The structural integrity of DNA – the molecular carrier of the genetic code – is constantly under attack. One of the most important external hazards for DNA is UV radiation from the sun. This radiation may be absorbed by the crucial building blocks of DNA, their bases. The resulting electronic excitation can trigger photochemical reactions. The most frequent of these photoreactions is the photoaddition of an excited thymine to another thymine adjacent on the DNA strand (see Figure 1). The reaction results in cyclobutane pyrimidine dimer (CPD). In a collaboration with Wolfgang Zinth’s and Bern Kohler’s group, the CDP formation was studied by femtosecond UV pump IR probe spectroscopy.1-3 The experiments show unequivocally that the reaction occurs in less than 1 ps (= 10-12 s). This implies that it proceeds with an excited singlet and not a triplet state as a precursor. The reaction, thus, follows the Woodward-Hoffman rules stating that [2+2] photoadditions are “allowed” and therefore fast.
Another important photo-lesion formed by two adjacent thymines is the (6-4) photo-product. Excitation of its pyrimidinone moiety may trigger secondary photo-reaction yielding a Dewar isomer (Figure 2). By time resolved spectroscopy our group showed that the photophysics4 and photochemistry5 of pyrimidinones strongly depend on the molecular environment.
Under environmental stress certain bacteria – among them very pathogenic ones - can transform into spores which are very resistant towards aggressive agents. The DNA of these spores exhibits a high UV tolerance and forms photo-lesions (spore photoproduct) not observed otherwise. The DNA of spores is located in its core which also contains large amounts of the calcium dipicolinate. The role of this salt in the UV resistance of the spore DNA is unclear. We performed the first photophysical characterization of calcium dipicolinate (CaDPA, see Figure 3) by time resolved spectroscopy.6 As inferred from femtosecond fluorescence measurements the initial excitation of this molecules is very short lived; it decays within ~ 100 fs (see Figure 3). This behavior is reminiscent to sunscreen molecules which rapidly convert electronic excitation into heat. Yet, as transient absorption experiments showed, the decay of the primary excitation results in the population of another excited state and finally the triplet state. Energy transfer from this triplet state to the ones of DNA seem feasible. Thus, calcium dipicolinate could act as sensitizer. Such a sensitizer might enhance the formation of the spore photoproduct.
DNA photochemistry can also involve natural and synthetic exogenous substances. A prominent example is the photoreaction of psoralens with DNA. Psoralens are natural compounds found in certain plants and are used in the light-dependent treatment of skin diseases like psoriasis (PUVA-therapy for psoralen and UV-A). Psoralens are DNA intercalators, i.e. they can insert themselves between the base pairs of DNA. Photo-excitation of such an intercalated psoralen may result in the binding to a thymine base. Thereby, a four-membered ring strongly resembling the CPD motif is formed (Figure 4). Using time resolved spectroscopy we could make two important findings concerning the PUVA process:
1. Intercalated psoralens like AMT (see structure in Figure 4) are prone to photo-induced electron transfer (PET) reactions.7 In this PET psoralens act as acceptors and the DNA base guanine as a donor.8 The PET occurs with characteristic times of a few picoseconds generating a radical pair. This pair recombines with several tens of picoseconds whereby the starting material is recovered. The PET, thus, causes a rapid dissipation of the excitation energy and suppresses the photo-addition of psoralens to DNA.
2. A corollary of the PET finding is that the photo-addition of psoralens to DNA is best studied in guanine free DNA. We, thus, conducted experiments on AMT intercalated into adenine, thymine only DNA. The UV pump IR probe experiments were performed in the lab of Wolfgang Zinth.9 The recorded spectro-temporal behavior shows unequivocally that the addition occurs on the microsecond time scale and therefore via a triplet state. The cyclobutane ring forms in a two-step process (see Figure 5). First, one single bond between AMT and thymine is formed within 1-6 µs. The resulting triplet bi-radical then recombines and forms a second single bond to yield the final photoproduct. This takes ~ 50 µs. The kinetics of this photoaddition is, thus, in stark contrast to the CPD formation in DNA, which is a concerted reaction occurring in a few 100 fs. Presently, we are developing design criteria for better PUVA agents based on these findings.
1. Schreier, W. J.; Schrader, T. E.; Koller, F. O.; Gilch, P.; Crespo-Hernandez, C. E.; Swaminathan, V. N.; Carell, T.; Zinth, W.; Kohler, B., Thymine dimerization in DNA is an ultrafast photoreaction. Science 2007, 315 (5812), 625-629.
2. Schreier, W. J.; Kubon, J.; Regner, N.; Haiser, K.; Schrader, T. E.; Zinth, W.; Clivio, P.; Gilch, P., Thymine Dimerization in DNA Model Systems: Cyclobutane Photolesion Is Predominantly Formed via the Singlet Channel. J. Am. Chem. Soc. 2009, 131 (14), 5038-5039.
3. Schreier, W. J.; Gilch, P.; Zinth, W., Early events of DNA photodamage. Annu. Rev. Phys. Chem. 2015, 66, 497-519.
4. Ryseck, G.; Villnow, T.; Hugenbruch, S.; Schaper, K.; Gilch, P., Strong impact of the solvent on the photokinetics of a 2(1H)-pyrimidinone. Photochem. Photobiol. Sci. 2013, 12 (8), 1423-1430.
5. Micheel, M.; Torres Ziegenbein, C.; Gilch, P.; Ryseck, G., Pyrimidinone: versatile Trojan horse in DNA photodamage? Photochem. Photobiol. Sci. 2015, 14 (9), 1598-1606.
6. Mundt, R.; Torres Ziegenbein, C.; Fröbel, S.; Weingart, O.; Gilch, P., Femtosecond Spectroscopy of Calcium Dipicolinate—A Major Component of Bacterial Spores. J. Phys. Chem. B 2016, 120 (35), 9376-9386.
7. Fröbel, S.; Reiffers, A.; Torres Ziegenbein, C.; Gilch, P., DNA Intercalated Psoralen Undergoes Efficient Photoinduced Electron Transfer. J. Phys. Chem. Lett. 2015, 6 (7), 1260-1264.
8. Fröbel, S.; Levi, L.; Ulamec, S. M.; Gilch, P., Photoinduced Electron Transfer between Psoralens and DNA: Influence of DNA Sequence and Substitution. ChemPhysChem 2016, 17, 1377–1386.
9. Diekmann, J.; Gontcharov, J.; Fröbel, S.; Torres Ziegenbein, C.; Zinth, W.; Gilch, P., The Photoaddition of a Psoralen to DNA Proceeds via the Triplet State. J. Am. Chem. Soc. 2019, 141 (34), 13643-13653.