HETEROCYCLES

Paper | Special issue | Vol. 79, No. 1, 2009, pp. 549-563
Received, 28th August, 2008, Accepted, 15th October, 2008, Published online, 16th October, 2008.
DOI: 10.3987/COM-08-S(D)14

■ An Efficient Synthesis of Procyanidins Using Equimolar Condensation of Catechin and/or Epicatechin Catalyzed by Ytterbium Triflate

Yoshihiro Mohri, Masayoshi Sagehashi, Taiji Yamada, Yasunao Hattori, Keiji Morimura, Yasunori Hamauzu, Tsunashi Kamo, Mitsuru Hirota, and Hidefumi Makabe*

Shinshu University, 8304 Minamiminowa-mura, Kamiina-gun, Nagano 399-4598, Japan

Abstract

Stereoselective synthesis of catechin and epicatechin dimers under intermolecular condensation of equimolar amount of catechin derivatives catalyzed by Yb(OTf)3. The coupled products were successfully converted to procyanidins B1, B2, B3, and B4, respectively. Procyanidins B1, B2, B3, and B4 could be used as standard compounds for identifying the polyphenols in natural source.

INTRODUCTION
†Dedicated to the late Professor John W. Daly for his outstanding contribution to natural product chemistry.
Proanthocyanidins are known as condensed or noncondensed hydrolysable tannins.1 These condensed tannins can be found in the vegetables kingdom.2 In particular, they exist in grape seeds and skins and red wines. Many biological activities, mainly a powerful free-radical scavenging activity, have been reported for flavonoids, and their investigation is increasingly important. Especially, procyanidins were paid attention since the relationship between procyanidin content and vasoactive properties of red wine have been reported by Coder and co-workers in 2006.3 Tannin extracts from plants give various types of polyphenol. Because their identification as well as purification is extremely difficult, further studies of proanthocyanidins remains. Recently, to obtain procyanidin oligomers in pure state, synthetic efforts were devoted.4 However, efficient syntheses are very limited because the formation of the intermolecular C-4-C-8 bond has some problems. The typical synthetic methods are as follows. The first example is nucleophilic addition of C-8 lithiated nucleophile onto a C-4 protected ketocatechin as a substrate.5 This reaction generally proceeds with the regioselective and oligomerization control demands of the coupling reaction, however, it does not satisfy the stereochemical requirement of the newly formed C-4 asymmetric center. The next is the nucleophilic substitution method which needs to use nucleophilic partner in large excess (3.0-4.5 eq.) to prevent further oligomerization. Thus the efficient synthetic method to prepare procyanidin dimers has some restrictions, although recent advance was made in the regio and stereoselective reaction.4 Until now, only a few attempts to prepare procyanidin dimers under stoichiometric conditions have been reported in the literature. The first example is an intramolecular coupling of monomeric units bound by a temporary diester link.6 This method is suitable for synthesizing procyanidin B1 (1) and B3 (3), however, it suffers from low yield of condensation for synthesizing B2 (2) and B4 (4). The second is reported by E. Fouquet and co-workers.7 They synthesized procyanidin dimers based on the intermolecular nucleophilic substitution of C-4 activated and C-8 halogenated monomer to prevent further oligomerization using TiCl4 as a Lewis acid. This reaction needs large excess of TiCl4. Quite recently, Oyama and co-workers reported the efficient synthesis of procyanidin B3. They use 4-acetoxy-3-O-acetyl-perbenzylcatechin as an electrophile. They made success to reduce the amount of nucleophile up to 1.5 eq.4e In the course of our research, we have developed a very simple and efficient intermolecular synthesis of procyanidin dimers.8 The key step is a coupling reaction between equimolar amounts of tetra-benzylated monomer 5a (nucleophile) and a C-4 activated monomer 6a (electrophile) using 1.0 eq. of rare earth metal Lewis acid such as Yb(OTf)3 (Figure 1).

RESULTS AND DISCUSSION
We chose tetrabenzylated catechin 5a, a nucleophilic unit, prepared by the Kawamoto’s procedure 9 and
electrophile unit 6a prepared by the Saito’s method.10 Equimolar condensation of 5a and 6a at rt was examined using various Lewis acids including rare earth metal in CH2Cl2 (Table 1).

The first attempt at the coupling reaction was conducted with equimolar amounts of the protected catechin 5a and the acetylated substrate 6a to obtain α-7, which is the precursor of procyanidin B3 (3). Typical Lewis acids, such as TiCl4 and BF3•Et2O gave sluggish results. These reactions required a large excess of the nucleophile at low temperature in order to limit the reaction of the activated monomer with itself or with the dimeric product leading in both cases to oligomeric side products.9,10 The next attempt at the coupling reaction was conducted with late transition metals as Lewis acids. Among Ag, Cu, and In, especially AgBF4 gave a good selectivity with moderate chemical yield.11 We further paid attention to rare metal Lewis acids such as Sc and La. While Sc gave poor stereoselectivity, La afforded high selectivity although the chemical yield was 34%. This result encouraged us to replace La to Yb. The reaction furnished good selectivity with 64% yield. When reaction time was longer than 0.5 h, further condensed products were observed. The catalytic amount of Yb(OTf)3 (10 mol%) also afforded coupled product in 42% yield at 91:9 ratio of the desired product. This result indicates that this reaction could be carried out using catalytic amount of Yb(OTf)3. Interestingly, Gd(OTf)3 and Lu(OTf)3 did not give any condensed product. The reported condensation reaction between catechin nucleophile 5a and catechin electrophile 6a required large amount excess of catechin nucleophile 5a to obtain desired dimer in high yield.10,12 Using large excess amount of nucleophile is a big problem because composition of desired coupled product is only a small part in the reaction system and it is necessary to get rid of large amount of starting material. Optimized equimolar condensation is extremely important for an efficient synthesis of catechin dimers. Next, we examined the condensation of the combination of catechin nucleophile 5a and epicatechin nucleophile 5b with catechin electrophile 6a and/or epicatechin electrophile 6b using Yb(OTf)3 as a Lewis acid. In each case, the reaction worked well. As to the stereoselectivty, however, the epicatechin nucleophile 5b gave a little bit poor results compared to catechin nucleophile 5a. In case of tri-benzylated phloroglucinol, the stereoselectivity of 11 showed 75:25 ratio.13 Some stereochemical requirement seems to be necessary to get high selectivity (Scheme 1).

Finally, condensed compounds α-7, β-8, β-9, and α-10 were subjected to the hydrolysis of the acetate with K2CO3 in MeOH followed by debenzylidation by Pd(OH)2 in THF-MeOH-H2O catalyzed hydrogenolysis to give procyanidins B1 (1)-B4 (4). All the spectral data for 1-4 were similar to those of the reported values (Scheme 2).4a, 4d, 6a

To characterize procyanidins in daily diet and evaluate their biological activities, it is necessary to obtain pure procyanidins. Synthetic procyanidins can be used as procyanidin standards. Recently, Hamauzu and co-workers reported the antiulceractive activity by apple phenolics in case of HCl/ethanol-induced ulcers.15 Thus, the synthesized authentic samples 1-4 were used to clarify the structure of procyanidins from juice of apple. As shown in Figure 2 and 3, procyanidin B2 was identified as a major constituent of procyanidin dimer by HPLC analysis (Figures 2, 3).16

Conclusion
In conclusion, we have synthesized procyanidins B1-B4 (1-4) based on a Yb(OTf)3 catalyzed equimolar condensation. The synthesis of various procyanidin oligomers according to the above-mentioned method and the structural identification of polyphenols from natural source are in progress.

EXPERIMENTAL
General
All melting points were uncorrected. 1H and 13C NMR spectra were measured with a Bruker DRX 500 FT-NMR spectrometer in CDCl3 at 500 and 125 MHz, respectively. Chemical shifts were relative to tetramethylsilane as an internal standard. The coupling constants were given in Hz. Mass spectra were obtained on JEOL JMS-700 mass spectrometer. IR spectra were recorded with JASCO FT-IR 480 Plus infrared spectrometer. Optical rotations were determined with a JASCO DIP-1000 polarimeter.

Representative procedure for Yb(OTf)3 catalyzed condensation; [4, 8’]-2, 3-trans-3, 4-trans : 2’, 3’-trans-Octa-O-benzyl-3-O-acetyl-bi-(+)-catechin (α-7). To a solution of nucleophile 5a (190 mg, 0.263 mmol) and electrophile 6a (171 mg, 0.263 mmol) in CH2Cl2 (10 mL) under an argon atmosphere was added Yb(OTf)3 (163 mg, 0.263 mmol). After the resulting mixture had been stirred for 2 h, the reaction was quenched with water. The mixture was extracted with Et2O, and the combined organic layer were washed with brine, dried over MgSO4, and concentrated. The crude product was purified with silica gel chromatography (hexane:AcOEt:CH2Cl2 = 4:1:2) to give diastereomeric mixture α-7 and β-7 (226 mg, 64%) as a colorless oil. 1H NMR analysis of diacetate derivative showed more than 98:2 ratio of α-7 and β-7.4b, 9 The selectivity was determined by 1H NMR analysis of C-3 position of diacetate derivative of α-7 (5.80 and 5.83 ppm) and β-7 (5.53 and 5.58 ppm) according to the reported procedure.4b Further purification with preparative TLC (hexane:AcOEt:CH2Cl2 = 4:1:2) gave α-7. [α]D24 −118 (c 1.36, CHCl3). IR (film) νmax cm-1: 3523, 3087, 2909, 2870, 1731, 1607, 1511, 1498, 1454, 1428, 1373, 1306, 1264, 1231, 1139, 1113, 1063, 1027, 911, 849, 809, 735, 697, 606. 1H NMR (500 MHz, CDCl3, 1:1 mixture of rotational isomers): δ = 7.49-7.12 (40H, m), 6.98-6.67 (5.5H, m), 6.43 (0.5H, dd, J = 1.7, 8.3 Hz), 6.25 (0.5H, s), 6.23 (0.5H, d, J = 2.3 Hz), 6.20 (0.5H, d, J = 2.3 Hz), 6.14 (0.5H, d, J = 2.3 Hz), 6.11 (0.5H, d, J = 2.3 Hz), 5.99 (0.5H, s), 5.98 (0.5H, t, J = 9.6 Hz), 5.86 (0.5H, t, J = 9.6 Hz), 5.22-4.54 (18.5H, m), 3.93 (0.5H, m), 3.58 (0.5H, m), 3.37 (0.5H, d, J = 8.8 Hz), 3.04 (0.5H, dd, J = 5.7, 16.2 Hz), 2.86 (0.5H, dd, J = 5.0, 16.5 Hz), 2.73 (0.5H, dd, J = 7.0, 16.5 Hz), 2.35 (0.5H, dd, J = 9.6, 16.1 Hz), 2.17 (0.5H, m), 1.62 (1.5H, s), 1.54 (1.5H, s), 1.33 (0.5H, d, J = 2.3 Hz). 13C NMR (125 MHz, CDCl3, 1:1 mixture of rotational isomers): δ = 158.20, 158.10, 157.79, 156.77, 156.72, 156.54, 155.89, 155.82, 155.54, 153.69, 152.53, 149.25, 149.04, 148.99, 148.94, 148.88, 148.84, 148.61, 137.95, 137.36, 137.31, 137.28, 137.26, 137.23, 137.07, 137.03, 136.97, 136.84, 136.59, 131.78, 131.59, 130.92, 128.51, 128.48, 128.44, 128.40, 128.36, 128.33, 128.31, 128.28, 128.11, 128.04, 127.96, 127.82, 127.76, 127.69, 127.60, 127.52, 127.47, 127.46, 127.43, 127.33, 127.28, 127.26, 127.19, 127.17, 127.09, 126.97, 126.85, 121.27, 120.92, 120.30, 120.05, 114.96, 114.79, 114.61, 114.45, 114.19, 114.07, 113.67, 110.74, 110.27, 108.29, 107.94, 102.34, 102.25, 94.91, 94.85, 94.42, 94.33, 91.24, 91.19, 80.64, 80.19, 80.10, 79.86, 71.57, 71.50, 71.35, 71.27, 71.15, 70.39, 70.26, 70.01, 69.98, 69.76, 68.84, 67.75, 35.40, 35.16, 28.62, 26.49, 20.65, 20.40. FAB-HRMS: Calcd for C88H77O13 [M+H]+, 1341.5364; found, 1341.5381.

[4, 8’]-2, 3-cis-3, 4-trans : 2’, 3’-trans-Octa-O-benzyl-3-O-acetyl-(−)-epicatechin-(+)-catechin (β-8).
In the same manner as prepared α-7, compounds 5a (72 mg, 0.11 mmol) and 6b (80 mg, 0.11 mmol) gave α-8 and β-8 (92 mg, 62%, α-8:β-8 = 9:91) in 62% yield. Further purification with preparative TLC (hexane:AcOEt:CH2Cl2 = 4:1:2) gave pure β-8.4d [α]D18 +66.0 (c 1.47, CHCl3). IR (film) νmax cm-1: 3568, 3062, 3031, 2927, 2870, 1740, 1594, 1513, 1498, 1454, 1425, 1376, 1331, 1265, 1216, 1119, 1073, 1028, 910, 851, 809, 737, 697, 625. 1H NMR (500 MHz, CDCl3, 0.71:0.29 mixture of rotational isomers): δ = 7.43-6.75 (43.29H, m), 6.51-6.49 (0.71H, m), 6.30 (0.71H, s), 6.22 (0.29H, s), 6.18 (0.29H, s), 6.12 (0.29H, s), 6.03 (0.71H, d, J = 2.0 Hz), 5.54 (0.71H, d, J = 2.0 Hz), 5.52 (0.71H, s), 5.45 (0.71H, s), 5.37 (0.29H, s), 5.26 (0.29H, s), 5.08-4.51 (20.58H, m), 3.78 (0.71H, dd, J = 9.1, 16.0 Hz), 3.65 (0.71H, d, J = 9.1 Hz), 3.24 (0.71H, dd, J = 6.5, 16.8 Hz), 2.68 (0.29H, dd, J = 9.6, 16.5 Hz), 1.69 (2.13H, s), 1.63 (0.29H, br, OH), 1.53 (0.87H, s), 1.27 (0.71H, d, J = 12.4 Hz, OH). 13C NMR (125 MHz, CDCl3, 0.71:0.29 mixture of rotational isomers): δ = 169.06, 158.07, 156.46, 156.12, 156.02, 155.56, 154.37, 149.33, 149.04, 148.85, 148.69, 137.39, 137.32, 137.22, 137.07, 132.18, 130.44, 128.93, 128.61, 128.58, 128.44, 128.40, 128.37, 128.31, 128.17, 128.07, 127.93, 127.72, 127.67, 127.56, 127.50, 127.42, 127.35, 127.31, 127.22, 127.19, 127.17, 127.09, 127.03, 120.63, 119.81, 114.76, 114.66, 114.19, 113.67, 112.10, 110.66, 104.47, 104.13, 93.62, 92.85, 91.51, 81.78, 79.18, 74.53, 72.09, 71.49, 71.35, 71.09, 70.86, 70.64, 70.35, 69.98, 69.63, 69.29, 68.67, 33.31, 29.67, 29.09, 20.73. HRFABMS calcd for C88H76O13Na [M+Na]+; 1363.5183; found 1363.5138.

[4, 8’]-2, 3-cis-3, 4-trans : 2’, 3’-cis-Octa-O-benzyl-3-O-acetyl-bi-(−)-epicatechin (β-9). In the same manner as prepared α-7, compounds 5b (36 mg, 0.05 mmol) and 6b (33 mg, 0.050 mmol) gave α-9 and β-9 (51 mg, 76%, α-9:β-9 = 12:88). Further purification with preparative TLC (hexane:AcOEt:CH2Cl2 = 4:1:2) gave pure β-9.4d [α]D18 +39.1 (c 2.59, CHCl3). IR (film) νmax cm-1: 3528, 3092, 2914, 2875, 1736, 1612, 1516, 1503, 1459, 1433, 1378, 1311, 1269, 1236, 1143, 1118, 1068, 1032, 916, 854, 814, 740, 702, 611. 1H NMR (500 MHz, CDCl3, 0.82:0.18 mixture of rotational isomers): δ = 7.43-6.80 (43H, m), 6.44 (0.82H, d, J = 8.1 Hz), 6.30 (0.82H, s), 6.27 (0.18H, d, J = 1.92 Hz), 6.23 (0.18H, s), 6.19 (0.18H, s), 6.12 (0.18H, d, J = 1.0 Hz), 6.01 (0.82H, br), 5.68 (0.82H, br), 5.63 (0.82H, br), 5.51 (0.82H, br), 5.37 (0.18H, br), 5.33 (0.18H, br), 5.17-4.75 (20.18H, m), 4.64 (0.82H, d, J = 10.9 Hz), 4.46 (0.82H, d, J = 11.3 Hz), 4.21-4.04 (0.36H, m), 3.88 (0.82H, br), 3.02-2.88 (2H, m), 1.70 (2.46H, s), 1.65 (0.18H, d, J = 4.55 Hz, OH), 1.49 (0.82H, br, OH), 1.35 (0.54H, s). 13C NMR (125 MHz, CDCl3, 0.82:0.18 mixture of rotational isomers): δ = 158.33, 158.05, 156.67, 156.54, 156.02, 155.45, 155.28, 154.43, 149.05, 148.92, 148.71, 148.62, 148.29, 137.41, 137.37, 137.33, 137.30, 137.19, 137.10, 136.96, 132.16, 131.27, 128.60, 128.57, 128.51, 128.47, 128.45, 128.38, 128.14, 128.01, 127.92, 127.83, 127.79, 127.72, 127.67, 127.64, 127.52, 127.50, 127.43, 127.35, 127.32, 127.26, 127.18, 127.14, 126.98, 126.72, 125.85, 119.99, 119.84, 119.53, 118.91, 115.19, 114.82, 114.60, 113.83, 113.66, 112.58, 110.60, 104.69, 102.16, 94.77, 94.04, 93.35, 92.98, 92.75, 91.55, 79.07, 78.37, 74.94, 74.75, 72.18, 71.54, 71.43, 71.36, 71.23, 71.17, 70.59, 70.16, 69.99, 69.81, 69.58, 69.22, 66.56, 66.34, 65.81, 33.32, 28.73, 20.74, 20.29. HRFABMS calcd for C88H77O13 [M+H]+; 1341.5364; found 1341.5391.

[4, 8’]-2, 3-trans-3, 4-trans : 2’, 3’-cis-Octa-O-benzyl-3-O-acetyl-(+)-catechin-(−)-epicatechin (α-10). In the same manner as prepared α-7, compounds 5b (42 mg, 0.064 mmol) and 6a (46 mg, 0.064 mmol) gave α-10 and β-10 (56 mg, 59%, α-10:β-10 = 98:2). Further purification with preparative TLC (hexane:AcOEt:CH2Cl2 = 4:1:2) gave pure β-10.4d [α]D18 −78.0 (c 0.52, CHCl3). IR (film) νmax cm-1: 3568, 3062, 3031, 2869, 1741, 1607, 1511, 1454, 1429, 1373, 1265, 1227, 1139, 1113, 1028, 911, 849, 736, 697, 619. 1H NMR (500 MHz, CDCl3, 0.67:0.33 mixture of rotational isomers): δ = 7.44-6.77 (44.65H, m), 6.60 (0.33H, dd, J = 1.5, 8.3 Hz), 6.23-6.21 (2.01H, m), 6.13 (0.67H, d, J = 2.3 Hz), 5.92-5.90 (0.67H, m), 5.80-5.76 (0.67H, m), 5.22-4.47 (29.37H, m), 4.08 (0.33H, br), 3.91(0.67H, br), 3.59 (0.67H, br), 2.99-2.91 (0.67H, m), 2.80 (0.67H, d, J = 16.8 Hz), 2.56 (0.67H, dd, J = 4.5, 17.1 Hz), 1.57 (2.01H, s), 1.50 (0.99H, s), 1.44 (0.33H, d, J = 6.3 Hz), 1.36 (0.67H, d, J = 5.8 Hz). 13C NMR (125 MHz, CDCl3, 0.67:0.33 mixture of rotational isomers): δ = 169.21, 158.30, 158.14, 157.82, 156.66, 156.31, 156.22, 156.05, 155.78, 153.21, 149.06, 149.02, 148.94, 148.81, 148.47, 137.43, 137.39, 137.36, 137.31, 137.29, 137.25, 137.19, 137.13, 137.08, 136.94, 136.56, 132.45, 131.03, 131.93, 128.67, 128.55, 128.51, 128.49, 128.47, 128.38, 128.33, 128.11, 128.07, 127.90, 127.88, 127.84, 127.77, 127.68, 127.62, 127.58, 127.52, 127.47, 127.44, 127.34, 127.28, 127.25, 127.11, 127.02, 126.88, 121.28, 120.92, 119.75, 119.03, 115.11, 114.83, 114.78, 114.01, 113.52, 111.10, 110.59, 108.48, 108.05, 102.17, 100.48, 94.92, 94.34, 91.61, 91.39, 80.19, 79.94, 78.24, 73.26, 71.75, 71.46, 71.38, 71.28, 71.22, 71.15, 70.87, 70.66, 70.36, 70.23, 70.19, 70.01, 69.95, 69.63, 66.79, 65.86, 35.30, 35.12, 28.59, 20.64, 20.39. HRFABMS calcd for C88H76O13Na [M+Na]+; 1363.5183; found 1363.5146.

[4, 8’]-2, 3-cis-3, 4-trans : 2’, 3’-trans-Octa-O-benzyl-(−)-epicatechin-(+)-catechin (12). To a solution of β-8 (78 mg, 0.057 mmol) in MeOH (10 mL) was added K2CO3 (156 mg, 1.10 mmol). After being stirred for 12 h at 60 oC, the mixture was diluted with H2O and extracted with diethyl ether. The organic layer was washed with water, brine, and dried with MgSO4. The solvent was evaporated and the residue was purified with preparative TLC (hexane:AcOEt: CH2Cl2 = 4:1:2) to afford 12 (60 mg, 82%) as an amorphous solid.6a [α]D17 +54.6 (c 1.03, CHCl3). IR (film) νmax cm-1: 3563, 3457, 3031, 2908, 1727, 1594, 1513, 1497, 1454, 1423, 1379, 1265, 1214, 1118, 1072, 1027, 910, 851, 808, 736, 696, 625. 1H NMR (500 MHz, CDCl3, 0.71 : 0.29 mixture of rotational isomers): δ = 7.49-7.10 (36.29H, m), 7.06-6.87 (6H, m), 6.79-6.78 (1H, m), 6.75-6.72 (1H, m), 6.49 (0.71H, dd, J = 1.7, 8.1 Hz), 6.34 (0.71H, s), 6.21 (0.29H, d, J = 2.2 Hz), 6.17 (0.29H, s), 6.05 (0.29H, d, J = 2.2 Hz), 6.02 (0.71H, d, J = 2.2 Hz), 5.54 (0.71H, d, J = 2.1 Hz), 5.40 (0.71H, s), 5.31 (0.29H, s), 5.12-4.82 (16H, m), 4.67 (0.29H, br), 4.64-4.60 (1H, m), 4.51 (0.71H, d, J = 11.3 Hz), 4.40 (0.29H, d, J = 12.0 Hz), 4.02 (0.71H, d, J = 4.8 Hz), 3.87 (0.29H, d, J = 4.2 Hz), 3.76-3.71 (0.71H, m), 3.65-3.62 (1H, m), 3.24 (0.71H, dd, J = 6.5, 16.8 Hz), 3.17 (0.29H, dd, J = 5.6, 16.4 Hz), 2.68 (0.29H, dd, J = 9.4, 16.3 Hz), 2.58 (0.71H, dd, J = 9.7, 16.7 Hz), 1.76 (0.71H, d, J = 6.01 Hz), 1.61 (0.29H, br), 1.43 (0.71H, br), 1.25 (0.29H, br). 13C NMR (125 MHz, CDCl3, 0.71 : 0.29 mixture of rotational isomers):δ = 158.28, 158.08, 157.74, 156.97, 156.85, 155.90, 155.82, 155.60, 155.15, 154.42, 152.91, 149.12, 148.96, 148.78, 148.72, 148.63, 148.47, 137.34, 137.29, 137.25, 137.22, 137.20, 137.15, 137.06, 137.01, 136.97, 136.90, 132.61, 132.50, 130.97, 130.27, 128.61, 128.56, 128.55, 128.53, 128.45, 128.42, 128.40, 128.36, 128.19, 128.17, 128.10, 128.05, 127.93, 127.73, 127.68, 127.57, 127.48, 127.43, 127.35, 127.31, 127.21, 127.16, 127.14, 127.11, 127.08, 126.96, 126.89, 126.75, 120.52, 120.16, 119.95, 119.62, 114.95, 114.51, 113.98, 113.88, 113.54, 112.92, 112.07, 111.19, 104.57, 104.26, 104.19, 102.69, 94.34, 93.46, 93.18. 93.00, 92.60, 91.47, 81.58, 81.39, 75.56, 75.38, 72.28, 71.74, 71.44, 71.40, 71.32, 71.23, 71.15, 71.07, 70.76, 70.55, 70.24, 69.89, 69.57, 69.43, 69.11, 68.62, 68.31, 35.78, 35.73, 29.06, 27.75.

[4, 8’]-2, 3-cis-3, 4-trans : 2’, 3’-cis-Octa-O-benzyl-bi-(−)-epicatechin (13). In the same manner as prepared 12, compound β-9 (48 mg, 0.034 mmol) gave 13 (40 mg, 90%).4d [α]D18 +34.9 (c 2.18, CHCl3). IR (film) νmax cm-1: 3572, 3067, 3036, 2933, 2874, 1737, 1611, 1515, 1498, 1454, 1431, 1382, 1269, 1220, 1182, 1112, 1067, 1031, 915, 851, 809, 741, 697, 624. 1H NMR (500 MHz, CDCl3, 0.6:0.4 mixture of rotational isomers): δ = 7.43-6.83 (42.8H, m), 6.80 (0.6H, d, J = 8.2 Hz), 6.44 (0.6H, s), 6.34 (0.6H, s), 6.22 (0.4H, d, J = 2.3 Hz), 6.17 (0.4H, s), 6.07 (0.4H, d, J = 2.2 Hz), 6.00 (0.6H, d, J = 2.2 Hz), 5.70 (0.6H, d, J = 2.2 Hz), 5.52 (0.6H, s), 5.33 (0.4H, s), 5.20-4.86 (15.8H, m), 4.79 (1.2H, s), 4.62 (1H, d, J = 11.2 Hz), 4.47 (0.6H, d, J = 11.3), 4.38 (0.4H, d, J = 12.0 Hz), 4.32 (0.4H, br), 4.06 (1H, br), 3.96 (0.4H, br), 3.86 (0.6H, d, J = 3.7 Hz), 3.06-2.87 (2H, m), 1.77 (0.6H, br), 1.69 (0.4H, d, J = 5.3 Hz), 1.50 (0.6H, br), 1.44 (0.4H, br). 13C NMR (125 MHz, CDCl3, 0.6:0.4 mixture of rotational isomers): δ = 158.08, 158.05, 157.87, 157.10, 157.04, 156.56, 156.48, 155.91, 155.86, 155.50, 155.04, 154.41, 149.20, 149.13, 148.73, 148.63, 148.50, 148.36, 148.26, 148.14, 137.39, 137.35, 137.32, 137.30, 137.26, 137.24, 137.19, 137.14, 136.98, 136.94, 132.67, 132.59, 131.16, 131.10, 128.58, 128.56, 128.54, 128.49, 128.47, 128.42, 128.38, 128.34, 128.30, 128.15, 128.12, 127.98, 127.96, 127.88, 127.83, 127.69, 127.66, 127.56, 127.50, 127.48, 127.44, 127.34, 127.32, 127.23, 127.17, 127.14, 127.12, 127.00, 126.97, 126.95, 126.90, 126.75, 126.59, 119.85, 119.76, 118.84, 118.76, 118.63, 115.15, 114.99, 114.86, 114.53, 114.32, 113.49, 112.61, 112.43, 111.65, 111.28, 111.14, 104.52, 104.42, 102.35, 102.26, 94.48, 93.93, 93.31, 93.18, 93.12, 91.50, 78.87, 78.81, 78.05, 75.64, 75.61, 72.39, 72.12, 71.56, 71.43, 71.32, 71.20, 70.93, 70.80, 70.55, 70.46, 69.98, 69.90, 69.84, 69.75, 69.52, 69.47, 69.11, 69.03, 66.50, 65.16, 35.83, 35.74, 28.64.

[4, 8’]-2, 3-trans-3, 4-trans : 2’, 3’-trans-Octa-O-benzyl-bi-(+)-catechin (14). In the same manner as prepared 12, compound α-7 (214mg, 0.155 mmol) gave 14 (169 mg, 84%).9 [α]D24 −127.5 (c 1.00, CHCl3). IR (film) νmax cm-1: 3567, 3062, 3031, 2928, 2869, 1732, 1606, 1510, 1498, 1454, 1426, 1377, 1264, 1215, 1177, 1112, 1062, 1026, 910, 850, 809, 736, 697, 623. 1H NMR (500 MHz, CDCl3, 0.67:0.33 mixture of rotational isomers):δ = 7.49-7.15 (40H, m), 7.04-6.47 (6H, m), 6.31 (0.67H, s), 6.23 (0.33H, d, J = 2.3 Hz), 6.20 (0.67H, d, J = 2.3 Hz), 6.13 (1H, d, J = 2.4 Hz), 6.04 (0.33H, d, J = 2.3 Hz), 5.20-4.48 (18H, m), 4.32 (0.67H, m), 4.20 (0.33H, m), 3.75-3.70 (1H, m), 3.67 (1H, d, J = 8.5 Hz), 3.20 (0.33H, dd, J = 5.9, 16.4 Hz), 3.08 (0.67H, dd, J = 5.6, 16.2 Hz), 2.68 (0.33H, dd, J = 9.4, 16.4 Hz), 2.42 (0.67H, dd, J = 9.1, 16.2 Hz). 13C NMR (125 MHz, CDCl3, 0.67:0.33 mixture of rotational isomers):δ = 158.00, 157.73, 156.98, 156.84, 155.58, 155.53, 153.86, 152.85, 149.28, 149.16, 149.04, 148.66, 137.69, 137.32, 137.30, 137.18, 137.14, 137.04, 136.70, 131.92, 131.74, 128.57, 128.53, 128.51, 128.47, 128.41, 128.38, 128.35, 128.33, 128.15, 128.06, 127.87, 127.84, 127.82, 127.78, 127.75, 127.71, 127.67, 127.61, 127.56, 127.51, 127.46, 127.44, 127.27, 127.23, 127.22, 127.11, 127.08, 127.05, 121.28, 120.82, 120.69, 120.11, 115.20, 115.02, 114.98, 114.70, 114.24, 113.90, 113.71, 112.16, 108.70, 108.53, 102.52, 94.97, 94.18, 91.89, 91.59, 82.06, 81.76, 81.27, 80.66, 73.42, 73.30, 71.38, 71.24, 71.21, 71.16, 71.04, 70.37, 70.13, 70.02, 69.98, 69.91, 68.49, 68.41.

[4, 8’]-2, 3-trans-3, 4-trans : 2’, 3’-cis-Octa-O-benzyl-(+)-catechin-(−)-epicatechin (15). In the same manner as prepared 12, compound α-10 (53 mg, 0.038 mmol) gave 15 (46mg, 93%).6a [α]D19 −96.9 (c 1.70, CHCl3). IR (film) νmax cm-1: 3567, 3063, 3032, 2912, 2870, 1741, 1607, 1512, 1454, 1427, 1379, 1266, 1218, 1107, 1058, 1027, 910, 849, 811, 735, 697, 624. 1H NMR (500 MHz, CDCl3, 0.71:0.29 mixture of rotational isomers) :δ = 7.45-6.78 (48H, m), 6.47 (0.29H, d, J = 8.3 Hz), 6.22-6.21 (1H, m), 6.19 (0.71H, d, J = 2.1 Hz), 6.12 (0.71H, d, J = 2.1 Hz), 5.99 (0.29H, s), 5.18 (0.71H, d, J = 12.1 Hz), 5.13 (2H, d, J = 5.6 Hz), 5.10 (1.71H, s), 5.08 (1H, s), 5.05-4.47 (12.87H, m), 4.27 (1H, dd, J = 8.8, 18.0 Hz), 4.09 (0.29H, br), 3.88 (0.71H, br), 3.79 (0.71H, s), 3.01 (0.29H, d, J = 17.1 Hz), 2.92 (0.29H, d, J = 4.6 Hz), 2.87 (0.71H, d, J = 17.3 Hz), 2.59 (0.71H, dd, J = 4.4, 17.1 Hz), 1.69-1.39 (2H, m). 13C NMR (125 MHz, CDCl3, 0.71:0.29 mixture of rotational isomers) :δ = 158.21, 158.09, 158.00, 157.81, 156.95, 156.84, 156.56, 156.13, 156.04, 155.49, 153.66, 152.89, 149.25, 149.16, 149.13, 149.04, 148.99, 148.78, 148.33, 137.66, 137.44, 137.41, 137.34, 137.30, 137.27, 137.24, 137.22, 137.19, 137.16, 137.13, 137.09, 136.99, 136.84, 136.70, 132.17, 131.93, 131.87, 131.09, 128.56, 128.55, 128.52, 128.48, 128.44, 128.42, 128.40, 128.37, 128.34, 128.13, 128.09, 128.04, 127.86, 127.85, 127.74, 127.71, 127.63, 127.59, 127.55, 127.52, 127.44, 127.25, 127.23, 127.21, 127.13, 127.11, 127.08, 121.29, 120.93, 119.95, 118.79, 115.31, 115.05, 114.98, 114.61, 114.00, 113.78, 113.17, 112.08, 111.74, 108.84, 108.28, 102.25, 100.88, 95.41, 94.96, 94.44, 94.16, 92.17, 91.57, 82.09, 81.86, 77.45, 77.25, 73.19, 72.52, 71.86, 71.50, 71.35, 71.24, 71.20, 71.13, 71.01, 70.37, 70.25, 70.15, 70.04, 69.99, 69.95, 69.73, 66.29, 65.96, 37.46, 37.06, 29.67, 28.85, 28.29, 22.66.

Procyanidin B1 (1). Compound 12 (60 mg, 0.046 mmol) and Pd(OH)2 on carbon (20wt%, 12 mg) in THF-MeOH-H2O (20:1:1, 10 mL) was stirred for 48 h under the H2 atmosphere. After the reaction had been completed the mixture was filtered and the solvent was evaporated. The residue was purified with ODS cartridge column chromatography (MeOH : H2O = 3:7) afforded 1 (20 mg, 76%) as a colorless solid. Mp 184.5-185.0 oC (decomp.); [α]D20 +43.3 (c 0.27, EtOH); 1H NMR (500 MHz, CD3OD) :δ = 2.57-2.63 (1H, m), 2.77 (1H, m), 3.94 (1H, m), 3.98-4.12 (1H, m), 4.56-4.60 (1H, m), 4.80 (1H, m), 5.10 (1H, m), 5.86-5.93 (3H, m), 6.69-6.84 (5H, m), 6.90 (1H, m); 13C-NMR (125 MHz, CD3OD) δ = 27.73, 30.20, 37.22, 68.64, 73.19, 77.13, 79.02, 79.28, 79.54, 82.49, 95.95, 96.40, 97.05, 101.32, 115.13, 115.40, 115.96, 116.16, 119.45, 132.90, 145.57, 145.87, 145.95, 155.73, 156.11, 157.77. HRFABMS calcd for C30H26O12Na [M+Na]+; 601.1322; found 601.1263. 6a,14

Procyanidin B2 (2). In the same manner as prepared 1, compound 13 (40 mg, 0.031 mmol) gave 2 (11 mg, 58%) as a colorless solid. Mp 194.5-195.0 oC (decomp.); [α]D20 +29.3 (c 0.16, EtOH); 1H NMR (500 MHz, CD3OD) :δ = 2.71-2.81 (1H, m), 2.84-2.93 (1H, m), 3.91 (1H, m), 4.09-4.27 (1H, m), 4.62 (1H, br. s), 4.93 (1H, m), 5.05 (1H, m), 5.91-5.94 (3H, m), 6.70-6.81 (4H, m), 6.89 (1H, br. s), 7.09 (1H, br. s); 13C-NMR (125 MHz, CD3OD) :δ = 29.29, 29.74, 37.21, 67.05, 67.54, 73.56, 77.16, 79.03, 79.29, 79.55, 95.98, 96.23, 96.50, 100.15, 100.63, 115.34, 115.39, 116.00, 119.47, 127.34, 132.13, 132.34, 145.68, 145.84, 145.93, 146.00, 156.55, 157.42, 157.74, 158.04. HRFABMS calcd for C30H26O12Na [M+Na]+; 601.1322; found 601.1299. 4d, 14

Procyanidin B3 (3). In the same manner as prepared 1, compound 14 (169 mg) gave 3 (60 mg, 58%) as a colorless solid. Mp 218-219 oC (decomp.); [α]D27 −181 (c 0.29, EtOH); 1H NMR (500 MHz, CD3OD, 0.67:0.33 mixture of rotational isomers) :δ = 2.49 (0.67H, dd, J = 8.0, 16.2 Hz), 2.59 (0.33H,dd, J = 7.4, 16.1 Hz), 2.76 (0.67H, dd, J = 5.5, 16.2 Hz), 2.82 (0.33H, dd, J = 5.6, 16.1 Hz), 3.78 (0.67H, m), 4.08 (0.33H, m), 4.26 (1H, d, J = 9.7 Hz), 4.35 (1H, dd, J = 7.9, 9.6 Hz), 4.41 (1H, d, J = 7.8 Hz), 4.54 (0.67H, d, J = 7.3 Hz), 4.75 (0.33H, d, J = 7.2 Hz), 5.79 (0.67H, d, J = 2.4 Hz), 5.82 (0.33H, d, J = 2.4 Hz), 5.85 (0.33H, J = 2.3 Hz), 5.89 (0.67H, d, J = 2.4 Hz), 5.95 (0.33H, s), 6.08 (0.67H, s), 6.26 (0.67H, dd, J = 1.8, 8.2 Hz), 6.48 (0.67H, dd, J = 1.9, 8.2 Hz), 6.58 (0.67H, d, J = 1.9 Hz), 6.68 (1.33H, d, J = 8.2 Hz), 6.74 (0.67H, d, J = 1.9 Hz), 6.75 (0.67H, dd, J = 1.9, 8.2 Hz), 6.78 (0.33H, dd, J = 8.2, 1.9 Hz), 6.80 (0.33H, dd, J = 8.2, 1.9 Hz), 6.96 (0.67H, d, J = 1.9 Hz); 13C-NMR (125 MHz, CD3OD, 0.71:0.29 mixture of rotational isomers) :δ = 28.51, 28.79, 38.64, 68.63, 68.94, 73.74, 82.50, 83.00, 83.98, 84.14, 96.18, 96.34, 96.96, 97.41, 97.64, 100.62, 102.34, 107.25, 107.30, 108.24, 108.40, 115.28, 115.58, 116.01, 116.13, 116.20, 116.28, 116.49, 119.96, 120.23, 120.70, 121.09, 131.93, 132.45, 132.68, 145.51, 145.65, 145.82, 146.17, 146.52, 154.93, 155.11, 155.68, 155.80, 155.90, 155.99, 157.15, 157.31, 157.42, 158.68, 159.94. HRFABMS calcd for C30H25O12 [M−H]<συπ>−</συπ>; 577.1346; found 577.1358. 4b

Procyanidin B4 (4). In the same manner as prepared 1, compound 15 (46 mg, 0.035 mmol) gave 4 (10 mg, 51%) as a colorless solid.6a Mp 178.5-179.5 oC (decomp.); [α]D19 −177 (c 0.097, EtOH); 1H NMR (500 MHz, CD3OD, 1:1 mixture of rotational isomers) :δ = 2.70 (0.5H, dd, J = 2.3, 17.1 Hz), 2.81-2.95 (1.5H, m), 4.06 (0.5H, m), 4.23 (0.5H, m), 4.31-4.32 (1H, m), 4.42 (0.5H, d, J = 9.7 Hz), 4.47 (0.5H, dd, J = 3.0, 5.0 Hz), 4.57 (0.5H, dd, J = 7.9, 9.6 Hz), 4.63 (0.5H, d, J = 7.9 Hz), 4.81 (0.5H, s), 4.93 (0.5H, s), 5.80 (0.5H, d, J = 2.3 Hz), 5.85 (0.5H, d, J = 2.3 Hz), 5.90 (0.5H, J = 2.4 Hz), 5.94 (0.5H, d, J = 2.4 Hz), 5.96 (0.5H, s), 6.10 (0.5H, s), 6.42-6.46 (1H, m), 6.62 (0.5H, d, J = 8.2 Hz), 6.67 (0.5H, d, J = 1.9 Hz), 6.70 (0.5H, d, J = 1.9 Hz), 6.72 (0.5H, d, J = 8.2 Hz), 6.79 (1H, dd, J = 1.9, 8.1 Hz), 6.87 (1H, d, J = 8.1 Hz), 6.99 (0.5H, d, J = 1.9 Hz), 7.09 (0.5H, d, J = 1.8 Hz); 13C-NMR (125 MHz, CD3OD, 1:1 mixture of rotational isomers) :δ = 29.42, 30.12, 38.83, 38.91, 67.45, 67.85, 73.86, 79.98, 80.11, 83.93, 84.10, 96.25, 96.54, 97.24, 97.69, 97.80, 99.59, 101.59, 107.22, 107.46, 108.32, 108.76, 114.90, 115.35, 116.02, 116.05, 116.13, 116.39, 116.51, 119.23, 120.32, 120.56, 121.25, 131.78, 132.35, 132.50, 132.65, 145.64, 145.71, 146.01, 146.18, 146.52, 155.43, 155.87, 155.94, 156.38, 156.45, 157.25, 157.33, 157.40, 157.57, 158.60, 158.75. HRFABMS calcd for C30H26O12Na [M+Na]+; 601.1322; found 601.1367.6a

Food materials. The apple juice was made from “Fuji (Malus domestica)” apples. The fresh was cut into small pieces, frozen in liquid N2 and freeze-dried. Then, the samples were ground to powdered from using a mixer and stored in a desiccator for further use.

Preparation of fruit phenolic fraction. Before the extraction of phenolics, the freeze-dried flesh powder (10 g) was mixed with petroleum ether in a beaker, stirred and filtered through filter paper on a Büchner funnel to remove lipids (100 mL×5 times). The phenolics were then extracted from the residue with 60% (v/v) aqueous acetone (100 mL×2 times) in the same manner. The 60% acetone solution was evaporated until all the organic solvent was removed. The aqueous solution of the extracts was applied onto a Sep-Pak Vac 20 cc (5 g) C18 cartridge column which was preconditioned with MeOH (10 mL) and 0.1% (v/v) TFA in water. The column was washed with 0.1% TFA solution (40 mL) and phenolics were eluted with MeOH (20 mL). The methanol solution was added to water and evaporated, and the resultant aqueous solution was frozen and then freeze-dried to obtain semi-purified phenolic powder. It was analyzed using HPLC for evaluation of phenolic composition.

HPLC analysis of procyanidins B1-B4 (1-4). Chromatographic separation was carried out on a Luna 5µ C18 column (150 × 4.6 mm, Phenomenex, Inc., Torrance, CA., USA) with a security guard cartridge (4 × 4.6 mm) at 40 °C. Solvents were 0.1% trifluoroacetic acid (A) and 0.1% trifluoroacetic acid in acetonitrile (B). The gradient program began with 5% B and was changed to obtain 15% B at 30 min, 32% B at 35 min, 40% B at 45 min, and 75% B at 50 min. The 75% B was maintained until 65 min. The flow rate was 1.0 mL/min and the injection volume was 20 µL. Detection was performed at 280 nm.

ACKNOWLEDGEMENTS
We thank Geol Cosmetics. Co., LTD for financial support.

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