The rising costs and complexities of drug discovery need sustainable and innovative approaches in pharmaceutical development. β-amino acids, as critical building blocks in medicinal chemistry, often pose synthetic challenges due to limitations in conventional organic synthesis. This thesis addresses these challenges by exploring enzyme-catalysed methods for the greener synthesis of pharmaceutical intermediates, emphasising sustainability and efficiency.
This thesis focuses on the sustainable synthesis and applications of β-amino acids, and their role in improving the stability of therapeutic peptides, particularly PEPITEM, a peptide known to regulate immune cell trafficking. Using engineered aspartase B enzymes mutants (B19 and F29) from Bacillus sp. YM551, two key β-amino acids, (R)-β-amino butyric acid and (S)-β-phenylalanine, were successfully synthesised. The B19 variant performed better, achieving a conversion efficiency of around 75% and an isolated yield of approximately 67% with high purity (99%). In contrast, the F29 variant gave lower conversion (<22%) and yield (<5%) but still demonstrated good purity (99%) after purification.
A major part of this work involved optimising the derivatisation of these β-amino acids into Fmoc-protected forms. Despite initial challenges, the reaction was successfully scaled from milligram to gram scale without compromising yield or purity, showing clear potential for practical and commercial use. These Fmoc-β-amino acids were then applied in both manual and automated solid-phase peptide synthesis (SPPS) of PEPITEM analogues. This process provided valuable insights into how resin selection, coupling efficiency, and cleavage conditions can significantly affect the successful synthesis of challenging peptide sequences.
The most impactful part of the project centred on improving the stability of PEPITEM. The native peptide degrades rapidly in vivo, with a reported half-life of less than 2 min. To address this, β-amino acids were introduced at key sites identified through computational cleavage enzyme prediction tools and verified by experimental approach. The resulting analogues showed a dramatic increase in stability, remaining intact for up to 72 h when exposed to digestive enzymes (pepsin and proteinase K), even under varying pH and enzyme concentrations.
While biological activity of the modified peptide was not tested, the success of similar β-amino acid containing drugs like bestatin, sitagliptin, and paclitaxel suggests that this backbone modification is likely to be compatible with receptor binding and bioactivity. Altogether, this project presents a practical and environmentally friendly approach to the synthesis of β-amino acids and their applications in therapeutic peptide development.
