Regioselective hydrolysis of pentaacetyl catechin and epicatechin by porcine liver esterase

Article abstract of DOI:10.1016/S0960-894X(03)00024-6

3,5,7,3′,4′-Pentaacetyl catechin was selectively hydrolyzed to either 3,7,3′,4′-tetraacetyl catechin or 3-acetyl catechin depending upon the duration of hydrolysis. A similar result was also obtained in the epicatechin series.

Full text of DOI:10.1016/S0960-894X(03)00024-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1083–1085
Regioselective Hydrolysis of Pentaacetyl Catechin and Epicatechin
by Porcine Liver Esterase
Amit Basak,* Subrata Mandal and Saibal Bandyopadhyay
Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, India
Received 8 November 2002; revised 28 December 2002; accepted 7 January 2003
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Abstract—3,5,7,3 ,4 -Pentaacetyl catechin was selectively hydrolyzed to either 3,7,3 ,4 -tetraacetyl catechin or 3-acetyl catechin
depending upon the duration of hydrolysis. A similar result was also obtained in the epicatechin series.
#
2003 Elsevier Science Ltd. All rights reserved.
The procyanidin oligomers have attracted considerable
attention in recent years due to their diverse pharmaco-
around d 2.2 disappeared. The hydrolysis was free from
any epimerization at C-2 as confirmed by the appear-
ance of only the signals for the expected product; no
other extraneous peaks were seen in the NMR spec-
trum. Moreover 3 could be reacetylated back to the
starting acetate 1. Similar result was obtained in the
epicatechin series (Scheme 2). The yield of the deacetyl-
ated product was almost quantitative (98%).
1
logical properties in addition to their continuing
2
3
industrial use. While developing a general method for
their synthesis in a stereo and region controlled man-
ner,4 we encountered a problem of deacetylation of
per acetylated catechin or epicatechin or their oligomers
by hydrolysis. Although deacetylation, especially of
phenolic hydroxyl is a simple base mediated hydrolysis,
the situation, in these systems, is much more compli-
cated than expected. The stereochemical integrity at C-2
ꢀ6
Having established the deprotection of all the phenolic
hydroxyl groups without any epimerization and in high
yields, we then turned our attention to the possibility of
selective deacetylation of the phenolic hydroxyl groups
if the hydrolysis was carried out for a short time. With
this in mind, the reaction was allowed to proceed for
one hour only. The product was isolated along with
some unconverted starting material and the NMR
showed that only one phenolic acetate group had been
hydrolyzed. The yield of the mono deacetylated pro-
duct, based on recovered starting material, was 70%.
The upfield shift of C-6 and C-8 hydrogens indicated
that the hydrolysis of either C-5 or C-7 acetate had
taken place. There was no change in the chemical shift
of the protons present in the aromatic ring C. To con-
firm exactly which acetate got hydrolyzed, the methyl
ether 7 was prepared by treatment with diazomethane in
ether for 36 h. The strong NOE (ꢁ5%) observed for
only one aromatic hydrogen unambiguously pointed
out that the C-5 acetate was hydrolyzed by PLE. If the
hydrolysis had taken place at C-7, then the methyl ether
should have shown NOE for both C-6 and C-8 hydro-
gens. The epicatechin penta acetate also behaved simi-
larly; the C-5 hydrogen was hydrolyzed in preference to
the other acetates as confirmed by NOE experiments.
7
is lost during the hydrolysis as shown in Scheme 1.
Moreover for regiospecific coupling, one needs a differ-
entially protected catechin or epicatechin to activate the
C-6 or C-8 position. Herein, we report an enzymatic
solution to both the problems. The method will help to
synthesize higher oligomers with free phenolic hydroxyl
groups in a regiocontrolled manner.
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,5,7,3 ,4 -Pentaacetyl catechin (1), prepared by acetyla-
3
8
tion of (+) catechin with acetyl chloride and pyridine,
was treated with PLE (isolated as acetone powder from
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fresh pig liver in our laboratory) in a mixture of ace-
tone and phosphate buffer at pH 8.0. When the reaction
was continued for 24 h, hydrolysis of all the phenolic
acetates took place giving 3-acetyl catechin (3) as the
only product. This was revealed by the appearance of a
three proton singlet at d 1.75 corresponding to the ali-
phatic acetyl methyl while the signals for all the methyls
corresponding to the phenolic acetates appearing at
*Corresponding author. Tel.:+91-3222-83300; fax:+91-3222-82252;
e-mail: absk@chem.iitkgp.ernet.in
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960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00024-6

1084
A.Basak et al ./ Bioorg.Med.Chem.Lett.13 (2003) 1083–1085
Scheme 1.
Scheme 2.
Additional evidence for the hydrolysis of C-5 acetate
was obtained by the appearance of weak NOE for the
C-4 hydrogens in both the series when the methyl signal
was irradiated as shown in Scheme 3.
layer was dried, filtered and evaporated to leave the
3-acetyl catechin or as a brownish solid that is more
than 90% pure. However, the material could be purified
further by chromatography on silica gel using ethyl
acetate as eluent. Yield: 130 mg (98%); d (200 MHz,
H
In conclusion, we have developed an enzymatic
method for the deprotection of phenolic acetates in
catechin or epicatechin series in regiospecific manner.
The same method can also be applied for complete
deprotection of phenolic acetates without any epi-
merization.
dMSO-d ) 8.34 (1H, bs, Ar-OH), 8.13 (1H, bs, Ar-OH),
0
6
7.93 (2H, bs, Ar-OH), 6.96 (1H, s, H-2 ), 6.75 (2H, s, H-
0
0
5 , 6 ), 6.01 (1H, d, J=2.4 Hz, H-8), 5.92 (1H, d, J=2.4
Hz, H-6), 5.32 (1H, m, H-3), 4.96 (1H, s, H-2), 2.93
(1H, dd, J=4.6, 17.4 Hz, H-4), 2.73 (1H, J=2.2,
17.4 Hz, H-4), 1.81 (3H, s, 3-OAc); mass (CI) 333
+
(MH ), 291. 3-Acetyl epicatechin (4) was prepared in
the same way in 97% yield starting from pentacetyl
epicatechin (2).
Selected Experimental Procedure and Spectral Data
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Preparation of 3-acetyl (+) catechin (3). 3,5,7,3 ,4 -Pen-
taacetyl catechin (1) (200 mg, 0.4 mmol) was dissolved
in acetone (10 mL). Phosphate buffer (pH=8.0, 20 mL)
was added followed by PLE as crude acetone powder
Preparation of 3,7,3 ,4 -tetraacetyl (+) catechin (5).
Procedure as described for the preparation of 3; the
hydrolysis was allowed to proceed for 1 h; the com-
pound 5 was isolated pure by column chromatography
ꢀ
1
(
100 mg) and the mixture was stirred at room tempera-
ture for 24 h. It was then filtered through Celite and the
filtrate was extracted with ethyl acetate. The organic
as brown solid; yield 70%; umax (KBr, cm ) 3422,
2929, 2859, 1751, 1614, 1505, 1434, 1375, 1213, 1118,
1033, 904, 831, 732, 611; d (200 MHz, CDCl ) 7.14–
H
3
0
0
0
7
.23 (3H, m, H-2 , 5 , 6 ), 6.30 (1H, s, H-8), 6.20 (1H, s,
H-6), 5.14 (1H, t, J=5.8 Hz, H-3), 5.05 (1H, d, J=6.2
Hz, H-2), 2.80–2.74 (2H, m, H-4), 2.27 (9H, s, 7, 2 , 3 -
+
0
OAc), 1.99 (3H, s, 3-OAc); mass (CI) 459 (MH ), 417,
0
279, 181.
0
0
Preparation of 3,7,3 ,4 -tetraacetyl (ꢀ) epicatechin (6).
Procedure as described for the preparation of 5; the
hydrolysis was allowed to proceed for 1 h; the com-
pound 6 was isolated pure by column chromatography
ꢀ1
as brown solid; yield 70%; umax (KBr, cm ) 3436, 2926,
Scheme 3.

A.Basak et al ./ Bioorg.Med.Chem.Lett.13 (2003) 1083–1085
1085
2
9
859, 1765, 1615, 1505, 1434, 1379, 1213, 1118, 1050,
09, 827, 740, 616; d (200 MHz, CDCl ) 7.35–7.20
References and Notes
H
3
0
0
0
1. (a) Haslam, E. In The Flavonoids; Harborne, J. B. T. J.,
Mabry, H., Eds.; Advances in Research; Academic Press: New
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(
6
3H, m, H-2 , 5 , 6 ), 6.32 (1H, d, J=4 Hz, H-8),
.20 (1H, d, J=4 Hz, H-6), 5.5 (1H, s, H-3), 5.06
0 0
(
1H, s, H-2), 2.98 (2H, m, H-4), 2.29 (9H, s, 7, 2 , 3 -
+
OAc), 1.91 (3H, s, 3-OAc); (CI) 459 (MH ), 417,
75.
3
0
0
Preparation of 5-O-methyl-3,7,3 ,4 -tetraacetyl (+) cate-
chin (7). A solution of 5 in dry ether was treated
with excess of ethereal diazomethane and was kept at
room temperature for 36 h. Evaporation followed by
chromatography of the residue afforded 7 as a
gummy solid (60%); d (200 MHz, CDCl ) 7.27–7.14
(
1
2
H
3
0
0
0
(
3H, m, H-2 , 5 , 6 -), 6.37 (1H, d, J=2 Hz, H-8), 6.23
(
3
1H, d, J=2 Hz, H-6), 5.28 (1H, dd, J=11.6, 5.9 Hz, H-
), 5.11 (1H, d, J=6.2 Hz, H-2), 3.77 (3H, s, OMe),
.86 (1H, dd, J=17.2, 5.2 Hz, H-4), 2.71 (1H, dd,
3
2
J=17.2, 2.2 Hz, H-4), 2.29, 2.27 (9H, s, 7, 2 , 3 -
4
¨
0
0
Am.Chem.Soc. 1999, 121, 12073.
5. Kawamoto, H.; Nakatsubo, F.; Murakami, K. Mokuzai
Gakkashi 1991, 37, 741.
+
OAc), 1.97 (3H, s, 3-OAc); mass (CI) 473 (MH ),
4
31, 279.
6
1
7
. Steynberg, P. J.; Nel, R. J. J.; Ferreira, D. Tetrahedron
998, 54, 8153.
. Burger, J. F. W.; Styenberg, J. P.; Young, D. A.; Brandt,
Acknowledgements
E. V. J.Chem.Soc ., Perkin Trans.1 1989, 671. Styenberg, J. P.;
Burger, J. F. W.; Young, D. A.; Brandt, E. V.; Steenkamp,
The author wishes to thank M&M Mars, USA, for
financial assistance. We are also grateful to Professor C.
A. Townsend, Johns Hopkins University, Baltimore,
U.S.A., for helpful discussions. S.M. thanks CSIR,
Government of India, for fellowship.
J. A.; Ferreira, D. J.Chem.Soc ,. Perkin Trans.1
. Kawamoto, H.; Nakatsubo, F.; Murakami, K. Synth.
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1988, 3323.
8