Topology and Excited State Multiplicity as Controlling Factors in the Carbazole-Photosensitized CPD Formation and Repair

Photosensitized thymine<>thymine (Thy<>Thy) formation and repair can be mediated by carbazole (Cbz). The former occurs from the Cbz triplet excited state via energy transfer, while the latter takes place from the singlet excited state via electron transfer. Here, fundamental insight is provided into the role of the topology and excited state multiplicity, as factors governing the balance between both processes. This has been achieved upon designing and synthesizing different isomers of trifunctional systems containing one Cbz and two Thy units covalently linked to the rigid skeleton of the natural deoxycholic acid. The results shown here prove that the Cbz photosensitized dimerization is not counterbalanced by repair when the latter, instead of operating through-space, has to proceed through-bond.


INTRODUCTION
Solar light arriving at Earth is essential for humans, but at the same time, it is responsible for serious deleterious effects. Although UVB radiation represents only a minor sunlight component, it is associated to melanoma skin cancer, since it can be absorbed by the thymine (Thy) or cytosine (Cyt) nucleobases. As a consequence, formation of cyclopyrimidine dimers, (CPDs) such as Thy<>Thy, 1 Thy<>Cyt, 2,3 or Cyt<>Cyt, as well as pyrimidine (6−4) pyrimidone adducts and their related Dewar isomers, are observed. 4,5 The Thy<>Thy dimers are the photoproducts obtained with higher yields (likely because Cyt exhibits the highest energy triplet among the DNA bases) 6 and also the most biologically significant. 7−9 In addition, the effects of UVA should not be disregarded, in particular when they can be mediated by photosensitizers absorbing in this region. A limited number of photosensitizers have been employed to investigate this DNA photodamage, including non-steroidal anti-inflammatory drugs, fluoroquinolones, ketones, pyridopsoralenes, or p-aminobenzoic acid derivatives. 10−12 The reported quantum yields for photosensitized dimerization range from 10 −5 to 10 −2 . Formation of Thy<>Thy dimers is thought to proceed through an initial triplet−triplet energy transfer step (TTET). 13,14 Efficient TTET requires, in principle, a donor chromophore with a high intersystem crossing quantum yield, a triplet energy above that of Thy, and a long triplet lifetime. In general, Thy<>Thy dimer formation follows an exponential distance dependence as expected from a Dexter-type TTET mechanism. 15,16 Nevertheless, alternative mechanisms involving the participation of triplet triplexes have been demonstrated to play a role in the photosensitized formation of Thy<>Thy mediated by benzo-phenone (Bzp). 17 Moreover, recent examples have demonstrated the generation of 3 Thy* at long (non-bonding) distance through-bond (TB), in intramolecular systems in which the photosensitizer and the nucleobase are separated by a rigid hydrocarbon bridge. 18 In prokaryotes, yeast, and plants, CPDs are repaired by photolyases. They act through a light-dependent single-electron transfer mechanism 19,20 with quantum yields for the repair of Thy<>Thy as high as 0.7−0.98. 21 The redox-active flavin adenine dinucleotide (FAD) cofactor plays a pivotal role in the photorepair activity of photolyases, since its fully reduced and protonated form (FADH − ) can be directly excited to reach its singlet excited state ( 1 FADH − *) or, more efficiently, via energy transfer from an antenna chromophore present in the medium. Then, the excited 1 FADH − * transfers one electron to the CPD and leads to the dimer radical anion, inducing the spontaneous cleavage of the cyclobutane, finally giving rise to the restored pyrimidines. The limiting factor for the repair efficiency seems to be the back electron transfer from the dimer radical anion and the electron donor. 22 Model compounds, which mimic the performance of the CPD-photolyase, have been reported to achieve the CPD photosensitized repair. 22−27 Among them, the activity of the carbazole (Cbz) chromophore has been tested upon incorporation of an artificial Cbz-nucleoside into a DNA duplex 28 or in covalently linked Cbz-thymine dimer compounds. 29 This activity relies on the lifetime (ca. 19 ns) and redox potential (E* Cbz 29 of the Cbz singlet excited state (in the order of the values reported for flavin derivatives), 30 making it able to produce the e − transfer to the Thy<>Thy (E Thy<>Thy / Thy<>Thy ·− = −2.2 V vs SCE). 29 Surprisingly, carbazoles can also mediate photosensitized CPD formation in DNA, although the efficiency of this process is lower than expected from the Cbz photophysical properties. 31 As a matter of fact, Cbz can be excited selectively in the presence of Thy, and also, Cbz exhibits a moderate intersystem crossing quantum yield (0.36), 32 a relatively high triplet energy (70.2 kcal mol −1 ), 32 a ππ* triplet configuration 33 (free from the problems associated with the competitive hydrogen abstraction by benzophenone), 34 and a reasonably long-living triplet excited state. Moreover, how far the energy migrates in DNA to eventually produce photodamage is still a matter of concern. 35,36 With this background, our aim was to control the balance between DNA damage and repair by the Cbz chromophore. In order to achieve this goal, we have designed appropriate trifunctional intramolecular systems (see Scheme 1). Here, the through-space (TS) TTET mediated by an intramolecular Cbz would result in the formation of Thy<>Thy, in such a way that photosensitized repair should necessarily happen TB. For this purpose, two Thy units and a Cbz will be anchored to the rigid skeleton provided by deoxycholic acid (DCA), preparing different diastereoisomers to evaluate the influence of the topology on the involved processes. The predominance of TTET for Thy<>Thy formation or the e − transfer for Thy<>Thy repair will be modulated through the absence or presence of oxygen in the atmosphere of the reaction media, which will favor the prevalence of the triplet or the singlet excited states of Cbz.

RESULTS AND DISCUSSION
2.1. Synthesis. Two new dyads derived from DCA have been synthesized (Scheme 1), containing the Cbz chromophore at the lateral chain and the Thy units at 3α + 12α (2) or the Cbz at 3α and two Thy moieties at 12α + the lateral chain (6). The developed synthetic strategy started with esterification of DCA with Cbz-CH 2 CH 2 OH to yield 1. Then, in the presence of an excess of Thy-CH 2 CO 2 H, the positions 3α and 12α were esterified providing 2. To prepare the derivative with the two Thy moieties at 12α and at the lateral chain, initially, the carboxyl group at DCA was reduced to the corresponding alcohol (3), and the Thy at the lateral chain was covalently attached using Thy-CH 2 CO 2 H to give 4. The following step was the introduction of the chromophore at 3α to yield 5. Subsequent treatment with ThyCH 2 CO 2 H provided 6. In summary, new derivatives in which different combinations of Thy units and distances to the chromophore have been designed, synthesized, and fully characterized ( 1 H and 13 C NMR and exact mass) to investigate the influence of the topology on the photosensitized formation of Thy<>Thy dimers and eventually in their photosensitized repair.

Photosensitized Thy<>Thy Dimer Formation.
Initially, diluted anaerobic solutions (4.4 × 10 −5 M in 4CH 3 CN:1H 2 O) of 2 or 6 were submitted to steady-state photolysis. Irradiation was performed at λ max = 350 nm and monitored after different irradiation times attending at the changes in the spectra at 260 nm, where the Thy chromophore has a maximum ( Figure 1 top for 2, middle for 6, and Figure S5.1 for the control experiments under aerobic conditions). The controls Thy (as ThyCH 2 CO 2 H), Cbz (as Cbz-CH 2 CH 2 OH), and the intermolecular 1Cbz:2Thy mixture showed a slight decrease in the absorbance at 260 nm ( Figure 1 and Figure S5.1 bottom); nevertheless, the intramolecular systems were clearly more reactive, although their reaction pattern was slightly different.
For preparative purposes, more concentrated deaerated solutions of the two dyads (2 and 6) in acetonitrile (8.3 × 10 −4 M and 1.7 × 10 −3 M, respectively) were independently irradiated (λ max = 350 nm), and only one Thy<>Thy dimer was isolated in each case, in 99 and 68% yields, respectively (Scheme 2). These photoproducts were characterized by 1 H and 13 C NMR spectroscopy, together with an exact mass. More specifically, for the case of 7, upon photosensitized [2 + 2] cycloaddition, the olefinic protons of the two Thy units at 6.96 and 6.98 ppm moved to the cyclobutane protons at 4.13 ppm and ca. 4.57 ppm. The corresponding 13 C-NMR signals moved from 140.7 and 140.5 ppm to 66.4 and 64.6 ppm in the case of the CHs and from 111.0 and 110.5 to 46.0 and 45.9 ppm for the quaternary carbons. Nevertheless, to unambiguously determine the stereochemistry of photoproduct 7 as the one shown in Scheme 2, the ester in the lateral chain was hydrolyzed using titanium(IV) isopropoxide and in situ converted into the benzyl ester (9) (see Section S4 in the Supporting Information). 37 Full characterization of compound 9 resulted to be coincident with the 3α,12α-Thy<>Thy-DCABn, previously reported by our group. 17 Analogously, in the case of 8, the olefinic protons of the two Thys at 5.53 and 6.99 ppm moved to 4.02 and 4.11 ppm upon formation of the cyclobutane ring. The 13 C NMR signals corresponding to the characteristic double bond of the Thys moved from 140.2 and 140.0 ppm to 66.3 and 65.9 ppm for the CHs and from 111.7 and 110.6 to 46.2 and 45.5 ppm for the quaternary carbons. We found difficulties in the NOEDIFF experiments due to the NOE zero zone fulfilled by these molecules as a result of their high molecular mass (917.46 g mol −1 ). Nevertheless, the photoproduct was found to be a transsyn Thy<>Thy, the structure of which was unambiguously established by crystal data (Figure 2, see also the video in the Supporting Information, section S3). A new compound analogous to 6 but without Cbz (11) was synthesized starting from 4 to evaluate the role of Cbz in the formation of Thy<>Thy upon 350 nm irradiation (see Sections S4 and S5). Analog 11 resulted to be unreactive upon prolonged irradiation times at 350 nm, under a deaerated atmosphere, confirming the active role of Cbz in the Thy<>Thy formation. Moreover, a compound analogous to 2 without Cbz has already been described and its irradiation only produced the Thy<>Thy dimer in the presence of the absorbing benzophenone. 17 2.3. Photophysics of the TTET in the Photosensitized Thy<>Thy Dimer Formation. The feasibility of the intermolecular TTET from the triplet of Cbz to Thy was investigated by LFP. 32,38 The transient absorption spectrum obtained after laser pulse excitation (λ exc = 308 nm) of CbzCH 2 CO 2 H showed a maximum at 420 nm. Thus, the decay of the characteristic 3 Cbz* obtained upon excitation at 308 nm was recorded upon the addition of one, two, and three equivalents of thymidine (Thd) (to achieve the required concentration), and these data were fitted to a first-order exponential equation (Figure 3, top). The corresponding lifetimes were fitted to a Stern−Volmer relationship, and the value for the intermolecular quenching constant was obtained from the slope of the linear fitting (k qT = 4.9 × 10 8 M −1 s −1 ). This low value for the quenching constant indicates that intermolecular TTET is efficient. Next, we investigated the TB vs TS nature of the TTET from Cbz to the Thy units in the dyads ( Figure 3, bottom). This was done by comparing the lifetime of the signals recorded at 420 nm upon selective excitation of Cbz at 308 nm in 5, 2, and 6. The decay corresponding to 5 was fitted to a first-order exponential equation, and the determined lifetime was 4.7 μs; by contrast, the triplet lifetime of 2 and 6 could not be accurately determined. In fact, in both cases, the amplitude of the signal observed just after the laser pulse was very low compared to the one of 5, which could be attributed to a very efficient quenching of the singlet excited state (see below), together with very efficient TS-TTET, giving rise to the 3 Thy* that has a very low extinction coefficient at 420 nm. 18 2.4. Photosensitized Thy<>Thy Dimer Repair. The photosensitized dimer repair was investigated upon selective irradiation of the Cbz chromophore (λ max = 350 nm) in the two dimers 7 and 8, by monitoring changes of UV−visible spectra, steady-state and time-resolved fluorescence spectroscopy, and HPLC analysis (Figures 4 and 5, top and bottom for 7 and 8, respectively). Furthermore, to avoid subsequent formation of Thy<>Thy dimers, the prevalence of the Cbz singlet excited state ( 1 Cbz*) was favored over the 3 Cbz* by performing the experiments under air, although the Thy<>Thy competes with O 2 for the 1 Cbz* (see Figure S6.2 for quenching by O 2 , k qS = 1.7 × 10 10 M −1 s −1 ).
When aerated solutions of 7 and 8 were independently irradiated (λ max = 350 nm), a remarkable increase in the absorbance at ∼260 nm was observed in both cases (Figure 4A,D, respectively). This increase could safely be attributed to the opening of the cyclobutane ring, giving rise to the two free Thy units. These changes were accompanied by a decrease in the fluorescence emission (steady-state and time-resolved, Figure  4B,C,E,F), which indicates that the free intramolecular Thy units have higher quenching capability of the singlet excited state of carbazole than the Thy<>Thy moieties. In fact, the efficiency of the TS intramolecular quenching of 1 Cbz* by Thy at 12α can be determined from the lifetimes of 1 Cbz* in 2 (pink trace in Figure 4C) and 6 (green trace in Figure 4F), and the lifetime of 1 Cbz* under air ( Figure S6.2). From the corresponding values of 4.8 ns for 2, 1.7 ns for 6, and 11.4 ns for 1 Cbz*, the intramolecular TS quenching values in 2 and 6 are k Sq = 1.2 × 10 8 s −1 and 5.0 × 10 8 s −1 , respectively, much higher than the intermolecular quenching of 1 Cbz* by Thy at the employed concentration (k qS × [Thy] ca. 2.1 × 10 4 s −1 , see Figure S6.3). This is not surprising since quenching of 1 Cbz* by Thy<>Thy is likely happening TB since the probability of the three units being together TS is very low, 17 while as soon as the Thy<>Thy are repaired, quenching of the Thy unit at 12α happens TS. Scheme 2. Irradiation (λ max = 350 nm) in Deaerated CH 3 CN of 2 (Top) to Give 7 (>99%) and 6 (Bottom) to Give 8 (68%) Figure 2. X-ray crystal structure (thermal ellipsoids drawn at the 50% probability level) of the Thy<>Thy 8 resulting from irradiation (λ max = 350 nm) of 6 in CH 3 CN under N 2 , and the detail of the cyclobutane fragment. CCDC 2159900 contains the supplementary crystallographic data for this paper. These data are provided free of charge by The Cambridge Crystallographic Data Centre.
The Journal of Organic Chemistry pubs.acs.org/joc Article Furthermore, the topology of the dimers plays again a role in the quenching of 1 Cbz*, and therefore, in the efficiency of the photosensitized repair, with 8 being more reactive than 7.
A further piece of evidence for the photosensitized Thy<>Thy repair was obtained by monitoring in parallel the evolution of the irradiation by HPLC ( Figure 5A,C). Interestingly, in both cases, irradiation of 7 or 8 led to the opening of the cyclobutane ring; however, while for 8 the conversion was practically quantitative ( Figure 5D), for 7, it seems that a photoequilibrium was obtained, likely due to the concomitant photosensitized dimer formation ( Figure 5B).

Computational
Results. The participation of a TS mechanism in the formation of 3 Thy* vs TB mechanism in the photosensitized repair was further investigated upon determining the chromophore−chromophore distances in compounds 2, 6, 7, and 8 by using molecular dynamics at 298 K (see Section S7 and geometries file in the Supporting Information). Since in a previous study 18 the effect of solvent was demonstrated to be crucial, here, the simulations include explicit solvent molecules, that is, a 4:1 mixture of acetonitrile:water. For each compound, 300,000 configurations were produced and their chromophore− chromophore distances employed to prepare the histograms are shown in Figure 6, and the configuration of the analyzed molecules is presented in Figure 7. The conformational analysis of 2 and 6 shows that the distances Cbz-Thy are lower than 10 Å (ca. 90% frequencies) in the case of 2, while for 6, only ca. 50% of dyads show distances <10 Å (Figure 6 left). These results support the TS-TTET proposed mechanism in the photo-  The Journal of Organic Chemistry pubs.acs.org/joc Article sensitized dimerization and are in agreement with the higher reactivity observed for 2 vs 6 (see Figure 1). Moreover, the chromophore−chromophore (Thy<>Thy-Cbz) distances <10 Å in 7 and 8 have frequencies lower than 16% (Figure 6 right), in agreement with the likely TB mechanism operating for the photosensitized repair. Moreover, the higher speed of the photosensitized TB-repair found in the case of 8 compared to 7 ( Figure 5) could be the result of a more favored overlap between the LUMO of the CBz* and the σ* of the spacer bonds.
Overall, opening of Thy<>Thy in 8 in aerated 4CH 3 CN:1H 2 O results quantitatively into 6, due to the high distance between Thy-Cbz in 6 that prevents the subsequent TS-TTET in an aerated atmosphere. Conversely, opening of Thy<>Thy in 7 results into 2, in which the low distance Cbz-Thy allows an efficient TS-TTET even under an aerated atmosphere. As a result of the opposite trends, different equilibrium compositions are found as observed in Figure 5, upon 180 min irradiation.

Mechanistic Proposal.
We have demonstrated that Cbz covalently attached to the skeleton of DCA together with two Thy units can act as an efficient photosensitizer to produce Thy<>Thy dimers that could be repaired depending on the reaction conditions (Scheme 3). Thus, Cbz can be selectively excited, in the presence of Thy, to its singlet excited state, which is efficiently quenched by O 2 and by TS electron transfer to the Thy unit in 12α. The thermodynamics of this electron transfer is favorable (E Thy/Thy ·− = −1.34 V, 38 E Cbz ·+ /Cbz = 1.12 V vs SCE, 29 and E1 Cbz* = 3.63 eV). 39 Nevertheless, this pathway would be an energy-wasting channel, which regenerates the initial dyad upon back electron transfer. Still, the 3 Cbz* is reached upon intersystem crossing. In the absence of oxygen, this excited

CONCLUSIONS
In rigid bile acid-derived systems, TS triplet energy transfer from 3 Cbz* to Thy gives rise to photosensitized Thy<>Thy formation. In general, when photorepair can also occur TS, the efficiency of this process via electron transfer from 1 Cbz* to CPDs converts Thy<>Thy formation into a residual DNA photodamage. Conversely, we have demonstrated that if geometrical constraints prevent the dimer and the Cbz units from being close enough to each other for the electron transfer to happen TS, the repair should happen TB. This is a much less efficient mechanism, which results in enhanced prospects of Thy<>Thy photodamage.

Synthesis of 3.
A stirred solution of DCA was converted into DCA-Bn following the procedure previously described in the literature. 8 Then, a stirred suspension of LiAlH 4 (0.33 g, 9.13 mmol) in anhydrous THF (8.5 mL) was cooled to −10°C, treated with a solution of DCA-Bn (1.54 g, 3.19 mmol) in anhydrous THF (6 mL), and then the reaction mixture was refluxed overnight (70°C). Afterward, the reaction was quenched with saturated aqueous NH 4 Cl solution (5 mL), redissolved with EtOAc, poured into aqueous HCl 1 M, and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO 4 , and concentrated. Purification by column chromatography (SiO 2 , EtOAc) gave 3 as a colorless solid (1.05 g, 87%); 1 H NMR (300 MHz, CDCl 3