Stereoselective Nucleoside Deuteration for NMR Studies of DNA

Abstract

A procedure has been elaborated for stereoselective deuterium substitution of one of the diastereotopic 5′-protons in 2′-deoxynucleotides. The synthetic scheme uses the reduction of the 5-oxosugar derivative with deuterated Alpine-Borane. The resulting deuterosugar is converted into pyrimidine nucleosides and incorporated into DNA using standard protocols. Comparison of 2D NMR spectra of the fully protonated and partially deuterated duplexes allowed us to assign diastereotopic 5′ protons, increasing the number of experimental restraints used for structure determination.

Structure and stability of DNA containing an aristolactam II-dA lesion: implications for the NER recognition of bulky adducts

“…We realized that NOE interactions involving the H5′ and H5″ protons of T17 could provide additional experimental restraints for establishing its conformation with greater confidence. In order to obtain stereospecific assignments of the T17-H5′ and T17-H5″ resonances and to use their NOE distance information fully, we employed our recently proposed isotope-labeling strategy ( 14 ) to prepare an ALII-dA duplex having T17-H5″ selectively deuterated. Comparison of the NOESY spectra of the deuterated and nondeuterated duplexes ( Supplementary Figure S3 ) yielded the unambiguous assignment of both diastereotopic 5′ protons and allowed the computation of five additional NOE restraints, including two key distances involving the ALII-H4 proton.…”

“…Two other duplexes having the lesion in the context of mutational hotspots ( 13 ), namely d[(CTCCTXGGTTG)•(CAACCTAGGAG)] (TXG duplex) and d[(ATGACXGAAAC)•(GTTTCTGTCAT)] (CXG duplex), were also synthesized and partially characterized. Preparation of oligonucleotides containing the ALII-dA lesion has been reported elsewhere ( 11 , 14 ). Non-modified oligonucleotides were prepared following standard solid-phase synthesis methods and purified by ion-pair reverse-phase HPLC ( 15 ).…”