Stereoselective Syntheses of All Stereoisomers of Lariciresinol and

Nov 9, 2011 - 0. ,9-triol, (А)-. Lariciresinol (1). The title compound was synthesized according to the ..... tion mixture of benzyl ether 19 (76 mg,...
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Stereoselective Syntheses of All Stereoisomers of Lariciresinol and Their Plant Growth Inhibitory Activities Hisashi Nishiwaki, Mitsuko Kumamoto, Yoshihiro Shuto, and Satoshi Yamauchi* Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan ABSTRACT: All stereoisomers of lariciresinol were synthesized to examine the effect of stereochemistry on plant growth. Configuration of benzylic 7-positions was constructed through SN1 or SN2 intramolecular etherification. 8- and 80 -position configurations were established from the starting material except for all cis stereoisomers, the 8-position configurations of which were achieved by employing stereoselective hydroboration. ()-Lariciresinol and its 7S,8S,80 R stereoisomer inhibited the root growth of Italian ryegrass to 5155% relative to the negative control, whereas other stereoisomers had less effect. These results demonstrate that the stereochemistry of lignans is one of the important factors influencing their inhibitory activity. KEYWORDS: lariciresinol, lignan, plant growth inhibitory, lettuce, Italian ryegrass

’ INTRODUCTION Among natural plant products, the lignan family is one of the most attractive chemical groups because of not only their original structures, including C6C3 units with asymmetric carbon atoms, but also their activities to various biological species including plants, insects, and microorganisms and anticancer activity.1,2 Various types of lignans have been isolated from plants to evaluate their bioactivity, and many reports of their syntheses have been published. Numerous studies on lignans are still being conducted; for example, a novel lignan with a unique epoxy structure was recently discovered.3 We have also analyzed the structureactivity relationship (SAR) of various lignans using enantiomerically pure compounds to demonstrate clear differences in biological activity depending on their absolute structures.46 In the field of natural products chemistry, the collation of stereoisomer libraries is necessary to study the relationship between stereochemistry and biological activity. ()- and (+)-lariciresinol, 1 and 2, respectively (Figure 1), are trisubstituted tetrahydrofuran lignans that are biosynthesized by plants.7 ()-Lariciresinol reportedly shows plant growth inhibitory activity, and (()-lariciresinol inhibits lettuce germination.810 However, the effect of the compound’s stereochemistry on its activity has not been evaluated, probably due to the difficulty in isolating enantiomerically pure lariciresinol.11,12 To elucidate the role of stereochemistry in the bioactivity of lariciresinol, we developed a synthetic pathway to all stereoisomers of lariciresinol, 18 (Figure 1). In addition, their plant growth regulatory activity was evaluated. This is the first report on the syntheses of all stereoisomers of lariciresinol and the relationship between lariciresinol stereochemistry and plant regulatory activity. ’ MATERIALS AND METHODS Chemicals. ()- and (+)-lariciresinol were synthesized according to a previously described method.13 To synthesize stereoisomers of lariciresinol other than 1 and 2, lactone 914 or 13 was used as the starting material (Figure 2). Reagents used for the syntheses were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo Chemical Industry Co, Ltd. (Tokyo, Japan), r 2011 American Chemical Society

and Aldrich Chemical Co. (Milwaukee, WI). Melting points were uncorrected. Optical rotations were measured on a Horiba SEPA-200 instrument. NMR data were obtained using a JNM-EX400 spectrometer. EI and FABMS data were measured with a JMS-MS700 V spectrometer. The silica gel used was Wakogel C-300 (Wako, 200300 mesh). HPLC analysis for compounds 18 was performed with Shimadzu LC6AD and SPD-6AV instruments. The column used for HPLC analyses was a 250 mm  4.6 mm i.d., 5 μm, CHIRALCEL AD-H (DAICEL Chemical Industries, Ltd., Tokyo, Japan). The numbering of compounds follows the nomenclature of lignans.15 (7R,8S,8 0 S)-3,30 -Dimethoxy-7,90 -epoxylignane-4,4 0 ,9-triol, ()Lariciresinol (1). The title compound was synthesized according to the described method:13 >99% ee (AD-H, iso-PrOH/hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR11 min). (7S,8R,80 R)-Stereoisomer, (+)-lariciresinol (2): >99% ee (AD-H, isoPrOH/hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR10 min. (70 S,8R,80 S)-4,40 -Dibenzyloxy-3,30 -dimethoxy-90 -(triisopropylsilyloxy) lignano-9,70 -lactone (17). A reaction mixture of alcohol 95 (0.43 g, 0.78 mmol), imidazole (0.26 g, 3.82 mmol), and TIPSCl (0.63 mL, 2.94 mmol) in DMF (1 mL) was stirred at room temperature for 20 h before additions of H2O and EtOAc. The organic solution was separated, washed with brine, and dried (Na2SO4). Concentration followed by silica gel column chromatography with EtOAc/hexane (1:4, v/v) gave silyl ether 17 (0.47 g, 0.66 mmol, 85%) as a colorless oil: [α]20D +33 (c 2.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.881.02 (21H, m, CH(CH3)2  3), 2.64 (1H, m, H-80 ), 2.922.96 (1H, overlapped, H-8), 2.95 (1H, dd, J = 13.6, 5.0 Hz, H-7a), 3.10 (1H, dd, J = 13.6, 4.4 Hz, H-7b), 3.14 (1H, dd, J = 10.3, 6.9 Hz, H-90 a), 3.25 (1H, dd, J = 10.3, 6.2 Hz, H-90 b), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 5.11 (2H, s, OCH2Ph), 5.12 (2H, s, OCH2Ph), 5.35 (1H, d, J = 7.8 Hz, H-70 ), 6.68 (1H, dd, J = 8.3, 2.1 Hz, H-6), 6.71 (1H, dd, J = 8.3, 2.1 Hz, H-60 ), 6.75 (1H, d, J = 2.1 Hz, H-2), 6.80 (1H, d, J = 2.1 Hz, H-20 ), 6.81 (1H, d, J = 8.3 Hz, H-5), 6.82 (1H, d, J = 8.3 Hz, H-50 ), 7.277.30 (2H, m, OCH2PhH), 7.327.36 (4H, m, OCH2PhH), 7.407.43 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 11.7 (CH, CH(CH3)2  3), 17.7 (CH3, CH(CH3)2  3), 35.3 (CH, C-80 ), 44.1 (CH, C-8), 46.5 (CH2, C-7), 55.96 (CH3, OCH3), Received: August 11, 2011 Revised: November 8, 2011 Accepted: November 9, 2011 Published: November 09, 2011 13089

dx.doi.org/10.1021/jf203222w | J. Agric. Food Chem. 2011, 59, 13089–13095

Journal of Agricultural and Food Chemistry

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Figure 1. ()- and (+)-lariciresinol and their stereoisomers.

Figure 2. Synthesis scheme of lariciresinol stereoisomers. 56.10 (CH3, OCH3), 62.1 (CH2, C-90 ), 71.1 (CH2, OCH2Ph  2), 81.1 (CH, C-70 ), 109.7, 112.7, 113.9, 114.1, 118.2, 121.2, 127.21, 127.23, 127.8, 127.9, 128.5, 128.9, 130.9, 136.9, 137.2, 147.2, 147.9, 149.6, 149.8, 178.6; MS (EI) m/z 710 (M+, 41), 91 (100); HRMS (EI) m/z calcd for C43H54O7Si (M+) 710.3639, found 710.3633.

(70 R,8S,80 R)-(17): [α]20D 30 (c 2.0, CHCl3). (7S,8S,80 R)-4,40 -Dibenzyloxy-3,30 -dimethoxy-9-(triisopropylsilyloxy)-7, 90 -epoxylignane (18). To a solution of lactone 17 (2.38 g, 3.34 mmol) in CH2Cl2 (15 mL) was added DIBAL-H (6.68 mL, 1 M in toluene, 6.68 mol) at 70 °C. After 2 h of stirring at 70 °C, 1 M aqueous HCl 13090

dx.doi.org/10.1021/jf203222w |J. Agric. Food Chem. 2011, 59, 13089–13095

Journal of Agricultural and Food Chemistry solution was added. The organic solution was separated, washed with saturated aqueous NaHCO3 solution, dried (Na2SO4), and concentrated to give crude hemiacetal. To an ice-cooled solution of crude hemiacetal in EtOH (15 mL) was added NaBH4 (0.38 g, 10.1 mmol). After the reaction mixture was stirred at room temperature for 1 h, saturated aqueous NH4Cl solution was added. The mixture was concentrated, and then the mixture was dissolved in EtOAc and H2O. The organic solution was separated, washed with brine, and dried (Na2SO4). Concentration gave crude diol. To an ice-cooled solution of crude diol and pyridine (1.15 mL, 14.2 mmol) in CH2Cl2 (15 mL) was added p-TsCl (0.59 g, 3.10 mmol). After the reaction solution was stirred at room temperature for 44 h, H2O was added. The organic solution was separated, washed with 1 M aqueous HCl solution and saturated aqueous NaHCO3 solution, and dried (Na2SO4). Concentration followed by silica gel column chromatography with EtOAc/hexane (1:6, v/v) gave epoxylignane 18 (1.66 g, 2.38 mmol, 71%, three steps) as a colorless oil: [α]20D 29 (c 2.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.930.97 (21H, m, CH(CH3)2  3), 2.33 (1H, m, H-8), 2.582.63 (2H, m, H-70 a, H-80 ), 2.96 (1H, dd, J = 18.1, 9.5 Hz, H-70 b), 3.19 (1H, dd, J = 10.2, 6.0 Hz, H-9a), 3.26 (1H, dd, J = 10.2, 8.9 Hz, H-9b), 3.63 (1H, dd, J = 8.8, 6.3 Hz, H-90 a), 3.86 (3H, s, OCH3), 3.87 (3H, s, OCH3), 4.15 (1H, dd, J = 8.8, 6.5 Hz, H-90 b), 5.07 (1H, d, J = 7.1 Hz, H-7), 5.12 (4H, s, OCH2Ph  2), 6.67 (1H, dd, J = 8.1, 1.8 Hz, H-6), 6.73 (1H, dd, J = 8.1, 1.8 Hz, H-60 ), 6.74 (1H, d, J = 1.8 Hz, H-2), 6.80 (1H, d, J = 8.1 Hz, H-5), 6.82 (1H, d, J = 8.1 Hz, H-50 ), 6.83 (1H, d, J = 1.8 Hz, H-20 ), 7.277.31 (2H, m, OCH2PhH), 7.337.37 (4H, m, OCH2PhH), 7.417.45 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 11.8 (CH, CH(CH3)2  3), 18.0 (CH3, CH(CH3)2), 39.4 (CH, C-8), 43.7 (CH, C-80 ), 51.4 (CH2, C-70 ), 55.9 (CH3, OCH3), 56.0 (CH3, OCH3), 63.3 (CH2, C-9), 71.2 (CH2, OCH2Ph  2), 73.1 (C-90 ), 81.8 (CH, C-7), 110.1, 112.5, 114.0, 114.2, 118.4, 120.6, 127.27, 127.29, 127.7, 127.8, 128.47, 128.51, 132.9, 133.8, 137.3, 137.4, 146.7, 147.1, 149.4, 149.6; MS (EI) m/z 696 (M+, 26), 91 (100); HRMS (EI) m/z calcd for C43H56O6Si 696.3846, found 696.3857. (7R,8R,80 S)-(18): [α]20D +30 (c 2.4, CHCl3). (7S,8S,80 R)-4,40 -Dibenzyloxy-3,30 -dimethoxy-7,90 -epoxylignan-9-ol (19). To a solution of silyl ether 18 (0.11 g, 0.16 mmol) in THF (10 mL) was added (n-Bu)4NF (0.16 mL, 1 M THF solution, 0.16 mmol). The resulting reaction solution was stirred at room temperature for 1 h before additions of EtOAc and saturated aqueous CuSO4 solution. The organic solution was separated, washed with saturated aqueous NaHCO3 solution, and dried (Na2SO4). Concentration followed by silica gel column chromatography with EtOAc/hexane (1:1, v/v) gave alcohol 19 (76 mg, 0.14 mmol, 88%) as colorless crystals: mp 113114 °C; [α]20D 40 (c 1.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.651.85 (1H, br, OH), 2.28 (1H, m, H-8), 2.48 (1H, m, H-80 ), 2.65 (1H, dd, J = 13.8, 8.6 Hz, H-70 a), 2.84 (1H, dd, J = 13.8, 6.6 Hz, H-70 b), 3.26 (2H, d, J = 6.3 Hz, H-9), 3.57 (1H, dd, J = 8.7, 7.7 Hz, H-90 a), 3.86 (3H, s, OCH3), 3.87 (3H, s, OCH3), 4.18 (1H, dd, J = 8.7, 7.4 Hz, H-90 b), 5.03 (1H, d, J = 7.1 Hz, H-7), 5.11 (4H, s, OCH2Ph), 6.67 (1H, dd, J = 8.1, 1.8 Hz, H-6), 6.74 (1H, d, J = 1.8 Hz, H-2), 6.77 (1H, dd, J = 8.3, 1.8 Hz, H-60 ), 6.81 (1H, d, J = 8.1 Hz, H-5), 6.85 (1H, d, J = 8.3 Hz, H-50 ), 6.88 (1H, d, J = 1.8 Hz, H-20 ), 7.277.30 (2H, m, OCH2PhH), 7.337.36 (4H, m, OCH2PhH), 7.417.43 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 38.9 (CH, C-8), 43.3 (CH, C-80 ), 51.1 (CH2, C-70 ), 56.03 (CH3, OCH3), 56.05 (CH3, OCH3), 62.5 (CH2, C-9), 71.1 (CH2, OCH2Ph), 71.2 (CH2, OCH2Ph), 73.1 (CH, C-90 ), 81.7 (CH, C-7), 109.8, 112.6, 114.1, 114.3, 118.1, 120.6, 127.30, 127.32, 127.79, 127.83, 128.50, 128.51, 132.3, 133.3, 137.1, 137.3, 146.7, 147.4, 149.7, 149.8; MS (EI) m/z 540 (M+, 44), 149 (52), 121 (66), 91 (100); HRMS (EI) m/z calcd for C34H36O6 540.2512, found 540.2497. (7R,8R,80 S)-(19): [α]20D +40 (c 1.8, CHCl3). (7S,8S,80 R)-3,30 -Dimethoxy-7,90 -epoxylignane-4,40 ,9-triol (3). A reaction mixture of benzyl ether 19 (76 mg, 0.14 mmol) and 5% Pd/C (0.10 g)

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in EtOAc (10 mL) was stirred under H2 gas at ambient temperature for 12 h before filtration. The filtrate was concentrated, and then the residue was applied to silica gel column chromatography with EtOAc/hexane (2:1, v/v) to give the 7S,8S,80 R isomer, 3 (44 mg, 0.12 mmol, 86%), as colorless crystals: mp 6566 °C; [α]20D 44 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.24 (1H, t, J = 2.1 Hz, OH), 2.29 (1H, m, H-8), 2.48 (1H, m, H-80 ), 2.66 (1H, dd, J = 13.8, 8.6 Hz, H-70 a), 2.84 (1H, dd, J = 13.8, 6.7 Hz, H-70 b), 3.263.29 (2H, m, H-9), 3.58 (1H, dd, J = 8.7, 7.7 Hz, H-90 a), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 4.19 (1H, dd, J = 8.7, 7.4 Hz, H-90 b), 5.04 (1H, d, J = 7.2 Hz, H-7), 5.70 (1H, br s, PhOH), 5.80 (1H, br s, PhOH), 6.69 (1H, dd, J = 8.1, 1.8 Hz, H-6), 6.70 (1H, d, J = 1.8 Hz, H-2), 6.79 (1H, dd, J = 8.1, 1.8 Hz, H-60 ), 6.82 (1H, d, J = 8.1 Hz, H-5), 6.85 (1H, d, J = 1.8 Hz, H-20 ), 6.87 (1H, d, J = 8.1 Hz, H-50 ); 13C NMR (100 MHz, CDCl3) δ 39.0 (CH, C-8), 43.5 (CH, C-80 ), 51.1 (CH2, C-70 ), 55.9 (CH3, OCH3), 56.0 (CH3, OCH3), 62.6 (CH2, C-9), 73.0 (CH2, C-90 ), 81.8 (CH, C-7), 108.6 (C-2), 111.3 (C-20 ), 114.46 (C-5), 114.50 (C-50 ), 118.9 (C-6), 121.3 (C-60 ), 131.0 (C-1), 131.9 (C-10 ), 144.1 (C-40 ), 145.0 (C-4), 146.6 (C-30 ), 146.7 (C-3); MS (EI) m/z 360 (M+, 62), 137 (100); HRMS (EI) m/z calcd for C20H24O6 360.1573, found 360.1548; >99% ee (AD-H, iso-PrOH/ hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR11 min). (7R,8R,80 S)-Stereoisomer (4): [α]20D +45 (c 1.0, CHCl3); >99% ee (AD-H, iso-PrOH/hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR 14 min). (7R,8S,80 R)-4,40 -Dibenzyloxy-3,30 -dimethoxy-9-(triisopropylsilyloxy)-7, 0 9 -epoxylignane (20). A reaction solution of crude diol prepared from lactone 17 (0.13 g, 0.18 mmol) and 10-camphorsulfonic acid (6.0 mg, 0.026 mmol) was stirred at room temperature for 37 h before the addition of a few drops of Et3N, and then the mixture was concentrated. The residue was applied to silica gel column chromatography with EtOAc/hexane (1:4, v/v) to give epoxylignane 20 (62 mg, 0.089 mmol, 49% from lactone) as a colorless oil: [α]20D +4 (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.001.10 (21H, m, CH(CH3)2), 1.94 (1H, m, H-8), 2.562.64 (2H, m, H-80 and H-70 a), 2.83 (1H, dd, J = 17.6, 10.0 Hz, H-70 b), 3.66 (1H, dd, J = 10.1, 4.1 Hz, H-9a), 3.72 (1H, dd, J = 10.1, 5.0 Hz, H-9b), 3.81 (1H, dd, J = 8.7, 5.5 Hz, H-90 a), 3.85 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.91 (1H, dd, J = 8.7, 5.5 Hz, H-90 b), 4.73 (1H, d, J = 8.2 Hz, H-7), 5.12 (2H, s, OCH2Ph), 5.15 (2H, s, OCH2Ph), 6.62 (1H, dd, J = 8.1, 1.9 Hz, H-6), 6.69 (1H, d, J = 1.9 Hz, H-2), 6.78 (1H, d, J = 8.1 Hz, H-5), 6.85 (2H, s, H-20 and H-50 ), 6.95 (1H, s, H-60 ), 7.277.29 (2H, m, OCH2PhH), 7.317.38 (4H, m, OCH2PhH), 7.437.44 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 11.9 (CH, CH(CH3)2), 18.1 (CH3, CH(CH3)2), 39.4 (CH, C-8), 43.7 (CH, C-80 ), 55.88 (CH2, C-70 and CH3, OCH3), 55.98 (CH3, OCH3), 62.3 (CH2, C-9), 71.1 (CH2, OCH2Ph), 71.2 (CH2, OCH2Ph), 73.1 (CH2, C-90 ), 83.0 (CH, C-7), 110.0, 112.4, 114.0, 114.1, 118.5, 120.6, 127.3, 127.8, 128.5, 133.8, 135.6, 137.3, 137.4, 147.5, 149.6, 149.7; MS (EI) m/z 696 (M+, 58), 431 (50), 121 (49), 91 (100); HRMS (EI) m/z calcd for C43H56O6Si 696.3847, found 696.3839. (7S,8R,80 S)-(20): [α]20D 4 (c 2, CHCl3). (7R,8S,80 R)-4,40 -Dibenzyloxy-3,30 -dimethoxy-7,90 -epoxylignan-9-ol (21). To a solution of silyl ether 20 (0.22 g, 0.32 mmol) in THF (20 mL) was added (n-Bu)4NF (0.33 mL, 1 M in THF, 0.33 mmol). After the reaction solution had been stirred at room temperature for 1 h, EtOAc and saturated aqueous CuSO4 solution were added. The organic solution was separated, washed with saturated aqueous NaHCO3 solution and brine, and dried (Na2SO4). Concentration followed by silica gel column chromatography with EtOAc/hexane (1:2, v/v) gave alcohol 21 (0.17 g, 0.31 mmol, 97%) as a colorless oil: [α]20D +8 (c 0.04, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.67 (1H, br s, OH), 1.98 (1H, m, H-8), 2.47 (1H, m, H-80 ), 2.66 (1H, dd, J = 13.8, 8.6 Hz, H-70 a), 2.77 (1H, dd, J = 13.8, 6.8 Hz, H-70 b), 3.59 (2H, d, J = 5.4 Hz, H-9), 3.83 (1H, dd, J = 8.9, 5.9 Hz, H-90 a), 3.84 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.94 (1H, dd, J = 8.9, 7.3 Hz, H-90 b), 4.60 (1H, d, J = 7.9 Hz, H-7), 5.10 (2H, s, OCH2Ph), 5.13 (2H, s, OCH2Ph), 6.62 (1H, dd, J = 8.1, 2.0 Hz, H-6), 13091

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Journal of Agricultural and Food Chemistry 6.69 (1H, d, J = 2.0 Hz, H-2), 6.79 (1H, d, J = 8.1 Hz, H-5), 6.84 (2H, s, H-20 and H-50 ), 6.96 (1H, s, H-60 ), 7.277.30 (2H, m, OCH2PhH), 7.337.36 (4H, m, OCH2PhH), 7.417.43 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 39.3 (CH, C-8), 44.1 (CH, C-80 ), 55.4 (CH2, C-70 ), 56.0 (CH3, OCH3), 56.1 (CH3, OCH3), 62.8 (CH2, C-9), 71.1 (CH2, OCH2Ph), 71.2 (CH2, OCH2Ph), 73.0 (CH2, C-90 ), 83.9 (CH, C-7), 110.0, 112.6, 114.0, 114.3, 118.5, 120.7, 127.27, 127.32, 127.8, 128.50, 128.52, 133.4, 135.3, 137.2, 137.3, 146.7, 147.7, 149.7, 149.8; MS (EI) m/z 540 (M+, 93), 91 (100); HRMS (EI) m/z calcd for C34H36O6 540.2512, found 540.2523. (7S,8R,80 S)-(21): [α]20D 8 (c 0.5, CHCl3). (7R,8S,80 R)-3,30 -Dimethoxy-7,90 -epoxylignane-4,40 ,9-triol (5). A reaction mixture of benzyl ether 21 (47 mg, 0.087 mmol) and 5% Pd/C (0.10 g) in EtOAc (20 mL) was stirred under H2 gas at the ambient temperature for 12 h before filtration. The filtrate was concentrated, and then the residue was applied to silica gel column chromatography with EtOAc/hexane (1:3, v/v) to give the 7R,8S,80 R stereoisomer, 5 (24 mg, 0.067 mmol, 77%), as a colorless oil: [α]20D +8 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.72 (1H, br s, OH), 1.99 (1H, m, H-8), 2.49 (1H, m, H-80 ), 2.67 (1H, dd, J = 13.8, 8.6 Hz, H-70 a), 2.79 (1H, dd, J = 13.8, 6.8 Hz, H-70 b), 3.63 (2H, d, J = 5.5 Hz, H-9), 3.84 (1H, dd, J = 8.8, 5.9 Hz, H-90 a), 3.85 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.95 (1H, dd, J = 8.8, 7.3 Hz, H-90 b), 4.60 (1H, d, J = 7.9 Hz, H-7), 5.60 (1H, br s, PhOH), 5.70 (1H, br s, PhOH), 6.65 (1H, d, J = 2.0 Hz, H-2), 6.66 (1H, dd, J = 8.2, 2.0 Hz, H-6), 6.82 (1H, d, J = 8.2 Hz, H-5), 6.85 (1H, dd, J = 7.8, 1.9 Hz, H-60 ), 6.88 (1H, d, J = 7.8 Hz, H-50 ), 6.91 (1H, d, J = 1.9 Hz, H-20 ); 13C NMR (100 MHz, CDCl3) δ 39.4 (CH, C-8), 44.2 (CH, C-80 ), 55.5 (CH2, C-70 ), 55.9 (CH3, OCH3), 56.0 (CH3, OCH3), 62.8 (CH2, C-9), 73.0 (CH2, C-90 ), 84.1 (CH, C-7), 108.8 (C-2), 111.3 (C-20 ), 114.3 (C-5), 114.4 (C-50 ), 119.2 (C-6), 121.4 (C-60 ), 132.1 (C-1), 134.0 (C-10 ), 144.1 (C-40 ), 145.2 (C-4), 146.5 (C-30 ), 146.7 (C-3); MS (EI) m/z 360 (M+, 85), 137 (100); HRMS (EI) m/z calcd for C20H24O6 360.1573, found 360.1570; >99% ee (AD-H, iso-PrOH/ hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR8 min). (7S,8R,80 S)-(6): [α]20D 8 (c 2, CHCl3); >99% ee (AD-H, isoPrOH/hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR7 min). (7R,80 R)-4,40 -Dibenzyloxy-3,30 -dimethoxy-7,90 -epoxylign-8-ene (22). To an ice-cooled solution of alcohol 21 (0.17 g, 0.31 mmol) and Et3N (0.060 mL, 0.43 mmol) in CH2Cl2 (5 mL) was added MsCl (0.033 mL, 0.43 mmol). After 1 h of stirring at room temperature, H2O and CH2Cl2 were added. The organic solution was separated and dried (Na2SO4). Concentration gave crude mesylate. A reaction mixture of crude mesylate, NaI (0.13 g, 0.87 mmol), and DBU (0.084 mL, 0.56 mmol) in DMF (5 mL) was stirred at 80 °C for 2 h before additions of H2O and CHCl3. The organic solution was separated and dried (Na2SO4). Concentration followed by silica gel column chromatography with EtOAc/hexane (1:1, v/v) gave alkene 22 (0.10 g, 0.19 mmol, 61%) as a colorless oil; [α]20D 2 (c 3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 2.70 (1H, dd, J = 13.8, 10.1 Hz, H-70 a), 2.86 (1H, dd, J = 13.8, 6.0 Hz, H-70 b), 3.05 (1H, m, H-80 ), 3.85 (3H, s, OCH3), 3.853.90 (2H, overlapped, H-90 ), 3.90 (3H, s, OCH3), 4.75 (1H, d, J = 1.9 Hz, H-9a), 4.97 (1H, d, J = 1.9 Hz, H-9b), 5.12 (2H, s, OCH2Ph), 5.15 (2H, s, OCH2Ph), 5.20 (1H, s, H-7), 6.66 (1H, dd, J = 8.3, 1.8 Hz, H-6), 6.72 (1H, d, J = 1.8 Hz, H-2), 6.81 (1H, d, J = 8.3 Hz, H-5), 6.86 (2H, s, H-20 and H-50 ), 6.93 (1H, s, H-60 ), 7.297.31 (2H, m, OCH2PhH), 7.347.37 (4H, m, OCH2PhH), 7.427.44 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 39.5 (CH, C-80 ), 45.8 (CH2, C-70 ), 55.9 (CH3, OCH3), 56.0 (CH3, OCH3), 71.0 (CH2, OCH2Ph), 71.1 (CH2, OCH2Ph), 71.7 (CH2, H-90 ), 84.0 (CH, C-7), 107.6, 111.0, 112.7, 113.6, 114.1, 119.8, 120.8, 127.19, 127.22, 127.7, 128.5, 133.1, 134.0, 137.2, 146.6, 147.9, 149.5, 149.6, 154.4; MS (EI) m/z 522 (M+, 9), 91 (100); HRMS (EI) m/z calcd for C34H34O5 522.2406, found 522.2424. (7S,80 S)-(22): [α]20D +2 (c 5, CHCl3). (7R,8R,80 R)-4,40 -Dibenzyloxy-3,30 -dimethoxy-7,90 -epoxylignan-9-ol (23). To an ice-cooled solution of alkene 22 (0.10 g, 0.19 mmol) in THF

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(5 mL) was added BH3 3 SMe2 (0.021 mL, 0.22 mmol). After the reaction solution had been stirred at room temperature for 1 h, saturated aqueous NaHCO3 solution (1 mL) and 30% aqueous H2O2 solution (1 mL) were added. The resulting mixture was stirred at room temperature for 1 h, and then CHCl3 and H2O were added. The organic solution was separated and dried (Na2SO4). Concentration followed by silica gel column chromatography with EtOAc/hexane (1:2, v/v) gave alcohol 23 (0.10 g, 0.18 mmol, 95%) as a colorless oil: [α]20D +33 (c 0.47, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.75 (1H, br s, OH), 2.40 (1H, m, H-8), 2.70 (1H, dd, J = 15.3, 11.7 Hz, H-70 a), 2.882.93 (2H, m, H-80 and H-70 b), 3.49 (1H, dd, J = 11.4, 3.7 Hz, H-9a), 3.55 (1H, dd, J = 11.4, 5.1 Hz, H-9b), 3.83 (1H, dd, J = 8.3, 6.6 Hz, H-90 a), 3.86 (6H, s, OCH3  2), 3.98 (1H, dd, J = 8.3, 7.8 Hz, H-90 b), 5.09 (1H, d, J = 5.4 Hz, H-7), 5.12 (2H, s, OCH2Ph), 5.13 (2H, s, OCH2Ph), 6.70 (1H, dd, J = 8.2, 1.8 Hz, H-6), 6.76 (1H, d, J = 1.8 Hz, H-2), 6.81 (1H, d, J = 8.2 Hz, H-5), 6.846.89 (2H, m, H-50 and H-60 ), 6.91 (1H, d, J = 1.4 Hz, H-20 ), 7.277.31 (2H, m, OCH2PhH), 7.337.37 (4H, m, OCH2PhH), 7.427.44 (4H, m, OCH2PhH); 13C NMR (100 MHz, CDCl3) δ 34.0 (CH, C-8), 44.1 (CH, C-80 ), 47.9 (CH2, C-70 ), 55.9 (CH3, OCH3), 59.4 (CH2, C-9), 71.0 (CH2, OCH2Ph), 71.1 (CH2, OCH2Ph), 72.4 (CH2, C-90 ), 83.0 (CH, C-7), 109.1, 112.2, 114.1, 114.2, 117.5, 120.2, 127.2, 127.3, 127.7, 127.8, 128.5, 132.3, 133.7, 137.0, 137.2, 146.5, 147.3, 149.6, 149.8; MS (EI) m/z 540 (M+, 82), 91 (100); HRMS (EI) m/z calcd for C34H36O6 540.2512, found 540.2515. (7S,8S,80 S)-(23): [α]20D 31 (c 0.40, CHCl3). (7R,8R,80 R)-3,30 -Dimethoxy-7,90 -epoxylignane-4,40 ,9-triol (7). A reaction mixture of benzyl ether 23 (26 mg, 0.048 mmol) and 5% Pd/C (0.10 g) in EtOAc (10 mL) was stirred under H2 gas at ambient temperature for 12 h before filtration. The filtrate was concentrated, and then the residue was applied to silica gel column chromatography with EtOAc/hexane (1:3, v/v) to give the 7R,8R,80 R) stereoisomer, 7 (17 mg, 0.048 mmol, 100%) as a colorless oil: [α]20D +35 (c 0.39, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.62 (1H, br. s, 9-OH), 2.43 (1H, m, H-8), 2.73 (1H, m, H-80 ), 2.902.98 (2H, m, H-70 ), 3.54 (1H, dd, J = 11.7, 3.3 Hz, H-9a), 3.61 (1H, dd, J = 11.7, 5.5 Hz, H-9b), 3.85 (1H, dd, J = 8.3, 8.3 Hz, H-90 a), 3.88 (3H, s, OCH3), 3.89 (3H, s, OCH3), 4.01 (1H, dd, J = 8.3, 8.3 Hz, H-90 b), 5.11 (1H, d, J = 5.5 Hz, H-7), 5.53 (1H, s, PhOH), 5.63 (1H, s, PhOH), 6.726.74 (2H, m, H-2 and H-6), 6.846.86 (2H, m, H-5 and H-60 ), 6.916.93 (2H, m, H-20 and H-50 ); 13C NMR (100 MHz, CDCl3) δ 34.1 (CH, C-8), 44.3 (CH, C-80 ), 48.0 (CH2, C-70 ), 55.88 (CH3, OCH3), 55.93 (CH3, OCH3), 59.5 (CH2, C-9), 72.4 (CH2, C-90 ), 83.1 (CH, C-7), 107.9 (C-2), 111.0 (C-20 ), 114.4 (C-5), 114.6 (C-50 ), 118.4 (C-6), 120.9 (C-60 ), 131.1 (C-1), 132.4 (C-10 ), 143.9 (C-40 ), 144.8 (C-4), 146.5 (C-30 ), 146.7 (C-3); MS (EI) m/z 360 (M+, 83), 137 (100); HRMS (EI) m/z calcd for C20H24O6 360.1573, found 360.1561; >99% ee (AD-H, iso-PrOH/hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR 24 min). (7S,8S,80 S)-(8): [α]20D 35 (c 0.62, CHCl3); >99% ee (AD-H, isoPrOH/hexane = 1:2 (v/v), 1 mL/min, 280 nm, tR15 min). Evaluation of Plant Growth Regulatory Activity. The plant growth regulatory activity of all stereoisomers of lariciresinol was evaluated using lettuce (Lactuca sativa L. Green-wave (Takii Seed Co. Ltd., Kyoto, Japan)) and Italian ryegrass (Lolium multiflorum Lam. Wase-fudo (Takii Seed Co. Ltd.)). A sheet of filter paper (diameter = 90 mm) was placed in a 90 mm Petri dish and wetted with 500 μL of test sample solution dissolved in acetone at concentrations from 6.0  103 to 6.0  108 M. After the filter paper had dried, 3 mL of water was poured into the dish to adjust the final concentration from 1.0  103 to 1.0  108 M. Thirty seeds of each plant were placed on the filter paper, and the Petri dishes were sealed with Parafilm. The Petri dishes were then incubated in the dark at 20 °C. The lengths of roots and shoots were measured after 3 days for lettuce and after 5 days for ryegrass by using an ordinary ruler. The root and shoot lengths of the control were ca. 3 and 1 cm for lettuce and 4 and 3 cm for ryegrass, respectively. We compared 13092

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the root and shoot lengths of the plants induced by each compound at different concentrations (lettuce, 1  103 M; ryegrass, 1  1031  108 M). Data are presented as percentage differences from the control; positive and negative values present stimulation and inhibition of plant growth, respectively. Experiments were performed in triplicate or more for each sample (n = 37). Statistical analyses were conducted by oneway ANOVA followed by Tukey’s multiple-comparison test using PRISM software (Graphpad Software, San Diego, CA), and the values of p < 0.05 were considered to be statistically significant. The germination rates of ryegrass seeds when treated with natural compounds 1 and 2 at a concentration of 1  103 M were recorded 3 days after seeding. The germination rate of ryegrass seeds treated with 3 (1  103 M) was also recorded because this compound has the highest potency against ryegrass growth.

’ RESULTS AND DISCUSSION The synthetic scheme is described in Figure 2. For the syntheses of lariciresinol stereoisomers 3, 5, and 7, lactone 913 was employed as the starting material. After protection of the primary hydroxy group as triisopropylsilyl ether, the resulting lactone 17 was reduced to diol by successive diisobutylaluminum hydride and NaBH4 reductions. The resulting diol was converted to the 7S tetrahydrofuran intermediate 18 as a single isomer with the retention of benzylic stereochemistry by treatment with p-toluenesulfonyl chloride and pyridine. On the other hand, exposure of the diol to a catalytic amount of 10-camphorsulfonic acid gave the 7R tetrahydrofuran intermediate 20 via the benzyl cation at the 7-position. The peak of 8-H of 20 (1.94 ppm) was observed in a higher field than that of compound 18 (2.33 ppm) because of the shielding effect, suggesting the presence of 7,8-cis18 and 7,8-trans-20. This SN1-type reaction gave the stable 7R,8S isomer as a single isomer rather than the sterically hindered 7S,8S isomer. Deprotection of 18 and 20 by treatment with (n-Bu)4NF followed by hydrogenolysis in the presence of Pd/C gave stereoisomers 3 and 5, respectively. To obtain the all-cis stereoisomer 7, stereoselective hydroboration of olefin 22 was employed. Olefin 22 was prepared from (7R,80 R)-hydroxymethyltetrahydrofuran intermediate 21 by reaction with methanesulfonyl chloride and triethylamine followed by treatment with 1,8-diazabicyclo[3.3.0]undec-7-ene and NaI. Because of the effect of the configurations of the 7- and 80 -positions, hydroboration using BH3 3 SMe2 stereoselectively proceeded to give the all-cis tetrahydrofuran intermediate 23 as a single isomer. Finally, hydrogenolysis in the presence of Pd/C gave the all-cis stereoisomer 7. Stereoisomers 4, 6, and 8 were synthesized from lactone 13 according to the same process. The differential NOE experiment was performed to confirm their stereochemistry (Figure 3). After irradiation of 7-H (5.04 ppm) of 3, NOE effects were observed at two 70 benzylic protons (2.66 and 2.84 ppm). Furthermore, on irradiation of 7-H (4.60 ppm) of 5, NOE effects were observed at 9-H2 (3.63 ppm) and 80 -H (2.49 ppm). In the case of stereoisomer 7, correlation between 7-H (5.11 ppm) and 80 -H (2.73 ppm) was observed by irradiation of 7-H. NOE effects were observed between neither 7-H and 9-H2 nor 7-H and 70 -H2. The plant growth regulatory activity of all prepared stereoisomers of lariciresinol 18 for lettuce and Italian ryegrass was evaluated, as shown in Figure 4. When ()-1, one of the natural compounds, was applied at the concentration of 1 mM, the growth rates of lettuce roots and shoots relative to the negative control were 8.2 and 13.3%, respectively (Figure 4A), suggesting that the compound was ineffective for lettuce growth. When applied at the same concentration, enantiomer (+)-2, another

Figure 3. Differential NOE of lariciresinol 1 and its stereoisomers 3, 5, and 7. Only the important correlations for determining the configuration are illustrated. Ar = 4-hydroxy-3-methoxyphenyl.

natural compound of lariciresinol, also had no significant effect on the growth of lettuce roots and shoots (14.4 and 22.0%, respectively). When ryegrass was treated with 1 and 2 at the concentration of 1  103 M, 1 suppressed root growth to 44.3%, although it showed lower inhibitory activity toward shoots (17.1%), and 2 suppressed root growth to 29.3%, indicating that 2 was relatively less potent than 1 (Figure 4B,C). These results suggest that the sensitivity of ryegrass to lariciresinol is higher than that of lettuce and that the plant regulatory activity of lignans for ryegrass roots depends on their stereochemistry. We examined whether the activity of 1 and 2 for ryegrass would change relative to concentration. Dilution of the sample solution to concentrations from 104 to 106 M slightly stimulated ryegrass root growth (up to 20%). The growth regulatory activity of 38, stereoisomers of lariciresinol other than natural compounds, was evaluated to investigate the effect of the stereochemistry of lariciresinol isomers on their activity. The growth rates of lettuce roots and shoots treated with 13 and 58 were between 20.7 and 1.6%, respectively, indicating that most of the diastereomers were ineffective for lettuce growth (Figure 4A). For the 7R,8R,80 S isomer, 4, the growth rate of lettuce shoots was 32.3%, which was slightly higher but not entirely effective. For ryegrass (Figure 4B,C), the 7S,8S,80 R isomer, 3, was found to be equipotent to natural compound 1 (7R,8S,80 S isomer). When 3 was applied at 1  103 M, the growth rate of ryegrass roots was 48.2%, and at concentrations from 104 to 107 M, root growth was stimulated up to 20%. On the other hand, enantiomer 4 (7R,8R,80 S isomer) was less potent than 3. Compounds 5 and 6 showed no adverse effect on ryegrass growth at each concentration examined. Compounds 7 and 8, the 7R,8R,80 R and 7S,8S,80 S isomers, respectively, suppressed the growth rate of ryegrass roots to around 30% at 1  103 M, but the absolute values of the rate 13093

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Figure 4. Effects of all stereoisomers of lariciresinol on growth of test plants: (A) root (black bars) and shoot (white bars) lengths of L. sativa when treated with each compound at the concentration of 1  103 M (the numbers of experiments are shown in parentheses); (B) root and (C) shoot lengths of L. multiflorum when treated with each compound at the concentration from 1  103 to 1  108 M (from left to right) (the number in parentheses is the number of experiments at 1  103 M; for all other concentrations, the number of experiments is three). Data are presented as the mean ( standard error of the mean. Asterisks indicate that the difference in data between sample treatment and the control is statistically significant (one-way ANOVA, Tukey post-test, p < 0.05).

decreased to below 20% by a series of dilution. When 7 and 8 were applied, the growth rates of ryegrass shoots were below 23%. Among 18, 1 and 3 significantly inhibited the root growth of ryegrass relative to the control (p < 0.05, one-way ANOVA, Tukey post test), suggesting the importance of both 8S absolute and 7,80 -trans relative configurations for higher inhibitory activity. Some lignans as well as ()-1 reportedly inhibit the germination of lettuce and peanut seeds.9,16 This prompted us to measure the germination rate of ryegrass seeds treated with natural compounds 1 and 2 as well as 3, which had the highest potency. The seed germination rates achieved with 13 were 80.0 ( 7.91% (n = 3), 72.5 ( 6.49% (n = 3), and 72.5 ( 2.20% (n = 3), respectively. On the other hand, the germination rate was 90.3 ( 1.40% (n = 3) when the sample was not treated with these compounds, suggesting that these compounds suppress germination to some extent. In addition, relative to the negative control, the growth rates of germinated grass roots were 57.2 ( 4.00% (n = 3), 40.7 ( 1.81% (n = 3), and 35.4 ( 6.49% (n = 3) for compounds 13, respectively. These results suggested that potent lignans 1 and 3 not only delayed the germination

of grass seeds but also suppressed root growth, thus inducing the apparent root growth inhibitory activity. (+)-Lariciresinol 2, the potency of which for seed germination and root elongation was comparable with that of the highest potency compound, 3, did not exhibit significant inhibitory activity; however, the reason for this remains unknown. Oliva et al.17 reported that the inhibition of root growth should be related to the influence of either cell division or cell elongation and that some aryltetralin lignans affected mitotic microtubular formation. Furthermore, lariciresinol was recently reported to change the membrane permeability of fungi, exhibiting antifungal activity.18 On the other hand, the biosynthetic pathway of lignans from coniferyl alcohol to matairesinol via lariciresinol has been elucidated in some plants, and some enzymes in the pathway have also been identified and crystallized.1922 It has also been reported that each pinoresinol/lariciresinol reductase Lu-1 and Lu-2 of Linum usitatissimum enantiospecifically recognizes 1 and 2, respectively.23 In addition, it has been reported that lariciresinol affected carbon and nitrogen metabolism, reducing glutamate synthase activity to inhibit the germination of lettuce seed 13094

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Journal of Agricultural and Food Chemistry by lariciresinol.24 In this study, the activities of stereoisomers of lariciresinol differed from each other, suggesting the involvement of receptors or enzymes. For 1 and 3, not only the apparent inhibition activity at 1  103 M but also low stimulation activity at lower concentrations was observed, which might suggest hormonal action. Although there are many possibilities for the mode of action of the plant regulatory activity of lariciresinol, it needs to be clarified. Recently, new stereoisomers of tetrahydrofuranoid lignans were isolated,25 leading us to expect the discovery of novel stereoisomers of lariciresinol from the nature. In addition to natural compounds 1 and 2, 38, which have not been identified as natural compounds to the best of our knowledge, are expected be effective in elucidating the mode of action of lariciresinol. In summary, we have established the synthetic pathways of all stereoisomers of lariciresinol for the first time. This synthetic study has enabled us to compare the biological activities of all lariciresinol stereoisomers; the results suggest the importance of both the 8S absolute configuration and the 7,80 -trans relative configuration for high inhibitory activity.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +81-89-946-9846. Fax: +81-89-977-4364. E-mail: [email protected]. Funding Sources

We are grateful to Marutomo Co. for financial support.

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