Selective Triazenation Reaction (STaR) of Secondary Amines for Tagging Monomethyl Lysine Post‐Translational Modifications

Abstract

Lysine monomethylation (Kme) is an impactful post‐translational modification (PTM) responsible for regulating biological processes and implicated in diseases, thus there is great interest in identifying these methylation marks globally. However, the progress in this area has been challenging because the addition of a small methyl group on lysine leads to negligible change in the bulk, charge, and hydrophobicity. Herein, we report an empowering chemical technology selective triazenation reaction, which we term “STaR”, of secondary amines using arene diazonium salts to achieve highly selective, rapid, and robust tagging of Kme peptides from a complex mixture under biocompatible conditions. Although the resulting triazene‐linkage with Kme is stable, we highlight the efficient decoupling of the triazene‐conjugate to afford unmodified starting components under mild conditions when desired. Our work establishes a unique chemoselective, traceless bioconjugation strategy for the selective enrichment of Kme PTMs.

Rapid Arene Triazene Chemistry for Macrocyclization

“…We also exposed triazene cyclic peptide 2b to sodium dithionite (5 equiv), and the cyclic peptide 2b was stable for 2 h without any degradation, which is in contrast to diazo compounds formed between the Tyr side chain and diazonium ions, which underwent complete degradation in 5 min. We have also shown the orthogonality of our approach by exposing triazene cyclic peptide 2a to piperidine used in Fmoc-SPPS, and no degradation of 2a for 12 h was observed as analyzed by HPLC and MS (Supporting Information Figure S20) …”

“…Inspired by observation of the facile reaction of the arene diazonium ion with a secondary amine to form a stable triazene, − we reasoned that the arene diazonium ion might serve as an attractive starting point, owing to the ease and flexibility of introducing it inside the peptide by the incorporation of commercially available p -amino phenylalanine (pAF) in the peptide chain . Arene diazonium ions would then chemoselectively react with secondary amines such as N-terminal proline or monomethyl lysine at pH 7.5 to generate a stable macrocycle with a triazene moiety at the site of cyclization (Figure ).…”

“…Reversibility of Triazene Cyclic Peptides: Response to External Stimuli. Based on our recent study with diazonium ions for labeling monomethyl lysine, 27 we hypothesized that triazene cyclic peptides will respond to a change in the pH by the protonation of the triazene. We hypothesized that at low pH, cyclic traizene will undergo protonation followed by the opening of the triazene ring to unchanged starting linear peptide with diazonium ions and N-terminal proline.…”

“…We have also shown the orthogonality of our approach by exposing triazene cyclic peptide 2a to piperidine used in Fmoc-SPPS, and no degradation of 2a for 12 h was observed as analyzed by HPLC and MS (Supporting Information Figure S20). 27 To evaluate the potential of triazene cyclic peptides for biological applications, we examined the proteolytic stability of a cyclic peptide in comparison with its linear counterpart. Linear peptide PGRAFKTAQS(pAF) 1q and corresponding triazene cyclic adduct 2q were incubated with trypsin, which hydrolyzed peptide bonds at the C-terminal side of lysine and arginine.…”