Syntheses of 4'-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4'-thiocytosine.

A facile synthetic route for the 4'-thioribonucleoside building block (4'S)N (N = U, C, A and G) with the ribose O4' replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case.

Scaleable and efficient synthesis of 2'-deoxy-2'-N-phthaloyl nucleoside phosphoramidites for oligonucleotide synthesis.

2'-Deoxy-2'-N-phthaloyl nucleosides were prepared from arabino nucleosides by triflate displacement with phthalimide in the presence of DBU. The corresponding phosphoramidites suitable for automated oligonucleotide synthesis were also synthesized. The scalability of described procedures was demonstrated on a 100-g scale preparation of 2'-deoxy-2'-amino-C phosphoramidite.

Synthesis of N-acetyl-D-galactosamine and folic acid conjugated ribozymes.

To evaluate potential improvement in tissue specific targeting and cellular uptake of therapeutic ribozymes, we have developed three new phosphoramidite reagents. These reagents can be used in automated solid-phase synthesis to produce oligonucleotide conjugates containing N-acetyl-D-galactosamine (targeting hepatocytes) and folic acid (targeting tumor). N-Acetyl-D-galactosamine was attached through a linker to both 2'-amino-2'-deoxyuridine and D-threoninol scaffolds, and these conjugates were converted to phosphoramidite building blocks.

The development of a monoclonal antibody specific for a 2(')-C-allyl modification of uridine, and its use in the localization of ribozymes in vivo.

Ribozymes are catalytically active RNA molecules that cleave other RNA molecules in a sequence-specific fashion, with significant turnover. The successful design and synthesis of ribozymes with modifications to increase their stability in biological fluids, while maintaining catalytic activity, has been instrumental in moving this technology from the laboratory into clinical trials. With the entry of ribozymes into the clinical setting, the need has arisen for reagents and/or assays to detect these drugs in tissues.