A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation

Mohammad Rashidian, Mohammad M. Mahmoodi, Rachit Shah, Jonathan K. Dozier, Carston R Wagner, Mark D Distefano

Research output: Contribution to journalArticle

79 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)333-342
Number of pages10
JournalBioconjugate Chemistry
Volume24
Issue number3
DOIs
StatePublished - Mar 20 2013

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Hydrazones
Oximes
Ligation
Aniline
Catalysts
Proteins
Ketones
Aldehydes
Kinetics
Labels
Immobilized Proteins
Ciliary Neurotrophic Factor
Imines
Fluorophores
Catalysis
Phenylalanine
Reaction kinetics
Solubility
Labeling
aniline

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A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation. / Rashidian, Mohammad; Mahmoodi, Mohammad M.; Shah, Rachit; Dozier, Jonathan K.; Wagner, Carston R; Distefano, Mark D.

In: Bioconjugate Chemistry, Vol. 24, No. 3, 20.03.2013, p. 333-342.

Research output: Contribution to journalArticle

Rashidian, Mohammad ; Mahmoodi, Mohammad M. ; Shah, Rachit ; Dozier, Jonathan K. ; Wagner, Carston R ; Distefano, Mark D. / A highly efficient catalyst for oxime ligation and hydrazone-oxime exchange suitable for bioconjugation. In: Bioconjugate Chemistry. 2013 ; Vol. 24, No. 3. pp. 333-342.
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