Veronika Šolínová

Chiral analysis of anti-acquired immunodeficiency syndrome drug, 9-(R)-[2-(phosphonomethoxy)propyl]adenine (tenofovir), and related antiviral acyclic nucleoside phosphonates by CE using β-CD as chiral selector

A new CZE method has been developed for chiral analysis of an important anti‐acquired immunodeficiency syndrome drug, 9‐(R)‐[2‐(phosphonomethoxy)propyl]adenine ((R)‐PMPA, tenofovir), and six related antiviral acyclic nucleoside phosphonates using β‐CD as a chiral selector. The influence of the composition, concentration and pH of the BGE and the type and concentration of chiral selector on enantiomer resolution was investigated. Complete separations of (R,S)‐enantiomers of PMPA with very good resolution (Rs=1.50–3.64) were achieved within a short time (4–15 min) in 20–50 mM sodium borate or sodium tetraborate BGEs, pH 10.0, at 20 mg/mL concentration of β‐CD. (R,S)‐enantiomers of five similar PMPA analogs containing purine bases (adenine, diaminopurine or guanine) and hydroxyl or fluor substituents at C3 carbon atom of propyl chain were baseline separated within 10–17 min in 35 mM sodium tetraborate BGE, pH 10.0, at 20 mg/mL β‐CD concentration. Another important antiviral used by acquired immunodeficiency syndrome patients, derived from pyrimidine base cytosine, 1‐(S)‐[3‐hydroxy‐2‐(phosphonomethoxy)propyl]cytosine (cidofovir), and the (R)‐enantiomer of this drug were successfully separated in 50 mM sodium tetraborate BGE, pH 10.5, at 20 mg/mL β‐CD concentration within 45 min. Using the UV‐absorption detection at 206 nm, the concentration detection limits of the analyzed acyclic nucleoside phosphonates were determined in the submicromolar to micromolar range (0.15–2.51 μg/mL level).

Acyclic Nucleoside Bisphosphonates: Synthesis and Properties of Chiral 2-Amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines

2-Amino-4,6-bis[(phosphonomethoxy)alkoxy]pyrimidines bearing two equal or different achiral or chiral phosphonoalkoxy chains have been prepared either by aromatic nucleophilic substitution of 2-amino-4,6-dichloropyrimidine or by alkylation of 4,6-dihydroxy-2-(methylsulfanyl)pyrimidine with appropriate phosphonate-bearing building block. Alkylation of 4,6-dihydroxy-2-(methylsulfanyl)pyrimidine proved to be the method of choice for efficient preparation of variety of bisphosphonates. The enantiomeric purity of selected compounds was determined by capillary electrophoresis. Antiviral activity of bisphosphonates is discussed.

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 pKas 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 m2V−1s−1, and ionic mobilities of peptides with charge number ±1 reached values (6.5–12.9) × 10−9 m2V−1s−1.

Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with β-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 β‐cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)‐enantiomers of ANPs‐based antiviral drugs and their derivatives with βCD 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 βCD. The apparent binding constants of the complexes of (R,S)‐enantiomers of ANPs with βCD 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 βCD 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 βCD concentration in the BGE. The complexes of (R,S)‐enantiomers of ANPs with βCD 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 β-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 β-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.

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.

Capillary electrophoresis in classical and carrier ampholytes-based background electrolytes applied to separation and characterization of gonadotropin-releasing hormones

Capillary zone electrophoresis (CZE) in classical buffer-based background electrolytes (BGEs) and carrier ampholytes-based capillary electrophoresis (CABCE) using narrow pH cuts of carrier ampholytes (CA) as constituents of quasi-isoelectric BGEs have been applied to separation and characterization of synthetic human and salmon gonadotropin-releasing hormones (GnRH) and their derivatives and fragments. The selectivity, separation efficiency, resolution and speed of CZE and CABCE analyses have been compared within a wide pH range of the BGEs (3.50-9.75) using two mixtures of structurally related GnRH peptides as model analytes. A baseline separation of mixture 1 (human GnRH and its three fragments) was achieved in CA-based BGEs at pH 3.5 and in both classical and CA-based BGEs within the pH range 7.00-9.75. Full separation of mixture 2 (salmon GnRH, its two fragments and human GnRH fragment) was obtained in both types of BGEs at acidic pH values 3.5 and 4.00 and at neutral pH 7.00. In addition to the separation of related GnRHs, their effective electrophoretic mobilities were determined and from the dependences of mobilities on pH, the isoelectric points (pI) of analyzed peptides were estimated. The pI values obtained by CABCE were in a good agreement with those determined by CZE in classical BGEs but in some cases rather different from those predicted by theoretical calculations.