Purification, Stereoisomeric Analysis and Quantification of Sex Pheromone Precursors in Female Whole Body Extracts from Pine Sawfly Species

Abstract

A GC-MS method to analyze the stereoisomeric composition of chiral secondary alcohols found in whole body extracts of pine sawfly females was developed. The tested alcohols were derivatized with optically pure (S)-2-acetoxypropionyl chloride prior to GC-MS analysis. Baseline separation was obtained for all sixteen stereoisomers of 3,7,9-trimethyltridecan-2-ol and for the four 3-methylpentadecan-2-ol stereoisomers. For 3,7-dimethyltridecan-2-ol, 3,7-dimethyltetradecan-2-ol and 3,7-dimethylpentadecan-2-ol baseline separation was obtained for 6 of the possible 8 stereoisomers. When a mixture of 16 stereoisomers of 3,7,11-trimethyltridecan-2-ol was tested, baseline separation of 7 peaks out of 16 possible was obtained. The investigated alcohols are pheromone precursors for some pine sawfly species that are severe defoliators of pine. Females from several Diprion, Neodiprion, Macrodiprion, Microdiprion, and Gilpinia species emit esters of such secondary alcohols as sex pheromones that attract males for mating. To quantify the small amounts of the precursor alcohol and its stereoisomeric composition found in whole body extracts from female pine sawflies, a purification method was optimized. An extract of 20 females of D. pini contained about 8 ng of (2S,3R,7R)-3,7-dimethyltridecan-2-ol per female, and three extracts of 18, 20, and 90 females of N. sertifer contained between 5 and 13 ng of (2S,3S,7S)-3,7-dimethylpentadecan-2-ol per female.

Stereoisomeric Analysis of 6,10,14-Trimethylpentadecan-2-ol and the Corresponding Ketone in Wing Extracts from African Bicyclus Butterfly Species

“…To determine the stereochemistries of the 6,10,14-trimethylpentadecan-2-ol and 6, 10,14-trimethylpentadecan-2-one, the pooled alcohol fraction was derivatized with (R)-trans-chrysanthemoyl chloride according to Bång et al (2012) or with (S)-2-acetoxypropionyl chloride according to Bång et al (2011). The pooled ketone fraction with 6,10,14-trimethylpentadecan-2-one was first reduced to alcohol with LiAlH 4 before derivatization, as described above.…”

“…Stereochemical Analysis Improved conditions were developed for separation of (R)-trans-chrysanthemoyl (Bång et al 2012) and (S)-2-acetoxypropionyl derivatives (Bång et al 2011) of 6,10,14-trimethylpentadecan-2-ol in GC/MS analyses. Equipment for the GC/MS analyses was as above.…”

“…However, even a small amount (<0.2 %) of the (2S, 3R,7R)-stereoisomer inhibits the attractiveness (Anderbrant 1999;Anderbrant et al 2000). This example emphasizes the need for analytical methods that effectively separate stereoisomers of biologically-active compounds (Bång et al 2011(Bång et al , 2012 in order to interpret and understand the results of bioassays.…”

“…The general approach to separate stereoisomers of secondary alcohols is to use pre-column derivatization, resulting in chiral or non-chiral derivatives in combination with chiral or non-chiral columns. However, combinations of different derivatives and columns often have been necessary to separate all the stereoisomers satisfactorily (Bång et al 2011;Bergström et al 1995;Wassgren et al 1992). Preparation of all eight stereoisomers individually is normally necessary to be able to determine the retention time of each stereoisomer, and this involves laborious and time consuming synthetic work.…”

“…In this study, we used GC/MS to determine the stereoisomeric composition of 6,10,14-trimethylpentadecan-2ol and 6,10,14-trimethylpentadecan-2-one in wing extracts from seventeen species of African Bicyclus butterflies. We improved the conditions for the GC/MS analysis of (R)-transchrysanthemoyl (Bång et al 2012) and (S)-2-acetoxypropionyl derivatives (Bång et al 2011) of 6,10,14-trimethylpentadecan-2-ol, including the identification of the elution order of each stereoisomer of the alcohol.…”