Organometallic methodologies for nucleic acid synthesis

A new, general preparation of 2'-5'-linked oligoadenylates has been developed, which relies on: (1) chemoselective 0phosphorylation of N-unblocked nucleosides via hydroxyl activation by Grignard reagents, (2) one-pot construction of internucleotide linkage using bifunctional phosphorylating agents, and (3) selective production of a 3',5'-di-O-protected adenosine. This solution-phase synthesis allows large-scale preparation of a wide range of related oligomers. Palladium chemistry coupled with allylic protection of functional groups leads to the development of an efficient solid-phase synthesis of DNA oligomers via a phosphoramidite approach. Ally1 groups on internucleotide linkages and allyloxycarbonyl groups on amino moieties of nucleobases are removable all at once on the solid supports by exposure to a palladium(0) complex and nucleophiles. This procedure has been utilized for synthesis of a DNA 60mer of unprecedented high purity. In addition, this marks the realization of the first efficient preparation of solid-anchored DNA oligomers. Oligonucleotides are one of the most significant classes of compounds from both the academic and industrial point of view. The chemical synthesis, however, poses an array of problems in the transformation and isolation of products, and high overall efficiency is secured only by using a combination of truly appropriate methods. Organometallic chemistry, though exerting general intense influence on contemporary organic synthesis, has seldom been used in nucleic acids synthesis, perhaps because of the multifunctional, polar nature of the substrates. We demonstrate here the potent utility of metallo-organic methodologies in this area by describing a solution-phase synthesis of some short-chain oligomers having unusual structures and a solid-phase preparation of a long-sequence DNA. GENERAL SYNTHESIS OF 2'-5'-LINKED OLIGOADENYLATES In solution-phase chemistry, we selected as targets oligoadenylates, 13, possessing 2'5'-internucleotide bonds (2-5As). This unusual class of compounds, which are produced from ATP by 2-5A synthetase induced by dsRNA in interferon-treated cells, play a major role in antiviral and antiproliferative actions of interferons (ref. 1 and 2). The 2-5A family exhibits a wide variety of biological activity, depending on the structures, and certain structural modifications are known to enhance their potency. Development of an efficient chemical synthesis has been strongly required, since the eldsting methods have three common drawbacks to practical synthesis: namely, (1) lack of chemoselectivity in phosphorylation of adenosine requiring undesirable protection/deprotection of the 6amino group, (2) tedious isolation of the adenosine 2'-phosphodiester intermediate before the internucleotide-linkage formation, and (3) nonselective protection of 2',3'unblocked adenosine generally yielding a mixture of comparable amounts of the 2'and 3'-O-protected products, which needs chromatographic separation unsuitable for largescale synthesis (ref. 3). We have cleared these impediments and opened a new, general way to 2-5A derivatives. The success relies mainly on the highly selective preparation of a 3',5'-di-O-protected adenosine and the chemoselective, one-pot formation of the internucleotide bonds by condensation of a bifunctional phosphorylating agent and Nunprotected nucleosides via hydroxyl activation.