Separation of enilconazole enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes by using NMR spectroscopy

Author: Ann Gogolashvili
Co-authors: Ann Gogolashvili, Elene Tatunashvili, Lali Chankvetadze, Tamas Sohajda, Julianna Szeman, Antonio Salgado, Bezhan Chankvetadze
Keywords: Chirality, Enantiomer, Capillary electrophoresis, Nuclear magnetic resonance spectroscopy, Migration order, Chiral separation
Annotation:

The chirality of a molecule may considerably affect its physiological activity. Many compounds of biological and pharmacological interest are chiral. Approximately 40% of the drugs in use are known to be chiral and only about 25% are administered as pure enantiomers. Enantiomers differ from each other from the viewpoint of their absorption, distribution, protein binding and receptor affinity. The living body with its numerous homochiral compounds being amazingly chiral selector, will interact with each racemic drug differently and metabolize each enantiomer by a separate pathway to generate different pharmacological activity. Thus, one isomer may produce the desired therapeutic activities, while the other may be inactive or, in worst cases, produce undesired or toxic effects. The administration of pure, pharmacologically active enantiomers is therefore of immense importance. Thanks to a wide range of modern technologies for chiral separation, US Food and Drug Administration (FDA) recently recommends the assessments of each enantiomer activity for racemic drugs in body and promotes the development of new chiral drugs as single enantiomers. Capillary electrophoresis is one of the youngest method for separation of enantiomers, established as very popular tool in this field. The major reasons for such a popularity of CE is its own unique mechanism for separation of enantiomers. In addition, CE is highly efficient, very flexible, cost-effective, environmentally friendly miniaturized technique. CE is performed in the capillary format, typically 25- to150 μm inner diameter (id), which are usually filled only with buffer. Use of the capillary has numerous advantages, particularly with respect to the detrimental effects of Joule heating (heat generated when a electric current flows through a conductor such as a buffer filled capillary. The high electrical resistance of the capillary enables the application of very high electrical fields (100 to 500 V/cm) with only minimal heat generation. The use of thehigh electrical fields results in short analysis times and high efficiency and resolution, often in excess of 1000000 theoretical plates. The numerous separation modes offer different separation mechanisms and selectivities, minimal sample volume requirements (1 to 50 nL), on-capillary detection, and the potential for quantitative analysis and automation. Originally considered primarily for the analysis of biological macromolecules, CE has proved useful for separations of compounds such as amino acids, chiral drugs, vitamins, pesticides, inorganic ions, organic acids, dyes, surfactants, peptides and proteins, carbohydrates, oligonucleotides and DNA restriction fragments, and even whole cells and virus particles. Various experimental and theoretical tools can be harnessed for understanding of the nature of those intermolecular forces which are involved in noncovalent selector-selectand binding and enantioselective recognition. However, none of these methods alone is able to provide a conclusive (more or less realistic) vision on chiral recognition that would enable to predict the best chiral selector or analyte from the viewpoint of recognition power. Some separation and non-separation methods fit to, and extend each other perfectly for chiral analysis. For instance, separations of enantiomers in CE are performed in homogenous solution in a single phase. These conditions can be perfectly mimicked in NMR spectroscopic experiments. Thus, the information gained on stereoselective intermolecular interactions with these two techniques is very complementary and thus, this tandem represents very powerful tool for better understanding of enantioselective recognition mechanisms on the molecular level. Over last 25 years various NMR spectroscopic experiments have been used for obtaining information on stoichiometry, binding constants and structure of selector-selectand complexes responsible for separation of enantiomers observed in CE experiments. However, as stated above there is still long way to go before one becomes able to identify the intermolecular forces involved in selector-selectand binding and chiral recognition and predict separation result based on the structure and nature of selector, selectand and separation medium. Particularly valuable information on chiral recognition in intermolecular interactions may be provided by gaining insight about the forces leading to a reversal of recognition. It seems that the enantiomer elution order (EEO) in HPLC mostly indicates a reversal of the recognition pattern in selector-selectand interactions, while this is not always the case in CE. In this technique, the mobility-related phenomena may also be responsible for an enantiomer migration order (EMO) reversal. Sometimes significant structural differences can be detected between the chiral analyte-chiral selector intermolecular complexes with opposite recognition pattern. However, in other cases just minor differences are observed between the structures of complexes while the recognition pattern is opposite. Thus, despite significant efforts in this direction it is still necessary to accumulate as many examples as possible of opposite enantiomer recognition pattern, study the most likely structure of these complexes and on the next step to calculate/compute forces involved in selector-selectand binding and enantioselective recognition. This approach may enable prevailing the forces and interactions governing enantioselective recognition in intermolecular complexes of cyclodextrins with chiral guest compounds. With this knowledge, the forces and interactions might be adjusted by changing the structure/nature of CD-type chiral selector, as well as the medium for interaction. Such knowledge may also allow predicting the separation results based on the structure of the chiral analyte, chiral selector and separation conditions. In the present study, the enantiomer migration order (EMO) of enilconazole in the presence of various cyclodextrins (CDs) was investigated by capillary electrophoresis (CE). Opposite EMO of enilconazole were observed when β-CD or the sulfated heptakis(2-O-methyl-3,6-di-O-sulfo)-β-CD (HMDS-β-CD) were used as the chiral selectors. Nuclear Magnetic Resonance (NMR) spectroscopy was used to study the mechanism of chiral recognition between enilconazole enantiomers and those two cyclodextrins. Based on rotating frame nuclear Overhauser (ROESY) experiments, the structure of an inclusion complex between enilconazole and β-CD was derived, in which (+)-enilconazole seemed to form a tighter complex than the (-)-enantiomer. This correlates well with the migration order of enilconazole enantiomers observed in CE. No evidence of complexation between enilconazole and HMDS-β-CD could be gathered due to lack of intermolecular NOE interactions. Most likely the interaction between enilconazole and HDMS-β-CD leads to formation of shallow external complex that is sufficient for separation of enantiomers in CE but cannot be evidenced based on ROESY experiment. Thus, in this case CE documents the presence/existence of intermolecular interactions which are at least very difficult to be evidenced/confirmed by other instrumental techniques.

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