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While glycosyltransferases are restrictively expressed in invertebrate model organisms, little is known of their glycan end products. One such restrictively expressed glycoepitope was localized to sensory and epithelial cells of leech and Caenorhabditis elegans using the Lan3-2 monoclonal antibody. A biological function for the neural Lan3-2 epitope was previously determined in the leech. Here we report on the chemical structure of this mannosidic epitope harvested from whole Hirudo medicinalis. Crude glycans were liberated from glycoproteins by hydrazinolysis. Re-N-acetylated glycans were subjected to immunoaffinity purification. The affinity-purified glycans were fractioned by size chromatography into oligosaccharides and polysaccharides. Lan3-2 oligosaccharide structure was characterized by gas chromatography of alditol acetates, methylation analysis, 500 MHz 1H NMR spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem MS-MS of permethylated derivatives. The predominant components of the Lan3-2 oligosaccharide fraction were a series of linear β-(1,4)-linked mannose polymers. The homologous expression of the Lan3-2 epitope in C. elegans will facilitate the exploration of its glycosylation pathway. Other invertebrates expressing the Lan3-2 epitope are Planaria dugesia, Capitella sp. I and Lumbriculus variegatus. The glycoepitope was not detected in the diploblastic animals Hydra littoralis and Aptaisia sp. or in deuterostomes.

We previously demonstrated that adrenergic and histaminergic receptors have an ascorbic acid (vitamin C) binding site on the first extracellular loop, immediately adjacent to the aminergic binding site. Binding of ascorbate to this site strongly potentiates any sub-maximal dose of an adrenergic or histaminergic compound, significantly increasing its duration of activity. We report here the successful synthesis of a tethered compound that mimics the combined effects of a mixture of ascorbate with norepinephrine. The tethered compound uses a four-unit polyethylene linker to tether ascorbate to norepinephrine. The tethered compound is about tenfold less effective than norepinephrine in stimulating rabbit aortic smooth muscle, but has a very significantly enhanced duration of activity compared with norepinephrine alone and comparable to a mixture of norepinephrine and ascorbate. Additional ascorbate does not enhance the tethered compound's effects and we demonstrate that the compound binds to a synthetic peptide spanning the ascorbate binding site of the receptor. These experiments strongly suggest that the compound binds to both the adrenergic binding site and the ascorbate binding site simultaneously. Tethered compounds with linkers of other lengths did not have these properties. We believe that the synthesis of enhanced adrenergic and histaminergic drugs by tethering them to potentiators such as ascorbate will permit a new class of potential drugs to be created with high specificity and long duration of activity. Drug Dev Res 69:242–250, 2008. © 2008 Wiley-Liss, Inc.

1-Deoxynojirimycin (DNJ) and 1-deoxymannojirimycin (DMJ) were synthesized through a concise and practicable pathway. A strategy of using carbohydrate derivatives bearing two leaving groups in the preparation of azasugars by di-N-alkylation of amines was developed. The strategy involved the selective partial protection of dibromo alditols.

A unique 1,3-dioxonium cation facilitated Ritter-type reaction was discovered when glycerol dipivaloates were treated with strong acid in acetonitrile. The stereoselectivity and regioselectivity of this new Ritter-type reaction were further explored by using erythritol tripivaloate and 1,2,4-butanetriol dipivaloate as the substrates. Several protected aminopolyols were generated as important chiral building blocks. This reaction was employed to synthesize a protected aminosugar lactone.

[structure: see text] The preparation of three new chiral thiazines from ribose is described. Two of these are dihydroxytetrahydro-1H-pyrrolo[2,1-c][1,4]benzothiazines with iminopentitol substructures corresponding to the L-lyxo and D-ribo configurations. They were designed to present a favorable transition-state mimic for the inhibition of ribosidases. This new thiazine class opens the way to the development of new inhibitors to carbohydrate processing enzymes of therapeutic importance such as nucleoside hydrolases and purine nucleoside phosphorylases.

Trihydroxy-2-thiaquinolizidines, a new class of bicyclic dideoxy-iminohexitol glycosidase inhibitor derivatives with nominally the d -gluco, l -ido, d -manno and l -gulo configurations were synthesized. X-ray analyses indicated that the preferred conformation for d -gluco and d -manno derivatives was a flat trans-fused system. Unlike deoxynojirimycin, the compound with d -gluco configuration was selective for α-glucosidases (yeast and rice) and showed no inhibitory activity towards β-glucosidase (almond), α-galactosidase (green coffee beans), α-galactosidase (E. coli) and α-mannosidase (jack bean), while the l -ido derivative was specific for β-glucosidase (almond).

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.

The important d -glucose and d -glucose 6-phosphate analogues 1,5-anhydro- d -glucitol and 1,5-anhydro- d -glucitol 6-phosphate were prepared from methyl- d -glucoside in high yield and purity. Protecting of the hydroxyl groups as their allyl ether followed by reductive cleavage of the glycosidic linkage with triethylsilane formed the protected anhydroglucitol. No ring rearrangement or ring contraction was observed during the reduction step. Using the PdCl 2 –CuCl 2 –activated charcoal system, the allyl ether bond was cleaved with a low loading of the catalyst (0.0025 equiv per allyl group). 1,5-Anhydro- d -glucitol 6-phosphate was prepared by the phosphylation of 1,5-anhydro- d -glucitol.

Here we present a one-pot procedure for the synthesis of a library of carbohydrate compounds belonging to four classes of glycosidase inhibitors: glycosides, aldonic acid lactams, aldonic acid lactones and dideoxy-iminoalditols. The complex mixture was separated into individual components that were screened for activity against α- and β-glucosidases. Among these were two 2- O -methyl glycosides that reduced the rate of the enzymatically-catalyzed hydrolysis of the p -nitrophenyl glucosides used in the assay. Relative inhibition indices normalized to the inhibition observed with deoxynojirimycin under similar conditions were determined. This methodology provides a straightforward, general and time-efficient way to synthesize, test and analyze an extensive number of glycosidase inhibitor candidates from several carbohydrate compound classes in one scheme. The direct reference to deoxynojirimycin gives an immediate indication of the concentration range in which a significant inhibitory activity can be obtained.

A simple high-yield three-step route to O -tritylated optically active 5-hydroxymethyl oxazolidinones from optically active 3-hydroxy-γ-butyrolactones is described. The key intermediate is the 4- O -trityl ether of homochiral 3,4-dihydroxybutyramide, which is obtained in quantitative yield from 3-hydroxy-γ-butyrolactone by treatment with ammonia. It is readily transformed to the oxazolidinone by Hoffmann rearrangement in a two-phase system. The carbonyl group in the oxazolidinone is derived from C-1 of the amide, and a separate carbonylation reaction is not required. Oxazolidinones are key compounds in drug synthesis especially in the areas of antibacterials and behavior disorder therapy.

Very long lipid tubules with uniform widths, spirals, and spirals nested in tubular superstructures are formed rapidly from a lipid analogue made by acylating 1- (N,N-1-dimethylamino)ethyl-2,3-dihydroxybutyramide with two molecules of pentacosa-10,12-diynoic acid. The high propensity of this molecule for tubule formation is attributed to the large carbonyl dipole in the headgroup. The tubules formed from the pure lipid are hollow and can be gold-plated by vapor deposition without collapsing. The superstructures can also be metallized by adsorption of nickel(II) or copper(II) ions followed by reduction with borohydride. The nickel-metallized tubules can be oriented in very modest magnetic fields. Tubules can be over 1000 μm in length and have a wide diameter. The unusual length, stability, and ease of preparation of these tubules should be of great utility in the construction of microstructures.

(R)-3-Hydroxy-4-trimethylaminobutyric acid ( l -carnitine) and (R)-4-amino-3-hydroxybutyric acid (GABOB) are two compounds with a very high level of medical significance. They can be prepared from (R)-3-hydroxy-γ-butyrolactone which is not readily available in significant quantities. The corresponding (S)-lactone is available in large quantities but attempts at inverting the stereochemistry of the hydroxyl group lead to elimination to give the furanone. Here we describe a straightforward route to these two compounds, starting from (S)-3-hydroxy-γ-butyrolactone by adding a highly oxidized carbon at one end whilst removing one carbon from the other, thus switching the functional group priorities. In this method, the lactone is transformed to an (R)-4-cyano-3-hydroxybutyric acid ester which is then converted to an acyl hydrazide by treatment with hydrazine. This stable, crystalline hydrazide has not been described before. It is readily converted to (R)-4-amino-3-hydroxybutyronitrile, a precursor of l -carnitine and GABOB, by Curtius rearrangement under conditions that do not result in deamination.

Phosphatidyl ethanolamine is one of the most common naturally occurring phospholipids. The presence of an amino group also makes it one of the most straightforward to functionalize. Unfortunately h...

Two new, very accessible, chiral, self-assembling phospholipid analogues containing diacetylenic units in the middle of their acyl chains were prepared by very simple and highly efficient routes. Films were cast and then polymerized by activation with UV light or doped with iodine. X-ray diffraction, NMR spectroscopy measurements, and molecular mechanics analyses using the MM3 force field indicated that the acyl chains are closely packed in a parallel fashion. The new molecules and the polymerized films derived from them were characterized by a variety of physical methods including confocal polarized light microscopy, atomic force microscopy, and near-infrared spectroscopy. These analytical methods showed that the unpolymerized molecules are able to form lamellar systems with very-long range order. They formed very uniform, extremely flat, thin films which are readily polymerized to give extensively conjugated systems which absorbed well out into the near-infrared region unlike typical polydiacetylenes. T...

Glycosyl diacylglycerols are excellent lipids for the formation of both bi- and monolayer lamellar systems but they are generally not commercially available, and the synthesis of optically pure gly...

The stereoselective addition of the 2-hydroxyl group of glucose to the mercurated vinyl group of 2-alkenyl glycosides followed by hydride reduction and removal of the saccharide fragment was used to prepare enantiomerically pure 1,2-dihydroxy alkanes. Diols of (R) or (S) configuration can be synthesized from (α)-glycosides or the (β) form respectively. Demercuration with chloride ion led to the insertion of a halo group adjacent to the new chiral center thus allowing for the possibility of further functionalization.

Steroids were isolated from the blood-sucking leech species Hirudo medicinalis and their structure was studied with one- and two-dimensional NMR spectroscopy (DQF-COSY and HMQC), GC-MS and ESI-MS spectrometry. Fractionating leech lipid using silicic acid chromatography led to the isolation of cholesterol in an early chloroform-eluted peak. Only minor traces of cholest-4-en-3-one, 4 beta-methylcholesterol, and sitosterol were present. The subsequent acetone-eluted fraction contained steroidtriols that were further purified by preparative TLC; these included cholest-7-ene-3,5,6 triol, cholest-4,7-diene-3,6,15 triol and to a lesser amount, cholestane-3,5,6 triol. A developmental study on cholesterol content in the leech showed that it is also the principal steroid in embryonic and freshly hatched leeches prior to feeding. The abundance of cholesterol, comprising approximately 5% of the total leech lipid, suggests that H. medicinalis, a blood sucking leech, has adapted itself fully to its mammalian host in terms of its steroid content. It remains to be seen whether lipids are directly transferred from the host to the parasite or whether leeches have evolved mechanisms to synthesize their own steroids.

To maintain an optimum dynamic range, membranes of living systems must have the ability to regulate their translational and vibrational motion in the face of environmental changes that might offset them. This is done through structural modifications of the lipids. Sarcina ventriculi was used as a case study to explore membrane structural reorganizations which allow some organisms to adapt to extreme environmental changes. It is capable of a variety of unusual and dramatic chemical processes including lipid alkyl chain tail-to-tail and lipid head-to-head coupling. There is also interlipid headgroup transfer or shuffling. The tail-to-tail coupling activity is capable of joining foreign (exogenously added) hydrocarbon chains to the native chains. The adaptative processes occur dynamically and instantaneously and render this organism tolerant to low and high pH, moderately high temperatures, the presence of organic solvents, and a wide spectrum of antibiotics at concentrations as high as 200 μg/mL. Chemical a...

Lowering oxygen tension in free-living Bradyrhizobium japonicum resulted in a dramatic switch of membrane chemistry in which phosphatidylcholine, the predominant lipid in aerated cultures, was no longer synthesized and phosphatidylethanolamine became the major lipid. Besides this change, phosphatidylinositol, a typical plant lipid rarely found in bacteria, was also synthesized.

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.

A facile three-step sequence for the preparation of anhydroalditols starting from commodity carbohydrates is presented.

We have examined the diversity of glycoconjugates and cellular lipids of Rhizobium "hedysari" IS123, a bacterial symbiont that specifically nodulates the drought-tolerant forage legume Hedysarum coronarium. IS123 develops a complete capsule consisting of a loose fibrillar network of ruthenium-red-staining acidic polymers and produces two different exopolysaccharides (EPS). EPS-A contains glucose, galactose, mannose, and a noncarbohydrate substitution tentatively identified as a lactyl ester. The composition of EPS-B, which includes glucose and galactose, as well as O-acetyl, pyruvyl, and succinyl substituents, is very similar to that of the EPS-II described in Rhizobium meliloti. IS123 also makes an O-acetylated heterooligosaccharide and unsubstituted β-1,2-glucans. The cellular fatty acid composition of IS123 is dominated by 18:1 and also includes 14:0, 16:0, 16:1, 3OH-16:0, 17:0Δ, 18:0, 3OH-18:0, and 19:0Δ. Phospholipids of IS123 include phosphatidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethyl phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.Key words: Rhizobium, capsule, exopolysaccharides, oligosaccharides, lipids.

A new β-1-O-acyl-β-1,2-diglucosyl glycoside has been isolated from the membranes of the gram positive bacterium Sarcina ventriculi. Its levels are regulated by environmental conditions such as pH and temperature. The structure was determined by 2-D-NMR spectroscopy and by mass spectrometry.

Lipopolysaccharides from Gram-negative bacteria interact with the mammalian immune system to trigger a cascade of physiological events leading to a shock syndrome which results in the death in over 70% of cases of severe shock. It is known that the supramolecular structures of lipopolysaccharide aggregates are critical contributors to their biological activities. Despite this, the molecular basis for the formation if the regular hexagonal plates and arrays observed in lipopolysaccharide films and suspensions is unknown. Since these structures are two dimensional, it is unlikely that X-ray crystallographic methods will shed much light on their detailed structure. Knowing this structure is important since it is becoming increasingly likely that the insertion of the lipopolysaccharide hydrocarbon chains in the target host cell membrane may be involved in triggering host responses. This work describes the three-dimensional structure of the lipopolysaccharide lipid A moiety. The structure was obtained by a combination of molecular mechanics calculations and nuclear magnetic resonance spectroscopy. This involved calculation of the dihedral angle between the two glucosamine residues of the lipid A molecule from coupling constants and measuring critical interresidue NOE values. The study also takes into account information from X-ray powder diffraction and electron microscopy studies.

Sarcina ventriculi, an anaerobic Gram-positive bacterium, adapts to increasing temperature, the presence of organic solvents, or the lowering of the pH of its growth medium by joining the tails of membrane lipids from opposite sides of the bilayer, forming transmembrane, bifunctional fatty acid species. Since this is done to offset the increase in membrane mobility caused by these perturbations, it is of interest to determine whether the motional (dynamic) properties of membrane lipid alkyl chains are conserved. In this study, conservation of the motional time scales of the alkyl chains of total membrane lipids from Sarcina ventriculi cells grown at different pH values was demonstrated using proton nuclear magnetic resonance (NMR) spectroscopy. The NMR longitudinal relaxation times (T1) of the protons in the bulk methylene groups were measured for lipids from cells grown at pH 3.0 and 7.0. These measurements indicated that the temperature profile of the T1 relaxation behavior for the methylene protons from these two different preparations was the same. Analysis of the data from T1 measurements indicated that the thermal barrier for relaxation is the same in both lipid systems. This is only true if the pH of the sample on which the measurement is being made is adjusted to the same value as that at which the corresponding cells were cultured. It is clear from this latter observation that the state of protonation of the lipid head groups is a contributor to the overall motional freedom of the membrane lipid components. The correlation times (tau c) of characteristic lipid alkyl chain motion were estimated to be approximately 10(-10) s.(ABSTRACT TRUNCATED AT 250 WORDS)

The general view on Rhizobium chitolipooligosaccharides (CLOS) is that they are made in very low levels as diffusible molecules and are primarily secreted by the bacteria into the extracellular milieu where they interact with the host. However, the structural and predicted physicochemical properties of these amphiphilic molecules led us to postulate that they should normally be targeted to bacterial membranes after synthesis. Thus, we analyzed membrane lipid extracts of Rhizobium leguminosarum bv. trifolii wild-type strain ANU843 cells and the corresponding culture supernatants for CLOS-type glycolipids. As predicted, fractionation of the membrane extracts from pelleted cells led to the isolation of a diverse family of CLOS in high yield (> or = 15 mg/L of culture), whereas all attempts to isolate CLOS from the corresponding culture supernatant failed. Structural analyses reveal that the membrane CLOS of ANU843 consist of a complex mixture of O-acetylated or non-O-acetylated chito- tri-, -tetra-, and pentasaccharides bearing an N-acyl moiety at the nonreducing glucosamine residue. cis-Vaccenic acid was the predominant acyl substituent (> 70%), but several other saturated, unsaturated, and 3-hydroxy fatty acids were found in the CLOS glycolipids. Membrane accumulation of CLOS in ANU843 is promoted by the presence of 4',7-dihydroxyflavone and pSym nod genes. Potential host-selective biological activity host-selective biological activity of the purified membrane CLOS fraction from ANU843 was indicated by its ability to elicit meristems resembling rudimentary nodule primordia in the root cortex of axenic seedlings of the host legume, white clover, but not of the nonhost legumes hairy vetch or alfalfa.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

In order to test the role of the supramolecular structure of lipopolysaccahrides (LPS) in determining endotoxicity, tow synthetic inhibitors, E11 and E12, which are structurally unrelated to lipid A, were designed, synthesized and examined for their ability to inhibit the biological activities of LPS. Both molecules were capable of inhibiting the activity of lipopolysaccharides in standard assays. The syntheses and aspects of the biological activities are described here.

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 β-isomer of C-glycoside, ethyl 2-acetamino-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosylacetate( 3 ) was prepared stereoselectively in two steps by reaction of 2-acetamino-2-deoxy-3,4,6-tri-O-acetyl-α-D-glucopyranosyl chloride( 4 ) with potassium diethylmalonate and 18-crown-6, followed by decarboxylation.

In order to test the role of the supramolecular structure of lipopolysaccahrides (LPS) in determining endotoxicity, tow synthetic inhibitors, E11 and E12, which are structurally unrelated to lipid A, were designed, synthesized and examined for their ability to inhibit the biological activities of LPS. Both molecules were capable of inhibiting the activity of lipopolysaccharides in standard assays. The syntheses and aspects of the biological activities are described here.

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 stereoselective addition of the 2-hydroxyl group of glucose to the mercurated vinyl group of 2-alkenyl glycosides followed by hydride reduction and removal of the saccharide fragment was used to prepare enantiomerically pure 1,2-dihydroxy alkanes. Diols of (R) or (S) configuration can be synthesized from (α)-glycosides or the (β) form respectively. Demercuration with chloride ion led to the insertion of a halo group adjacent to the new chiral center thus allowing for the possibility of further functionalization.

A short, efficient synthesis of (R)-3-hydroxytetradecanoic acid, a key component of bacterial endotoxins, using (R)-oxirane acetic acid ethyl ester as the source of chirality is described. The method is general and can be used in the preparation of other chiral 3-hydroxy acids.

Phosphatidyl ethanolamine is one of the most common naturally occurring phospholipids. The presence of an amino group also makes it one of the most straightforward to functionalize. Unfortunately h...

Here we present a one-pot procedure for the synthesis of a library of carbohydrate compounds belonging to four classes of glycosidase inhibitors: glycosides, aldonic acid lactams, aldonic acid lactones and dideoxy-iminoalditols. The complex mixture was separated into individual components that were screened for activity against α- and β-glucosidases. Among these were two 2- O -methyl glycosides that reduced the rate of the enzymatically-catalyzed hydrolysis of the p -nitrophenyl glucosides used in the assay. Relative inhibition indices normalized to the inhibition observed with deoxynojirimycin under similar conditions were determined. This methodology provides a straightforward, general and time-efficient way to synthesize, test and analyze an extensive number of glycosidase inhibitor candidates from several carbohydrate compound classes in one scheme. The direct reference to deoxynojirimycin gives an immediate indication of the concentration range in which a significant inhibitory activity can be obtained.

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.

An extracellular metabolite purified from Rhizobium trifolii ANU843 was established as N-acetylglutamic acid (GluNAc) by 1H NMR and Fourier transform IR spectroscopy, gas chromatography/mass spectrometry of its methylated product, and organic synthesis. TLC analyses indicated that extracellular accumulation of GluNAc by R. trifolii ANU843 grown in defined BIII culture medium was dependent on induction of its bacterial nodulation (nod) genes and the positive regulatory gene nodD on its symbiotic plasmid. 1H NMR analyses showed less GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843. GluNAc induced three morphological responses on axenic roots of white clover seedlings: (i) root hair branching; (ii) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which developed into nodule-like primordia. These biological activities of extracellular GluNAc from R. trifolii ANU843 were confirmed with authentic standards of GluNAc. These results indicate that extracellular accumulation of N-acetylglutamic acid is linked to flavone-dependent metabolism involving nodD, nodL, and nodM in R. trifolii ANU843. This constitutes the first report on the structure of a nod-dependent extracellular signal from R. trifolii that can affect root hair and nodule development in white clover and whose biological activity on this host has been confirmed with authentic standards.

While glycosyltransferases are restrictively expressed in invertebrate model organisms, little is known of their glycan end products. One such restrictively expressed glycoepitope was localized to sensory and epithelial cells of leech and Caenorhabditis elegans using the Lan3-2 monoclonal antibody. A biological function for the neural Lan3-2 epitope was previously determined in the leech. Here we report on the chemical structure of this mannosidic epitope harvested from whole Hirudo medicinalis. Crude glycans were liberated from glycoproteins by hydrazinolysis. Re-N-acetylated glycans were subjected to immunoaffinity purification. The affinity-purified glycans were fractioned by size chromatography into oligosaccharides and polysaccharides. Lan3-2 oligosaccharide structure was characterized by gas chromatography of alditol acetates, methylation analysis, 500 MHz 1H NMR spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem MS-MS of permethylated derivatives. The predominant components of the Lan3-2 oligosaccharide fraction were a series of linear beta-(1,4)-linked mannose polymers. The homologous expression of the Lan3-2 epitope in C. elegans will facilitate the exploration of its glycosylation pathway. Other invertebrates expressing the Lan3-2 epitope are Planaria dugesia, Capitella sp. I and Lumbriculus variegatus. The glycoepitope was not detected in the diploblastic animals Hydra littoralis and Aptaisia sp. or in deuterostomes.