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Originally published In Press as doi:10.1194/jlr.D300027-JLR200 on December 1, 2003

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Journal of Lipid Research, Vol. 45, 567-573, March 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

Chemical synthesis of (22E)-3{alpha},6ß,7ß-trihydroxy-5ß-chol-22-en-24-oic acid and its taurine and glycine conjugates

: a major bile acid in the rat

Genta Kakiyama*, Takashi Iida1,*, Atsushi Yoshimoto*, Takaaki Goto{dagger}, Nariyasu Mano{dagger}, Junichi Goto{dagger}, Toshio Nambara{dagger}, Lee R. Hagey§ and Alan F. Hofmann§

* Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan
{dagger} Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980-8578, Japan
§ Department of Medicine, University of California, San Diego, La Jolla, California 92093-0813

Published, JLR Papers in Press, December 1, 2003. DOI 10.1194/jlr.D300027-JLR200

1 To whom correspondence should be addressed. e-mail: takaiida{at}chs.nihon-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A method for the synthesis of {Delta}22-ß-muricholic acid ({Delta}22-ß-MCA), (22E)-3{alpha},6ß,7ß-trihydroxy-5ß-chol-22-en-24-oic acid, and its taurine and glycine conjugates ({Delta}22-ß-muricholyltaurine and {Delta}22-ß-muricholylglycine) is described. The key intermediate, 3{alpha},6ß,7ß-triformyloxy-23,24-dinor-5ß-cholan-22-al, was prepared from ß-muricholic acid (ß-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) {Delta}22-ß-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of {Delta}22-ß-MCA.

These synthetic reference compounds are now available for investigation of the metabolism of ß-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-2H or 3H-ß-MCA.

Abbreviations: DEPC, diethylphosphorocyanide; EI, electron ionization; ESI, electrospray ionization; EtOAc, ethyl acetate; HR-MS, high-resolution mass spectra; IR, infrared; LR-MS, low-resolution mass spectra; ß-MCA, ß-muricholic acid; {Delta}22-ß-MCA, {Delta}22-ß-muricholic acid; NIM, negative ion mode; PIM, positive ion mode; UDCA, ursodeoxycholic acid; {Delta}22-UDCA, {Delta}22-ursodeoxycholic acid

Supplementary key words unsaturated bile acid • tauro-{Delta}22-ß-muricholic acid • {Delta}22-ß-muricholyltaurine • glyco-{Delta}22-ß-muricholic acid • {Delta}22-ß-muricholylglycine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
When the chemical structure of the common natural C24 bile acids was deduced, it was assumed that the C5 side chain of all natural C24 bile acids was saturated. However, in 1973, while analyzing urinary bile acids from bile duct ligated rats, Danielsson (1) noted a bile acid with chromatographic behavior slightly different from that of ß-muricholic acid (ß-MCA; 3{alpha},6ß,7ß-trihydroxy-5ß-cholan-24-oic acid) and with two fewer mass units by mass spectroscopy. He proposed that this bile acid was ß-MCA with a double bond in the side chain. Similar observations were made by Kern et al. (2), who characterized the biliary bile acids of rats in whom cholestasis was induced by ethynylestradiol. In 1980, Kuriyama et al. (3) used gas chromatography-mass spectrometry to show that an unsaturated derivative of ß-MCA was present as a minor constituent in the biliary bile acids of the healthy Wistar rat. In 1993, Davis and Thompson (4) used 1H- and 13C-NMR to establish the chemical structure of the unsaturated bile acids as {Delta}22-ß-muricholic acid [{Delta}22-ß-MCA; (22E)-3{alpha},6ß,7ß-trihydroxy-5ß-chol-22-en-24-oic acid], and Thompson, Davis, and Morris (5) reported that its plasma concentration in female Fischer rats exceeded that of ß-MCA. This observation was confirmed for Sprague-Dawley rats in 1996 by Rodrigues et al. (6). Subsequently, this group reported that the concentration of {Delta}22-ß-MCA exceeded that of ß-MCA in liver homogenates (7), although no correction was made for entrapped blood, in which the bile acid concentration was likely to be much higher than in hepatocytes.

In the early studies in which {Delta}22-ß-MCA was identified in plasma or bile, it was inferred that the {Delta}22-ß-MCA had been formed by the enteric flora. This assumption was based on the finding that the compound disappeared from bile after prolonged biliary drainage (1, 8) as well as on reports from the Eyssen group that this acid was formed in vitro by bacteria (9) and in vivo in gnotobiotic rats (10). Kayahara et al. (11) confirmed and extended this finding by using gnotobiotic rats to show that several bacterial species form {Delta}22-ß-MCA. This group, working independently of the Eyssen group, also established its chemical structure by 1H- and 13C-NMR spectroscopy.

The first evidence that {Delta}22-ß-MCA could also be formed in the liver was presented by Thompson, Davis, and Morris (5), who in 1993 showed that {Delta}22-ß-MCA could be formed by rat liver slices. Two years later, Setchell et al. (12) provided additional indirect evidence for the formation of {Delta}22-ß-MCA in hepatocytes by showing that ursodeoxycholic acid (UDCA; 3{alpha},7ß-dihydroxy-5ß-cholan-24-oic acid) was converted to {Delta}22-UDCA when incubated with rat peroxisomes.

Thus, the consensus of the work to date is that {Delta}22-ß-MCA can be formed not only by the enteric flora of the distal intestine but also by hepatocyte peroxisomal enzymes, at least in the rat. In the rat, the compound is present in plasma, bile, and intestinal content in both conjugated and unconjugated forms (5), suggesting that it is absorbed from the distal intestine after formation by bacteria and then conjugated with taurine or glycine during transport through the hepatocyte; the conjugates then undergo enterohepatic circulation. However, {Delta}22-ß-MCA is sometimes absent in fecal bile acids of the rat despite being present in intestinal content (10, 11), indicating that intestinal bacteria can both desaturate and saturate the side chain of ß-MCA.

No studies have been performed on the metabolism of {Delta}22-ß-MCA because of the absence of synthetic material. Kihira and Hoshita (13) reported the synthesis of the {Delta}22 derivatives of the common natural bile acids (cholic, deoxycholic, and chenodeoxycholic acids and UDCA) but did not extend their work to ß-MCA, presumably because of the difficulty of the synthesis. Our laboratory has previously developed an improved synthesis of ß-MCA and its congeners ({alpha}- and {omega}-muricholic acid) (14). In this paper, we report the chemical synthesis of {Delta}22-ß-MCA [1a] and its taurine [1b] and glycine [1c] conjugates ({Delta}22-ß-muricholyltaurine and {Delta}22-ß-muricholylglycine). Chemical structures are shown in Fig. 1 .



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  		Fig. 1. Structures of {Delta}22-ß-muricholic acid ({Delta}22-ß-MCA) and its taurine and glycine conjugates [1a–1c].

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Melting points (mp) were determined on a micro hot-stage apparatus and are uncorrected. Infrared (IR) spectra were obtained on a Bio-Rad FTS-7 FT-IR spectrometer (Philadelphia, PA) as KBr discs. 1H- and 13C-NMR spectra were obtained on a JEOL JNM-EX 270 FT NMR instrument (Tokyo, Japan) at 270 and 68.80 MHz, respectively, with CDCl3 containing 10% CD3OD or CD3OD as the solvent; chemical shifts were expressed as {delta} ppm relative to tetramethylsilane. Low-resolution mass spectra (LR-MS) were recorded on a JEOL JMS-303 mass spectrometer with electron ionization (EI) at 70 eV under the positive ion mode (PIM). LR-MS was also obtained on a JEOL JMS-LCmate equipped with electrospray ionization (ESI) under the negative ion mode (NIM). High-resolution mass spectra (HR-MS) were recorded on a JEOL JMS-LCmate double-focusing magnetic mass spectrometer equipped with an ESI probe under the PIM or the NIM. HR-MS were also obtained on a JEOL JMS-700 mass spectrometer with an EI probe under the PIM. Sep-Pak Vac tC18 cartridges (adsorbent weight, 5 g) were purchased from Waters Associates (Milford, MA). Thin-layer chromatography was performed on precoated silica gel (0.25 mm layer thickness; E. Merck) using hexane-ethyl acetate (EtOAc)-acetic acid mixtures (80:20:1~20:80:1, v/v/v) or EtOAc-methanol-acetic acid mixtures (7:2:1, v/v/v) as the developing solvents.

3{alpha},6ß,7ß-Triformyloxy-5ß-cholan-24-oic acid [4]
A solution of ß-MCA [3] (1.0 g, 2.4 mmol), prepared from chenodeoxycholic acid [2] (see Results and Discussion), in 99% formic acid (7 ml) containing 60% perchloric acid (100 mg) was stirred at 50°C for 1.5 h. Acetic anhydride (3 ml) was added slowly with ice-bath cooling, and the mixture was poured into water. The reaction product was extracted with CH2Cl2, and the combined extract was washed with water, dried with anhydrous calcium sulfate (Drierite), and evaporated to dryness. The residue was recrystallized from benzene-hexane as colorless crystals: yield, 1.21 g (100%); mp, 90–92°C. IR (KBr), {nu}maxcm-1: 1,713 (C=O). 1H-NMR (CDCl3), {delta}: 0.72 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 4.85 (1H, brm, 3ß-H), 5.06 (1H, dd, J = 11.1 and 3.8 Hz, 7{alpha}-H), 5.14 (1H, m, 6{alpha}-H), 7.93, 8.02, and 8.10 (each 1H, s, 3{alpha}-, 6ß-, and 7ß-OCHO). LR-MS (EI-PIM), m/z: 492 (M+, 4%), 446 (M-HCOOH, 5%), 418 (9%), 400 (M-2HCOOH, 80%), 372 (39%), 354 (M-3HCOOH, 90%), 339 (27%), 299 (100%), 272 (22%), 253 [M-3HCOOH-side chain (S.C.), 66%], 213 (22%), 211 (M-3HCOOH-S.C.-ring D, 18%), 159 (31%). HR-MS (EI-PIM), calculated for C27H40O8 [M]+: 492.2723; found, m/z: 492.2692.

3{alpha},6ß,7ß-Triformyloxy-24-nor-5ß-chol-22-ene [5]
To a solution of the triformate [4] (1.0 g, 2.0 mmol) in dry benzene (18 ml), lead tetraacetate (1.44 g, 3.24 mmol), cuprous acetate (0.1 g, 0.55 mmol), and dry pyridine (0.1 ml) were added successively, and the mixture was refluxed for 12 h. Most of benzene was evaporated under reduced pressure, and the residual solution was poured onto a column of silica gel (30 g). Elution with benzene-EtOAc (9:1, v/v) afforded the title compound [5], which recrystallized from ethanol-water as colorless crystals: yield, 450 mg (49%); mp, 71–73°C. IR (KBr), {nu}maxcm-1: 1,728 (C=O). 1H-NMR (CDCl3), {delta}: 0.74 (3H, s, 18-CH3), 1.04 (3H, d, J = 6.5 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 4.77–4.87 (3H, brm, 3ß- and 23-H), 5.06 (1H, dd, J = 11.1 and 4.0 Hz, 7{alpha}-H), 5.13 (1H, m, 6{alpha}-H), 5.65 (1H, m, 22-H), 7.92, 8.02, and 8.10 (each 1H, s, 3{alpha}-, 6ß-, and 7ß-OCHO). LR-MS (EI-PIM), m/z: 446 (M+, 2%), 431 (M-CH3, 9%), 418 (18%), 400 (M-HCOOH, 4%), 389 (16%), 372 (16%), 354 (M-2HCOOH, 37%), 343 (33%), 326 (31%), 308 (M-3HCOOH, 14%), 299 (69%), 271 (21%), 253 (M-3HCOOH-S.C., 100%), 211 (M-3HCOOH-S.C-ring D, 11%), 197 (8%), 159 (21%). HR-MS (EI-PIM), calculated for C26H38O6 [M]+: 446.2669; found, m/z: 446.2665.

3{alpha},6ß,7ß-Triformyloxy-24-nor-5ß-cholan-22{xi},23-diol [6]
To the 24-nor-22-ene [5] (1.38 g, 3.0 mmol) dissolved in tert-butyl alcohol-tetrahydrofuran-water (34.5 ml; 7:2:1, v/v/v) was added N-methylmorpholine N-oxide (2 ml) and osmium tetroxide (30 mg, 0.12 mmol), and the mixture was allowed to stand at room temperature for 12 h. The reaction product was extracted with CHCl3, and the combined extract was washed with water, dried with Drierite, and evaporated to a dark brown oily residue. Chromatography of the oil on a column of silica gel (50 g) and elution with benzene-EtOAc (6:4–1:9, v/v) afforded the desired 24-nor-22{xi},23-diol [6] as viscous oil, which resisted crystallization attempts: yield, 950 mg (66%). IR (KBr) {nu}maxcm-1: 1,713 (C=O), 3,427 (OH). 1H-NMR (CDCl3), {delta}: 0.72 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 3.65–3.75 (3H, brm, 22{xi}- and 23-H), 4.81 (1H, brm, 3ß-H), 5.09–5.13 (2H, m, 6{alpha}- and 7{alpha}-H), 7.93, 8.02, and 8.10 (each 1H, s, 3{alpha}-, 6ß-, and 7ß-OCHO). LR-MS (EI-PIM), m/z: 462 (M-H2O, 2%), 444 (M-2H2O, 5%), 434 (M-HCOOH, 8%), 416 (M-H2O-HCOOH, 15%), 403 (48%), 388 (M-2HCOOH, 22%), 357 (69%), 342 (M-3HCOOH, 10%), 329 (39%), 311 (100%), 299 (32%), 281 (34%), 253 (M-3HCOOH-S.C., 29%), 211 (M-3HCOOH-S.C.-ring D, 18%), 159 (26%), 107 (25%). HR-MS (ESI-PIM), calculated for C26H40O8Na [M+Na]+: 503.2621; found, m/z: 503.2632.

3{alpha},6ß,7ß-Triformyloxy-23,24-dinor-5ß-cholan-22-al [7]
To a magnetically stirred solution of sodium periodate (NaIO4) (1.5 g, 7.0 mmol) dissolved in methanol (14 ml) and water (7 ml), a solution of the 24-nor-22{xi},23-diol [6] (900 mg, 1.9 mmol) in methanol (15 ml) was added, and the mixture was stirred at room temperature for 12 h. The reaction product was extracted with CHCl3, and the combined extract was washed with water, dried with Drierite, and evaporated to dryness. Although the residual oil [7] was found to be homogeneous according to TLC, it resisted crystallization attempts: yield, 800 mg (95%). IR (KBr), {nu}maxcm-1: 1,726 (C=O). 1H-NMR (CDCl3), {delta}: 0.77 (3H, s, 18-CH3), 0.96 (3H, d, J = 6.8 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 4.81 (1H, brm, 3ß-H), 5.07 (1H, dd, J = 11.3 and 3.5 Hz, 7{alpha}-H), 5.14 (1H, m, 6{alpha}-H), 7.92, 8.02, and 8.10 (each 1H, s, 3{alpha}-, 6ß-, and 7ß-OCHO), 9.57 (1H, d, J = 3.0 Hz, 22-CHO). LR-MS (EI-PIM), m/z: 448 (M+, 3%), 416 (6%), 402 (M-HCOOH, 7%), 374 (M-HCOOH-CHO, 17%), 356 (M-2HCOOH, 93%), 328 (M-2HCOOH-CHO, 70%), 310 (M-3HCOOH, 42%), 282 (M-3HCOOH-CHO, 20%), 272 (37%), 253 (M-3HCOOH-S.C., 45%), 211 (M-3HCOOH-S.C.-ring D, 4%), 159 (41%), 111 (100%). HR-MS (EI-PIM), calculated for C25H36O7 [M]+: 448.2461; found, m/z: 448.2459.

Methyl (22E)-3{alpha},6ß,7ß-triformyloxy-5ß-chol-22-en- 24-oate [8]
To a stirred solution of the 23,24-dinor-22-aldehyde [7] (370 mg, 0.6 mmol) in dry benzene (28 ml), methyl (triphenylphosphoranylidene)acetate (500 mg, 1.5 mmol) was added, and the mixture was refluxed for 12 h under a stream of N2. After cooling at room temperature, most of the solvent was evaporated under reduced pressure, and the reaction product was poured onto a column of silica gel (20 g). Elution with benzene-EtOAc (95:5, v/v) gave the title compound [8] as viscous oil, which resisted crystallization attempts: yield, 350 mg (84%). IR (neat), {nu}maxcm-1: 1,186 (C-O), 1,651 (C=C), 1,715, 1,732 (C=O). 1H-NMR (CDCl3), {delta}: 0.75 (3H, s, 18-CH3), 1.09 (3H, d, J = 7.0 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 3.72 (3H, s, COOCH3), 4.80 (1H, brm, 3ß-H), 5.06 (1H, dd, J = 10.8 and 3.8 Hz, 7{alpha}-H), 5.14 (1H, m, 6{alpha}-H), 5.74 (1H, d, J = 15.7 Hz, 23-H), 6.82 (1H, dd, J = 15.7 and 8.9 Hz, 22-H), 7.91, 8.02, and 8.10 (each 1H, s, 3{alpha}-, 6ß-, and 7ß-OCHO). LR-MS (EI-PIM), m/z: 504 (M+, 2%), 473 (M-CH3O, 2%), 458 (M-HCOOH, 1%), 412 (M-2HCOOH, 11%), 366 (M-3HCOOH, 7%), 343 (34%), 299 (M-2HCOOH-S.C., 30%), 271 (11%), 253 (M-3HCOOH-S.C., 50%), 211 (M-3HCOOH-S.C.-ring D, 6%), 159 (12%), 114 (100%). HR-MS (EI-PIM), calculated for C28H40O8 [M]+: 504.2723; found, m/z: 504.2722.

(22E)-3{alpha},6ß,7ß-Trihydroxy-5ß-chol-22-en-24-oic acid [1a] and its methyl ester
A solution of the triformyloxy-22-ene ester [8] (500 mg, 0.99 mmol) in 5% methanolic KOH (5 ml) was refluxed for 6 h. After evaporation of most of the solvent, the residue was dissolved in water and then acidified with 10% H2SO4 with ice-bath cooling. The precipitated solid was filtered off, washed with water, and recrystallized from aqueous methanol to give the desired {Delta}22-ß-MCA as colorless prisms: yield, 370 mg (92%); mp, 205–209°C. IR (KBr), {nu}maxcm-1: 1,707 (C=O), 3,342 (OH). 1H-NMR (CD3OD), {delta}: 0.73 (3H, s, 18-CH3), 1.09 (3H, s, 19-CH3), 1.11 (3H, d, J = 6.5 Hz, 21-CH3), 3.49–3.61 (3H, brm, 3ß-, 6{alpha}, and 7{alpha}-H), 5.72 (1H, d, J = 15.4 Hz, 23-H), 6.86 (2H, dd, J = 15.4 and 6.8 Hz, 22-H). 13C-NMR (CDCl3+10% CD3OD), {delta}: 12.1 (C-18), 19.1 (C-21), 20.6 (C-11), 25.2 (C-19), 26.9 (C-15), 28.2 (C-16), 29.4 (C-2), 33.6 (C-10), 35.0 (C-1), 35.3 (C-4), 38.2 (C-8), 39.4 (C-2), 39.5 (C-9), 39.7 (C-12), 43.7 (C-13), 47.2 (C-5), 54.1 (C-17), 55.3 (C-14), 70.4 (C-3), 73.0 (C-7), 75.4 (C-6), 118.7 (C-23), 155.6 (C-22), 169.2 (C-24). LR-MS (ESI-NIM), m/z: 405 ([M-H]-, 100%), 111 (42.3%). HR-MS (ESI-NIM), calculated for C24H37O5 [M-H]-: 405.2641; found, m/z: 405.2682.

A solution of the free acid [1a] (100 mg, 0.25 mmol) and p-toluenesulfonic acid (30 mg) in methanol (5 ml) was left overnight at room temperature. After evaporation of most of the solvent, the reaction product was extracted with EtOAc. The combined extract was washed with saturated brine, dried with Drierite, and evaporated to dryness. The resulting C-24 methyl ester, although homogeneous according to TLC, resisted crystallization attempts: yield, 93 mg (86%). IR (neat), {nu}maxcm-1: 1,651 (C=C), 1,715 (C=O), 3,364 (OH). 1H-NMR (CDCl3), {delta}: 0.73 (3H, s, 18-CH3), 1.10 (3H, s, 19-CH3), 1.10 (3H, d, J = 6.5 Hz, 21-CH3), 3.44 (1H, dd, J = 10.3 and 3.8 Hz, 7{alpha}-H), 3.51 (1H, brm, 3ß-H), 3.57 (1H, m, 6{alpha}-H), 3.72 (3H, s, COOCH3), 5.75 (1H, d, J = 16.2 Hz, 23-H), 6.90 (1H, dd, J = 16.2 and 8.1 Hz, 22-H). 13C-NMR (CDCl3), {delta}: 12.4 (C-18), 19.4 (C-21), 20.7 (C-11), 25.4 (C-19), 27.1 (C-15), 28.4 (C-16), 29.9 (C-2), 33.8 (C-10), 35.4 (C-1), 35.4 (C-4), 38.5 (C-8), 39.6 (C-2), 39.6 (C-9), 39.8 (C-12), 44.0 (C-13), 47.2 (C-5), 51.4 (COOCH3), 54.1 (C-17), 55.3 (C-14), 71.0 (C-3), 73.4 (C-7), 75.5 (C-6), 118.6 (C-23), 154.9 (C-22), 167.5 (C-24). LR-MS (EI-PIM), m/z: 420 (M+, 2%), 402 (M-H2O, 100%), 384 (M-2H2O, 52%), 369 (M-2H2O-CH3, 14%), 366 (M-3H2O, 4%), 347 (10%), 305 (22%), 289 (M-H2O-S.C., 22%), 271 (M-2H2O-S.C., 80%), 253 (M-3H2O-S.C., 53%), 229 (11%), 211 (M-3H2O-S.C.-ring D, 9%), 175 (12%), 147 (20%), 114 (52%). HR-MS (EI-PIM), calculated for C25H40O5 [M]+: 420.2876; found, m/z: 420.2893.

Tauro-(22E)-3{alpha},6ß,7ß-trihydroxy-5{gamma}-chol-22-en-24-oic acid sodium salt [(22E)-3{alpha},6ß,7ß-trihydroxy-5ß-chol-22-en-24-oyl taurine] [1b]
To a magnetically stirred solution of the nonamidated {Delta}22-ß-MCA [1a] (100 mg, 0.25 mmol) in dry N,N-dimethylformamide (DMF) (9 ml) were successively added powdered taurine (80 mg, 0.64 mmol), diethylphosphorocyanide (DEPC) (75 µl), and anhydrous Et3N (150 µl), and the resulting suspension was stirred at room temperature for 60 min. The reaction mixture was adjusted to pH 12–14 with 1 M NaOH and then to pH 8–9 with 10% HCl. The resulting solution was diluted with water (90 ml), passed through a preconditioned Sep-Pak Vac tC18 cartridge, and eluted successively with water (20 ml), 20% methanol (20 ml), and 50% methanol (25 ml). The last fraction, which contains the desired component, was evaporated under reduced pressure, and the residue was recrystallized from methanol-ether to give the taurine-conjugated {Delta}22-ß-MCA sodium salt [1b] as colorless crystals: yield, 82 mg (62%); mp, 283–285°C. IR (KBr), {nu}maxcm-1: 1,624 (C=C), 1,664 (C=O), 3,385 (OH). 1H-NMR (CD3OD), {delta}: 0.75 (3H, s, 18-CH3), 1.10 (3H, s, 19-CH3), 1.11 (3H, d, J = 7.0 Hz, 21-CH3), 3.03 (2H, t, J = 7.0 Hz, CH2S), 3.48 (1H, dd, J = 10.0 and 3.8 Hz, 7{alpha}-H), 3.55 (1H, brm, 3ß-H), 3.62 (1H, m, 6{alpha}-H), 5.83 (1H, d, J = 15.1 Hz, 23-H), 6.70 (2H, dd, J = 15.1 and 8.9 Hz, 22-H). 13C-NMR (CD3OD) {delta}: 12.9 (C-18), 20.2 (C-21), 21.9 (C-11), 26.1 (C-19), 28.1 (C-15), 29.5 (C-16), 30.6 (C-2), 34.9 (C-10), 36.4 (CH2N), 36.5 (C-1), 39.4 (C-8), 40.7 (C-20), 41.1 (C-9 and C-12), 44.9 (C-13), 49.1 (C-5), 51.5 (CH2S), 55.8 (C-17), 56.9 (C-14), 71.7 (C-3), 74.2 (C-7), 77.1 (C-6), 122.2 (C-23), 151.8 (C-22), 168.9 (C-24). LR-MS (ESI-NIM), m/z: 512 ([M-H]-). HR-MS (ESI-NIM), calculated for C26H41NO7S [M-H]-: 512.2682; found, m/z: 512.2665.

Glyco-(22E)-3{alpha},6ß,7ß-trihydroxy-5ß-chol-22-en-24-oic acid [(22E)-3{alpha},6ß,7ß-trihydroxy-5ß-chol-22-en-24-oyl glycine] [1c]
To a magnetically stirred solution of {Delta}22-ß-MCA [1a] (120 mg, 0.29 mmol) in dry DMF (4 ml), glycine methyl ester hydrochloride (100 mg, 0.65 mmol), DEPC (120 µl), and Et3N (0.5 ml) were added successively, and the resulting suspension was stirred at room temperature for 1 h. The reaction product was extracted with EtOAc, and the combined extract was washed with water, dried with Drierite, and evaporated to dryness. The residue was then refluxed in 5% methanolic KOH (10 ml) for 30 min. Most of the solvent was evaporated under reduced pressure, and the hydrolysis product dissolved in water (5 ml) was acidified by 5% H2SO4 with ice-bath cooling. After stirring for 10 min at room temperature, the precipitated solid was filtered, washed with water, and dried to give the glycine-conjugated {Delta}22-ß-MCA [1c], which was recrystallized from 1,4-dioxane-ether as colorless crystals: yield 120 mg (89%); mp, 234–235°C. IR (KBr), {nu}maxcm-1: 1,624 (C=C), 1,666, 1,732 (C=O), 3,375 (OH). 1H-NMR (as the methyl ester in CDCl3), {delta}: 0.73 (3H, s, 18-CH3), 1.10 (3H, d, J = 7.0 Hz, 21-CH3), 1.10 (3H, s, 19-CH3), 3.76 (2H, d, J = 4.1 Hz, CH2N), 3.78 (1H, s, COOCH3), 5.77 (1H, d, J = 15.4 Hz, 23-H), 6.06 (1H, t, J = 8.9 Hz, -NH-), 6.76 (2H, dd, J = 15.4 and 6.22 Hz, 22-H). 13C-NMR (as the methyl ester in CDCl3), {delta}: 12.3 (C-18), 19.5 (C-21), 20.7 (C-11), 25.4 (C-19), 27.1 (C-15), 28.5 (C-16), 29.3 (C-2), 33.8 (C-10), 35.4 (C-1), 35.6 (C-4), 39.4 (C-2), 39.6 (C-9), 39.8 (C-12), 41.3 (-CH2COOCH3), 43.9 (C-13), 47.2 (C-5), 52.4 (-CH2COOCH3), 54.2 (C-17), 55.3 (C-14), 71.0 (C-3), 73.5 (C-7), 75.5 (C-6), 120.5 (C-23), 151.2(C-22), 166.2 (C-24), 170.6 (-CH2COOCH3). LR-MS (ESI-NIM), m/z: 462 ([M-H]-). HR-MS (ESI-NIM), calculated for C26H40O6N [M-H]-: 462.2855; found: m/z, 462.2827.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The synthetic route to {Delta}22-ß-MCA [1a] is shown in Fig. 2 . The key starting compound, ß-MCA [3], was prepared according to the established procedures (14) reported previously by us from chenodeoxycholic acid [2] as follows: chenodeoxycholic acid [2] was converted to its 3{alpha}-cathyloxy-7-oxo ester; bromination of the resulting ketone with bromine in the presence of HBr afforded the 6{alpha}-bromo-3{alpha}-cathyloxy-7-oxo ester; reduction of the bromo-ketone with Zn(BH4)2 gave 6{alpha}-bromo-3{alpha}-cathyloxy-7{alpha}-hydroxy ester; treatment of the bromohydrin in acetic acid with zinc powder yielded 3{alpha}-cathyloxy-{Delta}6 ester; ß-face cis-dihydroxylation of the {Delta}6 ester with osmium tetroxide-N-methylmorpholine N-oxide afforded the 3{alpha}-cathyloxy-6ß,7ß-dihydroxy ester, which in turn was hydrolyzed to give the corresponding free acid [3] (total yield of 30% from chenodeoxycholic acid [2]).



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  		Fig. 2. Synthetic route to unconjugated {Delta}22-ß-MCA [1a].

 
Two methods have been reported for the introduction of a (22E)-double bond in the bile acid side chain (C4H8COOH) of ß-MCA [3]. The first method involves an ene reaction with (Z)-ethylidene-({Delta}17(20))-steroids and methyl propiolate (HC{equiv}CCOOCH3) under the presence of ethyl aluminum dichloride (EtAlCl2) or diethyl aluminum chloride as Lewis acid-catalyzed conditions (15, 16). The second method consists of condensation of (20S)-20-methyaldehyde pregnane derivatives with a Wittig reagent, (carbomethoxymethylene)triphenylphosphorane [(C6H5)3P=CHCOOCH3] (17). Of the two methods, the latter appeared to be much more practical than the former, because of easy preparation of the key intermediate, (20S)-23,24-dinoraldehyde [7].

Based on the above assumption, our next effort was directed to the preparation of the 24-nor-triformyloxy-22-ene intermediate [5] from ß-MCA [3]. Oxidative decarboxylation of the ß-MCA triformate derivative [4] with lead tetraacetate and cuprous acetate in refluxing pyridine (18) yielded 24-nor-triformyloxy-22-ene [5] in an isolated yield of 46% after chromatographic purification on a column of silica gel. The triformate [4] was obtained in nearly quantitative yield by treatment of [3] with 99% formic acid in the presence of 60% perchloric acid (19).

Vicinal dihydroxylation of the unsaturated 22-ene [5] in tert-butyl alcohol-tetrahydrofuran-water mixtures with osmium tetroxide and N-methylmorphorine N-oxide produced exclusively an epimeric mixture (22R and 22S) of vicinal 22{xi},23-diols [6], without being accompanied by a simultaneous hydrolysis of the formyloxy protecting groups at the C-3, C-6, and C-7 positions, as indicated by the 13C-NMR spectrum. The one-step dihydroxylation is much more simple and straightforward than the two-step procedure reported by Kihira and Hoshita (13) and Kihira and others (20, 21), in which epoxidation of the 22-ene [5] with hydrogen peroxide and subsequent methanolic KOH cleavage of the resulting epoxide were used. When the triformyloxy-22{xi},23-diol [6] (both epimers) was subjected to oxidation with NaIO4 in methanol-water mixtures (13), both the 22R- and 22S-epimers were quantitatively converted into the 23,24-dinor-triformyloxy-22-aldehyde [7] in an excellent yield of 92%.

Subsequent Wittig reaction of the triformyloxy-22-aldehyde [7] in benzene with a reagent, methyl (triphenylphosphoranylidene)acetate, led to the formation of the (E)-isomer of the 3{alpha},6ß,7ß-triformyloxy-22-ene methyl ester [8]. The resulting reaction product, which consisted essentially of a single component, was purified by passing through a column of silica gel and eluting with a mixture of benzene-EtOAc (95:5, v/v). Alkaline hydrolysis of 3{alpha},6ß,7ß-triformyloxy-22-ene methyl ester [8] with 5% methanolic KOH followed by acidification with 10% H2SO4 resulted in the simultaneous hydrolysis of the methyl ester at C-24 and the formyloxy protecting group at the C-3, C-6, and C-7 positions to give the desired (unconjugated) {Delta}22-ß-MCA [1a]. Esterification of [1a] using p-toluenesulfonic acid in methanol yielded the corresponding methyl ester derivative. In the {Delta}22-ß-MCA [1a] and its methyl ester, the stereochemical configuration of the {Delta}22-double bond as an E-form was determined on the basis of a large coupling constant (J, 15–16 Hz) arising from a doublet signal of the 23-H in the 1H-NMR (13, 22) and of characteristic olefinic carbon signal pairs appearing at ~119 and 155 ppm, respectively assigned to C-23 and C-22, in the 13C-NMR (4, 11).

The unconjugated {Delta}22-ß-MCA [1a] was then transformed into the corresponding taurine- and glycine-amidated conjugates. Condensation of the carboxyl group at C-24 in the {Delta}22-ß-MCA [1a] with the amino group of taurine (or of glycine methyl ester) was successfully attained by the use of DEPC as a coupling reagent and Et3N as a catalyst (23). The N-acylamidation reaction involves the formation of intermediary acyl phosphates as carboxyl-activated intermediates by the interaction of carboxylic acid with DEPC in the presence of Et3N and subsequent coupling with amines to give the corresponding amides in one step. The condensation reaction proceeded cleanly and rapidly by simple mixing of the unconjugated {Delta}22-ß-MCA [1a], taurine (or glycine methyl ester), DEPC, and Et3N in DMF at room temperature for 60 min. After the amidation reaction, sample clean up, crystallization, and/or hydrolysis were effected as follows. Because the free sulfonic acid form of tauro-{Delta}22-ß-MCA [1b] is highly hygroscopic, it was converted to the sodium salt by treatment with NaOH and then purified and isolated as a crystalline product by passing through a prepacked Sep-Pac Vac tC18 cartridge for reversed-phase solid extraction, eluting with 50% aqueous methanol. On the other hand, the glycine methyl ester form of glyco-{Delta}22-ß-MCA [1c] was hydrolyzed with methanolic KOH, followed by acidification with H2SO4 to give the free acid form of glyco-{Delta}22-ß-MCA [1c] as a crystalline product.

The 1H-NMR spectrum of tauro-{Delta}22-ß-MCA [1b] exhibited a triplet signal (J, 7 Hz) at 3.03 ppm arising from CH2S protons, whereas that of glyco-{Delta}22-ß-MCA [1c] showed a doublet signal (J, 4.1 Hz) at 3.76 ppm and a triplet signal (J, 8.9 Hz) at 6.06 ppm, attributable to CH2N and NH, respectively. The 13C-NMR spectrum of synthetic tauro-{Delta}22-ß-MCA [1b] is very compatible with that reported for the natural compound that was isolated from serum of female rats treated with {alpha}-naphthylisothiocyanate (4) as well as from bile, small and large intestinal content, and feces of gnotobiotic rats monocontaminated with E. coli, B. longum, B. vulgatus, C. ramosum, P. productus, or L. gasseri (11). Particularly noteworthy NMR features of the amidated {Delta}22-ß-MCA are carbon signals occurring at 36.4 (CH2N), 51.5 (CH2S), and 168.9 (C-24) ppm in the tauro-{Delta}22-ß-MCA [1b] and at 41.3 (-CH2COO-), 166.2 (C-24), and 170.6 (-CH2COO-) ppm in the glyco-{Delta}22-ß-MCA [1c]. Figure 3 shows ESI-NIM mass spectra of unconjugated {Delta}22-ß-MCA acid and its taurine and glycine conjugates (Fig. 3A–C). Each of the spectra is very simple, giving only a [M-H]- as the principal fragment ion.



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  		Fig. 3. Electrospray ionization-negative ion mode mass spectra of unconjugated and taurine and glycine conjugates of {Delta}22-ß-MCA [A–C].

 
Although the occurrence of {Delta}22-ß-MCA in rat is well established (see introduction), there have been no reports of {Delta}22-ß-MCA occurring in the mouse, a species in which ß-MCA is a dominant primary bile acid. ß-MCA has been detected in trace amounts in the urine of a cholestatic patient (24) and was also reported to be formed from administered UDCA in another cholestatic patient, based on its appearance in urinary bile acids during UDCA ingestion (25). As yet, {Delta}22-ß-MCA has not been reported to occur in humans. Even when ß-MCA was administered to human volunteers, {Delta}22-ß-MCA was not identified as a metabolite (26). When rats ingest UDCA, {Delta}22-UDCA is a major metabolite formed in the liver and possibly also by the enteric flora (12). Yet, the formation of {Delta}22-UDCA has not been observed in the biliary bile acids (27), urinary bile acids (25), or liver biopsies (7) of patients with liver disease who have received UDCA as therapy.

The availability of {Delta}22-ß-MCA [1a] as well as its taurine [1b] and glycine [1c] conjugates should facilitate the characterization of its metabolism and physiological properties. {Delta}22 -ß-MCA can also be used as a substrate for reductive deuteration or tritiation, thereby forming 22,23-2H-ß-MCA or 22,23-3H-ß-MCA. The latter should be useful for transport studies (28). The tritium atoms of 22,23-3H label of cholic acid and its principal metabolite deoxycholic acid have been shown to be stable in vivo during enterohepatic cycling in human (29), confirming the view (12) that bacterial desaturation of the side chain of these two bile acids does not occur. The stability of the label of 22,23-3H-ß-MCA during enterohepatic cycling is uncertain, because of the ability of the enteric flora to desaturate the side chain of this bile acid (11). Indeed, the availability of 22,23-3H-ß-MCA should permit the metabolism of ß-MCA by bacteria and by hepatic enzymes to be characterized.

Bile acids with a {Delta}22 side chain occur only rarely in the bile of other vertebrates. In a survey of the biliary bile acids of some 900 vertebrate species (30), only some caviomorph rodents were found to have {Delta}22 bile acid as their dominant biliary bile acid. For example, the mountain paca, a large South American rodent, was found to have 3{alpha}-hydroxy-7-oxo-5ß-chol-22-en-24-oic acid (31) as its major biliary bile acid.


   ACKNOWLEDGMENTS
 
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.

Manuscript received September 22, 2003 and in revised form October 24, 2003. and in re-revised form November 18, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
  1. Danielsson, H. 1973. Effect of biliary obstruction on formation and metabolism of bile acids in rat. Steroids. 22: 567–577.[CrossRef][Medline]

  2. Kern, F., H. Eriksson, T. Curstedt, and J. Sjövall. 1977. Effect of ethynylestradiol on biliary excretion of bile acids, phosphatidylcholines, and cholesterol in bile fistula rat. J. Lipid Res. 18: 623–634.[Abstract]

  3. Kuriyama, K., Y. Ban, T. Nakashima, and T. Murata. 1980. Simultaneous determination of biliary bile acids in rat: electron impact and ammonia chemical ionization mass spectrometric analysis of bile acids. Steroids. 34: 717–728.

  4. Davis, D. G., and M. B. Thompson. 1993. Nuclear magnetic resonance identification of the taurine conjugate of 3{alpha},6ß,7ß-trihydroxy-5ß,22-cholen-24-oic acid (tauro-{Delta}22-ß-muricholate) in the serum of female rats treated with {alpha}-naphthylisothiocyanate. J. Lipid Res. 34: 651–661.[Abstract]

  5. Thompson, M. B., D. G. Davis, and R. W. Morris. 1993. Taurine conjugate of 3{alpha},6ß,7ß-trihydroxy-5ß,22-cholen-24-oic acid (tauro-{Delta}22-ß-muricholate): the major bile acid in the serum of female rats treated with {alpha}-naphthylisothiocyanate and its secretion by liver slices. J. Lipid Res. 34: 553–561.[Abstract]

  6. Rodrigues, C. M. P., B. T. Kren, C. J. Steer, and K. D. R. Setchell. 1996. Formation of {Delta}22-bile acids in rats is not gender specific and occurs in the peroxisome. J. Lipid Res. 37: 540–550.[Abstract]

  7. Setchell, K. D. R., C. M. P. Rodrigues, C. Clerici, A. Solinas, A. Morelli, C. Gartung, and J. Boyer. 1997. Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei. Gastroenterology. 112: 226–235.[CrossRef][Medline]

  8. Eriksson, H., W. Taylor, and J. Sjövall. 1978. Occurrence of sulfated 5{alpha}-cholanoates in rat bile. J. Lipid Res. 19: 177–186.[Abstract]

  9. Eyssen, H., G. De Pauw, J. Stragier, and A. Verhulst. 1983. Cooperative formation of {omega}-muricholic acid by intestinal microorganisms. Appl. Environ. Microbiol. 45: 141–147.[Abstract/Free Full Text]

  10. Robben, J., P. Caenepeel, J. V. Eldere, and E. Eyssen. 1988. Effects of intestinal microbial bile salt sulfatase activity on bile salt kinetics in gnotobiotic rats. Gastroenterology. 94: 494–502.[Medline]

  11. Kayahara, T., T. Tamura, Y. Amuro, K. Higashino, H. Igimi, and K. Uchida. 1994. {Delta}22-ß-Muricholic acid in monoassociated rats and conventional rats. Lipids. 29: 289–296.[Medline]

  12. Setchell, K. D. R., H. Yamashita, C. M. P. Rodrigues, N. C. O'Connell, B. T. Kren, and C. J. Steer. 1995. {Delta}22-Ursodeoxycholic acid, a unique metabolite of administered ursodeoxycholic acid in rats, indicating partial ß-oxidation as a major pathway for bile acid metabolism. Biochemistry. 34: 4169–4178.[CrossRef][Medline]

  13. Kihira, K., and T. Hoshita. 1985. Synthesis of {alpha},ß-unsaturated C24 bile acids. Steroids. 46: 767–774.[CrossRef][Medline]

  14. Iida, T., T. Momose, T. Tamura, T. Matsumoto, F. C. Chang, J. Goto, and T. Nambara. 1989. Potential bile acid metabolites. 14. Hyocholic and muricholic acid stereoisomers. J. Lipid Res. 30: 1267–1279.[Abstract]

  15. Batcho, A. D., D. E. Berger, and M. R. Uskokovic. 1981. C-20 stereospecific introduction of a steroid side chain. J. Am. Chem. Soc. 103: 1293–1295.[CrossRef]

  16. Dauben, W. G., and T. Brookhart. 1981. Stereocontrolled synthesis of steroid side chains. J. Am. Chem. Soc. 103: 237–238.[CrossRef]

  17. Vanderah, D. J., and C. Djerassi. 1978. Marine natural products. Synthesis of four naturally occurring 20ß-H cholanoic acid derivatives. J. Org. Chem. 43: 1442–1448.[CrossRef]

  18. Carlson, C. L., D. T. E. Belobaba, A. F. Hofmann, and Y. Wedmid. 1977. 24-Nor-5ß-chol-22-enes derived from the major bile acids by oxidative decarboxylation. Steroids. 30: 787–793.[CrossRef][Medline]

  19. Tserng, K-Y., and P. D. Klein. 1977. Formylated bile acids: improved synthesis, properties, and partial deformylation. Steroids. 29: 635–648.[CrossRef][Medline]

  20. Kihira, K., T. Kuramoto, and T. Hoshita. 1976. New bile alcohols: synthesis of (22R)- and (22S)-5ß-cholestane-3{alpha},7{alpha},12{alpha},22,25-pentols. Steroids. 27: 383–393.[CrossRef][Medline]

  21. Kihira, K., Y. Morioka, and T. Hoshita. 1981. Synthesis of (22R and 22S)-3{alpha},7{alpha},22-trihydroxy-5ß-cholan-24-oic acids and structure of haemulcholic acid, a unique bile acid isolated from fish bile. J. Lipid Res. 22: 1181–1187.[Abstract]

  22. Lai, C. K., C. Y. Byon, M. Gut, D. Mostowicz, and W. G. Anderson. 1984. Deuterium and carbon-13 labeled cholic, chenodeoxycholic and ursodeoxycholic acids (1). J. Labelled Compd. Radiopharm. 21: 627–637.[CrossRef]

  23. Momose, T., T. Tsubaki, T. Iida, and T. Nambara. 1997. An improved synthesis of taurine- and glycine-conjugated bile acids. Lipids. 32: 775–778.[Medline]

  24. Almè, B., A. Bremmelgaard, J. Sjövall, and P. Thomassen. 1977. Analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometry. J. Lipid Res. 18: 339–362.[Abstract]

  25. Meng, L-J., H. Reyes, J. Palma, I. Hernandez, J. Ribalta, and J. Sjövall. 1997. Effects of ursodeoxycholic acid on conjugated bile acids and progesterone metabolites in serum and urine of patients with intrahepatic cholestasis of pregnancy. J. Hepatol. 10: 29–40.

  26. Sacquet, E., M. Parquet, M. Riottot, A. Raizman, B. Nordlinger, and R. Infante. 1985. Metabolism of ß-muricholic acid in man. Steroids. 45: 411–426.[CrossRef][Medline]

  27. Combes, B., R. L. Carithers, W. C. Maddrey, S. Munoz, G. Garcia-Tsao, G. F. Bonner, J. L. Boyer, V. A. Luketic, M. L. Shiffman, M. G. Peters, H. White, R. K. Zetterman, R. Risser, S. S. Rossi, and A. F. Hofmann. 1999. Biliary bile acids in primary biliary cirrhosis: effect of ursodeoxycholic acid. Hepatology. 29: 1649–1654.[CrossRef][Medline]

  28. Sorscher, S., J. Lillienau, J. L. Meinkoth, J. H. Steinbach, C. D. Schteingart, J. Feramisco, and A. F. Hofmann. 1992. Conjugated bile acid uptake by Xenopus laevis oocytes induced by microinjection with ileal poly A+ mRNA. Biochem. Biophys. Res. Commun. 186: 1455–1462.[CrossRef][Medline]

  29. Duane, W. C., C. D. Schteingart, H-T. Ton-Nu, and A. F. Hofmann. 1996. Validation of [22,23-3H]-cholic acid as a stable tracer through conversion to deoxycholic acid in human subjects. J. Lipid Res. 37: 431–436.[Abstract]

  30. Hagey, L. R. 1992. Bile Acid Biodiversity in Vertebrates: Chemistry and Evolutionary Implications. PhD Dissertation. University of California, San Diego, CA.

  31. Hagey, L. R., K. Takagi, C. D. Schteingart, H-T. Ton-Nu, and A. F. Hofmann. 1994. A hydroxy-oxo C24 bile acid with an unsaturated side chain is the predominant biliary bile acid of the mountain paca, a large rodent (Abstract). Hepatology. 20: A666.[CrossRef]


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