Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3β,7β-dihydroxy-5-cholen-24-oic acid, and related compounds: Unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

Article abstract of DOI:10.1016/j.steroids.2005.07.008

The chemical synthesis of 3β,7β-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to

Full text of DOI:10.1016/j.steroids.2005.07.008

steroids 7 1 ( 2 0 0 6 ) 18–29
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Chemical synthesis of the 3-sulfooxy-7-N-
acetylglucosaminyl-24-amidated conjugates of
3
␤,7␤-dihydroxy-5-cholen-24-oic acid, and related
compounds: Unusual, major metabolites of bile acid
in a patient with Niemann-Pick disease type C1
a,∗
a
a
a
b
Takashi Iida , Genta Kakiyama , Yohei Hibiya , Shohei Miyata , Takehiko Inoue ,
b
c
c
c
c
Kohsaku Ohno , Takaaki Goto , Nariyasu Mano , Junichi Goto , Toshio Nambara ,
Alan F. Hofmann
d
a
Department of Chemistry, College of Humanities and Sciences, Nihon University, Setagaya, Sakurajousui, Tokyo 156-8550, Japan
Division of Child Neurology, Faculty of Medicine, Tottori University, Nishi-machi, Yonago 683-8504, Japan
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 981-8578, Japan
Division of Gastroenterology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0813, USA
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemical synthesis of 3␤,7␤-dihydroxy-5-cholen-24-oic acid, triply conjugated by sul-
furic acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24,
is described. These are unusual, major metabolites of bile acid found to be excreted in the
urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3␤-
hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved
were: (1) ␤-d-N-acetylglucosaminidation at C-7 of methyl 3␤-tert-butyldimethylsilyloxy
Received 17 June 2005
Received in revised form 8 July 2005
Accepted 28 July 2005
Available online 28 September 2005
(TBDMSi)-7␤-hydroxy-5-cholen-24-oate with 2-acetamido-1␣-chloro-1,2-dideoxy-3,4,6-tri-
Keywords:
Unsaturated bile acid
Unusual bile acid multi-conjugates
Electrospray ionization mass
spectrometry
O-acetyl-d-glucopyranose in the presence of CdCO3 in boiling toluene; (2) sulfation at C-
3 of the resulting 3␤-TBDMSi-7␤-GlcNAc with sulfur trioxide-trimethylamine complex in
pyridine; and (3) direct amidation at C-24 of the 3␤-sulfooxy-7␤-GlcNAc conjugate with
glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-
4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-
conjugated bile acids were characterized by liquid chromatography-mass spectrometry with
an electrospray ionization probe under the positive and negative ionization modes.
© 2005 Elsevier Inc. All rights reserved.
Tandem mass spectrometry
Abbreviations:
NP-C1, Niemann-Pick disease type
C1
GlcNAc, ␤-d-N-acetylglucosamine
␣-acetochloroglucosamine,
2
3
-acetamido-1␣-chloro-1,2-dideoxy-
,4,6-tri-O-acetyl-d-glucopyranose
m.p., melting point
∗
Corresponding author. Tel.: +81 3 3329 1151; fax: +81 3 3303 9899.
E-mail address: takaiida@chs.nihon-u.ac.jp (T. Iida).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.07.008

steroids 7 1 ( 2 0 0 6 ) 18–29
19
IR, infrared
1
H-NMR, proton nuclear magnetic
resonance
GC/MS, gas
chromatography/mass
spectrometry
LC/MS, liquid
chromatography/mass
spectrometry
LR-MS, low-resolution mass
spectra
HR-MS, high-resolution mass
spectra
MS/MS, tandem mass
spectrometry
EI, electron ionization
APCI, atmospheric pressure
chemical ionization
ESI, electrospray ionization
PIM, positive ion mode
NIM, negative ion mode
TLC, thin layer chromatography
DMT-MM,
4
-(4,6-dimethoxy-1,3,5-triazin-2-
yl)-4-methylmorpholinium
chloride
Me4Si, tetramethylsilane; EtOAc
ethyl acetate
Et2O, diethyl ether
DMF, N,N’-dimethylformamide
1
.
Introduction
ones [1]. The carboxyl group at C-24 in bile acids is ami-
dated with glycine or taurine and the hydroxy group at C-3 is
conjugated with sulfuric acid or ␤-d-glycopyranose (i.e., glu-
curonic acid or glucose) to form the corresponding sulfate
Bile acids in biological material are known to exist in single-
and/or double-conjugated form, rather than in unconjugated
Fig. 1 – Structures of 7-oxygenated 3␤-hydroxy-ꢀ5 bile acid multi-conjugates.

2
0
steroids 7 1 ( 2 0 0 6 ) 18–29
or glycosidic conjugates. The specific, selective conjugation
of 7␤-hydroxylated bile acids with ␤-d-N-acetylglucosamine
dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose (␣-acetochloro-
glucosamine) was prepared from N-acetylglucosamine
according to a previous paper [9]. Reversed-phase prepacked
Sep-Pak® tC18 cartridges (adsorbent weight, 5 g) were available
from Waters Assoc. (Milford, MA, USA); they were washed
with 50 ml methanol and 100 ml distilled water prior to use.
All other reagents and solvents used were of analytical grades.
Melting points (m.p.) were determined on a micro hot-
stage apparatus and are uncorrected. IR spectra were obtained
in KBr discs on a Shimadzu FTIR-8300 spectrometer. 1H-NMR
spectra were recorded on a JEOL JNM-EX 270 FT instrument
(
GlcNAc) has also been reported in connection with hepato-
biliary diseases [2–5].
Meanwhile, Niemann-Pick disease type C1 (NP-C1) is a
fatal autosomal recessive neurovisceral disorder in humans
and in animals, characterized clinically by progressive neu-
rodegeneration in the central nervous system, and by hep-
atosplenomegaly [6,7]. Biochemically, NP-C1 is characterized
by the intracellular accumulation of unesterified cholesterol,
sphingomyelin, glycosphingolipids, glycolipids, and other
lipids within the endosomal/lysosomal system in various tis-
sues, particularly in visceral organs.
(Tokyo, Japan) at 270 MHz, with CDCl3 or CH OD containing
3
0.1% Me4Si as the solvent; chemical shifts are expressed
Recently, Alvelius et al. [8] have reported that an NP-
C1 patient with hepatosplenomegaly, mild signs of cholesta-
sis, hepatic inflammation, and extramedullary erythropoiesis,
together with chronic airway disease, excreted abnormal
amounts of a new type of unusual 7-oxygenated bile acid
multi-conjugates in urine. The structures of the unusual bile
acids were elucidated by a combined use of electrospray ion-
ization (ESI) mass spectrometry and gas chromatography-
mass spectrometry (GC-MS). After enzymatic and solvolytic
removal of the conjugating moieties, these acids were shown
to have a 3␤-hydroxy-ꢀ5 structure and to carry either a
hydroxy or oxo group at C-7. They were sulfated at C-3, and
either nonamidated, or amidated with glycine or taurine at C-
as ı ppm relative to Me4Si. Electron ionization (EI) low-
resolution mass spectra (LR-MS) were determined on a JEOL
JMS-303 mass spectrometer at 70 eV. High-resolution mass
(HR-MS) spectra were measured by using a JEOL JMS-LCmate
double-focusing magnetic mass spectrometer equipped with
an electrospray ionization (ESI) probe or an atmospheric
pressure chemical ionization (APCI) probe under the positive
ion mode (PIM) or the negative ion mode (NIM); a mixture
of methanol-water (1:1, v/v) was used as the mobile phase.
HR-MS spectra were also obtained on a JEOL JMS-700 mass
spectrometer with an EI probe under the PIM. Tandem mass
spectrometric (MS/MS) analysis was carried out using a
LCMS-IT-TOF tandem liquid chromatography-mass spec-
trometer (LC/ESI-MS/MS) (Shimadzu, Kyoto, Japan) equipped
with an ESI-PIM probe; a sample solution was dissolved
in methanol-water (1:1, v/v), and a mixed solution of 0.1%
formic acid in water and 0.1% formic acid in acetonitrile
was used as the mobile phase. Normal-phase (NP) thin-layer
chromatography (TLC) was performed on pre-coated silica
gel plates (0.25 mm layer thickness; E. Merck, Darmstadt, Ger-
many) using hexane-EtOAc-AcOH mixtures (80:20:1–40:60:1,
v/v/v) or EtOAc-methanol-AcOH (8:1:1–7:2:1, v/v/v) as the
developing solvent. Reversed-phase (RP) TLC was carried
out on pre-coated RP-18F254S plates using methanol-water
mixtures (5:5-6:4, v/v) as the developing solvent.
2
4. Part of the 7-hydroxy acid, presumably the 7␤-hydroxylated
one, was also conjugated with N-acetylhexosamine, proba-
bly GlcNAc, at the 7-hydroxy group. Based on previous data
concerning the effect of 3␤-hydroxy-ꢀ5 bile acids on bile acid
transport, Alvelius et al. [8] suggested that the formation of
such unusual bile acid multi-conjugates was responsible for
the neonatal cholestasis in the NP-C1 patient.
A more direct proof had to await chemical synthesis and
the demonstration of the identity of isolated compounds with
synthetic ones. The availability of authentic synthetic speci-
mens could also serve to establish a reliable method for ana-
lyzing the effects of the multi-conjugated bile acids in urine
with regard to metabolic disorders in humans, and could serve
to clarify the physiological significance of the pathway, the
region in which these conjugates are synthesized, the enzyme
catalyzing the conjugation, and the dynamics of the conju-
gates.
2.2.
Synthesis
2.2.1. Methyl 3␣,6␣-ditosyloxy-5␤-cholan-24-oate (3)
To a solution of methyl hyodeoxycholate 2 (2.0 g, 4.9 mmol)
in dry pyridine (12 ml) a solution of tosyl chloride (2.0 g,
10.5 mmol) in dry pyridine (6 ml) was added, and the mixture
was stirred at room temperature for 2 days. Ice chips were
added gradually to the mixture, and the precipitated solid was
filtered, washed with 10% HCl and water, and recrystallized
from benzene-hexane to give the ditosylate 3 in the form of
We herein report the chemical synthesis of the multi-
conjugates of 3␤,7␤-dihydroxy-5-cholen-24-oic acid and 3␤-
hydroxy-7-oxo-5-cholen-24-oic acid as authentic reference
compounds (Fig. 1), in order to shed light on an unknown
abnormality in the bile acid synthesis and metabolism of a
patient with NP-C1.
◦
colorless needles: yield, 3.36 g (95%); m.p., 166–167 C (lit. [10]
1
65–167 C). IR, ꢁmax cm : 1732 (C O), 3040 (Ar H). 1H-NMR
◦ −1
2
.
Experimental
(
CDCl3), ı: 0.59 (3H, s, 18-CH3), 0.80 (3H, s, 19-CH3), 0.88 (3H, d,
J = 6.5 Hz, 21-CH3), 2.17, 2.46 (each 3H, s, 3␣- and 6␣-C6H4CH3),
3.66 (3H, s, COOCH3), 4.30 (1H, brm, 3␤-H), 4.79 (1H, brm,
2
.1.
Materials and instruments
6␤-H), 7.34 and 7.75 (each 4H, m, 3␣- and 6␣-C6H4CH3). LR-
Methyl hyodeoxycholanoate (methyl 3␣,6␣-dihydroxy-5␤-
cholan-24-oate) was purchased from Tokyo Kasei Kogyo Co.,
Ltd. (Tokyo, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM) was obtained from
Tokuyama Co., Ltd. (Tokyo, Japan). 2-Acetamido-1␣-chloro-1,2-
MS (EI-PIM), m/z: 370 (M–2TsOH, 100%), 355 (M–2TsOH–CH3,
22%), 255 [M–2TsOH–S.C. (side chain, 115 u), 23%], 249 (33%),
213 [M–2TsOH–S.C.-ring D (42 u), 14%]. HR-MS (APCI-NIM),
−
calculated for C39H53O8S2 [M−H] : 713.3182; found, m/z:
713.3175.

steroids 7 1 ( 2 0 0 6 ) 18–29
21
1H-NMR (CDCl3), ı: 0.69 (3H, s, 18-CH3), 0.95 (3H, d, J = 6.5 Hz,
21-CH3), 1.20 (3H, s, 19-CH3), 3.69 (1H, brm, 3␣-H), 5.70 (1H, brs,
6-H). LR-MS (EI-PIM), m/z: 374 (M–CH3, 100%), 356 (M–CH3–H2O,
65%), 341 (M–H2O–2CH3, 51%), 289 (44%), 263 (68%), 255
[M–CH3–H2O–S.C. (101 u), 22%], 213 (M–CH3–H2O–S.C.-ring D,
2
.2.2. Methyl 3␤-hydroxy-5-cholen-24-oate (4) and its
acetate (5)
A solution of the ditosylate 3 (3.36 g, 4.7 mmol) and CH3COOK
(
360 mg, 3.6 mmol) dissolved in water (3 ml) and N,N’-
dimethylformamide (DMF; 20 ml) was refluxed for 24 h. The
solution was cooled at room temperature, with ice chips added
gradually. The precipitated solid was filtered off and washed
with water. The crude solid was recrystallized from aqueous
methanol to give an analytical pure sample of the ꢀ5 ester
+
29%). HR-MS (EI-PIM), calculated for C24H36O4 [M] : 388.5492;
found, m/z: 388.2614.
The acid 7a (1.86 g, 4.8 mmol) was then esterified by the
usual p-toluenesulfonic acid (p-TsOH)-methanol method to
give the corresponding methyl ester (7b), which was recrystal-
lized in quantitative yield from aqueous methanol in the form
4
in the form of colorless needles: yield, 1.76 g (96%); m.p.,
◦
◦
−1
1
3
39–141 C [lit. (10) m.p., 143–144 C]. IR, ꢁmax cm : 1717 (C O),
489 (O H). 1H-NMR (CDCl3), ı: 0.68 (3H, s, 18-CH3), 0.93 (3H,
◦
of colorless amorphous solids: yield, 1.92 g; m.p., 138–141 C.
IR, ꢁmax cm 1: 1672 (C O), 3475 (O H). 1H-NMR (CDCl3), ı:
0.69 (3H, s, 18-CH3), 0.93 (3H, d, J=6.2 Hz, 21-CH3), 1.20 (3H,
s, 19-CH3), 3.60 (1H, brm, 3␣-H), 3.67 (3H, s, COOCH3), 5.67
−
d, J = 6.5 Hz, 21-CH3), 1.01 (3H, s, 19-CH3), 3.52 (1H, brm, 3␣-H),
3
3
.66 (3H, s, COOCH3), 5.35 (1H, m, 6-H). LR-MS (EI-PIM), m/z:
88 (M , 100%), 370 (M–H2O, 62%), 355 (M–H2O–CH3, 40%), 303
+
+
(
40%), 277 (70%), 255 (M–H2O–S.C., 27%), 213 (M–H2O–S.C.-ring
(1H, brs, 6-H). LR-MS, m/z: 402 (M , 100%), 369 (M–H2O–CH3,
+
D, 36%). HR-MS (EI-PIM): calculated for C25H40O3 [M] : 388.5908;
found, m/z: 388.2977.
15%), 287 [M–S.C. (115 u), 13%], 269 (M–H2O–S.C., 5%). HR-MS
+
(EI-PIM), calculated for C25H38O4 [M] : 402.5762; found, m/z:
The ester (4) was converted into the corresponding acetate
402.2770.
(5) by the usual acetic anhydride-pyridine method, which was
recrystallized from acetone in the form of colorless thin plates:
yield, 1.85 g (95%); m.p., 147–150 C (lit. [10] m.p., 155–156 C).
2.2.5. Methyl 3␤-tert-butyldimethylsilyloxy-7-
oxo-5-cholen-24-oate (8)
To a solution of the 3␤-hydroxy-ꢀ ester 7 (800 mg, 2.0 mmol)
◦
◦
IR, ꢁmax cm 1: 1681 (C C), 1731 (C O). H-NMR (CDCl3), ı: 0.68
3H, s, 18-CH3), 0.93 (3H, d, J = 6.2 Hz, 21-CH3), 1.02 (3H, s, 19-
CH3), 2.03 (3H, s, 3␤-OCOCH3), 3.66 (3H, s, COOCH3), 4.60
−
1
5
(
in anhydrous DMF (6 ml) and pyridine (0.5 ml), imida-
zole (1.68 g) and tert-butyldimethylsilyl chloride (TBDMSiCl;
840 mg, 5.6 mmol) were added with ice-bath cooling at
(
(
1H, brm, 3␣-H), 5.37 (1H, m, 6-H). LR-MS (EI-PIM), m/z: 370
M–AcOH, 100%), 355 (M–AcOH–CH3, 15%), 255 (M–AcOH–S.C.,
◦
10 C, and the mixture was left to stand at room temper-
1
7%), 249 (20%), 213 (M–AcOH–S.C.-ring D, 10%). HR-MS (APCI-
ature for 30 min. The reaction product was extracted with
CH2Cl2 (30 ml), and the combined extract was washed with
water, dried with Drierite, and evaporated. The residue was
recrystallized from aqueous methanol to give the 3␤-tert-
butyldimethylsilyloxy (TBDMSi) derivative 8 in the form of col-
−
NIM), calculated for C27H41O4 [M−H] : 429.3005; found, m/z:
4
29.2971.
2
.2.3. Methyl 3␤-acetoxy-7-oxo-5-cholen-24-oate (6)
To a magnetically stirred suspension of the 3␤-acetoxy-ꢀ
ester 5 (1.8 g, 4.1 mmol), pyridinium dichromate (PDC; 4.6 g,
2 mmol) and Celite (4 g) in dry benzene (35 ml), and 70%
5
◦
orless amorphous solids: yield, 996 mg (97%); m.p., 179–181 C.
IR, ꢁmax cm 1: 1668, 1747 (C O), 3475 (O H). 1H-NMR (CDCl3),
ı: 0.06 [6H, s, –Si(CH3)2C(CH3)3], 0.68 (3H, s, 18-CH3), 0.89
[9H, s, –Si(CH3)2C(CH3)3], 0.93 (3H, d, J = 6.8 Hz, 21-CH3), 1.18
(3H, s, 19-CH3), 3.60 (1H, brm, 3␣-H), 3.67 (3H, s, COOCH3),
−
1
tert-butylhydroperoxide (t-BHPO; 3.5 ml, 27 mmol) were added
gradually with ice-bath cooling; the whole mixture was stirred
at room temperature for 24 h. After filtration on Celite, the
mother liquor was evaporated under reduced pressure to give
a dark brown residue, which was passed through a short col-
umn on silica gel (10 g). Elution with EtOAc-benzene (95:5, v/v)
gave the title compound (6), which was recrystallized from
ethanol in the form of colorless needles: yield, 1.3 g (70%); m.p.,
+
5.67 (1H, brs, 6-H). LR-MS (EI-PIM), m/z: 516 (M , 2%), 501
(M–CH3, 4%), 459 [M–C(CH3)3, 100%], 427 (30%). HR-MS (EI-
+
PIM), calculated for C31H52O4Si [M] : 516.8396; found, m/z:
516.3635.
2.2.6. Methyl 3␤-tert-butyldimethylsilyloxy-7␤-
hydroxy-5-cholen-24-oate (9)
67–169 C. IR, ꢁmax cm : 1664, 1735 (C O). 1H-NMR (CDCl3),
◦
−1
1
ı: 0.69 (3H, s, 18-CH3), 0.93 (3H, d, J = 6.2 Hz, 21-CH3), 1.21 (3H,
s, 19-CH3), 2.05 (3H, s, 3␤-OCOCH3), 3.67 (3H, s, COOCH3),
To a freshly prepared solution of Zn(BH4)2 in Et2O (18 ml), a
solution of the 3␤-TBDMSi-7-oxo ester 8 (910 mg, 1.8 mmol)
in benzene (9 ml) was added dropwise under N2. After fur-
ther stirring at room temperature for 2 h, the mixture was
poured into water, and the organic layer was washed with
10% acetic acid and water, dried with Drierite, and evapo-
rated to dryness. The residue was recrystallized from aque-
ous methanol to give the 3␤-TBDMSi-7␤-hydroxy ester 9 in
the form of colorless amorphous solids: yield, 730 mg (80%);
4
4
2
.72 (1H, brm, 3␣-H), 5.71 (1H, brs, 6-H). LR-MS (EI-PIM), m/z:
44 (M , 2%), 384 (M–AcOH, 100%), 369 (M–AcOH–CH3, 8%),
+
69 (M–AcOH–S.C., 18%), 227 [M–AcOH–CH3–S.C.-part of ring
+
D (27 u), 8%]. HR-MS (EI-PIM), calculated for C27H4O5 [M] :
4
44.6116; found, m/z: 444.2876.
2
.2.4. 3␤-Hydroxy-7-oxo-5-cholen-24-oic acid (7a) and its
methyl ester (7b)
The usual alkaline hydrolysis of the 7-oxo-ꢀ ester-acetate
(810 mg, 1.8 mmol) with 5% methanolic NaOH, followed by
acidification with 10% HCl, gave 3␤-hydroxy-7-oxo-5-cholen-
4-oic acid (7a), which was recrystallized from aqueous
methanol in the form of colorless needles: yield, 710 mg
◦
−1
1
m.p., 92–94 C. IR, ꢁmax cm : 1738 (C O), 3304 (O H). H-NMR
(CDCl3), ı: 0.05 [6H, s, –Si(CH3)2C(CH3)3], 0.69 (3H, s, 18-CH3),
0.89 [9H, s, –Si(CH3)2C(CH3)3], 0.93 (3H, d, J = 6.8 Hz, 21-CH3),
1.04 (3H, s, 19-CH3), 3.49 (1H, brm, 3␣-H), 3.67 (3H, s, COOCH3),
3.83 (1H, brm, 7␣-H), 5.24 (1H, brs, 6-H). LR-MS (EI-PIM), m/z:
5
6
2
+
518 (M , 11%), 500 (M-H2O, 9%), 461 (70%), 386 (M–TBDMSiOH,
◦
−1
(
100%); m.p., 237–239 C. IR, ꢁmax cm : 1683 (C O), 3286 (O H).
100%), 369 (M–TBDMSiOH–H2O, 63%), 327 (29%). HR-MS (EI-

2
2
steroids 7 1 ( 2 0 0 6 ) 18–29
+
PIM), calculated for C31H54O4Si [M] : 518.3806; found, m/z:
18.3791.
2.2.9. 3␤-Sulfooxy-7␤-(2-acetamido-2-deoxy-␤-d-gluco-
pyranosyl)-5-cholen-24-oic acid disodium salts (1a)
5
To a solution of the 3␤-hydroxy-7␤-GlcNAc ester-triacetate
11 (95 mg, 0.13 mmol) in dry pyridine (5 ml), sulfur trioxide-
2
.2.7. Methyl 3␤-tert-butyldimethylsilyloxy-7␤-(2-
trimethylamine complex (95 mg, 0.83 mmol) was added, and
the mixture was stirred at room temperature for 1 h. The
reaction mixture was poured into ice-cooled petroleum ether
(25 ml) and the precipitated solid was collected by filtration.
After being washed with petroleum ether, the solid product
was dissolved in 5% methanolic NaOH (10 ml), and the solu-
tion was stirred overnight at room temperature. The result-
ing solution was adjusted to pH 8 with 10% HCl, diluted
with water (90 ml), and loaded onto a Sep-Pak® tC18 car-
tridge. The cartridge was washed with water (20 ml) and 20%
methanol (20 ml). The desired 3␤-sulfate-7␤-GlcNAc disodium
salt (1a) was eluted with methanol (20 ml) and recrystallized
from methanol-EtOAc in the form of colorless amorphous
acetamido-2-deoxy-3,4,6-tri-O-acetyl-␤-d-
glucopyranosyl)-5-cholen-24-oate (10)
To a solution of the 3␤-TBDMSi-7␤-hydroxy ester 9 (500 mg,
0
1
.96 mmol) in anhydrous toluene (25 ml), 2-acetamido-
␣-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose
(
2
500 mg, 1.4 mmol), freshly prepared CdCO3 (500 mg,
.9 mmol), and molecular sieves (4 A˚ , 500 mg) were added,
and the resulting suspension was azeotropically refluxed
with stirring. After 2 and 4 h, additional amounts of the
halosugar (500 mg, 1.4 mmol) and CdCO3 (500 mg) were added
in several portions, and the mixture was further refluxed for
8
h. The precipitate was removed by filtration and washed
◦
−1
with toluene. The combined mother liquor was evaporated
down to dryness under reduced pressure, and the residue
was subjected to column chromatography on activated neu-
tral alumina (activity II, 120 g). Elution with EtOAc-benzene
solids: yield, 78 mg (85%); m.p., 234–236 C. IR, ꢁ
cm
:
max
1637, 1648, 1654 (C O), 3294 (O H). 1H-NMR (CD OD), ı: 0.70
3
(3H, s, 18-CH ), 0.96 (3H, d, J = 6.5 Hz, 21-CH ), 1.04 (3H, s, 19-
3
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
CH ), 3.38–3.88 (6H, brm, 3 -, 4 -, 5 -, 6 -, and 7␣-H), 4.15 (1H,
3
(
2:8–4:6, v/v) and recrystallization of the eluate from acetone-
brm, 3␣-H), 4.52 (1H, d, J = 8.1 Hz, 1 -H), 5.76 (1H, brs, 6-H). LR-
ꢀ
hexane gave the 3␤-TBDMSi-7␤-GlcNAc triacetate 10 in the
form of colorless amorphous solids: yield, 57 mg (7%); m.p.,
MS (ESI-NIM), m/z: 672 [M (=M−2Na+2H)−H, 100%], 497 (7%),
ꢀ
460 (9%), 389 [M –SO -part of GlcNAc (204 u), 15%], 278 (8%),
3
07–210 C. IR, ꢁmax cm : 1748 (C O). 1H-NMR (CDCl3), ı:
.06 [6H, s, –Si(CH3)2C(CH3)3], 0.64 (3H, s, 18-CH3), 0.88 [9H, s,
◦
−1
2
0
97 (HSO , 13%). HR-MS (ESI-NIM), calculated for C32H50NO12
[M−2Na+2H−H] : 672.3054; found, m/z: 672.3041.
S
4
−
–
Si(CH3)2C(CH3)3], 0.92 (3H, d, J = 5.9 Hz, 21-CH3), 1.02 (3H, s,
9-CH3), 1.92, 2.02, 2.03, 2.07 (each 3H, s, COCH3), 3.49 (1H,
brm, 3␣-H), 3.67 (3H, s, COOCH3), 3.83 (1H, brm, 7␣-H), 4.13
1
2.2.10. 3␤-Sulfooxy-7␤-(2-acetamido-2-deoxy-␤-d-
glucopyranosyl)-5-cholen-24-oyl glycine disodium
salts (1b)
To a magnetically stirred solution of the 3␤-sulfate-7␤-GlcNAc
disodium salt 1a (38 mg, 0.054 mmol) in DMF (4 ml), DMT-
MM (43 mg, 0.16 mmol) and triethylamine (190 ␮l) were added,
and the mixture was stirred at room temperature for 10 min.
Glycine methyl ester hydrochloride (40 mg, 0.32 mmol) was
then added to the mixture, which was further stirred for
3 h. After adding 5% methanolic NaOH (4 ml) and stirring for
ꢀ
ꢀ
ꢀ
ꢀ
(
4H, brm, 2 -, 5 - and 6 -H), 4.78 (1H, d, J = 8.1 Hz, 1 ␣-H), 5.02,
ꢀ
ꢀ
5
.32 (each 1H, m, 3 - and 4 -H), 5.44 (1H, brs, 6-H), 5.47 (1H, brs,
NH). LR-MS (EI-PIM), m/z: 790 (M–C(CH3)3, 24%), 517 [M-part
of sugar moiety (S.M.) (330 u), 55%], 500 [M–S.M. (346 u)–H,
4
5%], 369 (M–TBDMSiOH–S.M., 100%), 346 (S.M., 77%), 330
(
(
part of S.M., 84%), 286 (S.M.–AcOH, 38%), 259 (23%). HR-MS
APCI-NIM), calculated for C45H72NO12Si [M−H] : 846.4824;
−
found: m/z, 846.4834.
3
h at room temperature, the alkaline solution was adjusted
2
.2.8. Methyl 3␤-hydroxy-7␤-(2-acetamido-2-deoxy-3,4,6-
to pH 8, diluted with water (30 ml), and loaded onto a Sep-
Pak® tC18 cartridge, which was washed successively with
water (20 ml) and 20% methanol (20 ml). Elution with 50%
methanol gave the title compound (1b), which was recrystal-
lized from methanol-hexane in the form of colorless amor-
tri-O-acetyl-␤-d-glucopyranosyl)-5-cholen-24-oate (11)
To a solution of the 3␤-TBDMSi-7␤-GlcNAc triacetate 10
(
(
120 mg, 0.14 mmol) in ethanol (12 ml), p-toluenesulfonic acid
100 mg) was added. After being left to stand at room temper-
◦
ature for 1 h, the solution was neutralized with 5% NaHCO3,
and the solvent was evaporated. The residue was extracted
with CHCl3, and the combined extract was washed with water,
dried with Drierite, and evaporated to dryness. Recrystalliza-
tion of the residue from aqueous methanol gave the title
compound (11) in nearly quantitative yield in the form of color-
phous solids: yield, 39 mg (93%); m.p., 212–216 C. IR, ꢁmax
cm 1: 1653 (C O), 3293 (O H). 1H-NMR (CD OD), ı: 0.70 (3H,
−
3
s, 18-CH ), 0.98 (3H, d, J = 6.2 Hz, 21-CH ), 1.04 (3H, s, 19-CH ),
3
3
3
ꢀ
ꢀ
ꢀ
ꢀ
3.15-3.90 (6H, brm, 3 -, 4 -, 5 -, 6 - and 7␣-H), 3.74 (2H, brs,
ꢀ
CH N ), 4.15 (1H, brm, 3␣-H), 4.54 (1H, d, J = 8.4 Hz, 1 -H),
2
5.77 (1H, brs, 6-H), 7.82 (1H, brm, CONH ). LR-MS (ESI-NIM),
◦
ꢀ
ꢀ
less amorphous solids: yield, 103 mg; m.p., 207–210 C. IR, ꢁmax
cm 1: 1747 (C O), 3290 (O H). 1H-NMR (CDCl3), ı: 0.65 (3H, s,
m/z: 729 [M (=M−2Na+2H)−H, 100%], 672 (5%), 631 (M –H SO ,
2
4
−
ꢀ
6%), 460 (5%), 433 (7%), 410 (M –H SO –GlcNAc–H, 8%), 389
ꢀ
(10%), 364 (15%), 254 [M –H SO –S.C. (158 u)-GlcNAc, 12%], 97
2
4
2
4
1
2
8-CH3), 0.92 (3H, d, J = 5.9 Hz, 21-CH3), 1.03 (3H, s, 19-CH3), 1.92,
.02, 2.03, 2.08 (each 3H, s, COCH3), 3.49 (1H, brm, 3␣-H), 3.67
(HSO , 37%). HR-MS (ESI-NIM), calculated for C34H53N O13S
4
2
−
(
3H, s, COOCH3), 3.83 (1H, brm, 7␣-H), 3.66, 3.83, 4.19 (4H, each
[M−2Na+2H−H] : 729.3268; found, m/z: 729.3261.
ꢀ
ꢀ
ꢀ
ꢀ
m, 2 -, 5 - and 6 -H), 4.78 (1H, d, J = 8.1 Hz, 1 ␣-H), 5.03, 5.33 (each
ꢀ
ꢀ
1
H, m, 3 - and 4 -H), 5.46 (1H, brs, 6-H), 5.50 (1H, m, NH). LR-MS
2.2.11. 3␤-Sulfooxy-7␤-(2-acetamido-2-deoxy-␤-d-
glucopyranosyl)-5-cholen-24-oyl taurine disodium salts
(1c)
The 3␤-sulfate-7␤-GlcNAc disodium salt 1a (33 mg,
0.046 mmol) in DMF (4 ml) was treated with DMT-MM
(
EI-PIM), m/z: 386 (M–S.M.–H, 100%), 368 (M–S.M.–H–H2O, 51%),
2
53 [M–S.M.–H2O–S.C. (115 u)–H, 10%], 249 (27%). HR-MS (APCI-
−
NIM), calculated for C39H58NO12 [M−H] : 732.3959; found, m/z:
7
32.3942.

steroids 7 1 ( 2 0 0 6 ) 18–29
23
ꢀ
(
36 mg, 0.13 mmol) and triethylamine (160 ␮l), followed by
(M –H2SO4–S.C.–CH3-part of ring D, 16%). HR-MS (ESI-NIM),
−
taurine (33 mg, 0.26 mmol), as described for the prepara-
tion of 1b. After being processed analogously, the reaction
mixture was adjusted to pH 10 with 10% aqueous NaOH,
then to pH 8 with 10% HCl, diluted with water (30 ml), and
loaded onto a Sep-Pak® tC18 cartridge, which was washed
with water and 10% methanol. Elution with 30% methanol
afforded the desired triple-conjugate 1c as the disodium
salt, which was recrystallized from methanol-CHCl3 in the
form of colorless amorphous solids: yield, 32 mg (85 %); m.p.,
calculated for C26H38NO8S [M−2Na+2H−H] : 524.2318; found,
m/z: 524.2331.
2.2.14. 3␤-Sulfooxy-7-oxo-5-cholen-24-oyl taurine
disodium salts (12c)
The 3␤-sulfate-7-oxo-ꢀ disodium salt 12a (100 mg, 0.2 mmol)
5
was treated with DMT-MM (100 mg, 0.37 mmol) and tau-
rine (100 mg, 0.81 mmol), followed by 10% aqueous NaOH,
as described for the preparation of 1c. After being pro-
cessed analogously, the product was passed through a Sep-
Pak® tC18 cartridge and washed successively with water and
20% methanol. Elution with 60% methanol gave the double-
conjugate 12c, which was recrystallized from methanol-
EtOAc in the form of colorless amorphous solids: yield,
69–175 C. IR, ꢁmax cm : 1654 (C O), 3285 (O H). 1H-NMR
◦
−1
1
(
1
CD3OD), ı; 0.69 (3H, s, 18-CH3), 0.96 (3H, d, J = 6.5 Hz, 21-CH3),
.04 (3H, s, 19-CH3), 3.16-3.92 (6H, brm, 3 -, 4 -, 5 -, 6 - and
ꢀ
ꢀ
ꢀ
ꢀ
7
␣-H), 2.98 (2H, t, J = 6.8 Hz, CH2S ), 3.59 (2H, t, J = 6.8 Hz,
CH2N ), 4.26 (1H, brm, 3␣-H), 4.53 (1H, d, J = 8.1 Hz, 1 -H),
ꢀ
◦
−1
5
.76 (1H, s., 6-H), 7.88 (1H, brm, CONH ). LR-MS (ESI-NIM),
109 mg (90%); m.p., 177–181 C. IR, ꢁmax cm : 1543 (C C),
1666 (C O). 1H-NMR (CD OD), ı: 0.73 (3H, s, 18-CH ), 0.98
ꢀ
−
ꢀ
m/z: 801 ([M (=M−Na+H)−H] , 60%), 779 (M −Na+H−H,
3
3
ꢀ
ꢀ
1
2
5%), 699 (M −Na+H−H−SO3, 37%), 680 (M –Na–H2SO4–H,
3 3
(3H, d, J = 6.2 Hz, 21-CH ), 1.25 (3H, s, 19-CH ), 2.95 (2H, t,
ꢀ
9%), 490 (20%), 478 (M –Na–SO3–GlcNAc–H, 22%), 460
J = 6.8 Hz, CH2S ), 3.59 (2H, m, CH N ), 4.26 (1H, brm, 3␣-
H), 5.68 (1H, brs, 6-H), 7.87 (1H, brm, CONH ). LR-MS (EI-PIM),
2
ꢀ
(
M –Na–SO4–GlcNAc–H, 59%), 389 (100%), 278 (73%), 97 (HSO4,
ꢀ
5
7%). HR-MS (ESI-NIM), m/z: calculated for C34H54N2O14NaS2
m/z: 397 (3%), 370 [M (=M−Na+H)−SO –taurine–Na, 72%], 355
3
ꢀ
ꢀ
[
M−Na+H−H]: 801.2914; found, m/z: 801.2889.
(M –SO4–taurine–CH –Na, 8%), 311 (14%), 269 [M –H SO4–S.C.
3
2
ꢀ
230 u), 18%], 227 (M –H2SO4–S.C.–CH3-part of ring D, 9%). HR-
(
MS (ESI-NIM), calculated for C26H39NO NaS [M−Na+H−H]−:
2
.2.12. 3␤-Sulfooxy-7-oxo-5-cholen-24-oic acid disodium
9
2
salts (12a)
596.1964. found; m/z: 596.1980.
5
The 3␤-hydroxy-7-oxo-ꢀ ester 7 (580 mg, 1.5 mmol) was
treated with sulfur trioxide-trimethylamine complex, fol-
lowed by 5% methanolic NaOH, as described for the prepa-
ration of 1a. After passing through a Sep-Pak® tC18 car-
tridge and being washed with H2O and 20% methanol, the
crude 3␤-sulfate-7-oxo-ꢀ5 disodium salt 12a was eluted with
3.
Results and discussion
According to the recent findings of Avelius et al. [8], a
patient with NP-C1 excreted abnormal amounts of the
multi-conjugates of unusual 7-oxygenated ꢀ -bile acids in
5
6
0% methanol. Recrystallization from methanol-EtOAc gave
the analytical pure 12a in the form of colorless amorphous
urine. These conjugates were shown to have the parent 3␤-
hydroxy-ꢀ -cholenoic acid structure, and to carry an oxygen-
◦
−1
5
solids: yield, 512 mg (70%); m.p., 228–232 C. IR, ꢁmax cm
542 (C C), 1671 (C O). 1H-NMR (CD3OD), ı: 0.73 (3H, s, 18-
CH3), 0.97 (3H, d, J = 6.2 Hz, 21-CH3), 1.25 (3H, s, 19-CH3), 4.25
:
1
containing function (hydroxy or oxo group) at C-7. The
hydroxy groups at the C-3 and -7 positions were sulfated
and N-acetylamino-glucosylated, respectively, while the car-
boxyl group at C-24 was either unconjugated or amidated with
glycine or taurine (1a–1c). The abnormal excretion of the anal-
ogous conjugates of nonamidated and glycine- and taurine-
amidated 3␤-hydroxy-7-oxo-5- cholen-24-oic acid 3-sulfates
(
[
2
1H, brm, 3␣-H), 5.69 (1H, brs, 6-H). LR-MS (EI-PIM), m/z; 370
ꢀ
ꢀ
M
(=M−2Na+2H)−H2SO4, 100%], 355 (M –H2SO4–CH3, 10%),
ꢀ
ꢀ
69 [M –H2SO4–S.C. (101 u), 23%], 227 (M –H2SO4–CH3–S.C.-part
of ring D, 12%). HR-MS (ESI-NIM), calculated for C24H35O7S
−
[
M−2Na+2H−H] : 467.2103; found, m/z: 467.2102.
(
12a–12c) has also been recorded [8]. Therefore, these new and
2
.2.13. 3␤-Sulfooxy-7-oxo-5-cholen-24-oyl glycine
unusual bile acid multi-conjugates seem to be specific mark-
ers of NP-C1. The synthetic outline of the targeted bile acid
conjugates is shown in Figs. 2–4.
disodium salts (12b)
5
The 3␤-sulfate-7-oxo-ꢀ disodium salt 12a (100 mg, 0.2 mmol)
was treated with DMT-MM (100 mg, 0.37 mmol) and glycine
methyl ester hydrochloride (100 mg, 0.82 mmol), followed
by 5% methanolic NaOH, as described for the prepara-
tion of 1b. After being processed analogously, the product
was passed through a Sep-Pak® tC18 cartridge and washed
successively with water and 20% methanol. Elution with
A key intermediate in our synthesis is methyl 3␤-TBDMSi-
7␤-hydroxy-5-cholen-24-oate (9), which was prepared from
methyl hyodeoxycholate (2) in 8 steps (see Fig. 2). Thus, the
ester 2 was tosylated with tosyl chloride in pyridine to give the
corresponding 3,6-ditosylate (3) in nearly quantitative yield.
When 3 was treated with boiling DMF in the presence of
CH3COOK for 24 h, simultaneous inversion at the C-3 position
and elimination at the C-6 position took place to give methyl
3␤-hydroxy-5-cholen-24-oate (4) [11], which in turn was acety-
lated by the usual acetic anhydride-pyridine method to yield
the corresponding 3␤-acetoxy-ꢀ5 ester (5). Allylic oxidation
of 5 with t-BHPO-PDC [12] afforded the corresponding 7-oxo
compound (6) in good isolated yield. The reaction with the
oxidant system proceeded faster and more cleanly than with
conventional chromium (VI) complexes [13]. Alkaline hydrol-
6
0% methanol gave the double-conjugate 12b, which was
recrystallized from methanol-EtOAc in the form colorless
◦
amorphous solids: yield, 89 mg (70%); m.p., 239–242 C. IR,
ꢁ
max cm 1: 1654 (C O). 1H-NMR (CD3OD), ı: 0.73 (3H, s,
−
1
8-CH3), 0.98 (3H, brs, 21-CH3), 1.23 (3H, s, 19-CH3), 3.72
(
2H, d, J = 6.5 Hz, CH2N ), 4.24 (1H, brm, 3␣-H), 5.67 (1H,
ꢀ
brs, 6-H). LR-MS (EI-PIM), m/z: 427 [M (=M−2Na+2H)−H2SO4,
ꢀ
ꢀ
4
6%], 409 (19%), 370 (M –SO3–glycine, 100%), 352 (M –H2SO4-
ꢀ
glycine, 12%), 311 (54%), 269 [M –H2SO4–S.C. (158 u), 40%], 227

2
4
steroids 7 1 ( 2 0 0 6 ) 18–29
ysis of 6 with methanolic KOH, followed by re-esterification
of the resulting 3␤-hydroxy-7-oxo-ꢀ5 acid (7a) with p-TsOH
in methanol, gave methyl 3␤-hydroxy-7-oxo-5-cholen-24-oate
be essential for the preparation of the desired bile acid multi-
conjugates, because of the limited solubility of an intermedi-
ary conjugate in many organic solvents. As a result of prelimi-
nary experiments, the following order of conjugation reactions
with 9 was found to be suitable: (1) N-acetylglucosaminidation
at C-7; (2) sulfation at C-3; and (3) amidation at C-24
(see Fig. 3).
(7b). Subsequent protection of the 3␤-hydroxy group in 7b with
TBDMSiCl and imidazole in DMF gave the corresponding 3␤-
TBDMSi-7-oxo-ꢀ5 ester (8) in nearly quantitative yield.
A preliminary experiment revealed that the reduction of
the 7-oxo group in 8 with NaBH4 afforded a mixture of the
corresponding epimeric 7␣-/7␤-ols, accompanied by a small
amount of their C-24 hydrolyzed products, from which the
desired 7␤-hydroxy ester (9) was separated by tedious chro-
matographic purification. However, when Zn(BH4)2 in ether
At
first,
␤-d-N-acetylglucosaminidation
was
car-
ried out by the Koenigs-Knorr reaction of
9
with
␣-acetochloroglucosamine as a glycosyl donor, and a stoichio-
metric amount of freshly prepared CdCO3 as an activator, in
the presence of molecular sieves under azeotropic conditions
in refluxing toluene [18,19]. The condensed 3␤-TBDMSi-7␤-
GlcNAc methyl ester-triacetate (10) was isolated in fairly
low yield (7%), probably owing to the steric hindrance of
the axially-oriented 18- and 19-methyl groups. However, no
undesirable by-product was formed at all. Attempting the
Koenigs-Knorr reaction using either silver (I) oxide (Ag2O) or
mercury (II) cyanide [Hg(CN)2] [20,21] as an activator also gave
10 in less than 5% yield. Nevertheless, the Koenigs-Knorr
condensation with CdCO3 seems profitable, as a mixture of 9
and 10 are easily separated on a short column of silica gel, and
9 can be efficiently recovered. Subsequent deprotection of the
TBDMSi group in 10 was attained with p-TsOH in methanol at
room temperature for 1 h to afford the 3␤-hydroxy-7␤-GlcNAc
methyl ester-triacetate (11) in quantitative yield. The pres-
[
14,15] was used as the reducing agent, the reaction pro-
ceeded more cleanly and stereoselectively than with NaBH4,
and the 3␤-TBDMSi-7␤-hydroxy ester (9) was isolated in 77%
yield by direct recrystallization, without the need for a chro-
matographic purification. In addition, less basic Zn(BH4)2 also
left the C-24 ester group intact during the reduction at C-7.
The stereochemical configuration at the 7␤-hydroxy group in
1
9
was confirmed by the H-NMR signals appearing at 3.83 ppm
(
[
7␣-H) as a multiplet and at 5.24 ppm (6-H) as a broad singlet
16,17]. The overall yield of the key intermediate (9) based on
2
was 47%.
Our next effort was directed at finding a suitable order
of conjugation reactions with 9, as well as at removing the
protecting groups in both the steroid and sugar moieties. In
particular, the order of the conjugations with 9 appeared to
5
ence of the GlcNAc moiety attached to the ꢀ -steroid nucleus
Fig. 2 – Synthetic route to intermediary methyl 3␤-hydroxy-7-oxo-5-cholen-24-oate (7b) and methyl
␤-TBDMSi-7␤-hydroxy-5-cholen-24-oate (9) from methyl hyodeoxycholate (2).
3

steroids 7 1 ( 2 0 0 6 ) 18–29
25
Fig. 3 – Synthetic route to nonamidated and glycine- and taurine-amidated 3␤-sulfooxy-7␤-GlcNAc conjugates of
3
␤,7␤-dihydroxy-5-cholen-24-oic acid (1a–1c) from 9.
Fig. 4 – Synthetic route to nonamidated and glycine- and taurine-amidated conjugates of
␤-sulfooxy-7-oxo-5-cholen-24-oic acid (12a–12c) from 7b.
3

2
6
steroids 7 1 ( 2 0 0 6 ) 18–29
by a ␤-d-glycosidic linkage was confirmed by the 1H-NMR
spectra, judging from the signals appearing at 3.83 ppm (7␣-H)
the crystalline 1a (as the 3,24-disodium salt) in satisfactory
yield (85%).
ꢀ
as a broad multiplet; 4.78 ppm (anomeric 1 ␣-H) as a doublet
Finally, the formation of carboxamides at C-24 was car-
ried out by the direct condensation of the carboxylate sodium
salt at C-24 in 1a, and the amino group in glycine or taurine.
Exploratory experiments revealed that the direct amidation
of the carboxylate (1a) with glycine methyl ester hydrochlo-
ride in the presence of either N-ethylcarbonyl-2-ethoxy-1,2-
dihydroquinoline or diethylphosphorylcyanide [24,25] as a
coupling agent was unsatisfactory (yield: 60–70%). However,
when a versatile coupling agent, DMT-MM [26], was used, the
amidation reaction proceeded nearly quantitatively, requiring
only the mixing of 1a (1 eq.), glycine methyl ester hydrochlo-
ride (3 eq.), DMT-MM (6 eq.), and triethylamine in DMF at
room temperature for 3 h. Activation of the carboxylate salt
by DMT-MM and subsequent condensation of the resulting
acyloxytriazine with glycine methyl ester hydrochloride pro-
ceeded selectively, leading to the formation of the methyl
ester derivative of the 24-glyco-3␤-sulfooxy-7␤-GlcNAc (1b),
which in turn was hydrolyzed with methanolic NaOH. Since
ꢀ
ꢀ
ꢀ
(
J, 8.1 Hz) [22]; 1.92–2.08 ppm (acetyl methyls at C-2 , -3 , -4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
and -6 ) as four singlets; and 3.66–5.33 ppm (2 -, 3 -, 4 -, 5 -,
ꢀ
and 6 -H) as five multiplets.
The 3␤-hydroxy group in 11 was then sulfated with sulfur
trioxide-trimethylamine complex in pyridine at room temper-
ature for 3 h [23]. The sulfation reaction proceeded cleanly and
rapidly under mild conditions. The crude product was sub-
sequently hydrolyzed with methanolic NaOH to remove the
protecting groups at the C-24 methyl ester on the side chain in
ꢀ
ꢀ
the aglycone moiety, and the acetoxyl groups at C-3 , C-4 , and
ꢀ
C-6 in the sugar moiety. To purify and isolate the resulting 3␤-
sulfate-7␤-GlcNAc double-conjugate (1a) as a crystalline form,
the reaction mixture was adjusted to pH 8 with HCl, and loaded
onto a Sep-Pak® tC18 cartridge for the reversed-phase solid
extraction. The cartridge was washed successively with water
and 20% aqueous methanol to remove excess reagents, con-
taminants, and inorganic salts. Elution with methanol gave
Fig. 5 – LC-MS spectra of 3␤-sulfooxy-7␤-GlcNAc double-conjugate of 3␤,7␤-dihydroxy-5-cholen-24-oic acid disodium salt
1a).
(

steroids 7 1 ( 2 0 0 6 ) 18–29
27
the resulting free triple-conjugate was extremely hygroscopic,
the hydrolyzed solution was adjusted to pH 8 with HCl, and
the mixture was loaded onto a Sep-Pak® tC18 cartridge, which
upon elution with 50% aqueous methanol gave the desired
ary 3-monosulfate (12a) (see Fig. 4). As expected, the double-
and triple-conjugates (12b–12c and 1a–1c) thus prepared were
highly hydrophilic and readily soluble in water. However, they
were insoluble in many organic solvents, with methanol and
DMF as exceptions.
The 1H-NMR data for 1b, 1c, 12b, and 12c are indicative of
the formation of the sulfonate and carboxamide. An appre-
ciable downfield shift (ca. 4.2 ppm) of the 3␣-H (broad mul-
tiplet) was observed for the sulfo-conjugates by comparison
to data for 11 and the 3␣-H resonating at 4.15–4.26 ppm [23].
The glyco-conjugates 1b and 12b showed the broad singlet
signal at 3.74 ppm and the doublet signal at 3.72 ppm, both
1
(
b as the crystalline disodium salt, in excellent isolated yield
93%).
Transformation of 1a into the triple-conjugate of the 24-
tauro-3␤-sulfooxy-7␤-GlcNAc disodium salt (1c) was similarly
achieved by a slight modification of the procedure for 1b. Thus,
1
a was treated with taurine, DMT-MM, and triethylamine in
DMF. After the condensation reaction, the resulting solution
was adjusted to pH 8 with aqueous NaOH, and then loaded
onto a Sep-Pak® tC18 cartridge. Elution with 30% aqueous
methanol gave the analytically pure 1c in an isolated yield of
due to CH NH , and the broad multiplet signal at 7.82 ppm,
2
arising from CONH [24,25]. On the other hand, the tauro-
8
5%.
By combining of the procedures of sulfation and amida-
conjugates (1c and 12c) were identified by the appearance of
two triplet signals at 2.95-2.98 ppm ( CH2S ) and 3.59 ppm
tion, as described above, 3␤-hydroxy-7-oxo-5-cholen-24-oic
acid doubly conjugated with sulfuric acid at C-3, and with
glycine (12b) or taurine (12c) at C-24 (i.e. the major metabo-
lites in the NP-C1 patient [8]), were analogously prepared
from the corresponding unconjugate 7b, via the intermedi-
(
(
CH N ), and by a broad multiplet signal at 7.87–7.88 ppm
CONH ).
The LC/MS spectra with ESI-PIM or ESI-NIM of 1a–1c
2
(Figs. 5–7) provided confirming evidence for their structures,
always giving molecular-related ions as the major fragment
Fig. 6 – LC-MS spectra of 24-glyco-3␤-sulfooxy-7␤-GlcNAc triple conjugate of 3␤,7␤-dihydroxy-5-cholen-24-oic acid
disodium salt (1b).

2
8
steroids 7 1 ( 2 0 0 6 ) 18–29
Fig. 7 – LC-MS spectra of 24-tauro-3␤-sulfooxy-7␤-GlcNAc triple conjugate of 3␤,7␤-dihydroxy-5-cholen-24-oic acid
disodium salt (1c).
−
ions. In both the ESI-PIM and ESI-NIM, the fragmentation pat-
molecule [(M−Na+H)−H] at m/z 801, accompanied by an
ion at m/z 97, and a characteristic ion at m/z 460 arising
terns of 1a and 1b were similar to each other, while the pattern
1
of 1c differed. Thus, in the MS/MS with ESI-PIM, the MS spec-
from the elimination of the GlcNAc and OSO Na residues
3
tra of 1a and 1b exhibited an intense protonated molecule
from M.
+
[
M+H] at m/z 718.2782 and at m/z 775.3034, respectively; these
The relationship between the formation of the abnormal
bile acids and the metabolic defect in NP-C1 is not known.
Since a NP-C1 patient with cholestasis excretes significant
amounts of the unusual multi-conjugated bile acids 1a–1c and
12a–12b in the urine [8], these metabolites are expected to be
specific markers of the disease. Further studies on the biolog-
ical and physiological significance of the unusual metabolites
are now being conducted in our laboratory.
2
ions were selected as precursor ions in the MS spectra, and
the occurrence of the base peak at m/z 244 in each spectrum
indicated the presence of a GlcNAc moiety. On the other hand,
the MS spectrum of 1c showed an adducted molecule [M+Na]+
at m/z 847.2705; the selection of the ion as a precursor ion gave
the MS2 spectrum, in which several fragment ions associated
with the elimination of GlcNAc and/or OSO3Na residues from
M occurred at m/z 727, 626, and 506.
1
Meanwhile, the ESI-NIM spectra of 1a and 1b gave a depro-
−
tonated molecule [(M−2Na+2H)−H] at m/z 672 and at m/z
7
29, respectively, comprising the base peak in each spec-
Acknowledgments
trum. The presence of the OSO3Na moiety was confirmed
by the appearance of an ion at m/z 97. The ESI-NIM spec-
trum of 1a was essentially identical to that measured by
the collision-induced dissociation method for the 3-sulfo-
We would like to express our thanks to Shimadzu Co. for the
LC/ESI-MS/MS measurements. This work was supported in
part by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Sciences, Sports, and Culture of Japan for 2004-
2005 and Nihon University Multidisciplinary Research Grant
for 2005–2006.
7
-GlcNAc conjugate of a dihydrocholenoic acid identified
in the urine of a NP-C1 patient [8]. On the other hand,
the spectrum of 1c with ESI-NIM exhibited a deprotonated

steroids 7 1 ( 2 0 0 6 ) 18–29
29
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