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The full structure of the lipopolysaccharide core of bacteria Shigella flexneri types 2a and 5b, the causative agents of bacillary dysentery (shigellosis), was established by chemical methods, high-resolution electrospray ionization mass spectrometry, and two-dimensional NMR spectroscopy. The structure of the O-antigen repeating unit and the configuration and position of the linkage between the O-antigen and the core were determined in the lipopolysaccharide of S. flexneri type 2a.

A programme SCAN was elaborated for the 13C NMR--based structural analysis of regular polysaccharides, which represents our earlier programme (Lipkind G.M. et al. Carbohydr. Res. 1988. V. 175. No 1. P. 59-75) modified for IBM-PC-compatible personal computers. SCAN was successfully applied for the structural elucidation of 24 polysaccharides of bacterial origin. Optimal combinations of the computer-assisted method with other approaches for analysis of carbohydrates and scopes of application of the programme are discussed.

Mild acid degradation of lipopolysaccharide from Pseudomonas aeruginosa O(3a), 3d, 3f (Lányi classification) afforded O-specific polysaccharide containing N-acetyl-D-fucosamine, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic and D-mannuronic acid as well as O-acetyl groups. On the basis of O-deacetylation, selective cleavage with anhydrous fluoride, chemical transformation of the oligosaccharides obtained (hydrolysis or reductive deamination of the acetamidino group into acetamido or ethylamino group, respectively) and analysis by 13C NMR spectroscopy, it was concluded that the polysaccharide is built up mainly by trisaccharide repeating units of types A and B in the ratio approximately 2:1: (Formula: see text). The units of both types most probably enter the same polymeric chain. If so, such a hybrid structure can be accounted for by incompleteness of epimerization at C5 of the acetamidino derivative of mannuronic acid at the polymer level in the course of biosynthesis of this polysaccharide.

An extracellular polysaccharide from Arthrobacter globiformis is composed of N-acetyl-D-glucosamine, N-acetyl-D-fucosamine, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid and O-acetyl groups in the ratio 1:1:2:1. On the basis of solvolysis with anhydrous hydrogen fluoride, which resulted in a tetrasaccharide fragment, and analysis by 1H and 13NMR spectroscopy, it was concluded that the polysaccharide has the following structure: (formula; see text).

The following structure of the repeating unit of the Proteus hauseri O-specific polysaccharide was established on the basis of monosaccharide composition and 13C NMR data of the polysaccharide and products of its Smith degradation and partial cleavage with hydrogen fluoride: (Formula: see text).

On the basis of acid hydrolysis, methylation, Smith degradation, selective cleavage with anhydrous hydrogen fluoride, and 13C NMR analysis, the repeating unit of the O-specific polysaccharide of Citrobacter O32 was concluded to have the following structure: (Formula: see text). The repeating unit of the Salmonella arizonae O64 O-specific polysaccharide has the same structure lacking the O-acetyl group.

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.

O-Specific polysaccharide built up of trisaccharide repeating units containing 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid (ManNAcAmA), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (Man(NAc)2A), N-acetyl-D-fucosamine (FucNAc), and O-acetyl group was obtained on mild acid hydrolysis of P. aeruginosa O25 (Wokatsch classification) lipopolysaccharide. Basing on de-O-acetylation of polysaccharide with aqueous triethylamine accompanied by hydrolysis of acetamidino group to acetamido group, as well as on the 1H and 13C NMR data, the following structure of the repeating unit of the polysaccharide was established: (Formula: see text) P. aeruginosa O25 polysaccharide has the same carbohydrate skeleton as that of P. aeruginosa O3a,b (Lányi classification) and differs from the latter only by the presence of the O-acetyl group at position 4 of N-acetylfucosamine.

The following structure of the repeating unit of the Proteus mirabilis O27 O-specific polysaccharide was established: (formula; see text) where (formula; see text) is N-glucopyranuronoyl-L-lysine, (formula; see text) is N-galactopyranuronoyl-L-alanine. The polysaccharide was parially solvolysed with anhydrous HF and the resulting dephosphorylated tri- and tetrasaccharide with N-acetylglucosamine at the reducing end were studied by means of 1H and 13C NMR spectroscopy and (for methylated derivative of trisaccharide) mass-spectrometry. Smith degradation of the polysaccharide afforded linear polymer, and its structure was investigated by 13C NMR spectroscopy. The position of the ethanolamine phosphate group was determined by means of the analysis of the phosphorylation effects in the 13C NMR spectra of the linear and branched polysaccharides.

O-Specific polysaccharide, obtained by mild acid degradation of the Proteus mirabilis 03 lipopolysaccharide, was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N-(2-hydroxyethyl)-D-alanine. The structure of the polysaccharide was determined by methylation, the Smith degradation and computer-assisted analysis of the 13C NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was elucidated by using 1H and 13C NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative), optical rotation and CD spectrum data and comparison with the synthetic sample. The repeating unit of P. mirabilis 03 O-specific polysaccharide is shown to have the following structure: (formula; see text)

O-Specific polysaccharide chain of Pseudomonas aeruginosa immunotype 7 lipopolysaccharide is composed of 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid (GulNAcAmA), 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (ManN2Ac2A), and N-acetyl-D-fucosamine (FucNAc). On solvolysis with anhydrous hydrogen fluoride, the polysaccharide afforded a trisaccharide containing all its components. Borohydride reduction of the trisaccharide in boric acid solution resulted in conversion of reducing fucosamine into fucosaminitol, whereas in water the reduction was accompanied by reductive deamination of acetamidino function into ethylamino group. On hydrolysis with aqueous triethylamine, acetamidino group gave acetamido group. Analysis of the trisaccharides thus obtained by 1H NMR spectroscopy (including nuclear Overhauser effect), 13C NMR spectroscopy, and fast-atom bombardment mass spectrometry allowed the determination of the structure of the unusual uronic acid derivative and the following structure of the polysaccharide repeating unit: -4)-alpha-L-GulNAcAmA-(1-4)-beta-D-ManN2Ac2A-(1-3)-alpha-D-+ ++FucNAc-(1-.

O-Specific side chain of P. aeruginosa immunotype 3 lipopolysaccharide is composed of N-acetyl-D-fucosamine (FucNAc), 2,3-diacetamido-2,3-dideoxy-L-guluronic acid (GulN2Ac2A) and 3-acetamidino = 2-acetamido = 2,3 = dideoxy = D-mannuronic acid (ManNAcAmA). The latter sugar is identified on the basis of solvolysis with anhydrous hydrogen fluoride, 13C NMR spectroscopy and fast-atom bombardment mass spectrometry analysis, as well as of reactions of acetamidino function (alkaline hydrolysis to acetamido group and reductive deamination to ethylamino group). Earlier, in the course of investigation of P. aeruginosa O3 lipopolysaccharides, the structure of 1-methyl-2-imidazoline was erroneously ascribed to the acetamidino group. The following structure was established for the repeating unit of immunotype 3 polysaccharide which is identical to P. aeruginosa O3(a),3c polysaccharide: ----4)-beta-D-ManNAcAmA-(1----4)-alpha-L-GulN2Ac2A-(1----3)- beta-D-FucNac-(1----.

The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).

O-Specific polysaccharide chain of P. aeruginosa 013 (Lányi) lipopolysaccharide is composed of N-acetyl-D-quinovosamine (QuiNAc), acetamidino derivative of L-fucosamine (FucNAm), and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso nic acid (Sug). On solvolysis with HF in methanol, the polysaccharide afforded methylglycosides of a disaccharide and a trisaccharide both containing fucosamine and ulosonic acid derivatives. Chemical transformations (alkaline hydrolysis, reductive deamination, acetylation accompanied by intramolecular acylation of acetamidino group by ulosonic acid), 1H and 13C NMR analysis and mass spectral data proved the following structure of the trisaccharide unit of the polysaccharide: -8)-beta-Sug-(1-3)-alpha-L-FucNAm-(1-3)-alpha-D-QuiNAc -(1-

On mild acid degradation on lipopolysaccharides of seven Pseudomonas aeruginosa immunotypes, O-specific polysaccharides were obtained and their structures established. A peculiar feature of the polysaccharides is the presence of various, mostly acidic, mono- and diaminosugars, many of which have not previously been found in nature. The absence of serological cross-reactions (inhibition of passive haemagglutination) between lipopolysaccharides of seven immunotypes correlates with the absence of any common oligosaccharide fragments in their O-specific chains. The data obtained revealed structural and serological interrelations between O-antigens of seven immunotypes and P. aeruginosa O-serotypes, and showed that immunotypes 1 and 7 should be included into the serological classification scheme as individual O-serotypes.