Tryptophan C-mannosylation is the only type of protein glycosylation that involves the formation of a carbon-carbon bond between sugar and polypeptide. This unusual co-translational modification is installed on nascent polypeptides within the lumen of the endoplasmic reticulum by the DPY19 enzymes using dolichol phosphate mannose as glycosyl donor. While this modification is found on a diverse collection of secreted proteins, we are only just beginning to understand its biological function(s).
Recent studies suggest that tryptophan C-mannosylation plays an important role in stabilising proteins. However, quantitating the impact of C-mannosylation on the stability of protein and peptide fold has been difficult, owing to a dearth of methods for preparing pure glycoforms. The main barriers to realizing this goal are the production of Trp(Man) in sufficient quantities to facilitate automated solid phase peptide synthesis (SPPS) and the absence of methods for installing the modification at a late stage in peptide synthesis.
To address this issue, we developed unique Ni-catalyzed cross-coupling conditions that utilize photocatalysis or a Hantzsch ester photoreductant to couple glycosyl halides with (hetero)aryl bromides, thereby enabling the α-C-mannosylation of 2-bromo-tryptophan, peptides thereof, and (hetero)aryl bromides more generally. These developments enabled the first automated solid-phase peptide syntheses (SPPS) of C-mannosylated glycopeptides. We used this technology to perform the first verified synthesis of Carmo-HrTH-I, a C-mannosylated insect hormone, and also probe the preferred epitopes of an antibody created to map the C-glycome. During this study we discovered that the tryptophan C-mannosylation undergoes facile anomerization in the presence of strong acids, which can result in protecting group challenges when performing SPPS. To obviate these issues, we further optimized our photoredox/Ni dual catalysis method to enable the late-stage C-mannosylation of peptides bearing 2-bromo tryptophan. This reaction has a broad tolerance to many functional groups and has been used to prepare many glycopeptides.