Insights into the mechanism and catalysis of oxime coupling chemistry at physiological pH

Abstract

The dynamic covalent-coupling reaction involving α-effect nucleophiles has revolutionized bioconjugation approaches, due to its ease and high efficiency. Key to its success is the discovery of aniline as a nucleophilic catalyst, which made this reaction feasible under physiological conditions. Aniline however, is not so effective for keto substrates. Here, we investigate the mechanism of aniline activation in the oxime reaction with aldehyde and keto substrates. We also present carboxylates as activating agents that can promote the oxime reaction with both aldehyde and keto substrates at physiological pH. This rate enhancement circumvents the influence of α-effect by forming H-bonds with the rate-limiting intermediate, which drives the reaction to completion. The combination of aniline and carboxylates had a synergistic effect, resulting in a ∼14-31-fold increase in reaction rate at pD 7.4 with keto substrates. The biocompatibility and efficiency of carboxylate as an activating agent is demonstrated by performing cell-surface oxime labeling at physiological pH using acetate, which showed promising results that were comparable with aniline.

Keywords: coupling reaction; ketones; kinetics; oxime; reaction mechanisms.