Enhanced anti-influenza A virus activity of (-)-epigallocatechin-3-O-gallate fatty acid monoester derivatives: Effect of alkyl chain length

Article abstract of DOI:10.1016/j.bmcl.2008.02.020

A series of fatty acid monoester derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) were prepared by one-pot lipase-catalyzed transesterification. The introduction of long alkyl chains enhanced anti-influenza A/PR8/34 (H1N1) virus activity 24-fold relative to native EGCG.

Full text of DOI:10.1016/j.bmcl.2008.02.020

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 4249–4252
Enhanced anti-influenza A virus activity of (ꢀ)-epigallocatechin-
3-O-gallate fatty acid monoester derivatives:
Effect of alkyl chain length
Shuichi Mori,a Shinya Miyake,a Takayoshi Kobe,a Takaaki Nakaya,b
Stephen D. Fuller,c Nobuo Katoa and Kunihiro Kaihatsua,*
aThe Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
bResearch Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
cDivision of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive,
Oxford OX3 7BN, UK
Received 20 September 2007; revised 1 February 2008; accepted 7 February 2008
Available online 10 February 2008
Abstract—A series of fatty acid monoester derivatives of (ꢀ)-epigallocatechin-3-O-gallate (EGCG) were prepared by one-pot lipase-
catalyzed transesterification. The introduction of long alkyl chains enhanced anti-influenza A/PR8/34 (H1N1) virus activity 24-fold
relative to native EGCG.
ꢀ 2008 Elsevier Ltd. All rights reserved.
A major polyphenol component of green tea, (ꢀ)-epigal-
OR
2'
OR
OR
locatechin-3-O-gallate (EGCG; 1), has received much
attention due to its various biological activities such as
antiviral,1 antimicrobial,2 and anticancer.3 In particular,
the anti-influenza activity of 1 has been investigated by
several groups;1a–c the results show that 1 has the most
potent antiviral activity among tea polyphenols. How-
ever, relatively high concentrations of 1 were required
to observe significant antiviral activity, probably due
to the compound’s poor lipid membrane permeability,4
low chemical stability,5 and rapid metabolism.5
B
8
2
3
RO
O
6'
C
A
6
O
4
2''
OR
OR
OR
1 : R = H
2 : R = Ac
O
D
6''
OR
Figure 1. Structure of EGCG (1) and EGCG-peracetate (2).
We hypothesized that the introduction of straight-chain
fatty acids to the phenolic hydroxyl groups of 1 would
further enhance its anti-influenza virus activity.
Several strategies for increasing these biological proper-
ties have been investigated. For example, the introduc-
tion of alcoxyl groups to 1 improved lipid-membrane
permeability4 and metabolic stability,6 and peracetyla-
tion of 1 (Fig. 1) increased its chemical stability under
physiological conditions.7 Furthermore, the elimination
of hydroxyl groups from the A-ring of 1 moderately en-
hanced anti-influenza virus activity.8
A series of EGCG fatty acid monoester derivatives (3–7)
were prepared by lipase-catalyzed transesterification
(Scheme 1). EGCG-monoesters modified with butanoyl,
octanoyl, lauroyl, palmitoyl, and eicosanoyl groups are
represented as EGCG-C4, EGCG-C8, EGCG-C12,
EGCG-C16, and EGCG-C20, respectively. Although 1
has been reported to interfere with the catalytic activity
of lipases,9 we succeeded in preparing 3–7 by lipase-cat-
alyzed transesterification in polar organic solvents such
as N,N-dimethylformamide or acetonitrile.10,11 In con-
trast, the transesterification did not proceed in nonpolar
organic solvents such as tetrahydrofuran or diisopropy-
lether. The key to success with this approach might be
Keywords: Epigallocatechin gallate; Influenza virus; Lipase-catalyzed
esterification; Antivirus agent.
*
Corresponding author. Tel.: +81 6 6879 8471; fax: +81 6 6879
8474; e-mail: kunihiro@sanken.osaka-u.ac.jp
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.020

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S. Mori et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4249–4252
OH
OR1
OR2
OH
OH
HO
O
HO
O
Vinyl acylate
OH
O
O
Lipase
-Dimethylformamide
OH
OH
OH
OH
OR3
N,
N
O
O
57 ºC
1
OH
OH
OR4
3a : R2=R3=R4=H, R1=CO(CH)2CH3
3b : R1=R3=R4=H, R2=CO(CH)2CH3
3c : R1=R2=R4=H, R3=CO(CH)2CH3
3d : R1=R2=R3=H, R4=CO(CH)2CH3
5a : R2=R3=R4=H, R1=CO(CH)10CH3
5b : R1=R3=R4=H, R2=CO(CH)10CH3
5c : R1=R2=R4=H, R3=CO(CH)10CH3
5d : R1=R2=R3=H, R4=CO(CH)10CH3
7a : R2=R3=R4=H, R1=CO(CH)18CH3
7b : R1=R3=R4=H, R2=CO(CH)18CH3
7c : R1=R2=R4=H, R3=CO(CH)18CH3
7d : R1=R2=R3=H, R4=CO(CH)18CH3
4a : R2=R3=R4=H, R1=CO(CH)6CH3
4b : R1=R3=R4=H, R2=CO(CH)6CH3
4c : R1=R2=R4=H, R3=CO(CH)6CH3
4d : R1=R2=R3=H, R4=CO(CH)6CH3
6a : R2=R3=R4=H, R1=CO(CH)14CH3
6b : R1=R3=R4=H, R2=CO(CH)14CH3
6c : R1=R2=R4=H, R3=CO(CH)14CH3
6d : R1=R2=R3=H, R4=CO(CH)14CH3
Scheme 1. Preparation of EGCG monoester derivatives by lipase-catalyzed transesterifications.
enhancement of the conformational flexibility of the
lipase and elimination of nonspecific interactions
between the lipase and 1 in polar organic solvents.
Our lipase method affords 3–7 in 35–39% yield, whereas
a conventional chemical method12 affords them in less
than 23% yield. As shown in Scheme 1, EGCG-monoes-
ters (3–7) are composed of a mixture of four regioisom-
ers, each with acyl groups at either the meta-position or
para-position of the B- or D-ring. We confirmed the
ratio of each EGCG-monoester regioisomer by 1H
NMR spectroscopy; the results are summarized in Table
1. Interestingly, this lipase-catalyzed method afforded
B-ring modified esters as the major products, whereas
the conventional chemical method afforded D-ring mod-
ified esters as the major products. The purification of
each regioisomer was not accomplished as they have
very similar chemical properties. However, the lipase
method afforded EGCG-monoesters in a consistent
ratio of regioisomers, regardless of the alkyl chain length
(Table 1).
All compounds (1–7) inhibited virus infection in a dose-
dependent manner (Fig. 2). The EC50 values of each
compound are summarized in Table 2 (cell-based antivi-
ral effect) and show that the antiviral activities of
EGCG-monoesters were enhanced in an alkyl chain
length-dependent manner. In particular, the EC50 of
EGCG-C16 was approximately 4 lM and its inhibitory
effect was 24-fold higher than 1. This remarkable
enhancement in antiviral activity can be attributed to
the high efficiency of cellar uptake of EGCG-C16 as a
result of its improved cell membrane permeability. In
contrast, EGCG-C20 and EGCG-peracetate exhibited
lower antiviral activities compared to EGCG-C16, pre-
sumably due to decreased water solubility resulting from
their increased hydrophobicity.
The antiviral activity of EGCG-monoesters may vary
depending on the position of the acyl groups. Therefore
we prepared mixtures of EGCG-monopalmitate, com-
posed of four regioisomers (6a–d) in different propor-
tions, by both the lipase-catalyzed (EGCG-C16) and
We investigated the antiviral protective effects of 1,
EGCG-peracetate (2),13 and EGCG-monoesters (3–7)
against influenza A/PR8/34 (H1N1) infection in MDCK
cells. Briefly, a monolayer of MDCK cells was transfec-
ted with 1–7 2 h prior to infection with the virus. The
cell monolayer was rinsed to remove remaining EGCG
derivative in the cell culture medium, then the virus
was introduced. The antiviral activities of each sample
were assessed by the plaque formation assay.14
100
EGCG
EGCG-C4
80
EGCG-C8
EGCG-C12
EGCG-C16
60
EGCG-C20
40
20
0
Table 1. Mixture ratio of regioisomers in EGCG monoester deriva-
tives prepared by lipase-catalyzed transesterifications
Compound
Regioisomer ratio
B/Da
EGCG-C4
EGCG-C8
3a:3b:3c:3d
35:44:8:12
35:39:6:20
30:39:9:22
38:35:7:20
38:36:8:19
20:18:18:44
79/21
74/26
69/31
73/27
73/27
38/62
4a:4b:4c:4d
5a:5b:5c:5d
6a:6b:6c:6d
7a:7b:7c:7d
6a:6b:6c:6d
0
50
100
150
EGCG-C12
EGCG-C16
EGCG-C20
EGCG-C16Chem
Concentration of EGCG derivative (μM)
Figure 2. Inhibitory effects of EGCG monoester derivatives prepared
by lipase-catalyzed transesterification on plaque formation in the cell
based inhibition assay. Each data point represents the mean SD
from at least three independent experiments.
b
a Ratio of B- and D-ring modified EGCG monoester derivatives.
b Prepared by a conventional chemical method.

S. Mori et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4249–4252
Table 2. Anti-influenza effects and cytotoxicities of EGCG derivatives
4251
a
Entry
CC50 (lM)
Cell-based antiviral effect
Direct antiviral effect
b
b
EC50 (lM)
SIc
EC50 (lM)
SIc
EGCG
275.0 ( 6.0)
309.0 ( 4.0)
195.0 ( 9.0)
42.0 ( 3.9)
86.2 ( 12.5)
318.0 ( 55.0)
NDd
94.60 ( 11.10)
63.70 ( 10.10)
39.00 ( 3.10)
5.81 ( 0.87)
2.91
4.85
5.01
7.23
21.40
4.75
—
0.3910 ( 0.0560)
0.8320 ( 0.0910)
0.6200 ( 0.0910)
0.1180 ( 0.0230)
0.0204 ( 0.0069)
2.2500 ( 0.1900)
8.4900 ( 3.3500)
703
363
276
353
4230
127
—
EGCG-C4
EGCG-C8
EGCG-C12
EGCG-C16
EGCG-C20
EGCG-peracetate
4.02 ( 0.48)
66.90 ( 8.30)
77.00 ( 12.60)
a CC50 represents the concentration of compound required to reduce cell viability by 50% relative to the control well without test compound.
b EC50 represents the concentration of compound required to reduce plaque number by 50% relative to the control well without test compound.
c SI (Selectivity index) is the ratio of CC50 to EC50
.
d CC50 of EGCG-peracetate was not determined due to its low water solubility.
conventional chemical methods (EGCG-C16Chem). The
antiviral activities of these two sets of products were
examined. However, no distinct difference in antiviral
Acknowledgment
This study was supported by a Grant for Industrial
Technology Research (Financial support to young
researchers), from the New Energy and Industrial Tech-
nology Development Organization (NEDO).
activity was apparent (EC50 of EGCG-C16Chem
=
4.12 lM). This result suggested that the position of the
acyl groups of EGCG derivatives does not affect their
antiviral activities.
References and notes
The cytotoxicities of each EGCG derivative to MDCK
cells, given as CC50, are summarized in Table 2.15
EGCG-C12 and EGCG-C16 exhibited relatively higher
cytotoxicities than other derivatives. However, their
antiviral activities were significantly enhanced and there-
by resulted in increased selectivity index values.
1. For example, see: (a) Song, J.-M.; Lee, K.-H.; Seong,
B.-L. Antiviral Res. 2005, 68, 66; (b) Imanishi, N.; Tuji,
Y.; Katada, Y.; Maruhashi, M.; Konosu, S.; Mantani,
N.; Terasawa, K.; Ochiai, H. Microbiol. Immunol. 2002,
46, 491; (c) Nakayama, M.; Suzuki, K.; Toda, M.;
Okubo, S.; Hara, Y.; Shimamura, T. Antiviral Res.
1993, 21, 289; (d) Yamaguchi, K.; Honda, M.; Ikigai,
H.; Hara, Y.; Shimamura, T. Antiviral Res. 2002, 53, 19.
2. For example, see: (a) Taguri, T.; Tanaka, T.; Kouno, I. Biol.
Pharm. Bull. 2006, 29, 2226; (b) Yoda, Y.; Hu, Z.-Q.; Zhao,
W.-H.; Shimamura, T. J. Infect. Chemother. 2004, 10, 55.
3. For example, see: (a) Chen, D.; Daniel, K. G.; Kuhn, D.
J.; Kazi, A.; Bhuiyan, M.; Li, L.; Wang, Z.; Wan, S. B.;
Lam, W. H.; Chan, T. H.; Dou, Q. P. Front. Biosci. 2004,
9, 2618; (b) Ahn, W. S.; Huh, S. W.; Bae, S. M.; Lee, I. P.;
Lee, J. M.; Namkoong, S. E.; Kim, C. K.; Sin, J. I. DNA
Cell Biol. 2003, 22, 217.
4. Tanaka, T.; Kusano, R.; Kouno, I. Bioorg. Med. Chem.
Lett. 1998, 8, 1801.
5. Hong, J.; Lu, H.; Meng, X.; Ryu, J.-H.; Hara, Y.; Yang,
C. S. Cancer Res. 2002, 62, 7241.
6. Kida, K.; Suzuki, M.; Matsumoto, N.; Nanjo, F.; Hara,
Y. J. Agric. Food Chem. 2000, 48, 4151.
7. Lam, W. H.; Kazi, A.; Kuhn, D. J.; Chow, L. M. C.;
Chan, A. S. C.; Dou, Q. P.; Chan, T. H. Bioorg. Med.
Chem. 2004, 12, 5587.
In addition, we studied the direct interaction between
EGCG derivatives and influenza viral particles.16 Each
EGCG derivative was incubated with influenza virus
solution for 30 min prior to infection, followed by the
post-inoculation procedure described above. All com-
pounds inhibited virus infection at much lower concen-
trations compared to the CC50 and the EC50 obtained
from the cell-based antiviral assay (Table 2, direct anti-
viral effect). Interestingly, EGCG-C12 and -C16 also
exhibited remarkable enhancement of their direct anti-
viral effect in a manner similar to their cell-based anti-
viral effect. To clarify if these enhanced antiviral effects
were simply brought about by their detergent effects,
we investigated the antiviral effect of conventional
non-ionic detergents, n-dodecyl-b-D-maltoside and sor-
bitan monopalmitate. However, both detergents did
not exhibit apparent direct antiviral effects up to
10 lM (data not shown). A possible mechanism to ex-
plain the enhanced antiviral effect of EGCG derivatives
is that the acyl portion increases the accessibility of
EGCG to the viral membrane as well as the cell
membrane.
8. Furuta, T.; Hirooka, Y.; Abe, A.; Sugata, Y.; Ueda, M.;
Murakami, K.; Suzuki, T.; Tanaka, K.; Kan, T. Bioorg.
Med. Chem. Lett. 2007, 17, 3095.
9. For example, see: (a) Nakai, M.; Fukui, Y.; Asami, S.;
Toyoda-Ono, Y.; Iwashita, T.; Shibata, H.; Mitsunaga, T.;
Hashimoto, F.; Kiso, Y. J. Agric. Food Chem. 2005, 53,
4593; (b) Ikeda, I.; Tsuda, K.; Suzuki, Y.; Kobayashi, M.;
Unno, T.; Tomoyori, H.; Goto, H.; Kawata, Y.; Imaiz-
umi, K.; Nozawa, A.; Kakuda, T. J. Nutr. 2005, 135, 155;
(c) Juhel, C.; Armand, M.; Pafumi, Y.; Rosier, C.;
Vandermander, J.; Lairon, D. J. Nutr. Biochem. 2000,
11, 45.
In conclusion, we prepared a series of EGCG fatty acid
monoester derivatives using lipase-catalyzed transesteri-
fication and demonstrated that the introduction of long
acyl groups, such as lauroyl or palmitoyl, to EGCG
drastically enhanced its anti-influenza virus activity.
Our simple and robust methodology should expand
the utility of EGCG, an abundant natural tea ingredi-
ent, as a novel anti-influenza agent.
10. Synthesis of EGCG monoester derivatives by lipase-
catalyzed transesterification: as an example, experimental

4252
S. Mori et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4249–4252
procedure for synthesis of EGCG-C16 is described here.
14. Evaluation of cell-based antiviral inhibitory effects of EGCG
derivatives on influenza A/PR8/34 (H1N1): Opti-MEM
containing 0.2% dimethyl sulfoxide and each sample at
different concentration was added to a confluent mono-
layer Madin–Darby canine kidney (MDCK) cells in a 6-
well plate, followed by incubation for 2 h at 37 ꢁC in 5%
CO2. Then, after the solution was removed from each well,
the cell sheets were washed by D-PBS and infected with
influenza virus (MOI = 2.5 · 10ꢀ4) in DMEM containing
0.2% BSA. After 1 h for virus adsorption at room
temperature, the solution was removed and the cell sheets
were washed twice with D-PBS. Then, overlay medium
Lipase PL from Alcaligenes sp. (500 mg) was added to
100 mL N,N-dimethylformamide solution including 1 (1 g,
2.18 mmol) and vinyl palmitate (0.925 g, 3.27 mmol). This
mixture was stirred for 1.5 h at 57 ꢁC. After filtration of
reaction mixture, the solvent was removed by vacuum
pump and the residue was purified by silica gel column
chromatography to give the mixture of EGCG-mono-
1
palmitate regioisomers (EGCG-C16). H NMR (400 MHz,
CD3CN)
d 0.88 (3H, t, J = 6.8 Hz, –COCH2CH2
(CH2)12CH3 of 6a–6d), 1.27 (26H, m, –COCH2CH2
(CH2)12CH3 of 6a–6d), 1.67 (2H, tt, J = 14.6, 7.9 Hz,
–COCH2CH2(CH2)12CH3 of 6a–6d), 2.58 (2H, m,
–COCH2CH2(CH2)12CH3 of 6a–6d), 2.82 (1H, m, H-4 of
6a–6d), 2.97 (1H, m, H-4 of 6a–6d), 5.03 (1H, m, H-2 of
6a–6d), 5.49 (1H, m, H-3 of 6a–6d), 5.98 (2H, m, H-6 and
H-8 of 6a–6d), 6.49 (0.4H, s, 20 and 60 of 6d), 6.50 (0.14H,
s, 20 and 60 of 6c), 6.56 (0.7H, s, 20 and 60 of 6b), 6.74
(0.38H, d, J = 2.04 Hz, 20 or 60 of 6a), 6.82 (0.38H, d,
J = 1.96 Hz, 20 or 60 of 6a), 6.82–6.6.70 (7H, br s, –OH),
6.88 (0.76H, s, 200 and 600 of 6a), 6.90 (0.7H, s, 200 and 600 of
6b), 6.92 (0.14H, s, 200 and 600 of 6c), 7.09 (0.2H, d,
J = 1.96 Hz, 200 or 600 of 6d), 7.21 (0.2H, d, J = 2.00 Hz, 200
or 600 of 6d); ESI-MS m/z 719 C38H48O12Na (M+Na)+.
(DMEM containing 0.8% Oxoid agar No. 1, 6.0 · 10ꢀ4
%
trypsin, and 0.2% BSA) was added to each well. After
incubating for about 2 days at 37 ꢁC in 5% CO2, the cell
sheets were fixed with 5% glutaraldehyde and the overlay
agarose was removed. Then, the cell sheets were stained
with methylene blue solution. Plaques formed on each well
were counted and the inhibitory effects of each sample on
virus infection were evaluated.
15. Evaluation of cytotoxicities of EGCG derivatives on MDCK
cells: MDCK cells (1.5 · 104 cells/well) were cultured in a
96-well plate for 4–5 h. Opti-MEM containing each
sample at different concentration was added to each well
and the cell cultures were incubated for 2 h. After sample
solutions were removed and the cell cultures were washed
with D-PBS, DMEM containing 0.2% BSA was added to
each well. The cell cultures were incubated for 24 h at
37 ꢁC in 5% CO2. Then, after the solution was removed
and the cell cultures were washed with D-PBS, 100 lL
MTT solution was added to each well. After incubation
for 1 h, the optical densities at 490 nm of each well were
measured by 96-well plate reader.
16. Evaluation of direct antiviral inhibitory effects of EGCG
derivatives on influenza A/PR8/34 (H1N1): Each EGCG
derivative was mixed with influenza virus in Opti-MEM
containing 0.2% DMSO. After incubating for 30 min at
room temperature, the mixed solution was applied to
confluent monolayer MDCK cells in a 6-well plate
(MOI = 2.5 · 10ꢀ4). The following procedure was the
same as the cell-based inhibition assay described above.
11. Kaihatsu, K.; Kobe, T.; Fuller, S. D.; Kato, N. PCT Int.
Appl. 304788, 2006.
12. Synthesis of EGCG-C16 by a conventional chemical
method: Palmitoyl chloride (216 mg, 0.785 mmol) was
dropped in 50 mL tetrahydrofuran including 1 (300 mg,
0.654 mmol) and triethylamine (0.850 mmol). Reaction
mixture was stirred for 24 h at 0 ꢁC to room temperature.
After removed the solvent, the residue was purified by
silica gel column chromatography to give the mixture of
EGCG-monopalmitate regioisomers (EGCG-C16).
13. Synthesis of 2: EGCG-peracetate (2) was synthesized by
pyridine-catalyzed acetylation of EGCG using acetic
anhydride according to the following literature: Kohri,
T.; Nanjo, F.; Suzuki, M.; Seto, R.; Matsumoto, N.;
Yamakawa, M.; Hojo, H.; Hara, Y.; Desai, D.; Amin, S.;
Conaway, C. C.; Chung, F.-L. J. Agric. Food Chem. 2001,
49, 1042.