Photo-DHEA - A functional photoreactive dehydroepiandrosterone (DHEA) analog

Article abstract of DOI:10.1016/j.steroids.2011.01.009

The steroid hormone dehydroepiandrosterone (DHEA) has beneficial effects on vascular function, survival of neurons, and fatty acid metabolism. However, a specific receptor for DHEA has not been identified to date. Here, we describe the synthesis of a phot

Full text of DOI:10.1016/j.steroids.2011.01.009

Steroids 76 (2011) 502–507
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Photo-DHEA—A functional photoreactive dehydroepiandrosterone
(DHEA) analog
Gustav Waschatko1, Elzbieta Kojro, Michael Zahnow, Katja Gehrig-Burger∗
Institute of Pharmacy and Biochemistry, University of Mainz, Becherweg 30, 55099 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The steroid hormone dehydroepiandrosterone (DHEA) has beneficial effects on vascular function, survival
of neurons, and fatty acid metabolism. However, a specific receptor for DHEA has not been identified to
date. Here, we describe the synthesis of a photoreactive DHEA derivative (Photo-DHEA). In Photo-DHEA,
typical characteristics of DHEA are conserved: (i) a “planar” tetracyclic ring system with a ꢀ5 double
bond, (ii) a 3␤-hydroxyl group, and (iii) a keto group at C17. In cell-based assays, Photo-DHEA showed
the same properties as DHEA. We conclude that Photo-DHEA is suitable for radioiodination to yield a tool
for the identification of the elusive DHEA receptor.
Received 25 November 2010
Received in revised form 17 January 2011
Accepted 24 January 2011
Available online 1 February 2011
Keywords:
DHEA
Dehydroepiandrosterone
Photoaffinity labeling
Receptor
© 2011 Elsevier Inc. All rights reserved.
G protein-coupled receptor
1. Introduction
CMO (8), Scheme 1) in order to retain an unaltered DHEA core
structure with a hydroxyl group at C3 in ␤-configuration, a ꢀ5
The steroid dehydroepiandrosterone (Fig. 1(1)) is produced by
the adrenals and the central nervous system [1]. Besides its role as
a precursor of androgens and estrogens, DHEA has been reported
to be neuroprotective [2] and to have beneficial effects on dia-
betes, atherosclerosis, and obesity [3–5]. Declining DHEA and DHEA
sulfate (DHEAS) levels during aging is associated with pathophysi-
ological effects such as neuronal degeneration [6]. Although several
DHEA activated intracellular pathways have been characterized,
a specific DHEA binding receptor has not been identified to date.
Photoreactive steroid analogs are appropriate tools for analysis or
identification of steroid binding proteins. The formation of cova-
lent bonds between the photoreactive steroid and the protein upon
irradiation allows the application of denaturating preparative and
analytical methods such as SDS polyacrylamide gelelectrophoresis
and mass spectrometry.
double bound and a keto group at C17. DHEA-CMO was used
successfully for the synthesis of the fluorescent DHEA analog DHEA-
Bodipy (Fig. 1(3)), which we introduced previously as a tool to
study intracellular trafficking and localization of DHEA [7]. As
photoreactive group, we employed the aryl azide 4-azidosalicylic
acid that forms a nitrene upon irradiation which can react with
appropriate nucleophiles such as primary amines, and cysteinyl or
histidyl residues of proteins. In Photo-DHEA, DHEA-CMO is con-
nected with 4-azidosalicoyl-ethylenediamine by an amide bond.
The long spacer between DHEA and the aryl azide provides a flexi-
ble link between the steroid hormone and the photoreactive group.
Thereby, potentially interfering effects of the photoreactive aryl
azide on the functions of DHEA should be minimized. Moreover, a
certain flexibility between the photoreactive group and the steroid
is beneficial because this might enable the labeling of several amino
acids at the ligand binding site [8]. Aryl azides are the most com-
monly used photoactivatable reagents because they are easy to
synthesize, relatively stable, have high extinction coefficients, and
a relatively short half-life upon irradiation [9].
Here, we report the synthesis of the photoreactive DHEA-
analog Photo-DHEA (Fig. 1(2)). As starting compound we chose
5-androsten-3␤-ol-7,17-dione-7-O-carboxymethyloxime (DHEA-
The biological functionality of Photo-DHEA was confirmed by
two cell-based assays: displacement of DHEA-Bodipy from spe-
cific DHEA binding-sites on neuronal cells and induction of cAMP
synthesis by Photo-DHEA in liver cells. Our results indicate that
radioiodinated Photo-DHEA should be a useful tool for photoaffin-
ity labeling and identification of DHEA receptor(s).
∗
Corresponding author. Tel.: +49 06188 990519; fax: +49 06131 3925348.
E-mail addresses: katja.gehrig@gmx.de,
kburger@uni-mainz.de (K. Gehrig-Burger).
1
Present address: Max-Planck-Institute for Polymer Research, Ackermannweg
10, 55128 Mainz, Germany.
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.01.009

G. Waschatko et al. / Steroids 76 (2011) 502–507
2. Experimental
503
2.1. General methods
NMR spectra were recorded on Bruker AC-300 instrument in
CDCl3 at 300 MHz unless otherwise indicated. Chemical shift val-
ues are given in ı (ppm) and coupling constants are given in Hz.
ESI mass spectra were recorded on Micromass Q-TOF Ultima API
instrument. IR spectra were recorded on Bruker Tensor 27 instru-
ment. Analytical thin layer chromatography (TLC) was performed
on 0.5 mm thick silica gel 60 F254 plates. Compounds were revealed
by UV light (ꢁ = 254 nm) or by staining with ninhydrin. Commercial
reagents were used without further purification unless otherwise
noted.
Fig. 1. DHEA (1), Photo-DHEA (2), and DHEA-Bodipy (3).
2.2. Chemical synthesis
All synthesis steps were performed under dim light. The syn-
thetic pathways are depicted in Scheme 1.
2.2.1. 4-Azidosalicoyl-N-Boc-ethylenediamine (6)
Reaction and purification of 6 were performed according to
Bidasee et al. [10]. 500 mg (1.81 mmol) of 5 was dissolved in dioxane
(4 ml). 320 mg (2.0 mmol) of N-Boc-ethylenediamine (4) and 2.5 ml
triethylamine were added. The reaction mixture was stirred under
N2 at room temperature for 3.5 h. The mixture was evaporated, the
solid was dissolved in 10 ml of chloroform and chromatographed
on a silica gel column (33 cm, Ø 2.5 cm) with chloroform/methanol
(99/1) to afford 408.05 mg (1.3 mmol, 70%) of 6 as colorless crystals.
1H NMR (CDCl3, 300 MHz), ı [ppm]: 1.41 (s, 9H, tert-Butyl),
3.41–3.42. (t, 2H, CH2–N), 3.46–3.48. (t, 2H, CH2–N), 5.06 (t, 1H,
–OH), 6.46 (dd, 1H, ar), 6.58 (d, 1H, ar), 7.44 (d, 1H, ar), 7.84
(s, –NH–) IR (nujol) ꢂ˜ [cm−1] = 2120 (azide) (compare: IR (KBr) ꢂ˜
[cm−1] = 2130 (N3, 7) [11], IR (nujol) ꢂ˜ [cm−1] 2102 (azide, N-(4-
azido-salicyloyl-␤)-alanine ethyl ester)) [10].
TLC: detection UV and ninhydrin (yellow spot). Rf: 0.2
(CHCl3/MeOH (99:1, v/v)), 1.0 (ethylacetate).
2.2.2. 4-Azidosalicoyl-ethylenediamine hydrochloride (7)
400 mg (1.24 mmol) of 6 were dissolved in 8 ml of HCl/methanol
(1/4), stirred under N2 at room temperature for 20 h, and allowed
to stand at 4 ◦C [12]. The resulting crystals were filtered, washed
with diethylether and dried to afford 237.1 mg (0.9 mmol, 74%) of
7 as colorless crystals.
1H NMR (CD3OD, 300 MHz), ı [ppm]: 2.97–3.01 (t, 2H, CH2–N),
3.51–3.57 (q, 2H, CH2–NH), 6.63 (d, 1H, ar), 6.65–6.69 (dd, 1H, ar),
7.97 (d, 1H, ar), 9.04–9.07 (m, –NH–). Note: lack of the tert-butyl
singlet (1.41 ppm) indicates successful N-Boc deprotection.
TLC: detection UV and ninhydrin (red-purple spot). Rf: 0.3 (ethy-
lacetate).
2.2.3. Photo-DHEA (2) (4-azido-2-hydroxy-N-(2-(2-((E)-
((3S,8R,9S,10R,13S,14S)-3-hydroxy-10,13-dimethyl-17-oxo-
3,4,9,11-tetrahydro-1H-cyclopenta[a]phenanthren-
7(2H,8H,10H,12H,13H,14H,15H,16H,17H)-
ylidene)aminooxy)acetamido)ethyl)benzamide)
To a solution of 200 mg (0.53 mmol) of DHEA-CMO (8) in 1.5 ml
of DMF, 124 mg (1.08 mmol) of 9 and 104 mg (0.54 mmol) of EDC
were added similar to [13]. The reaction mixture was stirred under
N2 at room temperature for 22 h. 136 mg (0.53 mmol) of 7 in
0.5 ml of DMF and 80 ␮l of triethylamine were added. The reac-
tion mixture was stirred at room temperature for 24 h, filtrated and
diluted with H2O. The mixture was extracted with ethylacetate.
The combined organic layers were washed with saturated NaCl
solution, dried with Na2SO4, filtered and evaporated. The result-
ing yellow oil was chromatographed on a silica gel column (33 cm,
Scheme 1. Synthesis of Photo-DHEA.

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G. Waschatko et al. / Steroids 76 (2011) 502–507
Fig. 2. Photo-DHEA competes with DHEA-Bodipy (green) for DHEA binding-sites on the plasma membrane of neuronal PC12 cells. PC12 cells were incubated simultaneously
with 1 ␮M DHEA-Bodipy 30 ␮M steroid as indicated. Pictures of DHEA-Bodipy were taken 2 min after addition of the steroids. The scale bar represents 10 ␮m. Arrow,
plasma membrane; N, nucleus; C, cytoplasm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Ø 2.5 cm) with ethylacetate and rechromatographed with ethy-
lacetate/aceton (3/2) to afford 290.8 mg (0.50 mmol, 95%) of 2 as
colorless solid.
1640 containing 10% horse serum, 5% fetal calf serum (FCS),
2 mM glutamine, and penicillin/streptomycin. HepG2 cells were
grown in phenol red-free DMEM high glucose supplemented
with 10% FCS, 2 mM glutamine, and penicillin/streptomycin.
All cell lines were cultured at 37 ◦C, 5% CO2, and 100%
humidity.
1H NMR (CDCl3, 300 MHz), ı [ppm]: 0.86 (s, 3H, C18-methyl),
1.14 (s, 3H, C19-methyl), 1.45–2.61 (m, 18H, remaining C–H of the
steran), 3.52–3.68 (m, 6H, –NH–CH2–CH2–NH–), 4.54 (s, 2H, CH2-
Oximino), 6.49 (m, 1H, C6–H), 6.52 (d, 1H, ar, J = 2.1 Hz), 6.59 (d,
1H, ar, J = 2.4 Hz), 6.81–6.91 (m, 1H, –NH–), 7.44–7.47 (d, 1H, ar,
J = 8.4 Hz), 7.83–7.90 (m, 1H, –NH–).
2.3.2. Live-cell imaging of PC12 cells
For live-cell imaging, PC12 cells were seeded on 18 mm cover-
slips coated with poly-l-lysine plus collagen in 3.5 cm cell culture
dishes. 24 h before the experiment, cells were cultivated in phe-
nol red-free medium containing charcoal-stripped FCS, glutamine,
and penicillin/streptomycin. For live cell imaging, the coverslip was
mounted on a thermostated chamber (37 ◦C) on an Axiovert S100
microscope (Zeiss). For displacement experiments, PC12 cells were
incubated with 1 ␮M DHEA-Bodipy 30 ␮M pregnenolone, DHEA
or Photo-DHEA. Pictures were taken at an excitation wavelength of
ꢁ = 490 nm for DHEA-Bodipy. Fluorescence emission was measured
using a Q505LP filter. Cells were always checked for autofluores-
cence.
13C NMR (75.5 MHz) ı 220.72, 174.39, 172.70, 170.13, 170.10,
162.82, 162.74, 158.25, 158.23, 156.40, 145.66, 128.00, 112.53,
111.13, 111.12, 111.10, 111.08, 111.06, 110.02, 109.91, 107.80,
77.48, 77.05, 76.63, 72.57, 70.63, 53.77, 49.82, 47.85, 47.83, 45.96,
42.12, 40.97, 39.08, 38.52, 37.13, 36.61, 36.44, 35.30, 31.52, 31.00,
30.54, 29.18, 25.38, 24.87, 20.75, 20.07, 17.97, 13.88, 13.83, 13.81.
ESI MS m/z: 601.30 [M+Na]+, 1179.52 [2M+Na]+.
TLC: detection UV and ninhydrin (brown spot). Rf: 0.3 (ethylac-
etate), 0.6 (ethylacetate/acetone (3:2, v/v)).
HPLC elution profile: see Supporting Information.
To determine the time course of the photolysis of Photo-DHEA,
8.5 ␮g of Photo-DHEA in 1 ml of ethanol/methanol (1/32) were irra-
diated at ꢁ > 335 nm using a mercury arc lamp (200 W). When the
absorbance at ꢁmax = 265 nm was monitored, it decreased by 50%
only 20 s after UV irradiation. After 3 min the absorbance became
almost equivalent to the basal level (see Supporting Information).
2.3.3. GloSensorTM cAMP assay
For the GloSensorTM cAMP assay (Promega), HepG2 cells
were seeded on 96 well-plates coated with poly-l-lysine in
culture medium without antibiotics. At 90% confluency, cells
were transiently transfected with pGloSensorTM-20F cAMP plas-
mid. In brief, 100 ng (0.1 ␮l) plasmid was added to 50 ␮l
serum-free culture medium per well. 1 ␮l LipofectamineTM 2000
(Invitrogen) was added to 50 ␮l serum-free culture medium.
Diluted plasmid and diluted LipofectamineTM were combined
and incubated at room temperature for 20 min. Cells were
2.3. Biology
2.3.1. Cell culture
PC12 rat pheochromocytoma cells were cultured on poly-l-
lysine coated cell culture dishes in phenol red-free RPMI medium

G. Waschatko et al. / Steroids 76 (2011) 502–507
505
Fig. 3. (A) In HepG2 liver cells, DHEA binds to and activates a putative G protein-coupled receptor which in turn activates an adenylyl cyclase via the G␣s subunit of a
heterotrimeric G protein. The adenylyl cyclase synthesizes cAMP which can be monitored by a GloSensorTM cAMP assay (luminescence in (B) and (C)). The plant diterpene
forskolin is a direct activator of adenylyl cyclases. (B, left) Both, 30 ␮M DHEA and 30 ␮M Photo-DHEA induce the biosynthesis of the second messenger cAMP in human
HepG2 liver cells. (C, left) The effect of DHEA and Photo-DHEA is additive to the partial stimulation of the adenylyl cyclase by 1 ␮M of forskolin. Values correspond to the
mean of three samples. (B and C, right) For each treatment, areas below the curves of three samples were calculated and expressed as the mean SD. Statistical differences
between the mean values were determined by One Way-ANOVA/Bonferroni t-test. (***P < 0.001: vehicle versus 1 ␮M of forskolin, DHEA 1 ␮M of forskolin, and Photo-
DHEA 1 ␮M of forskolin, respectively; **P < 0.01: 1 ␮M of forskolin versus 1 ␮M of forskolin + DHEA, ***P < 0.001: 1 ␮M of forskolin versus 1 ␮M of forskolin + Photo-DHEA;
n.s.: not significant). Vehicle: DMSO (0.01%) + ethanol (0.1%). PM: plasma membrane.
incubated with the DNA/transfection complex (100 ␮l/well)
at 37 ◦C in
CO2 incubator. 5 h after transfection, medium
3. Results and discussion
a
was replaced by 100 ␮l phenol red-free medium containing
To determine whether Photo-DHEA is a functional photore-
active DHEA analog, two cell-based assays were employed. (i)
Displacement of a functional fluorescent DHEA-derivative (DHEA-
Bodipy) from rat pheochromocytoma (PC12) cells and (ii) induction
of cyclic adenosine monophosphate (cAMP) synthesis in human
liver (HepG2) cells.
charcoal-stripped FCS, glutamine, and penicillin/streptomycin.
Cells were incubated overnight at 37 ◦C in
a CO2 incuba-
tor. Then, cells were incubated in 90 ␮l/well of equilibration
medium (DMEM, 2 mM glutamine, 10 mM Hepes (pH 7.4), 10%
charcoal-stripped FCS, 2% GloSensorTM cAMP Reagent) at room
temperature for 2 h. 10 ␮l of the compound of interest in
medium (DMEM, 2 mM glutamine, 10 mM Hepes (pH 7.4), 10%
charcoal-stripped FCS) was added and luminescence was mea-
sured repeatedly from individual wells in a FLUOstar OPTIMA
microplate reader (BMG LABTECH GmbH) to record the kinetic
response.
3.1. Photo-DHEA and DHEA bind rapidly to the plasma
membrane of PC12 cells
DHEA has been reported to protect neuronal PC12 cells from
programmed cell death (apoptosis) which normally occurs upon

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G. Waschatko et al. / Steroids 76 (2011) 502–507
serum withdrawal [14]. DHEA exerts this neuroprotective effect
upon binding to specific plasma membrane binding sites [15].
Recently, we introduced DHEA-Bodipy, a functional fluorescent
DHEA-derivative suitable for live cell imaging of intracellular DHEA
transport and localization [7]. DHEA-Bodipy binds rapidly (within
2 min) and specifically to the plasma membrane of living PC12 cells
as could be shown by co-localization experiments with the plasma
membrane marker CellMaskTM Deep Red and displacement exper-
iments with different steroids [7]. Here, we demonstrate that both,
Photo-DHEA and DHEA (5-androsten-3␤-ol-17-one), compete with
DHEA-Bodipy for DHEA-binding sites at the plasma membrane of
PC12 cells (Fig. 2). This suggests a direct interaction between Photo-
DHEA and the putative DHEA receptor. In contrast, pregnenolone
(5-pregnen-3␤-ol-20-one), which differs from DHEA at position
C17, does not compete with DHEA-Bodipy for binding to PC12 cells
(Fig. 2).
PC12 cells have been demonstrated to possess saturable high-
affinity DHEA binding sites [15]. The C17-keto group of DHEA
interacts specifically with the putative DHEA receptor whereas
the C3-␤-hydroxy function is not necessary for plasma mem-
brane binding of DHEA in these cells [7,15]. Recently, Olivo et al.
developed a derivative of DHEA with both a photoreactive ben-
zophenone group and a biotin group at position C17 [16]. Although
this DHEA derivative lacks the 17-keto function of DHEA it specifi-
cally labeled plasma membranes of bovine aortic endothelial cells
(BAEC) [17]. In contrast to PC12 cells, DHEA binding sites of BAEC
tolerate large modifications at position C17 of DHEA whereas the
hydroxyl function at position C3 of DHEA is mandatory [18]. This
indicates that there might be species and/or cell type specific
receptor subtypes for DHEA or, in general, multiple DHEA recep-
tors from different protein families. Sex hormones differ mainly at
positions C3 and C17, underlining the importance to leave these
positions unchanged when developing fluorescent or photoreac-
tive steroid analogs. We chose DHEA-7-CMO for derivatization
with the photoreactive aryl azido group in order to retain an unal-
tered DHEA core structure with a long spacer as a flexible link
between the steroid hormone and the photoreactive group. There-
fore, Photo-DHEA introduced in this work should be able to label
DHEA receptors with very stringent requirements for the DHEA
structure.
adenylyl cyclase in liver cells probably via a G protein-coupled
pathway.
4. Conclusion
In spite of the numerous results concerning the mechanisms
of DHEA action, it is still a matter of debate whether there
is a single, specific receptor for DHEA or whether the biolog-
ical actions of DHEA involve multiple receptors [3,22]. Using
a neuronal and a hepatic cell line, we have shown here that
Photo-DHEA is a cell biologically active DHEA derivative. Radioio-
dination of Photo-DHEA at position C5 of the photoreactive group
4-azidosalicylic acid [23] could be achieved by incubation of Photo-
DHEA with Na[125I] in the presence of the iodination reagent
IODO-GEN (1,3,4,6-tetrachloro-3␣-6␣-diphenylglycoluril) which
can be coated onto a reaction vessel as a solid-phase oxidant
[24]. Radioactive iodination of Photo-DHEA should yield a valu-
able photoreactive DHEA analog suitable for photoaffinity labeling
studies for detection and identification of the elusive DHEA recep-
tor(s). Receptor identification is crucial for our understanding of
the plethora of physiological and pathophysiological mechanisms
DHEA is involved in.
Acknowledgements
We thank Sebastian Moschel (group of Prof. Detert) and Florian
Karch (group of Prof. Hoffmann-Röder) for 1H NMR and Paul Böhm
(group of Prof. Frey) for 13C NMR. We thank Professor Gerald Gimpl
for helpful comments and discussions. This work was supported by
the BMBF (JP2003G, Germany).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2011.01.009.
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