MECHANISM AND STEREOCHEMISTRY OF THE ENZYME-CATALYZED FORMATION OF A 2,2-DIMETHYLCHROMENE RING FROM A PRENYLATED PHENOL - CONVERSION OF ROT-2'-ENONIC ACID INTO DEGUELIN BY DEGUELIN CYCLASE

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'-(CH3)-H-3]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'-C-13]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 (delta(c) 28.20): (6'S) -deguelin has delta(c) 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 l'-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 beta-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)-[l'-H-3]rot-2'-enonic acid. The (6aS,12aS,1'R)-[1'-H-3]-counterpart was made by first preparing [4'-H-3]deguelin by syn-elimination from the sulfoxide formed from (6aS,12aS,4'R,5'S) -5'-phenylthio-4',5'-dihydro- [4'-H-3] deguelin. Addition of benzenesulfenyl chloride to the [4'-H-3]deguelin, followed by a sequence parallelling that above, using unlabelled sodium cyanoborohydride, gave the required (6aS,12aS,1'R)-[1'-H-3]rot-2'-enonic acid. Enzymic conversion of each [H-3]-labelled rot-2'-enonic acid into deguelin along with a [C-14]-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.