Synthesis And Functions Of Phosphine-containing Unnatural Amino Acids,Peptides And Proteins

In the long process of evolution,life on Earth has introduced phosphorus（P）into many important biological functional molecules,such as nucleic acid,phosphoproteins,phospholipid and so on.Due to the oxygen-rich environment of the earth,these phosphorus mainly exist in the form of+5 valence.Because phosphorus has rich electronic structure,valence and functional properties,scientists not only synthesized and modified phosphoproteins by chemical methods for biomedical function research,but also designed and synthesized numerous artificial small phosphine ligands with+3valence,which complexed with transition metals to form powerful homogeneous catalysts.This thesis focuses on how to efficiently synthesize phosphonic acid mimics of natural phosphothreonine and their functional applications,and how to effectively introduce oxygen-sensitive phosphine ligands into specific sites of proteins in life and to realize the preparation of new artificial metal proteins in aqueous air,a series of work has been carried out,and the following major progress has been made.In this thesis,starting from the simple and easily available valine derivatives,a new route for the synthesis of phospho-threonine non-hydrolysis mimic CH₂-p Thr with high efficiency,simplicity and stereoselectivity was developed by palladium-catalyzed C-H bond activation method,and phosphonic acid polypeptide inhibitors were prepared from the synthesized monomers,which can not only effectively resist the hydrolysis of phosphatase.The binding force to the targeted protein 14-3-3ζis stronger than that of natural phosphorylated peptides,which provides a powerful tool for the study and regulation of protein phosphorylation in vivo.In this thesis,a kind of borane-protected derivative P3BF containing phosphine phenylalanine was designed and synthesized,the unnatural amino acid is stable in aqueous and aerobic conditions.Using genetic codon expansion technique,on the basis of evolutionary screening of orthogonal aminoacyl-t RNA synthetase specifically recognizing P3BF,P3BF was successfully inserted into different specific sites of the target protein-green fluorescent protein（sf GFP）in E.coli,and the high resolution crystal structure of sf GFP containing unnatural phosphine-boron（P-B）bond（1.95?）was successfully obtained and analyzed.In this thesis,a new"one-pot"strategy which can directly convert phosphine-boron compound P3BF to phosphine-metal complex in aqueous and aerobic conditions is developed.this strategy mainly uses cyclic palladium metal complex to react with P3BF,which is simple,mild,easy to operate and does not need anaerobic treatment,which provides a new scheme for the site-directed chemical conversion of proteins and the synthesis of phosphine-containing metal proteins.The mechanism of"deboronation and palladium coordination"in this step was studied,and the possible reaction path was put forward.On this basis,the phosphine-boron unnatural amino acid P3BF was inserted into a specific site of a class of Lmr R proteins that could be dimerized to form a special hydrophobic cavity structure,and using the"one-pot"strategy,artificial metalloproteins containing phosphine-cyclic palladium complexes were successfully constructed under aqueous and air conditions.The new strategy of chemical biosynthesis of phosphine-containing metalloproteins lays an important foundation for the construction and functional application of artificial metalloenzymes.