Selective pH-Responsive Conjugation between a Pair of De Novo Discovered Peptides

There is a demand for site-selective peptide/protein conjugation chemistry that is fully reversible in a stimulus-responsive manner. The contemporary methods for site-selective protein modification enable the preparation of homogeneous protein–small molecule conjugates, which are indispensable for drug delivery and chemical biology purposes, but such chemistries are usually irreversible. In contrast, the existing reversible protein labeling techniques are generally not site-selective. Here, we report an mRNA display-enabled de novo discovery of a pair of peptides which selectively react with each other to form a conjugate that is stable under neutral conditions (pH 7.5) but rapidly dissociates into the constituents at pH 10. A Cys thiol of peptide CP1 rapidly reacts (k₁ = 340 M^–1·s^–1) with the isothiocyanate moiety in partner ITC6 to furnish a stable dithiocarbamate (t_1/2 = 6 h at pH 7.5). We show that the pH-responsive nature of the reaction (conjugate’s t_1/2 = 5 min at pH 10) can be leveraged to utilize ITC6 (1) as a pull-down handle to selectively isolate CP1 from cell lysates and (2) as a temporary protecting group to protect CP1 from nonspecific Cys labeling reagents such as iodoacetamide. Altogether, the chemistry developed here complements the existing approaches and is applicable in a variety of chemical biology settings where selective reversible reactions are needed.