Synthesis of Phosphorodithioate DNA via Sulfur-Linked, Base-Labile Protecting Groups1

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Abstract
Phosphorodithioate DNA, a new and potentially useful DNA analog with a deoxynucleoside-OPS2O-deoxynucleoside internucleotide linkage, was synthesized from deoxynucleoside 3‘-phosphorothioamidites having a variety of thioesters and thiocarbonates as base-labile phosphorus protecting groups. The major challenge in the synthesis of this DNA analog was to derive a reaction pathway whereby activation of deoxynucleoside 3‘-phosphorothioamidites occurred rapidly and in high yield under conditions that minimize Arbuzov rearrangements, exchange reactions, unwanted oxidation to phosphorothioates, and several other side reactions. Of the various phosphorus protecting groups examined for this purpose, a thorough evaluation of these parameters led to the conclusion that β-(benzoylmercapto)ethyl was preferred. Synthesis of phosphorodithioate DNA began by preparing deoxynucleoside 3‘-phosphorothioamidites from the appropriately protected deoxynucleoside, tris(pyrrolidino)phosphine, and ethanedithiol monobenzoate via a one-flask synthesis procedure. These synthons were activated with tetrazole and condensed with a deoxynucleoside on a polymer support to yield the deoxynucleoside thiophosphite. Subsequent steps involved oxidation with sulfur to generate the completely protected phosphorodithioate triester, acylation of unreacted deoxynucleoside, and removal of the 5‘-protecting group. Yields per cycle were usually 97−98% with 2−5% phosphorothioate contamination as determined by 31P NMR. By using deoxynucleoside 3‘-phosphorothioamidites and deoxynucleoside 3‘-phosphoroamidites, deoxyoligonucleotides having phosphorodithioate and the natural phosphate internucleotide linkages in any predetermined order can also be synthesized.