Resistance To Protease Degradation Of The Peptide Design, Synthesis And Metabolism And Biological Activity Study

Based on the catalytic mechanisms of proteases and the structure character of protease inhibitors, it has been hypothesized that adequate anti-protease degradation ability of peptides could be obtained by introducing the functional groups such as amino and ureido groups to the suitable sites of side chain. These functional groups probably competitively combined with the active site of a protease by hydrogen bonds, and occupied the position of amide bond to be degraded, then blocked or slowed the transition state between the peptide amide bond and active site of the protease. So hydrolysis of the peptide amide bond would be inhibited.In our previous work, LHRH antagonist was selected as model peptide. The functional groups were introduced on the side chains of selected amino acids at position 5 or 6 and N-terminal of LHRH antagonist. A series of deca-peptides were designed and synthesized. It was found that: the peptides with functional groups such as amino, acetyl amino and ureido groups showed better bioactivities in vivo and longer half-lives in pancreas protease mixture. These results seemed to favor previous hypothesis of long acting peptide. In order to further verify this hypothesis, the following works have been done in this thesis:1. The functional groups were introduced together at positions 5, 6 and N-terminal of LHRH antagonists. Bioactivities of synthesized LHRH analogues were evaluated using a testosterone levels test model in rat. All of the analogues showed bioactivity of inhibition of testosterone secretion (500μg/kg); but none showed longer duration than analogues independently with functional groups at position 5 or 6 and N-terminal.2. In order to study the interaction mechanism of protease and the functional groups, novel model peptides as substrates of chymotrypsin and trypsin were designed and synthesized. Protease degradation half-lives of 14 model hexa-peptides were evaluated used chymotrypsin and trypsin in vitro. The degraded fragments in chymotrypsin were analyzed by LC-MS. It was found that: introduction of carboxyl and amide groups as the side chain of aliphatic amino acids could prolong the half-lives of model peptides in chymotrypsin, and introduction of amino, acetyl amino and ureido groups on the side chain of aromatic amino acids could prolong the half-lives of model peptides in trypsin.