Studies on the porcine liver esterase-catalyzed hydrolysis of pentaacetyl catechin and epicatechin: Application to the synthesis of novel dimers and trimers

Article abstract of DOI:10.1016/j.bmcl.2008.07.058

Porcine liver esterase-catalyzed hydrolysis of 3,5,7,3′,4′-pentaacetylated catechin was studied. The selectivity of the enzyme in hydrolyzing the acetate moiety is time dependent. Careful control of the duration of hydrolysis makes it possible to isolate the differentially protected catechins. Similar result was also obtained in the epicatechin series. These results are important for elaboration of epicatechin or catechin into different derivatives with defined regiochemistry. These include novel dimeric and trimeric architectures.

Full text of DOI:10.1016/j.bmcl.2008.07.058

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4900–4903
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies on the porcine liver esterase-catalyzed hydrolysis of pentaacetyl
catechin and epicatechin: Application to the synthesis of novel dimers
and trimers
*
Amit Basak , Sanket Das, Shrabani Bisai
Department of Chemistry, Indian Institute of Technology, IIT Road, Kharagpur, West Bengal 721 302, India
a r t i c l e i n f o
a b s t r a c t
Porcine liver esterase-catalyzed hydrolysis of 3,5,7,30,40-pentaacetylated catechin was studied. The selec-
tivity of the enzyme in hydrolyzing the acetate moiety is time dependent. Careful control of the duration
of hydrolysis makes it possible to isolate the differentially protected catechins. Similar result was also
obtained in the epicatechin series. These results are important for elaboration of epicatechin or catechin
into different derivatives with defined regiochemistry. These include novel dimeric and trimeric
architectures.
Article history:
Received 6 March 2008
Revised 18 June 2008
Accepted 12 July 2008
Available online 17 July 2008
Keywords:
PLE
Ó 2008 Elsevier Ltd. All rights reserved.
Hydrolysis
Catechin
Epicatechin
Dimer
Trimer
The diverse array of pharmacological properties1 as well as the
continuing industrial use2 is the main reasons why procyanidin
oligomers have had considerable attention of scientists in recent
years. As a result, efforts are ongoing to synthesize structures with
more than one monomer unit (catechin or epicatechin), both nat-
ural and designed ones. We encountered a problem of deacetyla-
tion of per acetylated catechin or epicatechin or their oligomers
by hydrolysis while attempting to develop3 a general method for
their synthesis in a region and in a stereocontrolled manner.4–6
While deacetylation of phenolic esters is a simple base-mediated
hydrolysis, the situation, in these systems, is much more compli-
cated in flavonoid systems. The stereochemical integrity at C-2 is
lost7 in the process, as shown in Scheme 1. Moreover, for regio-
specific coupling, one needs differentially protected catechin or
epicatechin derivatives. The free phenolic hydroxyl group acts as
an activator of the aromatic ring to which it is attached. Earlier
we have reported the preparation of 3,7,30,40-tetraacetoxy cate-
chin/epicatechin by a Porcine Liver Esterase (PLE)-mediated hydro-
lysis of pentaacetylated monomers.8 In this account, we describe
the isolation of differentially acetylated catechin/epicatechin. We
have shown that by controlling the time of hydrolysis, tetra and
diacetates can be obtained in decent yields and in regiochemically
pure forms. Only the triacetates were obtained as a mixture of
regioisomers. Isolation of these partially protected catechin/epicat-
echin derivatives will help to synthesize higher oligomers in a reg-
iocontrolled manner preserving their stereochemical integrity,
which is a great challenge to the synthetic chemist.
In our earlier work,8 we had shown that PLE-catalyzed hydroly-
sis of pentaacetyl catechin or epicatechin led to 3,7,30,40-tetraacetyl
derivatives or 3-acetyl derivatives depending upon the duration of
hydrolysis (1 or 24 h, respectively) (Scheme 2). Moreover, the
OH
OH
O
O
HO
O
HO
O
R1
R2
R1
R2
OH
O
OH
OH
HO
O
R1
R2
OH
* Corresponding author. Tel.: +91 3222283300; fax: +91 3222282252.
E-mail address: absk@chem.iitkgp.ernet.in (A. Basak).
Scheme 1. Base-catalyzed epimerization.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.058

A. Basak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4900–4903
4901
chin, while the other was 3,7,40-triacetoxy isomer. Unfortunately,
these compounds could not be separated even by HPLC. When
the hydrolysis was allowed to proceed up to 18 h, only one diacet-
oxy compound was isolated (Scheme 3) whose structure was as-
signed as the 3, 7-diacetoxy catechin/epicatechin on the basis of
NMR studies. The upfield shift of both 20- and 50-protons pointed
out the hydrolysis of 30 and 40-acetoxy groups. No shift was ob-
served for the protons in ring A. All these results are summarized
in Table 1. The formation of similar hydrolysis products from both
catechin and epicatechin acetates ruled out the influence of C-3
acetate on the course of hydrolysis.
One application of this highly regioselective hydrolysis is man-
ifested in the synthesis of novel catechin and epicatechin-based di-
meric and trimeric molecules. Thus the tetraacetoxy catechin,
obtained from the corresponding pentaacetate by regioselective
PLE-catalyzed hydrolysis as described, when treated with mesitoyl
chloride (0.34 equiv) and triethyl amine (0.34 equiv) in methylene
OAc
OAc
OH
OH
O
AcO
O
HO
R1
R2
R1
R2
OAc
OH
1A-B
2A-B
OAc
OAc
O
AcO
R1
R2
For A R1 = OAc, R2 = H
For B R1 = H, R2 = OAc
OH
3A-B
a PLE, acetone-phosphate buffer, pH 8.0, 24 h
b PLE, acetone-phosphate buffer, pH 8.0, 1 h
Scheme 2. PLE-catalyzed hydrolysis for short and long duration.
Table 1
Result of PLE-catalyzed hydrolysis
hydrolysis was shown to be free from any epimerization. The spe-
cific question that we wanted to address is whether the phenolic
acetates are hydrolyzed in a sequential manner and whether it is
possible to isolate products corresponding to di or tri acetates if
the hydrolysis is carried out for intermediate duration. For this,
we needed to monitor the reaction at different time points.
When the reaction was allowed to proceed up to 9 h, a mixture
of triacetoxy compounds was isolated. The same mixture of com-
pounds was also isolated if the tetraacetoxy isomer was hydro-
lyzed separately for 8 h. The upfield shift of ring C-protons
indicated that the hydrolysis had taken place either at C-40 or C-
30. One of the compounds was 3,7,30-triacaetoxy catechin/epicate-
Duration of
hydrolysis (h)
Major product
Isolated
yield (%)
Ratioa of
penta:tetra:tri:di:mono
acetates
1
Tetraacetoxy
70
48
50
97
1:5:1:nd:nda
1:5:10:2:nd
nd:nd:6:10:2
Mostly nono
catechin/epicatechin
Triacetoxy catechin/
epicatechin
Diacetoxy catechin/
epicatechin
9
18
24
3-Acetoxy catechin/
epicatechin
a
Ratio determined by HPLC (ODS column, 15% H2O in MeOH as the mobile phase;
nd means not determined.
OAc
OAc
AcO
O
OAc
OAc
1A-B
a
c
b
OAc
OH
OAc
OH
AcO
O
AcO
O
OAc
3A-B
OAc
OH
OH 4A-B
OAc
OH
OH
OAc
+
AcO
O
AcO
O
OAc
OAc
OH
6A-B
5A-B
OH
a : PLE,acetone-phosphate buffer, pH 8.0, 1hr
b : PLE,acetone-phosphate buffer, pH 8.0, 9 hrs
c : PLE,acetone-phosphate buffer, pH 8.0, 18 hrs
Scheme 3. Time dependent PLE-catalyzed hydrolysis.

4902
A. Basak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4900–4903
chloride, furnished the novel C3-symmetric trimer 7A in 85% yield
as a white solid (Scheme 4). The structure of 7A was unequivocally
established by thorough analysis of 1H, 13C NMR, and mass (MAL-
DI) spectral data. In a similar way, the epicatechin acetate-based
trimer 7B has also been prepared and characterized. In order to ex-
plore the possibility whether these trimers can act as dendrimeric
core, 7A and 7B were subjected to PLE-catalyzed hydrolysis. How-
ever, the hydrolysis led to a complex mixture of products demon-
strating poor selectivity.
The various dimeric catechin or epicatechins 8A–10A and 8B–
10B were prepared by treating 2 mol equivalent of the tetraacetate
with 1 mol equivalent of phthaloyl/isophthaloyl or terephthaloyl
chloride. The linked dimers were obtained in 90% yield after Silica
gel column chromatography.
In conclusion, we have developed an enzymatic method for
the sequential deprotection of phenolic acetates in catechin or
epicatechin series in regiospecific manner without any loss of
stereochemical integrity. The synthesis of novel catechin and epi-
catechin acetate-based dimeric and trimeric systems demon-
strated the utility of corresponding partially acetylated
derivatives. Currently, we are studying the antioxidant activity
of these novel polyphenols and the results will be reported in
due course.
Selected experimental procedure and spectral data. General
hydrolysis procedure: 3,5,7,30,40-Pentaacetyl 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 pow-
der9 (100 mg) and the mixture was stirred at room temperature for
1–24 h. It was then filtered through celite and the filtrate was ex-
tracted with ethyl acetate. The organic layer was dried, filtered,
and evaporated to leave a brownish solid from which the major
products were purified by Si-gel chromatography.
3,7,30-Triacetyl (+) catechin and 3,7,40-triacetyl (+) catechin (mix-
ture) (5A and 6A): dH (200 MHz, CDCl3) 6.79–7.05 (3H, m, H-20, 50,
60), 6.29 (1H, d, J = 1.7 Hz, H-8), 6.14 (1H, d, J = 2.0 Hz, H-6), 6.10
(1H, d, J = 2.1 Hz, H-6), 5.25 (1H, t, J = 5.0 Hz, H-3), 5.15 (1H, d,
J = 4.7 Hz, H-2), 5.08 (1H, d, J = 5.4 Hz, H-2), 2.44–2.60 (2H, m, H-
4), 2.38 (3H, s, 30-OAc or 40-OAc), 2.27 (3H, s, 7-OAc), 2.0 (3H, s,
3-OAc), 1.98 (3H, s, 3-OAc); Mass (ESI) 416 (M+), 374, 355, 313.
3,7-Diacetyl (+) catechin (4A): dH (200 MHz, CDCl3) 6.79–6.81
(2H, m, H-20, 60), 6.69–6.74 (1H, m, H-50), 6.29 (1H, d, J = 2.0 Hz,
H-8), 6.09 (1H, d, J = 2.2 Hz, H-6), 5.26 (1H, t, J = 5.3 Hz, H-3),
5.09 (1H, d, J = 5.2 Hz, H-2), 2.30–2.57 (2H, m, H-4), 2.28 (3H, s,
7-OAc), 2.0 (3H, s, 3-OAc); Mass (ESI) 374 (M+), 355, 313.
3,7,30-Triacetyl (ꢀ) epicatechin(10) and 3,7,40-triacetyl (ꢀ) epicat-
echin (11) (mixture) (5B and 6B): dH (200 MHz, CDCl3) 6.79–7.11
OAc
AcO
O
AcO
OAc
OAc
COCl
R1
OAc
R1
R2
R2
O
O
AcO
O
OAc
O
OAc
ClOC
Et3N, DCM,O oC, 2h
COCl
O
R1
R2
O
O
OH
O
3A-B
O
AcO
R2
R1
7A-B
For A R1 = OAc, R2 = H
For B R1 = H, R2 = OAc
OAc
OAc
AcO
OAc
OAc
AcO
OAc
O
OAc
AcO
O
AcO
O
R2
OAc
R1
O
R2
O
R1
R1
R2
O
O
O
O
OAc
O
OAc
O
OAc
O
O
O
O
O
O
R2
R1
O
R1
R2
R1
R2
AcO
8A-B
OAc
9A-B
AcO
OAc
10A-B
AcO
OAc
Scheme 4. Synthesis of dimeric and trimeric architecture.

A. Basak et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4900–4903
4903
(3H, m, H-20, 50, 60), 6.31 (1H, bs, H-8), 6.17 (1H, bs, Hz, H-6), 5.39
(1H, bs, H-3), 4.95 (1H, bs, H-2), 2.91 (2H, bs, H-4), 2.43 (3H, s, 30-
OAc or 40-OAc), 2.27 (3H, s, 7-OAc), 1.89 (3H, s, 3-OAc), 1.80 (3H, s,
3-OAc); Mass (ESI) 416 (M+), 374, 355.
dd, J = 5.2, 16.8 Hz), 2.73 (2H, dd, J = 6.4, 16.8 Hz), 2.29 (12H, s),
2.28 (6H, s), 2.02 (6H, s); dC (100 MHz, CDCl3) 170.0, 169.0,
168.1, 164.5, 154.4, 149.9, 149.2, 142.0, 141.9, 136.0, 135.9,
132.1, 131.0, 129.5, 125.7, 124.5, 123.6, 121.8, 110.4, 108.8,
108.1, 77.6, 68.1, 24.0, 21.0, 20.8, 20.5; Mass (ESI) 1047 (MH+);
HRMS Calcd for C54H46O22+H+ 1047.2544. Found 1047.2540.
Isophthalic acid bis-[3,7-diacetoxy-2-(3,4-diacetoxy-phenyl)-chro-
man-5-yl] ester (epicatechin) (9B): dH (400 MHz, CDCl3) 8.94 (1H, s),
8.44 (2H, dd, J = 1.6, 8.0 Hz), 7.71 (1H, t, J = 7.6 Hz), 7.29–7.19 (6H,
m), 6.73 (4H, s), 5.41 (2H, bs), 5.16 (2H, s), 3.09 (2H, dd, J = 4.4,
18.0 Hz), 2.94 (2H, d, J = 18.0 Hz), 2.30 (18H, s), 1.92 (6H, s); dC
(100 MHz, CDCl3) 170.5, 169.0, 168.1, 163.2, 155.2, 149.8, 149.0,
142.0, 141.9, 136.0, 135.2, 132.0, 129.3, 129.1, 124.5, 123.3,
121.9, 110.0, 108.9, 108.5, 76.7, 66.8, 26.2, 21.0, 20.8, 20.5; Mass
(ESI) 1047 (MH+), 1069 (MNa+).
Terephthalic acid bis-[3,7-diacetoxy-2-(3,4-diacetoxy-phenyl)-
chroman-5-yl] ester (catechin) (10A): dH (400 MHz, CDCl3) 8.28
(4H, s), 7.28 (2H, bs), 7.18 (4H, m), 6.75 (2H, s) 6.73 (2H, s), 5.29
(2H, m), 5.20 (2H, m), 2.92 (2H, dd, J = 4.8, 17.6 Hz), 2.71 (2H, dd,
J = 6.0, 17.6 Hz), 2.30 (18H, s), 2.28, 2.0 (6H, s); dC (100 MHz, CDCl3)
170.1, 169.0, 168.1, 163.0, 154.4, 149.9, 149.2, 142.0, 136.0, 133.3,
130.4, 124.3, 123.7, 121.7, 110.2, 108.8, 108.0, 77.5, 68.0, 23.8, 21.1,
21.0, 20.9; Mass (ESI) 1047 (MH+), 1069 (MNa+).
Terephthalic acid bis-[3,7-diacetoxy-2-(3,4-diacetoxy-phenyl)-
chroman-5-yl] ester (epicatechin) (10B): dH (400 MHz, CDCl3) 8.31
(4H, s), 7.37 (2H, d, J = 1.6 Hz), 7.27 (2H, dd, J = 1.6, 8.4 Hz), 7.21
(2H, d, J = 8.4 Hz, 6.75 (4H, s), 6.75 (2H, s), 5.41 (2H, bs), 5.20
(2H, s), 3.05 (2H, dd, J = 4.4, 17.2 Hz), 2.94 (2H, d, J = 17.2 Hz),
2.30 (18H, s), 1.93 (6H, s); dC (100 MHz, CDCl3) 170.3, 169.0,
168.1, 168.0, 163.1, 155.1, 149.8, 149.6, 142.0, 141.9, 135.7,
133.3, 130.4, 124.3, 123.2, 122.0, 109.7, 108.8, 108.4, 77.2, 66.5,
26.1, 21.0, 20.7, 20.6; Mass (ESI) 1047 (MH+), 1069 (MNa+); HRMS
Calcd for C54H46O22+H+ 1047.2544. Found 1047.2549.
3,7-Diacetyl (ꢀ) epicatechin (4B): dH (200 MHz, CDCl3) 6.95 (1H,
s, H-50), 6.83 (1H, s, H-20, 60), 6.33 (1H, d, J = 2.02 Hz, H-8), 6.19 (1H,
d, J = 2.16 Hz, H-6), 5.42 (1H, bs, H-3), 4.97 (1H, s, H-2), 2.97 (2H, m,
H-4), 2.28 (3H, s, 7-OAc), 1.92 (3H, s, 3-OAc); Mass (ESI) 374 (M+).
General method for the synthesis of dimers and trimers: To a solu-
tion of tetraacetyl catechin/epicatechin (1 equiv) in CH2Cl2
(15 mL), mesitoyl chloride (0.34 equiv) or phthaloyl/isophthaloyl/
terepthaloyl chloride (0.5 equiv) was added followed by Et3N
(0.34 equiv or 0.5 equiv, respectively). The mixture was stirred
for 2 h at 0 °C. It was then poured into water and extracted with
CH2Cl2. The organic layer was dried and evaporated. The residue
upon Si-gel chromatography (hexane/EA 2:1) furnished the trimers
or dimers as white solids (85–90%).
Benzene-1,3,5-tricarboxylic acid tris-[3,7-diacetoxy-2-(3,4-diacet-
oxy-phenyl)-chroman-5-yl] ester (catechin) (7A): dH (200 MHz,
CDCl3) 9.13 (3H, s, Ar-H), 7.29–7.17 (3H, m, H-60, 50, 20) 6.72 (2H,
ABq, J = 1.8 Hz, H-8, 6), 5.29–5.16 (2H, m, H-3, 2), 2.89–2.75 (2H,
m, H-4), 2.27 (9H, s, 7, 30, 40-OAc), 1.96 (3H, s, 3-OAc); dC
(50 MHz, CDCl3) 170.04, 168.83, 167.99, 162.06, 154.59, 150.05,
149.14, 142.07, 136.33, 135.97, 130.64, 124.38, 123.70, 121.78,
110.48, 108.76, 108.32, 68.07, 24.07, 21.06, 20.89, 20.59; Mass
(MALDI) 1548.1447 (M+ +18).
Benzene-1,3,5-tricarboxylic acid tris-[3,7-diacetoxy-2-(3,4-diacet-
oxy-phenyl)-chroman-5-yl] ester (epicatechin) (7B): dH (200 MHz,
CDCl3) 9.18 (3H, s, Ar-H), 7.38 (1H, s, H-20), 7.25 (2H, Abq,
J = 8.2 Hz, H-60, 50) 6.75 (2H, Abq, J = 2.2 Hz, H-8, 6), 5.43 (1H, bs,
H-3), 5.17 (1H, bs, H-2), 3.09–2.98 (2H, m, H-4), 2.30 (9H, s, 7, 30,
40-Oac), 1.99 (3H, s, 3-Oac); dC (50 MHz, CDCl3) 170.32, 168.92,
168.03, 168, 162.08, 155.19, 149.43, 142, 141.89, 136.37, 135.72,
130.68, 124.23, 122, 109.8, 108.77, 66.46, 26.18, 21.04, 20.76,
20.59; Mass (MALDI) 1548.1225 (M+ +18).
Acknowledgments
Phthalic acid bis-[3,7-diacetoxy-2-(3,4-diacetoxy-phenyl)-chro-
man-5-yl] ester (catechin) (8A): dH (400 MHz, CDCl3) 8.03 (2H, m),
7.69 (2H, m), 7.27 (6H, m), 6.74 (2H, bs) 6.70 (2H, bs), 5.26 (2H,
m), 5.14 (2H, d, J = 6.4 Hz), 2.94 (2H,dd, J = 5.2, 16.8 Hz), 2.74 (2H,
d, J = 16.8 Hz), 2.28 (s, 18H), 1.94 (s, 6H); dC (100 MHz, CDCl3)
170.0, 169.0, 168.1, 164.5, 154.4, 149.9, 149.2, 142.0, 141.9,
136.0, 135.9, 132.1, 131.0, 129.5, 125.7, 124.5, 123.6, 121.8,
110.4, 108.8, 108.1, 77.6, 68.1, 24.0, 21.1, 20.8, 20.6; Mass (ESI)
1047 (MH+); HRMS Calcd for C54H46O22+H+ 1047.2544. Found
1047.2547.
Phthalic acid bis-[3,7-diacetoxy-2-(3,4-diacetoxy-phenyl)-chro-
man-5-yl] ester (epicatechin) (8B): dH (400 MHz, CDCl3) 8.02 (2H,
m), 7.65 (2H, m), 7.37–7.19 (6H, m), 6.74 (2H, d, J = 4.4 Hz,) 6.70
(2H, d, J = 4.4 Hz), 5.36 (2H, bs), 5.18 (2H, s), 3.06 (2H,dd, J = 4.4,
18.0 Hz), 2.50 (2H, d, J = 18.0 Hz), 2.29 (12H, s), 2.26 (6H, s), 1.89
(6H, s,); dC (100 MHz, CDCl3) 170.4, 169.01 168.1, 168.0, 164.7,
155.1, 149.7, 149.5, 141.9, 141.8, 136.0, 135.9, 132.2, 131.2,
131.0, 130.1, 129.5, 129.0, 124.4, 123.2, 122.0, 110.0, 108.8,
108.4, 77.6, 66.5, 26.0, 21.0, 20.7, 20.6; Mass (ESI) 1047 (MH+),
1069 (MNa+).
The authors (S.D.) are grateful to CSIR, Government of India, for
fellowship. A.B. is grateful to CSIR for the financial support. DST, In-
dia, is thanked for the grant to purchase 400 MHz Bruker NMR un-
der the IRPHA program.
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Isophthalic acid bis-[3,7-diacetoxy-2-(3,4-diacetoxy-phenyl)-chro-
man-5-yl] ester (catechin) (9A): dH (400 MHz, CDCl3) 8.91 (1H, s),
8.41 (2H, d, J = 8.0 Hz), 7.68 (1H, t, J = 7.6 Hz), 7.28–7.18 (6H, m),
6.73 (4H, s) 6.70), 5.26 (2H, m), 5.18 (2H, d, J = 6.0 Hz), 2.92 (2H,