Selective\npH-Responsive Conjugation between a Pair\nof De Novo Discovered Peptides

There\nis a demand for site-selective peptide/protein conjugation\nchemistry that is fully reversible in a stimulus-responsive manner.\nThe contemporary methods for site-selective protein modification enable\nthe preparation of homogeneous protein–small molecule conjugates,\nwhich are indispensable for drug delivery and chemical biology purposes,\nbut such chemistries are usually irreversible. In contrast, the existing\nreversible protein labeling techniques are generally not site-selective.\nHere, we report an mRNA display-enabled <i>de novo</i> discovery\nof a pair of peptides which selectively react with each other to form\na conjugate that is stable under neutral conditions (pH 7.5) but rapidly\ndissociates into the constituents at pH 10. A Cys thiol of peptide\nCP1 rapidly reacts (<i>k</i><sub>1</sub> = 340 M<sup>–1</sup>·s<sup>–1</sup>) with the isothiocyanate moiety in partner\nITC6 to furnish a stable dithiocarbamate (<i>t</i><sub>1/2</sub> = 6 h at pH 7.5). We show that the pH-responsive nature of the reaction\n(conjugate’s <i>t</i><sub>1/2</sub> = 5 min at pH\n10) can be leveraged to utilize ITC6 (1) as a pull-down handle to\nselectively isolate CP1 from cell lysates and (2) as a temporary protecting\ngroup to protect CP1 from nonspecific Cys labeling reagents such as\niodoacetamide. Altogether, the chemistry developed here complements\nthe existing approaches and is applicable in a variety of chemical\nbiology settings where selective reversible reactions are needed.