Synthesis and application of a photoaffinity analog of dehydroepiandrosterone (DHEA)

Article abstract of DOI:10.1016/j.bmcl.2009.12.019

We have synthesized an analog of dehydroepiandrosterone (DHEA, 1) containing both a benzophenone (BP) and a biotin (Bt) group (DHEA-BP-Bt, 8). Compound 8 was prepared by functionalization on C-17 of 1. Biocytin was reacted with 4-benzoylbenzoic acid and t

Full text of DOI:10.1016/j.bmcl.2009.12.019

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1153–1155
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and application of a photoaffinity analog
of dehydroepiandrosterone (DHEA)
a
b,
Horacio F. Olivo a, , Nury Perez-Hernandez , Dongmin Liu , Mary Iruthayanathan b, Brianne O’Leary c,
*
Laurie L. Homan b, Joseph S. Dillon b,c,
*
a Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, USA
b Division of Endocrinology, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, USA
c Research Service, Department of Veterans Affairs Medical Center, Iowa City, Iowa, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized an analog of dehydroepiandrosterone (DHEA, 1) containing both a benzophenone
(BP) and a biotin (Bt) group (DHEA–BP–Bt, 8). Compound 8 was prepared by functionalization on C-17
of 1. Biocytin was reacted with 4-benzoylbenzoic acid and the product was condensed with 1 containing
a diamine–hexane linker. We detected specific protein bands of approximately 55, 80, and 150 kDa by
SDS–PAGE analysis of vascular endothelial cell plasma membranes which had been photoirradiated in
the presence of 8.
Received 7 October 2009
Revised 1 December 2009
Accepted 3 December 2009
Available online 6 December 2009
Keywords:
DHEA
Ó 2009 Elsevier Ltd. All rights reserved.
Photoaffinity
Biotin
Benzophenone
Chemical probe
There is extensive epidemiological evidence that plasma concen-
trations of the adrenal steroid dehydroepiandrosterone (5-andros-
ten-3b-ol-17-one, DHEA, 1) are related to vascular function.1–5
Hormonal steroids activate well characterized intracellular recep-
tors, but may also activate cellular signaling by binding to receptors
expressed at the plasma membrane.6 However, there is no known
receptorfor1 and themolecularmechanismsunderlyingitsputative
vascular benefits are unknown.7 We and others have shown that 1
binds to specific, high affinity, plasma membrane sites on vascular
endothelial and other cells, to activate cellular signaling in a G-pro-
tein-dependent manner.8–10 Interaction of 1 with this plasma mem-
brane receptor has been proposed as a potential mechanism of its
vascular effects.8 Characterization of the putative receptor involved
in this pathway is crucial to advance these studies.
characterize receptors expressed in the plasma membrane, includ-
ing both G-protein coupled receptors11,12 and receptor tyrosine ki-
nases.13,14 Furthermore, biotin modified steroids have been used to
study intracellular receptors for these hormones.15–18 The biotinyl-
ated ligands rapidly bind to both their cognate receptor and to
immobilized avidin, allowing efficient affinity capture of the recep-
tor–ligand complex by avidin chromatography. To date there are
few publications detailing the use of biotinylated steroids to iden-
tify plasma membrane bound steroid receptors.19
While the use of biotinylated ligands for receptor isolation has
been a great advance in this field, the solubilization of plasma
membrane proteins, required for receptor purification, can signifi-
cantly decrease the affinity of the receptor for the biotinylated li-
gand and impair the ability to isolate low abundance receptors
by avidin chromatography. Modification of ligands to include phot-
oirradiation-activated cross-linking groups can facilitate extensive
solubilization without loss of ligand receptor interaction after
cross-linking.20,21 We therefore took advantage of photoaffinity
cross-linking and avidin–biotin affinity techniques, to design and
synthesize a novel photoactivatable biotinylated analog of 1, in or-
der to isolate the high affinity plasma membrane DHEA binding
site.
Many investigators have successfully used the high affinity of
avidin for biotin in protocols to affinity purify hormonal receptors
by initially biotinylating the cognate ligand. Thus, biotinylated
derivatives of the natural ligands have been extensively used to
* Corresponding authors. Address: Room 319 PHAR, University of Iowa College of
Pharmacy, Iowa City, IA 52242, USA. Tel.: +1 319 353 8849 (H.F.O); 200 Hawkins
Drive, Room E421 GH, University of Iowa, Iowa City, IA 52242, USA. Tel.: +1 319 353
7712; fax: +1 319 353 7850 (J.S.D).
E-mail addresses: horacio-olivo@uiowa.edu (H.F. Olivo), joseph-dillon@uiowa.
edu (J.S. Dillon).
A convergent approach was designed to prepare the DHEA li-
gand carrying a benzophenone-containing photoaffinity label and
biotin,22 attached at the 17 position of 1 based on our previous
structure–activity data.8 A six-carbon linker was attached to 1 by
Present address: Department of Human Nutrition, 1880 Pratt Dr., 1120B Building
XV, Virginia Tech Research Corporation Center, Blacksburg, VA 24060, USA.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.019

1154
H. F. Olivo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1153–1155
O
R
NH
O
O
O
O
N
N
H
N
OH
a
b
HO
HO
HO
DHEA (1)
2
3 R= BOC
4 R= H
c
Figure 1. Synthesis of 17-(O-carboxymethyl)oxime derivative of 1. Reagents and conditions: (a) carboxymethylamine, pyridine, 60 °C; (b) N-Boc-diaminohexane, DEAC,
diisopropylethylamine, DMF; (c) HCl in dioxane.
a carboxymethyl oxime group. Treatment of 1 with carboxymeth-
ylamine in pyridine delivered the 17-(O-carboxymethyl)oxime
derivative 2, Figure 1. The carboxylic acid 2 was then coupled with
N-Boc-hexyldiamine to give amide 3. N-Boc de-protection was car-
ried out under acidic conditions to deliver amine hydrochloride 4.
4-Benzoylbenzoic acid 5 was activated with N-hydroxysuccinimide
to give the O-succinimide ester 6, Figure 2. Biocytin was then trea-
ted with sodium hydroxide and the activated benzophenone 6 was
added to give compound 7 (designated benzophenone–biotin or
BP–Bt).22 Finally, coupling of the DHEA amine 4 and the biotinyla-
ted benzophenone 7 gave the desired photoaffinity ligand 8, (des-
ignated DHEA–benzophenone–biotin or DHEA–BP–Bt), Figure 3.
Characterization of all intermediates by 1H and 13C NMR spectros-
copy confirmed the structure of the compounds.
exposed to UV light for 15 min, and separated by SDS-PAGE. Pro-
tein bands of approximately 55, 80, and 150 kDa were seen in sam-
ples incubated with 8. This labeling was DHEA specific since it was
competed by 1 and no such labeling was seen in the absence of UV
cross linking or when samples were incubated with 7 rather than 8,
Figure 4. These data demonstrate that compound 8 can efficiently
and specifically identify the DHEA binding protein(s) and suggest
that 8 will be suitable for isolating the DHEA receptor by avidin–
agarose chromatography.
In conclusion, the aim of this study was to develop a reagent to
facilitate isolation of the putative plasma membrane DHEA recep-
tor. We and others have shown that this receptor has high affinity
for DHEA (Kd = 48.7 pM), is expressed on plasma membranes, and
is linked to the activation of Gi proteins.8,10 We have synthesized
a novel analog of 1 containing both a Bt group and a photoactivat-
able BP group. This novel photoaffinity ligand (8) was prepared by
functionalization on C-17 of 1. Biocytin was reacted with 4-ben-
zoylbenzoic acid and the product was condensed with 1 containing
We next tested this novel analog of 1 in a photolabeling strategy
to detect the plasma membrane DHEA binding protein by PAGE
and chemiluminescence. Solubilized plasma membranes, prepared
from bovine aortic endothelial cells, were incubated with 8,
O
HN
NH
O
O
O
HN
O
O
S
a
b
HO
N O
O
O
HO
O
N
5
6
H
O
O
7
Figure 2. Synthesis of compound 7. Reagents and conditions: (a) N-hydroxysuccinimide, DEAC, CH2Cl2, 93%; (b) biocytin, NaOH, H2O–DMF, 82%.
O
HN
NH
O
HN
O
S
a
4
+ 7
O
H
N
O
N
N
H
N
H
O
O
HO
DHEA benzophenone-biotin 8
Figure 3. Synthesis of compound 8. Reagents and conditions: (a) HOBt, Hunig’s base, EDAC, DMF, 40%.

H. F. Olivo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1153–1155
1155
Government. The studies were also supported by Grants from the
American Heart Association and the NIH (AG018928). We thank
Dr. Rebeca Lopez-Marure for critical reading of the manuscript.
*
*
*
120
90
52
Supplementary data
–
–
+
8
+ +
+
+
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.12.019.
40nM 20µM
–
–
–
–
1
– – –
7
References and notes
+ + + +
UV
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Figure 4. Compound 8 binds specifically to plasma membrane proteins of BAEC.
Plasma membranes were photoirradiated in the presence of 8, 7, and 1, as indicated.
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affinity ligand were determined by nuclear magnetic resonance
and mass spectroscopy. We have demonstrated that 8 can be used
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This material is based upon work supported by the Department
of Veterans Affairs, Veterans Health Administration, Office of Re-
search and Development, Biomedical Laboratory Research and
Development Program. The contents do not represent the views
of the Department of Veterans Affairs or the United States