Aristolochene synthase-catalyzed cyclization of 2-fluorofarnesyl-diphosphate to 2-fluorogermacrene A.
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.
Miller
David J
DJ
School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff, CF10 3AT, UK.
Yu
Fanglei
F
Allemann
Rudolf K
RK
D001665:Binding Sites; D002384:Catalysis; D003500:Cyclization; D008401:Gas Chromatography-Mass Spectrometry; D006845:Hydrocarbons, Fluorinated / Q000737:chemistry / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D010407:Penicillium / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism*; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D045788:Sesquiterpenes, Germacrane / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
D006845:Hydrocarbons, Fluorinated; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D045788:Sesquiterpenes, Germacrane; C471077:germacrene A; C004808:farnesyl pyrophosphate; D007535:Isomerases; C060050:aristolochene synthase