Mycobacterium alvei (ω-1)-methoxy mycolic acids: Absolute stereochemistry and synthesis.

Cells of Mycobacterium alvei are known to contain a unique set of mycolic acids with a (ω-1)-methoxy group; although the various enzymes in the biosynthesis of other types of mycolic acid have been widely studied, the biosynthetic route to this substituent is unclear. We now define the stereochemistry of the (ω-1)-methoxy fragment as R, and describe the synthesis of a major R-(ω-1)-methoxy-mycolic acid and its sugar esters, and of two natural M. alvei diene mycolic acids.

Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters ("cord factor") form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram-scale synthesis of four stereo-isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn-methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.

First access to a mycolic acid-based bioorthogonal reporter for the study of the mycomembrane and mycoloyltransferases in Corynebacteria.

In this study, we report the first synthesis of an alkyne-based trehalose monomycolate probe containing a ß-hydroxylated fatty acid and an α-branched chain similar to those of the natural mycolic acid. We demonstrate its utility for the labeling of the mycomembrane of Corynebacteria as well as for the study of mycoloyltransferases.

The synthesis of mycobacterial dimycoloyl diarabinoglycerol based on defined synthetic mycolic acids.

Complex mixtures of natural dimycoloyl diarabinoglycerols isolated from mycobacteria have been shown to be both potent immune signalling agents and potentially valuable antigens in the serodiagnosis of mycobacterial infections. We now report the highly stereocontrolled synthesis of diacyl l-glycerol-(1'→1)-ß-d-arabinofuranosyl-α-d-arabinofuranosides based on simple fatty acids and single defined synthetic mycolic acids. NMR analysis confirmed that the synthetic core was identical to that in natural mixtures.

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.

Glycerol mycolates from synthetic mycolic acids.

R- and S-Glycerol mycolates derived from single synthetic α-, keto- and methoxy-mycolic acids are described.

Mycolates of Mycobacterium tuberculosis modulate the flow of cholesterol for bacillary proliferation in murine macrophages.

The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB.

Synthesis and Monolayer Behaviors of Novel Hybrid Corynomycolic Acids Containing Semifluoroalkyl Groups.

In this work, novel hybrid-type corynomycolic acids [hybrid-OH and hybrid-COOH, with semifluoroalkyl groups (Rf-(CH2)n-: Rf = C4F9, n = 6 and Rf = C6F13, n = 3) located on the carbon atoms attached to the hydroxyl and carboxylic acid groups (C-OH and C-COOH), respectively] were successfully synthesized. The behaviors and formation of hybrid corynomycolic acid monolayers at the air-water interface were investigated by surface tension and surface pressure-area (π-A) measurements to clarify the effects of the Rf chain length, position of the semifluoroalkyl group, and surfactant molecule stereochemistry. Compared to dialkyl corynomycolic acid, both the critical micelle concentration (CMC) and the surface tension at the CMC (γCMC) of hybrid corynomycolic acids were reduced by the presence of the Rf group. With respect to the surface tension versus log concentration (γ vs. log C) isotherms, all syn-isomers of the hybrid-OH and hybrid-COOH acids showed two break points, while the anti-isomers showed only one break point. These different isotherms can be explained in terms of the steric repulsion between the two hydrophilic groups (OH and COO(-)), which depend on the stereochemistry of the surfactant. No effect of the location of the semifluoroalkyl group was observed. With respect to the formation of a monolayer film, four parameters-the lift-off area (AL), zero-pressure molecular area (A0), maximum of the Gibbs elastic modulus [EG (max)], and monolayer collapse pressure (πc)-were measured. Both AL and A0 of all hybrid corynomycolic acids were larger than the corresponding dialkyl acids due to the bulky and rigid Rf groups. Interestingly, syn- and anti-hybrids had almost identical isotherms on compression, although the values of πc of anti-hybrids were higher than those of syn-isomers. In addition, the values of EG (max) of hybrid-COOHs were slightly larger than those of the corresponding hybrid-OHs. Using the nascent soap method (agent-in-oil method), we found that anti-F4C6-OH (a hybrid corynomycolic acid) is a promising emulsifier for a ternary system comprising octane, water, and perfluoropolyether oil.

Thiol modified mycolic acids.

Patient serum antibodies to mycolic acids have the potential to be surrogate markers of active tuberculosis (TB) when they can be distinguished from the ubiquitously present cross-reactive antibodies to cholesterol. Mycolic acids are known to interact more strongly with antibodies present in the serum of patients with active TB than in patients with latent TB or no TB. Examples of single stereoisomers of mycolic acids with chain lengths corresponding to major homologues of those present in Mycobacterium tuberculosis have now been synthesised with a sulfur substituent on the terminal position of the α-chain; initial studies have established that one of these binds to a gold electrode surface, offering the potential to develop second generation sensors for diagnostic patient antibody detection.

Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis.

Mycolic acids constitute the waxy layer of the outer cell wall of Mycobacterium spp. and a few other genera. They are diverse in structure, providing a unique chromatographic foot-print for almost each of the more than 70 Mycobacterium species. Although mainly esterified to cell wall arabinogalactan, trehalose or glucose, some free mycolic acid is secreted during in vitro growth of Mycobacterium tuberculosis. In M. tuberculosis, α-, keto- and methoxy-mycolic acids are the main classes, each differing in their ability to attract neutrophils, induce foamy macrophages or adopt an antigenic structure for antibody recognition. Of interest is their particular relationship to cholesterol, discovered by their ability to attract cholesterol, to bind Amphotericin B or to be recognised by monoclonal antibodies that cross-react with cholesterol. The structural elements that determine this diverse functionality include the carboxylic acid in the mycolic motif, as well as the nature and stereochemistry of the two functional groups in the merochain. The functional diversity of mycolic acid classes implies that much information may be contained in the selective expression and secretion of mycolic acids to establish tuberculosis after infection of the host. Their cholesteroid nature may relate to how they utilize host cholesterol for their persistent survival.

Structure-function relationships of the antigenicity of mycolic acids in tuberculosis patients.

Cell wall mycolic acids (MA) from Mycobacterium tuberculosis (M.tb) are CD1b presented antigens that can be used to detect antibodies as surrogate markers of active TB, even in HIV coinfected patients. The use of the complex mixtures of natural MA is complicated by an apparent antibody cross-reactivity with cholesterol. Here firstly we report three recombinant monoclonal scFv antibody fragments in the chicken germ-line antibody repertoire, which demonstrate the possibilities for cross-reactivity: the first recognized both cholesterol and mycolic acids, the second mycolic acids but not cholesterol, and the third cholesterol but not mycolic acids. Secondly, MA structure is experimentally interrogated to try to understand the cross-reactivity. Unique synthetic mycolic acids representative of the three main functional classes show varying antigenicity against human TB patient sera, depending on the functional groups present and on their stereochemistry. Oxygenated (methoxy- and keto-) mycolic acid was found to be more antigenic than alpha-mycolic acids. Synthetic methoxy-mycolic acids were the most antigenic, one containing a trans-cyclopropane apparently being somewhat more antigenic than the natural mixture. Trans-cyclopropane-containing keto- and hydroxy-mycolic acids were also found to be the most antigenic among each of these classes. However, none of the individual synthetic mycolic acids significantly and reproducibly distinguished the pooled serum of TB positive patients from that of TB negative patients better than the natural mixture of MA. This argues against the potential to improve the specificity of serodiagnosis of TB with a defined single synthetic mycolic acid antigen from this set, although sensitivity may be facilitated by using a synthetic methoxy-mycolic acid.

The synthesis of a major alpha'-mycolic acid of Mycobacterium smegmatis.

The synthesis of (2R,3R,Z)-2-docosyl-3-hydroxytetracont-21-enoic acid, a significant alpha'-mycolic acid of Mycobacterium smegmatis and other mycobacteria is reported.

Synthesis of a tetrasaccharide phosphate from the linkage region of the arabinogalactan-peptidoglycan complex in the mycobacterial cell wall.

The synthesis of dibenzyl 6-O-naphthylmethyl-2,3,5-tri-O-benzoyl-beta-D-galactofuranosyl-(1-->5)-2,3-di-O-benzoyl-6-O-benzyl-beta-D-galactofuranosyl-(1-->4)-3-O-benzyl-2-O-pivaloyl-alpha-l-rhamnopyranosyl-(1-->3)-2-acetamido-2-deoxy-4,6-di-O-benzoyl-alpha-D-glucopyranosyl phosphate (1), a protected form of the tetrasaccharide phosphate of the linkage region of the arabinogalactan-peptidoglycan complex in the mycobacterial cell wall, has been accomplished. Key steps include the coupling of four monosaccharide building blocks with complete stereoselectivity by glycosylations employing thioglycosides, 2'-carboxybenzyl glycosides, and glycosyl fluorides as glycosyl donors. The alpha-glycosyl phosphate linkage was also stereoselectively elaborated by reaction of a tetrasaccharide hemiacetal with tetrabenzyl pyrophosphate in the presence of a base.

Mycobacterium tuberculosis beta-ketoacyl-ACP reductase: alpha-secondary kinetic isotope effects and kinetic and equilibrium mechanisms of substrate binding.

Beta-ketoacyl-ACP reductase catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein to generate beta-hydroxyacyl-acyl carrier protein and NADP+, the second step of the fatty acid elongation system type II of bacteria, plants, and apicomplexan organisms. Here, a modified and more efficient purification protocol is reported for recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP reductase (MabA). The increase in alpha-secondary deuterium kinetic isotope effect values measured at pH 10 as compared to those obtained at pH 7 points to isotope- and pH-sensitive steps occurring concomitantly. Equilibrium and kinetic fluorescence studies demonstrate positive cooperativity in binding of NADPH to MabA, with two forms of free enzyme in solution. Equilibrium dialysis shows no cooperativity in acetoacetyl-CoA binding to the enzyme. Moreover, modest affinity loss occurs when the substrates bind to the monomer as compared to the dimer of MabA. A mechanism of substrate binding to MabA is proposed on the basis of the experimental data.

A novel synthesis of succinic acid type Gemini surfactant by the functional group interconversion of Corynomicolic acid.

2,3-Bis(undecyl)succinic acid, succinic acid type Gemini surfactant, was successfully synthesized from Corynomicolic acid by the functional interconversion of OH of Corynomicolic acid to COOH via (1) mesylation of OH of syn-isomer, (2) elimination of methanesulfonate group using base to E-alpha,beta-unsaturated ester, (3) Michael addition of CN(-) with 18-crown-6 and (4) hydrolysis. In most hydrolysis conditions, a mixture of syn- and anti-2,3-bis(undecyl)succinic acid was obtained, but hydrolysis in 75% H(2)SO(4) gave only anti-2,3-bis(undecyl)succinic anhydride. After converting a mixture of syn- and anti-2,3-bis(undecyl)succinic acids to corresponding acid anhydrides, syn- and anti-anhydride isomers were separated by column chromatography, and by following hydrolysis syn- and anti-2,3-bis(undecyl)succinic acid was selectively prepared. Based on the surface tension measurement, the effect of stereochemistry on surface tension isotherms was discussed in terms of the hydrophobic interaction between two alkyl groups and the electrostatic repulsion between two hydrophilic COO(-) groups.

Effect of the synthesized mycolic acid on the biodegradation of diesel oil by Gordonia nitida strain LE31.

The dynamics of diesel oil biodegradation were previously investigated at initial substrate concentrations of 1000 to 20,000 ppm using Gordonia nitida isolated from wastewater. Following the gas chromatogram profiles of diesel oil degradation, diesel oil with concentrations of up to 15,000 ppm was efficiently degraded by this strain. At a concentrations of 20,000 ppm, however, the degradation by this strain was not effective. The enhancement of the biodegradation of diesel oi1 (at 15,000 and 20,000 ppm) by a synthetic mycolic acid biosurfactant (at 9, 90 and 900 ppm) was also investigated. In G. nitida inoculated cultures, the degradation of diesel oil was enhanced by the biosurfactant. For comparison, diesel oil degradation in batch incubations was measured after the addition of rhamnolipid and other surfactants. Synthetic mycolic acid enhanced the degradation to a greater extent than any other surfactant tested. Additionally, it was demonstrated that the degradation-enhancing property of synthetic mycolic acid was similar to that of rhamnolipid and Tween 80.

Synthesis and properties of methyl 5-(1'R,2'S)-(2-octadecylcycloprop-1-yl)pentanoate and other omega-19 chiral cyclopropane fatty acids and esters related to mycobacterial mycolic acids.

A 23-26-carbon chain length range of omega-19 (1'R,2'S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1'R,2'S)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated omega-19 cyclopropane fatty acids or esters. The 24-carbon omega-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2-3%) was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38-55%) stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon acids.

The synthesis of a single enantiomer of a major alpha-mycolic acid of Mycobacterium tuberculosis.

We report a synthesis of a single enantiomer of a dicyclopropane containing mycolic acid from Mycobacterium tuberculosis; this method can be simply varied to modify the chain lengths or the absolute stereochemistry of either cyclopropane.

Acción del Agua del Volcán Copahue (Neuquén, Argentina) sobre la morfología de las microbacterias / Action of the Copahue Volcano (Neuquén, Argentine) water of mycobateria morphology

Se efectuó un estudio de la acción del Agua del Volcán Copahue (AVC), Neuquén, Argentina, sobre 15 cepas de microbacterias Mycobacterium tuberculosis: M. bovis y los mycobacterum no tuberculosos ("Atipicos"), poniendo especial interés en los que forman "cuerdas". Se utilizó AVC con su pH l,3 y se la llevó a pH 6,5. En los bacilos que resistieron la acción del agua hasta el momento de la coloración, se advirtieron elementos más o menos alterados. Al aumentar el tiempo de contacto se llegó a la destrucción total, observándose en algunos casos muy pocos bacilos aislados, material deteriorado y formas granulares. Estas alteraciones fueron mucho más marcadas en las suspensiones que en el líquido del sedimento con el AVC; en las primeras directamente no se llegó a reparar la formación de cuerdas en ningun momento aun en bacilos que deberían formarla. Su acción no estaría asociada a una reacción bioquímica responsable en la síntesis de la pared celular, como la transpeptidación. El AVC actuaría sobre la síntesis del ácido micólico y se trataría de un agente bacteriolítico. Además se realizaron estudios en el "Laboratorio de tratamiento de imágenes", INEUCI (Instituto de Neurociencia), CONICET