The 'cord factor' from Corynebacterium diphtheriae was fractionated by thin-layer chromatography of the trimethylsilyl derivatives into three main components: the true 'cord factor' B1 which has been described previously and two compounds, B2 and B3, containing 3-0x0-acyl esters. These products were analyzed by mass spectrometry (field desorption/cationization and electron impact of the trimethylsilyl derivatives) and by chemical degradation.Compound B3 is a 6,6'-bis(3-oxo-acyl)-cc-~-trehalose. The 3-0x0-acyl groups are mainly saturated species, branched at C-2, containing 24, 26, 28, 30 or 32 carbon atoms. The main component of B3 is the 6,6'-bis(3-oxo-2-tetradecyl-octadecanoyl)-cc-~-trehalose.Compound B2 is a 6,6'-mixed ester of cc-D-trehalose with the preceding 3-0x0 acids and with corynomycolic acids. These latter contain an even number of carbon atoms from 24 to 32 and are a mixture of saturated, mono-unsaturated and di-unsaturated species. The main compound of B2 is 6-(3-oxo-2-tetradecyl-octadecanoyl)-6'-(3-hydroxy-2-tetradecyl-octadecanoyl)-cc-~-trehalose.The metabolic implications of this structural study, together with results already published, suggest that condensation of the common fatty acids by a Claisen-like process via the 0x0 ester pathway may not be the only way for the synthesis of corynomycolic acids.In previous papers, it was shown that both whole cells and cell-free extracts of Corynehacteriunz diphtheriae catalyzed the transformation of palmitic acid into C32 3-0x0 esters [1,2] by a Claisen-like condensation reaction. By pulse-labelling experiments the first condensation product has been detected [l], and further isolated and identified as 6-0-(3-0x0-2-tetradecyl-octadecanoy1)-cc-D-trehalose [3]. Some other compounds containing 3-0x0-ester groups were also detected: after labelling of whole cells by means of [l -'4C]palmitic acid, radioactive palmitone was isolated from products of alkaline hydrolysis of lipid fractions. For instance, the 'cord factor' fraction, which was an apparently homogenous fraction by chromatography, gave a small quantity of palmitone after hydrolysis, together with a large amount of corynomycolic acid. This work describes an exhaustive fractionation of 'cord factor' preparation and the isolation of new 3-0x0-ester-containing compounds. A structural study of the 'true' cord factor isolated from this fraction, namely a mixture of 6,6'-bis corynomycoloyl)-sc-D-trehaloses (compound B1 in Fig. 1) Ahhreviotion. Me3Si, trimethylsilyl. differing by the chain length and the unsaturation degree, was presented elsewhere [4].
MATERIALS AND METHODS
Physical MethodsThe infrared spectra were obtained with a Perkin Elmer 177 instrument.The mass spectra were measured on a Varian MAT 31 1A mass spectrometer equipped with a combined electron impact/field ionization/field desorption ion source. For field desorption measurements, the cationization method with caesium iodide [5] was used, by the two-step procedure : a carbon-activated tungstenwire emitter was first dipped into a 0.5 ...