The Photoaddition of a Psoralen to DNA Proceeds via the Triplet State.

Psoralens are natural compounds that serve in the light dependent treatment of certain skin diseases (PUVA therapy). They are DNA intercalators that upon photoexcitation form adducts with thymine bases. For one psoralen derivative, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), the photoreactions are characterized here by nanosecond UV-vis and IR absorption spectroscopy. The triplet state of AMT is identified as the reactive one. On the 1-10 µs time scale this local triplet state transforms into a triplet biradical bearing one single bond between the addends. Within ∼50 µs this biradical forms the final adduct featuring a cyclobutane ring. This kinetic behavior is in stark contrast to the closely related photoaddition of two thymine moieties within the DNA. Origins of the differences are discussed.

Triplet-Induced Lesion Formation at CpT and TpC Sites in DNA.

UV irradiation induces DNA lesions particularly at dipyrimidine sites. Using time-resolved UV pump (250 nm) and mid-IR probe spectroscopy the triplet pathway of cyclobutane pyrimidine dimer (CPD) formation within TpC and CpT sequences was studied. The triplet state is initially localized at the thymine base but decays with 30 ns under formation of a biradical state extending over both bases of the dipyrimidine. Subsequently this state either decays back to the electronic ground state on the 100 ns time scale or forms a cyclobutane pyrimidine dimer lesion (CPD). Stationary IR spectroscopy and triplet sensitization via 2'-methoxyacetophenone (2-M) in the UVA range shows that the lesions are formed with an efficiency of approximately 1.5 %. Deamination converts the cytosine moiety of the CPD lesions on the time scale of 10 hours into uracil which gives CPD(UpT) and CPD(TpU) lesions in which the coding potential of the initial cytosine base is vanished.

2'-Methoxyacetophenone: An Efficient Photosensitizer for Cyclobutane Pyrimidine Dimer Formation.

Stationary and time-resolved experiments show that 2'-methoxyacetophenone (2-M) is an interesting compound for the investigation of triplet states in thymine samples. Time-resolved emission experiments show that the fluorescence lifetime of 2-M is 660 ps. A similar time constant of 680 ps is found in transient IR experiments. The data indicate efficient intersystem crossing (≈97%) from the fluorescent singlet state to the triplet state. The lifetime of the triplet state of 2-M dissolved in D2O at room temperature and ambient oxygen concentration is 400 ns. 2-M has a strong absorption in the UV-A range and can photosensitize the triplet state of a thymidine dinucleotide with light at a wavelength of 320 nm. The experiments show that 2-M is well-suited for time-resolved experiments on the triplet-sensitizing process.

Quantum Yield of Cyclobutane Pyrimidine Dimer Formation Via the Triplet Channel Determined by Photosensitization.

UV-induced formation of the cyclobutane pyrimidine dimer (CPD) lesion is investigated by stationary and time-resolved photosensitization experiments. The photosensitizer 2'-methoxyacetophenone with high intersystem crossing efficiency and large absorption cross-section in the UV-A range was used. A diffusion controlled reaction model is presented. Time-resolved experiments confirmed the validity of the reaction model and provided information on the dynamics of the triplet sensitization process. With a series of concentration dependent stationary illumination experiments, we determined the quantum efficiency for CPD formation from the triplet state of the thymine dinucleotide TpT to be 4 ± 0.2%.