Most quantum mechanical studies of triterpene synthesis have been done on small models. We calculated mPW1PW91/6-311+G(2d,p)//B3LYP/6-31G* energies for many C30H51O+ intermediates to establish the first comprehensive energy profiles for the cationic cyclization of oxidosqualene to lanosterol, lupeol, and hopen-3beta-ol. Differences among these 3 profiles were attributed to ring strain, steric effects, and proton affinity. Modest activation energy barriers and the ample exothermicity of most annulations indicated that the cationic intermediates rarely need enzymatic stabilization. The course of reaction is guided by hyperconjugation of the carbocationic 2p orbital with parallel C-C and C-H bonds. Hyperconjugation for cations with a horizontal 2p orbital (in the plane of the ABCD ring system) leads to annulation and ring expansion. If the 2p orbital becomes vertical, hyperconjugation fosters 1,2-methyl and hydride shifts. Transition states leading to rings D and E were bridged cyclopropane/carbonium ions, which allow ring expansion/annulation to bypass formation of undesirable anti-Markovnikov cations. Similar bridged species are also involved in many cation rearrangements. Our calculations revealed systematic errors in DFT cyclization energies. A spectacular example was the B3LYP/6-311+G(2d,p)//B3LYP/6-31G* prediction of endothermicity for the strongly exothermic cyclization of squalene to hopene. DFT cyclization energies for the 6-311+G(2d,p) basis set ranged from reasonable accuracy (mPW1PW91, TPSSh with 25% HF exchange) to underestimation (B3LYP, HCTH, TPSS, O3LYP) or overestimation (MP2, MPW1K, PBE1PBE). Despite minor inaccuracies, B3LYP/6-31G* geometries usually gave credible mPW1PW91 single-point energies. Nevertheless, DFT energies should be used cautiously until broadly reliable methods are established.

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The genome of the model plant Arabidopsis thaliana encodes 13 oxidosqualene cyclases, 9 of which have been characterized by heterologous expression in yeast. Here we describe another cyclase, baruol synthase (BARS1), which makes baruol (90%) and 22 minor products (0.02-3% each). This represents as many triterpenes as have been reported for all other Arabidopsis cyclases combined. By accessing an extraordinary repertoire of mechanistic pathways, BARS1 makes numerous skeletal types and deprotonates the carbocation intermediates at 14 different sites around rings A, B, C, D, and E. This undercurrent of structural and mechanistic diversity in a superficially accurate enzyme is incompatible with prevailing concepts of triterpene biosynthesis, which posit tight control over the mechanistic pathway through cation-pi interactions, with a single proton acceptor in a hydrophobic active site. Our findings suggest that mechanistic diversity is the default for triterpene biosynthesis and that product accuracy results from exclusion of alternative pathways.

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