| Peptides are promising pro-drugs in many therapeutic fields,cyclization of peptides could promote their affinity,stability,and selectivity.Disulfide bonds constraining is specially noted among the many cyclization strategies.Disulfide bridges are the most important covalent for constraining the structure of many proteins and peptides in the post-translation modification,for example,there existing a couple of disulfide bonds in the structures of insulin family,conotoxins and defensins.Disulfide bonds are formed by quasi-spontaneous oxidation reaction of cysteine side chains with high efficiency and rare side reactions under oxidative condition,and no unnatural amino acid is needed during the course.These features make disulfide bond one of the most common cross-linker in cyclic peptide libraries,for instance,mono-/multi-cyclic peptide libraries generated from one or two and three disulfide bonds constraining have been constructed and screened for target proteins.Multi-cyclic peptides constrained by two or more disulfide bonds provided more structural diversity and have more restrict conformation and more rigid structure,thus show higher structure stability and higher resistance to proteases than mono-cyclic peptides.However,multiple disulfide bonds in peptides may undergo isomerization in the weakly reducing environment like biological fluids,and which would lead to the conversion of biological active multicyclic into their non-active isomeric forms,for example,peptide with four cysteine could oxidized to form three isomers,and usually,only one of these isomers is biologically active.Thus,precise disulfide pairing strategies are helpful and promising in the construction of multi-cyclic peptides.In the previous study of our group,by rationally manipulating the interplay between the’CXC’ motif and orthogonal pairing of Cys-Pen disulfide,we successfully synthesized fused and bridged bicyclic peptides which are stable for isomerization.In the study of this dissertation,we introduced this strategy to high throughput screening of bicyclic peptides,and reported several bicylic peptides which show high affinity to the target and exhibit no isomerization in the presence of biothiols.Three chapters are included in this dissertation and summarized as below.Chapter 1:We introduced kinds of peptides cyclization strategies and the properties of crosslinkers,a few examples of disulfide constrained peptide libraries and the general principal of phage display are also reviewed,we further summarized several technologies to restrict disulfide pairing.In the end,we proposed the objective,contents and significance of the research in this dissertation.Chapter 2:Successfully estabilished the platform of phage display screening system,constructed the phage encoded library of GCXCX5CX5C fomat peptides and screened against Keapl Kelch Domain and Plasma Kallikerin for iterative three rounds,several biologically active peptides were pulled out of the library and were in vitro synthesized to determine their binding affinities to the target.The characters of rapid isomerizaiton of XM-3 Isomer 1 and XM-10 Isomer 1 under weakly reducing environment were determined,and the affinities and proteolysis stabilities of two bicyclic peptides mentioned above with their linear and monocylic analogs were compared.Chapter 3:Two of four Cys in XM-2,XM-3 and XM-10 were substituted with Pen selectively,and after determined the affinities of Pen-substituted peptides,we found that some of them(XM-2 pi,XM-3 p2 and XM-10 p1)show identical(or slight higher)affinities to their four-Cys analogs,and what’s more,we observed no isomerization of XM-3 p2 and XM-10 p1 under weakly reducing environment.Finally,we analyzed the disulfide connectivities by 2D NMR spectroscopy.In the very end of this dissertation,the research contents were summarized and the prospection of the future work was discussed. |
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