posted on 2014-01-15, 00:00authored byMichaela Wendeler, Luba Grinberg, Xiangyang Wang, Philip
E. Dawson, Manuel Baca
The
conjugation of biomolecules by chemoselective oxime ligation
is of great interest for the site-specific modification of proteins,
peptides, nucleic acids, and carbohydrates. These conjugations proceed
optimally at a reaction pH of 4–5, but some biomolecules are
not soluble or stable under these conditions. Aniline can be used
as a nucleophilic catalyst to enhance the rate of oxime formation,
but even in its presence, the reaction rate at neutral pH can be slower
than desired, particularly at low reagent concentrations and/or temperature.
Recently, alternative catalysts with improved properties were reported,
including anthranilic acid derivatives for small molecule ligations,
as well as m-phenylenediamine at high concentrations
for protein conjugations. Here, we report that p-substituted
anilines containing an electron-donating ring substituent are superior
catalysts of oxime-based conjugations at pH 7. One such catalyst, p-phenylenediamine, was studied in greater detail. This
catalyst was highly effective at neutral pH, even at the low concentration
of 2 mM. In a model oxime ligation using aminooxy-functionalized PEG,
catalysis at pH 7 resulted in a 120-fold faster rate of protein PEGylation
as compared to an uncatalyzed reaction, and 19-fold faster than the
equivalent aniline-catalyzed reaction. p-Phenylenediamine
(10 mM) was also an effective catalyst under acidic conditions and
was more efficient than aniline throughout the pH range 4–7.
This catalyst allows efficient oxime bioconjugations to proceed under
mild conditions and low micromolar concentrations, as demonstrated
by the PEGylation of a small protein.