4-O-(1-carboxyethyl)-D-galactose. A new acidic sugar from the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain 49.

The structure of a new acidic sugar from the extracellular polysaccharide of Butyrivibrio fibrisolvens strain 49 was determined as 4-O-(1-carboxyethyl)-D-galactose on the basis of 13C-n.m.r. and 1H-n.m.r. spectroscopy, m.s. and chemical degradation studies.     

Structure of an acidic glycan present in the lipopolysaccharide extract from the reference strain for Serratia marcescens serogroup O18.

The lipopolysaccharide extract from the cell wall of the reference strain for Serratia marcescens serogroup O18 contained, in addition to a neutral glycan characterised previously, an acidic glycan. Acidity was contributed both by D-glucuronic acid and by 4-O-[(R)-1-carboxyethyl]-D-glucose (4-O-Lac-D-Glc). By using n.m.r. spectroscopy, methylation analysis, and chemical degradations, the repeating unit of the acidic glycan was identified as a branched hexasaccharide having the structure shown; an O-acetyl group also present was not located. The glycan is believed to define the O18 serogroup, but is probably not an integral component of the lipopolysaccharide. [formula: see text].     

A convenient synthetic route to the disaccharide repeating-unit of peptidoglycan

Glycosylation of the readily accessible benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha- D- glucopyranoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl chloride (2), using the silver triflate method in the absence of a base, afforded 65-70% of the fully protected [beta-D-GlcNPhth-(1----4)-MurNAc] methyl ester derivative 4, the structure of which was ascertained on the basis of 500-MHz 1H-n.m.r. data. 2,2'-Dideoxy-2,2'-diphthalimido-beta,beta-trehalose hexa-acetate was a by-product. Removal of the Phth group from 4, followed by acetylation, yielded 90% of the acetylated 1,6-di-O-benzyl derivative 5, which, on saponification and catalytic hydrogenation, afforded 2-acetamido-4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1- carboxyethyl]-2-deoxy-D-glucopyranose. Similarly, 5 was converted into the acetylated methyl ester derivative, which, on selective removal of the methyl ester group, gave benzyl 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-6-O-benzyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside. An alternative route for the preparation of 2 is described.     

Structural analysis of the specific capsular polysaccharide of Rhodococcus equi serotype 2

The specific capsular polysaccharide produced by Rhodococcus equi serotype 2 is a high-molecular-weight acidic polymer composed of D-glucose, D-mannose, D-glucuronic acid and 3-O-[(S)-1-carboxyethyl]-L-rhamnose in equimolar proportions. Structural analysis, employing a combination of chemical and n.m.r. techniques, established that the polysaccharide is composed of linear repeating tetrasaccharide units. (formula; see text) in which the beta-D-mannose residues carry O-acetyl groups at O-2 and O-3 to the extent of 1.7 mol equivalents. Unequivocal determination of the absolute chirality of the 3-O-[(S)-1-carboxyethyl]-alpha-L-rhamnose residues was achieved by chemical correlation with an authentic synthetic sample. The 1H and 13C-n.m.r. resonances of the native and O-deacetylated serotype 2 polysaccharides were fully assigned by homo- and heteronuclear chemical-shift correlation methods.     

New sugars from antigenic lipopolysaccharides of bacteria: identification and synthesis of 3-O-[(R)-1-carboxyethyl]-L-rhamnose, an acidic component of Shigella dysenteriae type 5 lipopolysaccharide.

A new acidic sugar, 3-O-[(R)-1-carboxyethyl]-L-rhamnose (1), has been identified as a constituent of the O-antigenic lipopolysaccharide of Sh. dysenteriae type 5. The structure of 1 has been established by physico-chemical methods and by synthesis. Alkylation of methyl 2,5-di-O-benzyl-alpha-L-rhamnofuranoside (6) with (S)- or (R)-2-chloropropionic acids, followed by removal of the protecting groups, afforded 3-O-[(R)-1-carboxyethyl]-L-rhamnose (9) and 3-O-[(S)-1-carboxyethyl]-L-rhamnose (10), respectively. The properties of 1 coincide with those of 9.     

The chemical structure of a fragment of Micrococcus lysodeikticus cell-wall.

A fragment of Micrococcus lysodeikticus cell-wall obtained by cetylpyridinium recipitation from the nondialyzable portion of the degradation products of egg-white lysozyme was studied by the periodate oxidation and methylation procedures. The fragment consists of a polysaccharide chain composed of about 40 repeating (1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranosyluronic acid)-(1 leads to 6)-O-(alpha-D-glucopyranosyl) residues with D-glucopyranosyl residues at both ends. The alpha-D-glucopyranose residue at the reducing end is linked to a phosphate group that is also linked to C-6 of a 2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl residue of a peptidoglycan chain composed of four repeating (1 leads to 4)-O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl] residues. The peptidoglycan chain has, as nonreducing group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, and, as reducing residue, a 2-acetamido-3-O-(D-1-carboxytheyl)-2-deoxy-beta-D-glucose residue.     

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).     

Gordonan, an acidic polysaccharide with cell aggregation-inducing activity in insect BM-N4 cells, produced by Gordonia sp

An acidic polysaccharide, termed gordonan, was isolated from the culture medium of Gordonia sp. as an inducer of cell aggregation in an insect cell line, BM-N4. Gordonan had an average molecular weight of 5 x 10(6) and its structure was identified as -->3)-4-O-(1-carboxyethyl)-beta-D-Manp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1--> mainly by acid hydrolysis experiments and NMR analysis. It induces cell aggregation at the concentration of 4 microg/ml. A partially hydrolyzed polysaccharide derived from gordonan with a molecular weight of 5 x 10(5) showed weak activity, while any fragment molecules with lower molecular weights prepared from gordonan showed no activity.     

Neutral sugar composition of extracellular polysaccharides produced by strains of Butyrivibrio fibrisolvens

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.     

Structure of the O-polysaccharide of Escherichia coli O150 containing 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose

An acidic O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Escherichia coli O150 and studied by sugar and methylation analyses, triflic acid solvolysis, Smith degradation, (1)H and (13)C NMR spectroscopy, including 2D ROESY, (1)H,(13)C HSQC, HMQC-TOCSY, and HMBC experiments. The polysaccharide was found to contain a regioisomer of N-acetylisomuramic acid, 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxy-d-glucose [d-GlcNAc4(Slac)]. The structure of its hexasaccharide repeating unit was established.     

Structural characterization of the released polysaccharide of desiccation-tolerant Nostoc commune DRH-1

The structure of the viscous extracellular polysaccharide (glycan) of desiccation-tolerant Nostoc commune DRH-1 was determined through chromatographic and spectroscopic methods. The polysaccharide is novel in that it possesses a 1-4-linked xylogalactoglucan backbone with D-ribofuranose and 3-O-[(R)-1-carboxyethyl]-D-glucuronic acid (nosturonic acid) pendant groups. The presence of D-ribose and nosturonic acid as peripheral groups is unusual, and their potential roles in modulating the rheological properties of the glycan are discussed. Nosturonic acid was present in the glycans of N. commune from diverse geographic locations, suggesting that this uronic acid is an integral component of this cosmopolitan anhydrophile.     

Structural studies of the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b

The extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b, when grown under strictly anaerobic conditions with glucose as carbohydrate source, has been studied by chemical and spectroscopic techniques. The results demonstrate that the polysaccharide consists of hexasaccharide repeating units with the following structure: [structure: see text] The isolated polysaccharide was found to be approximately 65% acetylated at O-2 of the 3-O-[(S)-1-carboxyethyl]-beta-D-Glcp residue. The absolute configuration of the 1-carboxyethyl groups was determined by circular dichroism.     

Synthesis and conformational analysis of the repeating units of bacterial spore peptidoglycan

Deprotection of the fully blocked disacharide allyl O-(2-amino-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranosyl-1',2-lactam)-(1-->4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside by selective de-O-allylation and parallel removal of the benzylidene and O-benzyl groups is described. The resulting beta-muramyl lactam-(1-->4)-GlcNAc disaccharide is characterised as the per-O-acetylated derivative by 1H and 13C NMR spectroscopy and X-ray structure analysis. Conformational analysis about glycosidic bond of repeating units of bacterial spore cortex is based on experimental data and molecular modelling.     

Jatropham derivatives and steroidal saponins from the bulbs of Lilium hansonii.

Two new jatropham derivatives and three new steroidal saponins were isolated from the fresh bulbs of Lilium hansonii, along with previously known compounds. The structures of the new compounds were elucidated, on the basis of spectroscopic data and chemical evidence, and by comparing them with those of known compounds, as (-)-5-hydroxy-3-methyl-3-pyrrolin-2-one (jatropham) 5-O-beta-D-glucopyranosyl-(1----3)-beta-D-glucopyranoside, (2S*,4R*)-1-(3-methyl-2-oxo-3-pyrrolinyl)-4-methyl-5-oxo-2-pyrr olidinecarboxyli c acid, 26-O-beta-D-glucopyranosyl-(25R)-5 alpha-furostan-3 beta,22 zeta-diol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside, (25R)-5 alpha-spirostan-3 beta,12 alpha-diol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside and (25R)-spirost-5-en-3 beta,12 alpha-diol 3-O-alpha-L-rhamnopyranosyl-(1----2)-O-[beta-D-glucopyranosyl-(1----4)]- beta-D-glucopyranoside, respectively. The stereostructure of jatropham dimer, the plain structure of which was presented previously, was confirmed by X-ray crystallographic analysis. The inhibitory activity on cyclic AMP phosphodiesterase of the steroidal saponins was evaluated.     

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H,(13)C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established:-->3)-alpha-D-GlcpNAc4(R-Lac)6Ac-(1-->2)-beta-D-GlcpA-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where L-6dTal and D-GlcNAc4(R-Lac) are 6-deoxy-L-talose and 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.     

Synthesis and mass spectra of 4-O-acetyl-1,5-anhydro-2,3,6-tri-O -(methoxycarbonylmethyl)-D-glucitol and the positional isomers of 4- O-acetyl-1,5-anhydro-di-O-(methoxycarbonylmethyl)-O-methyl-D-glucitol and 4-O-acetyl-1,5-anhydro-O-(methoxycarbonylmethyl)-di-O-methyl-D-glucitol .

Reductive cleavage of fully methylated, partially O-carboxymethylated cellulose had previously been shown to produce 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-, -2-O-(methoxycarbonylmethyl)-3,6-di-O-methyl-, -3-O-(methoxycarbonylmethyl)-2,6-di-O-methyl-, -6-O-(methoxycarbonylmethyl)-2,3-di-O-methyl-, -2,3-di-O-(methoxycarbonylmethyl)-6-O-methyl-, -2,6-di-O-(methoxycarbonylmethyl)-3-O-methyl-, -3,6-di-O-(methoxycarbonylmethyl)-2-O-methyl-, and -2,3,6-tri-O-(methoxycarbonylmethyl)-D-glucitol. Described herein is the independent synthesis of these derivatives, except for the first, which had been reported. In addition, their 1H-n.m.r. spectra, chemical-ionization (NH3) mass spectra, and electronionization mass spectra are tabulated.     

Lactones of methyl 3-O

Lactones of methyl 3-O-[(R)- and (S)-1-carboxyethyl]-alpha-D-gluco-, galacto- and manno-pyranoside were prepared by treatment of the sugar derivatives in acetic acid. The lactones were formed between the 1-carboxyethyl substituent and 2-OH or 4-OH in different proportions depending on the stereochemistry of the parent compounds. Relative formation rates in acetic acid-d4 and hydrolysis rates in buffered D2O solutions at pD 2.4, 4.6 and 7.4 were estimated. Hydrolysis of the formed lactones is relatively slow in D2O at pD 4.6, which permitted characterization of the lactones by 1H and 13C NMR spectroscopy in buffered D2O solutions. Hydrolysis of the lactones in 1 M aqueous NaOH at 80 degrees C gave no detectable isomerization of the alpha-carbon. The set of lactones formed from the 1-carboxyethyl substituted methyl glycosides used in this study showed large similarities in the NMR shifts (delta delta values). Deviations from the observed shift pattern were found for two lactones. Our findings strongly suggest that those two lactones differ from the rest by adopting a boat-like conformation, whereas the others adopt pseudo-chair conformations.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Structure of the O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Providencia alcalifaciens O32 containing N-acetylisomuramic acid

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O32 and studied by sugar and methylation analyses, solvolysis with triflic acid, 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. It was found that the polysaccharide has a branched tetrasaccharide repeating unit containing 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (D-GlcNAc3Slac, N-acetylisomuramic acid) with the following structure: [STRUCTURE: SEE TEXT]. Serological studies with O-antisera showed antigenic relationships between P. alcalifaciens O32 and O29 as well as several other Providencia and Proteus strains sharing putative epitopes on the O-polysaccharides.     

Synthesis of O-alpha-L-fucopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-2- acetamido-2-deoxy-D-glucopyranose (N-acetyl-3'-O-alpha-L-fucopyranosyllactosamine)

Methyl 2-O-benzyl-beta-D-galactopyranoside (6) was obtained in five, good yielding steps from methyl beta-D-galactopyranoside (1). Treatment of 1 with tert-butylchlorodiphenylsilane in N,N-dimethylformamide in the presence of imidazole afforded a 6-(tert-butyldiphenylsilyl) ether, which was converted into its 3,4-O-isopropylidene derivative (3). Benzylation of 3 with benzyl bromide-silver oxide in N,N-dimethylformamide, and subsequent cleavage of its acetal and ether groups then afforded 6. On similar benzylation, followed by the same sequence of deprotection, benzyl 2-acetamido-3,6-di-O-benzyl-4-O-[6-O-(tert-butyldiphenylsilyl)-3,4 -O- isopropylidene-beta-D-galactopyranosyl]-2-deoxy-alpha-D-glucopyranoside gave the 2-O-benzyl derivative (10). Compound 10 was converted into its 4,6-O-benzylidene acetal (11). Glycosylation (catalyzed by halide-ion) of 11 with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide afforded the fully protected trisaccharide derivative (13). Cleavage of the benzylidene and then the benzyl groups of 13 furnished the title trisaccharide (16). The structure of 16 was established by 13C-n.m.r. spectroscopy.