Electrophilic Affinity Labels and Fluorescent Conjugates for the Estrogen Receptor (Norhexestrol, Aziridine, Hexestrol, Fluorescein, Alkylator)
Zablocki, Jeffery Alan
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Permalink
https://hdl.handle.net/2142/70357
Description
Title
Electrophilic Affinity Labels and Fluorescent Conjugates for the Estrogen Receptor (Norhexestrol, Aziridine, Hexestrol, Fluorescein, Alkylator)
Author(s)
Zablocki, Jeffery Alan
Issue Date
1986
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, Organic
Abstract
We prepared several aziridine derivatives of the potent, non-steroidal estrogen hexestrol ((3R*,4S*)-3,4-bis(4-hydroxyphenyl)hexane)) in search of an estrogen agonist that achieves selective, efficient labeling of the estrogen receptor. The electrophilic agent, aziridine, was positioned at a side chain terminus which was linked to the bis-phenol binding component of hexestrol by various functionalities: ketone, ester, thioether, or methylene chain. The apparent competitive binding affinity of these derivatives for the estrogen receptor ranges from 1.8-25% of that of estradiol. Most of the norhexestrol aziridine derivatives bind to the receptor in a time-dependent, irreversible manner; however, the rate and efficiency of this binding varies widely with relatively small structural changes. This is consistent with the irreversible attachment requiring a precise alignment of activating and reacting residues in the binding site of the receptor. Nearly all of the norhexestrol aziridine derivatives retained the estrogenic activity of the parent hexestrol ligand as determined in human breast cancer cell growth experiments (MCF-7 cell line). Norhexestrol aziridine 34 ((6R*,7S*)-1-(N-aziridinyl)-6,7-bis(4-hydroxyphenyl)-5-nonanone) had the most potential as an estrogenic affinity labeling agent because of its agonistic properties, and its rapid, efficient irreversible binding to the receptor. Therefore, norhexestrol aziridine 34 was prepared in tritium labeled form (specific activity = 67 Ci/mmol), and was found to be an efficient, selective estrogenic affinity label of the estrogen receptor with 60% of the receptor labeled within 2 hours (55% selectivity, Az / ER , 12 nM/2 nM, lamb uterine cytosolic receptor).
As part of our development of a fluorimetric assay which determines estrogen receptor content in individual cells, we have prepared several estrogen-fluorophore conjugates in which we have systematically optimized both binding affinity and fluorescence properties by varying fluorophore and linking chain length. The estrogen-fluorophore conjugates prepared consist of hexestrol as the high affinity receptor binding component, connected by a thioether linkage to the following commercially available fluorophores: Nitrobenzoxadazole (NBD), fluorescein, and aminophthalimide.
NBD derivatives 107, 108, and fluorescein derivative 116 possess moderate affinity for the receptor and suitable fluorescence properties to be used as fluorescent probes in the development of a fluorimetric assay.
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