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Pavol Kovac - One of the best experts on this subject based on the ideXlab platform.
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mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of vibrio cholerae serotype ogawa and the recombinant tetanus toxin c fragment carrier
Journal of Mass Spectrometry, 2013Co-Authors: Farid Jahouh, Pavol Kovac, Peng Xu, Willie F Vann, Joseph BanoubAbstract:We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT–Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of Neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate–protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT–Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein. Copyright © 2013 John Wiley & Sons, Ltd.
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revealing the glycation sites in synthetic Neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of vibrio cholerae o1 serotype ogawa with the bsa protein carrier using lc esi qqtof ms ms
Journal of Mass Spectrometry, 2012Co-Authors: Farid Jahouh, Pavol Kovac, Rina Saksena, Joseph BanoubAbstract:In this manuscript, we present the determination of glycation sites in synthetic Neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the Neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic Neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein.
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determination of glycation sites by tandem mass spectrometry in a synthetic lactose bovine serum albumin conjugate a vaccine model prepared by dialkyl squarate chemistry
Rapid Communications in Mass Spectrometry, 2012Co-Authors: Farid Jahouh, Pavol Kovac, Joseph BanoubAbstract:RATIONALE Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. METHOD Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the Neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. RESULTS We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. CONCLUSIONS It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially. Copyright © 2012 John Wiley & Sons, Ltd.
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transcutaneous immunization with a synthetic hexasaccharide protein conjugate induces anti vibrio cholerae lipopolysaccharide responses in mice
Vaccine, 2009Co-Authors: Julianne E Rollenhagen, Pavol Kovac, Rina Saksena, Anuj Kalsy, Alaullah Sheikh, Mohammad Murshid Alam, Firdausi Qadri, Stephen B Calderwood, Edward T RyanAbstract:Antibodies specific for Vibrio cholerae lipopolysaccaride (LPS) are common in humans recovering from cholera, and constitute a primary component of the vibriocidal response, a serum complement-mediated bacteriocidal response correlated with protection against cholera. In order to determine whether transcutaneous immunization (TCI) with a V. cholerae neoglycoconjugate (CHO-BSA) comprised of a synthetic terminal hexasaccharide of the O-specific polysaccharide of V. cholerae O1 (Ogawa) conjugated with bovine serum albumin (BSA) could induce anti-V. cholerae LPS and vibriocidal responses, we applied CHO-BSA transcutaneously in the presence or absence of the immune adjuvant cholera toxin (CT) to mice. Transcutaneously applied neoglycoconjugate elicited prominent V. cholerae specific LPS IgG responses in the presence of CT, but not IgM or IgA responses. CT applied on the skin induced strong IgG and IgA serum responses. TCI with neoglycoconjugate did not elicit detectable vibriocidal responses, protection in a mouse challenge assay, or stool anti-V. cholerae IgA responses, irrespective of the presence or absence of CT. Our results suggest that transcutaneously applied synthetic V. cholerae neoglycoconjugate is safe and immunogenic, but predominantly induces systemic LPS responses of the IgG isotype.
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Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide Neoglycoconjugates.
FEMS Immunology & Medical Microbiology, 2006Co-Authors: Rina Saksena, Pavol Kovac, Terri K. Wade, William F. WadeAbstract:Ogawa hexasaccharide Neoglycoconjugates induce protective antibodies in mice. Similar Ogawa conjugates but with a longer linker that connects the carrier to shorter saccharides are immunogenic, but generally ineffective at inducing vibriocidal or protective antibodies. The efficacy of Ogawa hexasaccharide Neoglycoconjugates of different linker lengths were tested. The majority of mice given immunizations separated by a 14-day gap did not produce vibriocidal or protective antibodies. Mice immunized 28 days apart with immunogens containing the shortest or medium length linker, but not the longest, produced vibriocidal and protective antibodies. A nonprotective, priming dose of purified Ogawa LPS followed 5 days later with a booster of the Ogawa Neoglycoconjugates (di-, tetra-, or hexasaccharide) resulted in vibriocidal antibodies at day 10.
Joseph Banoub - One of the best experts on this subject based on the ideXlab platform.
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mapping the glycation sites in the neoglycoconjugate from hexasaccharide antigen of vibrio cholerae serotype ogawa and the recombinant tetanus toxin c fragment carrier
Journal of Mass Spectrometry, 2013Co-Authors: Farid Jahouh, Pavol Kovac, Peng Xu, Willie F Vann, Joseph BanoubAbstract:We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT–Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of Neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate–protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT–Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein. Copyright © 2013 John Wiley & Sons, Ltd.
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revealing the glycation sites in synthetic Neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of vibrio cholerae o1 serotype ogawa with the bsa protein carrier using lc esi qqtof ms ms
Journal of Mass Spectrometry, 2012Co-Authors: Farid Jahouh, Pavol Kovac, Rina Saksena, Joseph BanoubAbstract:In this manuscript, we present the determination of glycation sites in synthetic Neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the Neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic Neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein.
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determination of glycation sites by tandem mass spectrometry in a synthetic lactose bovine serum albumin conjugate a vaccine model prepared by dialkyl squarate chemistry
Rapid Communications in Mass Spectrometry, 2012Co-Authors: Farid Jahouh, Pavol Kovac, Joseph BanoubAbstract:RATIONALE Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. METHOD Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the Neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. RESULTS We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. CONCLUSIONS It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially. Copyright © 2012 John Wiley & Sons, Ltd.
Francisco Santoyogonzalez - One of the best experts on this subject based on the ideXlab platform.
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click multivalent homogeneous Neoglycoconjugates synthesis and evaluation of their binding affinities
European Journal of Organic Chemistry, 2009Co-Authors: Francisco Perezbalderas, Julia Moralessanfrutos, Fernando Hernandezmateo, Joaquin Isacgarcia, Francisco SantoyogonzalezAbstract:Mannose (α-Man)-containing Neoglycoconjugates possessing aliphatic-, aromatic-, and carbohydrate-centered architectures and differing in structural characteristics such as valency, topology, and nature of the linker have been synthesized using click chemistry that shows its value as an efficient and versatile methodology for accessing tailor-made multivalent Neoglycoconjugates. The binding behaviour of these glycomimics toward Concanavalin A (Con A) has been evaluated to determine the influence of the structural parameters. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
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click multivalent homogeneous Neoglycoconjugates synthesis and evaluation of their binding affinities eur j org chem 15 2009
European Journal of Organic Chemistry, 2009Co-Authors: Francisco Perezbalderas, Julia Moralessanfrutos, Fernando Hernandezmateo, Joaquin Isacgarcia, Francisco SantoyogonzalezAbstract:The cover picture shows two copper(I) click-based strategies for accessing multivalent neoglyconjugates by the coupling of complementary functionalized scaffolds and sugar derivatives. The methodology allows the easy preparation of structurally diverse homogeneous Neoglycoconjugates from which the influence of the structural parameters in the binding affinities toward Concanavalin A could be evaluated. Details are discussed in the article by F. Santoyo-Gonzalez et al. on page 2441ff.
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click multivalent heterogeneous Neoglycoconjugates modular synthesis and evaluation of their binding affinities
European Journal of Organic Chemistry, 2009Co-Authors: Mariano Ortegamunoz, Francisco Perezbalderas, Julia Moralessanfrutos, Fernando Hernandezmateo, Joaquin Isacgarcia, Francisco SantoyogonzalezAbstract:The efficient synthesis of structurally diverse, multivalent, heterogeneous Neoglycoconjugates by means of a series of different click-based strategies is described. The methodology is highly efficient and versatile allowing for easy access to a series of mannose (α-Man)-containing glycoconjugates differing in their valency, nature of the scaffold, nature of the constitutive sugars and length of the linker. The influence of those structural parameters on the binding affinities of these glycomimics toward Concanavalin A (Con A) was evaluated.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
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click multivalent Neoglycoconjugates as synthetic activators in cell adhesion and stimulation of monocyte machrophage cell lines
Organic and Biomolecular Chemistry, 2007Co-Authors: Mariano Ortegamunoz, Francisco Perezbalderas, Julia Moralessanfrutos, Fernando Hernandezmateo, Ma Dolores Girongonzalez, Natalia Sevillanotripero, Rafael Saltogonzalez, Francisco SantoyogonzalezAbstract:The efficient synthesis of fluorescent and non-fluorescent multivalent Neoglycoconjugates is described by means of the Cu(I) catalyzed azide–alkyne 1,3-dipolar cycloaddition (“click-chemistry”). A well-defined glycopolymer, glycocyclodextrin or glycocluster architecture displaying galactose or lactose epitopes has been chosen. Cellular assays using U-937 and RAW 264.7 monocyte/macrophage cells showed that these glycocompounds have the capability to act as synthetic activators mimicking the lipopolysaccharide (LPS) effects. Thus, the click compounds promote cell adhesion and stimulation of monocytes, measured as an increase in the amount of TNFα, facilitating their differentiation to macrophages.
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synthesis of multivalent Neoglycoconjugates by 1 3 dipolar cycloaddition of nitrile oxides and alkynes and evaluation of their lectin binding affinities
Tetrahedron, 2005Co-Authors: Francisco Perezbalderas, Fernando Hernandezmateo, Francisco SantoyogonzalezAbstract:The synthesis of multivalent Neoglycoconjugates by 1,3-dipolar cycloaddition of nitrile oxides and alkynes is reported. The nitrile oxides are generated in situ in the presence of alkynyl derivatives allowing the access to homo and hetero multivalent systems containing O- and C-linked glycosides and isoxazole bridges. The concanavalin A binding affinities of some of these neoglyconjugates bearing mannose residues were evaluated by enzyme-linked lectin essay (ELLA).
Rina Saksena - One of the best experts on this subject based on the ideXlab platform.
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revealing the glycation sites in synthetic Neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of vibrio cholerae o1 serotype ogawa with the bsa protein carrier using lc esi qqtof ms ms
Journal of Mass Spectrometry, 2012Co-Authors: Farid Jahouh, Pavol Kovac, Rina Saksena, Joseph BanoubAbstract:In this manuscript, we present the determination of glycation sites in synthetic Neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the Neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic Neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein.
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transcutaneous immunization with a synthetic hexasaccharide protein conjugate induces anti vibrio cholerae lipopolysaccharide responses in mice
Vaccine, 2009Co-Authors: Julianne E Rollenhagen, Pavol Kovac, Rina Saksena, Anuj Kalsy, Alaullah Sheikh, Mohammad Murshid Alam, Firdausi Qadri, Stephen B Calderwood, Edward T RyanAbstract:Antibodies specific for Vibrio cholerae lipopolysaccaride (LPS) are common in humans recovering from cholera, and constitute a primary component of the vibriocidal response, a serum complement-mediated bacteriocidal response correlated with protection against cholera. In order to determine whether transcutaneous immunization (TCI) with a V. cholerae neoglycoconjugate (CHO-BSA) comprised of a synthetic terminal hexasaccharide of the O-specific polysaccharide of V. cholerae O1 (Ogawa) conjugated with bovine serum albumin (BSA) could induce anti-V. cholerae LPS and vibriocidal responses, we applied CHO-BSA transcutaneously in the presence or absence of the immune adjuvant cholera toxin (CT) to mice. Transcutaneously applied neoglycoconjugate elicited prominent V. cholerae specific LPS IgG responses in the presence of CT, but not IgM or IgA responses. CT applied on the skin induced strong IgG and IgA serum responses. TCI with neoglycoconjugate did not elicit detectable vibriocidal responses, protection in a mouse challenge assay, or stool anti-V. cholerae IgA responses, irrespective of the presence or absence of CT. Our results suggest that transcutaneously applied synthetic V. cholerae neoglycoconjugate is safe and immunogenic, but predominantly induces systemic LPS responses of the IgG isotype.
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Length of the linker and the interval between immunizations influences the efficacy of Vibrio cholerae O1, Ogawa hexasaccharide Neoglycoconjugates.
FEMS Immunology & Medical Microbiology, 2006Co-Authors: Rina Saksena, Pavol Kovac, Terri K. Wade, William F. WadeAbstract:Ogawa hexasaccharide Neoglycoconjugates induce protective antibodies in mice. Similar Ogawa conjugates but with a longer linker that connects the carrier to shorter saccharides are immunogenic, but generally ineffective at inducing vibriocidal or protective antibodies. The efficacy of Ogawa hexasaccharide Neoglycoconjugates of different linker lengths were tested. The majority of mice given immunizations separated by a 14-day gap did not produce vibriocidal or protective antibodies. Mice immunized 28 days apart with immunogens containing the shortest or medium length linker, but not the longest, produced vibriocidal and protective antibodies. A nonprotective, priming dose of purified Ogawa LPS followed 5 days later with a booster of the Ogawa Neoglycoconjugates (di-, tetra-, or hexasaccharide) resulted in vibriocidal antibodies at day 10.
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effect of saccharide length on the immunogenicity of Neoglycoconjugates from synthetic fragments of the o sp of vibrio cholerae o1 serotype ogawa
Carbohydrate Research, 2005Co-Authors: Rina Saksena, Pavol Kovac, Terri K. Wade, William F. WadeAbstract:Abstract A synthetic hexasaccharide, identical to the terminal hexasaccharide of Ogawa LPS, coupled to bovine serum albumin induced protective antibodies in mice. To determine if there was a minimum saccharide length required for immunogenicity and efficacy, shorter (mono- to pentasaccharide) Neoglycoconjugates (CHO–BSA) were tested in mice. The Ogawa CHO–BSA was inoculated at either a constant mass but differing moles, or equal moles but differing masses. Humoral responses were essentially the same when mice received 9 μg of the carbohydrate (0.007 mM with the pentasaccharide) in each of the Neoglycoconjugates prepared from mono- through the pentasaccharide, or the same molar amount (0.007 mM), proportionally less by weight when going from the penta- to the monosaccharide. These data show that, within this dose range, the responses occurred virtually independently of the amount of immunogen. Humoral antibodies induced by these immunogens were generally not vibriocidal. Selected antisera induced by CHO–BSA immunogens were protective, but the ELISA titers of the sera were not predictive of the protective capacity. Purified, Ogawa LPS induced anti-Ogawa LPS IgM antibody titers similar to those induced by the Ogawa CHO–BSA conjugates. The anti-whole LPS sera were strongly vibriocidal, as were the previously reported sera induced by hexasaccharide conjugates. This suggests either that the shorter oligosaccharides lack a conformational epitope provided by the hexasaccharide or that the LPS has additional B cell epitopes or selects different B cells in the primary response.
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immunogens from a synthetic hexasaccharide fragment of the o sp of vibrio cholerae o 1 serotype ogawa
Tetrahedron-asymmetry, 2005Co-Authors: Rina Saksena, Jian Zhang, Pavol KovacAbstract:Abstract The known 5-(methoxycarbonyl)pentyl α-glycoside of the hexasaccharide of Vibrio cholerae O:1, serotype Ogawa 24 was newly prepared. The efficiency of construction of the hexasaccharide from the disaccharide glycosyl acceptor 6 and each of the two tetrasaccharide glycosyl donors 16, and 18, as an alternative to the iterative coupling of the disaccharide glycosyl donor 7 with 6, was evaluated. Compound 24 was treated with each of hydrazine hydrate, ethylenediamine, and hexamethylenediamine to give ligands 25, 27, and 29, respectively, equipped with different linkers. Reaction of the foregoing compounds with squaric acid diethyl ester, and the reactions of the thus formed monoesters 26, 28, and 30 with BSA were evaluated. The rate of formation of the corresponding monoester was higher with hydrazide 25 than with amines 27 and 29, whose reaction rates were virtually the same. Reactions of squaric acid derivatives 26, 28, and 30 with BSA were conducted under the same conditions (reaction temperature, ligand–BSA ratio, and concentration with respect to the hapten) and targeted for Neoglycoconjugates with 24–BSA ratio of ∼5. Monitoring the conversions by SELDI-TOF mass spectrometry in conjunction with the ProteinChip® system made it possible to conduct the conjugations in controlled fashion, and to terminate the reactions when the desired carbohydrate–protein ratios were reached. Products from 26, 28, and 30, Neoglycoconjugates 31–33, containing 4.9, 5.0, and 5.1 moles of 24/BSA, respectively, were obtained in 87–93% yields. When the conjugation started with the initial molar carbohydrate–protein ratio of 15:1, the final molar carbohydrate–BSA ratio reached after 14 days with ligands 26, 28, and 30 was 9.0, 11.0, and 9.1, respectively.
Paul Kosma - One of the best experts on this subject based on the ideXlab platform.
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comparative antigenicity of thiourea and adipic amide linked Neoglycoconjugates containing modified oligomannose epitopes for the carbohydrate specific anti hiv antibody 2g12
Bioconjugate Chemistry, 2019Co-Authors: Nino Trattnig, Patrick Mayrhofer, Renate Kunert, Lukas Mach, Ralph Pantophlet, Paul KosmaAbstract:Novel neoglycoproteins containing oligomannosidic penta- and heptasaccharides as structural variants of oligomannose-type N-glycans found on human immunodeficiency virus type 1 gp120 have been prepared using different conjugation methods. Two series of synthetic ligands equipped with 3-aminopropyl spacer moieties and differing in the anomeric configuration of the reducing mannose residue were activated either as isothiocyanates or as adipic acid succinimidoyl esters and coupled to bovine serum albumin. Coupling efficiency for adipic acid connected Neoglycoconjugates was better than for the thiourea-linked derivatives; the latter constructs, however, exhibited higher reactivity toward antibody 2G12, an HIV-neutralizing antibody with exquisite specificity for oligomannose-type glycans. 2G12 binding avidities for the conjugates, as determined by Bio-Layer Interferometry, were mostly higher for the β-linked ligands and, as expected, increased with the numbers of covalently linked glycans, leading to approxima...
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Comparative Antigenicity of Thiourea and Adipic Amide Linked Neoglycoconjugates Containing Modified Oligomannose Epitopes for the Carbohydrate-Specific anti-HIV Antibody 2G12
2018Co-Authors: Nino Trattnig, Patrick Mayrhofer, Renate Kunert, Lukas Mach, Ralph Pantophlet, Paul KosmaAbstract:Novel neoglycoproteins containing oligomannosidic penta- and heptasaccharides as structural variants of oligomannose-type N-glycans found on human immunodeficiency virus type 1 gp120 have been prepared using different conjugation methods. Two series of synthetic ligands equipped with 3-aminopropyl spacer moieties and differing in the anomeric configuration of the reducing mannose residue were activated either as isothiocyanates or as adipic acid succinimidoyl esters and coupled to bovine serum albumin. Coupling efficiency for adipic acid connected Neoglycoconjugates was better than for the thiourea-linked derivatives; the latter constructs, however, exhibited higher reactivity toward antibody 2G12, an HIV-neutralizing antibody with exquisite specificity for oligomannose-type glycans. 2G12 binding avidities for the conjugates, as determined by Bio-Layer Interferometry, were mostly higher for the β-linked ligands and, as expected, increased with the numbers of covalently linked glycans, leading to approximate KD values of 10 to 34 nM for optimized ligand-to-BSA ratios. A similar correlation was observed by enzyme-linked immunosorbent assays. In addition, dendrimer-type ligands presenting trimeric oligomannose epitopes were generated by conversion of the amino-spacer group into a terminal azide, followed by triazole formation using “click chemistry”. The severe steric bulk of the ligands, however, led to poor efficiency in the coupling step and no increased antibody binding by the resulting Neoglycoconjugates, indicating that the low degree of substitution and the spatial orientation of the oligomannose epitopes within these trimeric ligands are not conducive to multivalent 2G12 binding
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Chemical synthesis of the core oligosaccharide of bacterial lipopolysaccharide
Microbial Glycobiology, 2010Co-Authors: Paul KosmaAbstract:Publisher Summary Several in-depth reviews on the synthesis of higher-carbon bacterial sugars and of oligosaccharide structures of the core region of bacterial lipopolysaccharides (LPSs) have been published covering important aspects of chemical synthesis, protecting group strategies and preparation of Neoglycoconjugates. This chapter reviews major strategies toward the chemical synthesis of the core region of LPSs of Gram-negative bacteria illustrated by representative examples, including Pseudomonas aeruginosa, Neisseriaceae, Vibrio parahaemolyticus, and Haemophilus. The focus is on the synthesis of structural units and Neoglycoconjugates of the heptose- and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)-containing inner core region listed according to the corresponding bacterial species. Furthermore, the chapter discusses the synthesis of phosphate-substituted determinants as well as selected outer-core units. Synthetic work targeting larger LPS core fragments could be helpful in elucidating the physiological and immunochemical role of phosphate substituents in the heptose region.
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a novel strategy for the synthesis of Neoglycoconjugates from deacylated deep rough lipopolysaccharides
XXIst International Carbohydrate Symposium 2002, 2002Co-Authors: Sven Mullerloennies, Paul Kosma, Dieter Grimmecke, Lore Brade, Buko Lindner, Helmut BradeAbstract:We report a novel strategy for the preparation of Neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[α-Kdo-(2→4)-α-Kdo-(2→] and a Kdo trisaccharide [α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined Neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined Neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such Neoglycoconjugates a...
