Rapid, Hydrolytically Stable Modification of Aldehyde-Terminated Proteins and Phage Libraries

We describe the rapid reaction of
2-amino benzamidoxime (ABAO)
derivatives with aldehydes in water. The ABAO combines an aniline
moiety for iminium-based activation of the aldehyde and a nucleophilic
group (Nu:) ortho to the amine for intramolecular ring closure. The
reaction between ABAO and aldehydes is kinetically similar to oxime
formations performed under stoichiometric aniline catalysis. We characterized
the reaction by both NMR and UV spectroscopy and determined that the
rate-determining step of the process is formation of a Schiff base,
which is followed by rapid intramolecular ring closure. The relationship
between apparent rate constant and pH suggests that a protonated benzamidoxime
acts as an internal general acid in Schiff-base formation. The reaction
is accelerated by substituents in the aromatic ring that increase
the basicity of the aromatic amine. The rate of up to 40 M–1 s–1 between an electron-rich aldehyde and 5-methoxy-ABAO
(PMA), which was observed at pH 4.5, places this reaction among the
fastest known bio-orthogonal reactions. Reaction between M13 phage-displayed
library of peptides terminated with an aldehyde moiety and 1 mM biotin-ABAO
derivative reaches completion in 1 h at pH 4.5. Finally, the product
of reaction, dihydroquinazoline derivative, shows fluorescence at
490 nm suggesting a possibility of developing fluorogenic aldehyde-reactive
probes based on ABAO framework.