Synthesis, annealing properties, fluorine-19 NMR characterization, and detection limits of a trifluorothymidine-labeled antisense oligodeoxyribonucleotide 21 mer'
作者:William H. Gmeiner、Richard T. Pon、J. William Lown
DOI:10.1021/jo00011a028
日期:1991.5
The synthesis and characterization are described of trifluorothymidine groups incorporated into an antisense 21 mer designed to target gene sequences that encode serine proteases in T-lymphocytes. H-1 NMR titration studies on 3',5'-O-TPDS-trifluorothymidine (3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)trifluorothymidine) with 3',5'-O-TPDS-2'-deoxyadenosine provided clear evidence of normal Watson-Crick base pairing via detection of the imino proton signals. The chemical shift of the imino proton is moved ca. 0.5 ppm upfield relative to the position with the natural nucleoside. H-1 NMR also confirmed normal annealing in the 21 mer with its complement in the imino proton detection with the most notable difference being that six AT signals move upfield into the region characteristic of Watson-Crick GC base pairs. 1D-NMR experiments confirm that a single species exists in solution and CD studies indicate that the duplex formed with its complement adopts B-form geometry. The 1D-NMR experiments show that two of three ordinary methyl groups in the hybrid duplex exist in a single conformation while the third methyl group is predominantly in a single conformation. Molecular modeling of the duplex formed between the trifluorothymidine antisense sequence and complementary mRNA indicates a stable A-type helix in which the CF3 groups cause the thymidine base pairs to be displaced somewhat compared with the natural structure. The limits of F-19 NMR detection of the trifluorothymidine labeled 21 mer were determined to be ca. 10-mu-M at a 10:1 signal to noise ratio, i.e., satisfactory for projected in vivo NMR imaging studies.