VERIFICATION OF STRUCTURE AND DIRECTION OF BIOSYNTHESIS OF LONG-CHAIN METHYL-BRANCHED ALCOHOLS AND VERY LONG-CHAIN METHYL-BRANCHED ALCOHOLS IN PUPAE OF THE TOBACCO HORNWORM, MANDUCA-SEXTA

Deuterium labeling and chloride derivatization, followed by CGC-MS, demonstrated a difference between the long-chain methyl-branched alcohols (LMA, C25-C32; minor components) and the very long-chain methyl-branched alcohols (VLMA, C38- > C44; major components) in the positions of the methyl branches relative to the primary hydroxyl group, which indicated a difference in their biosynthesis. The substitution of the hydroxyl group by chloride is a new approach to determining the structure of methyl-branched alcohols. The chloride derivatives gave unequivocal mass spectra showing the position of the methyl branch(es) with respect to the chloride. Assuming that the primary hydroxyl group represented the terminal end of the alkyl chain during biosynthesis, the methyl branches in the VLMA were incorporated early during chain elongation. Conversely, the methyl branches in the LMA were incorporated late in chain elongation. Thus, previously reported structures of the LMA were mis-named because the methyl branch positions were determined by counting from the wrong end of the molecule; i.e. an alcohol previously designated as 15,19-dimethyloctacosan-1-ol was in fact 10,14-dimethyloctacosan-1-ol. Both the LMA and VLMA appear to be synthesized using acetate as a primer. [1-C-13]Propionate was incorporated early in chain elongation of the VLMA, but was incorporated late in chain elongation of the LMA, confirming the conclusions reached from the analysis of the mass spectra of the deuteride and chloride derivatives. The labeled carboxyl carbon of [1-C-13]propionate was located between the methyl branch methine and the primary hydroxyl group, confirming that the primary hydroxyl group was located on the terminal (elongating) end of the alkyl chain.