{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/22373"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/22373","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Design, synthesis, and biochemical evaluation of novel photoaffinity labeling reagents for the estrogen and progesterone receptors","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$H) tamoxifen aziridine, a known affinity label for ER. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluoro-substituted aryl azide analog in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone binding domain of this protein.","abstract_html":"A tetrafluoro-substituted aryl azide (TFAA) 1 and its protio analogue (PAA) 2, both photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared by direct coupling of the appropriately substituted 4-azidobenzoyl chloride with the electron rich C-3 of 2-(4-methoxyphenyl)-6-methoxybenzo (b) thiophene 6. This represents a rare example of aryl azide stability under Friedel-Crafts acylation conditions. TFAA 1 and PAA 2 have been prepared in high specific activity tritium-labeled form (19 Ci/mmol) and shown to undergo selective and efficient photocovalent attachment to ER from rat uterus. Both azides 1 and 2 demonstrate high binding affinity for ER as determined by both a competitive binding assay (relative binding affinities: estradiol = 100; TFAA = 9.3; PAA = 66) and a direct binding assay (K$\\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.","abstract_has_math":true,"creators":["Pinney, Kevin George"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Katzenellenbogen, John A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-05-07T13:37:46Z","date_published":"2011-05-07T13:37:46Z","updated_at":"2026-07-22T22:25:19Z","subjects":["Chemistry, Biochemistry","Chemistry, Organic"],"languages":["eng"],"rights":["Copyright 1990 Pinney, Kevin George"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114375","(UMI)AAI9114375"],"render_values":[{"text":"AAI9114375","href":null,"code":true},{"text":"(UMI)AAI9114375","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/22373","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Katzenellenbogen, John A."]},{"key":"dc:creator","label":"Author","values":["Pinney, Kevin George"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-05-07T13:37:46Z","10000-01-01","1990"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Chemistry, Biochemistry","Chemistry, Organic"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 1990 Pinney, Kevin George"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["AAI9114375","(UMI)AAI9114375","http://hdl.handle.net/2142/22373"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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"]},{"key":"dc:title","label":"Title","values":["Design, synthesis, and biochemical evaluation of novel photoaffinity labeling reagents for the estrogen and progesterone receptors"]}]}],"canonical_facts":{"dc:contributor":["Katzenellenbogen, John A."],"dc:creator":["Pinney, Kevin George"],"dc:date":["2011-05-07T13:37:46Z","10000-01-01","1990"],"dc:description":["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"],"dc:identifier":["AAI9114375","(UMI)AAI9114375","http://hdl.handle.net/2142/22373"],"dc:language":["eng"],"dc:rights":["Copyright 1990 Pinney, Kevin George"],"dc:subject":["Chemistry, Biochemistry","Chemistry, Organic"],"dc:title":["Design, synthesis, and biochemical evaluation of novel photoaffinity labeling reagents for the estrogen and progesterone receptors"],"dc:type":["text"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:19Z"}