With ongoing revelations of the role played by glycosylation in cancer and infectious disease, the demand for homogeneous glycopeptides is growing faster every year. Whether these molecules are used for therapeutic purposes, or to determine the role of the N- and O-linked glycans in the mechanism of action, structural homogeneity is paramount. Leveraging our knowledge of carbohydrate and peptide chemistry, we have delineated a process to synthesize glycopeptides with atomic control. While solving the technical challenges of bringing one of these molecules from the bench to clinical trial, we have optimized and validated our approach. Today, our technology represents what we believe is one of the most versatile and economical ways of producing homogeneous glycopeptides at a usable scale.
In order to access a functional cross-section of naturally occurring N-linked glycans, our team has developed reliable methods for the modular assembly of the three N-linked glycan subtypes (high-mannose, hybrid- and complex-). These methods have been scaled to produce multi-gram quantities of a high-mannose glycan at >95% purity for use in a Phase I clinical trial. A representative panel of the ever-expanding glycans we have synthesized is depicted below.
Our technology to produce synthetic glycosylated peptides relies on the paradigm of convergent N-linked glycopeptide assembly, where a glycan is coupled to an adequately protected peptide in solution. Used in conjunction with our gram-scale glycan production capabilities, this approach successfully yielded over a gram of a 30-mer glycopeptide harboring two high-mannose glycans intended for a Phase I clinical trial, with 98% purity. Even at this unprecedented preparative scale, this technology enables the incorporation of other post-translational modifications (O-glycosylation, phosphorylation, sulfation) or molecular handles (biotin, alkyne, azide), and is compatible with conformational stabilization elements (non-reducible disulfide bond surrogate, staple) used to coax our glycopeptides into the desired secondary structure.
The scope of our convergent N-linked glycopeptide assembly has also been extended to the synthesis of N-, O-linked glycopeptides which are observed in nature but are seldom reported in the scientific literature as synthetic homogeneous molecules. O-linked glycans functionalized on residues of interest (serine, threonine, tyrosine) are directly incorporated as a “cassette” in the peptide sequence during solid-phase synthesis. Once complete, the N-linked glycan is introduced as above to afford a peptide harboring N- and O-linked glycosylation motifs.