Syntheses and ligand-binding studies of 1α- and 17α-aminoalkyl dihydrotestosterone derivatives to human sex hormone-binding globulin

Article abstract of DOI:10.1016/S0039-128X(03)00092-8

We report on the syntheses of 1α- and 17α-aminoalkyl dihydrotestosterone (DHT) derivatives and the particularly high binding affinity of the 1α-aminohexyl ligand for human sex hormone-binding globulin (SHBG). The two 17α-aminopropyl-17β-hydroxy-5α -androstan-3-one (1) and 17α-aminocaproylamidoethyl-17β -hydroxy-5α-androstan-3-one (2) derivatives were synthesized via a 17β-spirooxirane intermediate in high yields. The 1α -aminohexyl-17β-hydroxy-5α-androstan-3-one compound (3) was obtained in a seven step synthesis using a copper-catalyzed conjugate addition of a ω-silyloxyhexyl Grignard reagent to 17β-benzoyloxy-5α -androst-1-en-3-one. All structures were elucidated based on ¹H NMR spectroscopy and mass spectral analyses. The three aminosteroid derivatives were tested as ligands for SHBG by competition experiments with tritiated testosterone as tracer under equilibrium conditions. The association constants of the two 17α-DHT derivatives were approximately 1×10 ⁷M^-1, whereas the 1α-DHT derivative showed a remarkably high binding affinity to SHBG with an association constant of 1.40×10⁹M^-1. These aminoalkyl derivatives, substituted either at the D-ring or the A-ring of the steroid skeleton, can be easily coupled onto a carboxymethylated solid state surface of a biosensor. Such a device lends itself to kinetic and thermodynamic studies aimed to provide a better understanding of the biospecific interaction of steroids with SHBG.

Full text of DOI:10.1016/S0039-128X(03)00092-8

Steroids 68 (2003) 629–639
Syntheses and ligand-binding studies of 1␣- and 17␣-aminoalkyl
dihydrotestosterone derivatives to human sex hormone-binding globulin
Hagen Hauptmanna, Jochen Metzgerb, Andreas Schnitzbauerb,
Claude Y. Cuilleronc, Elisabeth Mappusc, Peter B. Luppab,∗
Institute of Organic Chemistry, Universität Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany
a
b
Institute for Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar der Technischen Universität München,
Ismaninger Str. 22, D-81675 Munich, Germany
INSERM ERM 0322, Hoˆpital Debrousse, 29 rue Soeur Bouvier, F-69322 Lyon Cedex 05, France
c
Received 24 February 2003; received in revised form 23 April 2003; accepted 28 April 2003
Abstract
We report on the syntheses of 1␣- and 17␣-aminoalkyl dihydrotestosterone (DHT) derivatives and the particularly high binding affinity of
the 1␣-aminohexyl ligand for human sex hormone-binding globulin (SHBG). The two 17␣-aminopropyl-17␤-hydroxy-5␣-androstan-3-one
(1) and 17␣-aminocaproylamidoethyl-17␤-hydroxy-5␣-androstan-3-one (2) derivatives were synthesized via a 17␤-spirooxirane interme-
diate in high yields. The 1␣-aminohexyl-17␤-hydroxy-5␣-androstan-3-one compound (3) was obtained in a seven step synthesis using
a copper-catalyzed conjugate addition of a ␻-silyloxyhexyl Grignard reagent to 17␤-benzoyloxy-5␣-androst-1-en-3-one. All structures
1
were elucidated based on H NMR spectroscopy and mass spectral analyses. The three aminosteroid derivatives were tested as ligands
for SHBG by competition experiments with tritiated testosterone as tracer under equilibrium conditions. The association constants of the
two 17␣-DHT derivatives were approximately 1 × 107 M−1, whereas the 1␣-DHT derivative showed a remarkably high binding affinity to
SHBG with an association constant of 1.40 × 109 M−1
.
These aminoalkyl derivatives, substituted either at the D-ring or the A-ring of the steroid skeleton, can be easily coupled onto a
carboxymethylated solid state surface of a biosensor. Such a device lends itself to kinetic and thermodynamic studies aimed to provide a
better understanding of the biospecific interaction of steroids with SHBG.
© 2003 Elsevier Inc. All rights reserved.
Keywords: 1␣-Aminohexyl-17␤-hydroxy-5␣-androstan-3-one; 17␣-Aminopropyl-17␤-hydroxy-5␣-androstan-3-one; 17␣-Aminocaproylamidoethyl-
17␤-hydroxy-5␣-androstan-3-one; Human sex hormone-binding globulin; Steroid; 1H NMR
1. Introduction
molecule [8,9]. The two monomers of SHBG interact very
strongly with each other even in the absence of ligand [10].
The major physiological role of SHBG is the regulation
of bioavailability of T and E2 by controlling their respec-
tive metabolic clearance rates [11–14]. SHBG has also been
reported to exert cellular influences on the uptake of sex
steroids [15–17] in target cells and on signal transduction
[18,19]. Plasma SHBG levels vary considerably between in-
dividuals and are influenced by hormonal, metabolic, and
nutritional factors [1]. It is of particularly high clinical in-
terest that low serum levels of SHBG are found in women
suffering from disorders characterized by androgen excess
[1] and are also considered as a prognostic indicator for the
onset of type II diabetes mellitus, hyperthyroidism, and car-
diovascular disease [20,21]. Thus, the knowledge of factors
regulating SHBG levels, the understanding of the interaction
between steroid ligands and SHBG, and the measurement
Human sex hormone-binding globulin (SHBG),
a
93.4-kDa homodimeric glycoprotein produced by hepato-
cytes, is the major sex steroid-binding protein in plasma [1]
that binds testosterone (T), 5␣-dihydrotestosterone (DHT),
17␤-estradiol (E2), and related steroids with different affini-
ties [2–5]. The glycoprotein is a product of a single gene
located on the short arm of chromosome 17 [6,7]. Gly-
cosylation of the monomeric protein forms one O-linked
oligosaccharide at Thr-7 and two N-linked oligosaccha-
rides at Asn-351 and -367 in the C-terminal region of the
∗
Corresponding author. Tel.: +49-89-4140-4759;
fax: +49-89-4140-4875.
E-mail address: luppa@klinchem.med.tum.de (P.B. Luppa).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0039-128X(03)00092-8

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H. Hauptmann et al. / Steroids 68 (2003) 629–639
of protein-bound and free steroid fractions [22] are of great
endocrinological significance.
The polyclonal rabbit anti-SHBG antibody was from
Dako A/S, Glostrup, Denmark, and the secondary horseradish
peroxidase (HRP)-conjugated donkey anti-rabbit IgG poly-
clonal antibody (pab) was from Amersham Pharmacia. The
gel filtration LMW and HMW calibration kits and the Su-
perdex 200 HR10/30 column for size-exclusion chromatog-
raphy were from Amersham Pharmacia. PVDF membranes
(Immobilon-P, pore size 0.45 ␮m) and Microcon® centrifu-
gal filters were from Millipore, Bedford, MA, USA.
Optimal binding of steroids to SHBG is known to re-
quire a planar C19 steroid with a 17␤-hydroxyl group
and an electronegative functional group at C-3 [23]. The
recent crystal structure of the N-terminal recombinant hu-
man SHBG ligand-binding domain (LBD) complexed with
steroidal ligands [24] has revealed that each monomer con-
tains an LBD for a steroid molecule within the N-terminal
laminin G-like domain (G-domain). The C-3 oxygen atom
of bound DHT is anchored in the interior of the protein,
while rings A and B are completely buried. Substitutions
are well tolerated at the positions C-4, C-12␣, and C-17␣,
as expected from previous binding experiments [25–27].
Other modifications at several other ring positions have
been shown to significantly reduce the relative binding
affinities (RBA) [3]. However, the 1␣ position seems to be
qualified as a potential candidate for introducing a substitu-
tion, which may be deduced from the fact that mesterolone
(17␤-hydroxy-1␣-methyl-5␣-androstan-3-one) has a high
affinity for SHBG [26]. Moreover, recent site-directed muta-
genesis experiments have shown that dimerization-deficient
SHBG monomeric variants still contain a LBD with an
affinity and specificity indistinguishable from wild-type
SHBG [24,28].
2.2. Apparatus
Melting points (uncorrected) were determined on a Re-
ichert Thermovar and on a Büchi 510. 1H nuclear mag-
netic resonance (NMR) spectra were recorded in CDCl3 and
C5D5N on a Bruker Avance 400 (400 MHz), using Me4Si
as the reference signal. Data are reported in the following
form: chemical shift (multiplicity, coupling constant in Hz
if available, number of protons, position of proton). Infrared
(IR) spectra (in KBr or neat) were obtained on a Bio-Rad
Excalibur FTS 3000 MX spectrophotometer, only character-
istic IR bands (cm−1) are reported. Optical rotation values
were determined on a Perkin Elmer 241 polarimeter, c is
given in g per 100 cm−3. Mass spectrometry (MS) analyses
were performed on Finnigan MAT 95, MAT SSQ 700, as
well as on Varian MAT 112S and 311A mass spectrometers.
For size-exclusion chromatography, the ÄKTAFPLC system
from Amersham Pharmacia was used. Gel electrophoresis
was performed using the Mini-Protean 3 Cell, whereas pro-
tein transfer was realized using the Mini Trans-Blot Elec-
trophoretic Transfer Cell; both cells were purchased from
BioRad, Hercules, CA, USA.
To provide further physicochemical data on the thermo-
dynamics and kinetics of the interaction of androgen lig-
ands with SHBG by the way of highly sensitive real time
biosensor methodologies, our aim was first to synthesize
and determine the binding characteristics of DHT derivatives
modified at the 1␣ and 17␣ position by alkylamine spacers
sufficiently long enough to allow an unhampered access of
SHBG to the steroidal ligand after covalent coupling onto a
carboxymethylated solid state surface of a biosensor device.
2.3. Syntheses of the 1α- and 17α-DHT derivatives
2. Experimental
For general organic chemical procedures, see Mappus
et al. [29] and Hauptmann et al. [30].
2.1. Chemicals and reagents
2.3.1. 17α-Aminopropyl-17β-hydroxy-5α-androstan-
3-one (1)
Con A-Sepharose was purchased from Amersham Phar-
macia Biotech, Freiburg, Germany. DHT (17␤-hydroxy-
5␣-androstan-3-one), 5␣-androstan-3,17-dione, T (17␤-
hydroxy-4-androsten-3-one), and 3-amino-9-ethyl-carbazole
(AEC) were from Sigma, Deisenhofen, Germany. 9-Fluore-
nylmethoxycarbonyl (Fmoc)-ε-aminocaproic acid was pur-
chased from Bachem Biochemica, Heidelberg, Germany.
[1,2,6,7-3H]-T (3H-T, specific activity: 2.92 TBq/mmol)
was from Amersham Pharmacia Biotech. For column chro-
matography, silica gel (grain 0.063–0.200) from Merck and
neutral Al2O3 (aluminia N Super I, W 200) or basic Al2O3
(aluminia B Super I, W 200) from Woelm Pharma (Bad
Honnef, Germany) were used. Thin layer chromatography
(TLC) was performed using silica gel 60 F254 (0.2 mm) or
neutral Al2O3 60 F254 type E (0.2 mm), both materials from
Merck. All other laboratory chemicals were from Sigma.
A solution of 3,3-(dimethoxy)-17␣-aminopropyl-DHT
precursor (30 mg, 0.076 mmol) in 10 ml of CH2Cl2 contain-
ing 0.1 ml of aqueous 1 M HCl was stirred for 2 h at 22 ◦C.
Then, the reaction mixture was cooled to 4 ◦C, washed
with 1 ml of a saturated aqueous solution of NaHCO3 and
three times with 2 ml of water, and finally evaporated under
reduced pressure. Purification of the crude residue by TLC
on silica gel (ethyl acetate, then CHCl3/MeOH/NH4OH,
25:10:1) led to the final product 1 as a pale yellow oil
(16 mg, 60% yield). νmax (CHCl3): 1706 (3-CO); 1H NMR
(C5D5N). 0.93 (s, 3H, 19-CH3), 1.11 (s, 3H, 18-CH3), 3.45
(m, 2H, –CH2NH2); MS (ESI+, m/z, relative intensity %):
348 (MH+ 100%), 330 (M+ − H2O, 59%); high-resolution
MS (CI): found 348.2903, C22H37NO2 (MH) requires
348.2903; []D = +0.1◦ (CHCl3, c = 0.1).

H. Hauptmann et al. / Steroids 68 (2003) 629–639
631
2.3.2. 17α-Aminocaproylamidoethyl-17β-hydroxy-
5α-androstan-3-one (2)
3.19 (s, 3H, CH3–O), 3.21–3.32 (m, 1H, –CHHA–NHCO–),
3.62–3.72 (m, 1H, –CHHB–NHCO–), 6.67 (m, 1H,
–NH–CO–); MS (ESI, m/z, %): 493 (M + H+, 100%),
461 (M+ − 32, 58%); high-resolution MS (DCI, CH4):
found 493.4014, C29H53N2O4 (MH) requires 493.4005;
[␣]D = −0.2◦ (CH2Cl2, c = 0.85).
The 3,3-(dimethoxy)-17␣-aminoethyl-DHT derivative
1b was synthesized from 5␣-androstan-3,17-dione, accord-
ing to [29] with the following three key steps: epoxida-
tion, leading to 17␤-(2ꢀ-oxiran)-3,3-(dimethoxy)-5␣-and-
rostane, nucleophilic opening of the epoxide with cyanide
anion leading to 17␤-hydroxy-17␣-(cyanomethyl)-3,3-
(dimethoxy)-5␣-androstan-3-one, reduction with LiAlH4,
leading to the 17-ethylamine intermediate. 1b was puri-
fied by liquid–solid chromatography (LSC) on silica gel
using a CH2Cl2/EtOH/NH4OH mixture (10:10:1, v/v/v)
as eluent, giving the free amine as a pale resin in 69%
yield.
The 17-amino derivative 1b (100 mg, 0.26 mmol) was
suspended in 2 ml of dry CH2Cl2. Subsequently, the
Fmoc-protected 6-aminocaproic acid (101 mg, 0.29 mmol),
p-dimethylaminopyridine (DMAP) (6 mg, 0.05 mmol), and
N,Nꢀ-dicyclohexylcarbodiimide (DCC) (60 mg, 0.29 mmol)
were added to the reaction mixture and stirred at room
temperature for 24 h. The solvent was removed under
reduced pressure. The product was dissolved in a mix-
ture of CH2Cl2 and MeOH (9:1, v/v) and purified by
LSC on silica gel (CH2Cl2/MeOH, 9:1, v/v). The Fmoc
protected amide derivative 2a was obtained as a pale
resin (162 mg, 87% yield). νmax (KBr): 3411, 3331 (OH,
NH), 1704 (urethane CO), 1648 (amide I), 1539 (amide
II), 1110–1050 (H3C–O–C–); 1H NMR (CDCl3) δ: 0.84
(s, 3H, 18-CH3), 0.90 (s, 3H, 19-CH3), 2.14 (2H, t:
J = 7.3 Hz), 3.13 (s, 3H, CH3–O) and 3.19 (s, 3H,
CH3–O), 3.16–3.32 (m, 6H, –CH2–NH–CO–CH2–O;
CH3–O–C– and –CHHA–NHCO–), 3.59–3.71 (m, 1H,
Hydrolysis of the acetal group of compound 2b (50 mg,
0.1 mmol) was performed with 200 ␮l of 5N HCl in a
mixture of 2 ml tetrahydrofuran (THF) and 1 ml MeOH.
The mixture was stirred at room temperature for 3 h. After
evaporation of the solvent under reduced pressure, the de-
protected amine product 2 was recovered quantitatively as
a hydrochloride by lyophilization. The synthesis of 2 was
performed in an overall high yield of 68%. νmax (KBr):
3335 and 3191 (OH, NH), three broad bands at 3030, 2500,
2100 (NH3-), 1704 (3-CO), 1649 (amide I), 1551 (amide
1
II); H NMR (C5D5N) δ: 0.92 (s, 3H, 19-CH3), 1.10 (s,
3H, 18-CH3), 2.45 (2H, t: J = 7.3 Hz, CH3–CO–), 3.23
(2H, t: J = 7.3 Hz, –CH2–NH3+), 3.87–4.05 (m, 2H,
–CH2–NH–CO), 11.1 (s broad, 3H, –NH3+); MS (FAB,
m/z, %): 447 (MH+ − Cl, 100%), 429 (MH+ − Cl − H2O,
42%); elemental analysis as monohydrate: C27H49ClN2O4
(501.14), calcd C, 64.71, H, 9.86, N 5.59; found C, 63.95,
H, 9.75, N 5.29; [␣]D = +0.7◦ (CHCl3, c = 2.60).
2.3.3. 1α-Aminohexyl-17β-hydroxy-5α-androstan-
3-one (3)
2.3.3.1. 1α-TBSO-hexyl-3-oxo-5α-androstane-17β-yl ben-
zoate (3b). Synthesis started from the known compound
5␣-androst-1-en-3-one-17␤-yl benzoate (3a), which was
prepared according to the protocol from Pelc [31]. The
Grignard reagent dimethyl-t-butyl-silyloxy-hexyl-MgBr
(TBSO-n-hexyl MgBr) was synthesized as previously
described [32,33] by adding dropwise a solution of
TBSO-n-hexyl bromide (10 Eq.) in anhydrous THF to Mg
(11 Eq.) in THF under a N2 atmosphere at 50 ◦C. The re-
sulting clear solution was further diluted with solvent, and
after 1 h, cooled to −40 to −30 ◦C in an isopropanol/dry
ice-bath. A slurry of freshly prepared CuI (2 Eq.) in THF
was added. After 30 min, the enone 3a (200 mg, 0.51 mmol,
1 Eq.) in anhydrous THF was added dropwise over a period
of 50 min to the solution, which was maintained at −45
to −35 ◦C, giving a light lemon yellow suspension. After
stirring for a further 60 min, the color disappeared, and the
reaction mixture was quenched with glacial acetic acid at
−30 ◦C and then basified with a saturated NaHCO3 solu-
tion. The solvent was removed, and the residue extracted
with methyl-tert-butyl ether (MTBE) and washed with an
aqueous saturated NH4Cl solution. The crude oily extract
was first chromatographed on silica gel using CH2Cl2 as
eluent to separate the aliphatic byproducts and then sub-
jected to a LSC using petroleum ether (PE)/MTBE (1:1,
v/v) as eluent. The 1-silyloxyhexyl derivative 3b was iso-
lated as a colorless oil (199 mg, 64% yield). νmax (film):
1717 (3-CO), 1100, 836, 767 (all three Si–R); 1H NMR
–CHHB–NHCO–), 4.21 (1H, t: J
=
7.0 Hz, 9H of
the 9-alkylfluorene group), 4.39 (2H, d: J = 7.0 Hz,
O–CH2-fluorene), 4.89–4.94 (m, 1H, –NH–COCH2–), 6.48
(s broad, 1H, –CH2–NH–COCH2–), 7.40–7.77 (m, 8 H,
aromatic protons); MS (ESI, m/z, relative intensity %): 737
(M + Na+, 100%), 695 (M+ − 18, 56%); elemental analy-
sis: C44H62N2O6 (714.98), calcd C, 73.91, H, 8.74, N 3.92;
found C, 73.73, H, 8.86, N 3.70; [␣]D = −1.4◦ (CH2Cl2,
c = 1.22).
For cleavage of the Fmoc group, the amide derivative 2a
(120 mg, 0.17 mmol) was dissolved in a mixture of 1 ml
CH2Cl2 and 1 ml ethanolamine. After 1 h at room tem-
perature, the mixture was diluted with 5 ml CH2Cl2, and
ethanolamine was eliminated by three successive extractions
with 5 ml water. The combined organic layers were dried
with Na2SO4 and evaporated under reduced pressure. The
product was analyzed by TLC and then chromatographed
on silica gel (CH2Cl2/MeOH/NH4OH, 10:10:1, v/v/v).
After freeze drying, the amine intermediate 2b was ob-
tained (66 mg, 79% yield). νmax (KBr): 3350 (OH, NH),
1644 (amide I), 1556 (amide II), 1110–1050 (H3C–O–C–);
1H NMR (CDCl3) δ: 0.80 (s, 3H, 19-CH3), 0.84 (s, 3H,
18-CH3), 2.17 (2H, t: J = 7.3 Hz, –CH2–CO–NH–), 2.70
(2H, t: J = 6.6 Hz, –CH2–NH2), 3.14 (s, 3H, CH3–O),

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H. Hauptmann et al. / Steroids 68 (2003) 629–639
(CDCl3) δ: 0.04 (s, 6H, Me2–Si), 0.88 (s, 9H, tert-butyl-Si),
0.95 (s, 3H, 18-CH3), 1.14 (s, 3H, 19-CH3), 2.04–2.58
(m, 5H, 2␣, 2␤, 4␣, 4␤, 16␣ H), 3.58 (2H, t: J = 6.7 Hz,
–CH2–Si), 4.87 (1H, dd: J = 7.9 Hz and 9.1 Hz, 17␣-H),
7.4–7.6 (m, 5H, aromatic H); MS (DCI, NH3, m/z, %): 609
(MH+, 100%), 626 (M + NH4+, 62%); high-resolution MS
(DCI, CH4): found 609.4334, C38H61O4Si (MH) requires
609.4339; [␣]D = +51.5◦ (CH2Cl2, c = 5.55).
(HMPT) at room temperature. Eighty-eight milligrams
(1.35 mmol, 5 Eq.) of solid NaN3 were added, and the sus-
pension was stirred for 4 h. The light yellow reaction mix-
ture was diluted with 10 ml H2O and extracted three times
with MTBE. The organic layer was dried with Na2SO4 and
further purified by LSC using a PE/MTBE mixture (2:1,
v/v) as the eluent. The azidohexyl derivative 3e was isolated
as a pale yellow oil (124 mg, 89% yield). νmax (film): 2096
1
(N3), 1716 (3-CO), 1276 (C–O–C); H NMR (CDCl3) δ:
2.3.3.2. 1α-Hydroxyhexyl-3-oxo-5α-androstane-17β-yl be-
nzoate (3c). The silyl ether 3b (150 mg, 0.25 mmol, 1 Eq.)
was dissolved under N2 in 2 ml anhydrous THF, and 1 ml
of a tetrabutylammonium fluoride solution (TBAF) in THF
(4 Eq., 1 mmol/l) was added. The mixture was stirred at
room temperature for 2 h. Then, a saturated NaCl solution
was added, and the product was extracted three times with
MTBE. After removal of the solvent, purification by LSC,
using MTBE as eluent, led to the 1-hydroxyhexyl deriva-
tive 3c as colorless crystals, recrystallized from n-heptane
(117.5 mg, 95% yield). Melting point (m.p.): 118–120 ◦C.
νmax (KBr): 3463 (OH), 1715 (3-CO), 1276 (C–O–C);
1H NMR (CDCl3) δ: 0.95 (s, 3H, 18-CH3), 1.14 (s, 3H,
19-CH3), 2.05–2.58 (m, 5H, 2␣, 2␤, 4␣, 4␤, 16␣ H), 3.63
(2H, t: J = 6.6 Hz, –CH2–OH), 4.88 (1H, dd: J = 7.8 Hz
0.95 (s, 3H, 18-CH3), 1.14 (s, 3H, 19-CH3), 2.05–2.59 (m,
5H, 2␣, 2␤, 4␣, 4␤, 16␣ H), 4.88 (1H, dd: J = 7.9 and
9.1 Hz, 17␣-H), 7.41–7.59 (m, 5H, aromatic H); MS (EI,
70 eV, m/z, %): 519 (M•+, 5%), 491 (M•+ −N2, 58%), 476
(M•+ − HN3, 90%); high-resolution MS (EI, 70 eV): found
519.3461, C32H45N3O3 requires 519.3459; [␣]D = +55.4◦
(CH2Cl2, c = 1.90).
2.3.3.5. 1α-Azidohexyl-3,3-ethylenedioxy-5α-androstane-
17β-yl benzoate (3f). 123 mg (0.24 mmol, 1 Eq.) of the
azidohexyl derivative 3e were dissolved in 20 ml benzene.
Then, 300 mg of ethyleneglycol p.a. (4.8 mmol, 20 Eq.) and
PTSA (2 mg) were added. The emulsion was refluxed for
2 h using a Dean-Stark water separator. The reaction mix-
ture was treated with one drop Et3N and washed three times
with water. The benzene phase was dried with Na2SO4,
evaporated under reduced pressure, and further purified by
LSC using a PE/MTBE mixture (1:1, v/v) as the eluent. The
3,3-ethylene acetal derivative 3f was isolated as a viscous
oil (23 mg, 91% yield). νmax (film): 1716 (ester), 1277 (es-
and 9.1 Hz, 17␣-H), 7.42–7.59 (m, 5H, aromatic H); MS
(CI, NH3, m/z, %): 495 (MH+, 100%), 512 (M + NH4
,
+
95%); high-resolution MS (DCI, CH4): found 495.3472,
C32H47O4 (MH) requires 495.3474; [␣]D
(CH2Cl2, c = 4.53).
=
+51.5◦
1
ter C–O–C); H NMR (CDCl3) ␦: 0.93 (s, 3H, 18-CH3),
2.3.3.3. 1α-Methylsulfonyloxyhexyl-3-oxo-5α-androstane-
17β-yl benzoate (3d). 115 mg (0.23 mmol, 1 Eq.) of al-
cohol 3c were dissolved in 5 ml CH2Cl2, and freshly
distilled NEt3 (2 Eq.) was added at room temperature. A
solution of 30 mg mesyl chloride (0.25 mmol, 1.1 Eq.) in
2 ml CH2Cl2 was added dropwise. After stirring at room
temperature for 60 min, a saturated NaHCO3 solution was
added, and the reaction mixture was extracted three times
with CH2Cl2. The remaining yellow oil was further purified
by LSC using MTBE as the eluent. The mesylate 3d was
isolated as colorless crystals (119 mg, 90% yield). Recrys-
tallization was performed from n-heptane/CH2Cl2, m.p.:
113–116 ◦C. νmax (KBr): 1716 (3-CO), 1354 and 1175
1.14 (s, 3H, 19-CH3), 3.26 (2H, t: J = 7.0 Hz, –CH2–N3),
3.83–4.0 (m, 4H, –O–CH2–CH2–O), 4.87 (1H, dd: J = 7.7
and 9.1 Hz, 17␣-H), 7.42–7.58 (m, 5H, aromatic H); MS
(DCI, NH3, m/z, %): 564 (M+, 100%), 581 (M + NH4
,
+
83%); high-resolution MS (DCI, CH4): found 563.3714,
C34H49N3O4 requires 563.3723; [␣]D = +23.3◦ (CH2Cl2,
c = 1.25).
2.3.3.6. 1α-Aminohexyl-3,3-ethylenedioxy-17β-hydroxy-5α-
androstane (3g). LiAlH4 (115 mg, 15 Eq.) was added
under a N2 atmosphere to 10 ml of anhydrous THF. A sus-
pension of the azide 3f (116 mg, 0.2 mmol, 1 Eq.) in 3 ml
THF was added slowly to the LiAlH4 mixture at room
temperature and stirred for 20 h. The reaction mixture was
cooled in an ice-bath, and the excess of LiAlH4 was de-
stroyed by dropwise addition of 1 ml of 1N NaOH under
a nitrogen atmosphere and vigorous stirring. The super-
natant was decanted, the remaining slurry was extracted
three times with a total of 15 ml THF, and the solvent
was evaporated. The crude residue was dissolved in 1 ml
methanol, diluted with 3 ml of benzene, and the resulting
emulsion was freeze dried. The viscous crude product was
dissolved in MeOH/CH2Cl2 (1:1, v/v) and then subjected
to LSC using a CH2Cl2/MeOH/NH4OH mixture (10:10:1,
v/v). The 1-aminohexyl derivative 3g was isolated as a resin
(54 mg, 62% yield). νmax (film): 3364 and 3297 (NH2, OH);
1
(–SO2–O–); 1277 (C–O–C); H NMR (CDCl3) δ: 0.95 (s,
3H, 18-CH3), 1.14 (s, 3H, 19-CH3), 2.05–2.58 (m, 5H, 2␣,
2␤, 4␣, 4␤, 16␣ H), 3.00 (s, 3H, –O–SO2–CH3), 4.21 (2H,
t: J = 6.6 Hz), 4.89 (1H, dd: J = 7.8 and 9.1 Hz, 17␣-H),
7.42–7.58 (m, 5H, aromatic H); MS (CI, NH3, m/z, %):
573 (MH+, 31%), 590 (M + NH4+, 100%); high-resolution
MS (DCI, CH4): found 573.3255 (MH), C33H49O6S
(MH) requires 573.3250; [␣]D
c = 2.30).
=
+54.7◦ (CH2Cl2,
2.3.3.4. 1α-Azidohexyl-3-oxo-5α-androstane-17β-yl ben-
zoate (3e). 155 mg (0.27 mmol, 1 Eq.) of the mesylate 3d
was dissolved in 3 ml hexamethyl phosphoric acid triamide

H. Hauptmann et al. / Steroids 68 (2003) 629–639
633
1H NMR (CDCl3) δ: 0.74 (s, 3H, 18-CH3), 0.92 (s, 3H,
19-CH3), 2.68 (2H, t: J = 7.0 Hz, –CH2–NH2), 3.64 (1H,
t: J = 8.5 Hz, 17␣-H), 3.82–3.98 (m, 4H, –O–CH2–CH2–);
MS (DCI, NH3, m/z, %): 434 (MH+, 100%), high-resolution
MS (EI, 70 eV): found 433.3552, C27H47NO3 requires
433.3556; [␣]D = +18.0◦ (CH2Cl2, c = 1.15).
using 3-amino-9-ethyl-carbazole (AEC) as dye substrate.
SHBG-positive fractions were pooled and concentrated us-
ing Microcon centrifugal filters with a molecular weight
cut-off of 30 kDa. After adjustment to a concentration of
0.5 mg/ml, the purified SHBG was stored in aliquots at
−20 ◦C until analysis.
2.3.3.7. 1α-Aminohexyl-17β-hydroxy-5α-androstan-3-one
hydrochloride (3). Formation of the hydrochloride and
cleavage of the 3-acetal group were performed in two succes-
sive steps. First, the 3-ethylenedioxy-1-aminohexylderivative
3g (52 mg, 0.12 mmol, 1 Eq.) was dissolved in 1 ml MeOH,
and 6N HCl (1.5 Eq.) was added. Then, 3 ml of benzene
were added, and the solution was immediately freeze dried.
Secondly, the resulting dry foam was dissolved in 5 ml an-
hydrous acetone and maintained in the presence of catalytic
amounts of HCl gas at 40 ◦C for 24 h. Solvent and the
formed acetone ethylene acetal were removed in the high
vacuum. The 3-oxo-1-hexylamine hydrochloride end prod-
uct 3 was recovered quantitatively as a colorless rigid resin
2.5. Relative binding affinities of the 1α- and 17α-DHT
derivatives to SHBG
RBA of 1␣- and 17␣-DHT derivatives to SHBG were de-
termined under equilibrium conditions at 37 ◦C using Con
3
A-Sepharose as solid phase and H-T as tracer according
to the method of Dunn et al. [25]. The competition curves
were obtained in the presence of increasing concentrations
of each ligand. The purified homodimeric SHBG was used at
a constant concentration of 100 nM. SHBG was first prein-
cubated for 30 min with the Con A-Sepharose slurry at
room temperature. After centrifugation at 3000×g, the pel-
let was washed extensively and resuspended in PBS buffer.
Then, 3H-T (approximately 50,000 dpm) was added together
with various final concentrations (10–5000 nM) of the three
radioinert 1- and 17-aminoalkyl ligands. After incubation
for 90 min at 37 ◦C with shaking, the samples were cen-
trifuged at 3000 × g, and the supernatants were counted for
␤-radioactivity using opti-Fluor scintillation fluid (Packard
Instruments, Groningen, The Netherlands) in a 1219 Rack-
beta liquid scintillation counter from Wallac. Specific bind-
ing of the competitor was expressed as a percentage of
the maximum binding that was calculated by subtracting
non-specific binding (estimated in the presence of 100-fold
excess of T) from total binding. RBA to SHBG were calcu-
lated according to the following equation:
(51 mg, 27% overall yield). νmax (KBr): 3655 and 3383
1
=
(OH), 3030, 2400 and 2100 (NH3), 1705 (C O); H NMR
(CDCl3) δ: 0.76 (s, 3H, 18-CH3), 1.13 (s, 3H, 19-CH3),
2.92–3.06 (m, 2H, –CH2–NH3+), 3.67 (m, 1H, 17-H), 8.2
(s broad, 3H, –NH3); MS (DCI, NH3, m/z, %): 390 (MH+,
100%), 372 (MH+ − H2O, 7%), high-resolution MS (DCI,
CH4): found 390.3376, C25H44NO2 (MH+ − Cl) requires
390.3372; [␣]D = +20.0◦ (CH2Cl2, c = 1.22).
2.4. Size-exclusion chromatography of affinity purified
homodimeric SHBG
Purified human SHBG (lot #A0011105, isolated by affin-
ity chromatography from human pregnancy plasma) was
purchased from Fitzgerald Industries International, Concord,
MA, USA. A concentrated SHBG solution (5 mg/ml) was
prepared in bidistilled water from the supplied lyophilized
material. Subsequently, analytical size-exclusion chro-
matography was performed on a Superdex 200 HR10/30
column using the ÄKTAFPLC system, preequilibrated with
buffer solutions containing 5 mM Na2HPO4/NaH2PO4, pH
7.4, 50 mM K2SO4 and 1 mM CaCl2. Sample size was
0.25 ml at a flow rate of 0.2 ml/min, and UV monitoring was
performed at 280 nm. The column was calibrated with gel
filtration LMW and HMW standard proteins, which were
run under identical conditions. Gel chromatography frac-
tions of SHBG were run on a discontinuous sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
according to the method of Laemmli [34]. A stacking gel
and a 10% polyacrylamide separation gel were applied
under reducing conditions using a discontinuous buffer
system (0.1 M Tris base, pH 8.25, 0.1 M Tricine and 0.1%
SDS at the cathode and 0.2 M Tris, pH 8.9 at the anode).
The protein was electrotransferred to a PVDF membrane
(pore size 0.45 ␮m), probed with the specific anti-SHBG
pab, and visualized with HRP-conjugated secondary pab
RBA = (C0.5,T/C0.5,competitor) × 100 (with C0.5 repre-
senting the concentration of radioinert competitor required
3
to reduce H-T specific binding by 50%; RBA value for T
was set at 100%).
Determination of the association constant (Ka) of SHBG
for binding T was performed by adding unlabeled T in final
concentrations from 1 to 1000 nM. Subsequently, Ka was
evaluated from the slope of the Scatchard plot (y = dpm su-
pernatant/dpm total, x = nM 3H-T bound) and corresponded
to a value of 3.19 0.32 (S.D.) × 109 M−1 (n = 5). From
this, the association constants for the 1␣- and 17␣-DHT
derivatives were calculated using the following equation:
Ka
50% displacement) [26].
= Ka /((1 + R)/RBA) − R (with R = B/B0 at
T
competitor
3. Results and discussion
3.1. Syntheses
Syntheses of the 17␣-alkylamine derivatives of DHT
followed the synthetic pathway described in [29], whereas
for the 1␣-hexylamine DHT derivative, a route with a
copper-catalyzed conjugate addition of a Grignard reagent

634
H. Hauptmann et al. / Steroids 68 (2003) 629–639
OH
(CH2)3NH2
OH
(CH2)nNH2
n = 3
CH3O
CH3O
O
H
H
1a,b
1
Fmoc protected 6-aminocaproic acid
DMAP, DCC, CH2Cl2
n = 2
OH
(CH2)2NHCO(CH2)5NH - Fmoc
2b
H2N(CH2)2OH, CH2Cl2
aq. HCl, THF
1.
2.
CH3O
CH3O
2a
H
OH
(CH2)2NHCO(CH2)5NH2
.
2 HCl
O
H
Scheme 1. Synthetic pathway for 17␣-aminopropyl- and 17␣-aminocaproylamidoethyl-17␤-hydroxy-5␣-androstan-3-one.
to a 1-en-3-one functional group as the key reaction was
established.
was then cleaved to give the amine derivative 2b. Hydrol-
ysis of the 3-dimethoxy acetal protecting group led to the
final 17␣-substituted amine derivative 2, which was isolated
as the hydrochloride salt in a high overall yield (Scheme 1).
3.1.1. 17α-Aminopropyl-17β-hydroxy-5α-androstan-
3-one (1)
1
In a H NMR NOSY experiment on compound 2 (data
not shown), a NOE of the C-18␤ methyl to the 5ꢀ-methylene
protons of the 17␣-side chain was observed, suggesting the
existence of a hydrogen bond between the C-17␤ hydroxyl
and the amide carbonyl functions. Therefore, under these
experimental conditions (nonpolar organic solvent), the
aminocaproyl-amidoethyl side chain was assumed to have a
conformation in which it remained in close proximity to the
steroidal D-ring of compound 2, thus potentially inducing
steric hindrance around the 17-OH group and restricting
the through-space distance between C-17 and the terminal
amine.
The synthesis started from 5␣-androstan-3,17-dione,
which was converted to 3,3-(dimethoxy)-5␣-androstan-17-
one. Condensation with the dimethylsulfonium methylide
reagent [35,36] led to the formation of a 17␤-spirooxirane
intermediate as the major product with more than 90% yield
[37]. The epoxide ring was opened by an acetonitrile carban-
ion to give a 17␣-ethylcyanide group, which was reduced
by LiAlH4 to the 3,3-(dimethoxy)-17␣-aminopropyl-DHT
compound 1a. Acid hydrolysis of the 3-dimethoxy acetal
group of 1a led to the 17␣-aminopropyl-DHT derivative 1
(Scheme 1).
3.1.2. 17α-Aminocaproylamidoethyl-17β-hydroxy-
5α-androstan-3-one (2)
3.1.3. 1α-Aminohexyl-17β-hydroxy-5α-androstan-
3-one (3)
The 3,3-(dimethoxy)-17␣-aminoethyl-DHT precursor 1b
was synthesized from 5␣-androstan-3,17-dione according
to the procedure given in [29]. Condensation of the 17␣-
ethylamine group with Fmoc-protected 6-aminocaproic acid
was accomplished using the carbodiimide method to provide
the Fmoc protected amide derivative 2a. The Fmoc group
The synthetic route chosen for access to the 1␣-alkyl DHT
derivatives was established from the 17␤-benzoyloxy-5␣-
androst-1-en-3-one precursor (3a) [31]. Using a copper(I)-
mediated conjugate addition [38–40] of the ␻-silyloxyhexyl
Grignard reagent TBSO(CH2)6MgBr, the 1␣-alkyl-substi-
tuted derivative 3b was obtained as the only product in 74%

H. Hauptmann et al. / Steroids 68 (2003) 629–639
635
OBz
OBz
R
TBSO-(CH2)6-MgBr
TBAF
THF
40 ˚C
CuI, THF, -
3a
3b
O
O
R
H
H
R = (CH2)6-OTBS
OBz
OBz
R
NaN3
CH3SO2Cl
Et3N, CH2Cl2
HMPA
3d
3c
O
O
H
H
R =
R = (CH2)6-OH
H ) -OSO CH
(C
2
6
2
3
OBz
OBz
R
R
LiAlH4
THF
HO(CH2)2OH, PTSA
benzene, reflux
O
3e
O
3f
H
O
H
R = (CH2)6-N3
R = (CH2)6-N3
OH
OH
R
R
conc. HCl, acetone
O
.
3
HCl
3g
O
O
H
H
R = (CH2)6-NH2
R = (CH2)6-NH2
Scheme 2. Synthetic pathway for 1␣-aminohexyl-17␤-hydroxy-5␣-androstan-3-one.
yield. After removing the silyl group, mesylation of the ter-
minal hydroxyl group led to compound 3d. Substitution with
sodium azide gave the azide 3e. The 3-oxo group of this latter
compound was then protected as the ethylene acetal giving
the compound 3f. Reduction of the azide with LiAlH4 pro-
vided the amine derivative 3g. Finally, the protecting acetal
group was removed by transacetalization using gaseous HCl
and acetone, which also converted the target 1␣-aminohexyl
DHT molecule 3 into the hydrochloride salt (Scheme 2).
could be assigned by H–H correlated NMR spectroscopy in
the multiplet at 1.78–1.86 ppm. The coupling constants of
the A-ring protons (2␣, 2␤, 4␣, and 4␤, together with the
5␣ H) could be identified; the proton system may be consid-
ered as 2 ABX spectra (1␤, 2␣, 2␤ and 4␣, 4␤, 5␣), which
were resolved at first order, as shown in Fig. 1 for 3d. The
corresponding J values are summarized in Table 1.
3.2. Size-exclusion chromatography of affinity purified
SHBG
1
In the H NMR analyses for all 1␣-DHT derivatives, we
observed a typical signal pattern for the 2␣, 2␤, 4␣, and
4␤ protons with deducible coupling constants. The chemi-
cal shifts of these protons are in agreement with data de-
scribed for a series of 5␣-androstanes [41,42]. Kirk et al.
[41] found for 5␣-androstan-3-one the following chemical
shifts (δ, ppm): 1␣ H 1.34, 1␤ H 2.01, 2␣ H 2.27, 2␤ H
2.37, 4␣ H 2.06, 4␤ H 2.24. In our 1␣-DHT derivatives, we
found similar δ-values (e.g. for 3d: 2␣ H 2.31, 2␤ H 2.55,
4␣ H 2.08, 4␤ H 2.23). The chemical shift of the 1␤ proton
We used commercially available human SHBG, affinity
purified from pooled pregnancy sera. Although the purity of
this SHBG was high (>98% by SDS–PAGE), a further gel
chromatography was performed in order to further improve
the purity and to remove possible oligomeric forms as well
as bound steroidal ligands (mainly E2) in the SHBG prepa-
ration [43]. As shown in Fig. 2, a PAGE under denaturing
conditions showed the presence of two distinct monomers at

636
H. Hauptmann et al. / Steroids 68 (2003) 629–639
Fig. 1. 1H NMR spectrum (400 MHz; downfield portion) of 17␤-benzoyloxy-1␣-methyl-sulfonyloxyhexyl-5␣-androst-3-one (3d), giving a representative
overview of the signal pattern of the 2␣ (2.31 ppm), 2␤ (2.55), 4␣ (2.08) and 4␤ (2.23) protons of ring A.
molecular weights of approximately 50 and 47 kDa, as ex-
pected from the well established size heterogeneity of SHBG
attributed to variations in carbohydrate content [44]. Traces
of dimeric protein were seen at 90 kDa, while no other im-
purities could be detected.
tives 1 and 2 with either a short or a long spacer interact al-
most identically with SHBG. The calculated Ka values were
1.25 × 107 and 1.50 × 107 M−1, respectively. The 1␣-DHT
derivative 3, however, showed a much higher binding affin-
ity to SHBG, with a Ka value of 1.40 × 109 M−1. This rep-
3.3. Determination of binding affinities
The equilibrium measurements shown in Fig. 3 and data
presented in Table 2 depict that the two 17␣-DHT deriva-
Table 1
Splitting patterns and J values for the 2␣, 2␤, 4␣, and 4␤ protons of
the derivatives 3b–3d, obtained by 1H NMR analysis (for details, see
Section 3.1)
Proton
Compound
δ (ppm)
Splitting pattern
J (Hz)
2␣
3b
3c
3d
3e
2.33
2.32
2.31
2.32
dd
dd
dd
dd
15.0/2.0
15.0/2.0
15.0/2.0
15.0/2.3
2␤
4␣
4␤
3b
3c
3d
3e
2.54
2.55
2.55
2.55
dd
dd
dd
dd
15.0/5.6
15.0/5.6
15.0/5.7
15.0/5.6
3b
3c
3d
3e
2.10
2.09
2.08
2.08
ddd
ddd
ddd
ddd
15.0/4.3/2.0
15.0/4.4/2.0
15.0/4.4/2.1
15.0/4.5/2.2
Fig. 2. Western blot of affinity-purified native human SHBG after gel
electrophoresis in a 10% SDS polyacrylamide gel. Primary antibody:
rabbit anti-human SHBG pab; secondary antibody: HRP-labeled goat
anti-rabbit IgG pab; dye: 3-amino-9-ethylcarbazole. Left lane: molecular
weight marker; right lane: SHBG gel filtration preparation.
3b
3c
3d
3e
2.22
2.22
2.22
2.22
dd
dd
dd
dd
15.0/13.0
15.0/13.2
15.0/13.0
15.0/13.4

H. Hauptmann et al. / Steroids 68 (2003) 629–639
637
110
100
90
80
70
60
50
40
30
20
10
0
)
Steroid 1 (
Steroid 2 ( )
Steroid 3 ( )
10
100
1000
10000
Steroid concentration (nmol/l)
Fig. 3. Displacement of 3H-testosterone by the three unlabeled 1␣- and 17␣-aminoalkyl DHT derivatives from SHBG. The results are plotted as percent
3H-T bound to SHBG vs. unlabeled steroid concentrations; values are given as means 1 S.D. (n = 4, 4, 6). 1 (᭡), 2 (᭹), 3 (᭿).
Table 2
Binding affinities of the DHT derivatives 1–3 to SHBG, measured under equilibrium conditions
1 (n = 4)
RBA (%)
2 (n = 4)
RBA (%)
3 (n = 6)
RBA (%)
T (n = 5)
R
Ka (107 M−1
0.024 1.25
)
R
Ka (107 M−1
0.006 1.50
)
R
Ka (107 M−1
0.041 140 32
)
Ka (107 M−1
)
0.62
0.05 0.435
0.79 0.60
0.01 0.395
0.10 52.0
11.2 0.423
319.4
31.7
Results are given as means 1 S.D. RBA = relative binding activity; R = B/B0 at 50% displacement.
10
resents about half of the Ka value (3.19 × 109 M−1) deter-
mined in parallel for unsubstituted T (see Scatchard analysis
in Fig. 4), which is in good agreement with values given in
the literature [25,45–47].
8
6
4
2
0
These relatively high association constants, in particu-
lar, the unexpectedly high affinity of the 1␣-aminohexyl
DHT compound 3, which had a much larger 1-substituent
than the previously known 1␣-methyl substituted mes-
terolone ligands (vide infra), encouraged us to estab-
lish a biosensor system for ligand-binding studies of
the interaction of these 17␣- and 1␣-alkylamine DHT
derivatives with SHBG. These investigations using a
surface plasmon resonance (SPR) biosensor are pre-
sented elsewhere [48]. The approach is to immobilize the
amino DHT derivatives covalently onto a carboxymethyl
dextran-coated surface of the system’s flow cell by the
N-ethyl-Nꢀ-(3-diethyl-aminopropyl)-carbodiimide/N-hydro-
xysuccinimide (EDC/NHS) technique. Such biosensor mea-
surements may shed light on the thermodynamics and
kinetics of the interaction of steroids and other potential lig-
ands of the SHBG binding site. The access to immobilized
1␣- and 17␣-substituted DHT derivatives, displaying freely
available D- and A-rings, respectively, provides a potentially
useful tool for studying the interaction of the unsubstituted
parts of the steroid skeleton and for evaluating the role of
0
1
2
3
3H-testosterone-B (nmol/l)
Fig. 4. Scatchard analysis of 3H-testosterone binding to SHBG at 37 ◦C.

638
H. Hauptmann et al. / Steroids 68 (2003) 629–639
conformations of the covalent linker arms, which might
influence steroid positioning in the SHBG binding site.
[17] Pardridge WM. Selective delivery of sex steroid hormones to tissues
in vivo by albumin and sex hormone-binding globulin. Ann NY
Acad Sci 1988;538:173–92.
[18] Nakhla AM, Khan MS, Rosner W. Biologically active steroids
activate receptor-bound human sex hormone-binding globulin to
cause LNCaP cells to accumulate adenosine 3,5-monophosphate. J
Clin Endocrinol Metab 1990;71:398–404.
Acknowledgements
[19] Fissore F, Fortunati N, Comba A, Fazzari A, Gaidano G, Berta L,
et al. The receptor-mediated action of sex steroid-binding protein
(SBP, SHBG): accumulation of cAMP in MCF-7 cells under SBP
and estradiol treatment. Steroids 1994;59:661–7.
The authors are indebted to Ms. Manuela Meyer and Ms.
Monika Söder for their excellent technical assistance.
[20] Lim SC, Caballero AE, Arora S, Smakowski P, Bashoff EM, Brown
FM, et al. The effect of hormonal replacement therapy on the
vascular reactivity and endothelial function of healthy individuals
and individuals with type 2 diabetes. J Clin Endocrinol Metab
1999;84:4159–64.
[21] Skafar DF, Xu R, Morales J, Ram J, Sowers JR. Clinical review 91:
female sex hormones and cardiovascular disease in women. J Clin
Endocrinol Metab 1997;82:3913–8.
References
[1] Anderson DC. Sex-hormone-binding globulin. Clin Endocrinol
1974;3:69–96.
[2] Hammond GL. Molecular properties of corticosteroid binding
globulin and the sex-steroid binding proteins. Endocrine Rev
1990;11:65–79.
[22] Carlström K, Gershagen S, Rannevik G. Free testosterone and
[3] Westphal U. Steroid–protein interaction II: In: Monographs on
endocrinology, vol. 27. New York: Springer; 1986. p. 198–264.
[4] Petra PH. The plasma sex steroid-binding protein (SBP or SHBG).
A critical review of recent developments on the structure, molecular
biology, and function. J Steroid Biochem Mol Biol 1991;40:735–53.
[5] Joseph DR. Structure, function, and regulation of androgen
binding protein: sex hormone-binding globulin. Vitamins Hormones
1994;49:197–280.
[6] Hammond GL, Underhill DA, Rykse HM, Smith CL. The human sex
hormone binding globulin gene contains exons for androgen binding
protein and two other testicular messenger RNAs. Mol Endocrinol
1989;3:1869–76.
[7] Berubé D, Seralini G-E, Gagné R, Hammond GL. Localization of the
human sex hormone-binding globulin gene (SHBG) to the short arm
of chromosome 17 (17pl 2-p13). Cytogenet Cell Genet 1990;54:65–7.
[8] Walsh KA, Titani K, Takio K, Kumar S, Hayes R, Petra PH. Amino
acid sequence of the sex steroid binding protein of human blood
plasma. Biochemistry 1986;25:7584–90.
[9] Hammond GL, Underhill DA, Smith CL, Goping IS, Harley MJ,
Musto NA, et al. The cDNA-deduced primary structure of human
sex hormone binding globulin and location of its steroid binding
domain. FEBS Lett 1987;215:100–4.
testosterone/SHBG index in hirsute women:
a comparison of
diagnostic accuracy. Gynecol Obstet Invest 1987;24:256–61.
[23] Hammond GL, Bocchinfuso WP. Sex hormone-binding
globulin/androgen-binding protein: steroid-binding and dimerization
domains. J Steroid Biochem Mol Biol 1995;53:543–52.
[24] Grishkovskaya I, Avvakumov GV, Sklenar G, Dales D, Hammond
GL, Muller YA. Crystal structure of human sex hormone-binding
globulin: steroid transport by a laminin G-like domain. EMBO J
2000;19:504–12.
[25] Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones:
binding of 21 endogenous steroids to both testosterone-binding
globulin and corticosteroid-binding globulin in human plasma. J Clin
Endocrinol Metab 1981;53:58–68.
[26] Pugeat MM, Dunn JF, Nisula BC. Transport of steroid hormones:
interaction of 70 drugs with testosterone-binding globulin and
corticosteroid-binding globulin in human plasma. J Clin Endocrinol
Metab 1981;53:69–75.
[27] Grishkovskaya I, Avvakumov GV, Hammond GL, Catalano MG,
Muller YA. Steroid ligands bind human sex hormone-binding globulin
in specific orientations and produce distinct changes in protein
conformation. J Biol Chem 2002;277:45219–25.
[28] Avvakumov GV, Grishkovskaya I, Muller YA, Hammond GL.
Resolution of the human sex hormone-binding globulin dimer
interface and evidence for two steroid-binding sites per homodimer.
J Biol Chem 2001;276:34453–7.
[10] Strel’chyonok OA, Avvakumov GV. Specific steroid-binding
glycoproteins of human blood plasma: novel data on their structure
and function. J Steroid Biochem 1990;35:519–34.
[29] Mappus E, Chambon C, Fenet B, Rolland de Ravel M, Grenot C,
Cuilleron CY. Synthesis of (5-azido-2-nitrobenzoyl)amido, (4-azido-
2-nitrophenyl)amino, and (5-azido-2-nitro-3,4,6-trifluorophenyl)
amino derivatives of 17␣-methylamino-, 17␣-ethylamino-, and 17␣-
aminopropyl-5␣-dihydrotestosterone as reagents of different linker
lengths for the photo-affinity labeling of sex hormone binding
globulins and androgen receptors. Steroids 2000;65:459–81.
[30] Hauptmann H, Paulus B, Kaiser T, Herdtweck E, Huber E, Luppa
PB. Concepts for the syntheses of biotinylated steroids: part I.
Testosterone derivatives as immunochemical probes. Bioconjug Chem
2000;11:239–52.
[31] Pelc B. Steroid derivatives. XXVIII. Preparation of 1-methyl-
5␣-androstane derivatives. Collection Czech Chem Commun
1964;29:3089–95.
[32] Corey EJ, Venkateswarlu A. Protection of hydroxyl groups as
tert-butyl-dimethylsilyl derivates. J Am Chem Soc 1972;94:6190–1.
[33] Bowler J, Lilley TJ, Pittam JD, Wakeling AE. Novel steroidal pure
anti-estrogens. Steroids 1989;54:71–99.
[11] Bardin CW, Lipsett MB. Testosterone and androstenedione blood
production rates in normal women and women with idiopathic
hirsutism or polycystic ovaries. J Clin Invest 1967;46:891–902.
[12] Vermeulen AL, Verdonck L, Van der Straeten M, Orie M. Capacity
of the testosterone binding globulin in human plasma and influence
of specific binding of testosterone on its metabolic clearance rate. J
Clin Endocrinol Metab 1969;29:1470–80.
[13] Petra PH, Stanczyk FZ, Namkung PC, Fritz MA, Novy MJ. Direct
influence of the sex steroid-binding protein (SBP) of plasma on
the metabolic clearance rate of testosterone. J Steroid Biochem
1985;22:739–46.
[14] Plymate SR, Namkung PC, Matej LA, Petra PH. Direct effect of
plasma sex hormone binding globulin (SHBG) on the metabolic
clearance rate of 17␤-estradiol in the primate. J Steroid Biochem
1990;36:311–7.
[15] Bordin S, Petra PH. Immunocytochemical localization of the plasma
sex steroid-binding protein (SBP) in tissues of the adult male monkey,
Macaca nemestrina. Proc Natl Acad Sci USA 1990;77:5678–82.
[16] Strel’chyonok OA, Avvakumov GV, Survilo LI. A recognition
system for sex-hormone-binding protein–estradiol complex in human
decidual endometrium plasma membranes. Biochem Biophys Acta
1984;802:459–66.
[34] Laemmli UK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 1970;227:680–5.
[35] Corey EJ, Chaykovsky M. Dimethylsulfonium methylide, a reagent
for selective oxirane synthesis from aldehydes and ketones. J Am
Chem Soc 1962;84:3782–3.

H. Hauptmann et al. / Steroids 68 (2003) 629–639
639
[36] Ponsold K, Hübner M, Schade W, Oettel M, Freund R. Progestagene
vom Typ 17␣-CH2X-substituierter 19-Nortestosteronderivate. Phar-
mazie 1978;33:792–8.
[43] Lutz RA, Märki HH, Weder HG. Die Androgenbindung im
menschlichen Plasma und ihre physiologische Bedeutung. J Clin
Chem Clin Biochem 1977;15:57–67.
[37] Cook CE, Corley RC, Wall ME. Steroids. LXXIX. Synthesis and
reactions of oxiranes obtained from 3- and 17-ketosteroids. J Org
Chem 1968;33:2789–93.
[44] Danzo BJ, Bell BW, Black JH. Human testosterone-binding globulin
is a dimer composed of two identical protomers that are differentially
glycosylated. Endocrinology 1989;124:2809–17.
[38] Wiechert R, Inventor Schering AG, Germany, assignee. 3-Oxo-
1alpha-methyl steroids. Ger Offen 1152100.1960.
[39] Kocor M, Kroszczynski W, Pietrzak J, Cynkowski T. Steroids, part
XLI. Michael addition of diethyl malonate to steroidal 3-keto-1,4,6-
trienes. Polish J Chem 1979;53:149–55.
[40] Adamczyk M, Chen YY, Johnson DD, Reddy RE. A stereoselective
synthesis of 1␣-(3ꢀ-carboxypropyl)-4-androsten-17␤-ol-3-one: prepa-
ration of immunoreagents for quantifi-cation of testosterone by fluore-
scence polarization immunoassay. Tetrahedron 1997;53:12855–66.
[41] Kirk DN, Toms HC, Douglas C, White KA, Smith KE, Latif S, et al.
A survey of the high-field 1H NMR spectra of the steroid hormones,
their hydroxylated derivatives, and related compounds. J Chem Soc
Perkin Trans 1990;2:1567–94.
[42] Schneider HJ, Buchheit U, Becker N, Schmidt G, Siehl U. 1H
NMR analyses, shielding mechanisms, coupling constants, and
conformations in steroids bearing halogen, hydroxy, oxo groups, and
double bonds. J Am Chem Soc 1985;107:7027–39.
[45] Grenot C, Montard de A, Blachère T, Ravel de MR, Mappus
E, Cuilleron CY. Characterization of Met-139 as the photolabeled
amino acid residue in the steroid binding site of sex hormone
binding globulin using D6 derivates of either testosterone or estradiol
as unsubstituted photoaffinity labeling reagents. Biochemistry
1992;31:7609–21.
[46] Martin ME, Haourigui M, Pelissero C, Benassayag C, Nunez EA.
Interactions between phytoestrogens and human sex steroid binding
protein. Life Sci 1996;58:429–36.
[47] Vigersky RA, Kono S, Sauer M, Lipsett MB, Loriaux DL. Relative
binding of testosterone and estradiol to testosterone-estradiol-binding
globulin. J Clin Endocrinol Metab 1979;49:899–904.
[48] Metzger J, Schnitzbauer A, Meyer M, Söder M, Cuilleron
CY, Hauptmann H, Ligand-binding analysis of 1␣- and 17␣-
dihydrotestosterone derivatives to homodimeric sex hormone-binding
globulin. Biochemistry 2003, submitted for publication.