Periodic Report Summary 2 - GLYCOSURF (Surface-Based Molecular Imprinting for Glycoprotein Recognition)
Glycans, which exist in either as membrane-bound glycoconjugates or as secreted molecules, are intimately involved in various physiological and pathophysiological events. Yet, selective glycan recognition remains a daunting task due to their inherent diversity and complexity. Antibodies are widely used as receptor sites in the detection, quantification and purification of many proteins including clinically relevant glycoproteins. However, antibodies suffer from poor stability, need special handling and require a complicated, costly production procedure. Furthermore, the peculiarities of intracellular machinery, which is utilised in the commercial production of antibodies, is not ideally suited for the production of high affinity antibodies against carbohydrate-based antigens. For all of these reasons, more robust synthetic alternatives are sought. In this project, new synthetic platforms are being developed for highly specific recognition of a broad spectrum of glycoproteins and glycan structures. Initially studies were focus on the development of a platform for glycoprotein recognition. A hierarchical bottom-up route exploiting reversible covalent interactions with boronic acids and so-called click chemistry for the fabrication of glycoprotein selective surfaces that surmount current antibody constraints was devised. The self-assembled and imprinted surfaces, containing specific glycoprotein molecular recognition nanocavities, confer high binding affinities, nanomolar sensitivity, exceptional glycoprotein specificity and selectivity with as high as 30 fold selectivity for prostate specific antigen (PSA) over other glycoproteins. This synthetic, robust and highly selective recognition platform can be used in complex biological media and be recycled multiple times with no performance decrement. More recently, efforts are being focus on recognizing different glycoforms of the same glycoprotein and glycans with different structures. In order to recognize different glycoforms of the same protein, surface hydrogels and molecular imprinting are being combined. Molecular imprints within ultra-thin hydrogels were synthesised from a pre-prepared surface using atom transfer radical polymerisation (ATRP). Studies are underway to separate the different glycoforms of RNase B present in standard samples of pure, commercially available lypholized protein using size exclusion chromatography. Imprints of particular glycoforms will be then performed and demonstration of glycoform specificity will follow. A different strategy is also being pursued to distinguish with high specificity different glycan structures. It relies on the formation of high-yield, complex glycan-synthetic carbohydrate receptor assemblies and precise generation of surface-confined templated binding sites. For the later studies, benzoboroxoles are being employed as carbohydrate receptors. In contrast to their boronic acids analogous, benzoboroxoles can bind non-reducing hexopyranosides at pH values compatible with biological systems.