Microwave assisted synthesis and solid-state characterization of lithocholyl amides of isomeric aminopyridines

Microwave (MW) assisted synthesis and solid state structural characterizations of novel lithocholyl amides of 2-, 3-, and 4-aminopyridine are reported. It is shown that the MW technique is a proper method in the preparation of N-lithocholyl amides of isomeric aminopyridines. It offers many advantages compared to conventional heating. The molecular and crystal structures as well as the polymorphic and hydrated forms of prepared conjugates with their thermodynamic stabilities have been characterized by means of high resolution liquid- and solid-state NMR spectroscopy, single crystal and powder X-ray diffraction, and thermogravimetric analysis. Owing to the many biological functions of bile acids and amino substituted nitrogen heterocycles, knowledge of the crystal packing of these novel conjugates may have relevance for potential pharmaceutical applications.     

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled His4CYP98A3 is expressed at yields of 2-4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His4CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.     

Studies on flavonoid metabolism. Biliary and urinary excretion of metabolites of (+)-[U-14C]catechin

1. The biliary and urinary excretion of (+)-[U-(14)C]catechin was studied in normal male rats after a single injection of the flavonoid. 2. In rats large amounts of radioactivity (33.6-44.3% of the dose in 24h) were excreted in the bile as two glucuronide conjugates [one of which was a (+)-catechin conjugate] and three other unconjugated metabolites. 3. Excretion of radioactivity in the urine when the bile duct was not cannulated amounted to 44.5% of the dose. 4. In both the urine and bile the new metabolites showed maximum excretion in the (1/2)-1(1/2)h after intravenous injection of [(14)C]catechin. 5. The metabolites m-hydroxyphenylpropionic acid, p-hydroxyphenylpropionic acid, delta-(3-hydroxyphenyl)-gamma-valerolactone and delta-(3,4-dihydroxyphenyl)-gamma-valerolactione originate from the action of the intestinal micro-organisms on the biliary-excreted metabolites of (+)-catechin. These phenolic acid and lactone metabolites are then reabsorped and excreted in the urine. 6. It is proposed that, depending on the route of administration of (+)-catechin, there exists an alternative pathway, involving biliary excretion, for the metabolism of (+)-catechin.     

Syntheses and structural study of bile acid amidoalcohols

Preparation, structural and thermoanalytical characterization of fourteen N-hydroxyalkyl 5beta-cholan-24-amides have been performed in this study. The utilized techniques include liquid state and CP-MAS (13)C NMR spectroscopy, thermogravimetry, differential scanning calorimetry, and also powder and single crystal X-ray crystallography. The results were discussed and compared to each other and also to previous findings on similar compounds. One pure hydrate form was obtained. Six new single crystal structures were determined, including one hydrated chloroform solvate. Decomposition temperatures were found to correlate with the side chain length, and the number of the hydroxyl groups. The spatial direction of the groups in the steroid skeleton was also found to be relevant in predicting the thermal properties of bile acid amidoalcohols studied.     

Biliary excretion of foreign compounds. Benzene and its derivatives in the rat

1. The extent of the excretion in the bile of the rat of benzene and 21 of its simple derivatives was studied. 2. Some 16 compounds of molecular weight less than 200, and including neutral molecules (benzene and toluene), aromatic acids, aromatic amines and phenols, were injected in solution intraperitoneally into biliary-cannulated rats. Metabolites in the bile were identified and estimated. The extent of biliary excretion of these compounds was low, i.e. 0–10% of the dose in 24hr., and most appeared in the bile mainly as conjugates. 3. The biliary excretion of six conjugates of molecular weight less than 300, including three glycine conjugates, one sulphate conjugate, one glucuronic acid conjugate and two acetyl derivatives, was low (less than 3% of the dose). 4. It is concluded that simple benzene derivatives of molecular weight less than about 300 are poorly excreted in rat bile.     

The enzymatic formation of novel bile acid primary amides

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.     

Synthesis and characterization of pH-sensitive conjugate of isoniazid - methoxypoly(ethylene glycol)-b-poly(l-lysine)

The present paper deals with the preparation and characterization of a conjugate of isoniazid (INH) with the block copolymer methoxypoly(ethylene glycol)-b-poly(l-lysine) (mPEG-b-PLL). The structure of the conjugate (mPEG-b-PLL-INH) was verified by means of (1)H NMR, GPC, infrared spectroscopy, elemental analysis and powder X-ray diffraction. The conjugate contains six l-lysine units with five INH molecules, which are attached by means of pH-sensitive amidine bond. Under in vitro conditions, the conjugate is hydrolyzed and isoniazid is released (pH 4; 37°C; t(1/2) ≈10h).     

Determination of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate in human serum by gas chromatography-mass spectrometry

The glycine conjugate of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate labeled with deuterium at the C-2, -4, and -23 positions were synthesized. A highly sensitive and specific quantitative assay of the bile acid has been developed by selected ion monitoring in gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives using the deuterium labeled conjugates as internal standards. Calibration curves for the bile acid and its sulfate exhibited a linear relationship over the range of 0.01-100 micrograms/ml in human serum.     

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of ¹³C- and ¹⁵N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis¹³C,¹⁵N-labeled His₄CYP98A3 is expressed at yields of 2-4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His₄CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.     

Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy

The atomic resolution structure of Pf1 coat protein determined by solid-state NMR spectroscopy of magnetically aligned filamentous bacteriophage particles in solution is compared to the structures previously determined by X-ray fiber and neutron diffraction, the structure of its membrane-bound form, and the structure of fd coat protein. These structural comparisons provide insights into several biological properties, differences between class I and class II filamentous bacteriophages, and the assembly process. The six N-terminal amino acid residues adopt an unusual "double hook" conformation on the outside of the bacteriophage particle. The solid-state NMR results indicate that at 30 degrees C, some of the coat protein subunits assume a single, fully structured conformation, and some have a few mobile residues that provide a break between two helical segments, in agreement with structural models from X-ray fiber and neutron diffraction, respectively. The atomic resolution structure determined by solid-state NMR for residues 7-14 and 18-46, which excludes the N-terminal double hook and the break between the helical segments, but encompasses more than 80% of the backbone including the distinct kink at residue 29, agrees with that determined by X-ray fiber diffraction with an RMSD value of 2.0 A. The symmetry and distance constraints determined by X-ray fiber and neutron diffraction enable the construction of an accurate model of the bacteriophage particle from the coordinates of the coat protein monomers.     

Liver-specific drug targeting by coupling to bile acids.

Bile acids are selectively taken up from portal blood into the liver by specific transport systems in the hepatocyte plasma membrane. Therefore, studies were performed to evaluate the potential of bile acids as shuttles to deliver drugs specifically to the liver. The alkylating cytostatic drug chlorambucil and the fluorescent prolyl-4-hydroxylase inhibitor 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-oxaproline-Gly were covalently linked via an amide bond to 7 alpha, 12 alpha,-dihydroxy-3 beta- (omega-aminoalkoxy)-5-beta-cholan-24-oic acid. The chlorambucil-bile acid conjugates S 2521, S 2539, S 2567, and S 2576 inhibited Na(+)-dependent [3H]taurocholate uptake in a concentration-dependent manner both into isolated rat hepatocytes and rabbit ileal brush border membrane vesicles, whereas the parent drug chlorambucil showed no significant inhibitory effect. The chlorambucil-bile acid conjugates were able to prevent photoaffinity labeling of bile acid binding proteins in rat hepatocytes by the photolabile [3H]7,7-azo derivative of taurocholic acid indicating their bile acid character. The chlorambucil-bile acid conjugate S 2577 was able to alkylate proteins demonstrating the drug character conserved in the hybrid-molecules. Liver perfusion experiments revealed a secretion profile of the chlorambucil-bile acid conjugate S 2576 into bile very similar to taurocholate compared to chlorambucil which is predominantly excreted by the kidney. 4-Nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-oxaproline-Gly- t-butylester (S 4404), a fluorescent peptide inhibitor of prolyl-4-hydroxylase, was not transported in intact form from portal blood into bile in contrast to its bile acid conjugate S 3744; about 25% of the peptide-bile acid conjugate S 3744 was secreted in intact form into bile within 40 min compared with less than 4% of the parent oxaprolylpeptide S 4404. In conclusion, these studies reveal that modified bile acid molecules can be used as "Trojan horses" to deliver a drug molecule specifically into the liver and the biliary system. This offers important pharmacological options for the development of liver-specific drugs.     

Bile acid-cysteamine conjugates: structural properties, gelation, and toxicity evaluation

Design, synthesis, and characterization of six novel bile acid-cysteamine conjugates together with investigation of their structural studies, gelation properties, and preliminary toxicity evaluation, are reported. Solid state properties of selected compounds were studied by means of X-ray diffraction and (13)C CPMAS NMR spectroscopy. N-(2-thioethyl)-3α,7α,12α-trihydroxy-5β-cholan-24-amide was shown to exhibit (pseudo)polymorphism, and a single crystal structure of its non-stoichiometric hydrate is reported herein. Cholyl and dehydrocholyl derivatives bearing three functionalities in their steroidal backbone were shown to undergo self-assembly leading to gelation in certain organic solvents. Preliminary morphology studies of the formed gels by scanning electron microscopy (SEM) were performed. The standard model mouse fibroblast cell line together with the MTT and NR tests were utilized for evaluating the toxicity of the prepared compounds. Lithocholyl, ursodeoxycholyl, and dehydrocholyl derivatives turned out to be relatively non-toxic in the conditions studied.     

Structural requirements of the ASBT by 3D-QSAR analysis using aminopyridine conjugates of chenodeoxycholic acid

The human apical sodium-dependent bile acid transporter (ASBT) is a validated drug target and can be employed to increase oral bioavailability of various drug conjugates. The aim of the present study was to investigate the chemical space around the 24-position of bile acids that influences both inhibition and uptake by the transporter. A series of 27 aminopyridine and aminophenol conjugates of glutamyl-chenodeoxycholate were synthesized and their ASBT inhibition and transport kinetics (parametrized as K(i), K(t), and J(max)) measured using stably transfected ASBT-MDCK cells. All conjugates were potent ASBT inhibitors. Monoanionic conjugates exhibited higher inhibition potency than neutral conjugates. However, neutral conjugates and chloro-substituted monoanionic conjugates were not substrates, or at least not apparent substrates. Kinetic analysis of substrates indicated that similar values for K(i) and K(t) implicate substrate binding to ASBT as the rate-limiting step. Using 3D-QSAR, four inhibition models and one transport efficiency model were developed. Steric fields dominated in CoMFA models, whereas hydrophobic fields dominated CoMSIA models. The inhibition models showed that a hydrophobic or bulky substitute on the 2 or 6 position of a 3-aminopyridine ring enhanced activity, while a hydrophobic group on the 5 position was detrimental. Overall, steric and hydrophobic features around the 24 position of the sterol nucleus strongly influenced bile acid conjugate interaction with ASBT. The relative location of the pyridine nitrogen and substituent groups also modulated binding.     

Solid-state structure of N-o-, N-m-, and N-p-nitrophenyl-2,3,4-tri-O-acetyl-β-D-xylopyranosylamines

Comprehensive structural analyses were performed for N-o-, N-m-, and N-p-nitrophenyl-2,3,4-tri-O-acetyl-β-D-xylopyranosylamines. Single-crystal X-ray diffraction data were collected and revealed that one compound under investigation undergoes temperature-dependent polymorph transitions (crystal structures of three polymorphs were obtained). The number of molecules in the independent part of the crystal unit cells was in agreement with the number of resonances in solid-state (13)C NMR spectra. Therefore, the compounds exist as single polymorphs at room temperature, as confirmed by powder X-ray diffraction measurements. Significant differences in (13)C chemical shifts between solution and solid-state NMR for selected carbon atoms confirmed the existence of intra- and/or intermolecular interactions.     

Calcium binding by bile acids: in vitro studies using a calcium ion electrode

In this study, we compared in vitro calcium binding by the taurine and glycine conjugates of the major bile acids in human bile: cholic (CA), chenodeoxycholic (CDCA) and deoxycholic (DCA) acids, together with the cholelitholytic bile acids ursodeoxycholic (UDCA) and ursocholic (UCA) acids. At physiological total calcium (CaTOT) (1-15 mM) and bile acid (BA) (10-50 mM) concentrations, all the bile acids caused concentration-dependent falls in [Ca2+], suggesting calcium binding. Except for glycine-conjugated CDCA, all the other calcium-bile acid complexes were soluble in 150 mM NaCl. The calcium binding affinities followed the pattern: dihydroxy (CDCA, UDCA and DCA) greater than trihydroxy (CA and UCA) bile acids, and glycine conjugates greater than taurine conjugates. The glycine conjugate of UDCA, which increases during UDCA treatment, had the highest calcium binding affinity. Ten-20 mM phospholipid modestly increased calcium binding by CA conjugates, but not by CDCA, UDCA, and DCA conjugates. Phospholipid also prevented the precipitation of glyco-CDCA in the presence of calcium. Bile acid-calcium biding was pH-independent over the range 6.5-8.5. The different calcium binding affinities of the major biliary bile acids may partly explain their varying effects on biliary calcium secretion. The results also suggest that neither precipitation of calcium-bile acid complexes nor impaired calcium binding by bile acids is important in the pathogenesis of human calcium gallstone formation.     

The hydrolysis of bile acid conjugates

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroid oxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 12- oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing oxo groups was slow. Borohydride reduction of oxoacids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinations at this time.     

Solution and solid-state structure of N-acetamido-3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosylamine

High resolution 1H NMR and 13C NMR spectroscopic and single crystal X-ray structural analyses of N-acetamido-3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosylamine (1), a minor product of azidonitration reaction of 3,4,6-tri-O-acetyl galactal, are reported. The solution phase studies of 1 reflect that the compound exists in 4C1 conformation with cis-orientation of the substituents at C-1 and C-2. The solid-state structure of 1 reveals that a molecule of water is entrapped in the solid state of 1 and this water molecule serves to mediate N-H...O and C-H...O interactions.     

Bile salts of the green turtle Chelonia mydas (L.)

1. Bile salts of the green turtle Chelonia mydas (L.) were analysed as completely as possible. 2. They consist of taurine conjugates of 3 alpha, 7 alpha, 12 alpha, 22 xi-tetrahydroxy-5 beta-cholestan-26-oic acid (tetrahydroxysterocholanic acid) and 3 alpha 12 alpha, 22 xi-trihydroxy-5 beta-cholestan-26-oic acid, with minor amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5beta-cholan-24-oic acid (cholic acid), 3alpha, 12 alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid) and possibly other bile acids. 3. Cholic acid and deoxycholic acid represent the first known examples of bile acids common to chelonians and other animal forms: they may indicate independent evolution in chelonians to C24 bile acids. 4. The discovery of a 7-deoxy C27 bile acid is the first evidence that C27 bile acids or their conjugates have an enterohepatic circulation.     

Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion

We present the de novo resonance assignments for the crystalline 33 kDa C-terminal domain of the Ure2 prion using an optimized set of five 3D solid-state NMR spectra. We obtained, using a single uniformly ¹³C, ¹⁵N labeled protein sample, sequential chemical-shift information for 74% of the N, Cα, Cβ triples, and for 80% of further side-chain resonances for these spin systems. We describe the procedures and protocols devised, and discuss possibilities and limitations of the assignment of this largest protein assigned today by solid-state NMR, and for which no solution-state NMR shifts were available. A comparison of the NMR chemical shifts with crystallographic data reveals that regions with high crystallographic B-factors are particularly difficult to assign. While the secondary structure elements derived from the chemical shift data correspond mainly to those present in the X-ray crystal structure, we detect an additional helical element and structural variability in the protein crystal, most probably originating from the different molecules in the asymmetric unit, with the observation of doubled resonances in several parts, including entire stretches, of the protein. Our results provide the point of departure towards an atomic-resolution structural analysis of the C-terminal Ure2p domain in the context of the full-length prion fibrils.