Biomaterials 31 (2010) 4179–4185

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Relationship between the tautomeric structures of curcumin derivatives

and their Ab-binding activities in the context of therapies for
Alzheimer’s disease
Daijiro Yanagisawa a, b, Nobuaki Shirai c, Tomone Amatsubo a, b, Hiroyasu Taguchi a, Koichi Hirao c,
Makoto Urushitani a, Shigehiro Morikawa d, Toshiro Inubushi e, Masanari Kato f, Fuminori Kato f,
Kyuya Morino f, Hirohiko Kimura f, Ichiro Nakano f, Chikako Yoshida f, Takashi Okada f, Mitsuo Sano f,
Yoshiko Wada g, Ken-nosuke Wada g, Akitsugu Yamamoto g, Ikuo Tooyama a, *
a
Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520-2192, Japan
b
Japan Science and Technology Agency (JST), JST Innovation Satellite Shiga, 2-1 Uchidehama, Otsu 520-0806, Japan
c
Industrial Research Center of Shiga Prefecture, 232 Kamitoyama, Ritto 520-3004, Japan
d
Department of Fundamental Nursing, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520-2192, Japan
e
Biomedical MR Science Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520-2192, Japan
f
Central Research Institute, Ishihara Sangyo Kaisya Ltd., 2-3-1 Nishi-shibukawa, Kusatsu 525-0025, Japan
g
Nagahama Institute of Bioscience and Technology, 1266 Tamura-cho, Nagahama 526-0829, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Curcumin, which can exist in an equilibrium between keto and enol tautomers, binds to b-amyloid (Ab)
Received 28 October 2009 fibrils/aggregates. The aim of this study was to assess the relationship between the tautomeric structures
Accepted 29 January 2010 of curcumin derivatives and their Ab-binding activities. Curcumin derivatives with keto-enol tautom-
Available online 23 February 2010
erism showed high levels of binding to Ab aggregates but not to Ab monomers. The binding activity of
the keto form analogue of curcumin to Ab aggregates was found to be much weaker than that of cur-
Keywords:
cumin derivatives with keto-enol tautomerism. The color of a curcumin derivative with keto-enol
Alzheimer’s disease
tautomerism, which was substituted at the C-4 position, changed from yellow to orange within
Amyloid-b
Curcumin 30 min of being combined with Ab aggregates in physiological buffer. This resulted from a remarkable
Keto-enol tautomerism increase in the enol form with extended conjugation of double bonds upon binding. These findings
Amyloid detection suggest that curcumin derivatives exist predominantly in the enol form during binding to Ab aggregates,
and that the enolization of curcumin derivatives is crucial for binding to Ab aggregates. The keto-enol
tautomerism of curcumin derivatives may be a novel target for the design of amyloid-binding agents
that can be used both for therapy and for amyloid detection in Alzheimer’s disease.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction aggregate leads gradually to synaptic and neuritic compromise,

Alzheimer’s disease (AD) is characterized by progressive cogni-
tive impairment that is a consequence of neuronal and synaptic
losses and neurotransmitter deficits. The pathological features of AD
are extracellular plaques and intracellular neurofibrillary tangles,
which consist of b-amyloid (Ab) and hyperphosphorylated tau,
respectively [1,2]. Studies on genotype-to-phenotype conversions in
familial AD support the amyloid hypothesis: cerebral Ab accumu-
lation is the primary event in AD, and a chronic imbalance between
the production and clearance of Ab with a tendency to misfold and
* Corresponding author. Tel.: þ81 77 548 2328; fax: þ81 77 548 2402.
E-mail address: kinchan@belle.shiga-med.ac.jp (I. Tooyama).
including tau tangle formation [2,3]. Particularly, Ab oligomers and
protofibrils show strong neurotoxicity. Therefore it is of great
importance to be able to detect Ab oligomers and protofibrils in the
early diagnosis and therapeutics of AD.
In order to detect Ab oligomers and protofibrils, many
researches have tried to develop antibodies and chemicals that
have affinity specific for Ab aggregates. These include derivatives of
thioflavin, Congo red, benzoxazole and curcumin [4–11]. Among
them, curcumin is of great interest to us because it is purified from
food and shows superior safety property. Curcumin inhibited
aggregation and fibril formation of Ab by binding to small Ab
species [12]. Curcumin is also reported to reduce amyloid pathology
in Alzheimer transgenic mice [13]. When we tested the amyloid-
binding activity of a particular curcumin derivative (compound

0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.01.142
4180 D. Yanagisawa et al. / Biomaterials 31 (2010) 4179–4185
9), a dramatic color change occurred after adding Ab aggregates
into the solution (Fig. 1 and mmc1 Supplementary Video 1). In
contrast, no color change was observed after the addition of Ab
monomers to the solution of compound 9. We assume that struc-
tural conversion of curcumin derivatives occurs during binding to
Ab aggregates, and that this conversion causes the color change.
Therefore, in the present study, we investigated the relationship
between structural conversion of curcumin derivatives, focusing on
the keto-enol tautomerism, and their amyloid-binding activities.
2. Materials and methods
2.1. General methods
NMR spectra were determined using a 270-MHz NMR spectrometer (JNM-GX270;
JEOL, Tokyo, Japan). The 1H and 13C NMR spectra were referenced to tetramethylsilane
as the internal standard. Chemical shifts of the 19F NMR signals were referenced to C6F6
at 163 ppm as an external standard [4]. UV–visible absorption spectra were recorded
between 250 nm and 750 nm using a UV–visible spectrophotometer (UV-2500PC;
Shimadzu, Kyoto, Japan) in a rectangular quartz cuvette with 1.0-cm optical path length.
The slit width was 2 nm, and the scan speed was set at high.
2.2. Synthesis of curcumin derivatives
Curcumin derivatives (7–11) were prepared by condensing acetylacetone and its
derivatives (4–6) with appropriate aldehydes (1–3), using previously described
procedures [14–16]. Compounds 10 and 11 were methylated with methyl iodide in
the presence of potassium carbonate to give compounds 13 and 14, respectively.
Compound 12, which is a mimetic of the keto form of the curcumin derivatives, was
generated by hydrolysis of compound 14 in an acidic medium. The synthetic path-
ways are depicted in Scheme 1 and Supplementary Methods.
Fig. 1. Color changes of curcumin derivatives after the addition of Ab aggregates to the
solution. Representative photographs show the time course of the color change for
compound 9. Two hundred microliters of a 40-mM solution of compound 9, which is
a curcumin derivative substituted at the C-4 position, were mixed with 0.043 mg of
monomeric or aggregated Ab in 50 mM phosphate buffer (pH 7.5) that contained
100 mM NaCl. Subsequently, the mixture was placed at room temperature for 0 (a), 3
(b), 10 (c), and 30 min (d). The upper row in a shows samples without compound 9,
and the lower row in a shows samples that contain compound 9 at 0 min. The solution
of compound 9 without Ab (left) shows a yellowish color at all the time-points. In the
presence of Ab aggregates (right), the color of compound 9 gradually but dramatically
changes to reddish-orange over 30 min. In contrast, no color change is evident for
30 min after the addition of Ab monomers to the solution (middle).
2.3. Preparation of Ab aggregates
Ab (1-40) (Peptide Institute, Osaka, Japan) was dissolved in dimethyl sulfoxide
(DMSO) at a concentration of 2 mM. Then, 5 mL of 2 mM Ab solution was diluted to
100 mM in 50 mM phosphate buffer (pH 7.5) that contained 100 mM NaCl. One
microliter of the seed, which was prepared by sonicating preformed Ab aggregates
for 30 min, was added to 100 mM Ab solution. The mixture was then incubated at
30  C for 20 h. The Ab aggregates were stored at 4  C before use.
2.4. Thioflavin T fluorescence assay
Thioflavin T (ThT) (Kd ¼ 2 mM for Ab [8]), a well-known agent that binds to Ab
aggregates [6,8,9], was diluted to a concentration of 5 mM in 50 mM glycine-NaOH (pH
8.5), and the solution was stored at room temperature in the dark before use. For the
ThT fluorescence assay, 2 mL of aggregated Ab solution and curcumin derivatives
were placed in the bottom of each well of a black 96-well non-binding plate (Greiner
Bio-One, Frickenhausen, Germany). Then, 200 mL of 5 mM ThT solution were added to
each well. The fluorescence of ThT was measured with a FlexStation (Molecular
Devices, Sunnyvale, CA, USA) at an excitation wavelength of 440 nm and emission
wavelength of 490 nm with cut-off wavelength of 475 nm. The binding activities to
Ab aggregates of the curcumin derivatives, expressed as the half-maximal inhibitory
concentrations (IC50) with regard to ThT fluorescence, were estimated using
GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
2.5. Determination of pKa values
We prepared 40 mM solutions of the curcumin derivatives in 45.8%, 65.9%, and
99.5% methanol that contained a small volume of 1.0 M HCl, 0.1 M acetate buffer (pH
3.5–5.0), 0.1 M phosphate buffer (pH 6.0–8.0), 0.1 M borate buffer (pH 9.0–9.5) or
1.0 M NaOH in the 0.15–5.0 mM concentration range, to adjust the pH. The pH value of
each solution was measured with a pH meter when the UV–visible absorption
spectrum of the solution was measured. The pKa value was estimated according to
the procedure of Albert and Serjeant [17]. The absorbances at various pH values were
recorded at the analytical wavelength at which the greatest difference between the
absorption of the neutral species and the ionized species was observed. Then, the
pKa value was calculated.
2.6. Estimations of the keto and enol forms of curcumin during binding to Ab
aggregates
Aggregates of Ab (0.22 mg) were incubated in 50 mM phosphate buffer (pH 7.5)
that contained 100 mM NaCl, together with 2.5 mL of 5 mM curcumin derivative in
Protein LoBind Tubes (Eppendorf, Hamburg, Germany) for 30 min at 30  C. The
mixture was centrifuged at 3000  g for 10 min at 15  C. After several washings, the
supernatant was removed, and the precipitate was re-suspended in 55 mL of 50 mM
phosphate buffer (pH 7.5) that contained 100 mM NaCl. Fifty microliters of the
suspension were transferred into a well of a UV-transparent 96-well microplate
(UV-Star Microplate; Greiner Bio-One), and the UV–visible absorption spectrum was
measured between 250 nm and 750 nm in a microplate reader (Power Wave X;
BioTek Instruments, Winooski, VT, USA). The suspension in the microplate was then
collected into a tube. After centrifugation, the UV–visible absorption spectrum of an
aliquot of 50 mL collected from the top of the supernatant was measured. To estimate
the levels of the keto and enol forms of the curcumin derivatives under the binding
condition to Ab aggregates, we performed a computational analysis of the compo-
nents of the spectra using linear least-squares fitting.
3. Results
3.1. Binding activity of curcumin derivatives to Ab aggregates
We used electron microscopy to investigate whether the amyloid
structure of the Ab aggregates was altered by the curcumin deriva-
tives. No alterations to the amyloid structure were observed either in
the presence or absence of the curcumin derivatives (Supplementary
Fig. 1). Based on this observation, we used a ThT competition study to
estimate the binding activities of the curcumin derivatives. Compe-
tition curves for the curcumin derivatives in the ThT competition
study were plotted (Supplementary Fig. 1), and the IC50 values were
estimated. The IC50 values of the curcumin derivatives were as
follows: for curcumin (compound 7 with CF3 replaced by CH3),
0.20 mM; for compound 7, 0.26 mM; and for compound 9, 0.44 mM.
Although accurate IC50 values for compounds 12 and 13 could not be
obtained due to their low solubilities in aqueous buffers, their IC50
values should be >20 mM. These results indicate that the keto forms of
 D. Yanagisawa et al. / Biomaterials 31 (2010) 4179–4185 4181

Scheme 1. Synthetic pathways and structures of curcumin derivatives.

the curcumin derivatives significantly decrease binding activity to the
Ab aggregates.
3.2. Extent of enolization
In the 1H NMR spectrum of compound 9 in d6DMSO, two
significant signals were detected at d 4.65 ppm (t, J ¼ 7 Hz) and
d 17.90 ppm (s) (Table 1). The former signal corresponds to the C-4
proton of the keto form, while the latter signal corresponds to the OH
of the enol form (the large downfield shift indicates strong hydrogen
bonding). This indicates that both forms of curcumin exist in DMSO.
Similar observations were made by Pedersen and colleagues for
curcumin derivatives substituted at the C-4 position [18]. The 19F
NMR spectrum clearly showed two singlets at d 58.09 ppm and
d 57.96 ppm, and the extent of enolization, as calculated from the
integrated intensity of the two signals, was 44.9  2.4%. In a more
hydrophobic solvent, such as CDCl3, this value increased to almost
100%. A signal at d 16.22 ppm (accompanying a sharp singlet at
d 6.09 ppm, which was attributed to the C-4 proton) was observed
for compound 7 in the 1H NMR spectrum. The signal integrals
indicate that compound 7 exists predominantly in the enol form in
DMSO. Similarly, the enol form was predominant in CD3OD. This
4182 D. Yanagisawa et al. / Biomaterials 31 (2010) 4179–4185

Table 1
Chemical shifts in the 1H NMR and 19
F NMR spectra and the extent of enolization of the curcumin derivatives.

Compound Solvent Form Chemical shifts (ppm) Extent of

enolizaton (%)
OH H (at C-4) F
7 d6DMSO Enol 16.22 6.09 (s) 57.67 Almost 100%
7 CD3OD Enol – 6.01 (s) 60.44 Almost 100%
8 d6DMSO Enol 17.57 – 57.67 61.5  0.4
Keto – 4.57 (q, J ¼ 7 Hz) 57.81
9 d6DMSO Enol 17.90 – 57.65 44.9  2.41
Keto – 4.65 (t, J ¼ 7 Hz) 57.78
9 CDCl3 Enol 17.52 – 59.08 Almost 100%
7 99.5% methanol Enol – – 60.29 Almost 100%
7 80% methanol Enol – – 60.17 94.6  0.1
Keto – – 60.22
7 50% methanol Enol – – 59.97 86.9  0.4
Keto – – 60.03
9 99.5% methanol Enol – – 60.26 52.0  0.3
Keto – – 60.34
9 80% methanol Enol – – 60.19 31.6  1.8
Keto – – 60.25
9 50% methanol Enol – – 59.98 13.3  0.3
Keto – – 60.13

The values for the extent of enolization are expressed as the mean  standard deviation for three independent experiments. In compound 7 in d6DMSO, CD3OD, and 99.5%
methanol and compound 9 in CDCl3, the values for the extent of enolization were expressed as almost 100%, since the signals for the keto form were not detectable in these
experiments.

finding was further confirmed by the 13C NMR spectrum
(Supplementary Fig. 2). However, the ratio of the enol form to keto
form decreased in aqueous media as the water content increased
(Table 1). From these NMR data, we conclude that: 1) enolization of
curcumin derivatives is enhanced in hydrophobic media; 2) enoli-
zation is reduced in polar media, such as aqueous solutions; and 3)
a substituent at the C-4 position restrains the enolization.
3.3. UV–visible spectra and pKa values of curcumin derivatives
in an aqueous methanol solution
In order to determine the (keto or enol) forms of the curcumin
derivatives upon binding to the Ab aggregates, it is essential to
obtain typical UV–visible absorption spectra of the neutral species
and the anions of the keto form and enol form (Fig. 3). Compound
12 was selected as a typical keto form for the curcumin derivatives,
since enolization is not possible for this compound. As it was
difficult to obtain compounds fixed in the enol form, compound 7
was used as the typical enol form for the curcumin derivatives. This
compound exists almost exclusively in the enol form in methanol,
as mentioned above (Table 1). Therefore, the UV–visible spectrum
of compound 7 in 99.5% methanol was assumed to be representa-
tive of the enol form of curcumin derivative. The ionization
constants and UV–visible absorption spectra of compounds 7 and
12 in aqueous methanol are summarized in Table 2.
The apparent pH value of a methanol-containing solution is
slightly different from the true pH value of an aqueous solution
[19]. Nevertheless, the apparent pH value is consistent for solutions
that contain a certain amount of methanol, and it is used for
comparisons of acidity between solutions that contain the same
amount of methanol [19]. Even though it is difficult to compare the
apparent pKa value 8.1 of compound 7 with its true value in water,
it is important to know the pKa value in aqueous methanol so as to
obtain a typical spectrum for the enol form of the curcumin
derivatives.
The pKa values of compounds 7 and 12 in 99.5% methanol were
8.1 and 8.3, respectively (Table 2). Therefore, to obtain typical UV–
visible absorption spectra of the neutral species, we used
compound 12 in 99.5% methanol at pH 4.2 for the keto form and
compound 7 in 99.5% methanol at pH 4.5 for the enol form. To
obtain typical UV–visible absorption spectra for the anions, we
used compound 12 in 99.5% methanol at pH 10.8 for the keto form
and compound 7 in 99.5% methanol at pH 11.2 for the enol form.
The UV–visible absorption spectrum of compound 12 in 99.5%
methanol at pH 4.2 was analogous to that of compound 13, which is
an analogue of the neutral species of the keto form. This strongly
supports the notion that compound 12 in 99.5% methanol at pH 4.2
is typical of the neutral species of the keto form.
It is worth mentioning the colors of each of the four species of
compounds 7 and 12 in 99.5% methanol (Table 2 and Fig. 2).
Compound 7 showed a reddish-orange color in basic media (enol-
anion, lmax ¼ 482.5 nm, log 3 ¼ 4.79 M1 cm1) (Fig. 2e) and was
pale-yellowish in acidic media (enol-OH, lmax ¼ 407 nm,
log 3 ¼ 4.72 M1 cm1) (Fig. 2d). In contrast, compound 12 was
colorless in acidic media (keto-OH, lmax ¼ 326 nm,
log 3 ¼ 4.59 M1 cm1) (Fig. 2b) and yellowish in basic media (keto-
anion, lmax ¼ 396.5 nm, log 3 ¼ 4.64 M1 cm1) (Fig. 2c). The
combination of UV–visible spectra and the colors of the solutions of
the curcumin derivatives leads to the conclusion that only the anion
of the enol forms of the curcumin derivatives have a reddish color,
while the other species are colorless or yellowish in solution. Based
on these results, we hypothesize that the dramatic color change to
the reddish coloration (Fig. 1 and Supplementary Video 1) is due to
enolization and a subsequent increase in the level of the anion of
the enol form of the compound when it is bound to Ab aggregates.
3.4. Binding forms of curcumin derivatives
To test the above hypothesis, we estimated the proportions of
the four species of compound 9 before and after binding to Ab
aggregates using a linear least-squares fitting based on the typical
spectrum for each of the four species (Fig. 2). For this calculation,
we used the UV–visible spectrum of compound 7 in 99.5% meth-
anol as that of the enol form of compound 9. This assumption is
appropriate because the binding site on the aggregate should have
a hydrophobic environment. The results of the calculations are
listed in Fig. 3. From these data, we conclude that compound 9
exists predominantly in the keto form in the aqueous buffer (pH
7.5), and that this compound undergoes enolization when it binds
to the Ab aggregates.
4. Discussion
In order to bind to Ab aggregates, compounds need to be
coplanar and have a double-bond conjugation of certain length.
 D. Yanagisawa et al. / Biomaterials 31 (2010) 4179–4185 4183

a
Keto-OH Enol-OH
O O OH O

HO OH HO OH
OCF3 OCF3 OCF3 OCF3

Keto-anion Enol-anion
O O OH O

O- O- O- O-
OCF3 OCF3 OCF3 OCF3

b d
3 3
Absorbance

Absorbance
2 2

1 1

0 0
250 350 450 550 650 750 250 350 450 550 650 750
Wavelength (nm) Wavelength (nm)

c e
3 3
Absorbance

Absorbance

2 2

1 1

0 0
250 350 450 550 650 750 250 350 450 550 650 750
Wavelength (nm) Wavelength (nm)
Fig. 2. Typical UV–visible absorption spectra and colors of four species of curcumin derivatives. (a) Scheme for the equilibrium states of the four species of curcumin derivatives.
(b–e) Typical UV–visible absorption spectra and colors of the four species. UV–visible absorption spectra were determined for 40 mM of compounds 7 (d and e) and 12 (b and c) in
99.5% methanol that contained 0.15 mM HCl for the neutral species of the keto form (b) and the enol form (d), and in 99.5% methanol that contained 5 mM NaOH for the anions of the
keto form (c) and the enol form (e). The spectral data (lmax and log 3) values for the four species are listed in Table 2.

Reinke and Gestwicki outlined the optimal linker length and flex-
ibility (or rigidity) of two phenyl groups of curcumin for binding to
Ab aggregates, in what is termed the ‘‘Goldie-Locks model’’ [9]. The
enol forms of curcumin derivatives conform to these parameters
and fit this model, since in the enol form, the molecule retains
coplanarity and extends the double-bond conjugation through six-
membered hydrogen bonding at the center of curcumin. Therefore,
the enol form has strong binding activities for Ab aggregates, while
the keto form, in which the conjugation is broken and/or shortened,
has very low binding activities for Ab aggregates.
The ionization constants of the curcumin derivatives, 7 and 12
are much lower than those of curcumin (pKa 9.30 in 50% v/v
methanol [20]) owing to the electron-withdrawing effects of the
trifluoromethoxy groups on the benzene ring. Therefore, more than
half of the molecules of the curcumin derivatives with tri-
fluoromethoxy groups are expected to be ionized, even in buffers at
physiological pH (pH 7.5), for both tautomers (keto and enol forms).
Spectral analyses of the curcumin derivatives (Fig. 3) also showed
that more than 80% of compound 9 existed in the anion form in
50 mM phosphate buffer that contained 100 mM NaCl (pH 7.5).
Curcumin derivatives substituted at the C-4 position had lower
enol to keto ratios in aqueous solutions, and these solutions were
pale-yellow in color. However, the color of the solution changed
drastically to reddish-orange when the Ab aggregates were added
to the solution (Fig. 1 and Supplementary Video 1). We demon-
strate that this phenomenon is related to the predominance of the
enol form, as seen for compound 9 when it bound to the Ab
aggregates. The keto form of compound 9 in aqueous buffer
changed to the enol form when it bound to Ab aggregates that
were added to the solution, with the consequence that the keto
form was depleted. However, the color of the solution of
compound 9 did not change when mixed with the Ab monomer,
indicating that this compound does not bind to the monomeric
form of Ab. Thus, these phenomena are detected by changes in the
4184 D. Yanagisawa et al. / Biomaterials 31 (2010) 4179–4185

a b
0.8 Compound 9
Aβ

Absorbance
0.6 Aβ+Compound 9

0.4

0.2

0
250 350 450 550 650 750
Wavelength (nm)

Fig. 3. Proportions of the four species of compound 9 before and after binding to Ab aggregates. (a) UV–visible absorption spectrum of 20 mM of compound 9 in 50 mM phosphate buffer
(pH 7.5) that contained 100 mM NaCl, and UV–visible absorption spectrum of compound 9 after binding to Ab aggregates. There is no absorption peak in the visible region of the spectrum
of the Ab aggregates alone. (b) Estimations using a linear least-square fitting of the proportions of the four species of compound 9 before and after binding to the Ab aggregates.

coloration of the solutions. The binding activities of the curcumin Curcumin have been reported to cross the blood–brain barrier,
derivatives to Ab aggregates may be explained by the hydrophobic bind to amyloid plaques, block Ab fibrillation, and reduce amyloid
environment that is formed when aggregation takes place [21,22]. pathology in the amyloid precursor protein (APP)-transgenic
This environment should present functional groups that are suit- mouse, which is generally used as a model of AD [12]. Therefore,
able for hydrogen bonding to the phenolic hydroxyl group on the curcumin is a potential therapeutic drug for AD. In addition, cur-
benzene ring. Therefore, curcumin derivatives tend to be enolized cumin derivatives have been synthesized as positron emission
in this hydrophobic environment and bind to the aggregates tomography (PET) or near-infrared probes for in vivo amyloid
through hydrogen bond formation. However, curcumin derivatives imaging for the diagnosis of AD [10,23]. Although all of these
do not have similar binding activities for the Ab monomer, since studies report that curcumin and its derivatives exhibit high-
the monomeric form does not establish a similar hydrophobic affinity binding to Ab aggregates, it has not been elucidated how
environment. curcumin and its derivatives bind to Ab aggregates. In the present
Similar to compound 9, compound 8 having a methyl group at study, we reveal that the enol forms of curcumin derivatives are
the C-4 position, also exhibited a color change to reddish-orange when remarkably increased after binding to Ab aggregates, which
the Ab aggregates were added to the solution (see Supplementary suggests that the enol forms of the curcumin derivatives are the
Fig. 3). However, an aqueous solution of compound 7, which is a cur- predominant binding species. This finding provides a novel strategy
cumin derivative without a substituent at the C-4 position, retained its for the design of curcumin derivatives that target amyloid-binding
orange coloration even when it was mixed with the Ab aggregates, activities for use as therapeutic drugs or diagnostic tools in AD.
although the enol form should have been enhanced in the solution
that contained the Ab aggregates (see Supplementary Figs. 4 and 5).
5. Conclusion
This may be explained by the facts that a considerable amount of the
anion of the enol form already exists even in aqueous solutions
In the present report, we describe the relationship between the
without Ab aggregates, and that our eyes are not sufficiently sensitive
tautomeric structures of curcumin derivatives and their Ab-binding
to recognize a slight increase in orange-red coloration after the addi-
activities. Curcumin derivatives substituted at the C-4 position tend
tion of Ab aggregates.
to retain the keto form in polar media, such as aqueous solutions, and
have a pale-yellow color in solution. However, the color of this
Table 2 solution dramatically changes to reddish-orange when Ab aggre-
pKa values and UV–visible absorption spectra data for the curcumin derivatives. gates are added to the solution (no color change is observed
Compound Solvent pKa UV–vis. data
following the addition of Ab monomer). This color change is
attributed to the enolization of the b-diketone moiety of the cur-
lmax (nm) log 3 (M1 cm1) pH
cumin derivative, since the enol form of a curcumin derivative has
12 45.8% methanol 7.3 327 4.55 3.3
a reddish-orange color. The enol form of the derivative, which
393 4.62 11.6
12 65.9% methanol 7.7 327.5 4.59 3.4
should be present even in aqueous solutions albeit at low levels, is
395 4.64 11.9 bound to Ab aggregates, and the keto form changes to the enol form
12 99.5% methanol 8.3 326 4.59 4.2 with an equilibrium state between the keto and enol forms. The
396.5 4.64 10.8 percentage of the enol form increases, and eventually the enol form
13 99.5% methanol – 322 4.61 3.5
becomes the predominant form of curcumin in the solution. Our
322 4.61 10.9
7 99.5% methanol 8.1 407 4.72 4.5 findings suggest that the enolization of curcumin derivatives is
482.5 4.79 11.2 crucially involved in binding to Ab aggregates, and that curcumin
To obtain UV–visible spectra typical of the four species, we selected compound 12 as
derivatives exist predominantly in the enol form during binding to
a typical keto form and compound 7 as a typical enol form. For these compounds, the Ab aggregates. The keto-enol tautomerism of curcumin derivatives
UV–visible spectra at pH values sufficiently different from the pKa value (i.e., 2.5 pH may represent a novel target for the design of amyloid-binding
units above or below the pKa value) were defined as the typical spectra of the agents that can be used for both therapy and amyloid imaging in AD.
neutral species and anion. Compound 12 in 99.5% methanol that contained 0.15 mM
HCl (pH 4.2) was designated as the neutral species of the keto form, and compound
12 in 99.5% methanol that contained 5 mM NaOH (pH 10.8) was designated as the Acknowledgments
anion of the keto form. Similarly, a UV–visible absorption spectrum typical of the
neutral species was derived using compound 7 in 99.5% methanol that contained
0.15 mM HCl (pH 4.5), and the typical spectrum of the anion of the enol form was This study was supported by the JST Practical Application
obtained using compound 7 in 99.5% methanol that contained 5 mM NaOH (pH 11.2). Research Program.
 D. Yanagisawa et al. / Biomaterials 31 (2010) 4179–4185
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the on-line version, at doi:10.1016/j.biomaterials.2010.01.142.
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