¹HNMR Assignment and Melting Temperature Study of Cis-Syn and Trans-Syn Thymine Dimer Containing Duplexes of d(CGTATTATGC)·d(GCATAATACG)

The preparation and spectroscopic characterization of duplex decamers containing site-specific cis-syn and trans-syn thymine dimers are described. Three duplex decamers, d(CGTATTATGC)·d-(GCATAATACG), d(CGTAT[c,s]TATGC)·d(GCATAATACG), and d(CGTAT[t,s]TATGC)·d(GCATAATACG), were prepared by solid-phase phosphoramidite synthesis utilizing cis-syn and trans-syn cyclobutane thymine dimer building blocks (Taylor et al., 1987; Taylor & Brockie, 1988). NMR spectra (500 MHz 2D ¹H and 202 MHz 1D ³¹P) were obtained in “100%” D₂O at 10 °C, and 1D exchangeable ¹H spectra were obtained in 10% D₂O at 10 °C. ¹H NMR assignments for H5, H6, H8, CH₃, H1′, H2′, and H2″ were made on the basis of standard sequential NOE assignment strategies and verified in part by DQF COSY data. Comparison of the chemical shift data suggests that the helix structure is perturbed more to the 3′-side of the cis-syn dimer and more to the 5′-side of the trans-syn dimer. Thermodynamic parameters for the helix ⇄ coil equilibrium were obtained by two-state, all or none, analysis of the melting behavior of the duplexes. Analysis of the temperature dependence of the T5CH₃ ¹H NMR signal gave ΔH = 44 ± 4 kcal and ΔS = 132 α 13 eu for the trans-syn duplex. Analysis of the concentration and temperature dependence of UV spectra gave ΔH = 64 ± 6 kcal and ΔS = 178 α 18 eu for the parent duplex and ΔH = 66 ± 7 kcal and ΔS = 189 ± 19 eu for cis-syn duplex. It was concluded that photodimerization of the dTpdT unit to give the cis-syn product causes little perturbation of the DNA whereas dimerization to give the trans-syn product causes much greater perturbation, possibly in the form of a kink or dislocation at the 5′-side of the dimer.