UV irradiation of nucleic acids: formation, purification and solution conformational analysis of the ‘6-4 lesion’ of dTpdT

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Abstract
Irradiation of dTpdT with 300 kJ/m2 of 254 nm produces numerous photoproducts, one of which labeled dT6pd4T[l] was purified by HPLC. dT6pd4T has a UV spectrum (H2O, pH 7) with λmax = 326 nm and λmin = 265 nm, and a P-31 NMR resonance at -3.46 ppm (normal dTpdT occurs at -4.01 ppm; TMP, 30°C). 2-D COSY NMR spectra facilitated proton resonance assignments and 2-D NOESY spectra aided analysis of spatial orientation. Carbon-13 and proton-coupled P-31 NMR spectra of dT6pd4T were also obtained. These analyses indicate: (1) C5=C6 of dT6p- is saturated and the -pd4T base 1s more aromatic; (2) the dT6p- base possesses a configuration of 5R, 6S; (3) dT6p- and -pd4T have anti-type glycosidic conformations; (4) furanose conformation of dT6p- is mainly C3′-endo and that of -pd4T exists in a C3′-endo C3′-exo; (5) exocyclic bonds γ(C5′-C4′), β(05′-C5′) and ɛ(C3′-03′) are non-classical rotamers; (6) dihedral angle about ɛ(C3′-03′) is smaller relative to dTpdT.