Cytidine-5 -phosphate hydrolysis

Cytosine was isolated from hydrolysis of calf thymus in 1894 and by 1903 its structure was known and it had been synthesized from 2-ethylthiopyrimidin-4(3H)-one. The acid hydrolysis of ribonucleic acid gives nucleotides, among which are two cytidylic acids, 2 -and 3 -phosphates of cytidine further hydrolysis gives cytidine itself, i.e. the 1-/3-D-ribofuranoside of cytosine, and thence cytosine. The deoxyribonucleic acids likewise yield deoxyribonucleotides, including cytosine deoxyribose-5 -phosphate, from which the phosphate may be removed to give cytosine deoxyriboside and thence cytosine. 

The structure shown was confirmed by the identification of cytidine 5 -phosphate and N-acetylneuraminic acid after mild acidic hydrolysis, and by the stability of the derivative on treatment with sodium borohydride. Comparison of the optical rotation of the starting derivative182 (44), the products of its hydrolysis,182 and the anomeric... 

A good source of uncommon bases is tRNA. It provides substrates for studying the effect of base on the rate of hydrolysis. Baev et al. (62) showed that V2-dimethylguanylyl-(3 -5 )-cytidine-3 phosphate (G2m-pCp) was hydrolyzed much slower than the usual GpCp. Venkstern (63) reported that Tp was hydrolyzed very slowly. Naylor et al. (64) found that Cp was hydrolyzed with half the rate of CpU. The same group of workers introduced (64, 65) a chemical block on uridine and pseudo-uridine residues by reacting RNA with l-cyclohexyl-3-(2-morpho-liny]-(4)-ethyl)-carbodiimide metho-p-toluene sulfonate. The modification of the uridine residues completely blocked the action of venom exonuclease and also blocked the action of pancreatic RNase. 

With cytidine cyclic phosphate as a substrate the step 2 process alone is measured. The procedure of Crook et al. (395) relies on the increase in absorbance at 286 nm which occurs on hydrolysis of the cyclic phosphate ring. Cleavage of the ring also results in proton release which is the basis of the titrimetric procedure described by Stark and Stein (132). The assays employing C>p are usually carried out in 0.35 M NaCl. The ionic strength for maximum activity is much higher for this substrate than for RNA. A brief summary of the above procedures is given by Klee (396). 

Figure 9. Proposed transition state in the ribonuclease A-catalyzed hydrolysis of cytidine 2 -3 -phosphate. Two histidine residues of the enzyme participate in catalysis.

Acid hydrolysis of cytidine ribitol pyrophosphate led to a series of products, depending on the conditions of hydrolysis. L-Ribitol 1-phosphate (n-ribitol 5-phosphate), isomeric ribitol phosphates formed by acid-catalyzed migration of the phosphate group from the terminal position, and 1,4-anhydro-DL-ribitol were isolated. 

The structures of these cytidine nucleotides were confirmed by synthesis. Condensation of 1,2-0-isopropylidene-L-glyceritol 3-phosphate with cytidine 5-phosphate in the presence of dicyclohexylcarbodiimide, followed by careful acid hydrolysis of the isopropylidene group, yielded cytidine glyceritol pyrophosphate identical with the natural product. 

Eftink, M. R. and Biltonen, R. L. (1983) Energetics of Ribonuclease A Catalysis. 1. pH, Ionic Strength, and Solvent Isotope Dependence of the Hydrolysis of Cytidine Cyclic 2, 3 Phosphate, Biochemistry 22,... 

A uridine phosphate (5), obtained by treatment of uridine 5 -(a-D-glucopyranosyl pyrophosphate) with ammonia, imdergoes hydrazinolysis to D-ribose 5-phosphate (2) and 3-pyrazolone, which establishes the structure of (S) as uridine 5 -phosphate. - (Hydrazinolysis of uridine to pyrazolone, with the liberation of the sugar moiety, had been described, and has served as a useful tool in the determination of the position of attachment of the sugar moiety to the aglycon. ) The 5 -phosphates of adenosine, guanosine, cytidine, and uridine were obtained by enzymic hydrolysis of ribonucleic acid with phosphodiesterases from snake venom and from other sources (such as Streptomyces aureu ). 

Adenylic acids o and b were each converted by trifluoroacetic anhydride into the same 2 3 -cyclic phosphate (13, R = adenin-9-yl) by Brown, Magrath, and Todd. These authors also applied this method to the synthesis of 2 3 -cyclic phosphates (13) of cytidine, uridine, and guanosine. Hydrolysis of these cyclic phosphates gave the corresponding a and b nucleotide mixtures. Markham and Smith found that, during hydrolysis of ribonucleic acid with pancreatic ribonuclease, the 2 3 -cyclic phosphates of the pyrimidine nucleosides are formed as intermediates leading to the ribonucleoside phosphates b. The also showed that 2 3 -cyclic phosphates (13) are formed by very mild, alkaline hydrolysis of ribonucleic acid. The discoveries of these a and b isomers of mononucleotides from... 

The possibility that, during the alkaline hydrolysis of ribonucleic acid, tri-esters (18) - could be intermediates was ruled out on mechanistic groxmds."f > Furthermore, it was shown that the tri-ester, uridine 2 3 -(methyl phosphate) (18, R = CH3), synthesized by methylation of uridine 2 3 -cychc phosphate with diazomethane, is resistant to ribo-nuclease. f However, it has been observed that, when the benzyl ester of cytidylic acid 6 is partially hydrolyzed in acid, a significant proportion of the o isomer of this phosphoric diester is formed." ) In addition, the dinucleoside phosphates, such as adenylyl-(3 —>5 )-cytidine (14a), are also isomerized to the corresponding (2 - 5 )-dinucleoside phosphates by treatment with acid. " These studies suggest that cyclic triesters (or derivatives thereof) may be intermediates in isomerization of these phospho-diesters in acidic mediiun. 

As pyrimidine nucleosides are rather stable under acidic conditions, they may be phosphorylated with a mixture of 85% phosphoric acid and phosphorus pentaoxide ( polyphosphoric acid )- The 2, 3 -0-isopropylidene acetals of uridine and of cytidine were treated with polyphosphoric acid for two hours at 60°. After acid hydrolysis, the 5 -phosphates of these nucleosides were obtained in good yield. 2, 3 -0-Benzylidenecytidine was phosphorylated similarly to cytidine 5 -phosphate. The polyphosphoric acid method was also used to convert the 2, 3 -0-isopropylidene acetals of 5-bromouridine, 3-methyluridine, and A -methyl- and A -dimethyl-cytidine into their respective nucleoside 5 -phosphates. 

It remains to be explained why treatment of the protected nucleotide (153) with ammonium hydroxide produces cytidine 2 -phosphate only, whereas similar treatment of (156) gives a mixture of the 2 - and 3 -isomers. A possible explanation may reside in differences in the lability of the benzoyl group on 0-2 or 0-3 of ribonucleosides, the 2 -benzoate being the more labile to ammonium hydroxide. Such a hypothesis is in accordance with the observation that partial debenzoylation of iV -benzoyl-tri-0-benzoylcytidine yields a mixture containing (152), but not (155). It had been noted that, in ribonucleosides, the 2 -hydroxyl group is more acidic than the 3 - and 5 -hydroxyl groups. It would be expected that 2 -0-acyl derivatives of ribonucleosides should be relatively more susceptible to alkaline hydrolysis than the 3 - or 5 -0-acyl derivatives. 

If this hypothesis is correct, it is surprising that, in the acid hydrolysis of (175) and of cytidine 3 5 -cyclic phosphate, the presence of nucleotides of type (177) or (181), or both, was not observed. Neither was the presence of D-xylose or n-arabinose noted in the hydrolyzates. It may... 

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