Stereochemistry of erythro- and threo-syringylglycerol-8-O-4'-(sinapyl alcohol) ethers and their enzymatic formation with optical activity in Eucommia ulmoides

Abstract

In this study an enzymatic system, horseradish peroxidase (HRP)-hydrogen peroxide was used as catalysts for the enzymatic formation of enantiospecific syringulglycerol-8-O-4' (sinapyl alcohol) ethers (SGSEs) from enzyme preparations of Eucommia ulmoides with sinapyl alcohol (SA) as a monolignol precursor. Reversed phase HPLC analysis of the erythro- and threo- SGSE showed that the ratio of erythro: threo was 47: 53. Both isomers were isolated by HPLC and their structural confirmation was done by ^sup 1^H NMR spectra. Chiral column HPLC analysis of the erythro- and threo-SGSE showed that their enantiomeric compositions were as follows: (+)- erythro: (-)-erythro = 46.7:53.3 (6.6% e.e), and (+)-threo: (-)-threo = 45.2: 54.8 (9.6% e.e). To elucidate the stereochemistry of erythro and threo- SGSEs, we have determined absolute configurations of the four stereoisomers, (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo- SGSEs as (7R, 8S), (7S, 8R), (7S, 8S), and (7R, 8R), respectively, by Mosher's method through the ^sup 1^H NMR spectroscopy of (+)-(R)-α-methoxy- α- trifluoromethylphenylacetate (MTPA) esters of α, γ and γ' positions. [PUBLICATION ABSTRACT]

Headnote

Abstract: In this study an enzymatic system, horseradish peroxidase (HRP)-hydrogen peroxide was used as catalysts for the enzymatic formation of enantiospecific syringulglycerol-8-O-4' (sinapyl alcohol) ethers (SGSEs) from enzyme preparations of Eucommia ulmoides with sinapyl alcohol (SA) as a monolignol precursor. Reversed phase HPLC analysis of the erythro- and threo- SGSE showed that the ratio of erythro: threo was 47: 53. Both isomers were isolated by HPLC and their structural confirmation was done by ^sup 1^H NMR spectra. Chiral column HPLC analysis of the erythro- and threo-SGSE showed that their enantiomeric compositions were as follows: (+)- erythro: (-)-erythro = 46.7:53.3 (6.6% e.e), and (+)-threo: (-)-threo = 45.2: 54.8 (9.6% e.e). To elucidate the stereochemistry of erythro and threo- SGSEs, we have determined absolute configurations of the four stereoisomers, (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo- SGSEs as (7R, 8S), (7S, 8R), (7S, 8S), and (7R, 8R), respectively, by Mosher's method through the ^sup 1^H NMR spectroscopy of (+)-(R)-α-methoxy- α- trifluoromethylphenylacetate (MTPA) esters of α, γ and γ' positions.

Key words: Stereochemistry, SGSE, erythro, threo, MTPA, soluble enzyme, Eucommia ulmoides

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Introduction

Arylglycerol-β-aryl ethers are one of the most important substructures in lignin as well as lignans and neolignans. The biosynthesis of lignin has been advanced; phenolic phenylpropanoides occur lignin via oxidative processes catalyzed by enzymes such as peroxidase, which exchange the phenols into phenoxy radicals by an electron abstraction which is followed by C-C and carbon-oxygen bond formation (Ayres and Loike, 1990). Formation of chiral centers has been occurring in this circumstance. In addition biosynthesis of neolignans, especially syringyl 8-O-4'has a little known in contrast to guaiacyl one. Enantiomerically pure, (±)-α-oxoguaiacylglycerol- β-(vanillic acid) ether which formed from bio-degradation and α-ketonised racemate guaiacylglycerol- β-(vanillic acid) ethers have been reported by Katayama and others (Katayama et al., 1986; Katayama and Sogo, 1989). Recently Katayama et al. (2005) determined absolute configuration of the four stereoisomers, (+)-erythro-, (-)-erythro-, (+)- threo-, and (-)-threo-GGCEs as (7R, 8S), (7S, 8R), (7S, 8S), and (7R, 8R), respectively, by Mosher's method (Dale et al., 1969; Dale and Mosher, 1973; Yamaguchi, 1985). They suggested that (+)-erythro-GGCE was formed by the selective water addition to the (8R)- enantiomer of the racemic quinonemethide. Lignans and neolignans (except for 9,9?-deoxy ones) are biosynthesized from coniferyl alcohol (which contains 4-hydroxy-3-methoxyphenyl group, so-called guaiacyl group) or sinapyl alcohol (which contains 4-hydroxy-3,5- dimethoxyphenyl group, so-called syringyl group). Biosynthesis of guaiacyl lignans/neolignans from coniferyl alcohol have been studied much more than that of syringyl lignans/neolignans derived from sinapyl alcohol. Especially there were no studies on syringyl-8-O-4? neolignans information. Therefore, Lourith et al. (2005) found the formation of optically active erythro and threo- SGSEs by feeding experiments using radiolablled SA and the excised shoots of E. ulmoides as well as by the incubation of a mixture of SA and CA with an insoluble enzyme preparation from the plant. Recently, it have been reported (Alam et al., 2009) that incubation of SA with a cell-free extract (a soluble enzyme preparation) of E. ulmoides in the presence of hydrogen peroxide (H2O2) gave (-)-erythro- and (-)-threo-SGSEs, whereas incubation of SA with a cell-wall residue (an insoluble enzyme preparation) in the absence of H2O2 (+)-erythro- and (-)-threo-SGSEs. Tuning the enzymatic reaction to give dimers, and the use of metal complexes which mimic the enzymatic reaction allows the observation of the stereochemical course at the stereocenters and gives information related to the mechanism of lignin formation in nature. To clarify the formation mechanism of SGSE, stereochemistry of the four stereoisomers, (+)- erythro-, (-)-erythro-, (+)-threo-, and (-)-threo, of SGSEs, were required. However, the absolute configurations were unknown. In this paper, we report determination of their absolute configurations by Mosher's method [1H NMR spectroscopy of (R)-(+)-(MTPA) esters of SGSEs] and the related empirical rules (Katayama et al., 2000). In this study an enzymatic system, horseradish peroxidase (HRP)-hydrogen peroxide was used as catalysts for the enzymatic formation of enantiospecific syringulglycerol-8-O-4' (sinapyl alcohol) ethers (SGSEs) from enzyme preparations of E. ulmoides with sinapyl alcohol (SA) as a monolignol precursor. The (-)-erythro isomer was more favored than the (- )-threo one, with optical activity.

Materials and Methods Instrumentation and chromatography materials

All reagents and solvents were reagent grade. Analytical and preparative thin-layer chromatography (TLC) was done by using of plates precoated with Merck silica gel 60 F-254 (0.25mm and 0.5mm thickness, respectively). NMR spectra (600 MHz) were measured on a JEOL JNM - DELTA - 600 FT - NMR spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts and coupling constants (J) were expressed as δ (in ppm) and Hz, respectively. Analytical high performance liquid chromatography (HPLC) was carried out on a Jasco PU-2089 equipped with a Jasco UV-2075 plus Intelligent UV/VIS detector and a Shimadzu chromatopac C-R7A plus using a reversed phase column (TSKGEL, ODS-80Ts, column No E9479). Compounds were separated at a flow rate of 1.0 ml/min using the following linear gradient solvent system: MeOH with 3% AcOH in H2O (v/v) starting with isocratic elution at 25:75 (or 23:77) which was held for 10 min, and then linearly increased to 32:68 (or 28:72) within 5 (or3) min. This elution condition was then held for the remainder of the analysis. Chiral analysis was performed on a Daicel Chiralcel OD (H) column (250 x 46 mm) eluted with EtOH/n-hexane (23:77) (v/v) at a flow 1.0ml/min (for erythro SGSE) rate and of 0.8 ml/min (for threo-SGSE). All detection condition was at 280nm.

Plant materials

Eucommia ulmoides (Tochu) plants (Figure 1) were obtained from Sanyo Nouen Inc. and maintained at Faculty of Agriculture, Kagawa University, Japan.

Chemical synthesis

Monolignol (sinapyl alcohol) is the major precursor of syringyl lignans and neolignans (SGSE) was synthesized by the literature methods (Lourith et al., 2005; Tanahashi et al., 1976) and their diastereomer (erythro and threo SGSEs) were separated by TLC, subsequently quantification was done by high-performance liquid chromatography (HPLC) and their structures were determined by 1H NMR, 13C NMR. Enantiomeric separation of erythro and threo SGSEs has been done by chiral column HPLC as (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo-SGSEs for transformed there to (+)-MTPA esters derivatives. Diazomethane (CH2N2) has been prepared by the method of Bore and Backer (Bore and Backer, 1963) using carbitol with ρ-tolylsulfonyl meththyl nitrosamide in ether (Et2O). Methylation of the phenolic hydroxyl group of SGSEs and tri-(R)- MTPA esters were prepared by a method described in the literature (Dale and Mosher, 1973; Katayama et al., 2000).

Preparation of (+)-erythro-3,4,5- trimethoxyphenylglycerol-8-O-4'-(sinapyl alcohol) ethers

To a stirred solution of (+)-erythro-SGSE (4.6 mg, 10.5384μmol) in MeOH (0.5ml), a yellow color of freshly prepared diazomethane (CH2N2) solution (3ml) was added at 0o C temperature with a small size of staraba. Reaction condition was observed by analytical TLC [benzene/acetone = 1:1 (x1)] after 30min interval. The reaction was quenched by the addition of a small amount of granulated Na2SO4. The reaction mixture was then filtered over Na2SO4 with short column and wash by CH2Cl2. The filtered solution was then evaporated to dryness in vacuo. The residue was purified by preparative TLC [benzene/acetone = 1:1 (x1)] to give (+)- erythro-3,4,5-trimethoxyphenylglycerol-8-O- 4'-(sinapyl alcohol) ethers (3.48 mg, yield 96.90%). Structure was determined by 1H NMR spectra. Other stereoisomers [(-)-erythro, (+)-threo and (-)-threo-SGSEs] also converted to 3,4,5-trimethoxyphenylglycerol-8-O-4'- (sinapyl alcohol) ethers by the similarly to above one and confirmed by 1H NMR spectra.

4-O-methyl ether of (+)-erythro-SGSE

1H NMR delta (CDCl3): 3.923 ( s, 7-OH), 3.871 (s, 9-OH), 3.830 (s, 9'-OH), 3.850 [(exchangeable), 6H, s, 3 & 5-OCH3], 3.907 [(exchangeable), 6H, s, 3? & 5?-OCH3], 3.822 (3H, s, 4-OCH3 ), 4.123 (1H, m, 8-H), 5.040 (1H, d, J=8.82, 7-H), 6.590 (1H, d, J=15.96, 7'-H), 6.365 (1H, m, 8'-H), 4.357 (2H, d, J=4.68, 9'-H), 6.569 [(exchangeable), 2H, s, 2 & 6-H], 6.687 [(exchangeable), 2H, s, 2? & 6?- H].

4-O-methyl ether of (-)-erythro-SGSE

1H NMR delta (CDCl3): 3.923 ( s, 7-OH), 3.871 (s, 9-OH), 3.490 (o, 9'-OH), 3.851 [(exchangeable), 6H, s, 3 & 5-OCH3], 3.907 [(exchangeable), 6H, s, 3? & 5?-OCH3], 3.821 (3H, s, 4-OCH3 ), 4.128 (1H, m, 8-H), 5.039 (1H, d, J= 8.52, 7-H), 6.510 (1H, d, J=16.04, 7'-H), 6.344 (1H, m, 8'-H), 4.345 (2H, dd, J = 12.24, 7.50, 9'-H), 6.568 [(exchangeable) 2H, s, 2 & 6-H], 6.687 [(exchangeable) 2H, s, 2' & 6'- H ]

4-O-methyl ether of (+)-threo-SGSE

1H NMR delta (CDCl3): 2.691 ( s, 7-OH), 2.612 (s, 9-OH), 2.656 (d, J = 3.54, 9'-OH), 3.872 [(exchangeable), 6H, s, 3 & 5-OCH3], 3.925 [(exchangeable), 6H, s, 3? & 5?-OCH3], 3.830 (3H, s, 4-OCH3 ), 4.314 (1H, m, 8-H), 5.037 (1H, d, J=8.52, 7-H), 6.568 (1H, d, J =15.66, 7'-H), 6.321 (1H, dt, J = 15.81, 5.22, 8'-H), 4.352 (2H, d, J=4.38, 9'-H), 6.665 [(exchangeable), 2H, s, 2 & 6-H ], 6.703 [(exchangeable), 2H, s, 2' & 6'-H].

4-O-methyl ether of (-)-threo-SGSE

1H NMR delta (CDCl3): 2.690 ( s, 7-OH), 2.611 (s, 9-OH), 2.655 (d, J=5.22, 9'-OH), 3.871 [(exchangeable), 6H, s, 3 & 5-OCH3 ], 3.924 [(exchangeable), 6H, s, 3' & 5'-OCH3 ], 3.830 (3H, s, 4-OCH3 ), 4.3103 (1H, m, 8-H), 5.039 (1H, d, J=8.52, 7-H), 6.567 (1H, d, J=15.90, 7'-H), 6.320 (1H, dt, J=15.90, 5.41, 8'-H), 4.347 (2H, d, J=5.22, 9'-H), 6.664 [(exchangeable), 2H, s, 2 & 6-H ], 6.703 [(exchangeable), 2H, s, 2' & 6'-H].

Preparation of (+) -erythro-tri-(R)-MTPA esters of 3,4, 5-trimethoxyphenylglycerol-8- O-4'-(SA) ether

To a stirred solution of (+)-erythro-3,4,5- trimethoxyphenylglycerol-8-O-4'-(sinapyl alcohol) ethers (3.48mg, 7.66μmol) in 2ml of dry CH2Cl2 with DCC (18.96mg, 91.88μmol), (R)-MTPA esters (16.96mg, 71.90μmol) and DMAP (7.94mg, 65.00μmol) in 3ml of dry CH2Cl2 were added at 35oC temperature. After stirring for 15h under N2 atmosphere reaction mixture was cold at room temperature. The mixture was filtered by KIRIYAMA filter, washed with CH2Cl2 and then concentrated under reduced pressure by rotary evaporator. The resulting residue was purified by preparative TLC (100% CH2Cl2) to afford (+) - erythro-tri-(R)-MTPA esters of 3,4,5- trimethoxyphenylglycerol-8-O-4'-(SA) ether (4.1mg,48.43%). Other stereoisomers also converted to tri-(R)-MTPA esters of 3,4,5- trimethoxyphenylglycerol-8-O-4'-(sinapyl alcohol) ethers by the similarly to above one and determination was done by 1H NMR spectra.

(+) -Erythro-tri-(R)-MTPA esters of 3, 4, 5- trimethoxyphenylglycerol-8-O-4'-(sinapyl alcohol) ether

1H NMR delta (Chloroform D+TMS ): 3.6779 (3H, s, 7-MTPA-OCH3), 3.4229 (3H, s, 9-MTPA-OCH3), 3.5834 (3H, d, J=0.84, 9'- MTPA-OCH3), 3.6157 [(exchangeable),6H, s, 3 & 5 -OCH3], 3.6422 [(exchangeable),6H, s, 3' & 5' -OCH3], 3.7951 (3H, s, 4-OCH3), 4.86315 (1H, dd, J=11.94, 6.72 , 9a-H), 4.41125 (1H, dd, J=12.12, 3.30, 9b-H), 4.7312 (1H, m, 8-H), 6.1613 (1H, d, J=3.24, 7-H), 6.2045(1H, dt, J=15.66, 6.60, 8'-H), 6.5927(1H, d, J= 15.66, 7'-H), 4.9665 (2H, d, J=6.6, 9'-H), 6.2826 [(exchangeable),2H, s, 2 & 6 -H], 6.5311 [(exchangeable),2H, s, 2' & 6'-H].

(-) -Erythro-tri-(R)-MTPA esters of 3, 4, 5- trimethoxyphenylglycerol-8-O-4'-(SA) ether

1H NMR delta (Chloroform D+TMS ): 3.3699(3H, s, 7-MTPA-OCH3), 3.5489 (3H, s, 9-MTPA-OCH3), 3.5741 (3H, s, 9'-MTPAOCH3), 3.5951 [(exchangeable),6H, s, 3 & 5 - OCH3], 3.7645 [(exchangeable),6H, s, 3' & 5' - OCH3], 3.8144 (3H, s, 4-OCH3), 4.6255 (1H, dd, J=11.94, 3.42, 9a-H), 4.3910 (1H, dd, J=11.91, 4.53, 9b-H), 4.8043 (1H, m, 8-H), 6.12405 (1H, d, J=6.3, 7-H), 6.1435 (1H, dt, J=15.36,6.6,8'-H), 6.52985 (1H, d, J= 12.36, 7'-H), 4.9400 (2H, d, J=6.54, 9'-H), 6.4154 [(exchangeable),2H, s, 2 & 6 -H], 6.6136 [(exchangeable),2H, s, 2' & 6'-H].

(+) -Threo-tri-(R)-MTPA esters of 3, 4, 5- trimethoxyphenylglycerol-8-O-4'-(SA) ether

1H NMR delta (Chloroform D+TMS ): 3.4582(3H, s, 7-MTPA-OCH3), 3.4916(3H, s, 9-MTPA-OCH3), 3.5772 (3H, d, J=1.08 9'- MTPA-OCH3), 3.6010 [(exchangeable),6H, s, 3 & 5 -OCH3], 3.7667 [(exchangeable),6H, s, 3' & 5' -OCH3], 3.8546 (3H, s, 4-OCH3), 3.9211 (1H, dd, J=12.24, 3.96, 9a-H), 4.5168 (1H, dd, J=12.09, 3.57, 9b-H), 4.7845 (1H, dt, J=3.84, 6.3,8-H), 6.2966 (1H, dt, J=6.3, 7-H), 6.1718 (1H, dt, J= 15.96, 6.6, 8'-H), 6.5635 (1H, d, J= 15.66, 7'-H), 4.9523 (2H, dd, J=6.6, 1.08, 9'-H), 6.4739 [(exchangeable),2H, s, 2 & 6 -H], 6.6428 [(exchangeable),2H, s, 2' & 6'- H].

(-) -Threo-tri-(R)-MTPA esters of 3, 4, 5- trimethoxyphenylglycerol-8-O-4'-(SA) ether

1H NMR delta (Chloroform D+TMS ): 3.7178 or 3.6409 (3H, s, 7-MTPA-OCH3), 3.6409 or 3.4999 (3H, s, 9-MTPA-OCH3), 3.5851 (3H, s, 9'-MTPA-OCH3), 3.5068 [(exchangeable),6H, s, 3 & 5 -OCH3], 3.6643 [(exchangeable),6H, s, 3' & 5' -OCH3], 3.8057 (3H, s, 4-OCH3), 4.7625-4.7052 (3H, m, 8- H,9a-H & 9b-H), 6.3179 (1H, d, J=8.76, 7-H), 6.2070 (1H, dt, J=15.72, 6.51, 8'-H), 6.5983 (1H, d, J= 15.66, 7'-H), 4.9675 (2H, d, J=6.6, 9'-H), 6.3710 [(exchangeable),2H, s, 2 & 6 - H], 6.5394 [(exchangeable),2H, s, 2' & 6'-H].

Enzyme preparations

A soluble enzyme preparation [cell-free extracts with potassium phosphate (K-Pi) buffer] was prepared by the method of Katayama and Kado (Katayama and Sogo, 1989). All manipulations were carried out at 40C unless otherwise stated. Defoliated young shoot of E ulmoides (12-44 cm height, 58 g) were washed by tap water and distillation water wiped, sectioned (1-2 mm), frozen in liquid nitrogen, and reduced to a powder by means of a mortar and pestle. The resulting powder was homogenized with (polyvinyl polypyrilidon) PVPP (11.44 g), acid washed sea sand (22.88 g) and K-Pi buffering containing 10 mM (dithiothreitol) DTT (150 ml). The homogenate was filtered (Whatman GFA glass fiber filter). The filtrate (2.5 ml) was applied onto PD-10 column (Pharmacia, Sephadex G-25 M) equilibrated with K-Pi buffer. The excluded fraction (3.5 ml) was collected and used as crude enzyme.

Enzyme assay

Each assay mixture (5.75ml × 2) consisted of the soluble enzyme (5.0 ml) from defoliated young shoots of E. ulmoides, 0.43 mM of H2O2 (10 mM, 0.25 ml), and 2.6 mM of SA (30 mM, in 0.5ml of K-Pi buffer). After incubation at 300C for various time intervals, glacial AcOH (0.5 ml) was added. The assay mixture was then extracted three times with EtOAc (30 mlX3). The EtOAc solutions were combined, washed twice with saturated brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The resulting EtOAc extract was subjected to preparative TLC (7% MeOH in CH2Cl2 × 2) to isolate SGSE (a mixture of erythro and threo forms), which was then reconstituted in MeOH (100μl) and filtered through filter. An aliquot (10μl) of the filtrate was applied to C18 column reversed-phase HPLC. The erythro and threo diastereomers (Figure 2) were quantified as well as separated by reverse-phase column HPLC. Each diastereomer was then subjected to chiral column HPLC to quantify the enantiomers of SGSEs.

Results and Discussion

A mixture of (±)-erythro- and (±)-threo- SGSEs was prepared by dehydrogenative dimerization of SA with FeCl3 in dioxane-H2O (10:1) and purified by preparative TLC. (±)- Erythro-SGSE and (±)-threo-SGSE were separated carefully by preparative TLC and identified by 1H NMR acetone-d6 (data not shown) and HPLC using authentic samples (Lourith et al., 2005). The diastereomeric ratio (erythro:threo = 6:4) was determined by reverse phase HPLC. Their enantiomers were separated by chiral column HPLC to afford (+)- and- (-)-erythro-SGSEs, and (+)-and (-)- threo-SGSEs. Each phenolic hydroxyl group of the four stereoisomers was methylated with diazomethane (Figure 2A) separately. The resulting four stereoisomers were converted to 7, 9, 9?-tri-(R)-MTPA esters of 3,4,5- trimethoxyphenylglycerol-8-O-4?-(sinapyl alcohol) (Figure 2B) ethers and their structures were confirmed by 1H NMR spectra. According to the definition of erythro and threo diastereomers, absolute configuration of erythro SGSE and its 4-O-methyl ether is (7R, 8S) or (7S, 8R), and that of threo SGSE and its 4-O-methyl ether is (7R, 8R) or (7S, 8S).

According to the Mosher's method (Dale et al., 1969; Dale et al., 1973; Yamaguchi et al., 1985), because a preferred conformation of the MTPA esters has α-CH3, the >C=O of the MTPA ester, and the benzyl C-H in an eclipsed arrangement, the (R)-MTPA-OCH3 of (R)- MTPA ester of an (7S)-secondary benzyl alcohol [(R, 7S)-MTPA ester] is located on aromatic (e.g. 3,4,5-trimethoxyphenyl) ring, and the C8-H of the X group is on the benzene ring of the MTPA moiety, that is, the (R)- MTPA-OCH3 and the C8-H receive shielding effects. In contrast, the (R)-MTPA-OCH3 of (R)-MTPA ester of an (7R)-secondary benzyl alcohol [(R, 7S)-MTPA ester] is located not on the aromatic ring but on the C8-H, and the benzene ring is on the aromatic ring, that is the (R)-MTPA-OCH3 and the C8-H have no shielding effect. Therefore, the 1H NMR chemical shift (δs) of the (R)-MTPA-OCH3 of the (R, 7S)-MTPA ester is upfield relative to that (δR) of the (R)-MTPA-OCH3 of the (R, 7R)-MTPA ester. The Δδ value in the Mosher's method was defined as the absolute value of the difference in the 1H chemical shifts of the peak between the diastereomers, |δS-δR|. [Note: the relations between (R)- and (S)-isomers and between (+)- and (-)-isomers are enantiomers, whereas the relations between (R)-MTPA ester of (R)-isomer and that of (S)-isomer and between (R)-MTPA ester of (+)-isomer and that of (-)-isomer are diastereomers.

Furthermore, the authors' empirical rules (Katayama et al., 2000 and 2005) on the 1H NMR chemical shifts of arylglycerol-β(8-O- 4?)-aryl ethers are as below. Rule-1, the Δδ value of 7-MTPA-OCH3s were larger than those of 9-MTPA-OCH3s, because one diastereomer's 7-MTPA-OCH3s are on the aromatic rings and receive the shielding effect, and the others are not on the rings nor have the shielding effect. Such shielding effect was not expected for 9-MTPA-OCH3s. Rule-3, Katayama et.al observed that 7S-H of the tri- (R)-(+)-MTPA esters of (-)-erythro-3,4,5- trimethoxyphenylglycerol-8-O-4'-(sinapyl alcohol) ether that have the shielding effect gave J = 6.30(6) (Hz), whereas 7R-H of the (+)-erythro-3,4,5-trimethoxyphenylglycerol-8- O-4'-(sinapyl alcohol) ether that have not the effect gave J = 3.24(4), and that 7S-H of (+)- threo-3,4,5-trimethoxyphenylglycerol-8-O-4'- (sinapyl alcohol) ether that have the shielding effect gave J = 6.30(7), 7R-H of the (-)-threo- 3,4,5-trimethoxyphenylglycerol-8-O-4'- (sinapyl alcohol) ether that have not the effect gave J = 8.76(8.0-8.6). Rule-4, the 9?- MTPAOCH3s have the smallest Δδ, almost 0, among the three (7, 9, and 9?) MTPA-OCH3s, because the 9?- MTPA-OCH3 groups are located farthest from the two chiral centers, 7-C and 8- C, among the three MTPA-OCH3 groups [Δδ (7)> Δδ (9)> Δδ (9?) ~ 0].

Erythro SGSE

Figure 3 showed that, tri-(R)-MTPA esters and 4-O-methyl ether of (-)-erythro-SGSE have 1H NMR peaks of MTPA-OCH3 markedly upfield, it was suggested that the peak was due to α-MTPA-OCH3 with (7S)- configuration, and thus tri-(R)-MTPA esters and 4-O-methyl ether of (+)-erythro-SGSE have (7R)-configuration. The assignments in Table 1 were consistent with rules 1, 3, and 4 as follows. The Δδ values for 7-MTPA-OCH3 in tri-(R)-MTPA esters and 4-O-methyl ether of (+)-erythro and (-)-erythro-SGSEs (|δR -δS|) is 0.308ppm which are apparently larger than that of 9-MTPA-OCH3: 0.126ppm in tri-(R)- MTPA esters and 4-O-methyl ether of (-)- erythro and (+)-erythro-SGSEs (|δS -δR|).

The tri-(R)-MTPA esters and 4-O-methyl ether of (-)-erythro-SGSEs coupling constant (J = 6.30) of Cα-H is larger than that of tri-(R)- MTPA esters and 4-O-methyl ether of (+)- erythro-SGSEs coupling constant (J = 3.24) of Cα-H.

On the other hand, the Δδ values for 9?- MTPA-OCH3 in tri-(R)-MTPA esters and 4-Omethyl ether of (-)-erythro and (+)-erythro- SGSEs (|δS -δR|) is 0.009ppm, that mean almost 0.

Thus it was established that the 7-MTPAOCH3 of tri-(R)-MTPA esters and 4-O-methyl ether of (-)-erythro-SGSE was effected by the shielding effect of the part of 3,4,5- trimethoxyphenylglycerol-8-O-4?-(sinapyl alcohol) ether, whereas that of neither tri-(R)- MTPA esters and 4-O-methyl ether of (+)- erythro-SGSE was affected. Consequently, the C7 of tri-(R)-MTPA esters and 4-O-methyl ether of (-)-erythro-SGSE have an (S)- configuration, whereas the C7 of tri-(R)-MTPA esters and 4-O-methyl ether of (+)-erythro- SGSE have an (R)-configuration. So, the absolute configuration of (+)-erythro- and (-)- erythro-SGSEs are (7R, 8S) and (7S, 8R) respectively.

Threo SGSE

Figure 3 also showed that, tri-(R)-MTPA esters and 4-O-methyl ether of (+)-threo-SGSE have 1H NMR peaks of MTPA-OCH3 markedly upfield, it was suggested that the peak was due to α-MTPA-OCH3 with (7S)- configuration, and thus tri-(R)-MTPA esters and 4-O-methyl ether of (-)-erythro-SGSE have (7R)-configuration. The assignments in Table 1 were consistent with rules 1, 3, and 4 as follows. The Δδ values for 7-MTPA-OCH3 in tri-(R)-MTPA esters and 4-O-methyl ether of (-)-threo-and (+)-threo-SGSEs (|δR -δS|) is 0.259ppm which are apparently larger than that of 9-MTPA-OCH3: 0.149ppm in tri-(R)-MTPA esters and 4-O-methyl ether of (-)-threo- and (+)-threo-SGSEs (|δR -δS|).

On the other hand, the Δδ values for 9?- MTPA-OCH3 in tri-(R)-MTPA esters and 4-Omethyl ether of (-)-threo- and (+)-threo-SGSEs (|δR -δS|) is 0.008ppm, that also, mean almost 0.

The tri-(R)-MTPA esters and 4-O-methyl ether of (-)-threo-SGSEs coupling constant (J = 8.76) of Cα-H is larger than that of tri-(R)- MTPA esters and 4-O-methyl ether of (+)- threo-SGSEs coupling constant (J = 6.3) of Cα-H.

Thus it was established that the 7-MTPAOCH3 of tri-(R)-MTPA esters and 4-O-methyl ether of (+)-threo-SGSE was effected by the shielding effect of the part of 3,4,5- trimethoxyphenylglycerol-8-O-4?-(sinapyl alcohol) ether, whereas that of neither tri-(R)- MTPA esters and 4-O-methyl ether of (-)- threo-SGSE was affected. Consequently, the C7 of tri-(R)-MTPA esters and 4-O-methyl ether of (+)-threo-SGSE have an (S)- configuration, whereas the C7 of tri-(R)-MTPA esters and 4-O-methyl ether of (-)-threo-SGSE have an (R)-configuration. So, the absolute configuration of (+)-threo- and (-)-threo- SGSEs are (7S, 8S) and (7R, 8R) respectively.

According to this assignment, it could be concluded the absolute configuration of four stereoisomer of SGSEs (Figure 4), (+)-erythro- , (-)-erythro-, (+)-threo-, and (-)-threo-SGSEs were determined as (7R, 8S), (7S, 8R) , (7S, 8S) and (7R, 8R), respectively.

Enzymatic formation of erythro- and threo- SGSE

Incubation of a soluble enzyme preparation of Eucommia ulmoides with sinapyl alcohol (SA) in the presence of H2O2 gave erythro- and threo- syringylglycerol-8-O- 4?-(sinapyl alcohol) ethers (SGSEs) (Fig.2). The identification was achieved by 1H NMR and HPLC. Erythro-SGSE: 1H NMR (acetoned6): δ 3.71 (1H, s, H-9a), 3.77 [1H, overlapping (o), 9-OH], 3.82 (6H, s, A-OCH3), 3.83-3.88 (2H, o, 9?-OH & H-9b), 3.90 (6H, s, B-OCH3), 4.22 (2H, dd, J = 5.61, 4.15, H- 9?),4.72 (1H, d, J = 4.39, 7-OH), 4.87 (1H, m, H-8), 4.99 (1H, d, J = 4.64, H-7), 6.36 (1H, dt, J = 15.85, 5.29, H-8?), 6.58 (1H, d, J = 15.85, H-7?), 6.73 (2H, s, H-2 & 6), 6.82 (2H, s, H-2? & 6?), 7.51 (1H, s, 4-OH). Threo-SGSE: 1H NMR (acetone-d6): δ 3.3 (1H, s, 9-OH), 3.66 (1H, m, H-9a), 3.74 (1H, m, H-9b), 3.81(6H, s, A-OCH3), 3.82-3.88 (1H, o, 9?-OH), 3.92 (6H, s, B-OCH3), 3.99 (2H, dd, J = 3.41, H-8), 4.0 (2H, dd, J = 6.95, 3.56, H-9?), 4.23 (1H, d, J = 3.66, 7-OH), 4.98 (1H, d, J = 6.83, H-7), 6.36 (1H, dt, J = 15.85, 5.29, H-8?), 6.54 (1H, d, J = 16.1, H-7?), 6.78 (2H, s, H-2 & 6), 6.82 (2H, s, H-2? & 6?), 7.44 (1H, s, 4- OH). The SGSEs were diastereoselectively formed in erythro isomer with 53% d.e. at 60 min (Table 2). Quantification of the four stereoisomers, (+)- erythro-, (-)-erythro-, (+)-threo-, and (-)-threo- SGSEs demonstrated that they were in a percentage of 23.8 %, 28.8 %, 22.1 %, and 25.3 %, respectively (Fig. 4). Both products, (-)- erythro- and (-)-threo-SGSEs have optical activity with 10 % e.e. and 7 % e.e., respectively, at 60 min.

The Diastereoselective formation of syringyl-8-O-4?-neolignans with optical activity from two sinapyl alcohols (Lourith et al., 2005). This result recognized our present study report. The observation of soluble enzyme preparation, to preference for different enantiomers of the erythro isomer suggests that different enzymes regulate the 8-O-4? coupling of sinapyl alcohol in E. ulmoides.

Conclusions

The stereochemistry of four stereoisomer of SGSEs, (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo-SGSEs were determined as (7R, 8S), (7S, 8R), (7S, 8S) and (7R, 8R), respectively. On the other hand erythro-SGSE was formed more selectively from threo-SGSE, with soluble enzyme preparation by the incubation of SA in the presence of H2O2. The soluble enzyme preparation catalyzed the diastereoselective formation of erythro- and threo-SGSEs with optical activity.

References

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