## 4 Conclusions  
In this paper, a carefully theoretical exploration of the reaction mechanism for the photoconversion of DHSAN into SAN is reported. To this aim, the DHSAN photophysical properties, in terms of electronic transitions, singlet-triplet energy gaps and spin-orbit coupling constants were computed by means of DFT and TD-DFT, in order to ascertain its ability to generate the excited state of molecular oxygen, essential for in situ SAN formation. The plausible formation of both SAN iminium and alkanolamine forms present at physiological pH were examined. The characterized free energy surface proved the exergonicity of the drug generation in both forms, which is realized overcoming very low activation energy barriers. From here, two different modes for inducing cells apoptosis by SAN were taken into consideration: the intercalation into DNA helix and the capability to generate the cytotoxic species \({}^{1}\)O\({}_{2}\) through photoirradiation. The outcomes of the computational analysis presented here show that the intercalation process, explored by means of MD simulations, provokes conformational changes into DNA structure that ultimately might compromise its main functions, leading to cell death. From the other hand, it was seen that an improvement in the photophysical properties of SAN upon intercalation occurs. Since the wavelength of the radiation activating DHSAN falls outside the ideal therapeutic window, the possibility of TPE was taken into consideration. On the basis of the TPA calculated parameters it is possible to hypothesize that the efficacy of DHSAN can be increased by TPE in the range 550-850 nm. However, additional biological studies should be carried out to ascertain whether DHSAN could be a two-photon activatable drug by irradiation with long wavelength. This option is of particular interest aiming at reaching deep tumour tissues and could open the way to a new SAN-based therapeutic strategy. The following are available online at [[https://www.mdpi.com/article/10.3390/app12031095/s1](https://www.mdpi.com/article/10.3390/app12031095/s1)]([https://www.mdpi.com/article/10.3390/app12031095/s1](https://www.mdpi.com/article/10.3390/app12031095/s1)), Developed parameters for SAN. Figure S1: Natural Frontier Orbitals (NTOs), Highest Occupied Transition Orbital (HOTO), and Lowest Unoccupied Transition Orbitals (LUTO) for the most important DHSAN electronic transition computed for TPE. Figure S2: B3 LYP/6-311G** optimized structures of stationary points along the oxidative dehydrogenation reaction of DHSAN in water. Figure S3: DNA length change due to the intercalation of the SAN measured from the pre-terminal (one before the last) base pairs center of mass, taking the drug-free DNA as a reference. Figure S4: RMSD plots of DNA, four residues around the intercalated SAN obtained by DNA-SAN dynamics. Figure S5: RMSD plots of the four residues around the singuainarine taking the same residues in a drug-free DNA dynamics as a reference. Figure S6: Optimized geometries of the representative structures extracted from the most populated cluster selected from MD simulations that include SAN and the eight surrounding nucleotides for all the investigated intercalated positions. Figure S7: Contour plots of the key molecular orbitals for intercalated adducts extracted from MD simulations. Figure S8: Contour plots of the key molecular orbitals of SAN. Table S1: TD-DFT Benchmark computed at B3 LYP-D3/6-311G** level in water for SAN and in THF for DHSAN. Table S2: B3 LYP/6-311G** Vertical excitation energies, \(\Delta\)E (eV), \(\lambda_{\text{max}}\) (nm), oscillator strengths, \(f\), and main transitions computed in water for SAN and in THF for DHSAN. Table S3: Triplet States energies and MO contribution computed in water for SAN and THF for DHSAN. Table S4: MMPBSA terms for the binding of SAN into DNA different positions (kcal mol\({}^{-1}\)). Table S5: Calculated binding energy (kcal mol\({}^{-1}\)) values for each 25 ns of molecular dynamics simulation and for the total time trajectory 0-100 ns.