STEREOCONTROLLED SYNTHESIS OF P-CHIRAL ANALOGS OF OLIGONUCLEOTIDES

From the very beginning of oligonucleotide chemistry, natural oligonucleotides were obtained, at least transiently, as a mixture of P-chiral diastereoisomers due to protection of internucleotide diester phosphate residues required in most oligonucleotide synthesis methods. The 'phosphodiester' method developed by Khorana is a prominent exception. The chirality at the phosphorus, present in different variations of 'phosphotriester' methods, was eliminated after hydroxyl phosphate deprotection. Soon thereafter, P-chiral oligonucleotide derivatives were obtained for their own molecular features an utilized for diverse applications in the fields of molecular biology, enzymology, biochemistry, biophysical chemistry, and biomedical sciences. Due to nonstereoselectivity of standard methods of oligonucleotide synthesis, they were usually obtained as a random mixture of diastereoisomers. The emergence of the antisense technology added a new dimension to the present and future applications of P-chiral nucleic acid fragments and painfully brought out the need for stereocontrolled methods of their synthesis. One of the main factors underlying such a demand is a proven influence of absolute stereochemistry at phosphorus of P-chiral oligonucleotides upon their physicochemical properties. The other factor concerns future clinical trials of antisense oligonucleotides since, according to the legislative procedures, an 'as exact as possible' definition of material submitted for clinical studies is required. There are two main ways to solve the problems caused by P-chirality of oligonucleotide analogues, while still retaining the advantages resulting from internucleotide linkage modification. One way is to avoid the asymmetry at the phosphorus atom. The phosphorodithioate modification is the best known example. There are also many types of 'dephospho' analogues. Another possibility is to develop an efficient and cost-effective 'chirotechnology' of P-chiral, P-tactic oligonucleotide derivatives. There are many pro and contra achiral oligonucleotide analogues modified within the internucleotide linkage. It may be of interest, however, to mention that although phosphorodithioate oligonucleotides may act in a squence-nonspecific manner, the recent NMR and melting temperature studies demonstrate decrease duplex stability of phosphorodithioates if used as antisense agent. The present methods of stereocontrolled P-chiral oligonucleotide synthesis are founded on two qualitatively different approaches. One involves de novo internucleotide linkage formation, and the second is based on modification of the existing internucleotide bond. It should be pointed out that stereoselective methods of formation and stereospecific or stereoselective methods of modification of internucleotide linkage are extremely dependent on the structure of reacting components, and that their application is so far limited to synthesis of dimers. The only successful stereocontrolled elongation of the oligonucleotide chain can be noted for the methods based on stereospecific, de novo, formation of the internucleotide linkage. The stereospecific automated approaches to P-chiral oligonucleotide synthesis, if available, are methods of choice. It must be realized, however, that as high as 99% stereospecificity of a single step of synthesis does not allow one to get diastereoisomerically pure oligonucleotide, as opposed to the enzymatic methodology utilizing nucleoside 5'-O-(1-thiotriphosphate). The diastereoisomeric purity of the oligomer can be described by the expression (x + y)(n), x = portion of isomer R(p), y = portion of isomer S(p) in the product of individual coupling, n = total number of couplings. On the contrary, the stereospecific 'in solution' methods of P-tactic oligonucleotide synthesis may allow separation of any traces of undesired isomer, if present, after each coupling step, leading to theoretically 100% diastereoisomerically pure oligomer. Although these methods of stereocontrolled synthesis of P-chiral oligonucleotides suffer from several constraints, they do allow in some cases for synthesis of the unique models for further study of the relationship between stereochemistry at phosphorus and their physicochemical and biological properties. If the demand for P-tactic oligonucleotides persists, improved methods may soon emerge. © 1993 Academic Press, Inc.