The Studies On Novel Cell-penetrating Peptides Based Onα-aminoxy Acids

Cell-penetrating peptides (CPPs) are short peptide sequences with the ability to cross plasma membranes, therefore can be used as vectors for transport of various exogenous molecules without permeability into living cells, including small chemical molecules, peptides, proteins, nucleic acids and nanoparticles. More than 20 CPPs have been successfully applied in the intracellular delivery of membrane-impermeable molecules, however, seldom of them can be employed in vivo:1) most of them were consisted of natural amino acids, and easily degraded by intracellular protease or enzymes in blood, resulting in low stability; 2) endosomal entrapment was always occurred to CPPs containing natural amino acids at low concentrations, which disrupted its release into cytoplasm or induced lysosomal degradation after being transported to lysosomes, hindering the exertion of their biological functions. To solve these problems, unnatural amino acids were introduced into the constructs of CPPs, and CPPs containing unnatural amino acids not only showed higher stability than its corresponding natural amino acids-based analogues, but also entered cells in direct translocation way and produced diffuse distribution. As novel unnatural amino acids, a-aminoxy acids are of high stability towards protease and liable to form secondary structures, therefore, peptides containingα-aminoxy acids may be potential CPPs for application.In this thesis, firstly, we designed and synthesized series of novel CPPs based on a-aminoxy acids, and their secondary structures and biological properties were then studied; secondly, a permeable molecular probe based on CPP was designed and synthesized, and its cellular permeability was studied. The main contents and results were summarized as follows:1) CPPs based on a-aminoxy alanine were designed and synthesized, and studies on their secondary structures and biological properties indicated that these peptides formed amphiphilic secondary structures and produced diffuse distribution in cells, but showed low cellular uptake efficiency.2) CPPs based on a-aminoxy lysine were designed and synthesized, and studies on their biological properties indicated that a hybid peptide containing a-aminoxy lysine showed high stability and low toxicity; further study on its cellular uptake mechanism indicated that this hybrid peptide entered cells in direct membrane translocation way and produced diffuse distribution in cells.3) A permeable molecular probe based on CPP was designed and synthesized, and studies on its cellular permeability indicated that CPPs can promote the cellular uptake of membrane-impermeable molecular probe.Introduction of a-aminoxy acids into constructs of CPPs, not only provided new ideas for design of novel CPPs, also gave more understanding into cellular uptake mechanism of CPPs, and these studies on a-aminoxy acids based CPPs will support their biological applications technically and theoretically.