Energy and spectroscopic parameters of neutral and cations isomers of the CnH2 (n = 2-6) families using high-level ab-initio approaches.

Cationic species, previously detected from ion-induced desorption of solid methane by plasma desorption mass spectrometry (PDMS), and neutral species, are investigated using high-level ab-initio approaches. From a set of 25 cationic and 26 neutral structures belonging to CnH2 (n = 2-6) families, it was obtained the energy, rotational constants, harmonic vibrational frequency, charge distribution and excitation energies. The ZPVE-corrected energies, at CCSD(T)-F12; CCSD(T)-F12/RI/(cc-pVTZ-F12, cc-pVTZ-F12-CABS, cc-pVQZ/C) (n = 2-5) and CCSD(T)/cc-pVTZ (n = 6) levels, reveal that the topology of the most stable isomer vary with n and the charge. Out of 674 harmonic frequencies, those with maximum intensity are generally in the 3000-3500 cm-1 range. Analysis of 169 vertical transition energies calculated with the EOM-CCSD approach, suggest three C6H2 species as potential carriers of the diffuse interstellar bands (DIB). Systematic comparison of properties between neutral and cationic species can assist in the structural description of complex matrices.

Quantification of cresols in liquid smoke samples employing liquid-liquid extraction with low-temperature purification and analysis by gas chromatography-mass spectrometry.

Liquid smoke is a food additive and cresols are among its chemical constituents, potentially toxic to human health. Thus, the objective of this study was to develop a method to quantify cresols in liquid smoke. First, the liquid-liquid extraction with low temperature purification (LLE-LTP) was validated for cresols in water, as there are no cresol-free liquid smoke samples. Analyzes were performed by gas chromatography coupled to mass spectrometry in full scan mode. LLE-LTP was subsequently applied in five commercial samples of liquid smoke. Validation results showed that the proposed extraction method was selective for cresols, linear in the range of 0.5 to 35 mg L-1, limit of quantification of 0.5 mg L-1, recovery rate between 90% and 104% and relative standard deviation lower than 10%. The quantification of cresols in liquid smoke samples ranged from 3.0 to 38.3 mg L-1 and the concentration of these chemical contaminants in liquid smoke remained constant for at least 21 days at 25 °C.

Preparative Fractionation of Brazilian Red Propolis Extract Using Step-Gradient Counter-Current Chromatography.

Propolis is a resinous bee product with a very complex composition, which is dependent upon the plant sources that bees visit. Due to the promising antimicrobial activities of red Brazilian propolis, it is paramount to identify the compounds responsible for it, which, in most of the cases, are not commercially available. The aim of this study was to develop a quick and clean preparative-scale methodology for preparing fractions of red propolis directly from a complex crude ethanol extract by combining the extractive capacity of counter-current chromatography (CCC) with preparative HPLC. The CCC method development included step gradient elution for the removal of waxes (which can bind to and block HPLC columns), sample injection in a single solvent to improve stationary phase stability, and a change in the mobile phase flow pattern, resulting in the loading of 2.5 g of the Brazilian red propolis crude extract on a 912.5 mL Midi CCC column. Three compounds were subsequently isolated from the concentrated fractions by preparative HPLC and identified by NMR and high-resolution MS: red pigment, retusapurpurin A; the isoflavan 3(R)-7-O-methylvestitol; and the prenylated benzophenone isomers xanthochymol/isoxanthochymol. These compounds are markers of red propolis that contribute to its therapeutic properties, and the amount isolated allows for further biological activities testing and for their use as chromatographic standards.

The role of water in reactions catalysed by hydrolases under conditions of molecular crowding.

Under macromolecular crowding (MC) conditions such as cellular, extracellular, food and other environments of biotechnological interest, the thermodynamic activity of the different macromolecules present in the system is several orders of magnitude higher than in dilute solutions. In this state, the diffusion rates are affected by the volume exclusion induced by the crowders. Immiscible liquid phases, which may arise in MC by liquid-liquid phase separation, may induce a dynamic confinement of reactants, products and/or enzymes, tuning reaction rates. In cellular environments and other crowding conditions, membranes and macromolecules provide, on the whole, large surfaces that can perturb the solvent, causing its immobilisation by adsorption in the short range and also affecting the solvent viscosity in the long range. The latter phenomenon can affect the conformation of a protein and/or the degree of association of its protomers and, consequently, its activity. Changes in the water structure can also alter the enzyme-substrate interaction, and, in the case of hydrolytic enzymes, where water is one of the substrates, it also affects the reaction mechanism. Here, we review the evidence for how macromolecular crowding affects the catalysis induced by hydrolytic enzymes, focusing on the structure and dynamics of water.

Weak noncovalent interactions in two positional isomers of acrylonitrile derivatives: inputs from PIXEL energy, Hirshfeld surface and QTAIM analyses.

A single crystal X-ray diffraction analysis was performed on two positional isomers (m-tolyl and p-tolyl) of acrylonitrile derivatives, namely, (Z)-3-(4-(pyridin-2-yl) phenyl)-2-(m-tolyl) acrylonitrile (1) and (Z)-3-(4-(pyridin-2-yl)phenyl)-2-(p-tolyl) acrylonitrile (2). Compound 1 crystallized in the monoclinic P21/n space group with two crystallographically independent molecules. Compound 2 also possesses two crystallographically independent molecules and crystallized in the triclinic P-1 space group. The Hirshfeld surface analysis revealed that, in both isomers, intermolecular H⋅⋅⋅H/C/N contacts contribute significantly to the crystal packing. More than 40% of the contribution arises from intermolecular C-H⋅⋅⋅C(π) contacts. In both compounds, the relative contribution of these contacts is comparable, indicating that the positional isomeric effects are marginal. The structures in which these isomers are arranged in the solid state are very similar, and the lattice energies are also comparable between the isomers. The Coulomb-London-Pauli-PIXEL (CLP-PIXEL) energy analysis identified the energetically significant dimers. The strength of the intra- and intermolecular interactions was evaluated using the quantum theory of atoms in molecules approach. The UV-Vis absorbance in three different solvents (chloroform, ethanol, and ethyl acetate) for isomers 1 and 2 are very similar. This result is in good agreement with the time-dependent density-functional theory (TD-DFT) calculations.

Exploring the electronic structure of [RuF5NO]2- ion by the generation of a metastable state (MS1).

Some mononitrosyl complexes of transition metals exhibit one or two metastable states (linkage isomers, MS1 and MS2) when irradiated at low temperatures with appropriate wavelengths. In this work, the generation of metastable state one (MS1) (or Ru-ON linkage isomer) in K2[RuF5NO].H2O at 77 K was studied by sample excitation using laser light in a wide range of wavelengths. The effects of irradiation was monitored by infrared spectroscopy. ν(NO) in the ground state was shifted by -161 cm-1 when the complex was excited to MS1, a magnitude similar to that observed in other transition metal nitrosyls for a such state. We report on the excitation and deactivation of metastable states by using a wide variety of laser lines. A novel method for exploring the electronic structure of [RuF5NO]2- through the generation of MS1 is proposed. For this purpose, a sample was carefully irradiated with the same intensity of light for all laser lines in the spectral region 260-1064 nm. The integrated area under the ν(NO)MS1 band was used as a measure of MS1 population. The profile peaks of the MS1 population (ν(NO)MS1 band area) vs. the irradiation wavelength fit well with those of the electronic spectrum of the [RuF5NO]2- ion in an aqueous solution. The onset temperature for MS1 decay in K2[RuF5NO].H2O, at approximately 180 K, is slightly lower than the average reported for other ruthenium-nitrosyl systems.

Production of Two Isomers of Sphaeralcic Acid in Hairy Roots from Sphaeralcea angustifolia.

The Sphaeralcea angustifolia plant is used as an anti-inflammatory and gastrointestinal protector in Mexican traditional medicine. The immunomodulatory and anti-inflammatory effects have been attributed to scopoletin (1), tomentin (2), and sphaeralcic acid (3) isolated from cells in suspension cultures and identified in the aerial tissues of the wild plant. The hairy roots from S. angustifolia established by infecting internodes with Agrobacterium rhizogenes were explored to produce active compounds based on biosynthetic stability and their capacity to produce new compounds. Chemical analysis was resumed after 3 years in these transformed roots, SaTRN12.2 (line 1) produced scopoletin (0.0022 mg g-1) and sphaeralcic acid (0.22 mg g-1); instead, the SaTRN7.1 (line 2) only produced sphaeralcic acid (3.07 mg g-1). The sphaeralcic acid content was 85-fold higher than that reported for the cells in the suspension cultivated into flakes, and it was similar when the cells in suspension were cultivated in a stirring tank under nitrate restriction. Moreover, both hairy root lines produced stigmasterol (4) and ß-sitosterol (5), as well as two new naphthoic derivates: iso-sphaeralcic acid (6) and 8-methyl-iso-sphaeralcic acid (7), which turned out to be isomers of sphaeralcic acid (3) and have not been reported. The dichloromethane-methanol extract from SaTRN7.1 hairy root line had a gastroprotective effect on an ulcer model in mice induced with ethanol.

HPTLC-based fingerprinting: An alternative approach for fructooligosaccharides metabolism profiling.

Fructans are categorized as fructose-based metabolites with no more than one glucose in their structure. Agave species possess a mixture of linear and ramified fructans with different degrees of polymerization. Among them, fructooligosaccharides are fructans with low degree of polymerization which might be approachable by high performance thin layer chromatography (HPTLC). Thus, this study used two emblematic Agave species collected at different ages as models to explore the feasibility of HPTLC-based fingerprinting to characterize fructooligosaccharides (FOS) production, accumulation, and behavior through time. To do so, high performance anion exchange was also used as analytical reference to determine the goodness and robustness of HPTLC data. The multivariate data analysis showed separation of samples dictated by species and age effects detected by both techniques. Moreover, linear correlations between the increase of the age in agave and their carbohydrate fraction was established in both species by both techniques. Oligosaccharides found to be correlated to species and age factors, these suggest changes in specific carbohydrate metabolism enzymes. Thus, HPTLC was proven as a complementary or stand-alone fingerprinting platform for fructooligosaccharides characterization in biological mixtures. However, the type of derivatizing reagent and the extraction color channel determined the goodness of the model used to scrutinize agavin fructooligosaccharides (aFOS).

Enzymes and pathways in microbial production of 2,3-butanediol and 3-acetoin isomers.

2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.

Impact of Covalent Modifications on the Hydrogen Bond Strengths in Diaminotriazine Supramolecules.

Melamine (M) is a popular triamine triazine compound in the field of supramolecular materials. In this work, we have computationally investigated how substituents can be exploited to improve the binding strength of M supramolecules. Two types of covalent modifications were studied: the substitution of an H atom within an amine group -NHR, and the replacement of the whole -NH2 group (R=H, F, CH3 and COCH3 ). Through our dispersion-corrected density functional theory computations, we explain which covalent modification will show the best self-assembling capabilities, and why the binding energy is enhanced. Our charge density and molecular orbital analyses indicate that the best substituents are those that generate a charge accumulation on the endocyclic N atom, providing an improvement of the electrostatic attraction. At the same time the substituent assists the main N-H⋅⋅⋅N hydrogen bonds by interacting with the amino group of the other monomer. We also show how the selected group notably boosts the strength of hexameric rosettes. This research, therefore, provides molecular tools for the rational design of emerging materials based on uneven hydrogen-bonded arrangements.

Comparative measurement of D- and L-lactic acid isomers in vaginal secretions: association with high-grade cervical squamous intraepithelial lesions.

PURPOSE: Identification of low-cost protocols to identify women at elevated susceptibility to develop cervical intra-epithelial abnormalities would aid in more individualized monitoring. We evaluated whether quantitation of the D- and L-lactic acid isomers in vaginal secretions could differentiate women with normal cervical epithelia from those with a low (LSIL) or high (HSIL) grade squamous intraepithelial lesion or with cervical cancer. METHODS: Vaginal samples, collected from 78 women undetgoing cervical colposcopy and biopsy, were tested for pH, bacterial composition by Gram stain (Nugent score) and concentrations of D- and L-lactic acid by a colorimetric assay. RESULTS: Subsequent diagnosis was 23 women with normal cervical epithelium, 10 with LSIL, 43 with HSIL and 2 with cervical cancer. Vaginal pH and Nugent score were comparable in all subject groups. The concentration of L-lactic acid, but not D-lactic acid, as well as the L/D-lactic acid ratio, were significantly elevated (p < 0.01) in women with HSIL and cervical cancer. CONCLUSION: Comparative measurement of vaginal D- and L-lactic acid isomers may provide a low-cost alternative to identification of women with an elevated susceptibility to cervical abnormalities.

Simultaneous Detection of Dihydroxybenzene Isomers Using Electrochemically Reduced Graphene Oxide-Carboxylated Carbon Nanotubes/Gold Nanoparticles Nanocomposite.

An electrochemical sensor based on electrochemically reduced graphene oxide (ErGO), carboxylated carbon nanotubes (cMWCNT), and gold nanoparticles (AuNPs) (GCE/ErGO-cMWCNT/AuNPs) was developed for the simultaneous detection of dihidroxybenzen isomers (DHB) hydroquinone (HQ), catechol (CC), and resorcinol (RS) using differential pulse voltammetry (DPV). The fabrication and optimization of the system were evaluated with Raman Spectroscopy, SEM, cyclic voltammetry, and DPV. Under optimized conditions, the GCE/ErGO-cMWCNT/AuNPs sensor exhibited a linear concentration range of 1.2-170 µM for HQ and CC, and 2.4-400 µM for RS with a detection limit of 0.39 µM, 0.54 µM, and 0.61 µM, respectively. When evaluated in tap water and skin-lightening cream, DHB multianalyte detection showed an average recovery rate of 107.11% and 102.56%, respectively. The performance was attributed to the synergistic effects of the 3D network formed by the strong π-π stacking interaction between ErGO and cMWCNT, combined with the active catalytic sites of AuNPs. Additionally, the cMWCNT provided improved electrocatalytic properties associated with the carboxyl groups that facilitate the adsorption of the DHB and the greater amount of active edge planes. The proposed GCE/ErGO-cMWCNT/AuNPs sensor showed a great potential for the simultaneous, precise, and easy-to-handle detection of DHB in complex samples with high sensitivity.

Analytical tools for urocanic acid determination in human samples: A review.

Urocanic acid is a chromophore found in the skin that has been identified as an important immunosuppressant and carcinogenesis mediator through its photoisomerization from trans to cis form induced by ultraviolet radiation. Research on analytical methods that explore urocanic acid isomerization is indispensable to fully understand the deleterious effects mediated by this biomarker. In this context, the current relevant analytical methods for determination of these isomers in human samples are summarized in this review. The methods presented here are applicable to human samples collected by noninvasive methods (or minimally invasive), encompassing an array of analytical techniques, including high-performance capillary electrophoresis, confocal Raman spectroscopy, gas chromatography, high-performance liquid chromatography, and mass spectrometry, among others. Developed high-performance liquid chromatography methods have proven to be advantageous, allowing noninvasive collections for in vivo analysis and the confocal Raman, specially, for real-time analysis. Among all these methods, high-performance liquid chromatography is the most investigated one with mass spectrometry or ultraviolet detector, and the mass spectrometry detector being the most studied in the last years, demonstrating high sensitivity, very low detection limits, and accurate identification, especially for clinical investigations.

The impact that ß-citronellol isomers have on the biofilm formation of Candida yeasts.

Infections associated with biofilms developed by Candida spp. are becoming a great problem due to its resistance against the immune response of the host and the action of antifungal agents. Hence, finding substances that can inhibit the development of biofilms increases the likelihood that these compounds one day can become good antifungals applied in the clinic. The aim of this study was to evaluate the effect of ß-citronellol enantiomers on the biofilm formation by Candida albicans and Candida tropicalis isolated from bloodstream infections. Inhibition was evaluated by reading microplates treated with different concentrations of R-(+)-ß-citronellol, S-(-)-ß-citronellol and amphotericin B, compared to negative control, in spectrophotometer at 590 nm. All tested concentrations of ß-citronellol enantiomers inhibited the biofilm formation of Candida. However, it is still necessary to evaluate the behavior of these isomers on mature biofilms, so that they can become more viable as antifungal therapeutical agents.

How do nitrated lipids affect the properties of phospholipid membranes?

Biological membranes are under constant attack of free radicals, which may lead to lipid nitro-oxidation, producing a complex mixture of nitro-oxidized lipids that are responsible for structural and dynamic changes on the membrane. Despite the latter, nitro-oxidized lipids are also associated with several inflammatory and neurodegenerative diseases, the underlying mechanisms of which remain elusive. We perform atomistic molecular dynamics simulations using several isomers of nitro-oxidized lipids to study their effect on the structure and permeability of the membrane, as well as the interaction between the mixture of these products in the phospholipid membrane environment. Our results show that the stereo- and positional isomers have a stronger effect on the properties of the membrane composed of oxidized lipids compared to that containing nitrated lipids. Nevertheless, nitrated lipids lead to three-fold increase in water permeability compared to oxidized lipids. In addition, we show that in a membrane consisting of combined nitro-oxidized lipid products, the presence of oxidized lipids protects the membrane from transient pores. Is well stablished that plasma application and photodynamic therapy produces a number of oxidative species used to kill cancer cells, through membrane damage induced by nitro-oxidative stress. This study is important to elucidate the mechanisms and the molecular level properties involving the reactive species produced during that cancer therapies.

Density functional prediction of non cubic isomers of cubane and nitrated cubanes.

Density functional molecular orbital calculations are used to show the existence of two non-cubic isomeric structures of cubane and nitrated cubane. The isomers are shown to have no imaginary frequencies and are thus at a minimum on the potential energy surface. The calculations indicate the isomers are slightly more stable than the cubic structures.

(R)-(+)-ß-Citronellol and (S)-(-)-ß-Citronellol in Combination with Amphotericin B against Candida Spp.

The enantiomers (R)-(+)-ß-citronellol and (S)-(-)-ß-citronellol are present in many medicinal plants, but little is understood about their bioactivity against Candida yeasts. This study aimed to evaluate the behavior of positive and negative enantiomers of ß-citronellol on strains of Candida albicans and C. tropicalis involved in candidemia. The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined. The evaluation of growth kinetics, mechanism of action, and association studies with Amphotericin B (AB) using the checkerboard method was also performed. R-(+)-ß-citronellol and S-(-)-ß-citronellol presented a MIC50% of 64 µg/mL and a MFC50% of 256 µg/mL for C. albicans strains. For C. tropicalis, the isomers exhibited a MIC50% of 256 µg/mL and a MFC50% of 1024 µg/mL. In the mechanism of action assay, both substances displayed an effect on the fungal membrane but not on the fungal cell wall. Synergism and indifference were observed in the association of R-(+)-ß-citronellol and AB, while the association between S-(-)-ß-citronellol and AB displayed synergism, additivity, and indifference. In conclusion, both isomers of ß-citronellol presented a similar profile of antifungal activity. Hence, they can be contemplated in the development of new antifungal drugs providing that further research is conducted about their pharmacology and toxicity.

Theoretical study of the gas-phase thermolysis reaction of 3,6-dimethyl-1,2,4,5-tetroxane. Methyl and axial-equatorial substitution effects.

Organic peroxides are interesting compounds with a broad range of properties from antimalarial and antimicrobial activities to explosive character. In this work the gas-phase thermolysis reaction mechanism of the 3,6-dimethyl-1,2,4,5-tetroxane (DMT) is studied by DFT calculations, considering axial-axial, axial-equatorial, and equatorial-equatorial position isomers. The critical points of the singlet (S) and triplet (T) potential energy surfaces (PES) are calculated. Three mechanisms are considered: i) S-concerted, ii) S-stepwise, and iii) T-stepwise. The first intermediate of the reaction through S-stepwise-PES is a diradical open structure, o, yielding, as products, two molecules of acetaldehyde and one of O2 in the S state. The S-stepwise-mechanism gives exothermic reaction energies (Er) in the three position isomers. The S-concerted mechanism yields very high activation energies (Ea) in comparison with those of the S-stepwise mechanism. In the T-stepwise mechanism, a triplet open structure (T-o) is first considered, yielding an Er 12 kcal mol-1 more exothermic than that of the S-mechanisms. The S-o and T-o are similar in structure and energies; therefore, a crossing from the S- to T-PES is produced at the o intermediate as a consequence of a spin-orbit coupling. The highest Ea is the first step after o intermediate, and thus it is considered the rate limiting step. Therefore, the Er at the T-PES is more in agreement with the Er of the exothermic experimental diperoxide products. Ea, Er, and O···O distances are studied as a function of the number of methyl groups and the position isomerization.

Coordination numbers in hydrated Cu(II) ions.

The potential energy surface of [Cu(H2O)n]2+ clusters with n = 12, 16, and 18 was explored by using a modified version of the simulated annealing method. Such exploration was carried out by using the PM7 semiempirical method to obtain around 100,000 isomers, which provide candidates to be optimized with PBE0-D3, M06-2X, and BHLYP exchange-correlation functionals coupled with the 6-311++G** basis set. These methods based on the Kohn-Sham approach delivered isomers with coordination numbers of 4, 5, and 6. The analysis used to obtain coordination numbers was based on geometrical parameters and the quantum theory of atoms in molecules (QTAIM) approach. Our methodology found only one isomer with fourfold coordination and its probabilities to appear in these clusters are quite small for high temperatures. The procedure used in this article predicts important populations of fivefold and sixfold coordination clusters, in fact, the fivefold coordination dominates for PBE0-D3 and BHLYP methods, although the sixfold coordination starts to be important when the number of water molecules is increased. The nature of axial and equatorial contacts is discussed in the context of the QTAIM and the noncovalent interaction index (NCI), which gives a clear classification of such orientations. Also, these methods suggest a partial covalent interaction between the Cu2+ and water molecules in both positions; equatorial and axial.

Effect of CYP2D6 Poor Metabolizer Phenotype on Stereoselective Nebivolol Pharmacokinetics.

BACKGROUND: Nebivolol is a drug available as a racemate of d-nebivolol (SRRR) and lnebivolol (RSSS). In human liver microsomes, CYP2D6 mainly catalyses the metabolism of lnebivolol, while CYP2C19 catalyses the metabolism of d-nebivolol. Nebivolol stereoselective pharmacokinetics has been described only for extensive metabolizers (EM). OBJECTIVE: To describe the stereoseletive nebivolol pharmacokinetics in CYP2D6 poor metabolizers (PM) and to assess whether the phenotype has an impact on nebivolol pharmacokinetics. METHODS: Three healthy volunteers PM phenotyped (ratios of 20.1, 220 and 244 for the 8 h urinary excretion of metoprolol/alfa-hydroxymetoprolol) received a single oral dose of racemic nebivolol and sequential blood samples were collected between zero (predose) and 48 h. RESULTS: The obtained data were compared to 22 EM subjects with normal kidney function enrolled in our previous study. For both isomers, Cmax, Tmax and AUC0-48 were significantly greater in the PM group compared to the EMs (p = 0.006 - 0.001). For d-nebivolol, Cmax, Tmax and AUC0-48 were, on average, 5.9, 2.7 and 15.0 larger in PMs. The corresponding values for l-nebivolol were 4.4, 2.7 and 17.5. CONCLUSION: The decline in plasma concentration of both nebivolol isomers in PM phenotypes, especially those with MR of 220 and 244, which indicate minimal or absent CYP2D6 activity, points to alternative mechanisms for nebivolol elimination. Collectively, our results are the first to show the significant impact of CYP2D6 PM phenotype on the metabolic disposition and in vivo exposure to both nebivolol isomers.