3,4-Seco-12α-hydroxy-5β-cholan-3,4,24-trioic Acid, a Novel Secondary Bile Acid: Isolation from the Bile of the Common Ringtail Possum (Pseudocheirus peregrinus) and Chemical Synthesis

The major bile acids present in gallbladder bile of the common ringtail possum (Pseudocheirus peregrinus), an Australian marsupial, were isolated by preparative HPLC and identified by NMR and by comparison with synthetic standards. The major compound present (52%) was 3α,12α-dihydroxy-7-oxo-5β-cholan-24-oic acid (7-oxodeoxycholic acid), about three fourths conjugated with taurine. Also present was 3α,7β,12α-trihydroxy-5β-cholan24-oic acid (20%; ursocholic acid) largely in unconjugated form. In addition, 3,4-seco-12α-hydroxy-5β-cholan-3,4,24trioic acid was present in unconjugated form and constituted 8% of biliary bile acids. Proof of the structure of this novel 3,4-seco acid was obtained by its chemical synthesis from deoxycholic acid via an intermediary 3β,4β-dihydroxy derivative that was then oxidatively cleaved with sodium periodate. As all primary bile acids have a hydroxyl or oxo substituent at C-7, the absence of such in the seco-bile acid suggests that it is a secondary bile acid, synthesized by bacterial enzymes present in the intestine. 3,4-Seco-12α-hydroxy-5β-cholan-3,4,24-trioic Acid, a Novel Secondary Bile Acid: Isolation from the Bile of the Common Ringtail Possum (Pseudocheirus peregrinus) and Chemical Synthesis


Introduction
Bile acids (C 24 and C 27 ) and bile alcohols (C 27 ) are the end products of cholesterol metabolism that have multiple physiological functions. After their synthesis bile acids and bile alcohols are made water soluble by "conjugation" with glycine or taurine for bile acids and with sulfate for bile alcohols. In the liver, bile acids stimulate bile flow and solubilize biliary cholesterol. In the small intestine, bile acids solubilize dietary lipids, and in the large intestine, modulate water and electrolyte movement [1,2]. In addition, in the past decade, bile acids have been shown to also possess potent and important signaling properties [3]. Bile acids modulate the expression of multiple genes via the nuclear receptor FXR (farnesoid X receptor) that is activated by bile acids. Bile acids also activate the TGR5 (transmembrane G proteincoupled receptor 5) and thereby modulate intracellular events. Agents that activate FXR and/or TGR5 are in clinical development for the treatment of cholestatic liver disease and nonalcoholic steatohepatitis.
Bile acid structure varies widely in vertebrates. Haslewood proposed that bile acid structure provides additional phenotypic information for the establishment of phylogenetic relationships [4], and we have extended his pioneering work in a series of papers [5][6][7].
We report here the biliary bile acid composition of an Australian marsupial, the common ringtail possum (Pseudocheirus peregrinus) (Figure 1), a member of the Pseudocheiridae family of the order Diprotodontia. In particular, we report the presence of a novel 3,4-seco-bile acid whose structure was confirmed by chemical synthesis from deoxycholic acid. We also report that the biliary bile acids of the possum appear to be largely secondary bile acids, formed from primary bile acids by bacterial enzymes. Finally, we relate the biliary bile acid composition of the possum to marsupial phylogeny.

H and 13 C NMR analysis of isolated compounds
NMR spectra were recorded at 23°C in CDCl 3 or pyridine-d 5 on a JEOL ECA-500 instrument using 500.2 MHz for 1 H and 125. 8 MHz for 13 C. The 1 H and 13 C resonance assignments were made using a combination of Two-Dimensional (2D) homonuclear ( 1 H-1 H) and heteronuclear ( 1 H-13 C) shift-correlated techniques, which include 1 H-1 H COSY correlation, 1 H Nuclear Overhauser And Exchange Spectroscopy (NOESY), 1 H detected heteronuclear multiple quantum coherence (HMQC; 1 H-13 C coupling), and 1 H detected heteronuclear multiple bond connectivity (HMBC; long-range 1 H-13 C coupling) experiments. These 2D-NMR spectra were recorded using standard pulse sequences and parameters recommended by the manufacturer. The 13 C distortion less enhancement by polarization transfer (DEPT; 135°,90°, and 45°) spectra were also measured to determine the exact 13 C signal multiplicity and to differentiate between CH 3 , CH 2 , CH, and C based on their proton environments.

HPLC-ELSD analysis of gallbladder bile of the common ringtail possum
The Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) apparatus used was a JascoLC-2000 plus HPLC system, which consisted of two PU-2085 high-pressure pumps, an MX-2080-32 solvent mixing module, a DG-980-50 degasser, and a CO-2060 column heater with a ChromNAV data processing system (Tokyo, Japan). A Capcell Pack type C 18 AQ RP-column (3.0 mm × 150 mm I.D.; particle size, 5 μm; Shiseido, Tokyo, Japan) was employed and kept at 37°C. An Alltech 2000ES Evaporative Light-Scattering Detector (ELSD; Deerfield, IL, USA) was used under the following conditions: The flow rate of purified compressed air used as a nebulizing gas was 2.2 L/min and the temperature of the heated drift was 80.9°C. The mobile phase used was a mixture of 15 mM-ammonium acetate/acetic acid buffer solution (pH 5.0) and methanol (38:62, v/v); the flow rate was kept at isocratic conditions of 0.4 mL/min during the analysis.

Isolation of major biliary bile acids of common ringtail possum by preparative HPLC
The isopropanol solution of the common ringtail possum was evaporated under a stream of N 2 , and the residue was dissolved in water (1.5 mL). The aqueous solution was centrifuged for 10 min at 2000 rpm, and the supernatant solution was recovered; the procedure was repeated three times for the residue. The total volume of the combined supernatant solution (4.5 mL) was adjusted to 15 mL by diluting with water. The solution was passed through a preconditioned Sep-Pak ® tC18 cartridge (10 g; Waters, Milford, MA, USA). After the cartridge was washed with successively with water (50 mL) and then with 15 mM ammonium acetate/acetic acid buffer solution (pH 5.0) containing 5,10,15,20,25,30,35,40,45, 50 ~ 80, and 100% methanol. After evaporation of the solvent from each of the fractions, the residues were dried by lyophilization. The residues were then dissolved in methanol and combined supernatant liquids were filtered with a Mini-Uni Pre membrane filter (pore size, 0.45 μm; Whatman, NJ, USA).
Individual, major bile acids were isolated by preparative HPLC, which consisted of a Hitachi L-7100 pump, a Refraction Index (RI)-102 detectors, and a type 30V column heater. For simultaneous separation of unconjugated and glycine-and taurine-amidated bile acids, RP-HPLC separation was carried out by isocratic elution modes on a Capcell Pak type C 18 AQ RP-column (10 mm×250 mm I.D.; particle size, 5 μm) using a mixture of 15 mM ammonium acetate/acetic acid buffer (pH 5.0) and methanol (35:65, v/v) as the mobile phase at a flow rate of 3.0 mL/min. The 65% methanol fractions, which contained each of compounds A, B, C, D, and F, were collected by evaporation of the solvent, followed by vacuum freeze-drying. Figure 3 shows the HPLC-ELSD result that was obtained; the identities of individual peaks A ~ H are discussed in the Results section.
To a magnetically stirred solution of of compound 2 (31 mg, 55 μmol), prepared from DCA in 4 steps [10], in acetone (2 mL) was added a solution of sodium periodate (NaIO 4 , 50 mg) dissolved in water (1.5 mL). After the mixture was stirred at room temperature for 2 h, the reaction product was extracted with EtOAc. The combined extract was washed with water to neutrality, dried with Drierite, and evaporated to an oily residue. To a solution of the residue dissolved in acetone (2 mL) was added three drops of Jones reagent, and the mixture was stirred at room temperature for 30 min. After adding a few drops of 2-propanol, the reaction product was extracted with CH 2 Cl 2 . The combined organic layer was washed with water, dried with Drierite, and evaporated to give an oily residue. Chromatography of the residue on a column of silica gel (1.0 g) and elution with hexane-EtOAc-acetic acid (150:50:1, v/v/v) afforded the title compound (3) Figure 3 shows a representative HPLC-ELSD chromatogram of the bile acid composition in the gallbladder bile of the common ringtail possum. Table 1 gives the RRTs of each peak (A ~ H) observed using HPLC-ELSD as well as their HR-LC/ESI-MS data. Identification of the major peaks was made by a direct comparison with authentic reference compounds prepared in our laboratory. Thus, peak E (16.5% of total bile acids) was found to be cholyl taurine (3α,7α,12α-trihydroxy-5βcholan-24-oyl taurine); peak G (2.4%) was unconjugated cholic acid (3α,7α,12α-trihydroxy-5β-cholan-24-oic acid; CA); and peak H (1.3%) was chenodeoxycholyl taurine (3α,7α-dihydroxy-5β-cholan-24-oyl taurine).
The structure of the unknown compound F (7.9%) was then subjected to further analysis. By HR-LC/ESI-MS analysis, peak F showed m/z 437.2539, indicating the deprotonated molecule ion [M-H]of C 24 H 37 O 7 . This observation strongly suggested that the unknown F was a novel bile acid having three carboxyl groups, i.e., a seco bile acid. Table 2 shows the 1 H and 13 C NMR spectral data for naturally occurring compound F as well as that of synthetic 3,4-seco-12α-dihydroxy-5βcholan-3,4,24-trioic acid (1). The 1 H and 13 C NMR spectral patterns of both the compounds were essentially identical. Thus, the 18-, 19-, and 21-CH 3 signals in the both 1 H NMR spectra were observed at 0.71 (singlet; s), 0.99 (s) and 1.17 (doublet) ppm, along with the 12β-H at 4.16 ppm (multiplet). Furthermore, these compounds showed three characteristic signals arising from carboxyl groups at 176.4, 176.5 and 177.2 ppm and at 72.3 ppm due to the 12β-H bearing a 12α-hydroxyl group in the 13 C NMR spectra.
In order to determine the position of the three carboxyl groups in compound F, the HMBC spectrum was measured as shown in Figure 5. The three peaks occurred at 176.4, 176.5, and 177.2 ppm were correlated with the 2α-/2β-H 2 , 23-H, and 5β-H, respectively, thus suggesting that the carboxyl groups are probably situated at the C-3, C-4, and C-24 positions. The validity of such assignments was further confirmed by measuring the 1 H-1 H COSY spectrum of compound F ( Figure 6). The correlation peaks were only observed between the 2α-/2β-H 2 and 1α-/1β-H 2 . Similar couplings were also observed between the 5β-H and 6α-/6β-H 2 . However, no correlation peak was observed between the 3β-H and 2α-/2β-H 2 or the 5β-H and 4α-/4β-H 2 .

Biological aspects
The common ringtail possum (Pseudocheirus peregrinus) is an Australian marsupial (Figure 1). It lives in a variety of habitats (forests, dense scrub and suburban gardens) and eats a variety of leaves of both native and introduced plants, as well as flowers and fruits. The possum is coprophagic, producing two types of feces, one of which is eaten (see below). This behavioral characteristic is also observed in rabbits [13] and both genera have biliary bile acids that are predominantly secondary.
Our study shows that biliary bile acids in the common ringtail possum differ from those of most mammals in at least two ways (Table  3). First, a seco-bile acid was present. As all primary bile acids have a hydroxyl-or oxo-substituent at C-7, it is likely that the seco-bile acid is a secondary bile acid, formed by bacterial enzymes from Deoxycholic Acid (DCA) in the intestine. Second, the majority of bile acids (80%) appear to be secondary bile acids that have been generated from primary bile acids by bacterial enzymes.
The intermediates in the formation of the seco-bile acid are unknown. The four rings of the bile acid structural platform are generally considered to be stable in vertebrates. However, environmental bacteria have at least two pathways for opening the B ring [14]. We can speculate that the opening of the A ring occurs by an enzymatic pathway that parallels the Baeyer-Villiger oxidation reaction. In this reaction a bile acid with a 3-oxo functional group in the A ring is converted to a pair of regioisomers -3-oxa-4-one-4α-homo-and 3-one-4-oxa-4α-homo. Each of these regioisomers could serve as a precursor for the synthesis of the 3,4-seco bile acid. Other instances where the A ring has been opened are found in 3,4-secoterpenoids [15] and in steroids degraded by the thermophilic fungus Myceliophthora thermophila [16] as well as in steroids mediated by Steroidobacter denitrificans [17].
We propose the following sequence of events to explain the biliary bile acid composition of the possum. The dominant primary acid synthesized is cholic acid which is conjugated with taurine in the liver. In the intestine, cholyltaurine undergoes bacterial deconjugation. The liberated cholic acid is absorbed in part but a fraction in the intestine undergoes oxidation at C-7 by bacterial dehydrogenases to form 7-oxodeoxycholic acid (7-oxo-DCA; 3α,12α-dihydroxy-7-oxo-5β-cholan-24-oic acid), a fraction of which is absorbed. In the hepatocyte, the 7-oxo-DCA undergoes partial reduction to form cholic acid [18]. In addition, in the intestine, some of the 7-oxo compound is reduced by bacterial enzymes to form ursocholic acid (3α,7β,12α-trihydroxy-5βcholan-24-oic acid), which in turn is absorbed. As a hydrophilic bile acid, it may well be incompletely unconjugated during passage through the hepatocyte [19]. We cannot exclude the possibility that 7-oxo-DCA may also be a primary bile acid.
Ursocholic acid has been reported to be a major bile acid (10%) in a patient with cholesterol gallstones [20] as well as in a mouse model of cystic fibrosis where it constituted 25% of biliary bile acids [21]. In both instances, ursocholic acid was considered to be a secondary bile acid. Ursocholic acid has also been reported to be present in the urine [22] and feces [23] of healthy subjects.
The possum is known to engage in coprophagy [24,25]. Feces consist of two types of pellets, the one containing undigested residue, and the other, a "soft" pellet contains cecal content that is likely to include bile acids. The possum ingests the soft pellets, and as a result, colonic content including bile acids is exposed to the vast absorptive surface and the microbiome of the small intestine. Bile acid metabolism in the possum appears to be similar to that of the rabbit whose bile contains predominantly DCA [13].
Our paper confirms previous work attesting the diversity of bile acid structures to be found in Australian marsupials. The 1α-hydroxy derivative (1α-OH-CDCA) of Chenodeoxycholic Acid (CDCA) was shown to be the major bile acid in the Australian opossum Trichosurus vulpecula (Lesson), and dubbed vulpecholic acid [26,27]. This bile acid was also identified in the biliary bile acids of the spotted cuscus (Phalanger maculatus), and 1β-hydroxy-CDCA was identified in the biliary bile acids of the feather-tailed glider (Acrobates pygmaeus) [6].

Phylogenetic aspects
It is generally believed that the marsupials (Metatheria) are an old and relatively less advanced lineage that spilt away from their sister group of placental mammals (Eutheria) at some point deep in geologic time. The structures of bile salts found in the bile of marsupials do not support this idea. Primitive extant mammals still alive today (Paenungulates) utilize a mixture of C 27 bile alcohols. The supposedly older and even more primitive marsupials should also feature a similar suite of bile salts. Instead, what is found is a series of derived C 24 bile acids. It is apparent that the switch from bile alcohols to bile acids, and the utilization of taurine for conjugation had already occurred in marsupials far earlier in time than the last common precursor of marsupials and eutherian mammals (estimated to be more than 100 million years ago). Currently, the marsupial lineage is an active site of bile acid evolution, with different species exhibiting new and structurally unique bile salts as noted above [1,2,5,6].