Synthesis and Applications of Triazole- and Triazine-containing Amino Acids

Through their use as mimics of post-translational modifications (PTMs) and in bioorthogonal chemical reporting strategies, unnatural amino acids (UAAs) are vital tools for studying biological systems. τ-Phosphotriazolylalanine I can act as a non-hydrolysable analogue of phosphohistidine and is compatible with the Fmoc-strategy for peptide synthesis. Peptides containing either I or an alternative phosphoamino acid were synthesised and used to demonstrate the selectivity of the SH2 domain of the growth factor receptor-bound 2 protein (a phosphotyrosine binding protein) towards τ-phosphohistidine. In addition, peptides containing I were ineffectively used to study the binding interaction between a histidine-phosphorylated protein, phosphoenolpyruvate synthase and its cognate regulatory protein, YdiA. It was concluded that mimicking the primary structure of one protein through peptide generation was not sufficient to study this protein-protein interaction. As a result, third generation τ phosphotriazolylalanine II was synthesised, which has the potential to be genetically incorporated into proteins using amber suppression.
Bioorthogonal reactions can be used to selectively derivatise probes onto biomolecules. Although there are a number of chemical reactions that have been used for this purpose, many are limited in terms of biocompatibility and synthetic accessibility of bioorthogonal reagents. Therefore, a novel bioorthogonal reaction was developed based on the cycloaddition of 1,2,4-triazines to strained dienophiles. An accessible and robust synthesis to novel 1,2,4-triazin-3-yl-linked amino acid III was devised and its reactivity towards a range of strained dienophiles was investigated. It was determined that III reacted readily with bicyclononyne at 37 °C with a second order rate constant between 0.3 - 0.5 ×10-3 M-1 s-1, depending on solvent mix. The utility of III towards late stage functionalisation of probe molecules was also demonstrated through generation of a fluorescent probe containing III. This triazine probe was used to demonstrate cycloaddition of triazine to a strained dienophile in vitro.