title: Stereochemistry and deuterium isotope effects associated with the cyclization-rearrangements catalyzed by tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases, and the chimeric CH4 hybrid cyclase.
abstract: Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH4 hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D2O, together with gas chromatography-mass spectrometry (GC-MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH3-->CH2 eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25-4.64 for the CH3-->CH2 eliminations; (3) a significant intermolecular KIE (1.33+/-0.03) in competitive cyclizations of unlabeled FPP and FPP-d6 to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D2O into the 1beta position of epiaristolochene; (5) stereoselective eliminations of the 1beta and 9beta protons in formation of epiaristolochene and its delta(1(10)) isomer epieremophilene by TEAS and CH4; and (6) predominant loss of the 1alpha proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH4. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.
authors.affiliation: Department of Chemistry, University of Illinois, 600 South Mathews Ave., Urbana, IL 61801, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Schenk
authors.forename: Courtney M
authors.initials: CM
authors.lastname: Starks
authors.forename: Kathleen Rising
authors.initials: KR
authors.lastname: Manna
authors.forename: Joe
authors.initials: J
authors.lastname: Chappell
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: Robert M
authors.initials: RM
authors.lastname: Coates
mesh_terms: D019755:Carbon-Carbon Lyases / Q000737:chemistry*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry*; D032304:Hyoscyamus / Q000201:enzymology*; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D012717:Sesquiterpenes / Q000737:chemistry*; D013237:Stereoisomerism; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D012717:Sesquiterpenes; D003903:Deuterium; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase; D019731:Carbon-Nitrogen Ligases