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

Abstract

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.T he use of chemical signals is highly developed within insects, with semiochemicals mediating a wide variety of inter-and intraspecific behaviors. Volatile pheromones, such as sex and aggregation pheromones, are the most well-known types, but insects also use nonvolatile cuticular lipids as contact pheromones (1-4). The cuticular lipids consist of a complex blend of n-and methyl-branched alkanes, alkenes, and lesser amounts of more polar compounds such as esters and alcohols. The lipid layer acts primarily as a waterproofing barrier (5), but individual lipid components have evolved secondary roles as signals that mediate a variety of behaviors and physiological changes (1, 2, 6). For example, solitary insects use cuticular hydrocarbons (CHCs) to identify the species and sex of mates (7, 8) whereas, in social insects, CHCs mediate identification of nestmates (9, 10), recognition of castes, and task allocation within the colony (11). Social insect queens also use CHCs to signal fecundity and dominance status within the colony, inhibiting development of workers into reproductives (12)(13)(14).Determining the roles of specific CHCs as signals has been hindered by three interlinked problems. First, CHCs typically consist of a large number of compounds, which can be difficult to isolate in pure form to test their bioactivities. Specifically, CHCs have very similar polarity and so are not separable by liquid chromatography on silica gel or other polar chromatographic m...

Cuticular Hydrocarbon Profiles Differentiate Tropical Fire Ant Populations (Solenopsis geminata, Hymenoptera: Formicidae)

“…In S. geminata workers, there were 16 alkanes and 12 alkenes with 17 – 35 carbon chain lengths as shown in Table . Methyl‐alkanes in S. geminata workers should be all the ( R )‐enantiomer . In the dark form, major peaks 2 , 7 , 8 , 12 , 13 , 25 were heneicosane, ( Z )‐9‐tricosene, tricosane, ( Z )‐9‐pentacosene, pentacosane, hentriacontene, respectively.…”

Biological activity of the enantiomers of 3-methylhentriacontane, a queen pheromone of the antLasius niger

“…It was unexpected that the primer and releaser effects would appear to be mediated by different enantiomers, or that the (S)-enantiomer would be active at all, because, as noted in the Introduction, a recent study has suggested that the large majority of methyl-branched hydrocarbons in insect cuticular lipids are likely to have the (R)-configuration (Bello et al, 2015). However, our results are consistent with the study by Sharma et al (2015), who showed that the sensilla present on the antennae of Camponotus ants respond to both (R)-and (S)-enantiomers and that ants are able to discriminate between these enantiomers in behavioral tests.…”

“…For the olive fly, Dacus oleae, males and females produce a racemic mixture of their spiroacetal pheromone olean, but the sexes respond differently to the two enantiomers (Haniotakis et al, 1986). Unfortunately, the technology does not yet exist to determine the enantiomeric ratio of the ∼100 ng quantities of 3-MeC 31 that are present on the cuticle of a L. niger queen by analytical chemistry methods (Bello et al, 2015). Thus, our interpretation of the results described above remains somewhat speculative, until the enantiomer or blend of enantiomers produced by L. niger queens can be conclusively determined.…”

“…Thus, the goal of our study was to test the bioactivity of the (R)-and (S)-enantiomers of the queen pheromone of L. niger as inhibitors of ovary development in workers, and as modulators of aggression by workers. A recent study showed that in 36 monomethyl-branched cuticular hydrocarbons (MBCHs) isolated from species from nine different orders of insects, the stereochemistry of the MBCHs was conserved, with all of the compounds having the (R)-configuration regardless of chain length or methyl branch position (Bello et al, 2015). Thus, we hypothesized that the 3-MeC 31 queen pheromone of L. niger has the (R)-configuration.…”

Bromotyrosine-derived metabolites from an Indonesian marine sponge in the family Aplysinellidae (Order Verongiida)

“…In general though methyl branched fatty acids display small specific rotations, which decrease as the branch point is moved to the middle of the chain. 30 Therefore, based on Van't Hoff's rule of optical superposition, the contribution of the sidechain to the overal specific rotation will be mininal for 6 and 7 . Thus, while the configuration of the sidechain remains unassigned, the configuration of spirocycohexadienylisoxazoline cores in 6 and 7 is proposed as 1 S ,6 R as well.…”