Construction of Multivalent Homo- and Heterofunctional ABO Blood Group Glycoconjugates Using a Trifunctional Linker Strategy.

The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with 4 copies of 1 or 2 separate glycan epitopes, providing 4-8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15-18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive N-hydroxysuccinimide (NHS)-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after the removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing the attachment to the NHS-activated poly(ethylene glycol) (PEG) scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne-azide cycloaddition. Using a known tetravalent PEG scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose and the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, 3'-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target B-cell receptors and CD22 receptors on B cells, supporting the application of these reagents for investigating cellular response to carbohydrate antigens of the ABO blood group system.

Epitope mapping of histo blood group antigens bound to norovirus VLPs using STD NMR experiments reveals fine details of molecular recognition.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be critical for the infection process. Therefore, we have determined binding epitopes of synthetic type 1 to 6 blood group A- and B-tetrasaccharides binding to GII.4 human Norovirus virus like particles (VLPs) using STD NMR experiments. So far, little information is available from crystal structure analysis studies on the interactions of the reducing-end sugars with the protruding domain (P-domain) of the viral coat protein VP1. Here, we show that the reducing-end sugars make notable contacts with the protein surface. The type of glycosidic linkage, and the identity of the sugar at the reducing end modulate HBGA recognition. Most strikingly, type 2 structures yield only very poor saturation transfer indicating impeded binding. This observation is in accordance with previous mass spectrometry based affinity measurements, and can be understood based on recent crystal structure data of a complex of highly homologous GII.4 P-dimers with H-type 2 trisaccharide where the N-acetyl group of the reducing N-acetyl glucosamine residue points towards a loop comprising amino acids Q390 to H395. We suggest that in our case, binding of type 2 A- and B-tetrasaccharides leads to steric conflicts with this loop. In order to identify factors determining L-Fuc recognition, we also synthesized GII.4 VLPs with point mutations D391A and H395A. Prior studies had suggested that these residues, located in a second shell around the L-Fuc binding site, assist L-Fuc binding. STD NMR experiments with L-Fuc and B-trisaccharide in the presence of wild type and mutant VLPs yield virtually identical binding epitopes suggesting that these two mutations do not significantly alter HBGA recognition. Our study emphasizes that recognition of α-(1→2)-linked L-Fuc residues is a conserved feature of GII.4 noroviruses. However, structural variation of the HBGA core structures clearly modulates molecular recognition depending on the genotype.

Conjugation of A and B Blood Group Structures to Silica Microparticles for the Detection of Antigen-Specific B Cells.

Silica microparticles were functionalized with A and B blood group carbohydrate antigens (A type I, A type II, B type I, and B type II) to enable the detection and monitoring of ABO antigen-specific B cells. Microparticles were prepared via the Stöber synthesis, labeled with an Alexafluor fluorescent dye, and characterized via TEM and fluorescence microscopy. The silica microparticles were functionalized with (3-aminopropyl)trimethoxysilane (APTMS), followed by the use of an established fluorenylmethyloxycarbonyl (Fmoc)-protected PEG-based linker. The terminal Fmoc moiety of the PEG-based linker was then deprotected, yielding free amino groups, to which the A and B antigens were coupled. The carbohydrate antigens were synthesized with a p-nitrophenol ester to enable conjugation to the functionalized silica microparticles via an amide bond. The number of free amine groups available for coupling for a given mass of PEG-functionalized silica microparticles was quantified via reaction with Fmoc-glycine. The antigen-functionalized microparticles were then evaluated for their specificity in binding to A and B antigen-reactive B-cells via flow cytometry, and for blocking of naturally occurring antibodies in human serum. Selective binding of the functionalized microparticles to blood group-reactive B cells was observed by flow cytometry and fluorescence microscopy. The modular approach outlined here is applicable to the preparation of silica microparticles containing any carbohydrate antigen and alternative fluorophores or labels. This approach therefore comprises a novel, general platform for screening B cell populations for binding to carbohydrate antigens, including, in this case, the human A and B blood group antigens.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner.

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.

The overall architecture and receptor binding of pneumococcal carbohydrate-antigen-hydrolyzing enzymes.

The TIGR4 and SP3-BS71 strains of Streptococcus pneumoniae each produce family 98 glycoside hydrolases, called Sp4GH98 and Sp3GH98, respectively, which have different modular architectures and substrate specificities. Sp4GH98 degrades the Lewis(Y) antigen and possesses three C-terminal family 47 carbohydrate-binding modules (CBMs) that bind to this substrate. Sp3GH98 degrades the blood group A/B antigens and has two N-terminal family 51 CBMs that are of unknown function. Here, we examine the complex carbohydrate-binding specificity of the family 51 CBMs from Sp3GH98 (referred to as CBM51-1 and CBM51-2), the structural basis of this interaction, and the overall solution conformations of both Sp3GH98 and Sp4GH98, which are shown to be fully secreted proteins. Through glycan microarray binding analysis and isothermal titration calorimetry, CBM51-1 is found to bind specifically to the blood group A/B antigens. However, due to a series of relatively small structural rearrangements that were revealed in structures determined by X-ray crystallography, CBM51-2 appears to be incapable of binding carbohydrates. Analysis of small-angle X-ray scattering data in combination with the available high-resolution X-ray crystal structures of the Sp3GH98 and Sp4GH98 catalytic modules and their CBMs yielded models of the biological solution structures of the full-length enzymes. These studies reveal the complex architectures of the two enzymes and suggest that carbohydrate recognition by the CBMs and the activity of the catalytic modules are not directly coupled.

Synthesis and NMR studies on the ABO histo-blood group antigens: synthesis of type III and IV structures and NMR characterization of type I-VI antigens.

The ABO histo-blood group antigens are best known for their important roles in solid organ and bone marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type III and IV antigens with a 7-octen-1-yl aglycone. Also described is an NMR study of the ABO type I to VI antigens, which were carried out to probe differences in overall conformation of the molecules. These NMR investigations showed very little difference in the (1)H chemical shifts, as well as (1)H-(1)H coupling constants, across all compounds, suggesting that these ABO subtypes adopt nearly identical conformations in solution.

Biocompatible carbohydrate-functionalized stainless steel surfaces: a new method for passivating biomedical implants.

A convenient method for passivating and functionalizing stainless steel is described. Several methods of coating stainless steel (SS) samples with silica were investigated and of these methods, a thin (less than 15 nm thick) layer of silica created by atomic layer deposition (ALD) was found to give superior performance in electrochemical testing. These interfaces were then used as a platform for further functionalization with molecules of biological interest. Specifically, the SS samples were functionalized with biologically significant carbohydrates [N-acetyl-D-glucosamine (GlcNAc) and D-galactose (Gal)] that contain trialkoxysilane derivatives as chemical handles for linking to the surface. The presence and biological availability of these moieties on the silica coated SS were confirmed by XPS analysis and an enzyme-linked lectin assay (ELLA) using complementary lectins that specifically recognize the surface-bound carbohydrate. This method has the potential of being adapted to the functionalization of stainless steel biomedical implants with other biologically relevant carbohydrates.

Synthesis of ABO histo-blood group type I and II antigens.

The ABO histo-blood group system is one of the most clinically important antigen families. As part of our overall goal to prepare the entire set of the A, B and H type I-VI antigens for a range of biochemical investigations, we report herein the synthesis of the type I and II antigens with a 7-octen-1-yl aglycone. This linker was chosen to facilitate not only the future conjugation of the antigens to a protein or solid support but also the synthesis of the H type I and II octyl glycosides for enzyme kinetic studies.

Glycosyl iodides. History and recent advances.

The use of glycosyl iodides as an effective method for the preparation of glycosides has had a recent resurgence in carbohydrate chemistry, despite its early roots in which these species were believed to be of limited use. Renewed interest in these species as glycosylating agents has been spurred by their demonstrated utility in the stereoselective preparation of O-glycosides, and other glycosylic compounds. This review provides a brief historical account followed by an examination of the use of glycosyl iodides in the synthesis of oligosaccharides and other glycomimetics, including C-glycosylic compounds, glycosyl azides and N-glycosides.

The synthesis of 2-, 3-, 4- and 6-O-alpha-D-glucopyranosyl-D-galactopyranose, and their evaluation as nutritional supplements for pre-term infants.

Four methods have been screened for the synthesis of some alpha-D-glucopyranosides, with the recently reported (Mukaiyama) combination of 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl iodide and triphenylphosphine oxide being the most successful, especially in the diastereoselectivity exhibited. The alpha-D-glucopyranosides so obtained have been deprotected to yield 2-, 3-, 4- and 6-O-alpha-D-glucopyranosyl-D-galactopyranose. Only the last disaccharide showed any hydrolysis by alpha-glycosidases but this success was not emulated by mucosal extracts from the small intestine of the pig.