Unsymmetrical dendrimers, containing both mannose binding units and coumarin fluorescent units, have been prepared using click chemistry and shown to be highly efficient, dual-purpose recognition/detection agents for the inhibition of hemagglutination.

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.

The Cu(I)-catalyzed ATRP and azide-alkyne cycloaddition reactions together provide a versatile method for the synthesis of end-functionalized glycopolymers and their attachment to a suitably modified viral protein scaffold.

Summary The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high mannose glycans on the surface envelope spike of HIV suggesting that the “glycan shield” defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semi-synthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Qβ and cowpea mosaic virus (CPMV). The Qβ glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, Abs against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titres of anti-mannose Abs that do not cross-react with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV.

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.

Cell surface receptors that recognize glycans as ligands mediate a vast range of events in cellular biology through low-affinity, multivalent (simultaneous multipoint) binding interactions. 1 The development of ligand-based probes of these receptors must therefore address both the challenges of glycan synthesis and the need for multivalent platforms. A polyvalent display of glycan ligands is usually achieved by synthesis of the desired glycan with a linker containing a functional group at the reducing end, allowing conjugation to a polyvalent platform bearing the complementary reactive group. 2 An alternative approach of "immobilizing" glycan reaction precursors to the platform of choice and "building out" from such surfaces to make polyvalent oligosaccharides would provide a way to make complex and expensive oligosaccharides precisely placed, as has recently been demonstrated using gold nanoparticles. 3 Here we report the implementation of this strategy for labeling the exterior surface of a virus capsid, taking advantage of the compatibility of the protein particles with glycosyltransferase enzymes and chemoenzymatic reaction conditions. 4 The resulting structures comprise multivalent arrays of glycan ligands favorable for binding to a cell surface glycan receptor in a highly specific manner.Our target to probe the viability of on-virus oligosaccharide synthesis was CD22 (Siglec-2), a receptor specifically expressed on B cells and B lymphoma cells that is involved in regulation of B cell signaling. The natural ligand of CD22 is Siaα2-6Gal, which is found at the termini of N-linked glycans on B and T cells. 5 CD22 is bound by ligands presented by neighboring molecules on the same B cell surface, an interaction "in cis" that promotes strong binding even though the ligand-receptor pair has low intermolecular affinity (K d = ca. 0.1-0.2 mM). 6 Binding of other cells to the CD22 receptor is thereby inhibited unless the competing sialosides are presented in an effective polyvalent fashion.The 9-biphenyl carbonyl (BPC) derivative 7 of the natural sialoside ligand, Siaα2-6Galβ1-4GlcNAc, was selected as our target ligand for construction of multivalent capsid-based structures. 5 GlcNAc, the intermediate Galβ1 HHS Public AccessAuthor manuscript J Am Chem Soc. Author manuscript; available in PMC 2017 June 13. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript sialoside were prepared as O-linked azidoethyl derivatives 1, 2, and 3, respectively ( Figure 1). 4c,8 The conveniently accessible azide groups were used to array the carbohydrates on alkyne-decorated polyvalent particles by the Cu I -catalyzed azide-alkyne cycloaddition (CuAAC) reaction. 9 Two particles were employed: the cowpea mosaic virus (CPMV) 10 and bacteriophage Qβ coat protein, 11 both icosahedral structures approximately 30 nm in diameter. 12 Alkyne groups were introduced by acylation of surface lysine side chains with alkynyl N-hydroxysuccinimide ester 4. 13 Wild-type Qβ, having more subunits per particle (180) than CPMV (60) and...

Tetra- and hexasaccharides were arrayed on the exterior surface of cowpea mosaic virus by using a copper-catalyzed azide-alkyne cycloaddition reaction. Inoculation of chickens with these virus conjugates gave rise to large quantities of polyclonal anti-glycan IgY antibodies that displayed excellent avidity and specificity on analysis with printed glycan microarrays. Avian IgY antibodies are produced in significantly higher yield than is possible for mouse or rabbit IgG, and exhibit reduced cross reactivity with native mammalian proteins. For a tri-LacNAc antigen, affinity purification against immobilized mono-LacNAc was necessary to provide a set of antibodies with specific binding properties. Comparable performance was observed for the virus-based polyclonal versus a commercial monoclonal antibody raised against the globo-H tetrasaccharide; this highlights the utility of the glycan microarray for both quality control and rapid in-depth analysis. Virus-carbohydrate conjugates are promising candidates for development in diagnostic and immunotherapeutic applications.

Carbohydrate-protein binding is important to many areas of biochemistry. Back-scattering interferometry (BSI) is shown here to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSI. Dose-response curves, generated within several minutes and highly reproducible in multiple wash/measure cycles, provided adsorption coefficients that showed mannose to bind to concanavalin A with 3.7 times greater affinity than glucose, in line with literature values. Galactose was found to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were far higher than monovalent glycans, with increases of 60-200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Qβ. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and differs in its sensitivity to the number of binding events rather than changes in mass. Its operational simplicity, generality, and the near-native conditions under which the target binding proteins are immobilized make it an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.