Trehalose from cord factor

In order to determine which of the hydroxyl groups of trehalose are es-terified with mycolic acid, cord factor was methylated and the methylated cord-factor was then saponified the hexa-O-methyltrehalose thus obtained was hydrolyzed by dilute acid, and from this, a tri-O-methyl-D-glucose resulted which gave only one spot on paper chromatograms this proved that cord factor is symmetrical, that is, that, on each D-glucose moiety, the mycolic acid is esterified with the same-numbered hydroxyl group. 

The deacetylation of this product with sodium methoxide or hydroxyl-amine (mycolic acid esters react very slowly with hydroxylamine) gives, in rather low yield, a 6,6 -di-0-(3-hydroxy-x-methoxymycolanoyl)trehalose, m.p. 40-42°, [a]D -f 132°, CissHsvoOi 10 CH2 (9b), with an infrared spectrum identical with that of the natural cord-factor the biological activity of this compound was indistinguishable from that of natural cord-factor. ... 

Cord Factor Trehalose 6,6-dimycolaie 6.6 -di-0-mycolyl -a,a-trehalose (6-O-mycolyl -dr-D-glucopyranos-yl) 6-0 mycolyl-ot-D-glucopyranoside Toxic glycolipjds responsible for the cord formation and the leukotoxic effect of virulent bacilli. The term cord factor is widely used for the natural mixture of trehalose dimycolates produced by virulent Mycobacteria, Nocardia, Corynebacteria and attenuated BCG, q.v. For precise designation, the strain from which the preparation was isolated must be mentioned. First isolated from Mycobacterium tuberculosis H. Bloch, J. Exp. 

The presence of trehalase in the tubercle bacillus was discovered by Bloch and SuUman in 1945. Optimal hydrolysis of a,a-trehalose by the enzyme occurred in acid to neutral solutions. Since a,a-trehalose is not utilized by the bacteria so rapidly as is n-glucose, it was inferred that the bacillus only metabolizes the a, -trehalose after the trehalase has converted it into D-glucose. Trehalosamine (see p. 220) has an antimycobac-terial effect. This effect is antagonized by a,a-trehalose. One mole of trehalosamine as inhibitor removes 0.337 mole of Q ,a-trehalose from the surface of the enzyme. Total inhibition of mycobacterial trehalase is not readily produced. Cord factor was also isolated from wax D of a BCG strain of M. tuberculosis in 1959 by Nojima. A list of fatty acid esters of a, a-trehalose is given in Table I. 

Disaccharides to have been examined are two D-ribofuranosyl-D-ribitols produced from two bacterial capsular polysaccharides, and several aa-trehalose derivatives prepared as model bacterial cord factors [F.D. cationization with cesium iodide gave intense (M + Cs) ions]. The same group of workers have also reported on this method applied to the natural cord factor of Corynebacterium diphtheriae, and they found at least 22 species differing in the chain lengths of the two trehalose acyl substituents. A further report describes the determination of the sites of substitution of the acyl groups.Daunosamine-containing disaccharides have been examined by negative ion, positive ion, chemical ionization, and electron-impact methods. 

The cord factor from Mycobacterium tuberculosum could be permethylated with methyl iodide and sodium hydride in a diethyl ether-dimethylformamide mixture in the presence of a molecular sieve.It was confirmed that the mycolic acids were esterified at the 6,6 positions of the aa-trehalose molecule. Using 4-palmitoyl-D-glucose as a model compound, it was verified that acyl migration did not occur in this system. 

The cell walls of M. lepraemurium have been harvested from the livers of moribund rats that had been infected with the above organism. The walls contained a cord factor (oa-trehalose-6,6-dimycolate) that was similar in structure to the well-characterized cord factor from M. tuberculosumThis is the first unequivocal identification of a cord factor as a product of in vivo derived mycobacteria. 

The purification procedure of 6,6 -esters of OMf-trehalose from Coryne-bacterium diphtheria has been modified and the products were analysed by m.s. of the permethylated derivative.The constituent fatty acids have been extensively examined and the detailed structure of the major acids have been confirmed by a variety of techniques. Novel glycolipids in the cord factor fraction from C. diphtheria were a 6,6 -bis(3-oxo-acyl)-o a -D-trehalose and a 6, 6 -mixed ester of aa-trehalose with the 3-oxo-acyl group and a corynomycolic acid. The metabolic implications of these structural studies suggest that condensation of the common fatty acids by a Claisen-like process via the oxo-ester pathway may not be the only pathway for the synthesis of the cord factors. 

In the permethylatlon analysis of trehalose glycolipids such as cord factor and 6- -mycolyl-a,a-trehalose, various standard reagents proved unsatisfactory due to ester cleavage, but the method of Ciucanu and Kerek (Vol.18, p.50 powdered NaOH-Mel-DMSO) gave excellent results, only lower acyl groups such as acetyl being cleaved. 6,6 -Di- -methyl-a,a-trehalose was obtained from the... 

An important example of a carbohydrate ester is the so-called cord factor from Mycobacteria spp. This contains an ester of the disaccharide, trehalose, with two molecules of a complex acid, mycolic acid. The latter is a general term embracing a whole series of fatty acids containing 60-90 carbons. They are hydroxy fatty acids which differ in their degree of unsaturation and chain branching (Section 1.9). In the example given in Fig. 2.6 the mycolic acid is the 60-carbon compound found in Mycobacterium smeg-matis. 

The structure of the cord factor from Mycobacterium smegmatis has been confirmed as (j ,a trehalose 6,6 -dimycolate (CieeHajsOis). The a,a trehalose was identified by paper chromatography and the positions of ester linkages at 0-6 and 0-6 was established by C n.m.r. spectroscopy. 

In addition to the wall-bound mycolic acids three other important lipids are loosely associated with the murein layer. The st of these is the cord factor , so called since it was associated with the formation of cords , parallel rows forming characteristic serpentine strands demonstrable when smears of liquid grown cultures of mycobacteria are examined under the microscope. The cord factor of M. tuberculosis can be readily extracted from viable bacteria with organic solvents and identified as trehalose-6,6 -dimycolate. The mycosides constitute the second group of readily extractable lipids, gycosidically linked to the para position of a phenol... 

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