Direct Photoredox Synthesis of N-Linked Glycoproteins

The structure of N-linked glycans regulates protein folding, stability, conformation, and fundamental cellular functions. However, current biosynthesis methods yield heterogeneous glycoforms, and chemical total synthesis is prohibitively step-intensive, significantly hindering functional studies of glycan structure-function relationships, preventing detailed studies of the functions of N-glycans on proteins. To overcome these limitations, a novel, streamlined strategy for the direct single-step synthesis of glycoproteins with structurally defined N-glycans has been developed. This method utilizes visible-light-mediated photoredox catalysis in phosphate-buffered saline (PBS) to enable the straightforward addition of N-glycosyl carbamoyl radicals, generated from N-glycosyl-1,4-dihydropyridines (DHPs), to the dehydroalanine (Dha) double bond on proteins. Under mild conditions, this radical addition provides direct access to well-defined N-linked glycoproteins, such as small ubiquitin-related modifier 2 (SUMO2), phosphate-transport protein (PstS), interleukin-1 alpha (IL-1α), and β-lactoglobulin with two disulfide bonds. This streamlined approach promises accessible, well-defined glycoproteins for high-throughput structure-function studies.