Computation-Guided Rational Design of a Peptide Motif That Reacts with Cyanobenzothiazoles via Internal Cysteine–Lysine Relay

Abstract

Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments.

Proximity‐driven, Regioselective Chemical Modification of Peptides and Proteins

“…Recently, Brocchini and co‐workers demonstrated that a His‐Gly‐His tag could be conveniently modified with a bis‐sulfone reagent to site‐specifically alkylate various positions of interferon (IFN) with a 10 kDa PEG chain (Figure 6, panel B) [89] . In another recent example, Bertozzi and co‐workers reported the computational‐guided discovery of an 11‐amino acid tag (GGHPDPCPKGG) that undergoes a S‐to‐N transfer reaction to covalently modify Lys with a 2‐cyanobenzothiazole reagent (Figure 6, panel C) [90] . The conformation of this peptide tag is engineered to place the Cys and Lys residues in proximity, promoting the intramolecular transfer in a mechanism similar to native chemical ligation.…”

“…[89] In another recent example, Bertozzi and co-workers reported the computational-guided discovery of an 11-amino acid tag (GGHPDPCPKGG) that undergoes a S-to-N transfer reaction to covalently modify Lys with a 2-cyanobenzothiazole reagent (Figure 6, panel C). [90] The conformation of this peptide tag is engineered to place the Cys and Lys residues in proximity, promoting the intramolecular transfer in a mechanism similar to native chemical ligation.…”

Single‐Molecule Observation of Intermediates in Bioorthogonal 2‐Cyanobenzothiazole Chemistry

“…In addition, it is not clear whether alternative elimination pathways exist, stemming from the tetrahedral intermediate. Amidines, the products of thiolate expulsion, have been detected after CBT treatment of peptide sequences containing either N‐terminal cysteine or internal cysteine and lysine residues . Reversion from the tetrahedral intermediate back to the thioimidate through ring opening has not been reported.…”

Faculty Opinions recommendation of Single-Molecule Observation of Intermediates in Bioorthogonal 2-Cyanobenzothiazole Chemistry.

“…Amidines,t he products of thiolate expulsion, have been detected after CBT treatment of peptide sequences containing either N-terminal cysteine [13] or internal cysteinea nd lysine residues. [14] Reversion from the tetrahedral intermediate back to the thioimidate through ring opening has not been reported.…”