Mechanism and stereochemistry of the enzyme-catalysed formation of a 2,2-dimethylchromene ring from a prenylated phenol: conversion of rot-2′-enonic acid into deguelin by deguelin cyclase

Abstract

The stereochemistry of the enzymic conversion of rot-2′-enonic acid into deguelin, mediated by deguelin cyclase, has been studied. Using both an enzyme preparation and seedlings of Tephrosia vogellii, it is shown that (6aS,12aS,5′R/S)-5′-hydroxy-4′,5′-dihydro[6′,6′-C3H3]deguelin is not an acceptable intermediate: no evidence for other oxygenated intermediates was found.The (pro-R)- and (pro-S)-6′-methyl groups of deguelin were identified by synthesis from [(E)-4′-13C]rot-2′-enonic acid. Addition of benzeneselenenyl chloride gives two diastereoisomeric 5′-(phenyl selenides) of 4′,5′-dihydrodeguelin which are separated and their stereochemistry established by X-ray crystallography. Elimination of selenoxide from the (5′S)-stereoisomer then gives (6′R)-deguelin (δc 28.20): (6′S)-deguelin has δc 28.52.Although a chemical conversion of [4′-13C]rot-2′-enonic acid into labelled deguelin produces a 1:1 distribution of label between the (pro-R)- and (pro-S)-6′-methyls, the enzyme-mediated conversion results unexpectedly in a 76% incorporation into the (pro-R)- and 24% into the (pro-S)-form. The stereochemistry of the removal of the key 1′-hydrogens in rot-2′-enonic acid was therefore examined.Addition of benzenesulfenyl chloride to deguelin gave a highly reactive chloro sulfide by syn-addition through attack from the less hindered β-face of the molecule. Treatment with sodium cyanoborohydride displaced the reactive chlorine with complete inversion to give (6aS,12aS,5′S)-5′-phenylthio-4′,5′-dihydrodeguelin. Ring-E scission of the latter proceeded satisfactorily using sodium naphthalenide only after reduction of the 1,2-carbonyl to the alcohol: periodinane oxidation then produced rot-2′-enonic acid. Replacement of the unlabelled cyanoborohydride by cyanoborotritide gave the desired (6aS,12aS,1′S)-[1′-3H]rot-2′-enonic acid. The (6aS,12aS,1′R)-[1′-3H]-counterpart was made by first preparing [4′-3H]deguelin by syn-elimination from the sulfoxide formed from (6aS,12aS,4′R,5′S)-5′-phenylthio-4′,5′-dihydro-[4′-3H] deguelin. Addition of benzenesulfenyl chloride to the [4′-3H]deguelin, followed by a sequence parallelling that above, using unlabelled sodium cyanoborohydride, gave the required (6aS,12aS,1′R)-[1′-3H]rot-2′-enonic acid.Enzymic conversion of each [3H]-labelled rot-2′-enonic acid into deguelin along with a [14C]-labelled monitor, shows that a 73% loss of (pro-4′S-H) in rot-2′-enonic acid correlates with a 76% attainment of a (pro-6′R-Me) in deguelin, whilst a 27% loss of (pro-4′R-H) in the former correlates with a 24% attainment of a (pro-6′S-Me) in the latter. The possible enzymic mechanism of the reaction is discussed and related to a similar mechanism we have suggested for the enzymic formation of rotenone from rot-2′-enonic acid.