STEREOCHEMISTRY OF THE PROTON ELIMINATION IN THE FORMATION OF (-ALPHA-PINENE AND (-)-ALPHA-PINENE BY MONOTERPENE CYCLASES FROM SAGE (SALVIA-OFFICINALIS)())

The three pinene synthases (cyclases) from common sage (Salvia officin alis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pine ne, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pine ne and (+)-beta-pinene (cyclase III), in addition to smaller amounts o f monocyclic and acyclic monoterpene olefins. (1R)-4-H-2(1)-and (1S)-4 -H-2(1)-labeled geranyl pyrophosphates were prepared and used to exami ne the stereochemistry of the C3-proton elimination from the pinyl cat ion intermediates in the formation of the alpha-pinene enantiomers. Ma ss spectrometric analysis of the biosynthetic products derived from th e chirally deuterated substrates revealed that cyclase I and cyclase I II removed the C4-proR-hydrogen of the substrate (C3 proton trans to t he dimethyl bridge of the pinyl nucleus) with a stereoselectivity exce eding 94% in the formation of (+)-alpha-pinene. Similarly, cyclase II removed the C4-proS-hydrogen of the substrate (CS-trans proton of the corresponding pinyl cation) with a stereoselectivity exceeding 78% in the formation of (-)-alpha-pinene. The stereoselectivity of these C3-a xial hydrogen eliminations is rationalized on the basis of a stereoche mical model for the electrophilic isomerization-cyclization reaction s equence catalyzed by the pinene cyclases. The changes in the overall r ates of olefin biosynthesis by these enzymes and in the product ratios resulting from deuterium substitution also permitted confirmation of isotopically sensitive branching in pinene biosynthesis and allowed th e observation of primary kinetic isotope effects in isolation. (C) 199 4 Academic Press, Inc.