Efficient and selective photoaffinity labeling of the estrogen receptor using two nonsteroidal ligands that embody aryl azide or tetrafluoroaryl azide photoreactive functions

Biochemistry. 1991 Mar 5;30(9):2421-31. doi: 10.1021/bi00223a018.


3-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-6-hydroxy-2-(4- hydroxyphenyl)benzo[b]thiophene 1 (tetrafluoroaryl azide, TFAA) and its protio analogue 3-(4-azidobenzoyl)-6- hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene 2 (protioaryl azide, PAA), photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared in high specific activity tritium-labeled form (19 Ci/mmol) and shown to undergo selective and efficient photocovalent attachment to ER from rat uterus. Both azides 1 and 2 demonstrate high binding affinity for ER as determined by both a competitive binding assay (relative binding affinities: estradiol = 100; TFAA = 9.3; PAA = 66) and a direct binding assay (Kd: estradiol = 0.24 nM; TFAA = 2.64 nM; PAA = 0.37 nM). When unlabeled TFAA and PAA are irradiated at greater than 315 nm, they demonstrate site-specific photoinactivation of ER that reaches 43% and 55%, respectively, by 30 min. Specific photocovalent attachment to ER can be effected by irradiation of the tritium-labeled azides; the covalent attachment efficiency is good (1 = 20-30%, 2 = ca. 25%) and the selectivity of ER labeling is high. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of a major component at Mr 60,000 and a minor species at Mr 46,000, the same two species that are labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. The ER-specific antibodies H222Sp gamma and D547Sp gamma show a clean precipitation of only these two species. In the MCF-7 human breast cancer cell line, PAA is a full estrogen agonist in terms of stimulation of cell proliferation and induction of progesterone receptor. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluorosubstituted aryl azide analogue in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone-binding domain of this protein.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels / chemical synthesis*
  • Animals
  • Azides / chemical synthesis*
  • Azides / metabolism
  • Binding, Competitive
  • Cytosol / metabolism
  • Female
  • Kinetics
  • Ligands
  • Rats
  • Receptors, Estrogen / metabolism*
  • Sheep
  • Thiophenes / chemical synthesis*
  • Thiophenes / metabolism
  • Uterus / metabolism


  • Affinity Labels
  • Azides
  • Ligands
  • Receptors, Estrogen
  • Thiophenes
  • 3-(4-azido-2,3,5,6-tetrafluorobenzoyl)-6-hydroxy-2-(4-hydroxyphenyl)benzo(b)thiophene
  • 3-(4-azidobenzoyl)-6-hydroxy-2-(4-hydroxyphenyl)benzo(b)thiophene