DOI: 10.1002/chem.

201101129

On the Determination of the Stereochemistry of Semisynthetic Natural Product Analogues using Chiroptical Spectroscopy: Desulfurization of Epidithiodioxopiperazine Fungal Metabolites
Fanny Cherblanc,[a] Ya-Pei Lo,[a] Ewoud De Gussem,[b, c, d] Laura Alcazar-Fuoli,[e] Elaine Bignell,[e] Yanan He,[f] Nadine Chapman-Rothe,[g] Patrick Bultinck,[c, d] Wouter A. Herrebout,[b, d] Robert Brown,[g] Henry S. Rzepa,[a] and Matthew J. Fuchter*[a]
Abstract: Isolation and semisynthetic modification of the fungal metabolite chaetocin gave access to a desulfurized analogue of this natural product. Detailed chiroptical studies, comparing experimentally obtained optical rotation values, electronic circular dichroism spectra, and vibrational circular dichroism spectra to computationally simulated ones, reveal the desulfurization of chaetocin to unambiguously proceed with retention of configuraKeywords: circular dichroism configuration determination desulfurization natural products vibrational spectroscopy tion. Consideration of the plausible mechanisms for this process highlighted inconsistencies in the stereochemical assignment of related molecules in the literature. This in turn allowed the stereochemical reassignment of the natural product analogue dethiodehydrogliotoxin.

Introduction
Although the structure and function of complex naturally isolated compounds and their synthetic analogues continue to fascinate and inspire, structural misassignments are often encountered. Indeed, in 2005, Nicolaou and Snyder present-

[a] F. Cherblanc, Y.-P. Lo, Prof. H. S. Rzepa, Dr. M. J. Fuchter Department of Chemistry Imperial College London, South Kensington Campus London SW7 2AZ (UK) Fax: (+ 44) 207-594-5805 E-mail: m.fuchter@imperial.ac.uk [b] E. De Gussem, Prof. W. A. Herrebout Department of Chemistry, University of Antwerp Groenenborgerlaan 171, 2020 Antwerp (Belgium) [c] E. De Gussem, Prof. P. Bultinck Department of Inorganic and Physical Chemistry Ghent University (UGent), Krijgslaan 281 (S-3) 9000 Gent (Belgium) [d] E. De Gussem, Prof. P. Bultinck, Prof. W. A. Herrebout European Centre for Chirality, www.chiralitycentre.eu [e] Dr. L. Alcazar-Fuoli, Dr. E. Bignell Centre for Molecular Microbiology and Infection Imperial College London, Armstrong Road London SW7 2AZ (UK) [f] Dr. Y. He BioTools, Inc., 17546 Bee Line Highway (SR 710) Jupiter, FL 33458 (USA) [g] Dr. N. Chapman-Rothe, Prof. R. Brown Ovarian Cancer Action Centre Department of Surgery and Cancer Imperial College London Hammersmith Hospital Campus, London, W12 ONN (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201101129.

ed 50 examples (out of well over 300 found) of structural revisions published between 1990 and 2004.[1] Of these, many revisions related to errors in stereochemical assignment including amphidinolide A,[2a] glabrescol,[2b] tolyporphin A,[2c] himastatin,[2d] trunkamide A,[2e] and antillatoxin.[2f] Ultimately, for each of these cases, it was the total synthesis of the natural product that enabled the structural reassignment. Although total synthesis, in the absence of crystals suitable for X-ray crystallographic analysis, can provide unambiguous proof of the stereochemistry of such complex molecular architectures, it requires a huge amount of effort and resource. Clearly, suitable spectroscopic techniques to inform on stereochemistry are an important alternative. Chiroptical spectroscopy uses refraction, absorption, or emission of anisotropic radiation and therefore can be used for identifying the relative and absolute stereochemistry of a substance. Optical rotation at a fixed wavelength, optical rotatory dispersion (ORD), and electronic circular dichroism (ECD) are standard techniques associated with electronic transitions,[3] whereas the more recent techniques of vibrational circular dichroism (VCD) and Raman optical activity (ROA) focus on vibrational transitions.[4] Although the use of optical rotation, optical rotatory dispersion (ORD), and electronic circular dichroism (ECD) in the determination of stereochemistry is not new, traditionally correlative methods are applied, comparing obtained chiroptical spectra to those acquired from related molecular frameworks. While this is a common practice, such qualitative comparisons can in some cases lead to a significant chance of error. To combat such error, an alternative approach is to compare the experimentally obtained spectra with theoretical simulations for the same molecule. Although this idea (at least for optical rota-

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tion, ORD and ECD) was pioneered by Kuhn, Kirkwood, and others more than 70 years ago,[5] it is only relatively recently that computational power has enabled suitably accurate quantum-chemical simulations of the chiroptical spectra of larger molecules to enable unambiguous comparison. In line with our interests in natural products that target pathways in epigenetic gene regulation,[6] we became interested in the natural product chaetocin (1, Scheme 1), a reported histone methyltransferase inhibitor.[7] Chaetocin belongs to the 3,6-epidithio-diketopiperazine (ETP) class of toxic fungal metabolites[8] and, like other fungal metabolites of the ETP class, exhibits a broad range of antibacterial and cytostatic activity, including a remarkable cytotoxicity against HeLa cells.[9] Since the broad cytotoxicity of the ETPs is due to the presence of the disulfide bridge of the 3,6-epidithio-diketopiperazine, which can inactivate proteins by crosslinking or lead to reactive oxygen species (ROS) generation, or zinc chelation,[8, 10, 11] we reasoned that access to a semisynthetic derivative bearing an unreactive thioether linkage (2, Scheme 1), would be a valuable analogue to probe the chemical biology of this natural product. Such an analogue should be readily accessible by desulfurization of the natural product. Towards this end, the use of triphenylphosphine to desulfurize a variety of ETP natural products has been previously reported,[12] however the mechanism of this reaction and therefore the stereochemistry of the product has been under some debate. In the 1970s, Safe and Taylor reported that desulfurization of dehydrogliotoxin (3, Scheme 1) to the bridged-monosulfide (4, Scheme 1) proceeded with inversion of configuration.[12a] This conclusion was based on the observation that the ECD curve of this derivative exhibited an opposite sign of the Cotton effect compared with the natural product. It was later argued that this was mechanistically unfeasible by Sammes, who suggested the curves are not comparable.[13] Subsequent studies by Sato and Hino on synthetic ETP derivatives observed bridged-monosulfide derivatives en route to novel dimeric species for which they invoked a mechanism involving retention of configuration.[14] Further confusion came from detailed studies by Ottenheijm and co-workers, who revealed that an (R,R)-ETP analogue (compound 5) was desulfurized to give the S,S bridged-monosulfide (compound 6), with inversion at both centers.[12b] The stereochemistry of the product was determined by NMR spectroscopy, ECD and X-ray data. From this they drew the conclusion that the sign of the ECD curves of the bridged-monosulfide products in comparison to the parental ETP natural product was in fact a good criterion to determine stereochemistry. Barbier and co-workers however, determined that conversion of sirodesmin PL (compound 7) to its bridged-monosulfide derivative (compound 8) proceeded with retention of configuration, based on chemical derivatization studies and X-ray analysis of a diacetyl derivative.[12c] They also found that, despite the reaction proceeding with retention, the ECD curves of the natural product (7) and its desulfurized derivative exhibit Cotton effects of opposite signs and an appreciable shift of the maximum wavelengths. They therefore cautioned against the use of ECD data as a criterion of absolute configuration determination of the bridgehead carbon atoms in related molecules. In light of these conflicting results in the literature, and the ambiguous stereochemistry of the desulfurized derivative of chaetocin (1), assumed to be the same as the parental natural product,[9c] we decided to prepare this derivative and compare several chiroptical techniques to their theoretical predictions. We envisaged that such studies would not only unambiguously confirm the stereochemistry of this natural product analogue, but also allow further analysis of the mechanistic understanding of this reaction, especially in terms of stereochemical outcome.

Results and Discussion

To perform semisynthetic modifications on the ETP core of chaetocin (1), significant quantities of this metabolite were required. Chaetomium virescens var. thielavioideum was cultured on solid complete media (5 plates) for two weeks and then extracted with dichloromethane. After purification by column chromatography, milligram quantities of a solid were obtained. 1H NMR spectroscopy and LC-MS analysis were consistent with that of chaetocin. A larger scale preparation gave, in a reproducible manner, approximately 200 mg of chaetocin, purified by column chromatography and trituration. The spectroscopic data from this material were analogous to an authentic sample obtained commer-

Scheme 1. Structural assignments of ETP analogues.

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cially. Desulfurization of chaetocin using triphenylphosphine under conditions previously described for the synthesis of a scabrosin ester derivative[15] was then attempted. Pleasingly, desulfurization using triphenylphosphine in dichloromethane gave the ring-contracted derivative 2 in good yield (93 %, Scheme 1). In terms of the stereochemical course of this reaction, as highlighted above, one could envisage either inversion of the sulfur bridge relative to the parental molecule or retention (Figure 1). The symmetry inherent to the chaetocin

ment over our previously recommended CAM-B3LYP functional for chiro-optical properties (see the Supporting Information for more details on the optimization).[18] The measured optical rotation for our isolated analogue 2 was + 484 (c = 0.0039, CHCl3, 25 8C). Correspondingly, the calculated optical rotations (using a 6-311GACHTUNGRE(d,p) basis set) were + 467 for 2 a, + 357 for 2 b, and + 259 for 2 c. The significant difference in these predicted values gave us confidence for an effective assignment and indeed, the calculated value for 2 a was very close to that of our experimental analogue. This suggested the reaction had proceeded with retention of ETP stereochemistry. To further confirm this assignment and to assess the suitability of these methods to determine the configuration of what is a rather large molecule, we also undertook ECD and VCD analysis. The ECD spectra were again computed using time-dependent DFT procedures with a continuum solvation correction (CPCM) appropriate for methanol. At first, the wB97XD functional using the 6-311GACHTUNGRE(d,p) basis was selected for the calculations. The experimental ECD curve obtained was compared to the calculated ones for the three possible stereoisomers (Figure 2).

Figure 1. Possible stereoisomers for chaetocin desulfurized analogue calculated using Gaussian 09[16] at the wB97XD/6-311 + + GACHTUNGRE(d,p) level of theory.

Figure 2. ECD curves of predicted (right y axis) and experimental (left y axis) spectra for chaetocin monosulfide 2. The predicted curves were shifted by + 20 nm and convoluted with a line width 0.30 eV. The calculations were done at wB97XD/6-311GACHTUNGRE(d,p) level of theory; c: experimental results; ~: calcd. S,S S,Sstructure 2a; *: calcd. R,R S,Sstructure 2b; *: calcd. R,R R,Rstructure 2c.

framework limits the possible diastereoisomers to those where both ETP centers (i.e., the stereochemistry at C11/ C15 and C11/C15) had undergone retention (S,S S,Sstructure 2 a), one had undergone retention and one inversion (R,R S,Sstructure 2 b) or both had undergone inversion (R,R R,Rstructure 2 c). Upon inspection of the structures, it became rapidly apparent that commonly used techniques such as NMR spectroscopy would struggle to differentiate between these stereoisomers and therefore we turned to comparison of calculated and experimental properties such as optical rotation, ECD, and VCD analysis. Simulations were carried out using Gaussian 09[16] at a density functional level. The functional selected was wB97XD on the basis of its extensive testing against small molecules,[17] representing a small but significant improve-

Since the ECD curve of 2 c was absolutely inconsistent with the experimental one in terms of the sign of the Cotton effect at around 280 nm, this diastereoisomer was excluded immediately. The ECD curve of 2 a on the other hand, shows two positive Cotton effects (245 and 287 nm), which is in good agreement with the experimental spectrum. The ECD curve of 2 b is similar to that of 2 a, and only differs by a small negative Cotton effect at 270 nm. Since we felt this result was not sufficiently conclusive, we decided to use a larger basis set to obtain more accurate results. The augmented 6-311 + + GACHTUNGRE(d,p) basis set was selected as giving the best match to the experimental bandwidths (Figure 3). The experimental ECD curve of chaetocin monosulfide exhibited one negative (222 nm) then two positive (250, 305 nm) Cotton effects. The predicted ECD curve for 2 a, at this

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Figure 3. ECD curves of predicted (right y axis) and experimental (left y axis) spectra for chaetocin monosulfide 2. The predicted curves were shifted by + 20 nm and convoluted with a line width 0.24 eV. The calculations were done at wB97XD/6-311 + + GACHTUNGRE(d,p) level of theory; c: experimental results; ~: calcd. S,S S,Sstructure 2a; *: calcd. R,R S,Sstructure 2b; *: calcd. R,R R,Rstructure 2c.

level of theory, also had one negative followed by two positive (247, 288 nm) Cotton effects. At this level of theory, the ECD curve for 2 b had a clear pattern of negative (222 nm), positive (246 nm), negative (272 nm), positive (289 nm) Cotton effects and therefore was excluded. Taken together this analysis is once again consistent with the reaction proceeding with retention of ETP stereochemistry. To further validate the ability of these basis sets to predict ECD spectra for the chaetocin framework, analogous simulations were carried out on the parental natural product 1, confirming the accepted stereochemistry of the natural product (see the Supporting Information, Figure S18). Finally, a VCD analysis was undertaken. Although the use of VCD in stereochemical assignment for natural products is less common, it is emerging as a useful technique in this regard.[19] Once again, comparison of an ab initio simulated spectrum with the experimental counterpart should show the same sign and magnitude for the rotational strength of each normal mode. However, due to the approximations made in the simulations, a detailed quantitative analysis is required to ensure correct assignment. The experimental and theoretical IR and VCD spectra of product 2 are shown in Figure 4 and Figure 5. Simulated IR/VCD spectra are scaled to compensate for the overestimation of the vibrational frequencies in the harmonic approximation. Usually, the frequency scale factor lies roughly between 0.96 and 1.00.[20] This scale factor is chosen in such a manner that the simulated IR spectrum gives reasonable visual agreement with the experimental one. This is done for each diastereoisomer separately. For all three diastereoisomers (2 a, 2 b, and 2 c), an acceptable agreement for the IR spectra is apparent (Figure 4). The strong feature at l = 1245 cm1 is absent in all three calculated spectra, however, the spectrum of 2 a is the only one reproducing the strong absorption observed at l = 1320 cm1. The simulated VCD spectra were scaled using the same scale factors that were used for the IR spectra. For comparison of the calculated VCD spectra with the experimental spectrum, a thorough visual inspection was performed, correlating peaks in the IR spectrum to peaks in the VCD spec-

Figure 4. Comparison of the calculated (wB97XD/6-311GACHTUNGRE(d,p)) and experimental IR spectra of compounds 2, 2 a, 2 b, and 2 c.

Figure 5. Calculated (wB97XD/6-311GACHTUNGRE(d,p)) and experimental VCD spectra of compounds 2, 2 a, 2 b, and 2 c.

trum. This was done for seven important features in the VCD spectrum, numbered 17 in Figure 4 and Figure 5. Features 1 and 2 are not predicted for structures 2 a, 2 b, or 2 c. The remaining features (37) are only correctly predicted for structure 2 a. All attempts at matching the features for 2 b and 2 c gave very poor agreement, or a mirror-image relationship, which is obviously impossible (since this would involve the unnatural stereochemistry of the chaetocin-core at C2/C3 and C2/C3). Since structure 2 a gave by far the most satisfactory agreement, the purely visual VCD analysis was also supportive that desulfurization occurred with retention of stereochemistry. In addition, whereas usually the peak of the carbonyl stretch (around 1700 cm1) is not included in this type of analysis (since the important solvent

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effects for carbonyl stretch cannot always be predicted correctly by continuum solvent fields),[21] the nice agreement in this region between the experimental spectra and the calculated one for structure 2 a reinforces the conclusion of retention of stereochemistry. To ensure the visual interpretation of the VCD data was not subject to human bias, we further used an algorithm developed to assess how good the manual assignment was in light of previous successful assignments.[22] This algorithm is currently implemented in the CompareVOA program[23] and allows one to establish a confidence level using a Neighborhood Similarity (NS) measure for the absolute configuration assignment made by visual interpretation. First the scale factor is determined for the computed IR spectra by optimizing the similarity with the experimental spectrum constraining the scale factor to lie within 0.96 and 1.02. Given this scale factor, the similarity is computed between the calculated VCD spectra and the experimental one. This similarity measure S has a value between 0 and 1 and expresses the degree of agreement between theory and experiment. The enantiomeric similarity index D, gives the absolute difference between the values of S for both enantiomers of a given stereoisomer. Hence, D is a measure for the discriminative power of a VCD analysis and subsequently a high quality VCD spectrum is characterized by a high value of D. These quantities are then compared to a database of previous state-of-art successful assignments to calculate a confidence level (see the Supporting Information, S19). The results of the analysis for compound 2 a are shown in Table 1 (see the Supporting Information, Table 11 for 2 b/2 c values).
Table 1. Calculated quantities describing the similarity between the calculated and experimental IR and VCD spectra. Optimal scaling factor 2a 0.966 IR similarity [%] 84.9 S for best enantiomer [%] 59.4 D 39.8 Confidence level [%] 94

species, in which they invoked a mechanism involving retention of configuration (Scheme 2).[14] Following nucleophilic cleavage of the disulfide bond by triphenylphosphine (to give 10),[24, 25] they proposed elimination of triphenylphosphine sulfide to give species 11, which could undergo ringclosing to give monosulfide 12 a with retention of stereochemistry. Although this mechanism appears satisfactory, it cannot explain the results of Ottenheijm and co-workers, who unambiguously showed that ETP analogue 5 (see Scheme 1) was desulfurized to give the (S,S)-monosulfide 6, with inversion at both centers.[12b] Such a result would require epimerization at the thiolate centre. Ottenheijm and co-workers instead proposed that the phosphonium salt 10 is stable to elimination, and that, in the case of analogue 5, the thiolate centre can epimerize through ring-opening of the dioxopiperazine ring, conformational rearrangement and ring-closing (10!13!13!14), followed by SN2-type displacement of the triphenylphosphine sulfide leaving group. Such SN2-type displacement of the phosphonium intermediate has precedence in desulfurization chemistry,[24] particularly using aminophosphines.[25] Consistent with this mechanism, studies by Barbier and co-workers determined that conversion of sirodesmin PL to its monosulfide derivative 8 (see Scheme 1) proceeded with retention of configura-

The results shown in Table 1 indicate that there is good agreement for the IR spectrum with a physically acceptable scale factor. CompareVOA indicates good similarity for the VCD spectrum of 2 a. For the other possible diastereoisomers, the highest similarity was found for the chemically impossible enantiomeric structure. Compared to a database of previous successful assignments,[23] the confidence level is computed to be very high (94 %). Since all three comparative chiroptical techniques are in agreement, we assign the stereochemistry of the obtained desulfurized chaetocin to that of structure 2 a (Figure 1), that is, retention of configuration of C11/C15 and C11/C15. In light of the fact that this result is only the third unambiguous assignment of stereochemistry for the desulfurization of ETP natural products, it warrants a further analysis of the mechanistic course of this reaction. Studies by Sato and Hino on synthetic ETP derivatives observed monosulfide derivatives en route to novel dimeric

Scheme 2.

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tion.[12c] They highlighted that the pendent alcohol functionality adjacent to the leaving group in the ETP natural product could participate in the stereochemical course of the reaction. Indeed, it is plausible that such a neighboring group effect occurs through an SN2-type displacement of the triphenylphosphine sulfide leaving group by the hydroxyl group (15!16), followed by an SN2-type displacement of the oxirane by the thiolate anion (16!17). This double inversion mechanism would of course result in net retention of stereochemistry. Since, like sirodesmin PL, chaetocin contains a comparable pendent alcohol functionality, it is plausible that an analogous mechanism is in operation for its desulfurization, that is, double inversion (15!17), resulting in net retention. Indeed, this is consistent with our assignment of stereochemistry. Such a comparison, however, is in conflict with the studies of Safe and Taylor, who reported that desulfurization of dehydrogliotoxin (3, Scheme 1) proceeded with inversion of configuration,[12a] despite the fact that this molecule also contains an analogous pendent alcohol functionality. In light of the fact that their assignment of inversion was based on comparison of the ECD curve of the bridgedmonoACHTUNGREsulfide derivative to ECD curve of the parental natural product, which, as stated above, is not a reliable method for unambiguous analysis, it remained possible that they had misassigned the stereochemistry of the product. We therefore calculated the predicted ECD curves for dehydrogliotoxin (3) and dethiodehydrogliotoxin (4) and compared them to those obtained by Safe and Taylor. Since dehydrogliotoxin (3) and dethiodehydrogliotoxin (4) are significantly smaller than chaetocin (1), and therefore more computationally accessible, an extensive survey of functionals and basis sets was carried out to examine which quantitatively predict the experimental spectra (see the Supporting Information for details). At the wB97XD/6-311 + + GACHTUNGRE(d,p) level of theory, the accurate reproduction of the experimentally obtained spectra is seen for the natural product 3 (see the Supporting Information, Figure S17).[12a] However, the desulfurized derivative 4 in fact appears to have the opposite stereochemistry to that reported by Safe and Taylor (S,S) (Figure 6): Our results show that the calculated spectra for (R,R)-dethiodehydrogliotoxin (epi-4, Scheme 1) is in qualitative and quantitative agreement with the reported literature spectra, whereas the spectrum calculated for (S,S)dethiodehydrogliotoxin (4) does not match at all.[12a] We therefore propose that the assignment of stereochemistry by Safe and Taylor was incorrect and in fact dethiodehydrogliotoxin (4) has the opposite stereochemistry (R,R) to that shown in Scheme 1 (i.e., epi-4). Our computer simulation suggests that, even though comparison of the ECD curves for highly related molecular frameworks seems apparently a quite reasonable assumption, it can be quite untrustworthy. It is clear that the desulfurization of dehydrogliotoxin proceeds with retention at both carbon centers. In light of this proposed reassignment, it would seem that the desulfurization of dehydrogliotoxin (3) also proceeds with retention, following the trend of chaetocin (1) and siro-

Figure 6. ECD spectra of predicted (at the wB97XD/6-311 + + GACHTUNGRE(d,p) level of theory, scaled by 5 and shifted by + 15 nm, convoluted line width 0.25 eV) and literature reported spectra of dethiodehydrogliotoxin[12a] 1,4-dioxane; *: calcd. R,R-dethiodehydrogliotoxin; *: calcd. S,S-dethiodehydrogliotoxin; c: lit. dethiodehydrogliotoxin.

desmin PL (7); a plausible consequence of the pendent alcohol functionality present in the frameworks of these natural products. It is also apparent that synthetic derivative 5 remains the only ETP analogue to unambiguously undergo inversion of stereochemistry upon desulfurization with phosphine derivatives, and that this analogue does not have a pendent nucleophile to facilitate a double inversion. It therefore remains to be seen whether other ETP natural products[8] undergo such desulfurization with inversion or retention, but we would predict that a suitably positioned pendent nucleophile would enable conversion to the product with retention. In absence of such a group, the precise mechanism and therefore stereochemistry is unclear. Indeed, despite demonstrating the possibility of inversion in this reaction (5!6), Ottenheijm and co-workers comment that their inversion mechanism (see Scheme 2) holds only when the epidithiodioxopiperazine nucleus is condensed to no more than one ring.[12b] In support of this statement, they highlight the low reactivity of an acetyl derivative of aranotin (18) towards desulfurization.[26] Disappointingly however, the stereochemistry of the desulfurized product 18 was not reported by the authors. Far more recent studies would provide a similarly interesting example; the desulfurization of the scabrosin esters (19).[15] Unfortunately, once again, the authors made no attempt to determine the stereochemistry of the product.

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Conclusion
We have experimentally isolated and semisynthetically modified the fungal metabolite chaetocin to access a desulfurized derivative. Comparative assessment of experimental and calculated chiroptical properties of this molecule has allowed unambiguous characterization of the stereochemistry of this complex molecule. Furthermore, led by a mechanistic hypothesis and confirmed by simulation of ECD spectra, we have reassigned the previously reported product dethiodehydrogliotoxin. As a final concluding note, we are inclined to comment on the use of chiroptical spectroscopic techniques to determine absolute and relative stereochemistry. We would recommend that such experimental techniques are always accompanied by suitably accurate quantum-chemical simulations of the chiroptical spectra to ensure accuracy of the assignments. Where possible, at least two different techniques (for example optical rotation and ECD) should also be used to increase the level of certainty of the assignment.

(ESI) m/z 697 [M + H] + ; HRMS (ESI) m/z calcd (%) for C30H29N6O6S4 : 697.1031 [M + H] + ; found: 697.1021. Synthesis of chaetocin monosulfide (2): PPh3 (28 mg, 0.11 mmol) was added to a solution of chaetocin (1, 20 mg, 0.029 mmol) in CH2Cl2 (5 mL) and the resulting mixture was stirred for 2 h at room temperature. The solvent was removed in vacuo and the resulting pink solid was purified by column chromatography (CH2Cl2/EtOAc, 60:40). Monosulfide 2 was obtained as a white solid (17 mg, 93 %). M.p. 210212 8C (dec.); [a]20 = + 484 (c = 0.0039 in CHCl3); 1H NMR (400 MHz, CDCl3) d = 7.47 D (d, J = 7.8 Hz, 2 H), 7.22 (td, J = 7.8, 0.7 Hz, 2 H), 6.91 (td, J = 7.8, 0.7 Hz, 2 H), 6.74 (d, J = 7.8 Hz, 2 H), 5.46 (s, 2 H), 4.89 (s, 2 H), 4.13 (m, 4 H), 3.63 (d, J = 14.9 Hz, 2 H), 3.00 (s, 6 H), 2.85 (m, 2 H), 2.49 ppm (d, J = 14.9 Hz, 2 H); 13C (100 MHz, CDCl3) d = 174.9, 173.5, 149.2, 130.2, 128.1, 124.9, 120.3, 110.6, 80.7, 79.4, 78.5, 63.6, 59.1, 31.6, 28.3 ppm (dimeric structure); IR (neat) 3388, 2921, 1712, 1468, 1322, 753 cm1; UV/Vis (CHCl3): lmax (A): 243.4 (0.49), 298 (0.21) nm, CD (MeOH): lmax (mdeg) = 217 (0), 223.5 (43.7), 230 (0), 248.5 (+ 184.9), 285 (+ 59.5), 304 (+ 81.6), 350 (+ 0.8) nm; LCMS: Rt = 4.77 min; MS (ESI) m/z 633 [M + H] + ; HRMS (ESI) m/z calcd. for C30H29N6O6S2 : 633.1590 [M + H] + ; found: 633.1598. IR and VCD spectroscopy: The experimental spectra of compound 2 were recorded for a solution in [D6]DMSO with a concentration of 6.0 mg/0.2 mL, a 100 mm path-length cell equipped with BaF2 windows. The collection time was set to 10 h; the instrument was optimized at 1400 cm1. ECD spectroscopy: The experimental spectra were recorded on a Applied Photophysics Chirascan spectrometer (T = 22 8C, l = 180260 nm, step = 0.5 nm, band width = 1 nm, time per point = 1 s).

Experimental Section
Computational methods: Geometries of all species were optimized using Gaussian 09,[16] with the inclusion of a continuum solvent field (SCRF) appropriate for the experiment (methanol for 1 and 2; 1,4-dioxane for 3 and 4). Optical rotation calculations (589 nm, sodium d line) were computed at the wB97XD/6311GACHTUNGRE(d,p) level. Electronic circular dichroism spectra were calculated with a solvent field using time-dependent DFT procedures for 3 and 4, using seven different functionals (B3LYP, wB97XD, CAM-B3LYP, M06, M062X, LC-BLYP) and seven basis sets (631GACHTUNGRE(d,p), 631 + GACHTUNGRE(d,p), 631 + + GACHTUNGRE(d,p), 6311GACHTUNGRE(d,p), 6311 + GACHTUNGRE(d,p), 6311 + + GACHTUNGRE(d,p) and cc-PVACHTUNGRE(T+d) + +). The combination wB97XD/6311 + + GACHTUNGRE(d,p) was selected as giving the best overall match to the experimental line shapes. The computed spectra of 3 and 4 for the other methods are included in the supporting information for comparison. The ECD spectra of 1 and 2 were initially simulated at wB97XD/6 311GACHTUNGRE(d,p) and at the larger basis set 6311 + + GACHTUNGRE(d,p) level for more accurate intensities. VCD simulations were performed at the wB97XD/6 311GACHTUNGRE(d,p) level of theory with the inclusion of a continuum solvent field (DMSO). Isolation of chaetocin (1): Chaetomium virescens var. thielavioideum (CBS 623.80) was cultured at room temperature ( % 22 8C) in complete media (2.5 % glucose, 0.5 % yeast extract), according to Pontecorvo et al.[27] containing 1 % vitamin solution, 1 % glucose as carbon source and 5 mm ammonium tartrate as a nitrogen source. Conidia stocks were prepared from agar surfaces of 14 day old cultures and preserved in sterile distilled water at 4 8C. A conidial suspension was then inoculated in ten 1 L bottles slants with solid complete media and incubated for three weeks at room temperature. The cultures were then extracted with CH2Cl2 (2 500 mL per bottle) for 24 h and 48 h with orbital shaking. The resulting mixtures were then filtered, dried over MgSO4, and concentrated in vacuo. The extracts were then purified by column chromatography (CH2Cl2/MeOH, 98:2). The resulting solid was further purified by trituration in hexanes with sonication to afford 210 mg of chaetocin. [a]20 = + 542 (c = 0.82 in CHCl3); 1H NMR (400 MHz, CDCl3) d = 7.42 D (d, J = 7.5 Hz, 2 H), 7.25 (t, J = 7.5 Hz, 2 H), 6.92 (t, J = 7.5 Hz, 2 H), 6.74 (d, J = 7.5 Hz, 2 H), 5.25 (s, 2 H), 5.24 (s, 2 H), 4.25 (dd, J = 12.5, 6.0 Hz, 2 H), 4.17 (dd, J = 12.5, 9.4 Hz, 2 H), 3.83 (d, J = 15.0 Hz, 2 H), 3.28 (dd, J = 9.4, 6.0 Hz, 2 H), 3.08 (s, 6 H), 2.74 ppm (d, J = 15.0 Hz, 2 H); 13C (100 MHz, CDCl3) d = 165.6, 162.8, 149.1, 130.4, 127.4, 125.1, 120.4, 110.7, 80.5, 75.7, 73.3, 60.6, 59.8, 39.2, 27.3 ppm; IR (neat) 3383, 3336, 1670, 1066, 749 cm1; CD (MeOH): lmax (mdeg) = 237 (+ 434), 263 (0), 272 (42), 284 (0), 304 (+ 78), 350 (+ 1) nm; LCMS: Rt = 5.62 min; MS

Acknowledgements
We would like to acknowledge Cancer Research UK (studentship for F.C.) for funding (grant C21484A6944), Prof. Kurt Drickamer for access to the ECD spectrometer (BBSRC grant BB/C510859/1 for the instrument) and Frank Trundle (Biotools) for assistance with VCD.

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Received: April 12, 2011 Published online: September 5, 2011

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