| Summary: | Achieving catalyst-free, site-selective modification of proteins in water is a significant challenge in chemical biology. Issues of residue specificity, site-selectivity, reagent stability, and reaction rate are pervasive. To address these challenges, we developed a matched peptide pair termed the reactive peptide interface (RPI). This interface consists of two peptides: a nucleophilic, cysteine-containing peptide and an electrophilic, perfluoroarylated peptide. The unique sequences of these peptides enhance the reaction rate of a nucleophilic aromatic substitution reaction between these two peptides. Potential favorable non-covalent interactions between these two peptides could facilitate the rapid reaction rate and the site-selectivity of the system through molecular recognition. This peptide interface allows for rapid Cys arylation with a k = 152 ± 3 M⁻¹ s⁻¹ and enabled the site-selective modification of a miniprotein and an antibody in cell lysate.
Developing the technology to achieve selective lysine chemistries has progressed more slowly, in part due to lysine’s lower reactivity compared to cysteine. The diversity present in synthetic peptide libraries has been used to discover small peptide motifs that react preferentially with a specific electrophile. These relatively small motifs have shown promise in the site-selective labeling of biomolecules under mild aqueous conditions. This approach can be taken to discover peptide sequences that enhance the reactivity of an embedded lysine. We report various lysine-containing motifs that enable selective lysine acylation with moderately reactive oxygen esters. This conjugation approach forms stable amide bonds and allows for conjugation of cargo at lysine.
Finally, peptide library diversity greatly increases the probability of finding a hit candidate with the desired properties you are screening for. We turn to mRNA display, an in vitro selection technique that enables the screening of trillions of peptide sequences for desired functions. We show our efforts into establishing an mRNA display platform in our laboratory to discover novel binders and reactive sequences.
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