Stereochemical Assignment of Chiral Phosphotriester Analogues with Alu I Sites

Abstract

The stereochemistry of the diastereomers of a DNA duplex with the 2,2,2-trichloro-1,1,- dimethylethyl (TCDME) phosphotriester backbone substitution has been assigned by the use of 2D NMR spectroscopy. The duplex [G₁G₂A₃A₄G₅p(TCDME)C₆T₇A_gG₉G₁₀]- [C₁₁C₁₂T₁₃A₁₄G]5C₁₆T₁₇T₁₈C₁₉C₂₀] is a substrate of the restriction endonuclease Alu I, with placement of the TCDME group at the G₅-C₆ cleavage site of one strand. The stereochemical orientation of the TCDME group in relation to the structure of the double helix regulates the ability of Alu I to hydrolyze the complementary recognition site. The phosphotriester group of the isomer 1 duplex blocks cleavage of the complementary strand, while that of the isomer 2 duplex allows cleavage to proceed. Within the phosphotriester recognition site, no hydrolysis is detected nor is any seen when the single-stranded DNA substrate is utilized.

Data from the 2D NOESY spectra demonstrate that both DNA duplexes retain basic B-form geometry. The isomer 1 duplex shows NOE cross-relaxation from the protons of the two methyl groups of the TCDME modification (1.99, 2.00 ppm) to the G5 H3′ (5.30 ppm), G5 H4′ (4.53 ppm), and C6 H5′/H5″ (4.52, 4.62 ppm) protons. The isomer 2 duplex shows NOE cross-relaxation from the methyl protons of the TCDME modification (2.01, 2.03 ppm) to the C6 H6 (7.15 ppm), C6 H4′ (4.30 ppm), C6 H5′/H5″ (4.48, 4.62 ppm), G5 H3′ (5.26 ppm), and G5 H4′ (4.48 ppm) protons. Thus the NOE cross-relaxation between the methyl protons of the TCDME modification and the C6 H6 and C6 H4′ protons in isomer 2 is not found in the spectra of the isomer 1 duplex. These NMR data confirm the stereochemical assignment of the chirality of the TCDME phosphotriester group in isomer 1 as the Sp configuration and in isomer 2 as the Rp configuration. The Sp isomer features the TCDME group pointing away from the helix, while the Rp isomer shows the TCDME group pointing towards the major groove. Thus through the use of 2D NMR techniques, the stereochemistry of chiral phosphotriester linkages may be assigned in chemically modified DNA.