RNA molecules participate in many fundamental cellular processes either as a carrier of genetic information or as a catalyst, and hence, RNA has received increasing interest both as a chemotherapeutic agent and as a target of chemotherapy. In addition the dual nature of RNA has led to the RNA-world concept, i.e. an assumption that the evolution at an early stage of life was based on RNA-like oligomers that were responsible for the storage and transfer of information and as catalysts maintained primitive metabolism. Accordingly, the kinetics and mechanisms of the cleavage of RNA phosphodiester bonds have received interest and it is hoped they will shed light on the mechanisms of enzyme action and on the development of artificial enzymes. The major mechanistic findings concerning the cleavage by small molecules and ions and their significance for the development of efficient and biologically applicable artificial catalysts for RNA hydrolysis are surveyed in the present perspective.

Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters.

A Brønsted graph with a convex break at pK_a (Lg) = 12.58 provides compelling evidence for an intermediate in the alkaline cyclisation of uridine 3′-phosphate esters. The transient pentacoordinated oxyphosphorane dianion intermediate collapses to reactant and cyclic uridine 2′,3′-monophosphate faster than it can pseudo-rotate and isomerise to the 2′-isomer.

Oligonucleotides are usually prepared in lab scale on a solid support with the aid of a fully automated synthesizer. Scaling up of the equipment has allowed industrial synthesis up to kilogram scale. In spite of this, solution-phase synthesis has received continuous interest, on one hand as a technique that could enable synthesis of even larger amounts and, on the other hand, as a gram scale laboratory synthesis without any special equipment. The synthesis on a soluble support has been regarded as an approach that could combine the advantageous features of both the solution and solid-phase syntheses. The critical step of this approach is the separation of the support-anchored oligonucleotide chain from the monomeric building block and other small molecular reagents and byproducts after each coupling, oxidation and deprotection step. The techniques applied so far include precipitation, extraction, chromatography and nanofiltration. As regards coupling, all conventional chemistries, viz. phosphoramidite, H-phosphonate and phosphotriester strategies, have been attempted. While P(III)-based phosphoramidite and H-phosphonate chemistries are almost exclusively used on a solid support, the “outdated” P(V)-based phosphotriester chemistry still offers one major advantage for the synthesis on a soluble support; the omission of the oxidation step simplifies the coupling cycle. Several of protocols developed for the soluble-supported synthesis allow the preparation of both DNA and RNA oligomers of limited length in gram scale without any special equipment, being evidently of interest for research groups that need oligonucleotides in large amounts for research purposes. However, none of them has really tested at such a scale that the feasibility of their industrial use could be critically judged.

Several chimeric ribo/2′-O-methylribo oligonucleotides were synthesized and their hydrolytic cleavage studied in the presence of Mg²⁺, Zn²⁺, Pb²⁺ and the 1,4,9-triazacyclododecane chelate of Zn²⁺ (Zn²⁺[12]aneN₃) to evaluate the importance of RNA secondary structure as a factor determining the reactivity of phosphodiester bonds. In all the cases studied, a phosphodiester bond within a 4–7 nt loop was hydrolytically more stable than a similar bond within a linear single strand, but markedly less stable than that in a double helix. With Zn²⁺ and Zn²⁺[12]aneN₃, the hydrolytic stability of a phosphodiester bond within a hairpin loop gradually decreased on increasing the distance from the stem. A similar but less systematic trend was observed with Pb²⁺. Zn²⁺- and Pb²⁺-promoted cleavage was observed to be considerably more sensitive to the secondary structure of the chain than that induced by Zn²⁺[12]aneN₃. This difference in behaviour may be attributed to bidentate binding of uncomplexed aquo ions to two different phosphodiester bonds. Mg²⁺ was observed to be catalytically virtually inactive compared with the other cleaving agents studied.