A simple method for the small scale synthesis and solid-phase extraction purification of steroid sulfates

Article abstract of DOI:10.1016/j.steroids.2014.09.006

Steroid sulfates are a major class of steroid metabolite that are of growing importance in fields such as anti-doping analysis, the detection of residues in agricultural produce or medicine. Despite this, many steroid sulfate reference materials may have limited or no availability hampering the development of analytical methods. We report simple protocols for the rapid synthesis and purification of steroid sulfates that are suitable for adoption by analytical laboratories. Central to this approach is the use of solid-phase extraction (SPE) for purification, a technique routinely used for sample preparation in analytical laboratories around the world. The sulfate conjugates of sixteen steroid compounds encompassing a wide range of steroid substitution patterns and configurations are prepared, including the previously unreported sulfate conjugates of the designer steroids furazadrol (17β-hydroxyandrostan[2,3-d]isoxazole), isofurazadrol (17β-hydroxyandrostan[3,2-c]isoxazole) and trenazone (17β-hydroxyestra-4,9-dien-3-one). Structural characterization data, together with NMR and mass spectra are reported for all steroid sulfates, often for the first time. The scope of this approach for small scale synthesis is highlighted by the sulfation of 1 μg of testosterone (17β-hydroxyandrost-4-en-3-one) as monitored by liquid chromatography-mass spectrometry (LCMS).

Full text of DOI:10.1016/j.steroids.2014.09.006

Steroids 92 (2014) 74–80
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
A simple method for the small scale synthesis and solid-phase extraction
purification of steroid sulfates
⇑
Christopher C. Waller, Malcolm D. McLeod
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2014
Received in revised form 27 August 2014
Accepted 22 September 2014
Available online 5 October 2014
Steroid sulfates are a major class of steroid metabolite that are of growing importance in fields such as
anti-doping analysis, the detection of residues in agricultural produce or medicine. Despite this, many
steroid sulfate reference materials may have limited or no availability hampering the development of
analytical methods. We report simple protocols for the rapid synthesis and purification of steroid sulfates
that are suitable for adoption by analytical laboratories. Central to this approach is the use of solid-phase
extraction (SPE) for purification, a technique routinely used for sample preparation in analytical labora-
tories around the world. The sulfate conjugates of sixteen steroid compounds encompassing a wide range
of steroid substitution patterns and configurations are prepared, including the previously unreported
sulfate conjugates of the designer steroids furazadrol (17b-hydroxyandrostan[2,3-d]isoxazole), isofuraza-
drol (17b-hydroxyandrostan[3,2-c]isoxazole) and trenazone (17b-hydroxyestra-4,9-dien-3-one). Struc-
tural characterization data, together with NMR and mass spectra are reported for all steroid sulfates,
often for the first time. The scope of this approach for small scale synthesis is highlighted by the sulfation
Keywords:
Sulfate ester
Steroid sulfate
Sulfation
Solid-phase extraction
Anti-doping
of 1
lg of testosterone (17b-hydroxyandrost-4-en-3-one) as monitored by liquid chromatography–mass
spectrometry (LCMS).
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
A range of methods have been developed to access steroid
sulfates including the reaction of the parent steroid with sulfate
salts and acetic anhydride [14], chlorosulfonic acid [15], amine
complexes of sulfur trioxide [16–19], sulfuric acid and carbodii-
mides [20], sulfamic acid [21], or more recently by novel sulfuryl
imidazolium salts [22,23]. These reactions however, whilst
effective in affording the desired sulfate compounds, generally
require significant chemical expertise and may also require harsh
or hazardous conditions [15,19,20], specialised reagents [22,23],
or complicated purification methods. These factors make small
scale synthesis of steroid sulfates for analytical purposes a some-
what challenging undertaking. Simple synthetic access to steroid
sulfates would facilitate the identification of metabolites and assist
in the development of methods targeting these analytes. In this
paper we report a general method for the small scale synthesis
and purification of steroid sulfate compounds for anti-doping
research and other analytical applications that is suitable for adop-
tion by analytical laboratories. The method takes advantage of a
rapid purification by solid-phase extraction (SPE), a technique
familiar to analytical laboratories but with untapped potential in
chemical synthesis.
Steroid sulfates are a major class of phase II steroid metabolite
that are of growing importance in fields such as anti-doping anal-
ysis [1], the detection of residues in agricultural produce [2] and
medicine [3]. This is in part driven by improvements in liquid chro-
matography mass spectrometry (LCMS) technology that empower
the direct detection of phase II conjugates [4,5] but also arises
due to the important information unveiled by a thorough analysis
of phase II metabolism [6,7]. In the field of anti-doping science the
analysis of human sulfate metabolites can afford greater retrospec-
tivity for the detection of steroidal agents [8–10] and also serve as
markers to distinguish between steroids of exogenous and endog-
enous origin [11–13]. Although there are a range of reliable
approaches to analyse for phase II metabolites in both humans
and animals, the range of available steroidal sulfate reference
materials is incomplete and the ability to rapidly make and manip-
ulate steroid sulfates as standards or reference materials has limi-
tations [1]. This may mean that significant steroid sulfate markers
cannot be quantified or even identified [11,13].
⇑
Corresponding author. Tel.: +61 2 6125 3504; fax: +61 2 6125 8114.
E-mail address: malcolm.mcleod@anu.edu.au (M.D. McLeod).
http://dx.doi.org/10.1016/j.steroids.2014.09.006
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

C.C. Waller, M.D. McLeod / Steroids 92 (2014) 74–80
75
of 38% aqueous ammonium acetate (26.3 mM): 62% methanol. The
column and sample modules were set to 30 °C and 4 °C
respectively.
2. Experimental
2.1. Materials
2.3. Chemical synthesis
Chemicals and solvents including sulfur trioxide pyridine com-
plex (SO3Ápy), sulfur trioxide triethylamine complex (SO3ÁNEt3) and
1,4-dioxane, were purchased from Sigma–Aldrich (Castle Hill,
Australia) and were used as supplied unless otherwise stated.
2.3.1. General method for the small scale steroid sulfation reaction
with purification by SPE
A solution of SO3Ápy (10.0 mg, 62.8 mmol) in DMF (100
l
L) was
Androsterone (3
(3b-hydroxy-5 -androst-17-one), etiocholanolone (3
5b-androst-17-one), methandriol (17 -methylandrost-5-ene-
3b,17b-diol) and testosterone (17b-hydroxyandrost-4-en-3-one)
were obtained from Steraloids (Newport RI, USA). Estra-4,9-dien-
3,17-one was obtained from AK Scientific (Union City CA, USA).
Dehydroepiandrosterone (3b-hydroxyandrost-5-en-17-one) was
a
-hydroxy-5
a
-androst-17-one), epiandrosterone
added to a solution of steroid (1.0 mg) in 1,4-dioxane (100
lL) and
a
a-hydroxy-
the resulting solution was then stirred in a capped vial at room
temperature for 4 h. The reaction was then quenched with water
(1.5 mL) and subjected to purification by SPE. An Oasis WAX SPE
cartridge (6 cc) was pre-conditioned with methanol (5 mL) fol-
lowed by water (15 mL). The reaction mixture (1.7 mL) was then
loaded onto the cartridge and eluted under a positive pressure of
nitrogen at a flow rate of approximately 2 mL minÀ1 with the fol-
lowing solutions: formic acid in water (2% v/v, 15 mL), water
(15 mL), methanol (15 mL) and saturated aqueous ammonia solu-
tion in methanol (5% v/v, 15 mL). The methanolic ammonia frac-
tion was concentrated in vacuo to yield the desired steroid
sulfate as the corresponding ammonium salt.
a
obtained from BDH (Poole, UK). Lithocholic acid (3
cholan-24-oic acid) was obtained from L. Light & Co. (Colnbrook,
UK). Epitestosterone (17 -hydroxyandrost-4-en-3-one) was syn-
a-Hydroxy-5b-
a
thesised from testosterone using literature methods [24]. MilliQ
water was used in all aqueous solutions and in the liquid chroma-
tography mobile phase. Liquid chromatography (gradient) grade
methanol was obtained from Merck (Kilsyth, Australia) and was
used for preparing the liquid chromatography mobile phase and
steroid standard solutions. N,N-Dimethylformamide (DMF) and
aqueous ammonia solution were obtained from Chem-Supply
(Gillman, Australia). Formic acid was obtained from Ajax Chemi-
cals (Auburn, Australia). Ethyl formate and methanol were distilled
separately from calcium hydride under a nitrogen atmosphere
before use. Chlorosulfonic acid [CAUTION!] was distilled under a
nitrogen atmosphere before use. Tetrahydrofuran was distilled
from sodium wire before use. Solid-phase extraction (SPE) was
performed using Waters (Rydalmere, Australia) Oasis weak anion
exchange (WAX) 6 cc cartridges (186004647).
2.3.2. General method for the small scale steroid sulfation reaction
with conversion determined by 1H NMR analysis
A steroid sulfation reaction was performed as per Section 2.3.1
above. A modified SPE protocol eluting with only formic acid in
water (2% v/v, 15 mL), water (15 mL) and saturated aqueous
ammonia solution in methanol (5% v/v, 15 mL), followed by con-
centration of the methanolic ammonia fraction yielded a mixture
containing both the starting steroid and the corresponding steroid
sulfate as the ammonium salt. A 1H NMR spectrum was obtained
and integration of a suitable signal (typically C3-H or C17-H) of
both steroid and steroid sulfate provided a ratio of the two com-
pounds which was used to determine the percent conversion of
the sulfation reaction. The mixture was then subjected to a second
SPE purification using the conditions outlined in Section 2.3.1 to
yield pure steroid sulfate as the corresponding ammonium salt.
2.2. Instruments
Melting points were determined using a SRS Optimelt MPA 100
melting point apparatus and are uncorrected. Optical rotational
were determined using
a
Perkin–Elmer 241MC polarimeter
2.3.3. General method for the steroid sulfation reaction with
purification by recrystallisation
(sodium D line, 298 K) in the indicated solvents. 1H and 13C nuclear
magnetic resonance (NMR) spectra were recorded using either
Varian 400 MHz, Bruker Ascend 400 MHz, Bruker Avance
400 MHz or Bruker Avance 600 MHz spectrometers at 298 K using
deuterated methanol solvent unless otherwise specified. Data is
reported in parts per million (ppm), referenced to residual protons
or 13C in deuterated methanol solvent (CD3OD: 1H 3.31 ppm, 13C
49.00 ppm) unless otherwise specified, with multiplicity assigned
as follows: br = broad, s = singlet, d = doublet, dd = doublet of dou-
blets, t = triplet, q = quartet, m = multiplet. Coupling constants J are
reported in Hertz. Low-resolution mass spectrometry (LRMS) and
high-resolution mass spectrometry (HRMS) were performed using
positive electron ionisation (+EI) on a Micromass VG Autospec
mass spectrometer or negative electrospray ionization (ÀESI) on
a Micromass ZMD ESI-Quad, or a Waters LCT Premier XE mass
spectrometer. Reactions were monitored by analytical thin layer
chromatography (TLC) using Merck Silica gel 60 TLC plates (7:2:1
ethyl acetate:methanol:water, unless otherwise specified) and
were visualised by staining with a solution of potassium perman-
ganate [KMnO4 (3 g), K2CO3 (20 g), NaOH (0.25 g), H2O (305 mL)],
with heating as required. Liquid chromatography mass spectrome-
try (LCMS) was performed using an Agilent Technologies Infinity
1260 LC system equipped with an Agilent 1290 HTS LC Injector
Sulfation was performed by minor modification of known liter-
ature procedures [17]. A solution of steroid (100 mg) in pyridine
(1 mL) was added drop-wise to solid SO3ÁNEt3 or SO3Ápy (1.1–
1.6 equiv). The resulting solution was stirred at room temperature
for 20 h unless otherwise specified. The reaction was then added to
diethyl ether (20 mL) and the resulting precipitate was collected by
filtration. The crude steroid sulfate salt was recrystallised from
refluxing dichloromethane/diethyl ether, washed with small por-
tions of cold diethyl ether and finally dried in vacuo to yield the
desired steroid sulfate as its corresponding triethylammonium or
pyridinium salt.
2.3.4. Testosterone 17-sulfate, ammonium salt 1a [17]
A solution of testosterone (1.0 mg, 3.47
(100
L) was treated with a solution of SO3Ápy (10.0 mg, 62.8
18.1 equiv) in DMF (100 L) and purified by SPE as per Section
lmol) in 1,4-dioxane
l
lmol,
l
2.3.1 to yield the title compound 1a as a white solid. Performing
the sulfation reaction as per Section 2.3.2 showed full conversion.
Rf 0.34; dH (400 MHz): 5.71 (s, 1H, C4-H), 4.34 (t, J 8.6 Hz, 1H,
C17-H), 2.53-0.95 (m, 19H), 1.24 (s, 3H, C18-H3), 0.87 (s, 3H,
C19-H3); dC (100 MHz): 202.4 (C3), 175.2 (C5), 124.1 (C4), 87.9
(C17), 55.4, 55.1, 51.3, 43.8, 40.0, 37.7, 36.8, 34.7, 33.9, 32.8, 29.1,
24.3, 21.6, 17.7 (C18), 12.0 (C19); LRMS (ÀESI): m/z 367 (100%,
[C19H27O5S]À); HRMS (ÀESI): found 367.1579, [C19H27O5S]À
requires 367.1579. Copies of the 400 MHz 1H NMR, 100 MHz 13C
and an Agilent 6120 Quadrupole detector. Injections (10
resolved with an Agilent Zorbax SP-C18 UPLC column (2.1 mm Â
50 mm, 1.8 m, 600 bar) with an isocratic mobile phase consisting
lL) were
l

76
C.C. Waller, M.D. McLeod / Steroids 92 (2014) 74–80
NMR and ÀESI LRMS are reproduced in the Supporting informa-
tion. Experimental details, data and spectra for the steroid sulfate
ammonium salts 2–13a presented in Table 1 and Scheme 2 are
reported in the Supporting information.
(400 MHz): 5.71 (s, 1H, C4-H), 4.23 (t, J 8.4 Hz, 1H, C17-H), 3.10 (q,
J 7.3 Hz, 6H, NCH2CH3), 2.55–0.90 (m, 19H), 1.32 (t, J 7.4 Hz, 9H,
NCH2CH3), 1.24 (s, 3H, C18-H3), 0.87 (s, 3H, C19-H3); dC
(100 MHz): 202.4 (C3), 175.2 (C5), 124.1 (C4), 87.8 (C17), 55.4,
51.3, 47.9 (NCH2CH3), 43.9, 40.0, 37.7, 36.8, 34.7, 33.9, 32.8, 29.2,
24.3, 21.7, 17.7 (C18), 12.1 (C19), 9.2 (NCH2CH3), one carbon over-
lapping or obscured; LRMS (ÀESI): m/z 367 (100%, [C19H27O5S]À);
HRMS (ÀESI): found 367.1579, [C19H27O5S]À requires 367.1579.
Copies of the 400 MHz 1H NMR, 100 MHz 13C NMR and ÀESI LRMS
are reproduced in the Supporting information. Experimental details,
data and spectra for the steroid sulfate triethylammonium salts 2–5,
2.3.5. Testosterone 17-sulfate, triethylammonium salt 1b [17]
Testosterone (239 mg, 0.83 mmol) in pyridine (2 mL) was trea-
ted with solid SO3ÁNEt3 (236 mg, 1.30 mmol, 1.6 equiv) and stirred
for 2 h as per Section 2.3.3 to yield the title compound 1b (155 mg,
40%) as an off white solid. Rf 0.34; mp 164–166 °C (lit [17] 158–
163 °C); [
a]
25 + 58 (c 10, CHCl3) (lit [17] [
a]
D
25 + 64 [CHCl3]); dH
D
Table 1
Synthesis of steroid sulfates using SPE purification.
Entrya
Steroid sulfate
Conversion (%)b
(Yield %)c
Entrya
Steroid sulfate
Conversion (%)b
(Yield)c
1ad,e
>98 (94)
(40)
(43)
7ad,e
>98
(63)
1bd,e,f
1cd,e,f
7bd,e,f
2ad,e
>98
(41)
8ad,e
>98
2bd,e,f
3ad,e
97
(21)
9ad,e
>98
(76)
3bd,e,f
9bd,e,f
4ad,e
>98
(54)
10ad,e
11ad,e
12ad,e
>98
>98
>98
4bd,e,f
5ad,e
>98
(42)
5bd,e,f
6ad,e
>98
(55)
6cd,e,f
a
b
c
d
e
f
Steroid sulfate prepared as: a, ammonium salt; b, triethylammonium salt; or c, pyridinium salt.
Conversion based on 400 MHz 1H NMR integration.
Isolated yield of pure steroid sulfate.
The 400 or 600 MHz 1H NMR and ESI MS is provided in the Supporting information.
The 100 or 150 MHz 13C NMR spectrum is provided in the Supporting information.
Conditions: steroid (1 equiv), SO3ÁNEt3/SO3Ápy (1.1–1.6 equiv), pyridine, RT, 2–20 h.

C.C. Waller, M.D. McLeod / Steroids 92 (2014) 74–80
77
7 and 9b presented in Table 1 are reported in the Supporting
information.
m/z 349 (100%, [C18H21O5S]À), 269 (40%), 97 (20%, [HSO4]À), 80 (25%,
[SO3]À); HRMS (ÀESI): found 349.1110, [C18H21O5S]À requires
349.1110. Copies of the 400 MHz 1H NMR, 100 MHz 13C NMR and
ÀESI LRMS are reproduced in the Supporting information.
2.3.6. Testosterone 17-sulfate, pyridinium salt 1c [25]
Testosterone (216 mg, 0.75 mmol) in pyridine (2 mL) was trea-
ted with solid SO3Ápy (134 mg, 0.84 mmol, 1.1 equiv) as per Section
2.3.3 to yield the title compound 1c (145 mg, 43%) as an off white
2.3.9. Estradiol (estra-1,3,5(10)-triene-3,17b-diol) 3-sulfate,
ammonium salt 15a
solid. Rf 0.34; mp 140–145 °C (lit [25] 138–140 °C); [
a]
25 + 52 (c
D
A solution of estrone 3-sulfate, ammonium salt 14a (derived
10, CHCl3) (lit [26] [
a]
D
25 + 200 [c 10, MeOH]); dH (400 MHz): 8.88
from estrone, 5.0 mg, 18.5
lmol) in methanol (100
lL) was slowly
(m, 2H, o-pyridinium), 8.68 (m, 1H, p-pyridinium), 8.14 (m, 2H, m-
pyridinium), 5.71 (s, 1H, C4-H), 4.30 (t, J 8.6 Hz, 1H, C17-H), 2.55–
0.86 (m, 19H), 1.24 (s, 3H, C18-H3), 0.86 (s, 3H, C19-H3); dC
(100 MHz): 202.4 (C3), 175.1 (C5), 148.5 (o-pyridinium), 143.0 (p-
pyridinium), 128.9 (m-pyridinium), 124.2 (C4), 87.9 (C17), 55.4,
51.3, 43.8, 40.1, 37.7, 36.7 (2 C), 32.7, 32.6, 29.2, 24.3, 21.7, 17.7
(C18), 12.1 (C19), one carbon overlapping or obscured; LRMS (ÀESI):
m/z 367 (100%, [C19H27O5S]À), 97 (20%, [HSO4]À); HRMS (ÀESI) found
367.1579, [C19H27O5S]À requires 367.1579. Copies of the 400 MHz
1H NMR, 100 MHz 13C NMR and ÀESI LRMS are reproduced in the
Supporting information. Experimental details, data and spectra for
etiocholanolone 3-sulfate pyridinium salt 6c presented in Table 1
is reported in the Supporting information.
added to solid sodium borohydride (7 mg, 185
l
mol 10.0 equiv)
with cooling on ice. After the vigorous reaction had subsided the
reaction was capped, allowed to warm to room temperature and
stirred for 4 h. The reaction was quenched by the slow addition
of water (3 mL), adjusted to pH 7 (universal indicator strips) by
addition of aqueous hydrochloric acid (0.1 M, ꢀ2 mL) and then
purified by SPE as per Section 2.3.1 to yield the title compound
15a as a white solid. Performing the reduction reaction above with
SPE purification as per Section 2.3.2 showed full conversion. Rf
0.54; dH (400 MHz): 7.23 (d, J 8.4 Hz, 1H, C1-H), 7.08–6.98 (m,
2H, C2-H and C4-H), 3.67 (t, J 8.6 Hz, 1H, C17-H), 2.89–2.83 (m,
2H, C6-H2), 2.40–1.15 (m, 13H), 0.78 (s, 3H, C18-H3); dC
(100 MHz): 151.7 (C3), 138.8, 138.1, 127.0, 122.5, 119.7, 82.5
(C17), 51.4, 45.5, 44.4, 40.3, 38.0, 30.7, 30.6, 28.4, 27.5, 24.0, 11.7
(C18); LRMS (ÀESI): m/z 351 (100%, [C18H23O5S]À); HRMS (ÀESI):
found 351.1266, [C18H23O5S]À requires 351.1266. Copies of the
400 MHz 1H NMR, 100 MHz 13C NMR and ÀESI LRMS are repro-
duced in the Supporting information.
2.3.7. Estrone (3-hydroxyestra-1,3,5(10)-triene-17-one) 3-sulfate,
ammonium salt 14a [23]
Chlorosulfonic acid [CAUTION! Corrosive. Use in an efficient
fume hood.] (10
lL, 150.0
lmol, 8.1 equiv) was slowly added to a
solution of estrone (5.0 mg, 18.5
l
mol) in pyridine (50 L) with
l
cooling on ice. After the vigorous reaction had subsided, the reac-
tion was capped, allowed to warm and then stirred at room tem-
perature for 20 h. The reaction was quenched by the slow
addition of water (1.5 mL) and then purified by SPE as per Section
2.3.1 to yield the title compound 14a as a yellow-brown solid. Per-
forming the sulfation reaction above with SPE purification as per
Section 2.3.2 showed 65% conversion. Rf 0.49; mp 150–155 °C; dH
(400 MHz): 7.25 (d, J 8.4 Hz, 1H, C1-H), 7.08–7.01 (m, 2H, C2-H
and C4-H), 2.95–2.87 (m, 2H, C6-H2), 2.55–1.40 (m, 13H), 0.92 (s,
3H, C18-H3); dC (100 MHz): 223.7 (C17), 151.8 (C3), 138.7, 137.6,
127.0, 119.8, 112.5, 51.7, 45.5, 39.7, 36.7, 32.8, 30.5, 27.6, 27.0,
23.3, 22.5, 14.2 (C18); LRMS (ÀESI): m/z 349 (100%, [C18H21O5S]À),
269 (40%), 97 (20%, [HSO4]À), 80 (25%, [SO3]À); HRMS (ÀESI): found
349.1110, ([C18H21O5S]À) requires 349.1110. Copies of the
400 MHz 1H NMR, 100 MHz 13C NMR and ÀESI LRMS are repro-
duced in the Supporting information.
2.3.10. Estrone 2-sulfonate, ammonium salt 16a
Employing conditions developed by Dusza, Joseph and Bern-
stein [19], solid estrone (5.0 mg, 18.5
lmol) was mixed with solid
SO3Ápy (15 mg, 94.2 mol, 5.1 equiv) and heated at 180 °C for
l
15 min in an oil bath. The resulting brown melt was allowed to
cool, quenched with water (1.5 mL) and then purified by SPE as
per Section 2.3.1 to yield the title compound 16a as a yellow-brown
solid. Performing the sulfation reaction above with SPE purification
as per Section 2.3.2 showed full conversion. Rf 0.36; mp 180–
183 °C; dH (400 MHz): 7.57 (s, 1H, C1-H), 6.59 (s, 1H, C4-H),
2.90–2.83 (m, 2H, C6-H2), 2.55–1.40 (m, 13H), 0.92 (s, 3H, C18-
H3); dC (100 MHz): 223. 7 (C17), 152.8, 142.7, 132.3, 127.3, 125.3,
117.7, 51.6, 45.1, 39.7, 36.7, 32.7, 30.3, 27.5, 26.9, 22.5, 14.3
(C18), one carbon overlapping or obscured; LRMS (ÀESI): m/z 349
(100%, [C18H21O5S]À), 97 (30%, [HSO4]À), 80 (20%, [SO3]À); HRMS
(ÀESI): found 349.1097, [C18H21O5S]À requires 349.1110. Copies
of the 400 MHz 1H NMR, 100 MHz 13C NMR and ÀESI LRMS are
reproduced in the Supporting information.
2.3.8. Estrone 3-sulfate, pyridinium salt 14c [25,26]
Chlorosulfonic acid [CAUTION! Corrosive. Use in an efficient
fume hood.] (620 lL, 9.33 mmol, 5.0 equiv) was added drop-wise
to a rapidly stirring solution of estrone (505 mg, 1.87 mmol) in pyr-
idine with cooling on ice. After the vigorous reaction subsided the
reaction was allowed to warm to room temperature and then stir-
red for 20 h. The reaction was then added to aqueous potassium
hydroxide solution (0.1 M, 30 mL) and extracted into ethyl acetate
(4 Â 50 mL) and 3:1 chloroform-isopropanol solution (4 Â 40 mL).
The combined organic extracts were dried with magnesium sulfate
and evaporated to dryness. The crude steroid sulfate salt was
recrystallised as per Section 2.3.3 to yield the title compound 14c
(199 mg, 25%) as a white solid. Rf 0.45; mp 165–169 °C (lit [26]
3. Results and discussion
3.1. Sulfation reaction conditions
Of the variety of conditions available in the literature, the appli-
cation of sulfur trioxide amine complexes appeared to offer the
greatest utility due to their commercial availability, ease of
i) SO3.py (10 mg)
DMF (100 µL)
1,4-dioxane
170–175 °C); [a] a]
25 + 79 (c 10, CHCl3) (lit [26] [ 25 + 84 [c 0.96,
D
D
OH
OSO3 NH4
(100 µL)
CHCl3]); dH (400 MHz): 8.88 (m, 2H, o-pyridinium), 8.68 (m, 1H, p-
pyridinium), 8.12 (m, 2H, m-pyridinium), 7.24 (d, J 8.4 Hz, 1H, C1-
H), 7.07–7.01 (m, 2H, C2-H and C4-H), 2.95–2.90 (m, 2H, C6-H2),
2.55–1.40 (m, 13H), 0.92 (s, 3H, C18-H3); dC (100 MHz): 224.1
(C17), 151.5 (C3), 148.2 (o-pyridinium), 142.8 (p-pyridinium),
138.2, 137.5, 128.9 (m-pyridinium), 127.2, 120.0, 112.7, 51.7, 45.4,
39.8, 36.8, 32.9, 30.3, 27.5, 27.0, 23.2, 22.6, 14.3 (C18); LRMS (ÀESI):
RT, 4 h
ii) SPE
O
O
testosterone (1 mg)
1a (>98% conversion)
Scheme 1. Small scale synthesis and purification of steroid sulfates.

78
C.C. Waller, M.D. McLeod / Steroids 92 (2014) 74–80
handling, reasonable stability to residual moisture and mild reac-
tion conditions as opposed to competing methods [14–23]. In our
hands sulfur trioxide pyridine complex could be briefly weighed
in the laboratory without special precautions. A solution of sulfur
trioxide pyridine complex in DMF (100 mg mLÀ1) was used for
the sulfation reactions and when stored in a sealed vial at 4 °C
maintained activity for 2 weeks. In contrast to typical steroid sulfa-
tion reactions which use pyridine as the reaction solvent [16–18],
DMF and 1,4-dioxane were used instead to maintain compatibility
with the SPE protocol (Section 2.3.1) and to reduce toxicity and
odour concerns. Our reaction protocol involved treatment of a
solution of steroid in 1,4-dioxane with a solution of excess sulfur
trioxide pyridine complex in DMF, followed by quenching with
water and SPE as shown in Scheme 1, below. Under these condi-
tions testosterone (1 mg) could be reliably converted to testoster-
one 17-sulfate 1a with >98% conversion. On a larger scale (10 mg)
synthesis of testosterone 17-sulfate 1a the isolated yield (94%)
showed reasonable concordance with this high conversion. These
conditions proved quite general for the synthesis of a wide range
of secondary alcohol-derived steroid sulfates 1–13a (Table 1,
Scheme 2). Modified conditions were required for the synthesis
of estrone 3-sulfate derivatives (Section 3.5 below).
be the case with rare or expensive steroids or isotope-labeled sam-
ples. To circumvent this problem we adopted SPE which has been
routinely used in anti-doping laboratories for the extraction of ste-
roids and other compounds from biological matrices such as blood
and urine [27–29]. Rather surprisingly, despite the extensive appli-
cation of SPE in chemical analysis, it has not been widely used for
the preparation of steroid sulfates, and where employed, typically
forms only part of the purification process [30–32].
Our SPE purification protocol adopted Oasis WAX cartridges
which contain a mixed-mode polymeric/weak anion exchange
resin allowing fractionation of the anionic steroid sulfate from
any residual neutral steroidal alcohol and other non-volatile reac-
tion components. Cartridges were pre-conditioned with methanol
followed by water then loaded directly with the quenched reaction
mixture. Washing the cartridge sequentially with aqueous formic
acid solution (2% v/v), water and finally methanol eluted any resid-
ual steroidal alcohol and other reaction components. A final elution
with saturated aqueous ammonia in methanol (5% v/v) afforded
the desired steroid sulfate as the ammonium salt after removal
of eluant under reduced pressure. Alternatively, the methanolic
ammonia fraction could be dried at 60 °C for 1 h under stream of
nitrogen to give the steroid sulfate, albeit with trace amounts of
ammonium formate observable in the 1H NMR spectrum. During
development, this SPE protocol was shown to cleanly and effi-
ciently separate an equimolar mixture of testosterone and testos-
terone 17-sulfate 1a. Purification of reactions conducted on 5 mg
of steroidal alcohol or less were readily conducted using a single
500 mg resin (6 cc) SPE cartridge, with larger scale synthesis
requiring purification in parallel.
To further confirm the identity of these steroid sulfate ammo-
nium salts prepared by small scale synthesis and SPE, a range of
known reference compounds were also prepared using larger scale
sulfation and recrystallisation [17] as either the triethylammonium
salts 1–5, 7 and 9b or pyridinium salts 1 and 6c (Table 1). These
materials allowed for comparison of the different salts by NMR
and MS as detailed below (Section 3.3).
The SPE protocol outlined above resulted in the efficient purifi-
cation of all steroid sulfates investigated with the exception of the
lithocholic acid 3-sulfate 9a. In this case the lithocholic acid start-
ing material contains a carboxylate sidechain that can also interact
with the anion exchange resin leading to co-elution with lithochol-
ic acid sulfate 9a in the methanolic ammonia wash. In this
instance, washing the cartridge with formic acid in methanol (2%
v/v, 15 mL) was employed to elute lithocholic acid, with lithocholic
acid sulfate 9a then eluted cleanly by methanolic ammonia in the
final step.
Given the typically small scale of the synthesis, determining the
mass of product and hence chemical yield with precision was not
feasible. To address this we elected to monitor the conversion of
starting material to product by 1H NMR integration. Omitting the
methanol wash step in the SPE method (Section 2.3.1) resulted in
the elution by methanolic ammonia of a combined fraction con-
taining both free steroid and steroid sulfate. This was then sub-
jected to 400 MHz 1H NMR integration of selected steroidal
protons in both the starting material and product allowing for
the determination of reaction conversion as reported in Table 1.
For the secondary steroidal alcohols studied, sulfation occurred
with 97% conversion or greater. This contrasted with the larger
scale sulfation reactions that employed purification by recrystalli-
sation, which afforded moderate 21–76% isolated yields (Table 1,
entries 1–7 and 9b/c).
3.2. Solid-phase extraction purification
Traditionally one or more recrystallizations or chromatographic
separations are required to purify steroid sulfate compounds to an
acceptable standard for analytical use [16–18]. This is not practical
however when utilising small quantities of material, as often might
i) SO3.pyridine
DMF
OH
OSO3 NH4
1,4-dioxane
RT, 4 h
ii) SPE
HO
HO
HO
estradiol
13a (>98% conversion)
i) ClSO3H
pyridine
0 °C–RT
20 h
O
X
Y
ii) SPE
NH4O3SO
estrone
14a X,Y=O (65% conversion)
14c (25% yield)
i) NaBH4
MeOH, RT, 4 h
ii) SPE
15a X=OH, Y=H
3.3. NMR and MS analysis of steroid sulfates
(>98% conversion)
For each steroid investigated, (Table 1, Scheme 2), conducting
the reaction on a 1–2 mg scale afforded sufficient pure steroid sul-
fate to conduct 400 or 600 MHz 1H NMR analysis. The compounds
obtained by this sulfation protocol were of high purity (>95%) as
assessed by this technique (copies of NMR spectra for each steroid
sulfate are reproduced in the Supporting information). For the sec-
ondary steroidal alcohols studied, sulfation resulted in a 0.58–
0.74 ppm downfield shift of the oxymethine proton at the reaction
site, as expected based on electronic considerations. For the pheno-
i) SO3.pyridine
O
O
180 °C
15 min
O3S
HO
NH4
ii) SPE
HO
estrone
16a (>98% conversion)
Scheme 2. Small scale sulfation of estrogenic steroids.

C.C. Waller, M.D. McLeod / Steroids 92 (2014) 74–80
79
lic substrates estrone and estradiol, sulfation similarly resulted in a
0.50 ppm downfield shift of the protons ortho to the sulfate ester
and a smaller downfield shift of 0.18 ppm for H1 relative to the
parent steroid. Overlay of the 1H NMR spectra derived from the ste-
roid sulfate ammonium salts 1–7, 9a and 14a prepared by small
scale synthesis and SPE with the triethylammonium salts 1–5, 7,
and 9b, or pyridinium salts 1, 6 and 14c, prepared by large scale
synthesis and recrystallisation showed identical signals for the
protons of the steroidal nucleus, further supporting the identity
of these compounds. Further, SPE purification of testosterone 17-
sulfate, pyridinium salt 1c by the general method (Section 2.3.1)
afforded testosterone 17-sulfate, ammonium salt 1a which was
identical in all respects with that obtained directly from testoster-
one using the direct small scale synthesis (Section 2.3.4).
Sulfate conjugates of the estrogens were therefore attractive
targets for synthesis. Attempted sulfation of estrone under stan-
dard conditions (Section 2.3.1) failed to afford the desired estrone
3-sulfate, reflecting significant differences in reactivity for aliphatic
versus phenolic hydroxyl groups. This is despite literature reports
describing the formation of phenolic sulfates under similar condi-
tions [17,18]. This observation proved useful for the selective sul-
fation under standard conditions (Section 2.3.1) of estradiol to
afford estradiol 17-sulfate 13a in which reaction occurs at the sec-
ondary alkyl C17 hydroxyl group (Scheme 2) [18].
A range of conditions were investigated in efforts to effect sul-
fation of estrone on a small scale. Employing an excess of chloro-
sulfonic acid [CAUTION] in pyridine gave estrone 3-sulfate 14a
with 65% conversion [15]. Fortunately, these conditions were com-
patible with our existing SPE method (Section 2.3.1) providing the
sulfate in pure form. A 0.50 ppm downfield shift the H2 and H4
protons were observed in the 1H NMR spectrum consistent with
sulfation at H3. To extend this chemistry, sodium borohydride
reduction of the resulting estrone 3-sulfate 14a was used to selec-
tively afford estradiol 3-sulfate 15a after SPE purification.
During this study we found that the literature contained little or
no characterization data for several of the steroid sulfate ammo-
nium salt products that were prepared in the screen. To address
this, we scaled up our syntheses of these steroid sulfates 3–6, 8,
13 and 15a specifically those derived from androstanolone (17b-
hydroxy-5a-androstan-3-one), androsterone, epiandrosterone,
estradiol, etiocholanolone and methandriol, obtaining full 1H, 13C
NMR, and MS data for each. Further, both the small and large scale
synthesis provided significant additional 1H, 13C NMR, and MS
data, as well as spectra for the majority of steroid sulfates shown
in Table 1. The designer steroids furazadrol (17b-hydroxyandro-
stan[2,3-d]isoxazole), isofurazadrol (17b-hydroxyandrostan[3,2-
c]isoxazole) and trenazone (17b-hydroxyestra-4,9-dien-3-one)
gave rise to previously un-reported sulfate conjugates 10–12a that
were also prepared on a scale suitable for full characterization.
Mass spectrometry was also used to characterize the steroid
sulfates prepared. In each case the steroid sulfate, ammonium salts
exhibited satisfactory purity and identity by ÀESI LRMS and HRMS
respectively. Further the steroid sulfate ammonium salts 1–7, 9
and 14a prepared by small scale synthesis and SPE showed identi-
cal, albeit simple, ÀESI LRMS behavior with the triethylammonium
salts 1–5, 7 and 9b, or pyridinium salts 1, 6 and 14c, prepared by
large scale synthesis and recrystallisation.
During efforts to obtain estrone 3-sulfate we also investigated
the so-called ‘‘fusion’’ method whereby a neat mixture of the ste-
roid and sulfur trioxide pyridine complex are heated together at
180 °C, thereby conducting the sulfation reaction in the melt
[19]. This method led to the unexpected formation of the isomeric
steroid derivative estrone 2-sulfonate 16a, a product of electro-
philic aromatic substitution presumably promoted by the high
reaction temperature. The 400 MHz 1H NMR spectrum showed
two singlets at d 7.57 and 6.59 consistent with the proposed struc-
ture. This result suggests some caution is warranted in applying
the fusion method for the synthesis of aromatic sulfates.
3.6. Sulfation on the microgram scale
Finally, we wished to explore the suitability of this approach to
the synthesis of analytical quantities of material by applying this
protocol to a microgram scale synthesis of testosterone 17-sulfate
1a. Success with such small quantities would have important
applications for the sulfation of rare and expensive steroid
compounds such as novel designer steroids or isotope labeled com-
pounds [38] where significant time, resource or financial costs are
involved in obtaining sufficient quantities of material for analysis.
In addition, it raised the prospect of generating phase II steroid sul-
fates from phase I steroid metabolites isolated from biological sam-
ples. When the protocol was applied to a series of reactions with
3.4. Sulfation of secondary steroidal alcohols
The small scale synthesis and purification of steroid sulfates
proved highly effective for a wide variety of secondary steroidal
alcohol substitution patterns and configurations (Table 1,
Scheme 2). For methandriol (Table 1, entry 8a), which possesses
both secondary 3b- and tertiary 17b-hydroxyl groups, mono-sulfa-
tion was observed to take place regioselectively at the secondary
testosterone (100–1
the product steroid sulfate by single ion monitoring (SIM, m/z
À367) even at the lowest concentration trialled (1 g), and near-
quantitative conversion (1.28 0.04 testosterone sulfate,
lg), analysis by LCMS allowed detection of
hydroxyl group to cleanly give methandriol 3-sulfate 8a.
A
0.74 ppm downfield shift the H3 proton was observed in the 1H
NMR spectrum consistent with sulfation at C3 as expected based
on steric considerations. The sulfation of tertiary hydroxyl groups
has been reported on a larger scale with sulfur trioxide pyridine
complex [33] or the more reactive chlorosulfonic acid reagent
[34]. The tertiary alkyl sulfate esters so derived are typically unsta-
ble and undergo elimination, rearrangement or hydrolysis under
ambient conditions [33,35,36]. The sulfation of lithocholic acid
which possesses both a secondary 3b-hydroxyl group and a car-
boxylic acid also proceeded without incident using a modified
SPE method.
l
l
g
95 3% HPLC yield, mean sem, n = 3) to the desired sulfate was
still observed at these levels. These results highlight the power of
this approach for the small scale synthesis of steroid sulfate com-
pounds for analytical purposes.
4. Conclusion
Employing a small scale sulfation protocol with purification by
SPE, sixteen steroid sulfates have been synthesised on a milligram
scale with near-complete conversion, including the previously
unreported sulfate conjugates of the designer steroids furazadrol,
isofurazadrol and trenazone. The scope of this approach for small
3.5. Sulfation of estrogenic steroidal alcohols
Although estrogenic steroids are not typically considered candi-
dates for use as performance enhancing substances in sport, they
may offer indirect advantages [37]. Additionally, these compounds
and their in vivo metabolites play host a wide range of biological
functions that have important applications in medical science [3].
scale synthesis is highlighted by the sulfation of 1 lg of testoster-
one as monitored by LCMS. This quick and simple protocol employs
commercially available consumables and reagents and allows the
rapid and efficient synthesis and purification of steroid sulfate

80
C.C. Waller, M.D. McLeod / Steroids 92 (2014) 74–80
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Acknowledgements
We thank the Australian Research Council (LP120200444) for
financial support. We thank Dr Bradley Stevenson for his assistance
with LCMS.
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Appendix A. Supplementary data
Supplementary data (experimental procedures, characterisation
data, NMR and MS spectra for all compounds) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.steroids.2014.09.006.
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