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Background: Vibrio parahaemolyticus is a Gram-negative bacterium widely distributed in marine environments and a well-recognized invertebrate pathogen frequently isolated from seafood. V. parahaemolyticus may also spread into humans, via contaminated, raw, or undercooked seafood, causing gastroenteritis and diarrhea. Methods: A Nuclear Magnetic Resonance (NMR)-based detection system was used to detect pathogenic levels of this microorganism (105 CFU/ml) with Molecular Mirroring using iron nanoparticles coated with target-specific biomarkers capable of binding to DNA of the target microorganism. The NMR system generates a signal (in milliseconds) by measuring NMR spin–spin relaxation time T2, which correlates with the amount of microorganism DNA. Results: Compared with conventional microbiology techniques such as real-time PCR (qPCR), the NMR biosensor showed similar limits of detection (LOD) at different concentrations (105–108 CFU/ml) using two DNA extraction methods. In addition, the NMR biosensor system can detect a wide range of microorganism DNAs in different matrices within a short period of time. Conclusion: NMR biosensor represents a potential tool for diagnostic and quality control to ensure microbial pathogens such as V. parahaemolyticus are not the cause of infection. The “hybrid” technology (NMR and nanoparticle application) opens a new platform for detecting other microbial pathogens that have impacted human health, animal health and food safety. Keywords: Nuclear magnetic resonance (NMR), Vibrio parahaemolyticus, Food safety, Aquaculture, Biosensors

N-Linked glycosylation is not only present in eukaryotes but also occurs in archaea and bacteria and is mainly characterized by the β-glucosylamine linkage to the asparagine (GlcNAcβAsn). Earlier crystallographic studies aimed at understanding the structural significance of the linkage region constituents revealed that N-glycosidic torsion, ϕN is influenced considerably by variation in the glycan part as compared to the aglycon moiety. The ϕN value observed for XylβNHAc deviated maximum as compared to that of the model compound, GlcNAcβNHAc. The present work was undertaken to assess the influence of ribose on the N-glycosidic torsions and molecular assembly. Several ribopyranosyl alkanamides have been synthesized and crystal structures of three of them have been solved. A comprehensive crystal structure analysis of ribosyl alkanamides along with xylosyl and arabinosyl alkanamides showed the wide range of deviations in their ϕN values as compared to the negligible deviation shown by hexopyranosyl alkanamides. This study revealed the importance of C5-hydroxymethyl group and hydroxyl group configurations at C2, C3, and C4 stereocentres in controlling the N-glycosidic torsions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Triazole-linked diversely functionalized divalent glycoconjugates were synthesized using Cu(I) catalyzed [3+2] cycloaddition reaction of the desired azides and alkynes. A series of per-O-acetylated glycopyranosyl azides and per-O-acetylated glucopyranosyl azidoacetamide were reacted with nitro- and cyano-functionalized dialkyne building blocks as precursors of α- and β-amino acids, respectively. To introduce more conformational flexibility in the divalent molecules, peptoid-based triazole-linked divalent glycoconjugate was also synthesized., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The torsion angle around the N-glycoprotein linkage region (GlcNAc-Asn) is an important factor for presenting sugar on the cell surface which is crucial for many biological processes. Earlier studies using model and analogs showed that this important torsion angle is greatly influenced by substitutions in the sugar part. In the present work, uronic acid alkanamides and triazole derivatives have been designed and synthesized as newer analogs of N-glycoprotein linkage region to understand the influence of the carboxylic group on linkage region torsion as well as on molecular packing. Crystal structure of N-(β-D-galacturonopyranosyl)acetamide is solved with the space group of P22121. Comparison of the torsion angle and molecular packing of this compound with N-(β-D-galactopyranosyl)acetamide showed that changing the C6-hydoxymethyl group to the carboxylic acid group has minimum influence on the N-glycosidic torsion angle, ΦN and significant influence on the molecular packing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The linkage region, GlcNAcβAsn, is conserved in all eukaryotic N-glycoproteins. As a logical extension of a research endeavor aimed at understanding the structural significance of GlcNAc and Asn as the linkage region constituents, the newer analogs GlcNAcβNHBu and (GlcNAcβ(1-4)GlcNAc)alkanamides have been synthesized to assess the influence of aglycon as well as additional GlcNAc on the linkage region. X-ray crystallographic analysis of the GlcNAcβNHBu and (GlcNAcβ(1-4)GlcNAc)βNHBu is described. Comparative analysis of these structures with those of reported models and analogs shows that the deviation in N-glycosidic torsion, ϕN among the GlcNAc alkanamides is negligible (<2°) whereas (GlcNAcβ(1-4)GlcNAc)βNHBu deviates by ∼15° as compared to GlcNAcβNHBu. Under the influence of the molecular packing, the conformation around the C1'-C2' bond deviates from anti to gauche in (GlcNAcβ(1-4)GlcNAcβNHBu. Interestingly, C2-acetamido group in (GlcNAcβ(1-4)GlcNAc)NHBu orients differently as compared to GlcNAc alkanamides and this orientation was found to be almost similar to β-N,N'-diacetylchitobiose trihydrate. The bifurcated anti-parallel pattern involving N-H⋯O and C-H⋯O hydrogen bonds, a hallmark feature of the N-glycoprotein models, GlcNAcβNHAc and GlcNAcβAsn, is absent in both the title alkanamides. This is the first report on the crystal structure analysis of chitobiosyl alkanamide as analog of the N-glycoprotein linkage region, (GlcNAcβ(1-4)GlcNAc)βAsn., (Copyright © 2013. Published by Elsevier Ltd.)

N-Glycoprotein linkage region constituents namely 2-deoxy-2-acetamido-β-D-glucopyranose (GlcNAc) and asparagine (Asn) are conserved among all eukaryotes. Earlier crystallographic studies on the linkage region conformation revealed that among all the models and analogs of the N-glycoprotein linkage region, XylβNHAc showed maximum deviation in the ϕN value as compared to the value reported for the model compound, GlcNAcβNHAc. In order to understand the effect of another pentopyranose, viz., arabinose, on the N-glycosidic torsion angles and molecular assembly, three arabinopyranosyl alkanamides were synthesized and their X-ray crystal structures elucidated. A comparative analysis of the N-glycosidic torsion, ϕN of the three analogs revealed the greater rotational freedom around the C1-N1 bond as compared to the GlcNAc derivatives. Molecular assembly of propionamido and chloroacetamido derivatives is characterized by the presence of anti-parallel bilayers of the molecules. This unique molecular assembly is hitherto unknown in all other models and analogs of N-glycoprotein linkage region. This study reveals that N-glycosidic torsions are influenced by the glycan as well as molecular packing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

GlcNAcbetaAsn linkage is conserved in the N-glycoproteins of all eukaryotes. l-Glutamine (Gln), which is a one carbon higher homolog of Asn, is never glycosylated. X-ray crystallographic study of several beta-1-N-acetamido- and propionamido derivatives of monosaccharides has earlier shown that the N-glycosidic torsion, Phi(N), is influenced to a larger extent by the structural variation of the sugar part than that of the aglycon moiety. In order to examine the influence of the carbohydrate pendent groups on the conformational preference of the N-glycosidic linkage with respect to Phi(N,) several models and analogs with gluco and manno configuration have been studied in the present work by computational chemistry. The crystal structure of XylbetaNHPr is reported here and its molecular packing compared with related analogs. The conjunction of combining Crystallographic and computational studies allows to demonstrate the strong influence that the group at C2, and environmental factors particularly inter- and intramolecular interactions involving regular hydrogen bonds and the weak C-H...O contacts, have on the energy preference of the Phi(N) torsion angle.

H-Beta zeolite, a microporous solid acid, is demonstrated to be an efficient catalyst for the selective deprotection of cyclic as well as acyclic O-isopropylidene sugar acetals derived from d-glucose, D-xylose, D-mannose, and D-mannitol in aqueous MeOH at room temperature. A notable observation is the conversion of d-mannitol triacetonide into 1,2:3,4-di-O-isopropylidene-D-mannitol (48%) and 3,4-O-isopropylidene-D-mannitol (36%) brought about in 6h by H-beta zeolite and the non-occurrence of any hydrolysis in the case of H-ZSM-5 catalyzed reaction in 24h under the same conditions.

Zeolite-catalyzed glycosylation of long-chain alcohols, using the inexpensive and readily available peracetylated beta-D-gluco- and galactopyranoses as glycosyl donors under solvent free conditions, has been explored for the first time. Among the various forms (H-, Na-, Fe- and Zn) of beta zeolite examined as catalysts in the reaction of 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose with cetyl alcohol, Fe-beta zeolite gave the maximum yield of 63% of cetyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside and cetyl 3,4,6-tri-O-acetyl-alpha-D-galactopyranoside. Fe-beta Zeolite-catalyzed glycosylation was found to be general affording the title compounds in each case in a moderate yield, but with a good stereoselectivity. The yield of synthetically valuable acetylated long-chain alkyl 1,2-cis C2-hydroxy-glycopyranosides obtained in the present single-step procedure is considerably higher than that of the previously reported multi-step method employing the Stork silicon tether approach.

Reported is an attractive and environmentally friendly method for the synthesis of the title compounds in moderate yield using inexpensive 1,2,3,4,6-penta-O-acetyl-beta-D-gluco- and galactopyranoses as sugar donors, five different phenols as acceptors and H-beta zeolite as the catalyst. The yield (23-28%) of aryl 3,4,6-tri-O-acetyl-alpha-D-glycopyranosides obtained in this single-step procedure is considerably higher than that obtained using previously reported methods. Treatment of an orthoacetate, 3,4,6-tri-O-acetyl-[1,2-O-(1-p-fluorophenoxyethylidene)]-alpha-D-glucopyranose, with p-fluorophenol under the same solvent-free reaction conditions also led to the formation of the title compounds in similar yield and composition. X-ray crystallographic analysis of phenyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranoside and p-fluorophenyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranoside showed that the molecular packing is stabilized by C-H...O, C-H...pi and C-H...F interactions, in addition to regular hydrogen bonding patterns.

The linkage region constituents, namely, 2-acetamido-2-deoxy-beta-D-glucopyranose and asparagine are conserved in the N-glycoproteins of all the eukaryotes. The present work is aimed at understanding the reasons for the occurrence of GlcNAc and Asn as the linkage region constituents. A total of six sugar amides have been designed as models and analogs of the linkage region and their crystal structures have been solved. This is the first report on the X-ray crystallographic investigation of the effect of systematic changes in the linkage sugar as well as its aglycon moiety on the N-glycosidic torsion, psi(N) (O5-C1-N1-C1(')). This also forms the first report on the crystal structure of a model of L-RhabetaAsn, a variant linkage found in the surface layer glycoprotein of Bacillus stearothermophillus. Among the models and analogs examined, the acetamido derivatives of Man and Xyl, the linkage sugars of O-glycoproteins, show a psi(N) value of -114.5 degrees and -121.2 degrees, respectively, deviating maximum from the value of -89.8 degrees reported for the model compound GlcNAcbetaNHAc. The L-Rha and Gal derivatives also show noticeable deviations. The psi(N) values, -89.5 degrees and -91.0 degrees, of the propionamide derivatives of Glc and GlcNAc (analogs of GlcbetaGln and GlcNAcbetaGln, respectively) agree well with those (-93.8 degrees and -89.8 degrees ) reported for their corresponding acetamide derivatives suggesting Gln could serve as well as Asn as the linkage region amino acid. However, the rotational freedom about the additional C-C bond would lead to altered rigidity of the linkage region. An analysis of packing reveals that the molecular assembly of these compounds is driven by different infinite and finite chains of hydrogen bonds. The double pillaring of hydrogen bonds involving the amide groups at C1 and C2 is seen as a unique packing feature characteristic of beta-1-N-acyl derivatives of GlcNAc. Based on the findings of the present study, it is speculated that the linkage region constituents of the eukaryotic N-glycoproteins appear to fulfill three essential structural requirements: rigidity, planarity, and linearity and these are met by the trisaccharide core and Asn at the linkage region.

Equilibrium tautomeric mixtures of several mono- and disaccharides are obtained in anhydrous form, without the use of water, by reacting the commercially available reducing sugars with ammonia gas in dry methanol, followed by the concentration of the resultant solution to dryness. Mutarotation and hydrolysis of the initially formed glycosylamine in the resultant medium account for the transformation. Equilibrium anomeric mixtures enriched in the beta-form of commercially available sugars such as alpha-D-glucose and alpha-lactose have not only vastly increased solubility, but are also synthetically valuable as these can be readily converted to the methyl/benzyl/trimethylsilyl ether and other derivatives for further transformations.

Capillary electrophoresis (CE) with fluorescence detection was used to determine the concentration of 4-methylumbelliferone liberated from 4-methylumbelliferyl-beta-D-glucuronide by beta-glucuronidase. Enzyme substrate saturation kinetics were studied in buffer and the pH range for the enzyme reaction was optimized. A linear relationship of initial enzyme reaction velocity as a function of peak area of enzyme product was obtained for enzyme activity ranging from 1 to 100 units. The beta-glucuronidase activity in urine was next determined. Freshly collected urine samples were dialyzed, the retentate was incubated with 4-methylumbelliferyl-beta-D-glucuronide, boiled and centrifuged. The supernatant was separated by CE in an uncoated capillary with 0.1 M sodium acetate buffer by applying a voltage of 12 kV. The product of the enzymatic reaction, 4-methylumbelliferone, was detected by fluorescence, facilitating the determination of as little as one unit of beta-glucuronidase activity in a 0.5-h incubation time, with an error of less than +/-5%.

Concanavalin A (Con A) and agglutinins from the pea (PSA), lentil (LCH), and fava bean (VFA) constitute a group of D-mannose/D-glucose binding legume lectins. In addition to their sugar binding specificity, these lectins also contain sites that bind hydrophobic ligands. The present study explores a class of nonpolar binding sites reportedly present adjacent to the carbohydrate binding site in PSA, LCH, and VFA. A series of 2-O- and 3-O-substituted nitrobenzoyl and nitrobenzyl derivatives of methyl alpha-D-glucopyranoside and methyl alpha-D-mannopyranoside were synthesized. Evaluation of their binding to Con A, PSA, LCH, and VFA was carried out by the technique of hapten inhibition of precipitation reaction. The hapten inhibition assay results reveal that the presence of a methyl or methylene group at the O-2 or O-3 position of the sugar is essential for hydrophobic interaction with PSA, LCH, and VFA. The substitution of methyl by nitrobenzyl leads to enhanced binding (1.7-16.7 times for the 2-O-substituted compounds and 7.9-40.5 times for the 3-O-substituted compounds) with the m-nitrobenzyl group contributing to maximum binding. A hydrophobic interaction is also involved between Con A and 2-O-nitrobenzyl derivatives, resulting in enhanced binding, but the corresponding 3-O-isomers bind poorly due probably to steric reasons. These results may be rationalized on the basis of the recently published X-ray data of Con A and VFA. The nitrobenzyl derivatives, after transformation to their azido analogs, have potential applications in the photoaffinity labeling of these lectins.

A new method is described for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates. The reducing ends of various mono-, di-, tri-, and tetra-saccharides were conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (a fluorescent and negatively charged compound) by reductive amination using sodium cyanoborohydride. The sugar conjugates were purified by preparative gradient polyacrylamide gel electrophoresis followed by a newly developed technique involving their semi-dry transfer to positively charged nylon membranes and elution with sodium chloride. The structures of a monosaccharide- and trisaccharide-conjugate were established by f.a.b.-m.s. and 2D n.m.r. Seven linear oligosaccharide-fluorescent conjugates were treated sequentially with exoglycosidases and with endoglycosidases. Analysis of the products by gel electrophoresis provided sequence information. These methods may be useful for sequencing oligosaccharides that are chemically or enzymically (endoglycosidase) released from glycoproteins, glycolipids, and proteoglycans.

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.