13089-23-1

The β-N-acetylhexosaminidase of Aspergillus oryzae catalyses the formation of 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-glucopyranose (di-N-acetylchitobiose) and 2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-glucopyranose from p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside and 2-acetamido-2-deoxy-D-glucopyranose.The ratio of the two disaccharides is time-dependent.The ratio of (14)- to (16)-isomers is a maximum (ca. 9:1) at the point of disappearance of the glycosyl donor.If left to evolve, the ratio changes to 92:8 in favour of the (16)-isomer.Either the (14)- or the (16)-isomer can be isolated by treating the appropriately enriched disaccharide mixture with the β-N-acetylhexosaminidase of Jack bean (Canavalia ensiformis) or the β-N-acetylhexosaminidase of A. oryzae, respectively.Di-N-acetylchitobiose is an efficient donor of 2-acetamido-2-deoxy-D-glucopyranosyl units in reactions catalysed by the N-acetylhexosaminidase of A. oryzae.Di-N-acetylchitobiose itself acts as acceptor to give tri-N-acetylchitotriose .As the trisaccharide accumulates it, in turn, acts as acceptor giving tetra-N-acetylchitotetraose .The product mixture consisting of mono-, di-, tri-, and tetra-saccharides is conveniently separated by charcoal-Celite chromatography. Keywords: Glycosidase; N-acetylhexosaminidase; Chitooligosaccharides; Enzymatic synthesis.

β-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-β-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that β-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-β-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 m concentration of the salt with p-aminophenyl 1-thio-β-D-glucuronic acid-CH Sepharose 4B. Furthermore, the adsorbed enzyme was eluted by the addition of urea as well as ethylene glycol which are known as reagents which weaken hydrogen bonding. The results suggested that the interaction between the enzyme and the adsorbents with an amide bonding may be affected by the electrostatic force in the adsorbents under a high concentration of salt, although the electrostatic force decreases under the high concentration of salt. We also investigated whether or not the adsorbed enzyme was eluted by sodium cholate, cholic acid and triton X-100 known as hydrophobic reagents. It was assumed from the results of these chromatographies that the presence of amide bonding in adsorbents with glycosides as the ligand may be essential for the adsorption of the enzyme and that the glycosidic parts of the ligands have an effect on adsorption, however, it may not be essential for adsorption.

The present invention includes novel compositions and methods for treating comprising a compound with the Formula I: where n = 0-5; X = NH, O, S, CH2; Y = Phenyl, a phenyl group substituted with at least one methyl, a phenyl group substituted with at least one nitro, a phenyl group substituted with at least one nitrogen, a phenyl group substituted with at least one boron, aryl, substituted aryl, heteroaryl, four to six membered cycloalkyl, four to six membered heterocycloalkyl; R = H, C(O)R2, SO2R2; R1 = H, C(O)R2, SO2R2; R2 = Ethyl, methyl, isopropyl, n-propyl, t-butyl, n- butyl, NH2, NR3R4; R3, R4 = Ethyl, methyl, isopropyl, n-propyl, t-butyl, n-butyl, three to six membered cycloalkyl and Z = NH, O, S, CH2 or none, wherein the amount of the compound is selected to either inhibit or activate the immune response.

Protein O-glycosylation is a universal post-translational modification and plays essential roles in many biological processes. Recently we reported a technology termed cellular O-glycome reporter/amplification (CORA) to amplify and profile mucin-type O-glycans of living cells growing in the presence of peracetylated Benzyl-α-GalNAc (Ac3GalNAc-α-Bn). However, the application and development of the CORA method are limited by the properties of the precursor benzyl aglycone, which is relatively inert to further chemical modifications. Here we described a rapid parallel microwave-assisted synthesis of Ac3GalNAc-α-Bn derivatives to identify versatile precursors for cellular O-glycomics. In total, 26 derivatives, including fluorescent and bioorthogonal reactive ones, were successfully synthesized. The precursors were evaluated for their activity as acceptors for T-synthase and for their ability to function as CORA precursors. Several of the precursors possessing useful functional groups were more efficient than Ac3GalNAc-α-Bn as T-synthase acceptors and cellular O-glycome reporters. These precursors will advance the CORA technology for studies of functional O-glycomics.

The present invention relates generally to compositions and methods comprising abiotic, synthetic polymers with affinity and specificity to proteins. The synthetic polymers are an improvement over biological agents by providing a simpler, less expensive, and customizable platform for binding to proteins. In one embodiment, the compositions and methods relate to synthetic polymers with affinity and specificity to vascular endothelial growth factor (VEGF). In one embodiment, the compositions are useful for treating diseases and disorders related to the overexpression of VEGF. In one embodiment, the compositions are useful for treating cancer. In one embodiment, the compositions are useful for detecting VEGF levels from biological samples. In one embodiment, the compositions are useful for detecting overexpression of VEGF from biological samples. In one embodiment, the compositions are used to diagnose cancer.

A versatile glycosynthase (TnG-E338A) with strikingly broad substrate scope has been developed from Thermus nonproteolyticus β-glycosidase (TnG) by using site-directed mutagenesis. The practical utility of this biocatalyst has been demonstrated by the facile generation of a small library containing various oligosaccharides and a steroidal glycoside (total 25 compounds) in up to 100 % isolated yield. Moreover, an array of eight gluco-oligosaccharides has been readily synthesized by the enzyme in a one-pot, parallel reaction, which highlights its potential in the combinatorial construction of a carbohydrate library that will assist glycomic and glycotherapeutic research. Significantly, the enzyme provides a means by which glycosynthase technology may be extended to combinatorial chemistry.

2,2-Bipyridines having β-lactoside, β-d-glucoside, β-d-galactoside, and N-acetyl-β-d-glucosaminide were prepared and then, complexed with ferrous ion to afford trivalent glycoclusters having tris-bipyridine ferrous complex cores. Each glycocluster provides a dynamic combinatorial library composed of four diastereomeric stereoisomers (Δmer, Δfac, Λmer, and Λfac) whose ratios depend on their relative stabilities. CD spectral analyses of these glycoclusters showed that various cations (Na+, Mg2+, K+ or Ca2+) enriched Δ-forms of the glycocluster having β-lactosides and N-acetyl-β-d-glucosaminides possibly by cations-induced intramolecular carbohydrate-carbohydrate interactions.

The synthetic procedures for a large-scale preparation of o- and p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside are described. The synthetic pathway employs the glycosylation of phenol with ManNAc oxazoline, followed by nitration of the aromatic moiety yielding a separable mixture of the o- and p-nitrophenyl derivative in a 2:3 ratio.

An efficient method for the large-scale preparation of α-N-acetylglycosaminides, such as fluorogenic TN-antigen probes and an α-GalNAc-Ser derivative, has been achieved using FeCl3-catalyzed one-step condensation between commercially

The syntheses of 4-nitrophenyl β-glycosides of the 3-thio and 4-thio analogues of the two principal 2-acetamido-2-deoxy-hexoses found in living systems, GlcNAc and GalNAc, are described. While synthesis of the 4-thio analogues could be achieved via nucleophilic displacements of sulfonate derivatives with thioacetate, problems with neighbouring group acetamido participation necessitated the use of sulfamidate intermediates for the 3-thio analogues. These 3- and 4-thio analogues are employed in the chemo-enzymatic synthesis of thio-oligosaccharide analogues of structures present in glycosaminoglycans, glycoproteins and glycolipids.

Synthetic routes for the preparation of new sugar nitriles 8-10 derived from 2-acetamido-2-deoxy-β-D-glucopyranosides bearing a cyano group at the C-5 or C-6 position are presented. In an attempt to prepare the glycosyl azide 10 by treatment of tosylate 23 with KCN/DMF at 60°C, an intramolecular 1,3-dipolar cycloaddition reaction occurred to give the highly constrained nonisolable tetrazole 24, which was readily converted into the imino-azido compound 25 through an azido-tetrazole tautomerism. Compounds 8 and 10 were found to be poorer substrates of fungal β-N-acetylhexosaminidases than compound 9 and none of these compounds was accepted as substrates of the nitrilase or nitrile hydratase. Docking of the nitriles 8-10 in the active site of the β-N-acetylhexosaminidase from Aspergillus oryzae gave interaction energies comparable with the natural substrate. Based on these data, which indicate strong binding of these compounds (8 > 9 > 10) to the active site, it has been proposed that some cyano derivatives may act as competitive inhibitors of β-N-acetylhexosaminidases. This hypothesis is consistent with enzyme inhibition experiments which showed strong inhibitory properties of compound 9 (KI = 0.37 mM) and in particular of compound 8 (KI = 7.6 μM). Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Glycosylation of phenols with α-D-glucosaminyl chloride peracetate catalyzed by polyethylene glycol (PEG) was carried out in a solid-liquid phase transfer system at room temperature. The results were compared with those previously obtained for the catalys

The crown ether-catalyzed glycosylation of phenol, 4-methoxyphenol, and 4-nitrophenol was studied under phase transfer conditions in solid-liquid system. The asymmetric dibenzocrown esters are superior to [3.3]dibenzo-18- crown-6 and 15-crown-5 in the cat

The title compounds were synthesized by the selective reduction of the azido group in 4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranoside (8) and 4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-mannopyranoside (11), and by subsequent acetylation. Compound 8 was prepared by opening of the epoxide ring in methyl 2,3-anhydro-4,6-O-benzylidene-α-D-glucopyranoside (1) with sodium azide, followed by inversion of the configuration at C-3 in the resulting altropyranoside and glycosidation with 4-nitrophenol.

Glycosylation of various phenols with α-D-glucosaminyl chloride peracetate in a solid phase-liquid system catalyzed by crown compounds was studied. The highest yields of aryl β-glycosides were observed at room temperature in acetonitrile using anhydrous p

We have succeeded in achieving a facile access to 4-nitrophenyl and 4-methylumbelliferyl-α-sialosides. To sialosyl chlorides which are fully protected with acetyl groups as glycosyl donors, the action of phenols with diisopropylethylamine in acetonitrile brought about high yield as well as facile product isolation.

Four N-acetyl-β-D-glucosaminides, 4-amino-2,6-dibromophenyl (1a), 4-amino-2,6-dichlorophenyl (1b), 4-amino-2-chlorophenyl (1c) and 4-aminophenyl N-acetyl-β-D-glucosaminides (1d) were synthesized. Substrates lac were hydrolyzed by N-acetyl-β-D-glucosaminid

The glycosyl chloride 1 and bromides 2 and 3 were stereospecifically transformed into p-nitrophenyl glycosides by phase transfer catalysis; these glycohydrolase substrates were reduced and N-acryloylated to afford Michael acceptors which reacted with amine functions of proteins.

Starting from chloride 1, a series of para-substituted aryl 2-acetamido-2-deoxy-β-D-glucopyranosides were prepared using phase transfer catalysis conditions with tetrabutylammonium hydrogen sulfate in 1 M sodium hydroxide and methylene chloride at room temperature.Zemplen de-O-acetylation afforded the unprotected glycosides.Optimization of reaction conditions was evaluated.Several functional group manipulations were effected to widen the number and nature of the para-substituents. Key words: phase transfer catalysis, aryl 2-acetamido-2-deoxy-β-D-glucopyranosides.