The first syntheses of single enantiomers of the major methoxymycolic acid of Mycobacterium tuberculosis

Mycolic acids (Scheme 1) are major constituents of the cell envelope of Mycobacterium tuberculosis and other mycobacteria, some of which are pathogenic to animals and humans.1 and 2 Their presence is thought to be linked with the resistance of these organisms to most current antibiotics and other chemotherapeutic agents.3 Mycolic acids can be divided into two parts, meromycolate and mycolic motif (Scheme 1). The latter is common to each mycobacterial mycolic acid, except for minor variations in the length of the chain(d)We thank Prof. D. E. Minnikin (Univ. of Birmingham) for providing a sample of natural methoxymycolic acid extracted from M. tuberculosis

The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies

Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto- MA, each with specific biological functions and conformational characteristics. Antibodies in tuberculosis (TB) patient sera respond differently towards the three MA classes and were reported to cross-react with cholesterol. To understand the antigenicity and cholesterol cross-reactivity of MA, we generated three different chicken -derived phage-displayed single- chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA, but the first recognized all three classes of chemically synthetic MAs, the second only the two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity was investigated after grafting each of the three scFv types onto two configurations of constant chain domains±CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol was found only with CH2-4 versions, notably those two that were also able to recognize the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore seems to be determined by the trans-keto MA subclass. The significantly weaker binding to cholesterol in comparison to MA confirms the potential TB diagnostic application of these antibodies.S1 Fig. Sequences of gallibody clones produced by antibody engineering. 12) Anti-MA 12, 16) Anti-MA 16, 18) Anti-MA 18, CH1-4 = full length constant region, CH2-4 = truncated constant region, VH = variable heavy chain, VL = variable light chain.S2 Fig. SDS-PAGE analysis illustrating gallibody purification using Ni-NTA affinity columns. A) 12CH1-4, B) 16CH1-4, C) 18CH1-4, D) 12CH2-4, E) 16CH2-4, F) 18CH2-4. Gel lanes 1) Marker, 2) Culture supernatant, 3) Flow through 1, 4) Flow through 2, 5) Washes, 6) Elution 1, 7) Elution 2, 8) Elution 3, 9) Elution 4. Successful purification is demonstrated by the comparable thickness of the 67 kDa band obtained with the culture supernatant (2) and the elutions (6±9).S1 Dataset. Experimental data used for producing Figs 3 and 4.S2 Dataset. Experimental data used for producing Fig 5.S3 Dataset. Experimental data used for producing Fig 6.The Council for Scientific and Industrial Research (CSIR) parliamentary grants (YL) and the National Research Foundation of South Africa for the grants, unique grant numbers: 99386 (HR), 88622, 80577 (YL) and TTK1206281756 (LN).http://www.plosone.orgam2018Biochemistr

Synthesis of Complex Sugar Mycolates of Mycobacterium Tuberculosis

Mycobacteria are present in many environments and complex mixtures of sugar esters and mycolic acids are present in their cell wall structure. This complicated mixture is thought to be responsible for their high resistance to known antibiotics and chemotherapeutic treatments. Mycolic acids are high molecular weight α-alkyl-branched β-hydroxy long-chain fatty acids, have 60-90 carbon atoms, and various classes of mycolic acids are made by different species of mycobacteria. Sugar esters of mycolic acids associated with the cell wall of mycobacteria have very interesting toxic and immunological properties, and thus could be useful for the control and treatment of mycobacterial infections. The main objectives of this thesis will be discussed in three parts. The main target of the first part involved the first synthesis of a single enantiomer of the glycolipid di-mycolyl di-arabino glycerol (DMAG) (I), which has interesting toxicological and immunological properties. This was achieved by a successful synthesis of the glycan moiety of DMAG with the L-stereochemistry of the glycerol component, followed by the successful esterification of the glycan di-arabino glycerol with three normal fatty acids, a model mycolic acid, and five different mycolic acids. The NMR spectra of the synthetic isomer of the DMAG penta-acetate analogue, in the sugar region, matched very well those reported for the peracetate formed from the natural mixtures, confirming the stereochemistry of the arabinose units and establishing the absolute stereochemistry of the glycerol unit. An efficient route to prepare the DMAG glycan with excellent β-selectivity and in excellent yield was achieved. The second part entailed the first preparation of di-mycolyl tri-arabino glycerol (DMTAG) (II), which involved the synthesis of the donor moiety part according to literature methods with slight modifications, and the new arabino glycerol acceptor. The coupling of the donor and the acceptor to prepare the desired glycan was carried out using known coupling conditions. After the success in synthesising this tetra-saccharide, a model glycolipid was prepared through esterification with a normal fatty acid. Furthermore, a series of three DMTAG compounds were prepared, based on two common classes of mycolic acids. The final part of this project was the synthesis of glycerol mycolates (GroMM) (III), which have interesting adjuvant properties in vaccines, by coupling five common classes of synthetic mycolic acids with the S-glycerol stereoisomer. One model GroMM was prepared from a simple fatty acid. These compounds were prepared to study whether the stereochemistry of the glycerol component (R & S) has any effect on their biological activities. Initial studies of the biological activity of the synthetic DMAGs showed that some could be used to distinguish serum from cattle infected with bovine TB from uninfected cattle, and that they selectively activate THP-1 cells. In contrast, initial ELISA results with the synthetic GroMM showed little response to serum from patients with active TB