5-hydroxymethyl cytosine

Hydroxymethylcytosine (967) was isolated only in 1952 from the T-even bacteriophages of Escherichia coli, in which it occurs instead of cytosine in the 2-deoxyribonucleic acid (65MI21304). Of several syntheses described, the most convenient is probably that beginning with ethyl 4-amino-2-methylthiopyrimidine-5-carboxylate which is reduced by LAH to 4-amino-2-methylthiopyrimidin-5-ylmethanol followed by hydrolysis to 5-hydroxymethyl-cytosine (967) (B-68MI21302, B-68MI21306). 

For various reasons, the generalizations mentioned above must be regarded as strictly provisional. Analyses utilizing formic acid indicate the presence of more than one phosphorus atom per purine or pyrimidine residue. This discrepancy, it is pointed out, could equally well result from an apparent deficiency of bases, due to error in the analytical technique.160 It is also necessary to consider that some nucleic acids are now known to contain more bases than was previously realized. Thus, 5-(hydroxymethyl)-cytosine is present in various viruses,181-182 and 5-methylcytosine occurs in various animal and plant deoxyribonucleic acids but is absent from those of microbial origin.17-160-1M- 184- 186 Certain microbial deoxyribonucleic acids also contain 6-methylaminopurine.186a Various bacteriophage deoxyribonucleic acids have been found to contain a component which is believed to consist of a D-glucoside186b of 5 -(hydroxymethyl)cytidylic acid. 

The most important pyrimidine derivatives are those upon which biological organisms depend. Cytosine 1018 and uracil 1019 are found in ribonucleic acid (RNA) in the form of their ribonucleotides, cytidine 1020 and uridine 1021, while in deoxyribonucleic acid (DNA), cytosine and thymine 1022 are found in the form of their 2 -deoxyribonucleotides, 2 -deoxycytidine 1023 and thymidine 1024. 5-Methylcytosine 1025 is also found to a small extent (c. 5%) in human DNA in the form of its 2 -deoxyriboside 1026, and 5-(hydroxymethyl)cytosine-2 -deoxyriboside 1027 has also been detected in smaller amounts <2005CBI1>. Many variants of cytosine and uracil can be found in RNA including orotic acid 1028 in the form of its ribonucleotide orotidine 1029. Other pyrimidine derivatives to have been isolated from various biological sources include 2 -deoxyuridine 1030, alloxan 1031, and toxopyrimidine (pyramine) 1032 (Figure 2). 

Setlow and Carrier57 report that 5-hydroxymethyl cytosine does not form a photohydrate. 

It was concluded39 from these studies that the two nucleotides obtained by enzymic hydrolysis of the deoxyribonucleic acid of T2 bacteriophage are 2-deoxy-5 -(hydroxymethyl)cytidylic acid and a glucose derivative thereof. It was also concluded that the glucose residue is affixed to the pyrimidine portion of the nucleotide and nucleoside of 5-(hydroxymethyl)-cytosine and, on the basis of the near identity of the spectra of the two nucleotides of 5-(hydroxymethyl)cytosine, it was suggested that the hexose is attached to the hydroxymethyl group of the pyrimidine. 

The discovery of the presence of glucose in the nucleoside of 5-(hydroxy-methyl)cytosine was as unexpected as the original finding of the presence36 of 5-(hydroxymethyl)cytosine and the absence of cytosine.36 As Cohen suggests,84 other viruses contain cytosine they therefore lack the hydroxy -... 

The assignment of the 2-deoxy-D-er ypurine nucleosides of T6 nucleic acid liberated, on weak acid hydrolysis, a deoxypentose having the same R value as the deoxypentose of thymus deoxyribonucleic acid.37 In addition, the glucose-free nucleotide of 5-(hy-droxymethyl)cytosine39 gave a positive Dische test76 for deoxypentose. 

The infected system was shown to contain deoxymidine 5-phosphate this observation is interesting, since this nucleotide has not yet been shown to be present in normal cells. The nucleotide is presumably used for the synthesis of thymidine 5-triphosphoric acid. Detectable quantities of 5-(hydroxymethyl)cytosine or of 5-(hydroxymethyl)cytosine nucleotides were not present, despite the fact that this base is a normal constituent of the phage deoxyribonucleic acid. Explanations for this observation are that (a) the amount present in the acid-soluble fraction at any given moment is too small for detection by the methods of anal3rsis employed, or (b) the newly synthesized 5-(hydroxymethyl)cytosine is directly incorporated into deoxyribonucleic acid. 

The bases always present in DNA are adenine, guanine, cytosine, and thymine, while in RNA the bases are adenine, guanine, cytosine, and uracil. Trace amounts of other bases are occasionally present in DNA and RNA. For example, DNA may contain 5-hydroxymethyl cytosine and several N-methyl purines. The tRNA may contain several unusual bases such as certain methyl purines or hydroxymethyl pyrimidines. 

The deoxyribonucleic acids of some bacterial viruses (T2, T4, and T6) are unique in that they contain the base 5-(hydroxymethyl)cytosine... 

The D-glucopyranosyl residues are transferred from uridine 5-(a-D-gluco-pyranosyl pyrophosphate) to the hydroxyl group of 5-(hydroxymethyl)-cytosine residues in deoxyribonucleic acid. " Unpolymerized 5 -(hydrox-ymethyl)cytidine and its 5-mono- or tri-phosphate do not function as D-glucopyranosyl acceptors. 

Many natural products are derived from pyrimidine. The pyrimidine bases thymine, cytosine and uracil (22-24) are important as building-blocks for the nucleic acids. 5-Methylcytosine (obtained from hydrolytic extracts of tubercle bacilli) and 5-(hydroxymethyl)cytosine (obtained from the bacteriophage of Escherichia coli) are rarer. 

Hydroxymethyl)cytosine has been found in the DNA of the T2, T4 and T phages of . coli [277, 278]. A glycosyl-substituted 5- hydroxymethyl)-2 -deoxycytidylic acid was isolated from a T2, -f-phage [279]. 

Other bases are also present in the DNA and RNA polymers, but to a much lesser extent. Examples include 5-methyl cytosine (present in the DNA of some plants and bacteria), 5-hydroxymethyl cytosine (present in the DNA of T-even phages bacterial viruses) and 4-thiouracil (present in some bacterial tRNA). 

Hydroxymethyl- cytosine Pyrimidine 2,6-Dihydroxy-5-hydroxymethyl 2,6-Dihydroxy-5-hydroxy-methylcytidine See cytidine S -phosphate... 

The more recent finding that the virus deoxycytidylic acid was in fact a deoxyribonucleotide of 5-hydroxymethyl cytosine further supports Weed and Cohen s conclusion (322b). 

The four bases mentioned account for most of those found in the DNA of higher forms of life. 5-methylcytosine forms the principal exception 25 per cent of the cytosine in the DNA of plants is in this form, but animals have much less, and bacteria have only 0--2 per cent (Vanyushin et al. 1968). Bacterial and viral DNAs sometimes contain other methylated bases, such as 6 -methyladenine, 2-methyladenine, or 5-hydroxymethyl-uracil. In some phages, all cytosine is replaced by 5-hydroxymethyl-cytosine, made by a virus-induced enzyme in the bacterial host (Cohen, 1963). 

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