Robert Linhardt | Rensselaer Polytechnic Institute (original) (raw)
Papers by Robert Linhardt
We announce the availability of the 5.023-Mbp high-quality draft assembly of the Escherichia coli... more We announce the availability of the 5.023-Mbp high-quality draft assembly of the Escherichia coli strain Nissle 1917 (serovar O6:K5:H1) genome. Short genomic segments from this important probiotic strain have been available in public databases, but the full genome sequence has remained inaccessible. Thus, high-coverage, whole genome sequencing of E. coli Nissle 1917 is presented herein. Reannotation and metabolic reconstruction will enable comparative genomics analysis and model-guided predictions of genetic manipulations leading to increased production of the K5 capsular polysaccharide known as N-acetyl heparosan, a precursor to the anticoagulant pharmaceutical heparin.
Wereport the 4.682-Mbp high-quality draft assembly of the Escherichia coli strain ATCC 23502 (ser... more Wereport the 4.682-Mbp high-quality draft assembly of the Escherichia coli strain ATCC 23502 (serovar O5:K4:H4, also known as NCDC U1-41) genome. This uropathogenic strain, commonly referred to as E. coli K4, produces a glycosaminoglycan-like capsular polysaccharide with a backbone similar in structure to unsulfated chondroitin, a precursor to the nutraceutically and potentially pharmaceutically valuable compound chondroitin sulfate. Metabolic reconstruction of this genome will enable prediction of genetic engineering strategies leading to increased chondroitin production.
Journal of Biological Chemistry, 1998
Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfat... more Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfated polysaccharides. The nature of the SLPI-polysaccharide interaction, explored with affinity chromatography, indicated that this interaction was sensitive to the charge and type of polysaccharide. Dextran and chondroitin had the lowest affinity for SLPI, followed by dermatan, heparan, and dextran sulfates. While heparin bound SLPI tightly, the highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin oligosaccharides, prepared using heparin lyase I were SLPI-affinity fractionated. Surprisingly, undersulfated heparin oligosaccharides bound SLPI with the highest affinity, suggesting the importance of free hydroxyl groups for high affinity interaction. Isothermal titration calorimetry was used to determine the thermodynamics of SLPI interaction with a low molecular weight heparin, an undersulfated decasaccharide and a tetrasaccharide. The studies showed 12-14 saccharide units, corresponding to molecular weight of ϳ4,800, were required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore, an undersulfated decasaccharide was able to bind SLPI tightly (K d ϳ13 nM), resulting in its activation and the inhibition of neutrophil elastase and pancreatic chymotrypsin. The in vitro assessment of heparin and the decasaccharide and tetrasaccharide using stopped-flow kinetics suggested that heparin was the optimal choice to study SLPI-based in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.
FEBS Letters, 1999
The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied u... more The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a v vH of 3 330 kcal/mole corresponding to a dissociation constant (K d ) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12^16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a K d of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of s 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.
Analytical biochemistry, Jan 15, 2002
Journal of Virology, 2006
Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and ... more Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003).
The Journal of biological chemistry, Jan 19, 2007
The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous ... more The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interactio...
Nature Chemical Biology, 2014
Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to tr... more Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.
European Journal of Organic Chemistry, 2009
α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazol... more α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition ("click chemistry"). Our approach is to generate nonnatural N-glycosides of sialic acid that are resistant to neuraminidase-catalyzed hydrolysis as opposed to the natural Oglycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89 % yield. A disaccharide mimic of sialic acid has also been prepared using the [a] Scheme 1. Use of a peracetylated (3) or unprotected (1) α-sialic acid azide donor. www.eurjoc.org 2612 Scheme 2. Reductive amination of 2g using a lysine residue.
The Journal of Organic Chemistry, 2008
An improved understanding of the biological activities of heparin requires structurally defined h... more An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5′-diphosphoiduronic acid (UDP-IdoA) and uridine 5′-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5′-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.
Bioorganic & Medicinal Chemistry, 2009
Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of ol... more Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC 50 = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47 phox and p22 phox . To that end, while apocynin was unable to block the interaction of his-tagged p47 phox with a surface immobilized biotinalyted p22 phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC 50 = 1.6 μM). These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase.
Carbohydrate Research, 2007
Glycosylation in room temperature ionic liquid is demonstrated using unprotected and unactivated ... more Glycosylation in room temperature ionic liquid is demonstrated using unprotected and unactivated donors. Modest yields of simple benzyl glycosides and disaccharides of glucose, mannose and N-acetylgalactosamine were obtained in 1-ethyl-3methylimidazolium benzoate with Amberlite IR-120 (H + ) resin or p-toluenesulfonic acid as promoters.
Journal of Biological Chemistry, 2007
The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous ... more The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). ...
Tetrahedron: Asymmetry, 2011
Two carba-sugar containing pseudodisaccharide diastereomers have been synthesized from a racemic ... more Two carba-sugar containing pseudodisaccharide diastereomers have been synthesized from a racemic bicyclooctene derivative. The determination of the absolute configurations was carried out by means of CD measurements, CD calculations and X-ray diffraction. The compounds showed moderate competitive inhibitory effects on chondroitin AC-I lyase.
Journal of the American Chemical Society, 2008
We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a unifor... more We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly labeled [ 13 C, 15 N]N-acetylheparosan (-GlcA(1,4)GlcNAc-) prepared from E. coli K5. Glycosaminoglycan (GAG) precursors and heparin were formed from N-acetylheparosan by the following steps: chemical N-deacetylation and N-sulfonation leading to N-sulfoheparosan (-GlcA (1,4)GlcNS-); enzyme-catalyzed C 5 -epimerization and 2-O-sulfonation leading to undersulfated heparin (-IdoA2S(1,4)GlcNS-); enzymatic 6-O-sulfonation leading to the heparin backbone (-IdoA2S(1,4)GlcNS6S-); and selective enzymatic 3-O-sulfonation leading to the anticoagulant heparin, containing the GlcNS6S3S residue. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution structure of [ 13 C, 15 N]N-acetylheparosan, precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13 C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labeled heparin was indistinguishable from heparin derived from animal tissues and is a novel reagent for studying the interaction of heparin with proteins.
Several natural polyketides (PKs) have been associated with important pharmaceutical properties. ... more Several natural polyketides (PKs) have been associated with important pharmaceutical properties. Type III polyketide synthases (PKS) that generate aromatic PK polyketides have been studied extensively for their substrate promiscuity and product diversity. Stilbene synthase-like (STS) enzymes are unique in the type III PKS class as they possess a hydrogen bonding network, furnishing them with thioesterase-like properties, resulting in aldol condensation of the polyketide intermediates formed. Chalcone synthases (CHS) in contrast, lack this hydrogen-bonding network, resulting primarily in the Claisen condensation of the polyketide intermediates formed. We have attempted to expand the chemical space of this interesting class of compounds generated by creating structure-guided mutants of Vitis vinifera STS. Further, we have utilized a previously established workflow to quickly compare the wild-type reaction products to those generated by the mutants and identify novel PKs formed by using XCMS analysis of LC-MS and LC-MS/MS data. Based on this approach, we were able to generate 15 previously unreported PK molecules by exploring the substrate promiscuity of the wild-type enzyme and all mutants using unnatural substrates. These structures were specific to STSs and cannot be formed by their closely related CHS-like counterparts.
ChemInform, 2004
Sialic acids (or ulosonic acids) are a family of acidic ketoses (including neuraminic acid, KDN a... more Sialic acids (or ulosonic acids) are a family of acidic ketoses (including neuraminic acid, KDN and KDO) that are found at the non-reducing terminus of many glycoconjugates. These saccharide residues are recognized ligands of protein lectins and are removed in the first step in glycoconjugate catabolism. Moreover, sialic acid containing carbohydrates, such as glycolipid gangliosides (i.e. GM3), glycopeptides (i.e. Tn)
Capillary Electrophoresis of Carbohydrates, 2003
We announce the availability of the 5.023-Mbp high-quality draft assembly of the Escherichia coli... more We announce the availability of the 5.023-Mbp high-quality draft assembly of the Escherichia coli strain Nissle 1917 (serovar O6:K5:H1) genome. Short genomic segments from this important probiotic strain have been available in public databases, but the full genome sequence has remained inaccessible. Thus, high-coverage, whole genome sequencing of E. coli Nissle 1917 is presented herein. Reannotation and metabolic reconstruction will enable comparative genomics analysis and model-guided predictions of genetic manipulations leading to increased production of the K5 capsular polysaccharide known as N-acetyl heparosan, a precursor to the anticoagulant pharmaceutical heparin.
Wereport the 4.682-Mbp high-quality draft assembly of the Escherichia coli strain ATCC 23502 (ser... more Wereport the 4.682-Mbp high-quality draft assembly of the Escherichia coli strain ATCC 23502 (serovar O5:K4:H4, also known as NCDC U1-41) genome. This uropathogenic strain, commonly referred to as E. coli K4, produces a glycosaminoglycan-like capsular polysaccharide with a backbone similar in structure to unsulfated chondroitin, a precursor to the nutraceutically and potentially pharmaceutically valuable compound chondroitin sulfate. Metabolic reconstruction of this genome will enable prediction of genetic engineering strategies leading to increased chondroitin production.
Journal of Biological Chemistry, 1998
Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfat... more Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfated polysaccharides. The nature of the SLPI-polysaccharide interaction, explored with affinity chromatography, indicated that this interaction was sensitive to the charge and type of polysaccharide. Dextran and chondroitin had the lowest affinity for SLPI, followed by dermatan, heparan, and dextran sulfates. While heparin bound SLPI tightly, the highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin oligosaccharides, prepared using heparin lyase I were SLPI-affinity fractionated. Surprisingly, undersulfated heparin oligosaccharides bound SLPI with the highest affinity, suggesting the importance of free hydroxyl groups for high affinity interaction. Isothermal titration calorimetry was used to determine the thermodynamics of SLPI interaction with a low molecular weight heparin, an undersulfated decasaccharide and a tetrasaccharide. The studies showed 12-14 saccharide units, corresponding to molecular weight of ϳ4,800, were required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore, an undersulfated decasaccharide was able to bind SLPI tightly (K d ϳ13 nM), resulting in its activation and the inhibition of neutrophil elastase and pancreatic chymotrypsin. The in vitro assessment of heparin and the decasaccharide and tetrasaccharide using stopped-flow kinetics suggested that heparin was the optimal choice to study SLPI-based in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.
FEBS Letters, 1999
The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied u... more The interaction of heparin with heparin binding growth-associated molecule (HB-GAM) was studied using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). ITC studies showed that, in solution, heparin bound HB-GAM with a v vH of 3 330 kcal/mole corresponding to a dissociation constant (K d ) of 460 nM. The stoichiometry of interaction was 3 moles of HB-GAM per mole of heparin, corresponding to a minimum heparin binding site for HB-GAM of 12^16 saccharide residues. Kinetic measurements of heparin interaction with HB-GAM made by SPR afforded a K d of 4 nM, suggesting considerably tighter binding when HB-GAM was immobilized on a surface. Affinity chromatography of a sized mixture of heparin oligosaccharides, having a degree of polymerization (dp) of s 14 saccharide units, on HB-GAM-Sepharose demonstrated that oligosaccharides having more than 18 saccharide residues showed the tightest interaction.
Analytical biochemistry, Jan 15, 2002
Journal of Virology, 2006
Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and ... more Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003).
The Journal of biological chemistry, Jan 19, 2007
The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous ... more The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interactio...
Nature Chemical Biology, 2014
Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to tr... more Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.
European Journal of Organic Chemistry, 2009
α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazol... more α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition ("click chemistry"). Our approach is to generate nonnatural N-glycosides of sialic acid that are resistant to neuraminidase-catalyzed hydrolysis as opposed to the natural Oglycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89 % yield. A disaccharide mimic of sialic acid has also been prepared using the [a] Scheme 1. Use of a peracetylated (3) or unprotected (1) α-sialic acid azide donor. www.eurjoc.org 2612 Scheme 2. Reductive amination of 2g using a lysine residue.
The Journal of Organic Chemistry, 2008
An improved understanding of the biological activities of heparin requires structurally defined h... more An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5′-diphosphoiduronic acid (UDP-IdoA) and uridine 5′-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5′-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.
Bioorganic & Medicinal Chemistry, 2009
Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of ol... more Enzymatic oxidation of apocynin, which may mimic in vivo metabolism, affords a large number of oligomers (apocynin oxidation products, AOP) that inhibit vascular NADPH oxidase. In vitro studies of NADPH oxidase activity were performed to identify active inhibitors, resulting in a trimer hydroxylated quinone (IIIHyQ) that inhibited NADPH oxidase with an IC 50 = 31 nM. Apocynin itself possessed minimal inhibitory activity. NADPH oxidase is believed to be inhibited through prevention of the interaction between two NADPH oxidase subunits, p47 phox and p22 phox . To that end, while apocynin was unable to block the interaction of his-tagged p47 phox with a surface immobilized biotinalyted p22 phox peptide, the IIIHyQ product strongly interfered with this interaction (apparent IC 50 = 1.6 μM). These results provide evidence that peroxidase-catalyzed AOP, which consist of oligomeric phenols and quinones, inhibit critical interactions that are involved in the assembly and activation of human vascular NADPH oxidase.
Carbohydrate Research, 2007
Glycosylation in room temperature ionic liquid is demonstrated using unprotected and unactivated ... more Glycosylation in room temperature ionic liquid is demonstrated using unprotected and unactivated donors. Modest yields of simple benzyl glycosides and disaccharides of glucose, mannose and N-acetylgalactosamine were obtained in 1-ethyl-3methylimidazolium benzoate with Amberlite IR-120 (H + ) resin or p-toluenesulfonic acid as promoters.
Journal of Biological Chemistry, 2007
The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous ... more The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). ...
Tetrahedron: Asymmetry, 2011
Two carba-sugar containing pseudodisaccharide diastereomers have been synthesized from a racemic ... more Two carba-sugar containing pseudodisaccharide diastereomers have been synthesized from a racemic bicyclooctene derivative. The determination of the absolute configurations was carried out by means of CD measurements, CD calculations and X-ray diffraction. The compounds showed moderate competitive inhibitory effects on chondroitin AC-I lyase.
Journal of the American Chemical Society, 2008
We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a unifor... more We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly labeled [ 13 C, 15 N]N-acetylheparosan (-GlcA(1,4)GlcNAc-) prepared from E. coli K5. Glycosaminoglycan (GAG) precursors and heparin were formed from N-acetylheparosan by the following steps: chemical N-deacetylation and N-sulfonation leading to N-sulfoheparosan (-GlcA (1,4)GlcNS-); enzyme-catalyzed C 5 -epimerization and 2-O-sulfonation leading to undersulfated heparin (-IdoA2S(1,4)GlcNS-); enzymatic 6-O-sulfonation leading to the heparin backbone (-IdoA2S(1,4)GlcNS6S-); and selective enzymatic 3-O-sulfonation leading to the anticoagulant heparin, containing the GlcNS6S3S residue. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution structure of [ 13 C, 15 N]N-acetylheparosan, precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13 C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labeled heparin was indistinguishable from heparin derived from animal tissues and is a novel reagent for studying the interaction of heparin with proteins.
Several natural polyketides (PKs) have been associated with important pharmaceutical properties. ... more Several natural polyketides (PKs) have been associated with important pharmaceutical properties. Type III polyketide synthases (PKS) that generate aromatic PK polyketides have been studied extensively for their substrate promiscuity and product diversity. Stilbene synthase-like (STS) enzymes are unique in the type III PKS class as they possess a hydrogen bonding network, furnishing them with thioesterase-like properties, resulting in aldol condensation of the polyketide intermediates formed. Chalcone synthases (CHS) in contrast, lack this hydrogen-bonding network, resulting primarily in the Claisen condensation of the polyketide intermediates formed. We have attempted to expand the chemical space of this interesting class of compounds generated by creating structure-guided mutants of Vitis vinifera STS. Further, we have utilized a previously established workflow to quickly compare the wild-type reaction products to those generated by the mutants and identify novel PKs formed by using XCMS analysis of LC-MS and LC-MS/MS data. Based on this approach, we were able to generate 15 previously unreported PK molecules by exploring the substrate promiscuity of the wild-type enzyme and all mutants using unnatural substrates. These structures were specific to STSs and cannot be formed by their closely related CHS-like counterparts.
ChemInform, 2004
Sialic acids (or ulosonic acids) are a family of acidic ketoses (including neuraminic acid, KDN a... more Sialic acids (or ulosonic acids) are a family of acidic ketoses (including neuraminic acid, KDN and KDO) that are found at the non-reducing terminus of many glycoconjugates. These saccharide residues are recognized ligands of protein lectins and are removed in the first step in glycoconjugate catabolism. Moreover, sialic acid containing carbohydrates, such as glycolipid gangliosides (i.e. GM3), glycopeptides (i.e. Tn)
Capillary Electrophoresis of Carbohydrates, 2003