Analyses of biologically active steroids: antitumor active OSW-1 and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry.

Naturally occurring constituents of biological or pharmaceutical interest often exist in the form of glycosides or conjugates. Mass spectral investigations of these compounds require soft ionization techniques if information on molecular mass, sugar sequence, or conjugate content is desired. In this study, matrix-assisted laser desorption/ionization (MALDI) quadrupole ion trap (QIT) time-of-flight tandem mass spectrometry (TOF-MS(n)) was used to identify both OSW-1, an acetylated cholestane diglycoside showing antitumor activity, and the cardiotonic steroid, bufotoxin. Each molecular-related ion was identified, and subsequent collision-induced dissociation experiments in which a molecular-related ion was selected as a precursor ion produced the characteristic product ions that are essential for structural elucidation. OSW-1 and its analogue with a modified side chain, thienyl OSW-1, were synthesized, and bufotoxins, i.e., marinobufotoxin and its homologue, marinobufagin 3-pimeloylarginine ester, were isolated from toad venom. On MALDI-TOF-MS, sodium-adduct [M+Na](+) ions were observed in the steroid glycosides, although protonated [M+H](+) ions were relatively more abundant than sodium-adduct [M+Na](+) ions in the bufotoxins. On the basis of tandem MS results, we propose key fragmentation pathways. The sugar moiety or side chain from the precursor ion was eliminated in OSW-1. However, characteristic product ions originating from the cleavage of the side chain with an ester formation were observed in the bufotoxins. Post-source decay (PSD) on MALDI-TOF-MS is also described when evaluating alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as a matrix to obtain useful ions required for the identification of compound.

A direct method for the separation and quantification of bile acid acyl glycosides by high-performance liquid chromatography with an evaporative light scattering detector.

A direct method for the separation and quantification of a series of bile acid acyl glycosides using high-performance liquid chromatography coupled to an evaporative light scattering detector (HPLC-ELSD) is described. Complete separation of each of 15 bile acid acyl 24-alpha-glucosides and their 24-beta-anomers and 24-beta-galactosides was achieved by the stepwise gradient elution mode on a C18 column using a mixture of acetonitrile-methanol (8:2, v/v) and 1% aqueous acetic acid as the mobile phase. 24-beta-Galactosides were always eluted faster than the corresponding 24-beta-glucosides, which eluted after the corresponding 24-alpha-anomers. Calibration curves of different 24-beta-galactosides were linear over a range of 0.2-40 nmol of injected amount and the detection limits (S/N > 3) were from 0.08 to 0.1 nmol. The present HPLC-ELSD method may provide an insight into the separation and quantification of the biologically interesting neutral bile acids.

Identification of a novel bile acid in swans, tree ducks, and geese: 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid.

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by (1)H and (13)C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3alpha,7alpha,15alpha-Trihydroxy-5beta-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15alpha-hydroxy-chenodeoxycholic acid.

Nuclear magnetic resonance spectroscopy of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid multi-conjugates: unusual bile acid metabolites in human urine.

Complete 1H and 13C resonance assignments were made for a new type of 3 beta,7 beta-dihydroxy-5-cholen-24-oic acid doubly conjugated with sulfuric acid at C-3 and N-acetylglucosamine at C-7 and its glycine- and taurine-amidated triple-conjugates by the combined use of several homonuclear and heteronuclear shift-correlated 2D NMR techniques. The effects of sulfation at C-3, N-acetylglucosaminidation at C-7, and aminoacyl amidation at C-24 on the 1H and 13C chemical shifts and signal multiplicity were clarified. The shielding data serving to characterize each of the bile acid multi-conjugates are also discussed.

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1.

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.

Chemical synthesis of 24-beta-D-galactopyranosides of bile acids: a new type of bile acid conjugates in human urine.

A method is reported for the preparation of the C-24 carboxyl-linked beta-D-galactopyranosides of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids, two of which were recently identified as a novel type of the metabolites of bile acids excreted in human urine. Direct esterification (galactosidation) of the unprotected bile acids with 2,3,4,6-tetra-O-benzyl-D-galactopyranose in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent and subsequent hydrogenolysis of the resulting benzyloxy-protected bile acid 24-beta-D-galactopyranosides over 10% palladium on charcoal under atmospheric pressure afforded the title compounds. The structures of the bile acid acyl galactosides were confirmed by measuring several (1)H-(1)H and (1)H-(13)C shift correlated 2D NMR.

Capillary gas chromatographic separation of bile acid acyl glycosides without thermal decomposition and isomerization.

A direct method for the capillary gas chromatographic (cGC) separation of the acyl glycosides of bile acids was successfully attained. The free acyl glycosides were derivatized to their complete trifluoroacetyl (TFA) derivatives with N-methyl-bis(trifluoroacetamide). The highly volatile TFA derivatives were chromatographed on a short-length (10 m), narrow-bore (0.1 mm) capillary column coated with a thin film (0.1 microm) of 5% phenyl polysilphenylene-siloxane at a column temperature below 280 degrees C. Each exhibited a single, well-separated peak of the theoretical shape without any accompanying peaks due to the thermal decomposition and isomerization. The bile acid 24alpha-glucosides were always eluted faster than the corresponding 24beta-glucosides, which eluted before the corresponding 24beta-galactosides. The method could be usefully applied to biosynthetic and metabolic studies of bile acid acyl glycosides in biological materials.

The remote-oxyfunctionalization of unactivated carbons in (5 beta)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by ruthenium-porphyrin and HBr: a direct lactonization at C-20.

Remote-oxyfunctionalization induced by 2,6-dichloropyridine N-oxide (DCP N-oxide) as an oxygen donor and a (5,10,15,20-tetramesitylporphyrinate) ruthenium(II) carbonyl complex (Ru-porphyrin) and HBr as catalysts was examined for a series of methyl ester-peracetylated derivatives of (5 beta)-3-oxobile acids. Using the DCP-N-oxide/Ru-porphyrin/HBr system, 5 beta-hydroxylation predominated for the substrates having a 12-acetoxyl substituent due to steric hindrance, but the presence of a 7-acetoxyl substituent decreased the reactivity of the 5 beta-position allowing for the competitive (20S)-20-oxyfunctionalization, subject to electronic constraints. A variety of novel 5 beta-hydroxylation and (20S)-24,20-gamma-lactonization products, as well as their double-oxyfunctionalization and dehydration products, were obtained in one-step. The alkaline hydrolysis of the gamma-lactones gave the corresponding stereoselective (20S)-20-hydroxy-carboxylic acids.

Synthesis of 3alpha,7alpha,14alpha-trihydroxy-5beta-cholan-24-oic acid: a potential primary bile acid in vertebrates.

A method for the synthesis of 3alpha,7alpha,14alpha-trihydroxy-5beta-cholan-24-oic acid which is a possible candidate of bile acid metabolite in vertebrates was developed. The principal reactions involved were 1). stereoselective remote-hydroxylation of methyl ursodeoxycholate diacetate with dimethyldioxirane, 2). site-selective protection at C-3 by tert-butyldimethylsilylation of the resulting 3alpha,7alpha,14alpha-trihydroxy ester, 3). oxidation of the diol with pyridinium dichromate adsorbed on activated alumina, 4). stereoselective reduction of the 7-ketone with zinc borohydride, and 5). cleavage of the protecting group at C-3 with p-toluenesulfonic acid. A facile elimination of the 14alpha-hydroxy group under an acidic or neutral condition is also described. The synthetic reference compound is now available for comparison with unidentified biliary bile acids detected in vertebrate bile.

Chemical synthesis of (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid and its taurine and glycine conjugates: a major bile acid in the rat.

A method for the synthesis of Delta(22)-beta-muricholic acid (Delta(22)-beta-MCA), (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Delta(22)-beta-muricholyltaurine and Delta(22)-beta-muricholylglycine) is described. The key intermediate, 3 alpha,6 beta,7 beta-triformyloxy-23,24-dinor-5 beta-cholan-22-al, was prepared from beta-muricholic acid (beta-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)-beta-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)-beta-MCA. These synthetic reference compounds are now available for investigation of the metabolism of beta-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-(2)H or (3)H-beta-MCA.

Biomimetic oxidation of unactivated carbons in steroids by a model of cytochrome P-450, oxorutheniumporphyrinate complex.

Biomimetic oxidation of unactivated carbons for structurally different steroids was studied with a model of cytochrome P-450, oxorutheniumporphyrinate complex, which is generated in situ by 2,6-dichloropyridine N-oxide as an oxygen donor and (5,10,15,20-tetramesitylporphyrinate) ruthenium(II) carbonyl complex and HBr as catalysts. The O-insertion positions depended significantly on specific structural features of the substrates to give novel and remote-oxygenated steroids in one step. The electrophilic oxorutheniumporphyrinate attacked predominantly allylic and benzylic beta-carbons adjacent to a pi-bond and/or less hindered, electron-rich tert-methine carbons in the substrates to give regio- and stereoselectively the corresponding oxo and/or hydroxy derivatives.

An improved method for the capillary gas chromatographic derivatization of polyhydroxylated steroids having tert-hydroxyl groups.

An improved method for a suitable derivatization of polyhydroxylated steroids having one or two tert-hydroxyl groups at the 5beta-, 14alpha-, 17alpha-, 24-, and/or 25-positions by capillary gas chromatography (CGC) is described. By using trimethylsilyl triflate as a silylating reagent and 2,6-lutidine as a catalyst, each of 5beta-cholane and 5alpha-cholestane series of steroids was successfully transformed into trimethylsilyl (TMS) ether derivatives to give a single CGC peak under mild conditions. More bulky triethylsilyl (TES) etherification of 14alpha- and 17alpha-hydroxy compounds provided multiple CGC peaks arising from completely- and/or incompletely-derivatized TES ethers accompanied by their thermal elimination products.

Functionalization of unactivated carbons in 3alpha,6- and 3alpha,24-dihydroxy-5beta-cholane derivatives by dimethyldioxirane.

Direct remote functionalization of unactivated carbons by dimethyldioxirane (DMDO) was examined for 3alpha,6- and 3alpha,24-dihydroxy-5beta-cholane derivatives. DMDO oxidation of stereoisomeric methyl 3alpha,6-diacetoxy-5beta-cholanoates caused the direct, unexpected 14alpha- and 17alpha-hydroxylations, in analogy with that of the 5alpha-H analogs, regardless of the differences in stereochemical configuration of the A/B-ring junction and of the acetoxyl groups at C-3 and C-6. On the other hand, the ester derivatives of 3alpha,24-dihydroxy-5beta-cholane with DMDO were transformed into the corresponding 5beta-, 14alpha-, and 17alpha-hydroxy compounds, whereas the ether derivatives yielded the 5beta-hydroxy, 3-oxo, and C-24 oxidized products, accompanied by their dehydrated ones.

Potential bile acid metabolites. 25. Synthesis and chemical properties of stereoisomeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids.

Epimeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids and some related compounds were synthesized from chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), respectively. The key reaction involved one-step remote oxyfunctionalization of unactivated methine carbons at C-17 of CDCA and at C-14 of UDCA as their methyl ester-peracetate derivatives with dimethyldioxirane (DMDO). After dehydration of the resulting 17alpha- and 14alpha-hydroxy derivatives with POCl(3) or conc. H(2)SO(4), the respective Delta(16)- and Delta(14)-unsaturated products were subjected to hydration via hydroboration followed by oxidation to yield the 3,7,16- and 3,7,15-triketones, respectively. Stereoselective reduction of the respective triketones with tert-butylamine-borane complex afforded the epimeric 3alpha,7alpha,16- or 3alpha,7alpha,15-trihydroxy derivatives exclusively. A facile formation of the corresponding epsilon-lactones between the side chain carboxyl group at C-24 and the 16alpha- (or 16beta-) hydroxyl group in bile acids is also clarified.

Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides and their chemical properties.

A facile and efficient synthesis of the carboxyl-linked glucosides of bile acids is described. Direct esterification of unprotected bile acids with 2,3,4,6-tetra-O-benzyl-D-glucopyranose in pyridine in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent afforded a mixture of the alpha- and beta-anomers (ca. 1:3) of the 1-O-acyl-D-glucoside benzyl ethers of bile acids, which was separated effectively on a C18 reversed-phase chromatography column (isolated yields of alpha- and beta-anomers are 4-9% and 12-19%, respectively). Subsequent hydrogenolysis of the alpha- and beta-acyl glucoside benzyl ethers on a 10% Pd-C catalyst in acetic acid/methanol/EtOAc (1:2:2, by vol) at 35 degrees C under atmospheric pressure gave the corresponding free esters in good yields (79-89%). Chemical specificities such as facile hydrolysis and transesterification of the acyl glucosides in various solvents were also discussed.