## 3 Results and Discussion
### Computational Calculations
#### 3.4.3 The Complex of [Cu\({}^{\rm II}\)(\(\beta\)-CD)(Ezm)\({}_{2}\)]\({}^{2+}\)  
Due to their stereo-isomeric difference, \(RSR\)-EZM is likely to bind at the entrance of the \(\beta\)-CD while \(SR\)-EZM tends to penetrate the cavity and coordinate with Cu. However, when the second EZM molecule enters the system, it remains challenging to clarify what the most stable configurations are and how they interact with each other. Based on the result of the MS/MS fragments, we can know that the second EZM molecule more easily broken up than the first one, suggesting that the interaction force between EZM and [Cu\({}^{\text{II}}\)(\(\beta\)-CD)(EZM)]\({}^{2+}\) is weaker compared to coordination. Normally, the H-bond is weaker compared with coordination bond. Therefore, the second EZM molecule has a strong possibility to bind with the first EZM molecule or the \(\beta\)-CD instead of Cu. The optimized structures of [Cu\({}^{\text{II}}\)(\(\beta\)-CD)(EZM)\({}_{2}\)]\({}^{2+}\) are shown in Figure 7. Unsurprisingly, H-bond becomes the main binding form for the \(RSR\)-EZM(Figure 7A). Both of the two drug molecules connected the \(\beta\)-CD with H-bond, whose lengths are 1.89 A and 1.86 A, respectively. To get a more stable configuration, there is another H-bond forming to link the two \(RSR\)-EZMs with a distance of 1.75 A. Copper ion coordinated with the oxygens on the \(\beta\)-CD, the bond lengths of which are 1.92 A, 1.92 A and 2.72 A, respectively, similar to the [Cu\({}^{\text{II}}\)(\(\beta\)-CD)(EZM)]\({}^{2+}\)(Figure 6A). However, compared to the complex containing only one drug molecule, the entrance size and shape has been enlarged. It is shown in Table 2 that the corresponding distances changed from 13.02 A, 12.27 A, 14.18 A, 5.38 A to 13.32 A, 12.9 A, 14.23 A,  
Interestingly, _SRS_-EZM can stabilize in two forms (Figure 7B,C). The optimized structures illustrate that one of the drug molecules would coordinate with the Cu(II) ion and the other would connect the \(\beta\)-CD with the H-bond. In Figure 7B, the Cu(II) ion form a coordinate covalent bond with one oxygen atom on the _SRS_-EZM with a bond length of 2.03 A. The remaining coordinate bonds involved the oxygens on the \(\beta\)-CD with a longer bond length of 2.15 and 2.16 A. Except that, the two _SRS_-EZMs both interacted with the \(\beta\)-CD through four H-bonds. The _SRS_-EZM not only coordinated with copper ion, but also formed two H-bonds with the length of 1.74 A and 1.76 A, respectively. The other drug molecule binds at the other side with 1.76 A and 1.77 A H-bonds. The two drugs have no interactions between each other. In general, the _SRS_-EZM ternary complex owns a lower binding energy than _RSR_-EZM (\(\sim\)14.46 kcal/mol lower), indicating that the former is more stable, which is consistent with the result of the MS spectra. The MD simulation was also performed to investigate the relative binding strength of the two EZM with the [Cu\({}^{\Pi}\)(\(\beta\)-CD)\({}^{2+}\) complex. The most stable structures of [Cu\({}^{\Pi}\)(\(\beta\)-CD)(EZM)\({}_{2}\)\({}^{2+}\) derived from the DFT results were taken as the initial model. The snapshots taken at different simulation time from the trajectory are shown in Figure 8. For both RSR and SRS complex, the drug molecules can stay close to the \(\beta\)-CD when the temperature gradually increased up to 500 K within 500 ps, indicating a reasonable initial structure suggested by the DFT calculation. Afterwards, the RSR complex started to break up at 570 ps and one of the drug molecules moved away from the \(\beta\)-CD, while the other still stayed inside. In contrast, the SRS complex shows obvious stability within the 1000 ps run, which is consistent with the previous results, revealing that the [Cu\({}^{\Pi}\)(\(\beta\)-CD)(_SRS_-EZM)\({}^{2}\)\({}^{2+}\) is more stable than [Cu\({}^{\Pi}\)(\(\beta\)-CD)(_RSR_-EZM)\({}^{2}\)\({}^{2+}\). MD simulations can provide auxiliary information for clarifying the enantiomers separation mechanism.