Three photoaffinity ligands (PALs)for the human serotonin transporter(hSERT) were synthesized based on the selective serotonin reuptakeinhibitor (SSRI), (S)-citalopram (1).The classic 4-azido-3-iodo-phenyl group was appended to either theC-1 or C-5 position of the parent molecule, with variable-length linkers,to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (Ki= 24–227 nM) for hSERT, as assessedby [3H]5-HT transport inhibition. When tested against Ser438ThrhSERT, all three PALs showed dramatic rightward shifts in inhibitorypotency, with Kivalues ranging from 3.8to 9.9 μM, consistent with the role of Ser438 as a key residuefor high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adductionto hSERT by all ligands, but the reduced (S)-citalopraminhibition of labeling by [125I]15comparedto that by [125I]22and [125I]26suggests differences in binding mode(s). These radioligandswill be useful for characterizing the drug–protein bindinginteractions for (S)-citalopram at hSERT. [ABSTRACT FROM AUTHOR]