Efficient Chemistry Synthesis And Oxidative Folding Studies Of Centipede Toxin RhTx And Spider Toxin GsMTx4

Peptides are biomolecules composed of α-amino acids through amide peptide bonds,which play important roles in various physiological and pathological activities.Peptide based drugs have been widely used in the treatment of various diseases such as tumors,cardiovascular diseases and diabetes etc.Peptide based drugs are mainly obtained by chemical synthesis,and solid-phase peptide synthesis(SPPS)is the most widely used method.Centipede toxin peptide RhTx is a toxin peptide isolated from the venom of Chinese red-headed centipede.RhTx contains 27 amino acid residues and two pairs of disulfide bonds.As an agonist,RhTx could specifically target the outer pore region of the capsaicin receptor TRPV1 channel and promote the opening of TRPV1 through allosteric regulation,ultimately inducing severe pain.RhTx has become an effective tool to study the activity and structures of TRPV1.Spider toxin peptide GsMTx4 is a toxin peptide isolated from the venom of Grammostola spatulata spider.GsMTx4 contains 34 amino acid residues and three pairs of disulfide bonds,which is the only reported inhibitor specifically targeting Piezo.Mutations in the Piezo channel are associated with a variety of inherited human diseases involving mechanical conduction.RhTx and GsMTx4 are important molecular tools to study the physiological mechanism of TRPV1 and Piezo channels,as well as the target based rational drug design.Nevertheless,the natural abundance of RhTx and GsMTx4 is low,and it is difficult for traditional protein recombinant expression methods to obtain sufficient target peptides.The chemical synthetic method is an effective strategy to obtain tens of milligrams of RhTx and GsMTx4.The recently developed Oxyma/DIC condensation system has the advantages of inhibiting racemization,high coupling efficiency,low cost and high operation safety etc,which facilitate the future wide application of Oxyma/DIC in peptide synthesis.Establishing the optimal reaction ratio of coupling reagents under high temperature conditions is critical for the Oxyma/DIC based SPPS.Moreover,establishing the fast and efficient SPPS utilizing the Oxyma/DIC condensation system has important theoretical significance and application value.Disulfide bonds play a central role in maintaining the structure of RhTx and GsMTx4,and the oxidative folding of disulfide bonds is the key step in peptide synthesis.Exploring the efficiency and yield of three mainstream oxidative folding strategies has great reference significance for the preparation of disulfidecontaining peptides.The first part of this thesis achieved the efficient synthesis and in vitro folding of RhTx.Moreover,the efficiency and yield of three mainstream oxidative folding strategies was systematically evaluated.1.Through the robust HCTU/DIEA based condensation system,three linear peptides of RhTx were synthesized under normal temperature conditions:RhTx-1(linear peptide with four free thiol groups of Cys side chains),RhTx-2(linear peptide with 1.3-Cys side chain thiol groups protected by the Acm group),RhTx-3(linear peptide with 2.4-Cys side chain thiol groups protected by the Acm group).2.Explore the optimal reaction ratio of the Oxyma/DIC condensation system under high temperature conditions,and synthesize six linear RhTx peptides: RhTx-4(linear peptide with 1.3-Cys side chain thiol groups protected by the Acm group)and five linear peptides(RhTx-5,RhTx-6,RhTx-7,RhTx-8 and RhTx-9)in which the side chain thiol groups of four Cys are all exposed.3.In order to study the correctness of RhTx oxidative folding and the effect of different oxidative folding strategies on peptide folding efficiency and yield,the onestep oxidative folding,two-step oxidative folding and one-pot oxidative folding strategies were conducted on the synthetic linear RhTx,affording the target RhTx.The second part of this thesis achieved the efficient synthesis and in vitro folding of GsMTx4.Strikingly,the optimal ratio of Oxyma/DIC condensation system under high temperature for SPPS is established.1.Using the HCTU/DIEA based SPPS,the linear peptide GsMTx4-1 was first synthesized under normal temperature conditions.2.The optimal reaction ratio of the Oxyma/DIC condensation system under high temperature conditions was studied,affording three linear peptides(ie.GsMTx4-2,GsMTx4-3 and GsMTx4-4)of GsMTx4.3.All four linear peptides were independently folded by one-step oxidative folding strategy,giving the target GsMTx4.Analytical RP-HPLC and ESI-MS were used to evaluate the efficiency and yield of refolding process.Meanwhile,the CD and patch clamp activity test were conduct to evaluate the structure and biological activity of the synthetic target GsMTx4.The results of this thesis indicated that: 1.Under the condition of 50℃,the isolated yields of RhTx and GsMTx4 were not significantly improved by increasing the reaction ratio of Oxyma or DIC,indicating that Fmoc-amino acid: Oxyma: DIC=4 eq : 4 eq : 8 eq was the best reaction ratio,which could achieve the rapid condensation of amino acids.2.The isolated yield of two-step oxidative folding strategy(40%～45%)was higher than that of both one-step oxidative folding strategy(30%～40%)and one-pot oxidative folding strategy(10%～25%).One-pot oxidative folding strategy may induced the misfolding of disulfide bonds containing peptides.Besides,there is no significant difference in the process of two-step oxidative folding of linear peptides with 1.3-Cys or 2.4-Cys side chain thiol groups protected by the Acm.3.After separation,the purity of all linear peptides and target peptides could reach more than 95%.4.Analytical RP-HPLC,ESI-MS,CD,and activity evaluation indicated that three different oxidative folding strategies could achieved the correct folding of RhTx.Moreover,no significant difference was detected in the structure and activity of RhTx prepared by different synthetic and refolding conditions.5.The analytical RP-HPLC and ESI-MS tests indicated the folding of linear GsMTx4 prepared by different synthetic conditions could be achieved by the one-step oxidative folding strategy,affording the target GsMTx4,and the isolated yield is 25% ～ 30%.The CD and activity evaluation indicated that there is no difference in the activity and structure of four final GsMTx4.6.The CD and activity evaluation together showed that disulfide bonds play a vital role in maintaining the structure and activity of RhTx and GsMTx4.In summary,through exploring the optimal ratio of the Oxyma/DIC based SPPS,this thesis stablished the robust high temperature SPPS strategy for peptide synthesis.The refolding efficiency and yield of one-step oxidative folding,two-step oxidative folding and oxidative folding strategies were systematically studied.Through the combination of high efficient synthetic strategy and refolding method,RhTx and GsMTx4 could be prepared with ideal yield.This thesis not only provides key molecular tools for the study of TRPV1 and Piezo channels,but also provides important references for the synthesis of peptide based drugs,especially disulfide-containing peptides.