A5–4 PYRIMIDINE‐PYRIMIDONE PHOTOPRODUCT PRODUCED FROM MIXTURES OF THYMINE AND 4‐THIOURIDINE IRRADIATED WITH 334 nm LIGHT

Abstract

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and 1H- and 13C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(beta-D-erythro-pentofuranosyl)-4'-pyrimidin-2'- one]dihydrothymine (O6hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90 degrees C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

The (5‐4) and (6‐4) Adducts of 1‐Methylthymine and Their Dewar Valence Isomers

“…We also demonstrate that each of these three adducts, when irradiated by UVB light, is transformed into a mixture of two diastereomeric Dewar valence isomers. Finally, we present evidence that irradiation of thymidine under the same conditions produces two (5‐4) type adducts, as well as the previously characterized (6‐4) adducts (28–30). (The numbering system is displayed in the structure for IIIb ; asterisks in the various structures in Scheme 2, including those at bridgehead nitrogens, denote chiral centers.…”

“…After irradiation of an aqueous mixture of Thy and 4‐thiouridine at 335 nm, a diastereomeric pair of stable (5‐4) adducts was isolated and characterized by Blazek et al. (25). These products were indicated to be secondary thermal reaction products, arising via replacement of a 6SH group in the originally formed (5‐4) adducts by a 6OH.…”

“…There is evidence that such products involving Thy can be formed, although not with both reaction partners being major DNA or RNA nucleobases, nucleosides or closely related compounds. After irradiation of an aqueous mixture of Thy and 4thiouridine at 335 nm, a diastereomeric pair of stable adducts was isolated and characterized by Blazek et al (25). These products were indicated to be secondary thermal reaction products, arising via replacement of a 6SH group in the originally formed adducts by a 6OH.…”

Photoinduced Fluorescent Cross-Linking of 5-Chloro- and 5-Fluoro-4-thiouridines with Thymidine

“…The photoadducts 3a,b are structurally distinct from a 5-4 pyrimidine-pyrimidone photoadduct formed by irradiation of 4SU in the presence of T , as well as similar 5-4 and/or 6-4 bipyrimidine products formed upon irradiation of 4SU in the presence of other pyrimidine bases . Furthermore, the specificity and the overall yield of 3a and 3b are significantly larger than for photoadducts of 4SU with pyrimidine nucleobases.…”

“…The 4-thiouracil (4SU) chromophore and its derivatives have attracted considerable interest in recent years as efficient photo-cross-linking agents in the studies of the structure of nucleic acids and their interactions with proteins. − The use of 4-thiouridine and 4-thiothymidine as structural probes , indicates that a range of cross-links is possible, including interstrand cross-links . To identify the covalent bonds that form and to better understand the cross-linking mechanism, a number of experiments involving photoirradiation of thiouridine have been conducted in solution in the presence of common nucleosides or using dinucleotides. − In agreement with the results of 4SU photo-cross-linking experiments with DNA, it has been demonstrated that 4SU forms mixed photoadducts with all nucleobases with thymidine being the best acceptor. , The (5-4) or (6-4) bipyrimidine compounds were identified as major photoproducts of irradiation of 4SU in the presence of pyrimidine nucleosides. Non-native 5-halogenated pyrimidines have been studied in parallel to the 4-thiopyrimidines as probes of nucleoprotein interactions as well as DNA structure…”