SUMMARY Glycoproteins constitute approximately 70% of all therapeutic proteins approved for clinical use in the United States. Although there are various technologies available to manufacture homogenous materials, even the most successful strategies fail to produce only one (of a few) glycoform. This limitation represents a major challenge that can affect efficacy, safety, and the costs of production. In this project, we will develop a broadly applicable technology that can eliminate the inherent limitations associated with in vivo and ex vivo glycoprotein production systems. In aim 1, we will invent and optimize a chemical bioconjugation reaction that will furnish glycoproteins containing the native glycan linkage at the Asn residue as a single glycoform. In aim 2, we will (a) test the new glycoconjugation method in diversification of small proteins and (b) demonstrate its generality in a model system containing high-mannose glycans. Taken together, the new technology will eliminate the safety, regulatory, and IP issues currently associated with glycoprotein production, manipulation, and characterization.

PROJECT NARRATIVE Protein glycosylation, one of the most common forms of post-translational modification, affects various chemical, physical, and biological features of proteins. In this proposal, we describe the development of a chemical technology to produce homogeneous N-linked glycoproteins by harnessing the unique reactivity of radical intermediates generated under photochemical or thermal conditions. If successful, this method will have a transformative impact how glycoproteins are produced and used in clinical practice.