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Tripterygium regelii is a rich source of triterpenoids, containing many types of triterpenes with high chemical diversity and interesting pharmacological properties. The cDNA of the multifunctional oxidosqualene cyclase (TrOSC, GenBank accession number: MH161182), consisting of a 2289 bp open reading frame and coding for 762 amino acids, was cloned from the stems and roots of Tripterygium regelii. Phylogenetic analysis using OSC genes from other plants suggested that TrOSC might be a mixed-amyrin synthase. The coding sequence was cloned into the expression vector pYES2 and transformed into the yeast Saccharomyces cerevisiae. The resulting products were analysed by GC-MS. Surprisingly, although it showed 76% sequence identity to lupeol synthase from Ricinus communis, TrOSC was found to be a multifunctional triterpene synthase producing both α- and β-amyrin, the precursors of ursane and oleanane type triterpenes, respectively. qRT-PCR analysis revealed that the transcript of TrOSC accumulated mainly in roots and stems. Taken together, our findings contribute to the knowledge of key genes in the pentacyclic triterpene biosynthesis pathway.

The enzymatic polycyclization reactions catalyzed by oxidosqualene (OXSQ) cyclases (OSCs) proceed with complete regio- and stereospecificity, leading to the formation of new C-C bonds and chiral centers and to the generation of diverse polycyclic sterols and triterpenoids. The diverse structural array is remarkable, and approximately 150 different carbon frameworks have been found. Detailed investigations on squalene-hopene cyclase (SHC) and lanosterol synthase (LaS) have been reported, but progress in the study of β-amyrin synthase, which is ubiquitously found in plants, has lagged in comparison. In the past several years, remarkable advances in β-amyrin biosynthetic studies have been made. In this review, the catalytic mechanism and substrate recognition of β-amyrin synthase, as revealed by site-directed mutagenesis and substrate analog experiments, are outlined and compared with those of LaS and SHC to highlight the features of β-amyrin synthase.

β-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on β-amyrin synthase are very limited as compared with those of squalene-hopene cyclase and lanosterol synthase. This study was conducted to elucidate the function of the F474 residue of Euphorbia tirucalli β-amyrin cyclase, which is highly conserved in the superfamily of oxidosqualene cyclases. Nine site-specific variants with Gly, Ala, Val, Leu, Met, Tyr, Trp, His, and Thr were constructed. We isolated 9 products from these mutants in addition to β-amyrin and determined the chemical structures. The Gly and Ala mutants produced significantly larger amounts of the bicyclic products and a decreased amount of β-amyrin, indicating that the F474 residue was located near the B-ring formation site. Surprisingly, the Ala variant produced (9βH)-polypoda-7,13,17,21-tetraen-3β-ol and (9βH)-polypoda-8(26),13,17,21-tetraen-3β-ol, which are generated from a chair-boat folding conformation. This is the first report describing the conformational change from the chair-chair into the chair-boat folding conformation among the reported mutagenesis studies of oxidosqualene cyclases. Substitution with aliphatic amino acids lacking π-electrons such as Val, Leu, and Met led to a significantly decreased production of bicyclic compounds, and in turn exhibited a higher production of β-amyrin. Furthermore, the Leu and Met variants exhibited high enzymatic activities: ca. 74% for Leu and ca. 91% for Met variants as compared to the wild-type. These facts unambiguously demonstrate that the major role of Phe474 is not to stabilize the transient cation via cation-π interaction, but is to confer the appropriate steric bulk near the B-ring formation site, leading to the completion of the normal polycyclization pathway without accumulation of abortive cyclization products.

Reduction of 3β-acetoxy-12α,13α-epoxyoleanan-3β-yl acetate (β-amyrin acetate epoxide)(5) with lithium–ethylamine affords β-amyrin (17%), oleanane-3β,12α-diol (3; R1= R2= H)(50%) and 13α-oleanane-3β,12α-diol(6; R1= R2= H)(27%). On photolysis 3β-acetoxyoleanan-12α-yl nitrite (3; R1= Ac, R2= NO) affords the 27-oxime (10), which has been further transformed into the aldehyde (9; R1= H, R2= OH) and the corresponding lactone (11; R1R2= O). The latter had previously been identified erroneously as oleanolic lactone acetate (12; R1= Ac, R2R3= O).

The hydride shifts accompanying 2,3-epoxysqualene cyclisation to lanosterol in yeast and to β-amyrin in peas have been checked using 2,3-epoxy[11,14-3H2]squalene. The results support the Ruzicka–Eschenmoser hypothesis and not a plausible alternative which was considered. The synthesis of 2,3-epoxy[11,14-3H2]squalene by two routes is described.

18α-Olean-12-ene, which is in acid-catalysed equilibrium with olean-13(18)-ene (δ-amyrene), has been converted in several steps into olean-12-ene (β-amyrene). Transformation of this hydrocarbon into its 11α-hydroxyderivative and photolysis of the nitrite of the latter gave 11α-hydroxy-1-oximino-olean-12-ene. This oxime has been hydrolysed and further processed to furnish olean-12-en-1-one. Several methods have been developed to change the 1-oxo into a 3-oxo-function and vice versa. Since olean-12-en-3-one has already been reduced to β-amyrin and olean-13(18)-ene has been synthesised, our work constitutes a synthesis of β-amyrin.Related studies on the photolysis of 11β-hydroxyolean-12-ene nitrite and on the interrelationship of the 1- and 3-oxo-groups in the lanostane series are also recorded.

β-Amyrin has been converted into 3β-acetoxy-13-hydroxyoleanane. Photolysis of the nitrite of this alcohol and further standard transformations gave oleanolic acid lactone acetate and thence oleanolic acid. This conversion of β-amyrin into oleanolic acid represents, in the formal sense, a total synthesis of the latter.By a similar series of reactions ursolic acid has been obtained from α-amyrin.

Treatment of 19β-hydroxyolean-11,13(18)-dien-3β-yl acetate with boron trifluoride etherate yields 11-oxo-α-amyrin acetate, together with olean-9(11),12,18-trien-3β-yl acetate and an unidentified trienyl acetate.

A specifically tritiated specimen of the bicyclic epoxide (11) undergoes enzymic cyclization in a homogenate from Pisum sativum to give β-amyrin (5) without randomization of label.