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We report a novel glycan array architecture that binds the mannose-specific glycan binding protein, concanavalin A (ConA), with sub-femtomolar avidity. A new radical photopolymerization developed specifically for this application combines the grafted-from thiol-(meth)acrylate polymerization with thiol-ene chemistry to graft glycans to the growing polymer brushes. The propagation of the brushes was studied by carrying out this grafted-to/grafted-from radical photopolymerization (GTGFRP) at >400 different conditions using hypersurface photolithography, a printing strategy that substantially accelerates reaction discovery and optimization on surfaces. The effect of brush height and the grafting density of mannosides on the binding of ConA to the brushes was studied systematically, and we found that multivalent and cooperative binding account for the unprecedented sensitivity of the GTGFRP brushes. This study further demonstrates the ease with which new chemistry can be tailored for an application as a result of the advantages of hypersurface photolithography.

Spatially encoded glycan microarrays promise to rapidly accelerate our understanding of glycan binding in myriad biological processes, which could lead to new therapeutics and previously unknown drug targets. Here, we bring together a digital micromirror device, microfluidic introduction of inks, and advanced surface photochemistry to produce multiplexed glycan microarrays with reduced feature diameters, an increased number of features per array, and precise control of glycan density at each feature. The versatility of this platform was validated by printing two distinct glycan microarrays where, in the first, different glycans were immobilized to create a multiplexed array and, in another, the density of a single glycan was varied systematically to explore the effect of surface presentation on lectin-glycan binding. For lectin binding studies on these miniaturized microarrays, a microfluidic incubation chip was developed that channels multiple different protein solutions over the array. Using the multiplexed array, binding between eight lectin solutions and five different glycosides was determined, such that a single array can interrogate the binding between 40 lectin-glycan combinations. The incubation chip was then used on the array with varied glycan density to study the effects of glycan density on lectin binding. These results show that this novel printer could rapidly advance our understanding of critical unresolved questions in glycobiology, while simultaneously increasing the throughput and reducing the cost of these experiments.

Synthetic carbohydrate receptors (SCRs) that selectively recognize cell-surface glycans could be used for detection, drug delivery, or as therapeutics. Here we report the synthesis of seven new C2h symmetric tetrapodal SCRs. The structures of these SCRs possess a conserved biaryl core, and they vary in the four heterocyclic binding groups that are linked to the biaryl core via secondary amines. Supramolecular association between these SCRs and five biologically relevant C1 -O-octyloxy glycans, α/s-glucoside (α/s-Glc), α/s-mannoside (α/s-Man), and s-galactoside (s-Gal), was studied by mass spectrometry, 1 H NMR titrations, and molecular modeling. These studies revealed that selectivity can be achieved in these tetrapodal SCRs by varying the heterocyclic binding group. We found that SCR017 (3-pyrrole), SCR021 (3-pyridine), and SCR022 (2-phenol) bind only to s-Glc. SCR019 (3-indole) binds only to s-Man. SCR020 (2-pyridine) binds s-Man and α-Man with a preference to the latter. SCR018 (2-indole) binds α-Man and s-Gal with a preference to the former. The glycan guests bound within their SCR hosts in one of three supramolecular geometries: center-parallel, center-perpendicular, and off-center. Many host-guest combinations formed higher stoichiometry complexes, 2:1 glycan⋅SCR or 1:2 glycan⋅SCR, where the former are driven by positive allosteric cooperativity induced by glycan-glycan contacts.

Interactions between cell surface glycans and glycan binding proteins (GBPs) have a central role in the immune response, pathogen-host recognition, cell-cell communication, and a myriad other biological processes. Because of the weak association between GBPs and glycans in solution, multivalent and cooperative interactions in the dense glycocalyx have an outsized role in directing binding affinity and selectivity. However, a major challenge in glycobiology is that few experimental approaches exist for examining and understanding quantitatively how glycan density affects avidity with GBPs, and there is a need for new tools that can fabricate glycan arrays with the ability to vary their density controllably and systematically in each feature. Here, we use thiol-ene reactions to fabricate glycan arrays using a recently developed photochemical printer that leverages a digital micromirror device and microfluidics to create multiplexed patterns of immobilized mannosides, where the density of mannosides in each feature was varied by dilution with an inert spacer allyl alcohol. The association between these immobilized glycans and FITC-labeled concanavalin A (ConA) - a tetrameric GBP that binds to mannosides multivalently - was measured by fluorescence microscopy. We observed that the fluorescence decreased nonlinearly with increasing spacer concentration in the features, and we present a model that relates the average mannoside-mannoside spacing to the abrupt drop-off in ConA binding. Applying these recent advances in microscale photolithography to the challenge of mimicking the architecture of the glycocalyx could lead to a rapid understanding of how information is trafficked on the cell surface.

Synthetic carbohydrate receptors could serve as agents for disease detection, drug delivery, or even therapeutics, however, they are rarely used for these applications because they bind weakly and with a preference towards the all-equatorial glucosides that are not prevalent on the cell surface. Herein the binding of 8 receptors with 5 distinct octyloxy pyranosides, which was measured by mass spectrometry and by 1 H NMR titrations in CD2 Cl2 at 298 K, is reported, providing binding affinities that vary from ≈101 -104 m-1 . Although the receptors are promiscuous, 1 shows selectivity for β-Man at a ratio of 103:1 β-Man:β-Gal, receptors 2-4 and 6 have preference for α-Man, 5 is selective for β-Gal, and 10 prefers α-Glc (Man=mannose; Gal=galactose, Glc=glucose). A variety of 1D and 2D NMR, and computational techniques were used to determine the thermodynamic binding parameters (ΔHo and ΔSo ) and the structure of the host-guest complex, revealing that dimeric receptor 10 binds β-Man with increased enthalpy, but a larger entropic penalty than 1. The first-principles modelling suggests that 10⋅β-Man forms an inclusion-type complex where the glycan engages both monomeric subunits of 10 through H-bonding and C-H⋅⋅⋅π interactions. Like natural glycan-binding proteins, these receptors bind pyranosides by accessing multivalent and cooperative interactions, and these studies suggest a new approach towards biomimetic synthetic carbohydrate receptors, where conformational flexibility and promiscuity are incorporated into design.