Biomimetic Synthesis of Resveratrol Trimers Catalyzed by Horseradish Peroxidase

Biotransformation of trans-resveratrol and synthetic (±)-ε-viniferin in aqueous acetone using horseradish peroxidase and hydrogen peroxide as oxidants resulted in the isolation of two new resveratrol trimers (3 and 4), one new resveratrol derivative (5) with a dihydrobenzofuran skeleton, together with two known stilbene trimers (6 and 7), and six known stilbene dimers (8–13). Their structures and relative configurations were identified through spectral analysis and possible formation mechanisms were also discussed. Among these oligomers, trimers 6 and 7 were obtained for the first time through direct transformation from resveratrol. Results indicated that this reaction is suitable for the preparation of resveratrol oligomers with a complex structure.


Introduction
Stilbenes are a class of plant polyphenols that can be divided into two categories, namely, monomeric and oligomeric stilbenes. Resveratrol oligomers possess novel structures and exhibit various biological activities, such as anticarcinogenesis, anti-inflammation, and tyrosinase activity inhibition. These oligomers can be used to treat cancer, AIDS, bacterial infections, and other diseases. Some oligostilbenes exhibit more potent bioactivities than their monomers do [1][2][3].
Various resveratrol oligomers exist in nature, especially in grapevine. However, the structures of minor stilbene oligomers have yet to be elucidated because sufficient amounts of these minor components are difficult to isolate for subsequent structural characterization. In recent years, some researchers focused their attention on the synthesis of these oligomers and total synthetic routes of numerous resveratrol oligomers, including dimers, trimers, and tetramers, have been reported in literature [4][5][6][7][8][9][10], but long reaction steps render these approaches unsuitable for specific preparations of the complex oligomers. Therefore, biomimetic synthesis is still a concise and practical alternative for the preparation of oligostilbenes with intricate structures. The natural biotransformation of stilbene oligomers in nature can be simulated in vitro by transformation with biological enzymes, unorganized fermentation, metal oxidants, light, acids and alkali [2,11]. In combination with diverse separation methods, transformation can induce the accumulation of large amounts of minor compounds. Using this approach, a number of known natural stilbene oligomers and new stilbene oligomers have been obtained, and their structures have also been identified successfully [11]. The oxidative coupling of resveratrol, including its analogs, has been examined under different conditions since ε-viniferin was first isolated in 1977 by Langcake and Pryce [12]. Nevertheless, the in vitro biocatalyzed oxidation of stilbenes has rarely been explored, and studies on biocatalyzed  In this study, semisynthetic (±)-ε-viniferin (2) under the procedure shown in Figure 2 was used as the starting material for biotransformation. According to the method reported in our previous paper [17], trans-resveratrol was subjected to an oxidative coupling reaction in aqueous methanol using FeCl 3 ·6H 2 O as an oxidant, and this procedure was performed in combination with column chromatography on silica gel. Thus, 2 with 13.5% yield was produced.

Synthesis of 2 with Resveratrol as a Starting Material and HRP and Hydrogen Peroxide-Catalyzed Biotransformation of 1 and 2
In this study, semisynthetic (±)-ε-viniferin (2) under the procedure shown in Figure 2 was used as the starting material for biotransformation. According to the method reported in our previous paper [17], trans-resveratrol was subjected to an oxidative coupling reaction in aqueous methanol using FeCl3·6H2O as an oxidant, and this procedure was performed in combination with column chromatography on silica gel. Thus, 2 with 13.5% yield was produced. Successively, biotransformation of 1 and 2 in aqueous acetone catalyzed by horseradish peroxidase and hydrogen peroxide generated a major product peak 8 and a complicated mixture ( Figure S30 in Supplementary Materials), which resulted in the isolation and identification of four resveratrol trimers (3, 4, 6, and 7, where 3 and 4 are new ones), one new resveratrol derivative 5, and six known dimers (8)(9)(10)(11)(12)(13) (Figure 1). Their structures and stereochemistry were elucidated by analyzing spectroscopic data.

Probable Coupling Reaction Mechanisms
Considering the obtained results, a mechanism on how different trimeric derivatives formed were proposed. HRP-catalyzed biotransformation is presumed on the basis of radical reaction [13,14,29]. Induced by hydrogen peroxide, resveratrol, and (±)-ε-viniferin were dehydrogenated and rearranged to form different radicals ( Figure 6). Afterward, these HRP-catalyzed radicals were combined to produce different dimers and trimers. The coupling of one radical D and one radical A, and subsequent tautomeric rearrangement and intramolecular nucleophilic attack to the intermediate quinone yielded the dihydrofuran trimers 4 and 7 (Figure 7). Consequently, the formation of 3 and 6 can be easily explained by the coupling of one radical D with one radical B and subsequent addition of a water molecule to the intermediate quinone ( Figure 8). Furthermore, 5 may be formed through the oxidation of (±)-ε-viniferin by H 2 O 2 and HRP, but an appropriate account for the reactivity cannot be established with this evidence. In addition, the formation mechanisms of the dimers obtained in this work are the same as those reported in our previous paper [26,30,31].

Probable Coupling Reaction Mechanisms
Considering the obtained results, a mechanism on how different trimeric derivatives formed were proposed. HRP-catalyzed biotransformation is presumed on the basis of radical reaction [13,14,29]. Induced by hydrogen peroxide, resveratrol, and (±)-ε-viniferin were dehydrogenated and rearranged to form different radicals ( Figure 6). Afterward, these HRP-catalyzed radicals were combined to produce different dimers and trimers. The coupling of one radical D and one radical A, and subsequent tautomeric rearrangement and intramolecular nucleophilic attack to the intermediate quinone yielded the dihydrofuran trimers 4 and 7 (Figure 7). Consequently, the formation of 3 and 6 can be easily explained by the coupling of one radical D with one radical B and subsequent addition of a water molecule to the intermediate quinone ( Figure 8). Furthermore, 5 may be formed through the oxidation of (±)-ε-viniferin by H2O2 and HRP, but an appropriate account for the reactivity cannot be established with this evidence. In addition, the formation mechanisms of the dimers obtained in this work are the same as those reported in our previous paper [26,30,31].

Materials and Instrumentation
Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained on a JASCOP650 spectrometer (JASCO). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission, Thermo Electron Corporation, Madison, WI, USA). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1 H and 125 or 150 MHz for 13 C, respectively, on Varian INOVA 500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation,

Probable Coupling Reaction Mechanisms
Considering the obtained results, a mechanism on how different trimeric derivatives formed were proposed. HRP-catalyzed biotransformation is presumed on the basis of radical reaction [13,14,29]. Induced by hydrogen peroxide, resveratrol, and (±)-ε-viniferin were dehydrogenated and rearranged to form different radicals ( Figure 6). Afterward, these HRP-catalyzed radicals were combined to produce different dimers and trimers. The coupling of one radical D and one radical A, and subsequent tautomeric rearrangement and intramolecular nucleophilic attack to the intermediate quinone yielded the dihydrofuran trimers 4 and 7 (Figure 7). Consequently, the formation of 3 and 6 can be easily explained by the coupling of one radical D with one radical B and subsequent addition of a water molecule to the intermediate quinone ( Figure 8). Furthermore, 5 may be formed through the oxidation of (±)-ε-viniferin by H2O2 and HRP, but an appropriate account for the reactivity cannot be established with this evidence. In addition, the formation mechanisms of the dimers obtained in this work are the same as those reported in our previous paper [26,30,31].

Materials and Instrumentation
Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained on a JASCOP650 spectrometer (JASCO). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission, Thermo Electron Corporation, Madison, WI, USA). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1 H and 125 or 150 MHz for 13 C, respectively, on Varian INOVA 500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, Karlsruhe, Germany), in acetone-d6 or methanol-d4, with solvent peaks as references. ESI-MS and

Probable Coupling Reaction Mechanisms
Considering the obtained results, a mechanism on how different trimeric derivatives formed were proposed. HRP-catalyzed biotransformation is presumed on the basis of radical reaction [13,14,29]. Induced by hydrogen peroxide, resveratrol, and (±)-ε-viniferin were dehydrogenated and rearranged to form different radicals ( Figure 6). Afterward, these HRP-catalyzed radicals were combined to produce different dimers and trimers. The coupling of one radical D and one radical A, and subsequent tautomeric rearrangement and intramolecular nucleophilic attack to the intermediate quinone yielded the dihydrofuran trimers 4 and 7 (Figure 7). Consequently, the formation of 3 and 6 can be easily explained by the coupling of one radical D with one radical B and subsequent addition of a water molecule to the intermediate quinone ( Figure 8). Furthermore, 5 may be formed through the oxidation of (±)-ε-viniferin by H2O2 and HRP, but an appropriate account for the reactivity cannot be established with this evidence. In addition, the formation mechanisms of the dimers obtained in this work are the same as those reported in our previous paper [26,30,31].

Materials and Instrumentation
Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained on a JASCOP650 spectrometer (JASCO). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission, Thermo Electron Corporation, Madison, WI, USA). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1 H and 125 or 150 MHz for 13

Materials and Instrumentation
Optical rotations were measured on P2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were obtained on a JASCOP650 spectrometer (JASCO). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission, Thermo Electron Corporation, Madison, WI, USA). 1D and 2D NMR spectra were acquired at 500 or 600 MHz for 1 H and 125 or 150 MHz for 13 C, respectively, on Varian INOVA 500 MHz, or Bruker AVANCE III HD 600 MHz (Bruker Corporation, Karlsruhe, Germany), in acetone-d 6 or methanol-d 4 , with solvent peaks as references. ESI-MS and HR-ESI-MS data were measured using an AccuToFCS JMST100CS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA, USA). Column chromatography (CC) was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China). HPLC separation was performed on an instrument consisting of a Waters 515 pump and a Waters 2487 dual λ absorbance detector (Waters Corporation, Milford, MA, USA) with a YMC semi-preparative column (250 × 10 mm i.d.) packed with C18 (5 µM). TLC was carried out with glass precoated silica gel GF254 plates (Qingdao Marine Chemical, Inc., Qingdao, China). Spots were visualized under UV light or by spraying with 7% H 2 SO 4 in 95% EtOH followed by heating.

Synthesis of Compoud 2
The solution of FeCl 3 ·6H 2 O (380 g, 1.43 mol) in H 2 O (100 mL) was added to a solution of 1 (300 g, 1.32 mol) in methanol (500 mL) under stirring at room temperature, and the mixture was stirred for 60 h at room temperature. After removing of methanol in vacuo, water was added to the mixture, and the mixture was extracted with EtOAc. Subsequently, the obtained organic layer was washed with brine and water, dried over anhydrous Na 2 SO 4 for 24 h, then concentrated in vacuo to give a residue, which was further chromatographed on silica gel column with CHCl 3 -MeOH (15:1, v/v) as eluent to provide unreacted resveratrol 130 g and product 2 (23 g, yield 13.5%).

Conclusions
The HRP-catalyzed biotransformation of 1 and 2 produced various resveratrol stilbene oligomers, including dimers, trimers, and tetramers. In this reaction mixture, four resveratrol trimers (3, 4, 6, and 7), one new resveratrol derivative (5) with a dihydrofuran skeleton, and six dimers (8)(9)(10)(11)(12)(13) were isolated and identified. Among these compounds, 3 and 4 were newly identified in our study. The raceme nature of the dimers was indicated by the zero values of their optical rotations, and this finding suggested that a radical mechanism was involved in HRP-catalyzed biotransformation. Our study favored the enzymatic biotransformation of stilbenes by HRP as a prominent method to produce oligomeric stilbenes for research activity. Considering that these new compounds may occur naturally as minor constituents, we observed that our reference data provided a basis for the detection of the presence of these stilbene oligomers in future investigations. Oligostilbenes were reported to show various activities [1,2]. Therefore, these products should be further examined, and results will be reported in our future research.