Chiral recognition mechanism studies of Tyr-Arg-Phe-Lys-NH2 tetrapeptide on crown ether-based chiral stationary phase.

Even though chiral recognition for crown-ether CSPs is generally understood, on a molecular level, exact mechanisms for the resolution are still unclear. Furthermore, short peptide analytes often contain multiple amino moieties capable of binding to the crown ether selector. In order to extend the understanding in chiral recognition mechanisms, polar organic mode separation of Tyr-Arg-Phe-Lys-NH2 tetrapeptide llll/dddd enantiomers on S- and R-(3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 stationary phases was studied with 50-mM perchloric acid in methanol as mobile phase. Deviation from the generally acceptable 1:1 stoichiometry was supported by mass spectroscopy analysis of the formed complexes between tetrapeptide enantiomer and crown ether selectors, which revealed adducts possessing 1:1, 1:2, and 1:3 stoichiometry. Further investigation of complexation induced shifts by NMR indicated on different binding mechanisms between llll/dddd enantiomers of Tyr-Arg-Phe-Lys-NH2 and crown ether selectors. Enantioselective proton shifts were observed in studied tetrapeptide tyrosine and phenylalanine residues exclusively for llll enantiomer upon binding with S-(3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 selector (and dddd enantiomer with R-(3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 selector), indicating that these two amino acid residues contribute to chiral recognition. The obtained results were in agreement with the LC data.

Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor.

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.

Application of hydrophilic interaction chromatography for simultaneous separation of six impurities of mildronate substance.

The possibility of separating the impurities of mildronate, an antiischemic drug, by hydrophilic interaction chromatography (HILIC) was investigated on different polar stationary phases (silica, amino, cyano and zwitterionic sulfobetaine). The investigations have shown that HILIC is a useful alternative to reversed phase and ion-pair chromatography. The impact of HILIC separation conditions (acetonitrile content, buffer pH in mobile phase) on retention and selectivity has been systematically studied. Importance of these factors was found to be dependent on the structural properties of solutes. A HILIC method using a zwitterionic sulfobetaine stationary phase was developed and validated to determine six impurities in the drug substance. The method was validated in terms of specificity, limit of quantitation, limit of detection, linearity, accuracy and precision.

Isocratic, simultaneous normal-phase LC separation of four impurities for a stavudine synthesis control.

The isocratic separation of impurities for a stavudine synthesis control has been studied by liquid chromatography (LC). The investigations have shown that isocratic reversed-phase LC conditions were unable to achieve a good separation. It was established that isocratic normal-phase LC system with common silica column as a stationary phase and the mixture of ethyl acetate with 4% (v/v) of ethylene glycol as a mobile phase is useful in simultaneous determination of four impurities for a stavudine synthesis control.