2026-09-21T22:13:20Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/22373 2023-07-10 col_2142_5131 col_2142_14789 com_2142_5130 com_2142_14788 com_2142_8903

Katzenellenbogen, John A. Pinney, Kevin George 2011-05-07T13:37:46Z 2011-05-07T13:37:46Z 10000-01-01 1990 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. Made available in DSpace on 2011-05-07T13:37:46Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9114375.pdf: 7514210 bytes, checksum: ef8efa8c44740c133a372506a7eaf6f4 (MD5) Previous issue date: 1990 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:57:11Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:26:48-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only AAI9114375 (UMI)AAI9114375 http://hdl.handle.net/2142/22373 eng Copyright 1990 Pinney, Kevin George Chemistry, Biochemistry Chemistry, Organic Design, synthesis, and biochemical evaluation of novel photoaffinity labeling reagents for the estrogen and progesterone receptors text Chemistry Chemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.