3, 5 -phosphodiester bond

The fundamental problem of oligodeoxyribonucleotide synthesis is the efficient formation of the intemucleotidic phosphodiester bond specifically between C-3 and C-5 positions of two adjacent nucleosides. Any functional group (NH of nucleic base the other OH of deoxy-... 

The discovery of nbozymes (Section 28 11) in the late 1970s and early 1980s by Sidney Altman of Yale University and Thomas Cech of the University of Colorado placed the RNA World idea on a more solid footing Altman and Cech independently discovered that RNA can catalyze the formation and cleavage of phosphodiester bonds—exactly the kinds of bonds that unite individual ribonucleotides in RNA That plus the recent discovery that ribosomal RNA cat alyzes the addition of ammo acids to the growing peptide chain in protein biosynthesis takes care of the most serious deficiencies in the RNA World model by providing precedents for the catalysis of biologi cal processes by RNA... 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

FIGURE 9.25 Teichoic acids are covalently linked to the peptidoglycan of Grampositive bacteria. These polymers of (a, b) glycerol phosphate or (c) ribitol phosphate are linked by phosphodiester bonds. 

FIGURE 11.29 The vicinal—OH groups of RNA are susceptible to nucleophilic attack leading to hydrolysis of the phosphodiester bond and fracture of the polynucleotide chain DNA lacks a 2 -OH vicinal to its 3 -0-phosphodiester backbone. Alkaline hydrolysis of RNA results in the formation of a mixture of 2 - and 3 -nucleoside monophosphates. 

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. 

FIGURE 14.23 RNA splicing in TetraAjimejta rRNA matnradon (a) the gnanosine-mediated reaction involved in the antocatalytic excision of the Tetrahymena rRNA intron, and (b) the overall splicing process. The cyclized intron is formed via nncleophilic attack of the 3 -OH on the phosphodiester bond that is 15 nncleotides from the 5 -GA end of the spliced-ont intron. Cyclization frees a linear 15-mer with a 5 -GA end. 

Heterocycles as agents for the cleavage of phosphodiester bonds in RNA 98CRV961. 

The therapeutic utility of systemically administered ASON had been limited by their short plasma half life (sometimes even less than 3 min). This is due to their sensitivity to nuclease digestion. When the first-generation ASON were chemically modified, e.g., by replacing the oxygen in the phosphodiester bond with sulfur (phosphorothiorate) they obtained an increased stability in biological fluids while their antisense effect has been maintained. First-generation agents can be delivered via intravitreal injection, parenterally, by topical cream, enema, and inhaled aerosol. These antisense... 

Staphylococcal nuclease (SNase) is a single-peptide chain enzyme consisting of 149 amino acid residues. It catalyzes the hydrolysis of both DNA and RNA at the 5 position of the phosphodiester bond, yielding a free 5 -hydroxyl group and a 3 -phosphate monoester... 

The overall catalytic rate constant of SNase is (see, for example, Ref. 3) kcat — 95s 1 at T = 297K, corresponding to a total free energy barrier of Ag at = 14.9 kcal/mol. This should be compared to the pseudo-first-order rate constant for nonenzymatic hydrolysis of a phosphodiester bond (with a water molecule as the attacking nucleophile) which is 2 x 10 14 s corresponding to Ag = 36 kcal/mol. The rate increase accomplished by the enzyme is thus 101S-1016, which is quite impressive. 

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. 

While formation of a dinucleotide may be represented as the elimination of water between two monomers, the reaction in fact strongly favors phosphodiester hydrolysis. Phosphodiesterases rapidly catalyze the hydrolysis of phosphodiester bonds whose spontaneous hydrolysis is an extremely slow process. Consequently, DNA persists for considerable periods and has been detected even in fossils. RNAs are far less stable than DNA since the 2khydroxyl group of RNA... 

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. 

The base sequence or primary structure of a polynucleotide can be represented as shown below. The phosphodiester bond is represented by P or p, bases by a single letter, and pentoses by a vertical line. 

Where all the phosphodiester bonds are 5 more compact notation is possible ... 

Mononucleotides linked by 3 — 5 -phosphodiester bonds form polynucleotides, directional macromolecules with distinct 3 - and 5 - ends. For pTpGpTp or TGCATCA, the 3 - end is at the left, and all phosphodiester bonds are 3 —> 5. ... 

Figure 35-6. A segment of a ribonucleic acid (RNA) molecule in which the purine and pyrimidine bases— guanine (G), cytosine (C), uracii (U), and adenine (A)—are held together by phosphodiester bonds between ribo-syl moieties attached to the nucieobases by N-giycosidic bonds. Note that the polymer has a polarity as indicated by the iabeied 3 -and 5 -attached phosphates.

DNA consists of four bases— A, G, C, and T— which are held in linear array by phosphodiester bonds through the 3 and 5 positions of adjacent de-oxyribose moieties. 

The process of RNA synthesis in bacteria—depicted in Figure 37-3—involves first the binding of the RNA holopolymerase molecule to the template at the promoter site to form a PIC. Binding is followed by a conformational change of the RNAP, and the first nucleotide (almost always a purine) then associates with the initiation site on the 3 subunit of the enzyme. In the presence of the appropriate nucleotide, the RNAP catalyzes the formation of a phosphodiester bond, and the nascent chain is now attached to the polymerization site on the P subunit of RNAP. (The analogy to the A and P sites on the ribosome should be noted see Figure... 

Schulz, W. G. Nieman, R. A. Skibo, E. B. Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[l,2-a] benzimidazoles, small molecules capable of causing sequence specific phosphodiester bond hydrolysis. Proc. Natl. Acad. Sci. USA 1995, 92, 11854-11858. 

Other problems arise when modified oligonucleotides are synthesized. Oligonucleotides are most commonly synthesized today for pharmaceutical purposes in the form of phosphorothioates (PS), in which sulfurization of the phosphodiester bond has taken place (Figure 1). 

Unlike other enzymes that we have discussed, the completion of a catalytic cycle of primer extension does not result in release of the product (TP(n+1)) and recovery of the free enzyme. Instead, the product remains bound to the enzyme, in the form of a new template-primer complex, and this acts as a new substrate for continued primer extension. Catalysis continues in this way until the entire template sequence has been complemented. The overall rate of reaction is limited by the chemical steps composing cat these include the chemical step of phosphodiester bond formation and requisite conformational changes in the enzyme structure. Hence there are several potential mechanisms for inhibiting the reaction of HIV RT. Competitive inhibitors could be prepared that would block binding of either the dNTPs or the TP. Alternatively, noncompetitive compounds could be prepared that function to block the chemistry of bond formation, that block the required enzyme conformational transition(s) of turnover, or that alter the reaction pathway in a manner that alters the rate-limiting step of turnover. 

In contrast, the hairpin ribozyme (HPR) [107, 108], which catalyzes the reversible, site-specific phosphodiester bond cleavage of an RNA substrate, is unique in that the chemical steps of the reaction do not require involvement of a divalent metal ion [107-111]. This lack of an explicit metal ion requirement [112] makes the hairpin ribozyme an ideal target for theoretical studies aimed to characterize the contribution of generalized solvation provided by the solvated ribozyme on catalysis. 

B r/(5 -CCGp-(9-PhCl-3 )—the 5 -OH function is free, and in the ligation step, it attacks the activated phosphate group of A in a nucleophilic manner, thus forming the required 3 -5 -phosphodiester bond between A and B. The 3 end of B is inactivated by means of an o-chlorophenylphosphate protecting group. 

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