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Isolation and determination of absolute configurations of insect-produced methyl-branched hydrocarbons

Significance Methyl-branched hydrocarbons (MBCHs) are widely used for inter- and intraspecific communication by insects. MBCHs can exist as two or more stereoisomers, but it remains unknown which stereoisomer(s) insects produce, nor is it clear whether insects can distinguish MBCH stereoisomers. To address these questions, we developed a generic method for isolation of MBCHs (and other lipids) from a range of insect species. Polarimetric analyses of 36 isolated MBCHs showed that their stereochemistry is conserved across nine orders of Insecta, with all compounds having the (R)-configuration, regardless of methyl branch position or chain length. These results will enable further explorations of the role of stereochemistry in contact chemical communication, and the molecular mechanisms involved in recognition of hydrocarbons. Although the effects of stereochemistry have been studied extensively for volatile insect pheromones, little is known about the effects of chirality in the nonvolatile methyl-branched hydrocarbons (MBCHs) used by many insects as contact pheromones. MBCHs generally contain one or more chiral centers and so two or more stereoisomeric forms are possible for each structure. However, it is not known whether insects biosynthesize these molecules in high stereoisomeric purity, nor is it known whether insects can distinguish the different stereoisomeric forms of MBCHs. This knowledge gap is due in part to the lack of methods for isolating individual MBCHs from the complex cuticular hydrocarbon (CHC) blends of insects, as well as the difficulty in determining the absolute configurations of the isolated MBCHs. To address these deficiencies, we report a straightforward method for the isolation of individual cuticular hydrocarbons from the complex CHC blend. The method was used to isolate 36 pure MBCHs from 20 species in nine insect orders. The absolute stereochemistries of the purified MBCHs then were determined by digital polarimetry. The absolute configurations of all of the isolated MBCHs were determined to be (R) by comparison with a library of synthesized, enantiomerically pure standards, suggesting that the biosynthetic pathways used to construct MBCHs are highly conserved within the Insecta. The development of a straightforward method for isolation of specific CHCs will enable determination of their functional roles by providing pure compounds for bioassays.