Phosphodiester chemistry

Many examples of catalytic nucleic acids obtained by in vitro selection demonstrate that reactions catalyzed by ribozymes are not restricted to phosphodiester chemistry. Some of these ribozymes have activities that are highly relevant for theories of the origin of life. Hager et al. have outlined five roles for RNA to be verified experimentally to show that this transition could have occurred during evolution [127]. Four of these RNA functionalities have already been proven Its ability to specifically complex amino acids [128-132], its ability to catalyze RNA aminoacylation [106, 123, 133], acyl-transfer reactions [76, 86], amide-bond formation [76,77], and peptidyl transfer [65,66]. The remaining reaction, amino acid activation has not been demonstrated so far. 

Accordingly, protocols for activating monoesters such as adenosine-5 -phosphate would soon be developed and these breakthroughs would eventually pave the way for H. Khorana and his colleagues pioneering work on phosphodiester chemistry and sequence-defined synthetic oligonucleotides at the University of Wisconsin, Madison. 

The most common trityl anchor was monomethoxytrityl (Figure 19.5), employed in the phosphodiester method on PS-1% DVB by Melby and Strobach [146], on macroporous PS by Roster and Cramer [159] and on PTFE grafted polystyrene by Potapov d. al. [167] A dimethoxytrityl linker was used by Roster and Cramer for the phosphodiester chemistry on popcorn PS [144] and by Belagaje and Brush [155] for their original adaptation of the phosphotriester method for the synthesis in 5 -to-3 direction (Figure 19.5). Roster has also described a trityl anchor linked to a silica gel support [185]. Similar linkers have been exploited for liquid-phase oligonucleotide synthesis (Section 19.4). 

The phosphorothiolate linkage was used by Sommer and Cramer [255] for the thymidine 5 -phosphorothioate attachment to chloromethylated PS resin (5% cross-linking) in the early days of phosphodiester chemistry (Figure 19.9). In DMF at 60 °C after 6h of reaction with a stoichiometric amount of the nucleoside, a substitution level of about 35% of the initial chlorine has been obtained. The phosphorothiolate linkage between the oligonucleotide and the resin was split by treatment with iodine solution ( 5 mg ml.1) in 75% aqueous pyridine at ambient temperature for 22 h. 

Many DNAzymes catalyse RNA ligation reactions to yield linear, branched, and lariat-type reaction products. The ligation of DNA strands as well as the phosphorylation of DNA or RNA oligonucleotides was described. Some notable extensions beyond phosphodiester chemistry include a photoreversion reaction, a deglycosylation, porphyrin metalation, nucleopeptide bond formation, and finally a Diels-Alder reaction. This latter reaction is essentially the same studied by the Jaschke lab with RNA as a catalyst (as discussed below in more detail), and the information published to-date indicates that the catalytic proficiency of DNA and RNA enzymes for Diels-Alder reactions is very similar. 

In this last section, I will discnss stmcture and mechanism of one artificial ribozyme in more detail. This ribozyme, selected in my laboratory, is the only RNA catalyst for small-molecule chemistry with a known spatial stmcture, and due to extensive studies, it is arguably the best-characterized artificial ribozyme known to-date. These data provide for the first time an insight into how a small RNA can accelerate reactions different from phosphodiester chemistry, and what stractural prerequisites are required. 

Quinone methides have been shown to be important intermediates in chemical synthesis,1 2 in lignin biosynthesis,3 and in the activity of antitumor and antibiotic agents.4 They react with many biologically relevant nucleophiles including alcohols,1 thiols,5-7 nucleic acids,8-10 proteins,6 11 and phosphodiesters.12 The reaction of nucleophiles with ortho- and /iara-quinone methides is pH dependent and can occur via either acid-catalyzed or uncatalyzed pathways.13-17 The electron transfer chemistry that is typical of the related quinones does not appear to play a role in the nucleophilic reactivity of QMs.18... 

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. 

G. Pourceau, A. Meyer, J.-J. Vasseur, and F. Morvan, Combinatorial and automated synthesis of phosphodiester galactosyl cluster on solid support by click chemistry assisted by microwaves, J. Org. Chem., 73 (2008) 6014—6017. 

FIGURE 26-15 Splicing mechanism of group II introns. The chemistry is similar to that of group I intron splicing, except for the identity of the nucleophile in the first step and formation of a lariatlike intermediate, in which one branch is a 2, 5 -phosphodiester bond. 

Fig. 10.3 Effect of oligonucleotide chemistry on permeability in the rat intestine in situ [54]. P=0 designates phosphodiester ASO backbone chemistry P=S designates phosphorothioate ASO backbone chemistry.

The chemistry of the elongation reaction catalyzed by DNA polymerase I is shown in Figure 13. The enzyme catalyzes the nucleophilic attack of the 3 -0H terminus of the primer molecule on the a-phosphorus of the deoxyribonucleoside triphosphate to form a new phosphodiester bond with release of pyrophosphate. Elongation of the DNA chain proceeds in the 5 +3 direction at a rate of approximately ten nucleotides per second per molecule of DNA polymerase I. It is thought that the reaction is processive, in that many nucleotide units are added without release of the enzyme from the template. 

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