Synthesis and characterization of radiolabeled 17β-estradiol conjugates

Article abstract of DOI:10.1002/jlcr.1864

The use of radioactive tracers for environmental fate and transport studies of emerging contaminants, especially for those that are labile, offers convenience in tracking study compounds and their metabolites, and in calculating mass balances. The aim of

Full text of DOI:10.1002/jlcr.1864

Research Article
Received 24 June 2010,
Revised 20 October 2010,
Accepted 24 November 2010
Published online 17 February 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1864
Synthesis and characterization of radiolabeled
17b-estradiol conjugates
Ã
Suman L. Shrestha,a Xuelian Bai,b David J. Smith,c Heldur Hakk,c
Francis X. M. Casey,b Gerald L. Larsen,c and G. Padmanabhana
The use of radioactive tracers for environmental fate and transport studies of emerging contaminants, especially for those
that are labile, offers convenience in tracking study compounds and their metabolites, and in calculating mass balances.
The aim of this study was to synthesize radiolabeled glucuronide and sulfate conjugates of 17b-estradiol (17b-E2). The
conjugates 17b-[4-14C]estradiol-3-glucuronide ([14C]17b-E2-3-G) and 17b-[4-14C]estradiol-17-sulfate ([14C]17b-E2-17-S) were
synthesized utilizing immobilized enzyme and chemical syntheses, respectively. Microsomal proteins from the liver of a
phenobarbital induced pig (Sus scrofa domestica) were harvested and used to glucuronidate [14C]17b-E2. Synthesis of
[
E2, sulfation at C-17 position, and subsequent deblocking to yield the desired synthetic product. Successful syntheses of
14C]17b-E2-17-S consisted of a three-step chemical process – introducing a blocking group at the C-3 position of [14C]17b-
[
14C]17b-E2-3-G and [14C]17b-E2-17-S were achieved as verified by liquid chromatography, radiochemical analyses,
quadrupole-time-of-flight (QTOF) mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy.
Radiochemical yields of 84 and 44% were achieved for 17b-E2-3-G and 17b-E2-17-S, respectively. Synthetic products
were purified using high-performance liquid chromatography and radiochemical purities of 98% or greater were obtained.
Keywords: 17b-estradiol; immobilized enzyme; conjugation; glucuronide; sulfate; steroid hormone
function in the detections of ‘free’ steroidal estrogens in the
environment. Although, estrogen conjugates are biologically
Introduction
inactive,24 they can potentially be cleaved by microbial enzymes
Medical research has used radiolabeled estrogenic compounds
to study breast and uterine cancers,1 estrogenic receptors,2 and
as imaging agents in breast tumors.3 More recently though,
the radiolabeled hormonal compounds (e.g. [14C]estradiol,
[14C]estrone, and [14C] testosterone4–8 and 6,7-3H-estradiol6)
have been used to study the fate and transport of steroids in the
environment. Exposure to exogenous reproductive hormones
has been associated with adverse effects in certain aquatic9–11
and terrestrial12–14 species. Human waste treatment and animal
feeding operations (AFOs) are sources of estradiol (E2), estrone
(E1), and estriol (E3) to the environment. Estradiol is the most
potent of these natural estrogens.15–18
to form the more potent parent compound.24
Conjugation reactions are a common vertebrate mechanism
in which hormones, drugs, toxicants, and non-nutritive organic
molecules are eliminated.25 During conjugation a charged, polar
moiety is attached to a hydrophobic compound (e.g. estrogen),
which increases its water solubility and excretion in urine or bile.
Estrogens are typically conjugated with glucuronic or sulfuric
acid at the C-3 and/or the C-17 positions24 (Schemes 1 and 2).
Glucuronidation of estrogen is catalyzed by uridine 50-diphospho-
glucuronosyltransferase (UGT) enzymes in the endoplasmic
reticulum and sulfation is catalyzed by cytosolic sulfotransferases
(SULTs).26
The environmental fate of estradiol conjugates has not been
extensively studied, possibly because radiolabeled conjugates
are not commercially available. The availability of radiolabeled
conjugated hormones would enable studies to be conducted
that would improve the understanding of the fate and transport
of these labile compounds in the environment. The objective of
Laboratory studies suggest estrogens should have little to no
mobility and should not persist in the environment because they
bind rapidly and strongly to soil and degrade within hours.4,8,19
Field studies, however, have indicated that estrogens are present
in the environment at frequencies and concentrations that imply
they are moderately mobile and persistent.20,21 Estrogen
conjugates, which have different water solubilities, sorption
coefficients, and degradation rates relative to their ‘free’ estrogen
counterparts, may offer insights into why steroidal estrogens are
frequently detected in the environment.20 Swine (sus scrofa
domesticus), poultry (gallus domesticus), and cattle (bos taurus)
excrete 96, 69, and 42%, respectively, of the estrogens as
conjugates.22 In fact, appreciable amounts of 17b-estradiol
(17b-E2) in conjugated forms have been measured in swine
manure slurry (liquid urine and feces) from AFO manure storage
lagoons.23 Conjugates form a major portion of total environ-
mental estrogen loading from AFOs and might play a significant
aDepartment of Civil Engineering, North Dakota State University (NDSU), Fargo,
ND 58105, USA
bDepartment of Soil Science, NDSU, Fargo, ND 58105, USA
cBiosciences Research Laboratory, USDA-ARS, Fargo, ND 58102, USA
*Correspondence to: Heldur Hakk, USDA ARS Biosciences Research Laboratory,
1605 Albrecht Blvd, Fargo, ND 58102-2765, USA.
E-mail: Heldur.Hakk@ARS.USDA.GOV
J. Label Compd. Radiopharm 2011, 54 267–271
Copyright r 2011 John Wiley & Sons, Ltd.

S. L. Shrestha et al.
Scheme 1. Glucuronidation of the hydroxyl group at C-3 of 17-b-estradiol by uridine 50-diphospho-glucuronosyltransferase (UGT).
Scheme 2. Chemical synthesis of [14C]17b-estradiol-17-sulfate conjugate from [14C]17b-estradiol.
this paper is to provide a method to synthesize carbon-14- Use Committee guidelines. The hog was intramuscularly (2 d)
labeled 17b-E2-3-G and 17b-E2-17-S.
then intraperitoneally dosed (2 d) with approximately 20 mg/kg
phenobarbital for 4 consecutive days, after which, the hog
was euthanized. The liver was homogenized and microsomes
were isolated via differential centrifugation. Proteins were
solubilized and immobilized onto Sepharose beads27 and were
stored in a 1:1 suspension with 0.1 M Tris buffer (pH 7.4) at 41C
until use.
Experimental
Materials
[14C]17b-E2 (55 mCi/mmol) (1) was purchased from American
Radiolabeled Chemicals (St Louis, MO). Unlabeled 1, UDP
glucuronic acid, magnesium chloride, ethanol, potassium phos-
phate monobasic, potassium phosphate dibasic, potassium hydro-
xide, hydrochloric acid, ethyl acetate, pyridine, sodium hydroxide,
chlorosulfonic acid, and acetic acid were obtained from Sigma-
Aldrich. Triethylamine (Fluka); benzoyl chloride (Bayer); trisodium
phosphate (Mallinkrodt, Paris, KY) were obtained from other
sources. Acetonitrile (ACN) was obtained from EMD Chemicals
(Gibbstown, NJ). Scintillation fluid EcoLiteTM was obtained from MP
Biomedicals (Santa Ana, CA). SPE cartridges Bond ElutTM C18 (6 g,
20 mL) and Sep-Paks Vac C18 were obtained from Varian (Harbor
City, CA) and Waters (Milford, MA), respectively.
Liquid scintillation counting
Radioactivity was quantitated with a Packard 1900 CA scintilla-
tion analyzer (Downers Grove, IL), and samples were dissolved in
EcoLiteTM scintillation cocktail.
High-performance liquid chromatography
Analytical high-performance liquid chromatography (HPLC) for 2
was performed using a Waters 600E System Controller and
pump (Milford, MA), equipped with a Jasco FP 920 fluorescence
detector (Jasco, Easton, MD) with the following conditions:
Phenomenex-C18, 4.6 ꢀ 250 mm, 5 mm; A: 10% ACN in 50 mM
Uridine 50-diphospho-glucuronosyltransferase (UGT)
A castrated, cross-bred hog weighing 24.4 kg was used as the ammonium acetate (pH 4.5), B: 90% ACN in 50 mM ammonium
source of the UGT enzymes, following USDA Animal Care and acetate (pH 4.5); gradient: 20–100% B, 29 min, 100% B, 3 min
www.jlcr.org
Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 267–271

S. L. Shrestha et al.
hold, 1.0 mL/min, excitation and emission wavelengths of 280 Synthesis of 17b-[4-14C]estradiol-17-sulfate (5)
and 312 nm, respectively. Prep-HPLC was performed on Jones
Chromatography-C18, 10 ꢀ 250 mm, 5 mm; A: 5% ACN in 50 mM
ammonium acetate (pH 4.5), B: 90% ACN in water; isocratic 85%
solvent A, 15% solvent B; 4.7 mL/min.
[14C]17b-estradiol-3-benzoate (3)
Radiolabeled 1 (259.5 mg, 0.95 mmol, 47.7 mCi) was mixed with
For 5, analytical HPLC was performed on a Gilson System
unlabeled 1 (11.43 mg, 42 mmol) in ethanol and the solvent was
evaporated.28 The residue was re-dissolved in 2 mL of acetoni-
45NC Gradient Analytical instrument (Gilson Medical Electronics,
Middleton, WI) equipped with a variable wavelength UV
detector with the following conditions: Radial-Pak-C18,
trile, and 13 mL triethylamine and 11 mL benzoyl chloride
(13.3 mg, 94.7 mmol) were added; the reaction mixture was
8 ꢀ 100 mm (Waters Associates, Milford, MA); A: 10:90 metha-
nol/water, B: 90:10 methanol/water; gradient: 20% B to 100% B,
28 min., 4 min hold; 1.0 mL/min 220 nm. HPLC for 3 was
conducted using following conditions: Radial-Pak-C18,
8 ꢀ 100 mm; A: 10:90 methanol/water, B: 90:10 methanol/water;
gradient: 20% B to 100% B, 30 min, 15 min hold; 1.0 mL/min, UV
220 nm.
stirred at room temperature for 2 h and subsequently dried
under a stream of nitrogen. To the white residue, 4 mL of 0.1 M
trisodium phosphate solution was added and the mixture was
sonicated for 30 min resulting in a light yellow suspension. The
suspension was extracted with ethyl acetate (3 mL ꢀ 3), and
the organic solvent was evaporated under a stream of nitrogen.
The residue (3) was dissolved in ethyl acetate (3 mL) and water
(1 mL) for further purification using HPLC. The yield of 3 was
59.3% and radiochemical purity was 98%. 13C NMR (MeOH-d4)d:
165.94, 150.13, 139.49, 139.38, 134.86, 130.99, 130.99, 129.83,
129.83, 127.47, 122.63, 119.79, 82.45, 51.33, 45.55, 44.34, 40.14,
37.99, 30.69, 30.56, 28.27, 27.48, 24.04, 11.68. 1H NMR (MeOH-d4)
d(aromatic A-ring protons): 7.44 (d), 6.94 (d), 6.89 (d); d(benzoate
protons): 8.14 (d), 7.66 (dd), 7.54 (dd). LC/MS-QTOF:
M-H = 375.21, m/z 361.21, 356.85, 334.82, 332.82.
Mass spectral analysis
Negative ion LC/MS was performed with a Waters Alliance 2695
HPLC (Symmetry-C18, 2.1 ꢀ 100 mm; A: 40% ACN in water,
B: 60% ACN in water; gradient: 40–100% B, 10 min, 5 min hold,
0.2 mL/min), and a Waters Micromass QTOF (API-US in an
ES-mode, MassLynx software, FWHM: 6500, source temperature
1201C, desolvation temperature 3501C, cone voltage 35 V,
capillary voltage 2500 V, collision energy 5 eV for sulfate and
20 eV for glucuronide conjugates.
[14C]17b-estradiol-3-benzoate-17-sulfate (4)
Sulfur trioxide-pyridine complex was synthesized in-house29 by
adding chlorosulfonic acid (138 mL, 2.07 mmol) with stirring to
dry pyridine (1.66 mL) at 01C. The solution was allowed to warm
to room temperature, followed by dilution with dry pyridine
(623 mL). 3 was dissolved into 1.1 mL of pyridine, and the
solution was heated to 501C, to which sulfur trioxide-pyridine
complex, also heated to 501C, was added. The mixture was
stirred for 30 min at 501C followed by solvent evaporation under
nitrogen, addition of water (4 mL), and adjusting to pH 8 (1 M
NaOH). The mixture was partially purified with a Sep-Paks Vac
C18 cartridge and 4 eluted with methanol. 13C NMR (MeOH-d4)d:
166.95, 150.11, 139.42, 139.27, 134.95, 130.85, 130.85, 129.89,
129.89, 127.52, 122.64, 119.84, 88.22, 50.78, 45.41, 44.23, 40.29,
NMR spectra
A Bruker AM-400 spectrometer (Billerica, MA) operating at either
1
400.13 MHz or 100.61 MHz was used to record the H and 13C
NMR spectra, respectively. 1H NMR spectra were run in fully
coupled mode with 128 scans and an acquisition time of
3.9713 s. 13C NMR spectra were run in CPD mode, with 64K
scans obtained with an acquisition time of 1.307 s. The chemical
shifts for the NMR spectra for 1 were 13C NMR (MeOH-d4)d:
155.84, 138.8, 132.32, 127.22, 116.05, 113.72, 82.49, 51.26,
45.34, 44.35, 40.5, 38.00, 30.72, 30.68, 28.83, 27.53, 24.03,
11.71; 1H NMR (MeOH-d4)d(aromatic A-ring protons): 7.06 (d),
6.53 (d), 6.47 (s).
1
37.98, 30.53, 29.22, 28.18, 27.47, 24.11, 12.24. H NMR (MeOH-
d4)d(aromatic A-ring protons): 7.30 (d), 6.90 (d), 6.86 (s);
d(benzoate protons): 8.13 (d), 7.66 (dd), 7.52 (dd). LC/MS-QTOF:
Synthesis of 17b-[4-14C]estradiol-3-glucuronide (2)
Five millilitres of 0.1 M phosphate buffer (pH 7.4) was added to M-H = 455.10, m/z 351.12.
20 mL of pre-rinsed microsomal proteins immobilized on
Sepharose beads. Forty microlitres of 2.63 M magnesium 17b-[4-14C]estradiol-17-sulfate (5)
chloride, 63 mg of UDP glucuronic acid (5 mM final concentra-
Hydrolysis30 of 4 was accomplished with 5% NaOH in methanol
tion), and 164.7 mg of [14C] labeled 1 (0.60 mmol; 33 mCi; dissolved
in 567 mL ethanol) and 6477 mg of unlabeled 1 (23.78 mmol,
dissolved in 540 mL ethanol) were added to the reaction flask.
The reaction flask was slowly stirred on a Roto-Vap (Bu¨chi,
Flawil, Switzerland) without vacuum at 371C for 24 h determined
a priori. The aqueous fraction was collected by filtration. 2 was
partially purified on a Bond ElutTM C18 SPE cartridge precondi-
tioned with ACN and nanopure water by eluting with 20:80
ACN-water. The final radiochemical purity was 99% obtained
after preparative HPLC. 13C NMR (MeOH-d4)d: 176.52, 156.99,
135.66, 127.20, 117.96, 115.41, 102.65, 82.47, 77.71, 76.68, 74.74,
73.59, 51.26, 45.41, 44.32, 40.34, 38.97, 37.97, 30.69, 30.04, 28.40,
27.51, 24.00, 11.67. 1H NMR (MeOH-d4)d(aromatic A-ring
protons): 7.18 (d), 6.87 (d), 6.81 (s). LC/MS-QTOF: M-H = 447.21,
m/z 271.17, 175.03, 113.02.
(5mL) added to 4, stirring for 1 h at room temperature, then
neutralization with 10% acetic acid, and evaporation under
nitrogen. After purification by HPLC, 21mCi (18.9 mmol; 7.1 mg;
98% pure) of 5 was obtained (overall yield: 44%). 13C NMR
(MeOH-d4)d: 155.89, 138.76, 132.54, 127.26, 116.04, 113.76, 88.19,
50.78, 45.3, 44.24, 40.34, 38.00, 30.71, 29.22, 28.48, 27.48, 24.10,
12.19. 1H NMR (MeOH-d4)d(aromatic A-ring protons): 7.06 (d), 6.53
(d), 6.47 (s). LC/MS-QTOF: M-H = 351.07, m/z 96.96.
Results and discussion
Synthesis of 17b-[4-14C]estradiol-3-glucuronide (2)
A one-step enzymatic synthesis of 2 is described that permitted
regioselective attachment of a glucuronide acid moiety to 1.
J. Label Compd. Radiopharm 2011, 54 267–271
Copyright r 2011 John Wiley & Sons, Ltd.
www.jlcr.org

S. L. Shrestha et al.
As the reaction occurred in a buffered solution, reaction induce hepatic bilirubin UGTs,37 which show a strong selectivity
progress (Scheme 1) could be readily followed by reversed- for phenolics.38
phase HPLC. The radiolabeled parent peak (1) at 27.57 min
Product yield of 2 was 84%, and was attributed to the
dropped steadily in intensity, while the increase in peak intensity induction of UGT’s by phenobarbital.39 The same microsomal
at 5.55 min occurred for the desired product (2) (Figure 1). proteins also were active at glucuronidating hydroxylated
The reaction was essentially complete by 24 h. C-18 SPE polybrominated diphenyl ether metabolites, triclosan, and
purification yielded a radiochemical purity of 95%; semiprepara- ractopamine hydrochloride.
tive HPLC improved radiochemical purity to 99%. LC/MS-QTOF
analysis of 2 showed ions at m/z 447.21, 271.17, 175.03, and Synthesis of 17b-[4-14C]estradiol-17-sulfate (5)
113.02, representing the molecular ion of 2 and ions of 1,
The synthesis of 5, presented in Scheme 2, was initiated by
blocking the more reactive C-3 hydroxyl in 1, which was
glucuronic acid, and a glucuronide fragment, respectively.
To determine the site of conjugation, 13C nuclear magnetic
accomplished with
intermediate 3.
a 59.3% product yield to form the
resonance (NMR) spectra of 1 and 2 were compared with each
other and with literature values of 131,32 and bisphenol A
glucuronide.33 Glucuronidation was indicated by the presence
of an additional six carbons in the 13C NMR spectrum of 2; and
the site of conjugation was indicated by the downfield shift
of C-3 from 132.32 to 135.66 ppm in the spectrum of 2. Chemical
The negative ion LC/MS analysis of 3 resulted in a molecular
ion at 375.21, a methyl loss fragment at 361.21, and a water loss
at 356.85. Losses of propanyl and propenyl groups were
consistent with fragments at m/z 332.82 and 334.82, respec-
tively. 1H NMR analysis of 1 and 3 indicated shifts in protons
ortho- and meta- to C-3 occurred in 3 relative to 1 (6.53–6.94;
7.06–7.44; 6.47–6.89 ppm). Benzoate protons were present at
8.14, 7.54, and 7.66 ppm of 3. The 13C NMR spectrum confirmed
that the blocking had occurred at C-3 because carbons ortho-
and meta- to C-3 of 3 were shifted downfield relative to their
chemical shift position in 1 (113.72–119.79; 132.32–134.86;
116.05–122.63 ppm).
1
shifts in the H NMR spectrum of 2 were also consistent with
glucuronidation at C-3. For example, protons ortho and meta to
C-3 were shifted downfield 6.53–6.87, 7.06–7.18, and
6.47–6.81 ppm for 1 and 2, respectively. In addition, as one of
the most diagnostic components of a sugar conjugated
spectrum, an anomeric singlet at 4.30 ppm also confirms the
formation of 2. Chemical shift assignments for the C-17
remained invariant for 1 and 2.
The formation of 4 was confirmed by a molecular ion at
455.09 in the LC/MS spectrum, and was accompanied by a
prominent fragment at m/z 351.12, which was consistent with a
benzoate fragment loss. Sulfation at C-17 was suggested by
significant downfield chemical shifts for the C-17 proton
(3.67–4.31 ppm) and carbon (82.45–88.22 ppm) in the 1H and
13C NMR spectra of 3 and 4, respectively. A radiochemical purity
of 95% was achieved and was considered satisfactory for the
next step.
Diglucuronide conjugation was theoretically possible due to
two hydroxyl groups in 1, one a phenolic in the A-ring, and the
other an aliphatic on the D-ring. However, only one site of
conjugation was expected because enzyme-catalyzed reactions
are usually regiospecific and stereospecific.34 UGT enzymes are
divided into two distinct subfamilies, UGT1 and UGT2.35,36
Phenobarbital treatment of hepatoma cell lines is known to
The purification of the final product (5) yielded 21 mCi
(18.9 mmol; 7.1 mg) of 98% radiochemical purity. The formation
of 5 was confirmed by a molecular ion at 351.07 and the sulfate
moiety ion at m/z 96.96 in the LC/MS spectrum of 5. 13C NMR
analyses of 5 indicated a significant upfield chemical shift of C-3
relative to 4 (132.54 from 134.95 ppm), as well as for carbons
ortho to C-3 (116.04 from 122.64; 113.76 from 119.84 ppm).
Upfield shifts of the aromatic protons between 5 and 4 were
observed (6.53 from 6.90; 7.06 from 7.30; 6.47 from 6.86 ppm),
but no chemical shift difference was observed for the C-17
proton. Collectively the physical data provide convincing
evidence that sulfation of 1 had occurred at C-17.
The overall yield of 5 was 44%, which possibly could have
increased if the reaction conditions had been optimized;
however, optimization was not an immediate objective. The
radiochemical yield was satisfactory for the immediate needs of
the research program.
Conclusion
[14C]Radiolabeled 17b-E2-3-G and 17b-E2-17-S were successfully
synthesized using enzymatic and chemical approaches, respec-
tively, which permitted their use for laboratory scale fate and
transport experiments in soil–water systems. Though our
objective was measuring and modeling the movement of
endocrine disrupting compounds in the environment, these
studies are but a small portion of the potential studies in which
Figure 1. Progress of enzymatic synthesis of 17b-estradiol-3-glucuronide with time
and the concurrent consumption of 17b-estradiol.
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Copyright r 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 267–271

S. L. Shrestha et al.
radiolabeled conjugates could be used. As glucuronidation and
sulfation are the major conjugation pathways in vertebrates
for not only steroid hormones, but other xenobiotics,40 we
hypothesize that radiolabeled glucuronides and sulfates of other
emerging contaminants can also be synthesized following the
approaches provided in this paper, or with appropriate
modifications of them.
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Acknowledgements
The authors thank Colleen Pfaff, Dee Anne Ellig, Barb Magelky,
Mike Giddings, Grant Harrington, Jason Holthusen (USDA-ARS),
and Nathan Derby (Soil Science Department, NDSU) for their
support in the laboratory work and Glenn Wittenberg (USDA-
ARS) for his IT support. This research was based upon work
supported by the National Science Foundation under Grant No.
0730492. Any opinions, findings, and conclusions or recommen-
dations expressed in this material are those of the authors and
do not necessarily reflect the views of the National Science
Foundation. The use of trade, firm, or corporation names in this
publication is for the information and convenience of the reader.
Such use does not constitute an official endorsement or
approval by the United States Department of Agriculture or
the Agricultural Research Service of any product or service to
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