Design Of Structurally Complex Peptides Based On Novel Precise Disulfide Pairing Strategy

Peptides have been widely used in the diagnosis and treatment of diseases mainly from its advatanges such as high affinity,high targeting selectivity,sequence diversity and so on.Cyclic peptides have special ring structure and conformational constraint as compared to their linear peptide precursors.Among the methods for cyclization of peptides,the formation and reduction of disulfide bond are highly efficient and no side reactions.Thus,it is the most common ways to use multiple disulfide bonds for the construction of mulcyticlic peptides in natue.The disulfide bonds are not only crucial to defining and stabilizing the structures of peptides,but also are considered to be important determinant that increases structural diversity of disulfide-rich multicyclic peptides by the formation of different cysteine pattern.However,a peptide containing many cysteines the number of possible disulfde isomers is large(For example,for a six-cysteine-containing peptide,after the oxidation,there will be 15 possible isomers formed in theory).Precise disulfide pairing in nature,atifical synthesis and computational design is driven by the primary sequence.That needs plenties of amino acid residues to encode the oxidative folding information,which leads to these amino acid residues haven't role in function.Thus,Precise disulfide pairing depends on sequence,not only limited the structural diversity of disulfide pepitdes,but also limited the functional diversity of disulfide peptides.To challenge the exisiting problem of disulfide-rich,we consider studying the chemistry problem of disulfide pairing is a breakthrough.Based on this,in this thesis we proposed the new concept of the orthogonal disulfide pairing for guiding the oxidative folding information.This thesis includes five chapters summarized as the following:Chapter 1:We introduced the classifications,characteristics and biological applications of disulfide-rich peptides,and then summarized the existing methods for the design of the disulfide-rich peptide.Based on these reviews,the research ideas and significance of this thesis were also proposed.Chapter 2;Oxidative folding and disulfide isomers are one of the main reasons that limited the development and utilization of disulfide-rich peptides.Our study found that disulfide pairing between cysteine(Cys)and penicillamine(Pen)is orthogonal.Based on this,we proposed the new concept of orthogonal Cys-Pen pairing for guiding the oxidative folding of peptide.Based on this concept,when the model peptides-containing one CXPen/PenXC motif,which can successfully force the oxidative folding of peptides into specific bicyclic isomers.The method developed in this chapter can provide new idea for the design and synthesis of multicyclic peptides.Chapter 3:Tricyclic peptides typically have higher structural rigidity and richer sequence/structure information.However,the precise disulfide pairing of disulfide-rich tricyclic peptides have nerver been breakthrough.In this chapter,we strategically introduced CXPen/PenXC motif,Pen and non-natural dithiol amino acid(Dtaa)into peptide chain,which can successfully reduce the oxidative folding products from a total of 15 isomers to the 2.Compared with natural disulfide-rich peptide scaffolds,the tricyclic peptide scaffolds developed in this chapter have many advatanges such as precise disulfide pairing,the high oxidative folding yield,the stability of isomer,the tolerance of disulfide isomer,the tolerance of sequence randomized and so on.Thus,these tricyclic scaffolds would fit well with the grafting of bioactive peptides and then used to design bioactive multicyclic peptides.Chapter 4:Although the combination of the effect of Dtaa and orthogonal Cys-Pen pairing can be used to encode the oxidative folding information effectively,we found that it is difficult to use this method to design topologically-formidable tricyclic peptides.In this chapter,we found that the orthogonality between two CXPen/PenXC have great ability to guide the oxidative folding of peptides.According to the orthogonality between two CXPen/PenXC,we can obtain arbitrary the isomers of disulfide-rich tricyclic peptides,which includs two topologically-formidable isomers with disulfide connectivities(1-4,2-5,3-6 and 1-6,2-5,3-4).Furthermore,two topologically-formidable isomers have highly constrained conformation,tolerance of sequence randomized,the stability of isomer and so on.Thus,this method can be easily used to design constrained tricyclic peptides.Chapter 5:Disulfide-rich dimer are a kind of peptides with novel structure.However,peptide-containing even number cysteine tend to form intramolecular disulfide pairing without sequence encoding information and in low concentration.Our study discovered that directing the pairing of intermolecular Cys-Pen disulfide bonds in the present of selenocystine.We further combine the chemical method of intermolecular pairing,de novo protein design and directed evolution for the design and systhesis of structurally complex dimer peptides with specific function.Compared with natural disulfide-rich dimer peptides,the disulfide-rich dimer peptides developed in this chapter have many advantanges such as more stable structure,the tolerance of sequence randomized and so on.Thus,the combinational method in this chapter can be used in the development of disulfide-rich dimer peptides with structurally stable and diverse.Finally,the study contents of this thesis were summarized and the further research works was discussed.