Site-Specific Modification of Proteins through N-Terminal Azide Labeling and a Chelation-Assisted CuAAC Reaction

Abstract

Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The Nterminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.

Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification

“…It is therefore likely that N‐terminal modification will not affect the inherent function of wild‐type proteins or enzymes in most cases . Since an N‐terminal α‐amino group has a lower p K a value than the ϵ‐amino group of the Lys side chain, a reagent targeting the N‐terminal amino acid is expected to be particularly effective for modification of this site . For example, Francis and co‐workers, and other researchers have reported a site‐specific and one‐step N‐terminal α‐amino group modification Scheme using 2‐pyridinecarbaldehyde derivatives …”

“…In biological systems, pyridoxal phosphate is an important cofactor which is involved in post‐translational modification of an amino group in peptides and proteins . Specific N‐terminal modification of a protein with 2‐pyridylcarbaldehyde, a nitrogen‐containing heteroaromatic compound with a formyl group which is similar to the pyridoxal phosphate cofactor, was reported to proceed via formation of a 4‐imidazolidinone ring involving two nitrogen atoms of the N terminus and adjacent residues . On the basis of this knowledge, we began our investigation by examining a series of five‐membered nitrogen‐containing heteroaromatic aldehydes as N‐terminal specific modification reagents.…”

Herding cats: Label-based approaches in protein translocation through nanopore sensors for single-molecule protein sequence analysis

“…The main drawback of NHS esters for N-terminal specific labeling is minimizing the cross reactivity with lysines. For example, RNase A, which has 10 lysine residues, had 2 and 5 simultaneous modifications at pH 6.5 and 7.5, respectively, when reacted with 2-azidoacetic acid NHS ester ( Inoue et al., 2019 ).…”

“…More recently, other groups have also performed similar or revised approaches for N-terminal specific modification ( Chen et al., 2017 ; Deng et al., 2020 ; Inoue et al., 2019 ; Li et al., 2018 ; Onoda et al., 2020 ). In 2019, Inoue et al.…”

“…In 2019, Inoue et al. reported one-step N-terminal specific modification using 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives ( Inoue et al., 2019 ). This study showed that 6AMPC provides excellent conversions toward various peptides and proteins (e.g., 95% for Angiotensin I and >90% for RNase A).…”