## 3 Results and Discussion
### Structures and Dynamics of SAN Intercalation into DNA  
As underscored above, the various pharmacological effects exhibited by SAN including its anticancer activity, can involve SAN strong capability to bind DNA, as one of the most important mechanisms. A large number of studies have been dedicated to this aspect [53], and we are going to illustrate the outcomes of MD simulations we have carried out with the aim of obtaining more detailed information about the intercalation process. Intercalative binding is one of three different modes, together with electrostatic and groove binding, by which molecules can be non-covalently attached to DNA. The stacking effect as well as hydrogen bonding with surrounding nucleotides are, generally, prominent in determining the intercalation strength that, in turn, can induce DNA conformational and functional changes ultimately leading to cell apoptosis. For cationic intercalators, it is reported that the stabilization energy is considerably larger than for uncharged species [59]. Due to its planar structure and aromaticity, intercalation of SAN, in its iminium cationic  
Figure 2: B3 LYP-D3/6-311G(d,p) free energy profile for oxidative dehydrogenation reaction of DHSAN. form, is very likely. Therefore, MD simulations were performed manually putting the molecule into the adopted B-DNA dodecamer model considering different positions across the major groove of the helix between two base pairs in analogy with our previous studies [60; 61; 62]. The examined DNA sites are shown in Figure 3A. Due to the asymmetric structure of SAN, two different orientations of the molecule in positions labeled **2** and **4** were examined (Figure 3B). The labels used for the studied sites of intercalation are: **1** where the drug is stacked within the cytosine-guanine (CG) and guanine-cytosine (GC) base pairs, **2** corresponds to the drug stacked within adenine-thymine (AT) and adenine-thymine (AT) base pairs, **3** for adenine-thymine (AT) and thymine-adenine (TA), and **4** for thymine-adenine (TA) and cytosine-guanine (CG) base pairs stacking. The two different orientations of SAN were included adding the labels a and b to both **2** and **4** positions. Trajectories simulating all the arrangements were extended up to 100 ns and more detailed information concerning the outcomes of the MD simulations can be found in Figures S3-S5 of the Supplementary Materials. The plot of the DNA length change, calculated as the distance between the center of mass of the two pre-terminal base pairs of the helix and taking drug-free DNA as a reference, shows that intercalation causes a lengthening of about 3 A for each position. This elongation is mainly due to the increased spacing between the base pairs at the intercalation sites. Plots of the Root Mean Square Deviation (RMSD) for every intercalation site give an indication of the conformational distortion caused by intercalation with respect to the unperturbed DNA structure. Oscillations are more evident when the whole structure is taken into consideration caused by the flexibility of the terminal base pairs and consequent temporary breaking of the established H-bonds. Binding due to the intercalation appears to be stable over the whole simulation time. The binding free energies, BEs, calculated by means of the MM-PBSA approach over the full  
Figure 3: (**A**) Schematic representation of SAN intercalated at the four positions, labelled **1–4**, of the B-DNA dodecamer; (**B**) its possible orientations within **2** and **4** positions; (**C**) MMPBSA calculated binding energies (kilocalories per mole) for all the considered intercalated positions. trajectories, are listed in panel C of Figure 3. These values are not very different amongst them and span in a range from 20.4 to 21.6 kcal mol\({}^{-1}\) with an uncertainty comprised between 2.1 and 2.4 (Table S4).