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Title:Design, synthesis, and biochemical evaluation of novel photoaffinity labeling reagents for the estrogen and progesterone receptors
Author(s):Pinney, Kevin George
Doctoral Committee Chair(s):Katzenellenbogen, John A.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Chemistry, Organic
Abstract: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$H) tamoxifen aziridine, a known affinity label for ER. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluoro-substituted aryl azide analog in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone binding domain of this protein.
DU41165, a retroprogestin (9$\beta$,10$\alpha$) embodying a fluorine-substituted dienone system, has been prepared in high specific activity tritium-labeled form (4 Ci/mmol) and shown to be a high affinity ligand for the progesterone receptor (PgR) and a highly selective labeling reagent for PgR. The binding affinity of DU41165 for PgR was determined by both a competitive binding assay and a direct binding assay (Scatchard analysis) to be 1.6-2.2-times higher than that of the high affinity synthetic progestin promegestone (R5020). In radiolabeled form, ($\sp3$H) DU41165 demonstrates specific covalent attachment with an efficiency of 5-7%. SDS-polyacrylamide gel electrophoresis of photoattached ($\sp3$H) DU41165 confirms that there is covalent labeling of both the B subunit (M$\sb{\rm r}$ = 118,000), and the A subunit (M$\sb{\rm r}$ = 88,000) of PgR in a molar ratio of approximately 1:3. In tissue distribution studies in estrogen-primed immature rats, ($\sp3$H) DU41165 shows uterus-to-muscle ratios of 15 at 1 h, and 18-71 between 2 and 6 h, suggesting that it may be a very promising candidate for selective imaging of PgR-positive breast tumors.
Issue Date:1990
Rights Information:Copyright 1990 Pinney, Kevin George
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9114375
OCLC Identifier:(UMI)AAI9114375

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