Cyclic phosphodiester

DNA is not susceptible to alkaline hydrolysis. On the other hand, RNA is alkali labile and is readily hydrolyzed by dilute sodium hydroxide. Cleavage is random in RNA, and the ultimate products are a mixture of nucleoside 2 - and 3 -monophosphates. These products provide a clue to the reaction mechanism (Figure 11.29). Abstraction of the 2 -OH hydrogen by hydroxyl anion leaves a 2 -0 that carries out a nucleophilic attack on the phosphorus atom of the phosphate moiety, resulting in cleavage of the 5 -phosphodiester bond and formation of a cyclic 2, 3 -phosphate. This cyclic 2, 3 -phosphodiester is unstable and decomposes randomly to either a 2 - or 3 -phosphate ester. DNA has no 2 -OH therefore DNA is alkali stable. 

The phenyl group can be removed by catalytic hydrogenation to yield the cyclic phosphodiester (31). 

The discrepancy between the behavior of arabinose- and xylose-3, 5-(hydrogen phosphates) probably reflects the difference which exists in the steric arrangement of these two cyclic phosphodiesters. In the xylo-furanose derivative (89), the hydroxyl group on C-3 and the primary hydroxyl group are cis the cyclic phosphate is easily formed and perfectly strainless. In arabinofuranose these same groups are in position trans and, although an apparently strainless molecule can be constructed from models, it is probable that in this case the main ring of the compound will be that formed by the phosphodiester, with a concomitant tendency for the compound to assume the aldehydo form. Objection to... 

Nucleoside triphosphates have high group transfer potential and participate in covalent bond syntheses. The cyclic phosphodiesters cAMP and cGMP function as intracellular second messengers. 

Hydrolysis of RNA by alkali or pancreatic RNase leads initially to fragments which terminate in 2, 3 -cyclic phosphodiesters. Micrococcal nuclease, on the other hand, gives rise to fragments terminating in 3 -phos-phomonoester groups which facilitate their isolation, and this enzymic hydrolysis has been used to prepare 3 -ribodinucleotides. ... 

The phosphodiester substrates used for the model studies described herein are shown in Figure 8. ApA and 2, 3 -c-UMP represent several diribonucleotides and cyclic 2, 3 -cyclic ribonucleotides. 

The enthalpies of hydrolysis of glycoside cyclic phosphodiesters have been measured42 by flow microcalorimetry, using a phosphohydrolase from Enterobacter aerogenesiz as catalyst. This phosphohydrolase can hydrolyse a wide variety of phosphodiesters, which enables the enthalpies of hydrolysis of glycoside cyclic phosphodiesters to be compared with those of acyclic and monocyclic phosphodiesters. It was found42 that the phosphohydrolase cleaves the 3 - and 5 -ester bonds with similar enthalpies, which are less negative (—11.1 0.2 kcal mol-1) than the value (-13.2 0.4 kcal mol-1) that had been reported previously.44... 

A series of diaquatetraaza cobalt(III) complexes accelerated the hydrolysis of adenylyl(3 -50adenosine (ApA) (304), an enhancement of 10 -fold being observed with the triethylenetetramine complex (303) at pH 7. The pentacoordinated intermediate (305), which is formed with the complex initially acting as an electrophilic catalyst, then suffers general acid catalysis by the coordination water on the Co(III) ion to yield the complexed 1,2-cyclic phosphate (306), the hydrolysis of which occurs via intracomplex nucleophilic attack by the metal-bound hydroxide ion on the phosphorus atom. Neomycin B (307) has also been shown to accelerate the phosphodiester hydrolysis of ApA (304) more effectively than a simple unstructured diamine. 

Phosphate diesters resist acid hydrolysis, and they are only inefficiently hydrolyzed in alkaline solutions. The exception is any five-membered ring containing a phos-phodiester, such as that formed upon alkaline treatment of RNA. Such five-membered cyclic phosphodiesters are rapidly hydrolyzed in alkaline solutions. 

CHIRAL ATP PSEUDOROTATION Nucleoside 2, 3 -cyclic phosphodiesters, RIBONUCLEASE (RNase)... 

The covalent backbone of DNA and RNA is subject to slow, nonenzymatic hydrolysis of the phosphodiester bonds. In the test tube, RNA is hydrolyzed rapidly under alkaline conditions, but DNA is not the 2 -hydroxyl groups in RNA (absent in DNA) are directly involved in the process. Cyclic 2, 3 -monophosphate nucleotides are the first products of the action of alkali on RNA and are rapidly hydrolyzed further to yield a mixture of 2 -and 3 -nucleoside monophosphates (Fig. 8-8). 

Hydrolysis of cAMP cAMP is rapidly hydrolyzed to 5-AMP by cAMP phosphodiesterase, one of a family of enzymes that cleave the cyclic 3 5 -phosphodiester bond. 5-AMP is not an intracellular signalling molecule. Thus, the effects of neurotransmitter- or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed. [Note Phosphodiesterase is inhibited by methylxanthine derivatives, such as theophylline and caffeine.3]... 

The mechanism involves participation of the free 2 -OH of the ribose groups and formation of cyclic 2, 3 -phosphates and is similar to that of pancreatic ribo-nuclease (Chapter 12). Because deoxyribose lacks the free 2 -OH, the phosphodiester linkages in DNA are quite stable in base. 

AH fungal RNases (T, T , Ni, Ui, and U2) treated in this section catalyze the reaction shown in Fig. 1. The first step (phosphate transfer) is the cleavage of the phosphodiester bond between the 3 and 5 positions of the ribose moities in the RNA chain with the formation of nucleoside 2, 3 -cyclic phosphates and oligonucleotides with 2, 3 -cyclic phosphate at 3 terminal. The nature of the phosphodiester bonds to be cleaved depends on the base specificity of the enzyme. This phosphoryl transfer step is reversible. In the second step (hydrolysis), these terminal cyclic phosphate groups are hydrolyzed with the formation of corresponding 3 -phosphates. Because the first-step is usually faster than the second step, more or less accumulation of the cyclic phosphate may be observed. 

A curious observation by Podder and Tinoco (47) that G-(2, 5 )-G bond was synthesized by RNase Tx from G-cyclic-p led Egami and Inoue (unpublished) to reinvestigate the phosphotransferase activity at various temperatures. At 100°, unlike at 36°, G-(2, 5 )-G3 -p is split to produce G3 -P, and G-(3, 5 )-G3. p is attacked in quite a different way at 100° than it is at 36°. This may result from the altered action of partly heat-denatured RNase Tx. Native RNase Tx is specific, however, to the internucleotide 3, 5 -phosphodiester bonds at normal temperature. 

Holy and Sorm (49, 50) observed that RNase T2, like RNase Ti, attacks 9-(/3-D-ribofuranosyl) and 9-(a-L-lyxofuranosyl) derivatives but not 9-(j8-L-ribofuranosyl) and 9-(a-D-lyxofuranosyl) derivatives. Also, like RNase RNase T2 is quite inactive on the phosphodiester bonds of the nucleotide with 2 -0-methyl ribose, such as 2 -0-methyl guanylic acid (96) or 2 -0-methyl cytidylic acid (86). Thus, the action of RNase T2 is in good accord with that of RNase Ti and RNase A on sugar specificity, which may be a common property throughout all RNases, producing 3 -phosphate via 2, 3 -cyclic phosphate. 

Ribonuclease U2 is a novel enzyme found in the culture broth of Ustilago sphaerogena (7, 106). Ribonuclease U2 splits, practically specifically, the phosphodiester bonds of purine nucleotides in RNA with the intermediary formation of purine nucleoside 2, 3 -cyclic phosphates, indicating the specificity is complementary to that of pancreatic RNase A (106). Like RNase N, RNase U2 very slowly hydrolyzes the intermediate, nucleoside 2, 3 -cyclic phosphate, to 3 -nucleotides (80, 106). Thus, RNase U2 is a useful tool, not only for the analysis of nucleotide sequences of RNA (90, 92, 107, 108) but also for the synthesis of various oligonucleotides containing adenylyl or guanylyl residue (30) (T. Koike, T. Uchida, and F. Egami, unpublished). 

Enzymes are available from a variety of sources which split the phosphodiester bond of nucleoside 2, 3 - and 3, 5 -cyclic phosphates. The ability of the ribonucleases to hydrolyze ribonucleoside 2, 3,-cyclic phosphates to the corresponding 3 -phosphates is well known. During... 

Thermodynamically, step 1 is easily reversible while step 2 is not. The reverse reaction of step 1 consists of the formation of a phosphodiester from a pyrimidine cyclic phosphate and a primary alcohol, either a simple aliphatic alcohol or a nucleoside or nucleotide (385-387). The... 

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