5-Methyl cytidine

In higher eukaiyotes, most of the chromosomal DNA carries 5-methyl-cytidine residues located in CpG sequence motives. There is a close correlation between transcriptional inactivation and methylation. On the other hand, considerable evidence shows that regions of DNA that are actively engaged in transcription lack 5-methyl-cytidine nucleotides in CpG motivs. Hence DNA methylation is a means how cells regulate gene expression. DNA methylation which is catalyzed by DNA methyltransferases is the best characterized epigenetic mechanism. 

An essential instrument for the suppression of transcription activity in heterochromatin, as well as for the differential regulation in euchromatin, is the methylation of DNA on the C5 atom of cytidine in the CpG sequence (Fig. 1.43). CpG sequences occur imevenly distributed in the genome. They may be concentrated in CpG islands. Higher eucaryotes possess a characteristic distribution pattern of 5-methyl cytidine (m C), which remains intact upon cell division. Mechanisms must therefore exist to ensure that the methylation pattern is precisely retained in the daughter cells following cell division. A methyl transferase that carries out hemi-methylation in the CpG sequences (Fig. 1.43) is responsible for the inheritance of the methylation pattern. The methyl group is derived from S-adenosyl methionine. The preferential substrates for the hemi-methylation are DNA sequences in which the complementary strand is already methylated. Such a hemi-methylation occurs, for example, shortly after replication of the sequence. 

Fig. 1.43. The methylation of DNA 5-methyl-cytidine and maintenance methylation. a) The methylation of cytidine residues on DNA is catalyzed by a methyl transferase that employs S-ade-nosine methionine as a methyl group donor. The peferable substrate for the methyl transferase are hemi-methylated CpG sequences. 5-aza-cytidine is a specific inhibitor of methyl transferses. b) The methylation pattern of DNA remains intact upon DNA replication and is passed on to the daughter cells. The newly synthesized strands are unmethylated immediately after DNA rephca-tion. The methyltransferase uses the previously methylated parent strand as a matrix to methylate the CpG sequences of the newly synthesized strand.

The substituents at the 5 or 6 position of the pyrimidine base have no relation to the susceptibility to RNase T2. Madison and Holley 93) have shown that RNase T2 is able to split the phosphodiester bonds of pseudouridylic acid and 5,6-dihydrouridylic acid. Recently, Saneyoshi et al. 86) have found a new minor constituent, Vp, with 5-methyl cytidine 3 -phosphate and other mononucleotides on the two-dimensional paper chromatogram of RNase T2 exhaustive digest of E. coli tRNAVal later the structure of V was proved to be uridine-5-oxyacetic acid 94). 

This difficulty may be surmountable in view of the finding of Behe and Felsenfeld (Xi) that 5-methyl cytidine substituted for C in d(GC) causes the salt concentration of the midpoint of the B to Z transition to be greatly reduced. This has enabled us to follow... 

Related methods are being applied to the determination of the secondary structure of RNA molecules665 666 and to the study of interactions with proteins. For example, treatment with dimethyl sulfate under appropriate conditions methylates bases that are not paired, giving largely 1-methyladenosine and 3-methyl-cytidine.667... 

Figure 29-2. Sequence and projection of the conformation of the phenyl alanine specific f-RNAP " of yeast. D, Dihydrouridine DiMeG, 2,2-dimethylguanosine 2 OMeC, 2 methyl cytidine ij/, pseudo-uridine (base bonded to the sugar group via C ).

Here we illustrate our efforts towards the optimization of 2 -methyl-cytidine prodrugs employing a kinase-bypass approach based on 5 -phosphoramidate prodrugs (McGuigan). 

Among derivatives based on the stucture of biological compound, one may recall studies on the photochemistry of 4 -azido-2 -deoxy-2 -methyl-cytidine, and on 6-azidopurine ribonucleoside in water. 

A-Acetyl-9-deoxy-9-fluoroneuraminic acid (591) was prepared by treatment of a protected 6-hydroxyl precursor with A, A-diethylaminosulfur trifluoride (DAST) or through condensation of 2-acetamido-2,6-dideoxy-6-fluoro-D-mannopyranose with potassium di(/ >r/-butyl) oxaloacetate. Compound 591 is a substrate for cytidine monophosphate (CMP)-sialic acid synthetase, giving rise to CMP-5-A-acetyl-9-deoxy-9-fluoroneuraminic acid, which is cytotoxic against tumor cells. 5-A-Acetyl-3-fluoroneuraminic acids 592-594 were prepared through fluorine (or acetyl hypofluorite) addition (in AcOH) to methyl 5-acetamido-4,7,8,9-tetra-0-acetyI-2,6-anhy-dro-2,3,5-trideoxy-D- /ycm>D- a/arto-non-2-enopyranosate. Compound 592 was found to be a potent neuraminidase inhibitor. 

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.)...

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