RESUMEN
In this study, we describe the experimental variables influencing enantioseparation of twelve ß-blockers when analyzed under polar-organic, reversed-phase and hydrophilic interaction liquid chromatography conditions on a column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) as chiral stationary phase. Regarding polar-organic mode, two component mobile phases consisting of methanol, ethanol or acetonitrile with the addition of basic additives such as diethylamine, triethylamine, mono-ethanolamine, ethylendiamine or trifluoroacetic acid/diethylamine mixture were evaluated. Studies of retention at different temperatures were also performed. In reversed-phase mode, mixtures consisting of methanol or acetonitrile with either aqueous boric acid-borate buffer or sodium hydrogen carbonate-carbonate buffer solutions were compared aiming to study the influence of organic modifier as well as buffer type and pH on resolution. In addition, a systematic evaluation of the retention factors of ß-blockers enantiomers in hydro-organic eluents containing acetonitrile in presence of diethylamine as additive was carried out by increasing progressively the water content, in order to check for retention dependencies indicative of the interplay of both hydrophilic interaction liquid chromatography and reversed-phase modes.
Asunto(s)
Antagonistas Adrenérgicos beta/análisis , Antagonistas Adrenérgicos beta/aislamiento & purificación , Amilosa/análogos & derivados , Cromatografía Liquida , Cromatografía de Fase Inversa , Fenilcarbamatos/química , Acetonitrilos/química , Amilosa/química , Interacciones Hidrofóbicas e Hidrofílicas , Estereoisomerismo , Agua/químicaRESUMEN
In this work, the use of different solvents and temperatures was explored, aiming to evaluate their influence on the enantioseparation of pesticides by HPLC in polar-organic conditions, employing a column containing immobilized amylose tris(3-chloro-5-methylphenyl-carbamate). The chiral separation of seventeen different pesticides widely used as herbicides, fungicides, insecticides and precursors were studied. The mobile phases included methanol, ethanol, iso-propanol, n-propanol and acetonitrile; either pure or containing additives such as diethylamine, trifluoroacetic acid, formic acid, acetic acid or mixtures thereof. We studied the influence of these eluents on chiral separation of those pesticides in terms of retention factor, enantioselectivity, enantioresolution and peak symmetry. Regarding temperature influence, evaluated within the range 5 - 40 °C, nearly all the compounds decreased their retention and selectivity factors with the increase in temperature, although the effect was dependent on the mobile phase solvent. Moreover, estimation of thermodynamic parameters was performed based on linear van´t Hoff plots.
Asunto(s)
Amilosa/análogos & derivados , Compuestos Orgánicos/química , Plaguicidas/química , Plaguicidas/aislamiento & purificación , Fenilcarbamatos/química , Temperatura , Amilosa/química , Cromatografía Líquida de Alta Presión , Estándares de Referencia , Solventes/química , EstereoisomerismoRESUMEN
The substitution of serine and threonine residues in nucleocytoplasmic proteins with 2-acetamido-2-deoxy-ß-D-glucopyranose (O-GlcNAc) residues is an essential post-translational modification found in many multicellular eukaryotes. O-glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase (O-GlcNAcase) hydrolyzes O-GlcNAc residues from post-translationally modified serine/threonine residues of nucleocytoplasmic protein. O-GlcNAc has been implicated in several disease states such as cancer, Alzheimer's disease, and type II diabetes. For this paper, a model of the human O-GlcNAcase (hOGA) enzyme based on the X-ray structures of bacterial Clostridium perfringens (CpNagJ) and Bacteroides thetaiotaomicrometer (BtOGA) homologues has been generated through molecular homology modeling. In addition, molecular docking, molecular dynamics (MD) simulations, and Linear Interaction Energy (LIE) were employed to determine the bind for derivatives of two potent inhibitors: O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-methyl-R-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NAG-thiazoline), with hOGA. The results show that the binding free energy calculations using the Linear Interaction Energy (LIE) are correlated with inhibition constant values. Therefore, the model of the human O-GlcNAcase (hOGA) obtained here may be used as a target for rational design of new inhibitors.
Asunto(s)
Acetilglucosamina/análogos & derivados , Proteínas Bacterianas/química , Simulación del Acoplamiento Molecular , Oximas/química , Fenilcarbamatos/química , Tiazoles/química , beta-N-Acetilhexosaminidasas/química , Acetilglucosamina/química , Proteínas Bacterianas/antagonistas & inhibidores , Bacteroides/química , Bacteroides/enzimología , Sitios de Unión , Clostridium perfringens/química , Clostridium perfringens/enzimología , Cristalografía por Rayos X , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Cinética , Ligandos , Unión Proteica , Conformación Proteica , Homología Estructural de Proteína , Termodinámica , beta-N-Acetilhexosaminidasas/antagonistas & inhibidoresRESUMEN
The enzymatic kinetic resolution of tert-butyl 2-(1-hydroxyethyl) phenylcarbamate via lipase-catalyzed transesterification reaction was studied. We investigated several reaction conditions and the carbamate was resolved by Candida antarctica lipase B (CAL-B), leading to the optically pure (R)- and (S)-enantiomers. The enzymatic process showed excellent enantioselectivity (E > 200). (R)- and (S)-tert-butyl 2-(1-hydroxyethyl)phenylcarbamate were easily transformed into the corresponding (R)- and (S)-1-(2-aminophenyl)ethanols.
Asunto(s)
Candida/enzimología , Proteínas Fúngicas/química , Lipasa/química , Compuestos Organometálicos/síntesis química , Compuestos de Organoselenio/síntesis química , Fenilcarbamatos/síntesis química , Telurio/química , Biocatálisis , Esterificación , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Oxidación-Reducción , Fenilcarbamatos/química , Solventes , EstereoisomerismoRESUMEN
(±)-Licarin A (1), a neolignan obtained by the oxidative coupling reaction of isoeugenol, had in this study its enantiomers resolved. A novel, quick and efficient enantiomeric resolution of 1 was directly performed by chiral high-performance liquid chromatography (HPLC-PDA) protocol (CHIRALPACK(®) AD column; 9:1 (v/v) n-hexane:2-propanol; 1.0 mL/min). This method provided a chromatogram profile with a well-resolved peak separation. After isolation of each enantiomer with ee>99.9%, they were analysed in a polarimeter. Compound 2, which showed a retention time (t(r)) of 12.13 min, was the (+)-enantiomer and compound 3 (t(r)=18.90 min) was the (-)-enantiomer.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Lignanos/química , 2-Propanol/química , Amilosa/análogos & derivados , Amilosa/química , Eugenol/análogos & derivados , Eugenol/química , Hexanos/química , Lignanos/aislamiento & purificación , Fenilcarbamatos/química , EstereoisomerismoRESUMEN
The enzyme O-glycoprotein 2-acetamino-2-deoxy-beta-d-glucopyranosidase (O-GlcNAcase) is responsible for the removal of N-acetylglucosamine moieties from 2-acetamido-2-deoxy-beta-D-glucopyranose (O-GlcNAc) residues of serine/threonine residues of modified proteins. We herein present results of hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations applied to the study of the interactions established between a bacterial Clostridium perfringens homologue (CpNagJ) and PUGNAc, a potent known inhibitor of this enzyme. Electrostatic binding free energy and energy term decomposition have been computed for the wild-type CpNagJ and several mutants: D297N, D298N, Y335F, N390A, N396A, D401A, and W490A. The theoretical results have been compared with recently experimental data based on crystallographic and mutation studies on the same system. Our results reveal that, first, there is a strong interaction between Asp401, Asp298, and Asp297 residues and the PUGNAc inhibitor; and, second, the electrostatic substrate binding free energy is higher in wild-type, N390A and W490A mutants than in D297N, D298N, Y335F, N396A, and D401A ones, in accordance with the experimental results. Finally, both our theoretical predictions and the experimental data are compatible with a substrate-assisted reaction mechanism, involving two conserved aspartate residues.
Asunto(s)
Acetilglucosamina/análogos & derivados , Clostridium perfringens/enzimología , Simulación de Dinámica Molecular , Oximas/química , Fenilcarbamatos/química , beta-N-Acetilhexosaminidasas/química , Acetilglucosamina/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Mutagénesis Sitio-Dirigida , Unión Proteica , Teoría Cuántica , Electricidad Estática , Termodinámica , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
O-glycoprotein 2-acetamino-2-deoxy-beta- d-glucopyranosidase ( O-GlcNAcase) hydrolyzes 2-acetamido-2-deoxy-beta- d-glucopyranose ( O-GlcNAc) residues of serine/threonine residues of modified proteins. O-GlcNAc is present in many intracellular proteins and appears to have a role in the etiology of several diseases including cancer, Alzheimer's disease, and type II diabetes. In this work, we have carried out molecular dynamics simulations using a hybrid quantum mechanics/molecular mechanics approach to determine the binding of two potent inhibitors, PUGNAc and NAG, with a bacterial O-GlcNAcase. The results of these simulations show that Asp-401, Asp-298, and Asp-297 residues play an important role in the protein-inhibitor interactions. These results might be useful to design compounds with more interesting inhibitory activity on the basis of its three-dimensional structure.
Asunto(s)
Acetilglucosamina/análogos & derivados , Inhibidores Enzimáticos/química , Modelos Moleculares , Oximas/química , Fenilcarbamatos/química , Teoría Cuántica , Tiazoles/química , beta-N-Acetilhexosaminidasas/antagonistas & inhibidores , beta-N-Acetilhexosaminidasas/química , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Acetilglucosamina/farmacología , Sitios de Unión , Biocatálisis/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Conformación Molecular , Oximas/metabolismo , Oximas/farmacología , Fenilcarbamatos/metabolismo , Fenilcarbamatos/farmacología , Unión Proteica , Protones , Electricidad Estática , Termodinámica , Tiazoles/metabolismo , Tiazoles/farmacología , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Liquid chromatography is known as one of the most flexible, efficient and cost-effective methods to resolve racemic mixture in order to attend the growing demand of the pharmaceutical industry for pure enantiomeric compounds. Cellulose tris(3,5-dimethylphenylcarbamate) is frequently used as a stationary phase for enantiomeric separations because of its attractive properties, including high enantioselectivity, high loading capacity and good mechanical stability. In this study, we investigated the usefulness of cellulose tris(3,5-dimethylphenylcarbamate) as the stationary phase and of ethanol and hexane mixtures as the mobile phases for the chromatographic separation of potential pharmaceutical intermediates. Using adsorption equilibrium data, we determined the optimal operational conditions for the separation of the N-Boc-4-[p-chloro-phenyl]-2-pyrrolidone enantiomers - a baclofen precursor - in a semi-preparative scale simulated moving bed unit. This unit was used to obtain high purity enantiomers on a scale of 1g/day. The outlet streams were analyzed by an on-line system that consisted of a UV-vis spectrophotometric unit, a polarimeter, and HPLC. Enantiomeric purities of up to 97% were obtained for the raffinate stream and up to 90% for the extract stream.
Asunto(s)
Baclofeno/síntesis química , Celulosa/análogos & derivados , Cromatografía Líquida de Alta Presión/métodos , Fenilcarbamatos/química , Pirrolidinonas/aislamiento & purificación , Celulosa/química , Cromatografía Líquida de Alta Presión/instrumentación , Etanol , Hexanos , EstereoisomerismoRESUMEN
The paper presents the study of some physicochemical properties of 2-, 3-, 4-alkoxyphenylcarbamic acid derivatives with various substituted N-phenylpiperazin-1-yl moiety in the basic part of the molecule. Elemental analysis, melting point, solubility, surface activity, dissociation constant and some lipophilicity parameters i.e.--partition coefficient, capacity factor obtained from HPLC, and R(M) values from reversed-phase thin-layer chromatography were determined.