Capillary electrophoresis separation of the new anti‐AIDS agents 9‐(2‐phosphonylmethoxyethyl)adenine and 9‐(2‐phosphonylmethoxyethyl)‐2,6‐diaminopurine in mixtures with some monoribonucleotides or the most common deoxynucleotides

Abstract

The present work describes an electrophoretic method for the separation and determination of the new antivirals, 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(2-phosphonylmethoxyethyl)-2,9-diaminopurine (PMEDAP) in model mixtures with some monoribonucleotide isomers (3'-AMP, 2'-CMP, 3'-CMP, 3'-GMP, 2'-GMP, 3'-UMP, 5'-GMP, and 5'-UMP) or with the most common deoxynucleotides (dCMP, dCDP, dCTP, dTMP, dTDP, dTTP, dGMP, dGDP, dGTP, dAMP, dADP, dATP). A fused-silica capillary tube, 75 microm ID, 67.8 cm total length (60.3 cm length to the detector), with detection at 210 nm was employed. A hydrodynamic injection for 10 s (1.5 psi vacuum) was utilized to introduce the sample, and 30 kV voltage was applied for the separation. The complete separation of PMEA and PMEDAP from the mononucleotide isomers and deoxynucleotide mixtures is possible in less that 10 min and 25 min, respectively, using 20 mM borate buffer, pH 9.9, with the addition of 10 mM beta-cyclodextrin. Efficiencies of more than 120 000 and resolution higher than 1.9 were reached for each of the compounds studied. This capillary electrophoretic procedure opens the possibility for future determination of PMEA and PMEDAP in cell pool samples.

Electrophoretic behaviour of oligonucleotides and mono-, di- and triphosphate nucleotides by capillary zone electrophoresis

“…All injections were made in triplicate unless otherwise stated. Lyophilised standard mixtures of polydeoxythymidylic acids of chain lengths between 12 and 18 nucleotides (pd(T) [12][13][14][15][16][17][18] ) were purchased (Pharmacia, Uppsala, Sweden) and reconstituted in very high purity water (18 MO/cm). Aqueous solutions of individual pd(T) 5,6,7,8,9,10 and pd(T) 11 (at 5 optical density (OD) units/ mL) were prepared in the Department of Biochemistry, Queens Medical Centre, University of Nottingham, Nottingham, UK.…”

“…Aqueous solutions of individual pd(T) 5,6,7,8,9,10 and pd(T) 11 (at 5 optical density (OD) units/ mL) were prepared in the Department of Biochemistry, Queens Medical Centre, University of Nottingham, Nottingham, UK. A 200 mL standard mixture of 2.5 OD units/ mLpd(T) [5][6][7][8][9][10][11][12][13][14][15][16][17][18] was prepared by mixing appropriate quantities of the purchased standards.…”

“…Significant improvement in the between and within-run repeatability of nucleotides would be expected with particular application to improving the determination of nucleotides in complex matrices such as biological samples. Figure 5 shows the electropherogram for the separation of a mixture of polydeoxythymidylic acids between 5 and 18 nucleotides in chain length (pd(T) [5][6][7][8][9][10][11][12][13][14][15][16][17][18] ) using the standard conditions for those to separate the nucleotides in Fig. 1.…”

“…5 that the mixture could be separated up to and including pd(T) 11 . However, the larger chain lengths of pd(T) [12][13][14][15][16][17][18] coelute, identified as the large single peak. This apparent limit in resolving power for the mixture using this method can be explained in terms of the separation mechanism for this mode of capillary electrophoresis.…”

“…Nevertheless, good separations of a variety of nucleotide mixtures have been achieved in practice [9][10][11][12]. However, the underlying physicochemical properties which are responsible for the electrophoretic behaviour of small nucleic acids have been less well studied.…”