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Synthesis of nitrogen-containing bicyclic sesquiterpenes as potential transition state inhibitors of aristolochene synthase

Al-Lami, Naeemah 2013. Synthesis of nitrogen-containing bicyclic sesquiterpenes as potential transition state inhibitors of aristolochene synthase. PhD Thesis, Cardiff University.
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Aristolochene synthase from Penicillium roqueforti(PR-AS) is sesquiterpene synthase that catalyses the Mg2+-dependent conversion of farnesyl diphosphate FDP to (+)-aristolochene. Through the use of site directed mutagenesis, fluorinated FDPs and an aza-analogue of the eudesmane cation, the reaction was previously shown to involve germacrene A and eudesmane cation as intermediates. The subsequent series of rearrangements that transform the eudesmane cation to (+)-aristolochene have not been investigated previously. To probe the carbocationic nature of these 1,2-hydride and methyl shifts, new aza-analogues were designed to mimic the geometric and electrostatic properties of postulated carbocation intermediates in the catalytic mechanism of PR-AS. Here is described the synthesis of both enantiomers of 10-aza-eremophilane in enantiomerically pure from the common precursor (4S)-limonene oxide and their analysis as inhibitors of PR-AS. The synthesis of (7R,4S,5S)-10-aza-eremophilane cation was accomplished in 8 steps, starting from a known keto ester that in turn was obtained by degradation of (-)-limonene oxide. An identical synthetic protocol was repeated from (4R)-limonene oxide to give the enantiomer of 10-aza-eremophilane cation. Inhibition studies with compound (7R,4S,5S)-10-aza-eremophilane indicated that this ammonium salt acted as a moderate competitive inhibitor of PR-AS (Ki = 38 μM), and showed that eremophilane cation is likely a true intermediate on the pathway from FDP to aristolochene during PR-AS catalysis. The inhibition potency of 10-aza-eremophilane was increased by the addition of diphosphate PPi (Ki = 2.9 μM). This synergetic kinetic effect suggests that the possible involvement of PPi as a stabilizing anion for the eremophilane carbocation in PR-AS biosynthesis. Inhibition studies of the enantiomer of (7R,4S,5S)-10-aza-eremophilane cation, (7S,4R,5R)-10-aza-eremophilane cation, which has incorrect stereocenteres, with PR-AS indicated that this ammonium salt was a poor inhibitor of PR-AS (Ki = 1.03 mM). The data obtained for this compound highlight the chiral environment of the active site of PR-AS, and more importantly supports the postulate that terpene synthases form a product-like contour at their active site that steers the carbocationic cascade catalyzed by PR-AS toward the production of a single enantiomer. iv In the second part of the present work, progress was made towards the stereoselective synthesis of 5-aza-eudesmane cation. This teriary amine is a structural mimic of the 5-eudesmane carbocation, another putative intermediate in the reaction cascade catalysed by PR-AS. However, this tertiary amine was not obtained with desired stereochemistry, nevertheless, two diastereoisomers of the desired compound were obtained.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Date of First Compliant Deposit: 30 March 2016
Last Modified: 01 Aug 2018 15:38

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