A Cysteine Reversible Protecting Strategy Based On Rac-2-Br-DMNPA To Facilitate Chemical Protein Synthesis

Chemical protein synthesis(CPS)enables the straightforward incorporation of a limitless range of coded or non-coded amino acids into a protein molecule,at any position in the protein’s polypeptide chain and in any number and combination,which is an important method for obtaining high-purity precisely modified proteins in large quantities and with high efficiency.Solid phase peptide synthesis(SPPS)and native chemical ligation(NCL)represent the two key technologies for protein chemical synthesis which have been applied to the chemical synthesis of various homogeneous proteins.To improve the yield,more efficient multi-segment NCL strategies and protecting-group-removal strategy have been developed in a one-pot fashion.However,most of the thiol protecting groups need to be introduced during the SPPS process,limiting their vast applicability,and the accumulation of reagents in multi-step reactions leads to difficulties in subsequent purification and separation.Therefore,a series of thiol caging agents have been discovered to achieve the late-stage protection of thiol groups,and based on this,Solid-phase Chemical Ligations(SPCL)have been developed to improve the efficiency of synthesis by separating the reaction substrate from the target product through a washing step.Nevertheless,the current SPCL has fewer types of linker and requires the presence of strong acids,bases or metal reagents to removal,which may lead to side reactions of sensitive groups of peptide amino acid side chains.Meanwhile,the utilization of the classic photolabile orthoNitrobenzyl(o-NB)derivatives as the Cysteine(Cys)thiol caging reagent for proteins synthesis has been exploited.The o-NB derivative with a diversified carboxylate terminal could be effectively used to accommodate late-stage protein thiol protection and rapid removal under UV irradiation,but the direct application of these o-NB class molecules in CPS has not yet been explored in depth.In this work,we developed a reversible Cys thiol caging reagent based on o-NB derivatives,rac-2-Br-2-(4,5-dimethoxy-2-nitrophenyl)-acetic acid(rac-2-Br-DMNPA),for the late-stage peptide thiol protection and efficient photolysis deprotection.We explored the capability this thiol-protection strategy to assist the one-pot chemical synthesis of chlorotoxin(CTX)(2-36)by dividing CTX(2-36)into three segments and using one-pot-two-step NCL combined with rac-DMNPA and Fmoc removal reaction to realize the efficient chemical synthesis of CTX(2-36).More importantly,we further exploring the capability of rac-2-BrDMNPA to serve as a traceless SPCL linker for traceless SPCL strategy,which has good compatibility with reactions such as NCL,peptide thioester conversion,and 9-fluorenylmethyloxycarbonyl(Fmoc)and Thiazolidinyl(Thz)deprotection to achieve ligation of multiple peptides segments without purification in each step.Finally,we combined this strategy with semi-synthesize to obtain a selenoprotein F(Sel F)(63-134)(U65C/A75C)peptide fragment via SPCL.In summary,we developed a caging reagent,rac-2-Br-DMNPA,for the reversible protection of Cys thiol groups,using it to assist the one-pot chemical synthesis of CTX(2-36)and realizing the photolysis removal of this protecting group under mild conditions.Moreover,we expanded the utility of rac-2-Br-DMNPA as a traceless SPCL bifunctional linker,which provides an effective method for protein solid-phase chemical synthesis and a powerful technical support for obtaining challenging proteins.