Synthesis and Evaluation of Novel Amino Acids for Carbohydrate Conjugation

Glycosylation is a common eukaryotic post-translational modification of peptides and proteins. However, elucidating the relationship between carbohydrate structure and glycoprotein function is challenging. Contributing significantly to this challenge is the arduousness of glycoprotein synthesis, making the preparation of glycoform libraries for biological evaluation impractical.

This thesis investigated proposed new methodologies of preparing O- and N-glycosylated peptides and proteins by preparing and evaluating novel amino acid derivatives. Firstly, it was investigated whether native O-glycosylation can be achieved by utilising O-substituted aminooxy serine derivatives, envisioned to react with reducing carbohydrates and then cyclise to form oxazolidines. To this end, aminooxy serine derivatives were synthesised in six steps from ᴅ-serine and overall yields of 9-26%. With the aminooxy serine derivatives prepared, reactions of the oxyamines with N-acetylglucosamine (GlcNAc) were attempted. However, there was no product formation observed under the reaction conditions used. In addition, the aminooxy serine derivatives were found to degrade under the mildly acidic reaction conditions. In contrast, alcohol or thiol functionalised oxyamines, which lacked ester groups, did react with GlcNAc to give β-pyranosides or thioaminals, respectively. These results highlighted that the aminooxy serine derivatives may be challenging to use for glycan conjugation.

The second part of this thesis explores new methodology for the synthesis of N-glycosylated peptides and proteins based on amide formation by reacting acylsilanes with oxyamines. For this purpose, an asymmetric organocatalysed Mannich-type reaction was developed to prepare a protected acylsilane functionalised amino acid. The novel acylsilane functionalised amino acid was successfully reacted with a simple diethylcarbamoyl oxyamine, forming the expected amide. Mosher amide analysis of the amide derivative of the acylsilane demonstrated that the asymmetric Mannich-type reaction had led to the formation of the desired enantiomer with an e.e. of 86%. Finally, experiments were undertaken to assess whether this acylsilane functionalised amino acid derivative could be employed to form amides with oxime derivatives of GlcNAc.