Concise synthesis of catechin probes enabling analysis and imaging of EGCg

Article abstract of DOI:10.1039/c0cc03676e

A concise synthesis of APDOEGCg (3) was accomplished. Due to the reactivity of its amine group, the compound could be easily converted to the fluorescein probe 21 and immunogen probe 22 efficiently. We then demonstrated the usefulness of the probes for im

Full text of DOI:10.1039/c0cc03676e

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Concise synthesis of catechin probes enabling analysis and imaging
of EGCgw
Atsushi Yoshida, Yasuo Hirooka, Yusuke Sugata, Mariko Nitta, Tamiko Manabe,
Shunsuke Ido, Kouki Murakami, Repon Kumer Saha, Takashi Suzuki, Motohiro Ohshima,
Akira Yoshida, Kunihiko Itoh, Kosuke Shimizu, Naoto Oku, Takumi Furuta,z
Tomohiro Asakawa, Toshiyuki Wakimotoy and Toshiyuki Kan*
Received 6th September 2010, Accepted 15th November 2010
DOI: 10.1039/c0cc03676e
A concise synthesis of APDOEGCg (3) was accomplished. Due
to the reactivity of its amine group, the compound could be easily
converted to the fluorescein probe 21 and immunogen probe
22 efficiently. We then demonstrated the usefulness of the probes
for imaging studies and the generation of antibodies.
(–)-Epigallalocatechin gallate (EGCg) (1), which is a major
constituent of green tea extract,1 has received special attention
for its antitumor,2 antiviral,3 and other important bioactivities.3
Due to these promising bioactivities,4 EGCg and its derivatives
are expected to constitute lead compounds for drug development.
Thus, the imaging and/or analysis of the dynamics of 1 by
probe molecules will be essential for future drug development.
However, there are few reports on the preparation of
catechin probes.5 Although conversion of 1 to a probe
molecule would be an excellent strategy, the direct selective
incorporation of a probe unit into 1 has been difficult due to
the structural instabilities of 1 and the lack of appropriate
tethering functional groups. Over the course of our synthetic
investigations into 1,6 we found that the synthetic derivative 2
possessed a more potent anti-influenza infection activity than
natural 1.6a Inspired by this finding, we began a synthesis of
the EGCg probe precursor 3 (6-(5-aminopentyl)-5,7-deoxy-
epigallocatechin gallate: APDOEGCg), which contains linkers
and reactive amino groups as shown in Fig. 1.7 In this
communication we report on the synthesis of probe precursor
3 and its conversion to catechin probe molecules 21 and 22.
Our retrosynthetic analysis for 3 is described in Scheme 1. In
our preliminary investigation into the incorporation of a linker
unit in the DOEGCg derivatives, we attempted a Suzuki–
Miyaura coupling8 of the catechin skeletons with and without
Fig. 1 Structures of EGCg (1), DOEGCg (2), APDOEGCg (3) and
APDOGCg (4).
Scheme 1 Our synthetic strategy for the EGCg probe precursor 3.
the Mitsunobu reaction with our Ns amide (2-nitrobenzene-
sulfonamide)9,10 under neutral reaction conditions. The
separation of cis and trans isomers of the dihydrobenzopyran
ring was readily accomplished by incorporating the gallate
unit,6b and the dihydrobenzopyran ring of 5 was constructed
by cyclization under acidic conditions from the diol 6 through
the cationic intermediate.
As shown in Scheme 2,11 condensations of the A- and B-ring
were accomplished by the Julia–Kocien
´
ski reaction12 between
a
gallate ester, but we could not produce the target
compounds. We decided to incorporate the linker unit into a
cyclization precursor 7a, which was prepared by a condensation
reaction of 8 and 9. Incorporation of a reactive amino group at
the terminal position of the linker was found to be suitable for
a phenyltetrazole (PT)-sulfone 8 and an aldehyde 9 to provide
7a as a single isomer in 87% yield. The observed Z-selective
reactivity13 was similar to that reported in a previous synthesis.6b
The subsequent incorporation of a linker group into 7a was
performed by the Suzuki–Miyaura coupling reaction.8 After
hydroboration of MOM-protected 4-pentenol 10 with 9-BBN,
alkyl borate 11 was subjected to cross-coupling reactions
without purification. Upon treatment of a mixture of borate
11 and 7a with catalytic quantities of PdCl2(dppf) and NaOH
in THF, the coupling reaction proceeded smoothly to give 12
in high yields. After simultaneous deprotection of the MOM
and TBS ether, incorporation of the amino group into 13 was
accomplished by the Mitsunobu reaction14,15 using Cbz and
School of Pharmaceutical Sciences, University of Shizuoka and Global
COE Program, 52-1 Yada, Suruga-ku, Shizuoka-shi,
Shizuoka 422-8526, Japan. E-mail: kant@u-shizuoka-ken.ac.jp;
Fax: +81 (0)54-264-5746; Tel: +81 (0)54-264-5745
w Electronic supplementary information (ESI) available: Experimental
Procedures and NMR spectral data. See DOI: 10.1039/c0cc03676e
z Present address: Institute for Chemical Research, Kyoto University,
Uji, Kyoto 611-0011, Japan.
y Present address: Graduate School of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
c
1794 Chem. Commun., 2011, 47, 1794–1796
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Scheme 3 Conversion of 3 to the fluorescein probe 21.
the original compound. Contrary to expectations, the
sustained activities of 3 and 4 demonstrated that incorporation
of probe units and/or tags into 3 and 4 via terminal amino
groups did not result in the loss of biological activity.5d
Encouraged by these results, we turned our attention to the
preparation of the probe molecules from precursor 3. Reactive
amine 3 possessed an advantage for the incorporation of a
probe unit furnished without the need for protection of the
phenolic hydroxy groups. A preliminary study of probe
molecule preparation was demonstrated by the synthesis of
the fluorescein probe, which permitted in vivo imaging under
physiological conditions. Among several fluorescein variants,
we selected a reliable photophore, Tokyogreen (TG).21 As
shown in Scheme 3, a condensation of the probe precursor 3
and TG activated ester 2011 provided the fluorescein probe
21.11
Once the desired probe 2122 had been synthesized, its
usefulness for imaging studies was assessed using HUVECs
(human umbilical vein endothelial cells).23 After incubation of
21 with HUVECs for 3 h, the fluorescence of 21 was imaged
under a fluorescence microscope. As shown in Fig. 2, the
strong fluorescence observed in the cells indicated that the
fluorescence probe 21 will be useful for elucidating the
dynamics of EGCg (1) cellular uptake, intracellular transport,
and metabolism. Our group is currently undertaking further
fluorescence imaging studies.
Scheme 2 Synthesis of EGCg probe precursors 3. Reagents and
conditions: (a) LHMDS, 9, THF, 0 1C, 87%; (b) 9-BBN, THF,
50 1C; (c) 11, PdCl2(dppf)ꢀCH2Cl2, 3 M NaOH aq., THF, reflux;
(d) conc. HCl, MeOH, 60 1C, 89%; (e) NsNHCbz, DMEAD, PPh3,
toluene, 72%; (f) OsO4, NMO, acetone/H2O, 72%; (g) TsOHꢀH2O,
toluene, 60 1C, 89%; (h) EDCI, DMAP, toluene, 95%; (i) PhSH,
Cs2CO3, MeCN, 92%; (j) Separation; (k) Pd(OH)2, H2, THF/MeOH,
89%. TBS = tert-butyldimethylsilyl, PT = phenyltetrazole; MOM =
methoxymethyl, Cbz = carboxybenzyl; Ns = o-nitrobenzenesulfonyl,
Bn = benzyl, LHMDS = lithiumhexamethyl disilazane, 9-BBN =
9-borabicyclo[3.3.1]nonane, dppf = 1,10-bis (diphenylphosphino)-
ferrocene, DMEAD = di-2-methoxyethyl-azodicarboxylate, Ts =
p-toluenesulfonyl, EDCI
=
1-ethyl-3-(3-dimethyl-aminopropyl)-
carbodiimide, DMAP = 4-dimethylaminopyridine.
Ns-amide9 (Ns strategy).10 A racemic mixture of 3 and 4 was
prepared by dihydroxylation of the cis-olefin 14 with OsO4
and NMO to give the diol 15.16 Upon treatment of 15 with
TsOH, the regioselective cyclization reaction proceeded
smoothly to provide the desired dihydrobenzopyran 16 as a
1 : 1 mixture.17 After incorporation of the gallate derivative
17, deprotection of the Ns groups of 18 was performed by
treatment with a thiol and base.18 Separation of 19a and 19b19
was readily accomplished by silica gel column chromatography.
Finally, deprotection of all benzyl and Cbz groups under
hydrogenolysis conditions afforded 3 and 4 from 19b and
19a, respectively.
Next we focused on the generation of antibodies specific to
EGCg, which would be useful for immunological detection of
subcellular and tissue localization. Furthermore, enzyme-
linked immunosorbent assays (ELISA) with color or fluorescence
endpoints would be useful for quantitating trace amounts of
EGCg in serum. The probe precursor 3 was conjugated to a
hapten, which enabled the generation of EGCg antibodies.24,25
As shown in Scheme 4, conjugation of 3 to the carrier protein
With the desired EGCg derivatives in hand, we evaluated
the inhibitory activities of 3 and 4 against influenza virus
infection. As shown in Table 1, APDOEGCg (3) and
APDOGCg (4) showed potent inhibition of the infectivity of
the influenza virus A/Memphis/1/71 (H3N2) toward MDCK
cells, yielding IC50 values of 4.18 and 4.40 mM, respectively.11
These values were higher than those of the natural 1 and
synthetic 2.20 The biological activity of a molecule usually
decreases when a probe unit and/or linker group is attached to
Table 1 Inhibition of influenza A viral infectivity toward
MDCK cells
Compound
Complement inhibition IC50a/mM
EGCg (1)
66.3 (ꢁ9.21)
9.05 (ꢁ2.26)
4.18 (ꢁ4.29)
4.40 (ꢁ2.36)
DOEGCg (2)
APDOEGCg (3)
APDOGCg (4)
a
Values are reported as the mean of three experiments, and the
standard deviation is given in parentheses.
Fig.
2
Fluorescence microscopy image of HUVECs incubated
with 21.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 1794–1796 1795

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I. Nakanishi, K. Ohkubo, Y. Obara, A. Tada, K. Imai, A.
Ohno, A. Nakamura, T. Ozawa, S. Urano, S. Saito,
S. Fukuzumi, K. Anzai, N. Miyata and H. Okuda, Chem. Commun.,
2009, 6180.
6 (a) T. Furuta, Y. Hirooka, A. Abe, Y. Sugata, M. Ueda,
K. Murakami, T. Suzuki, K. Tanaka and T. Kan, Bioorg. Med.
Chem. Lett., 2007, 17, 3095; (b) Y. Hirooka, M. Nitta, T. Furuta
and T. Kan, Synlett, 2008, 3234; (c) Y. Aihara, A. Yoshida,
T. Furuta, T. Wakimoto, T. Akizawa, M. Konishi and T. Kan,
Bioorg. Med. Chem. Lett., 2009, 19, 4171.
7 For our investigation of amide bond-mediated conjugation of
ligand molecules and probe units, see: (a) T. Kan, Y. Kita,
Y. Morohashi, Y. Tominari, S. Hosoda, H. Natsugari,
T. Tomita, T. Iwatsubo and T. Fukuyama, Org. Lett., 2007, 9,
2055; (b) T. Kan, Y. Tominari, Y. Morohashi, H. Natsugari,
T. Tomita, T. Iwatsubo and T. Fukuyama, Chem. Commun.,
2003, 2244.
8 For a review of the Suzuki–Miyaura coupling reaction, see:
N. Miyaura and A. Suzuki, Chem. Rev., 1995, 95, 2457.
9 T. Fukuyama, M. Cheung and T. Kan, Synlett, 1999, 1301.
10 For a review of Ns-strategy, see: (a) T. Kan and T. Fukuyama,
J. Synth. Org. Chem. Jpn., 2001, 59, 779; (b) T. Kan and
T. Fukuyama, Chem. Commun., 2004, 353.
11 Detailed experimental procedures and spectral data are described
in the ESIw.
´
12 (a) P. R. Blakemore, W. J. Cole, P. J. Kocienski and A. Morley,
Synlett, 1998, 26; (b) For a review of the modified Julia reaction,
see: P. R. Blakemore, J. Chem. Soc., Perkin Trans. 1, 2002, 2563;
(c) For Z-selective Julia olefination, see: M.-E. Lebrun,
P. L. Marquand and C. Berthelette, J. Org. Chem., 2006, 71, 2009.
13 A dramatic reversal in stereochemistry was observed by changing
the combination of sulfone and aldehyde; see ESIw.
Scheme 4 Conjugation of 3 to the HSA carrier protein.
(HSA: human serum albumin) was performed by using
glutaraldehyde as a cross-linker26 to give 22. The immunogen
22 was mixed in a saline solution with Freund’s complete
adjuvant and was injected into mice. After several weeks, the
mice were sacrificed, and the venous blood was collected. Sera
were separated by centrifugation and used for subsequent
experiments.11
In summary, we have developed a novel EGCg derivative
that includes terminal amino groups without loss of activity.
The introduced amino groups were useful for the development
of a variety of probe molecules. The fluorescein probes and
antibodies show promise for visualizing localization on the
cellular and organ scale, respectively. These probe molecules
are promising tools for investigations into the localization and
target sites of EGCg.
This work was financially supported by a project of the
Shizuoka Prefecture and Shizuoka City Collaboration of
Regional Entities for the Advancement of Technological
Excellence, the Japan Science and Technology Agency (JST),
and a Grant-in-Aid for Scientific Research on Priority from
the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan. We also thank Professors
Tsutomu Nakayama, Masahiko Hara, and Takeshi Ishii
(Department of Food and Nutritional Science, University of
Shizuoka) for valuable discussion.
14 For a review of Mitsunobu reactions, see: (a) O. Mitsunobu,
Synthesis, 1981, 1; (b) D. L. Hughes, Org. React., 1992, 42, 335.
15 For a DMEAD mediated Mitsunobu reaction, seeT. Sugimura and
K. Hagiya, Chem. Lett., 2007, 36, 566.
16 Optically active 15 was obtained by employment with an AD-mix
oxidation of 14: see ESIw.
17 Recently, we have developed a stereoselective synthetic method for
cis and trans dihydrobenzopyrane ring, respectively, by utilizing a
similar participation of benzyl cation with a gallate carbonyl
group: see ESIw.
18 (a) W. Kurosawa, T. Kan and T. Fukuyama, Organic Synthesis,
Wiley, New York, 2002, Vol. X, p. 482; (b) T. Fukuyama,
C.-K. Jow and M. Cheung, Tetrahedron Lett., 1995, 36, 6373.
19 For a detailed explanation of the selectivity see ESIw.
20 For a similar enhancement of anti-influenza activity by incorporation
of fatty acid into EGCg, see: S. Mori, S. Miyake, T. Kobe,
T. Nakaya, S. D. Fuller, N. Kato and K. Kaihatsu, Bioorg. Med.
Chem. Lett., 2008, 18, 4249.
Notes and references
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22 The purpose of fluorescence-labeling is to investigate the trafficking
of EGCg. Although a significant decrease in the inhibitory activity
of 21 towards influenza virus infection was observed, we confirmed
that incorporation of TG into APDOEGCg (3) did not affect the
intracellular trafficking in HUVECs. The detailed results of this
experiment, see in S. Piyaviriyakul, K. Shimizu, T. Asakawa,
T. Kan, P. Siripong and N. Oku, Biol. Pharm. Bull., in press.
23 S. Yamakawa, T. Asai, T. Uchida, M. Matsukawa, T. Akizawa
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c
1796 Chem. Commun., 2011, 47, 1794–1796
This journal is The Royal Society of Chemistry 2011