Chemical synthesis of disaccharides of the specific phenolic glycolipid antigens from Mycobacterium leprae and of related sugars

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3,-di-O-methyl-L -rhamnopyranose, which is the nonreducing disaccharide of the haptenic trisaccharide of the Mycobacterium leprae-specific, phenolic glycolipid I, O-(6-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamn opyranose, the nonreducing end of the specific, phenolic glycolipid III, and the nonhaptenic O-beta-(D-glucopyranosyl)-(1----4)-2,3-di-O-methyl-L-rhamnopyranose++ +, were synthesized in relatively good yield from 3-O-methyl-D-glucose, or D-glucose, and L-rhamnose via Koenigs-Knorr reactions. These disaccharides can be used as precursors in the synthesis of the trisaccharide unit of phenolic glycolipid I and of neoglycoconjugates suitable for the serodiagnosis of leprosy.     

Location of acyl groups of trehalose-containing lipooligosaccharides of mycobacteria

A variant of a Mycobacterium sp. originating in a patient with Crohn's disease, but not necessarily implicated in the disease, provided a simple version of a newer class of species-specific surface glycolipids, the trehalose-containing lipooligosaccharides. A combination of high-resolution 1H nuclear magnetic resonance, methylation, ethylation, and absolute configurational analysis established the structure of the oligosaccharide unit as beta-D-Glcp(1----3)-alpha-L-Rhap(1----3)-alpha-D-Glcp(1----1)-alph a-D-Glcp (where Glc is glucose, Rha is rhamnose, and p is pyranosyl), and gas chromatography-electron impact mass spectrometry allowed identification of the fatty acyl esters as primarily 2,4-dimethyltetradecanoate. The relative simplicity of the glycolipid combined with the application of a mild methylation procedure and californium-252 plasma desorption mass spectrometry allowed recognition of three such acyl residues on the 3-, 4-, and 6-hydroxyl positions of the terminal glucosyl residue of the trehalose unit. Thus, the glycolipid is decidedly amphipathic yet is clearly not membranous. This observation leads to speculation about the role of these novel lipooligosaccharides in contributing to the outer segment of the hydrophobic barrier of the cell wall of certain mycobacteria.     

Structure and antigenicity of the major specific glycolipid antigen of Mycobacterium leprae

Earlier we reported on the presence of a specific phenolic glycolipid (Phenolic Glycolipid-I) in Mycobacterium leprae, and in infected armadillo tissues (Hunter, S. W., and Brennan, P. J. (1981) J. Bacteriol. 147, 728-735). It had an inherent oligosaccharide, composed of 3-O-Me-rhamnose, 2,3-di-O-Me-rhamnose, and 3,6-di-O-Me-glucose, glycosidically linked to the phenol substituent. The structure of the oligosaccharide has now been determined, by partial acid hydrolysis, permethylation, 1H NMR, and 13C NMR as: 3,6-di-O-Me-Glcp(1 beta leads to 4)2,3-di-O-Me-Rhap(1 alpha leads to 2)3-O-Me-Rhap1 alpha leads to phenol (assuming that the glucose substituent is in the D-enantiomeric configuration, and the two methylated rhamnoses are L). Acid hydrolysis of deacylated Phenolic glycolipid-I yielded a phenolic phthiocerol "core," and mass spectrometry and proton NMR of the permethylated core suggested the following structure: (formula, see text) Combined gas-liquid chromatography-mass spectrometry showed three tetramethyl branched "mycocerosic" acids, C30, C32 and C34, with molecular weights (as methyl esters) of 466, 494, and 522, respectively. These are esterified to the hydroxyl functions of the branched glycolic chain. Evidence is also presented that the glycolipid is immunologically active, reacting with rabbit antisera to M. leprae and with sera from lepromatous leprosy patients.     

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.     

Chemical synthesis of the trisaccharide unit of the species-specific phenolic glycolipid from Mycobacterium leprae

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, the haptenic trisaccharide of the Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) antigen, and related trisaccharides, were synthesized by allylation of O-2 of benzyl 4-O-benzyl-alpha-L-rhamnopyranoside using phase-transfer catalysis, methylation of the product, deallylation, and coupling to O-(2,4-di-O-acetyl-3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3- di-O-methyl-L-rhamnopyranosyl bromide or related disaccharides. Anomeric mixtures of the trisaccharide derivatives were separated by preparative t.l.c., deacetylated, and hydrogenolyzed, to give the pure trisaccharides. It had already been demonstrated that only those trisaccharides containing an intact, terminal 3,6-di-O-methyl-beta-D-glucopyranosyl unit are effective in inhibiting the specific binding between PGL-I and anti-PGL-I immunoglobulin M antibodies in human lepromatous leprosy sera.     

A characteristic phenolic glycolipid antigen from Mycobacterium haemophilum

A characteristic phenolic glycolipid was detected, by thin layer chromatography, in non-polar lipid extracts of nine representative strains of Mycobacterium haemophilum . The lipid was a specific antigen that reacted strongly with serum raised against whole cells of M. haemophilum. Sera from six other mycobacterial strains were tested but only that from Mycobacterium kansasii give a weak reaction.     

New-found phenolic glycolipids in Mycobacterium bovis BCG. Presence of a diglycosylated glycolipid

A crude phenolic glycolipid extract from Mycobacterium bovis bacille Calmette-Guerin (BCG) was fractionated until homogeneity at the intact level into four phenolic glycolipids called B, B-1, B-2, and B-3 according to their polarity. The apolar one, which is the most abundant was assigned to the well-known mycoside B. The B-2 and B-3 phenolic glycolipids were purified by direct-phase high performance liquid chromatography using a 5 micron Spherisorb column but were only recovered in small amounts (3 mg). A linear gradient of 0-20% methanol in chloroform was used. The B-1, B-2, and B-3 glycolipids were subjected to suitable modern analytical techniques selected for their potential to elucidate the structure at the intact level. Desorption chemical ionization-mass spectrometry allowed the molecular mass of B-3 to be determined as 1652 Da for the major homolog establishing the molecular formula as C103H192O14. Thus, the B-3 polar phenolic glycolipid contained two deoxyhexoses, one molecule of phenolphthiocerol esterified by two molecules of mycocerosic acid. Using two-dimensional 1H NMR (correlated chemical shift and nuclear Overhauser effect spectroscopy) at the intact level the B-3 oligosaccharide structure was determined as an alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap. This is the first report of a diglycosylated phenolic glycolipid in a nonpathogenic mycobacteria. The disaccharide unit, the antigenic determinant, appears to be characteristic of M. bovis BCG. This polar glycolipid B-3 and the apolar ones, B-1 and B-2, were reactive in enzyme-linked immunosorbent assay against serum from rabbit hyperimmunized with M. bovis BCG.     

Stereochemistry of 2,3-alkanediols obtained from the Harderian gland of Mongolian gerbil (Meriones unguiculatus)

The stereochemistry of the alcohol moieties of 2,3-alkanediol diacyl esters obtained from the Harderian gland of the Mongolian gerbil was investigated. There were five major 2,3-alkanediols, C14-C22 (even carbon numbers), all having the erythro configuration as determined by GC-MS analysis of their isopropylidene derivatives in comparison with synthetic erythro- and threo-2,3-hexadecanediols. 13C-NMR spectroscopy of the synthetic materials showed distinct differences of chemical shift at the C-1, C-3, and C-4 carbons, from which the native 2,3-alkanediols were definitely determined to be in the erythro series. The absolute configurations of the C-2 and C-3 asymmetric centers were assigned as 2S and 3R, respectively, based on known 2S,3R-octanediol.     

Absolute stereochemistry of the strevertenes

The CD exciton chirality method was applied to determine the absolute stereochemistry of the strevertenes, antifungal pentaene macrolides produced by Streptoverticillium sp. LL-30F848. The CD difference spectrum of strevertene A methyl ester 15-dimethylaminobenzoate showed a positive couplet between the dimethylaminobenzoate and the pentaene chromophores, and therefore established the 15R configuration. Thus, by considering the relative configurations of the remaining stereogenic centers as derived from X-ray crystallography and ROESY experiments, the absolute stereochemistry of the strevertenes is established as 2R, 3S, 5S, 7S, 11R, 13R, 14R, 15R, 26S and 27R.     

A novel mannose containing phenolic glycolipid from Mycobacterium kansasii

Using high-performance liquid chromatography, a new kind of phenolic glycolipid quantitatively minor, called phenolic glycolipid-II, was isolated from a lipidic fraction of Mycobacterium kansasii. The structure was determined by fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy, as: 2,4-di-O-Me-alpha-D-Manp(1----3) 4-O-Ac-2-O-Me-alpha-L-Fucp(1----3)2-O-Me- alpha-L-Rhap(1----3) 2,4-di-O-Me-alpha-L-Rhap 1----phenolphthiocerol dimycocerosate. Phenolic glycolipids I and II differ only by their distal monosaccharide hapten which is 2,6-dideoxy-4-O-Me-alpha-D-arabinohexopyranosyl and the 2,4-di-O-Me-alpha-D-mannopyranosyl, respectively. This sugar appears to be characteristic and apparently unique in the Mycobacterium genus. Moreover, phenolic glycolipids I and II constitute with the lipooligosaccharides two classes of antigens of M. kansasii.     

Novel type-specific lipooligosaccharides from Mycobacterium tuberculosis

Mycobacterium tuberculosis (strain Canetti) is characterized by the presence of two novel glycolipids of the alkali-labile, trehalose-containing lipooligosaccharide class. Their structures were established by permethylation, partial acid hydrolysis, infrared and high-field NMR spectroscopy, and electron-impact and fast atom bombardment mass spectrometry of the native glycolipids and hydrolysis products. The trehalose substituent is unique in that it is methylated at the 6'-position. The structure of the simpler of the two glycolipids is 2-O-Me-alpha-L-Fucp(1----3)-beta-D-Glcp(1----3)-2-O-Me- alpha-L-Rhap(1----3)-2-O-Me-alpha-L- Rhap(1----3)-beta-D-Glcp(1----3)-4-O-Me-alpha-L-Rhap(1----3) -6-O-Me-alpha-D- Glc. Further glycosylation of the octaglycosyl unit of this nonantigenic glycolipid by an incompletely defined N-acyl derivative of a 4-amino-4,6-dideoxy-Galp residue results in the second, highly antigenic nonasaccharide-containing glycolipid. Application of two-dimensional proton correlation spectroscopy demonstrated that the fatty acyl substituents are located on the 2,3,6 and 3,4,6 hydroxyl groups of the terminal glucosyl unit in the proportions of 2:3. Gas chromatography/mass spectrometry and optical rotation measurement allowed identification of the fatty acyl esters as primarily 2L-, 4L-dimethylhexadecanoate, 2L-,4L-,6L-,8L-tetramethyloctadecanoate, and 2-methyl-3-hydroxyeicosanoate. The relationship of these glycolipids to different morphological forms of M. tuberculosis and to virulence is discussed.     

Convergent synthesis of higher-order oligosaccharides corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci group A. A branched hexasaccharide hapten.

A convergent synthesis of a hexasaccharide corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococci Group A is described. The strategy relies on the preparation of a key branched trisaccharide unit alpha-L-Rhap-(1----2)-[beta-D-GlcpNAc-(1----3)]-alpha-L-Rhap which functions both as a glycosyl acceptor and donor. The hexasaccharide is obtained after only three glycosylation reactions. This fully functionalized unit can serve, in turn, as a glycosyl acceptor or donor for the synthesis of higher-order structures. Deprotection gives a hexasaccharide for use as a hapten in immunochemical studies. The characterization of all compounds by high resolution 1H- and 13C-n.m.r. spectroscopy is also described.     

Diglycosyl phenol phthiocerol diester of Mycobacterium leprae

A diglycosyl phenol phthiocerol diester that had not been previously detected was isolated from M. leprae-infected armadillo tissues. Spectroscopy methods allowed the elucidation of its structure. The diglycoside was a 2,3-di-O-methylrhamnopyranosyl (alpha 1----2)3-O-methylrhamnopyranosyl (alpha 1-linked to the phenolic hydroxyl of phthiocerol dimycocerosates). It differs from the major phenolic glycolipid (PGL I) only by the absence of the terminal 3,6-di-O-methylglucopyranosyl unit. The diglycoside could be an intermediate in the synthesis of the latter antigen or a degradative product in the host detoxification process.     

Synthesis of a selectively protected trisaccharide building block of the capsular polysaccharide of Streptococcus pneumoniae types 6A and 6B

4-Methoxybenzyl 2,4-di-O-benzyl-3-O-[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-benzyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L-rhamnopyranoside (22), a building block for the alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap fragment of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B [----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-( 1----X)-D- RibOH-(5-P----]n (6A, X = 3; 6B, X = 4) has been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside was coupled with 4-methoxybenzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside in ether, using methyl triflate as promoter. The resulting alpha-D-Glcp-(1----3)-alpha-L-Rhap derivative was deallylated with KOBut in N,N-dimethylformamide followed by 0.1M HCl in 9:1 acetone-water. The product was coupled with 3,4,6-tri-O-acetyl-2-O-allyl-alpha,beta-D-galactopyranosyl trichloroacetimidate in ether, using trimethylsilyl triflate, to yield 19. Deacetylation, benzylation, and deallylation then gave 22.     

Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes.

On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.     

Absolute configuration of the unique 2,6-dideoxy-4-O-methyl-arabino-hexopyranose of the major phenolic glycolipid antigen from Mycobacterium kansasii

The antigenicity and the structure of the major phenolic glycolipid from Mycobacterium kansasii have been established. A monoacetylated tetrasaccharide structure was proposed for the oligosaccharide moiety in which the distal sugar, unique in nature, corresponds to 2,6-dideoxy-4-O-methyl-alpha-arabinohexopyranose. Its terminal position in the oligosaccharide part confers to this residue a key role in the antigen-antibody interaction. By improvement of the methanolysis procedure, this new kind of sugar was obtained in higher amounts than by hydrolysis of the glycolipid. Its 1H-NMR spectrum is presented and its optical rotatory power measurement agrees with a D absolute configuration while the deoxyhexoses involved in the glycolipid tetrasaccharide structure present the L absolute configuration.     

Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy

Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base.     

Monoclonal antibodies directed to chemically synthesized lactogangliotetraosylceramide, a leukemia-associated antigen having a novel branching structure

Murine leukemia cells (M1), in their undifferentiated state, have been characterized by the presence of cancer-associated lactoganglio-series glycolipids, one of which was identified as lactogangliotetraosylceramide (LcGg4) having a novel branching at the II-Gal of lactosylceramide through GlcNAc beta 1----3 and GalNAc beta 1----4 linkage, as shown below (Kannagi, R., Levery, S.B., and Hakomori, S. (1984) J. Biol. Chem., 259, 8444-8451): GalNAc beta 1----4 Gal beta 1----4Glc beta 1----1Cer GlcNac beta 1----3 Since this glycolipid is a very minor component, it has been difficult to obtain enough of the purified glycolipid for the preparation of a monoclonal antibody. We developed a method to chemically synthesize this glycolipid using a lactose unit, a ceramide unit, and two hexosamine donors as synthons and made the synthetic glycolipid available as an immunogen. The two monoclonal antibodies we obtained (YI328-18 and YI328-51, both IgG3) specifically recognized the novel branching structure and had no cross-reactivity with gangliotriaosylceramide or lactotriaosylceramide. Thus, the antibodies were found to be useful probes to detect lactogangliotetraosylceramide expressed in undifferentiated M1 leukemia cells, which disappears on induced differentiation. The results of this study indicate a new strategy to establish monoclonal antibody directed to novel minor glycolipid markers or their artificially designed analogs, employing chemically synthesized glycolipid antigens.     

Carbohydrate epitope structural elucidation by 1H-NMR spectroscopy of a new Mycobacterium kansasii phenolic glycolipid antigen.

The complete primary structure of the carbohydrate moiety of a new phenolic glycolipid antigen namely PheGl K-IV from Mycobacterium kansasii was successfully established from only one- and two-dimensional 1H-NMR data. Among the scalar two-dimensional techniques, correlated spectroscopy with a 45 degree mixing pulse and phase-sensitive double-quantum-filtered correlated spectroscopy were selected, combined with two-dimensional dipolar techniques (nuclear Overhauser effect). These techniques using milligram of quantities native PheGl K-IV allowed the following monoacetylated tetrasaccharide to be proposed for its carbohydrate part: 4-O-Me-alpha-Manp-(1----3)-4-O-Ac-2-O-Me-alpha-Fucp-(1----3) -2-O-Me-alpha-Rhap- (1----3)-2,4-di-O-Me-alpha-Rhap. The PheGl K-IV shares, with the other phenolic glycolipids isolated from M. kansasii (K-I, K-II), a common core assigned to the lipid aglycone glycosylated by the monoacetylated trisaccharide part. It differs in the structure of the distal monosaccharide residue.     

Further specific triglycosyl phenol phthiocerol diester from Mycobacterium tuberculosis

Mono- and two-dimensional 1H-NMR allowed the structural elucidation of a glycolipid belonging to the phenolic mycosides series: 2,3,4-tri-0-methyl fucopyranosyl (alpha 1----3) rhamnopyranosyl (alpha 1----3) rhamnopyranosyl (alpha 1----dimycocerosyl) phenol phthiocerol. It shares with the major phenolic glycolipid the two terminal sugar residues, suggesting its potential antigenicity. The glycolipid may also represent an intermediate in the biosynthesis of the major one.