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

5-Fluorouridine (FUrd) has been successfully converted to yl)-2'-O-(te rt-butyldimethylsilyl)-5-fluorouridine 3'-(cyanoethyl N,N-diisopropyl( phosphoramidite) by methods similar to those employed for the preparat ion of other ribonucleoside phosphoramidites. The desired product, as well as its precursors and the incorrect regioisomers, are prepared an d fully characterized by H-1 NMR, F-19 NMR, P-31 NMR, FAR-MS, UV absor bance, elemental analysis, and melting points. The purified material i s used for the production of RNA by solid-phase methods. Coupling yiel ds of 94% are obtained with this material. The resulting RNA is readil y purified and used for biophysical studies. The self-complemetary RNA decamer 5'-rGCGAAU(FU)CGC is prepared and purified. The material adop ts an A-form duplex in solution at neutral pH as characterized by CD s pectroscopy. The thermal stability of the duplex is similar to the par ent duplex oligoribonucleotide prepared with uridine instead of 5-fluo rouridine. The control duplex has a T-m of 53.8 and the FUrd substitut ed duplex has a T-m of 56.6 (xb0)degrees C as determined by UV hyperch romicity at 260 nm. The CD spectra and UV hyperchromicity data for the duplex oligoribonucleotide containing FUrd have a slight pH dependenc e indicating that ionization of FUrd has a slight but measurable impac t 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 oligoribo nucleotide as a consequence of the electron-withdrawing inductive effe ct of fluorine.