A Highly Efficient Catalyst for Oxime Ligation and Hydrazone–Oxime Exchange Suitable for Bioconjugation

Abstract

Imine-based reactions are useful for a wide range of bioconjugation applications. Although aniline is known to catalyze the oxime ligation reaction under physiological conditions, it suffers from slow reaction kinetics, specifically when a ketone is being used or when hydrazone-oxime exchange is performed. Here, we report on the discovery of a new catalyst that is up to 15 times more efficient than aniline. That catalyst, m-phenylenediamine (mPDA), was initially used to analyze the kinetics of oxime ligation on aldehyde- and ketone-containing small molecules. While mPDA is only modestly more effective than aniline when used in equal concentrations (~ 2-fold), its much greater aqueous solubility relative to aniline allows it to be used at higher concentrations, resulting in significantly more efficient catalysis. In the context of protein labeling, it was first used to site-specifically label an aldehyde-functionalized protein through oxime ligation, and its kinetics were compared to reaction with aniline. Next, a protein was labeled with an aldehyde-containing substrate in crude cell lysate, captured with hydrazide-functionalized beads and then the kinetics of immobilized protein release via hydrazone-oxime exchange were analyzed. Our results show that mPDA can release and label 15 times more protein than aniline can in 3 h. Then, using the new catalyst, ciliary neurotrophic factor, a protein with therapeutic potential, was successfully labeled with a fluorophore in only 5 min. Finally, a protein containing the unnatural amino acid, p-acetyl phenylalanine, a ketone-containing residue, was prepared and PEGylated efficiently via oxime ligation using mPDA. This new catalyst should have a significant impact on the field of bioconjugation, where oxime ligation and hydrazone-oxime exchange are commonly employed.

Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation

“…The discovery by Dawson and coworkers of aniline catalysis of the click reaction with α-effect nucleophiles such as hydrazine and aminooxy groups at pH 4–6 has made this popular process even more effective (Figure 2A) (Gaertner et al, 1992; Jencks, 1959; Sander and Jencks, 1968), with the organocatalytic process proceeding through a highly reactive Schiff base (Cornish et al, 1996; Dirksen and Dawson, 2008; Dirksen et al, 2006a; Dirksen et al, 2006b; Jencks, 1959; Rashidian et al, 2013; Ulrich et al, 2014). To improve biocompatibility and increase reaction rate at pH 7, substituted anilines (Dirksen and Dawson, 2008; Dirksen et al, 2006b; Rashidian et al, 2013; Wendeler et al, 2013) such as water-soluble 5-methoxyanthranilic, 3,5-diaminobenzoic (Crisalli and Kool, 2013b), and 2-aminobenzenephosphonic acid (Crisalli and Kool, 2013a) have been found to accelerate the condensation reaction by up to 40-fold at pH 7 as compared to the aniline-catalyzed reaction. While the electronic and acid/base properties of the nucleophilic reactants strongly influence the rate at biological pH (Kool et al, 2014), it must be remembered that all of these ligations are reversible, and that hydrazones are especially prone to dissociation at low concentrations (Dirksen et al, 2006a; Dirksen et al, 2006b; Kalia and Raines, 2008), whereas oximes are more stable (Kalia and Raines, 2008).…”

Synthesis of hydrophilic aminooxy linkers and multivalent cores for chemoselective aldehyde/ketone conjugation

“…These conjugation reactions can be conducted under mild aqueous conditions, which generally include the use of aniline 1,2 or other aromatic amines such as m -phenylenediamine (mPDA) 3 as a catalyst, and yield the oxime linkage (Figure 1). The oxime has been shown to be stable to hydrolysis, and is more stable than simple hydrazones, making it an attractive functional group for bioconjugation reactions involving aldehydes and ketones.…”

Borylated oximes: versatile building blocks for organic synthesis

“…11 As a result of their intrinsic hydrolytic stability, oximes are also desired functional moieties in bioconjugation chemistry. 13 …”