Chromatographic and mass spectrometric characterization of 3-O-benzoyl methyl ester derivatives of mycolic acid fractions from Corynebacterium pseudotuberculosis, C. diphtheriae and Rhodococcus rhodochrous

Abstract

A benzoyl group was attached to the 3-hydroxyl group of the methyl ester derivative of corynomycolic acid fraction isolated from Corynebacterium pseudotuberculosis. The infrared spectrum of the 3-O-benzoylated compound displayed a series of characteristic absorptions found at 1110, 1267 and 1603 cm-1 that confirmed the presence of a monosubstituted phenyl grouping. The 1H-NMR spectrum showed peaks representing protons of the aromatic ring at 7.4 ppm and 8.0 ppm. The UV spectrum revealed two absorption maxima: at 190 and 228 nm. The mass spectrum of the 3-O-benzoylated material exhibited the following peaks: (1) a prominent peak at m/z 105 of the benzoyl group that constituted the base peak; (2) peaks of methyl esters representing the α-hydrocarbon side chain plus carbon atoms C1 and C2 of the mycolic acid molecule; and (3) peaks of molecular ion minus benzoic acid and/or molecular ion minus benzoxyl group. When subjected to liquid chromatography (LC) on an octadecylsilane-silica gel column the 3-O-benzoylated methyl ester derivatives of the corynomycolic acid fraction were separated into their constituent homologous fractions corresponding to underivatized corynomycolic acids with the chain length C30, C32 and C34. Reversed phase HPLC of saturated and monounsaturated species of 3-O-benzoylated derivatives of the mycolic acid fraction from C. diphtheriae and Rhodococcus rhodochrous led to the separation of the corresponding homologous fractions. Mass spectrometry by electron impact mode identified both series of the homologous materials differing in mass by 28 units. © 1993.