Chem. Res. Toxicol. 1993,6, 561-567
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Identification and Structure Determination of a Third Cyclobutane Photodimer of Thymidylyl-(3’-.5’) -thymidine: The trans-syn-I1 Product Jeffrey L.-F.Kao,+ Sourena Nadji, and John-Stephen Taylor’ Department of Chemistry, Washington University, St. Louis, Missouri 63130 Received October 19, 1992 Photolysis of thymidylyl-(3’+5’)-thymidine (TpT) has been previously reported by many workers to lead to only two cyclobutane dimers, the cis-syn and tram-sp-I dimers. This is curious in light of the fact that photolysis of thymidylyl-(3’+5’)-deoxyuridine (TpdU), which has a hydrogen in place of a methyl group a t C6 of the 3’-thymidine, produces two trans-syn diastereomers. Recently, we discovered by way of X-ray crystallography that photolysis of the @,)-methyl phosphate ester of T p T leads to two trans-syn diastereomers, prompting us to reexamine the photochemistry of TpT. In this paper we show that sensitized photolysis of T p T also leads to the hitherto unknown tr~ns-syn-11diastereomer in 2% yield. We also report the solution-state lH NMR assignment of the t~ans-syn-11photodimer of T p T and its @,)-methyl phosphate ester by way of 2D homonuclear Hartmann-Hahn experiment, rotating frame nuclear Overhauser effect spectroscopy, and proton-detected 1H-31P correlation spectroscopy. Conformational analysis of three-bond ‘H-lH, 1H-31P,and l3C-3lP coupling constant data established a close similarity between the solution-state structures of the t~am-syn-11photodimer of T p T and its @,)-methyl phosphate ester, with the crystal structure of the methyl phosphate ester.
Introduction The major photoproduct of DNA is the cis-syn cyclobutane dimer of thymidylyl-(3’+5’)-thymidine (TpT)’ sites 2g which results from a photochemically allowed [2+2] cycloaddition reaction between the 5,6 double bonds of adjacent thymines (1,2) (Figure 1). The stereochemistry of this photoproduct (5‘-5R6R,3‘-5S6S) is dictated by the stereochemistry of the B DNA helix, the prevalent form of DNA in vivo, in which both thymidines are in the anti glycosyl conformation. In the conformationally less restricted dinucleotide TpT, a second cyclobutane dimer was found to be formed in addition to the cis-syn dimer (3). It was not until much later that this second cyclobutane dimer was established to have the trans-syn-I structure 3a (5’-5S6S, 3’-5S6S) (4-6). The trans-syn-I photoproduct of TpT formally results from a photodimerization reaction in which the 5‘-thymidine is in the syn conformation and the 3’4hymidine is in the anti conformation. It is curious that only one trans-syn diastereomer of TpT has been reported considering that photolysis of thymidylyl-(3’+5’)-deoxyuridine (TpdU) lb, the analog of TpT in which the 3’-thymine lacks a methyl group, produces the trans-syn-I1 diastereomer 4b (5’-5R6R,3’5R6R),in addition to the trans-syn-I diastereomer 3b (4). The formation of trans-syn dimers in photolyzed DNA has been established by the identification of the transsyn thymine dimer that is liberated upon acid hydrolysis of the glycosidic linkages. Chromatographic analysis of the acid hydrozylates indicates that trans-syn dimers are formed at a level of about 1/7 that of cis-syn dimers in
* To whom correspondence should be addressed.
Washington University High-ResolutionNMR Facility. Abbreviatiom: COSY, correlated spectroscopy;DMT, 4,4’-dimethoxytrityl; HOHAHA, homonuclear Hartmann-Hahn experiment; NOE, nuclearOverhausereffect;rms,rootmeansquare;ROESY,rotating frame nuclearOverhauser effectspectroscopy;TBDMS, tert-butyldimethyleilyk TMP, trimethyl phosphate; TpdU, thymidylyl-(3’+5’)-deoxyuridine; t
1
TpT,thymidylyl-(3’-+5’)-thymidine.
PN
TP
R1 R2 R3 R4
a H (-)
b H
(-) CH3 H H H
c d e f g DMT DMT H H DNA CH3 CH3 CH3 CH3 (-) CH3 CH3 CH3 CH3 CH3 TBDMS H TBDMS H DNA
Figure 1. Cyclobutane photodimers of TpT TpdU (R3 = H)and their derivatives.
(R3
= CH3) and
single-strand DNA, and at a much lower level (
