Ribose phosphorylation

Uridine is a nucleoside containing uracil. Uridine differs from uracil in containing a sugar (ribose). Phosphorylation of uridine produces a nucleotide found in RNA. 

Structure. The mononucleotides of RNA consist of ribose phosphorylated at C3, and linked by an N-gly-cosidic bond to one of four bases adenine, guanine, cytosine or uracil. Many other bases (chiefly methylated bases) also occur, but are less common (see Rare nucleic acid components). The mononucleotide units form a linear chain via 3, 5 phosphodiesler bonds (see Nucleic acids). Sequence analysis of RNA has become a standard technique. In many cases the amino acid sequences of proteins are predicted from the sequence of the corresponding mRNA (or DNA) because it is much easier to clone the nucleic acid than to isolate the protein. 

Clearly, further efforts to exploit the use of azg as a selective agent seemed unjustified. It was therefore decided to test 6-mercaptopurine (6-MP), since this substance is also known to undergo ribose phosphorylation through the action of HGPRT enzyme. After several dose-response experiments and some consternation, a standard concentration of 50 ig/ml of 6-MP was deemed suitable. As indicated by the data presented in Table 3, the coincidence survival which troubled us when azg was employed was not a problem. We then proceeded to investigate the efficacy of 6-MP as a selective agent in a forward mutational assay system at the HGPRT locus. 

Phosphate esters of glucose, fructose, and other monosaccharides are important metabolic intermediates, and the ribose moiety of nucleotides such as ATP and GTP is phosphorylated at the 5 -position (Figure 7.13). 

A more complicated reaction sequence has been used by Ukita and Nagasawa (59) in their synthesis of 2-deoxy D-ribose 5-phosphate (2-deoxy D-erythro-pentose 5-(dihydrogen phosphate)), (29). They phosphorylated a mixture of the anomeric methyl deoxyribofuranosides (24)... 

Generally speaking, the phosphorylated deoxysugars undergo the usual reactions of carbohydrates without complication. For instance, both 2-deoxy D-ribose 5-phosphate (52, 59) and 2-deoxy D-xylose 5-phosphate (2) can be reduced to the corresponding 2-deoxy d-erythro- (48) and 2-deoxy D-threo-pentitol 5-phosphates (49). 2-deoxy ribose 5-phosphate has also been oxidized (52) to the corresponding phosphorylated acid (50). 

In theory, periodate oxidation could have given a clear-cut answer as to the composition of the isomeric mixture of deoxy ribose phosphates. The 4-phosphate (73), devoid of vicinal diol groups, should be resistant to periodate the 3-phosphate (74) should reduce one and only one molar equivalent of the oxidant and yield one molar equivalent of both formaldehyde and the phosphorylated dialdehyde (75), whereas the 5-phosphate (76) could be expected to reduce one molar equivalent of periodate relatively rapidly, followed by a slower overoxidation reaction owing to the oxidation of malonaldehyde, formed as a result of the glycol cleavage. 

In fact, it has been found (52) that in unbuffered solution, at room temperature, authentic 2-deoxy ribose 5-phosphate reduces more than 4 molar equivalents of periodate, but. that there is no noticeable slowing down of the reaction rate after the reduction of the first molar equivalent. This may be owing to the fact that only the aldehydo form (76) of 2-deoxy ribose 5-phosphate has a free vicinal diol group as the acyclic 2-deoxy ribitol 5-phosphate reduces one molar equivalent of periodate quite fast (58), it is probable that the time-curve of periodate uptake by the phosphorylated sugar reflects the rate of formation of the aldehyde form from the furanose form. 

An early observation that 2 -d-3 -AMP was a more potent inhibitor of adenylyl cyclases than 2 -d-Ado suggested that the enzyme would accept substitutions at the 3 -ribose position and that phosphate was particularly well tolerated. This led to the generation of a family of 3 -phosphoryl derivatives of 2, 5 -dideoxyadenosine exhibiting ever greater inhibition with the addition of an increasing number of 3 -phosphoryl groups, the most potent of which is 2, 5 -dideoxyadenosine-3 -tetraphosphate (2, 5 -dd-3 -A4P Table 4) [5]. These constitute a class of inhibitors historically referred to as P -site ligands that caused inhibition of adenylyl... 

The water-soluble B vitamins supply important components of numerous coenzymes. Many coenzymes contain, in addition, the adenine, ribose, and phosphoryl moieties of AMP or ADP (Figure 7-2). Nicotinamide and riboflavin are components of the redox coenzymes... 

Figure 34-2 illustrates the intermediates and reactions for conversion of a-D-ribose 5-phosphate to inosine monophosphate (IMP). Separate branches then lead to AMP and GMP (Figure 34-3). Subsequent phosphoryl transfer from ATP converts AMP and GMP to ADP and GDP. Conversion of GDP to GTP involves a second phosphoryl transfer from ATP, whereas conversion of ADP to ATP is achieved primarily by oxidative phosphorylation (see Chapter 12).

Figure 13.1 Chemical structures of, and relationship between, adenosine and adenosine 5 -triphosphate (ATP). Adenosine contains an adenine ring and ribose component. Phosphorylation of the latter s termial (C5) hydroxy with three phosphate groups gives ATP...

Any discussion of the prebiotic phosphorylation of nucleosides must take into account the probably neutral or alkaline conditions in a prebiotic environment. Some model phosphorylating systems have been studied, for example, the synthesis of /S-o-ribofuranose 1-phosphate from ribose and inorganic phosphate in the presence of cyanogen. Sodium trimetaphosphate will phosphorylate cw-glycols in good yield under alkaline... 

DNA and RNA are formed of nucleotides. Each nucleotide or nucleoside is composed of a purine or pyrimidine base linked to the 1-position of a ribose sugar in the case of RNA and a 2 -deoxyribose sugar in the case of DNA.155 The 5 position is phosphorylated in the case of a nucleotide, while the nucleoside is not phosphorylated therefore, nucleotides are nucleoside phosphates. Phosphorylation can include one, two, or three phosphate groups. The most familiar example of a phosphorylated nucleotide is phosphorylated adenosine, which occurs as the mono-, di-, and triphosphate (AMP, ADP, and ATP, respectively) and is a principal means of energy storage in biological systems. 

The search for routes to phosphorylated nucleosides or phosphorylated ribose started, as mentioned in Sect. 4.7.3, with work done by Ponnamperuma (1963). The synthesis of ATP was carried out by phosphorylation of ADP using carbamoyl phosphate in the presence of Ca2+ ions as catalyst yields of up to about 20% were obtained (Saygin and Ellmauerer, 1984). 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to be phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduction of the ribose to the 2 -deoxyri-bose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled by reduction with NADPH by the enzyme thioredoxin reductase. 

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