Total Synthesis of Resvebassianol A, a Metabolite of Resveratrol by Beauveria bassiana

Resveratrol is a well-known dietary polyphenol because it has a variety of beneficial biological activities. The fungus Beauveria bassiana is one of the most frequently used microorganisms for the biotransformation of polyphenols. Recently, resvebassianol A (2), a glycosylated metabolite of resveratrol by B. bassiana, was isolated and structurally elucidated. It was demonstrated to exhibit antioxidant, regenerative, and anti-inflammatory activities with no cytotoxicity. Here, we report the first total synthesis of resvebassianol A, 4′-O-β-(4‴-O-methylglucopyranosyl)resveratrol (2), and its regiomer, 3-O-β-(4‴-O-methylglucopyranosyl)resveratrol (3). Key reactions include (i) the construction of a stilbene core via a novel Heck reaction of aryl halides and styrenes, and (ii) glycosylation with unnatural methylglucopyranosyl bromide. The glycosylation step was carefully optimized by varying the bases and solvents. Resveratrol metabolites 2 and 3 were obtained at 7.5% and 6.3% of the overall yield, respectively.

Despite their pharmacological activities, various in vivo studies have shown that the potential of polyphenols is impaired by their insolubility in water, ultraviolet light instability, poor intestinal absorption, short half-life, rapid clearance, low bioavailability, and rapid metabolism [21,22]. The introduction of a glycosyl moiety on polyphenols not only helps to enhance the solubility of substrates but also reduces their toxicity, which ultimately increases the activity of biosynthetic intermediates [23]. Moreover, the sugar moiety of polyphenol glycosides might play a major role in their absorption, resulting in an acceptable concentration in the circulatory streams [24]. Polyphenols are subjected to enzymatic oxidation by polyphenol oxidases in plants, during food processing, and also after human consumption, which can be protected by glycosylation [25]. The incorporation of sugar moieties into different types of pharmacophores, natural products, or prodrugs has been proven to improve anti-cancer activities [26].
The fungus Beauveria bassiana is the most frequently used biocatalyst and has been used to transform more than 300 bioactive compounds [29,30]. For instance, B. bassiana ATCC 7159 has been used for the biotransformation of curvularin and kaempferol, leading to the production of new metabolites resulting from 4-O-methyl glucosylation of the substrate, and was highly selective among different hydroxyl groups in the same molecule [30]. Recently, resvebassianol A (2) shown in Figure 1, was identified through biotransformation of resveratrol by B. bassiana and exhibited important pharmacological activities such as inhibition of inflammatory cytokine expression and cell rejuvenation. Moreover, compared with resveratrol, resvebassianol A proved to be less toxic and more stable [31].
Several synthetic approaches for the formation of glycosidic bonds to phenolic OH in resveratrol have been reported. Direct coupling of resveratrol with a bromo-glucuronide donor was performed by Wang et al. for the synthesis of two glycoconjugates [32]. Coupling of resveratrol with glucuronyl bromide was performed using silver carbonate as an activator, in order to produce glucuronide-conjugated resveratrol in low yield, possibly due to the low solubility of resveratrol in organic solvents. Lucas et al. synthesized resveratrol 3-O-β-D-glucuronide by coupling a trichloroacetimidate glycosyl donor with protected resveratrol using TMSOTf and BF3.OEt2 as promoters [3]. Learmonth also synthesized two glucuronide conjugates of resveratrol, in which palladium-catalyzed Heck coupling of an iodo-O-β-D-glucuronate derivative and its corresponding styrene was adopted [33].
The structural uniqueness and natural resource scarcity of resvebassianol A for biological evaluation prompted us to develop an efficient synthetic method for the metabolite. In this study, we report the total synthesis of resvebassianol A (2), a metabolite of resveratrol by B. bassiana, and its regiomer,

Chemical Reagents
All chemicals and solvents were reagent grade and were purchased from Sigma Aldrich (Saint Louis, MO, USA), TCI (Tokyo, Japan), and Alfa Aesar (Haverhill, MA, USA). All reagents were used directly without further purification.

Purification and Instrumentation
All reactions were carried out in an inert atmosphere in flame-dried glassware. Reactions were monitored by thin-layer chromatography using 0.25 mm silica gel plates and visualized using UV 254/286 nm. Flash chromatography was carried out using silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany) as the stationary phase. 1 H and 13 C NMR spectra were recorded using a 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) with deuterochloroform (CDCl 3 ), methanol-d4 (CD 3 OD), or DMSO-d6 (CD 3 ) 2 SO. Data for 1H NMR spectra are reported as chemical shifts (multiplicity, coupling constants, integration), and multiplicities are reported as s = singlet, d = doublet, t = triplet, q = quartet, septet = septet, m = multiplet and/or multiple resonances, number of protons, and coupling constant (J). High-resolution mass spectra (HRMS) were recorded using electrospray ionization (ESI) mass spectroscopy on a JEOL JMS-700 (FAB and EI) and an Agilent 6530 Q-TOF LC/MS/MS system (ESI). (13) Methyl α-D-glucopyranoside 8 (10 g, 51.5 mmol) was dissolved in anhydrous N,Ndimethylformamide (100 mL) under a N 2 atmosphere, p-toluene sulfonic acid (1.62 g, 9.4 mmol) was added, followed by the addition of benzaldehyde dimethyl acetal (9.2 mL, 61.8 mmol), and the solution was stirred under N 2 for 16 h. After completion of the reaction, triethylamine (4 mL) was added to the reaction, which was then diluted with ethyl acetate. The organic layer was subsequently washed with saturated sodium bicarbonate and brine, dried over Na 2 SO 4 , and concentrated in vacuo. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 30:1) to yield product 13 (12 g, 93%) as a white solid. 1 (14) Methyl 4,6-O-benzylidene-α-D-glucopyranoside 13 (11.5 g, 40.73 mmol) was dissolved in anhydrous N,N-dimethylformamide (100 mL) under a N 2 atmosphere. The solution was cooled to 0 • C in an ice bath, after which NaH (60% dispersion in mineral oil, 4 g, 163 mmol) was added, and the reaction was stirred for 1 h at room temperature. The solution was cooled to 0 • C, and benzylbromide (14.5 mL, 122 mmol) was added dropwise. The solution was stirred at room temperature overnight, after which methanol (10 mL) was added, and the mixture was concentrated under reduced pressure. The residue was dissolved in CH 2 Cl 2 (200 mL), washed with water (2 × 75 mL) and brine (1 × 75 mL), and dried over MgSO 4 . The residue was purified by silica gel column chromatography (ethyl acetate/n-hexane = 1:7) to yield product 14 (17.5 g, 92%) as a white solid compound. 1 (17.25 g, 38 mmol) was dissolved in anhydrous CH 2 Cl 2 (100 mL) under a N 2 atmosphere, and the solution was cooled to 0 • C. Et 3 SiH (30 mL, 186 mmol) and trifluoroacetic acid (14 mL, 186 mmol) were added, and the solution was stirred at 0 • C for 4 h. The reaction was quenched with Et 3 N and methanol. CH 2 Cl 2 was added, and the solution was washed with water and brine, dried over MgSO 4 , filtered, and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (ethyl acetate/n-hexane = 1:4) to afford product 15 (12 g, 91%) as a colorless oil. 1   NaH (60% dispersion in mineral oil, 1.5 g, 63 mmol) was added to a solution of methyl 2,3,6-tri-O-benzyloxy-α-D-glucopyranoside 15 (11.5 g, 25 mmol) in anhydrous N,Ndimethylformamide (100 mL) at 0 • C. The reaction mixture was stirred for 1 h at 0 • C, and methyl iodide (3.8 mL, 63 mmol) was added to the reaction mixture. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with methanol and ice-cold water and then extracted with ethyl acetate. The collected organic layers were washed with brine, dried with Na 2 SO 4 , and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (ethyl acetate/n-hexane = 1:7) to yield product 16 (11.2 g, 95%) as a viscous liquid. 1  Pd (10%)/C (3 g) was added to a solution of methyl 2,3,6-Tri-O-benzyloxy-4-O-methylα-D-glucopyranoside 16 (11 g, 22 mmol) in anhydrous methanol (100 mL), and the mixture was stirred under an atmosphere of hydrogen at room temperature for 24 h. The catalyst was filtered out, and the solvents were removed under reduced pressure. The crude residue was purified by silica gel column chromatography (ethyl acetate/n-hexane = 1:1) to afford the viscous product 17 (4.6 g, 95%). 1  Compound 17 (4.5 g, 22 mmol) was dissolved in acetic anhydride (25 mL, 217 mmol) and pyridine (25 mL, 217 mmol) and stirred at room temperature for 12 h. After completion of the reaction, pyridine and acetic anhydride were removed in vacuo. The residue was dissolved in CH 2 Cl 2 and washed with dilute HCl. The organic layer was collected, washed with brine, dried with MgSO 4 , and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography (ethyl acetate/n-hexane = 1:4) to afford the viscous product 18 (5.1 g, 71%). 1  (d, J = 6.0 Hz, 6H); 13

Retrosynthesis
Metabolite 2 and its regiomer 3 consist of a glycone attached to the aglycone moiety. The glycone moiety is 4-O-methyl glucopyranose, whereas the aglycone is a functional resveratrol featuring a stilbene core with a polyhydroxy group. Metabolite 2 is a structure in which 4-O-methylglucopyranose is attached to the 7-position hydroxyl group of resveratrol, whereas its regiomer 3 consists of a glycosyl moiety attached to the 3-position hydroxyl group of resveratrol. The synthesis of both metabolites involves a glycosylation reaction that introduces methylated glucose as the core reaction and the Heck reaction to form a stilbene skeleton [34]. Schemes 1 and 2 provide a retrosynthetic methodology for the synthesis of both metabolites 2 and 3. Stilbene moiety 2 and its regiomer 3 were constructed via palladiumcatalyzed Heck coupling. The rate-limiting step of the glycosylation reaction was performed with selectively protected compound 6 and commercially available iodophenol 7. Compound 10 was obtained from the glycosylation of compound 6 and styrene 12, which was synthesized from readily available dihydroxy benzaldehyde 9.
Metabolite 2 and its regiomer 3 consist of a glycone attached to the aglycone mo The glycone moiety is 4-O-methyl glucopyranose, whereas the aglycone is a functi resveratrol featuring a stilbene core with a polyhydroxy group. Metabolite 2 is a struc in which 4-O-methylglucopyranose is attached to the 7-position hydroxyl grou resveratrol, whereas its regiomer 3 consists of a glycosyl moiety attached to the 3-pos hydroxyl group of resveratrol. The synthesis of both metabolites involves a glycosyla reaction that introduces methylated glucose as the core reaction and the Heck reactio form a stilbene skeleton [34]. Schemes 1 and 2 provide a retrosynthetic methodology for the synthesis of both tabolites 2 and 3. Stilbene moiety 2 and its regiomer 3 were constructed via palladi catalyzed Heck coupling. The rate-limiting step of the glycosylation reaction was formed with selectively protected compound 6 and commercially available iodophen Compound 10 was obtained from the glycosylation of compound 6 and styrene 12, w was synthesized from readily available dihydroxy benzaldehyde 9. The glycone moiety is 4-O-methyl glucopyranose, whereas the aglycone is a functi resveratrol featuring a stilbene core with a polyhydroxy group. Metabolite 2 is a struc in which 4-O-methylglucopyranose is attached to the 7-position hydroxyl grou resveratrol, whereas its regiomer 3 consists of a glycosyl moiety attached to the 3-pos hydroxyl group of resveratrol. The synthesis of both metabolites involves a glycosyla reaction that introduces methylated glucose as the core reaction and the Heck reactio form a stilbene skeleton [34]. Schemes 1 and 2 provide a retrosynthetic methodology for the synthesis of both tabolites 2 and 3. Stilbene moiety 2 and its regiomer 3 were constructed via palladi catalyzed Heck coupling. The rate-limiting step of the glycosylation reaction was formed with selectively protected compound 6 and commercially available iodophen Compound 10 was obtained from the glycosylation of compound 6 and styrene 12, w was synthesized from readily available dihydroxy benzaldehyde 9.

Chemistry
The reaction commenced with the preparation of the glycosyl donor, 4-O-methylglycop yranosyl bromide 6, as shown in Scheme 3, which involves eight steps from commercially available methyl-α-D-glucopyranoside 8. Regioselective protection of 4, 6-diol from the starting material was accomplished by the introduction of a 4,6-O-benzylidene group using benzaldehyde dimethyl acetal under acidic conditions, yielding protected compound 13. 2,3-Di-O-benzylation of 13 generated 14 using NaH and BnBr. Further regioselective opening of the benzylidene ring of intermediate 14 was conducted with the help of triethylsilane (TES) and trifluoroacetic acid (TFA) to obtain alcohol 15 [35]. Methylation of compound 15 with NaH and MeI in N, N-dimethylformamide yielded product 16, followed by hydrogenolysis to yield product 17. Acetylation of the hydroxy groups of 17 was performed using pyridine and acetic anhydride to yield 18, followed by the replacement of an anomeric methoxy group with an acetoxy group using boron trifluoride diethyl etherate to yield 19. Finally, grafting of the anomeric acetoxy group was performed to incorporate bromine using HBr (33% in acetic acid) to yield acylated glycosyl bromide 6 at 73% [36]. As the final compound, 4-O-methylglucopyranosyl bromide 6, has poor chemical stability, it is suitable to obtain a large amount of acetate compound 19 and synthesize 6 immediately when necessary.

Chemistry
The reaction commenced with the preparation of the glycosyl donor, 4-O-methylgly copyranosyl bromide 6, as shown in Scheme 3, which involves eight steps from commer cially available methyl-α-D-glucopyranoside 8. Regioselective protection of 4, 6-diol from the starting material was accomplished by the introduction of a 4,6-O-benzylidene group using benzaldehyde dimethyl acetal under acidic conditions, yielding protected com pound 13. 2,3-Di-O-benzylation of 13 generated 14 using NaH and BnBr. Further regiose lective opening of the benzylidene ring of intermediate 14 was conducted with the help o triethylsilane (TES) and trifluoroacetic acid (TFA) to obtain alcohol 15 [35]. Methylation o compound 15 with NaH and MeI in N, N-dimethylformamide yielded product 16, fol lowed by hydrogenolysis to yield product 17. Acetylation of the hydroxy groups of 17 was performed using pyridine and acetic anhydride to yield 18, followed by the replace ment of an anomeric methoxy group with an acetoxy group using boron trifluoride di ethyl etherate to yield 19. Finally, grafting of the anomeric acetoxy group was performed to incorporate bromine using HBr (33% in acetic acid) to yield acylated glycosyl bromide 6 at 73% [36]. As the final compound, 4-O-methylglucopyranosyl bromide 6, has poo chemical stability, it is suitable to obtain a large amount of acetate compound 19 and syn thesize 6 immediately when necessary. 3,5. Dihydroxystyrene 12 and 3,5 diacetoxyystyrene 5 were synthesized from com mercially available 3,5-dihydroxy benzaldehyde 9 according to Scheme 4. Protection o the hydroxy group of 9 with TBDMS yielded 20, and the Wittig reaction yielded olefin 21 using methyltriphenylphosphonium bromide under basic conditions. The TBDMS group in intermediate 21 was removed using tetrabutylammonium fluoride (TBAF) to furnish dihydroxy styrene 12, and further acetylation of both hydroxy groups resulted in 12 a 70% yield. 3,5-Dihydroxystyrene 12 and 3,5 diacetoxyystyrene 5 were synthesized from commercially available 3,5-dihydroxy benzaldehyde 9 according to Scheme 4. Protection of the hydroxy group of 9 with TBDMS yielded 20, and the Wittig reaction yielded olefin 21 using methyltriphenylphosphonium bromide under basic conditions. The TBDMS group in intermediate 21 was removed using tetrabutylammonium fluoride (TBAF) to furnish dihydroxy styrene 12, and further acetylation of both hydroxy groups resulted in 12 at 70% yield.

Scheme 4. Synthesis of diacetoxystyrene 5 and dihydroxystyrene 12.
After obtaining 6 and 12, the next target was to synthesize substrates 4 and 10, w participate in the Heck reaction for the synthesis of the stilbene core.
We performed glycosylation of both iodophenol 7 and dihydroxystyrene 12 s rately with 4-O-methylglycopyranosyl bromide 6 under different reaction condition shown in Tables 1 and 2. Using Ag2CO3 in acetonitrile produced low-yield glycoside p ucts 4 and 10 up to 16% and 19%, respectively. We attempted to improve glycosyla using a phase transfer catalyst (TBAB) in a two-phase system (aqueous NaOH and K2 in CHCl3. Unfortunately, the reaction yielded trace amounts. The reaction was incomp and the substrate was recovered for reuse. Bromide compound 6 can be decomposed glycal by an alkaline water phase and phenoxide anion [37]. Therefore, excess use of w in the reaction lowers the yield of the compound. After utilizing several conditions (Ta 1 and 2), the glycosylation reaction under the phase transfer catalyst BnNBu3Cl and K as a base at room temperature yielded product 4 at 57% yield. The desired mono-g sylated product 10 was obtained at 40% yield along with the undesired di-glucosy product as a mixture, which was separated by column chromatography. Aryl iodide 7 was the choice for palladium-catalyzed Heck coupling because o greater reactivity over other aryl halides. After obtaining 6 and 12, the next target was to synthesize substrates 4 and 10, which participate in the Heck reaction for the synthesis of the stilbene core.
We performed glycosylation of both iodophenol 7 and dihydroxystyrene 12 separately with 4-O-methylglycopyranosyl bromide 6 under different reaction conditions, as shown in Tables 1 and 2. Using Ag 2 CO 3 in acetonitrile produced low-yield glycoside products 4 and 10 up to 16% and 19%, respectively. We attempted to improve glycosylation using a phase transfer catalyst (TBAB) in a two-phase system (aqueous NaOH and K 2 CO 3 ) in CHCl 3 . Unfortunately, the reaction yielded trace amounts. The reaction was incomplete, and the substrate was recovered for reuse. Bromide compound 6 can be decomposed into glycal by an alkaline water phase and phenoxide anion [37]. Therefore, excess use of water in the reaction lowers the yield of the compound. After utilizing several conditions (Tables 1 and  2), the glycosylation reaction under the phase transfer catalyst BnNBu 3 Cl and K 2 CO 3 as a base at room temperature yielded product 4 at 57% yield. The desired mono-glucosylated product 10 was obtained at 40% yield along with the undesired di-glucosylated product as a mixture, which was separated by column chromatography. After obtaining 6 and 12, the next target was to synthesize substrates 4 and 10, which participate in the Heck reaction for the synthesis of the stilbene core.
We performed glycosylation of both iodophenol 7 and dihydroxystyrene 12 separately with 4-O-methylglycopyranosyl bromide 6 under different reaction conditions, as shown in Tables 1 and 2. Using Ag2CO3 in acetonitrile produced low-yield glycoside products 4 and 10 up to 16% and 19%, respectively. We attempted to improve glycosylation using a phase transfer catalyst (TBAB) in a two-phase system (aqueous NaOH and K2CO3) in CHCl3. Unfortunately, the reaction yielded trace amounts. The reaction was incomplete, and the substrate was recovered for reuse. Bromide compound 6 can be decomposed into glycal by an alkaline water phase and phenoxide anion [37]. Therefore, excess use of water in the reaction lowers the yield of the compound. After utilizing several conditions (Tables  1 and 2), the glycosylation reaction under the phase transfer catalyst BnNBu3Cl and K2CO3 as a base at room temperature yielded product 4 at 57% yield. The desired mono-glucosylated product 10 was obtained at 40% yield along with the undesired di-glucosylated product as a mixture, which was separated by column chromatography.
Aryl iodide 7 was the choice for palladium-catalyzed Heck coupling because of its greater reactivity over other aryl halides.   The palladium-catalyzed Heck reaction between glycosylated compounds 4 and 5 under (Pd(OAc)2, BnEt3N + Cl − , Bu3N) in warm acetonitrile produced 22 at 55% yield. Deprotection of the acetyl protecting groups of 22 under a methanolic solution of sodium methoxide resulted in metabolite 2 at 85% yield.
The hydroxy group of glycosylated product 10 was protected by acetylation to yield 23. Similar to Scheme 5, the protected product 23 undergoes a palladium-catalyzed Heck reaction with 4-iodophenyl acetate 11 to build styrene compound 24 at 80% yield. Finally, basic hydrolysis of the acetyl protecting groups under a methanolic solution of sodium methoxide afforded another metabolite, 3, at 84% yield (Scheme 6).  Aryl iodide 7 was the choice for palladium-catalyzed Heck coupling because of its greater reactivity over other aryl halides.
The palladium-catalyzed Heck reaction between glycosylated compounds 4 and 5 under (Pd(OAc) 2 , BnEt 3 N + Cl − , Bu 3 N) in warm acetonitrile produced 22 at 55% yield. Deprotection of the acetyl protecting groups of 22 under a methanolic solution of sodium methoxide resulted in metabolite 2 at 85% yield.
The hydroxy group of glycosylated product 10 was protected by acetylation to yield 23. Similar to Scheme 5, the protected product 23 undergoes a palladium-catalyzed Heck reaction with 4-iodophenyl acetate 11 to build styrene compound 24 at 80% yield. Finally, basic hydrolysis of the acetyl protecting groups under a methanolic solution of sodium methoxide afforded another metabolite, 3, at 84% yield (Scheme 6).  The palladium-catalyzed Heck reaction between glycosylated compounds 4 and 5 under (Pd(OAc)2, BnEt3N + Cl − , Bu3N) in warm acetonitrile produced 22 at 55% yield. Deprotection of the acetyl protecting groups of 22 under a methanolic solution of sodium methoxide resulted in metabolite 2 at 85% yield.
The hydroxy group of glycosylated product 10 was protected by acetylation to yield 23. Similar to Scheme 5, the protected product 23 undergoes a palladium-catalyzed Heck reaction with 4-iodophenyl acetate 11 to build styrene compound 24 at 80% yield. Finally, basic hydrolysis of the acetyl protecting groups under a methanolic solution of sodium methoxide afforded another metabolite, 3, at 84% yield (Scheme 6).   The palladium-catalyzed Heck reaction between glycosylated compounds 4 and 5 under (Pd(OAc)2, BnEt3N + Cl − , Bu3N) in warm acetonitrile produced 22 at 55% yield. Deprotection of the acetyl protecting groups of 22 under a methanolic solution of sodium methoxide resulted in metabolite 2 at 85% yield.
The hydroxy group of glycosylated product 10 was protected by acetylation to yield 23. Similar to Scheme 5, the protected product 23 undergoes a palladium-catalyzed Heck reaction with 4-iodophenyl acetate 11 to build styrene compound 24 at 80% yield. Finally, basic hydrolysis of the acetyl protecting groups under a methanolic solution of sodium methoxide afforded another metabolite, 3, at 84% yield (Scheme 6).

Conclusions
In conclusion, an efficient total synthesis was performed for the preparation of resvebassianol A (2, a metabolite of resveratrol by Beauveria bassiana) and its regiomer (3) through glycosylation and a palladium-catalyzed Heck reaction. Resvebassianol A and regiomer 3 were synthesized in 11 and 12 linear steps, with overall yields of 7.5% and 6.3%, respectively. Incorporation of 4-O methyl glyosyl was performed through the glycosylation reaction and was optimized using a phase transfer catalyst with varying bases. This resulted in an elevated yield of up to 40% and 57%, respectively. Thus, this method can be helpful for the synthesis of metabolites that are difficult to obtain from plant sources and through microbial biotransformation. This strategy can also be used for the synthesis of other related metabolites.