Synthesis of 3- and 21-monosulfates of [2,2,3β,4,4-2H 5]-tetrahydrocorticosteroids in the 5β-series as internal standards for mass spectrometry

Article abstract of DOI:10.1016/j.steroids.2011.05.014

The 3- and 21-monosulfates of pentadeuterated 5β- tetrahydrocorticosteroides were synthesized, starting from cortisol and 11-deoxycotisol. The principal reactions used were (1) perdeuteration of the methylene groups adjacent to the 3-oxo group of 17,20:20,21-bismethylendioxy- 5β-3-ketosteroids with NaOD in CH₃OD followed by stereoselective reduction with NaBD₄, (2) sulfation of hydroxy groups with sulfur trioxide-trimethylamine complex, and (3) removal of the 17,20:20,21- bismethylendioxy group with hydrogen fluoride. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.

Full text of DOI:10.1016/j.steroids.2011.05.014

Steroids 76 (2011) 1232–1240
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of 3- and 21-monosulfates of [2,2,3b,4,4-2H5]-tetrahydrocorticosteroids
in the 5b-series as internal standards for mass spectrometry
a
a
Shigeo Ikegawa a, , Kaori Nagae , Takayuki Mabuchi , Rika Okihara a,1, Maki Hasegawa a,2
,
⇑
Toshie Minematsu a, Takashi Iida b, Kuniko Mitamura a
a Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
b Department of Chemistry, College of Humanities & Sciences, Nihon University, Sakurajousui, Setagaya-ku, Tokyo 156-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 April 2011
Received in revised form 25 May 2011
Accepted 30 May 2011
Available online 25 June 2011
The 3- and 21-monosulfates of pentadeuterated 5b-tetrahydrocorticosteroides were synthesized, starting
from cortisol and 11-deoxycotisol. The principal reactions used were (1) perdeuteration of the methylene
groups adjacent to the 3-oxo group of 17,20:20,21-bismethylendioxy-5b-3-ketosteroids with NaOD in
CH3OD followed by stereoselective reduction with NaBD4, (2) sulfation of hydroxy groups with sulfur tri-
oxide–trimethylamine complex, and (3) removal of the 17,20:20,21-bismethylendioxy group with hydro-
gen fluoride. The labeled compounds can be used as internal standards in liquid chromatography/mass
spectrometry assays for clinical and biochemical studies.
Keywords:
Multiple deuteration
Hydrogen–deuterium exchange
Sodium borodeuteride
Internal standard
Ó 2011 Elsevier Inc. All rights reserved.
Electrospray ionization
Mass spectrometry
1. Introduction
8]. Tetrahydro-11-deoxycortisol (THS) and its 5a-stereoisomer
(allo-THS) which are tetrahydro-reduced metabolites of 11-deoxy-
cortisol, a biosynthetic precursor of cortisol, are also excreted in ur-
ine as conjugated forms. Measurement of the urinary excretion of
glucocorticoids (GCs) and their metabolites provides a non-inva-
sive, integrated evaluation of adrenocortical GC secretion, enabling
the diagnosis of GC hypersecretion as occurs in Cushing’s syn-
drome and the metabolic syndrome [9–14].
Glucocorticoids (cortisol and cortisone in man) are synthesized
in and secreted from the zona fasciculata of the adrenal grand,
under the control of the adrenocorticotropic hormone (ACTH, also
known as corticotropin) which is secreted from the anterior lobe of
the pituitary gland [1–3]. They are metabolized by several
enzymes, including irreversible inactivation by A-ring reductases
(5
a
- and 5b-reductase). They also undergo reversible activation
Stable isotope dilution mass spectrometry is widely accepted as
the most accurate and specific method for estimation of the small
amounts of endogenous and synthetic steroids in biological fluids.
We have previously reported a highly sensitive and specific liquid
chromatography (LC)/electrospray ionization (ESI)–linear ion trap
mass spectrometry (MS) method using a deuterium-labeled inter-
nal standard for the direct measurement of 12 tetrahydrocorticos-
teroid glucuronides in human urine [14]. In addition, we have
reported the synthesis of the 18 sulfated conjugates of tetrahydro-
corticosteroids as reference standards [15]. The availability of such
standards should permit the identification and measurement of all
of the sulfate conjugates found in urine using LC/ESI-MS and in
turn would permit assessment of the dynamics of their formation
and disposal. One significant drawback of such sulfate conjugate
analysis, however, is the lack of stable isotope labeled internal
standards.
and inactivation mediated by 11b-hydroxysteroid dehydrogenase
that converts active cortisol to inactive cortisone as well as
converting tetrahydrocortisol (THF) to tetrahydrocortisone (THE)
[3,4]. Further metabolism such as sulfation or glucuronidation
(phase 2 biotransformation, conjugation) decreases the biological
activity of hormones, decreases protein binding in plasma, and
renders them substrates for organic anion transporters, thus
promoting their elimination in urine and bile. At least 90% of the
tetrahydro-derivatives of cortisol and cortisone metabolites are
excreted into the urine as sulfate or glucuronide conjugates [5–
⇑
Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6730 1394.
E-mail address: ikegawa@phar.kindai.ac.jp (S. Ikegawa).
Present address: Faculty of Pharmaceutical Sciences, Setsunan University, 45-1
1
Nagatoge-cho, Hirakata, Osaka 575-0101, Japan.
The present paper describes the chemical synthesis of the 3-
and 21-sulfated conjugates of tetrahydrocorticosteroids (THS,
2
Present address: Sumika Chemical Analysis Service, Ltd., 3-1-135 Kasugade-Naka,
Konohana-ku, Osaka 554-0022, Japan.
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.05.014

S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
1233
O
O
2.3. Chemical synthesis
2.3.1. 17,20:20,21-Bismethylenedioxy-4-pregnen-3-one (2)
CH2OH
OH
CH2OSO3H
OH
R
R
D
D
D
To a solution of 11-deoxycortisol (1, 2 g) in CHCl3 (25 ml) was
added 7 M HCl (40 ml) containing paraformaldehyde (4.5 g), and
the mixture was stirred at room temperature for 5 h. The CHCl3
was separated and the aqueous phase was extracted with CHCl3.
The combined extract was washed with 5% NaHCO3 and H2O, dried
over anhydrous Na2SO4, and evaporated to dryness. Purification of
the product by column chromatography on silica gel with toluene–
acetone (20:1, v/v) as an eluent and recrystallization of a homoge-
nous effluent from CH2Cl2–MeOH gave 2 as colorless prisms: yield,
1.6 g (71%); mp 255–257 °C (lit. [16], mp 254–255 °C). 1H NMR
(CDCl3) d: 0.87 (s, 3H, 18-CH3), 1.20 (s, 3H, 19-CH3), 3.98 and
4.01 (d, each 1H, J = 9.2 Hz, 21-CH2), 5.04 (s, 2H, OCH2O), 5.07
and 5.20 (s, each 1H, OCH2O), 5.73 (s, 1H, 4-H).
D
D
D
HO3SO
HO
H
H
D
D
D
D
THS-21-Sulfate: R1=H2
THF-21-Sulfate: R1=β-OH
THE-21-Sulfate: R=O
THS-3-Sulfate: R1=H2
THF-3-Sulfate: R1=β-OH
THE-3-Sulfate: R=O
Fig. 1. Structures of 3- and 21-monosulfate conjugates of [2,2,3b,4,4-d5]-tetrahy-
drocorticosteroids in the 5b-series.
THF, and THE) at C-2, C-3, and C-4 positions with deuterium atom
for use of isotope dilution mass spectrometry (Fig. 1).
2.3.2. 17,20:20,21-Bismethylenedioxy-5b-pregnan-3-one (3)
A solution of 2 (1 g) in pyridine (34 ml) was hydrogenated over-
night at room temperature in the presence of 5% Pd/CaCO3 catalyst
(2 g). After filtration of the catalyst on celite, the filtrate was con-
centrated to dryness under reduced pressure. Recrystallization of
the product from CH2Cl2–MeOH gave 3 as colorless plates: yield,
767 mg (76%); mp 182–185 °C (lit. [16], mp 180.5 °C). 1H NMR
(CDCl3) d: 0.85 (s, 3H, 18-CH3), 1.03 (s, 3H, 19-CH3), 3.98 and
4.01 (d, each 1H, J = 9.2 Hz, 21-CH2), 5.05, 5.06, 5.09, and 5.20 (s,
each 1H, 2Â OCH2O).
2. Experimental
2.1. Materials
11-Deoxycortisol (1) and cortisol (7) were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Silica gel plates
(Merck; F254) and silica gel (Merck; 70–230 mesh) were used for
analytical and column chromatography, respectively. 40% NaOD
in D2O (99.5 atom% D) and CH3OD (99.5 atom% D) were supplied
by Sigma-Aldrich Japan K. K. (Tokyo, Japan). NaBD4 (99% isotopic
purity) was obtained from Cambridge Isotope Laboratories, Inc.
(Andover, MA, USA). Acetonitrile and ammonium acetate of HPLC
grade were purchased from Nacalai Tesque, Inc. (Kyoto, Japan),
and distilled H2O of HPLC grade was purchased from Wako Pure
Chemical Industries, Ltd. An OasisÒ HLB cartridge (adsorbent
weight, 1 g) was provided by Waters Co. (Milford, MS, USA) and
successively conditions by washings with MeOH and H2O prior
to use. All other chemicals and solvents were analytical grade
and obtained from Nacalai Tesque Inc.
2.3.3. [2,2,3b,4,4-d5]-17,20:20,21-Bismethylenedioxy-3a-hydroxy-5b-
pregnane (4a)
To a solution of 3 (200 mg) in CH3OD (10 ml) and dry dioxane
(1.2 ml) was added 40% NaOD (1 ml) and D2O (1.2 ml), and the
mixture was stirred at 60 °C for 22 h under a gentle stream of N2
gas. After being cooled in an ice bath, NaBD4 (40 mg) was added
to the solution and the reaction mixture was stirred at room tem-
perature for 30 min. After addition of 10% AcOH to decompose the
excess reagents, the organic solvent was evaporated under reduced
pressure. The residue was diluted with EtOAc, washed with 5%
NaHCO3 and H2O, dried over anhydrous Na2SO4, and evaporated
to dryness. Purification of the crude product by column chroma-
tography on silica gel with n-hexane–EtOAc (5:1, v/v) as an eluent
and recrystallization of a homogeneous effluent from acetone–n-
hexane gave 4a as colorless plates: yield, 128 mg (64%); mp 180–
181 °C. Mixed mp with the non-deuterated compound (lit. [16],
mp 179–180 °C) showed no depression. 1H NMR (CDCl3) d: 0.74
(s, 3H, 18-CH3), 0.86 (s, 3H, 19-CH3), 3.90 and 3.93 (d, each 1H,
J = 9.4 Hz, 21-CH2), 4.97, 4.98, 5.01, and 5.12 (s, each 1H, 2Â
OCH2O).
2.2. Instruments
All melting points (mp) were determined on a micro hot-stage
apparatus and are uncorrected. 1H NMR spectra were recorded on
a JNM-AL400 (JEOL Ltd., Tokyo, Japan) at 400 MHz, with CDCl3 or
CD3OD containing 0.1% Me4Si as the solvent; chemical shifts
were expressed as d ppm relative to Me4Si. The following abbre-
viations are used: s = singlet, d = doublet, m = multiplet. The LC/
ESI-MS analyses were carried out using a Finnigan LTQ linear
ion-trap mass spectrometer (Thermo Fischer Scientific Inc., Wal-
tham, MA, USA) equipped with an ESI source and coupled to a
Paradigm MS4 pump (Michrom Bioresources Inc., Auburn, CA,
USA) and an autosampler (HTC PAL, CTC Analytics, Zwingen,
Switzerland). The ionization conditions for verifying the struc-
tures were as follows: ion source voltage, À4 kV; capillary tem-
perature, 270 °C; capillary voltage, À20 V; sheath gas (N2 gas)
flow rate, 50 arbitrary units; auxiliary gas (N2 gas) flow rate, 5
arbitrary units; tube lens offset voltage, À100 V. For collision in-
duced dissociation (CID) analysis, helium gas was used as the col-
lision gas. The normalized collision energy and the activation Q
value were set at 35% and 0.18, respectively. LC separations were
conducted on a reversed-phase (RP) semi-micro column, TSKgel
2.3.4. [2,2,3b,4,4-d5]-17a,21-dihydroxy-3a-sulfooxy-5b-pregnan-20-
one sodium salt ([2,2,3b,4,4-d5]-THS-3-sulfate sodium salt; 5)
To a solution of compound 4a (112 mg) in dry pyridine (3 ml)
was added sulfur trioxide–trimethylamine (SO3–TMA) complex
(60 mg) and the mixture was stirred at room temperature for
3 h. After evaporation of the solvent under reduced pressure, the
residue dissolved in a small amount of H2O was adjusted to pH 8
with 20% NaOH and loaded onto an OasisÒ HLB cartridge. After
being washed with H2O, elution with MeOH gave the intermediary
3a-sulfate (4b), which without isolation it was treated with 46%
hydrogen fluoride (3 ml) in EtOH (0.4 ml)–tetrahydrofuran
(0.4 ml) and kept at ice temperature for 7 h. After addition of
saturated Na2CO3 to adjust to pH 8, the resulting solution was
diluted with H2O and centrifuged at 3000 rpm for 10 min. The
supernatant was loaded onto an OasisÒ HLB cartridge. After being
washed with H2O, the desired sulfate was eluted with MeOH.
Purification of the product by column chromatography on silica
ODS-100S (5
l
m, 150 Â 2.0 mm I.D.) from Tosoh Co. (Tokyo, Ja-
pan) by a linear gradient elution: 15% solvent B (acetonitrile)
over 5 min and then 15% solvent B to 50% B against solvent A
(5 mM ammonium acetate buffer, pH 6.0) over 30 min at a flow
rate of 200 ll/min.

1234
S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
gel with CHCl3–MeOH (5:1, v/v) as an eluent and recrystallization
of a homogenous effluent from Et2O–MeOH gave 5 as colorless
amorphous substances: yield, 75 mg (58%); mp 195 °C (dec.).
Mixed mp with the non-deuterated compound [15] showed no
depression. 1H NMR (CD3OD) d: 0.60 (s, 3H, 18-CH3), 0.95 (s, 3H,
19-CH3), 4.28 and 4.64 (d, each 1H, J = 19.2 Hz, 21-CH2). ES-MS
analysis indicated d0 (0.00%), d1 (0.00%), d2 (0.00%), d3 (0.11%), d4
(8.52%), d5 (91.37%).
and 4.01 (d, each 1H, J = 9.4 Hz, 21-CH2), 4.40–4.43 (m, 1H, 11a-
H), 5.02, 5.03, 5.05 and 5.22 (s, each 1H, 2Â OCH2O), 5.68 (s, 1H,
4-H).
2.3.8. 17,20:20,21-Bismethylenedioxy-11b-hydroxy-5b-pregnan-3-
one (9)
The compound 8 (1.14 g) was hydrogenated with 10% Pd(OH)2/
C (114 mg) in pyridine (10 ml), as described for preparation of 3.
After being processed in an analogous manner, the product was
recrystallized from acetone–MeOH to give 9 as colorless needles:
yield, 770 mg (67%); mp 239–241 °C (lit. [21], 239–240 °C). 1H
NMR (CDCl3) d: 1.10 (s, 3H, 18-CH3), 1.27 (s, 3H, 19-CH3), 3.98
2.3.5. [2,2,3b,4,4-d5]-3
one (6a)
a-Acetoxy-17a,21-dihydroxy-5b-pregnan-20-
The compound 4a (123 mg) was acetylated with Ac2O (0.25 ml)
in pyridine (0.5 ml) at room temperature for 20 h. After addition of
H2O, the resulting solution was extracted with EtOAc. The organic
layer was washed with 5% HCl, 5% NaHCO3, and H2O, and dried
over anhydrous Na2SO4. Evaporation of the solvent under reduced
and 4.02 (d, each 1H, J = 8.8 Hz, 21-CH2), 4.33–4.38 (m, 1H, 11
a-
H), 5.04, 5.05, 5.06, and 5.23 (s, each 1H, 2Â OCH2O).
2.3.9. Reduction of 9 with sodium borohydride
pressure afforded the 3
a
-acetoxy derivative (4c), which without
To a solution of 9 (100 mg) in MeOH (0.3 ml)–tetrahydrofuran
(0.4 ml) was added NaBH4 (60 mg) and the mixture was stirred
at room temperature for 30 min. After addition of 10% AcOH to
decompose the excess reagent, the resulting solution was diluted
with EtOAc, washed with 5% NaHCO3 and H2O, dried over anhy-
drous Na2SO4, and evaporated to dryness. Purification of the crude
product by column chromatography on silica gel with n-hexane–
EtOAc (1:1, v/v) and recrystallization of a less polar effluent from
acetone–n-hexane gave 17,20:20,21-bismethylenedioxy-3b,11b-
dihydroxy-5b-pregnane (10a) as colorless plates: yield, 11 mg
(11%); mp 203–205 °C. 1H NMR (CDCl3) d: 1.07 (s, 3H, 18-CH3),
1.22 (s, 3H, 19-CH3), 3.96 and 4.02 (d, each 1H, J = 8.8 Hz, 21-
isolation it was treated with 46% hydrogen fluoride (3 ml) in EtOH
(0.5 ml)–tetrahydrofuran (0.5 ml) at ice temperature for 7 h. After
addition of saturated Na2CO3 to adjust the pH to 8, the resulting
solution was extracted with EtOAc. The organic layer was washed
with H2O, dried over anhydrous Na2SO4, and evaporated to dry-
ness. Purification of the crude product by column chromatography
on silica gel with n-hexane–EtOAc (1:1, v/v) as an eluent and
recrystallization of a homogenous effluent from acetone–n-hexane
gave 6a as colorless needles: yield, 67 mg (55%); mp 197–198 °C.
Mixed mp with the non-deuterated compound [17,18] showed
no depression. 1H NMR (CDCl3) d: 0.65 (s, 3H, 18-CH3), 0.93 (s,
3H, s, 19-CH3), 2.03 (s, 3H, OCOCH3), 4.30 and 4.66 (d, each 1H,
J = 20.0 Hz, 21-CH2).
CH2), 4.08–4.12 (m, 1H, 3a-H), 4.26–4.28 (m, 1H, 11a-H), 5.04,
5.05, 5.06, and 5.22 (s, each 1H, 2Â OCH2O). Recrystallization of a
more polar effluent from acetone–n-hexane gave 17,20:20,21-
2.3.6. [2,2,3b,4,4-d5]-3
a
,17
a
-Dihydroxy-21-sulfooxy-5b-pregnan-20-
bismethylenedioxy-3a,11b-dihydroxy-5b-pregnane (10b) as color-
one sodium salt ([2,2,3b,4,4-d5]-THS-21-sulfate sodium salt; 6c)
To a solution of 6a (39 mg) in dry pyridine (0.8 ml) was added
SO3–TMA complex (20 mg), and the mixture was stirred at room
temperature for 5 h. After evaporation of the solvent under re-
duced pressure, the residue was dissolved in a small amount of
H2O, the pH adjusted to pH 8 with 20% NaOH, and the solution
loaded onto an OasisÒ HLB cartridge. After being washed with
less plates: yield, 85 mg (85%); mp 216–218 °C. 1H NMR (CDCl3) d:
1.06 (s, 3H, 18-CH3), 1.18 (s, 3H, 19-CH3), 3.62–3.70 (m, 1H, 3b-H),
3.96 and 4.01 (d, each 1H, J = 8.8 Hz, 21-CH2), 4.26–4.28 (m, 1H,
11a-H), 5.04, 5.05, 5.06, and 5.22 (s, each 1H, 2Â OCH2O).
2.3.10. 3 -Acetoxy-17,20:20,21-bismethylenedioxy-11b-hydroxy-5b-
a
pregnan (10c)
H2O, elution with MeOH gave the intermediary 3
a
-acetoxy-21-
The compound 10b (80 mg) was acetylated with Ac2O (0.5 ml)
in pyridine (1 ml) at room temperature for 10 h. After addition of
H2O, the resulting solution was extracted with EtOAc. The organic
layer was washed with 5% HCl, 5% NaHCO3, and H2O, dried over
anhydrous Na2SO4, and evaporated to dryness. Recrystallization
of the product from acetone gave 10c as colorless pillars: yield,
89 mg (100%); mp 209–211 °C. 1H NMR (CDCl3) d: 1.00 (s, 3H,
18-CH3), 1.16 (s, 3H, 19-CH3), d: 1.99 (s, 3H, OCOCH3), 3.95–3.98
sulfate (6b), which without isolation it was hydrolyzed with 20%
NaOH (0.5 ml) in MeOH (1.5 ml) at room temperature for 1 h. After
evaporation of the solvent, the residue dissolved in H2O was loaded
onto an OasisÒ HLB cartridge. After being washed with H2O, the de-
sired sulfate was eluted with MeOH. Purification of the crude prod-
uct by column chromatography on silica gel with CHCl3–MeOH
(4:1, v/v) as an eluent and recrystallization of a homogeneous
effluent from Et2O–MeOH gave 6c as colorless amorphous sub-
stances: yield, 15 mg (33%); mp 190 °C (dec.). Mixed mp with the
non-deuterated compound [15] showed no depression. 1H NMR
(CD3OD) d: 0.62 (s, 3H, 18-CH3), 0.93 (s, 3H, 19-CH3), 4.82 and
5.16 (d, each 1H, J = 18.0 Hz, 21-CH2). ESI-MS analysis indicated
d0 (0.02%), d1 (0.01%), d2 (0.01%), d3 (0.05%), d4 (10.15%), d5
(89.76%).
(d, each 1H, J = 9.0 Hz, 21-CH2), 4.23–4.25 (m, 1H, 11a-H), 4.68–
4.76 (m, 1H, 3b-H), 5.02 (s, 2H, OCH2O), 5.04 and 5.20 (s, each
1H, OCH2O).
2.3.11. 3a-Acetoxy-17,20;20,21-bismethylenedioxy-5b-pregnan-11-
one (11a)
To a stirred solution of 10c (258 mg) in acetone (7 ml) was
added Jones’ reagent (2.1 ml), and the mixture was continued to
stir at room temperature for 30 min. After addition of MeOH to
decompose the excess reagent, the organic solvent was evaporated
under reduced pressure. The residue was dissolved in EtOAc,
washed with 5% NaHCO3 and H2O, and dried over anhydrous
Na2SO4. After evaporation of the solvent, the crude product was
purified by column chromatography on silica gel with n-hexane–
EtOAc (1:1, v/v) as an eluent to give 11a as colorless amorphous
substances: yield, 187 mg (83%). 1H NMR (CDCl3) d: 0.70 (s, 3H,
18-CH3), 1.09 (s, 3H, 19-CH3), 1.95 (s, 3H, OCOCH3), 3.87 and
3.91 (d, each 1H, J = 9.4 Hz, 21-CH2), 4.60–4.68 (m, 1H, 3b-H),
4.94, 5.00, 5.01, and 5.12 (s, each 1H, 2Â OCH2O).
2.3.7. 17,20:20,21-Bismethylenedioxy-11b-hydroxy-4-pregnen-3-one
(8)
Cortisol (7, 4 g) was treated with 7 M HCl (40 ml) containing
paraformaldehyde (4.5 g) in CHCl3 (40 ml), as described for prepa-
ration of 2. After being processed in an analogous manner, the
product was subjected to column chromatography on silica gel
with toluene–acetone (5:1, v/v) as an eluent. Recrystallization of
a homogenous effluent from acetone–MeOH gave 8 as colorless
needles: yield, 2.91 g (65%); mp 230–232 °C (lit. [19], mp 220–
223 °C from Et2O–MeOH and lit. [20], 222–227 °C from EtOAc).
1H NMR (CDCl3) d: 1.13 (s, 3H, 18-CH3), 1.45 (s, 3H, 19-CH3), 3.98

S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
1235
2.3.12. 17,20:20,21-Bismethylenedioxy-3a-hydroxy-5b-pregnan-11-
J = 9.0 Hz, 21-CH2), 4.24–4.25 (m, 1H, 11
a
-H), 5.05 (s, 2H, OCH2O),
one (11b)
5.06 and 5.22 (s, each 1H, OCH2O).
To a solution of 11a (168 mg) in MeOH (6.5 ml) was added 20%
NaOH (0.7 ml), and the mixture was stirred at room temperature
for 1 h. After evaporation of the solvent, the residue was diluted
with EtOAc, washed with H2O, dried over anhydrous Na2SO4, and
evaporated to dryness. Purification of the crude product by column
chromatography on silica gel with n-hexane–EtOAc (1:1, v/v) as an
eluent and recrystallization of a homogenous effluent from ace-
tone–n-hexane gave 11b as colorless needles: yield, 128 mg
(84%); mp 191–193 °C. 1H NMR (CDCl3) d: 0.77 (s, 3H, 18-CH3),
1.15 (s, 3H, 19-CH3), 3.60–3.67 (m, 1H, 3b-H), 3.94 and 3.97 (d,
each 1H, J = 9.2 Hz, 21-CH2), 5.01, 5.07, 5.08, and 5.19 (s, each
1H, 2Â OCH2O).
2.3.16. [2,2,3b,4,4-d5]-3
pregnan-20-one (13b)
a-Acetoxy-11b,17a,21-trihydroxy-5b-
The compound 12c (134 mg) was treated with 46% hydrogen
fluoride (3.5 ml) in EtOH (0.8 ml)–tetrahydrofuran (0.8 ml) at ice
temperature for 6 h, as described for 6a. After being processed in
an analogous manner, the crude product was purified by column
chromatography on silica gel with n-hexane–EtOAc (3:1, v/v) as
an eluent. Recrystallization of a homogeneous effluent from ace-
tone–n-hexane gave 13b as colorless needles: yield, 83 mg (68%);
mp 192–194 °C. Mixed melting point with the non-deuterated
compound [22] showed no depression. 1H NMR (CDCl3) d: 0.81
(s, 3H, 18-CH3), 1.12 (3H, s,19-CH3), 1.96 (3H, s, 3-OCOCH3), 4.22
and 4.58 (d, each 1H, J = 19.0 Hz, 21-CH2), 4.23–4.24 (m, 1H, 11a-
H).
2.3.13. [2,2,3b,4,4-d5]-17,20:20,21-Bismethylenedioxy-3
dihydroxy-5b-pregnan (12a)
a,11b-
To a solution of 9 (200 mg) in dry dioxane (1 ml)–CH3OD (3 ml)
were added 40% NaOD (1 ml) and D2O (1.4 ml), and the solution
was heated at 60 °C for 22 h under a gentle stream of N2 gas. After
cooling down, NaBD4 (40 mg) was added to the mixture, and the
resulting solution was stirred at room temperature for 30 min.
After addition of 10% AcOH to decompose the excess reagent, the
resulting solution was diluted with EtOAc, washed with 5% NaH-
CO3 and H2O, dried over anhydrous Na2SO4, and evaporated to dry-
ness. Purification of the crude product by column chromatography
on silica gel with n-hexane–EtOAc (2:1, v/v) as an eluent and
recrystallization of a homogeneous effluent from acetone–n-hex-
ane gave 12a as colorless needles: yield, 184 mg (90%); mp 217–
219 °C. Mixed mp with compound 10b showed no depression. 1H
NMR (CDCl3) d: 1.06 (s, 3H, 18-CH3), 1.17 (s, 3H, 19-CH3), 3.98
2.3.17. [2,2,3b,4,4-d5]-3a,11b,17a-trihydroxy-21-sulfooxy-5b-
pregnan-20-one sodium salt ([2,2,3b,4,4-d5]-THF-21-sulfate sodium
salt; 13d)
The compound 13b (73 mg) was treated with SO3–TMA com-
plex (40 mg) in dry pyridine (1.5 ml) at room temperature for
5 h, as described for 5. After being processed in an analogous man-
ner, the intermediary 21-sulfate (13c) obtained was then hydro-
lyzed with 20% NaOH (0.1 ml) in MeOH at room temperature for
20 min, as described for 6c. After evaporation of the solvent under
reduced pressure, the residue dissolved in H2O was loaded onto an
OasisÒ HLB cartridge and the cartridge was washed with H2O
(2 ml). The desired sulfate was eluted with MeOH. Purification of
the crude product by column chromatography on silica gel with
CHCl3–MeOH (3:1, v/v) as an eluent and recrystallization of a
homogenous effluent from Et2O–MeOH gave 13d as colorless gran-
ular: yield, 87 mg (95%); mp 186 °C. Mixed mp with the non-deu-
terated compound [15] showed no depression. 1H NMR (CD3OD) d:
and 4.01 (d, each 1H, J = 9.0 Hz, 21-CH2), 4.26–4.29 (m, 1H, 11
a-
H), 5.04, 5.05, 5.06, and 5.22 (s, each 1H, 2Â OCH2O).
2.3.14. [2,2,3b,4,4-d5]-11b,17a,21-trihydroxy-3a-sulfooxy-5b-
pregnan-20-one sodium salt ([2,2,3b,4,4-d5]-THF-3-sulfate sodium
salt; 13a)
0.83 (s, 3H, 18-CH3), 1.16 (s, 3H, 19-CH3), 4.24–4.25 (m, 1H, 11a-
H), 4.83 and 5.03 (each 1H, d, J = 18.0 Hz, 21-CH2). ESI-MS analysis
indicated d0 (0.01%), d1 (0.00%), d2 (0.00%), d3 (0.03%), d4 (4.20%), d5
(95.76%).
The compound 12a (67 mg) was treated with SO3–TMA com-
plex (80 mg) at room temperature, as described for preparation
of 4b. After being processed in an analogous manner, the interme-
diary 3-sulfate (12b) was treated with 46% hydrogen fluoride
(2.5 ml) in EtOH (0.3 ml)–tetrahydrofuran (0.3 ml) at ice tempera-
ture for 10 h, as described for 5. After being processed in an anal-
ogous manner, the crude product was subjected to column
chromatography on silica gel with CHCl3–MeOH (2:1, v/v) as an
eluent. Recrystallization of a homogeneous effluent from Et2O–
MeOH gave 13a as colorless granular: yield, 33 mg (53%); mp
198 °C (dec.). Mixed mp with the non-deuterated compound [15]
showed no depression. 1H NMR (CD3OD) d: 0.81 (s, 3H, 18-CH3),
2.3.18. [2,2,3b,4,4-d5]-17,20:20,21-bismethylendioxy-3a-hydroxy-5b-
pregnane-11-one (14b)
To a solution of 12c (74 mg) in CH2Cl2 (0.3 ml) were added
pyridinium chlorochromate (44 mg) and sodium acetate trihydrate
(4 mg) and celite (100 mg), and the reaction mixture was stirred at
room temperature for 1.5 h. After filtration through a short pad of
celite, the filtrate was evaporated to dryness. Purification of the
crude product by column chromatography with n-hexane–EtOAc
(3:1, v/v) as an eluent gave the intermediary 11-ketone 14a
(60 mg), which without isolation it was hydrolyzed with 30% NaOH
(0.2 m) in MeOH (1.8 ml) at room temperature for 1 h. After work-
up in the usual manner, the crude product was subjected to col-
umn chromatography on silica gel with toluene–acetone (4:1, v/
v) as an eluent. Recrystallization of a homogeneous effluent from
acetone–n-hexane gave 14b as colorless needles: yield, 50 mg
(75%); mp 193–195 °C. Mixed mp with compound 11b showed
no depression. 1H NMR (CDCl3) d: 0.76 (s, 3H, 18-CH3), 1.15 (s,
3H, 19-CH3), 3.92 and 3.98 (d, each 1H, J = 9.2 Hz, 21-CH2), 5.01,
5.07, 5.08, and 5.19 (s, each 1H, 2Â OCH2O).
1.17 (s, 3H, 19-CH3), 4.24–4.25 (m, 1H, 11a-H), 4.27 and 4.64 (d,
each 1H, J = 19.0 Hz, 21-CH2). ESI-MS analysis indicated d0
(0.00%), d1 (0.00%), d2 (0.01%), d3 (0.05%), d4 (2.09%), d5 (97.85%).
2.3.15. [2,2,3b,4,4-d5]-3a-Acetoxy-17,20:20,21-bismethylendioxy-
11b-hydroxy-5b-pregnane (12c)
The compound 12a (156 mg) was acetylated at C-3 with Ac2O
(0.5 ml) in pyridine (1 ml) at room temperature for 10 h. After
addition of H2O, the resulting solution was extracted with EtOAc.
The organic layer was washed with 5% HCl, 5%, NaHCO3, and
H2O, and dried over anhydrous Na2SO4. After evaporation of the
solvent under reduced pressure, the product was recrystallized
from acetone to give 12c as colorless pillars: yield 171 mg
(100%); mp 209–211 °C. Mixed mp with compound 10c showed
no depression. 1H NMR (CDCl3) d: 1.07 (s, 3H, 18-CH3), 1.18 (s,
3H, 19-CH3), 2.02 (s, 3H, OCOCH3), 3.97 and 4.01 (d, each 1H,
2.3.19. [2,2,3b,4,4-d5]-17a,21-dihydroxy-3a-sulfooxy-5b-pregnan-
11,20-dione ([2,2,3b,4,4-d5]-THE-3-sulfate sodium salt: 15a)
To a solution of 14b (40 mg) in dry pyridine (2 ml) was added
SO3–TMA complex (30 mg), and the mixture was stirred at room
temperature for 5 h. After evaporation of solvent under a gentle
stream of N2 gas, the residue was dissolved in a small amount of

1236
S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
H2O, the pH was adjusted to 8 with 20% NaOH, and the solution
was loaded onto an OasisÒ HLB cartridge. After being washed with
H2O, elution with MeOH gave the intermediary 3-sulfate (14c),
which without isolation it was treated with 46% hydrogen fluoride
(3 ml) in EtOH (0.3 ml)–tetrahydrofuran (0.3 ml) at ice tempera-
ture for 10 h, as described for 5. After being processed in an anal-
ogous manner, the crude product was purified by column
chromatography on silica gel with CHCl3–MeOH (4:1, v/v) as an
eluent. Recrystallization of a homogeneous effluent from MeOH
gave 15a as colorless needles: yield, 9.9 mg (22%); mp 177 °C.
Mixed mp with the non-deuterated compound [15] showed no
depression. 1H NMR (CDCl3) d: 0.54 (s, 3H, 18-CH3), 1.15 (s, 3H,
19-CH3), 4.24 and 4.63 (d, each 1H, J = 19.6 Hz, 21-CH2). ESI-MS
analysis indicated d0 (0.00%), d1 (0.00%), d2 (0.01%), d3 (0.06%), d4
(2.56%), d5 (97.37%).
the known synthetic methods [15] starting from commercial
sources of stable isotopically labeled cortisol and cortisone. Here,
the difficulties encountered are the low yields due to the multiple
reaction steps and the high cost of the stable isotopically labeled
steroids. Moreover, these synthetic methods are inadequate for
preparation of the multiduterated sulfates of THS due to the
unavailability of the stable isotopically labeled THS and 11-deoxy-
cortisol containing at least three deuterium atoms in their mole-
cules. As an alternative approach toward the final goal, we
undertook to introduce multiple deuterium into the active methy-
lene adjacent to the carbonyl group of the 5b-3-keto steroid deriv-
atives by a keto–enol exchange reaction in active solvent (D2O and
CH3OD) under alkaline condition followed by stereoselective
reduction with NaBD4 leading to the 3a-hydroxy steroids.
Frequently the dihydroxyacetone side chain is unstable or inert
to the reaction condition needed to modify the steroid nucleus. The
bismethylenedioxy (BMD) group represents the most suitable pro-
tecting group for the dihydroxyacetone side chain of corticoste-
roids. This group is stable to brief heating with NaOH in alcoholic
solution and Jones’ oxidation, and can be hydrolyzed in a high yield
by employing aqueous hydrofluoric acid at low temperature. We
therefore prepared 5b-3-keto BMD derivatives (3 and 9) as the
key compounds starting from 11-deoxycortisol (1) and cortisol
(7) by treatment with paraformaldehyde as a source of alcohol-free
formaldehyde in CHCl3 containing HCl followed by catalytic reduc-
2.3.20. [2,2,3b,4,4-d5]-3a-Acetoxy-17a,21-dihydroxy-5b-pregnane-
11,20-dione (15b)
The compound 14a (60 mg) was treated with 46% hydrogen
fluoride (4 ml) in EtOH (0.6 ml)–tetrahydrofuran (0.6 ml) at room
temperature for 8 h, as described for 5. After being processed in
an analogous manner, the crude product was subjected to column
chromatography on silica gel with toluene–acetone (3:1, v/v) as an
eluent. Recrystallization of a homogeneous effluent from EtOAc
gave 15b as colorless needles: yield 18 mg (47%); mp 189–
191 °C. Mixed melting point with the non-deuterated compound
[20] showed no depression. 1H NMR (CDCl3) d: 0.60 (s, 3H, 18-
CH3), 1.15 (s, 3H, 19-CH3), 2.03 (s, 3H, OCOCH3), 4.26 and 4.64 (d,
each 1H, J = 17.4 Hz, 21-CH2).
tion of conjugated
D
4-3-ketosteroids into their corresponding 5b-
3-ketosteroids with Pd(OH)2/C and/or Pd/CaCO3. Exchange reaction
of the compound 3 with 20% NaOD in CH3OD–dioxane followed by
reduction with NaBD4 gave [2,2,3b,4,4-d5]-3
(4a), which was effectively separated from the 3b-steroisomer by
column chromatography on silica gel. The 3 -hydroxy group in
a-hydroxy-5b-steroid
a
2.3.21. [2,2,3b,4,4-d5]-3a,17a-dihydroxy-21-sulfooxy-5b-pregnane-
11,20-dione sodium salt ([2,2,3b,4,4-d5]-THE-21-sulfate sodium salt:
15d)
The C-21 sulfate ester of compound 15b (25 mg) was prepared
using SO3–TMA complex (20 mg) in dry pyridine (1 ml) at room
temperature for 3 h, followed by alkaline hydrolysis of the result-
the resulting 4a was sulfated with SO3–TMA complex in pyridine.
The sulfation reaction proceeded cleanly and rapidly under mild
experimental condition. The resulting sulfated compound (4b)
was hydrolyzed with hydrogen fluoride to remove the BMD pro-
tecting group. To purify the crude 3-sulfate of [2,2,3b,4,4-d5]-THS
(5), it was loaded onto an OasisÒ HLB cartridge for reversed-phase
solid phase extraction. After the cartridge was washed with H2O to
remove excess reagents and inorganic salts, elution with MeOH
ing intermediary 3a-sulfooxy derivative (15c) with 20% NaOH
(0.15 ml) in MeOH (1.4 ml) at room temperature for 1 h, as de-
scribed for preparation of 6c. After being processed in an analogous
manner, the crude product was purified by column chromatogra-
phy on silica gel with CHCl3–MeOH (4:1, v/v) as an eluent. Recrys-
tallization of a homogeneous effluent from Et2O–MeOH gave 15d
as colorless granular: yield, 13 mg (44%). mp 192–193 °C (dec.).
Mixed mp with the non-deuterated compound [15] showed no
depression. 1H NMR (CD3OD) d: 0.56 (s, 3H, 18-CH3), 1.13 (s, 3H,
19-CH3), 4.73 and 5.02 (d, each 1H, J = 19.6 Hz, 21-CH2). ESI-MS
analysis indicated d0 (0.00%), d1 (0.00%), d2 (0.00%), d3 (0.00%), d4
(9.17%), d5 (90.83%).
yielded
a homogenous effluent, which was characterized as
[2,2,3b,4,4-d5]-THS-3-sulfate (5) after chromatographic purifica-
tion on silica gel.
Compound 4a was acetylated at C-3 to obtain 3-acetate (4c),
which in turn was hydrolyzed with hydrogen fluoride to afford
[2,2,3b,4,4-d5]-3
ment of 6a with SO3–TMA complex yielded [2,2,3b,4,4-d5]-3
acetoxy-17 -hydroxy-20-one-21-sulfate (6b). Subsequent alkaline
a-acetoxy-17a,21-dihydroxy-20-one (6a). Treat-
a-
a
hydrolysis at C-3 in 6b with 20% NaOH in MeOH resulted in the for-
mation of [2,2,3b,4,4-d5]-THS-21-sulfate (6c) (Fig. 2).
Our next synthetic targets were focused on the 3- and 21-
monosulfates of [2,2,3b,4,4-d5]-THF (13a and 13d) and
[2,2,3b,4,4-d5]-THE (15a and 15d). It is well known that the reduc-
3. Results and discussion
The use of stable isotopically labeled (13C, 2H, 15N, 18O) internal
standards for the LC/MS analysis offers major advantages that they
behave in an almost identical manner to the analytes through all
steps in the isolation and chromatographic procedure, thereby
allowing procedural losses to be compensated for. The difference
between the molecular masses of the analyte and isotopically la-
beled IS should be at least three mass units to avoid overlapping
in the monitored mass peaks. For obtaining the desired stable iso-
topically labeled sulfates of THF and THE, direct sulfation of com-
tion of 5b-3-ketosteroid with NaBH4 gives a predominance of 3
hydroxy steroids. In fact, when compound 9 was subjected to
NaBH4 reduction, the major isolated product was the 3 -hydroxy
steroids (10b). The -configuration at C-3 in 10b was confirmed
a-
a
a
by the axial 3b-H signal appearing at 3.62–3.70 ppm as a multiplet
in the 1H NMR spectrum. As the non-deuterated authentic samples,
compounds 10c, 11a, and 11b were prepared by the selective acet-
ylation at the 3
pyridine followed by Jones’ oxidation of the resulting 3
(10c) and subsequent alkaline hydrolysis of the obtained 3
a
-hydroxy group of compound 10b with Ac2O in
-acetate
-acet-
a
mercial sources of [9,11
a
,12-d3]-THF and [9,12,12,21,21-d5]-THE
a
with sulfating reagent such as SO3–TMA complex can be consid-
ered. However, lack of regioselective sulfation at 3- and 21-hydro-
xy group of these substrates are possible problems of this synthetic
approach. The other method that can be considered is to employ
oxy-11-ketone (11a).
Based on these findings, essentially identical procedures were
used to prepare the 3- and 21-monosulfates of [2,2,3b,4,4-d5]-
THF (13a and 13d) and [2,2,3b,4,4-d5]-THE (15a and 15d), starting

S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
1237
O
O
O
Reagents and conditions:
O
O
O
O
O
O
i) (CH2O)n, 7 M HCl, CH3Cl, r.t., 5 h.
ii) H2, 5% Pd/CaCO3, Pyridine, r.t..
iii) NaOD/MeOD/Dioxane/D2O, 60oC, 22 h.
iv) NaBD4, r.t., 30 min.
CH2OH
OH
i)
ii)
v) SO3--N+(CH3)3, py, r.t..
71%
76%
O
O
O
vi) 46% HF, EtOH-Tetrahydrofuran, 0oC, 7 h.
vii) (CH3CO)2O, Pyridine, r.t., 20 h.
viii) 20% NaOH, MeOH, r.t., 1 h.
H
1
2
3
iii), iv)
64%
O
O
O
O
O
O
CH2OH
OH
CH2OR2
OH
D
D
D
D
vi)
vi)
D
D
D
RO
D
D
58%
(4a 4b 5)
55%
NaO3SO
R1O
(4a 4c 6a)
D
D
D
H
D
H
H
D
D
5
4a: R = H
6a: R1 = Ac, R2 = H
v)
v)
vii)
33%
4b: R = SO3H
4c: R = Ac
6b: R1 = Ac, R2 = SO3H
6c: R1 = H, R2 = SO3Na
(6a 6b 6c)
viii)
Fig. 2. Synthetic route to 3- and 21-monosulfate conjugates of [2,2,3b,4,4-d5]-THS. Ac = –COCH3.
O
O
O
O
O
O
O
O
O
O
O
O
HO
H
O
H
HO
H
iv)
83%
RO
RO
HO
10b: R = H
11a: R = Ac
11b: R = H
10a
x)
100%
84%
ix)
10c: R = Ac
11%
iii)
85%
O
O
O
O
O
O
O
O
O
O
O
O
CH2OH
OH
CH2OR2
OH
53%
HO
HO
HO
O
O
HO
HO
(12a 12b 13a)
viii)
D
D
i)
ii)
67%
v), vi)
90%
D
D
D
D
65%
68%
O
O
RO
R1O
O
(12c 13b)
D
H
D
H
D
H
D
9
13a: R1 = SO3Na, R2 = H
13b: R1 = Ac, R2 = H
7
8
12a: R = H
vii)
ix)
100%
12b: R = SO3H
12c: R = Ac
vii)
x)
Reagents and conditions:
95%
13c: R1 = Ac, R2 = SO3H
13d: R1 = H, R2 = SO3Na
(13b 13c 13d)
i) (CH2O)n, 7 M HCl, CHCl3, r.t..
ii) H2, 10% Pd(OH)2/C, Pyridine, r.t..
iii) NaBH4, MeOH, r.t..
iv) Jones' reagent, acetone, r.t., 30 min.
v) NaOD/MeOD/Dioxane/D2O, 60oC, 22 h.
vi) NaBD4, r.t., 30 min.
vii) SO3--N+(CH3)3, Pyridine, r.t..
viii) 46% HF, EtOH-Tetrahydrofuran, 0oC.
ix) (CH3CO)2O, Pyridine, r.t..
x) 20% NaOH, MeOH, r.t..
xi)
O
O
O
O
O
CH2OR2
OH
22%
(14b 14c 15a)
O
O
H
D
D
D
viii)
D
D
D
47%
RO
R1O
(14a 15b)
D
D
H
D
D
xi) PCC, CH2Cl2, r.t., 1.5 h.
15a: R1 = SO3Na, R2 = H
15b: R1 = Ac, R2 = H
14a: R = Ac
14b: R = H
75%
x)
(12c 14a 14b)
vii)
x)
44%
vii)
15c: R1 = Ac, R2 = SO3H
15d: R1 = H, R2 = SO3Na
(15b 15c 15d)
14c: R = SO3H
Fig. 3. Synthetic route to 3- and 21-monosulfate conjugates of [2,2,3b,4,4-d5]-THF and [2,2,3b,4,4-d5]-THE. Ac = –COCH3.
from 17,20:20,21-bismethylendioxy-11b-hydroxy-5b-pregnan-3-
one (9) which was prepared from cortisol (7) in two steps, as out-
lined in Fig. 3.
5 Da at m/z 434.3 m/z for 3- and 21-sulfates of d5-THS, at m/z
450.4 for 3- and 21-sulfates of d5-THF, and at m/z 448.3 for 3-
and 21-sulfates of d5-THE, respectively (Figs. 4–6). The CID spectra
of the 3-sulfates derived from the steroidal moiety by the loss of
H2O and the cleavage of dihydroxyacetone side chain were shifted
5 Da, whereby [DSO4]À or [MÀHÀCH2O]À ion as the base peak with
modest peak corresponding to [MÀHÀH2O]À, [MÀHÀCH2O]À, and
[MÀHÀCH2COÀH2O]À ions were observed. In contrast, the CID
spectra of the 21-sulfates derived from the steroidal moiety by
The structures of the obtained sulfates were confirmed by 1H
and 13C NMR spectra along with ESI-MS and CID spectra. 1H and
13C NMR (data not shown) spectra were identical with those of
the non-deuterated compounds except for the disappearance of
3b-proton signal. The negative-ion ESI-MS spectra showed the m/
z values of the deprotonated molecule which were shifted to

1238
S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
m/z 404
m/z 374
-
-
O
[M-H]
434.3
[M-H-CH2O]
CH2OH
OH
404.3
100
100
D
D
D
+
+D
m/z 98
-H2O
-
H
434.3
-O3SO
[DSO4]
97.9
m/z 416
D
100
50
0
D
-
[M-H-CH2CO-H2O]
374.3
[2,2,3β,4,4-d5]-THS-3-Sulfate
50
50
435.3
436.3
Isotopic purity :
d0(0.00%), d1(0.00%), d2(0.00%),
d3(0.11%), d4(8.52%), d5(91.37%)
433.5
432.7
428.1 429.4 430.7
428 429 430 431 432 433 434 435 436
-
[M-H-H2O]
416.3
0
0
500
600
100
200
300
400
100
200
300
400
500
m/z
m/z
-
-
[M-H]
434.4
[M-H-CH2OSO3]
324.4
+
+H
100
100
m/z 324
m/z 97
-
O
CH2 OSO3
m/z 139
OH
434.3
D
100
50
0
D
D
-H2O
HO
-
[HSO4]
96.9
H
D
m/z 416
50
50
D
435.3
433.7
436.3
[2,2,3β,4,4-d5]-THS-21-Sulfate
437.3
429 430 431 432 433 434 435 436 437 438
429.5 430.6431.7 432.6
-
-
[M-H]
434.3
-
[COCH2OSO3H]
139.0
Isotopic purity :
d0(0.02%), d1(0.01%), d2(0.01%),
d3(0.05%), d4(10.15%), d5(89.76%)
[M-H-H2O]
416.4
0
0
100
200
300
m/z
400
500
500
600
100
200
300
400
m/z
Fig. 4. ESI-MS (left) and product ion spectra obtained by CID of [MÀH]À (right) of 3- and 21-monosulfate conjugates of [2,2,3b,4,4-d5]-THF. Proposed structures of major
fragments are depicted.
m/z 420
-
[M-H]
O
CH2OH
OH
-
450.3
[DSO4]
97.8
m/z 390
100
100
HO
D
D
+
+D
D
m/z 98
-H2O
450.4
m/z 432
-O3SO
100
50
0
H
D
D
-
[M-H-CH2O]
420.3
-
[M-H-CH2CO-H2O]
390.3
[2,2,3β,4,4-d5]-THF-3-Sulfate
50
50
-
[M-H-H2O]
432.3
451.2
Isotopic purity :
448.7449.7
445.5 446.6 447.6
445
446
447
448
449
450
451
d0(0.00%), d1(0.00%), d2(0.01%),
d3(0.05%), d4(2.09%), d5(97.85%)
0
0
100
200
300
m/z
400
500
500
600
100
200
300
400
m/z
m/z 324
-[O]
-
-
[M-H]
450.3
[M-H-CH2OSO3]
340.3
-H2O
+
+H
100
100
m/z 340
m/z 97
-
m/z 306
O
CH2 OSO3
m/z 139
HO
OH
450.4
D
D
100
50
0
D
HO
H
D
50
50
D
-
[M-H-CH2OSO3-16]
324.4
451.3
-
[M-H-CH2OSO3-16-H2O]
306.4
449.7
[2,2,3β,4,4-d5]-THF-21-Sulfate
444.6
445.5 446.6 447.6 448.7
-
[M-H]
450.3
445 446 447 448 449 450 451
-
[HSO4]
96.9
Isotopic purity :
d0(0.01%), d1(0.00%), d2(0.00%),
d3(0.03%), d4(4.20%), d5(95.76%)
-
[COCH2OSO3H]
139.0
0
0
100
200
300
400
500
500
600
100
200
300
400
m/z
m/z
Fig. 5. ESI-MS (left) and product ion spectra obtained by CID of [MÀH]À (right) of 3- and 21-monosulfate conjugates of [2,2,3b,4,4-d5]-THE. Proposed structures of major
fragments are depicted.

S. Ikegawa et al. / Steroids 76 (2011) 1232–1240
1239
m/z 418
-
[M-H]
448.3
O
CH2OH
OH
-
[DSO4]
97.9
m/z 388
100
100
O
D
D
D
+
+D
m/z 98
-H2O
448.3
-O3SO
m/z 430
100
50
0
H
D
D
[2,2,3β,4,4-d5]-THE-3-Sulfate
50
50
449.3
450.2
-
Isotopic purity :
[M-H-CH2CO-H2O]
388.2
-
[M-H-CH2O]
418.2
447.7
451.2
443 444 445 446 447 448 449 450 451 452
d0(0.00%), d1(0.00%), d2(0.01%),
d3(0.06%), d4(2.56%), d5(97.37%)
-
[M-H-H2O]
430.4
0
0
100
200
300
400
500
500
600
100
200
300
400
m/z
-
-
[M-H-CH2OSO3]
338.3
[M-H]
448.3
m/z 350
m/z 338
m/z 97
+
+H
100
100
50
0
-
O
CH2 OSO3
m/z 139
m/z 310
O
D
OH
448.3
100
50
0
D
D
-
-
[HSO4]
96.9
[M-H-COCH2OSO3]
HO
H
50
310.3
D
D
449.2
447.6
450.2
-
[M-H-H2SO4]
350.5
[2,2,3β,4,4-d5]-THE-21-Sulfate
451.2
443.5 444.5 445.7 446.7
443 444 445 446 447 448 449 450 451 452
-
[M-H]
448.3
Isotopic purity :
d0(0.00%), d1(0.00%), d2(0.00%),
d3(0.00%), d4(9.17%), d5(90.83%)
-
[COCH2OSO3H]
138.9
0
100
200
300
400
500
500
600
100
200
300
400
m/z
m/z
Fig. 6. ESI-MS (left) and product ion spectra obtained by CID of [MÀH]À (right) of 3- and 21-monosulfate conjugates of [2,2,3b,4,4-d5]-THS. Proposed structures of major
fragments are depicted.
the cleavage of dihydroxyacetone side chain were shifted
5 Da, whereby [MÀHÀCH2OSO3]À ion as the base peak with
modest or small peak corresponding to [MÀHÀCH2OSO3À16]À,
[MÀHÀCH2OSO3À16ÀH2O]À, [COCH2OSO3H]À and [HSO4]À ions
were observed. Thus, the results of CID analysis confirmed the
structures of [2,2,3b,4,4-d5]-THS, -THF, and -THE, which gave rise
to transition [MÀH]À ? [MÀHÀCH2O]À for THS-3-sulfate and
for 2007–2011. We thank Dr. Alan F. Hofmann, University of
California, San Diego for editing the manuscript.
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THF-
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Acknowledgements
This work was supported in part by Grant-in-Aids for Scientific
Research (C) (to. K.M., Grant 20590164) for 2008–2010, (to S.I.,
Grant 21590184) for 2009–2011, and (to T.I., Grant 21550091)
for 2009–2011 from the Japan Society for the Promotion of Science,
and Culture of Japan, and High-Tech Research Center Project for
Private Universities: matching fund subsidy from the Ministry
of Education, Culture, Sports, Science and Technology (to S.I.)
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