| Chiral analysis is a difficult and hot issue in pharmaceutical analysis at present.Capillary electrophoresis (CE) has unique advantages for chiral separation. However, thevital enantioseparation factor how to select the chiral selectors in CE has no systematictheoretical and methodological researches. Detailed mechanisms underlying the separationin the presence of CD as a chiral selector for many analytes remain unknown due to thelimitations of the experimental methods. This project has studied thestructure-enantioselective relationship between the chiral selectors, which are theβ-cyclodextrin and its derivatives, and the enantiomers, which are the representativestructure type of triazole antifungal activity of enantiomers. By means of computer-aidedmolecular modeling technology, the host-guest inclusion process between cyclodextrin andenantiomers was investigated and chiral recognition mechanisms were discussed. Thebinding energy of the inclusion was calculated by molecular mechanics. According to thesimulation data of molecular docking combined with the experimental results of chiralseparation by CE, the mathematical model was established by studying the structureenantioselective relationship and thus to predict the enantioseparation result.(1) The first CD-CZE method for the enantiomeric separations of iodiconazole, a newantifungal drug, and its structurally related triadimenol analogues has been developed andvalidated. It was found that hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was the mosteffective chiral selector. Furthermore, the influence of the concentration of HP-γ-CD,buffer pH, buffer concentration, temperature, and applied voltage was investigated, and themethod was validated. In this method, a high resolution (Rs=2.69) of iodiconazoleenantiomers with an analysis time of around16min was achieved. The study of the analytestructure-enantioseparation relationships showed that substitutions in the side chains,especially the N-methyl group in the side chains, had important influences on enantiomericseparation.(2) Computer-aided molecular modeling study of enantioseparation of iodiconazoleand structurally related triadimenol analogues by capillary electrophoresis: Chiralrecognition mechanism and mathematical model for predicting chiral separation. The effectof structural features of analytes on t and Rswas studied under the optimum separationconditions. The results showed that substitutions in the side chains, especially the N-methyl group in the side chains, had important influences on enantiomeric separation. Usingmolecular docking technique and binding energy calculations by Discovery Studio2.5/Sybyl/Gold software, the inclusion process between HP-γ-CD and enantiomers wasinvestigated and chiral recognition mechanisms were discussed. The results suggest thathydrogen bonding between fluorine at position4of the phenyl group beside the chiralcarbon and the hydroxyl group on the HP-γ-CD rim and face to face π-π interactionsbetween two phenyl rings highly contributed to the enantiorecognition process betweenHP-γ-CD and iodiconazole. The N-methyl group beside chiral carbon also played animportant role in enantiomeric separation. Additionally, the big difference in bindingenergy (E) highly contributed to good separation in the presence of HP-γ-CD chiralselector, which may be a helpful initial guide for chiral selector selection and predicting theresult of enantioseparation. Furthermore, the new mathematical equation established basedon the results of molecular mechanics calculations exhibited good capability in predictingchiral separation of these triadimenol analogues using HP-γ-CD mediated CE. Inconclusion, the molecular modeling technique provides us with a good perspective ofenantioseparation and serves as a useful method for studying chiral recognitionmechanisms and predicting chiral separation.(3) Chiral separation of seven new triadimenol antifungal active compounds bycapillary electrophoresis and chiral recognition mechanisms by computer-aided molecularmodeling techniques were studied. Eight neutral cyclodextrins were used as the chiralselectors. Only the heptakis-(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) exhibited a veryhigh enantioselectivity power to all the seven active compounds compared to the othertested CDs. The influences of the concentration of DM-β-CD, buffer pH, bufferconcentration, applied voltage, and temperature were investigated. The enantiomericseparation of seven active compounds and the best results were obtained in30mmol/LNaH2PO4buffer(adjust to pH2.2with H3PO4)containing30mmol/L DM-β-CD．Thevoltage was20kV and the temperature was20℃．The four active compounds werebaseline separated on this condition (Rs>1.60)．By means of computer-aided molecularmodeling software Discovery Studio2.5/Sybyl/Gold and binding energy calculations, theinclusion process between DM-β-CD and enantiomers was investigated and chiralrecognition mechanisms were discussed. The results suggested that the enantioseparationresult related to the difference of binding energy. And the good separation obtained in thepresence of the DM-β-CD chiral selector was due to the big binding energy difference. (4) Chiral separation of twelve new triadimenol antifungal active compounds bearingtert-butyl group by capillary electrophoresis were studied. Eight neutral cyclodextrins wereused as the chiral selectors. Both HP-γ-CD and heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) exhibited a very high enantioselectivity power to all the twelveactive compounds compared to the other tested CDs. The influences of the concentration ofHP-γ-CD or TM-β-CD, buffer pH, buffer concentration, applied voltage, and temperaturewere investigated, respectively. Under the optimum separation conditions, the twelveactive compounds were baseline separated and the resolutions of most compounds werebeyond2.00. The enantioseparation results of many compounds using TM-β-CD as thechiral selector were better than using HP-γ-CD as the chiral selector.(5) Molecular modeling study of chiral separation and recognition mechanism ofβ-adrenergic antagonists by capillary electrophoresis. Chiral separations of fiveβ-adrenergic antagonists (propranolol, esmolol, atenolol, metoprolol, and bisoprolol) werestudied by capillary electrophoresis using six CDs as the chiral selectors.Carboxymethylated-β-cyclodextrin (CM-β-CD) exhibited a higher enantioselectivity powercompared to the other tested CDs. The influences of the concentration of CM-β-CD, bufferpH, buffer concentration, temperature, and applied voltage were investigated. The goodchiral separation of five β-adrenergic antagonists was achieved using50mM Tris buffer atpH4.0containing8mM CM-β-CD with an applied voltage of24kV at20°C. In order tounderstand possible chiral recognition mechanisms of these racemates with CM-β-CD,host-guest binding procedures of CM-β-CD and these racemates were studied using themolecular docking software Autodock. The binding free energy was calculated using theAutodock semi-empirical binding free energy function. The results showed that the phenylor naphthyl ring inserted in the hydrophobic cavity of CM-β-CD and the side chain wasfound to point out of the cyclodextrin rim. Hydrogen bonding between CM-β-CD and theseracemates played an important role in the process of enantionseparation and a model of thehydrogen bonding interaction positions was constructed. The difference in hydrogenbonding formed with the–OH next to the chiral center of the analytes may help to increasechiral discrimination and gave rise to a bigger separation factor. In addition, the longer sidechain in the hydrophobic phenyl ring of the enantiomer was not beneficial forenantioseparation and the chiral selectivity factor was found to correspond to the differencein binding free energy. Molecular docking software AutoDock is also a useful method tostudy the enantioseparation and chiral recognition mechanism by studying the host-guest inclusion process and calculating binding free energy.In conclusion, the new building method, which will be more scientificly andefficiently to show the technical advantage of CE in chiral analysis, can be investigate theenantioseparation mechanism and forecast the result of the enantiomeric separation andprovide theoretical basis for chiral separation.
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