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

Pinney, Kevin George

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Description

Title

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

Author(s)

Pinney, Kevin George

Issue Date

1990

Doctoral Committee Chair(s)

Katzenellenbogen, John A.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Chemistry, Biochemistry
• Chemistry, Organic

Language

eng

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.

Type of Resource

text

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Copyright and License Information

Copyright 1990 Pinney, Kevin George

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