Chemical synthesis and biochemical characterization of a biotinylated derivative of 17β-estradiol with a long side chain covalently attached to its C-7α position

Article abstract of DOI:10.1016/j.steroids.2008.06.004

High-affinity biotinylated derivatives of 17β-estradiol (E₂) are of value for isolation of various estrogen-binding proteins (including estrogen receptors) and also for studying protein-protein interactions involving these proteins. In this study, we developed a simplified route for the chemical synthesis of a biotinylated derivative of E₂ (compound 7) with a side chain attached to its C-7α position. Compound 7, i.e., 7α-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol, could be readily synthesized from 6-keto-estradiol-3,17β-di-tetrahydropyranyl ether (compound 2, which can be prepared from E₂), with a final yield of 36%. In vitro receptor-binding assay confirmed that the synthesized affinity ligand has a high binding affinity for both human estrogen receptor α and β. When the affinity ligand (compound 7) was immobilized with avidin on an affinity column, it effectively bound human estrogen receptor α, and the receptor protein could be selectively eluted with a biotin-containing buffer. Using the same affinity ligand, prolyl 4-hydroxylase β-subunit (also known as protein disulfide isomerase) was identified as one of the high-affinity E₂-binding proteins in the whole cytosolic protein mixture prepared from MCF-7 human breast cancer cells. Computational molecular modeling analysis showed that compound 7 can bind to human estrogen receptor α in a similar manner as ICI-182,780 and raloxifene, and their binding energy values are also similar.

Full text of DOI:10.1016/j.steroids.2008.06.004

steroids 7 3 ( 2 0 0 8 ) 1252–1261
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Chemical synthesis and biochemical characterization of a
biotinylated derivative of 17␤-estradiol with a long side
chain covalently attached to its C-7␣ position
Xiang-Rong Jiang1,2, Pan Wang1, Xinmiao Fu1, Bao Ting Zhu∗
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center,
Kansas City, KS 66160, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High-affinity biotinylated derivatives of 17␤-estradiol (E2) are of value for isolation of
various estrogen-binding proteins (including estrogen receptors) and also for study-
ing protein–protein interactions involving these proteins. In this study, we developed
Received 23 October 2007
Received in revised form
12 June 2008
a
simplified route for the chemical synthesis of
a biotinylated derivative of E2
Accepted 12 June 2008
Published on line 22 June 2008
(compound 7) with side chain attached to its C-7␣ position. Compound 7, i.e.,
a
7␣-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol, could be readily synthesized from 6-
keto-estradiol-3,17␤-di-tetrahydropyranyl ether (compound 2, which can be prepared from
E2), with a final yield of 36%. In vitro receptor-binding assay confirmed that the synthesized
affinity ligand has a high binding affinity for both human estrogen receptor ␣ and ␤. When
the affinity ligand (compound 7) was immobilized with avidin on an affinity column, it
effectively bound human estrogen receptor ␣, and the receptor protein could be selectively
eluted with a biotin-containing buffer. Using the same affinity ligand, prolyl 4-hydroxylase
␤-subunit (also known as protein disulfide isomerase) was identified as one of the high-
affinity E2-binding proteins in the whole cytosolic protein mixture prepared from MCF-7
human breast cancer cells. Computational molecular modeling analysis showed that com-
pound 7 can bind to human estrogen receptor ␣ in a similar manner as ICI-182,780 and
raloxifene, and their binding energy values are also similar.
Keywords:
Biotinylated estradiol
Affinity ligand
Computational molecular modeling
Chemical synthesis
Estrogen receptor
Novel estrogen-binding proteins
© 2008 Elsevier Inc. All rights reserved.
1.
Introduction
as other E2-binding proteins [1–4]. Also, they are useful
for studying protein–protein interactions involving these
High-affinity biotinylated derivatives of 17␤-estradiol (E2)
are useful for isolation of estrogen receptors (ERs) as well
estrogen-binding proteins. The most commonly used estro-
gen receptor affinity ligands include two basic types: the
Abbreviations: E2, 17␤-estradiol; ER␣ and ER␤, estrogen receptor alpha and beta subtypes, respectively; NMR, nuclear magnetic reso-
nance; THP, tetrahydropyran; DMF, dimethylformamide; TEA, triethylamine; TMS, tetramethylsilane; t-BuOK, potassium t-butoxide; RAL,
raloxifene; PDI, protein disulfide isomerase.
The study was supported, in part, by a grant from the NIH (CA97109). Note that part of the work described in this paper was completed
w∗hen the authors were at the University of South Carolina, Columbia, SC 29208, USA.
Corresponding author at: Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical
Center, Room 4061 of KLSIC Building, 2146 W. 39th Street, Kansas City, KS 66160, USA. Tel.: +1 913 588 9842; fax: +1 913 588 8356.
E-mail address: BTZhu@kumc.edu (B.T. Zhu).
1
These authors contributed almost equally to this study.
Present address: PharmAgra Labs, Inc., 40 McLean Road, Brevard, NC 28712, USA.
2
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.06.004

steroids 7 3 ( 2 0 0 8 ) 1252–1261
1253
C-17␣-substituted derivatives that are commonly synthe-
sized from 17␣-ethynylestradiol, and the C-7␣-substituted
derivatives that are usually synthesized from E2 or estrone.
While the C-17␣-substituted derivatives have the advantage
of shorter steps for chemical synthesis, ligands of this group
generally have rather low binding affinity for ERs, because
C-17␣ substitution drastically reduces their binding affinity
[5,6]. In comparison, C-7␣-substituted affinity ligands retain
high affinity for ER binding [7,8], just like the C-7␣ sub-
stituted antiestrogens such as ICI-182,780 [9,10]. However,
C-7␣-substituted affinity ligands are generally more difficult to
make because of the long and difficult synthetic steps involved
[2–4,8,11].Recently, we have compared a number of methods
for the chemical synthesis of C-7␣-substituted E2 derivatives
[12]. Based on the improvements we recently made, we report
here a simplified route for the chemical synthesis of a new C-
7␣ biotinylated E2 derivative (structure shown in Scheme 1).
Based on the earlier studies showing that some of the sub-
Scheme 1 – The flow chart for the synthesis of 7␣-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol (compound 7) using E2
(compound 1) as the starting material. The reagents and the reaction conditions used are summarized below: (a) Swern
oxidation. (b) t-BuOK, THF, 0 ◦C, compound 9, −78 ◦C. (c) NaBH4, methanol. (d) 10% Pd(OH)2/C, H2, 45 psi, ethanol, 60 ◦C 8 h. (e)
Compound 13, Et3N, DMF, rt, 24 h. (f) AcOH:MeOH:H2O (4:2:1), rt, 24 h. (g) NaI, acetone, reflux, 12 h. (h) Isobutyl
chloroformate, tri-n-butylamine, DMF. (i) N-Hydroxysuccinimide, dicyclohexyl carbodiimide, DMF.

1254
steroids 7 3 ( 2 0 0 8 ) 1252–1261
stituted E2 derivatives with a long side chain attached to the
C-7␣ position usually retain ER␣-binding affinity, it was thus
expected that the new affinity ligand we synthesized likely
would also have ER-binding activity. The affinity ligand was
experimentally tested in this study to determine its binding
affinity for human ER␣ and ER␤ in vitro. In addition, we also
tested its usefulness for the isolation of human ER␣ protein
or other E2-binding proteins when it was used in combination
with an immobilized avidin column. To help better understand
its binding interactions with human ER␣, we used the compu-
tational molecular modeling tools to dock compound 7 and
two of its theoretical analogs with a shorter side chain to the
binding pocket of the receptor and compared its binding con-
formation with two well-known ER antagonists, ICI-182,780
and raloxifene (RAL).
Then, to the residue 150 mL hexane was added. After the solid
was removed by filtration, the filtrate was concentrated. Flash
chromatography (hexane as an eluent) afforded compound 9
(14.5 g, 98%).
2.1.4. Synthesis of 8-N-biotinylaminooctanoic acid
(compound 12)
8-N-Biotinylaminooctanoic acid, a known compound, was pre-
pared according to the procedures described by Baulieu and
co-workers [13]. Isobutyl chloroformate (160 ␮L) was added
to a solution of biotin (250 mg) in dimethylformamide (DMF,
20 mL) containing tri-n-butylamine (320 ␮L). After 10 min at
room temperature, the mixture was added to a suspension of
8-aminooctanoic acid (163 mg) in DMF (20 mL) at 5 ◦C and was
stirred for 2 h. The solvent was removed in vacuo. The crude
product was re-crystallized from ethanol to afford 220 mg
product in 56% yield. 1H NMR (DMSO-d6, 300 MHz,): 7.71 (1H,
t, J = 5.4 Hz), 6.41 (1H, s), 6.34 (1H, s), 4.28 (1H, m), 4.11 (1H, m),
3.06 (1H, m), 2.97 (2H, q, J = 12.6, 6.3 Hz), 2.80 (1H, dd, J = 12.3,
5.1 Hz), 2.55 (1H, d, J = 12.3 Hz), 2.15 (2H, t, J = 7.5 Hz), 2.02 (2H, t,
J = 7.2 Hz), 1.58–1.22 (17H, m). MS (TOF MS ES+, C18H31N3O4S, M:
385): 225, 386 (M+H+, base peak), 408 (M+Na+). HRMS: 386.2113
(calculated) and 386.2121 (observed).
2.
Experimental
2.1.
Chemical synthesis
2.1.1. Chemicals and reagents
Most of the commercially available reagents were obtained
from the Sigma–Aldrich Chemical Co. (St. Louis, MO) or ACROS
(through Fisher Scientific, Atlanta, GA), and were used directly
without additional steps of purification. 17␤-Estradiol (E2) was
purchased from Steraloids Inc. (Newport, RI). Tetrahydrofu-
ran (THF) was distilled over sodium/benzophenone ketyl and
methylene chloride was distilled over calcium hydride in our
laboratory prior to use.
[2,4,6,7,16,17-3H]E2 (specific activity of 123 Ci/mmol) was
obtained from PerkinElmer (Waltham, MA) and it was re-
purified using an HPLC method prior to use in the in vitro
receptor-binding assay. The recombinant human ER␣ and ER␤
proteins and bovine serum albumin (BSA) were obtained from
PanVera Corporation (Madison, WI). Hydroxylapatite (HAP)
was obtained from Calbiochem (San Diego, CA). UltraLink
immobilized monomeric avidin was obtained from Pierce
Biotechnology Inc. (Rockford, IL). The primary antibody (no.
sc-543) against ER␣ was purchased from Santa Crutz Biotech-
nology (Santa Cruz, CA). Human breast cancer cell line MCF-7
was obtained from ATCC (Manassas, VA). Protease inhibitor
cocktail was purchased from Sigma–Aldrich Chemical Com-
pany (St. Louis, MO).
2.1.5. Synthesis of N-hydroxysuccinimide ester of
8-N-biotinylaminooctanoic acid (compound 13)
N-Hydroxysuccinimide ester of 8-N-biotinylaminooctanoic
acid was prepared from 8-N-biotinylaminooctanoic acid
and N-hydroxysuccinimide by coupling with dicyclohexyl-
carbodiimide according to the procedures of Bayer and
Wilchek [14]. Dicyclohexyl carbodiimide (0.8 g) was added to
a solution of 8-N-biotinylaminooctanoic acid (1.54 g) and N-
hydroxysuccinimide (0.6 g) in 20 mL of DMF. The suspension
was stirred at room temperature for 24 h. The precipitate was
filtered, and the filtrate was maintained at 0 ◦C overnight. Any
additional precipitates were filtered, and the resulting filtrates
were evaporated under reduced pressure. The residue was
washed extensively with ether. The crude product was used
in next step without further purification.
2.1.6. Synthesis of 6-keto-7˛-(6-cyanohexyl)-estradiol-
3,17ˇ-di-tetrahydropyranyl ether (compound 3)
Using E2 (compound 1 in Scheme 1) as the starting material,
6-hydroxy-estradiol-3,17␤-di-tetrahydropyranyl ether (com-
pound 2a) was first prepared according to an earlier study [11].
After a solution of methylene chloride (70 mL) and oxalyl
chloride (3.6 mL, 41.9 mmol) in a 250-mL three-neck round-
bottom flask was cooled to −78 ◦C, DMSO (5.9 mL, 83 mmol,
in 15 mL methylene chloride) was added slowly to the stirred
oxalyl chloride solution and the reaction mixture was stirred
further for 5 min. Note that the addition of the DMSO solution
needs to be relatively slow so that the temperature of the reac-
tion mixture would not rise above −50 ◦C (usually controlled
between −50 and −60 ◦C), which is critical for this reaction.
After that, compound 2a (15.7 g, 34.5 mmol, in 30 mL methy-
lene chloride) was slowly added over 15 min, and the mixture
was stirred for an additional 25 min. Then trimethylamine
(TEA, 24 mL) was added and the reaction mixture was stirred
for 5 min and allowed to warm to room temperature. Water
(100 mL) was added and the aqueous phase was extracted with
2.1.2. Spectrometric analyses
Mass spectra were recorded with a VG70S analytical mass
spectrometer. An aliquot of the ethanol solution of the test
compound was used for direct-probe mass analysis. The
nuclear magnetic resonance (NMR) spectra of all test com-
pounds in deuterochloroform solution were recorded on a
Varian Mercury/VX 300 spectrometer with tetramethylsilane
(TMS) as internal standard (ı = 0) unless noted otherwise.
2.1.3. Synthesis of 7-iodoheptanenitrile (compound 9)
Under nitrogen, 10 g (52.4 mmol) of 7-bromoheptanenitrile
was added into a mixture of 39 g (262 mmol) sodium iodide
and 100 mL acetone. The mixture was refluxed over night.
Upon completion, the reaction mixture was allowed to cool to
room temperature. The organic solvent was removed in vacuo.

steroids 7 3 ( 2 0 0 8 ) 1252–1261
1255
methylene chloride (50 mL). The organic phase was combined,
washed with a saturated sodium chloride solution (100 mL),
and dried over anhydrous sodium sulfate. Flash chromatogra-
phy afforded compound 2 (14.6 g, 94% yield).
(1H, brS), 5.29 (1H, m), 4.59 (1H, m), 3.85 (2H, m), 3.67 (1H, t,
J = 8.4 Hz), 3.55 (1H, m), 3.43 (1H, m), 2.81 (2H, m), 2.21 (2H, m),
1.92–1.12 (38H, m), and 0.73 (3H, s). MS (TOF MS ES+, C35H55O4N,
M+ 553): 555 (M+1) (base peak). HRMS (M+1): 554.4209 (calcu-
lated) and 554.4208 (observed). Compound 5a. 1H NMR (300 Hz,
DMSO-d6): 9.15 (1H, brs), 8.20 (3H, brs), 7.07 (1H, d, J = 8.4 Hz),
6.57 (1H, d, J = 8.4 Hz), 6.51 (1H, s), 4.94 (1H, brs), 3.59 (1H, t,
J = 7.8 Hz), 2.77 (3H, m), 2.65 (1H, d, J = 16.8Hz), 2.27 (2H, m),
1.97–1.11 (21H, m), and 0.72 (3H, s). MS (C25H39NO2, M 385):
385 (base peak). HRMS (M): 385.2981 (calculated) and 385.2984
(observed).
To synthesize compound 3, a 1.0 M solution of potassium
t-butoxide (t-BuOK) in THF (20 mL, 20 mmol) was added to a
cooled solution (at 0 ◦C) of compound 2 (1.2 g, 2.67 mmol) in
THF (20 mL). The reaction mixture was stirred at 0 ◦C for 40 min
and then cooled to −78 ◦C. 7-Iodoheptanenitrile (compound
9, 2.0 g, 7.1 mmol) was added dropwise to the solution. The
reaction mixture was allowed to warm to 0 ◦C and stirred for
2 h, and then to room temperature and stirred overnight. The
reaction was quenched with water and extracted with ethyl
acetate. The organic phase was dried over sodium sulfate and
the solvent was evaporated in vacuo. Flash chromatography
(hexane:ethyl acetate = 4:1, v:v) afforded compound 3 in 62%
yield (0.93 g). 1H NMR (300 Hz, CDCl3): 7.61 (1H, brS), 7.15–7.25
(2H, m), 5.39 (1H, m), 4.62 (1H, m), 3.80 (2H, m), 3.68 (1H, t,
J = 8.4 Hz), 3.56 (1H, m), 3.42 (1H, m), 2.59 (1H, m), 2.39 (2H, m),
2.24 (2H, t, J = 7.2Hz), 1.16–2.02 (31H, m), and 0.73 (3H, s). MS
(Scan ES+, C35H49O5N, M+1 564): 564 (base peak), 586 (M+Na).
HRMS (M+1): 564.3689 (calculated) and 564.3682 (observed).
2.1.9. Synthesis of 7˛-{7-[8-(biotinamido)-octanamido]-
heptyl}-estradiol (compound 7)
The crude preparation containing mostly compound
5
(42 mg, 0.11 mmol) was dissolved in 10 mL DMF. Then
0.053 g (0.11 mmol) of N-hydroxysuccinimide ester of 8-N-
biotinylaminooctanoic acid (compound 13) and 47 ␮L of TEA
were added. The mixture was stirred at room temperature
for 24 h. The solvents were removed under high vacuum, and
the raw product was dissolved in a mixture of acetic acid,
methanol and water (4:2:1, v:v:v) and stirred for 24 h at room
temperature. The solvents were removed in vacuo, and the raw
product was purified by HPLC to afford compound 7.
2.1.7. Synthesis of 6-hydroxy-7˛-(6-cyanohexyl)-
estradiol-3,17ˇ-di-tetrahydropyranyl ether (compound 4)
1H NMR (300 Hz, DMSO-d6): 8.98 (1H, s), 7.72 (2H, m), 7.04
(1H, d, J = 8.7 Hz), 6.49 (1H, dd, J = 8.7, 2.7 Hz), 6.43 (1H, s), 6.41
(1H, d, J = 2.7 Hz), 6.36 (1H, s), 4.48 (1H, d, J = 4.8 Hz), 4.29 (1H,
m), 4.13 (1H, m), 3.53 (1H, m), 3.09 (1H, m), 3.0 (4H, m), 2.80
(2H, m), 2.62 (2H, m), 2.26 (2H, m), 2.0 (4H, m), 190–1.05 (38H,
m), and 0.66 (3H, s). MS (TOF MS ES+, C43H68N4O5S, M+ 752.5):
753.5 (M+1) (base peak). HRMS (M+1): 753.4988 (calculated) and
753.4982 (observed).
Compound
3 (0.51 g, 0.91 mmol) was dissolved in 20 mL
methanol. To this solution was added sodium borohydride
(0.69 g, 18.1 mmol). The reaction mixture was stirred at room
temperature for 4 h. The methanol was removed in vacuo.
The residue was then dissolved in water (20 mL). The aque-
ous solution was extracted with methylene chloride three
times (3 × 20 mL). Organic fractions were combined, dried over
sodium sulfate, and concentrated. The crude product was
purified by flash chromatography (ethyl acetate:hexane = 1:3,
v:v) to afford compound 4 (0.5 g, 98%). 1H NMR (300 Hz, CDCl3):
7.4 (1H, d, J = 2.4 Hz), 7.09 (1H, d, J = 8.8 Hz), 6.84 (1H, m), 5.35 (1H,
m), 4.81 (1H, m), 4.57 (1H, m), 3.84 (2H, m), 3.68 (1H, t, J = 7.6 Hz),
3.52 (1H, m), 3.42 (1H, m), 2.18–2.31 (4H, m), 1.87–2.07 (4H, m),
1.76 (4H, m), 1.20–1.65 (25H, m), and 0.73 (3H, s). MS (TOF MS
ES+, C35H51O5N, M+ 565): 540, 548, 566, 583 (M+NH4+) (base
peak), 588 (M+Na+). HRMS [588 (M+Na+)]: 588.3665 (calculated)
and 588.3660 (observed).
2.2.
ER˛- and ERˇ-binding assays
The ER␣ and ER␤ competitive binding assays were performed
according to the method described in our recent study [15].
The ER-binding buffer used for dilution of the receptor prepa-
rations consisted of 10% glycerol, 2 mM dithiothreitol, 1 mg/mL
BSA, and 10 mM Tris–HCl (pH 7.5). The ER␣ washing buffer con-
tained 40 mM Tris–HCl and 100 mM KCl (pH 7.4), but the ER␤
washing buffer contained only 40 mM Tris–HCl (pH 7.5). The
50% (v/v) hydroxylapatite (HAP) slurry was prepared by using
the 50 mM Tris–HCl solution (pH 7.4). The reaction mixture
contained 50 ␮L of varying concentrations of the test com-
pound in the ER-binding buffer, and 45 ␮L of [3H]E2 solution
(at 22.22 nM). Then 5 ␮L of ER␣ or ER␤ protein was added and
mixed gently. Nonspecific binding by [3H]E2 was determined
by addition of a 400-fold concentration of the nonradioactive
E2 (8 ␮M). The binding mixture was incubated at room tem-
perature for 2 h. At the end of the incubation, 100 ␮L of the
HAP slurry was added and the tubes were incubated on ice for
15 min with three times of brief vortexing. An aliquot (1 mL)
of the washing buffer was added, mixed, and centrifuged
at 10,000 g for 1 min, and the supernatants were discarded.
This wash step was repeated twice. The HAP pellets were
then resuspended in 200 ␮L ethanol, and the content was
transferred to scintillation vials for measurement of the 3H-
radioactivity in a liquid scintillation counter (Packard Tri-CARB
2900 TR; Downers Grove, IL). The data obtained from dupli-
2.1.8. Synthesis of
7˛-(7-aminoheptyl)-estradiol-3,17ˇ-di-tetrahydropyranyl
ether (compound 5)
Compound 4 (0.4 g, 0.71 mmol) and 10% palladium hydrox-
ide on carbon (400 mg) were added to 50 mL of ethanol. The
reaction vessel was evacuated of air and filled to a pres-
sure of 45 psi with hydrogen on a Parr apparatus, and then
shaken for 8 h at 60 ◦C. The mixture was then filtered through
celite, and the filtrate was evaporated under reduced pressure
to give a mixture of compound 5 and compound 5a [7␣-(7-
aminoheptyl)-estradiol] by a ratio of 5:1 based on 1H NMR (total
0.39 g, ∼100%). This mixture was not further purified for the
next synthetic step.
For spectrometric analysis, a fraction of the samples was
used for separation of compounds 5 and 5a by flash chro-
matography (hexane:ethyl acetate = 4:1, v:v). Compound 5. 1
NMR (300 Hz, CDCl3): 7.11 (1H, m), 6.77 (1H, d, J = 8.4 Hz), 6.68
H

1256
steroids 7 3 ( 2 0 0 8 ) 1252–1261
cate measurements were expressed as the % specific binding
of [3H]E2 versus the log molar concentration of the compet-
ing compound. The IC50 value (calculated using the SigmaPlot
software) represents the concentration of a test compound
required to reduce the [3H]E2 binding by 50%.
of the InsightII. The flexible docking procedures were carried
out using the Simulated Annealing Docking method in Affinity
module of InsightII. The binding pocket was defined to include
all residues within the 7-Å reach of the original ligand RAL. A
combination of Monte Carlo and simulated annealing methods
was used to explore all possible conformations of ICI-182,780
and compounds 7. One hundred conformations were obtained
and the one with the lowest potential energy was chosen for
further minimization using the Standard Dynamics Cascade pro-
tocol in the Discovery Studio. The backbone of the protein and
its key residues in the binding pocket (namely, E353, R394 and
H524) were fixed during the docking procedures.
For energy minimizations, the steepest descent method
was employed first to 10 kcal/(mol Å) root mean square (RMS)
energy gradient and followed by the Polak and Ribiere con-
jugate gradient method until the final convergence criterion
reached 0.01 kcal/(mol Å) RMS energy gradient. Then the
whole system was heated from 100 to 300 K in 2 ps and equi-
librated in 300 K for 100 ps. One hundred conformations were
collected in 20 ps production phase at 300 K. The conformation
with the lowest potential energy was further minimized and
used for Binding Energy Calculation protocol. The backbone of
the protein and its key residues in the binding pocket (namely,
E353, R394 and H524) were constrained during the simulation
process.
2.3.
Effectiveness of the affinity ligand (compound 7)
for isolation of human E2-binding proteins
In the first experiment, the human ER␣ protein was incubated
with compound 7 or biotin alone (at 10 ␮M) at 4 ◦C overnight,
and then the mixture was loaded onto a small column pre-
packed with UltraLink immobilized monomeric avidin. After
a washout with five-bed volume of 10 mM sodium phosphate
buffer (pH 7.4), a sequential elution using three-bed volume of
10 mM sodium phosphate buffer (pH 7.4) and two-bed volume
of 2 mM biotin-containing sodium phosphate was performed.
The fractions were then subject to 13% trichloroacetic acid pre-
cipitation, and the Western blotting of the precipitated ER␣
protein was performed.
In the second sets of experiments, MCF-7 cells were cul-
tured in the presence of 10 nM of E2, harvested in 10 mM
sodium phosphate buffer, and sonicated in the presence of
1 mM EDTA and a protease inhibitor cocktail. The whole
cytosolic protein extracts were then incubated with com-
pound 7 (at 10 ␮M) at 4 ◦C over-night before loaded onto a small
column pre-packed with UltraLink immobilized monomeric
avidin. After a washout with five-bed volume of 10 mM sodium
phosphate buffer (pH 7.4, containing 0.5 M NaCl), a sequential
elution using three-bed volume of 10 mM sodium phosphate
buffer (pH 7.4) and two-bed volume of E2-containing (20 ␮M)
sodium phosphate buffer was performed. After incubation
at 4 ◦C for 6 h, continuous elution with two-bed volume
E2-containing buffer was carried out to elute the tightly
bound proteins. The fractions were then subject to 13%
trichloroacetic acid precipitation and SDS-PAGE analysis. The
bands of interest were cut and sent to ProtTech (Norristown,
PA) for protein identification using the NanoLC–MS/MS pep-
tide sequencing technology.
3.
Results and discussion
3.1.
Chemical synthesis
Compound 2 was readily obtained in a high yield from
E2 according to a previously described procedure [11,12,17].
Compound 9 was prepared from compound 8 by react-
ing with sodium iodide refluxing in acetone overnight in
90% yield. Compound 2 was then reacted with compound
9 to give compound 3 in 60% yield using potassium tert-
butoxide in THF [11,18]. Compound
compound 4 using sodium borohydride in methanol, then
hydrogenolysis of compound under the conditions of
3 was reduced to
4
2.4.
Molecular docking of ligands into the binding
45 psi hydrogen, heating (to 60 ◦C), and palladium hydrox-
ide (used as catalyst) in ethanol was carried out to give
pocket of human ER˛
compounds
to 1). Here it is optional to separate compound 5a from
since the protecting groups could be removed later.
5 and 5a (with a ratio of approximately 5
Docking study was performed with InsightII modeling soft-
ware (Version 2005, Accelrys Inc. San Diego, CA) installed in a
Dell Precision 690 workstation with Red Hat Enterprise Linux
WS4.0 operating system (Red Hat Inc., Raleigh, NC). Energy
minimization and molecular dynamics simulation were per-
formed with Discovery Studio modeling program (Version 1.7,
Accelrys Inc., San Diego, CA) installed on the same computer.
CHARMm force field was used by Discovery Studio for molecular
dynamics simulation.
Because the crystal structure of human ER␣ in complex
with a full antagonist is not available, the crystallographic
structure of ER␣ in complex with RAL (PDB code: 1ERR [16]),
which is a partial agonist/antagonist, was thus used as a tem-
plate for the docking study of ICI-182,780 and our affinity
ligand (i.e., compound 7, structure shown in Fig. 3). The struc-
tures of ICI-182,780 and compound 7 were first built using the
Builder module and then minimized with the Discover module
5
This mixture was then reacted with N-hydroxysuccinimide
ester of 8-N-biotinylaminooctanoic acid (compound 13)
to give 7␣-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol-
3,17␤-di-tetrahydropyranyl ether (compound 6), followed by
removal of the THP protecting groups in a solution of acetic
acid, methanol and water (4:2:1, v:v:v) to give the final com-
pound 7 in 81% yield over two steps. In the hydrogenolysis
step, if the reaction time was prolonged to 24 h, compound 5
could be transformed to 5a completely. The advantage of this
process is that the removal of hydroxyl on the C-6 position, the
reduction of the nitrile to amine at the end of the side chain
and deprotection of THP at the C-3 and C-17␤ positions could
be achieved in one single step under the mild conditions.
Compared to other methods [2–4,7,8,11], this is a rel-
atively shorter route for the chemical synthesis of 7␣-(7-

steroids 7 3 ( 2 0 0 8 ) 1252–1261
1257
aminoheptyl)-E2 and also its analogs with different lengths
3.3.
Use of biotin-E2 ligand (compound 7) for the
of the side chain. The procedures also appear to be envi-
ronmental friendly and rather economic with regard to the
chemical reagents required and waste products generated.
From this compound, with one more step, C-7␣-biotinylated
E2 affinity ligands can be readily synthesized. It is of note
that this intermediate is not just limited to the synthesis of
biotinylated E2 affinity ligands, it can also be used for the syn-
thesis of other useful chemicals such as fluorescent probes for
estrogen-binding proteins [19].
isolation of E2-binding proteins
To determine the usefulness of the synthesized affinity lig-
and for isolation of E2-binding proteins, we first determined
whether it could isolate the recombinant human ER␣ protein.
The ER␣ protein was incubated with compound 7 or biotin and
then loaded onto an avidin-immobilized sepharose affinity
column. We found that the receptor protein could be selec-
tively eluted off the affinity column by a biotin-containing
buffer (compare lane 4 with lane 2 in Fig. 2A). These data
suggest the ability of the affinity ligand to specifically bind
human ER␣ and the affinity ligand-bound ER␣ can be selec-
tively eluted off the avidin-immobilized affinity column with
a biotin-containing buffer. It is of interest to note that biotin
itself (in the absence of an affinity ligand) also appeared to
increase ER␣ binding to the column, and the bound ER␣ was
selectively eluted off the column with a biotin-containing
buffer (compare lane 3 with lane 1 in Fig. 2A). This observa-
tion is intriguing, and it suggests that human ER␣ protein may
have a weak binding affinity for biotin. This possibility mer-
its further investigation. The lower bands with a molecular
weight of approximately 50 kDa in lanes 3 and 4 are probably
a degradation product of ER␣ based on the fact that this protein
could be selectively washed out by a biotin-containing buffer
(compare lanes 4 and 2) and could be stained with anti-ER␣
antibodies.
3.2.
In vitro ER-binding assays
In the present study, we also determined the relative bind-
ing affinity of compound 7 for the recombinant human ER␣
and ER␤ proteins (data summarized in Fig. 1). The IC50 values
of ICI-182,780 and RAL for human ER␣ were quite similar to
each other (25.1 and 22.4 nM, respectively). The IC50 values of
the synthesized affinity ligand (compound 7) for ER␣ and ER␤
were 90 and 100 nM, respectively. The apparent affinities of
this ligand for human ERs were slightly lower than those of
ICI-182,780.
Following the above test which confirmed that the newly-
synthesized affinity ligand is capable of binding human ER␣
and the affinity ligand–ER␣ complex can be selectively washed
off with a biotin-containing elution buffer, we then used this
affinity ligand to isolate various E2-binding proteins or those
proteins that may tightly bind to the E2-binding proteins from
the cytosolic protein extracts of MCF-7 human breast can-
cer cells. The mixture containing crude cytosolic lystates and
the affinity ligand was incubated at 4 ◦C overnight and then
directly applied to an avidin-immobilized affinity column. The
column was eluted with a buffer with or without 20 ␮M E2. The
eluted proteins were analyzed using SDS-gel electrophoresis.
Fig. 2B showed that several protein bands were stronger during
the two sequential elutions with an E2-containing buffer. The
first elution was done directly with the E2-containing buffer
without incubation, and the subsequent second elution was
done after incubation with the E2-containing buffer for 6 h at
4 ◦C.
Among the protein bands identified in the second elution,
two quantitatively major ones (bands b and d labeled with a
fat arrowhead in Fig. 2B) were further analyzed in the present
study for their structural compositions by NanoLC–MS/MS
analysis (Table 1). The corresponding bands in the elution con-
trol, i.e., bands a and c, were also analyzed for comparison.
Prolyl 4-hydroxylase ␤-subunit, a protein commonly known as
protein disulfide isomerase (PDI), was identified to be present
in band b with 33 peptide fragments compared to 14 peptides
identified in band a. Similarly, 11 peptide fragments of PDI
were identified in band d compared to none in band c. It is of
note that since the theoretical molecular weight of PDI is larger
than proteins in band d, it is likely that either a quantitatively
major degradation fragment of PDI or a truncated spicing vari-
ant of PDI that still retained E2-binding activity was present
Fig. 1 – Comparison of the relative binding affinity (IC50) of
compound 7 for human ER␣ and ER␤. The binding affinity
was also compared with those of E2, RAL and ICI-182,780
(summarized in the accompanying table). The relative
binding affinity of each chemical was determined by
measuring its inhibition of the binding of 10 nM [3H]E2 to
the recombinant human ER␣ and ER␤. Eight concentrations
(0.06, 0.24, 0.98, 3.9, 15.6, 62.5, 250, and 1000 nM) of each
competing ligand were tested. The IC50 values for the
competing ligands were calculated according to the
sigmoid inhibition curves.

1258
steroids 7 3 ( 2 0 0 8 ) 1252–1261
Fig. 2 – Use of compound 7 for the isolation of human ER␣ and other E2-binding proteins from human MCF-7 cells. (A)
Western blot analysis of the eluted fractions from affinity chromatography of human recombinant ER␣ protein after
incubation with either biotin alone (lanes 1 and 3) or compound 7 (lanes 2 and 4). The avidin-immobilized affinity column
was first eluted with biotin-free solution (lanes 1 and 2) and then followed by 2 mM biotin-containing solution (lanes 3 and
4). The proteins in the collected fractions were separated by SDS-PAGE electrophoresis. (B) SDS-PAGE analysis of potential
E2-binding proteins isolated from cytosolic proteins of MCF-7 cells. After incubation of crude cytosolic protein mixtures with
compound 7 or biotin, the mixture was loaded onto the affinity column and eluted sequentially with an E2-free elution
buffer (elution control), 20 ␮M E2-containing elution buffer (elution 1), or after incubation at 4 ◦C for 6 h with 20 ␮M E2 and
then eluted with the E2-containing buffer (elution 2). The small arrows label the proteins that appear to be preferentially
eluted off with an E2-containing buffer either during the first elution or during the second elution. The two quantitatively
major bands with a fat arrowhead were further analyzed with NanoLC–MS/MS for structural compositions. The data are
shown in Table 1.
Table 1 – NanoLC/MS-MS identification of potential proteins in the cytosol of MCF-7 human breast cancer cells that can
directly bind E2 or bind to E2-bidning proteins
Protein band in Fig. 2B
Number of peptides
Proteins identified
14
11
6
Prolyl 4-hydroxylase ␤-subunit (PDI) [190384 | gb | AAC13652.1]a
Pyruvate kinase [35505 | emb | CAA39849.1]
a
Chaperonin containing TCP1, subunit 6A [76827911 | gb | AAI06943.1]
CCT7 [48145555 | emb | CAG33000.1]
5
33
12
10
6
Prolyl 4-hydroxylase ␤-subunit (PDI) [190384 | gb | AAC13652.1]
Pyruvate kinase [35505 | emb | CAA39849.1]
b
c
60 kDa chaperonin (Protein Cpn60) (groEL protein) [2493643 | sp | P95800]
Chaperonin containing TCP1, subunit 6A [76827911 | gb | AAI06943.1]
18
10
3
Heat shock 70 kDa protein 8 isoform 2 variant [62896815 | dbj | BAD96348.1]
Thyroid autoantigen 70 kDa [57165052 | gb | AAW34364.1]
Heat shock 70 kDa protein 9B precursor [24234688 | ref | NP 004125.3]
23
11
6
Heat shock 70 kDa protein 8, isoform 1 [16741727 | gb | AAH16660.1]
Prolyl 4-hydroxylase ␤-subunit (PDI) [190384 | gb | AAC13652.1]
Thyroid autoantigen 70 kDa (Ku antigen) [57165052 | gb | AAW34364.1]
d
a
The protein access number is provided in the bracket following the protein name.

steroids 7 3 ( 2 0 0 8 ) 1252–1261
1259
Fig. 3 – Chemical structures of ICI-172,780, raloxifene (RAL), and compounds 7, 7a and 7b. For compound 7, n = 6. Note that
for the two putative analogs of compound 7, namely, compounds 7a and 7b, the length of their side chains were reduced
(n = 4 and 2, respectively).
in band d. In addition, several heat shock protein-70 kDa iso-
forms or their truncated splicing variants were identified in
the lower bands (c and d). Some of these proteins are known
to be tightly associated with ER␣.
It is of note that earlier studies have reported that protein
disulfide isomerase (PDI) has considerable E2-binding affinity
[20]. Recently, we have also selectively expressed the human
PDI and pyruvate kinase in Escherichia coli. We confirmed that
the recombinant PDI has selective E2-binding activity, but
pyruvate kinase was found to have no specific E2-binding
activity (unpublished data). Currently, we are in the process of
characterizing other protein bands shown in Fig. 2B that were
isolated by the affinity ligand, such as the 60-kDa chaperonin
by using the same approach. Lastly, it is of note that among the
proteins isolated by the affinity ligand, the amount of ER␣ pro-
tein was very low or barely detectable. The main reason was
that the ER␣-positive MCF-7 cells were cultured in a medium
supplemented with excessive amount of E2 (at 10 nM), and
under this estrogen-rich condition, ER␣ was expected to be
mostly translocated into the nuclei and the amount of the
ER␣ present in the cytosolic compartment would be low or
undetectable.
Fig. 4 – Interactions of raloxifene (RAL), ICI-182,780 and compound 7 with the ligand binding domain (LBD) of human ER␣.
(A) The three-dimensional structures of ER␣ LBD in complex with RAL (magenta), ICI-182,780 (purple) and compound 7
(white). The secondary structure of ER␣ (in ribbon) is colored from blue (N-terminus) to red (C-terminus). This figure is
drawn using the PyMOL software. (B) Key amino acid residues in ER␣ LBD that interact with ICI-182,780 and compound 7.
Amino acids are colored for their elements (white for hydrogen, red for oxygen, and blue for nitrogen). Compound 7 is
colored in magenta and ICI-182,780 in purple. The green dashes show hydrogen bonds between ligands and ER␣. This
figure is drawn using the Discovery Studio software. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of the article.)

1260
steroids 7 3 ( 2 0 0 8 ) 1252–1261
ER␤ in complex with ICI-164,384 (an analog of ICI-184,780)
3.4.
Computational modeling
[21], which provides support for the proposed binding struc-
ture based on our computational models. Our modeling data
also suggest that the C-11␤-substituted ER antagonists could
be more ideal than the C-7␣-substituted antagonists for ER
binding.
According to earlier studies of ICI-182,780 and its analogs
[9,10], it was empirically predicted that the designed affinity
ligands (compounds 7, 7a and 7b) most likely would function
as ER antagonists owing to their long C-7␣ side chains. To
provide a more scientific prediction of the ER␣-binding ability
of the affinity ligand, we conducted computational molecular
modeling analysis of its binding interactions with human ER␣
ligand binding domain, and its interactions were compared
with ICI-182,780 (Fig. 3).
3.5.
Conclusions
In the present study, we have synthesized
a
new 7␣-
biotinylated E2 analog as affinity ligand for human ER␣ and
ER␤ from 6-keto-estradiol-3,17␤-di-tetrahydropyranyl ether
(compound 2). The key step involves the hydrogenolysis of
compound 4 using palladium hydroxide on carbon as cata-
lyst to remove the 6-hydroxyl group and the simultaneous
reduction of nitrile to amine to yield 7␣-(7-aminoheptyl)-
estradiol-3,17␤-di-tetrahydropyranyl ether (compound 5). The
overall yield for the synthesis is approximately 36%. In vitro
receptor-binding assay confirmed that the synthesized affinity
ligand has a rather high binding affinity for both human ER␣
and ER␤. Using this affinity ligand, protein disulfide isomerase
was identified as one of the high-affinity E2-binding pro-
teins present in the cytosolic fraction of human MCF-7 cells.
Computational molecular modeling analysis also showed that
compound 7 can bind to human ER␣ in a similar way as
ICI-182,780 and RAL, and their binding energy values are com-
parable.
Our docking models showed that compound 7 and ICI-
182,780 can fit rather tightly inside the binding pocket in a very
similar manner (Fig. 4A), with their steroidal rings and espe-
cially their C-3 and C-17␤ hydroxyl groups overlapping with
each other extensively (Fig. 4B). Their hydroxyl groups form
hydrogen bonds with the amino acid residues E353, R394 and
H524 of ER␣ nearly in the same positions as those of E2. In addi-
tion to the hydrogen bonds formed between the C-3 or C-17␤
hydroxyl groups of compound 7 and ER␣, its side chain forms
an additional hydrogen bond with T347 of ER␣. We also cal-
culated the relative binding energy values (ꢀEbinding) for these
binding models (data summarized in Table 2). In addition, we
also docked two putative analogs of compound 7 (compounds
7a and 7b) with a shorter side chain (structures shown in
Fig. 3). The binding energy values for these two analogs did
not vary significantly compared to the value for compound 7
(Table 2).
Our docking structure of ICI-184,780 in complex with ER␣
revealed that this pure antagonist adopts a unique binding
mode inside the binding pocket by flipping their core steroidal
structure 180◦ along the C-3 and C-17␤ hydroxyl axis. In this
way, the long C-7␣ side chain is positioned toward the C-11␤
channel and then exits the ligand binding pocket in a simi-
lar manner as RAL. Interestingly, in this altered binding mode,
the characteristic hydrogen bonds of C-3 and C-17␤ hydroxyl
groups with the receptor-binding pocket basically remain
unchanged. Notably, although the crystallographic structure
of human ER␣ in complex with ICI-184,780 is not available at
present, the docking conformation of ICI-184,780 as developed
in this study matched the reported crystal structure of human
r e f e r e n c e s
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Compound 7
Compound 7a (n = 4)
Compound 7b (n = 2)
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