Chemical synthesis of 24-β-d -galactopyranosides of bile acids: a new type of bile acid conjugates in human urine

Chemistry and Physics of Lipids 134 (2005) 141–150

Chemical synthesis of 24-␤-d-galactopyranosides of bile acids: a new type of bile acid conjugates in human urine Genta Kakiyama a , Shinji Sadakiyo a , Takashi Iida a,∗ , Kumiko Mushiake b , Takaaki Goto b , Nariyasu Mano b , Junichi Goto b , Toshio Nambara b a

Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan b Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 981-8578, Japan Received 7 December 2004; received in revised form 14 December 2004; accepted 4 January 2005

Abstract A method is reported for the preparation of the C-24 carboxyl-linked ␤-d-galactopyranosides of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids, two of which were recently identified as a novel type of the metabolites of bile acids excreted in human urine. Direct esterification (galactosidation) of the unprotected bile acids with 2,3,4,6-tetra-Obenzyl-d-galactopyranose in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent and subsequent hydrogenolysis of the resulting benzyloxy-protected bile acid 24-␤-d-galactopyranosides over 10% palladium on charcoal under atmospheric pressure afforded the title compounds. The structures of the bile acid acyl galactosides were confirmed by measuring several 1 H–1 H and 1 H–13 C shift correlated 2D NMR. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Bile acid; Bile acid acyl 24-galactoside; Ester galactosidation; 2-Chloro-1,3,5-trinitrobenzene; Hydrogenolysis; 2D NMR

1. Introduction Many lipophilic biomolecules and drugs containing hydroxy and/or carboxyl groups are metabolized to their glycosidic conjugates in vivo (Faed, 1984). Unconjugated bile acids are transformed into the glycosidic-conjugated forms in vivo, in which there are two major conjugation pathways. One pathway ∗ Corresponding author. Tel.: +81 3 3329 1151; fax: +81 3 3303 9899. E-mail address: [email protected] (T. Iida).

involves the conjugation of a hydroxy group on the 5␤-steroid nucleus of bile acids with the 1 ␤-anomeric hydroxy group in a pyranose such as ␤-d-glucuronic acid, ␤-d-glucose, or ␤-d-N-acetylglucosamine to give the ether-linked glycosides (Back, 1976; Matern et al., 1984; Marschall et al., 1989). The other one is carboxyl-linked glycosides, also termed acyl or ester glycosides, which is formed by esterification between the C-24 carboxyl group on the side chain of bile acids and the anomeric hydroxy group in a pyranose (Radomi´nska-Pyrek et al., 1986; Shattuck et al., 1986; Ikegawa et al., 1999b). It is also documented that bile

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G. Kakiyama et al. / Chemistry and Physics of Lipids 134 (2005) 141–150

Fig. 1. Synthetic route to bile acid acyl galactosides and their structures.

acid acyl 24-glycosides are chemically unstable and/or active (Bolze et al., 2002) and irreversibly bind to proteins to produce protein-bonded adducts (Ikegawa et al., 1999a). During the course of our study on bile acid metabolites in human biological fluids, we have recently revealed the presence of a novel type of bile acid conjugates in human urine, which was tentatively assigned as acyl 24-␤-d-galactopyranosides of deoxycholic acid (3␣,12␣-dihydroxy-5␤-cholan-24-oic acid; DCA) and cholic acid (3␣,7␣,12␣-trihydroxy-5␤-cholan-24-oic acid; CA) (Goto et al., 2005). The existence of the bile acid acyl 24-galactosides was elucidated by the deconjugation of a glycosidic-conjugated fraction obtained from human urine by chemical cleavage, followed by chromatographic and mass spectrometric analyses of the liberated residues. Therefore, a more direct proof for their existence had to await chemical synthesis and demonstration of identity of the isolated compounds with synthetic ones. The availability of authentic specimens may also serve to establish a reliable method for the quantitative and qualitative analyses of bile acid acyl 24-galactosides in biological materials with regard to metabolic disorders in humans, as well as to clarify the physiological significance of the pathway, and the region where these conjugates are synthesized, the enzyme catalyzing the conjugation, and the dynamics of

the conjugates. We herein report the synthesis of the acyl 24-␤-d-galactopyranosides (1c–5c) of five prominent bile acids, lithocholic acid (LCA; 1a), DCA (2a), chenodeoxycholic acid (CDCA; 3a), ursodeoxycholic acid (UDCA; 4a), and CA (5a) (see Fig. 1) and on the spectroscopic properties.

2. Experimental 2.1. Materials and methods LCA, DCA, and CA were purchased from Wako Pure Industries, Ltd. (Osaka, Japan). CDCA and UDCA were kindly donated by Mitsubishi Pharma Corporation (Tokyo, Japan). 2,3,4,6-Tetra-O-benzyld-galactopyranose (TBGP) was available from Sigma Chemical Co. (St. Louis, MO, USA). 2-Chloro-1,3,5trinitrobenzene (CTNB) was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). All other chemicals and solvents used are of analytical reagent grade. All bile acid derivatives were dried by azeotropic distillation (benzene–CH2 Cl2 or methanol–CH2 Cl2 ) before use in reactions. Melting points (mp) were determined on a micro hot-stage apparatus and are uncorrected. Infrared (IR) spectra were obtained in KBr discs on a Shimadzu

G. Kakiyama et al. / Chemistry and Physics of Lipids 134 (2005) 141–150

FTIR-8300 spectrometer. Proton (1 H) and carbon (13 C) nuclear magnetic resonance (NMR) spectra were obtained on a JEOL JNM-EX 270 Fourier transform spectrometer (Tokyo, Japan) operating at 270 and 68.8 MHz for 1 H and 13 C, respectively, with CDCl3 or CD3 OD containing 0.1% tetramethylsilane (Me4 Si) as the solvent; chemical shifts are expressed as δ ppm relative to Me4 Si. Electron ionization (EI) low-resolution mass (LR-MS) spectra were determined on a JEOL JMS-303 mass spectrometer at 70 eV. High-resolution mass (HRMS) spectra were measured by using a JEOL JMSLCmate double-focusing magnetic mass spectrometer equipped with an electrospray ionization (ESI) probe under the positive ion mode (PIM) or the negative ion mode (NIM). HR-MS spectra were also obtained on a JEOL JMS-700 mass spectrometer with an EI probe under the PIM. The apparatus used for mediumpressure liquid chromatography (MPLC) consisted of a Shimamura YRD-880 refractive index detector and an uf-3040S chromatographic pump (Shimamura Tech., Tokyo, Japan): mixtures of benzene/EtOAc/acetic acid (20:80:1, by vol.) and methanol/EtOAc/water/acetic acid (75:10:15:1–80:10:10:1, by vol.) were employed as the mobile phases on normal-phase (NP) and on reversed-phase (RP) MPLC, respectively. NP thin-layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates (0.25 mm layer thickness; E. Merck, Darmstadt, Germany) using hexane/EtOAc/acetic acid mixtures (80:20:1–40:60:1, by vol.) as the developing solvent. RP-TLC was carried out on precoated RP-18 F254S plates using methanol/water/acetic acid mixtures (80:20:1–90:10:1, by vol.) as the developing solvent. Two-dimensional (2D) 1 H–1 H and 1 H–13 C shift correlated NMR spectra were recorded at 23 ◦ C for ca. 0.05 mM solutions in CD3 OD in a 5 mm tube on a JEOL ECA-600 instrument (600 and 149.4 MHz for 1 H and 13 C, respectively). 1 H and 13 C resonance assignments were made using a combination of 2D homonuclear (1 H–1 H) and heteronuclear (1 H–13 C) chemical shift techniques, which include 1 H–1 H COSY, long-range 1 H–1 H COSY, 1 H–1 H phase-sensitive nuclear Overhauser effect (PSNOE), 1 H–1 H homonuclear Hartmann–Harn (HOHAHA) (Ramamoorthy, A. and Chandrakumar, N. 1992), 1 H–13 C HETCOR and long-range 1 H–13 C HETCOR (COLOC) experiments. These 2D NMR spectra were recorded by using standard pulse sequences and parameters recommended by

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the manufacturer. The 13 C distortionless enhancement by polarization transfer (DEPT, 135, 90 and 45◦ ) spectra were also measured in order to determine the exact 1 H signal multiplicity and to differentiate among CH , 3 CH2 , CH and C. 2.2. General procedure for the esterification of bile acids by CTNB To a magnetically stirred solution of bile acid (1a–5a, 1.5 mmol), TBGP (1.1 g, 2 mmol) and molec˚ in dry pyridine (4 ml) was added ular sieves (2 g; 3 A) CTNB (515 mg, 2 mmol) in one portion. The mixture was stirred at room temperature for 5 h under a stream of N2 ; the reaction was monitored by TLC. To the mixture was added successively 8% NaHCO3 (10 ml), Et2 O (10 ml) and water (5 ml), and the whole mixture was stirred until a clear solution was obtained. The organic layer was washed with 5% HCl and saturated brine, dried with Drierite, and evaporated to dryness under reduced pressure. The residue was chromatographed on a column of silica gel (70–230 mesh, 50 g) eluting with benzene/EtOAc mixtures to give the crude esterified product, which was shown to be a mixture of ␣- and ␤-anomers (approximate ratio, 1:5–1:6). Purification of the mixture by NP-MPLC on silica gel 60 column (230–400 mesh, 100 g) or RP-MPLC on C18 -bonded silica gel column (50 ␮m, 100 g) gave the desired ␤-anomer of bile acid 24-galactoside benzyl ether (1b–5b). 2.2.1. 1-O-(24-Lithocholyl)-2,3,4,6-tetra-Obenzyl-␤-d-galactopyranose (1b) The crude esterified product was subjected to a column of silica gel, eluting with benzene/EtOAc/acetic acid (80:20:1, by vol.), followed by RP-MPLC on C18 -bonded silica gel eluting with methanol/EtOAc/H2 O/acetic acid (80:10:10:1, by vol.). Although this component was homogeneous according to TLC and 1 H NMR analyses, it was resisted recrystallization attempts: yield, 23%. IR, ␯max cm−1 : 1080 (C O), 1751 (C O), 2864, 2928 (C H), 3400 (O H). 1 H NMR (CDCl3 ), δ: 0.60 (3H, s, 18-CH3 ), 0.87 (3H, d, J = 6.2 Hz, 21-CH3 ), 0.91 (3H, s, 19CH3 ), 3.59 (1H, brm, 3␤-H), 3.50–3.72 and 3.92–4.00 (6H, m and brm, 2 -, 3 -, 4 -, 5 - and 6 -H), 4.36–5.00 (8H, m, 4×-CH2 C6 H5 ), 5.60 (1H, d, J = 8.4 Hz, 1 -

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H), 7.25–7.32 (20H, brm, 4×-CH2 C6 H5 ). LR-MS (EI), m/z: 431 (12%), 358 [M − benzylic sugar (B.S., 540u), 12%], 282 (9%), 253 (48%), 239 [M − B.S.–side chain (S.C., 101u) H2 O, 11%], 215 [M − B.S.–S.C.–ring D (42u), 13%], 181 (45%), 107 (C6 H5 CH2 O, 18%), 91 (C6 H5 CH2 , 100%). HR-MS (ESI-PIM), calculated for C58 H74 O8 Na [M + Na]+ : 921.5281; found, m/z: 921.5292. 2.2.2. 1-O-(24-Deoxycholyl)-2,3,4,6-tetra-Obenzyl-␤-d-galactopyranose (2b) The crude esterified product was subjected to a column of silica gel, eluting with benzene/EtOAc/acetic acid (30:70:1, by vol.), followed by RP-MPLC on C18 -bonded silica gel eluting with methanol/EtOAc/H2 O/acetic acid (75:10:15:1, by vol.). Although this component was homogeneous according to TLC and 1 H NMR analyses, it was resisted recrystallization attempts: yield, 24%. IR, νmax cm−1 : 1080 (C O), 1749 (C O), 2864, 2931 (C H), 3400 (O H). 1 H NMR (CDCl3 ), δ: 0.64 (3H, s, 18-CH3 ), 0.91 (3H, s, 19-CH3 ), 0.92 (3H, d, J = 5.9 Hz, 21-CH3 ), 3.59 (1H, brm, 3␤-H), 3.60–3.72 and 3.92–4.00 (6H, m and brm, 2 -, 3 -, 4 -, 5 - and 6 -H), 3.95 (1H, m, 12␤-H), 4.41–4.92 (8H, m, 4×-CH2 C6 H5 ), 5.58 (1H, d, J = 8.4 Hz, 1 H), 7.25–7.34 (20H, brm, 4×-CH2 C6 H5 ). LR-MS (EI), m/z: 358 (B.S.-2C6 H5 CH2 , 12%), 282 (15%), 253 (44%), 181 (41%), 107 (C6 H5 CH2 O, 18%), 91 (C6 H5 CH2 , 100%). HR-MS (ESI-PIM), calculated for C58 H74 O9 Na [M + Na]+ : 937.5230; found, m/z: 937.5249. 2.2.3. 1-O-(24-Chenodeoxycholyl)-2,3,4,6-tetraO-benzyl-␤-d-galactopyranose (3b) The crude esterified product was subjected to a column of silica gel, eluting with benzene/EtOAc/acetic acid (30:70:1, by vol.), followed by RP-MPLC on C18 -bonded silica gel eluting with methanol/EtOAc/H2 O/acetic acid (75:10:15:1, by vol.). Although the major fraction was homogeneous according to TLC and 1 H NMR analyses, it was resisted recrystallization attempts: yield, 12.7%. IR, νmax cm−1 : 1078 (C O), 1755 (C O), 2866, 2925 (C H), 3404 (O H). 1 H NMR (CDCl3 ), δ: 0.62 (3H, s, 18CH3 ), 0.88 (3H, d, J = 6.4 Hz, 21-CH3 ), 0.90 (3H, s, 19CH3 ), 3.45 (3H, brm, 3␤-H), 3.53–3.72 and 3.92–3.98 (6H, m and brm, 2 -, 3 -, 4 -, 5 - and 6 -H), 3.83

(1H, m, 7␤-H), 4.41–4.89 (8H, m, 4×-CH2 C6 H5 ), 5.58 (2H, d, J = 8.4 Hz, 1 -H), 7.24–7.32 (20H, m, 4×CH2 C6 H5 ). LR-MS (EI), m/z: 356 (M − B.S.–H2 O, 16%), 341 (M − S.C.–H2 O–CH3 , 8%), 253 (31%), 181 (33%), 91 (C6 H5 CH2 , 100%). HR-MS (ESI-PIM), calculated for C58 H74 O9 Na [M + Na]+ : 937.5230; found, m/z: 937.5223. 2.2.4. 1-O-(24-Ursodeoxycholyl)-2,3,4,6-tetra-Obenzyl-␤-d-galactopyranose (4b) The crude esterified product was subjected to an open column of silica gel, eluting with benzene/EtOAc/acetic acid (30:70:1, by vol.), followed by RP-MPLC on C18 -bonded silica gel eluting with methanol/EtOAc/H2 O/acetic acid (75:10:15:1, by vol.). Although the major fraction was homogeneous according to TLC and 1 H NMR analyses, it was resisted recrystallization attempts: yield, 15%. IR, νmax cm−1 : 1055 (C O), 1751 (C O), 2864, 2932 (C H), 3422 (O H). 1 H NMR (CDCl3 ), δ: 0.64 (3H, s, 18CH3 ), 0.88 (3H, d, J = 5.9 Hz, 21-CH3 ), 0.94 (3H, s, 19-CH3 ), 3.47–3.70 and 3.91–3.99 (6H, m and brm, 2 -, 3 -, 4 -, 5 - and 6 -H), 3.59 (1H, brm, 3␤-H), 3.59 (1H, brm, 7␣-H), 4.41–4.89 (8H, m, 4×-CH2 C6 H5 ), 5.58 (2H, d, J = 8.4 Hz, 1 -H), 7.24–7.36 (20H, m, 4×CH2 C6 H5 ). LR-MS (EI), m/z: 356 (M − B.S.–H2 O, 4%), 253 (16%), 181 (19%), 107 (C6 H5 CH2 O, 10%), 91 (C6 H5 CH2 , 100%). HR-MS (ESI-PIM), calculated for C58 H74 O9 Na [M + Na]+ : 937.5230; found, m/z: 937.5220. 2.2.5. 1-O-(24-Cholyl)-2,3,4,6-tetra-O-benzyl-␤d-galactopyranose (5b) The crude esterified product was subjected to a column of silica gel, eluting with benzene/EtOAc/acetic acid (30:70:1, by vol.), followed by NP-MPLC on silica gel column eluting with benzene/EtOAc/acetic acid (20:80:1, by vol.). Although the major fraction was homogeneous according to TLC and 1 H NMR analyses, it was resisted recrystallization attempts: yield, 36%. IR, νmax cm−1 : 1078 (C O), 1749 (C O), 2866, 2934 (C H), 3396 (O H). 1 H NMR (CDCl3 ), δ: 0.65 (3H, s, 18-CH3 ), 0.89 (3H, s, 19-CH3 ), 0.93 (3H, d, J = 6.2 Hz, 21-CH3 ), 3.45 (1H, brm, 3␤-H), 3.84 (1H, m, 7␤-H), 3.47–3.72 and 3.92–3.98 (6H, m and brm, 2 -, 3 -, 4 -, 5 - and 6 -H), 3.95 (1H, m, 12␤H), 4.41–4.89 (8H, m, 4×-CH2 C6 H5 ), 5.58 (2H, d, J = 8.1 Hz, 1 -H), 7.24–7.35 (20H, m, 4×-CH2 C6 H5 ).

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LR-MS (EI), m/z: 372 (M − B.S.–H2 O, 4%), 354 (M − B.S.–2H2 O, 4%), 271 (M − B.S.–S.C.–H2 O, 7%), 253 (M − B.S.–S.C.–2H2 O, 20%), 181 (18%), 107 (C6 H5 CH2 O, 5%), 91 (C6 H5 CH2 , 100%). HR-MS (ESI-PIM), calculated for C58 H74 O10 Na [M + Na]+ : 953.5179; found, m/z: 953.5167. 2.3. General procedure for the deprotection of benzyl ether in the sugar moiety by catalytic hydrogenation A suspension of bile acid acyl 24-galactoside benzyl ether (1b–5b, 0.74 mmol) and 10% palladium on charcoal (300 mg) in methanol/EtOAc/acetic acid (22 ml; 5:5:1, by vol.) was stirred under a hydrogen atmosphere at 35 ◦ C for 6 h; the reaction was monitored by NP- and RP-TLC. The catalyst was filtered off on Celite and the filtrate was evaporated to dryness under reduced pressure. Recrystallization of the oily residue from EtOAc gave an analytical pure bile acid acyl 24-galactoside (1c–5c) quantitatively as colorless crystals. 2.3.1. 1-O-(24-Lithocholyl)-O-␤-dgalactopyranose (1c) Yield, 100%; mp, 117–121 ◦ C. IR, νmax cm−1 : 1074 (C H), 1747 (C O), 2864, 2934 (C H), 3364 (O H). 1 H NMR (CD OD), δ: 0.65 (3H, s, 18-CH ), 0.90 3 3 (3H, s, 19-CH3 ), 0.92 (3H, d, J = 4.3 Hz, 21-CH3 ), 3.27 (1H, m, 4 -H), 3.48 (1H, m, 5 -H), 3.49 (1H, brm, 3␤-H), 3.61 (1H, m, 2 -H), 4.07 (1H, m, 3 -H), 5.39 (1H, d, J = 7.8 Hz, 1 -H). LR-MS (EI), m/z: 376 (12%,) 358 [M − galactose (Gal, 180u), 100%], 343 (M-GalCH3 , 22%), 304 (14%), 257 (M − Gal–S.C., 12 %), 248 (20%), 230 [M − Gal–S.C.–part of ring D (27u), 33%], 215 (M − Gal–S.C.–CH3 –part of ring D, 94%). HRMS (APCI-NIM), calculated for C30 H49 O8 [M − H]− : 537.3427; found, m/z: 537.3438. 2.3.2. 1-O-(24-Deoxycholyl)-O-␤-dgalactopyranose (2c) Yield, 100%; mp, 132–136 ◦ C. IR, νmax cm−1 : 1072 (C O), 1734 (C O), 2864, 2936 (C H), 3385 (O H). 1 H NMR: (see Table 1). LR-MS (EI), m/z: 374 (M − Gal, 20%), 356 (M-GalH2 O, 2%), 341 (M − Gal–H2 O–CH3 , 25%), 302 (10%), 273 (M − Gal–S.C., 87%), 255 (M − Gal– S.C.–H2 O, 100%), 248 (10%), 213 (M − Gal– S.C.–H2 O–CH3 –part of ring D, 17%), 201 (15%). HR-

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MS (APCI-NIM), calculated for C30 H49 O9 [M − H]− : 553.3376; found, m/z: 553.3358. 2.3.3. 1-O-(24-Chenodeoxycholyl)-O-␤-dgalactopyranose (3c) Yield, 100%; mp, 102–105 ◦ C. IR, νmax cm−1 : 1078 (C O), 1744 (C O), 2866, 2930 (C H), 3364 (C O). 1 H NMR (CD3 OD), δ: 0.69 (3H, s, 18-CH3 ), 0.92 (3H, s, 19-CH3 ), 0.96 (3H, d, J = 6.2 Hz, 21CH3 ), 3.31–3.88 (6H, m and brm, 2 -, 4 -, 5 -, 6 - and 7␤-H), 3.55 (1H, brm, 3␤-H), 4.09 (1H, m, 3 -H), 5.43 (1H, d, J = 7.8 Hz, 1 -H). LR-MS (EI), m/z: 392 (11%), 374 (M − Gal, 38%), 356 (M − Gal–H2 O, 100%), 341 (M − Gal–H2 O–CH3 , 48%), 302 (15%), 273 (M − Gal–S.C., 10%), 264 (26%), 255 (M − Gal–S.C.–H2 O, 29%), 246 (M − Gal–S.C.–part of ring D, 28%), 228 (M − Gal–S.C.–H2 O–part of ring D, 46%), 213 (M − Gal–S.C.–H2 O–CH3 –ring D, 41%). HR-MS (APCI-NIM), calculated for C30 H49 O9 [M − H]− : 553.3376; found, m/z: 553.3361. 2.3.4. 1-O-(24-Ursodeoxycholyl)-O-␤-dgalactopyranose (4c) Yield, 100%; mp, 124–128 ◦ C. IR, νmax cm−1 : 1076 (C H), 1744 (C O), 2866 and 2934 (C H), 3364 (O H). 1 H NMR (CD3 OD), δ: 0.71 (3H, s, 18-CH3 ), 0.96 (3H, d, J = 6.2 Hz, 21-CH3 ), 0.96 (3H, s, 19-CH3 ), 3.46 (2H, brm, 3␤- and 7␣-H), 3.53 (1H, dd, J = 10 and 3.5 Hz, 4 -H), 3.60–3.67 (2H, m, 2 - and 5 -H), 3.71 (2H, d, J = 5.7 Hz, 6 -H), 3.88 (1H, d, J = 2.9 Hz, 3 -H), 5.43 (1H, d, J = 8.1 Hz, 1 -H). LR-MS (EI), m/z: 392 (36%), 374 (M − Gal, 68%), 356 (M − Gal–H2 O, 100%), 341 (M − Gal–H2 O–CH3 , 46%), 302 (19%), 273 (M − Gal–S.C., 16%), 264 (44%), 255 (M − Gal–S.C.–H2 O, 50%), 246 (M − Gal–S.C.–part of ring D, 56%), 228 (M − Gal–H2 O–S.C.–part of ring D, 44%), 213 (M − Gal–H2 O–CH3 –S.C.–part of ring D, 43%). HR-MS (APCI-NIM), calculated for C30 H49 O9 [M − H]− : 553.3376; found, m/z: 553.3390. 2.3.5. 1-O-(24-Cholyl)-O-␤-dgalactopyranose (5c) Yield, 100%; mp, 167–170 ◦ C. IR, νmax cm−1 : 1074 (C O), 1734 (C O), 2837, 2940 (C H), 3354 (O H). 1 H NMR: (see Table 1). LR-MS (EI), m/z: 390 (M − Gal, 6%), 372 (M − Gal–H2 O, 91%), 354 (M − Gal–2H2 O,

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Table 1 and 13 C chemical shifts (δ ppm) of bile acid acyl galactosidesa

1H

DC-24-Gal (2c)b,c Type

C-24-Gal (5c)b,c 13 C

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6

CH2 CH2 CH CH2 CH CH2 CH2 CH CH C CH2 CH C CH CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH2 C CH CH CH CH CH CH2

36.45 28.40 72.54 37.20 43.63 28.40 28.57 29.91 34.82 35.30 29.91 74.01 47.57 49.29 24.85 31.07 48.05 13.20 23.70 36.55 17.60 31.80 31.94 177.77 96.51 71.30 70.08 74.91 77.51 62.21

1H

␣

␤

1.76 1.26

0.96 1.87 3.51(brm) 1.44 1.40 1.87 1.44 1.44

1.80 1.28

1.44 1.51

1.51 3.94(m)

1.61 1.09 1.55 1.84 0.70(s) 0.92(s) 1.42 1.00(d, 6.6) 1.38, 1.82(each, m) 2.31, 2.48(each, m)

Type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

CH2 CH2 CH CH2 CH CH2 CH2 CH CH C CH2 CH C CH CH2

1.60 1.44 0.70(s) 0.92(s) 1.42 1.00(d, 6.6) 1.38, 1.82(each, m) 2.31, 2.48(each, m)

5.43(d, 8.4) 3.66(dd, 9.6, 8.4) 3.87(d, 2.4) 3.53(dd, 9.6, 3.6) 3.62(t, 6.6) 3.71(d, 6.6)

LC-24-Gal (1c)d No.

13 C

Type

3.71(d, 6.6)

CH2 CH2 CH CH2 CH CH2 CH CH CH C CH2 CH C CH CH2 CH2 CH CH3 CH3 CH CH3 CH2 CH2 C CH CH CH CH CH CH2

36.49 28.60 72.89 40.48 43.21 35.85 69.05 41.03 27.89 35.90 29.59 74.01 47.50 43.00 24.22 31.19 48.01 12.99 23.15 36.62 17.66 31.84 31.96 177.76 96.14 71.32 70.08 74.94 77.53 62.21

CDC-24-Gal (3c)d 13 C

36.48 31.18 72.41 37.16 43.52 28.35 27.65 37.23 41.87 35.67 21.94 41.52 43.90 57.90 25.26

Type CH2 CH2 CH CH2 CH CH CH2 CH CH C CH2 CH C CH CH2

1H

␣

␤

1.79 1.32

0.96 1.87 3.36(brm) 1.64

2.25 1.39 1.51

1.94 3.79(m) 1.54 2.25

1.58

1.58 3.94(m)

1.98 1.11

1.74

1.85 0.71(s) 0.91(s) 1.42 1.01(d, 6.6) 1.39, 1.81(each, m) 2.32, 2.48(each, m)

0.71(s) 0.91(s) 1.42 1.01(d, 6.6) 1.39, 1.81(each, m) 2.32, 2.48(each, m)

5.43(d, 8.4) 3.66(dd, 10.2, 8.4) 3.87(d, 2.4) 3.52(dd, 10.2, 3.6) 3.61(t, 6.6) 3.71(d, 6.6)

3.71(d, 6.6)

UDC-24-Gal (4c)d 13 C

35.90 31.35 72.86 41.04 43.18 36.57 69.05 40.77 34.05 36.22 21.78 40.48 43.68 51.53 24.62

Type CH2 CH2 CH CH2 CH CH CH2 CH CH C CH2 CH C CH CH2

13 C

36.08 31.03 72.11 38.61 44.05 38.00 71.95 44.51 40.72 35.16 22.37 41.56 44.78 57.50 27.94

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147

Table 1 (Continued) LC-24-Gal (1c)d

CDC-24-Gal (3c)d

UDC-24-Gal (4c)d

No.

Type

13 C

Type

13 C

Type

13 C

16 17 18 19 20 21 22 23 24 1 2 3 4 5 6

CH2 CH CH3 CH3 CH CH3 CH2 CH2 C CH CH CH CH CH CH2

29.20 57.90 12.51 23.95 36.57 18.79 31.79 31.95 174.69 96.13 71.28 70.04 74.88 77.48 62.17

CH2 CH CH3 CH3 CH CH3 CH2 CH2 C CH CH CH CH CH CH2

29.90 57.30 12.18 23.39 36.64 18.85 31.94 31.95 174.71 96.15 71.30 70.08 74.91 77.52 62.21

CH2 CH CH3 CH3 CH CH3 CH2 CH2 C CH CH CH CH CH CH2

29.58 56.51 12.65 23.93 36.55 18.96 31.86 31.95 174.71 96.13 71.28 70.06 74.89 77.50 62.17

a b c d

Abbreviations used: s, singlet; d, doublet; t, triplet; dd, doublets of doublet; m, multiplet; brm, broad multiplet. Values in parentheses refer to coupling constants (J in Hz). Measured in CD3 OD at 600 MHz for 1 H and 149.4 MHz for 13 C NMR. Measured in CD3 OD at 68.8 MHz for 13 C NMR.

60%), 343 (40%), 339 (M − Gal–2H2 O–CH3 , 25%), 300 (17%), 271 (M − Gal–S.C.–H2 O, 100%), 253 (M − Gal–S.C.–2H2 O, 84%), 244 (M − Gal–S.C.–H2 O–part of ring D, 18%), 226 (M − Gal–S.C.–2H2 O–part of ring D, 35%). HR-MS (APCI-NIM), calculated for C30 H49 O10 [M − H]− : 569.3325; found, m/z: 569.3326.

3. Results and discussion Acyl glycosides are unstable and/or reactive metabolites that can be easily hydrolyzed and isomerized (by acyl migration) at physiological pH (Faed, 1984; Bolze et al., 2002). Until several years ago, the chemical syntheses of acyl glycosides have required multi-steps with several protections and deprotections, resulting in low overall yields (Compernolle, 1980). Recently, convenient and short-step procedures have been reported for the preparation of acyl glycosides (Smith III et al., 1986; Juteau et al., 1997). One procedure is an application of Mitsunobu’s esterification, that is condensation between carboxyl group in steroid substrates and an anomeric hydroxy group of protected pyranose in the presence of diethylazodicarboxylate (DEAD) and triphenylphosphine (TPP) as coupling agents (Goto et al., 1998). The other method involves a

similar condensation of carboxylic acids in pyridine with a protected pyranose by using 2-chloro-1,3,5trinitrobenzene (CTNB) as a condensing agent (Iida et al., 2002). The synthesis of bile acid acyl galactosides also requires d-galactose whose hydroxy groups at C2, -3, -4 and -6 are protected as benzyl ethers, since the benzyloxy groups are suitable for the subsequent deprotection reaction. Of the two condensing procedures, the latter one seemed to be more favorable, because of its much higher yield and easier work-up process. Attempted ester galactosidation of unprotected bile acids (1a–5a) in tetrahydrofuran with TBGP by Mitsunobu reaction (DEAD, 1.44 equiv. and TPP, 1.44 equiv.) at room temperature for 20 h was unsatisfactory: crude yield of the total esterified product was less than 35%, as judged by 1 H NMR and high-performance liquid chromatographic analyses. However, when CTNB (Takimoto et al., 1981) was used as a coupling agent in pyridine, the ester galactosidation proceeded smoothly and cleanly at room temperature for 5 h. In consequence, 1a–5a were converted to the corresponding crude acyl 24galactoside benzyl ether derivatives in 70–80% yield, after separation by open column chromatography on silica gel. No self-coupling with a hydroxy group on a steroid nucleus to form a bile acid dimer was observed. Each of the crude esterified products showed a single

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spot on both the NP-TLC and RP-TLC, but it was estimated to be a mixture of the ␣-/␤-anomers (approximate ratio, 1:5–1:6) of 24-galactosides, as evidenced by the appearance of anomeric proton signals (as doublets) resonating at 6.1 (1 ␤-H) and 5.4 ppm (1 ␣-H) in the 1 H NMR (see below). Isolation of the desired ␤-anomers (1c–4c) of the acyl 24-galactosides from the crude esterified products was accomplished by RP-MPLC on C18 -bonded silica gel, except for 5c, which was isolated by NP-MPLC on silica gel. To prevent the hydrolysis and/or isomerization of 1c–5c during the chromatographic purification, 1% acetic acid was added in the mobile phases. The isolated yield was 13–36%; in all the cases, the corresponding ␣-anomer was not isolated at all. The structures of acyl 24-␤-d-galactoside benzyl ethers were confirmed by the occurrence of aromatic proton signals appeared at 7.24–7.36 ppm and 1 ␣-H signal occurred at 5.58–5.60 ppm as a doublet (J = 8.1–8.4 Hz), indicating the presence of benzyloxy groups and ␤-dpyranose moiety (Iida et al., 2002). Deprotection of the benzyl groups in 1b–5b was effected by hydrogenolysis under controlled conditions (Iida et al., 2002). When the hydrogenolysis was carried out in the presence of 10% Pd on charcoal catalyst in methanol/EtOAc/acetic acid solvent (5:5:1, by vol.) at 35 ◦ C for 6 h, a single component of the desired acyl 24-␤-d-galactosides (1c–5c) were obtained in quantitative yield (∼100%), which recrystallized directly from EtOAc. The presence of acetic acid in the solvent was also essential to prevent the hydrolysis and/or isomerization during the catalytic hydrogenation. Again, the 1 H NMR spectra of 1c–5c showed 1 ␣-H signals at 5.39–5.43 ppm as a doublet (J = 7.8–8.4 Hz). Complete 1 H and 13 C signal assignments were carried out for 2c and 5c, which were excreted in human urine (Goto, et al., 2005), in order to further confirm their fine structures. The results are compiled in Table 1. The 13 C NMR assignments of the C-1–C-24 signals of the 5␤-steroid moiety were made on the basis of the corresponding unconjugated bile acids reported in a previous paper (Blunt and Stothers, 1977; Iida et al., 1983). The 1 H resonances attached to the skeletal carbon atoms were assigned by correlating the 13 C/1 H signals in the HETCOR spectra. Although the 1 H and 13 C resonances of the aglycone moiety in 2c and 5c differed from each other, both the compounds showed a very similar spectral pattern in the sugar region.

The combined use of the several 2D NMR techniques made possible to assign the 1 H and 13 C signals of the sugar moiety in 2c and 5c. Thus, in the 13 C NMR, the C-1 anomeric and C-6 methylene carbons could be readily characterized by the appearance of the signals resonated at a lower field of ca. 96 ppm and at a higher field of ca. 62 ppm, respectively (Kasai et al., 1979; Iida et al., 2003). Confirmation of the anomeric 1 ␣-H attached to the C-1 in the galactopyranose moiety was made on the 1 H NMR, because the 1 ␣-H (5.43 ppm, d, J = 8.4 Hz) signal in glycopyranosides usually occurs at much higher field than the corresponding 1 ␤-H (ca. 6.1 ppm) (Kasai et al., 1979; Hobley et al., 1996). Therefore, a signal appearing at 3.71 ppm as doublet (J = 6.6 Hz) in the 1 H–13 C HETCOR spectra was assigned to the 6 -H2 . The assignment of the remaining 1 H and 13 C signals in the sugar residue in 2c and 5c were made as follows. The correlation peaks observed for the 1 ␣-H/2 ␤-H in the 1 H–1 H COSY and HOHAHA spectra and the 1 ␣-H/C-2 in the 1 H-13 C COLOC spectra (Fig. 2) revealed that a 1 H signal at 3.66 ppm (dd, J = 9.6–10.2, 8.4 Hz) and a 13 C signal at 71.30–71.32 ppm are assigned to the 2 ␤H and C-2 , respectively. Analogously, the appearance of the cross peaks between 6 -H2 /5 ␣-H and 6 -H2 /C5 in the 2D NMR spectra indicates that a 1 H signal at 3.61–3.62 ppm and a 13 C signal at 77.51–77.53 ppm are assigned to the 5 ␣-H and the C-5 signals, respectively. Of further importance was the occurrence of the distinct cross peaks at 3.62 and 5.43 ppm in the PSNOE spectra, due to the 1,3-diaxial interaction between 1 ␣-H and 5 ␣-H (Agrawal, 1992). The correlation and/or the connectivity of 1 H/1 H or 1 H/13 C peaks in the 2D NMR also enabled the signal assignment of the 3 ␣-H (3.87 ppm, d, J = 2.4 Hz), 4 ␣-H (3.52–3.53 ppm, dd, J = 9.6–10.2, 3.6 Hz), C-3 (70.08 ppm) and C-4 (74.91–74.94 ppm). Again, the PSNOE spectra exhibited the cross peaks at 3.87 and 5.43 ppm, owing to the 1,3-diaxial 1 ␣-H/3 ␣H. The above 1 H and 13 C resonances and the signal multiplicities of the 24-␤-d-galactopyranosyl moiety in 2c and 5c much differed from those reported for the corresponding ␤-d-glucosides (Iida et al., 2003) and, therefore, may be useful for the structural determination of bile acid acyl glycosides present in human biological materials. The availability of bile acid acyl 24galactosides as authentic samples may be helpful for further studies on the metabolic disorders and physiological significance in human.

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149

Fig. 2. Partial 1 H–13 C COLOC spectrum of cholic acid 24-galactoside (5c).

Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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