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Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15α-hydroxylithocholic acid

2007; Elsevier BV; Volume: 48; Issue: 12 Linguagem: Inglês

10.1194/jlr.m700340-jlr200

ISSN

1539-7262

Autores

Genta Kakiyama, Hideyuki Tamegai, Takashi Iida, Kuniko Mitamura, Shigeo Ikegawa, Takaaki Goto, Nariyasu Mano, Junichi Goto, Peter Holz, Lee R. Hagey, Alan F. Hofmann,

Tópico(s)

Cholesterol and Lipid Metabolism

Resumo

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15α-hydroxylithocholic acid (3α,15α-dihydroxy-5β-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15α-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Δ14 intermediate. The synthesis of its C-15 epimer, 15β-hydroxylithocholic acid (3α,15β-dihydroxy-5β-cholan-24-oic acid), is also reported. The taurine conjugate of 15α-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15α-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15α-hydroxylation of lithocholic acid that was formed by bacterial 7α-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15α-hydroxylation as a novel detoxification mechanism for lithocholic acid. The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15α-hydroxylithocholic acid (3α,15α-dihydroxy-5β-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15α-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Δ14 intermediate. The synthesis of its C-15 epimer, 15β-hydroxylithocholic acid (3α,15β-dihydroxy-5β-cholan-24-oic acid), is also reported. The taurine conjugate of 15α-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15α-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15α-hydroxylation of lithocholic acid that was formed by bacterial 7α-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15α-hydroxylation as a novel detoxification mechanism for lithocholic acid. Bile acids are amphipathic end products of cholesterol metabolism that mediate numerous physiological functions in the liver, biliary tract, and intestine. Bile acids are of two types: C24 bile acids [with a C5 (isopentanoic acid) side chain] and C27 bile acids [with a C8 (isooctanoic acid) side chain]. The C24 and C27 bile acids, together with C27 bile alcohols, are the predominant chemical metabolites of cholesterol and are the major chemical form in which cholesterol is eliminated in most vertebrates (1Moschetta A. Xu F. Hagey L.R. van Berge-Henegouwen G.P. van Erpecum K.J. Brouwers J.F. Cohen J.C. Bierman M. Hobbs H.H. Steinbach J.H. et al.A phylogenetic survey of biliary lipids in vertebrates.J. Lipid Res. 2005; 46: 2221-2232Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). All primary C24 bile acids have a hydroxyl group at position C-3 (from cholesterol) and at C-7, as cholesterol 7α-hydroxylation is the rate-limiting step in bile acid biosynthesis. Thus, chenodeoxycholic acid (CDCA; 3α,7α-dihydroxy-5β-cholan-24-oic acid) is the root C24 bile acid. In the majority of vertebrates, hydroxylation of CDCA (or an intermediate in the synthesis of CDCA) occurs at an additional site on the steroid nucleus. The sites of such hydroxylation have been identified at C-1, C-4, C-5, C-6, C-12, C-15, and C-16 (for original references, see Ref. 2). We have suggested that the ability to form such trihydroxy bile acids by the hepatocyte may have evolved as a response to the acquisition of a bacterial flora in the cecum mediating dehydroxylation at C-7. Bacterial dehydroxylation of CDCA results in the formation of lithocholic acid, a 3α-monohydroxy secondary bile acid (3Hofmann A.F. Bile acids.in: Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schacter D.A. Shafritz D.A. In The Liver: Biology and Pathobiology. 3rd edition. Raven Press, New York1994: 677-718Google Scholar). Lithocholic acid is toxic when it accumulates in the enterohepatic circulation of bile acids (4Holsti P. Cirrhosis of the liver induced in rabbits by gastric instillation of 3-monohydroxycholanic acid.Nature. 1960; 186: 250Crossref PubMed Scopus (53) Google Scholar, 5Fischer C.D. Cooper N.S. Rothschild M.A. Mosbach E.H. Effect of dietary chenodeoxycholic acid and lithocholic acid in the rabbit.Am. J. Dig. Dis. 1974; 19: 877-886Crossref PubMed Scopus (85) Google Scholar, 6Palmer R.H. Hruban Z. Production of bile duct hyperplasia and gallstones by lithocholic acid.J. Clin. Invest. 1966; 45: 1255-1267Crossref PubMed Scopus (91) Google Scholar, 7Zaki F.G. Carey J.B. Hoffbauer F.W. Nwokolo C. Biliary reaction and choledocholithiasis induced in the rat by lithocholic acid.J. Lab. Clin. Med. 1967; 69: 737-748PubMed Google Scholar, 8Palmer R.H. Toxic effects of lithocholate on the liver and biliary tree.in: Taylor W. In The Hepatobiliary System. Fundamental and Pathological Mechanisms. Plenum Press, New York1976: 227-240Crossref Google Scholar, 9Zhang J. Huang W. Qatanani M. Evans R.M. Moore D.D. The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid- induced toxicity.J. Biol. Chem. 2004; 279: 49517-49522Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 10Kitada H. Miyata M. Nakamura T. Tozawa A. Honma W. Shimada M. Nagata K. Sinal C.J. Guo G.I. Gonzalez F.J. et al.Protective role of hydroxysteroid sulfotransferase in lithocholic acid-induced liver toxicity.J. Biol. Chem. 2003; 278: 17838-17844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 11Stedman C.A. Robertson G.R. Coulter S.A. Liddle C. Feed-forward regulation of bile acid detoxification by CYP3A4: studies in humanized transgenic mice.J. Biol. Chem. 2004; 279: 11336-11433Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 12Fickert P. Fuchsbichler A. Marschall H-U. Wagner M. Zollner G. Krause R. Zatloukal K. Jaeschke H. Denk H. Trauner M. Lithocholic acid feeding induces segmental bile duct obstruction and destructive cholangitis in mice.Am. J. Pathol. 2006; 168: 410-422Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), causing segmental bile duct obstruction and destructive cholangitis in the mouse (12Fickert P. Fuchsbichler A. Marschall H-U. Wagner M. Zollner G. Krause R. Zatloukal K. Jaeschke H. Denk H. Trauner M. Lithocholic acid feeding induces segmental bile duct obstruction and destructive cholangitis in mice.Am. J. Pathol. 2006; 168: 410-422Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and bile duct hyperplasia and choledocholithiasis in the rat (6Palmer R.H. Hruban Z. Production of bile duct hyperplasia and gallstones by lithocholic acid.J. Clin. Invest. 1966; 45: 1255-1267Crossref PubMed Scopus (91) Google Scholar, 7Zaki F.G. Carey J.B. Hoffbauer F.W. Nwokolo C. Biliary reaction and choledocholithiasis induced in the rat by lithocholic acid.J. Lab. Clin. Med. 1967; 69: 737-748PubMed Google Scholar). On the other hand, 7-dehydroxylation of a trihydroxy bile acid results in the formation of a dihydroxy bile acid; the dihydroxy bile acids formed by bacterial 7-dehydroxylation of the common natural bile acids are known to be less toxic than lithocholic acid when they accumulate in the enterohepatic circulation. We report here the structure of a novel dihydroxy bile acid, 15α-hydroxylithocholic acid, which is a major biliary bile acid in the wombat (Vombatus ursinus), a common Australian marsupial. We have confirmed its structure by direct synthesis of this bile acid from CDCA. The 15α-hydroxylithocholic acid occurs in bile as its taurine conjugate, which was also synthesized and shown to have identical chromatographic and spectroscopic properties as the compound isolated from bile. Wombat bile was obtained by aspiration of the gallbladder at autopsy from two wombats that died as a result of being struck by automobiles. The wombat cadavers had been transported to the Healesville Sanctuary (Victoria, Australia), where the autopsy disclosed no tissue abnormalities other than those caused by trauma. Bile samples were diluted in three volumes of isopropanol and mailed to the laboratory of T.I. for analysis. Melting point (mp) values were determined on a micro hot-stage apparatus and are uncorrected. Infrared (IR) spectra were obtained in KBr discs on a Jasco FT-IR 4100 spectrometer (Tokyo, Japan). 1H- and 13C-NMR spectra were obtained on a JEOL JNM-EX 270 FT instrument at 270 and 68.8 MHz, respectively; in this report, only values for critical functionalities are given. EI low-resolution (LR) mass spectra were determined on a JEOL JMS-303 mass spectrometer at 70 eV. High-resolution (HR) mass spectra were measured using a JEOL LCmate double-focusing magnetic mass spectrometer equipped with an EI or fast-atom bombardment (FAB) probe under the positive ion mode. HR-MS spectra were also obtained on a JEOL JMS-700 mass spectrometer with an EI or FAB probe under the positive ion mode. Normal-phase TLC was performed on precoated silica gel plates (0.25 mm layer thickness; E. Merck, Darmstadt, Germany) using hexane-ethyl acetate-acetic acid mixtures (40:60:1–60:40:1, v/v/v) as the developing solvent. Reverse-phase TLC was carried out on precoated RP-18F254S plates using methanol-water-acetic acid mixtures (90:10:1, v/v/v) as the developing solvent. Sep-Pak Vac tC18 cartridges (adsorbent weight, 5 g) for solid-phase extraction were purchased from Waters Associates (Milford, MA); they were washed successively with 50 ml of methanol and 50 ml of distilled water before use. CDCA was kindly supplied by Mitsubishi Pharma (Osaka, Japan). Cholyl taurine, chenodeoxycholyl taurine, ursodeoxycholyl taurine, deoxycholyl taurine, and lithocholyl taurine were from our laboratory collection. The apparatus used was a Jasco HPLC system (two PU-2085 high-pressure pumps, an MX-2080-32 solvent mixing module, and an HG-980-50 degasser; Tokyo, Japan) equipped with a Shimadzu C-R8A data-processing system (Kyoto, Japan). A Capcell Pak-type MGII column [250 mm × 3.0 mm inner diameter (ID); particle size, 5 μm; Shiseido, Tokyo, Japan] was used for analytical purposes. The column temperature was kept at 37°C using a Sugai u-620-type 30V column heater (Wakayama, Japan). An Alltech 2000ES evaporative light-scattering detector (Deerfield, IL) was used; the flow rate of purified compressed air as a nebulizing gas was 1.6 l/min, and the temperature of the heated drift tube was 82°C. For simultaneous separation of unconjugated, glycine-amidated, and taurine-amidated bile acids, an isocratic elution mode was used according to the procedure of Roda et al. (13Roda A. Cerrè C. Simoni P. Polimeni C. Vaccari C. Pistillo A. Determination of free and amidated bile acids by high-performance liquid chromatography with evaporative light-scattering mass detection.J. Lipid Res. 1992; 33: 1393-1402Abstract Full Text PDF PubMed Google Scholar). The mobile phase used was 65% (v/v) aqueous methanol containing 15 mM ammonium acetate adjusted to pH 5.4 with acetic acid, and the flow rate was kept at 400 μl/min. Figure 1 shows the HPLC result that was obtained; the identities of individual peaks are discussed in Results. The bile of the wombats was diluted with isopropanol (∼10 ml) and filtered, and the filtrate was evaporated under a nitrogen stream at 35°C. The residue was dissolved in methanol-water (1:9) (5 ml) and then applied to a preconditioned Sep-Pak tC18 cartridge (400 mg; Waters). After the cartridge was washed successively with water (2 ml) and 20% methanol (2 ml), the bile acid fraction was eluted with methanol. The methanol eluate was evaporated under a nitrogen stream at 35°C; the residue was then dissolved in 200 μl of methanol, and its individual bile acids were isolated by preparative HPLC. The apparatus consisted of a Jasco Gulliver series HPLC system (two PU-980 high-pressure pumps, an HG-980-31 solvent mixing module, and an HG-980-50 degasser). Reverse-phase chromatographic separation was carried out by stepwise gradient elution on a Capcell Pak C18-type MGII column (5 μm, 250 mm × 10 mm ID) using methanol-water mixtures as the mobile phase. The methanol composition was gradually increased at a flow rate of 2 ml/min as follows: 48% (0–15 min) → 50% (15.1–30 min) → 52% (30.1–45 min) → 54% (45.1–60 min). The 52% methanol fraction, which contained compound C (the unknown bile acid), was evaporated under a nitrogen stream at 35°C. LC-MS analyses of wombat bile components were performed on a JEOL JMS-LCmate double focusing magnetic mass spectrometer equipped with an ESI probe using the negative ion mode. Chromatographic separation was carried out on a YMC Pack Pro C18 column (3 μm, 100 × 2.0 mm ID; YMC, Kyoto, Japan) using a 20 mM ammonium acetate (pH 7)-methanol mixture (35:65, v/v) as the mobile phase at a flow rate of 200 μl/min. The mass detector was set to the following conditions: needle voltage, −2.5 kV; orifice 1 temperature, 150°C; desolvating plate temperature, 250°C; ring lens voltages, 30 V/100 V. Negative ion LC-ESI-MS/MS spectra of compounds C and 1d (see below) were obtained on a Finnigan LTQ (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with a Paradigm MS4 HPLC system (AMR, Inc., Tokyo, Japan). Chromatographic separation was carried out on a TSKgel ODS-100V (5 μm, 150 × 2.0 mm ID) using 5 mM ammonium acetate (pH 6)-acetonitrile mixtures as the mobile phase. A linear gradient was used: 30% CH3CN (0 min) → 80% CH3CN (30 min); the flow rate was kept constant at 200 μl/min. The mass detector was set as follows: capillary temperature, 270°C; sheath gas flow rate, 50 arbitrary units; auxiliary gas flow rate, 5 arbitrary units; source voltage, ±4 kV; capillary voltage, ±30 V; tube lens offset voltage, ±100 V. The synthetic scheme used to prepare 15α- and 15β-hydroxylithocholic acid (and their methyl esters) is shown in Fig. 2 (compounds 1–8). To a magnetically stirred solution of methyl chenodeoxycholate 3-cathylate (3; 4.4 g) in pyridine (50 ml), phosphoryl chloride (15 ml) was slowly added with ice bath cooling. After being allowed to stand overnight at room temperature, the mixture was poured into ice water (0°C) and extracted with CH2Cl2. The combined CH2Cl2 extract was washed with 10% HCl and then with water to neutrality, dried with Drierite, and evaporated to an oily residue, which, when treated with CH2Cl2-methanol, crystallized as colorless crystals: yield, 3.6 g (86%); mp 119–123°C [literature value (14Osawa R. Yamasaki K. Dehydration of bile acids and their derivatives. VII. Isomerization of methyl 3α-hydroxy-Δ7-cholenate to methyl 3α-hydroxy-Δ14-cholenate.Bull. Chem. Soc. Jpn. 1959; 32: 1302-1306Crossref Google Scholar) mp 120–121°C]. IR (KBr) νmaxcm−1; 1,738 (C=O). 1H-NMR (CDCl3) δ; 0.54 (3H, s, 18-CH3), 0.87 (3H, s, 19-CH3), 0.93 (3H, d, J = 6.2 Hz, 21-CH3), 1.29 (3H, t, J = 7.0 Hz, COOCH2CH3), 3.67 (3H, s, -COOCH3), 4.16 (2H, m, COOCH2CH3), 4.58 (1H, brm, 3β-H), 5.09 (1H, m, 7-H). 13C-NMR (CDCl3) δ; 11.8 (C-18), 14.2 (COOCH2CH3), 18.4 (C-21), 24.3 (C-19), 51.4 (COOCH3), 63.5 (COOCH2CH3), 77.3 (C-3), 115.2 (C-7), 137.2 (C-8), 154.6 (COOCH2CH3), 174.7 (C-24). LR-MS (EI) m/z: 370 (M-CtOH, 100%), 355 (M-CtOH-CH3, 49%), 255 [M-CtOH-CH3-side chain (S.C.)-ring D, 32%], 228 (M-CtOH-S.C.-part of ring D, 11%), 213 (M-CtOH-S.C.-ring D, 21%). The 3α-cathyloxy-Δ7 ester 4 (2.65 g) was hydrolyzed using 5% methanolic KOH (35 ml), followed by acidification with H2SO4 and recrystallization from methanol. The Δ7-unsaturated acid 5a was isolated as colorless needles: yield, 1.98 g (92%); mp 183–185°C [literature value (14Osawa R. Yamasaki K. Dehydration of bile acids and their derivatives. VII. Isomerization of methyl 3α-hydroxy-Δ7-cholenate to methyl 3α-hydroxy-Δ14-cholenate.Bull. Chem. Soc. Jpn. 1959; 32: 1302-1306Crossref Google Scholar) mp 183–184°C]. IR (KBr) νmaxcm−1; 1,708 (C=O), 3,352 (OH). 1H-NMR (CDCl3) δ; 0.55 (3H, s, 18-CH3), 0.86 (3H, s, 19-CH3), 0.95 (3H, d, J = 6.2 Hz, 21-CH3), 3.63 (1H, brm, 3β-H), 5.10 (1H, m, 7-H). 13C-NMR (CDCl3) δ; 11.9 (C-18), 18.4 (C-21), 24.5 (C-19), 71.4 (C-3), 115.4 (C-7), 137.2 (C-8), 179.3 (C-24). LR-MS (EI) m/z: 374 (M, 25%), 356 (M-H2O, 100%), 341 (M-H2O-CH3, 69%), 302 (16%), 255 [M-H2O-S.C., 32%], 228 (M-H2O-part of ring D, 14%), 213 (M-H2O-S.C.-ring D, 30%). HR-MS (EI), calculated for 374.2821 [M]; found m/z, 374.2799. The corresponding Δ7 ester 5b, prepared from 5a by the usual methanol and p-toluenesulfonic acid method, was recrystallized from methanol as colorless thin plates: yield, 60%; mp 88–92°C [literature value (14Osawa R. Yamasaki K. Dehydration of bile acids and their derivatives. VII. Isomerization of methyl 3α-hydroxy-Δ7-cholenate to methyl 3α-hydroxy-Δ14-cholenate.Bull. Chem. Soc. Jpn. 1959; 32: 1302-1306Crossref Google Scholar) mp 104–105°C]. IR (KBr) νmaxcm−1; 1,614 (C=C), 1,734 (C=O), 3,017 (=C-H), 3,337 (OH). 1H-NMR (CDCl3) δ; 0.54 (3H, s, 18-CH3), 0.85 (3H, s, 19-CH3), 0.94 (3H, d, J = 5.9 Hz, 21-CH3), 3.62 (1H, brm, 3β-H), 3.67 (3H, s, -COOCH3), 5.10 (1H, m, 7-H). 13C-NMR (CDCl3) δ; 11.9 (C-18), 18.4 (C-21), 24.4 (C-19), 51.4 (COOCH3), 71.3 (C-3), 115.4 (C-7), 137.2 (C-8), 174.7 (C-24). LR-MS (EI) m/z: 388 (M, 27%), 370 (M-H2O, 100%), 316 (M-H2O-ring A, 14%), 355 (M-H2O-CH3, 75%), 255 (M-H2O-S.C., 42%), 228 (M-H2O-S.C.-part of ring D, 18%), 213 (M-H2O-S.C.-ring D, 41%). HR-MS (EI), calculated for C25H40O3: 388.2977; found m/z, 388.2974. A solution of the Δ7 ester 5b (300 mg) in dry CHCl3 (10 ml) was prepared, and dry HCl gas was bubbled through it for 2 h in an ice bath. The CHCl3 solution was washed successively with water, with 5% NaHCO3 solution, and then again with water to neutrality. It was then dried with Drierite and evaporated to an oily residue, which, according to capillary GC analysis, consisted of a mixture of three components in an approximate ratio of 17:44:39 (%). The mixture was chromatographed on a column of 25% AgNO3-impregnated silica gel (30 g) (15Vroman H.E. Cohen C. Separation of sterol acetates by column and thin-layer chromatography.J. Lipid Res. 1967; 8: 150-152Abstract Full Text PDF PubMed Google Scholar). Elution with hexane-EtOAc (4:1 → 7:3, v/v) provided three well-separated fractions. The less polar fraction was identified as methyl 3α-hydroxy-5β-chol-8(9)-en-24-oate (9; Fig. 3), which resisted crystallization attempts: yield, 36 mg (12%); viscous oil [literature value (16Nakada F. Dehydration of bile acids and their derivatives. XIII. Isomerization of chola-7,11-dienic acid derivatives to 7,9-diene compounds and dehydration of 3α,12α-dihydroxy-chol-8(14)-enic acid.Steroids. 1963; 2: 403-416Crossref Scopus (12) Google Scholar)] mp 116–117°C). IR (KBr) νmaxcm−1; 1,737 (C=O), 3,420 (OH). 1H-NMR (CDCl3) δ; 0.87 (3H, s, 18-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 0.99 (3H, s, 19-CH3), 3.59 (1H, brm, 3β-H), 3.66 (3H, s, -COOCH3). 13C-NMR (CDCl3) δ; 19.3 (CH3), 23.6 (CH3), 28.6 (CH3), 51.3 (COOCH3), 70.9 (C-3), 130.1 and 131.7 (C-8 and C-9, respectively, or vice versa), 174.6 (C-24). LR-MS (EI) m/z: 388 (M, 27%), 370 (M-H2O, 100%), 355 (M-H2O-CH3, 59%), 316 (79%), 273 (M-S.C., 18%), 255 (M-H2O-S.C., 83%), 229 (M-H2O-S.C.-part of ring D, 17%), 213 (M-H2O-S.C.-ring D, 26%). HR-MS (EI), calculated for C25H40O3, 388.2977; found m/z, 388.2981. Continued elution gave a homogeneous viscous oil, which was characterized as methyl 3α-hydroxy-5β-chol-8(14)-en-24-oate (10; Fig. 3): yield, 115 mg (38%). IR (KBr) νmaxcm−1; 1,739 (C=O), 3,393 (OH). 1H-NMR (CDCl3) δ; 0.80 (3H, s, 18-CH3), 0.83 (3H, s, 19-CH3), 0.94 (3H, d, J = 6.2 Hz, 21-CH3), 3.64 (1H, brm, 3β-H), 3.66 (3H, s, -COOCH3). 13C-NMR (CDCl3) δ; 18.0 (C-18), 18.6 (C-21), 23.6 (C-19), 51.4 (COOCH3), 127.0 (C-8), 141.5 (C-14), 174.7 (C-24). LR-MS (EI) m/z: 388 (M, 35%), 370 (M-H2O, 100%), 355 (M-H2O-CH3, 78%), 316 (13%), 255 (M-H2O-S.C., 40%), 229 (M-H2O-S.C.-part of ring D, 23%), 215 (46%), 213 (M-H2O-S.C.-ring D, 39%). HR-MS (EI), calculated for C25H40O3, 388.2977; found m/z, 388.2993. The most polar fraction was crystallized from aqueous ethanol to give the desired methyl 3α-hydroxy-5β-chol-14-en-24-oate (6) as colorless amorphous solids; yield, 92 mg (31%); mp 140–141°C [literature value (14Osawa R. Yamasaki K. Dehydration of bile acids and their derivatives. VII. Isomerization of methyl 3α-hydroxy-Δ7-cholenate to methyl 3α-hydroxy-Δ14-cholenate.Bull. Chem. Soc. Jpn. 1959; 32: 1302-1306Crossref Google Scholar) mp 140–141°C]. IR (KBr) νmaxcm−1; 1,740 (C=O), 3,048 (=C-H), 3,255 (OH). 1H-NMR (CDCl3) δ; 0.89 (3H, s, 18-CH3), 0.92 (3H, d, J = 5.9 Hz, 21-CH3), 0.93 (3H, s, 19-CH3), 3.62 (1H, brm, 3β-H), 3.67 (3H, s, -COOCH3), 5.15 (1H, brs, 15-H). 13C-NMR (CDCl3) δ; 16.8 (C-18), 18.5 (C-21), 23.1 (C-19), 51.5 (COOCH3), 71.9 (C-3), 116.9 (C-15), 155.4 (C-14), 174.8 (C-24). LR-MS (EI) m/z: 388 (M, 9%), 370 (M-H2O, 21%), 355 (M-H2O-CH3, 11%), 273 (M-S.C., 57%), 255 (M-H2O-S.C., 100%), 208 (part of ring C+ring D+CH3+S.C., 13%). HR-MS (EI), calculated for C25H40O3, 388.2977; found m/z, 388.2961. To a stirred solution of the Δ14 ester (6) (30 mg) in dry tetrahydrofuran (THF) (900 μl), a solution of BH3/THF (1.0 mol/l, 390 μl) was added gradually, and the mixture was stirred at room temperature under a stream of N2. After 5 h, 3 N NaOH (300 μl) and then 30% H2O2 (300 μl) were added with ice-bath cooling, and the mixture was stirred overnight at room temperature. The resulting solution was acidified with 10% HCl, and the reaction product was extracted with EtOAc. The combined EtOAc layers were washed with saturated brine and evaporated to dryness. The oily residue was chromatographed on a reverse-phase C18-bonded silica gel (10 g; particle size, 50 μm). Elution with methanol-water (7:3, v/v) afforded the title compound 7, which was recrystallized from EtOAc-hexane as colorless amorphous solids: yield, 21 mg (75%); mp 102–105°C. IR (KBr) νmaxcm−1; 3,340 (OH). 1H-NMR (CDCl3) δ; 0.68 (3H, s, 18-CH3), 0.92 (3H, d, J = 7.0 Hz, 21-CH3), 0.94 (3H, s, 19-CH3), 3.61 (2H, t, J = 6.6 Hz, 24-H2), 3.63 (1H, brm, 3β-H), 3.94 (1H, brm, 15β-H). 13C-NMR (CDCl3) δ; 13.3 (C-18), 18.4 (C-21), 23.4 (C-19), 63.5 (C-24), 71.8 (C-3), 73.8 (C-15). LR-MS (EI) m/z: 360 (45%, M-H2O), 342 (96%, M-2H2O), 327 (M-2H2O-CH3, 44%), 288 (M-H2O-ring A, 30%), 273 (M-H2O-S.C., 86%), 255 (M-2H2O-S.C., 100%) 217 (ring A+B+C, 62%). HR-MS (FAB+), calculated for C24H42O3Na [M+Na], 401.3032; found m/z, 401.3045. Jones reagent (500 μl) was added gradually to a solution of the 3α,15α,24-triol 7 (30 mg) in acetone (4 ml) under 10°C, and the mixture was stirred for 30 min at room temperature. Methanol (500 μl) was added, and the oxidation product was extracted with EtOAc. The combined EtOAc extracts were washed with saturated brine, dried over Drierite, and evaporated to dryness. The oily residue was chromatographed using a column of C18-bonded silica gel (10 g; particle size, 50 μm). Elution with methanol-water (7:3, v/v) afforded the title dioxo acid 8a, which was recrystallized from EtOAc-hexane as colorless amorphous solids: yield, 22 mg (72%); mp 165–166°C. IR (KBr) νmaxcm−1; 1,705 (C=O, ketone), 1,736 (C=O, carboxyl group). 1H-NMR (CDCl3) δ; 0.78 (3H, s, 18-CH3), 1.02 (3H, d, J = 5.9 Hz, 21-CH3), 1.03 (3H, s, 19-CH3). 13C-NMR (CDCl3) δ; 13.0 (C-18), 18.5 (C-21), 22.5 (C-19), 177.9 (C-24), 212.9 (C-3), 214.9 (C-15). LR-MS (EI) m/z: 388 (M, 61%), 370 (19%), 318 (M-ring A, 10%), 287 (M-S.C., 44%), 259 (M-S.C.-part of ring D, 48%), 232 (M-S.C.-ring D, 15%), 215 (16%), 197 (S.C.+ring D+CH3, 100%). HR-MS (EI), calculated for C24H36O4 [M], 388.2614; found m/z, 388.2587. The free acid 8a, reesterified with methanol and p-toluenesulfonic acid and processed by the usual work-up, yielded the corresponding methyl ester. Recrystallization from EtOAc-hexane gave the dioxo ester 8b as colorless amorphous solids: yield, 91%; mp 110–112°C. IR (KBr) νmaxcm−1; 1,713 (C=O, ketone), 1,735 (C=O, ester). 1H-NMR (CDCl3) δ; 0.78 (3H, s, 18-CH3), 1.00 (3H, d, J = 6.2 Hz, 21-CH3), 1.03 (3H, s, 19-CH3), 3.67 (3H, s, COOCH3). 13C-NMR (CDCl3) δ; 12.9 (C-18), 18.5 (C-21), 22.5 (C-19), 51.6 (COOCH3), 174.1 (C-24), 212.8 (C-3), 214.9 (C-15). LR-MS (EI) m/z: 402 (M, 45%), 387 (M-CH3, 34%), 371 (20%) 329 (16%), 287 (M-S.C., 100%), 259 (M-S.C.-part of ring D, 54%), 211 (S.C.+CH3+ring D, 33%). HR-MS (EI), calculated for C25H38O4 [M+], 402.2770; found m/z, 402.2762. tert-Butylamineborane complex (30 mg) was added to a stirred solution of the 3,15-dioxo ester 8b (30 mg) in CH2Cl2 (3 ml), and the mixture was refluxed overnight. After cooling the mixture, 10% HCl (900 μl) was added, and the solution was stirred for 30 min. The CH2Cl2 layer was washed with 5% NaHCO3 and water, dried over Drierite, and evaporated to dryness. The oily residue, which consisted essentially of two components on normal-phase TLC, was chromatographed on a column of silica gel (10 g). Elution with EtOAc-hexane (1:1–3:2, v/v) provided two well-separated fractions. The less polar fraction was identified as methyl 3α,15β-dihydroxy-5β-cholanoate (2b), which recrystallized from aqueous methanol as colorless amorphous solids: yield, 17 mg (56%); mp 64–67°C. IR (KBr) νmaxcm−1; 1,739 (C=O), 3,409 (OH). 1H-NMR (CDCl3) δ; 0.92 (3H, d, J = 5.7 Hz, 21-CH3), 0.93 (3H, s, 19-CH3), 0.96 (3H, s, 18-CH3), 3.64 (1H, brm, 3β-H) 3.67 (3H, m, -COOCH3), 4.19 (1H, t, J = 5.7 Hz, 15α-H). 13C-NMR (CDCl3) δ; 14.6 (C-18), 18.2 (C-21), 23.3 (C-19), 51.5 (COOCH3), 70.2 (C-15), 71.7 (C-3), 174.6 (C-24). LR-MS (EI) m/z: 388 (M-H2O, 59%), 370 (M-2H2O, 42%), 355 (M-2H2O-CH3, 22%), 273 (M-H2O-S.C., 34%), 255 (M-2H2O-S.C., 33%), 217 (ring A+B+C, 43%), 208 (100%). HR-MS (EI), calculated for C25H40O3, 388.2977 [M]; found m/z, 388.2976. The more polar fraction was characterized as the desired 3α,15α-dihydroxy ester 1b, which resisted crystallization attempts: yield, 10 mg (33%). IR (KBr) νmaxcm−1; 1,737 (C=O), 3,350 (OH). 1H-NMR (CDCl3) δ; 0.68 (3H, s, 18-CH3), 0.90 (3H, d, J = 5.7 Hz, 21-CH3), 0.93 (3H, s, 19-CH3), 3.62 (1H, brm, 3β-H), 3.66 (3H, m, -COOCH3), 3.94 (1H, brm, 15β-H). 13C-NMR (CDCl3) δ; 13.3 (C-18), 18.1 (C-21), 23.4 (C-19), 51.5 (COOCH3), 71.7 (C-3), 73.6 (C-15), 174.6 (C-24). LR-MS (EI) m/z: 388 (M-H2O, 72%), 370 (M-2H2O, 42%), 355 (M-2H2O-CH3, 50%), 273 (M-H2O-S.C., 98%), 255 (M-2H2O-S.C., 88%), 217 (ring A+B+C, 41%), 208 (100%). HR-MS (EI), calculated for C25H40O3, 388.2977 [M]; found m/z, 388.2969. The 3α,15α-dihydroxy ester 1b (50 mg) was refluxed in 5% methanolic KOH (1 ml) for 1 h. Solvent was removed by evaporation, and the residue was dissolved in water and acidified with 10% H2SO4 with stirring and ice-bath cooling. The precipitate was collected by filtration, washed with water, and recrystallized from EtOAc as colorless amorphous solids of 3α,15α-dihydroxy acid 1a (analytically pure): yield, 46 mg (95%); mp 206–208°C. IR (KBr) νmaxcm−1; 1,703 (C=O), 3,245 (OH). 1H NMR (CD3OD) δ; 0.71 (3H, s, 18-CH3), 0.94 (3H, d, J = 5.9 Hz, 21-CH3), 0.96 (3H, s, 19-CH3), 3.54 (1H, brm, 3β-H), 3.84 (1H, brm, 15β-H). 13C NMR (CD3OD) δ; 13.7 (C-18), 18.6 (C-21), 21.8 (C-11), 24.0 (C-19), 27.6 (C-6), 28.3 (C-7), 31.2 (C-2), 31.9 (C-22), 32.2 (C-23), 35.7 (C-10), 36.1 (C-20), 36.6 (C-1), 36.9 (C-8), 37.2 (C-4), 41.6 (C-16), 41.7 (C-12), 42.0 (C-5), 43.5 (C-9) 44.9 (C-13), 54.8 (C-17), 64.0 (C-14), 72.4 (C-3), 74.2 (C-5) 178.0 (C-24). LR-MS (EI) m/z: 374 (M-H2O, 36%), 356 (M-2H2O, 100%), 341 (M-2H2O-CH3, 43%), 302 (M-H2O-ring A, 32%), 273 (M-H2O-S.C, 48%), 255 (M-2H2O-S.C., 60%), 217 (ring A+B+C, 51%). HR-MS (FAB+), calculated for C24H40O4Na, 415.2824 [M+Na]; found m/z, 415.2788. The 3α,15β-dihydroxy ester 2b (50 mg) was hydrolyzed with 5% methanolic KOH (1 ml) and processed as described for the preparation of 1a to yield the crude acid. Recrystallization from EtOAc gave the 3α,15β-dihydroxy acid 2a as colorless amorphous solids: yield, 44 mg (91%); mp 119–121°C [literature value (17Kulprecha S. Nihira T. Shimomura C. Yamada K. Nilubol N. Yoshida T. Taguchi H. 15β-Hydroxylation of lithocholic acid by Cunninghamella sp.Tetrahedron. 1984; 40: 2843-2846Crossref Scopus (10) Google Scholar) mp 185.5–186.5°C]. IR (KBr) νmaxcm−1; 1,710 (C=O), 3,321 (OH). 1H NMR (CD3OD) δ; 0.94 (3H, s, 18-CH3), 0.95 (3H, d, J = 7.6 Hz, 21-CH3), 0.98 (3H, s, 19-CH3), 3.53 (1H, brm, 3β-H), 4.16 (1H, brm, 15α-H). 13C NMR (CD3OD) δ; 15.2 (C-18), 18.8 (C-21), 21.8 (C-11), 24.0 (C-19), 26.8 (C-7), 28.3 (C-6), 31.2 (C-16), 32.0 (C-23), 32.3 (C-2), 33.0 (C-20), 35.9 (C-10), 36.4 (C-8), 36.6 (C-22), 37.3 (C-1), 42.1 (C-4), 42.2 (C-9), 42.9 (C-12), 43.6 (C-5 and C-13), 57.6 (C-17), 62.4 (C-14), 70.6 (C-15), 72.5 (C-3), 178.2 (C-24). LR-MS (FAB) m/z: 374 (M-H2O, 39%), 356 (M-2H2O, 54%), 341 (M-2H2O-CH3, 27%), 302 (M-H2O-ring A, 11%), 273 (M-H2O-S.C., 34%), 263 (38%), 255 (M-2H2O-S.C., 54%), 217 (M-H2O-S.C.-ring D, 100%). HR-MS (FAB+), calculated for C24H40O4Na, 415.2824 [M+Na]; found m/z, 415.2802. To a magnetically stirred solution of 3α,15α-dihydroxy acid 1a (30 mg) in dry dimethylformamide (3 ml), glycine methyl ester hydrochloride (25 mg), diethylphosphorocyanide (30 μl), and triethylamine (125 μl) were added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was extracted with EtOAc, and the combined extracts were washed with saturated brine and evaporated to dryness. The residue was then refluxed in 5% methanolic NaOH (4 ml) for 1 h, and the solution was adjusted to pH 9 by 10% HCl. Most of the solvent was evaporated under reduced pressure, and the residue was dissolved in water (5 ml). The aqueous solution was loaded onto a Sep-Pak Vac tC18 cartridge, which was washed successively with water (20 ml), 30% meth

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