Synthesis of bile acid 24-acyl glucuronides

Article abstract of DOI:10.1016/S0039-128X(97)00160-8

The synthesis of acyl glucuronides of common bile acids is described. By means of the Mitsunobu reaction employing diethylazodicarboxylate and triphenylphosphine, bile acids were condensed through the inherent C-24 carboxy group with benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate, which was prepared from 1-O-methyl-α-D-glucose. The separation and purification of the β2-anomers at the anomeric position of the sugar moiety were attained by preparative thin-layer chromatography and/or high-performance liquid chromatography on a column packed with phenyl-bonded silica using H₂O-MeOH as a mobile phase. The removal of the benyl group on the sugar moiety was achieved by catalytic hydrogenation with 10% palladium on carbon to yield the desired acyl glucuronides of bile acids. The structures of these acyl glucuronides were confirmed by proton nuclear magnetic resonance spectral properties.

Full text of DOI:10.1016/S0039-128X(97)00160-8

Synthesis of bile acid 24-acyl
glucuronides
Junichi Goto, Naoaki Murao, Junji Oohashi, and Shigeo Ikegawa
Faculty of Pharmaceutical Sciences, Tohoku University, Aobayama Sendai, 980-8578, Japan
The synthesis of acyl glucuronides of common bile acids is described. By means of the Mitsunobu reaction
employing diethylazodicarboxylate and triphenylphosphine, bile acids were condensed through the inherent C-24
carboxy group with benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate, which was prepared from 1-O-methyl-␣-D-
glucose. The separation and purification of the ␤-anomers at the anomeric position of the sugar moiety were
attained by preparative thin-layer chromatography and/or high-performance liquid chromatography on a column
packed with phenyl-bonded silica using H2O-MeOH as a mobile phase. The removal of the benzyl group on the
sugar moiety was achieved by catalytic hydrogenation with 10% palladium on carbon to yield the desired acyl
glucuronides of bile acids. The structures of these acyl glucuronides were confirmed by proton nuclear magnetic
resonance spectral properties. (Steroids 63:180–185, 1998) © 1998 by Elsevier Science Inc.
Keywords: bile acid; carboxyl-linked glucuronide; Mitsunobu reaction; benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate
acyl glucuronides, which have been identified as their
acetate-methyl ester derivatives by liquid chromatography
(LC)-atmospheric pressure chemical ionization (APCI)-
mass spectrometry (MS), in an incubation mixture of liver
microsomes from male rats. These observations strongly
imply the occurrence of similar acyl glucuronides of com-
mon bile acids in humans. Our continuing interests in the
biodynamics of bile acids in normal subjects and patients
with liver disease have led to the development of a reliable
method for the simultaneous determination of the bile acid
24-glucuronides. However the instability of acyl glucu-
ronides, for example, which are almost as susceptible to
transesterification and hydrolysis under mild conditions, has
prevented preparation of authentic specimens for use in
their direct identification and quantitation in biological ma-
terials. The present paper deals with the synthesis of the
titled compounds by selective coupling of the carboxy
group of bile acids with a D-glucuronic acid benzyl ester-
benzyl ether derivative employing the Mitsunobu reaction.13
Introduction
Bile acids are synthesized from cholesterol in hepatocytes,
followed by secretion into the small intestine, reabsorbed
from the ileum-proximal colon and then returned to the liver
via the portal vein. This enterohepatic circulation is respon-
sible for the low concentration of bile acids in the peripheral
blood and urine of healthy subjects. In hepatobiliary dis-
eases, disturbances of synthesis and clearance by the liver
and absorption by the intestine cause changes in the level
and the metabolic profile of bile acids in biological fluids.
The conjugation with D-glucuronic acid to produce glu-
curonides plays an important role in the elimination of
endogenous substances such as steroid hormones and bile
acids as well as drugs.1 The principal purpose of this me-
tabolism is believed to be transformation of lipophilic bio-
logical materials into strongly dissociated compounds
which are more soluble under physiological conditions. The
D-glucuronic acid moiety of UDP-glucuronic acid is easily
transformed not only to a hydroxy group but also to car-
boxy, amino, imino and sulfhydryl groups of various bio-
logical importance by hepatic glucuronyltransferases. It has
been believed that common bile acids are converted into the
ether-type glucuronides through the 3␣-hydroxy group on
the steroid nucleus.2–6 In the recent decade, it has also been
reported that unusual bile acids such as 24-nor lithocholic
acid are metabolized into carboxy-linked glucuronides, so
called acyl glucuronides.7–11 In a previous communica-
tion,12 we have disclosed the formation of common bile acid
Experimental
Material
Cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA) and
lithocholic (LCA) acids were purchased from Sigma (St. Louis,
Missouri, USA). Ursodeoxycholic acid (UDCA) was kindly do-
nated by Tokyo Tanabe Co. (Tokyo, Japan). Preparative thin-layer
chromatography (TLC) was performed using silica gel (Kiesel gel
60 F254, 20 cm ϫ 20 cm ϫ 0.5 mm, Merck KGaA, Darmstadt,
Germany). Melting points were taken on a micro hot-stage appa-
ratus and are uncorrected. Proton nuclear magnetic resonance (1H
NMR) spectra were recorded on Varian Gemini-2000 and Hitachi
FT-NMR R-3000 spectrometers at 300 MHz. Chemical shifts are
Address reprint requests to Junichi Goto, Ph.D., Faculty of Pharmaceutical
Sciences, Tohoku University, Aobayama Sendai, 980-77, Japan.
Received November 7, 1997; accepted December 1, 1997.
Steroids 63:180–185, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(97)00160-8

Synthesis of bile acid 24-acyl glucuronides: Goto et al.
given as the ␦ value with tetramethylsilane as the internal standard
(s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets;
m, multiplet). Column chromatography was performed using silica
gel (Kieselgel 60, 200–300 mesh, Merck KGaA) and Cosmosil
140C18-OPN (140 ␮m, Nacalai Tesque, Inc., Kyoto, Japan). The
apparatus used for semi-preparative high-performance liquid chro-
matography consisted of an injector (Rheodyne 7125, Catati, Cal-
ifornia, USA), a Hitachi model L-4200 UV-VIS detector (254 nm)
(Hitachi, Ltd., Tokyo) and a chromatographic pump (110B Solvent
Delivery Module, Beckman Instruments, Inc., California, USA). A
Cosmosil 5-Ph column (5 ␮m, 150 ϫ 4.6 mm i.d.) (Nacalai
Tesque, Inc.) was used at ambient temperature. LC/MS was carried
out using a Hitachi M-1000H quadrupole mass spectrometer with
electrospray ionization (ESI) and atmospheric pressure chemical
ionization (APCI) systems in the negative and positive ion detec-
tion modes, respectively. The mass spectrometer was set at a drift
voltage of Ϫ60 V for an ESI mode and at ϩ30 V for an APCI
mode with a nebulizer gas temperature of 130°C.
vacuo. The residue was subjected to column chromatography on
silica gel (100 g) using n-hexane-AcOEt (1:2, v/v) to give 4 (2.54
1
g, 85.3%) as a semicrystalline product. H NMR (CDCl3) ␦: 3.35
(3H, s, OCH3), 3.49–3.71 (5H, unresolved m, 2-, 4-, 5- and 6-H),
4.00 (1H, t, J ϭ 9.8 Hz, 3-H), 4.56–4.96 (7H, m, 1-H and 3 ϫ
CH2C6H5), 7.21–7.36 (15H, m, C(C6H5)3). Analysis calculated for
C28H32O6: C, 72.39; H, 6.94. Found: C, 72.18; H, 6.92.
Benzyl 1-O-methyl-2,3,4-tri-O-benzyl-␣-D-
glucopyranuronate (5)
To an ice-cooled solution of 4 (1.7 g) in acetone (10 mL), anhy-
drous chromic acid (3.7 g) in 17% H2SO4 (v/v, 11 mL) was added
dropwise, and the mixture was stirred for 30 min at room temper-
ature. After addition of MeOH (10 mL) to decompose the excess
reagents, the resulting solution was extracted with AcOEt, which
in turn was washed with H2O, dried on Na2SO4, and evaporated in
vacuo. To the crude product obtained in dimethylsulfoxide (5 mL)
were added NaHCO3 (850 mg) and benzylbromide (3.5 mL), and
the resulting mixture was stirred for 8 h at room temperature,
followed by extraction with AcOEt. The organic layer was washed
with H2O and saturated NaCl, dried on Na2SO4, and evaporated in
vacuo. The residue was subjected to column chromatography on
silica gel (50 g) using n-hexane-AcOEt (5:1, v/v) to give 5 (2.06 g,
98.8%) as a semicrystalline product. 1H NMR (CDCl3) ␦: 3.40
(3H, s, OCH3), 3.59 (1H, dd, J ϭ 3.6 and 9.6 Hz, 2-H), 3.71 (1H,
dd, J ϭ 9.0 and 9.9 Hz, 4-H), 3.98 (1H, t, J ϭ 9.3 Hz, 3-H), 4.21
(1H, d, J ϭ 9.9 Hz, 5-H), 4.62 (1H, d, J ϭ 3.6 Hz, 1-H), 4.42–5.20
(8H, m, 4 ϫ CH2C6H5), 7.23–7.33 (20H, m, 4 ϫ CH2C6H5).
Analysis calculated for C35H36O7: C, 73.92; H, 6.38. Found: C,
73.64; H, 6.23.
A Cosmosil 5C8 column (5 ␮m, 150 ϫ 4.6 mm i.d., Nacalai
Tesque, Inc.) was also used for the separation of bile acid 24-
glucuronide acetate-methyl esters at a flow rate of 1 mL/min
according to the method previously reported.12
1-O-Methyl-6-O-trityl-␣-D-glucopyranose (2)
A solution of 1-O-methyl-␣-D-glucose (1, 20 g) and triphenylm-
ethyl chloride (34.7 g) in anhydrous pyridine (200 mL) was stirred
at room temperature overnight. The resulting solution was diluted
with H2O and extracted with AcOEt. The organic layer was
washed successively with 5% HCl, 5% NaHCO3 and saturated
NaCl, dried on Na2SO4, and then evaporated in vacuo. Recrystal-
lization of the resulting product from acetone-hexane afforded 2
(42.2 g, 91.2%) as colorless needles. m.p. 97–99°C. 1H NMR
(CDCl3) ␦: 3.38–3.69 (6H, unresolved m, 2-, 3-, 4-, 5- and 6-H),
3.43 (3H, s, OCH3), 4.77 (1H, d, J ϭ 3.7 Hz, 1-H), 7.24–7.47
(15H, m, C(C6H5)3). Analysis calculated for C26H28O6 ⅐ 1/4H2O:
C, 70.81; H, 6.51. Found: C, 70.68; H, 6.36.
Benzyl 1-O-acetyl-2,3,4-tri-O-benzyl-D-
glucopyranuronate (6)
A mixed solution of H2SO4-AcOH (1:10, v/v) (2.8 mL) was added
dropwise to a cooled solution of 5 (1.6 g) in acetic acid (16 mL)
and acetic anhydride (2.6 mL), and the resulting solution was
stirred for 5 h at room temperature. The reaction mixture was
poured into ice water and extracted with AcOEt, which in turn was
washed successively with 5% NaHCO3, H2O and saturated NaCl,
dried on Na2SO4, and evaporated in vacuo. The residue obtained
was subjected to column chromatography on silica gel (50 g) using
n-hexane-AcOEt (6:1, v/v) to give 6 (1.36 g, 81.1%) as a semic-
1-O-Methyl-2,3,4-tri-O-benzyl-6-O-trityl-␣-D-
glucopyranose (3)
Benzylbromide (8.1 mL) and sodium hydride (1.65 g) were suc-
cessively added to a stirred solution of 2 (5 g) and tetrabutyl-
ammonium iodide (138 mg) in anhydrous tetrahydrofuran (20
mL), and the reaction mixture was further stirred at room temper-
ature for 24 h. After concentration of the solvent, the resulting
solution was diluted with H2O and extracted with AcOEt, which in
turn was washed with 5% HCl, 5% NaHCO3 and saturated NaCl,
dried on Na2SO4, and then evaporated in vacuo. The residue was
subjected to column chromatography on silica gel (100 g) using
n-hexane-AcOEt (5:1, v/v) to give 3 (6.9 g, 85.2%) as a semic-
rystalline product. 1H NMR (CDCl3) ␦: 3.23 (1H, dd, J ϭ 3.9 and
9.0 Hz, 2-H), 3.44 (3H, s, OCH3), 3.48 (1H, t, J ϭ 9.1 Hz, 4-H),
3.61 (2H, m, 6-H), 3.80 (1H, m, 5-H), 3.96 (1H, t, J ϭ 9.1 Hz,
3-H), 4.28 (1H, d, J ϭ 10.6 Hz), 4.62–4.91 (6H, m), 7.19–7.48
(30H, m, 3 ϫ CH2C6H5 and C(C6H5)3). Analysis calculated for
C47H46O6: C, 79.86; H, 6.56. Found: C, 79.75; H, 6.76.
1
rystalline product. H NMR (CDCl3) ␦: 2.03 (0.6H, s, OCOCH3
(␤-anomer)), 2.15 (2.4H, s, OCOCH3 (␣-anomer)), 3.71–3.81
(2.2H, unresolved m, 2- (␣- and ␤-anomer), 3- (␤-anomer), and
4-H (␣- and ␤-anomer), 3.92 (0.8H, t, J ϭ 9.3 Hz, 3-H (␣-
anomer)), 4.10 (0.2H, d, J ϭ 10.2 Hz, 5-H (␤-anomer)), 4.30
(0.8H, d, J ϭ 10.2 Hz, 5-H (␣-anomer)), 4.45–5.19 (8H, m, 4 ϫ
CH2C6H5), 5.65 (0.2H, d, J ϭ 7.7 Hz, 1-H (␤-anomer)), 6.34
(0.8H, d, J ϭ 3.7 Hz, 1-H (␣-anomer)), 7.06–7.35 (20H, m, 4 ϫ
CH2C6H5). Analysis calculated for C36H36O8: C, 72.46; H, 6.08.
Found: C, 72.57; H, 5.97.
Benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate (7)
2-Aminoethanol (0.3 mL) was added to a solution of 6 (1.7 g) in
AcOEt (20 mL) and dimethylsulfoxide (0.2 mL), and the mixture
was stirred at room temperature for 7 h. The resulting solution was
diluted with AcOEt, washed successively with 5% HCl, 5%
NaHCO3 and saturated NaCl, dried on Na2SO4, and evaporated in
vacuo. The crude product was subjected to column chromatogra-
phy on silica gel (50 g) using n-hexane-AcOEt (5:1, v/v) to give a
mixture of ␣- and ␤-anomer 7 (1.1 g, 71%), which was subjected
to crystallization from acetone-hexane to give 7 as colorless nee-
1-O-Methyl-2,3,4-tri-O-benzyl-␣-D-glucopyranose (4)
To a stirred solution of 3 (4.5 g) in EtOH (50 mL), concentrated
H2SO4 (2.6 mL) was added dropwise under ice-cooling. After
stirring for another 3 h at room temperature, the solution was
neutralized with 10% NaOH and concentrated in vacuo, followed
by extraction with AcOEt. The organic layer was washed with
H2O and saturated NaCl, dried on Na2SO4, and evaporated in
Steroids, 1998, vol. 63, April 181

Papers
Figure 1 Structure of bile acid acyl glucuronides and its related compounds.
1
dles. m.p. 123–125°C. H NMR (CDCl3) ␦: 3.45 (0.4H, dd, J ϭ
6.9 and 9.3 Hz, 2-H (␤-anomer)), 3.60 (0.6H, dd, J ϭ 3.3 and 9.1
Hz, 2-H (␣-anomer)), 3.66 (0.4H, t, J ϭ 9.9 Hz, 4-H (␤-anomer)),
3.76 (0.6H, t, J ϭ 9.2 Hz, 4-H (␣-anomer)), 3.86 (0.4H, t, J ϭ 9.6
Hz, 3-H (␤-anomer)), 3.94-4.03 (1.6H, unresolved m, 3- (␣-
anomer), 5-H (␣- and ␤-anomer)), 4.46–5.24 (9H, m, 1-H (␣- and
␤- anomer) and 4 ϫ CH2C6H5), 7.12–7.34 (20H, m, 4 ϫ
CH2C6H5). Analysis calculated for C34H34O7: C, 73.63; H, 6.18.
Found: C, 73.89; H, 5.84.
mL/min to give 8 (20.0 mg, 9.1%) as a semicrystalline product. 1H
NMR (CDCl3) ␦: 0.62 (3H, s, 18-H), 0.88 (3H, d, J ϭ 6.3 Hz,
21-H), 0.92 (3H, s, 19-H), 3.63 (1H, m, 2Ј- and 3␤-H), 3.73 (1H,
t, J ϭ 8.6 Hz, 4Ј-H), 3.85 (1H, t, J ϭ 9.3 Hz, 3Ј-H), 4.10 (1H, d,
J ϭ 9.3 Hz, 5Ј-H), 4.42–5.16 (8H, m, 4 ϫ CH2C6H5), 5.67 (1H, d,
J ϭ 7.8 Hz, 1Ј-H), 7.10–7.33 (20H, m, 4 ϫ CH2C6H5). Analysis
calculated for C58H72O9: C, 76.28; H, 7.95. Found: C, 76.09; H,
8.30.
Benzyl 1-O-(24-chenodeoxycholyl)-2,3,4-tri-O-benzyl-␤-D-gluco-
pyranuronate (9): The crude product was subjected to column chro-
matography on silica gel (5 g) using n-hexane-AcOEt (2:1, v/v) and
then semipreparative HPLC with a Cosmosil 5-Ph column using
H2O-MeOH (1:10, v/v) to give 9 (15.1 mg, 6.8%) as a semicrystalline
product. 1H NMR (CDCl3) ␦: 0.64 (3H, s, 18-H), 0.89 (3H, d, J ϭ 6.0
Hz, 21-H), 0.90 (3H, s, 19-H), 3.47 (1H, m, 3␤-H), 3.63 (1H, t, J ϭ
8.4 Hz, 2Ј-H), 3.73 (1H, t, J ϭ 8.7 Hz, 4Ј-H), 3.85 (2H, m, 3Ј- and
7␤-H), 4.10 (1H, d, J ϭ 9.5 Hz, 5Ј-H), 4.42–5.20 (8H, m, 4 ϫ
CH2C6H5), 5.67 (1H, d, J ϭ 7.7 Hz, 1Ј-H), 7.09–7.32 (20H, m, 4 ϫ
CH2C6H5). Analysis calculated for C58H72O10: C, 74.97; H, 7.81.
Found: C, 74.73; H, 8.13.
Benzyl 1-O-(24-ursodeoxycholyl)-2,3,4-tri-O-benzyl-␤-D-gluco-
pyranuronate (10): The crude product was subjected to column chro-
matography on silica gel (5 g) using n-hexane-acetone (5:1, v/v)
and then submitted to preparative TLC using n-hexane-acetone
(3:2, v/v) as a developing solvent. The spot corresponding to Rf
value of 0.45 was scraped off and the desired compound was
eluted with AcOEt to give 10 (25.2 mg, 11.3%) as a semicrystal-
line product. 1H NMR (CDCl3) ␦: 0.65 (3H, s, 18-H), 0.90 (3H, d,
J ϭ 6.3 Hz, 21-H), 0.94 (3H, s, 19-H), 3.56 (2H, m, 3␤- and
7␣-H), 3.63 (1H, t, J ϭ 8.4 Hz, 2Ј-H), 3.73 (1H, t, J ϭ 9.2 Hz,
4Ј-H), 3.84 (1H, t, J ϭ 9.0 Hz, 3Ј-H), 4,10 (1H, d, J ϭ 9.2 Hz,
Coupling reaction of bile acid with benzyl 2,3,4-tri-
O-benzyl-D-glucopyranuronate (7)
Diethylazodicarboxylate (43 ␮L) was added to a stirred solution of
7 (160 mg) and bile acid (0.24 mmol) in the presence of triph-
enylphosphine (60 mg) in anhydrous tetrahydrofuran (2 mL) under
an Ar gas stream. After stirring for another 3 h at room tempera-
ture, the solution was evaporated in vacuo and the residue obtained
was diluted with AcOEt, which in turn was washed successively
with 5% HCl, H2O and saturated NaCl, dried on Na2SO4, and
evaporated in vacuo. The crude product obtained was then purified
as follows.
Benzyl 1-O-(24-lithocholyl)-2,3,4-tri-O-benzyl-␤-D-glucopyra-
nuronate (8): The crude product was subjected to column chroma-
tography on silica gel (5 g) using n-hexane-AcOEt (7:1, v/v). The
residue obtained was then submitted to preparative TLC using
n-hexane-acetone (2:1, v/v) as a developing solvent. The spot
corresponding to Rf value of 0.45 was scraped off and the desired
compound was eluted with AcOEt. The residue was further puri-
fied by semipreparative HPLC with a Cosmosil 5-Ph column using
H2O-MeOH (1:10. v/v) as a mobile phase at a flow rate of 1
182 Steroids, 1998, vol. 63, April

Synthesis of bile acid 24-acyl glucuronides: Goto et al.
Figure 2 A typical high-performance liquid chromatogram of a synthetic product of benzyl 1-O-(24-chenodeoxycholyl)-2,3,4-tri-O-
benzyl-␤-D-glucopyranuronate. Conditions: column, Cosmosil 5-Ph; mobile phase, H2O-MeOH (1:10, v/v); flow rate, 1.0 mL/min;
detection, 260 nm.
5Ј-H), 4.42–5.17 (8H, m, 4 ϫ CH2C6H5), 5.67 (1H, d, J ϭ 7.8 Hz,
1Ј-H), 7.03–7.31 (20H, m, 4 ϫ CH2C6H5). Analysis calculated for
C58H72O10: C, 74.97; H, 7.81. Found: C, 74.25; H, 7.58.
Benzyl 1-O-(24-deoxycholyl)-2,3,4-tri-O-benzyl-␤-D-glucopy-
ranuronate (11): The crude product was subjected to column
chromatography on silica gel (5 g) using n-hexane-AcOEt (2:1,
v/v) and then on Cosmosil 140C18-OPN (8 g) using MeOH to give
11 (67.8 mg, 30.4%) as a semicrystalline product. 1H NMR
(CDC13) ␦: 0.65 (3H, s, 18-H), 0.91 (3H, s, 19-H), 0.94 (3H, d,
J ϭ 6.3 Hz, 21-H), 3.63 (2H, m, 2Ј- and 3␤-H), 3.73 (1H, t, J ϭ
8.6 Hz, 4Ј-H), 3.84 (1H, t, J ϭ 9.2 Hz, 3Ј-H), 3.96 (1, m, 1 2␤-H),
4.10 (1H, d, J ϭ 9.5 Hz, 5Ј-H), 4.41–5.15 (8H, m, 4 ϫ CH2C6H5),
5.67 (1H, d, J ϭ 7.7 Hz, 1Ј-H), 7.03–7.31 (20H, m, 4 ϫ
CH2C6H5). Analysis calculated for C58H72O10: C, 74.97; H, 7.81.
Found: C, 74.45; H, 8.18.
remove less polar compounds and then layered onto the top of
Cosmosil 140C18-OPN (5 g) packed on a sintered glass filter. After
washing with 30 mM AcOH, the adsorbates were eluted with 10
mM AcOH-EtOH (1:9, v/v) to give the desired compounds as
colorless semicrystalline substances.
1-O-(24-Lithocholyl)-␤-D-glucopyranuronic acid (13): m.p.
1
140–142°C. H NMR (CD3OD) ␦: 0.69 (3H, s, 18-H), 0.94 (3H,
s, 19-H), 0.95 (3H, d, J ϭ 6.6 Hz, 21-H), 3.37 (1H, t, J ϭ 10.0 Hz,
2Ј-H), 3.42 (2H, m, 3Ј- and 4Ј-H), 3.53 (1H, m, 3␤-H), 3.67 (1H,
d, J ϭ 10.6 Hz, 5Ј-H), 5.49 (1H, d, J ϭ 8.5 Hz, 1Ј-H). ESI-MS
(m/z): 551 ([M-H]Ϫ, 100%).
1-O-(24-Chenodeoxycholyl)-␤-D-glucopyranuronic acid (14):
m.p. 144–145°C. 1H NMR (CD3OD) ␦: 0.69 (3H, s, 18-H), 0.92 (3H,
s, 19-H), 0.96 (3H, d, J ϭ 6.6 Hz, 21-H), 3.35–3.52 (5H, unresolved
m, 2Ј-, 3Ј-, 4Ј-, 5Ј- and 3␤-H), 3.79 (1H, m, 7␤-H), 5.49 (1H, d, J ϭ
7.8 Hz, 1Ј-H). ESI-MS (m/z): 567 ([M-H]Ϫ, 100%).
Benzyl 1-O-(24-cholyl)-2,3,4-tri-O-benzyl-␤-D-glucopyranuro-
nate (12): The crude product was subjected to column chromatog-
raphy on silica gel (5 g) using n-hexane-acetone (2:1, v/v) and then
by semipreparative HPLC using H2O-MeOH (1:8, v/v) to give 12
1-O-(24-Ursodeoxycholyl)-␤-D-glucopyranuronic acid (15):
1
m.p. 121–123°C. H NMR (CD3OD) ␦: 0.71 (3H, s, 18-H), 0.96
(3H, s, 19-H), 0.96 (3H, d, J ϭ 6.6 Hz, 21-H), 3.34–3.57 (6H,
unresolved m, 2Ј-, 3Ј-, 4Ј-, 5Ј-, 3␤- and 7␣-H), 5.49 (1H, d, J ϭ 7.7
Hz, 1Ј-H). ESI-MS (m/z): 567 ([M-H]Ϫ, 100%).
1
(21 mg, 9.3%) as a semicrystalline product. H NMR (CDCl3) ␦:
0.66 (3H, s, 18-H), 0.89 (3H, s, 19-H), 0.95 (3H, d, J ϭ 6.3 Hz,
21-H), 3.45 (1H, m, 3␤-H), 3.63 (1H, t, J ϭ 8.2 Hz, 2Ј-H), 3.73
(1H, t, J ϭ 8.8 Hz, 4Ј-H), 3.84 (2H, m, 3Ј- and 7␤-H), 3.96 (1H,
m, 1 2␤-H), 4,10 (1H, d, J ϭ 9.5 Hz, 5Ј-H), 4.41–5.16 (8H, m, 4 ϫ
CH2C6H5), 5.67 (1H, d, J ϭ 7.7 Hz, 1Ј-H), 7.04–7.31 (20H, m,
4 ϫ CH2C6H5). Analysis calculated for C58H72O11: C, 73.70; H,
7.68. Found: C, 73.32; H, 7.72.
1-O-(24-Deoxycholyl)-␤-D-glucopyranuronic acid (16): m.p.
1
164–166°C. H NMR (CD3OD) ␦: 0.71 (3H, s, 18-H), 0.93 (3H,
s, 19-H), 1.00 (3H, d, J ϭ 6.2 Hz, 21-H), 3.36–3.69 (5H, unre-
solved m, 2Ј-, 3Ј-, 4Ј-, 5Ј- and 3␤-H), 3.95 (1H, m, 1 2␤-H), 5.50
(1H, d, J ϭ 7.7 Hz, 1Ј-H). ESI-MS (m/z): 567 ([M-H]Ϫ, 100%).
1-O-(24-Cholyl)-␤-D-glucopyranuronic acid (17): m.p. 159–
161°C. 1H NMR (CD3OD) ␦: 0.71 (3H, s, 18-H), 0.91 (3H, s,
19-H), 1.01 (3H, d, J ϭ 6.2 Hz, 21-H), 3.34–3.52 (5H, unresolved
m, 2Ј-, 3Ј-, 4Ј-, 5Ј- and 3␤-H), 3.80 (1H, m, 7␤-H), 3.95 (1H, m,
12␤-H), 5.50 (1H, d, J ϭ 7.7 Hz, 1Ј-H). ESI-MS (m/z): 583
([M-H]Ϫ, 100%).
Catalytic reduction
A mixture of 10% palladium on carbon (100 mg) and the benzyl
ester-benzyl ether derivatives of bile acid 24-glucuronides (8–12,
each 16 ␮mol) in AcOEt containing 1% AcOH (1 mL) was shaken
under a H2 atmospheric condition with a pressure of 5 Kbar at
room temperature for 12 h. The resulting solution was centrifuged
at 2600 rpm for 10 min and the supernatant was discarded. The
precipitate was washed several times with 1 mL of AcOEt to
Results and discussion
The synthesis of acyl glucuronides requires D-glucuronic
acid of which the hydroxy groups at C-2, 3 and 4 are
Steroids, 1998, vol. 63, April 183

Papers
Figure 3 Selected ion recording of bile acid 24-glucuronide acetate-methyl esters monitored with corresponding adduct ion [M ϩ
NH4]ϩ. Conditions: column, Sumichiral OA-2500 for DCA, UDCA and CA, and Cosmosil 5C8 for LCA and CDCA; mobile phase, 200 mM
ammonium acetate (pH 7.0)-MeOH (1:4, v/v); flow rate, 1.0 mL/min. De, newly prepared sample; St, authentic specimen.
suitably protected. In the previous communication, we re-
ported the synthesis of bile acid 24-glucuronide acetate-
methyl esters by the use of the Mitsunobu reaction employ-
ing methyl 2,3,4-tri-O-acetyl-D-glucopyranuronate.12 This
procedure is not satisfactory for the synthesis of the target
compounds, because of the instability of the produced acyl
glucuronides under basic conditions, under which the cleav-
age of the glycosyl linkage to give liberated free bile acids
easily occurs. Therefore, for this purpose, D-glucuronic acid
having groups easily removable under mild and non-
hydrolytic conditions not only on hydroxy moieties but also
on a carboxy moiety is really required. Recently, several
methods have been published employing a D-glucuronic
acid derivative protected with a benzyl,14 a trichloroethyl,15
an ␣-ethoxyethyl16 or an allyloxy group.16 Panfil et al.
reported a prominent method for the synthesis of lithocholic
acid 24-glucuronide using benzyl 2,3,4-tri-O-benzyl-D-
glucopyranuronate which was derived from benzyl 2,3,4-
tri-O-benzyl-1-O-methyl-D-glucopyranuronate.16 The ben-
zyl groups on the hydroxy and carboxy moieties were easily
removed by catalytic hydrogenation. The latter compound,
however, is not commercially available. Initial effort was
therefore directed to the synthesis of benzyl 2,3,4-tri-O-
benzyl-D-glucopyranuronate from methyl ␣-D-glucose em-
ploying the novel procedure (Figure 1). The primary hy-
droxy group at C-6 was protected with a trityl ether group
(2) by treatment with triphenylmethyl chloride in pyridine.
The protection of hydroxy groups at C-2, 3 and 4 to give a
benzyl ether derivative (3) was attained by the condensation
reaction of 2 with benzylbromide in the presence of sodium
hydride and tetrabutyl ammonium iodide. Upon treatment
with H2SO4, a trityl group of 3 was readily removed to
produce a 6-hydroxy derivative (4). Subsequent oxidation
of the primary hydroxy group with Jones reagent, followed
by esterification with benzyl bromide in the presence of
sodium bicarbonate in dimethylsulfoxide gave a benzyl
ester derivative (5). The acetolysis of a methyl glycidic
group with an equimolar amount of H2SO4 in a mixed
solvent of acetic acid and acetic anhydride yielded a mixture
of ␣- and ␤-anomers of an 1-O-acetyl derivative (6). The
hydrolysis with 2-aminoethanol in AcOEt containing 1%
dimethylsufoxide afforded a 3:2 mixture of ␣- and
␤-anomers of the desired benzyl 2,3,4-tri-O-benzyl-D-
glucopyranuronate (7) in a satisfactory yield.
The synthesis of acyl glucuronides of bile acids were
then undertaken. The condensation of a carboxy group of
bile acids with an anomeric hydroxy group of the glucuronic
acid derivatives (7) was achieved by the use of the Mit-
sunobu reaction employing triphenylphosphine and diethy-
lazodicarboxylate in tetrahydrofuran.17 No alternative cou-
pling with a hydroxy group on a steroid nucleus to form a
bile acid dimer was observed. In the previous paper, we
have disclosed that the ␤-anomer of bile acid 24-
glucuronides was produced only in rat liver microsomal
fractions.12 Advantageously, the condensation reaction gave
exclusive formation of the ␤-anomer with a small amount of
184 Steroids, 1998, vol. 63, April

Synthesis of bile acid 24-acyl glucuronides: Goto et al.
the ␣-anomer. Purification of the desired ␤-anomers of the
acyl glucuronides derived from lithocholic, chenodeoxy-
cholic and cholic acids were accomplished by semi-
preparative HPLC on a column packed with phenyl-bonded
silica using H2O-MeOH (1:8 or 1:10) as a mobile phase.
The ␤-anomers of deoxycholic and ursodeoxycholic acid
24-glucuronides could be separated and purified by prepar-
ative TLC. A typical high-performance liquid chromato-
gram of a mixture of ␣- and ␤-anomers of benzyl 1-O-(24-
chendeoxycholyl)-2,3,4-tri-O-benzyl-D-glucopyranuronate is
illustrated in Figure 2. The structural confirmation of the
isolated bile acid 24-glucuronide benzyl ester-benzyl ether
derivatives was performed by 1H NMR. The anomeric pro-
ton signal of the sugar moiety appeared at 5.67 ppm (J ϭ
7.7–7.8 Hz), indicating the ␤-glucuronosyl linkage. Finally,
the removal of the protecting groups was attained by hy-
drogenation with palladium on carbon in AcOEt containing
1% AcOH to give the acyl glucuronides. 1H NMR spectra of
these glucuronides also showed the anomeric proton signals
at 5.49–5.50 ppm (J ϭ 7.7–8.5 Hz). On the ESI-MS anal-
ysis, these bile acid 24-glucuronides exhibited a singly
charged intact ion [M-H]Ϫ at m/z 551 for lithocholic acid
24-glucuronide, 567 for chenodeoxycholic, deoxycholic and
ursodeoxycholic acid 24-glucuronides, and 583 for cholic
acid 24-glucuronide, respectively. It is noteworthy that bile
acid acyl glucuronides did not undergo acyl migration under
these reaction and purification conditions described above.
In the previous paper, we have reported the separatory
determination of bile acid 24-glucuronide acetate-methyl
esters by LC/APCI-MS.12 Accordingly, structural confirma-
tion was further carried out by this method with the aid of
authentic specimens. After conversion of bile acid 24-
glucuronides into their acetate-methyl esters in the manner
previously reported, the derivatives were subjected to an LC/
APCI-MS analysis with selected ion monitoring. The retention
times of peaks on the chromatogram were definitely identical
with those of corresponding authentic ␤-anomers (Figure 3).
No contamination of the ␣-anomer in the synthesized glucu-
ronides was observed.
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Acknowledgments
This work was supported in part by a grant in aid from the
Ministry of Education, Sciences, Sports and Culture of Japan.
Steroids, 1998, vol. 63, April 185