Construction Of Peptide Disulfide Bonds By MetSeO And Trans-[PtBr₂（CN）₄]^2-

In rencent years,disulfide bond-containing peptides have been attracted attentions as drugs for treating various diseases.Furthermore,some disulfide-containing peptides have been developed in clinical trials,which makes up for the shortcomings of small molecule drugs.The disulfide bond is an important structural motif that can stabilize a peptide’s second structure,increasing the biological and pharmaceutical activities of the molecule.The synthesis of disulfide bonds is an important step in the synthesis of peptide drugs,but some drawbacks such as long reaction time,low oxidation yield and side reactions on some amino acid residues limit the synthesis of disulfide bond-containing peptides.Therefore,expanding the effective and simple synthetic methods has become the research hotspots of disulfide bonds in synthetic peptides.MetSeO was synthesized and characterized by mass spectrometry and nuclear magnetic resonance.The reaction of MetSeO with reduced peptides to construct peptide disulfide bonds was studied in this work.The stoichiometry for the reaction between MetSeO and reduced peptide is 1:1.In different pH solutions,the reaction rate of the synthesis of disulfide bonds by MetSeO oxidation is fast,and the excellent oxidation yields were achieved（≥92%）.MetSeO is suitable for constructing disulfide bonds in peptide cocktails（yield≥95%）,and can also be used to construct oxytocin disulfide bonds on resin.Therefore,MetSeO is an efficient oxidant for constructing disulfide bonds in peptides.The kinetics for the reaction between MetSeO and DTT（a model-compound for the dicysteine-containing peptide）was studied by a stopped-flow spectrometer,which showed that the reaction is first-order in both[MetSeO]and[DTT].The values of p K_a1 and p K_a2 of MetSeO determined by titration method are 2.69±0.05 and 8.92±0.07,respectively.The reaction rate is determined by the pH value of the reaction medium and the protolytic constants of sulfhydryl groups.By taking all the above considerations into account,a reaction mechanism for the reaction is proposed.The sulfur atom of DTT attacks the selenium atom of MetSeO,generating the transient selenosulfide intermediate（formation of Se-S bond）,followed by the rapid reaction with another sulfur atom of DTT,leading to the formation of disulfide bond.The overall rate equation was deduced.Values of rate constants and equilibrium constants were calculated.The research mechanism is proposed that can help us gain insights into the reaction of the oxidative synthesis of disulfide bonds via MetSeO oxidation.TentaGel-MetSeO and PEG-MetSeO were synthesized and characterized by scanning electron microscope、X-ray photoelectron spectroscopy and inductively coupled plasma mass spectrometry.The reactions of TentaGel-MetSeO and PEG-MetSeO with different reduced peptides in various solutions were studied by HPLC.The results showed that the yield of disulfide bond generated from the PEG-MetSeO oxidation is higher than that of disulfide bond generated from the TentaGel-MetSeO oxidation.The reaction yield of disulfide bond synthesis by PEG-MetSeO is≥77%.PEG-MetSeO can be recycled for 10 times without the significant yield decrease.The reaction of trans-[PtBr₂（CN）₄]^2-with a peptide containing two acetylaminomethyl protected cysteine residues（Cys（Acm））was studied in this work.One molecule of trans-[PtBr₂（CN）₄]^2-reacted with two Cys（Acm）to give an intramolecular disulfide bond,and the reaction stoichiometry is 1:1.Kinetic studies show that this reaction is first-order in both[Pt（IV）]and[peptide].The reaction mechanism is proposed.In the mechanism,a bromine bridge between S atom in Cys（Acm）and an axial bromide ligaind of trans-[PtBr₂（CN）₄]^2-was formed,and then an two electron transfer process was occurred,resulting the remove of one Acm group to form a sulfenyl bromide intermediate,followed by reaction with another Cys（Acm）to form an intramolecular disulfide bond.Finally,combanition of MetSeO with trans-[PtBr₂（CN）₄]^2-,one-pot regioselective synthesis of two disulfide bonds inα-conotoxin SI was investigated in aqueous solution.The isolated yield ofα-conotoxin SI is 61%.