Structural and functional implications of the interaction between macrolide antibiotics and bile acids.

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC50 ) values.

Disentangling scalar coupling patterns by real-time SERF NMR.

Scalar coupling constants and signal splitting patterns in NMR spectra contain a wealth of short-range structural information. The extraction of these parameters from (1)H NMR spectra is often prohibited by simultaneous scalar coupling interactions with several other protons. Here we present a high-resolution NMR experiment where scalar coupling to only one selected signal is visible. All other couplings are removed from the spectrum. This real-time selectively refocused NMR experiment is achieved by spatially selective homonuclear broadband decoupling combined with selective refocusing during acquisition. It allows the unperturbed extraction of scalar coupling constants from the highly resolved acquisition dimension of NMR spectra.

Spectroscopic and quantum mechanical investigation of N,N'-bisarylmalonamides: solvent and structural effects.

The UV absorption spectra of ten N,N'-bisarylmalonamides have been recorded in the range 200-400 nm in a set of selected solvents. The solute-solvent interactions have been analyzed on the basis of the linear solvation energy relationship (LSER) concept proposed by Kamlet and Taft. The effects of substituents on the absorption spectra have been interpreted by correlating absorption frequencies with Hammett substituent constants. Furthermore, the experimental findings have been interpreted using the DFT CAM-B3LYP/6-311+G(d,p) method. Electronic energies have been calculated using the same method in combination with the implicit solvation model (conductor-like polarizable continuum model, CPCM) as well as with the explicit addition of two molecules of solvent.

Long wavelength absorbing carbostyrils as test cases for different TDDFT procedures and solvent models.

The vertical excitation energies of 3,4-dicyano-6-methoxy and 3,4-dicyano-6,7-dimethoxy carbostyril have been computed with different approximations for the time-dependent density functional theory (TD-DFT) procedure and with different implementations of the continuum solvation model COSMO. Different DFT functionals were tested in TD-DFT and Tamm-Dancoff approximations (TDA) for the excitation energies in the gas phase. TDA-B3LYP showed the best agreement with the experimental data. Then TDA-B3LYP computations were performed combined with the COSMO model of solvation comparing a linear response (LR) and a post-configuration interaction (CI) implementation of the fast solvent reorganization. The post-CI solvent model overestimates the π→π* transitions and strongly underestimates the n→π* transition. The TDA approximation in combination with the linear response implementation of the COSMO solvation model perfectly computes the experimental results. TDA-LR is the most reliable method for the computation of the vertical excitation energies in a solvent. Comparison with explicit solvent calculations shows there is only a minor effect on the energies of the electronic interaction of the solute with the solvent.

Phenol-quinone tautomerism in (arylazo)naphthols and the analogous Schiff bases: benchmark calculations.

Tautomerization energies of a series of isomeric [(4-R-phenyl)azo]naphthols and the analogous Schiff bases (R = N(CH3)2, OCH3, H, CN, NO2) are calculated by LPNO-CEPA/1-CBS using the def2-TZVPP and def2-QZVPP basis sets for extrapolation. The performance of various density functionals (B3LYP, M06-2X, PW6B95, B2PLYP, mPW2PLYP, PWPB95) as well as MP2 and SCS-MP2 is evaluated against these results. M06-2X and SCS-MP2 yield results close to the LPNO-CEPA/1-CBS values. Solvent effects (CCl4, CHCl3, CH3CN, and CH3OH) are treated by a variety of bulk solvation models (SM8, IEFPCM, COSMO, PBF, and SMD) as well as explicit solvation (Monte Carlo free energy perturbation using the OPLSAA force field).

Spectroscopic behavior of loratadine and desloratadine in different aqueous media conditions studied by means of TD-DFT calculations.

In this study, we explained the influence of the stepwise protonation of two antihistaminic drugs on their experimental absorption spectra. We demonstrated the capability of the TD-CAM-B3LYP method, combined with a polarizable continuum model, to produce good performance for the calculated spectra. The lowest energy transitions and the molecular orbital plots were analyzed in detail. The calculated UV spectra are proposed as potential alternatives to initialize the well-known MCR-ALS algorithm, especially when the spectra of the pure analytes are not available. Moreover, it can be a useful strategy for planning an experimental methodology oriented to multiway analysis when the drug species exhibit acid-base properties.

A computational study of base-catalyzed reactions of cyclic 1,2-diones: cyclobutane-1,2-dione.

The reaction of cyclobutane-1,2-dione with hydroxide was studied by a variety of ab initio (MP2, SCS-MP2, CCSD(T), CEPA/1) and density functional (M06-2X) methods. Three possible reaction paths of the initially formed tetrahedral adduct leading to either 1-hydroxycyclopropane-1-carboxylate (benzilic acid type rearrangement, path A), α-oxobutanoate (path B) or γ-oxobutanoate (path C) were considered. Although the latter two products show similar or even more negative Gibbs free energies of reaction than calculated for the benzilic acid type rearrangement, the Gibbs free energies of activation are substantially higher. According to the calculations, the only feasible reaction appears to be the formation of 1-hydroxycyclopropane-1-carboxylate, which is corroborated by previous experimental observations.

Unusual C=C bond isomerization of an α,ß-unsaturated γ-butyrolactone catalysed by flavoproteins from the old yellow enzyme family.

An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (k(cat) =0.076 s(-1) ) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN-mediated hydride addition onto exo-Cß, followed by hydride abstraction from endo-Cß', which is in line with the well-established C=C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene-reductases.

A substrate-driven approach to determine reactivities of α,ß-unsaturated carboxylic esters towards asymmetric bioreduction.

The degree of C=C bond activation in the asymmetric bioreduction of α,ß-unsaturated carboxylic esters by ene-reductases was studied, and general recommendations to render these "borderline-substrates" more reactive towards enzymatic reduction are proposed. The concept of "supported substrate activation" was developed. In general, an additional α-halogenated substituent proved to be beneficial for enzymatic activity, whereas ß-alkyl or ß-aryl substituents were detrimental for the reactivity of nonhalogenated substrates, and α-cyano groups showed little effect. The alcohol moiety of the ester functionality was found to have a strong influence on the reaction rate. Overall, activities were determined by both steric and electronic effects.

Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

Coordination of methanol clusters to benzene: a computational study.

Benzene-methanol cluster structures were investigated with theoretical chemistry methods to describe the microsolvation of benzene and the benzene-methanol azeotrope. Benzene-methanol (MeOH) clusters containing up to six methanol molecules have been calculated by ab initio [MP2/6-311++G(d,p)//MP2/6-31+G(d,p) + BSSE correction] method. The BSSE was found quite large with this basis set, hence, different extrapolation schemes in combination with the aug-cc-pVxZ basis sets have been used to estimate the complete basis set limit of the MP2 interaction energy [ΔE(MP2/CBS)]. For smaller clusters, n ≤ 3, DFT procedures (DFTB+, MPWB1K, M06-2X) have also been applied. Geometries obtained for these clusters by M06-2X and MP2 calculations are quite similar. Based on the MP2/CBS results, the most stable C(6)H(6)(MeOH)(3) cluster is characterized by a hydrogen bonded MeOH trimer chain interacting with benzene via π···H-O and O···H-C(benzene) hydrogen bonds. Larger benzene-MeOH clusters with n ≥ 4 consist of cyclic (MeOH)(n) subclusters interacting with benzene by dispersive forces, to be denoted by C(6)H(6) + (MeOH)(n). Interaction energies and cooperativity effects are discussed in comparison with methanol clusters. Besides MP2/CBS calculations, for selected larger clusters the M06-2X/6-311++G(d,p)//M06-2X/6-31+G(d,p) procedure including the BSSE correction was also used. Interaction energies obtained thereby are usually close to the MP2/CBS limit. To model the benzene-MeOH azeotrope, several structures for (C(6)H(6))(2)(MeOH)(3) clusters have been calculated. The most stable structures contain a tilted T-shaped benzene dimer interacting by π···H-O and O···H-C (benzene) hydrogen bonds with a (MeOH)(3) chain. A slightly less negative interaction energy results for a parallel displaced benzene sandwich dimer with a (MeOH)(3) chain atop of one of the benzene molecules.

Application of the Quantum Cluster Equilibrium (QCE) model for the liquid phase of primary alcohols using B3LYP and B3LYP-D DFT methods.

The Quantum Cluster Equilibrium (QCE) model was applied to the liquid phase for the first few members of the homologous series of unbranched aliphatic primary alcohols, methanol, ethanol, propan-1-ol, and butan-1-ol. Cluster structures and energies were calculated by density functional theory [B3LYP/6-311++G(2d,2p)]. For butan-1-ol the dispersion interaction was also considered with the B3LYP-D method. In agreement with previous findings, cyclic cluster structures are the most probable ones. In addition, weak C-H...O interactions as well as dispersion interactions between the longer alkyl chains were found to be important in the cluster formation. The reliability of the model was assessed by the calculated constant pressure heat capacity (C(p)) values. Larger deviations between theory and experiment were found for higher homologes (propan-1-ol, butan-1-ol) with the B3LYP method. When the B3LYP-D method was applied for butan-1-ol, adequate agreement was found between experimental and calculated C(p) values.

Reductive biotransformation of nitroalkenes via nitroso-intermediates to oxazetes catalyzed by xenobiotic reductase A (XenA).

A novel reductive biotransformation pathway for ß,ß-disubstituted nitroalkenes catalyzed by flavoproteins from the Old Yellow Enzyme (OYE) family was elucidated. It was shown to proceed via enzymatic reduction of the nitro-moiety to furnish the corresponding nitroso-alkene, which underwent spontaneous (non-enzymatic) electrocyclization to form highly strained 1,2-oxazete derivatives. At elevated temperatures the latter lost HCN via a retro-[2 + 2]-cycloaddition to form the corresponding ketones. This pathway was particularly dominant using xenobiotic reductase A, while pentaerythritol tetranitrate-reductase predominantly catalyzed the biodegradation via the Nef-pathway.

A highly efficient ADH-coupled NADH-recycling system for the asymmetric bioreduction of carbon-carbon double bonds using enoate reductases.

The asymmetric bioreduction of activated alkenes catalyzed by flavin-dependent enoate reductases from the OYE-family represents a powerful method for the production of optically active compounds. For its preparative-scale application, efficient and economic NADH-recycling is crucial. A novel enzyme-coupled NADH-recycling system is proposed based on the concurrent oxidation of a sacrificial sec-alcohol catalyzed by an alcohol dehydrogenase (ADH-A). Due to the highly favorable position of the equilibrium of ene-reduction versus alcohol-oxidation, the cosubstrate is only required in slight excess.

Hybrid QM/MM Calculations on the First Redox Step of the Catalytic Cycle of Bovine Glutathione Peroxidase GPX1.

The first reaction step of the redox cycle of bovine erythrocyte glutathione peroxidase from class 1 (GPX1) was investigated using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations using the ONIOM methodology. The reduction of hydrogen peroxide by the active-site selenocysteine in selenolate form assisted by the Arg177 residue was modeled based on a proposal from previous molecular dynamics simulations and pKa calculations (J. Chem. TheoryComput. 2010, 6, 1670-1681). The redox reaction is predicted as a concerted SN2 nucleophilic substitution with a concomitant proton transfer from Arg177 onto leaving hydroxide ion upon reduction of hydrogen peroxide. The height of the reaction barrier was predicted in range of 6-11 kcal mol(-1), consistent with an experimental rate constant of ca. 10(7) M(-1) s(-1). The proposed GPX1-Se(-)-Arg177H(+) mechanism for GPX1 is compared with the GPX3-SeH-Gln83 one proposed for human glutathione peroxidase from class 3 (GPX3) and with the solvent-assisted proton exchange mechanism proposed for GPX-like organic selenols. The structural and energetic parameters predicted by various density functional theory methods (B3LYP, MPW1PW91, MPW1K, BB1K, M05-2X, M06-2X, and M06) are also discussed.

Selective blockade of oxytocin and vasopressin V(1a) receptors in anaesthetised rats: evidence that activation of oxytocin receptors rather than V(1a) receptors increases sodium excretion.

BACKGROUND: Although it is known that moderate-to-high doses of the neurohypophysial hormones oxytocin and vasopressin are natriuretic, doubts remain over the identity of the receptors responsible. To address this issue, we have used highly selective antagonists of oxytocin and vasopressin receptors in animals with elevated endogenous circulating levels of the 2 hormones. METHODS: Rats were anaesthetised and prepared surgically for clearance studies, thereby raising plasma oxytocin and vasopressin concentrations. Sodium excretion, glomerular filtration rate and lithium clearance (an index of end-proximal fluid delivery) were measured: first during a control period, then after administration of the selective oxytocin receptor antagonist desGly-NH(2),d(CH(2))(5)[D-Trp(2),Thr(4),Dap(5)]OVT, the selective vasopressin V(1a) receptor antagonist d(CH(2))(5)[Tyr(Me)(2),Dab(5)]AVP, or vehicle alone. RESULTS: Absolute and fractional sodium excretion fell in rats given the oxytocin antagonist (by 32 and 27%, respectively, compared with corresponding values in vehicle-infused rats), but not in those given the V(1a) antagonist or vehicle. Antinatriuresis was associated with a small reduction in the ratio of sodium clearance to lithium clearance (an index of the fraction of distally delivered sodium that escapes reabsorption in the distal nephron). CONCLUSIONS: These results corroborate previous studies showing that activation of oxytocin receptors increases sodium excretion and imply that the natriuretic effect of elevated plasma vasopressin concentrations results from stimulation of oxytocin receptors.

Theoretical prediction of pKa values of seleninic, selenenic, sulfinic, and carboxylic acids by quantum-chemical methods.

Aqueous acid dissociation constants of substituted areneseleninic, areneselenenic, arenesulfinic, and benzoic acids are calculated by ab initio (MP2) and DFT (B3LYP) methods in combination with bulk solvation models (IEFPCM, CRSrad) from appropriate thermodynamic cycles. Mean absolute deviations (MAD) between experimental and calculated pK(a) values are quite large for basis sets without diffuse functions; however, trends are reasonably well described. Best agreement with experiment as described by MAD as well as correlation coefficient and slope of the correlation equation pK(a) = a*ΔG(calc)/RT ln(10) + b is obtained with the CPCM solvation model using the defaults optimized within COSMO-RS (CRSrad; MAD = 1.54, R(2) = 0.94, a = 0.83). Sulfenic (selenenic) acid tautomers are significantly more stable than the corresponding sulfoxide (selenoxide) forms.

Dibenzocyclooctadiene lignans from Magnolia and Talauma (Magnoliaceae): their absolute configuration ascertained by circular dichroism and X-ray crystallography and re-evaluation of previously published pyramidatin structures.

Twelve pyramidatins, i.e., dibenzocyclooctadiene-type lignans, together with Machilin G, were isolated from the dichloromethane extracts of aerial material of Talauma gloriensis, Magnolia fraseri, and Magnolia pyramidata (Magnoliaceae). These lignans contain a highly oxidized 7,9'-epoxy-2,2'-cyclolignane skeleton. Their structures were established using NMR spectroscopy (1D and 2D experiments) and mass spectrometry. The absolute configurations of five pairs of atropisomers (S(a)/R(a)-pyramidatins) and two single atropisomers (S(a)-pyramidatins) were determined by experimental and calculated circular dichroism (CD). In addition, the absolute configuration of (S(a))-3,3',4,4',5,5'-hexamethoxypyramidatin was confirmed using X-ray crystallography. Five pyramidatins, (R(a))-3,3',4,4',5,5'-hexamethoxypyramidatin, (R(a))-3,3'-dimethoxy-4,5:4',5'-bis(methylenedioxy)pyramidatin, (S(a))-3,3',4,5'-tetramethoxy-4,5-methylenedioxypyramidatin, (R(a))-3,3',4,5'-tetramethoxy-4,5-methylenedioxypyramidatin, and (R(a))-3,3',4,5-tetramethoxy-4',5'-methylenedioxypyramidatin are reported herein for the first time. In the current dataset, NMR values are in accordance with the observed and calculated CD values. These values are herein reported with particular reference to previously described data of pyramidatins, which have to be revised.

Derivatives of schisandrin with increased inhibitory potential on prostaglandin E(2) and leukotriene B(4) formation in vitro.

Four derivatives of schisandrin, a major dibenzo[a,c]cyclooctadiene lignan of Schisandra chinensis (Turcz.) Baillon were synthesized and structurally characterized by means of NMR and mass spectroscopy. Furthermore, axial chirality of the biphenyl system was determined by comparison of calculated with measured circular dichroism (CD) spectra. Three of the obtained derivatives showed a ring contraction during chemical modification. While the original lignans were inactive on the performed bioassays, the compounds which showed the cycloheptadiene skeleton revealed remarkable activities. For the inhibition of LTB(4) production the IC(50) values of aR-6,7-dihydro-6-(1'-hydroxyethyl)-3,9-dimethoxy-6-methyl-5H-dibenzo[a,c]cycloheptene-1,2,10,11-tetraol (6) and aR-6-(1'-iodoethyl)-1,2,3,9,10,11-hexamethoxy-6-methyl-5H-dibenzo[a,c]cycloheptene (8) were 4.2+/-0.3microM and 4.5+/-0.2microM, respectively. aR-6,7-Dihydro-6-(1'-hydroxyethyl)-6-methyl-5H-dibenzo[a,c]cycloheptene-1,2,3,9,10,11-hexaol (5) revealed dual inhibition on COX-2 (IC(50) 32.1+/-2.5microM) and on LTB(4) production (37.3+/-5.5% inhibition at 50microM).

Theoretical Study on the Redox Cycle of Bovine Glutathione Peroxidase GPx1: pKa Calculations, Docking, and Molecular Dynamics Simulations.

Three approaches of computational chemistry [quantum mechanics (QM) calculations, docking, and molecular dynamics (MD) simulations] were used to investigate the redox cycle of bovine erythrocyte glutathione peroxidase from class 1 (GPx1, EC 1.11.1.9). The pKa calculations for two redox states of the active-site selenocysteine of GPx1 (selenol, Sec45-SeH, and selenenic acid, Sec45-SeOH) were estimated using a bulk solvent model (B3LYP-IEFPCM and B3LYP-CPCM-COSMO-RS). The calculated pKa values of Sec45-SeH and Sec45-SeOH were corrected via a simple linear fit to a training set of organoselenium compounds, which consisted of aliphatic selenols and aromatic selenenic acids with available experimental pKa values. Based on docking calculations, binding sites for both molecules of the cofactor glutathione (GSH) are described. MD simulations on the dimer of GPx1 have been performed for all chemical states of the redox cycle: without GSH and with one or two molecules of GSH bound at the active site. Conformational analyses of MD trajectories indicate high mobility of the Arg177 and His79 residues. These residues can approach the vicinity of Sec45 and take part in the catalytic mechanism. On the basis of the calculated data, new atomistic details for a generally accepted mechanism of GPx1 are proposed.