Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-<i>threo</i>-Methylphenidate

The development of photoaffinity ligands for determining covalent points of attachment to the dopamine transporter (DAT) has predominantly focused on tropane-based compounds bearing variable-length linkers between the photoreactive group and the inhibitor pharmacophore. To expand the array of photoprobes useful for mapping inhibitor-binding pockets within the DAT, a compact nontropane ligand was synthesized featuring a photoreactive azide and iodine tag directly attached to the aromatic ring of (±)-<i>threo</i>-methylphenidate. (±)-<i>threo</i>-4-Azido-3-iodomethylphenidate [(±)-<b>6</b>; <i>K</i>_<i>i</i> = 4.0 ± 0.8 nM] displayed high affinity for hDAT. Moreover, a radioiodinated analogue of (±)-<b>6</b> demonstrated covalent ligation to the DAT in cultured cells and rat striatal membranes, thus suggesting the potential utility of this photoprobe in DAT structure–function studies