Among pentacyclic triterpenes commonly found in plants, γ-amyrin and isoursenol are seldom reported and considered rare in nature. It was hypothesized that these triterpenes are instead routinely overlooked due to inadequate spectral characterization. γ-Amyrin was prepared by HCOOH isomerization of α-amyrin, and isoursenol was isolated from products of a heterologously expressed oxidosqualene cyclase. With precise NMR and GC-MS data, a metabolomics strategy was used to identify isoursenol and γ-amyrin in a wide range of plants.

Combining bioengineering with chemical synthesis has enabled an efficient method for producing Δ7-dafachronic acid, a steroidal hormone associated with nematode germline longevity. Saccharomyces cerevisiae was engineered to produce 7,24-cholestadienol, a convenient starting material for a four-step synthesis of Δ7-dafachronic acid.

Plants allocate an estimated 15–25% of their proteome to specialized metabolic pathways that remain largely uncharacterized. Here, we describe a genome mining strategy for exploring such unknown pathways and demonstrate this approach for triterpenoids by functionally characterizing three cytochrome P450s from Arabidopsis thaliana. Building on proven methods for characterizing oxidosqualene cyclases, we heterologously expressed in yeast known cyclases with candidate P450s chosen from gene clustering and microarray coexpression patterns. The yeast cultures produced mg/L amounts of plant metabolites in vivo without the complex phytochemical background of plant extracts. Despite this simplification, the product multiplicity and novelty overwhelmed analytical efforts by MS methods. HSQC analysis overcame this problem. Side-by-side HSQC comparisons of crude P450 extracts against a control resolved even minor P450 products among ∼100 other yeast metabolites spanning a dynamic range of >10 000:1. HSQC and GC–MS then jointly guided purification and structure determination by classical NMR methods. Including our present results for P450 oxidation of thalianol, arabidiol, and marneral, the metabolic fate for most of the major triterpene synthase products in Arabidopsis is now at least partially known.

The triterpene product profile is reported for At5g36150 (PEN3), the last unexamined oxidosqualene cyclase in the reference plant Arabidopsis thaliana. PEN3 makes tirucalla-7,24-dien-3β-ol (∼85%) and several minor products. Also discussed are the unexpectedly facile convergent evolution of another Arabidopsis tirucalladienol synthase (LUP5), mechanistic origins of the 20S configuration, and active-site remodeling necessary to accommodate the 17α side chain. This work marks the first completed functional characterization of all triterpene synthases in a higher plant.

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-3β-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.

The triterpenoids are a large group of natural products derived from C30 precursors. Nearly 200 different triterpene skeletons are known from natural sources or enzymatic reactions that are structurally consistent with being cyclization products of squalene, oxidosqualene, or bis-oxidosqualene. This review categorizes each of these structures and provides mechanisms for their formation.The triterpenoids are a large group of natural products derived from C30 precursors. Nearly 200 different triterpene skeletons are known from natural sources or enzymatic reactions that are structurally consistent with being cyclization products of squalene, oxidosqualene, or bis-oxidosqualene. This review categorizes each of these structures and provides mechanisms for their formation.