Enzyme-catalyzed In Situ Cleavage And Cyclization Of Peptides On Solid Support

The Staphylococcus aureus sorting enzyme Soraste A?Srt A?is a transpeptidase that is responsible for anchoring proteins to the peptidoglycan layer of the bacterial cell wall.It specifically recognizes the conserved sequence LPXTG?X=any amino acid except for Cys?in the protein and cleaves the amide bond between T and G to form a thioester-enzyme intermediate with the substrate.The amino group on the oligo-glycine modified substrate acts as a nucleophile to attack the thioester-enzyme intermediate,forming a new amide bond with the substrate,and finally releasing the Srt A enzyme to complete the ligation between the protein and glycine nucleophilic compound.Srt A-mediated ligation?SML?can be used for both intermolecular and intramolecular transpeptidation,which is widely used in peptide or protein site-specific labeling,protein-protein fusion,antibody site-specific modification,peptide or protein cyclization,protein?enzyme?immobilization,and cell or viral particle site-specific labeling.Although Srt A has been widely used as a powerful ligation tool,many problems occurred in SML are still encountered:?1?Multi-step purification and lower yield;?2?Hydrolysis by-products are always detected in SML;?3?Oligomer by-products are easily generated in the cyclization reaction;?4?Especially in the synthesis of disulfide-bond cyclic peptides,such as cyclotides,cyclization is not compatible with disulfide oxidation;?5?Moreover,isomers are easily generated during formation of disulfide-bond.Therefore,the development of a new ligation strategy based on SML is very important for tackling the problem of SML and improving the yield of products.To overcome above problems,after anchoring the peptide containing LPXTG motif on solid support,Srt A directly recognized and cleaved the peptide to form intermediate,which was then ligated with oligo-glycine modified substrate or cyclized in situ to generate ligation product in high yield.The main results of this study are summarized as follows:?1?Synthesis of multivalent RGD?Arg-Gly-Asp;RGD?macrocyclic peptides.A linear peptide containing 1-3 RGD sequences was designed and synthesized by solid phase peptide synthesis?SPPS?via standard Fmoc chemistry.The linear peptides containing Srt A recognition sequence LPETGGS at C-terminus and N-terminus polyglycine residue[NH₂-G₇RGDLPETGGS-COOH,NH₂-G₄?RGD?₂LPETGGS-COOH,NH₂-G?RGD?₃LPETGG S]were cyclized in liquid phase system using SML.The yield of multivalent RGD macrocyclic peptides were about 38%to 46%.Cyclic peptides and linear peptides with different RGD were detected by cell adhesion competition inhibition experiments.The results showed that the linear peptide NH₂-G₄?RGD?₂LPETGGS-COOH and cyclic peptide cyclo-?G7RGDLPET?had good selective binding ability to integrin?v?3.?2?Srt A-mediated on-resin recognition,cleavage and in situ ligation with glycine-modified substrate.Srt A was firstly found to recognize a PEGA resin supported peptide donor containing a LPXTG motif and to ligate with a suitable acceptor containing oligo-glycine to afford conjugates in one-pot.This approach has solved problems,such as complicated purification,hydrolysis by-profuct and lower yield occurred in Srt A-mediated ligation in liquid phase.And this strategy was successfully employed to synthesize dual functional peptides?yield 52%-68%?,modify peptides with lipids?yield 61%?,biotin?yield71%?and PEG?yield 65%?,as well as protein N-terminal labeling in high efficiency?71%?.?3?Srt A-mediated on-resin peptide cleavage and in situ cyclization enables efficient synthesis of cyclopeptide and cyclotides in one-pot.Srt A could efficiently recognize bifunctional peptide containing C-terminal LPXTG motif and N-terminal oligo glycine residue associated on PEGA resin and perform enzymatic cleavage and in situ cyclization to generate cyclic peptides.This approach has solved problems,such as complicated purification,oligomerization by-profuct,isomer and lower yield occurred in Srt A-mediated cyclization in liquid phase.Antibacterial bovine lactoferricin peptide LFcinB_20-35 was successfully synthesized using this method in a yield of 67%.We have also demonstrated that this enzymatic in situ cyclization strategy on PEGA resin was compatible with disulfide bond formation and complex cyclotides containing one or two disulfide bond,such as sunflower trypsin inhibitors-1?SFTI-1?and?-conotoxin M?,were successfully synthesized in one-pot in a yield of 77%and 61%,respectively.?4?Srt A-mediated synthesis of RGD cyclic peptide library on PEGA resin in one pot.The NH₂-G₅RGDXVLPETGGS-COOH?X is 20 kinds of L-type amino acids and 19 kinds of D-type amino acids?sequence was synthesized on the PEGA resin by SPPS.The cyclic peptide was synthesized by Srt A-mediated on resin in situ cyclization in one-pot.Cyclic peptide library containing 39 RGD cyclic peptides was obtained after one step purification.Many drawbacks in traditional cyclic peptide libaray synthesis,such as complicated procedures and difficult formation of amide bond cyclic peptide skeleton were tackled by this strategy.The surface plasmon resonance technique?SPR?and the cell adhesion competitive inhibition assay were used to screen the binding ability of the RGD cyclic peptide to integrin??v?3 and?v?5?.The results indicated that cylic peptide c-K showed remarkable potency and selectivity to?v?3 integrin?IC50=3.5?M?.