Chemoselective Reagents for Covalent Capture and Display of Glycans in Microarrays

Abstract

Glycobiology has made very significant progress in the past decades. However, further progress will significantly depend on the establishment of novel methods for miniaturized, high‐throughput analysis of glycan–protein interactions. Robust solid‐phase chemical tools and new, chemoselective reagents for biologically meaningful display of surface‐immobilized glycans are likely to play a key role. Here we present four new bifunctional linkers that allow highly chemoselective capture of unprotected glycans in solution to form glycan‐linker conjugates for direct construction of glycan microarrays (glycochips). The bifunctional linkers carry O‐linked aminooxy moieties, some with N‐substituents at one end and an amino group at the other. In addition, they contain a substituted benzene ring for UV traceability and improved purification of glycan‐linker conjugates. NMR spectroscopic studies in solution proved that N‐substituted aminooxy linkers provided model glycan‐linker conjugates with the β‐glucopyranoside configuration, i.e. the ring‐closed form required for biological recognition. Then an ensemble of glycan‐linker conjugates were assembled from mannobiose, lactose, and N‐acetyl‐lactosamine and used for covalent printing of glycan microarrays. The stability of oximes were studied both in solution and on‐chip. In solution, two of the linkers provided glycan‐linker conjugates with a remarkable stability at pH 4 or higher, on‐chip this relative stability was upheld. Two of the linkers, with different properties, are recommended for the glycobiology toolbox for the construction of glycan microarrays from unprotected glycans.