Abstract
We developed a one-pot method for peptide cleavage from a solid support via the N,S-acyl shift of N-2-[thioethyl]glycine and transthioesterification using external thiols to produce cyclic peptides through native chemical self-ligation with the N-terminal cysteine. The feasibility of this methodology is validated by the syntheses of model short peptides, including a tetrapeptide, the bicyclic sunflower trypsin inhibitor SFTI-1, and rhesus Θ-defensin RTD-1. Synthesis of the whole peptide precursor can be fully automated and proceeds without epimerization or dimerization.
Highlights
We developed a one-pot method for peptide cleavage from a solid support via the N,S-acyl shift of N-2[thioethyl]glycine and transthioesterification using external thiols to produce cyclic peptides through native chemical self-ligation with the N-terminal cysteine
We observed that the reaction ofcysteamine with the solid support can be completed in 1− 2 h instead of overnight when using microwave-assisted heating at 75 °C
After the Fmoc-solid-phase peptide synthesis (SPPS) of the peptide precursors, we tested multiple conditions to study the rates of the N,S-acyl shift, transthioesterification, and intramolecular ligation in neutral and acidic conditions in the presence of various thiol additives
Summary
Our methodology is validated by the synthesis of diverse cyclic peptides containing cysteine residues, (Figure 1). After the Fmoc-SPPS of the peptide precursors, we tested multiple conditions to study the rates of the N,S-acyl shift, transthioesterification, and intramolecular ligation in neutral and acidic conditions in the presence of various thiol additives We compared the susceptibility for cleavage in two sequences: AAAAA, which has no N-terminal cysteine, and CPKA This experiment demonstrates that our device works well for peptide cyclization even without an additional thiol, it is less efficient for thioester formation in heterogeneous conditions (Figure S18). Presented studies suggest that both the following mechanisms take place: in the first step, the peptide thioester is released from the solid support as a result of the N,S-acyl transfer and transthioesterification, which undergoes the intramolecular NCL reaction to result in the formation of a homodetic cyclic peptide. The potential between the spray needle and the orifice was set to
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