Mechanism of Squalene Cyclization

It is generally accepted that the C-20 cation or its stabilized equivalent is an intermediate in the biosynthesis of certain triterpenes and sterols. To evaluate this hypothesis the biosynthesis of the protosterol fusidic acid was investigated. Fusidic acid was biosynthesized by incubating the fungus Fusidium coccineum on a medium containing (3RS,4R)-(2-14C, 4-3H)-mevalonic acid (MVA). The obtained fusidic acid was shown to contain 6 14C atoms and 4 tritium atoms. It was proven that tritium atoms were located at the critically important 9β and 13α positions. Evidence was also adduced for the location of the remaining 2 tritium atoms at the 5α and 24 positions. The results show that the tritium atoms derived from 4-pro-R position of MVA are located at the theoretically predicted positions. Also this provides support for the view that fusidic acid is biosynthesized by stabilization of the C-20 cation (protosterol numbering) or its stabilized equivalent by elimination of the C-17 proton without backbone rearrangement. The retention of six 14C-atoms in the antibiotic confirms the view that in this fungus the transformation of the parent 4-gem dimethyl protosterol to fusidic acid involves the loss of the 4β-methyl derived from 3'-of MVA. The absence of isotopic hydrogen at C-3, proven by us, indicates that the removal of the 4β-methyl probably proceeds through a 3-keto intermediate. The possible modes of stabilization of the C-20 cation and the formation of the C-17—C-20 double bond are discussed.