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Mechanism of Abietadiene Synthase Catalysis: Stereochemistry and Stabilization of the Cryptic Pimarenyl Carbocation Intermediates

2002; American Chemical Society; Volume: 124; Issue: 24 Linguagem: Inglês

10.1021/ja017734b

1943-2984

Matthew M. Ravn, Reuben J. Peters, Robert M. Coates, Rodney Croteau,

Microbial Natural Products and Biosynthesis

Abietadiene synthase (AS) catalyzes the complex cyclization−rearrangement of (E,E,E)-geranylgeranyl diphosphate (8, GGPP) to a mixture of abietadiene (1a), double bond isomers 2a−4a and pimaradienes 5a−7a as a key step in the biosynthesis of the abietane resin acid constituents (1b−4b) of conifer oleoresin. The reaction proceeds at two active sites by way of the intermediate, copalyl diphosphate (9). In the second site, a putative tricyclic pimaradiene or pimarenyl(+) carbocation intermediate of undefined C13 stereochemistry and annular double bond position is formed. Three 8-oxy-17-nor analogues of 9 (17 and 19a,b) and three isomeric 15,16-bisnorpimarenyl-N-methylamines (26a−c) were synthesized and evaluated as alternative substrates and/or inhibitors for recombinant AS from grand fir. The stereospecific cyclization of 8α-hydroxy-17-nor CPP (19a) to 17-normanoyl oxide (20a) and the higher inhibitory potency of the norpimarenylamine 26a (Ki = 0.1 nM) both suggest pimarenyl intermediates having the 13β methyl configuration and 8,14-double bond corresponding to sandaracopimaradiene (5a). The 2000-fold stimulation of inhibition by 26a in the presence of inorganic pyrophosphate indicates an important role for carbocation/OPP anion stabilization of the secondary sandaracopimaren-15-yl(+) ion. The failure of 8β-hydroxy-17-nor CPP (19b) to undergo enzymatic cyclization was taken as evidence that 9 is bound with a "coplanar" side chain conformation and that the SN' cyclization occurs on the 17α face. The routing of the sandarcopimara-15-en-8-yl carbocation toward various diterpenes in biogenetic schemes is attributed to differing conformations of ring C and/or orientations of the C13 vinyl group in the active sites of the corresponding diterpene cyclases.