Related Trisaccharide Research Articles

CST-II’s recognition domain for acceptor substrates in α-(2→8)-sialylations

CST-II is a bacterial sialyltransferase known for its ability to perform α-(2→8)-sialylations using GM3 related trisaccharide substrates. Previously, we probed the enzyme’s substrate specificity and developed an efficient synthesis for α-(2→8)-oligosialosides, and we suggested that CST-II could have a very small substrate recognition domain. Here we report our full studies on CST-II’s recognition feature for acceptor substrates. The current study further demonstrates the versatility of CST-II in preparing complex oligosaccharides that contain α-(2→8)-oligosialyl moieties.

Trisaccharide mimetics of the aminoglycoside antibiotic neomycin

A highly convergent approach for the chemical synthesis of eight structurally related trisaccharides that contain 3 to 5 amino groups has been described. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been employed to determine the dissociation constants (Kd) for the binding of the trisaccharides to a prototypical fragment of 16S ribosomal RNA. A compound that contained a 4,6-dideoxy-4-amino-beta-D-glucopyranoside moiety at C-3 displayed binding in the low micromolar range. It was found that small structural changes of the saccharides resulted in large differences in affinity. The described structure-activity relationship is expected to be valuable for the development of novel antibiotics that target rRNA.

Blackbody Infrared Radiative Dissociation of Nonspecific Protein–Carbohydrate Complexes Produced by Nanoelectrospray Ionization: The Nature of the Noncovalent Interactions

Gas-phase thermal dissociation experiments, implemented with blackbody infrared radiative dissociation (BIRD) and Fourier transform ion cyclotron resonance mass spectrometry, have been performed on a series of protonated and deprotonated 1:1 and protonated 1:2 protein-carbohydrate complexes formed by nonspecific interactions during the nanoflow electrospray (nanoES) ionization process. Nonspecific interactions between the proteins bovine carbonic anhydrase II (CA), bovine ubiquitin (Ubq), and bovine pancreatic trypsin inhibitor and several carbohydrates, ranging in size from mono- to tetrasaccharides, have been investigated. Over the range of temperatures studied (60-190 degrees C), BIRD of the protonated and deprotonated complexes proceeds exclusively by the loss of the carbohydrate in its neutral form. The rates of dissociation of the 1:1 complexes containing a mono- or disaccharide decrease with reaction time, suggesting the presence of two or more kinetically distinct structures produced during nanoES or by gas-phase processes. In contrast, the 1:1 complexes of the tri- and tetrasaccharides exhibit simple first-order dissociation kinetics, a result that, on its own, is suggestive of a single preferred carbohydrate binding site or multiple equivalent sites in the gas phase. A comparative analysis of the dissociation kinetics measured for protonated 1:1 and 1:2 complexes of Ubq with alphaTal[alphaAbe]alphaMan further supports the presence of a single preferred binding site. However, a similar analysis performed on the complexes of CA and alphaTal[alphaAbe]alphaMan suggests that equivalent but dependent carbohydrate binding sites exist in the gas phase. Analysis of the Arrhenius activation parameters (E(a) and A) determined for the dissociation of 1:1 complexes of CA with structurally related trisaccharides provides evidence that neutral intermolecular hydrogen bonds contribute, at least in part, to the stability of the gaseous complexes. Surprisingly, the E(a) values for the complexes of the same charge state are not sensitive to the structure (primary or higher order) of the protein, suggesting that the carbohydrates are able to form energetically equivalent interactions with the various functional groups presented by the protein. For a given protein-carbohydrate complex, the dissociation E(a) is sensitive to charge state, initially increasing and then decreasing with increasing charge. It is proposed that both ionic and neutral hydrogen bonds stabilize the nonspecific protein-carbohydrate complexes in the gas phase and that the relative contribution of the neutral and ionic interactions is strongly influenced by the charge state of the complex, with neutral interactions dominating at low charge states and ionic interactions dominating at high charge states.

Atomic Mapping of the Interactions between the Antiviral Agent Cyanovirin-N and Oligomannosides by Saturation-Transfer Difference NMR

The minimum oligosaccharide structure required for binding to the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by saturation-transfer difference (STD) NMR spectroscopy. Despite the low molecular mass of the protein (11 kDa), STD-NMR spectroscopy allowed the precise atomic mapping of the interactions between CV-N and various di- and trimannosides, substructures of Man-9, the predominant oligosaccharide on the HIV viral surface glycoprotein gp120. Contacts with mannosides containing the terminal Manalpha(1-->2)Manalpha unit of Man-9 were observed, while (1-->3)- and (1-6)-linked di- and trimannosides showed no interactions, demonstrating that the terminal Manalpha(1-->2)Manalpha structure plays a key role in the interaction. Precise epitope mapping revealed that, for Manalpha(1-->2)ManalphaOMe, Manalpha(1-->2)Manalpha(1-->3)ManalphaOMe, and Manalpha(1-->2)Manalpha(1-->6)ManalphaOMe, the protein is in close contact with H2, H3, and H4 of the nonreducing terminal mannose unit. In contrast, the STD-NMR spectrum of the CV-N/trisaccharide Manalpha(1-->2)Manalpha(1-->2)ManalphaOMe complex was markedly different, with resonances on all sugar units displaying equal enhancements, suggesting that CV-N is able to discriminate between the three structurally related trisaccharides.

Synthetic Study on Campylobacter jejuni Lipopolysaccharides: An Improved Synthesis of a Branched, Heptose‐Containing Trisaccharide Core Structure and Its Conversion into Ganglioside GD3 Related Hexasaccharide

AbstractThe first synthesis of the oligosaccharide skeleton of the Campylobacter jejuni lipooligosaccharide composed of a branched, heptose‐containing trisaccharide core structure and, including a substantially improved preparation of the core structure, the ganglioside GD3 related trisaccharide epitope is reported. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9).

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man(8) and Man(9). Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manalpha(1-2)Manalpha (which represents the terminal disaccharide of all three arms of Man(9)) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manalpha(1-2)Manalpha complex revealed that CVN recognizes the stacked conformation of Manalpha(1-2)Manalpha through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manalpha(1-2)Manalpha(1-2)Man, methyl Manalpha(1-2)Manalpha(1-3)Man and methyl Manalpha(1-2)Manalpha(1-6)Man with remarkable selectivity, and binds these trisaccharides with K(A) values ranging from 8.1x10(3)M(-1) to 6.6x10(6)M(-1). Site 2 is less selective in that it binds all three trisaccharides with similar K(A) values ranging from 1.7 to 3.7(+/-0.3)x10(5)M(-1), but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display alpha(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.

Crystal state and solution conformation of the B blood group trisaccharide alpha- l-Fucp-(12)-[alpha- d-Galp]-(13)]-beta- d-Galp-OCH3

The crystal structure of the human B blood group related trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)-beta-D-Galp-OCH3 (1) has been determined. The solution structure of 1 was studied by two-dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles phiH and psiH, derived from 3JCH coupling constants, and 10 inter-residue proton-proton distances. 3JCH could be accurately measured by a recently introduced two-dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement-derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo-Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related alpha-L-Fucp-(1-->2)-[6-deoxy-alpha-D-Galp]-(1-->3)-beta-D-Galp O-(CH2 )7CH3 trisaccharide (2).

Crystal state and solution conformation of the B blood group trisaccharide α‐l‐Fucp‐(1→2)‐[α‐d‐Galp]‐(1→3)]‐β‐d‐Galp‐OCH3

The crystal structure of the human B blood group related trisaccharide α‐l‐Fucp‐(1→2)‐[α‐d‐Galp]‐(1→3)‐β‐d‐Galp‐OCH3 (1) has been determined. The solution structure of 1 was studied by two‐dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles φH and ψH, derived from 3JCH coupling constants, and 10 inter‐residue proton–proton distances. 3JCH could be accurately measured by a recently introduced two‐dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement‐derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo‐Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related α‐l‐Fucp‐(1→2)‐[6‐deoxy‐α‐d‐Galp]‐(1→3)‐β‐d‐GalpO‐(CH2)7CH3 trisaccharide (2).

Carbohydrate haptens: 4-nitrophenyl 2-acetamido-2-deoxy-β- d-glucopyranosyl-(1 → 6)-α- d-mannopyranosyl-(1 → 6)-β- d-mannopyranoside and a related trisaccharide

Carbohydrate haptens: 4-nitrophenyl 2-acetamido-2-deoxy-β- d-glucopyranosyl-(1 → 6)-α- d-mannopyranosyl-(1 → 6)-β- d-mannopyranoside and a related trisaccharide

Molecular recognition XIII. The binding of the H-type 2 human blood group determinant by a winged bean (Psophocarpus tetragonolobus) acidic lectin

The winged bean acidic lectin (WBA II) binds the H-type 2 human blood group related trisaccharide (α-L-Fuc-(1c → 2b)-β-D-Gal-(1b → 4a)-β-D-GlcNAc-OMe, 1). Interactions that importantly contribute to the specificity of the complex formation are provided by CH2-6b, OH-4b, OH-3b, and OH-2c of 1. On the basis of the relative activities of the monodeoxy and mono-O-methyl derivatives of 1, the hydroxyl groups at the 3a, 6a, and 4c positions become located at the periphery of the combining site, whereas the CH3O-1a, NHAc-2a, and CH3-5c groups are more remote from the protein surface and fully exposed to bulk water. Whereas the WBA II lectin marginally recognizes the H-type 1 related trisaccharide (α-L-Fuc-(1c → 2b)-β-D-Gal-(1b → 3a)-β-D-GlcNAc-OMe, 23), the de-N-acetyl derivative (24) is bound 3.4 times more strongly than 1. This and other results, which are related to changes in interaction between the ligand and the apron of the combining site, are attributed to changes in hydration that lead to enthalpy–entropy compensation. In certain cases, the interactions are found to stabilize the complex.

Use of the shadow optical method of caustics to predict fatigue crack growth in compression: Leftheris, B. P. and Papazian, J. M. J. Eng. Mater. Technol. (Trans. ASME) (Oct. 1992) 114 (4) 399–405

Cyanovirin-N (CVN) is a novel cyanobacterial protein that selectively binds with nanomolar affinities the mammalian oligosaccharides Man8 and Man9. Consequently, CVN potently blocks HIV entry through highly avid carbohydrate-mediated interactions with the HIV-envelope glycoprotein gp120, and is under preclinical investigation as an anti-HIV microbicide. CVN contains two non-overlapping carbohydrate-binding sites that bind the disaccharide Manα(1-2)Manα (which represents the terminal disaccharide of all three arms of Man9) with low to sub-micromolar affinities. The solution structure of a 1:2 CVN:Manα(1-2)Manα complex revealed that CVN recognizes the stacked conformation of Manα(1-2)Manα through a deep hydrophilic-binding pocket on one side of the protein (site 2) and a semi-circular cleft on the other (site 1). With the prominent exception of the C1 hydroxyl group of the reducing mannopyranose ring, the bound disaccharide is positioned so that each hydroxyl group is involved in a direct or water-mediated hydrogen bond to the polar or charged side-chains comprising the binding pocket. Thus, to determine whether the next-most reducing mannopyranose ring will augment CVN affinity and selectivity, we have characterized by NMR and ITC the binding of CVN to three synthetic trisaccharides representing the full-length D1, D2 and D3 arms of mammalian oligomannosides. Our findings demonstrate that site 1 is able to discriminate between the three related trisaccharides methyl Manα(1-2)Manα(1-2)Man, methyl Manα(1-2)Manα(1-3)Man and methyl Manα(1-2)Manα(1-6)Man with remarkable selectivity, and binds these trisaccharides with KA values ranging from 8.1×103 M−1 to 6.6×106 M−1. Site 2 is less selective in that it binds all three trisaccharides with similar KA values ranging from 1.7 to 3.7(±0.3)×105 M−1, but overall binds these trimannosides with higher affinities than site 1. The diversity of pathogenic organisms that display α(1-2)-linked mannosides on their cell surfaces suggests a broad defensive role for CVN in its cyanobacterial source.

Synthesis and 13C NMR spectra of 2,3-di- O-glycosyl derivatives of methyl α- l-rhamnopyranoside and methyl α- d-mannopyranoside

The syntheses are described of 2,3-di- O-glycosyl derivatives of methyl α- l- rhamnopyranoside ( 1–5)and α- d-mannopyranoside ( 6–9). [Display omitted] X Y X Y 1 α- l-Rha p α- d-Glc p 6 α- l-Rha p α- d-Glc p 2 α- d-Glc p α- l-Rha p 7 α- d-Man p α- d-Glc p 3 α- l-Rha p β- d-Glc p 8 β- l-Fuc p β- l-Fuc p 4 α- l-Rha p β- l-Fuc p 9 α- l-Fuc p β- l-Fuc p 5 β- d-Glc p β- d-Gal p The deviation from additivity in 13C NMR spectra calculated for 1–9 were similar for stereochemicallyrelated trisaccharides.

Molecular recognition XII. The binding of the H human blood group determinants and congeners by a lectin of Galactiatenuiflora

The H-type 2 human blood group-related trisaccharide (α-L-Fuc-(1c → 2b)-β-D-Gal-(1b → 4a)-β-D-GlcNAc-OMe (52)) is bound by the anti-H lectin of Galactiatenuiflora very differently than by the lectin I of Ulexeuropaeus. The reason why the Galactia lectin binds the H-type 1 related trisaccharide (α-L-Fuc-(1c → 2b)-β-D-Gal-(1b → 3a)-β-D-GlcNAc-OMe (5)) more strongly and methyl α-L-fucopyranoside much more weakly than does the Ulex lectin is that, for the Galactia lectin, the hydroxyl groups at positions 3a, 3b, 4b, and 4c are indispensable to complex formation whereas it is the hydroxyl groups at positions 3b, 2c, 3c, and 4c which provide the key polar intractions in the case of the Ulex lectin. The H-type 2 (52)•Galactia lectin complex appears to have the hydroxyl groups at positions 6b, 2c, and 3c at or near the periphery of the combining site and the three key hydroxyl groups hydrogen bonded to the protein deep within the combining site and sheltered from water. The CH3O-1a, NHAc-2a, and CH2OH-6a groups likely remain in the aqueous phase remote from the surface of the protein.

Molecular recognition XI. The synthesis of extensively deoxygenated derivatives of the H-type 2 human blood group determinant and their binding by an anti-H-type 2 monoclonal antibody and the lectin 1 of Ulexeuropaeus

The relative potencies of a wide variety of deoxygenated derivatives of the methyl glycoside of α-L-Fuc-(1 → 2)-β-D-Gal-(1 → 4)- β-D-GlcNAc (the H-type 2 human blood group related trisaccharide) for the inhibition of the binding of an artificial H-type 2 antigen by the lectin I of Ulexeuropaeus confirmed the previous evidence that the key and productive interaction involves only the three hydroxyl groups of the α-L-fucose unit, the hydroxyl at the 3-position of the β-D-galactose residue, and the nonpolar groups in their immediate environment. Except for the acetamido group and the hydroxymethyl of the β-D-Gal unit, which stay in the aqueous phase, on complex formation the remaining three hydroxyl groups appear to come to reside at or near the periphery of the combining site since their replacement by hydrogen causes relatively small changes (< ± 1 kcal/mol) in the stability of the complex (ΔG0). Relatively much larger but compensating changes occur for the enthalpy and entropy terms, and these may arise primarily from the differences in the water structure about the periphery of the combining site and the oligosaccharide both prior to and after complexation. It is proposed that steric constraints lead to an ordered state of the water molecules hydrogen-bonded to the polar groups within the cleft formed by the key region of the amphiphilic combining site. Their release to form less ordered clusters of more strongly hydrogen-bonded water molecules in bulk solution would contribute importantly to the driving force for complexation. It is demonstrated that the surface used for the binding of H-type 2-OMe by a monoclonal anti-H antibody is virtually identical to that used by the Ulex lectin. Keywords: molecular recognition, H-type 2 blood group determinant and deoxygenated derivatives, lectin I of Ulexeuropaeus, anti-H-type 2 monoclonal antibody, enthalpy–entropy compensation.

Synthesis of O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-D- glucopyranose and two related trisaccharides containing the “lacto-N-biose II” unit

Condensation of 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-2,4,6-tri-O-acetyl-α-D-galactopyranosyl bromide (1) with benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside in boiling benzene and in the presence of mercuric cyanide afforded a trisaccharide that was O-deacetylated to give benzyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-β-D-galactopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy -α-D-glucopyranoside, the benzyl groups of which were cleaved by catalytic hydrogenolysis to furnish the title trisaccharide. Alternatively, bromide 1 was allowed to react with 2-acetamido-1,6-anhydro-3-O-benzyl-2-deoxy-β-D-glucopyranose in 1:1 benzene-nitromethane in the presence of mercuric cyanide, followed by O-deacetylation, column chromatography, and reacetylation to give O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-(2,4,6-tri-O-acetyl-β-D- galactopyranosyl)-(1→4)-2-acetamido-1,6-anhydro-3-O-benzyl-2-deoxy-β-D-glucopyranose. Acetolysis, followed by catalytic hydrogenation and subsequent O-deacetylation furnished the title trisaccharide. A similar condensation of bromide 1 with 4-nitrophenyl 2-acetamido-2-deoxy-4,6-O-(4-methoxybenzylidene)-β-D-glucopyranoside and 2-nitrophenyl 2-acetamido-2-deoxy-4,6-O-(4-methoxybenzylidene)-α-D-galactopyranoside produced two trisaccharide derivatives, the acetal groups of which were cleaved in hot, 60% aqueous acetic acid, and the resulting diol intermediates O-deacetylated to furnish the desired trisaccharides 4-nitrophenyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-β-D-galactopyranosyl)-(1→3)-2- acetamido-2-deoxy-β-D-glucopyranoside and 2-nitrophenyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D- galactopyranoside.

Chemical synthesis of the trisaccharide unit of the species-specific phenolic glycolipid from Mycobacterium leprae

O-(3,6-di- O-methyl-β- d-glucopyranosyl)-(1→4)- O-(2,3-di- O-methyl-α- l-rhamnopyranosyl)-(1→2)-3- O-methyl- l- rhamnopyranose, the haptenic trisaccharide of the Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) antigen, and related trisaccharides, were synthesized by allylation of O-2 of benzyl 4- O-benzyl-α- l-rhamnopyranoside using phase-transfer catalysis, methylation of the product, deallylation, and coupling to O-(2,4-di- O-acetyl-3,6-di- O-methyl-β- d-glucopyranosyl)-(1→4)-2,3-di- O-methyl- l-rhamnopyranosyl bromide or related disaccharides. Anomeric mixtures of the trisaccharide derivatives were separated by preparative t.l.c., deacetylated, and hydrogenolyzed, to give the pure trisaccharides. It had already been demonstrated that only those trisaccharides containing an intact, terminal 3,6-di- O-methyl-β- d-glucopyranosyl unit are effective in inhibiting the specific binding between PGL-I and unit-PGL-I immunoglobulin M antibodies in human lepromatous leprosy sera.

Oxidized saccharides as inhibitors of α-glucan synthesis by Streptococcus mutans glucosyltransferase

Specific inhibition by periodate-oxidized dextrans of the synthesis of α-glucan by S. mutans glucosyltransferase prompted a search for structurally related inhibitors that might be effective as anticaries agents. Clinical dextran derivatives in which from 5 to 50% of the d-glucose units were oxidized acted as potent and specific enzyme-inhibitors, as did 10%-oxidized derivatives of dextran fractions ranging in mol. wt from 10 4 to 2 x 10 6. Within these limits, differences in oxidation or molecular weight did not significantly affect the high inhibitory potency of the derivatives. In contrast, periodate oxidation of (1→6)-α-, (1→3)-α-, and (1→4)-α-linked oligosaccharides containing <∼15 d-glucose units, and of sucrose and structurally related trisaccharides, yielded derivatives that were poor inhibitors. Enzymic hydrolysis of oxidized dextrans caused a loss of their inhibitory power and indicated that, to act as specific inhibitors, oxidized molecules must contain at least 16 to 20 d-glucosyl residues. The similar, minimum size required in order that unoxidized oligosaccharides may act as efficient acceptors in the glucosyltransferase reaction suggests that the inhibitory potencies of oxidized derivatives may reflect their relative abilities to bind at the acceptor site of the enzyme.