METABOLISM OF PTEROSTILBENE BY BOTRYTIS CINEREA: INVOLVEMENT OF AN OXIDATIVE PROCESS LINKED TO LACCASE-LIKE STILBENE OXIDASE
A-C BREUIL, F CHOPIN, M ADRIAN, N PIRIO, R BESSIS and P JEANDET
1Laboratoire des Sciences de la Vigne, Institut Jules Guyot, Université de Bourgogne, BP 138, 21004 Dijon cedex, France;
2Laboratoire de Synthèse et Electrosynthèse des Organométalliques, Université de Bourgogne, BP 138, 21004 Dijon cedex, France; 3Laboratoire d'Oenologie, Université de Reims, BP 1039, 51687 Reims, France
Background and objectives
It has been shown previously that grapevine phytoalexins  could undergo degradation by a laccase-like stilbene oxidase  produced by Botrytis cinerea, the causal organism of grey mould.This enzyme is an ortho-para diphenol oxidase . In a recently published paper , we have characterized a resveratrol metabolite synthesized during this degradation process. This compound was identified as a pentaphenolic dehydrodimer of resveratrol with the molecular formula C28H22O6 and a trans-stilbenic skeleton. 1H-NMR, 13C-NMR and 1H-1H, 1H-13C correlations allowed us to determine its structure. In the same way we have studied the oxidation process of pterostilbene by laccase of Botrytis cinerea.
Materials and methods
The phytoalexin was incubated in a crude protein extract obtained from culture filtrates of the fungus (containing the stilbene oxidase activity). The final concentration of ethanol in the incubation medium was 10%. HPLC analysis coupling photodiode-array detection and fluorometry was used to determine the chromatographic and spectral properties of the pterostilbene metabolite. The compound was purified by semi-preparative TLC on silica-C18 (RP18 F2545, 10x20x0.25 cm, Merck) in methanol:water (75:25). 1H-NMR at 500 MHz and 1H-NMR-1H-NMR correlations were performed to assign protons.
Results and conclusions
Kinetics of pterostilbene degradation, analysed by HPLC, showed the appearance of a major fluorescent compound (wavelength exc=330 nm, wavelength em=374 nm) corresponding to the pterostilbene metabolite. UV spectroscopy data showed that this metabolite has absorption maxima between 308-336 nm and 281-313 nm and a high blue fluorescence characteristic of trans-stilbene. Its UV spectrum was very similar to that of trans-pterostilbene. The corresponding cis-isomer has a UV spectrum identical to that of cis-stilbenes (wavelength max=281 nm). These data suggested that the metabolite has kept, at least in part, a stilbenic skeleton. High-resolution EIMS examination (70 eV) (M+. calc. 510.20424, found 510.20608) allowed us to determine that its molecular formula was C32H30O6. 1H-NMR analysis showed the presence of two isomers of the pterostilbene metabolite: one major with a trans-ethylenic structure (J=16 Hz, for the ethylenic protons), one minor compound with a cis-ethylenic structure (J=12.2 Hz). Our studies on treatment of resveratrol and pterostilbene with laccase suggest that they both undergo a dimerization process without supplementary oxidation of the benzenic rings. It is also shown that the phenolic group situated in the 4 position of the stilbene moiety is involved in the dimerization process of stilbenes by the laccase of B. cinerea.
1. Langcake P, Cornford CA, Pryce RJ, 1979. Phytochemistry 18, 1025-1027.
2. Pezet R, Pont V, Hoang-Van K, 1991. Physiological Molecular Plant Pathology 39, 441-450.
3. Mayer AM, 1987. Phytochemistry 26, 11-20.
4. Breuil AC, Adrian M, Pirio N et al., 1997. Tetrahedron Letters, in press.