ES2819307B2 - CHIRAL IONIC LIQUIDS BASED ON L-CARNITINE ESTERS AND THEIR USE AS CHIRAL SELECTORS IN DUAL SYSTEMS FOR THE SEPARATION OF ENANTIOMERS BY CAPILLARY ELECTROPHORESIS - Google Patents
CHIRAL IONIC LIQUIDS BASED ON L-CARNITINE ESTERS AND THEIR USE AS CHIRAL SELECTORS IN DUAL SYSTEMS FOR THE SEPARATION OF ENANTIOMERS BY CAPILLARY ELECTROPHORESIS Download PDFInfo
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- ES2819307B2 ES2819307B2 ES201930903A ES201930903A ES2819307B2 ES 2819307 B2 ES2819307 B2 ES 2819307B2 ES 201930903 A ES201930903 A ES 201930903A ES 201930903 A ES201930903 A ES 201930903A ES 2819307 B2 ES2819307 B2 ES 2819307B2
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- chiral
- carnitine
- separation
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- anion
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- 238000000926 separation method Methods 0.000 title claims description 56
- 239000002608 ionic liquid Substances 0.000 title claims description 25
- PHIQHXFUZVPYII-ZCFIWIBFSA-N (R)-carnitine Chemical class C[N+](C)(C)C[C@H](O)CC([O-])=O PHIQHXFUZVPYII-ZCFIWIBFSA-N 0.000 title claims description 22
- 230000009977 dual effect Effects 0.000 title claims description 19
- 238000005251 capillar electrophoresis Methods 0.000 title claims description 17
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical compound C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 title description 11
- 235000001014 amino acid Nutrition 0.000 claims description 39
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- 230000004071 biological effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229940078469 dl- cysteine Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- QSZMZKBZAYQGRS-UHFFFAOYSA-N lithium;bis(trifluoromethylsulfonyl)azanide Chemical compound [Li+].FC(F)(F)S(=O)(=O)[N-]S(=O)(=O)C(F)(F)F QSZMZKBZAYQGRS-UHFFFAOYSA-N 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
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- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B57/00—Separation of optically-active compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/08—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
- C07C311/48—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups having nitrogen atoms of sulfonamide groups further bound to another hetero atom
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
DESCRIPCIÓNDESCRIPTION
LÍQUIDOS IÓNICOS QUIRALES BASADOS EN ÉSTERES DE L-CARNITINA Y SU IONIC CHIRAL LIQUIDS BASED ON L-CARNITINE ESTERS AND ITS
USO COMO SELECTORES QUIRALES EN SISTEMAS DUALES PARA LA USE AS CHIRAL SELECTORS IN DUAL SYSTEMS FOR
SEPARACIÓN DE ENANTIÓME ROS POR EL ECTROFORESIS CAPILARSEPARATION OF ENANTHYOMES BY CAPILLARY ECTROPHORESIS
SECTOR DE LA TÉCNICATECHNICAL SECTOR
La presente invención se enmarca en el campo de la obtención de líquidos iónicos y su uso en sistemas duales de selectores quirales para la separación de enantiómeros por Electroforesis Capilar.The present invention is framed in the field of obtaining ionic liquids and their use in dual systems of chiral selectors for the separation of enantiomers by Capillary Electrophoresis.
ESTADO DE LA TÉCNICASTATE OF THE ART
La Electroforesis Capilar (CE) es una técnica analítica con enorme potencial para la separación de enantiómeros debido a sus interesantes características como son su elevada eficacia, bajo consumo de disolventes y muestras, y posibilidad de añadir un selector quiral a la fase móvil para llevar a cabo una separación enantiomérica. El análisis quiral tiene en la actualidad un enorme interés debido a la distinta actividad biológica que pueden presentar los enantiómeros de un compuesto quiral. La necesidad de controlar la presencia de los diferentes enantiómeros en muestras de interés farmacológico, clínico, alimentario o medioambiental requiere del desarrollo de metodologías analíticas avanzadas sensibles que permitan determinar de forma individual dichos enantiómeros.Capillary Electrophoresis (CE) is an analytical technique with enormous potential for the separation of enantiomers due to its interesting characteristics such as its high efficiency, low consumption of solvents and samples, and the possibility of adding a chiral selector to the mobile phase to carry out perform enantiomeric separation. Chiral analysis is currently of enormous interest due to the different biological activity that the enantiomers of a chiral compound can present. The need to control the presence of the different enantiomers in samples of pharmacological, clinical, food or environmental interest requires the development of sensitive advanced analytical methodologies that allow these enantiomers to be individually determined.
La Cromatografía Electrocinética (EKC) es un modo de separación en el formato de la CE en el cual se añade un selector quiral a la fase móvil con el fin de que interaccione de forma diferencial con cada uno de los enantiómeros de un compuesto quiral dando lugar a su separación. Existe un gran número de selectores quirales que se pueden utilizar en EKC como son las ciclodextrinas, los antibióticos macrocíclicos, las proteínas, micelas poliméricas, éteres corona, etc. Ello confiere a EKC una extraordinaria versatilidad a la hora de llevar a cabo una separación quiral por esta técnica. La predicción del selector quiral más adecuado para llevar a cabo una separación quiral es difícil todavía a pesar del gran número de estudios que se han llevado a cabo para estudiar las interacciones analito-selector quiral por distintas técnicas. Por ello, en ocasiones, cuando no es posible encontrar un selector quiral adecuado para llevar a cabo una separación enantiomérica, puede ser de gran utilidad el uso de mezclas de selectores quirales constituyendo sistemas duales. En este sentido, los líquidos iónicos han demostrado un gran potencial como agentes que son capaces de originar importantes efectos sinérgicos cuando se utilizan en combinación con otros selectores quirales. De hecho, aunque los líquidos iónicos se han empleado como selectores quirales únicos en el medio de separación en EKC [1,2], la mayor parte de los trabajos llevados a cabo se han basado en el empleo de mezclas de líquidos iónicos con otros selectores quirales, principalmente con ciclodextrinas y antibióticos macrocíclicos [1, 2, 3].Electrokinetic Chromatography (EKC) is a separation mode in the CE format in which a chiral selector is added to the mobile phase in order for it to interact differentially with each of the enantiomers of a chiral compound, giving rise to to their separation. There are a large number of chiral selectors that can be used in EKC such as cyclodextrins, macrocyclic antibiotics, proteins, polymeric micelles, crown ethers, etc. This gives EKC extraordinary versatility when it comes to carrying out chiral separation by this technique. The prediction of the most suitable chiral selector to carry out a chiral separation is still difficult despite the large number of studies that have been carried out to study the analyte-chiral selector interactions by different techniques. For this reason, on occasions, when it is not possible to find a suitable chiral selector to carry out After enantiomeric separation, the use of mixtures of chiral selectors constituting dual systems can be very useful. In this sense, ionic liquids have shown great potential as agents that are capable of producing important synergistic effects when used in combination with other chiral selectors. In fact, although ionic liquids have been used as unique chiral selectors in the separation medium in EKC [1,2], most of the work carried out has been based on the use of ionic liquid mixtures with other selectors. chirals, mainly with cyclodextrins and macrocyclic antibiotics [1, 2, 3].
Los líquidos iónicos son sales orgánicas con puntos de fusión por debajo de 100 °C. Están formados por cationes orgánicos y voluminosos entre los cuales se encuentran cationes amonio, fosfonio, alquilimidazolio, piridinio, pirrolidinio y por aniones orgánicos o inorgánicos tales como hexafluorofosfato, tetrafluoroborato, triflato, etc.Ionic liquids are organic salts with melting points below 100 ° C. They are formed by bulky organic cations, among which are ammonium, phosphonium, alkylimidazolium, pyridinium, pyrrolidinium cations and by organic or inorganic anions such as hexafluorophosphate, tetrafluoroborate, triflate, etc.
Dentro de los líquidos iónicos se pueden destacar aquellos en los que o bien la parte catiónica, o la parte aniónica o ambas, son quirales, en cuyo caso se denominan líquidos iónicos quirales (CILs). Dentro del grupo de los CILs, se pueden mencionar aquellos cuya parte catiónica es un aminoácido quiral habiéndose descrito en la bibliografía la síntesis de diferentes CILs dentro de este grupo [2,4, 5].Within the ionic liquids, those in which either the cationic part, or the anionic part or both, are chiral, in which case they are called chiral ionic liquids (CILs). Within the group of CILs, mention can be made of those whose cationic part is a chiral amino acid, the synthesis of different CILs within this group having been described in the literature [2,4, 5].
Sin embargo, solo algunos de los CILs sintetizados y cuya parte catiónica es un aminoácido quiral, se han empleado en EKC como selectores quirales únicos o en sistemas duales con otros selectores quirales dando lugar a la separación enantiomérica de distintos compuestos (Tabla 1).However, only some of the CILs synthesized and whose cationic part is a chiral amino acid, have been used in EKC as single chiral selectors or in dual systems with other chiral selectors giving rise to the enantiomeric separation of different compounds (Table 1).
Tabla 1. CILs cuya parte catiónica es un aminoácido quiral y que han sido empleados como selectores quirales únicos o en sistemas duales de selectores quirales en EKC, o como ligandos quirales en CE de intercambio de ligandos.Table 1. CILs whose cationic part is a chiral amino acid and which have been used as single chiral selectors or in dual systems of chiral selectors in EKC, or as chiral ligands in CE of ligand exchange.
Abreviaturas: Ala: Alanina; Asn: Asparagina; Asp: Ácido aspártico; CF 3 CO 2- : trifluoroacetato, Cl-Dns: cloruro de dansilo; Ile: Isoleucina; Met: Metionina, NTÍ 2 : bis(trifluorometilsufonil)imida; Lac: Lactato; Phe: Fenilalanina, Phn: Fenilalaninamida , Pro: Prolina, Ser: Serina; Thr: Treonina, Tyr: Tirosina, Val: Valina. Abbreviations: Ala: Alanine; Asn: Asparagine; Asp: Aspartic acid; CF 3 CO 2- : trifluoroacetate, Cl-Dns: dansyl chloride; Ile: Isoleucine; Met: Methionine, NTÍ 2 : bis ( trifluoromethylsufonyl) imide; Lac: Lactate; Phe: Phenylalanine, Phn: Phenylalaninamide, Pro: Proline, Ser: Serine; Thr: Threonine, Tyr: Tyrosine, Val: Valine.
Las patentes CN105152950, CN105152949 y CN105061236 tratan de la síntesis de líquidos iónicos en los que la parte catiónica es un aminoácido, tal como tirosina, fenilalanina, lisina, leucina o alanina, que no coincide con el de la presente invención (éster de L-carnitina). En la solicitud de patente US2009145197 se citan otros aminoácidos como parte catiónica siendo uno de ellos la D-carnitinanitrilo que tampoco coincide con el éster de L-carnitina.Patents CN105152950, CN105152949 and CN105061236 deal with the synthesis of ionic liquids in which the cationic part is an amino acid, such as tyrosine, phenylalanine, lysine, leucine or alanine, which does not coincide with that of the present invention (L- ester). carnitine). In patent application US2009145197 other amino acids are mentioned as cationic part, one of them being D-carnitinanitrile, which also does not coincide with the L-carnitine ester.
Uno de los problemas resueltos por la presente invención es proporcionar un proceso de obtención de CILs derivados de ésteres de L-carnitina como selectores quirales, cuya síntesis no había sido previamente descrita, a partir de disoluciones acuosas de dichos ésteres con sales de plata, sódicas o potásicas derivadas de los correspondientes aniones inorgánicos u orgánicos de una manera sencilla y económica. One of the problems solved by the present invention is to provide a process for obtaining CILs derived from L-carnitine esters as chiral selectors, the synthesis of which had not been previously described, from aqueous solutions of said esters with silver, sodium salts. or potassium derived from the corresponding inorganic or organic anions in a simple and inexpensive way.
Los ClLs de la invención son útiles en la separación de enantiómeros de compuestos quirales de interés alimentario, farmacéutico, cosmético, agroquímico o medioambiental, y en el control de calidad de productos de estos sectores.The ClLs of the invention are useful in the separation of enantiomers of chiral compounds of food, pharmaceutical, cosmetic, agrochemical or environmental interest, and in the quality control of products from these sectors.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
Un primer aspecto de la presente invención se refiere a un líquido iónico quiral (CIL) no prótico basado en ésteres de la L-carnitina de estructura general IA first aspect of the present invention refers to a non-protic chiral ionic liquid (CIL) based on L-carnitine esters of general structure I
en la que:in which:
- la parte catiónica es asimétrica formada por un catión amonio diferentemente sustituido, en el que los sustituyentes no son todos iguales, derivado del correspondiente éster (-COOR) del aminoácido quiral L-carnitina. - R está seleccionado entre alquilo de 1 a 5 átomos de carbono, lineal o ramificado, sustituido o no sustituido, bencilo, fenilo sustituido o no sustituido, heterociclos así como heteroarilos,- the cationic part is asymmetric formed by a differently substituted ammonium cation, in which the substituents are not all the same, derived from the corresponding ester (-COOR) of the chiral amino acid L-carnitine. - R is selected from alkyl of 1 to 5 carbon atoms, linear or branched, substituted or unsubstituted, benzyl, substituted or unsubstituted phenyl, heterocycles as well as heteroaryls,
- la parte aniónica (A-) está formada por un anión inorgánico u orgánico.- the anionic part (A-) is formed by an inorganic or organic anion.
El grupo R según realizaciones particulares está seleccionado entre metilo, etilo, propilo, isopropilo, tert-butilo, n-butilo, pentilo, bencilo, fenilo, un grupo fenilo substituido mediante la introducción de un sustituyente o dos, iguales o diferentes, seleccionados entre, nitro, halo, alquil, alcoxi, hidroxi, trifluorometil, cianuro, heterociclos y heteroarilos. Entre los aniones inorgánicos el líquido iónico quiral puede estar seleccionado entre haluros (cloruros (Cl-), bromuro (Br-), Ioduro (I-)), hexafluorofosfato (PF6-) y tetrafluoroboratos (BF 4 -).The group R according to particular embodiments is selected from methyl, ethyl, propyl, isopropyl, tert-butyl, n-butyl, pentyl, benzyl, phenyl, a phenyl group substituted by introducing one or two substituents, the same or different, selected from , nitro, halo, alkyl, alkoxy, hydroxy, trifluoromethyl, cyanide, heterocycles, and heteroaryls. Among the inorganic anions, the chiral ionic liquid can be selected from halides (chlorides (Cl-), bromide (Br-), Iodide (I-)), hexafluorophosphate (PF6-) and tetrafluoroborates (BF 4 -).
Entre los aniones orgánicos el líquido iónico quiral puede ser acetato (AcO-), triflato(TfO-), taurinato, tetracianoborato (B(CN) 4 -), n-octil sulfato, docusato, L-lactato, amino ácidos, bis(trifluorometilsufonil)imida (NTf 2 ), bis(pentafluoroetilsulfonil)imida, anión de tricianometano, tris(pentafluoroetil)trifluorofosfato (FAP), así como otros aniones orgánicos descritos en las referencias [5] (Chira! ionio Liquids: A compendium of synthesis and applications) y [20] (Ionic liquids in analytical chemistry).Among the organic anions, the chiral ionic liquid can be acetate (AcO-), triflate (TfO-), taurinate, tetracyanoborate (B (CN) 4 -), n-octyl sulfate, docusate, L-lactate, amino acids, bis ( trifluoromethylsufonyl) imide (NTf 2 ), bis (pentafluoroethylsulfonyl) imide, tricyanomethane anion, tris (pentafluoroethyl) trifluorophosphate (FAP), as well as other organic anions described in references [5] ( Chira! ionio Liquids: A compendium of synthesis and applications) and [20] (Ionic liquids in analytical chemistry).
Según realizaciones particulares, la parte aniónica comprende un anión seleccionado entre un haluro, hexafluorofosfato o tetrafluoroborato, acetato, triflato, tetracianoborato, n-octil sulfato, docusato, L-lactato, amino ácidos, bis(trifluorometilsufonil)imida (NTf 2 ) o bis(pentafluoroetilsulfonil)imida.According to particular embodiments, the anionic part comprises an anion selected from a halide, hexafluorophosphate or tetrafluoroborate, acetate, triflate, tetracyanoborate, n-octyl sulfate, docusate, L-lactate, amino acids, bis (trifluoromethylsulfonyl) imide (NTf 2 ) or bis (pentafluoroethylsulfonyl) imide.
Como es bien conocido, la polaridad y los caracteres hidrofóbicos e hidrofílicos, propiedades físico-químicas, como conductividad, volatilidad, presión de vapor, estabilidad térmica, y su correspondiente miscibilidad en disolventes orgánicos como en agua de los diferentes líquidos iónicos reside fundamentalmente en la combinación que existe entre el catión y el anión seleccionado. De dichas combinaciones se obtienen diferentes CILs con diferentes propiedades como selectores quirales o como componentes de sistemas duales de selectores quirales [2].As is well known, the polarity and the hydrophobic and hydrophilic characters, physicochemical properties, such as conductivity, volatility, vapor pressure, thermal stability, and their corresponding miscibility in organic solvents as in water of the different ionic liquids resides fundamentally in the combination that exists between the cation and the selected anion. From these combinations, different CILs are obtained with different properties as chiral selectors or as components of dual systems of chiral selectors [2].
En una realización preferida de la presente invención, la parte catiónica del CIL es el éster metílico de L-carnitina y la parte aniónica es uno cualquiera de los aniones inorgánicos u orgánicos descritos anteriormente.In a preferred embodiment of the present invention, the cationic part of the CIL is the methyl ester of L-carnitine and the anionic part is any one of the inorganic or organic anions described above.
En otra realización preferida, el anión es bis(trifluorometilsufonil)imida (NTf 2 ).In another preferred embodiment, the anion is bis (trifluoromethylsufonyl) imide (NTf 2 ).
La presente invención también se refiere a un compuesto intermedio de fórmula general IIThe present invention also relates to an intermediate compound of general formula II
en la que X- es un anión haluro y "R” tiene el mismo significado que el indicado anteriormente para los compuestos de fórmula I. Preferentemente el anión X- es un cloruro.wherein X- is a halide anion and "R" has the same meaning as indicated above for the compounds of formula I. Preferably the anion X- is a chloride.
La presente invención también se refiere a un sistema dual para la separación de compuestos químicos que comprende un líquido iónico quiral definido anteriormente y un segundo componente que es un selector quiral. De mantera preferente, el selector quiral es una ciclodextrina.The present invention also relates to a dual system for the separation of chemical compounds that comprises a chiral ionic liquid defined above and a second component that is a chiral selector. Preferably, the chiral selector is a cyclodextrin.
La presente invención también se refiere a un procedimiento de obtención de los ClLs descritos y que comprende los siguientes pasos: The present invention also refers to a procedure for obtaining the ClLs described and comprising the following steps:
a) formación del compuesto intermedio de fórmula general II mediante una reacción de un haluro de L-carnitina (III), en medio ácido alcohólico a 40- 120°C hasta la desaparición del material de partida, durante un tiempo comprendido entre 3 a 24 horas y posterior eliminación del medio alcohólico,a) formation of the intermediate compound of general formula II by a reaction of a halide of L-carnitine (III), in alcoholic acid medium at 40-120 ° C until the disappearance of the starting material, for a time between 3 to 24 hours and subsequent elimination of the alcoholic medium,
b) opcionalmente, un intercambio del correspondiente anión haluro, preferentemente cloruro, en el derivado II obtenido en el paso (a) en medio acuoso, con una sal seleccionada entre una sal de plata, sales sódicas, sales potásicas y sales de litio derivadas del correspondiente anión inorgánico u orgánico representado como A- en la fórmula I, o con el ácido correspondiente a dicho anión.b) optionally, an exchange of the corresponding halide anion, preferably chloride, in derivative II obtained in step (a) in an aqueous medium, with a salt selected from a silver salt, sodium salts, potassium salts and lithium salts derived from corresponding inorganic or organic anion represented as A- in formula I, or with the acid corresponding to said anion.
A efectos de la presente invención, la reacción de intercambio aniónico se lleva a cabo mediante un proceso denominado metátesis aniónica, generando, de esta forma los correspondientes CILs. Esta etapa comprende el tratamiento de cantidades estequiométricas de la correspondiente sal de haluro (II) con una sal seleccionada entre sales de plata, sales sódicas, sales potásicas y sales de litio derivadas del correspondiente anión inorgánico u orgánico representado como A- en la fórmula I derivadas de los correspondientes aniones inorgánicos u orgánicos representados como A- en la fórmula I, tales como sales derivadas de bromuro (Br-), ioduro (I-)), hexafluorofosfato (PF6-)[16], derivados de tetrafluoroborato (BF 4 -), acetato (AcO-), triflato (TfO-), tetracianoborato (B(CN) 4 -), n-octil sulfato, Docusato, L-lactato, aminoácidos, bis(trifluorometilsufonil)imida (NTf 2 ) [17], bis(pentafluoroetilsulfonil)imida, acetato (CH 3 CO 2 -), trifluoroacetato (CF 3 CO 2 -),For the purposes of the present invention, the anion exchange reaction is carried out through a process called anionic metathesis, thus generating the corresponding CILs. This step comprises the treatment of stoichiometric amounts of the corresponding halide (II) salt with a salt selected from silver salts, sodium salts, potassium salts and lithium salts derived from the corresponding inorganic or organic anion represented as A- in formula I derived from the corresponding inorganic or organic anions represented as A- in formula I, such as salts derived from bromide (Br-), iodide (I-)), hexafluorophosphate (PF6 -) [16], tetrafluoroborate derivatives (BF 4 -), acetate (AcO-), triflate (TfO-), tetracyanoborate (B (CN) 4 -), n-octyl sulfate, Docusate, L-lactate, amino acids, bis (trifluoromethylsufonyl) imide (NTf 2 ) [17] , bis (pentafluoroethylsulfonyl) imide, acetate (CH 3 CO 2 -), trifluoroacetate (CF 3 CO 2 -),
- o con el correspondiente ácido libre del apropiado anión.- or with the corresponding free acid of the appropriate anion.
En la etapa a) el haluro de L-carnitina (III) puede ser de origen comercial, y es preferentemente cloruro.In step a) the L-carnitine (III) halide can be of commercial origin, and is preferably chloride.
"Medio ácido alcohólico” significa que se utiliza un alcohol, que puede ser cualquiera, y preferentemente está seleccionado entre metanol, etanol, isopropanol, n-butanol, tert- butanol, preferentemente metanol, y un ácido, que es preferentemente, ácido clorhídrico."Medium alcoholic acid" means that an alcohol is used, which can be any, and is preferably selected from methanol, ethanol, isopropanol, n-butanol, tert - butanol, preferably methanol, and an acid which is preferably hydrochloric acid.
El alcohol utilizado en el medio alcohólico en la etapa a) se elimina a presión reducida, comprendida entre 20 y 200 mbares, después de terminar la reacción.The alcohol used in the alcoholic medium in step a) is removed under reduced pressure, comprised between 20 and 200 mbar, after the reaction has ended.
Según realizaciones particulares, la temperatura de la reacción de la etapa a) está comprendida entre 40 y 100°C. Según realizaciones particulares adicionales, la temperatura de la reacción de la etapa a) está comprendida entre 40 y 90°C.According to particular embodiments, the reaction temperature of step a) is between 40 and 100 ° C. According to additional particular embodiments, the reaction temperature of step a) is between 40 and 90 ° C.
Según realizaciones particulares el tiempo de reacción de la etapa a) está comprendido entre 3 y 15 horas. Según realizaciones particulares adicionales, el tiempo de reacción de la etapa a) está comprendido entre 3 y 8 horas.According to particular embodiments, the reaction time of step a) is between 3 and 15 hours. According to additional particular embodiments, the reaction time of step a) is between 3 and 8 hours.
La reacción de metátesis aniónica de la etapa b) se lleva a cabo en medio acuoso por tratamiento de un equivalente del compuesto de fórmula general II con un equivalente de una sal de plata, sales sódicas, sales potásicas y sales de litio derivadas del correspondiente anión inorgánico u orgánico, preferentemente con sales de litio, a temperaturas comprendidas entre 15 y 50°C, más preferentemente entre 15°C y 30°C, aún más preferentemente a 20°C, generándose como subproducto de reacción agua o las correspondientes sales de haluro.The anionic metathesis reaction of step b) is carried out in an aqueous medium by treating one equivalent of the compound of general formula II with one equivalent of a silver salt, sodium salts, potassium salts and lithium salts derived from the corresponding anion. inorganic or organic, preferably with lithium salts, at temperatures between 15 and 50 ° C, more preferably between 15 ° C and 30 ° C, even more preferably at 20 ° C, generating as a by-product of reaction water or the corresponding salts of halide.
Realizaciones particulares del procedimiento se muestran representadas en el esquema siguiente:Particular embodiments of the procedure are shown represented in the following diagram:
A: NO3-, BF4-, NTF2-,A: NO3-, BF4-, NTF2-,
CH3COO-, CH3CH2OHCO2-, etc...CH3COO-, CH3CH2OHCO2-, etc ...
Algunas alternativas adicionales para la síntesis de líquidos iónicos quirales se describen por ejemplo en [15].Some additional alternatives for the synthesis of chiral ionic liquids are described for example in [15].
La presente invención se refiere también al uso de los líquidos iónicos quirales definidos anteriormente en la industria química, médica o farmacéutica. De forma preferente se refiere al uso de los ClLs en separación de productos quirales. De forma más preferente se refiere al uso de los CILs como selectores quirales, y más preferentemente como componentes de sistemas duales de selectores quirales en EKC para mejorar la resolución obtenida de los enantiómeros de compuestos quirales de interés. Los CILs se usan como componentes de un sistema dual constituido por otro selector quiral como segundo componente para la separación de compuestos quirales, más preferiblemente de aminoácidos. Los sistemas duales están constituidos por un CIL y otro selector quiral como puede ser una ciclodextrina.The present invention also relates to the use of the chiral ionic liquids defined above in the chemical, medical or pharmaceutical industry. Preferably refers to the use of ClLs in the separation of chiral products. Most preferably it relates to the use of CILs as chiral selectors, and more preferably as components of dual systems of chiral selectors in EKC to improve the obtained resolution of the enantiomers of chiral compounds of interest. CILs are used as components of a dual system consisting of another chiral selector as a second component for the separation of chiral compounds, more preferably amino acids. The dual systems are made up of a CIL and another chiral selector such as a cyclodextrin.
Según realizaciones particulares, la separación es una separación enantiomérica de aminoácidos por Electroforesis Capilar. Una realización concreta se refiere a la separación de los aminoácidos homocisteína y cisteína.According to particular embodiments, the separation is an enantiomeric separation of amino acids by Capillary Electrophoresis. A specific embodiment relates to the separation of the amino acids homocysteine and cysteine.
Según realizaciones particulares adicionales, el líquido iónico es [L-carnitina][NTf 2 ] y se encuentra a una concentración comprendida entre 1 y 20 mM en el proceso de separación.According to additional particular embodiments, the ionic liquid is [L-carnitine] [NTf 2 ] and is found at a concentration between 1 and 20 mM in the separation process.
Según realizaciones particulares adicionales el sistema dual comprende gammaciclodextrina en una concentración 2 mM.According to additional particular embodiments, the dual system comprises gammacyclodextrin in a concentration of 2 mM.
Según realizaciones particulares adicionales en el procedimiento de separación el sistema dual comprende tampón acetato pH 5,0, fosfato a pHs 6,0 y 7,0, o tampón borato a pH 9,0.According to additional particular embodiments in the separation process, the dual system comprises acetate buffer pH 5.0, phosphate at pH 6.0 and 7.0, or borate buffer at pH 9.0.
La CE es una técnica de separación conocida por cualquier experto en este tema que permite la separación de gran número de moléculas de distintas características y naturaleza. La separación se lleva a cabo en un capilar que contiene un medio electrolítico o tampón de separación, así como las moléculas a separar, por ejemplo, aminoácidos. La separación se basa en la distinta movilidad de los analitos con distinta relación masa/carga bajo la aplicación de un campo eléctrico que se origina al establecer una diferencia de potencial entre los extremos del capilar. La separación de los analitos se puede mejorar optimizando distintas variables experimentales como son la temperatura, el voltaje aplicado, el tampón de separación, entre otras. Esta técnica se caracteriza por un consumo muy reducido de reactivos, disolventes y muestras en relación a otras técnicas de separación. Por ello, se considera una técnica analítica limpia y respetuosa con el medioambiente. Los analitos se pueden detectar mediante distintos sistemas como son UV-Vis, fluorescencia, Espectrometría de Masas, etc.CE is a separation technique known to anyone skilled in this field that allows the separation of a large number of molecules of different characteristics and nature. The separation is carried out in a capillary containing an electrolyte medium or separation buffer, as well as the molecules to be separated, eg amino acids. The separation is based on the different mobility of the analytes with different mass / charge ratio under the application of an electric field that originates when establishing a potential difference between the ends of the capillary. The separation of analytes can be improved by optimizing different experimental variables such as temperature, applied voltage, separation buffer, among others. This technique is characterized by a very low consumption of reagents, solvents and samples compared to other separation techniques. Therefore, it is considered an analytical technique clean and respectful with the environment. Analytes can be detected by different systems such as UV-Vis, fluorescence, Mass Spectrometry, etc.
En el caso en el que los analitos se separan en base a su interacción diferencial con un selector quiral presente en el medio de separación, el modo de CE se denomina Cromatografía Electrocinética (EKC).In the case where the analytes are separated based on their differential interaction with a chiral selector present in the separation medium, the CE mode is called Electrokinetic Chromatography (EKC).
En una realización preferida de la presente invención, el líquido iónico es [L-Carnitina][NTf 2 ] y se encuentra a una concentración entre 1 mM y 20 mM en el tampón de separación en EKC, más preferiblemente la concentración es entre 1mM y 10 mM y más preferiblemente es de 5 mM.In a preferred embodiment of the present invention, the ionic liquid is [L-Carnitine] [NTf 2 ] and is at a concentration between 1mM and 20mM in the separation buffer in EKC, more preferably the concentration is between 1mM and 10 mM and more preferably 5 mM.
En una realización preferida de la presente invención, la separación por EKC se lleva a cabo en las condiciones experimentales siguientes: utilizando un voltaje de 20 kV, a una temperatura de 20°C; la muestra preferiblemente se inyecta mediante presión (inyección hidrodinámica), más preferiblemente a una presión de aproximadamente 50 mbar, y aún más preferente durante unos 4 segundos; además preferiblemente se emplea un capilar de 50 pm de diámetro interno y de 50 cm de longitud total con un tampón neutro.In a preferred embodiment of the present invention, the EKC separation is carried out under the following experimental conditions: using a voltage of 20 kV, at a temperature of 20 ° C; the sample is preferably injected by pressure (hydrodynamic injection), more preferably at a pressure of about 50 mbar, and even more preferably for about 4 seconds; furthermore, preferably a capillary with an internal diameter of 50 µm and a total length of 50 cm with a neutral buffer is used.
El tampón utilizado en la presente invención es cualquiera a un pH superior al pKa de los aminoácidos y conocido por cualquier experto en electroforesis capilar, preferiblemente a pH de entre 7,0 y 9,0 y más preferiblemente se emplea un tampón fosfato.The buffer used in the present invention is any at a pH higher than the pKa of the amino acids and known to anyone skilled in capillary electrophoresis, preferably at a pH between 7.0 and 9.0 and more preferably a phosphate buffer is used.
Los analitos a separar en la presente invención podrían ser aminoácidos proteicos y no proteicos.The analytes to be separated in the present invention could be protein and non-protein amino acids.
Mediante la separación, se pueden determinar individualmente los enantiómeros de un compuesto quiral, con aplicación al control de calidad de fármacos, alimentos u otras muestras.Through separation, the enantiomers of a chiral compound can be determined individually, with application to quality control of drugs, foods, or other samples.
En la descripción y reivindicaciones de la presente invención, la palabra "comprende” y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en este tema, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Con carácter ilustrativo se presentan a continuación las siguientes figuras y ejemplos que no se pretende que sean limitativos de la presente invención.In the description and claims of the present invention, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in this field, other objects, advantages and characteristics of the invention will emerge in part of the description and part of the practice of the invention. For illustrative purposes, the following figures and examples are presented below and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
Figura 1. Separación enantiomérica del aminoácido homocisteína (Hcy) empleando el sistema gamma-ciclodextrina [L-Carnitina][NTf 2 ] como selector quiral.Figure 1. Enantiomeric separation of the amino acid homocysteine (Hcy) using the gamma-cyclodextrin [L-Carnitine] [NTf 2 ] system as a chiral selector.
Figura 2. Separación enantiomérica del aminoácido proteico cisteína (Cys) empleando gamma-ciclodextrina como selector quiral.Figure 2. Enantiomeric separation of the protein amino acid cysteine (Cys) using gamma-cyclodextrin as a chiral selector.
Figura 3. Separación enantiomérica del aminoácido homocisteína (Hcy) empleando el sistema gamma-ciclodextrina [L-Carnitina][NTf 2 ] como selector quiral.Figure 3. Enantiomeric separation of the amino acid homocysteine (Hcy) using the gamma-cyclodextrin [L-Carnitine] [NTf 2 ] system as a chiral selector.
Figura 4. Separación enantiomérica del aminoácido proteico cisteína (Cys) empleando el sistema gamma-ciclodextrina [L-Carnitina][NTf 2 ] como selector quiral.Figure 4. Enantiomeric separation of the protein amino acid cysteine (Cys) using the gamma-cyclodextrin [L-Carnitine] [NTf 2 ] system as a chiral selector.
EJEMPLOSEXAMPLES
Reactivos, materiales y métodos instrumentales generalesGeneral instrumental reagents, materials and methods
Hidrocloruro de L-carnitina (98%, CAS: 6645-46-1), sal de litio derivada de bis(trifluorometano)sulfonamida (99,85%, CAS: 90076-65-6), Disolución 1,25 M de HCl en metanol y Metanol. Todos los reactivos se adquirieron en la compañía Sigma -AldrichQuímica. Todos los reactivos empleados se usaron sin ninguna purificación extra. Los espectros de resonancia magnética nuclear (RMN) se registraron empleando los disolventes deuterados (DMSO-Ó6 o CD 3 OD), como se indica en cada caso, en un espectrómetro Varian - 300 MHz o 500 MHz. Como patrón interno se empleó tetrametilsilano (TMS). Los desplazamientos químicos (5) se indican en partes por millón (ppm) y los disolventes no deuterados residuales se emplean como referencias internas para el protón (3,36 ppm para CD 3 OD y 2,49 para DMSO-da) y para el carbono (39,9 ppm para el DMSO-da). En 1H-RMN las constantes de acoplamiento (J) se indican en Hercios (Hz) y las multiplicidades se representan como se indica a continuación: s (singlete), d (doblete), t (triplete), m (multiplete), y br (como señal ancha). Los espectros de masas de baja resolución se obtuvieron empleando Agilent Technologies 6120 Quadrupole LC-MS con ionización mediante electrospray (ESI) con el sistema Agilent Technologies 1260 Infinity LC empleando la columna SeQuantZic-Hilic (Merck-Millipore) de dimensiones 150 mm x 4.6 mm, 5 pm. Se empleó el software de Agilent Technologies para procesar los datos. El espectro de masas (MS) se indica como la relación de unidades de masa /carga (m/z). El análisis se llevó a cabo empleando un flujo de 1mL/min manteniendo la temperatura de la columna a 25°C. El detector se fijó a 277,4 nm ± 16 nm. La fase móvil está formada por la fase móvil A (200 mM de acetato amónico, pH 5,2, sin EDTA) y fase móvil B (Acetonitrilo al 80% y una disolución de acetato amónico al 20%), empleando un método isocrático.L-carnitine hydrochloride (98%, CAS: 6645-46-1), lithium salt derived from bis (trifluoromethane) sulfonamide (99.85%, CAS: 90076-65-6), 1.25 M HCl solution in methanol and methanol. All reagents were purchased from Sigma-Aldrich Chemistry. All reagents used were used without any extra purification. Nuclear magnetic resonance (NMR) spectra were recorded using deuterated solvents (DMSO-O6 or CD 3 OD), as indicated in each case, in a Varian - 300 MHz or 500 MHz spectrometer. Tetramethylsilane ( TMS). The chemical shifts (5) are indicated in parts per million (ppm) and the residual undeuterated solvents are used as internal references for the proton (3.36 ppm for CD 3 OD and 2.49 for DMSO-da) and for the carbon (39.9 ppm for DMSO-da). In 1H-NMR the coupling constants (J) are indicated in Hertz (Hz) and the multiplicities are represented as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (as a wide signal). Low resolution mass spectra were obtained using Agilent Technologies 6120 Quadrupole LC-MS with electrospray ionization (ESI) with the Agilent Technologies 1260 Infinity LC system using the SeQuantZic-Hilic column (Merck-Millipore) with dimensions 150 mm x 4.6 mm , 5 pm. Agilent Technologies software was used to process the data. The mass spectrum (MS) is indicated as the ratio of units of mass / charge (m / z). The analysis was carried out using a flow of 1mL / min keeping the temperature of the column at 25 ° C. The detector was set at 277.4 nm ± 16 nm. The mobile phase consists of mobile phase A (200 mM ammonium acetate, pH 5.2, without EDTA) and mobile phase B (80% Acetonitrile and a 20% ammonium acetate solution), using an isocratic method.
El hidrógeno fosfato de sodio se obtuvo en Panreac Química S.A. (Barcelona, España). El hidróxido de sodio, ácido bórico, pentano, los aminoácidos DL-homocisteína (Hcy), L-homocisteína y el agente de derivatización cloruro de 9-fluorenilmetoxicarbonilo (FMOC) se adquirieron en Sigma-Aldrich Química (Madrid, España). El Acetonitrilo grado HPLC se obtuvo en Scharlau (Barcelona, España). El selector quiral gammaciclodextrina, así como los aminoácidos DL-cisteína (Cys) y L-cisteína obtuvieron en Fluka (Buchs, Suiza). Las disoluciones se realizaron con agua ultrapura purificada a través de un sistema Milli-Q de Millipore (Bedford, MA, USA). Todas las disoluciones se filtraron antes de ser inyectadas en el sistema de electroforesis capilar con filtros de jeringa de nylon de 0,45 pm de diámetro de poro adquiridos de Scharlau (Barcelona, España).Sodium hydrogen phosphate was obtained from Panreac Química S.A. (Barcelona, Spain). Sodium hydroxide, boric acid, pentane, the amino acids DL-homocysteine (Hcy), L-homocysteine, and the derivatizing agent 9-fluorenylmethoxycarbonyl chloride (FMOC) were purchased from Sigma-Aldrich Química (Madrid, Spain). Acetonitrile grade HPLC was obtained from Scharlau (Barcelona, Spain). The chiral selector gammacyclodextrin, as well as the amino acids DL-cysteine (Cys) and L-cysteine obtained from Fluka (Buchs, Switzerland). The solutions were made with purified ultrapure water through a Millipore Milli-Q system (Bedford, MA, USA). All solutions were filtered before being injected into the capillary electrophoresis system with 0.45 pm pore diameter nylon syringe filters purchased from Scharlau (Barcelona, Spain).
Preparación de las disoluciones de aminoácidosPreparation of amino acid solutions
Los aminoácidos se derivatizan para su posterior detección UV siguiendo un procedimiento descrito anteriormente [18]. De manera resumida, se mezclaron 200 pL de una disolución del agente derivatizante disuelto en Acetonitrilo (30 mM) con 200 pL de una disolución del aminoácido a estudiar (10 mM). La reacción se mantuvo a temperatura ambiente durante 2 minutos. El exceso del agente derivatizante se eliminó añadiendo 500 pL de pentano y la disolución final se diluyó 10 veces con agua Milli-Q antes de inyectarla en el sistema.Amino acids are derivatized for subsequent UV detection following a previously described procedure [18]. Briefly, 200 pL of a solution of the derivatizing agent dissolved in Acetonitrile (30 mM) was mixed with 200 pL of a solution of the amino acid to be studied (10 mM). The reaction was kept at room temperature for 2 minutes. The excess derivatizing agent was removed by adding 500 pL of pentane and the final solution was diluted 10 times with Milli-Q water before injecting into the system.
Electroforesis CapilarCapillary Electrophoresis
Los análisis se llevaron a cabo en un equipo de electroforesis capilar Agilent 7100 (Agilent Technologies, Waldbronn, Alemania) con un detector UV-DAD. Se emplearon capilares de sílice fundida (Polymicro Technologies, Phoenix, AZ, USA) con un diámetro interno de 50 pm y de 50 cm de longitud efectiva (58,5 cm longitud total). Entre inyecciones se realizó un acondicionamiento con 0,1M hidróxido de sodio (1 bar) durante 2 minutos, agua Milli-Q (1 bar) durante 1 minuto y tampón de separación (1 bar) durante 3 minutos. La separación se llevó a cabo utilizando una temperatura de 20 °C, aplicando un voltaje de 20 kV y detección UV a una longitud de onda de 210 nm con un ancho de banda de 4 nm. La muestra se inyectó por presión 50 mbar durante 4 s. Se utilizó un tampón fosfato 50 mM (pH 7,0) para la separación enantiomérica de los aminoácidos.The analyzes were carried out on an Agilent 7100 capillary electrophoresis equipment (Agilent Technologies, Waldbronn, Germany) with a UV-DAD detector. Fused silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) with an internal diameter of 50 pm and an effective length of 50 cm (58.5 cm total length) were used. Between injections, conditioning was carried out with 0.1M sodium hydroxide (1 bar) for 2 minutes, Milli-Q water (1 bar) for 1 minute and separation buffer (1 bar) for 3 minutes. The separation was carried out using a temperature of 20 ° C, applying a voltage of 20 kV and UV detection at a wavelength of 210 nm with a bandwidth of 4 nm. The sample was pressure injected at 50 mbar for 4 s. A 50 mM phosphate buffer (pH 7.0) was used for the enantiomeric separation of amino acids.
Síntesis y caracterización de líquidos iónicosSynthesis and characterization of ionic liquids
A modo de empleo se describen los siguientes ejemplos ilustrativos, que no pretenden ser limitantes:By way of use, the following illustrative examples are described, which are not intended to be limiting:
a) La preparación de haluros del éster metílico derivado de L-carnitina de acuerdo con el paso (a) del procedimiento de la presente invención (Ejemplo 1).a) The preparation of halides of the methyl ester derived from L-carnitine according to step (a) of the process of the present invention (Example 1).
b) La preparación del correspondiente CIL que contiene como anión bis(trifluorometilsulfonil)imida (NTf 2 ) (Ejemplo 2) a partir del cloruro del éster metílico de L-carnitina, obtenido en el paso a).b) The preparation of the corresponding CIL containing bis (trifluoromethylsulfonyl) imide (NTf 2 ) as anion (Example 2) from the L-carnitine methyl ester chloride, obtained in step a).
Los CILs sintetizados se han caracterizado por distintas técnicas entre las que se encuentran Resonancia Magnética Nuclear (RMN) de 1H y 13C, HPLC-MS y análisis elemental.The synthesized CILs have been characterized by different techniques, including 1H and 13C Nuclear Magnetic Resonance (NMR), HPLC-MS and elemental analysis.
EJEMPLO 1. Cloruro de (S)-2-hidroxi-4-trimetilamonio-butirato de metiloEXAMPLE 1. Methyl (S) -2-hydroxy-4-trimethylammonium-butyrate chloride
Una disolución de cloruro de L-carnitina (III) (0,40 g; 2,00 mmol, 1,0 eq.) en metanol (3 mL) se trató con una disolución 1,25 M de HCl en metanol (1 mL). La mezcla resultante se calentó a 80°C durante 3 horas. Cuando la reacción terminó, el disolvente se evaporó a vacío proporcionando el derivado cloruro de (S)-2-hidroxi-4-trimetilamonio-butirato de metilo (420 mg, 98%) como un sólido blanco. 1H-NMR (CD 3 OD) 5 (ppm): 4,25 (m, J = 6,0 Hz, 1H); 3,42 (s, 3H); 3,18 (d, J = 8,5 Hz, 2H); 2,95 (s, 9H); 2,30 (d, J = 8,5 Hz, 2H).A solution of L-carnitine (III) chloride (0.40 g, 2.00 mmol, 1.0 eq.) In methanol (3 mL) was treated with a 1.25 M solution of HCl in methanol (1 mL ). The resulting mixture was heated at 80 ° C for 3 hours. When the reaction was complete, the solvent was evaporated in vacuo to give the chloride derivative of methyl (S) -2-hydroxy-4-trimethylammonium-butyrate (420 mg, 98%) as a white solid. 1H-NMR (CD 3 OD) 5 (ppm): 4.25 (m, J = 6.0 Hz, 1H); 3.42 (s, 3H); 3.18 (d, J = 8.5 Hz, 2H); 2.95 (s, 9H); 2.30 (d, J = 8.5 Hz, 2H).
13C-RMN (CD 3 OD) 5 (ppm): 176,8; 62,5; 53,6; 53,5; 53,4; 50,9; 39,6. Análisis mediante HPLC-MS (modo positivo): tR = 1,023 min (m/z 176,2 (M+H)+).13C-NMR (CD 3 OD) 5 (ppm): 176.8; 62.5; 53.6; 53.5; 53.4; 50.9; 39.6. Analysis by HPLC-MS ( positive mode): tR = 1.023 min (m / z 176.2 (M + H) +).
EJEMPLO 2. CIL derivado del éster metílico de L-carnitina con el anión Bis(trifluorometano) sulfonamida [L-Carnitina][NTf2]. EXAMPLE 2. CYL derived from L-carnitine methyl ester with Bis (trifluoromethane) sulfonamide [L-Carnitine] [NTf 2 ] anion.
Una disolución de cloruro de (S)-2-hidroxi-4-trimetilamonio-butirato de metilo (358 mg; 1,68 mmol; 1,0 eq.) en agua destilada (2 mL) se trató con una disolución equimolar de la sal de litio derivada de bis (trifluorometano) sulfonamida ((Tf) 2 NLi) (482,8 g; 1,68 mmol, 1,0 eq.) en agua destilada (1 mL). La correspondiente mezcla de reacción se agitó a temperatura ambiente (25 °C) durante 3 horas. Después de este tiempo, en la mezcla de reacción se observó la formación de dos fases que se separaron mediante un embudo de extracción y se recogió la fase de abajo y se secó a vacío durante toda una noche (45°C, 150 mbar), proporcionando 361 mg (47%) de [L-Carnitina][NTf 2 ] como un aceite denso e incoloro. 1H-NMR (CD 3 OD) 5 (ppm) 4,25 (m, J = 6,0 Hz, 1H); 3,42 (s, 3H); 3,18 (d, J = 8,5 Hz, 2H); 2,95 (s, 9H); 2,30 (d, J = 8,5 Hz, 2H). Análisis mediante HPLC-MS (modo positivo): ír = 1,023 min (m/z 176,2 (M+H)+). Anal. Calcd. para C 10 H 18 N 2 O 7 S 2 F 6 : C, 26,3; H, 4; F, 25; N, 6,1; S, 14,1. Encontrado: C, 25,29; H, 4,14; N, 6,20; S, 14,40. Los ensayos de análisis elemental se realizaron empleando una dosis de oxígeno de 30.A solution of methyl (S) -2-hydroxy-4-trimethylammonium-butyrate chloride (358 mg, 1.68 mmol, 1.0 eq.) In distilled water (2 mL) was treated with an equimolar solution of the lithium salt derived from bis (trifluoromethane) sulfonamide ((Tf) 2 NLi) (482.8 g, 1.68 mmol, 1.0 eq.) in distilled water (1 mL). The corresponding reaction mixture was stirred at room temperature (25 ° C) for 3 hours. After this time, in the In the reaction mixture, the formation of two phases was observed, which were separated by means of an extraction funnel and the phase below was collected and dried under vacuum overnight (45 ° C, 150 mbar), yielding 361 mg (47%) of [L-Carnitine] [NTf 2 ] as a thick, colorless oil. 1H-NMR (CD 3 OD) 5 (ppm) 4.25 (m, J = 6.0 Hz, 1H); 3.42 (s, 3H); 3.18 (d, J = 8.5 Hz, 2H); 2.95 (s, 9H); 2.30 (d, J = 8.5 Hz, 2H). Analysis by HPLC-MS (positive mode): ír = 1.023 min (m / z 176.2 (M + H) +). Anal. Calcd. for C 10 H 18 N 2 O 7 S 2 F 6 : C, 26.3; H, 4; F, 25; N, 6.1; S, 14.1. Found: C, 25.29; H, 4.14; N, 6.20; S, 14.40. Elemental analysis tests were performed using an oxygen dose of 30.
Uso de los CILs basados en el éster metílico de la L-carnitina para la separación enantiomérica de aminoácidos por electroforesis capilarUse of CILs based on L-carnitine methyl ester for enantiomeric separation of amino acids by capillary electrophoresis
Se evaluó el uso de los CILs basados en el éster metílico de la L-carnitina en un sistema dual junto a la gamma-ciclodextrina para la separación enantiomérica de aminoácidos proteicos y no proteicos, más preferiblemente para los aminoácidos homocisteína y cisteína. Se obtuvo una mayor Rs enantiomérica con la adición de estos nanoaditivos al medio de separación. Las condiciones experimentales empleadas fueron las siguientes: capilar de 50 pm de diámetro interno y 50 cm de longitud efectiva (58,5 cm de longitud total); temperatura del capilar, 20 °C; voltaje aplicado, 20 kV; inyección por presión, 50 mbar durante 4 s; detección UV a 210 nm (4 nm de ancho de banda) y tampón de separación fosfato 50 mM a pH 7,0. Las concentraciones de los selectores quirales fueron de 2 mM para la gamma-ciclodextrina y 5 mM para el líquido iónico. En estas condiciones se obtuvieron dos picos correspondientes a los enantiómeros de los aminoácidos. Para intentar mejorar la separación de optimizaron algunas condiciones electroforéticas. Así, se probaron diferentes pH y tampones de separación. Se probaron: tampón acetato pH 5,0, fosfato pH 6,0 y 7,0 y tampón borato pH 9,0. La mejor separación se obtuvo para el tampón fosfato a pH 7,0. Además, se probaron diferentes concentraciones de la gamma-ciclodextrina en un rango de 1-15 mM obteniéndose la mejor separación a una concentración de 10 mM para la homocisteína y de 2 mM para la cisteína (Figuras 1 y 2). Con el objetivo de obtener una mayor resolución enantiomérica se añadió al medio de separación el líquido iónico [L-Carnitina][NTf 2 ]. Se probaron diferentes concentraciones de líquido iónico (1-20 mM) fijando la concentración de la gamma-ciclodextrina a 2 mM. Una concentración de 5 mM de [L-Carnitina][NTf 2 ] permitió la mejor separación de los aminoácidos (Figuras 3 y 4). The use of CILs based on the methyl ester of L-carnitine in a dual system together with gamma-cyclodextrin was evaluated for the enantiomeric separation of protein and non-protein amino acids, more preferably for the amino acids homocysteine and cysteine. A higher enantiomeric Rs was obtained with the addition of these nanoadditives to the separation medium. The experimental conditions used were the following: capillary with an internal diameter of 50 pm and an effective length of 50 cm (total length 58.5 cm); capillary temperature, 20 ° C; applied voltage, 20 kV; pressure injection, 50 mbar for 4 s; UV detection at 210 nm (4 nm bandwidth) and 50 mM phosphate separation buffer at pH 7.0. The concentrations of the chiral selectors were 2 mM for the gamma-cyclodextrin and 5 mM for the ionic liquid. Under these conditions, two peaks corresponding to the enantiomers of the amino acids were obtained. To try to improve the separation, some electrophoretic conditions were optimized. Thus, different pH and separation buffers were tested. The following were tested: acetate buffer pH 5.0, phosphate pH 6.0 and 7.0 and borate buffer pH 9.0. The best separation was obtained for the phosphate buffer at pH 7.0. In addition, different concentrations of gamma-cyclodextrin were tested in a range of 1-15 mM, obtaining the best separation at a concentration of 10 mM for homocysteine and 2 mM for cysteine (Figures 1 and 2). In order to obtain a higher enantiomeric resolution, the ionic liquid [L-Carnitine] [NTf 2 ] was added to the separation medium. Different ionic liquid concentrations (1-20 mM) were tested by setting the concentration of gamma-cyclodextrin at 2 mM. A concentration of 5 mM of [L-Carnitine] [NTf 2 ] allowed the best separation of amino acids (Figures 3 and 4).
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AGRADECIMIENTOSTHANKS
Los autores agradecen la financiación recibida del Ministerio de Economía y Competitividad (proyecto CTQ2016-76368-P) y de la Comunidad de Madrid y fondos europeos de los programas FSE y FEDER (proyecto S2018/BAA-4393, AVANSECAL-II-CM). The authors are grateful for the funding received from the Ministry of Economy and Competitiveness (project CTQ2016-76368-P) and the Community of Madrid and European funds from the FSE and FEDER programs (project S2018 / BAA-4393, AVANSECAL-II-CM).
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