Design,Synthesis And Application Of Precisely-Paired Disulfide-Rich Multicyclic Peptides

Peptides are important biomolecules in drug development with their high specificities to their targets.Peptide-based drugs have played a remarkable role in medical practice since the advent of insulin therapy in the 1920s.However,peptides have some disadvantages such as poor metabolic stability and poor oral bioavailability,which lead to relatively slow pace of development of peptide therapeutics.In nature,disulfide-rich cyclic peptides have high stabilities and a wide range of biological activities.Disulfide bond is a dynamic covalent bond,which is prone to rearrangement and isomerization during the oxidative folding of peptides.For synthesis of peptides containing multiple pairs of disulfide bonds,one of the challenges is how to achieve the precise paring of disulfide bonds.Naturally evolved and computer-designed disulfide-rich peptides usually rely on the primary sequence to guide the correct pairing of disulfide bonds.If a large number of amino acid residues are used to assist the precise paring of disulfide bonds rather than encode its function,which greatly limits the functional diversity of the peptide molecules.Therefore,we need to develop sequenceindependent cyclic peptide construction methods to lay the foundation for the development of new peptide-based drugs.This dissertation is divided into five chapters,the content is as follows:Chapter 1:Firstly,we reviewed the development and application of monocyclic peptides and multicyclic peptides,and then the disulfide-rich peptides in nature and the synthesis methods were introduced.Finally,we reviewed the development and application of site-specific incorporation of unnatural amino acids into proteins and mRNA display technology.Based on these reviews,we proposed the research ideas and significance of this dissertation.Chapter 2:We proposed a general and reliable strategy for the construction of isomerically pure tricyclic peptides.This strategy relies on the formation of regioselective bisthioether and orthogonal pairing of disulfide bonds.We designed and synthesized a range of structurally diverse tricyclic peptide scaffolds tolerate to extensive sequence manipulations.Chapter 3:The practicability of the tricyclic peptide construction strategy proposed in Chapter 2 was verified by the epitope grafting experiments.We grafted the epitope containing the RGD motif and the epitope for the Keap1 protein related to Nrf2 onto the designed mini-cyclic peptide scaffolds,and investigated the bioactivity of the grafted peptides and their proteolytic stability.Chapter 4:In this chapter,we explored and discovered a new motif(βPenC)and a dithiol amino acid(2SHPen)that can guide the precise pairing of disulfide bonds.They could replace CXPen/PenXC motifs that found in previous work,and provide more options for the construction of bicyclic and tricyclic peptides.Moreover,the new disulfide-directing motifs will increase the structural diversity of multicyclic peptides.Chapter 5:IT this chapter,the compatibility of βPenC motif and 2SHPen with ribosome expression system was investigated.It was demonstrated that the βPenC motif could be incorporated at the N-terminal of peptides,and 2SHPen can also be incorporated at internal position of peptides.This successful experiment shows that we can apply the designed disulfide-directing motifs to the design and synthesis of mRNA display multicyclic peptide libraries.Finally,the research contents of this dissertation were summarized and the further work was prospected.