P-azidoiodoacetanilide, a new short photocrosslinker that has greater cysteine specificity than p-azidophenacyl bromide and p-azidobromoacetanilide.

An important criterion for a protein modifying agent is its residue selectivity. We report the synthesis of a new photocrosslinking agent, p-azidoiodoacetanilide (AIA), which has a greater specificity to modify cysteine residues than the widely used p-azidophenacyl bromide (APB). Crosslinking of UmuD protein, which only has one cysteine, in the homodimer using APB or AIA resulted in 39% and 30% crosslinking, respectively; however, crosslinking of UmuD/C24A, a derivative with no cysteines, resulted in 16% crosslinked dimer using APB but only 2% using AIA. In addition, incorporation of [2-14C]APB into UmuD/C24A was 43% the amount of incorporation into wildtype UmuD, whereas incorporation of [2-14C]AIA into UmuD/C24A was only 13% the amount incorporated into wildtype UmuD. We also examined the cysteine specificity of p-azidobromoacetanilide (ABA) and found it to be less cysteine specific than AIA.