Lingquan Deng

Photo-click immobilization of carbohydrates on polymeric surfaces--a quick method to functionalize surfaces for biomolecular recognition studies.

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 alpha-D-mannopyranoside was synthesized and covalently attached to alkyne-functionalized polymeric surfaces using the techniques. The protein recognition properties of the carbohydrate-presenting surfaces were evaluated using quartz crystal microbalance biosensor instrumentation.

Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for Selective Carbohydrate-Protein Interaction Analyses

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 α-D-mannose, β-D-galactose, and N-acetyl-β-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.

Multivalent Glyconanoparticles with Enhanced Affinity to the Anti-Viral Lectin Cyanovirin-N

Low-mannose (LM) structures were coupled to gold nanoparticles (Au NPs) to amplify the affinity of LMs with Cyanovirin-N (CV-N) lectins and to study the structures of CV-N variants CVN(Q50C) and CVN(MutDB).

Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging

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.

Photogenerated carbohydrate microarrays to study carbohydrate-protein interactions using surface plasmon resonance imaging

A photochemical strategy to generate carbohydrate microarrays on flat sensor surfaces, and to study the protein-binding effects of these arrays by surface plasmon resonance imaging is described. The approach was validated using a panel of carbohydrate-binding proteins. The coupling agents, thiol-functionalized perfluorophenyl azides, allow the covalent attachment of underivatized carbohydrates to gold surfaces by a fast photochemical reaction. Carbohydrate microarrays composed of 3,6-di-O-(α-D-mannopyranosyl)-D-mannopyranose (Man3), 2-O-α-D-mannopyranosyl-D-mannopyranose (Man2), D-mannose (Man), D-glucose (Glc), and D-galactose (Gal) were constructed, and the binding studies were carried out in real-time using surface plasmon resonance imaging. Results showed that the immobilized carbohydrate ligands retained their binding affinities with lectins, the rank order of which was consistent with that of the free ligands in solution. The detection limit of Man3, Man2, Man, and Glc with the lectin Concanavalin A was measured to be 0.29 nM, 0.18 nM, 0.61 nM, and 3.1 nM, respectively. In addition, soybean agglutinin and Griffonia simplicifolia lectin II were tested on the array, and the results were consistent with the binding selectivity of these lectins with the carbohydrate ligands.

pH-Dependent Mutarotation of 1-Thioaldoses in Water. Unexpected Behavior of (2S)-D-Aldopyranoses

The pH-dependent mutarotation of 1-thioaldopyranoses in aqueous media has been investigated. Anomerization readily occurred at lower and neutral pH for all aldopyranoses studied, whereas mainly for (2S)-D-aldopyranoses at higher pH. 1-Thio-D-mannopyranose and 1-thio-D-altropyranose showed very strong pH dependence where the anomeric equilibrium ratios changed dramatically from a preference for the β-anomer at lower pH to the α-anomer at higher pH.

Sensing lectin-glycan interactions using lectin super-microarrays and glycans labeled with dye-doped silica nanoparticles

A new microarray platform, based on lectin super-microarrays and glycans labeled with dye-doped nanoparticles, has been developed to study glycan-lectin interactions. Glycan ligands were conjugated onto fluorescein-doped silica nanoparticles (FSNPs) using a general photocoupling chemistry to afford FSNP-labeled glycan probes. Lectins were printed on epoxy slides in duplicate sets to generate lectin super-microarrays where multiple assays could be carried out simultaneously in each lectin microarray. Thus, the lectin super-microarray was treated with FSNP-labeled glycans to screen for specific binding pairs. Furthermore, a series of ligand competition assays were carried out on a single lectin super-microarray to generate the dose-response curve for each glycan-lectin pair, from which the apparent affinity constants were obtained. Results showed 4-7 orders of magnitude increase in affinity over the free glycans with the corresponding lectins. Thus, the glycan epitope structures having weaker affinity than the parent glycans could be readily identified and analyzed from the lectin super-microarrays.

Perfluorophenyl azide immobilization chemistry for single molecule force spectroscopy of the concanavalin A/mannose interaction.

The versatility of perfluorophenyl azide (PFPA) derivatives makes them useful for attaching a wide variety of biomolecules and polymers to surfaces. Herein, a single molecule force spectroscopy (SMFS) study of the concanavalin A/mannose interaction was carried out using PFPA immobilization chemistry. SMFS of the concanavalin A/mannose interaction yielded an average unbinding force of 70-80 pN for loading rates between 8000 and 40,000 pN/s for mannose surfaces on aminated glass, and an unbinding force of 57 ± 20 pN at 6960 pN/s for mannose surfaces on gold-coated glass. Dynamic force spectroscopy was used to determine the dissociation rate constant, k(off), for this interaction to be 0.16 s(-1).