Extendable stapling of unprotected peptides by crosslinking two amines with o-phthalaldehyde

Methods for peptide stapling, or covalently linking amino acid residues to create a non-linear structure, mostly rely on cysteine residues, which imposes a significant practical limitation. Here the authors disclose a method to chemoselectively macrocyclize two free-amine-containing residues in mild, peptide-relevant conditions, using a commercially available reagent. Peptide modification methods that do not rely on the cysteine residue are underdeveloped, and their development could greatly expand the current toolbox for peptide chemistry. During the course of preliminary investigations into the classical ortho-phthalaldehyde (OPA)-amine-thiol condensation reaction, we found that in the absence of thiol, OPA readily condenses with two primary alkyl amines to form a class of underexplored isoindolin-1-imine compounds under mild aqueous conditions. From the intramolecular version of this OPA-2amines reaction, an efficient and selective methodology using mild reaction conditions has been developed for stapling unprotected peptides via crosslinking of two amino groups in both an end-to-side and side-to-side fashion. The stapling method is superfast and broadly applicable for various peptide substrates with the reacting amino groups separated by a wide range of different amino acid units. The macrocyclization reactions of selected substrates are completed within 10 seconds at 5 mM concentration and within 2 minutes at 50 mu M concentration. Importantly, the resulting cyclized peptides with an isoindolinimine linkage can be extended in a one-pot sequential addition manner with several different electron-deficient pi electrophiles, thereby generating more complex structures.

Automated summary

In this study, a method for macrocyclization of two free-amine-containing residues in mild conditions is disclosed. This method can efficiently generate complex peptide structures, expanding the toolbox for peptide chemistry.