Two kinds of tetravalent double-headed sialo-glycosides with short/long spacers between the Neu5Acα2,6Galβ1,4GlcNAc unit and ethylene glycol tetraacetic acid (EGTA) scaffold were found to be capable of binding to virus-like particles of Merkel cell polyomavirus (MCPyV-LP). The binding process and time course of interaction between the tetravalent ligand and MCPyV-LP were assessed by dynamic light scattering (DLS). On the addition of increasing concentrations of ligand to MCPyV-LP, larger cross-linked aggregates formed until a maximum size was reached. The binding was stronger for the tetravalent ligand with a short spacer than for that with a long spacer. The binding of the former ligand to the virus was observed to proceed in two stages during agglutination. The first step was the spontaneous formation of small aggregates comprising the cross-linked ligand-virus complex. In the second step, the aggregates grew successively larger by cooperative binding among the initially produced small aggregates. In transmission electron microscopy, the resulting complex was observed to form aggregates in which the ligands were closely packed with the virus particles. The cross-linked interaction was further confirmed by a simple membrane filtration assay in which the virus-like particles were retained on the membrane when complexed with a ligand. The assay also showed the effective capture of particles of pathogenic, infectious human polyomavirus JCPyV when complexed with a ligand, suggesting its possible application as a method for trapping viruses by filtration under conditions of virus aggregation. Collectively, these results show that the tetravalent glycocluster serves as a ligand not only for agglutinating MCPyV-LP but also for trapping the pathogenic virus.

mine (LacNAc) or related compo""ds were synthesized. First, sugars were collyerted into theiT corresponding t7glycosylamines with ammonium hydrogen carbonate. Then, the fi-glycosylamines were condensated with the carboxyl groups of poly(Lriglutamic acid). IVLGIycopolypeptides with different degrees of substitution of sugars were iselated by passage throllgh a column of Sephadex G-25. These synthetic polymers were used as model compounds in the analysis of oligosaccharide-lectin interactions. Interactions with some lectins were inyestigated by agar-gel double-difusion tests and in terms of inhibition of hemag-

An end-modified beta-D-galactosyl chitotetraose derivative [4(4)-O-beta-D-galactosyl-beta-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)(3)D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN)(3)D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)(3)D was specifically cleaved to Gal(GlcN)(2) and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GIcN)(3)D to lysozyme is more favorable than that of (GlcN)(4)D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)(3)D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GIcN)(3)D by lysozyme through a conjugated reaction involving beta-N-acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2-31 mu g of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible. (C) 2017 Elsevier Inc. All rights reserved.

Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.

A series of multivalent sialoglyco-conjugated nanoparticles were efficiently synthesized by using highly-branched α-glucuronic acid-linked cyclic dextrins (GlcA-HBCD) as a backbone. The sialoglycoside-moieties, with varying degrees of substitution, could be incorporated onto the preformed nanoparticles. These synthesized particles, which are highly soluble in aqueous solution, were shown to have a spherical nanostructure with a diameter of approximately 15nm. The interactions of the sialoglyco-nanoparticles (Neu5Acα2,6LacNAc-GlcA-HBCDs) with human influenza virus strain A/Beijing/262/95 (H1N1) were investigated using a hemagglutination inhibition assay. The sialoglyco-nanoparticle, in which the number of sialic acid substitution is 30, acted as a powerful inhibitor of virus binding activity. We show that both distance and multiplicity of effective ligand-virus formation play important roles in enhancing viral inhibition. Our results indicate that the GlcA-HBCD backbone can be used as a novel spherical nanocluster material for preparing a variety of glyco-nanoparticles to facilitate molecular recognition.

Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Å resolution crystal structure of the periplasmic i-type lysozyme inhibitor from Aeromonas hydrophila (PliI-Ah) in complex with the i-type lysozyme from Meretrix lusoria is reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key-lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme-inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key-lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs.

A novel alkyl spacer-conjugated derivative of P(k) trisaccharide (P(k)), one of the active receptors of Shiga toxins (Stxs; Stx1 and Stx2) produced by pathogenic Escherichia coli (STEC), was designed and synthesized by a combination of cellulase-mediated condensation from Trichoderma reesei and α1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae. The specific activity of N. gonorrhoeae LgtC was 66U/mg, which was 13-fold higher than that from N. meningitidis expressed in E. coli. 5-trifluoroacetamidopentyl-β-P(k) (TFAP-P(k)) was synthesized (yield of 86%, based on the amount of TFAP-lactose added) and its binding to Stx1a-B and Stx2a-B was evaluated. The dissociation constants (KDs) of Stx1a-B and Stx2a-B to the spacer-linked P(k), immobilized on a CM5 sensor chip, were 6.8×10(-6) M (kon=4.1×10(1)M(-1)S(-1), koff=2.8×10(-4)S(-1)) and 2.2×10(-5)M (kon=3.9×10(2)M(-1)S(-1), koff=8.6×10(-3)S(-1)), respectively. This result suggests that the monovalent P(k)-derivative, conjugated to a pentylamino group, represents a promising Stx-neutralizing agent. This cellulase-mediated condensation using cellulase and glycosyltransferase is a valuable tool for the synthesis of spacer-linked oligosaccharide.

Tri-N-acetylchitotriosyl moranoline, (GlcNAc)3-M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-β-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem. 288:6072-6082). The findings prompted us to examine the interaction of di-N-acetylchitobiosyl moranoline, (GlcNAc)2-M, with a family GH19 chitinase from moss, Bryum coronatum (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (Tm) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc)2-M, while the chitin dimer, (GlcNAc)2, elevated Tm only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc)3 and (GlcNAc)2-M with BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)2 was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using (15)N-labeled BcChi19A and BcChi19A-E61A revealed that both (GlcNAc)2 and (GlcNAc)2-M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc)2-M is markedly stronger than that of (GlcNAc)2 for both enzymes. However, (GlcNAc)2-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by BcChi19A (IC50 = 130-620 μM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc)2-M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc)2-M did not fit into the catalytic cleft (subsite -1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the BcChi19A-catalyzed hydrolysis.

1 Department of Chemistry and Biochemistry, Fukushima National College of Technology 30 Nagao, Iwaki, Fukushima 970―8034, Japan 2 Department of Bioscience, Graduate School of Science and Technology, Shizuoka University 836 Ohya, Suruga ward, Shizuoka 422―8529, Japan 3 Department of Advanced Bioscience, Kinki University 3327―204 Nakamachi, Nara 631―8505, Japan 4 Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) 1―1―1 Higashi, Tsukuba, Ibaraki 305―8566, Japan

Non-catalytic synthesis of 4-O-β-2-acetamido-2-deoxy-d-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-glucopyranose (GND) from chitin disaccharide, N,N′-diacetylchitobiose (GlcNAc)2, was achieved, with a maximum yield of 24.7% in high-temperature water at 120–220 °C and 25 MPa with a reaction time of 8–39 s.

4-O-beta-Di-N-acetylchitobiosyl moranoline (2) and 4-O-beta-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)(4), and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with K-i values of 2.01 x 10(-5) and 1.84 x 10(-6) M, respectively. From IT Canalysis, the binding of 3 was found to be driven by favorable enthalpy change (Delta H-r degrees), which is similar to those obtained for 2 and (GlcNAc)(4). However, the entropy loss (-T Delta S-r degrees) for the binding of 3 was smaller than those of 2 and (GlcNAc)(4). Thus the binding of 3 was found to be more favorable than those of the others. Judging from the K-d value of 3 (760 nM), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites -4 to -1 and the moranoline moiety adopts an undistorted C-4(1) chair conformation almost overlapping with the -1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G.J., Laine, R., and Withers, S. G. (2001) Nature 412, 835-838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.

The hemagglutinin (HA) of avian influenza viruses plays a very important role in the infection of host cells. In this study, the HA gene of the highly pathogenic avian influenza H5N1 virus was cloned and expressed in silkworm larvae. The expressed recombinant HA (rHA) was purified using fetuin-agarose chromatography and Superdex 200 10/300 GL gel filtration chromatography, and the identity of purified rHA was confirmed by SDS-PAGE and Western blot. Approximately 500 μg of purified rHA was obtained from a total of 30 silkworm larvae, suggesting the high efficiency of the silkworm expression system. The purified rHA bound to a rabbit polyclonal antibody against influenza A virus H5N1 (avian flu) HA, suggesting its antigenicity and potential application in vaccine development. Gel filtration chromatography showed that purified HA was present in the void volume fractions, indicating that rHA may form an oligomer. The rHA bound to poly{Neu5Acα2,3LacNAcβ-O[(CH₂)₅NHCO]₂(CH₂)₅NH-/γ-PGA}, which mimics an avian type receptor, but did not bind to γ-polyglutamic acid or human type receptor mimic, poly{Neu5Acα2,6LacNAcβ-O[(CH₂)₅NHCO]₂(CH₂)₅NH-/γ-PGA}, suggesting that it could be utilized as a blocking agent against infection by highly pathogenic influenza viruses.

Non-catalytic synthesis of 2-acetamido-2,3-dideoxy-D-erythro-hex-2-enofuranose (Chromogen I) and 3-acetamido-5-(1′,2′-dihydroxyethyl)furan (Chromogen III) from N-acetyl-D-glucosamine (GlcNAc) was achieved, with the highest yields of 23.0% and 23.1%, respectively, in high-temperature water at 120–220 °C and 25 MPa with a reaction time of 7–39 s.

Influenza A virus (IAV) recognizes two types of N-acetylneuraminic acid (Neu5Ac) by galactose (Gal) linkages, Neu5Acα2,3Gal and Neu5Acα2,6Gal. Avian IAV preferentially binds to Neu5Acα2,3Gal linkage, while human IAV preferentially binds to Neu5Acα2,6Gal linkage, as a virus receptor. Shift in receptor binding specificity of avian IAV from Neu5Acα2,3Gal linkage to Neu5Acα2,6Gal linkage is generally believed to be a critical factor for its transmission ability among humans. Surveillance of this shift of highly pathogenic H5N1 avian IAV (HPAI) is thought to be a very important for prediction and prevention of a catastrophic pandemic of HPAI among humans. In this study, we demonstrated that receptor binding specificity of IAV bound to sialo-glycoconjugates was sensitively detected by quantifying the HA gene with real-time reverse-transcription-PCR. The new assay enabled direct detection of receptor binding specificity of HPAIs in chicken clinical samples including trachea and cloaca swabs in only less than 4 h.

The artificial sulfated sialoglycopolypeptides (5 and 6) carrying the Neu5Acα2,3Galβ1,4 (6-sulfo) GlcNAc and Neu5Acα2,6Galβ1,4(6-sulfo)GlcNAc residues in the side chain were designed as hemagglutinin inhibitors against influenza viruses. 5-Trifluoroacetamidopentyl β-6-O-sulfo-N-acetylglucosaminide (5-TFAP-β-6SGN, 1) was first produced by β-N-acetylhexosaminidase-mediated transglycosylation using β-D-6-O-sulfo-GlcNAc-O-pNP (6SGN-β-pNP) as the donor and 5-trifluoroacetamido-1-pentanol as the acceptor. Galactosylation with glycosyltransferase was carried out to afford the key disaccharide, 6-Osulfated N-acetyllactosaminide derivative 2. After detrifluoroacetylation of 2, 6-O-sulfated N-acetyllactosaminide 3 with the 5-aminopentyl group at the reducing end was obtained in three steps in 62% yield. 6-O-Sulfated disaccharide 3 was then coupled with the carboxyl groups of γ-polyglutamic acid (γ-PGA) by a conventional BOP/HOBt chemistry, giving glycopolypeptide 4. Trans-sialylation of glycopolypeptide 4 with α2,3-sialyltransferase or α2,6-sialyltransferase gave the corresponding sulfated sialoglycopolypeptides 5 and 6, respectively. The binding affinity of sulfated sialoglycopolypeptides (5 and 6) to influenza virus hemagglutinin was examined using a hemagglutination inhibition assay. The sulfated α2,6-sialoglycopolypeptide (6) selectively inhibited hemagglutination mediated by human virus A/Aichi/2/68 (H3N2) and had a relative binding affinity for hemagglutinin of ca. 4.9 × 102-fold higher than that of the naturally occurring fetuin control. The sulfated α2,3-sialoglycopolypeptide (5) selectively inhibited hemagglutination mediated by avian virus A/Duck/HongKong/313/4/78 (H5N3) and displayed an approximately 1.2 × 103-fold higher affinity over fetuin. In addition, the binding affinity of 5 was slightly higher than that of the non-sulfated α2,3 sialoglycopolypeptide (7).

β-(1→6)-Glucanase from the culture filtrate of Penicillium multicolor LAM7153 was purified by ammonium sulfate precipitation, followed by cation-exchange and affinity chromatography using gentiotetraose (Gen4) as ligand. The hydrolytic mode of action of the purified protein on β-(1→6)-glucan (pustulan) was elucidated in real time during the reaction by HPAEC-PAD analysis. Gentiooligosaccharides (DP 2-9, Gen2-9), methyl β-gentiooligosides (DP 2-6, Gen2-6 β-OMe), and p-nitrophenyl β-gentiooligosides (DP 2-6, Gen2-6 β-pNP) were used as substrates to provide analytical insight into how the cleavage of pustulan (DP¯ 320) is actually achieved by the enzyme. The enzyme was shown to completely hydrolyze pustulan in three steps as follows. In the initial stage, the enzyme quickly cleaved the glucan with a pattern resembling an endo-hydrolase to produce a short-chain glucan (DP¯ 45) as an intermediate. In the midterm stage, the resulting short-chain glucan was further cleaved into two fractions corresponding to DP 15-7 and DP 2-4 with great regularity. In the final stage, the lower oligomers corresponding to DP 3 and DP 4 were very slowly hydrolyzed into glucose and gentiobiose (Gen2). As a result, the hydrolytic cooperation of both an endo-type and saccharifying-type reaction by a single enzyme, which plays a bifunctional role, led to complete hydrolysis of the glucan. Thus, β-(1→6)-glucanase varies its mode of action depending on the chain length derived from the glucan.

A novel and facile one-pot isomerization of aldo-disaccharides to keto-disaccharides is achieved by making use of deep sea water in sub-critical conditions. The resulting keto-disaccharides having pharmaceutical and food applications were obtained in modest yields of about 30–32% at the optimum reaction conditions (temperature: 180°C, reaction time: 5min). Detailed investigation of all major ions in deep sea water revealed that bicarbonate ions play a key role as a catalyst in this isomerization reaction.