Determination of the binding constants and ionic mobilities of diquat complexes with randomly sulfated cyclodextrins by affinity capillary electrophoresis.

The enantiomers of diquats (DQs), a new class of functional organic molecules, were recently separated by capillary electrophoresis (CE) with high resolution up to 11.4 within 5-7 min using randomly sulfated α-, ß-, and γ-cyclodextrins (CDs) as chiral selectors. These results indicated strong interactions between dicationic diquats and multiply negatively charged sulfated CDs (S-CDs). However, the binding strength of these interactions was not quantified. For that reason, in this study, affinity CE was applied for the determination of the binding constants and ionic mobilities of the complexes of DQ P- and M-enantiomers with CD chiral selectors in an aqueous medium. The non-covalent interactions of 10 pairs of DQ enantiomers with the above CDs were investigated in a background electrolyte (BGE) composed of 22 mM NaOH, 35 mM H3PO4, pH 2.5, and 0.0-1.0 mM concentrations of CDs. The average apparent binding constant and the average actual ionic mobility of the DQ-CD complexes were determined by nonlinear regression analysis of the dependence of the effective mobility of DQ enantiomers on the concentration of CDs in the BGE. The complexes were found to be relatively strong with the averaged apparent binding constants in the range 13 600-547 400 L/mol.

The separation of the enantiomers of diquats by capillary electrophoresis using randomly sulfated cyclodextrins as chiral selectors.

Diquats, derivatives of the widely used herbicide diquat, represent a new class of functional organic molecules. A combination of their special electrochemical properties and axial chirality could potentially result in their important applications in supramolecular chemistry, chiral catalysis, and chiral analysis. However, prior to their practical applications, the diquats have to be prepared in enantiomerically pure forms and the enantiomeric purity of their P- and M-isomers has to be checked. Hence, a chiral capillary electrophoresis (CE) method has been developed and applied for separation of P- and M-enantiomers of 11 new diquats. Fast and better than baseline CE separations of enantiomers of all 11 diquats within a short time 5-7 min were achieved using acidic buffer, 22 mM NaOH, 35 mM H3 PO4 , pH 2.5, as a background electrolyte, and 6 mM randomly sulfated α-, ß-, and γ-cyclodextrins as chiral selectors. The most successful selector was sulfated γ-cyclodextrin, which baseline separated the enantiomers of all 11 diquats, followed by sulfated ß-cyclodextrin and sulfated α-cyclodextrin, which baseline separated enantiomers of 10 and nine diquats, respectively. Using this method, a high enantiopurity degree of the isolated P- and M-enantiomers of three diquats with a defined absolute configuration was confirmed and their migration order was identified.

Capillary electrokinetic chromatography for studying interactions between ß-blockers and Intralipid emulsion.

Toxicity of ß-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of ß-blockers of different hydrophobicity's (log KD values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed ß-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log KD values of the ß-blockers. The binding and adsorption constants also show that less hydrophobic ß-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of ß-blockers is worth further investigation.

Covalent anionic copolymer coatings with tunable electroosmotic flow for optimization of capillary electrophoretic separations.

We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9  m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.

Chiral analysis of ß-alanyl-d,l-tyrosine and its derivatives and estimation of binding constants of their complexes with 2-hydroxypropyl-ß-cyclodextrin by capillary electrophoresis.

Chiral CE methods were developed for the elucidation of l- or d-configuration of tyrosine residue in antimicrobial dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) isolated from the hemolymph of larvae of fleshfly Neobellieria bullata and for the evaluation of enantiopurity of its synthetic isomers (ß-Ala-d-Tyr and ß-Ala-l-Tyr), and enantiomers of their amidated and acetylated derivatives, ß-Ala-d,l-Tyr-NH2 and N-Ac-ß-Ala-d,l-Tyr, respectively. Baseline separations were achieved for all three pairs of enantiomers: (i) for ß-Ala-d,l-Tyr in acidic background electrolyte composed of 32/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 2.5, and 20 mg/mL 2-hydroxypropyl-ß-cyclodextrin as chiral selector; (ii) for ß-Ala-d,l-Tyr-NH2 enantiomers in acidic background electrolyte consisting of 48/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 3.5, and 30 mg/mL 2-hydroxypropyl-ß-cyclodextrin; and (iii) for enantiomers of N-Ac-ß-Ala-d,l-Tyr in alkaline background electrolyte composed of 50/49 mM Na2 B4 O7 /NaOH, pH 10.5, and 60 mg/mL 2-hydroxypropyl-ß-cyclodextrin. From CE analyses of mixed samples of isolated ß-Ala-Tyr and synthetic standards ß-Ala-l-Tyr and ß-Ala-d-Tyr, it turned out that isolated ß-Ala-Tyr was pure l-enantiomer. In addition, the average apparent binding constants, Kb , and average actual ionic mobilities of the complexes of ß-Ala-d,l-Tyr and its above derivatives with 2-hydroxypropyl-ß-cyclodextrin were determined. These complexes were weak, with Kb values ranging from 11.2 to 79.1 L/mol. Their cationic mobilities were equal to (5.6-9.2) × 10-9 m2 /V/s, and anionic mobilities to (-1.3-1.6) × 10-9 m2 /V/s.

Nonaqueous capillary electrophoresis and quantum chemical calculations applied to investigation of acid-base and electromigration properties of azahelicenes.

Nonaqueous capillary electrophoresis (NACE) using methanol (MeOH) as a solvent of the BGEs and quantum mechanical density functional theory (DFT) have been applied to determine the thermodynamic acidity (ionization) constants (pKa ) of mono- and diaza[5]helicenes, mono- and diaza[6]helicenes, and their dibenzo derivatives in MeOH and water. First, the mixed acidity constants, pKa,MeOHmix${\rm{p}}K_{{\rm{a,MeOH}}}^{{\rm{mix}}}$ , of ionogenic pyridinium groups of azahelicenes and their derivatives in MeOH were obtained by nonlinear regression analysis of pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by NACE in a large series of methanolic BGEs within a wide conventional pH range (pHMeOH 1.6-12.0) and at ambient temperature (21-26°C) in a home-made CE device. Prior to mixed acidity constant calculation, the effective mobilities were corrected to reference temperature (25°C) and constant ionic strength (25 mM). Then, the mixed acidity constants were recalculated to the thermodynamic acidity constants pKa,MeOH by the Debye-Hückel theory of nonideality of electrolyte solutions. Finally, from the methanolic thermodynamic pKa,MeOH values, the aqueous thermodynamic pKa,H2O${\rm{p}}{K_{{\rm{a,}}{{\rm{H}}_{\rm{2}}}{\rm{O}}}}$ constants were estimated using the empirical relations between methanolic and aqueous acidity constants derived for structurally related pyridine derivatives. Depending on the number and position of the nitrogen atoms in their molecules, the analyzed azahelicenes were found to be weak to moderate bases with methanolic pKa,MeOH in the range 2.01-8.75 and with aqueous pKa,H2O${\rm{p}}{K_{{\rm{a,}}{{\rm{H}}_{\rm{2}}}{\rm{O}}}}$ in the range 1.67-8.28. The thermodynamic pKa,MeOH obtained by the DFT calculations were in a good agreement with those determined experimentally by NACE.

Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial ß-Alanyl-Tyrosine from Hemolymph of Fleshfly Neobellieria bullata.

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of ß-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of ß-Ala-Tyr and its admixtures have been converted into preparative purification of ß-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the ß-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

Covalent cationic copolymer coatings allowing tunable electroosmotic flow for optimization of capillary electrophoretic separations.

Electroosmotic flow (EOF) plays a pivotal role in optimization of capillary electrophoresis (CE) separations of (bio)molecules and (bio)particles. EOF velocity is directly related to analysis time, peak resolution and separation efficiency. Here, we report a concept of charged polymer coatings of the inner fused silica capillary wall, which allows anodic EOF with mobility ranging from 0 to ∼(30-40) × 10-9 m2V-1s-1. The capillary wall is modified by covalently bound cationic copolymer poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride) (PAMAPTAC) containing variable ratio of the charged monomer in the 0-60 mol. % interval. The EOF mobility showed minor variability with composition of background electrolyte (BGE) and pH in the 2-10 interval. The coatings were evaluated by CE-UV and nanospray CE-MS in the counter-EOF arrangement for a series of basic drug molecules in acetic acid based acidic BGE. Tunable EOF velocity was demonstrated as a useful tool for optimization of peak resolution, separation efficiency and migration time of analytes. Electrostatic repulsion of positively charged capillary surface was shown as beneficial for suppression of analyte adsorption, notably for hydrophobic cationic analytes.

Polyhalogenated Bicyclo[1.1.1]pentane-1,3-dicarboxylic Acids.

Herein we report the highly selective radical chlorination of 2,2-difluorobicyclo[1.1.1]pentane-1,3-dicarboxylic acid. Together with radical hydrodechlorination by TMS3SiH, four new bicyclo[1.1.1]pentane cages carrying two fluorine and one to three chlorine atoms in bridge positions have been obtained. The exact positions of all halogen atoms have been confirmed by X-ray diffraction. The acidity constants (pKa) for all new derivatives have been determined by capillary electrophoresis, and these experimental values show excellent agreement with pKas predicted by DFT methods. Extensive DFT calculations have been used to rationalize the selective formation of four out of nine possible F2Cl1-4 isomers of bridge-halogenated bicyclo[1.1.1]pentanes and to obtain relative strain energies for all possible isomers.

Micellar electrokinetic chromatography in the determination of triazoles in fruit peel.

Triazole fungicides (TAFs) are frequently used fungicides for various antifungal treatments of crops. Tre treatment is provided foliarly. However, some significant amount of TAFs may remain on/in fruits. We have developed a methodology for the determination of penconazole, tebuconazole and cyproconazole in tomato fruit peel. The extraction of TAFs was provided with chloroform (acidified with 0.1% acetic acid). In the electrokinetic chromatography, the mixed micellar pseudo-stationary phase was composed of anionic detergent sodium dodecyl sulphate (15 mM) and randomly highly sulphated gamma-cyclodextrin (17.5 mg/mL). The background electrolyte consisted of 100 mM phosphoric acid and 100 mM Tris in the mixed hydro-organic solvent water/methanol (80/20 v/v), apparent pH 4.8. Complete separation of penconazole, tebuconazole, and two diastereomers of cyproconazole with resolutions higher than 5.1 were achieved within a relatively short time of less than 17 min in the bare fused silica capillary of 425/500 mm total/effective lengths and 50/375 µm I.D./O.D. at separation voltage -15 kV (cathode at injection capillary end) and at constant capillary cassette temperature of 22°C. The TAFs were detected by a UV-spectrophotometric diode array detector set at 200 nm. The limits of detection and limits of quantification were in the range of 71-92 and 214-278 µg/kg of peel, respectively. Analyses of the peel extracts revealed that even 10 days after the last treatment, TAF concentrations were higher than the recommended maximum residue limits in both application ways, as individual as well as in the TAF binary or ternary mixtures.

Phosphofructokinases A and B from Mycobacterium tuberculosis Display Different Catalytic Properties and Allosteric Regulation.

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.

Determination of acidity constants, ionic mobilities, and hydrodynamic radii of carborane-based inhibitors of carbonic anhydrases by capillary electrophoresis.

Capillary electrophoresis (CE) has been applied for determination of the thermodynamic acidity constants (pKa ) of the sulfamidoalkyl and sulfonamidoalkyl groups, the actual and limiting ionic mobilities and hydrodynamic radii of important compounds, eight carborane-based inhibitors of carbonic anhydrases, which are potential new anticancer drugs. Two types of carboranes were investigated, (i) icosahedral cobalt bis(dicarbollide)(1-) ion with sulfamidoalkyl moieties, and (ii) 7,8-nido-dicarbaundecaborate with sulfonamidoalkyl side chains. First, the mixed acidity constants, pKamix , of the sulfamidoalkyl and sulfonamidoalkyl groups of the above carboranes and their actual ionic mobilities were determined by nonlinear regression analysis of the pH dependences of their effective electrophoretic mobility measured by capillary electrophoresis in the pH range 8.00-12.25, at constant ionic strength (25 mM), and constant temperature (25°C). Second, the pKamix were recalculated to the thermodynamic pKa s using the Debye-Hückel theory. The sulfamidoalkyl and sulfonamidoalkyl groups were found to be very weakly acidic with the pKa s in the range 10.78-11.45 depending on the type of carborane cluster and on the position and length of the alkyl chain on the carborane scaffold. These pKa s were in a good agreement with the pKa s (10.67-11.27) obtained by new program AnglerFish (freeware at https://echmet.natur.cuni.cz), which provides thermodynamic pKa s and limiting ionic mobilities directly from the raw CE data. The absolute values of the limiting ionic mobilities of univalent and divalent carborane anions were in the range 18.3-27.8 TU (Tiselius unit, 1 × 10-9 m2 /Vs), and 36.4-45.9 TU, respectively. The Stokes hydrodynamic radii of univalent and divalent carborane anions varied in the range 0.34-0.52 and 0.42-0.52 nm, respectively.

Multipodal insulin mimetics built on adamantane or proline scaffolds.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 µM) than proline-derived compounds (Kd values of 15-38 µM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.

Enantioselective resolution of side-chain modified gem-difluorinated alcohols catalysed by Candida antarctica lipase B and monitored by capillary electrophoresis.

An enzymatic alternative to the chemical synthesis of chiral gem-difluorinated alcohols has been developed. The method is highly effective and stereoselective, feasible at laboratory temperature, avoiding the use of toxic heavy metal catalysts which is an important benefit in medicinal chemistry including the synthesis of drugs and drug precursors. Candida antarctica lipases A and B were applied for the enantioselective resolution of side-chain modified gem-difluorinated alcohols, (R)- and (S)-3-benzyloxy-1,1-difluoropropan-2-ols (1a and 1b), compounds serving as chiral building blocks in the synthesis of various bioactive molecules bearing a gem-difluorinated grouping. The catalytic activity of these lipases was investigated for the chiral acetylation of 1a and 1b in non-polar solvents using vinyl acetate as an acetyl donor. The dependence of the reaction course on various substrate and enzyme concentrations, reaction time, and temperature was monitored by chiral capillary electrophoresis (CE) using sulfobutyl ether ß-cyclodextrin as a stereoselective additive of the aqueous background electrolyte. The application of CE, NMR, and MS methods has proved that the complex enzyme effect of Candida antarctica lipase B leads to the thermodynamically stable (S)-enantiomer 1b instead of the expected acetylated derivatives. In contrast, the enantioselective acetylation of racemic alcohol 1 was observed as a kinetically controlled process, where (R)-enantiomer 1a was formed as the main product. This process was followed by enzymatic hydrolysis and chiral isomerisation. Finally, single pure enantiomers 1a and 1b were isolated and their absolute configurations were assigned from NMR analysis after esterification with Mosher's acids.

Bridge-Chlorinated Bicyclo[1.1.1]pentane-1,3-dicarboxylic Acids.

Radical chlorination of bicyclo[1.1.1]pentane-1,3-dicarboxylic acid is highly selective, and up to four chlorine atoms can be introduced relatively easily without damage to the strained bicyclic cage. Combined with hydrodechlorination with TMS3SiH, direct chlorination provides access to five of the 15 possible chlorinated diacids. Their configuration has been established by X-ray diffraction. Their p Ka values have been measured by capillary electrophoresis and calculated at the B3LYP-D3BJ/6-311+G(d,p)-level. The results are in good agreement and reflect the expected trend, from 2.78 ± 0.08 and 4.14 ± 0.10 in the parent to 1.07 ± 0.03 and 2.31 ± 0.03 in the tetrachlorinated diacid. Strain energy relative to the parent diacid was calculated for all 15 chlorinated diacids and shows a dramatic increase with successive chlorination, due to nonbonded Cl-Cl repulsions.

Pressure assisted partial filling affinity capillary electrophoresis employed for determination of binding constants of human insulin hexamer complexes with serotonin, dopamine, arginine, and phenol.

A new method, pressure assisted partial filling affinity capillary electrophoresis, has been developed to study noncovalent molecular interactions of the hexamer of human insulin (HI) with biologically relevant ligands, basic phenolic neurotransmitters serotonin and dopamine, basic amino acid arginine, and very weakly acidic phenol, in alkaline aqueous media. The apparent binding constants, Kb, of the HI-ligand complexes were determined from the dependence of the effective migration time changes of the above ligands on the variable zone lengths of HI hexamer dissolved in the background electrolyte (BGE) and hydrodynamically introduced into the bare fused silica capillary close to the UV detector. The strong cationic electroosmotic flow (EOF) in alkaline BGEs, 40/40 mM Tris/tricine, pH 8.1, and 25/34 mM NaOH/tricine, pH 8.5, with EOF mobilities 52.0 × 10-9 and 58.0 × 10-9 m2V-1s-1, respectively, was reduced by the hydrodynamic counter flow induced by external pressure at the outlet capillary end to avoid expulsion of HI zone out of the capillary and to allow HI interaction with both cationic and anionic ligands inside the capillary. The HI hexamer interactions with the above ligands were found to be weak to moderately strong, with Kb values in the range 385-1314 L mol-1, and decreasing in the order HI-phenol > HI-dopamine > HI-serotonin > HI-arginine.

Investigation of the acid-base and electromigration properties of 5-azacytosine derivatives using capillary electrophoresis and density functional theory calculations.

Capillary electrophoresis (CE) and quantum mechanical density functional theory (DFT) were applied to the investigation of the acid-base and electromigration properties of important compounds: newly synthesized derivatives of 5-azacytosine - analogs of efficient antiviral drug cidofovir. These compounds exhibit a strong antiviral activity and they are considered as potential new antiviral agents. For their characterization and application, it is necessary to know their acid-base properties, particularly the acidity constants (pKa) of their ionogenic groups (the basic N3 atom of the triazine ring and the acidic phosphonic acid group in the alkyl chain). First, the mixed acidity constants (pKamix) of these ionogenic groups and the ionic mobilities of these compounds were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities. Effective mobilities were measured by CE in a series of background electrolytes in a wide pH range (2.0-10.5), at constant ionic strength (25mM) and constant temperature (25°C). Subsequently, the pKamix values were recalculated to thermodynamic pKa values using the Debye-Hückel theory. The thermodynamic pKa value of the NH+ moiety at the N3 atom of the triazine ring was found to be in the range 2.82-3.30, whereas the pKa of the hydrogenphosphonate group reached values from 7.19 to 7.47, depending on the structure of the analyzed compounds. These experimentally determined pKa values were in good agreement with those calculated by quantum mechanical DFT. In addition, DFT calculations revealed that from the four nitrogen atoms in the 5-azacytosine moiety, the N3 atom of the triazine ring is preferentially protonated. Effective charges of analyzed compounds ranged from zero or close-to-zero values at pH 2 to -2 elementary charges at pH≥9. Ionic mobilities were in the range (-16.7 to -19.1)×10-9m2V-1s-1 for univalent anions and in the interval (-26.9 to -30.3)×10-9m2V-1s-1 for divalent anions.

Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with ß-cyclodextrin by affinity capillary electrophoresis.

Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of (R,S)-enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector ß-cyclodextrin (ßCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)-enantiomers of ANPs-based antiviral drugs and their derivatives with ßCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0-25 mM) of ßCD. The apparent binding constants of the complexes of (R,S)-enantiomers of ANPs with ßCD were estimated from the dependence of effective electrophoretic mobilities of (R,S)-enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of ßCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on ßCD concentration in the BGE. The complexes of (R,S)-enantiomers of ANPs with ßCD have been found to be relatively weak - their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3-46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3-55.2 L/mol.

Enantiopurity analysis of new types of acyclic nucleoside phosphonates by capillary electrophoresis with cyclodextrins as chiral selectors.

CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogs bearing [(3-hydroxypropan-2-yl)-1H-1,2,3-triazol-4-yl]phosphonic acid, 2-[(diisopropoxyphosphonyl)methoxy]propanoic acid, or 2-(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6-diaminopurine, uracil, and 5-bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5-bromouracil-derived acyclic nucleoside phosphonate with a 2-(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51-2.94, within a reasonable time, 13-28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85, and 10.30, respectively) containing 20 mg/mL ß-cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5-bromouracil-derived acyclic nucleoside phosphonate with 2-(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20:40 mM Tris/phosphate, pH 2.20) using 60 mg/mL quaternary ammonium ß-cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range.

Determination of acidity constants and ionic mobilities of polyprotic peptide hormones by CZE.

CZE has been applied to determination of thermodynamic acidity constants (pKa ) of ionogenic groups and actual ionic mobilities of polyprotic peptides-synthetic human and salmon gonadotropin-releasing hormones and their derivatives and fragments. First, the mixed acidity constants, pKa,imix, of ionogenic groups, and actual ionic mobilities, mi , of gonadotropin-releasing hormone peptides were determined by nonlinear regression analysis of pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE in a series of BGEs within a broad pH range (1.80-12.10), at constant ionic strength (25 mM) and reference temperature (25°C). Second, the pKa,imix values were recalculated to thermodynamic pKa s using the Debye-Hückel theory. Thermodynamic pKa of carboxyl groups was estimated to be in the range of 2.5-3.3 for C-terminal amino acids of the above peptides, and 5.2 for glutamic acid in the middle of peptide chain; pKa of imidazolyl group of histidine residues was in the range of 5.7-6.8, pKa of N-terminal amino group of the peptide with free N-terminus was equal to 6.2, pKa of phenol group of tyrosine residues was in the range of 9.8-10.8, and pKa of guanidinyl group or arginine residues reached values 11.1-11.3, depending on the position of the residues in the peptide and on the amino acid sequence of the peptide. Absolute values of actual ionic mobilities of peptides with charge number ±2 were in the range (14.6-18.6) × 10(-9) m(2) V(-1) s(-1) , and ionic mobilities of peptides with charge number ±1 reached values (6.5-12.9) × 10(-9) m(2) V(-1) s(-1) .