Uridine cyclic 2 ,3 -phosphate hydrolysis

Treatment of 2 -deoxyuridine derivatives of type 16 with a polymer-supported fluoride at room temperature gave the 2,3 -anhydrosystems 17 in good yield. The 8,5 -cyclonucleoside 18 was prepared by intramolecular displacement of an 8-bromosubstituent, and the conformationally fixed uridine cyclic phosphate 19, and the corresponding 2,5 -compound, have been prepared to study the effect of the conformation on their hydrolysis by ribonuclease. ... 

Alkaline hydrolysis of uridine 5 - (a - D- glucopyranosyl pyrophosphate) results in the formation of uridine 5 -phosphate and a-D-glu-copyranose 1,2-cyclic phosphate24 (81). The reaction reaches completion after 30 min at 0° in concentrated aqueous ammonia, or after 2 min at 100° and pH 8.5. Partial conversion of the cyclic phosphate (81) into a-D-glucopyranosyl phosphate and D-glucose 2-phosphate occurs under conditions of elevated temperature. 

Ribonuclease Ua digestion of ApU has revealed reduced hydrolase activity in the second step of RNase U2 action (30). When 87.4% of ApU was readily degraded to produce uridine and adenosine 2, 3 -cyclic phosphate, no 3 -adenylic acid was detected. After exhaustive degradation of ApU, hydrolysis of adenosine 2, 3 -cyclic phosphate occurred and 3 -adenylic acid gradually appeared. 

The sugar configuration about the T, 2, 3, and 4 positions can be changed by synthesis. A variety of pyrimidine nucleoside cyclic phosphates have been made. Ukita et al. (425) prepared -D-lyxo-uridine 2 3 -cyclic phosphate (Fig. 21a). The configuration about the 2 and 3 positions is inverted and the two OH groups are now cis to the base rather than trans as in the D-ribose series. No hydrolysis at all of this compound was observed in the presence of RNase. However, both the cyclic phosphate and the free 2 (3 )-nucleotides inhibit the enzyme. The... 

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... 

Indeed, in the acid hydrolysis of uridine 3 5 -cyclic phosphate (175), reported by Khorana and coworkers, the 67% yield of uracil observed may represent the minimum amount of anhydronucleotide intermediates (176) and (180) which had formed, as uridine 5 -phosphate is, under these conditions, completely stable. In addition to cleavage to uracil, it would be expected that some of the anhydronucleotide (180) would also give rise to uridine 3 (2 )-phosphate. Furthermore, intermediate (176) may also generate l- 3-D-xylofuranosyluracil 5 -phosphate (177) and its arabino isomer (181), as well as uracil. Such a reaction has been observed by Yung and Fox in the acid hydrolysis of 2,3 -anhydro-l- 3-D-xylofuranosyl-uracil (182), which yielded the xylo- (183) and arabwo-nucleosides (184) and uracil. The formation of the arabino nucleoside (184) would arise by conversion of some of (182) into a 2, 3 -epoxide, which would be directly hydrolyzed to (183) and (184), or by conversion of this 2, 3 -epoxide into a 2,2 -anhydronucleoside, which would be hydrolyzed by acid to (184). Such mechanisms, applied to the anhydronucleotide (176), could give rise to (177) and (181). 

In 1970, Eckstein and co-workers reported the first stereochemical study of an enzyme-catalyzed hydrolysis of a phosphate ester, the hydrolysis of the endo isomer of uridine 2, 3 -cyclic phosphorothioate (enrfo-cyclic UMPS) (72) by ribonuclease A (RNase A) 13). The hydrolysis of RNA catalyzed either by base or by RNase A proceeds by a two-step mechanism in which the 2 -hydroxyl group of a nucleotide unit within an RNA molecule acts as a nucleophile on the 3 -phosphodiester bond to displace the 5 -hydroxyl group of the neighboring nucleoside to form a 2, 3 -cyciic phosphate intermediate. RNase A then catalyzes the hydrolysis of this cyclic phosphate, mimicked by Eckstein s endo-cyclic UMPS, to yield the ultimate 3 -mononucleotide product. 

A detailed investigation has been reported into the cleavage of 3 - 5 -uridyluridine to form the 2, 3 -cyclic phosphate, and its isomerization to 2 5 -uridyluridine. The hydrolysis of uridine 2 -, 3 - and 5 -phosphoromonothio-ates under acidic and neutral conditions has been investigated in mild acid only hydrolysis to uridine occurs, whilst at low pH desulfurization occurs in the cases of the 2 - and 3 -thioates. The same workers have also studied the kinetics of hydrolysis and desulfurization of the diastereomeric monothio-analogues of uridine 2, 3 -cyclic phosphate under neutral or acidic conditions desulfurization competes with phosphoester hydrolysis. The hydrolysis of the 2 -thionucleoside 3 -phosphate 267 (X=SH) has been studied the predominant reaction pathway at pH 13 is the formation of the 5-phosphate whilst at pH 7-10 mostly the 2, 3 -cyclic monothiophosphate was produced. The 2 -fluorocompound 267 (X=F), which has a C-3 -endo- conformation, underwent hydrolysis ten times faster than did the deoxycompound 267 PC=H). The kinetics of hydrolysis of thymidine 5 -boranomonophosphate (269) have been studied by NMR. It was found that 269 hydrolyses slowly to thymidine and [03P-BH3 ], with the latter hydrolysing even more slowly to phosphonate and boric acid. ... 

Diethyl pyrocarbonate has been used to convert uridine 2 - or 3 -phosphate into the 2, 3 -cyclic phosphate in high yield, and an adamantyl 2, 3 -cyclic phosphonate (421) was obtained when uridine reacted with I-adamantylphos-phonyl chloride. Phosphorylation of ribonucleosides with pyrophosphoryl chloride followed by hydrolysis in neutral solution afforded a simple synthesis of ribonucleoside 2, 3 -cyclic phosphate 5 -phosphates. Two phosphorylating agents mentioned earlier in this section can also yield 3, 5 -cyclic phosphates either by treatment of nucleoside 5 -(2-iV7V-dimethylamino-4-nitrophenyl phosphates) with acetic acid in pyridine (Scheme I4i) i, 83 qj. treatment of nucleoside 5 -(5 -methyl phosphorothioates) with iodine in pyridine. Another route to 3, 5 -cyclic phosphates involved cyclization of the nucleoside 5 -trichIoro-methylphosphonates (422) (obtained by the action of trichloromethylphosphonyl... 

Adenosine 2 - and 3 -monophosphate monomethyl and monoisopropyl esters undergo isomerization and hydrolytic cleavage to the 2, 3 -cyclic phosphate and the alcohol at comparable rates under acidic conditions, whilst at neutral pH, a pH-independent phosphate migration prevails.197 However the hydrolysis of the 2-chlorophenyl ester of uridine-3 -phosphate to 2-chlorophenol and uridine monophosphates is rapid compared with the phosphate migration under acidic conditions. 198 These studies have been extended to the interconversion and hydrolysis of 2 ,5 - and 3 ,5 -dinucleoside phosphates, where for adenosine systems, acid-catalysed depurination competed with phosphate migration and hydrolysis at pH<3.i99... 

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. 

When RNA is subjected to controlled alkaline hydrolysis eight nucleotides, two isomers each of AMP, GMP, cytosine monophosphate (CMP), and uridine monophosphate (UMP), are formed. These can be separated from one another on columns of anion exchange resins ISl), by paper chromatographic methods 1S2), and by fractional crystallization (iSS). The isomers are the 2 -phosphates ( a ) (XI) and 3 -phosphates ( b ) (XII) which arise from an intermediate cyclic 2, 3 -phosphate mononucleotide ester [Eq. (44)] (1S4-1S6). 

This chapter summarizes the techniques that have been developed for the syntheses and configurational analyses of phosphate mono- and diesters that are chiral only by virtue of substitution with the stable isotopes of oxygen O, 99.759% natural abundance O, 0.037% natural abundance and a nuclear spin of and K), 0.204% natural abundance. In addition, a few enzyme systems in which these techniques have been used are described. This approach, which dates back only to 1978, was not the first method to ascertain the stereochemical consequences of enzyme-catalyzed reactions at phosphorus. In 1970, Usher and Eckstein reported the seminal experiment in this field (Usher et al., 1970) the stereochemical course of the hydrolysis of the endo isomer of uridine 2, 3 -cyclic phosphorothioate catalyzed by... 

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