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

Article abstract of DOI:10.1021/jo00098a044

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 1H NMR, 19F NMR, 31P NMR, FAB-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 94percent 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 deg 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.