A structural and mechanistic study of π-clamp-mediated cysteine perfluoroarylation

Abstract

Natural enzymes use local environments to tune the reactivity of amino acid side chains. In searching for small peptides with similar properties, we discovered a four-residue π-clamp motif (Phe-Cys-Pro-Phe) for regio- and chemoselective arylation of cysteine in ribosomally produced proteins. Here we report mutational, computational, and structural findings directed toward elucidating the molecular factors that drive π-clamp-mediated arylation. We show the significance of a trans conformation prolyl amide bond for the π-clamp reactivity. The π-clamp cysteine arylation reaction enthalpy of activation (ΔH‡) is significantly lower than a non-π-clamp cysteine. Solid-state NMR chemical shifts indicate the prolyl amide bond in the π-clamp motif adopts a 1:1 ratio of the cis and trans conformation, while in the reaction product Pro3 was exclusively in trans. In two structural models of the perfluoroarylated product, distinct interactions at 4.7 Å between Phe1 side chain and perfluoroaryl electrophile moiety are observed. Further, solution 19F NMR and isothermal titration calorimetry measurements suggest interactions between hydrophobic side chains in a π-clamp mutant and the perfluoroaryl probe. These studies led us to design a π-clamp mutant with an 85-fold rate enhancement. These findings will guide us toward the discovery of small reactive peptides to facilitate abiotic chemistry in water.

Arylation Chemistry for Bioconjugation

“…Systematic mutation studies and structural characterization with solid‐state NMR suggest that the hydrophobic side chains in the π‐clamp interact with the perfluoroarenes. Importantly, the prolyl amide bond favors trans conformation, as evidenced by both NMR and computational studies …”

“…Even though the perfluoroaryl–thiol reactions showed good functional group tolerance in the modification of synthetic molecules (Figure ), their chemoselectivity in a bioconjugation setting had not been extensively studied. Pentelute developed a perfluoroaryl–thiol S N Ar reaction platform for bioconjugation . As a starting point, Pentelute in 2013 evaluated the reactions between various amino acids and commercial hexafluorobenzene and decafluorobiphenyl reagents .…”

Site‐Selective Cysteine–Cyclooctyne Conjugation

“…Tuning the reactivity of peptides has led to efficient and selective catalysts and tags . In particular, site‐selective reactions enabled by unique amino acid sequences have been discovered, including a tetracysteine tag that reacts with the biarsenical fluorescent dyes, a tetraserine tag that reacts with rhodamine‐derived bisboronic acid, sequence‐specific 2‐cyanobenzothiazole ligation, a p ‐(chloromethyl)benzamide‐reactive peptide and π‐clamp‐mediated cysteine perfluoroarylation . In light of these findings, we envisioned that a designer peptide sequence could be used to achieve site‐selective cysteine bioconjugation through a thiol–yne reaction.…”

Site‐Selective Cysteine–Cyclooctyne Conjugation

“…Tuning the reactivity of peptides has led to efficient and selective catalysts [23–25] and tags [26–33] . In particular, site-selective reactions enabled by unique amino acid sequences have been discovered, including tetracysteine tag that reacts with the biarsenical fluorescent dyes, [26,27] tetraserine tag that reacts with rhodamine-derived bisboronic acid, [28] sequence-specific 2-cyanobenzothiazole ligation, [29] p- (chloromethyl)benzamide-reactive peptide [30] and the the π-clamp-mediated cysteine perfluoroarylation.…”

“…In particular, site-selective reactions enabled by unique amino acid sequences have been discovered, including tetracysteine tag that reacts with the biarsenical fluorescent dyes, [26,27] tetraserine tag that reacts with rhodamine-derived bisboronic acid, [28] sequence-specific 2-cyanobenzothiazole ligation, [29] p- (chloromethyl)benzamide-reactive peptide [30] and the the π-clamp-mediated cysteine perfluoroarylation. [31–33] In light of these findings, we envisioned that a designer peptide sequence could be used to achieve site-selective cysteine bioconjugation by thiol-yne reaction. We focused on the thiol-yne reaction because the important DBCO reagents are widely used and commercial available.…”