Chiral Separation of Stilbene Dimers Generated by Biotransformation for Absolute Configuration Determination and Antibacterial Evaluation

Abstract

A series of complex stilbene dimers have been generated through biotransformation of resveratrol, pterostilbene, and the mixture of both using the enzymatic secretome of Botrytis cinerea Pers. The process starts with achiral molecules and results in the generation of complex molecules with multiple chiral carbons. So far, we have been studying these compounds in the form of enantiomeric mixtures. In the present study, we isolated the enantiomers to determine their absolute configuration and assess if the stereochemistry could impact their biological properties. Eight compounds were selected for this study, corresponding to the main scaffolds generated (pallidol, leachianol, restrytisol and acyclic dimers) and the most active compounds (trans-δ-viniferin derivatives) against a methicillin-resistant strain of Staphylococcus aureus (MRSA). To isolate these enantiomers and determine their absolute configuration, a chiral HPLC-PDA analysis was performed. The analysis was achieved on a high-performance liquid chromatography system equipped with a chiral column. For each compound, the corresponding enantiomeric pair was obtained with high purity. The absolute configuration of each enantiomer was determined by comparison of experimental and calculated electronic circular dichroism (ECD). The antibacterial activities of the four trans-δ-viniferin derivatives against two S. aureus strains were evaluated.

Keywords: Botrytis cinerea Pers.; ECD calculations; MRSA; Staphylococcus aureus; biotransformation; chiral-HPLC; enantiomers; stilbene dimers.

FIGURE 1

Structures of the four trans-δ-viniferin derivatives (1–4) and the four other selected compounds (5–8) representing the main scaffolds obtained by the biotransformation of resveratrol and pterostilbene with the enzymatic secretome of Botrytis cinerea Pers.

FIGURE 2

(A) UHPLC-PDA analysis of 1 using an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm i. d., 1.7 µm; Waters), and separation of its enantiomers 1a and 1b on HPLC-PDA using an Chiralpak^® IB N-5 column (250 × 4.6 mm i. d., 5 µm; Daicel). (B) UV spectra of compounds 1, 1a, and 1b. (C) Relative configuration determination by ROESY NMR experiment. The spectrum with the correlations is presented in Supplementary Figure S1. (D) Calculated ECD spectra. (E) ECD and specific rotation ([α]_D) analysis of compounds 1, 1a, and 1b. (F) Determination of the absolute configuration of compounds 1a and 1b.

FIGURE 3

(A) Relative stereochemistry of compounds 2, 3, and 4 defined by the NMR analysis. (B) specific rotation ([α]_D) and (C) and ECD analysis of compounds 2a, 2b, 3a, 3b, 4a, and 4b. (D) Absolute stereochemistry attribution based on calculated ECD spectra.

FIGURE 4

Relative configuration, experimental ECD spectra and calculated ECD spectra of the enantiomeric pairs of compounds 5–8. Experimental red curve matches with the calculated orange one, while the experimental blue curve matches with the experimental black curve. (A) pallidol-type 5. (B) acyclic dimer-type 6. (C) leachianol-type 7. (D) restrytisol-type 8.