Uridine separation from cytidine

The product, uridine, was separated from cytidine and allopurinol (internal standard) by chromatography on a Hypersil ODS column (4.6 mm X 100 mm, 5 pun). Hie mobile phase consisted of 100 mM ammonium acetate (adjusted to pH 5.0 with 6 M HC1) containing 1% (v/v) methanol and 1mA/ 1-octanesulfonic acid. The effluent was monitored at 262 nm. Uridine production was calculated with reference to a uridine standard, and after correction based on the concentration of the internal standard. 

Fig. 4.4.3. Separation of nucleobases and nucleosides on a LiChrosorb SI 100 column (0.46 x 25 cm). Eluent Dichloromethane/methanol/2.65 M formic acid (80 18 2). Peaks Thy, thymine Ura, uracil Ade, adenine Xan, xanthine Ado, adenosine Hyp, hypoxanthine Urd, uridine Gua, guanine Ino, inosine Xao, xanthosine Cyt, cytosine Guo, guanosine Cyd, cytidine. Reprinted from Ref. 4 with permission.

Bull seminal plasma also contains 5-nucleotidase activity. The enzyme has been purified from this source and found to split adenosine-5 -phosphate, uridine-5 -phosphate, cytidine-5 -phosphate, guanosine-5 -phosphate, and nicotinamide-ribose-5 -phosphate. Of several dozen other esters, only the desoxyribonucleotides have been found active as substrates. Snake venom contains a highly specific 5-nucleotidase which can be separated from phosphodiesterase by means of cellulose column chromatography. ... 

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.)...

Condensation of LXVIa (R = p-toluyl) with monomercurithymine (prepared from 1-acetylthymine) produces a mixture of acylated nucleoside anomers (LXVlb and LXVIc, R = thyminyl) which are separated and de-esterified to thymidine and a-thymidine. Similarly, reaction of N-acetylcytosinemercury (LV) with LXVIa (R = p-chlorobenzoyl) yields the a and ft anomers of acylated 2-deoxycytidine which, after separation and deacylation, are converted smoothly to 2-deoxycytidine (LXVlb, R = H, R = cytosinyl) and its a anomer (LXVIc). In like manner, 2-deoxy-5 -fluoro-uridine and -cytidine were synthesized, along with their... 

Fig. 10.20. Optimisation of temperature and voltage in separation of nucleosides. Column 250 mm x 100 pm i.d. packed with CEC Hypersil C18, 3 pm, Conditions mobile phase, (5 mM acetic acid, 3 mM TEA, pH 5)-acetonitrile, 92 8 (v/v) injection, 10 kV for 3 s. (A), 25°C and 20 kV (B), 25°C and 25 kV (C), 20°C 25 kV. Peak identification (in order of elution) 1, cytidine 7, thiourea 2, uridine 3, inosine 4, guanosine 5, thymidine 6, adenosine. Reproduced with permission from Helboe and Hansen [116].

Figure 9.89 Pyrimidine 5 -nucleotidase assay in undialyzed human erythrocyte lysate obtained by 1 5 dilution of packed cells with deionized water, (a) Separation of 1 nmol of CMP, cytidine, and uridine as the standards. Separation of the assay mixture, containing 50 mM Tris-HCl, pH 7.5, 0.2 mM CMP, 1 mM MgCl2, 1 mM DTT, and 20 fiL of lysate in 0.5 mL of total volume (b) at time zero and (c) after 30 minutes of incubation (c). (From Amici et al., 1994.)...

S ATP + deoxyadenosine <1-10> (<1> transfers a phospho group from specific nucleoside 5 -triphosphate donors to 5 -position of deoxyadenosine [1] <1> no activity with adenosine and guanosine [1] <2> no activity with cytidine, uridine, guanosine, deoxyguanosine and thymidine [3] <4> enzyme bears two separate but interacting active sites for deoxyadenosine and deoxycytidine kinase activity [5] <4> enzyme exists in two heterodimeric complexes, complex 1 deoxycytidine/deoxyadenosine kinase and complex II deoxyguanosine/deoxyadenosine kinase [6] <3> enzyme has both adenosine kinase and deoxyadenosine kinase activity [14]) (Reversibility <1-10> [1-9,11,12,15]) [1-9, 11, 12, 14, 15]... 

Fig. 2. Erythrocyte nucleotide formation from exogenous precursors. 0.5 ml aliquots of normal washed erythrocytes from fresh heparinised bloo was labelled for the times indicated wi h a) 20 jJiCi 5- H uridine (1.6 (imoles/l), b) 0 pCi 5- H cytidine (1.4 pmoles/1) or c) 0.5 pCi 2- C orotate (16 pmoles/1), and labelled nucleotides in the PGA extracts quantitated after separation on HPLC.

Fig. 1 shows such a separation in a PGA extract of H-Uridine labelled erythrocytes from a patient with pyrimidine 5 nucleotidase deficiency. Peaks corresponding in elution time to uridine and cytidine mono-, di-, and tri-phosphates can be seen. Label is present in all three uridine nucleotide peaks as well as in a fourth peak which corresponds in elution time in these and under other separation conditions with UDP-glucose (UDPG). 

The elegant ion-exchange separation techniques evolved in the recent years have enabled investigators to isolate from yeast, bacteria, and animal tissues, uridine 5 -di- (UDP) and -triphosphates (UTP) (134)y cytidine 5 -di- (CDP) and -triphosphates (CTP), and guanosine 5 -di- (GDP) and -triphosphates (GTP) (135), Application of the synthetic approaches developed for ADP and ATP has permitted the ready synthesis of uridine 5 -di- and -triphosphates (136) and could be applied to the other two pairs of nucleotides (CDP, CTP GDP, GTP). Although the functions of these latter compounds have not been elucidated, it seems probable that they are involved as coenzymes in transphosphorylation reactions similar to ATP. 

Figure 20.3 Separation of nucleotides and nucleosides on a silica gel layer. Compounds E, adenosine H, guanosine F, uridine G, cytidine A, adeno-sine-5-monophosphate B, uridine-5-monophosphate C, cystidine-5-mono-phosphate D, guanosine-5-monophosphate. [Reproduced with the permission of John Wiley and Sons, Inc., from Sleckman et al. (1985).]...

Fig. 24.30 (a) Synthetic steps towards a paira-ter/-butyl-calix[4]arene-bonded silica stationary phase [94]. (b) Chemical structure of para-tert-butyl-calix[8]arene-bonded silica gel stationary phase [95]. (c) Isocratic separation of nucleosides cytidine, uridine, guanosine and adenosine on an Aren Si 60 column with 0.02 M NaH2P04 (pH 3.5) as mobile phase (x-axis time in min y-axis absorbance at 254 nm) [93]. (Reprinted from Ref [93])... 

FIGURE 28.5 Typical chromatogram for separation of five nucleotides by ion-pair reversed-phase HPLC method. (1) cytidine 5 -monophosphate (CMP) (2) uridine 5 -monophosphate (UMP) (3) guanosine 5 -monophosphate (GMP) (4) inosine 5 -mono-phosphate (IMP) (5) adenosine 5 -monophosphate (AMP). The concentration of each nucleotide injected onto the column was 15 mg L h (Reprinted from Food Chem., 7A, Ferreira, I. M. P. L. V. O. et al., The determination and distribution of nucleotides in dairy products using HPLC and diode-array detection, 239-244, Copyright 2001, with permission from Elsevier.)... 

Fig. 17 a-c. Electrophoretic separations of RNA from salivary gland cell cytoplasm a, gastric caeci b and midgut c 2 days after precursor administration to the medium (25 pCi [ H]uridine and 25 pCi [ H]cytidine in 5 ml of culture medium for 20 h after which the animals were left in isotope-free medium for a further 48 h before killing). It can be seen that the late mRNA is only present in salivary gland cell cytoplasm. (After Edstrom and Tan-guay, 1974)... 

---