SYNTHESIS OF PHOSPHORODITHIOATE DNA VIA SULFUR-LINKED, BASE-LABILE PROTECTING GROUPS

Phosphorodithioate DNA, a new and potentially useful DNA analog with a deoxynucleoside-OPS2O-deoxynucleoside internucleotide linkage, was sy nthesized from deoxynucleoside 3'-phosphorothio-amidites having a vari ety of thioesters and thiocarbonates as base-labile phosphorus protect ing groups. The major challenge in the synthesis of this DNA analog wa s to derive a reaction pathway whereby activation of deoxynucleoside 3 '-phosphorothioamidites occurred rapidly and in high yield under condi tions that minimize Arbuzov rearrangements, exchange reactions, unwant ed oxidation to phosphorothioates, and several other side reactions. O f the various phosphorus protecting groups examined for this purpose, a thorough evaluation of these parameters led to the conclusion that b eta-(benzoylmercapto)ethyl was preferred. Synthesis of phosphorodithio ate DNA began by preparing deoxynucleoside 3'-phosphorothioamidites fr om the appropriately protected deoxynucleoside, tris(pyrrolidino)phosp hine, and ethanedithiol monobenzoate via a one-flask synthesis procedu re. These synthons were activated with tetrazole and condensed with a deoxynucleoside on a polymer support to yield the deoxynucleoside thio phosphite. Subsequent steps involved oxidation with sulfur to generate the completely protected phosphorodithioate triester, acylation of un reacted deoxynucleoside, and removal of the 5'-protecting group. Yield s per cycle were usually 97-98% with 2-5% phosphorothioate contaminati on as determined by P-31 NMR. By using deoxynucleoside 3'-phosphorothi oamidites and deoxynucleoside 3'-phosphoroamidites, deoxyoligonucleoti des having phosphorodithioate and the natural phosphate internucleotid e linkages in any predetermined order can also be synthesized.