SYNTHESIS OF 5'-O-(4,4'-DIMETHOXYTRITYL)-2'-O-(TERT-BUTYLDIMETHYLSILYL)-5-FLUOROURIDINE 3'-(CYANOETHYL N,N-DIISOPROPYLPHOSPHORAMIDITE) AND ITS USE IN THE SYNTHESIS OF RNA

5-Fluorouridine (FUrd) has been successfully converted to 5'-O-(4,4'-dimethoxytrityl)-2'-O-(tert-butyldimethylsilyl)-5-fluorouridine 3'-(cyanoethyl N,N-diisopropyl(phosphoramidite) by methods similar to those employed for the preparation of other ribonucleoside phosphoramidites. The desired product, as well as its precursors and the incorrect regioisomers, are prepared and fully characterized by H-1 NMR, F-19 NMR, P-31 NMR, FAR-MS, UV absorbance, elemental analysis, and melting points. The purified material is used for the production of RNA by solid-phase methods. Coupling yields of 94% are obtained with this material. The resulting RNA is readily purified and used for biophysical studies. The self-complemetary RNA decamer 5'-rGCGAAU(FU)CGC is prepared and purified. The material adopts an A-form duplex in solution at neutral pH as characterized by CD spectroscopy. The thermal stability of the duplex is similar to the parent duplex oligoribonucleotide prepared with uridine instead of 5-fluorouridine. The control duplex has a T-m of 53.8 and the FUrd substituted duplex has a T-m of 56.6 (xb0)degrees C as determined by UV hyperchromicity at 260 nm. The CD spectra and UV hyperchromicity data for the duplex oligoribonucleotide containing FUrd have a slight pH dependence indicating that ionization of FUrd has a slight but measurable impact on RNA duplex stability. 1D NMR spectroscopy of the imino hydrogens for this duplex in H2O solution confirms the formation of Watson-Crick base pairs. The imino hydrogen resonance for the FUrd-A base pair is moved downfield and broadened compared with a control duplex oligoribonucleotide as a consequence of the electron-withdrawing inductive effect of fluorine.