2-(7,13-Dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-3H]taurine and [1-14C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of 3H and 14C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

Tetrahydroxystearic acid-containing taurolipid isolated from Tetrahymena thermophila

The second taurine-containing lipid (taurolipid B) was found in cells of Tetrahymena thermophila. The lipid accounted for about 1.4% of the total lipids of the cells. The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolysis products were identified by mass and nuclear magnetic resonance spectrometry, as taurine, 2,3,7,13-tetrahydroxystearicacid and non-hydroxy fatty acids. By spin-decoupling analysis in nuclear magnetic resonance spectrometry, the structure of the taurolipid B was identified as 2-(3-acyloxy-2,7,13-trihydroxyoctadecanoyl)aminoethane-sulfonic acid. This structure shows that taurolipid B is a homologue of the first taurine-containing lipid (taurolipid A).     

2-(7,13-Dihydroxy-2-trans-octadecenoylamino) ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-³H]taurine and [1-¹⁴C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of ³H and ¹⁴C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

Identification of pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from Tetrahymena thermophila

A pentahydroxystearic acid-containing taurolipid (taurolipid C) was found in cells of Tetrahymena thermophila. The lipid accounted for about 1.2% of the total taurolipids of the cells. The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolyses products were identified by mass and nuclear magnetic resonance spectrometries, as taurine, non-hydroxy fatty acid and 2,3,7,12,13-pentahydroxystearic acd, a novel fatty acid. The NMR spectra of the intact and acetylated lipid showed that the carboxyl group of the pentahydrxyostearic acid was combined with the amino group of taurine, and the hydroxy group at C-3 was esterified with non-hydroxy fatty acids. From these results, the pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from T. thermophila was identified as 2-(3-acyloxy-2,7,12,13-tetrahydroxyoctadecanoylamino)ethanesulf oni c acid.     

Metabolism of (24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids and 27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid in guinea pigs

[7β-³H]-(24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids and [7β-³H]-27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid (C₂₇ and C₂₆ bile acids having the same nuclear configuration as cheno-deoxycholic acid and its precursor, 3α,7α-dihydroxy-5β-cholestan-26-oic-acid) were synthesized and administered intraperitoneally to bile fistula guinea pigs. The biliary bile acids formed were hydrolyzed and analyzed by thin layer chromatography, and the metabolites were identified by the inverse isotope dilution method. The results showed that both (24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids were not metabolized by the liver and were excreted unchanged as their taurine and glycine conjugates whereas 27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid was converted to chenodeoxycholic acid.     

The Constituents of Petasites japonicus: Structures of Fukiic Acid and Fukinolic Acid

A major polyphenol from P. japonicus, named fukinolic acid (XII), was isolated and its structure was investigated. Fukinolic acid afforded a new polyphenol named fukiic acid (I) and caffeic acid on hydrolysis in molar ratio 1:1. The structure of fukiic acid was established as 2,3-dihydroxy-4-(3′,4′-dihydroxyphenyl)-3-carboxybutyric acid (I), and its methyl derivatives were described.

Based on evidences presented, the structure for fukinolic acid was proposed as XII. It was further demonstrated that fukinolic acid caused the brown discoloration on action of a polyphenoloxidase from the plant tissues.     

Metabolism of 3α, 7α-dihydrexy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyi-5β-cholanoic acid in hamsters

The metabolic fate of the bile add analogs, 3α,7α-dihydroxy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, was investigated and compared with that of chenodeoxycholic acid in hamsters. Both bile acid analogs were absorbed rapidly from the intestine and excreted into bile at similar to that of chenodeoxycholic acid. In the strain of hamster studied, the biliary bile were conjugated with both glycine and taurine. After continuous intravenous infusion, chenodeoxycholic acid the analogs became the major bile acid constituents in bile. After oral administration of a single dose of these compounds, fecal analysis revealed the existence of unchanged material (25–35%) as well as considerable amounts of metabolites (65–75%). The major metabolites excreted into feces were more polar than the starting material and were tentatively identified as trifaydroxy-7-methyl compounds by radioactive thin-layer chromatography. However, monohydroxy compounds were also found in the fecal extracts. These results show that chenodeoxycholic acid and ursodeoxycholic acid with a methyl group at the 7-position are resistant to bacterial 7-dehydroxylation than the normally occurring bile acids and that a certain proportion of these analogs is hydroxylated to give the corespondiag trihydroxy compound(s), In a control experiment, about 5% of administered chenodeoxychoulic acid was metabolized to a trihydroxy feile acid, but most of the compound (95%) was transformed into lithocholic acid.     

Biological activities of isomers of 8,15-dihydroxyeicosatetraenoic acid

Chemically synthesized 8(S), 15(S)-dihydroxy-5,11-cis-9, 13-trans-eicosatetraenoic acid and 8(R), 15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid were inactive, in comparison to leukotriene B4, in a human polymorphonuclear leukocyte chemokinetic assay and a rat polymorphonuclear leukocyte aggregation assay.     

Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

Pathway of taurolipid B formation from exogenous taurolipid A by Tetrahymena thermophila

When Tetrahymena thermophila was incubated with taurolipid A isolated from T. pyriformis NT-1, the exogenously added taurolipid A was deacylated rapidly, and taurolipid B content in the cells was increased. The deacylated taurolipid A (lysotaurolipid A) content reached a maximum early on during incubation, and then declined. Taurolipid B and lysotaurolipid B contents in the cells were increased continuously during the incubation. These observations suggest that lysotaurolipid A was an intermediate for taurolipid B formation. When cells were incubated with lysotaurolipid A, newly formed lysotaurolipid B and taurolipid B were observed. Furthermore, when cells were incubated with lysotaurolipid B, only taurolipid B was newly formed. In contrast, newly formed lysotaurolipid B was observed when cells were incubated with exogenous taurolipid B. From the results, we have postulated the biosynthetic pathway of taurolipid B from exogenous taurolipid A in cells of Thermophila.     

Biliary bile acids in birds of the Cotingidae family: taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid

Three C(27) bile acids were found to be major biliary bile acids in the capuchinbird (Perissocephalus tricolor) and bare-throated bellbird (Procnias nudicollis), both members of the Cotingidae family of the order Passeriformes. The individual bile acids were isolated by preparative RP-HPLC, and their structures were established by RP-HPLC, LC/ESI-MS/MS and NMR as well as by a comparison of their chromatographic properties with those of authentic reference standards of their 12α-hydroxy derivatives. The most abundant bile acid present in the capuchinbird bile was the taurine conjugate of C(27) (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid, a diastereomer not previously identified as a natural bile acid. The four diastereomers of taurine-conjugated (24ξ,25ξ)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid could be distinguished by NMR and were resolved by RP-HPLC. The RRT of the diastereomers (with taurocholic acid as 1.0) were found to be increased in the following order: (24R,25R)<(24S,25R)<(24S,25S)<(24R,25S). Two epimers (25R and 25S) of C(27) 3α,7α-dihydroxy-5β-cholestan-27-oic acid were also present (as the taurine conjugates) in both bird species. Epimers of the two compounds could be distinguished by their NMR spectra and resolved by RP-HPLC with the (25S)-epimer eluting before the (25R)-epimer. Characterization of the taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid should facilitate their detection in peroxisomal disease and inborn errors of bile acid biosynthesis.     

Definition of total biosynthesis pathway of taurolipids in Tetrahymena cells

Taurine-combined fatty acids were found in the lipotaurine fraction of cells of Tetrahymena thermophila. Taurine and fatty acid moieties of the compounds were identified by nuclear magnetic resonance and gas chromatography-mass spectrometry, respectively. The molar ratio of fatty acid methyl esters and taurine in the hydrolysate of the lipotaurine fraction by methanolic hydrochloric acid hydrolysis, was 1.06:1.00. From the results, the structures of six taurine-combined fatty acids including lipotaurine in the fraction were identified. These structures suggest that the compounds are precursors of lipotaurine as an intermediate of taurolipids biosynthesis, and lipotaurine is biosynthesized via 2-(octadecanoylamino)ethanesulfonic acid and 2-(7-hydroxy-13-octadecenoylamino)ethanesulfonic acid. From the results of the present study and our previous studies, the total biosynthesis pathway of taurolipids is defined.     

Chemical constituents of Polygala tenuifolia roots and their inhibitory activity on lipopolysaccharide-induced nitric oxide production in BV2 microglia

A methanolic extract of the roots of Polygala tenuifolia (Polygalaceae) significantly attenuated nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. Five xanthones, 1-hydroxy-7-methoxyxanthone (1), 3,6-dihydroxy-1,2,7-trimethoxyxanthone (2), 1,3,6-trihydroxy-2,7-dimethoxyxanthone (3), 1,7-dihydroxy-2,3-dimethoxyxanthone (4) and 1,7-dihydroxy-3-methoxyxanthone (5), and five phenylpropanoids, 4-hydroxy-3-methoxypropiophenone (6), methyl 4-hydroxy-3-methoxycinnamic acid (7), 3,4,5-trimethoxycinnamic acid (8), 4-methoxycinnamic acid (9) and β-d-(3-O-sinapoyl) fructofuranosyl-α-d-(6-O-sinapoyl)glucopyranoside (10), were isolated from CHCl(3) fraction using bioactivity-guided fractionation. Among these compounds, compounds 1, 2, 4, 5 and 7 showed significant inhibitory effects on LPS-induced NO production in BV2 microglia cells at the concentration ranging from 10.0 to 100.0 μM.     

Bacterial and fungal oxidation of dibenzofuran.

Cunninghamella elegans and a mutant strain (B8/36) of Beijerinckia both oxidized dibenzofuran to 2,3-dihydroxy-2,3-dihydrodibenzofuran. The bacterial metabolite was extremely unstable and, in the presence of acid, was rapidly converted into a mixture of 2- and 3-hydroxydibenzofuran. In contrast, the 2,3-dihydroxy-2,3-dihydrodibenzofuran formed by C. elegans was stable and only yielded 2- and 3-hydroxydibenzofuran when heated under acidic conditions. The results suggest that Beijerinckia B8/36 and C. elegans form the respective cis- and trans-isomers of 2,3-dihydroxy-2,3-dihydrodibenzofuran. C. elegans also oxidized dibenzofuran to 2- and 3-hydroxydibenzofuran under conditions that would not lead to the dehydration of the trans-dihydrodiol. These observations implicate the initial formation of dibenzofuran- 2,3-epoxide in the fungal oxidation of dibenzofuran. Beijerinckia B8/36 also produced a second unstable dihydrodiol that was tentatively identified as cis-1,2-dihydroxy-1,2-dihydrodibenzofuran. This compound gave 2-hydroxydibenzofuran as the major dehydration product and the cis relative stereochemistry was suggested by the isolation and characterization of an isopropylidine derivative. A preparation of cis-naphthalene dihydrodiol dehydrogenase and cell extracts of the parent strain of Beijerinckia oxidized both bacterial dihydrodiols to catechols. Cell extracts prepared from C. elegans catalysed an analogous oxidation of trans-2,3-dihydroxy-2,3-dihydrodibenzofuran to 2,3-dihydroxydibenzofuran. The latter product was also isolated and identified from culture filtrates. The results suggest that bacteria and fungi utilize different mechanisms to initiate the oxidation of dibenzofuran.     

Hydroperoxide-dependent oxygenation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene by ram seminal vesicle microsomes. Source of the oxygen

Incubation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with ram seminal vesicle microsomes (RSVM) triggers the oxygenation of $\text{trans}$-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). The principal oxidation products are 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes which are non-enzymatic hydrolysis products of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. At short incubation times, an additional product is isolated which is identified as r-7,t-8,t-9-trihydroxy-c-10-methoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product appears to arise by solvolysis of the extracted diolepoxide during high performance liquid chromatography using methanol-water solvent systems. The incubation of ¹⁸O-labeled 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid with BP-7,8-diol and RSVM leads to very little incorporation of ¹⁸O into the stable solvolysis products (analyzed by gc-ms of their peracetates). Parallel incubations conducted with ¹⁶O-labeled hydroperoxide under an ¹⁸O₂ atmosphere indicate that the principle source of the epoxide oxygen is molecular oxygen.     

Structural studies on the acidic exopolysaccharide from Haloferax denitrificans ATCC 35960.

The structure of a linear, acidic exopolysaccharide isolated from the Archaeon Haloferax denitrificans ATCC 35960 has been determined using NMR spectroscopy. The sugar residues in the repeating unit of the polysaccharide were identified as Gal and GlcA2,3NAc after the assignment of the 1H and 13C resonances using COSY, HOHAHA, HMQC and HMQC-TOCSY experiments. The sequence of the residues in the polysaccharide was established from the inter-residue connectivities observed in the HMQC-NOESY plot. The only sugar released on acid hydrolysis was shown to be D-Gal by GLC analysis, while the absolute configuration of the acidic sugars was shown to be D by comparison of the carbon chemical shifts with those of model compounds. Partial acid hydrolysis yielded a tetrasaccharide, terminated by D-Gal at the reducing end, whose structure confirmed that of the repeating unit of the polysaccharide as-->4)-beta-D-GlcpA2,3NAc-(1-->4)-beta-D-GlcpA2, 3NAc-(1-->4)-alpha-D-GlcpA2,3NAc-(1-->3)-alpha-D-Galp- (1-->, where D-GlcpA2,3NAc is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic acid.     

Metabolism of prostaglandin D2 in the monkey.

[3H7]Prostaglandin D2 was biosynthesized and infused into an unanesthetized monkey. The urinary metabolites were isolated and subsequently identified by gas chromatography-mass spectrometry. Two pathways of prostaglandin D2 metabolism were identified and resulted in metabolites with prostaglandin D (3-hydroxycyclopentanone) and prostaglandin F (cyclopentane-1,3-diol) ring structures. The major prostaglandin D ring metabolite was identified as 9,20-dihydroxy-11,15-dioxo-2,3-dinorprost-5-en-1-oic acid. Nine other prostaglandin D ring metabolites were identified reflecting various combinations of metabolism by beta and omega oxidation, 15 dehydrogenation, and 13-14 reduction. In greater abundance were those prostaglandin D2 metabolites which had the prostaglandin F ring structure. The major prostaglandin D2 metabolite which had the prostaglandin F ring structure was identified as 9,11,15-trihydroxy-2,3-dinorprosta-5,13-dien-1-oic acid (dinor prostaglandin F2 alpha). Nine other metabolites with the prostaglandin F ring structure were identified, including prostaglandin F2 alpha itself. These, for the most part, were the structural counterparts of the metabolites with the prostaglandin D ring. Since many prostaglandin D2 metabolites were found to be identical with the metabolites of prostaglandin F2 alpha, quantitative determinations of prostaglandin F ring metabolites may not be a specific indicator of prostaglandin F2 alpha biosynthesis. Likewise, data involving the measurement of a biological effect of prostaglandin D2 must be re-examined to account for the possible contribution of prostaglandin F2 alpha, a metabolite of prostaglandin D2, to the biological response.     

Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 4-trifluoromethyl(TFM)-benzoate by 4-isopropylbenzoate grown Pseudomonas putida JT strains

Enzymes of the p-cymene pathway in Pseudomonas putida strains cometabolized the intermediate analogue 4-trifluoromethyl(TFM)benzoate. Three products, 4-TFM-2,3-dihydro-2,3-dihydroxybenzoate, 4-TFM-2,3-dihydroxy-benzoate and 2-hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic proterties.Certain TFM-substituted analogue metabolites of the p-cymene pathway were transformed at drastically reduced rates.Hammett type analysis of ring cleavage reactions of 4-substituted 2,3-dihydroxybenzoates revealed the negative inductive and especially mesomeric effect of substituents to be rate determining. Whereas decarboxylation of 3-carboxy-7-TFHOD was not affected by fluorine substitution the subsequent hydrolysis of 7-TFHOD proceeded very slowly. The negative inductive effect of the TFM-group probably inhibited heterolysis of the carbon bond between C₅ and C₆ of 7-TFHOD.Abbreviations DHB 1,2-Dihydroxy-2-hydrobenzoate - DHC 2,3-Dihydro-2,3-dihydroxybenzoate, this compound was termed DHC simply to distinguish it from the similar 1,2-dihydroxy-2-hydrobenzoate (DHB) as described in the preceeding paper (Engesser et al. 1988) - HMS 2-Hydroxymuconic semialdehyde - HOD 2-Hydroxy-6-oxohepta-2,4-dienoate - 7-TFHOD 2-Hydroxy-6-oxo-7,7,7-trifluorohepta-2,4-dienoate - TFM Trifluoromethyl This work was supported, in part, by the Gesellschaft für Strahlen- und Umweltforschung, Neuherberg/München, FRG     

Chemical synthesis of 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid: An internal standard for mass spectrometric analysis of the abnormal Δ-bile acids occurring in Niemann-Pick disease

In Niemann-Pick disease, type C1, increased amounts of 3β,7β-dihydroxy-5-cholenoic acid are reported to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC-MS/MS analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satisfactory internal standard, 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The key reactions involved were (1) the so-called “second order” Beckmann rearrangement (one-carbon degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6 of the ditosylate derivatives of the resulting 3α,6α-dihydroxy-24-nor-5β-cholanoic acid with potassium acetate in aqueous N,N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary 3β,7β-dihydroxy-24-nor-Δ⁵ derivative using sulfur trioxide-trimethylamine complex. Overall yield of the desired compound was 1.8% in 12 steps from HDCA.