Methods to rapidly functionalize specific polymeric surfaces with alkynes, which can subsequently be linked to azide-containing carbohydrates, are presented. The methods comprise two main concepts: azide photoligation and Cu-catalyzed azide-alkyne cycloaddition. 2-Azidoethyl-functionalized CL-D-mannopyranoside was synthesized and covalently attached to alkyne-functionalized polymeric surfaces using the techniques. The protein recognition properties of the carbollydrate-presenting surfaces were evaluated using quartz crystal microbalance biosensor instrumentation.

A photoclick method based on azide photoligation and Cu-catalyzed azide-alkyne cycloaddition has been evaluated for the immobilization of carbohydrates to polymeric materials. The biomolecular recognition properties of the materials have been investigated with regard to applicable polymeric substrates and selectivity of protein binding. The method was used to functionalize a range of polymeric surfaces (polystyrene, polyacrylamide, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and polypropene) with various carbohydrate structures (based on alpha-D-mannose, beta-D-galactose, and N-acetyl-beta-D-glucosamine). The functionalized surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance flow through system with a series of different carbohydrate-binding proteins (lectins). The method proved to be robust and versatile, resulting in a range of efficient sensors showing high and predictable protein selectivities.

A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S-and O-linked structures with intermediate linker length.

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.

The use of thioglycosides and other glycan derivatives with anomeric sulfur linkages is gaining increasing interest, both in synthesis and in various biological contexts. Herein, we demonstrate the occurrence and circumvention of anomerization during 1-S-glycosylation reactions, and present highly efficient and stereocontrolled syntheses of a series of photoprobe-thiosaccharide conjugates. Mutarotation of glycosyl thiols proved to be the origin of the anomeric mixtures formed, and kinetic effects could be used to circumvent anomerization. The synthesized carbohydrate conjugates were then evaluated by both solution- and solid-phase-based techniques. Both binding results showed that the S-linked glycosides interact with their cognate lectins comparably to the corresponding O-analogs in the present cases, thus demonstrating the reliability of the solid-support platform built upon our photo-initiated carbohydrate immobilization method for probing protein bindings, and showing the potential of combining these two means for studying carbohydrate-protein inter-actions.

Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds are presented. A glycosylation method was developed in which a sulfhydryl glycosyl acceptor was coupled to a 1-O-acetyl-glycosyl donor under Lewis acid catalysis. Final debenzylation of the S-linked dimannosides were accomplished through Birch reduction in high yields. The protein recognition properties of the synthesized dimannosides were then evaluated in a competition binding assay with the model lectin Con A, to investigate the effect of sulfur in the glycosidic bonds. Mannose-presenting surfaces were produced according to a previously reported Photo-Click immobilization method and the subsequent binding study was performed in an automated QCM flow through instrumentation. The recorded EC₅₀-values correlated well to previously reported binding affinities for the O-linked dimannosides. Results were in agreement with known binding affinities, where the S-analogs displayed slightly weaker binding and a positive apparent cooperativity.