Design, synthesis, and biochemical evaluation of novel photoaffinity labeling reagents for the estrogen and progesterone receptors

A tetrafluoro-substituted aryl azide (TFAA) 1 and its protio analogue (PAA) 2, both photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared by direct coupling of the appropriately substituted 4-azidobenzoyl chloride with the electron rich C-3 of 2-(4-methoxyphenyl)-6-methoxybenzo (b) thiophene 6. This represents a rare example of aryl azide stability under Friedel-Crafts acylation conditions. TFAA 1 and PAA 2 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 (K$\sb{\rm d}$: estradiol = 0.24 nM; TFAA = 2.64 nM; PAA = 0.37 nM). When unlabeled TFAA 1 and the corresponding PAA 2 are irradiated at $>$315 nm, they demonstrate site specific photoinactivation of ER that reaches 43% and 55%, respectively, by 30 min. Specific photocovalent attachment to ER can be effected by irradiation of the tritium-labeled azides; the covalent attachment efficiency is good (1 = 20-30%, 2 = 25-50%) and the selectivity of ER labeling is high. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of a major component at M$\sb{\rm r}$60,000 and a minor species at M$\sb{\rm r}$46,000, the same two species that are labeled by ($\sp3$H) tamoxifen aziridine, a known affinity label for ER. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluoro-substituted aryl azide analog 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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Pinney, Kevin George

Contributors dc:contributor

• Katzenellenbogen, John A.

• Chemistry, Biochemistry
• Chemistry, Organic

Statement dc:rights

• Copyright 1990 Pinney, Kevin George

Language dc:language

eng

Identifier

AAI9114375
(UMI)AAI9114375

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/22373