Structure and absolute configuration of an unsaturated anteiso fatty acid from Bacillus megaterium

Abstract

Gas chromatography in combination with electron ionization mass spectrometry (GC/EI-MS) was used to determine the fatty acids of a membrane lipid from Bacillus megaterium. Special attention was put on the structure and absolute configuration of a monoenoic fatty acid previously described in this sample. GC/EI-MS operated in the selected ion monitoring mode was used to determine twelve fatty acids in the bacterium. Methyl esters were prepared to verify the presence of a 14-methylhexadecenoic acid (a17:1) isomer. The position of the double bond of the a17:1 isomer and four further monoenoic fatty acids was elucidated by means of their picolinyl esters produced by the transesterification of the phospholipid. For the a17:1 isomer, the double bond was located between C-5 and C-6. Silver ion liquid chromatography was used to verify that the double bond was in cis-configuration. The bacterial 14-methylhexadec-5-enoic acid (a17:1Δ5) is chiral due to the stereogenic C-14 carbon. Initial enantioselective measurements were carried out with isomers of a17:1Δ5 which were available in form of racemic and ( S)-enantiopure cis- and trans-isomers of a17:1Δ12 previously synthesized. The cis-a17:1Δ12 enantiomers were partly resolved on a chiral stationary phase coated with 50% heptakis(6-O- tert.-butyldimethylsilyl-2,3-di- O-methyl)-β-cyclodextrin in OV-1701 (β-TBDM). However, resolution of the enantiomers of the trans-isomer of a17:1Δ12 failed. Only one peak was also observed for the a17:1Δ5 isomer from B. megaterium. Thus, it remained unclear whether the compound a17:1Δ5 was racemic or enantiopure in the sample. To clarify this point, we separated the cis-monoenoic fraction from the saturated fatty acids. Then, the monoenoic fraction was hydrogenated in order to transform a17:1Δ5 into 14-methylhexadecanoic acid (a17:0). This chiral fatty acid was known to be sufficiently enantioseparated on the β-TBDM column and was found to be ( S)-enantiopure in the sample. Hence, these measurements verified that the B. megaterium sample contained enantiopure ( S)-a17:1Δ5.