Synthesis and application of a new bifunctional amidination reagent

Methyl 4-hydroxy-3-nitrobenzimidate hydrochloride (I) is presented as a newly developed amidination reagent. It is synthesized in two steps from commercially available 4-hydroxybenzonitrile. Its incorporation into proteins can be easily determined spectroscopically. The activities of the three enzymes lactate dehydrogenase (pig heart), alkaline phosphatase (calf intestine) and tryptophan synthase (E. coli) were not greatly reduced after modification with the reagent. The nitro groups can be reduced by mild treatment with Na2S2O4 without alteration of the enzymatic activity. The newly formed aromatic amino groups can further be made to react at lower pH than the native amino groups.