Synthetic studies of proanthocyanidins. Part 5.1 Highly stereoselective synthesis and inhibitory activity of Maillard reaction of 3,4-trans catechin and epicatechin dimers, procyanidin B1, B2, B3, B4 and their acetates

Article abstract of DOI:10.3987/com-03-s(p)42

TMSOTf-catalyzed condensation of a potential electrophile with a nucleophile was achieved with a high level of 3,4-trans condensation, and we succeeded in the stereoselective synthesis of procyanidin B2 and its peracetate. Studies on the inhibitory activity of the Maillard reaction of four 3,4-trans series of catechin and epicatechin dimers, procyanidin B1, B2, B3 and B4, and their peracetates were also carried out.

Full text of DOI:10.3987/com-03-s(p)42

HETEROCYCLES, Vol. 62, 2004, pp. 479 - 489
Received, 31st July, 2003, Accepted, 10th September, 2003, Published online, 4th November, 2003
1
SYNTHETIC STUDIES OF PROANTHOCYANIDINS. PART 5.
HIGHLY STEREOSELECTIVE SYNTHESIS AND INHIBITORY
ACTIVITY OF MAILLARD REACTION OF 3,4-TRANS CATECHIN AND
EPICATECHIN DIMERS, PROCYANIDIN B1, B2, B3, B4 AND THEIR
ACETATES
1
2
2ꢀ
3
Akiko Saito, Noriyuki Nakajima, * Nobuyasu Matsuura, Akira Tanaka, and
2
Makoto Ubukata
1
Biotechnology Center, Toyama Prefecture
2
Biotechnology Research Center, Toyama Prefectural University
3
Department of Biosources Science, College of Technology, Toyama
Prefectural University
Kosugi, Toyama 939-0398, JAPAN e-mail: nori@pu-toyama.ac.jp
Abstract – TMSOTf-catalyzed condensation of a potential electrophile with a
nucleophile was achieved with a high level of 3,4-trans condensation, and we
succeeded in the stereoselective synthesis of procyanidin B2 and its peracetate.
Studies on the inhibitory activity of the Maillard reaction of four 3,4-trans series
of catechin and epicatechin dimers, procyanidin B1, B2, B3 and B4, and their
peracetates were also carried out.
Introduction
Nonenzymatic browning reactions by heating a mixture solution of sugar with protein or amino acid are
1
known as the Maillard reaction, which occurs extensively in food systems and in vivo. The reaction is
roughly divided into two stages, the early and late stages. 3-Deoxyglucosone (3DG) was generated from
Amadori compounds in the early stage, leading to generation of advanced glycation endproducts (AGEs),
which are characterized by fluorescence, a brown color, and intra- or intermolecular cross-linking. The
2
3
relationship of the Maillard reaction to diabetic complications has been noted in the field of sitology.
1
5
6
7
8
Under these circumstances, aminoguanidine (AG), aspirin, vitamin B6, taurine, quercetin and
9
components of tea extracts (catechin derivatives) , have been reported as important Maillard reaction
inhibitors.
We have studied a synthetic method of proanthocyanidin oligomers. Proanthocyanidins are known as
1
0-12
condensed tannins and/or oligomeric flavonoids,
and many biological activities, powerful antioxidant
1
3
14
15
activity, free-radical-scavenging activity and an anti-tumor-promoting effect, have been reported in
1
6
these compounds. In this paper we describe the synthesis of 3,4-trans-proanthocyanidin dimers as
shown in Figure 1, and the inhibitory activity of the Maillard reaction. Previously described TMSOTf-

OR
OR
OR
OR
RO
O
4
RO
O
4
OH
O
OR
OR
OR
OR
OR
OR
RO
OR
RO
O
OR
OR
OR
OR
1
: R=H, procyanidin B1
(-)-epicatechin-(+)-catechin]
Ac -1: R=Ac, procyanidin B1 decaacetate
2
: R=H, procyanidin B2
(-)-epicatechin-(-)-epicatechin]
Ac -2: R=Ac, procyanidin B2 decaacetate
[
[
1
0
1
0
OR
OR
OR
OR
RO
O
4
RO
O
4
OH
OR
OR
OR
OR
OR
OR
RO
OR
RO
O
O
OR
OR
OR
OR
3
a: R=H, procyanidin B3
,4-trans (+)-catechin-(4a→8)-(+)-catechin dimer
4
: R=H, procyanidin B4
[(+)-catechin-(-)-epicatechin]
Ac -4: R=Ac, procyanidin B4 decaacetate
3
Ac -3a: R=Ac, procyanidin B3 decaacetate
10
1
0
3
b: R=H, 3,4-cis (+)-catechin-(4b→8)-(+)-catechin dimer
Figure 1
catalyzed catechin and catechin condensation achieved high levels of 3,4-trans condensation in excellent
1
7-19
isolation yield to give procyanidin-B3 (3a).
We also applied TMSOTf-catalyzed condensation under
2
0
intramolecular conditions. 3,4-cis-Catechin-catechin dimer (3b), 3,4-trans-epicatechin-catechin dimer
(
1) and 3,4-trans-catechin-epicatechin dimer (4) were obtained in excellent to good yields. However, 3,4-
1
trans-epicatechin-epicatechin dimer (2) was not obtained under the intramolecular conditions. This paper
reports the synthesis of dimer (2) under TMSOTf-catalyzed intermolecular condensation and the
inhibitory activity of the Maillard reaction of all synthesized procyanidin dimers (1 ~ 4) and their acetyl
derivatives (Ac -1 to Ac -4).
1
0
10
TMSOTf-catalyzed condensation
TMSOTf-catalyzed condensations were attempted by the combinations of catechin and epicatechin
electrophiles (9 ~ 12) and nucleophiles (7 and 8). The 4-O-ethoxyethylated flavan-3,4-diol derivative was
used as the electrophile; the nucleophile was used in 4.5-fold excess to avoid higher oligomer formation.
To a solution of the nucleophile and the electrophile in CH Cl was added 1 equivalent of TMSOTf at
2
2
–
78°C, and the mixture was allowed to react for 5 min. In all cases, 3,4-trans dimerization products (13 ~
0) were obtained in moderate to excellent yield as a single isomer.
2

When the epicatechin electrophile (10) was condensed with tetra-O-benzylated catechin (8) in the
presence of TMSOTf (1.0 eq.) at –78°C, the 3,4-trans-octa-O-benzylated (-)-epicatechin-(4 b→8)-(+)-
catechin dimer (13) was obtained in 45 % yield. The reaction conditions were first checked using 3-O-
acetyl electrophile (10), because the neighboring group participation of the 3-O-acetyl moiety was
1
8
effective in attaining significant improvement in the selectivity. However, the epicatechin component
was less reactive than the catechin component; by removal of the 3-O-acetyl group, dimerization
1
proceeded smoothly to give dimer (14) in better 67 % yield. On the epicatechin and epicatechin
condensation, better yield was also obtained by using 3-hydroxy electrophile (9) in place of 3-O-acetyl
electrophile (10), epicatechin-epicatechin dimers (15) and (16) were selectively obtained in 75 % and
2
1
9
3 % yields, respectively. The catechin component was more reactive than the epicatechin component;
the condensation of 8 and 7 with 3-O-acetylated catechin derivative (12) proceeded smoothly to give the
1
8
1
corresponding catechin-catechin dimer (17) and catechin-epicatechin dimer (19) in 97 % and 90 %
2
2
yields, respectively.
Natural procyanidin B2 (2) was synthesized from dimer (16) by hydrogenolysis with Pd(OH) in
2
THF/MeOH/H O and following Sephadex® LH-20 and normal-phase HPLC purification (Kanto
2
1
Chemical Co., Mightysil Si 60, EtOAc) in 86% yield. All H-NMR peaks of 2 were broadened; therefore
1
the structure was confirmed as a decaacetyl compound (Ac -2), which was spectroscopically ( H-NMR,
1
0
1
3
C-NMR, MS) identical with the natural product, and also had specific rotation in good agreement with
2
3,24
the literature.
Syntheses of other B-type procyanidins (1, 3, 4) and their decaacetates (Ac -1, Ac -3,
10 10
1
17,25
1,25
Ac -4) are already achieved via 14, 18,
and 20.
1
0
Maillard reaction inhibitory activity
Inhibitory activity of the Maillard reaction of natural procyanidin dimers (1 ~ 4) and their acetates (Ac -1
1
0
2
6
~
Ac -4) was next determined using our reported method. The value of IC (50% inhibitory activity) of
10 50
all compounds is shown in Table 1. Inhibitory activity of procyanidins B1, 2, 3 and 4 (1-4) showed 30-72
mM. Their acetyl derivatives, Ac -1, Ac -2, Ac -3 and Ac -4, showed 7-40 mM. Because the IC value
1
0
10
10
10
50
2
6
of the Maillard reaction inhibitor aminoguanidine was 200 mM, synthesized dimers and their acetates are
candidates for a clinically useful Maillard reaction inhibitor. The phenolic hydroxy group of a catechin
9
monomer or oligomer seemed to be effective for the antioxidant activity. However, it is interesting that
effect of the phenolic hydroxy group and inhibitory activity against the Maillard reaction are not parallel;
inhibitory activity of acetyl derivatives (Ac -1 ~ Ac -4) was 6-1.5 times higher than that of phenol
1
0
10
derivatives (1 ~ 4). Further investigations on the structure and activity relationship of Maillard reaction
inhibition are now underway.
Conclusion
We have established a 3,4-trans natural procyanidin dimer synthesis based on the TMSOTf-catalyzed

OBn
OBn
OR
OR
OBn
OBn
OR
OR
BnO
O
RO
O
BnO
O
RO
O
OR
O(CH ) OEt
OH
OR
O(CH ) OEt
OH
OBn
OR
OBn
OR
2 2
2
2
5: R=H, 7: R=Bn
9: R=H,10: R=Ac
6: R=H, 8: R=Bn
11: R=H, 12: R=Ac
OBn
OBn
BnO
a
O
4
8
8
7
7
+ 10
+ 9
+ 10
+ 9
1
3: R =Ac, R =OH, R =H (45%)
1 2 3
OR1
O
OBn
Ac10-1
14: R =H, R =OH, R =H (67%)
1
1
2
1
2
3
OBn
BnO
5: R =Ac, R =H, R =OH (75%)
1 2 3
b
c
OBn 16: R =H, R =H, R =OH (93%)
Ac10- 2
1
2
3
R2
86 %
63 %
R3
OBn
OBn
BnO
O
OBn
1
1
1
2
7: R =Ac, R =OH, R =H (97%)
1 2 3
8
+ 12
d
e
a
4
8: R =H, R =OH, R =H
3
Ac10 - 3
Ac10 - 4
(
8 + 11)
+ 12
7 + 11)
OR1
O
OBn
OBn
1
2
3
9: R =Ac, R =H, R =OH (90%)
7
OBn
BnO
1
2
3
0: R =H, R =H, R =OH
4
(
1
2
3
R2
R3
OBn
reagents and conditions: a) TMSOTf, CH Cl , -78˚C, 5 min b) Pd(OH) -H , THF/MeOH/H O
2
2,
2
2
2
c) Ac O, TEA, DMAP, CH Cl d) KCN, 95%-EtOH, 98% e) DIBAL, CH Cl , -78°C, 88%
2
2
2
2
2
Scheme 1
Table 1. Inhibitory activity of catechin dimers and their acetylation derivatives
Compound
IC , mM
50
1
2
3
4
30
58
42
72
16
40
7
Ac -1
1
0
Ac -2
1
0
Ac -3
1
0
Ac -4
26
1
0
intermolecular condensation method. The inhibitory activity of the Maillard reaction of synthesized
dimers (1 ~ 4) and their acetates (Ac -1 ~ Ac -4) was also studied to find that these dimers have a high
1
0
10
level of Maillard reaction inhibitory activity.
EXPERIMENTAL
Maillard reaction inhibitory activity
Chemicals. BSA (fraction V) was obtained from Sigma Chemical Co. Ltd. (St. Louis, MO). All other

chemicals were purchased from Nakarai Tesque (Kyoto, Japan). Alkaline PBS was adjusted PBS to pH 10
with 0.25 N NaOH.
In vitro Glycation. The evaluation of in vitro glycation, the Maillard reaction was achieved by using the
2
6
previously described method. Briefly, 500 mL of reaction mixture was prepared containing 400 mg BSA,
2
00 mM glucose with or without 10 mL of inhibitor in 50 mM phosphate buffer, pH 7.4, and the reaction
mixture was heated at 60℃ on a heat block for indicated hours. A blank sample was kept at 4℃ until
measurement. After cooling, aliquots of 100 mL were transferred to new 1.5 mL plastic tubes, and 10 mL
of 100 % w/v TCA was added to each tube. The supernatant containing glucose and inhibitor was
removed after agitation and centrifugation (15,000 rpm, 4℃, 4 min.), then the precipitate of AGEs-BSA
was dissolved with 400 mL of alkaline PBS to serve for screening. The changes in fluorescence intensity
(
(
ex. 370 nm, em. 440 nm) based on AGEs were monitored by using spectrofluorometer RF-1500
Shimadzu, Japan).
The Measurement of the Maillard reaction inhibitory activity. Real inhibition activity was estimated
by subtracting the quenching effect from the apparent inhibitory activity. Apparent inhibitory activity was
calculated by the above method. Quenching effect was measured by using the same sample dissolved
with alkaline PBS after TCA treatment of the mixed solution of 2 mL of inhibitor solution and 100 mL of
the 30 hours incubated control solution.
Synthesis
1
Optical rotation was measured with a Horiba SEPA-300 spectrometer. H-NMR spectra were measured
with JEOL JNMLA400 spectrometer, and MS spectra were recorded with a JEOL JMS-AX500
instrument. Synthetic procyanidin B2 (2) was analyzed on a Mightysil® RP-18 GP column (Kanto
Chemical Co. Inc, Japan; 250 x 4.6 mm, 5 mm) using the solvents (A) 0.1% CF CO H in CH CN and (B)
3
2
3
0
.1% CF CO H in H O. Elusion was done with a linear gradient 5 to 100% A in 40 min (flow rate, 0.5
3 2 2
mL/min).
[
4,8]-2,3-cis-3,4-trans:2,3-trans-Octa-O-benzyl-(-)-3-O-acetyl-(-)-epicatechin-(+)-catechin (13). To a
solution of tetra-O-benzylated catechin (8) (101 mg, 0.16 mmol) and (2R,3R,4S)-3-acetoxy-5, 7, 3', 4'-
tetrabenzyloxy-4-(2’’-ethoxyethoxy)flavan (10) (27 mg, 0.035 mmol) in CH Cl (30 mL) was added
2
2
dropwise TMSOTf (0.07 mmol, 0.035 mmol, 0.5 M solution in CH Cl ) at –78˚C. After stirring for 5 min,
2
2
the pale yellow reaction mixture was quenched with sat. NaHCO . The solution was extracted with CHCl3
3
and the organic phase was washed with water and brine, and dried (Na SO ). Filtration, concentration and
2
4
silica gel column chromatography (hexane/EtOAc, 6/1~1.5/1) afforded 21 mg (45 %) of 13 as a colorless

2
7
1
amorphous solid: [a] = +62.2° (c 0.62, CHCl ); H-NMR (400 MHz, CDCl , 0.75 : 0.25 mixture of
D
3
3
rotational isomers ) major isomer: 7.43-6.73 (30.75H, m), 6.96 (0.75H, dd, J = 1.7, 8.3 Hz), 6.88 (0.75H,
d, J = 8.3 Hz), 6.76 (0.75H, d, J = 8.3 Hz), 6.73 (0.75H, d, J = 1.7 Hz), 6.49 (0.75H, dd, J = 1.7, 8.3 Hz),
6
.29 (0.75H, s), 6.03 (0.75H, d, J = 2.2 Hz), 5.54 (0.75H, d, J = 2.2 Hz), 5.52 (0.75H, br s), 5.46 (0.75H,
br s), 5.09-4.79 (10.5H, m), 4.71 (0.75H, br s), 4.64 (0.75H, d, J = 11.2 Hz), 4.51 (0.75H, d, J = 11.2 Hz),
3
2
.78 (0.75H, ddd, J = 6.6, 9.0, 9.5 Hz), 3.64 (0.75H, d, J = 9.0 Hz), 3.24 (0.75H, dd, J = 6.6, 16.6 Hz),
.58 (0.75H, dd, J = 9.5, 16.8 Hz), 1.69 (2.25H, s), 1.43 (0.75H, br s, OH); minor isomer: 7.43-6.73
(
11.5H, m), 6.22 (0.25H, d, J = 2.2 Hz), 6.19 (0.25H, s), 6.12 (0.25H, d, J = 2.2 Hz), 5.37 (0.25H, brs),
5
5
.26 (0.25H, br s), 5.16-4.46 (4.25H, m), 4.58 (0.25H, br s), 3.66-3.63 (0.25H, m), 3.19 (0.25H, dd, J =
1
3
.6, 15.9 Hz), 2.69 (0.25H, dd, J = 9.8, 15.9 Hz), 1.63-1.54 (0.25H, m, OH), 1.27 (0.75H, s); C-NMR
(
100 MHz, CDCl , 0.75 : 0.25 mixture of rotational isomers) major isomer: 169.3, 158.0, 156.4, 156.1,
3
1
1
8
56.0, 155.5, 154.4, 149.3, 149.0, 148.8, 148.6, 137.34, 137.28, 137.21, 137.17, 137.03, 137.01, 132.1,
30.4, 128.6-127.0 (Cx25), 120.6, 119.8, 114.6, 114.0, 113.5, 112.1, 110.6, 104.4, 104.1, 93.5, 92.8, 91.4,
1.7, 77.4, 74.5, 72.0, 71.4, 71.25, 71.22, 70.6, 70.2, 69.91, 69.56, 69.2, 68.6, 33.2, 29.1, 20.7; minor
isomer: 169.1, 158.2, 157.5, 156.0, 155.9, 154.5, 152.9, 148.8, 148.62, 148.58, 148.4, 137.34, 137.26,
37.21, 137.17, 136.9 (x2), 132.3, 132.0, 128.6-127.0 (Cx25), 120.7, 119.8, 114.5, 113.6, 113.5, 112.2,
11.4, 104.8, 103.2, 93.2, 92.3, 91.4, 81.1, 74.9, 72.3, 71.4, 71.3, 71.0, 70.9, 70.8, 70.1, 69.6, 69.2, 68.7,
1
1
6
2
1
+
+
7.9, 33.5, 29.7, 20.2; FAB-MS (m/z) 1365 (5), 1364 ([M+Na] , 12), 1344 (11), 1343 (20), 1342 ([M+H] ,
+
0), 1281 (50), 1190 (40), 631 (100); FAB-HRMS calcd for C H O [M+H] , 1341.5364; found,
8
8
77 13
341.5328.
[
4,8]-2,3-cis-3,4-trans:2,3-cis-Octa-O-benzyl-3-O-acetyl-bi-(-)-epicatechin (15). TMSOTf-catalyzed
condensation according to the above procedure using tetra-O-benzylated epicatechin (7) (101.3 mg,
0
0
.16 mmol) with (2R,3R,4S)-3-acetoxy-5,7,3',4'-tetrabenzyloxy-4-(2’’-ethoxyethoxy)flavan (10) (27 mg,
.035 mmol) in CH Cl (30 mL), TMSOTf (0.070 mL, 0.035 mmol, 0.5M solution in CH Cl ) at –78˚C
2
2
2
2
2
5
1
afforded 35 mg (75 %) of 15 as a colorless amorphous solid: [a]D = +25.6° (c 0.70, CHCl ); H-NMR
3
(
(
(
400 MHz, CDCl , 0.7 : 0.3 mixture of rotational isomers ) major isomer:ꢀ7.44-6.77 (30.8H, m), 6.80
3
0.7H, d, J = 8.3 Hz), 6.44 (0.7H, dd, J = 1.7, 8.3 Hz), 6.30 (0.7H, s), 6.01 (0.7H, d, J = 2.0 Hz), 5.67
0.7H, d, J = 2.0 Hz), 5.63 (0.7H, br s), 5.51 (0.7H, br s), 5.10-4.68 (10.5H, m), 4.64 (0.7H, d, J = 11.2
Hz), 4.44 (0.7H, d, J = 11.2 Hz), 4.05 (0.7H, s), 3.87 (0.7H, br s), 3.00-2.87 (1.4H, m), 1.70 (2.1H, s),
.48 (0.7H, br, OH); minor isomer:ꢀ7.44-6.77 (13.8H, m), 6.23 (0.3H, d, J = 1.9 Hz), 6.19 (0.3H, s), 6.12
0.3H, d, J = 1.9 Hz), 5.37 (0.3H, br s), 5.32 (0.3H, br s), 5.10-4.68 (4.8H, m), 4.64 (0.3H, d, J = 11.2 Hz),
.44 (0.3H, d, J = 11.2 Hz), 4.20-4.15 (0.3H, m), 3.00-2.87 (0.6H, m), 1.34 (0.9H, s), 1.21 (0.3H, br,
1
(
4
1
3
OH); C-NMR (100 MHz, CDCl , 0.7 : 0.3 mixture of rotational isomers ) major isomer:ꢀ169.0, 158.0,
3
1
56.6, 156.5, 156.0, 155.4, 154.4, 149.0, 148.6, 148.5, 148.2, 137.4, 137.34, 137.27, 137.17, 137.14,

1
1
3
1
32.1, 131.8, 131.2, 128.6-126.7 (Cx24), 119.9, 118.8, 114.74, 114.68, 114.5, 113.5, 112.2, 110.5, 104.7,
02.1, 93.9, 92.9, 91.4, 79.0, 77.2, 74.7, 72.1, 71.34, 71.26, 71.1, 70.5, 69.94, 69.88, 69.76, 69.1, 66.5,
3.2, 28.6, 20.8; minor isomer: 169.0, 158.3, 157.4, 156.5, 155.9, 155.7, 154.3, 148.8, 148.6, 148.4, 148.3,
37.34, 137.27, 137.14, 137.07, 136.8, 131.78, 131.70, 130.9, 128.6-126.9 (Cx24), 119.8, 119.7, 114.5,
113.7, 113.4 (x2), 111.5, 110.6, 104.8, 101.8, 94.5, 93.3, 92.6, 78.2, 76.8, 74.9, 72.4, 71.4, 71.3, 71.1, 70.5,
+
7
1
1
0.0, 69.9, 69.7, 69.5, 65.8, 33.4, 28.9, 20.3; FAB-MS (m/z) 1365 (30), 1364 ([M+Na] , 30), 1344 (21),
+
+
343 (30), 1342 ([M+H] , 40), 1281 (85), 1190 (56), 631 (100); FAB-HRMS calcd for C H O [M+H] ,
8
8
77 13
341.5364; found, 1341.5402.
[
4,8]-2,3-cis-3,4-trans:2,3-cis-Octa-O-benzyl-bi-(-)-epicatechin (16). TMSOTf-catalyzed condensation
according to the above procedure using tetra-O-benzylated epicatechin (7) (594 mg, 0.91 mmol) with
2R,3R,4S)-3-hydroxy-5,7,3',4'-tetrabenzyloxy-4-(2’’-ethoxyethoxy)flavan (9) (150 mg, 0.20 mmol) in
CH Cl (30 mL), TMSOTf (0.40 mL, 0.20 mmol, 0.5 M solution in CH Cl ) at –78˚C afforded 243 mg
(
2
2
2
2
3
0
1
(
93 %) of 16 as a colorless amorphous solid; [a]D = +40.6° (c 1.00, CHCl ); H-NMR (400 MHz, CDCl ,
3 3
0
.67 : 0.33 mixture of rotational isomers ) major isomer: 7.44-6.89 (28.81H, m), 6.88 (0.67H, d, J = 1.7
Hz), 6.80 (0.67H, d, J = 8.3 Hz), 6.43 (0.67H, dd, J = 1.7, 8.3 Hz), 6.34 (0.67H, s), 6.00 (0.67H, d, J = 2.2
Hz), 5.69 (0.67H, d, J = 2.2 Hz), 5.53 (0.67H, br s), 5.12-4.86 (8.71H, m), 4.80 (1.34H, s), 4.62 (0.67H, d,
J = 11.3 Hz), 4.46 (0.67H, d, J = 11.3 Hz), 4.10-4.04 (0.67H, m), 4.05 (0.67H, br s), 3.88-3.82 (0.67H, m),
2
1
.97 (0.67H, d, J = 18.0 Hz), 2.88 (0.67H, dd, J = 4.7, 18.0 Hz), 1.79 (0.67H, d, J = 5.6 Hz, OH), 1.47-
.45 (0.67H, br, OH); minor isomer: 7.44-6.89 (14.85H, m), 6.85 (0.33H, d, J = 8.3 Hz), 6.23 (0.33H, d, J
=
2.2 Hz), 6.18 (0.33H, s), 6.07 (0.33H, d, J = 2.2 Hz), 5.29 (0.33H, br s), 5.12-4.86 (4.95H, m), 4.63
(
(
(
0.33H, d, J = 12.0 Hz), 4.38 (0.33H, d, J = 12.0 Hz), 4.34-4.28 (0.33H, m), 4.10 (0.33H, br s), 3.98-3.94
0.33H, m), 3.04 (0.33H, d, J = 16.1 Hz), 2.99-2.94 (0.33H, m), 1.70 (0.33H, d, J = 6.1 Hz, OH), 1.32
1
3
0.33H, d, J = 7.8 Hz, OH); C-NMR (100 MHz, CDCl , 0.67 : 0.33 mixture of rotational isomers ) major
3
isomer: 158.1, 157.1, 156.5, 155.9, 155.0, 154.4, 148.7, 148.39, 148.37, 148.2, 137.27, 137.25 (x2),
1
1
2
1
37.20, 137.15 (x2), 132.6, 131.1, 128.6-126.7 (Cx27), 119.8, 118.8, 115.0 (x2), 113.4, 112.5, 111.2,
04.4, 102.3, 94.0, 93.1, 91.5, 78.8, 75.6, 72.4 71.3, 71.2, 70.5, 69.92, 69.90, 69.85, 69.0, 66.5, 35.8,
8.6; minor isomer: 158.3, 157.9, 156.8, 156.5, 155.5, 152.8, 149.1, 149.0, 148.9, 148.6, 137.38 137.35,
37.30, 137.1, 136.98, 136.94, 132.4, 131.2, 128.6-126.6 (Cx27), 119.9, 118.6, 114.9, 114.4, 114.1, 112.9,
1
2
1
11.6, 104.4, 101.7, 94.3, 93.3, 92.9, 78.1 77.2, 72.1, 71.5, 71.3, 70.8, 69.9, 69.7, 69.5, 65.1, 60.3, 35.9,
+
+
8.7; FAB-MS (m/z) 1302 (45), 1301 (97), 1300 ([M+H] , 100); FAB-HRMS calcd for C H O [M+H] ,
8
6
75 12
299.5259; found, 1299.5229.
[
4,8]-2,3-trans-3,4-trans:2,3-cis-Octa-O-benzyl-3-O-acetyl-(+)-catechin-(-)-epicatechin (19). TMSOTf-
catalyzed condensation according to the above procedure using tetra-O-benzylated epicatechin (7) (420 mg,

0
0
.65 mmol) with (2R,3S,4S)-3-acetoxy-5,7,3', 4'-tetrabenzyloxy-4-(2’’-ethoxyethoxy)flavan (12) (112 mg,
.14 mmol) in CH Cl (30 mL), TMSOTf (0.28 mL, 0.14 mmol, 0.5 M solution in CH Cl ) at –78˚C
2
2
2
2
3
0
1
afforded 169 mg (90 %) of 19 as a colorless amorphous solid: [a]D = -75.8° (c 1.00, CHCl ); H-NMR
3
(
(
400 MHz, CDCl , 0.67 : 0.33 mixture of rotational isomers ) major isomer: 7.46-6.72 (28.81H, m), 7.06
3
0.67H, d, J = 1.7 Hz), 6.77 (0.67H, d, J = 8.3 Hz), 6.73 (0.67H, dd, J = 1.7, 8.3 Hz), 6.213 (0.67H, d, J =
2
.2 Hz), 6.210 (0.67H, s), 6.13 (0.67H, d, J = 2.2 Hz), 5.79 (0.67H, dd, J = 9.5, 10.3 Hz), 5.21 (0.67H, d,
J = 12.0 Hz), 5.19-4.76 (8.04H, m), 5.16 (0.67H, d, J = 12.0 Hz), 4.85 (0.67H, d, J = 9.5 Hz), 4.70 (0.67H,
d, J = 10.3 Hz), 4.60 (0.67H, d, J = 10.3 Hz), 4.48 (0.67H, d, J = 10.3 Hz), 3.94-3.87 (0.67H, m), 3.56
(
(
0.67H, br s), 2.80 (0.67H, d, J = 17.3 Hz), 2.55 (0.67H, dd, J = 4.6, 17.3 Hz), 1.58 (2.01H, s), 1.28
0.67H, br, OH); minor isomer: 7.46-6.72 (14.19H, m), 6.76-3.73 (0.66H, m), 6.60 (0.33H, dd, J = 1.7,
8
.3 Hz), 6.24 (0.33H, d, J = 2.4 Hz), 6.22 (0.33H, d, J = 2.4 Hz), 5.93 (0.33H, dd, J = 9.6, 10.3 Hz), 5.90
(
(
0.33H, s), 5.19-4.55 (5.94H, m), 4.99-4.97 (0.33H, m), 4.78-4.76 (0.33H, m), 3.01-2.91 (0.66H, m), 1.50
1
3
0.99H, s), 1.37 (0.33H, br, OH); C-NMR (100 MHz, CDCl , 0.67 : 0.33 mixture of rotational isomers )
3
major isomer: 169.2, 158.1, 157.8, 156.6, 156.2, 155.7, 153.2, 149.0, 148.9, 148.7, 148.4, 137.4, 137.4,
37.23, 137.19, 137.13, 136.9, 136.7, 132.4, 128.7-127.0 (Cx26), 121.2, 119.0, 115.0, 114.5, 113.8, 113.3,
11.0, 108.4, 100.4, 94.8, 94.3, 91.3, 80.2, 76.9, 73.2, 71.4, 71.3, 71.2, 71.1, 70.2, 70.1, 70.0, 69.9, 65.8,
1
1
3
1
5.1, 28.5, 20.6; minor isomer: 169.1, 158.3, 158.1, 156.7, 156.3, 156.0, 153.4, 148.94, 148.85, 184.4,
41.9, 138.0, 137.31, 137.26, 137.2, 137.1, 136.9, 136.7, 130.8, 128.7-126.9 (Cx26), 120.9, 119.6, 114.6,
113.9, 113.8, 113.3, 110.1, 108.0, 102.1, 95.2, 94.7, 91.5, 79.9, 78.2, 77.2, 71.7, 71.1, 71.0, 70.8, 70.6,
+
7
0.3, 69.9, 69.6, 66.8, 35.2, 28.7, 20.4; FAB-MS (m/z) 1364 ([M+Na] , 12), 1344 (8), 1343 (15), 1342
+
+
(
[M+H] , 17), 1281 (41), 1190 (40), 631 (100); FAB-HRMS calcd for C H O [M+H] , 1341.5364;
88 77 13
found, 1341.5319.
Procyanidin B2 (2).ꢀA solution of 145 mg (0.11 mmol) of 16 in THF/MeOH/H O, 20/1/1 (20 mL) was
2
hydrogenated over 20% Pd(OH) /C (14 mg) for 12 h in a current of H at rt. Filtration and concentration
2
2
afforded a pale brown oil, which was purified by Sephadex® LH-20 column chromatography (EtOH) to
2
7
t
give pure procyanidin-B2 (2) (55 mg, 86 %) as a pale brown powder: HPLC R-2, 19.4 min; [a]
=
D
1
6
20
1
+
21.5° (c 0.58, EtOH) [lit., [a]
= +15.2° (c 1.2, EtOH)]; H-NMR (400 MHz, CD OD, all the NMR
78 3
5
peaks were broadened and the discrimination of rotational isomers was impossible) 7.10 (1H, br s), 6.90
1H, br s), 6.78-6.62 (4H, m), 6.05-5.80 (3H, m), 5.06-4.95 (1H, m), 5.00-4.90 (1H, m), 4.62 (1H, br s),
.35-4.20 (1H, m), 3.90 (1H, br s), 2.90-2.83 (1H, m), 2.82-2.70 (1H, m); FAB-MS (m/z) 602 (5), 601
(
4
+
+
+
(
[M+Na] , 13), 580 (3), 579 ([M+H] , 8), 176 (100); FAB-HRMS calcd for C H O Na [M+Na] ,
30 26 12
6
01.1322; found, 601.1359.
Decaacetylprocyanidin-B2 (Ac -2). Acetylation of 2 (25 mg, 0.043 mmol) with Ac O (81 mL, 0.86
1
0
2

mmol) and 4-(dimethylamino)pyridine (1 mg, 0.008 mmol) in Py (5 mL) afforded 27 mg (63 %) of Ac -2
1
0
2
6
1
as a colorless amorphous solid:[a]D = +24.7° (c 0.52, CHCl ); H-NMR (400 MHz, CDCl , 0.7 : 0.3
3
3
mixture of rotational isomers) major: 7.36 (0.7H, d, J = 2.0 Hz), 7.30-7.25 (0.7H, m), 7.18 (0.7H, d, J =
.5 Hz), 7.04 (0.7H, d, J = 8.5 Hz), 7.01 (0.7H, d, J = 2.0 Hz), 6.89 (0.7H, dd, J = 2.0, 8.5 Hz), 6.66 (0.7H,
s), 6.24 (0.7H, d, J = 2.2 Hz), 5.99 (0.7H, d, J = 2.2 Hz), 5.57 (0.7H, s), 5.17-5.16 (0.7H, m), 5.12-5.09
0.7H, m), 4.54 (0.7H, s), 4.46 (0.7H, d, J = 1.7 Hz), 2.93 (0.7H, dd, J = 4.9, 18.3 Hz), 2.86 (0.7H, d, J =
8.3 Hz), 2.36 (2.1H, s), 2.30-2.27 (10.5H, m), 2.19 (2.1H, s), 2.05 (2.1H, s), 1.99 (2.1H, s), 1.88 (2.1H,
8
(
1
s); minor isomer: 7.30-7.25 (0.6H, m), 7.22 (0.3H, dd, J = 2.0, 8.5 Hz), 7.19-7.16 (0.6H, m), 7.13 (0.3H,
d, J = 8.5 Hz), 6.77 (0.3H, d, J = 2.2 Hz), 6.63 (0.3H, d, J = 2.2 Hz), 6.58 (0.3H, s), 5.55-5.52 (0.3H, m,),
5
2
.40 (0.3H, br s), 5.34-5.32 (0.3H, m), 5.26 (0.3H, br s), 4.64 (0.3H, s), 3.07 (0.3H, dd, J = 4.4, 17.8 Hz),
.98 (0.3H, d, J = 17.8 Hz), 2.31 (0.9H, s), 2.30-2.27 (2.7H, m), 2.27 (0.9H, s), 2.25 (0.9H, s), 2.02 (0.9H,
1
3
s), 1.86 (0.9H, s), 1.74 (0.9H, s), 1.56 (0.9H, s); C-NMR (100 MHz, CDCl , 0.7 : 0.3 mixture of
3
rotational isomers) major: 170.4, 170.0, 169.7, 169.0, 168.9, 168.3, 168.2, 168.0, 167.87, 167.85, 155.3,
1
1
2
1
1
7
54.2, 149.1, 149.0, 147.9, 147.8, 142.0, 141.9, 141.8, 141.6, 136.5, 134.4, 125.0, 124.4, 123.1, 122.8,
22.4, 122.2, 116.7, 111.7, 111.5, 110.3, 108.6, 107.2, 77.2, 73.6, 71.0, 66.8, 33.9, 26.6, 21.2, 20.95,
0.88, 20.7, 20.64. 20.60 (x2), 20.5, 20.2, 19.9; minor isomer: 170.2, 170.0, 169.7, 169.0 (x2), 168.4,
68.11, 168.07, 167.97. 167.87, 154.9, 151.8, 149.9, 149.7, 148.5, 147.5, 142.1, 142.0, 141.9, 141.7,
36.6, 135.6, 124.3, 123.7, 123.2, 123.1, 121.8, 121.4, 117.5, 111.5, 110.9, 109.8, 109.0, 108.1, 76.7, 74.3,
0.5, 66.3, 34.1, 26.3, 21.2, 21.0, 20.9, 20.74, 20.72, 20.60, 20.56, 20.4, 20.2, 19.9; FAB-MS (m/z) 1023
+
+
(
[
5), 1022 (13), 1021 ([M+Na] , 26), 1000 ([M+H] , 1), 632 (100); FAB-HRMS calcd for C H O Na
50 46 22
+
M+Na] , 1021.2378; found, 1021.2363.
ACKNOWLEDGEMENT
We thank the Japan Society for the Promotion of Science (JSPS) for the Young Scientists Research
Fellowship (to A. S.).
REFERENCE AND NOTES
1
.
Synthetic studies of procyanidins. Part 4. See A. Saito, N. Nakajima, A. Tanaka, and M. Ubukata,
Heterocycles, 2003, 61, accepted.
2
3
4
5
6
7
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J. I. Malone, S. Lowitt, and W. R. Cook, Pediatr. Res., 1990, 27, 293.

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9
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Y. Morimitsu, K. Yoshida, S. Esaki, and A. Hirota, Biosci. Biotechnol. Biochem., 1995, 59, 2018.
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2
418.
0. J. B. Harborne, The Flavonoids: Advances in research from 1986, Chapman and Hall, London,
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1
1
1
1
1
1. J. B. Harborne and H. Baxter, The Handbook of Natural Flavonoids, John Wiley & Sons, NY, 1999.
2. D. Ferreira and X. -C. Li, Nat. Prod. Rep., 2000, 17, 193.
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69.
1
1
1
4. T. Ariga, I. Koshiyama, and D. Fukushima, Agric. Biol. Chem., 1988, 52, 2717.
5. J. Zhao, J. Wang, Y. Chen, and R. Agarwal, Carcinogenesis, 1999, 20, 1737.
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Chem. Soc., Perkin Trans. 1, 1972, 1387; R. Eastmond, J. Inst. Brew., 1974, 188, 423; H. Kolodziej,
Phytochemistry, 1985, 24, 2460; G. Fonknechten, M. Moll, D. Cagniant, G. Kirsch, and J. F. Muller,
J. Inst. Brew., 1983, 89, 423; S. Schleep, H. Friedrich, and H. Kolodziej, J. Chem. Soc., Chem.
Commun., 1986, 392; M. D. Barkley, R. W. Hemingway, and W. L. Mattice, J. Am. Chem. Soc.,
1
987, 109, 6614; H. Kolodziej, Phytochemistry, 1990, 955; T. Hatano and R. W. Hemingway, J.
Chem. Soc., Perkin Trans. 2, 1997, 37, 488; K. Weinges, H. Schick, and F. Rominger, Tetrahedron,
001, 57, 2327. Dimer synthesis: A. C. Fletcher, L. J. Porter, E. Haslam, and R. K. Gupta, J. Chem.
2
Soc., Perkin Trans. 1. 1977, 1682; H. Kawamoto, F. Nakatsubo, and K. Murakami, Mokuzai
Gakkaishi, 1991, 37, 488.
1
1
7. M-C. Pierre, C. Cheze, and J. Vercauteren, Tetrahedron Lett., 1997, 38, 5639.
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A. Saito, N. Nakajima, A. Tanaka, and M. Ubukata, Tetrahedron, 2002, 58, 7829.
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and E. V. Brandt, Tetrahedron, 1992, 48, 1743.
1
2
2
0. A. Saito. N. Nakajima, A. Tanaka, and M. Ubukata, Tetrahedron Lett., 2003, 44, 5449.
1. Previously, the Tückmantel group reported the epicatechin and epicatechin condensation. Under the
conditions, a mixture of a dimer, two trimers, and a tetramer were obtained in 53 %, 22 %, 4.5 %,
and 1.5 % yields, respectively. W. Tückmantel, A. P. Kozikowski, and L. J. Romaczyk, Jr., J. Am.
Chem. Soc., 1999, 121, 12073; A. P. Kozikowski, W. Tückmantel, and Y. Hu, J. Org. Chem., 2001,
6
6, 1287; A. P. Kozikowski, W. Tückmantel, and Y. Hu, J. Org. Chem., 2003, 68, 1641.
2
2. The neighboring group participation at C3-position of catechin electrophile was important for 3,4-
trans selective condensation. The condensation of 8 and 7 with 3-hydroxy catechin electrophile

1
8
17
(
11) gave a mixture of 3,4-trans and 3,4-cis dimer products in 2/1 and 8/1 ratio.
2
2
3. R. W. Hemingway, L. Y. Foo, and L. J. Porter, J. Chem. Soc., Perkin Trans. 1, 1982, 1209.
4. K. M. Lokman, E. Haslam, and M. P. Williamson, Magnetic Resonance in Chemistry, 1997, 35,
8
54.
1
7
2
2
5. The acetyl group of 17 was hydrolyzed with 10 equivalent of KCN in 95% EtOH to give 18 in
1
9
8 % yield. And the acetyl group of 19 was removed by DIBAL treatment to give 20 in 88 %
yield.
6. N. Matsuura, T. Aradate, C. Sasaki, H. Kojima, M. Ohara, J. Hasegawa, and M. Ubukata, J. Health
Science, 2002, 48, 520.