A homobifunctional rhodamine for labeling proteins with defined orientations of a fluorophore

The synthesis and characterization of a bifunctional rhodamine dye bearing 2-(iodoacetamido)ethyl substituents on the 3'- and 6'-nitrogen atoms is described. Aspects of the conversion of chloroacetamides to iodoacetamides are discussed, including a remarkably mild dehalogenation of an aromatic haloacetamide in the presence of NaI and camphorsulfonic acid. The bifunctional rhodamine has been designed for two-site, 1:1 labeling of proteins that contain two suitably disposed cysteine residues and is intended to constrain the orientation of the rhodamine absorption and emission dipoles in a predictable relationship to the protein structure.