Selective Acylation of Proteins at Gly and Lys in His Tags

Abstract

The chemical modification of proteins is of great importance in chemical biology, biotechnology, and for the production of modified biopharmaceuticals, as it enables introduction of fluorophores, biotin, half‐life extending moieties, and more. We have developed two methods that use poly‐His sequences to direct the highly selective acylation of proteins, either at the N‐terminus or at a specific Lys residue. For the former, we used an N‐terminal Gly‐His6 segment (Gly‐His tag) that directed acylation of the N‐terminal Nα‐amine with 4‐methoxyphenyl esters, resulting in stable conjugates. Next, we developed the peptide sequences Hisn‐Lys‐Hism (Lys‐His tags) that direct the acylation of the designated Lys Nϵ‐amine under mild conditions and with high selectivity over native Lys residues. Both the Gly‐His and Lys‐His tags maintain the capacity for immobilized metal ion affinity chromatography. We have demonstrated the robustness of these methods by attaching different moieties such as azides, fluorophores, and biotin to different proteins, including antibodies.

Precise and Controlled Modification of Proteins using Multifunctional Chemical Constructs

“…[1][2][3][4][5][6][7][8] Alternatively, modification of isolated proteins has resulted in superior treatments, as is clear from the recent surge in the developments of antibody-drug conjugates (ADCs). [9] For both avenues, approaches have been developed to incorporate a unique chemically [10,11] and/or enzymatically [12][13][14][15][16] reactive moiety in the protein. [17] In this concept paper, modification strategies for wild-type proteins are surveyed and guiding principles for an integrated toolbox for the modification of a range of wildtype proteins are derived.…”

Development and Recent Advances in Lysine and N-Terminal Bioconjugation for Peptides and Proteins

“…The high selectivity of the His tag acylation was obtained through specific base catalysis and proton transfer. The authors proposed that a histidine side chain of the His tag took part in deprotonation during the direct acylation reaction between the glycine α-amine and 68 [150]. After that, Brune, Tārs, and co-workers reported a fast site-selective α-amino acylation for N-terminal Gly-His-tagged proteins by using 6-azido-6-deoxy-glucono-1,5-lactone (6AGDL; commercially available) or azidogluconolactone 69 at neutral pH (7.5) in 2021 (Figure 8f) [151].…”