Phosphodiester phosphorylation

The ideal phosphorylating reagents for phosphodiester syntheses should meet the following criteria ... 

The 5 -phosphoryl group of a mononucleotide can es-terify a second —OH group, forming a phosphodi-ester. Most commonly, this second —OH group is the 3 -OH of the pentose of a second nucleotide. This forms a dinucleotide in which the pentose moieties are linked by a 3 —> 5 phosphodiester bond to form the backbone of RNA and DNA. 

Phosphodiester bonds link the 3 - and 5 -carbons of adjacent monomers. Each end of a nucleotide polymer thus is distinct. We therefore refer to the 5 - end or the 3 - end of polynucleotides, the 5 - end being the one with a free or phosphorylated 5 -hydroxyl. 

Figure 1. Phosphoiylation, dephosphorylation and cleavage of Ptdlns. PI3-K phosphorylates the inositol ring at D3, while PTEN dephosphotylates it at the same position. SHIP dephosphotylates 5-phosphotylated Ptdlns, PLOy hydrolyses the phosphodiester bond between the glycerol backbone and the inositol headgroup of Ptdlns (3,4)/ 2> releasing IP3.

Phospholipase C (PTC, EC 3.1.4.3) catalyzes the hydrolysis of the phosphodiester bond in phospholipids. It releases the second messenger molecule diacylglycerin (DAG) important in the signal transduction cascade and a phosphorylated headgroup . The active site of the enzyme contains three Zn ions with two of them in close proximity. Only few crystal structures are solved until now " . ... 

A zinc(II) complex 22a with an alcohol-pendent polyamine has been synthesized (23). The alcoholic OH deprotonates with pifa of 8.6 (determined by pH-metric titration), yielding 22b. Reaction of 22 (2 mM) with a phosphotriester diethyl(4-nitrophenyl) phosphate (0.1 mM) in 10 mM TAPS buffer (pH 8.6) at 25°C seemed to promote phosphoryl-transfer reactions to 23, just like acyltransferred intermediates 10 and 16a in the reactions between Znn-macrocyclic complexes with an alcohol pendent and NA (see Scheme 4). The pH dependence of the first-order rate constants gave a sigmoidal curve with an inflection point around the pKa value of 8.6. The hydrolysis of the substrate phosphotriester to the phosphodiester product diethyl phosphate thus seemed to... 

Extant ribozymes generally promote one of two types of reactions hydrolytic cleavage of phosphodiester bonds or phosphoryl transfers (Chapter 26). In both cases, the substrates of the reactions are also RNA molecules. The ribosomal RNAs provide an important expansion of the catalytic range of known ribozymes. Coupled to the laboratory exploration of potential RNA catalytic function (see Box 26-3), the idea of an RNA world as a precursor to current life forms becomes increasingly attractive. 

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. 

In contrast to the lack of specificity with respect to the nonphosphoryl part of the substrate or acceptor is the strict specificity for the phosphoryl residue. Phosphodiesters and triesters are not hydrolyzed nor are mixed esters of types (III) and (IV) 124). 

Scheme 15). The reaction pathway of (295) is similar to that of ribonucleotides but there are some differences noted in this paper for example, (295) hydrolyses 27-fold slower than its 2 -hydroxyl analogue.263 2,-Phosphorylated (298 R = OH) and 2 -thiophosphorylated (298 R = SH) dinucleotides were found to dephosphorylate readily at 90 °C in neutral aqueous solution to give UpU.264 The neighbouring 3 -5 phosphodiester function is thought to facilitate the 2 -dephosphorylation. 

Most naturally occurring ribozymes catalyze phosphoryl transfer reactions, where a sugar 2 -OH or 3 -OH attacks a phosphodiester linkage [121]. The two main classes are intramolecular ribozymes, where the sugar-OH nucleophile attacks its own 3 -phosphodiester (Figure 5.21a) and intermolecular ribozymes, where the nucleophile comes from a different RNA strand (Figure 5.21b). There is also evidence that the RNA catalyzes the peptide bond-forming aminoacyl transfer reaction in ribosomes [124,125]. 

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