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The beta(1?3) N-acetylgalactosaminyltransferase/UDP-N-acetylglucosamine C4 epimerase fusion protein was constructed and used in coupled enzymatic reactions to synthesize a variety of globotetraose and isoglobotetraose derivatives from the corresponding lactoside acceptors.

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beta-D-Galactosidase was purified 115-fold from a saline extract of papaya seeds by fractionation with ammonium sulfate, DEAE-Sephadex chromatography and gel-filtration on Sephadex G-75, G-150, and G-100.The purified beta-D-galactosidase (MW, 56,000 daltons) had an isoelectric point (pI) at pH 8,4 and the optimal pH for its activity was 3.5 to 4.5.The enzyme activity was inhibited by Cu(2+), Ag(1+), Hg(2+), Pb(2+), NaAsO2 and p-chloromercuribenzoate at concentrations of 1E-3 M.Among the various mono- and oligosaccharides tested, D-galactose, D-galacturonic acid, D-galactono-gamma-lactone and melibiose significantly inhibited the enzyme activities at concentrations of 2E-3 to 1E-2 M.The purified enzyme hydrolyzed p-nitrophenyl beta-D-galactoside (Km = 1.0E-3 M), methyl beta-D-galactoside (Km = 1.6E-2 M), aminoethyl beta-D-galactoside (Km = 3.3E-2 M) and lactose (Km = 9.1E-2 M). beta-(1–>3)-Linked galactotetraosyl-erythritol and asialo-glycopeptide isolated from fetuin were also hydrolyzed to the extent of 78 and 75percent, respectively, on the basis of their galactose contents. alpha-D-Mannosidase from papaya seeds was also purified 130-fold by ammonium sulfate fractionation, DEAE-Sephdex chromatography, gel-filtration on Sephadex G-150 and hydroxylapatite chromatography.The purified enzyme (MW, 156,000 daltons), consisting of two subunits (78,000 x 2), was inhibited by Hg(2+), Ag(1+), Cu(2+), p-chloromercuribenzoate, D-glucose, D-glucosamine and D-mannose at concentration of 1E-3 to 1E-2 M.The alpha-D-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannoside (Km = 5.6E-3 M), methyl alpha-D-mannoside (Km = 2.8E-2 M), alpha-D-mannosyl-D-mannitol (Km = 2.2E-2 M), alpha (1–>2)-linked D-mannobiosyl-D-mannitol (Km = 6.3E-3 M) and D-mannotriosyl-D-mannitol (Km = 5.3E-3 M).

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The synthesis of a series of six-membered 2-acetamido l-iminosugar C-alkyl and C-aryl glycosides is reported. A diastereoselective sugar-ring enlargement/C-alkylation (or arylation) /ring-contraction sequence applied to d-gluco- and d-manno-configured 2-acetamido azidolactols provides new l-iminosugar C-glycosides. The 1,2-trans stereocontrolled introduction of the pseudoanomeric substituent in these heterocycles strongly suggests a NHAc neighbouring-group participation. In their deprotected form, the nine iminosugars exhibit moderate hexosaminidases and beta-glucuronidase inhibition.

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A series of C3 O-functionalized 2-acetamido-2-deoxy-4-beta-d- glucuronides were synthesized to explore noncharge interactions in subsite 2 of the influenza virus sialidase active site. In complex with A/N8 sialidase, the parent compound (C3 OH) inverts its solution conformation to bind with all substituents well positioned in the active site. The parent compound inhibits influenza virus sialidase at a sub-muM level; the introduction of small alkyl substituents or an acetyl group at C3 is also tolerated.

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A versatile glycosynthase (TnG-E338A) with strikingly broad substrate scope has been developed from Thermus nonproteolyticus beta-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.

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From the methanolic extract of the leaves of Lansium domesticum, three new onoceranoid-type triterpenoids, lansium acids X?XII and a new cycloartane-type triterpene, lansium acid XIII, were isolated. The chemical structures of the isolated new compounds were elucidated on the basis of chemical/physicochemical evidence. For new onoceranoid-type triterpenoids, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. The isolated onoceranoid-type triterpenoids showed antimutagenic effects in the Ames assay against 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1).

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Glycosidase-mediated coupling of glycal acceptors with galactose donors affords beta-1,3-linked dissacharides that are versatile intermediates for further chemical and enzymatic manipulation.The utility of these disaccharides is demonstrated by the elaboration of these compounds into type I and type IV core disaccharides of carbohydrate-containing antigens.Further conversion of the type IV dissacharide to a mucin-type trisaccharide (Galbeta1,3(GlcNAcbeta1,6)GalNAc) was accomplished with the use of a beta-1,6-N-acetylglucosaminyltransferase.This combined use of enzymatic andchemical methodologies allows for the rapid assembly, with high regio- and stereoselectivity, of core oligosaccharides of biologically important glycoconjugates.

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Inexpensive and readily available sulfonic acids, p-toluenesulfonic acid, and sulfuric acid are versatile and efficient catalysts for the peracetylation of a broad spectrum of carbohydrate substrates in good yield and in a practical time frame. Three appealing features in sulfonic acid-catalyzed acetylation of free sugars were explored including (1) suppression of furanosyl acetate formation for d-galactose and l-fucose; (2) high yielding chemoselective acetylation of sialic acid under appropriate conditions; and (3) peracetylation of amino sugars with different amino protecting functions. Simple one-pot two step acetylation-thioglycosidation methods for the expeditious synthesis of p-tolyl per-O-acetyl thioglycosides were also delineated.

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Abstract: A continuous flow procedure for the synthesis of methyl glycosides (Fischer glycosylation) of various monosaccharides using a heterogenous catalyst has been developed. In-depth analysis of the isomeric composition was undertaken and high consistency with corresponding results observed under microwave heating was obtained. Even in cases where addition of water was needed to achieve homogeneity?a prerequisite for the flow experiments?no detrimental effect on the conversion was found. The scalability was demonstrated on a model case (mannose) and as part of the target-oriented synthesis of d-glycero-d-manno heptose, both performed on multigram scale.

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Analogues of the potent anti-influenza A and B compound, 4-guanidino-Neu5Ac2en, are described in which the stereochemically demanding C-6-glycerol side-chain is truncated.Syntheses of the one- and two-carbon side-chain analogues, of both 4-guanidino- and 4-amino-Neu5Ac2en, are presented, as well as the syntheses of analogues lacking any side-chain.Whilst complete removal of the C-6 side-chain abolishes activity, a stepwise increase in inhibition of influenza neuraminidase and influenza A and B in cell culture with increasing C-6 chain length is observed.The one-carbon, hydroxymethyl derivative retains significant activity to represent a suitable lead in the search for neuraminidase inhibitors of reduced stereochemical demand and synthetic complexity.

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