Synthesis and properties of DNA oligomers containing an O4-ethylated thymine. d(e4TpA), d(Ape4T), d(Ape4TpA), and d(Tpe4TpT)

Abstract

Syntheses via the phosphotriester method are described for DNA oligonucleotides containing O4-ethylated thymine (e4Thy) and adenine (Ade) bases, d(e4TpA), d(Ape4T), and d(Ape4TpA), for their unmodified analogs, d(TpA), d(ApT), and d(ApTpA), and for d(Tpe4TpT). 1H NMR chemical shift data obtained from 10 to 70 °C at 300 and 500 MHz show that these molecules form right-handed minihelices at low temperature in aqueous solution; the presence of the O4-ethyl group does not seem to have a drastic effect on the stacking geometry of the thymine base. The presence of right-handed stacking is confirmed by circular dichroic data obtained over a similar temperature range. Coupling constants for the sugar ring indicate that the e4Tp unit of d(e4TpA) does not show the shift towards the 3′-endo (N) pucker noted for the corresponding unit of d(e4TpT). A prominent quasimolecular ion peak [M – H]− is observed for the modified and unmodified molecules in the spectra obtained by Fast Atom Bombardment mass spectrometry (FAB-MS) operating in the negative ion mode, indicating that the labile O4-ethyl group is sufficiently stable to withstand the ionization process used in this method. In addition, a number of fragment ion peaks such as [O4-ethylthymine – H]− are observed that reveal the potential of FAB-MS for characterizing DNA oligomers modified by alkylating agents. Keywords: oligonucleotides, O-alkylation, stereochemistry, NMR, mass spectrometry.