The excited states Singlets and triplets of DNA or RNA bases were performed in vacuum. Table 1 displays the excitation energies and the probability coefficients of the six lowest excited singlet and triplet states of DNA or RNA bases calculated in vacuum at the B3LYP/Lanl2DZ level.

The singlet states of these bases have absorption bands that range from 190 nm to 300 nm. In addition, the UV-visible spectrum of these bases in vacuum indicates that they absorb between 130 nm and 300 nm in vacuum (Figure 3). Thus, these nitrogenous bases of DNA absorb all in the UV. Therefore, the use of any wavelength within the therapeutic window will not cause any alteration on these bases [17].

As for the triplet states, we note that these states have low oscillation forces except for a few states whose forces are significant. Actually, the states with very low values of oscillation forces are less likely to occur. Therefore, they will not be considered in our analyses. Only states with oscillation forces greater than or equal to 0.001 will be considered. Based on this principle, we have a diversification of the number of significant states from one base to another. For the adenine base, we have three significant states, the first of which is at 1306 nm; for the cytosine base, we have three significant states, the first of which is at 1644 nm. The guanine displays four significant states and the first one occurs at 1227 nm. For the thymine, four significant states are also observed, the first of which

is at 921 nm, and for the uracil base, three significant states are observed with the first at 999 nm. These triplet states have S² values very close to 2, which attests that these states are free of any contamination [18].

In this section, we first focused on the singlet states of these complexes. The singlet states provide information on the absorption bands of the molecules. We evaluated the six lowest-energy singlet states of the δ-OsCl₂(Azpy)₂ complex with a view to highlight the absorption bands that lie within the therapeutic window [17]. Secondly, we were interested in the triplet states. In fact, photosensitive reactions generally occur in the triplet excited state because of the much longer lifetime of this state compared to the singlet excited state. This very long lifetime of the triplet state considerably increases the probability of collision and thus of interaction and/or reaction with other molecules [19]. Therefore, photosensitized reactions will almost always involve the triplet state of a molecule. Besides, we have determined the excitation energies (ET1) of the six lowest energy triplet states of photosensitizers that are crucial in understanding the photosensitization mechanisms. The values of the energies of these singlet S and triplet T states of the photosensitizers were calculated by the TD-DFT method in vacuum and solvent. The different results are reported in Table 2. Only states with an oscillation force greater than or equal to 0.001 will be taken into account in the analysis of these results. Figure 4 shows the absorption spectrum of the δ-OsCl₂(Azpy)₂ complex in vacuum and solvent.

The determination of the six lowest energy singlet and triplet states was performed by the TDDFT method in vacuum and at the B3LYP/LANL2DZ level. As the photochemical reactions of this complex are carried out in biological milieu, it seems justified to evaluate the impact of the solvent on these triplet states. At this level, it should be noted that the solvents can be grouped into four categories which are: polar protic solvents, dipolar aprotic solvents, low-polar aprotic solvents and apolar aprotic solvents. In the coming work, we will use a sample of each of the above solvent types. The first of these solvents is water. It is a polar

protic solvent. It can form hydrogen bonds, it has a strong ionizing power and a high separating power. The second of these solvents is dimethyl sulfoxide (DMSO). It is a dipolar aprotic solvent. It cannot form hydrogen bonds, but its molecules act as dipoles. It is observed that opposite charges appear by mesomerism. It has a strong dipole moment and a high dielectric constant. The third of these solvents is diethylether. It is an aprotic solvent with a low polarity. It has a low dipole moment. It cannot form hydrogen bonds. The fourth solvent is benzene. It does not influence, or influences very little the reaction.

The results reported in Table 2 indicate that δ-OsCl₂(Azpy)₂ complex has its absorption bands beyond 400 nm. A portion of the absorption bands of these complexes is composed mainly of transitions t_2g → π* that are located in the therapeutic window [17]. In vacuum, the most significant state that belong to the therapeutic window is the excited state 3, which is located at 753.35 nm. In solvents, we observe a hypsochromic effect with respect to vacuum, which increases with the polarity of the solvent in the singlet states. This effect is accompanied by an increase in the energies of the singlet states of the complex.

As for the triplet states, δ-OsCl₂(Azpy)₂ indicates four significant states at 1708 nm, 1611 nm, 703 nm and 568 nm with energies of 0.7258 eV; 0.7695 eV; 1.7634 and 2.1808 eV respectively. These triplet states have S² values very close to 2, which attests that these states are devoid of any contamination [18]. In solvents, there is an increase of the energy in the triplet states. In water, the first triplet state is at 1689 nm with an energy of 0.7337 eV. A conservation of the number of significant states is noted, accompanied by a decrease in the oscillation forces. In DMSO, the first state is at 1690.76 nm. We also note an increase in the number of significant triplet states. For instance, the state of δ-Os goes from 3 significant triplets in water to 4 significant triplet states in DMSO. We believe that these changes are due to the dipole moment of the complex which interacts with those of the solvent molecules (DMSO). In diethylether, the δ-OsCl₂(Azpy)₂ complex admits its first significant triplet state at 1659 nm with an energy of 0.7473 eV. This complex retains the same number of significant triplet states as in DMSO. In benzene, δ-OsCl₂(Azpy)₂ provides with energies close to those obtained in diethylether. However, the values of these energies are higher than those obtained in water and DMSO. Besides, δ-OsCl₂(Azpy)₂ complex admits its first significant triplet state at 1674 nm with an energy of 0.7403 eV. It has three significant states in this solvent.

From the above, we can say that the polarity of the solvent influences the absorption and emission spectra. The transition from a polar medium such as water or DMSO to a less polar medium such as diethylether or apolar benzene is accompanied by a shift towards high energies. This shift is certainly due to the dipolar interactions between the complex and the solvent, also indicating the existence of a stabilized charge-transfer state CT in a polar medium. This result is in agreement with that found by the team of B. BENALI et al. who studied the effect of solvent polarity on the properties of excited electronic states of l,l-binaphthyl by UV-visible spectroscopy [20]. The order of magnitude of the energies of the first significant triplet states for the complex δ-OsCl₂(Azpy)₂ is as follows:

ET1 (vacuum) < ET1 (DMSO) < ET1 (water) < ET1 (benzene) < ET1 (diethylether).

By comparing the evolution of the energies of the first significant triplet states of this complex in the different solvents, we can conclude that the energies of the triplet states of the organometallic complexes are a function of the polarity of the solvent.

In order to determine the behavior of DNA or RNA bases following electronic exchanges, we determined the vertical electron affinity and vertical ionization potential of these bases. The results obtained are shown in Table 3.

VEA_so = Ea − Ep, VIP_so = Ec − Ep.

The values of the vertical VIP ionization potentials of the DNA or RNA bases indicate that Guanine is the most easily ionizable base because of its lowest VIP values in vacuum as well as in solvents. In vacuum, the VIP_so value of guanine is 7.91. This value decreases in a solvent. The main decrease is recorded in water. The reactivity of DNA or RNA nucleobases during ionization decreases in the following order: Guanine > Adenine > Cytosine > Uracil > Thymine. In addition, it is found that VIP_so of each base increases in solvents as follows the rank: vacuum > Benzene > diethylether > DMSO > water. So VIP_so values evolve in the opposite direction of the solvent polarity. Therefore, we can assume that polar solvents are favorable to the ionization of the DNA or RNA bases.

As for the VEA_so vertical electron affinity values, the order of reactivity of the bases during electron capture is as follows: Adenine < Guanine < Cytosine < Thymine < Uracil. Thus, the Uracil is the DNA base that has the greatest ease of electron acceptance. The VEA_so values indicate the following ranking with respect to the polarity of the solvent: VEA_so (vacuum) > VEA_so (Benzene) > VEA_so (diethylether) > VEA_so (Dmso) > VEA_so(water). The electronic affinity VEA_so evolves in the same direction as the polarity of the solvent. In order to evaluate the behavior of these bases in their triplet state, we determined the parameters VEA_T1 (vertical electronic affinity in the triplet state) and VIP_T1 (vertical ionization potential in the triplet state). VEA_T1 indicates the ability for the molecule to extract an electron in its immediate environment while VIP_T1 indicates the ability to yield an electron [21]. The results of these parameters are reported in Table 4.

VEA_T1 values indicate that the anion-forming reactions at the triplet state of DNA or RNA bases are thermodynamically favorable. In addition, the Thymine base is the most capable of receiving an electron in its immediate environment. Moreover, VEA_T1 values show that the anions of these bases are better stabilized in polar solvents [6]. When looking at the VIP_T1 values, Guanine is still the most easily ionizable base in each of the three media considered. These results agree with those obtained by the team of Liang Shen et al. [6].

As in the case of DNA bases, these same parameters were evaluated at the level of the photosensitizer complex. The results obtained are shown in Table 5 and Table 6.

Table 5 shows the VIP_so and VEA_so of δ-OsCl₂(Azpy)₂ in solvent and in vacuum. The lowest VIP_so values are found with polar solvents with values of 5.156 and 5.154 in water and DMSO respectively. Whereas the vertical electron affinity VEA_so, the best results are obtained in the aqueous phase with −3.907 hartree. These results also indicate that this complex reacts better in polar solvents.

Table 6 shows the VIP_T1 and VEA_T1 values of the δ-OsCl₂(Azpy)₂ complex in solvent and vacuum. The VEA_T1 values indicate that the reactions of triplet anion formation of the complex are thermodynamically favorable. In addition,

VEA_so = E_a− E_p, VIP_so = E_c− E_p.

VEA_T1 = VEA_so − E_T1, VIP_T1 = VIP_S0 − E_T1.

δ-OsCl₂(Azpy)₂ has the highest affinity in the aqueous phase. It is therefore more capable of stripping electrons from its environment in water. As far as VIP_T1 values are concerned, δ-OsCl₂(Azpy)₂ is still the most easily ionizable complex in water and in DMSO. The reactivity of this complex during an ionization according to the solvents considered is presented in the following order: DMSO > water > diethylether > benzene > vacuum.

From the abovementioned statement, we can say that the change in multiplicity increases the donor character of this molecule. This increase is certainly due to the rise in the number of unpaired electrons within the molecular edifice.

Here, azopyridine complex associates with DNA or RNA by the intercalation mode [22] [23] [24]. Considering this mode of association and the photosensitivity of the complex, irradiation of the latter must lead to several reactions that are bond to occur.

Firstly, this complex can react in its T1 triplet state with the bases of DNA or RNA by stripping them of an electron leading to their ionization state. In this case, the donor is the DNA or RNA base and the acceptor is the azopyridine complex. This reaction can be translated by Equation (3):

[ δ -OsCl 2 ( Azpy ) 2 ] ( T 1 ) + B → [ δ -OsCl 2 ( Azpy ) 2 ] · − + B + (3)

where B stands for DNA or RNA base.

This reaction is governed by the vertical electronic affinities in the VEA_T1 triplet state of the complex and the VIP vertical ionization potentials of the DNA bases. A negative sum of the two parameters indicates that this reaction is feasible [13]. Thus, we evaluated these parameters at the B3LYP/Lanl2DZ level in vacuum and in the presence of the solvents water, DMSO, diethylether and benzene. The results obtained are shown in Table 5 and Table 6. The various interactions following this reaction are recorded in Table 7.

Through Table 7, we can notice that the values are all positive. This indicates that no interaction following this reaction is thermodynamically favorable.

Second, the radical cation [δ-OsCl₂(Azpy)₂]^°+ of each photosensitizer complex can react with DNA or RNA bases by accepting an electron according to the Equation (4):

[ δ -OsCl 2 ( Azpy ) 2 ] · + + B → [ δ -OsCl 2 ( Azpy ) 2 ] ( S 0 ) + B · + (4)

This radical cation [ δ -OsCl 2 ( Azpy ) 2 ] · + of the photosensitizer complex can be generated by self-ionization reactions between the states T₁ and S₀ (Equation (5)) or the two T₁ states (Equation (6)).

[ δ - O s C l 2 ( A z p y ) 2 ] ( T 1 ) + [ δ - O s C l 2 ( A z p y ) 2 ] ( S 0 ) → [ δ - O s C l 2 ( A z p y ) 2 ] · + + [ δ - O s C l 2 ( A z p y ) 2 ] · − (5)

[ δ - O s C l 2 ( A z p y ) 2 ] ( T 1 ) + [ δ - O s C l 2 ( A z p y ) 2 ] ( T 1 ) → [ δ - O s C l 2 ( A z p y ) 2 ] · + + [ δ - O s C l 2 ( A z p y ) 2 ] · − (6)

Reaction 3 is governed by the sum VEA_T1 + VIP or VIP_T1 + VEA of the photosensitizer complex. A negative value of one of these sums indicates a thermodynamically favorable reaction. We have evaluated the different sums and the results are recorded in Table 8.

C-A: VEA_T1 (δ-OsCl₂(Azpy)₂) + VIP (Adenine); C-C: VEA_T1 (δ-OsCl₂(Azpy)₂) + VIP (Cytosine); C-G: VEA_T1 (δ-OsCl₂(Azpy)₂) + VIP (Guanine); C-T: VEA_T1 (δ-OsCl₂(Azpy)₂) + VIP (Thymine); C-U: VEA_T1 (δ-OsCl₂(Azpy)₂) + VIP (Uracil).

The values of the sums VEA_T1 + VIP and VIP_T1 + VEA are all positive, both in a vacuum and in a solvent. Therefore, reaction 3 is not thermodynamically favorable.

As for reaction 4, it is governed by the sum VEA_T1 + VIP_T1. We have determined the different values of this sum. The results are shown in Table 8. All the values are positive except for two values. They are obtained with the polar solvents water and DMSO. They reflect the interaction between two molecules in the T1 triplet state of the δ-OsCl₂(Azpy)₂ complex. These sums are −0.218 and −0.212 in water and DMSO respectively. We can assume that reaction 4 is therefore allowed in these solvents because of the negative values. These results indicate that only δ-OsCl₂(Azpy)₂ is likely to form the radical cation [ δ -OsCl 2 ( Azpy ) 2 ] · + . The formation of this cationic radical is also accompanied by the formation of an anionic radical [ δ -OsCl 2 ( Azpy ) 2 ] · − of this photosensitizer complex.

In photosensitization mechanism II, the photosensitizer must be in a triplet state. From this triplet T state, first the photosensitizer can react directly with oxygen by transferring its excess energy, which changes the oxygen ³O₂ to its singlet ¹O₂ state. This singlet oxygen ¹O₂ is a powerful oxidant that reacts with numerous cellular constituents such as saturated triacyl glycerols, membrane cholesterol, phospholipids, amino acids (histidine, tryptophan, methionine) and nucleic acids [25]. Due to its very short lifetime and high reactivity, ¹O₂ reacts at its site of formation in the cell [25]. The interaction of the photosensitizer in the triplet state with oxygen in the triplet basic state (³O₂) can be translated by the reaction translated by Equation (7).

[ δ - O s C l 2 ( A z p y ) 2 ] ( T 1 ) + O 3 2 → [ δ - O s C l 2 ( A z p y ) 2 ] ( S 0 ) + O 1 2 (7)

This reaction is highly dependent on the energy of the lowest triplet state of the photosensitizer. This is explained by the fact that a photosensitizer capable of producing singlet oxygen must have an energy gap between the fundamental singlet state and the lowest triplet state ∆E_S-T greater than the energy required to switch from the ³O₂ oxygen to its singlet ¹O₂ state [19]. This energy is experimentally estimated at 0.98 eV [18]. Theoretically, this energy is sometimes estimated at 0.91 eV [18] or 1.06 eV [26]. The singlet oxygen ¹O₂ is generated by a triplet-triplet energy transfer between the triplet ground state of the oxygen and the triplet excited state of the photosensitizer which is formed by inter-system conversion ISC [19]. This inter-system conversion can take place between two energy levels such as from S₁ to T_n or from T₁ to S₀ [19] [26] [27]. Therefore, we have evaluated the energies of the lowest triplet states of the azopydine complex in this work. The condition for the selection is the following: these energies of the triplet states T_n must be lower than those of their corresponding singlet states S₁. The energy gaps obtained after this selection are shown in the diagram in Figure 5.

The diagram shows a large energy gap between the singlet and triplet states. All singlet states have energies above 1.5 eV. As for the δ-OsCl₂(Azpy)₂ complex, it has three triplet states T₁, T₂ and T₃ whose energies are lower than that of the singlet state S₁. The highest values of the energies of the T₁ triplet states of δ-OsCl₂(Azpy)₂ complex are 0.747 eV in diethylether and 0.740 in benzene, respectively. As for the other T₂ and T₃ states, the highest energies are obtained in polar solvents. In water, the highest value is 1.699 eV and in DMSO it is 1.703 eV. So, we can conclude that δ-OsCl₂(Azpy)₂ is able to produce singlet oxygen in these two polar solvents by energy transfer. The photocytotoxic activity will depend on the combination of the quantum yields of formation of the singlet state S₁, the conversion of S₁ to T_n, the efficiency of energy transfer from T_n to oxygen and the abundant presence of O₂.

Secondly, the complex in the T₁ state can react with ³O₂ by electron transfer to generate the anionic superoxide radical ( O 2 · − ). This reaction can be translated by Equation (8).

[ δ - O s C l 2 ( A z p y ) 2 ] ( T 1 ) + O 3 2 → [ δ - O s C l 2 ( A z p y ) 2 ] · + + O 2 · − (8)

The condition for the realization of this reaction is that the sum of the ionization potential in the VIP_T1 triplet state of the complex and the adiabatic electron affinity of oxygen ³O₂ must be negative. Furthermore, the adiabatic electron affinity of ³O₂ oxygen is −0.59 eV in vacuum, −3.91 eV in water; −3.65 eV in DMSO; −3.14 eV in ether and −2.33 eV in benzene [6] [13] [28]. The results of the evaluation of this sum are shown in Table 9.

Table 9 shows that Equation (6) which translates the electron transfer between the photosensitizer and oxygen in the triplet state is not possible with the solvents considered. However, there is another possibility of production of the anionic superoxide radical O 2 · − . This is the electron transfer reaction between the anionic radical resulting from auto-ionization and oxygen in the triplet state. This reaction can be represented by Equation (9):

[ δ - O s C l 2 ( A z p y ) 2 ] · + + O 3 2 → [ δ - O s C l 2 ( A z p y ) 2 ] ( S 0 ) + O 2 · − (9)

This reaction is possible if the difference between the adiabatic electron affinity of oxygen ³O₂ and the vertical singlet state electron affinity of the photosensitizer is negative. Table 9 indicates one possible reaction. This is the reaction between the δ-OsCl₂(Azpy)₂ and oxygen in water. Hence, δ-OsCl₂(Azpy)₂ will be able to generate the anionic superoxide radical O 2 · − in water. The superoxide radical anion O 2 · − thus formed can react directly with different substrates or act as a precursor of other ROS following the Fenton reaction [13] [29] or by the Haber-Weiss reaction [13] [30] which will effectively amplify the photosensitizing activity of these azopyridine complexes. The different ROS formed during these last photochemical reactions (H₂O₂, O 2 · − , OH^-) represent powerful oxidants for a wide variety of biomolecules such as cholesterol or the side chains of certain amino acids (tryptophan, histidine and methionine) [31].