Search

The o -nitrophenyl group, a protecting group with latent activation potential, was used as a protecting group for the glycosidic position. It is stable to common conditions used in synthesis and can be activated for displacement and glycoside formation by an alcohol, using zinc chloride as a catalyst. Good to excellent yields of β-glycosides of the important amino sugar N -acetylglucosamine were obtained. A mechanism for the reaction is proposed.

Cerebrosides were isolated from the leech species, Hirudo medicinalis, and purified to homogeneity by silicic acid chromatography, followed by preparative thin-layer chromatography. Their structure was determined by spectroscopic and chemical methods. 1D and 2D 1H NMR spectroscopy, DQF–COSY and HMQC indicated that the head group consists of a single galactose residue in the β configuration. The galacto configuration was determined by the characteristic chemical shift, the spin–spin splitting and the multiplicity of the characteristic resonance of its equatorial H-4 proton, as well as by the splittings of the other ring protons. GC, GC–MS and fast-atom-bombardment mass spectrometry studies indicated that C24:0 and C22:0 are the major saturated fatty acid species. Unsaturated fatty acids present were C25:2, C27:2, C27:3, C28:3, C29:3, C30:3, C33:3. GC–MS indicated the presence of hydroxylated C27:2 and one other unidentified hydroxylated fatty acid. The cerebroside contained an unusual polyunsaturated sphingosine analogue, namely 2-amino-1,3-dihydroxydocosatriene.

We have isolated and characterized glycerol-3-yl 6-deoxy-6- C -sulfo- α - d -glucopyranoside (glyceryl α -sulfoquinovoside), a key metabolite involved in sulfoquinovosyl diacylglycerol (SQDG) biosynthesis in photosynthetic organisms, from the cellular metabolic pools of Rhizobium . The compound has not been found in any other non-photosynthetic organisms. This result suggests some commonalities between the biosynthesis of SQDG in Rhizobium and in plant organelles and photosynthetic organisms and further adds to the evolving picture that there is considerable gene overlap between Rhizobium and photosynthetic systems, including plants.

The structure of peptidoglycan extracted from the Gram-positive bacterium Sarcina ventriculi grown at pH 3 was characterized by amino acid analysis, mass spectrometry, and two-dimensional NMR spectroscopy. The basic muropeptide subunit consisted of an N -acetylglucosamine- β -1,4- N -acetylmuramic acid disaccharide substituted with an oligopeptide with the sequence Ala- iso Gln-A 2 pm(-Gly)-Ala. The dimeric muropeptide was also characterized as a cross-linked bis-disaccharide-penta-hexapeptide with the structure, GlcNAc-MurNAc-Ala- iso Gln-A 2 pm(-Gly)-Ala → GlcNAc-MurNAc-Ala- iso Gln-A 2 (-Gly)-Ala-Ala. These results are consistent with a structure proposed based on enzymatic degradation and chemical modifications but with no use of spectroscopic information [O. Kandler, D. Claus, and A. Moore, Arch. Mikrobiol. , 82 (1972) 140–146]. The cell wall of this organism is very tightly cross-linked and is much more rigid than that of most other Gram-positive bacteria. There is, however, a large degree of conservation in the general structure compared to peptidoglycan in other bacteria that are not well adaptable to extremes, indicating that the membrane plays a more important role in adaptation.

Increased activity of the main carbohydrate pathways (glycolysis, pentose phosphate, and hexosamine biosynthetic pathways) is one of the hallmarks of metabolic diseases such as cancer. Sulfoquinovosyl diacylglycerol is a sulfoglycolipid found in the human diet that possesses anticancer activity that is absent when its carbohydrate moiety (glucose 6-sulfonate or sulfoquinovose) is removed. This work used bacterial systems to further understand the metabolism of this sugar through three main carbohydrate processing pathways and how this could influence its biological activity. Using 13C NMR spectroscopy and enzyme assays, we showed that glucose 6-sulfonate cannot enter the pentose phosphate pathway, hence decreasing pentose and nucleotide biosyntheses. In glycolysis, glucose 6-sulfonate only provides one pyruvate per monosaccharide molecule, decreasing the flux of this pathway by half when compared to glucose 6-phosphate. Glucose 6-sulfonate can enter the hexosamine biosynthetic pathway by producing glucosamine 6-sulfonate, which is a reported antibacterial agent that competitively inhibits hexosamine production. All these interactions with carbohydrate routes might help explain the observed anticancer activity that glucose 6-sulfonate has in vitro. This adds to our knowledge of how vegetables rich in glucose 6-sulfonate can also act as metabolic inhibitors of pathways that are increased in metabolic diseases.

The structure of an unusual trisaccharide component isolated from the lipopolysaccharide (LPS) of a Tn5 mutant of Rhizobium leguminosarum biovar viciae VF39 which is defective in infection of its host plant has been elucidated. This mutant also appears to be defective in the synthesis of a tetrasaccharide component normally synthesized by the wild-type organism. The three glycosyl components are galactose, mannose, and 3-deoxy- d - manno -2-octulosonic acid (Kdo). Mannose is linked to the 5-position and galactose to the 7-position of the 3-deoxy-2-octulosonic acid residue (Kdo). Both hexosyl components are in the α-pyranosyl form. In the isolated molecule the octulosonic acid appears to be present as its γ-lactone. However, in the lipopolysaccharide molecule, it is most likely present in the pyranosyl form. The structure was determined by 1 H NMR spectroscopy and methylation analysis as well as by fast-atom-bombardment mass spectrometry of the peracetylated and per(trideuterio) acetylated oligosaccharides. Small amounts of the methylation analysis product of another tetrasaccharide different to normal tetrasaccharide component made by the wild-type organisms were detected. This indicates that in this mutant, there is a block in the synthesis of the normal tetrasaccharide component in addition to a switch in the synthesis of the LPS type.

The structural rationale, synthesis and evaluation of an inhibitor designed to block glucosamine synthesis by competitively inhibiting the action of glutamine: fructose-6-phosphate amidotransferase and subsequently reducing the transformation of any glucosamine-6-phosphate formed to UDP-N-acetylglucosamine are described. The inhibitor 2-acetamido-2,6-dideoxy-6-sulfo-D-glucose (D-glucosamine-6-sulfonate) is an analog of glucosamine-6-phosphate in which the phosphate group in the latter is replaced with a sulfonic acid group. The inhibitor is designed to function by three different modes which together reduce UDP-N-acetylglucosamine synthesis. This reduction was confirmed by evaluating the effect of the inhibitor on bacterial cell-wall synthesis and by demonstrating that it inhibits acetylation of glucosamine-6-phosphate competitively and by acting as a surrogate substrate. Inhibition of glucosamine production or suitably activated glucosamine in bacteria leads to disruption of the peptidoglycan structure, which results in softening, bulging, deformation, fragility and lysis of the cells. These modifications were documented by scanning electron microscopy for bacteria treated with the inhibitor. They were observed for inhibitor concentrations in the 20 mg/mL range for Escherichia coli and Bacillus subtilis and the 5 mg/mL range for Rhizobium trifolii.

The lipopolysaccharides (LPS) of a wild type strain of Rhizobium leguminosarum biovar viciae (strain VF-39) and two symbiotically defective Tn5 mutants (VF-39-32 and VF-39-86) have been studied. The LPS of the mutants reflected impaired synthesis of the O-antigen. In the LPS of one mutant, the core tetrasaccharide was lacking and in that of the other it was truncated to a disaccharide containing mannose and 3-deoxy- d - manno -oct-2-ulosonic acid (KdO). The latter mutant also synthesized an unusual carbohydrate component containing mannose, galactose, and an unidentified saccharide. The lipid A composition was similar to that found in other strains of R. leguminosarum biovar viciae . The O-antigen of the wild-type bacterium contained 2- O -methylfucose, fucose, 3,6-dideoxy-3-(methylamino)hexose, glucose, 2-amino-2,6-dideoxyhexose, and heptose. This study clearly defines a role for the bacterial LPS in the proper functioning of the Rhizobium legume symbiosis.

Attempted cleavage of the benzylidene group of 3,5-O-benzylidene-2,6,7-tri-O-trimethylacetyl-D-glycero-D-gulo-heptono-1,4-lactone with N-bromosuccinimide led to the formation of a 7-bromo derivative with a benzoyl group in the 3-position and trimethylacetyl groups in the 5 and 6 positions. Analysis of the coupling constants in the proton NMR spectra indicated that both trimethylacetyl groups had participated to shift the crowded electron-deficient center formed at the 5-position by the decomposition of the bromobenzylidene group to the much more accessible 7-position. The net result of this cascade rearrangement was retention at both the C-5 and C-6 positions. This is an interesting example of a cascade rearrangement under strict entropic and stereo control the synthetic utility of which is being investigated.

Imino sugars (also called azasugars), a class of compounds of which the 1,5-dideoxy and 1,5,6-trideoxyiminoalditols are members, are important glycosidase inhibitors with very high potential as drugs. Their potential therapeutic applications range from the treatment of diabetes to cancer and AIDS. We present here a general method for the preparation of such compounds with the d -gluco and d -galacto configurations starting from β- d -glycosides. The procedure is especially appealing because of its high stereoselectivity and straightforwardness. The key steps are the selective oxidation of the glycosides to hexulosonic acids and reduction of the oxime derivatives to lactams, which are further reduced to the target compounds. The C-6 position can be deoxygenated during the reduction if it bears an acetoxy group. Trideoxy imino sugars are then produced. Deacetylation prior to oxime reduction gives dideoxy compounds.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.

The structure of a family of unusual glucans from Sarcina ventriculi has been characterized by NMR spectroscopy, methylation analysis, and mass spectrometry. One is a trisaccharide containing a β -(1 → 3) and a β -(1 → 4)-linkage. The other is a hexasaccharide that is simply a 1,4-linked dimer of the trisaccharide unit. This is the first report of β-glucan biosynthesis in a Gram-positive organism. Their occurrence in these organisms supports an even more general link between their synthesis and the adaptability of bacteria.

The structure of the O-antigen chain of the lipopolysaccharide (LPS) of Rhizobium trifolii 4s has been determined by a combination of chemical and spectroscopic methods. The glycosyl components were found to be l -rhamnose, N -acetyl- d -glucosamine, and N -acetyl- d -mannosamine in 3:1:1 molar proportion, as determined by gas chromatography and gas chromatography-mass spectrometry of alditol acetate and persilylated ( R )-2-hydroxybutyl glycoside derivatives. The linkage positions and configurations of the glycosyl residues were obtained by 1D and 2D NMR spectroscopy. The polymer has a pentasaccharide repeating-unit containing rhammose and N -acetylglucosamine in the main chain and N -acetylmannosamine as the sole-side chain component. This latter residue is linked to a main-chain rhamnose residue. This result was suggested by NMR spectroscopy and confirmed by periodate oxidation. The sequence was deduced by 1D and 2D NMR NOE experiments and by partial hydrolysis studies. The repeating unit of the polysaccharide is shown. This constitutes the first complete structure of an O-antigenic chain of the lipopolysaccharide of any strain of Rhizobium trifolii .

A novel glycolipid was isolated by chloroform-methanol extraction of Rhizobium trifolii ANU843 cells. Compositional analysis, methylation studies, 1 H NMR and spectroscopies led to the identification of a diglycosyl diacylglyceride: 1,2-di- O -acyl-3- O -[α- d -glucopyranosyl-(1 → 3)- O -α- d -mannopyranosyl]glycerol. Iso-hexadecanoic and anteiso-heptadecanoic acids were the predominant fatty acids esterifying the glyceryl moiety, but a microheterogeneity in fatty acid composition was found, resulting in at least five distinct molecular species of the glycolipid. Although widespread in plants, animals and Gram-positive bacteria, glycosyl glycerides have been seldom reported in Gram-negative bacteria and this work is the first evidence of their occurrence in the bacterial family Rhizobiaceae .

The complete structure of the acidic, extracellular, capsular polysaccharide of Rhizobium trifolii 843 has been elucidated by a combination of chemical, enzymic, and spectroscopic methods, confirming an earlier proposed sugar sequence and assigning the locations of the acyl substituents. The polysaccharide was depolymerized by a lyase into octasaccharide units which were uniform in carbohydrate composition and linkage. These units also contained a uniform distribution of acetyl and pyruvic acetal [ O -(1-carboxyethylidene)] groups, and half of them were further acylated with d -3-hydroxybutanoyl groups. A much smaller proportion ( d -3-hydroxy-butanoyl group. The locations of the substituents were determined chemically and by J -correlated, 1 H-n.m.r. spectroscopy, proton nuclear Overhauser effect (n.O.e.)_ measurements, doubie-resonance 1 H-n.m.r. spectroscopy, and 13 C-n.m.r. spectroscopy. The composition and structure of the carbohydrate chain were determined by methylation analysis using g.l.c.-m.s. fast-atom-bombardment mass spectrometry, and n.m.r. studies on the reduced, deacylated oligomer. Structural studies were supplemented by n.m.r. analyses on the original polymer. The oligosaccharides were found to be branched octasaccharides with four sugar residues in each branch, and the carbohydrate sequence agreed well with that expected from earlier work. In the abbreviated sequence and structure ( 1a ), the sugar residues are labelled “ a ” through “ h ”. The main chain ( a–d ) is composed of a 4-deoxy-α- l - threo -hex-4-enopyranosyluronic acid group ( a ) that is linked to O-4 of a 3- O -acetyl- d -glucosyluronic acid residue ( b ) which is β-linked to O-4 of a d -glucosyl residue ( c ). Residue c is β-linked to O-4 of the branching d -linked to O-4 of a d -glucosyl residue ( d ). The side chain consists of a substituted d -galactosyl group ( h ) which is β-linked to O-3 of residue 9 of a β-(1→4)-linked d -glucose trisaccharide (fragment e–f–g ). The reducing end of the resulting tetrasaccharide ( e–f–g–h ) is β-linked to O-6 of the branching d -glucose residue ( d ). In the native polymer, this branching residue is α-linked to O-4 of the modified d -glucuronic acid residue ( a ) which is the unsaturated sugar in the oligomer. A small proportion of the O-2 atoms of the acetylated d -glucosyluronic acid residues is acetylated because of ester migration. The two terminal sugars ( g and h ) of the branch chain bear 4,6- O -(1-carboxyethylidene) groups. The d -galactosyl groups of half of the oligomers are acylated by d -3-hydroxybutanoyl groups at O-3. About 5% of the oligomers bear a second d -3-hydroxybutanoyl group at O-2 of the d -galactosyl group ( h ).

A direct synthetic route from methyl α- d -glucopyranoside to 3,6-dideoxy-3-(methylamino)hexoses having the d - gluco , d - galacto , and d - manno configurations has been developed. Methyl α- d -glucoside was converted into the 4,6- O -benzylidene-2,3,-di- O -tosyl derivative, which has then transformed into the 4- O -benzyl-6-deoxy 2,3-ditosylate ( 5 ) by successive reductive cleavage of the acetal ring, iodination, and reduction. The intermediate 5 was readily converted into the allo 2,3-epoxide, which yielded the pivotal intermediate methyl 4- O -benzyl-3,6-dideoxy-3-(methylamino)-α- d -glucopyranoside ( 7 ) by cleavage of the oxirane ring with methylamine. The amino compound 7 can be directly converted into the derivatized galacto and manno derivatives for mass-spectrometric identification by selective inversion at C-4 and C-2, respectively, followed by hydrolysis, reduction, and acetylation.