Studies Of Antitumor Iridium Hydrides Complexes And The Evolution Of Sortase A, Protein Modification With Unnatural Amino Acids

This paper mainly deals with three parts:1) the evolution of sortase A and its application in protein modification;2) the synthesis, biological evaluation and mechanism studies of iridium hydride complexes.;3) the application of protein modification based on unnatural amino acids (UAAs).Sortase A is one of the most powerful tool conducting protein modification and cell tagging, which can specifically recognize protein sequence LPTEG and break down peptide bond between T and G, and meanwhile catalyze the formation of new peptide bond between T and peptide and protein with N-terminal oligo-G. However, a bottleneck of applying sortase A to protein modification and cell tagging lies in the low catalyst efficiency. Thus, we set out to enhance the catalyst efficiency of sortase-A through evolutionary method. We tried several ways and finally established a screening system based on FRET effects of fusion fluorescent proteins and made optimization of screening conditions. We established mutant library through error-prone PCR combined with rational design based on protein structure. We obtained a series of mutants with high activity through screening, with which we did enzymatic activity tests. Finally, we obtained a mutant with more than100folds catalyst efficiency compared with wild type sortase A through protein directed evolution.Research of metal complexes has long been considered as important given that many successful medicines or precursors for antitumor are based on metal complexes. Nitrone (PBN), a kind of free radical trapper, has been recently reported with an antitumor activity. We obtained a series of nitrone iridium hydride complexes through reacting nitrone precursory ligand with iridium complex. We measured their antitumor activity in vitro and research carefully into the mechanism of antitumor activity of complex2f, the complex with the highest antitumor activity and we primarily concluded that the main mechanism may be that2f interacts with DNA, especially with adenine and cytimidine. Our work expanded the antitumor field of iridium complexes.Genetic coded unnatural amino acids incorporation is a newly emerging technique in chemical biology, through which a series of reactive groups can be introduced into protein of interest and thus provide them with new functions. We synthesized a series of unnatural amino acids for various purposes and tried expression of protein of interest with unnatural amino acids site specifically incorporated through suppression of amber codon in Escherichia coli(E. coli). Finally, two unnatural amino acids, L-Thr-ε-Lys and Bloc-Lys, can be incorporated with high efficiency. Then we conducted periodate oxidation and conjugation of protein incorporated with L-Thr-ε-Lys. We successfully conjugated mPEG derivatives and small molecules to proteins and completed site specific protein modification. Besides we also conducted thion-ene reaction to conjugate compounds containing thiol group with proteins with bloc group site specifically incorporated. This technique can be applied to site-specific protein modification, such as protein fluorescence tagging and protein PEGylation and bears potential application in biology and medicine research.