Chromatography nucleotide separation

Many schemes for fractionating nucleotides, nucleosides and bases on sulphonated polystyrene resins have been published. The main difficulty with these methods is variation between resin batches (e.g. Anderson et al. 1963). Nucleotide separations can be achieved on DEAE-cellulose (Whatman Data Sheet 13, 1967) and DEAE-Sephadex (Piers et al. 1965b) but these media do not seem to be widely used. Gel filtration columns will separate some nucleotide components. Ligand exchange chromatography and partition chromatography of nucleosides are useful for minor components. 

The selectivity of ion-exchange chromatography for nucleotide separations is significantly affected by the pH of the mobile phase and this can therefore be manipulated to achieve many separations. 

The Gp Rh-based sensor can also be used for quantitative analyses. This was demonstrated with the analytes ATP, cAMP, and PPi. The hydrolysis of ATP to give cAMP and PPi is catalyzed by adenylate cyclase (AC). AC is an important enzyme that is involved in many signaling pathways. Its activity is commonly measured by monitoring the conversion of [a- P]ATP to [a- P]cAMP using ion-exchange chromatography to separate the nucleotides. To show that the sensor can be used to measure simultaneously the concentrations of ATP and cAMP/PPi,... 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

In relation to separation of nucleotides, Hoffman61 found that adenine nucleotides interacted most strongly with cycloheptaamylose, presumably by inclusion of the base within the cavity of cyclodextrin. When epichlorohydrin-cross-linked cycloheptaamylose gel was used as a stationary phase for nucleic acid chromatography, adenine-containing compounds were retarded most strongly. 

Horvath, C. G., Preiss, B. A., and Lipsky, S. R., Fast liquid chromatography an investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers, Anal Chem., 39, 1422, 1967. 

A cyclic nucleotide, 2, 3 -bis(2-chloroethyl)aminophosphoryl-3 -amino-3 -deoxyadenosine (139) showing antitumor activity, was prepared in 40% yield starting from 3 -amino-3 -deoxyadenosine 133 by its phosphorylation with N,N-bis-(2-chloroethyl) amidophosphoryl dichloride 138. Both P-diastcrcomers separated by column chromatography exhibit activity against KB tumor cell cultures (Scheme 40) [71]. 

The bearing which these discoveries have had on the elucidation of the structure of ribopolynucleotides will be discussed later. It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography iB mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,66 or by deamination by a specific enzyme... 

In view of the difficulty of hydrolyzing the pyrimidine nucleosidic linkages, ribonucleic acids have been hydrolyzed to a mixture of purine bases and pyrimidine nucleotides which is then separated by paper chromatography.132, 163 164 This method has been employed extensively for the analysis of ribonucleic acids, and gives reproducible results,166 but it has not been used to any great extent for deoxyribonucleic acids, probably because, under these conditions of hydrolysis, they yield some pyrimidine deoxy-ribonucleoside diphosphates.166... 

TLC has similar applications to paper chromatography. The stationary phase is a coating, such as silica gel, on a glass or plastic plate. Depending on the TLC plate used, components may be separated based on differences in molecular weight, charge, or polarity (see Chapter 11). TLC with a 70% isopropyl alcohol mobile phase and a silica gel plate is an effective substitute for paper chromatography separation of amino acids. Nucleotides may be separated on a special silica gel plate and a 20% ethanol (in water) mobile phase. 

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