Background: Previously, we proposed a screening system for intracellular functional peptides; it combined a photolytic peptide array system with cell-permeable peptides (CPPs)1, 2. However, conjugating CPPs to functional peptides for intracellular delivery is responsible for reduced activity of functional peptides.
Objectives: In this study, we developed a synthetic method that can cross-link CPP and functional peptide by disulfide bond. The hetero-dimeric peptides can degrade in the intracellular reductive environment, and the peptide library constructed with hetero-dimers is applicable to a screening system for novel intracellular functional peptides excluding the influence of CPP.
Methods: As a first step, Fmoc-Lys(ivDde)-OH is linked on cellulose membrane. Photolytic linker, cysteine (C), CPP were synthesized to the main chain amine and photolytic linker, C, functional peptides were synthesized to the side chain amine. Disulfides were formed between the main chain and side chains by an oxidation reaction, and finally the CPP-functional peptide hetero-dimer was released from membrane by UV irradiation. The cell death inducing peptide (WELVVLGKL)3 used as model peptide were cross-linked with octaarginine which is one of the CPP by disulfide bond. Peptide synthesis was evaluated by Mass spectrometry. Cell death inducing activity of peptides was evaluated by cell based viability assay using HeLa cells.
Results: The mass spectrum of hetero-dimer of the cell death inducing peptide and CPP was selectively detected by oxidative treatment and decreased by reduction. The effective cell death was observed by the heterodimeric peptide’s treatment. Peptides library with 162 candidates was made by amino acids substitution and evaluated cell death inducing activity. We identified 6 peptides with significantly higher activity than that of the original one.
Conclusion: Novel synthetic method can synthesized hetero-dimeric peptides via disulfide bond and can be used to screen for intracellular functional peptides.