Determination of the absolute stereochemistry of methyl-branched hydrocarbons isolated from several orders of insects

Insect cuticular lipids are comprised of a complex mixture of methyl-branched hydrocarbons (MBCHs), n- hydrocarbons, long-chain olefins, and polar compounds as minor constituents. The primary function of the cuticular wax layer is to maintain water balance, but in many species, components of the cuticular lipids also serve as contact pheromones. The majority of insect-produced MBCHs are chiral, but despite the fact that they are ubiquitous components of insect cuticular lipids, no studies have attempted to determine whether they are biosynthesized enantiospecifically. This is mostly due to the minute optical rotations of MBCHs, which make enantiomeric analysis through polarimetry impractical due to the inadequate amounts (ng to μg) of hydrocarbons obtained from insects. Difficulties in isolation of individual MBCHs from the cuticular blend, and the lack of enantiopure MBCH standards, have also hindered the stereochemical analysis of insect-produced MBCHs.

Here we report methods for the identification of the absolute stereochemistry of monomethyl-branched hydrocarbons isolated from several orders of insects. The MBCHs were isolated by initial fractionation of the cuticular extracts with AgNO3 impregnated silica gel column chromatography into fractions containing saturated hydrocarbons, olefins, and polar compounds respectively. The n-hydrocarbons were separated from MBCHs by adsorption of the former into 5Å molecular sieves, and the enriched MBCHs were further fractionated with reverse- phase HPLC. The sign of the optical rotations of individual MBCHs were measured with an advanced laser polarimeter detector, which were then compared to the optical rotations of synthesized stereochemically pure MBCH standards to determine their absolute stereochemistries.