We are developing affinity labeling agents and fluorescent ligands as molecular probes for steroid receptors. I. In order to study the structure of the estrogen receptor and to probe for the gene sites with which it interacts, we will prepare new aziridine- substituted ligands for the estrogen receptor that will be estrogenic (agonistic) and are labeled with iodine-125 at high specific activity. Covalently-labeled receptor will be subjected to proteolysis to generate a 6kDa-peptide fragment that will be sequenced. This should enable the site of ligand binding and convalent attachment to be localized within the known amino acid sequence of the receptor. The gene labeling studies will be done as a collaboration and will involve receptor labeling with the I-125 labeled agonistic aziridine and then protein DNA cross- linking. II. Two approaches will be taken to improve the efficiency of steroid receptor photoaffinity labeling. Since the low efficiency of progesterone receptor photolabeling by R5020 (ca. 5-10%) may be due to the non-reactivity of the excited state, we will prepare R5020 analogs that are fluorine- substituted in a way that will switch the excited state to the more reactive state, thereby increasing the efficiency of radical pair generation and ligand-protein coupling. An acyl azide with a built-in triplet sensitizer will be prepared as an efficient photoaffinity label for the estrogen receptor. III. Three types of fluorescent estrogens will be prepared to enable receptor assay by ligand binding in individual viable breast cancer cells by flow cytometry or image-intensified fluorescence microscopy: Inherently fluorescent ligands based on 2-phenylindenes substituted with appropriate donors and acceptors; photofluorogenic estrogens based on cis-stilbazoles, and ligand conjugates with chelates of europium (III) and terbium (III) ions. The very long luminescence lifetimes of these lanthanide ion complexes may permit the use of the powerful technique of time resolution in receptor assays and microscopic imaging.