18531856 (2006). 20. Bakker, E. & Pretsch, E. Nanoscale potentiometry. Trends Anal. Chem. 27, 612618 (2008). 21. Horrocks, B. R. et al. Scanning electrochemical microscopy 19. Ion selective potentiostatic microscopy. Anal. Chem. 65, 12131224 (1993). 22. Shao, Y. et al. Nanometer-sized electrochemical sensors. Anal. Chem. 69, 16271634 (1997).
Figure 6 | Potentiometric response of a hydrated glass nanoelectrode to solution pH. Potential was measured versus a Ag/AgCl reference electrode. The glass nanoprobe was transferred sequentially between ten different buffer solutions, and each measurement was taken after the potential stabilized to within +1 mV.
to or smaller than that of the existing nanopipette-based potentiometric sensors20. It may also be used as a scanning probe for pH microscopy21, where its small size can help to signicantly improve spatial resolution. At the same time, glass nanosensors are not likely to replace conventional pH electrodes in routine analytical measurements because of their fragility and limited lifetime. The borosilicate glass used in this work may not be the best material for a pH sensor because its composition is different from conventional pH glass (for example, Corning 0150 glass), and it would not be suitable for the fabrication of macroscopic pH probes. A relatively slow response (minutes) can probably be improved by using more suitable glass for the preparation of pH nanosensors.
Methods
Chemicals. Ferrocenemethanol (FcCH2OH, 97%; Aldrich) was recrystallized twice from acetone. Hexaammineruthenium (III) chloride (99%) was obtained from Strem Chemicals. KNO3 , Li2SO4 and KCl (99 %, Aldrich) were used as supporting electrolytes. Aqueous solutions were prepared from deionized water (Milli-Q, Millipore). Twice-distilled HPLC grade DCE (Sigma-Aldrich) was used to prepare organic solutions. Potassium hexachloroiridate (III) (Alfa Aesar), H2SO4 (Aldrich) and CuSO4 (Mallinckrodt) were used as received. Electrodes. To prepare a dry glass-covered nanoelectrode, an annealed 25-mm platinum wire (Goodfellow) was pulled into a borosilicate capillary (Drummond; OD, 1.0 mm; ID, 0.2 mm) under vacuum with the help of a Sutter P-2000/G laser pipette puller, as described previously9,22. To form hydrated gel, a glass-covered platinum nanoelectrode was soaked in 6 M HCl solution overnight. As electrodes were removed from the HCl solution they were rinsed with water before measurements. To improve reproducibility, such electrodes were stored in aqueous solution between experiments. However, it was found that the hydrated gel remains on the electrode surface, even after being kept for several days in an oven at 100 8C. A two-electrode conguration was used for voltammetry and chronoamperometry, with either a commercial Ag/AgCl reference electrode or a Ag quasi-reference (DCE solutions). Instrumentation and procedures. Cyclic voltammograms were obtained using either an EI-400 bipotentiostat (Ensman Instruments) or a BAS-100B electrochemical analyser (Bioanalytical Systems). All experiments were carried out at room temperature (23+2 8C) inside a Faraday cage. Unless otherwise specied, CVs obtained at glass-covered electrodes show the second or subsequent potential cycles, which are essentially indistinguishable from one another (steady-state response), but are different from the rst potential sweep. pH measurements were carried out in the following solutions: HCl/KCl (pH 1), phthalate buffers (pH 24), acetate buffers (pH 36), phosphate buffers (pH 78) and carbonate buffers (pH 910). SEM images were obtained using a eld-emission scanning electron microscope (Zeiss Supra 55 VP) with no conductive coating applied to the nanoelectrodes.
Acknowledgements
The authors gratefully acknowledge support from the National Science Foundation (CHE0645958) and a grant from PSC-CUNY. The authors would like to thank H. Gafney, F. Laforge and A. Bard for helpful discussions and J. Morales (CCNY electron microscopy facility) for his help with SEM imaging.
J.V. performed the experiments. D.Z. conceived the experiments and developed analytical tools for nanoelectrode characterization. M.V.M. conceived and designed the experiments, analysed data and wrote the paper.
The authors declare no competing nancial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to M.V.M.
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Enhancement of anhydrous proton transport by supramolecular nanochannels in comb polymers

Yangbin Chen1, Michael Thorn2, Scott Christensen3, Craig Versek2, Ambata Poe1, Ryan C. Hayward3 *, Mark T. Tuominen2 * and S. Thayumanavan1 *
Transporting protons is essential in several biological processes as well as in renewable energy devices, such as fuel cells. Although biological systems exhibit precise supramolecular organization of chemical functionalities on the nanoscale to effect highly efcient proton conduction, to achieve similar organization in articial systems remains a daunting challenge. Here, we are concerned with transporting protons on a micron scale under anhydrous conditions, that is proton transfer unassisted by any solvent, especially water. We report that proton-conducting systems derived from facially amphiphilic polymers that exhibit organized supramolecular assemblies show a dramatic enhancement in anhydrous conductivity relative to analogous materials that lack the capacity for self-organization. We describe the design, synthesis and characterization of these macromolecules, and suggest that nanoscale organization of proton-conducting functionalities is a key consideration in obtaining efcient anhydrous proton transport.
fcient and selective transport of protons is critical both in biological contexts1 and in materials for renewable energy2. In biological systems, nature has optimized proton conduction on a nanometre scale by using secondary and tertiary structures of proteins to arrange precisely the appropriate side chains of amino acids, for example in the membrane protein M2 (refs 35). Although control of proton transfer on this scale is adequate for most biological processes, it is essential that efcient proton conduction be obtained on a micron scale for clean-energy applications6,7. In hydrogen fuel cells, for example, after oxidation of molecular hydrogen at the anode, the resulting protons must be transported across a selective membrane to reach the cathode and complete the conversion of chemical energy into electrical energy. The proton conductivity of this membrane, often called the protonexchange membrane or the polymer electrolyte membrane (PEM), has been one of the bottlenecks to achieving affordable fuel-cell technology. Naon, a poly(tetrauoroethylene)-based polymer with sulfonic acid groups arranged at intervals along the backbone, is one of the most widely used materials for this membrane8. The key to proton transport in Naon is thought to be nanochannels of sulfonic acid groups, through which hydrated protons can pass efciently911. Although a good proton conductor for hydrated protons, Naon suffers from poor conductivity in unassisted proton transfer, that is Grotthuss or anhydrous proton transfer12,13, which results in low conductivities at temperatures above the boiling point of water. PEMs with high proton conductivities at temperatures of 120200 8C are desirable, because operation at higher temperatures can increase fuel-cell efciency, reduce cost, simplify heat management and provide better tolerance of the catalysts against poisoning14. One approach to address this issue is to use amphoteric functional groups that allow anhydrous proton transport15,16, for example imidazole, which is a common motif in biological proton transport in the form of the amino acid histidine. Several groups have studied synthetic polymers that contain such amphoteric functional groups as candidates for high-temperature proton transfer1722. Although a number of interesting candidate materials were
1
identied, one avenue that was not explored in these anhydrous proton-conducting systems is the role of supramolecular organization in nanoscale ion-conducting channels. This is surprising because, in the context of hydrated proton-conducting systems, such as Naon911, and sulfonated block copolymers12,2326, as well as lithium-ion conducting supramolecular assemblies2729, it is well-established that the formation of nanoscale domains enriched in the ion-conducting materials is critical to the resulting macroscopic ionic conductivity. In this paper, we describe the molecular design and synthesis of a novel class of comb polymers with amphoteric proton-transfer functionalities that can self-assemble into organized supramolecular structures. We also show that very subtle changes in the monomer and analogous polymer provide solid-state structures that lack such nanoscale organization. By comparing these polymers, we show that the self-assembled structures yield a dramatic increase in proton conductivity (by as much as three orders of magnitude), presumably because of the locally increased concentration of proton-transport functionalities within the nanophase-separated domains. These results suggest that a careful consideration of macromolecular architecture and nanoscale assembly is critical to optimizing anhydrous proton transport in new materials for PEMs.
Results
To prepare polymers that form supramolecular assemblies with proton-transporting functionalities concentrated within nanoscale domains, we made use of comb polymer architectures (Fig. 1). One of our groups recently used this architecture to prepare amphiphilic comb polymers by attaching lipophilic and hydrophilic functionalities at the meta-positions of the benzene ring of each styrenic repeat unit30,31. Such polymers were shown to form assemblies of a micelle type in aqueous milieu and of an inverse-micelle type in apolar organic solvents. Thus, we hypothesized that similar polymers would also form nanoscale assemblies in the melt state. For this purpose, we designed a series of styrenic comb polymers in which one of the meta-positions contained a polar N-heterocyclic functionality capable of proton transport, and the other contained
Department of Chemistry, 2 Department of Physics, 3 Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 01003, USA. * e-mail: thai@chem.umass.edu; tuominen@physics.umass.edu; rhayward@mail.pse.umass.edu
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Figure 1 | Structures and synthesis of benzotriazole-based polymers. a, Benzotriazole-based polymers that only differ by the inclusion of a decyl chain. b, Synthetic scheme for benzotriazole-based polymers. This synthesis was modied slightly to prepare proton-conducting polymers without decyl chains and those that contain imidazole in place of benzotriazole. Details are given in the Supplementary Information. PCC pyridinium chlorochromate.
a non-polar alkyl chain to drive phase segregation. For example, polymer 1 (Fig. 1a) contains non-conducting decyl chains and the proton-conducting heterocyclic functionality benzotriazole. Although Liu et al.18 reported that triazole appreciably enhances proton conductivities with respect to imidazole (also benzimidazole has been studied20 as a proton-transporting functionality), we are not aware of any reports of benzotriazole used as a proton conductor. To test our premise that nanoscale phase segregation of 1 would facilitate proton transport, we synthesized the analogous polymer 2 that has no alkyl chains and therefore did not undergo nanoscale assembly. Syntheses of these polymers are exemplied with polymer 1 in Fig. 1b. To determine proton-conductivity values, polymer lms were drop-cast from solution onto a hole in a piece of Kapton tape and subsequently sandwiched between two electrodes to allow characterization by impedance spectroscopy, as described previously19. The Kapton tape determined the thickness of the polymer lm, which was therefore constant at 125 mm for all impedance measurements. Separate thermogravimetric analyses (TGAs) were conducted to verify that all polymers reported in this study were thermally stable up to at least 200 8C, which was the highest temperature investigated in the impedance measurements. Conductivities of the polymer samples were measured through several heating cooling cycles (40200 8C) under high vacuum and were found to be consistent from cycle to cycle, which eliminates any possible effects of residual solvent on the performance of these polymers. Proton conductivities for 1 and 2 measured as a function of temperature between 40 and 200 8C are shown in Fig. 2a. Both polymers show qualitatively similar non-Arrhenius increases in conductivity with temperature that are typical for anhydrous proton-conducting polymers. However, the conductivity of 1 ranges
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from 6 1026 S cm21 at ambient temperature to 1.3 1023 S cm21 at 200 8C, at least two orders of magnitude larger than the conductivity of 2 across the same temperature range, which varies from 4 1029 S cm21 at ambient temperature to 1.2 1025 S cm21 at 200 8C. As a benchmark, Naon membranes show room-temperature conductivities of 1022 to 1021 S cm21 when fully hydrated32, but at low humidity (below 5%) their conductivities were reported as 1027 to 1025 S cm21 (ref. 33). (Under our experimental conditions, measured conductivities of Naon were below the noise oor of the measurements of 1029 S cm21.) Thus, although the conductivities of our materials remain signicantly below those of Naon under ideal conditions, the dramatic increase in conductivity from 2 to 1 suggests an important design principle for optimizing proton transport under anhydrous conditions. At rst, it may seem surprising that the addition of a decyl chain to each repeat unit of a polymer could boost proton conductivity by two orders of magnitude. After all, the average density of protontransporting groups is lowered by the presence of the decyl chain; the benzotriazole unit makes up only 23 weight per cent (wt%) of 1 as compared to 34 wt% of 2. However, we hypothesized that the decyl chain renders the mixing of 1 with the amphoteric heterocycles incompatible, and so drives 1 to self-assemble and form nanoscale domains that contain enhanced local concentrations of benzotriazole, and thereby facilitates proton transport. To test this hypothesis, we characterized the structures of these polymers using small-angle X-ray scattering (SAXS). As shown in Fig. 2b, polymer 1 gave rise to scattering peaks that indicate self-assembled nanostructures, but the control polymer 2 yielded a completely featureless pattern that indicates a homogeneous phase-mixed structure. The rst-order scattering peak from 1 falls at q * 1.47 nm21, which corresponds to a real-space
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Decyl chain Spacer Benzotriazole
Figure 2 | Conductivity and SAXS results for benzotriazole polymers. a, Proton conductivity over a wide temperature range, in which the polymer that contains the decyl chain exhibits a much higher conductivity than that of its counterpart. b, SAXS proles of benzotriazole polymers that indicate ordering of the alkylated polymer. Curves are shifted vertically for clarity. I(q) is the azimuthally-averaged scattering intensity as a function of scattering wave-vector (q). c, An illustration of the proposed structure of 1, with two units arranged with the benzotriazoles head-to-head, which thus allows for hydrogen bonding. a.u. arbitrary units.
distance of d 4.3 nm, and a faint, although clearly resolvable, p second-order peak at 4q *. Although the structure cannot be determined unambiguously from these data, the presence of only a second-order peak suggests a lamellar structure with a repeat spacing of 4.3 nm. We estimate the fully stretched length of a monomer, from the tip of the decyl chain to the benzotriazole group, as 3 nm, and thus the observed repeat spacing is consistent with back-to-back stacking of polymer chains with some interdigitation of the decyl chains and/or benzotriazole group and spacer. A schematic of this proposed structure is shown in Fig. 2c, in which two repeating units arranged with the benzotriazoles head-to-head allow hydrogen bonding, with the alkyl groups pointed away from each other. Analysis of the rst-order peak revealed a width (full-width
at half-maximum, Dq) of 0.6 nm21 for polymer 1, which yielded a correlation length of 2p/Dq 10 nm, indicating that the size of ordered domains is small, with positional correlations that extend only over several repeat units. As demonstrated by Ikkala and co-workers34, self-assembly into anisotropic nanostructures yields orientation-dependent conductivity, and McGrath and co-workers have shown that continuity of nanoscale domains is critical to efcient proton transport in sulfonated polymers35. For our polymers, although the limited length of ordering precludes any considerations of the effects of orientation or dimensionality of nanoscale domains on conductivity, the nanoscale organization provided by supramolecular assembly clearly enhances anhydrous proton conductivity by at least two orders of magnitude
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Table 1 | Properties of polymers 1, 2, 7 and 8.
Polymer Decomposition onset (8 C) (5% 8 weight loss) 218 233 225 221 Tg (8 C) 8 55 67 61 71 N-heterocycle weight fraction (%) 23 34 14 20 Molecular weight (Mn) 25,000 25,000 23,000 24,000
NATURE CHEMISTRY
DOI: 10.1038/NCHEM.629
1 2 7 8
Glass-transition temperatures, decomposition onset temperatures, N-heterocycle weight fractions and molecular weight of polymers 1, 2, 7 and 8. Mn number-average molecular weight.
compared with that of the phase-mixed control polymer. The exact mechanism of enhancement is currently unclear, although we speculate that self-organization leads to interconnected channels of locally enriched benzotriazole concentrations that facilitate proton hopping through the membrane. To test for the possibility of disordering or nanoscale structural transitions at high temperature (which, in some cases, have yielded dramatic changes in ionic conductivity27), we carried out variable-temperature SAXS measurements over the range 40200 8C. Although the intensity of the
a
n-C10H21 O O O N H N NH
rst-order scattering peak decreased continuously with increasing temperature, its position and width remained nearly constant, which indicates that a similar level of nanoscale organization was present in these materials over the entire temperature range of interest (see Supplementary Information for details). An additional factor to consider when the proton conductivities of two polymer chains that bear the same functional group are compared is the glass-transition temperature (Tg), because the mobility of the polymer chain is well-known to inuence the rate of proton transport36. To test whether the difference in conductivity observed between 1 and 2 simply reects a decrease in Tg because the decyl chain is present, we carried out differential scanning calorimetry experiments. As summarized in Table 1, the Tg values of 1 and 2 were 55 8C and 67 8C, respectively. The modest difference in Tg between these polymers suggests that the mobility of the polymer backbone is not a major contributor to the difference in proton conductivities observed. As relatively high conductivity values of 1 were achieved at temperatures well above Tg , the mechanical properties of this polymer at such temperatures are not well-suited for application as PEMs. Although components with higher Tg values or semicrystalline components need to be incorporated to provide materials
b
102 103 (S cm1) 227 181 Temperature (C) 144 111 84 60 39 7 8
104 105 106 107 108 109
7 O O N H N NH
Conductivity
1010 2.0 2.2 2.4 2.6 2.8 3.0 3.2 1,000/T (K1)
1.4 1.2 1.0
q* = 1.64 nm1
7 8
Decyl chain
I(q) (a.u.)
0.8 0.6 0.4 0.2 0.0 1 2
3 q*
Spacer Imidazole
3 q (nm1)
Figure 3 | Results for imidazole-based polymers a, Structures of imidazole polymers that only differ by the inclusion of a decyl chain. b, Proton conductivity over a wide temperature range, which again demonstrates that the polymer with an added decyl chain has a much higher conductivity. c, SAXS proles of imidazole polymers, which show order in polymer 7 and a disordered polymer 8. Curves are shifted vertically for clarity. d, An illustration of the proposed structure of 7, which self-assembles into cylindrical domains.
506
NATURE CHEMISTRY
DOI: 10.1038/NCHEM.629
ARTICLES
Methods
TGA was carried out using a TA Instruments TGA 2950 thermogravimetric analyser with a heating rate of 10 8C min21 from room temperature to 500 8C under nitrogen. Glass-transition temperatures were obtained by differential scanning calorimetry using a TA Instruments Dupont DSC 2910. Samples were analysed at a heating rate of 10 8C min21 from 0 8C to 150 8C under a ow of nitrogen (50 ml min21). Electrochemical impedance data were obtained using a Solartron 1287 potentiostat and 1252A frequency response analyser in the range 0.1 Hz to 300 kHz. Measurements were conducted under vacuum at temperatures between 40 8C and 200 8C with a sinusoidal excitation root-mean-square voltage of 0.1 V. The sample thickness and contact surface area were controlled by a 125 mm thick Kapton tape with a 0.3175 cm diameter hole. SAXS measurements were carried out on an in-house beamline using a Rigaku rotating anode source to generate Cu Ka radiation (wavelength l 0.154 nm). Scattering patterns were collected on an image plate positioned a distance of 500 mm from the sample. All samples yielded isotropic patterns, and thus data were integrated to yield plots of intensity as a function of the magnitude of the scattering vector, q (4p/l )sin(u), where 2u is the total scattering angle. The actual scattering angles were calibrated using the known reection from silver behenate.
with adequate mechanical properties, we emphasize that the polymers described here provide proof-of-concept results and demonstrate the importance of nanoscale organization in anhydrous proton conductivity. If our hypothesis and conclusions are correct, this molecular design strategy should also work for other amphoteric heterocycles. To test the generality of our molecular design, we prepared polymers that contained imidazole as a proton-transporter functionality, which was also shown to be capable of anhydrous proton transfer1619 and has a very different dissociation constant (pKa) value in the protonated form as compared to that of benzotriazole. In analogy to 1 and 2, we synthesized polymers 7 and 8 with imidazole moieties (Fig. 3a). Alternating current impedance measurements revealed that polymer 7, with its decyl chain, exhibits dramatically higher conductivity than that of the corresponding control polymer 8, in this case by more than three orders of magnitude (Fig. 3b). The morphologies of these polymers were investigated using SAXS and revealed that 7 gave two well-dened scattering peaks, which indicates the presence of self-assembled nanostructures, but 8 showed no signs of structure (Fig. 3c). The second scatp tering peak for 7 falls at a position of 3q*, which clearly indicates a non-lamellar structure and suggests a hexagonal symmetry that probably corresponds to a structure of hexagonally packed cylinders (Fig. 3d). Tg values determined for these polymers (Table 1) are very similar, which once again reveals that mobility of the polymer backbone is not a signicant factor in the difference in proton conductivity of three orders of magnitude. We also tested random copolymers synthesized from a monomer disubstituted with decyl groups and another monomer disubstituted with N-heterocycles (1:1 ratio). These random copolymers also provided some extent of phase separation, but with nanostructures organized more poorly than those of the comb polymers and with only a single scattering peak for each. The conductivities of these random copolymers are generally signicantly greater than those of the unorganized control homopolymers 2 and 8, although somewhat less than those of the comb polymers 1 and 7 (see Supplementary Information). This further supports our conclusion that phase separation on a nanoscale is tied directly to the efciency of proton transport. In summary, we have designed, synthesized and characterized a new class of comb polymers for anhydrous proton transport. We have shown that: styrenic comb polymers that contain incompatible functionalities at opposite faces of the monomer units provide ordered nanostructures through self-assembly in the melt state; styrenic polymers that contain a non-conducting decyl group and a proton-conducting functionality on the meta-positions of the phenyl ring exhibit conductivities two-to-three orders of magnitude greater than those of polymers that contain only the conducting functionality, despite the lower overall content of proton transporter in the former; polymer backbone mobility is not a major contributor to the observed differences in proton conductivity in these systems; the high conductivities observed for the decyl-functionalized polymers correlate with the ability to form organized lamellar or hexagonal nanostructures that consist of domains with locally high concentrations of proton conductors that facilitate transport; this molecular design strategy works for two different protontransfer functionalities with substantially different pKa values, which suggests that the importance of nanochannel formation in proton conduction is a general phenomenon. Our work here indicates that careful consideration of polymer architecture and nanoscale morphology is a key element in the design of efcient anhydrous PEMs.
Received 16 November 2009; accepted 16 March 2010; published online 25 April 2010
References
1. Williams, R. J. P. Proton circuits in biological energy interconversions. Annu. Rev. Biophys. Biophys. Chem. 17, 7197 (1988). 2. Kreuer, K. D. Proton conductivity: materials and applications. Chem. Mater. 8, 610641 (1996). 3. Sass, H. J. et al. Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin. Nature 406, 649653 (2000). 4. Schnell, J. R. & Chou, J. J. Structure and mechanism of the M2 proton channel of inuenza A virus. Nature 451, 591560 (2008). 5. Stouffer, A. L. et al. Structural basis for the function and inhibition of an inuenza virus proton channel. Nature 451, 596600 (2008). 6. Carrette, L., Friedrich, K. A. & Stimming, U. Fuel cells fundamentals and applications. Fuel Cells 1, 539 (2001). 7. Steele, B. C. H. & Heinzel, A. Materials for fuel-cell technologies. Nature 414, 345352 (2001). 8. Mauritz, K. A. & Moore, R. B. State of understanding of Naon, Chem. Rev. 104, 45354585 (2004). 9. Diat, O. & Gebel, G. Proton channels. Nature Mater. 7, 1314 (2008). 10. Schmidt-Rohr, K. & Chen, Q. Parallel cylindrical water nanochannels in Naon fuel-cell membranes. Nature Mater. 7, 7583 (2008). 11. Elliott, J. A., Hanna, S., Elliott, A. M. S. & Cooley, G. E. Interpretation of the small-angle X-ray scattering from swollen and oriented peruorinated ionomer membranes. Macromolecules 33, 87088713 (2000). 12. Hickner, M. A., Ghassemi, H., Kim, Y. S., Einsla, B. R. & McGrath, J. E. Alternative polymer systems for proton exchange membranes (PEMs). Chem. Rev. 104, 45874612 (2004). 13. Rikukawa, M. & Sanui, K. Proton-conducting polymer electrolyte membranes based on hydrocarbon polymers. Prog. Polym. Sci. 25, 14631502 (2000). 14. Li, Q., He, R., Jensen, J. O. & Bjerrum, N. J. Approaches and recent development of polymer electrolyte membranes for fuel cells operating above 100 8C. Chem. Mater. 15, 48964915 (2003). 15. Kreuer, K. D. Fast proton conductivity: a phenomenon between the solid and the liquid state? Solid State Ionics 94, 5562 (1997). 16. Kreuer, K. D., Fuchs, A., Ise, M., Spaeth, M. & Maier, J. Imidazole and pyrazolebased proton conducting polymers and liquids. Electrochim. Acta 43, 12811288 (1998). 17. Scharfenberger, G. et al. Anhydrous polymeric proton conductors based on imidazole functionalized polysiloxane. Fuel Cells 6, 237250 (2006). 18. Zhou, Z., Li, S. W., Zhang, Y. L., Liu, M. L. & Li, W. Promotion of proton conduction in polymer electrolyte membranes by 1H-1,2,3-triazole. J. Am. Chem. Soc. 127, 1082410825 (2005). 19. Granados-Focil, S., Woudenberg, R. C., Yavuzcetin, O., Tuominen, M. T. & Coughlin, E. B. Water-free proton-conducting polysiloxanes: a study on the effect of heterocycle structure. Macromolecules 40, 87088713 (2007). 20. Persson, J. C. & Jannasch, P. Intrinsically proton-conducting benzimidazole units tethered to polysiloxanes. Macromolecules 38, 32833289 (2005). 21. Shogbon, C. B., Brousseau, J.-L., Zhang, H., Benicewicz, B. C. & Akpalu, Y. Determination of the molecular parameters and studies of the chain conformation of polybenzimidazole in DMAc/LiCl. Macromolecules 39, 94099418 (2006). 22. Subbaraman, R., Ghassemi, H. & Zawodzinski, T. A. Jr 4,5-Dicyano-1H-[1,2,3]triazole as a proton transport facilitator for polymer electrolyte membrane fuel cells. J. Am. Chem. Soc. 129, 22382239 (2007). 23. Won, J. et al. Fixation of nanosized proton transport channels in membranes. Macromolecules 36, 32283234 (2003). 24. Serpico, J. M. et al. Transport and structural studies of sulfonated styrene ethylene copolymer membranes. Macromolecules 35, 59165921 (2002).
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25. Shi, Z. & Holdcroft, S. Synthesis and proton conductivity of partially sulfonated poly([vinylidene diuoride-co-hexauoropropylene]-b-styrene) block copolymers. Macromolecules 38, 41934201 (2005). 26. Rubatat, L., Shi, Z., Diat, O., Holdcroft, S. & Frisken, B. J. Structural study of proton-conducting uorous block copolymer membranes. Macromolecules 39, 720730 (2006). 27. Cho, B. K., Jain, A., Gruner, S. M. & Wiesner, U. Mesophase structure mechanical and ionic transport correlations in extended amphiphilic dendrons. Science 305, 15981601 (2004). 28. Kishimoto, K. et al. Nano-segregated polymeric lm exhibiting high ionic conductivities. J. Am. Chem. Soc. 127, 1561815623 (2005). 29. Wanakule, N. S. et al. Ionic conductivity of block copolymer electrolytes in the vicinity of orderdisorder and orderorder transitions. Macromolecules 42, 56425651 (2009). 30. Savariar, E. N., Aathimankandan, S. V. & Thayumanavan, S. Supramolecular assemblies from amphiphilic homopolymers: testing the scope. J. Am. Chem. Soc. 128, 1622416230 (2006). 31. Basu, S., Vutukuri, D. R. & Thayumanavan, S. Homopolymer micelles in heterogeneous solvent mixtures. J. Am. Chem. Soc. 127, 1679417695 (2005). 32. Wintersgill, M. C. & Fontanella, J. J. Complex impedance measurements on Naon. Electrochim. Acta 43, 15331538 (1998). 33. Sanders, E. H. et al. Characterization of electrosprayed Naon lms. J. Power Sources 129, 5561 (2004). 34. Ruotsalainen, T. et al. Structural hierarchy in ow-aligned hexagonally selforganized microphases with parallel polyelectrolytic structures. Macromolecules 36, 94379442 (2003).
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35. Roy, A. et al. Inuence of chemical composition and sequence length on the transport properties of proton exchange membranes. J. Polym. Sci. B 44, 22262239 (2006). 36. Schuster, M. F. H. & Meyer, W. H. Anhydrous proton-conducting polymers. Annu. Rev. Mater. Res. 33, 233261 (2003).
Acknowledgements
This work was supported by the National Science Foundation through the Fueling the Future Center for Chemical Innovation at the University of Massachusetts Amherst (CHE-0739227). We thank W. de Jeu for discussions on the X-ray scattering results.
S.T. and Y.C. conceived the molecular design. S.T., R.H. and Mark T. planned the project. Y.C, Michael T., S.C. and C.V. carried out the experiments and analysed the data. Y.C. and A.P. synthesized the discussed compounds, Michael T. and C.V. measured ionic conductivities, and S.C. performed SAXS. Results were discussed by R.H., Mark T. and S.T. All authors contributed to writing the manuscript.
The authors declare no competing nancial interests. Supplementary information and chemical compound information accompany this paper at www.nature.com/ naturechemistry. Reprints and permission information is available online at http://npg.nature. com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to R.C.H., M.T.T. and S.T.
508
in your element
Is lithium the new gold?

Jean-marie tarascon ponders on the value of lithium, an element known for about 200 years, whose importance is now fast increasing in view of the promises it holds for energy storage and electric cars.
lthough it has been known for almost two centuries, lithium is suddenly making the news: it is the primary ingredient of the lithium-ion batteries set to power the next generation of electric vehicles and, as such, could become as precious as gold in this century 1. It is also non-uniformly spread within the Earths crust, sparking rumours that Andean South American countries could soon be the new Middle-East. Together, these factors set the scene for controversial debates about the available reserves24 and the anticipated demands1: if all cars are to become electric within 50 years, fears of a crunch in lithium resources and thus a staggering price increase such as that faced today with fossil fuels are permeating. With its atomic number of 3, lithium is located in the top left corner of the periodic table. It was Johann August Arfvedson, one of Jns Jakob Berzeliuss students, who first detected its presence in 1817 while analysing the mineral petalite (LiAlSi4O10), itself discovered in 1800. Berzelius called this new element lithos (Greek word for stone). Lithium, whose silvery-white colour tarnishes on oxidation when exposed to air, is the most electropositive metal (3.04 V versus a standard hydrogen electode), the lightest (M = 6.94 g mol1) and the least dense ( = 0.53 g cm3) solid element at room temperature, and is also highly flammable. Owing to this high reactivity, lithium is present only in compounds in nature either in brines or hard rock minerals and must be stored under anhydrous atmospheres, in mineral oil or sealed evacuated ampoules. Their particular physical, chemical and electrochemical properties make lithium and its compounds attractive to many fields. Apart from the recent advent of lithiumbased batteries, lithium niobate (LiNbO3) is an important material in nonlinear optics. Engineers use lithium in hightemperature lubricants, to strengthen alloys, and for heat-transfer applications. It is also
widespread in the fine chemical industry, as organo-lithium reagents are extremely powerful bases and nucleophiles used to synthesize many chemicals. Its effect on the nervous system has also made lithium attractive as a mood-stabilizing drug, and in nuclear research tritium (3H) is obtained by irradiating 6Li. Annual demand has therefore grown by 710%, currently reaching about 160,000 tons of lithium carbonate (Li2CO3) per year about 2025% of which is for the battery sector.
Energy storage, which should help mitigate the issues of pollution, global warming and fossil-fuel shortage, is becoming more important than ever, and Li-ion batteries are now the technology of choice to develop renewable energy technology and electric vehicles. They typically consist of a Li-containing positive electrode and a Li-free negative electrode, separated by a Libased electrolyte. From simple calculations, assuming a one-molar Li-based electrolyte and a 3.6 V LiMPO4 electrode (where M is Fe or Mn), the demand is estimated to be about 0.8 kg Li2CO3 per kWh and this number is not expected to decrease with recently developed batteries such as lithiumair or lithiumsulfur, which need an excess of lithium at the negative electrode to function properly. The fact that tritium might also be used with deuterium for nuclear fusion could increase demands. Extracting lithium from hard rocks is laborious and expensive, however, and most of that produced (roughly 83%) at present comes
from brine lakes and salt pans: salty water is first pumped out of the lake into a series of shallow ponds, then concentrated using solar energy into a lithium chloride brine, which is subsequently treated with soda to precipitate Li2CO3. Considerable amounts of lithium are present in sea water, but its recovery is trickier, and highly expensive. It is extremely difficult to estimate the worlds lithium reserves13 a debate typically fed by investors and venture capitalists. The present production of Li2CO3 is about half what would be needed to convert the 50 million cars4 produced every year into plug-in hybrid electric vehicles (with an electric motor powered by a 7 kWh Liion battery and a combustion engine). The demand becomes astronomic if we consider full electric vehicles which require an onboard battery of 40 kWh. These numbers bring fears of a potential Li shortage in a few decades, painting a dim picture. This alarming global situation will hopefully drive researchers to investigate new battery technologies5 and loosen our dependence on lithium. Fortunately, the situation improves if one also considers recycling the low melting point (180 C) of lithium metal and the very low water solubility of its fluoride, carbonate and phosphate salts make its recovery quite easy. Combining further brine exploitation with an efficient recycling process should be enough to match the demands of a propulsion revolution that would solely rely on Li-ion cells, lessening geopolitical risks.
JEAN-MAriE TArAscON is at the Laboratory of reactivity and solid-state chemistry, University of Picardie Jules Verne, F-80039 Amiens, France. e-mail: jean-marie.tarascon@sc.u-picardie.fr
References
1. Greene, L. Batteries & Energy Storage Technology 3741 (Spring issue, 2009) 2. Tahil, W. The Trouble With Lithium (Meridian International Research, 2006); http://go.nature.com/jhDqLH 3. Tahil, W. The Trouble With Lithium2 (Meridian International Research, 2008); http://go.nature.com/AWITRo 4. http://www.worldometers.info/cars/ 5. Armand, M. & Tarascon, J. M. Nature 451, 652657 (2008).
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