Biosynthesis of Monoterpenes: Regio- and Stereochemistry of (+)-3-Carene Biosynthesis

Abstract

Incubation of [1- 3H 1]geraniol with stem disks of Douglas fir ( Pseudotsuga menziesii) and incubation of [1- 3H 1]geranyl pyrophosphate with both a soluble enzyme extract from Douglas fir and a partially purified preparation of (+)-3-carene synthase from lodgepole pine ( Pinus contorta) resulted in the production of (+)-3-[ 3H] carene. Subsequent conversion of the product to car-3-en-5-one and to 4-isocaranone followed by base-catalyzed exchange of the α-hydrogens established that the 3H located at C1 in the geranyl substrate resided at C5 of (+)-3-carene. Incubation of the (+)-3-carene synthase preparation with ( S)-[5- 3H 1, 4- 14C]geranyl pyrophosphate resulted in the production of (+)-3-carene without loss of tritium, indicating that the 5- proR hydrogen is eliminated during cyclopropyl ring closure. Analysis of the conformational requirements for this 1,3 elimination involving the 5- proR hydrogen suggested that cyclopropyl ring formation occurs via a (4 S)-α-terpinyl cation derived from the anti-endo cyclization of a (3 S)-linalyl pyrophosphate intermediate. Kinetic analyses of the conversion of (1 Z, 3 R)-[1- 3H 1]linalyl pyrophosphate, (1 Z, 3 S)-[1- 3H 1]linalyl pyrophosphate and [1- 3H 1]geranyl pyrophosphate by (+)-3-carene synthase revealed that the velocity of the reaction with the (3 S)-linalyl enantiomer was 25-fold greater than the velocity with the (3 R)-enantiomer and twice that of the natural substrate, geranyl pyrophosphate, thereby confirming this stereo-chemical prediction and also indicating that the cyclization of the linalyl intermediate is faster than the coupled isomerization and cyclization of the geranyl substrate. From these results, a model that details the regio- and stereochemistry of the enzymatic conversion of geranyl pyrophosphate to (+)-3-carene is proposed.