Nucleoside separation

Separation of proteins Separation of nucleosides Separation of nucleic acid bases and their nucleosides... 

Figure 2.21 The effect of washing procedures on removal of debris (dextran sulfate) as measured by separation of nucleosides. Separations were carried out on a reversed-phase (C g) column with a mobile phase of 65 mM potassium phosphate (pH 3.6), 2% acetonitrile, and 1 mM tetra-n-butylammonium phosphate. The flow rate was 2 mL/ min the column was eluted isocratically and monitored at 254 nm. (A) Separation routinely achieved with AMP, ADP, and ATP. (B), Separation observed after clogging the column with 10 mAf dextran sulfate. (C) The separation observed after washing the column with 6 M urea. (D) The separation obtained after washing the column with toluene.

Mundry (1965) has also described the use of DEAE-Sephadex for the separation of ribonucleotides and nucleosides. Using a 1.1 x85 cm column and a 1.1-1 linear gradient from 0.04 M Tris-HCl pH 8.8 to 0.20 M Tris-HCl, 0.25 M NaCl pH 9.5 or a linear gradient from 0.04 M triethylamine carbonate pH 8.8 to 0.35 M triethylamine carbonate, 0.15 M NaCl pH 9.5 he has separated the 8 main ribonucleosides and nucleotides. The nucleoside separation is very sensitive to traces of salt and some difficulty with reproducibility was experienced. 

The general principles which have been outlined above are applicable to all nucleoside separations but it is instructive to consider specific examples drawn from the various areas in which these techniques are valuable. 

Keith (1995) presented reference maps of mobilities of modified ribonucleotides and nucleosides separated by two-dimensional TLC on cellulose. Paw and Misztal (1995) isolated cytosine, uracil, and their arabinosides from human plasma by solid-phase extraction and Adsobex minicolumns and separated them by horizontal and ascending TLC in various mobile phases. 

Diethylaminoethyl-cellulose (DE AE) is the functional group incorporated into the paper. It is an anion exchanger which is generally used to separate proteins and enzymes and similar materials but which is also used for nucleic acids, nucleotides, deoxynucleotides, and nucleosides. Separation on DEAE-cellulose is not as sharp as on PEI-cellulose but there is considerable data on the separation of nucleic acids on these layers. 

Dowex 50W-X4 1.1 0.80 Strongly acidic cation exchanger with S-DVB matrix for separation of amino acids, nucleosides and cations. Molecular weight exclusion is < 1400. 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Protective group chemistry for these amines has been separated from the simple amines because chemically they behave quite differently with respect to protective group cleavage. The increased acidity of these aromatic amines makes it easier to cleave the various amide, carbamate, and sulfonamide groups that are used to protect this class. A similar situation arises in the deprotection of nucleoside bases (e.g., the isobutanamide is cleaved with methanolic ammonia ), again, because of the increased acidity of the NH group. 

A wide variety of bases, nucleosides and nucleotides have been separated using porous layer bead ion exchangers. A representative chromatogram of the separation of ribonucleoside mono-phosphoric acids from the work of Smukler ( ) is shown in Figure 4. Recently, ion exchangers chemically bonded to small particle diameter (> 10 ym) silica have been successfully applied to the separation of nucleic acid constitutents (37). The rapid separations using such supports undoubtedly mean that they will find increasing use in the future. 

Figure 8.24 Separation of the major deoxyribonucleosides and their 5 - monophosphate deoxynucleotides on a strong cation exchange column (column one) and a reversed-phase column. The unseparated nucleosides. A, on the ion- exchange column were switched to the reversed-ptose column. Pe2dc Identification A = nucleosides, B d-CMP, C d-AMP, D - d-GJIP, E - d-CVD, P d-UKO, G THD, and H = d-AOO. (Reproduced with permission from ref. 298. Copyright Preston Publications, Inc.)...

Figure 12 Gradient separation of bases, nucleosides and nucleoside mono- and polyphosphates. Column 0.6 x 45 cm. Aminex A-14 (20 3 p) in the chloride form. Eluent 0.1 M 2-methyl-2-amino-l-propanol delivered in a gradient from pH 9.9-100 mM NaCl to pH 10.0-400 mM NaCl. Flow rate 100 ml/hr. Temperature 55°C. Detection UV at 254 nm. Abbreviations (Cyt) cytosine, (Cyd) cytidine, (Ado) adenosine, (Urd) uridine, (Thyd) thymidine, (Ura) uracil, (CMP) cytidine monophosphate, (Gua) guanine, (Guo) guanosine, (Xan) xanthine, (Hyp) hypoxanthine, (Ino) inosine, (Ade) adenosine, (UMP) uridine monophosphate, (CDP) cytidine diphosphate, (AMP) adenosine monophosphate, (GMP) guanosine monophosphate, (IMP) inosine monophosphate, (CTP) cytidine triphosphate, (ADP) adenosine diphosphate, (UDP) uridine monophosphate, (GDP) guanosine diphosphate, (UTP) uridine triphosphate, (ATP) adenosine triphosphate, (GTP), guanosine triphosphate. (Reproduced with permission of Elsevier Science from Floridi, A., Palmerini, C. A., and Fini, C., /. Chromatogr., 138, 203, 1977.)...

The identification of the HIV-1-specific non-nucleoside reverse transcriptase inhibitors (NNRTIs) as a separate class of HIV inhibitors was heralded by the discovery of the tetrahydroimidazo[4,5,1 -// .][ 1,4]benzo-diazepin-2(l //)-onc and -thione (TIBO) derivatives (Fig. 7) [58,59] and 1 -(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivatives (Fig. 8) [60,61]. The first TIBO derivatives (R82150, R82913) were the first NNRTIs [58] postulated to act as inhibitors of HIV-1 RT [59], For the HEPT derivatives it became evident that they also interact specifically with HIV-1 RT after a number of derivatives (i.e., E-EPU, E-EBU, and E-EBU-dM) had been synthesized that were more active than HEPT itself [62,63]. Following HEPT and TIBO, several other compounds, i.e., nevirapine, pyridinone, and bis(heteroaryl)piperazine (BHAP), were... 

The hydroxy groups of the pyrimido[l,6-c][l,3]oxazine 316 (R1 = R2 = H) were mono-tritylated with 4,4 -(MeO)2-trityl chloride in pyridine/NEt3. The mixture of the two products 316 (R1 =4,4 -(MeO)2-trityl R2 = H) and 316 (R1 = H R2 = 4,4 -(MeO)2-trityl) was coupled with nucleoside phosphoramidites, oxidized to protected dinucleosides and the isomers were separated <1999NN2031>. 

Singhal, R.P., Bajaj, R.K., Buess, G.M., Smoll, D.B., and Vakharia, V.N. (1980) Reversed-phase boro-nate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs. Anal. Biochem. 109, 1-11. 

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