Aristolochene Synthase‐Catalyzed Cyclization of 2‐Fluorofarnesyl‐Diphosphate to 2‐Fluorogermacrene A

Abstract

The mechanism of the conversion of (E,E)-farnesyl diphosphate (FPP, 1a) to aristolochene (6) catalyzed by aristolochene synthase from Penicillium roqueforti has been proposed to proceed through the neutral intermediate germacrene A (4a). However, much of the experimental evidence is also in agreement with a mechanism in which germacrene A is not an intermediate in the predominant mechanism that leads to the formation of aristolochene, but rather an off-pathway product that is formed in a side reaction. Hence, to elucidate the mechanism of FPP cyclisation the substrate analogue 2-fluoroFPP (1b) was synthesized, and upon incubation with aristolochene synthase was converted to a single pentane extractable product according to GC-MS analysis. On the basis of NMR analyses this product was identified as 2-fluorogermacrene A (4b). Variable temperature (1)H NMR spectroscopy indicated the existence of two conformers of 4b that were in slow exchange at -60 degrees C, while at 90 degrees C the two isomers gave rise to averaged NMR signals. In the major isomer (approximately 75%) the methyl groups on C3 and C7 were most likely in the down-down orientation as had been observed for other (E,E)-germacranes. This work suggests that after an initial concerted cyclisation of FPP to germacryl cation deprotonation leads to the formation of germacrene A, and provides compelling evidence that germacrene A is indeed an on-pathway product of catalysis by aristolochene synthase.

Sesquiterpene Synthase‐Catalysed Formation of a New Medium‐Sized Cyclic Terpenoid Ether from Farnesyl Diphosphate Analogues

“…Initial 1 H NMR spectroscopic analysis of the batch product at room temperature in CDCl 3 showed broad, poorly defined signals in some areas of the spectrum; this hindered a full assignment of the spectrum (Figure ). Slow exchange on the NMR timescale at room temperature has been observed previously for similar medium‐sized ring systems; hence variable‐temperature NMR spectra were measured between −50 °C and +50 °C. The structure was successfully elucidated at +50 °C.…”

Unearthing the roots of the terpenome

“…For example, modest catalytic promiscuity has recently been discovered for trichodiene synthase from F. sporotrichioides, which generates 11% alternative sesquiterpene products; multiple products are also detected when 2-fluorofarnesyl diphosphate and 4-methylfarnesyl diphosphate are used as substrates, and mutagenesis of metal binding residues alters product ratios [37,38]. In other examples, taxadiene synthase catalyzes the cyclization of 6-fluorogeranylgeranyl diphosphate to generate a mixture of 7-fluoroverticillenes [39], and aristolochene synthase catalyzes the cyclization of 2-fluorofarnesyl diphosphate to form 2-fluorogermacrene A [40]. 5-Epi-aristolochene synthase, for which the stereochemical details of the cyclization mechanism have been recently outlined [41], generates 25 sesquiterpene products [42], and mutagenesis studies targeting evolutionarily variable residues in and surrounding the active site show that product arrays can be altered by design [43].…”

X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

“…Section 1734 solely to indicate this fact. with the intact substrates FPP and 2-fluorofarnesyl diphosphate (2F-FPP) (12), and the inhibitor 12,13-difluorofarnesyl diphosphate (DF-FPP) (13). Differences observed in active site conformations and substrate conformations appear to be linked to differences in metal binding, analysis of which suggests a possible sequence for metal ion binding and conformational changes required for catalysis.…”

“…The syntheses of 2F-FPP and DF-FPP have been reported (12)(13)(14), and an additional synthesis of 2F-FPP is outlined in the supplemental materials. Recombinant A. terreus aristolochene synthase was expressed in Escherichia coli BL21(DE3)pLysS and purified as previously described (11,15).…”