Structural elucidation of a novel family of acyltrehaloses from Mycobacterium tuberculosis

Analysis of the lipids of Mycobacterium tuberculosis H37Rv, by both normal- and reverse-phase thin-layer chromatography, revealed a series of novel glycolipids based on 2,3-di-O-acyltrehalose. The structures of these acylated trehaloses were elucidated by a combination of gas chromatography-mass spectrometry, 1H, 13C, two-dimensional 1H-1H, and 1H-13C nuclear magnetic resonance spectrometry. The fatty acyl substituents were mainly of three types: saturated straight-chain C16-C19 acids; C21-C25 "mycosanoic acids"; and C24-C28 "mycolipanolic acids." Analysis of one of the major 2,3-di-O-acyltrehaloses by two-dimensional 1H-chemical shift correlated and 1H-detected heteronuclear multiple-bond correlation spectroscopy established that the C18 saturated straight-chain acyl group was located at the 2 position and that the C24 mycosanoyl substituent was at the 3 position of the same "right-hand" glucosyl residue. At least six molecular species differing only in their fatty acid content comprised this family of di-O-acylated trehaloses. We regard these acyltrehaloses as elemental forms of the multiglycosylated acyltrehaloses (the lipooligosaccharides) perhaps due to an inability of the majority of isolates of virulent tubercle bacilli to glycosylate core acyltrehaloses. The acyltrehaloses are minor but consistent components of virulent M. tuberculosis and apparently the basis of the specific serological activity long associated with its lipid fractions.