Enzyme-promoted oxidative cross-coupling for the synthesis of oxyresveratrol-related heterodimers

Horseradish peroxidase–H2O2-mediated oxidative cross-coupling reactions of oxyresveratrol 2-methyl ether and brominated isorhapontigenin or brominated resveratrol efficiently produce two 8-5’-coupled dihydrobenzofuran-type heterodimers. The LiAlH4-catalysed reductive debrominations of these cross-coupled dimeric intermediates provided the first synthesis of two unnatural oxyresveratrol-related heterodimers.


Introduction
Natural polyphenolic oligostilbenes possess complex structures and have diverse biological properties as potential therapeutic agents in human health. Most of them are the homooligomers of stilbene units including resveratrol (1), isorhapontigenin (2), piceatannol (3) and oxyresveratrol (4) (Figure 1). 1-9 Numerous efforts have been devoted to synthesizing these homooligomeric products, especially oligomers of 1 for investigating their structure-activity relationships, and significant achievements had been made over the past 40 years. [10][11][12][13][14] However, little attention has been paid to the small number of heterooligomers polymerized from different stilbene monomers, such as the heterodimers 5−8. 5,7,15 Only a few efforts to synthesize these dimers have been made so far, which included FeCl 3 ·6H 2 Ocatalysed oxidative cross-coupling reactions of stilbenes 1 and 2 conducted by Lin's group, 2 and enzyme-mediated biotransformation of the mixture of 1 and pterostilbene studied by Queiroz's team. 16 Both methods invariably afforded various complex homodimers and heterooligomers of two monomers in very low isolated yields, which resulted from the lack of regioselective control in the oxidative cross-coupling reactions of stilbenes. Recently, our group efficiently synthesized two resveratrol-related  17 based on our research on the regioselective biomimetic synthesis of natural homodimers of 1 or 2. [18][19][20][21][22][23][24] This preliminary success inspired us to prepare other natural stilbene heterooligomers such as oxyresveratrol-related heterodimers 6−8 using a similar synthetic strategy.
The key to synthesis of oxyresveratrol-related heterodimers 6−8 is the selection of stilbene precursors. We recently found that the direct oxidation of 4 promoted by different catalysts invariably led to a complex product mixture and the selective methyl protection of the reactive 2-OH in 4 remarkably improved the regioselectivity of the oxidative coupling reactions. 25 Hence, oxyresveratrol 2-methyl ether (9), as one of two hetero-monomers in the oxidative crosscoupling dimerization, should be more suitable than stilbene 4 for the synthesis of dimers 6−8.

Results and discussion
The oxidative coupling reactions of monomer 9 and stilbenes 1, 2 or 3 are believed to be the most concise approach to the heterooligomeric structures 6-8. We initially explored the direct cross-coupling reaction of 9 and 2 or 1 in the HRP-H 2 O 2 -aqueous acetone system, which resulted in complex and indistinguishable coupling product mixtures consisting of several homodimers and heterodimers of the two precursors. This result encouraged us to apply brominated isorhapontigenin 10 and brominated resveratrol 11 as the other precursor used with 9 to improve the regioselective control in the cross-coupling reactions.
As shown in Scheme 1, when monomer 9 was added slowly into an acetone-water solution of stilbene 10 and HRP-H 2 O 2 , a small amount of heterodimer 12 and a large quantity of homodimer 13 were formed as the major coupling products. Consistent with our earlier work, 5 the yield of heterodimer 12 was largely influenced by the molar ratios and the addition sequences of the two coupling precursors (9 and 10), as well as the amount and the addition rate of oxidant (HRP-H 2 O 2 ). Hence, we optimized the cross-coupling reaction conditions to increase the yield of 12. When HRP and 3% H 2 O 2 were added in batch within 20 min to a solution of equivalent amounts of 9 and 10 in acetone-water and the reaction continued for 1 h at room temperature until precursor 9 had disappeared, the heterodimer 12 with a 43% maximum yield and only a trace amount of homodimer 13 were isolated from the product mixture.
The dihydrobenzofuran skeleton of heterodimer 12 was established on the basis of 1 H and 13 C NMR spectroscopy as well as by HRMS. The connectivity of the two heteromonomer units in dimer 12 was further confirmed by X-ray crystallographic analysis. This is the first report on the X-ray crystal structure of benzofuran-type oligostilbenes ( Figure 2). The formation of the 8-5'-coupled heterodimer 12 verified the higher reactivity of the M 8 radical mesomer of stilbene 10 in comparison to that of 9, which preferentially participated in the cross-coupling dimerization under the optimized reaction conditions. Based on the above result, we further studied the oxidative cross-coupling reaction of 9 and 11 under the HRP-H 2 O 2 -catalysed conditions (Scheme 1). When equivalent amounts of precursors 9 and 11 were added simultaneously into the reaction system, the oxidation of 9 was obviously faster than that of 11 and mostly produced the homodimer 13, and a very small amount of the 8-5'-coupled heterodimer 14. Under the optimized reaction conditions, 1.5 equiv. of 9 and 3% H 2 O 2 were added dropwise into the reaction mixture within 30 min and stirring was continued for 1 h at room temperature until precursor 9 had disappeared. The homodimer 13 in 17% yield and heterodimer 14 in a 30% maximum yield were isolated from the product mixture. These results further confirmed that the yield of heterodimer 14 can be effectively increased by the optimization of the molar ratio and the addition sequence of precursors 9 and 11.
We next attempted to remove the bromine protecting groups and methyl groups from the heterodimeric intermediates 12 and 14 in order to synthesize several oligostilbenes. As shown in Scheme 2, the LiAlH 4 -catalysed reductive debromination reaction of dimer 12 in tetrahydrofuran at 50 °C smoothly led to unnatural product 15 in 65% yield. Under the same deprotection condition, the bromine atoms of dimer 14 were removed and the heterodimer 16 was obtained in 70% yield. The subsequent direct demethylation of 12 for the synthesis of natural 7 or 8 was not successful because of the insolubility of 12 in the BBr 3dichloromethane system. The global acetylation of 12 followed by deacetylation and demethylation in one-pot finally led to an inseparable product mixture.

Conclusion
In conclusion, we have investigated the biomimetic crosscoupling reactions of two different stilbene monomers under HRP-H 2 O 2 -acetone-H 2 O oxidative conditions. The key to the regioselective synthesis of heterodimers 12 and 14 lays first in the distinct difference in the reactivity of 2-methylated oxyresveratrol (9) and brominated isorhapontigenin 10 or brominated resveratrol 11 under the same HRP-H 2 O 2catalysed oxidative conditions, and second in the application of appropriate molar ratios and addition sequences of the two coupling precursors. The X-ray crystal structure of 12 was reported, which confirms the dihydrobenzofuran skeleton of the oligostilbenes. Two unnatural oligostilbenes 15 and 16 were synthesized by the reductive debromination reactions of heterodimer 12 or 14. The formation of dihydrobenzofuran-type heterodimers 15 and 16 confirmed that this regioselective synthetic strategy is applicable to the preparation of other stilbene heterooligomers.

General methods
Structural determinations of the isolated compounds were based on 1 H, 13 C NMR, NOESY, 1 H-1 H COSY and 1 H-1 H NOSEY spectra, and HRMS analysis. All NMR spectra were recorded on Varian Mercury 500 or 600 MHz instruments in the indicated solvent. HRMS spectra were measured on an Autostec-3090 mass spectrometer. All solvents were freshly purified and dried by standard techniques prior to use. Purification of products was performed by column chromatography on silica gel (200-300