Étude sur l'utilisation de liquides ioniques à base imidazolium pour l'extraction sélective de phosphopeptides

La phosphorylation des protéines constitue l’une des plus importantes modifications post-traductionnelles (PTMs) et intervient dans de multiples processus physiologiques tels, la croissance, la différenciation cellulaire, l’apoptose, etc. En dépit de son importance, l’analyse des phosphoprotéines demeure une tâche difficile en raison de leur nature dynamique (car la phosphorylation des protéines est un processus réversible) et de leur faible abondance relative. En effet, la détermination des sites de phosphorylation est souvent difficile car les phosphopeptides sont souvent difficiles à détecter par des méthodes d’analyse chromatographique classique et par spectrométrie de masse (MS). De récentes études ont démontré que les nombreuses méthodes d’enrichissement de phosphopeptides existantes ne sont pas complètes, et que le nombre total de phosphopeptides détectés ne chevauchent pas complètement ces méthodes. C’est pour cela qu’il existe une nécessité de combler les lacunes des méthodes d’enrichissement existantes afin d’avoir des analyses phosphoprotéomiques plus complètes. Dans cette étude, nous avons utilisé les liquides ioniques (LI), plus particulièrement les sels d’imidazolium, comme une technique d’enrichissement alternative, dans le but de favoriser une extraction sélective de phosphopeptides présents en solution. Les sels d’imidazolium ont donc été utilisés en raison de leurs propriétés physico-chimiques "facilement" ajustables selon la nature des substituants sur le noyau imidazolium et la nature de l’anion. Les sels de monoimidazolium et de bis-imidazolium possédant respectivement des chaînes linéaires à 4, 12 et 16 atomes de carbone et ayant différents anions ont été synthétisés et utilisés pour effectuer des extractions liquide-liquide et solide-liquide des phosphopeptides en solution. Dans un premier temps, des extractions liquide-liquide ont été réalisées en utilisant un liquide ionique (LI) ayant une chaine linéaire de 4 atomes de carbone. Ces extractions réalisées avec le bis(trifluoromethanesulfonyl) amide de 3-butyl-1-methylimidazolium (BMIM-NTf2) et l’hexafluorophosphate de 3-butyl-1-methylimidazolium (BMIM-PF6) n’ont pas montré une extraction notable du PPS comparativement au PN. Dans un deuxième temps, des extractions solide-liquide ont été réalisées en fonctionnalisant des particules solides avec des sels d’imidazolium possédant des chaines linéaires de 12 ou 16 atomes de carbone. Ces extractions ont été faites en utilisant un phosphopentapeptide Ac-Ile-pTyr-Gly-Glu-Phe-NH2 (PPS) en présence de 2 analogues acides non-phosphorylés. Il a été démontré que les sels d’imidazolium à chaine C12 étaient meilleurs pour extraire le PPS que les deux autres peptides PN (Ac-Ile-Tyr-Gly-Glu-Phe-NH2) et PE (Ac-Glu-Tyr-Gly-Glu-Phe-NH2) L’électrophorèse capillaire (CE) et la chromatographie liquide à haute performance couplée à la spectrométrie de masse (LC-MS) ont été utilisées pour quantifier le mélange des trois peptides avant et après extraction ; dans le but de mesurer la sélectivité et l’efficacité d’extraction de ces peptides par rapport à la composition chimique du liquide ionique utilisé.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.

Consumo de sustancias y noxas prenatales en madres de pacientes con síndrome de Möbius y Möbius Like

Introducción: El síndrome de Möbius y Möbius Like es una entidad poco frecuente caracterizada principalmente por parálisis congénita del VI y VII par craneal. Su etiología es poco conocida aunque se ha asociado a inductores del aborto. El objetivo de este estudio es describir factores anómalos, tóxicos o nocivos que hayan estado presentes en el embarazo de las madres de estos pacientes. Metodología: se realizó una encuesta auto-diligenciable a 15 madres de pacientes con el diagnóstico, indagando sobre condiciones anómalas y/o exposicionales del embarazo, el padre y el ambiente. Resultados: Las madres se encontraban entre los 16 y 34 años al momento de quedar embarazadas, en su mayoría eran solteras, estudiantes y sin planes de embarazo. Once en total usaron algún medicamento y/o sustancias durante la gestación; seis de ellas Misoprostol (40%). Las otras sustancias utilizadas incluyeron: alternativas, cigarrillo, alcohol, ibuprofeno, anticonceptivos, otros. Como anomalías del periodo prenatal se reportaron sangrado activo y/o amenaza de aborto, infección, exposición a químicos ambientales y enfermedad materna activa. Las condiciones paternas descritas fueron alcoholismo y/o drogadicción, enfermedad y edad ≥ 40 años en bajo porcentaje. Conclusión: El síndrome de Möbius y Möbius Like es una patología poco frecuente de la cual aún se debe seguir investigando sobre su etiología, para plantear posibles medidas de prevención.

Eficacia del diagnóstico prenatal no invasivo por células fetales libres en sangre materna 1990-2014. Revisión sistemática

ANTECEDENTES: El aislamiento de células fetales libres o ADN fetal en sangre materna abre una ventana de posibilidades diagnósticas no invasivas para patologías monogénicas y cromosómicas, además de permitir la identificación del sexo y del RH fetal. Actualmente existen múltiples estudios que evalúan la eficacia de estos métodos, mostrando resultados costo-efectivos y de menor riesgo que el estándar de oro. Este trabajo describe la evidencia encontrada acerca del diagnóstico prenatal no invasivo luego de realizar una revisión sistemática de la literatura. OBJETIVOS: El objetivo de este estudio fue reunir la evidencia que cumpla con los criterios de búsqueda, en el tema del diagnóstico fetal no invasivo por células fetales libres en sangre materna para determinar su utilidad diagnóstica.  MÉTODOS: Se realizó una revisión sistemática de la literatura con el fin de determinar si el diagnóstico prenatal no invasivo por células fetales libres en sangre materna es efectivo como método de diagnóstico.  RESULTADOS: Se encontraron 5,893 artículos que cumplían con los criterios de búsqueda; 67 cumplieron los criterios de inclusión: 49.3% (33/67) correspondieron a estudios de corte transversal, 38,8% (26/67) a estudios de cohortes y el 11.9% (8/67) a estudios casos y controles. Se obtuvieron resultados de sensibilidad, especificidad y tipo de prueba. CONCLUSIÓN: En la presente revisión sistemática, se evidencia como el diagnóstico prenatal no invasivo es una técnica feasible, reproducible y sensible para el diagnóstico fetal, evitando el riesgo de un diagnóstico invasivo.

Systematic studies on 3d-3d and 3d-4f multimetallic complexes with interesting magnetic and/or luminescent properties

324 p.

A tungsten complex containing a highly delocalized metal-ligand system

Nucleophilic attack of (triphenylphosphonio) cyclopentadienide on the dichlorodiazomethane-tungsten complex trans[ BrW(dppe)(2)(N2CCl2)]PF6 [dppe is 1,2-bis(diphenylphosphino) ethane] results in C-C bond formation and affords the title compound, trans-[W(C24H18ClN2P)Br(C26H24P2)(2)]PF6 center dot 0.6CH(2)Cl(2). This complex, bis[1,2- bis(diphenylphosphino)ethane] bromido{chloro[3-(triphenylphosphonio) cyclopentadienylidene] diazomethanediido} tungsten hexafluorophosphate dichloromethane 0.6-solvate, contains the previously unknown ligand chloro[3-(triphenylphosphonio) cyclopentadienylidene] diazomethane. Evidence from bond lengths and torsion angles indicates significant through-ligand delocalization of electron density from tungsten to the nominally cationic phosphorus(V) centre. This structural analysis clearly demonstrates that the tungsten-dinitrogen unit is a powerful pi-electron donor with the ability to transfer electron density from the metal to a distant acceptor centre through an extended conjugated ligand system. As a consequence, complexes of this type could have potential applications as nonlinear optical materials and molecular semiconductors.

Mono- and binuclear bipyridyl derivatives of the Mo(eta(3)-C3H5)(CO)(2) fragment: structural studies and fluxionality in solution

New mono- and binuclear complexes of the Mo(eta(3)-C3H5)(CO)(2) fragment, containing bipyridyl ligands (2,2'-bpy, 4,4'-Me-2-2,2'-bpy) as chelates, and mono- (4-CNpy, 4-Mepy, NCMe, Br) or bidentate nitrogen ligands (4,4'-bpy, bipyridylethylene, pyrazine) as terminal or bridging ligands, respectively, were prepared. The binuclear complex [{Mo(eta(3)-C3H5)(CO)(2)(2,2'-bpy)}(2)(mu-4,4'-bpy)][PF6](2) (2) was shown by X-ray diffraction to assemble in the crystal forming large channels with a rectangular section. A longer bridge, such as bipyridylethylene, led to a different structure (3). 4-CNpy behaved as monodentate ligand (4), coordinating through the pyridine nitrogen as a terminal ligand. NMR spectroscopy studies showed that the complexes exhibited a fluxional behavior in solution, the endo and exo forms of the more symmetrical equatorial isomers being usually present and interconverting in solution. The solid state structures of the complexes revealed a preference for the more symmetrical equatorial isomer, with the two chelate ligands in trans positions in the binuclear species. The rings tended to become parallel in the organized crystal. (C) 2003 Elsevier B.V. All rights reserved.

Supramolecular aggregates between carboxylate anions and an octaaza macrocyclic receptor

The 28-membered octaazamacrocycle Me-2[28]py(2)N(6) was used as a receptor for the molecular recognition of aromatic and aliphatic carboxylate substrates. The receptor-substrate binding behaviour of (H6Me2[28]py(2)N(6))(6+) with an aliphatic (-O2C(CH2)(n)CO2-, n=0 to 4) and an aromatic (phthalate, isophthalate, terephthalate, 4,4'-dibenzoate, benzoate, 3- and 4-nitrobenzoate) series of carboxylate anions was evaluated by H-1 NMR spectroscopy (carried out in DMSO-d(6) at 300 K). Two association constants were found for most of the studied cases, except for 3- and 4-nitrobenzoate for which only K-1 was determined. For oxalate, malonate, benzoate and dibenzoate anions only the beta(2) constants could be obtained. The values of the first association constant cover a range from 2.86 to 3.69 (log units), and the second stepwise constant from 2.15 to 2.89 (also in log units). No special selectivity was found but the highest values were determined for adipate and the lowest for the monoprotic 3- and 4-nitrobenzoates. Single crystal X-ray structures of H6Me2[28]py(2)N(6)(6+) with terephthalate, 1, and 4,4'-dibenzoate (2) were determined showing supramolecular entities with general formula (H6Me2[28]py(2)N(6)).(substrate)(2)(PF6)(2).4H(2)O. These anions are the building blocks of an extensive 3-D network of hydrogen bonds.

Carboxylate anions binding and sensing by a novel tetraazamacrocycle containing ferrocene as receptor

A tetraazamacrocycle containing ferrocene moieties has been synthesized and characterized. The tetraprotonated form of this compound was evaluated as a receptor (R) for anion recognition of several substrates (S), Cl-, PF6-, HSO4-, H2PO4- and carboxylates, such as p-nitrobenzoate (p-nbz(-)), phthalate (ph(2-)), isophthalate (iph(2-)) and dipicolinate (dipic(2-)). H-1 NMR titrations in CD3OD indicated that this receptor is not suitable for recognizing HSO4- and H2PO4-, but weakly binds p-nbz(-), and strongly interacts with ph(2-), dipic(2-), and iph(2-) anions forming 1 : 2 assembled species. The largest beta(2) binding constant was determined for ph(2-), followed by dipic(2-) and finally iph(2-). The effect of the anionic substrates on the electron-transfer process of the ferrocene units of R was evaluated using cyclic voltammetry (CV) and square wave voltammetry (SWV) in methanol solution and 0.1 mol dm(-3) (CH3)(4)NCl as the supporting electrolyte. Titrations of the receptor were undertaken by addition of anion solutions in their tetrabutylammonium or tetramethylammonium forms. The protonated ligand exhibits a reversible voltammogram, which shifts cathodically in the presence of the substrates. The data revealed kinetic constraints in the formation of the receptor/substrate entity for dipic(2-), ph(2-) and iph(2-) anions, but not for p-nbz(-). In spite of the slow kinetics of assembled species formation with the ph(2-) substrate, this anion provides the largest redox-response when the supramolecular entity is formed, followed by dipic(2-), iph(2-) and finally p-nbz(-) anions. This trend is in agreement with the H-1 NMR results and the values of the binding constants. Single crystal X-ray structures of the receptor with PF6-, ph(2-), iph(2-) and p-nbz(-) were carried out and showed that supermolecules with a RS2 stoichiometry are formed with the first three anions, but RS4 with p-nbz(-). In all cases the binding occurs outside the macrocyclic cavity via N-H center dot center dot center dot O=C hydrogen bonds for carboxylate anions and N - H center dot center dot center dot F hydrogen bonds for the PF6- anion, which is in agreement with the solution results. The macrocyclic framework adopts different conformations in order to interact with each substrate having Fe center dot center dot center dot Fe intramolecular distances ranging from 10.125(14) to 12.783(15) angstrom.

Imidazole-imidazole stacking in some inorganic complexes

Reaction of CuCl2 center dot 2H(2)O with the 1:1 condensate (L) of 2-(2-aminoethyl) pyridine and 1-methyl-2-imidazolecarboxaldehyde in methanol yields monomeric CuLCl2 center dot H2O (1). Recrystallisation of 1 from aqueous methanol medium containing excess of PF6- affords the 1D coordination polymer [CuLCl](n)(PF6)(n) (2). A chloride bridge results in the coordination polymer. A face-to-face interaction is observed between the imidazole rings in 2. The interaction influences the structure and magnetic properties of 2 markedly. The complex 2 is ferromagnetic with a J value of 1.79 +/- 0.01 cm (1). The imidazole fragments in 2 are coordinated to the metal. In mononuclear [HgL2 ''](ClO4)(2), where L '' is the 1:2 condensate of ethylenediamine and 1-methyl-2-imidazolecarboxaldehyde, the imidazolyl moieties are not under the direct influence of the metal. Here the imidazole-imidazole interaction is angular and more distant. (C) 2009 Elsevier B.V. All rights reserved.

Ruthenium (III) complexes of tetradentate NSNO pyridylthioazophenolates: Synthesis, spectral studies, crystal structure and redox properties

A family of ruthenium (III) complexes of tetradentate monobasic NSNO donor chelators (HL) have been synthesized and isolated in their pure form. On chromatographic separation, trans-dichloro and cis-dichloro ruthenium (111) complexes of pyridylthioazophenolates are eluted using 19:1 and 7:3 (v/v) DCM-MeOH mixtures, respectively. Both cis and trans isomers of the dark brown colored ruthenium (111) complexes, having the general formula of [Ru(L)Cl-2], have been characterized by elemental analyses, spectroscopic and other physico-chemical tools. The magnetic moments of both the cis- and trans-[Ru(L)Cl-2] complexes are in the range of 1.71-1.79 BM. One of the complexes, trans-[Ru(L1)Cl-2] (2a), has been subjected to single-crystal X-ray analysis which confirms that the chlorines are in mutually trans positions in the molecule. The EPR spectra of the cis-[Ru(L)Cl-2] complexes (1) in DMF are consistent with the fact that the complexes are low-spin octahedral with one unpaired electron having three different g values (g(x) not equal g(y) not equal g(z)) complexes are monomeric with an octahedral coordination sphere. The electrochemical studies of [Ru(L)Cl,] in DMF show a quasi-reversible voltammogram. The reduction potentials for the cis-isomers are comparatively lower than those of the corresponding trans isomers. On reaction with the bidentate bipyridyl ligand in the presence of AgNO3, the cis-[Ru(L)Cl-2] complexes (1) produce a series of complexes with the general formula [Ru(L)(bpy)(2)](PF6)(2) (3). which have also been characterized by elemental analyses, spectroscopic and other physico-chemical tools. (c) 2006 Elsevier Ltd. All rights reserved.

A new emissive Ru(II)N5O core

From the reaction of cis-Ru(1,10-phenanthroline)(2)Cl(2 center dot)2H(2)O with 2-picolinic acid in 1:1 molar ratio in degassed methanol-water mixture, [Ru(1,10-phenanthroline)(2)(2-picolinate)]PF6 center dot H2O (1) has been isolated as a red compound by adding excess of NH4PF6. Single crystal X-ray crystallography shows that the metal in 1 has an octahedral N5O coordination sphere. Complex 1 displays (MLCT)-M-1 bands in the 400-500 nm region in acetonitrile. Upon excitation at 435 nm, complex 1 gives rise to a broad emission band at 675 nm in acetonitrile at room temperature with a quantum yield of 0.0022. The energy of the MLCT state in 1 is estimated as 1.99 eV. Since, from cyclic voltammetry, the ground state potential of the Ru(II/III) couple in 1 is found to be 1.01 V vs NHE, the potential of the same couple in the excited state is calculated as -0.98 V vs NHE. The emissive state in 1 seems to be the triplet Ru(II) -> 1, 10-phenanthroline charge transfer state.

Acid dissociation of 2,2 '-dipyridylamine in non-aqueous medium when chelated to some Ru(II)N-4 cores

[Ru(2,2'-bipyridine)(2)(Hdpa)](BF4)(2) center dot 2H(2)O (1), [Ru(1,10-phenanthroline)(2)(Hdpa)] (PF6)(2) center dot CH2Cl2 (2) and [Ru(4,4,4',4'-tetramethyl-2,2'- bisoxazoline)(2)(Hdpa)] (PF6)(2) (3) are synthesized where Hdpa is 2,2'-dipyridylamine. The X-ray crystal structures of 1 and 2 have been determined. Hdpa in 1 and 2 is found to bind the metal via the two pyridyl N ends. Comparing the NMR spectra in DMSO-d(6), it is concluded that 3 has a similar structure. The pK(a) values (for the dissociation of the NH proton in Hdpa) of free Hdpa and its complexes are determined in acetonitrile by exploiting molar conductance. These correlate linearly with the chemical shift of the NH proton in the respective entities. (C) 2007 Elsevier B.V. All rights reserved.

A hemiacetalate complex of Ru(II)

Reaction of cis-Ru(bisox)(2)Cl-2, where bisox is 4,4,4',4'-tetramethyl-2,2'-bisoxazoline, with excess of pyridine-2-carboxaldehyde (py-2-al) in 1:1 (v/v) methanol-water mixture under nitrogen atmosphere and subsequent addition of excess of NH4PF6 give [Ru(bisox)(2)(py-2-al)](PF6)(2)center dot H2O (1). Refluxing of 1 in dehydrated methanol in presence of triethylamine yields the corresponding hemiacetalate complex: [Ru(bisox)(2) (pyridine-2-(alpha-methoxymethanolato))] PF6 center dot 1.5H(2)O (2). Both the complexes have been characterised by single crystal X-ray crystallography, FTIR and NMR. In cyclic voltammetry in acetonitrile at a glassy carbon electrode, 2 displays a quasireversible Ru(II/III) couple at 1.08 V versus NHE which is not observed in 1. A tentative mechanism is proposed for the conversion of 1 to 2. DFT calculations with the LanL2DZ basis set have been performed to investigate these observations theoretically. (C) 2008 Elsevier B.V. All rights reserved.