Potential bile acid metabolites. XVIII. Synthesis of stereoisomeric 3,6,12 alpha-trihydroxy-5 beta-cholanoic acids.

Two new 6-hydroxylated bile acids, 3 beta, 6 alpha, 12 alpha- and 3 beta, 6 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids, were synthesized from deoxycholic acid. In addition, their C-3 epimers, 3 alpha, 6 alpha, 12 alpha- and 3 alpha, 6 beta, 12 alpha-trihydroxy acids, were prepared by a new route. The principal reactions used were 1) 6 beta-hydroxylation of 3-methoxy-3,5-dienes with m-chloroperbenzoic acid in aqueous dioxane; 2) catalytic hydrogenation of the resulting 6 beta-hydroxy-3-oxo-4-enes to the 6 beta-hydroxy-3-oxo-5 beta compounds with palladium on calcium carbonate catalyst in ethanol; and 3) stereoselective reduction of appropriate 3-oxo derivatives with potassium tri-sec-butylborohydride and tert-butylamine-borane complex. The thin-layer chromatographic, gas-liquid chromatographic, and high performance liquid chromatographic mobilities, and 1H- and 13C-nuclear magnetic resonance spectroscopic data of the four stereoisomers are presented. With this work all the 6-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids in the 5 beta series are now known and have been synthesized.     

Potential bile acid metabolites. 16. Synthesis of stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids

New synthetic routes to the four possible stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids (and their methyl esters), one of which (3 alpha,6 alpha 7 beta,12 alpha) is new, and some related compounds are described. In addition, the 5 alpha-epimer of the new acid was obtained. The final products were obtained in high purity for use as reference compounds in the analysis of bile acids in human biologic samples. The results of analysis of the prepared stereoisomers by proton and carbon 13 nuclear magnetic resonance spectroscopies are briefly discussed along with the thin-layer and gas-liquid chromatographic properties.     

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 3β,7β-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.     

Synthesis of new bile acid analogues and their metabolism in the hamster: 3 alpha, 6 alpha-dihydroxy-6 beta-methyl-5 beta-cholanoic acid and 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid

This paper reports the chemical synthesis of two new bile acid analogues, namely, 3 alpha, 6 beta-dihydroxy-6 alpha-methyl-5 beta-cholanoic acid from 3 alpha-hydroxy-6-oxo-5 beta-cholanoic acid and describes their metabolism in the hamster. A Grignard reaction of the oxo acid with methyl magnesium iodide in tetrahydrofuran gave two epimeric dihydroxy-6-methyl-cholanoic acids which were separated as the methyl esters by silica gel column chromatography. The configuration of the 6-methyl groups was assigned by proton nuclear magnetic resonance spectroscopy and was supported by the chromatographic properties of the new compounds. The metabolism of the two new bile acid analogues was studied in the hamster. After intraduodenal administration of the 14C-labeled analogues into bile fistula hamsters, both compounds were absorbed rapidly from the intestine and secreted into bile. Intravenous infusion studies revealed that these compounds were efficiently extracted by the liver; the administered analogues became major biliary bile acids, present as either the glycine or taurine conjugates. These compounds are useful to study the effect of methyl-substituted bile acids on cholesterol and bile acid metabolism and may possibly possess cholelitholytic properties.     

Potential bile acid metabolites. 13. Improved routes to 3 beta, 6 beta- and 3 beta, 6 alpha-dihydroxy-5 beta-cholanoic acids

New synthetic routes to three possible stereoisomers of hyodeoxycholic (3 alpha, 6 alpha-dihydroxy-5 beta-cholanic) acid are described. The principal reactions involved were inversion at C-3 of 3 alpha-hydroxy-6-oxo derivatives with diethyl azodicarboxylate-triphenylphosphine-formic acid and with N,N-dimethylformamide, without allomerization to the more stable 5 alpha form. On the basis of physical and chromatographic data, previously reported 3 beta, 6 alpha-dihydroxy-5 beta-cholanic acid and its methyl ester are shown to be C-3 epimeric mixtures. The 13C nuclear magnetic resonance spectra were of key importance in characterizing the stereoisomers and estimating their purity.     

Nuclear magnetic resonance studies of amino acids and proteins. Rotational correlation times of proteins by deuterium nuclear magnetic resonance spectroscopy

We show that measurement of the spin-lattice (T1) and spin-spin (T2) relaxation times (or line widths) of irrotationally bound 2H nuclei in macromolecules undergoing isotropic rotational motion outside of the extreme narrowing limit (i.e., for the case omega 02 tau R2 much greater than 1) permits determination of both the rotational correlation time (tau R) of the macromolecule and the electric quadrupole coupling constant (e2qQ/h) of the 2H label. The technique has the advantage over 13C nuclear magnetic resonance (NMR) that no assumptions about bond lengths (which appear to the sixth power in 13C relaxation studies) or relaxation mechanisms need to be made, since relaxation will always be quadrupolar, even for aromatic residues at high field. Asymmetry parameter (eta) uncertainties are shown to cause negligible effects on tau R determinations, and in any case it is shown that both e2qQ/h and eta may readily be determined in separate solid-state experiments. By way of example, we report 2H NMR results on aqueous lysozyme (EC 3.2.1.17) at 5.2 and 8.5 T (corresponding to 2H-resonance frequencies of 34 and 55 MHz). Interpretation of the results in terms of the isotropic rigid-rotor model yields e2qQ/h values of approximately equal to 170 or approximately equal to 190 kHz, respectively, for the imidazolium and free-base forms of [epsilon 1-2H] His-15 lysozyme in solution, in excellent agreement with e2qQ/h values of approximately 167 and approximately 190 kHz obtained for the free amino acids in the solid state. In principle, the method may in suitable cases permit comparison between the dynamic structures of proteins in solution and in the crystalline solid state.     

Evaluation of base-pairing schemes for E. coli 5S RNA by 400 MHz proton nuclear magnetic resonance spectroscopy

The number of base pairs in the denatured “B” form of $\text{E. coli}$ 5S RNA has been determined directly from 400 MHz high resolution proton nuclear magnetic resonance spectroscopy. The experimental NMR spectrum from ?11.6 to ?14.5 ppm from a sodium 2,2-dimethyl-2-silapentane sulfonate reference can be simulated by a theoretical spectrum consisting of 33 Lorentzian lines of equal width (corresponding to 33 base pairs) at 26°C. This result is inconsistent with previously proposed secondary structures of 17 and 23 base pairs, but is readily adapted to the Luoma-Marshall cloverleaf secondary structure.     

Determination of the acid dissociation constants for WR-1065 by proton NMR spectroscopy.

The acid dissociation constants for N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were determined in D2O by 360- and 500-MHz NMR spectroscopy. Results obtained at 0.21 M initial ionic strength and 26 degrees C were corrected to 25 degrees C yielding pKD1 = 8.28 +/- 0.04, pKD2 = 9.88 +/- 0.07, and pKD3 = 11.58 +/- 0.03. Correction of these values for the effect of the deuterium isotope upon the ionization reaction yielded dissociation constants in water of pKH1 = 7.69 +/- 0.09, pKH2 = 9.35 +/- 0.09, and pKH3 = 11.10 +/- 0.08. Analysis of the changes in chemical shift with pD indicated that the first ionization occurs largely through ionization of the thiol group (approximately 67%) and to a lesser extent the secondary ammonium group (approximately 30%), whereas the third ionization involves mainly the secondary ammonium group (approximately 65%) and to a lesser extent the primary ammonium group (approximately 30%). Estimates of the microscopic pK values for WR-1065 were also obtained from the results.     

Nuclear magnetic resonance studies on pyridine dinucleotides. Carbon-13 assignments and structure determination of NADHX and the primary acid product of NADH.

The ¹³C spectra of β-NADH, NADHX, and the primary acid product of NADH were obtained and assigned. The conversion of the NADHX isomers to the two isomers of NADH acid product is demonstrated through the use of ¹³C-enriched compounds. The structure of NADHX is assigned as β-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide and the structures of the primary acid products of NADH are assigned as α-O^2′-6B-cyclotetrahydronicotinamide adenine dinucleotide and α-O^2′-6A-cyclotetrahydronicotinamide adenine dinucleotide.The structures of NADHX and the major isomer of the primary acid product, derived from studies of model compounds, are consistent with those proposed by Oppenheimer and Kaplan [Biochemistry (1974) 13, 4675, 4685]. However, the spectra of ¹³C-enriched primary acid product also demonstrated the existence of the A isomer which was not observed in the latter ¹H study. The A and B isomers were found to exist in the same ratio even when the primary acid product was formed directly from NADHX. This observation is discussed in terms of the previously proposed mechanism for the acid decomposition of NADH.     

Nuclear magnetic resonance determinations of permeation coefficients for maleic acid in phospholipid vesicles.

Lipid bilayer permeation coefficients for the neutral maleic acid molecule and the maleate monoanion have been determined by proton magnetic resonance techniques. Phosphatiydylcholine-cholesterol (2:1) unilamellar vesicles were prepared having an initial maleate anion concentration gradient stabilized by coupling to an impermeant potassium counterion. The coupling was released by addition of valinomycin, and the time evolution of external pH, internal pH, and maleate concentration followed using nuclear magnetic resonance areas and chemical shifts. Transport rate equations were numerically integrated to fit the date, yielding best fit permeation coefficients of 4 X 10(-9) and 4 X 10(-5) cm/s for maleate monoanion and maleic acid, respectively.     

The isolation of tetrahydroxy bile acids as methyl esters from human urine and their characterization by 1H- and 13C-nuclear magnetic resonance spectroscopy

Treatment with phenobarbital causes an increased urinary excretion of tetrahydroxylated bile acids in patients suffering from intrahepatic cholestasis. The main components were isolated from urine by means of column and thin-layer chromatography and were studied as methyl esters by nuclear magnetic resonance spectroscopy. The results obtained strongly support the contention that the main components are 1 beta-, 6 alpha- and 6 beta-hydroxylated derivatives of cholic acid.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Strategies for assignments.

Natural abundance 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes) of aqueous native proteins yield numerous narrow single carbon resonances of nonprotonated aromatic carbons. Techniques for the assignment of these resonances are presented. Each technique is applied to one or more of the following proteins: ferricytochrome c from horse heart and Candida krusei, ferrocytochrome c and cyanoferricytochrome c from horse heart, lysozyme from hen egg white, cyanoferrimyoglobins from horse and sperm whale skeletal muscle, and carbon monoxide myoglobin from horse. In all of the protein spectra we have examined, methine aromatic carbons give rise to broad bands. Studies of the narrow resonances of nonprotonated aromatic carbons of proteins are facilitated by removal of these broad bands by means of the convolution-difference method, preferably from spectra recorded under conditions of noise-modulated off-resonance proton decoupling. We present a summary of the chemical shift ranges for the various types of nonprotonated aromatic carbons of amino acid residues and hemes of diamagnetic proteins, based on our results for hen egg white lysozyme, horse heart ferrocytochrome c, horse carbon monoxide myoglobin, and carbon monoxide hemoglobins from various species...     

Systematic application of two-dimensional 1H nuclear-magnetic-resonance techniques for studies of proteins. 2. Combined use of correlated spectroscopy and nuclear Overhauser spectroscopy for sequential assignments of backbone resonances and elucidation of polypeptide secondary structures

This paper describes a new nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. To illustrate the practical application of the new procedure, two-dimensional correlated spectroscopy and two-dimensional nuclear Overhauser enhancement spectroscopy were used to obtain individual assignments for all the backbone protons of the beta-sheet secondary structures in the basic pancreatic trypsin inhibitor. First, combined connectivity diagrams of these two methods recorded in both 2H2O solution and H2O solution of the inhibitor were employed to obtain sequential, individual resonance assignments for the separate strands in the beta sheet. Second, a 2D nuclear Overhauser enhancement spectrum recorded with a long mixing time was used to determine how the separate, extended polypeptide strands are linked by hydrogen bonds in the sheet structures. By combination of these results with the identifications of the amino acid side-chain resonances described in the preceding paper, the beta-sheet structures can, without reference to data on the spatial structure obtained with other techniques, be localized in the amino acid sequence. This investigation confirms results on limited regions of the beta sheet in the inhibitor obtained previously with one-dimensional nuclear magnetic resonance experiments and demonstrates that the entire beta-sheet structure seen in single crystals of the inhibitor is preserved in aqueous solution.     

Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography.

The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.     

H-1, C-13, and F-19 nuclear magnetic resonance assignments of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate.

The molecular structure of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate was analyzed by 1H, 13C, and 19F nuclear magnetic resonance (NMR) techniques; two-dimensional NMR was used to assigned 1H and 13C resonances. The 1H NMR spectrum in deuterated chloroform shows three sharp singlets (delta = 0.74, 0.79, and 2.00 ppm) integrating for three protons each, an isolated triplet at 4.55 ppm integrating for one proton, and overlapping multiplets between 0.72 and 2.12 ppm integrating for 31 protons. The 13C spectrum shows 18 resonances between 10 and 55 ppm, and three additional resonances at 82.9, 124.0, and 171.5 ppm. The 19F[1H] spectrum shows two sets of doublets (observed 2J = 150 Hz) at 5.00 and -4.80 ppm. Multiplets arising from 19F-13C J-coupling provide the starting assignment for all resonances by means of 1H homonuclear correlation (COSY) and 1H-13C heteronuclear correlation spectroscopy.