Scalar, dipolar‐correlated and J‐resolved 2D‐NMR spectroscopy of the specific phenolic mycoside of Mycobacterium tuberculosis

Abstract

Two-dimensional chemical shift correlated (COSY>. nuclear Overhauser (NOESYj and J-resolved spectroscopy were used to determine the complete structure of the major triglycosyl dimycocerosyl phenol phthiocerol of the tubercle bacillus (strain Canctti) without any other analytical technique. The COSY spectrum of the native glycolipid allowed the composition of the trisaccharide and the location of one methoxyl group to be determined through the assignment of the resonances of the non-anomeric methine protons. Information with respect to the configuration of the sugar residues, the pyranose structure and the linkage sites of the trisaccharide were obtained by the analysis of the COSY spectrum of the peracetylated glycolipid. The NOESY spectrum confirmed the linkage sites through the inter-residue connectivity across glycosidic linkages and allowed the determination of the sequence of the oligosaccharidc and the configuration of its sugar residues from the through-space connectivities. The J-resolved spectroscopy was used to elucidate thc structure of the glycolipid and gave additional data showing the presence of 11 non-equivalent methyl groups and allowing the recognition of two other analogucs of the phenol phthiocerol derivative in the mixture. Thus, the non-destructive 2D-NMR spectroscopy was found to be satisfactory for the analysis of mycobacterial mycosides.Most mycobacteria are endowed with large quantities of glycolipids which contain within them small oligosaccharides of sufficient antigenicity to evoke antibodies of such exquisite specificity as to allow unequivocal identification of species or subspecies [l]. This concept w-as originally formulated by Smith et al. who gave the name 'mycosides' to type-specific glycolipids of mycobacteria limited in distribution to a single species or organism 121. These specific components are of three classes: the C-mycosides (glycopeptidolipidsj, the treh, '1 1 osecontaining lipooligosaccharides, and the phenolic glycolipids To determine the primary structure of the carbohydrate moiety ol' these glycolipids, modern spectroscopic techniques, in conjunction with elaborate derivatization of substances, have been widely used. To date, no secondary structural information (conformation), which is important for understanding thc biological activities of these substances, has been publishcrl. Furthermore, proton nuclear magnetic resonance ('€1-NMK) spectroscopy, which is very suitable for providing primary and secondary structural information on glycolipids [3], yielded only fragmentary data (composition and configuration of sugar residues) in studies on mycosides.Recently, we elucidated the complete primary structure of NMR and by application of a combination of compositional analysis, mass spectrometry and chemical degradations [4, 51. The spin-decoupling method in one-dimensional NMR was found to be a powerful method for assigning the rhamnosyl ring H-1 to H-6 chemical shifts, and for analyzing the linkage sites using peracetylated glycolipids [4, 51. However, the assign...

Analysis of the Phthiocerol Dimycocerosate Locus ofMycobacterium tuberculosis

“…The additional signal observed at 1.05 ppm in the spectrum of the compound with the lowest mobility on TLC corresponded to proton resonance of a methyl group in the ␣-position of a keto function (Fig. 4, signal g) (21). Altogether the NMR data clearly established that the pairs of lipid compounds examined belong to the DIM family and that both types of DIM (A and B) were produced by the three strains.…”

“…Proton assignments were validated previously by two-dimensional chemical shift-correlated and J-resolved spectroscopy (21). In addition to the broad signal resonance attributable to the polymethylenic (CH 2 ) protons (at 1.25 ppm), several terminal methyl (CH 3 ) proton resonances were detected at 0.8 -1.0 ppm (Fig.…”

“…The presence of a doublet at 1.15 ppm indicated the occurrence of a methyl group in the ␣-position of the fatty acyl residues (Fig. 4, signal f) (21); the proton that substituted at this position was detected at 2.55 ppm (Fig. 4, signal d).…”

Chemical characterization of the ester-linked 3-hydroxy fatty acyl-containing lipids in Mycobacterium tuberculosis

“…Two-dimensional chemical-shift correlated spectroscopy data were obtained by using the previously described parameters (12). MS was performed on a ZAB-MS reverse geometry mass spectrometer (VG Analytical).…”

NMR spectroscopic identification of cholesterol esters, plasmalogen and phenolic glycolipids as fingerprint markers of human intracranial tuberculomas

“…Moreover, breakdown of mycolates results in liberation of the free gaseous form of cyclopropane, which was present in 1 H NMR spectra of cerebrospinal fluid samples from patients with tuberculous meningitis (48). Mycobacterium species are known to possess various forms of phenolic glycolipids as integral membrane structural components (49,50), and may have a role in the inhibition of phagocytosis by macrophages (51). Tissue destruction during TB infection often leads to cheese-like formation of necrotic tissue (caseation) caused by membrane rupture of MTB during phagocytosis, which is unique to tuberculomas.…”