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An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O10 and studied after full acid hydrolysis and carboxyl reduction by 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H,13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY). It was found that the polysaccharide contains 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid, and L-altruronic acid, and the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [sequence: see text]

Based on acid hydrolysis, methylation, and one- and two-dimensional 1H- and 13C-NMR spectroscopy, including homonuclear and 1H, 13C heteronuclear correlation spectroscopy (COSY) and rotating-frame nuclear Overhauser effect spectroscopy (ROESY); it was found that the O-specific polysaccharide chain of the lipopolysaccharide of the bacterium Proteus mirabilis O30 is a hexosamino-glucuronan built up of tetrasaccharide repeating units having the following structure: [formula: see text] The degree of O-acetylation of 2-acetamido-2-deoxyglucose is about 70%.

The composition and structure of the O-specific polysaccharide chain of the bacterium Proteus mirabilis O26 lipopolysaccharide have been studied using one- and two-dimensional 1H- and 13C-NMR spectroscopy, including homonuclear correlation spectroscopy (COSY), H-detected 1H,13C heteronuclear multi-quantum coherence and rotating-frame nuclear Overhauser effect spectroscopy. It has been found that the polysaccharide is acidic due to the presence of D-galacturonic acid (D-GalA) residues, part of which are O-acetylated, while the other part form an amide with the alpha-amino group of L-lysine and is built up of tetrasaccharide repeating units having the following structure: [formula: see text] The P. mirabilis O26 polysaccharide is structurally similar to the O-specific polysaccharide of P. mirabilis O28 studied earlier by us and containing amides of D-galacturonic acid with L-lysine and L-serine.

Like some Pseudomonas cepacia serogroups studied earlier, serogroups C, I (Nakamura), O1 and O4 (Heidt) are characterized by the presence of at least two structurally different O-antigenic polysaccharide chains in cell-wall lipopolysaccharides. On the basis of acid hydrolysis, methylation, 1H- and 13C-NMR spectroscopy, including computer-assisted 13C-NMR-based analysis, the complete structures of the predominant polysaccharides of serogroups I (I), C and O4 (III) and the minor polysaccharides of serogroups I (II) and O1 (V) were established, and the structure of the predominant polysaccharide of serogroup O1 (IV) established earlier (Cox A. D., Wilkinson S. G.@Carbohydr. Res. 1990. V. 195. No 2. P. 295-301) was confirmed.

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.

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.

On the basis of non-destructive analysis by means of 1H and 13C NMR spectroscopy and calculation of specific optical rotation, it was concluded that O-specific polysaccharide of Pseudomonas cepacia strain IMV 4207 (serotype A) has the structure (I): (formula; see text) Two structurally different polysaccharides were found in the ratio of approximately 2.5:1 in P. cepacia strain IMV 598/2 which is serologically related to serotype A in Nakamura classification and serotype 2 in Heidt classification. The minor polysaccharide has the structure (I) whereas the major one possesses the structure (II) which is characteristic of the formerly studied O-specific polysaccharide of P. cepacia strain IMV 4137 belonging to serotype 2: ----4)-beta-D-Galp-(1----2)-alpha-L-Rhap-(1----.

On the basis of acid hydrolysis, methylation, 1H and 13C NMR analysis, and calculation of specific optical rotation, the following structures were established for O-specific polysaccharides of Pseudomonas cepacia serotypes B and E: ----3)-beta-D-Galf-(1----3)-alpha-D-Fucp-(1----serotype B ----3)-beta-D-GlcpNAc-(1----3)-alpha-D-Fucp-(1----serotype E A characteristic feature of the polysaccharides is the presence of D-fucose, rather rare for bacterial antigens.

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----.

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.

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-.