Site-selective Au(III)-mediated metalloenzyme conjugations

Herein, we introduce an operationally simple approach for the direct covalent modification of metalloenzymes via Au(III)-mediated S-arylation chemistry. Using a model class of rubredoxin metalloenzymes containing Fe or Ni in the tetrathiolate active site, we show that these biomolecules can be site-selectively tagged with a variety of abiotic functional groups by leveraging a cysteine residue positioned distal to the active site. The bioconjugation proceeds under mild aqueous conditions, rapidly, and in quantitative yields, and most importantly without perturbing the structural or functional integrity of the enzyme, which normally can occur through adventitious demetallation of the active site. Detailed structural studies, including the first reported X-ray crystal structure of a cysteine-arylated metalloprotein, illuminate a clear preference for regioselective arylation at solvent-exposed cysteine residues, offering a blueprint for predictable and programmable bioconjugation in complex biomolecules. This work demonstrates a unique power of “backgroundless” Au(III) arylation chemistry, rendering site-selective bioconjugation of some of the most delicate biomolecules.