Figure 1. Chemical structure of antirheumatic and antitumor drug methotrexate (MTX, top) and the macrocyclic host molecule tetrakis(androst-4-en-3-one-17α-ethinyl)-cavitand (TAC, bottom) (8β-H, 9α-H and 13α-H are omitted for clarity).

Figure 2. Absorption spectra of MTX (10 µM) and TAC (10 µM) (A) and fluorescence emission spectra of MTX (10 µM) in the absence and presence of TAC (0–30 µM) at 393.15 K (B) PL and a.u. reflect photoluminescence and arbitrary units, respectively.

Figure 3. Fluorescence emission spectra of MTX (10 µM) in the absence and presence of TAC (0–80 µM) at 393.15 K (A) and at 313.15 K (B) (λ_excitation = 390 nm). Arrows indicate increasing TAC concentrations (CPS reflects counts per s, colors only support the clarity).

The stability constants of this interaction can be calculated using the Benesi–Hildebrand method. To determine the thermodynamic parameters, the stability constants were calculated at temperatures 293.15 K, 296.48 K, 299.82 K, 303.15 K, 306.48 K, 309.82 K and 313.15 K. Considering that the DMSO solvent shows considerable Raman peaks overlapping the emission of MTX, two methods were applied to confirm the complex stabilities: either the HyperQuad code was applied using 10 selected wavelengths around the emission maxima (470–480 nm, see Figure 2.) or applying the Benesi–Hildebrand method, the right leg of the emission spectra is used, where the Raman scattering is negligible. In this case intensities obtained at 540 nm emission wavelength were applied to evaluate the data. Table 1. summarizes these results. These stability constants were then used to calculate the thermodynamic parameters of the interaction (Table 2.).

Table 1. Stability constants determined at different temperatures (BH reflects the Benesi-Hildebrand method).

Table 2. Thermodynamic parameters of the complex formation between TAC and MTX (ΔH and ΔS are the enthalpy change and entropy change, respectively).

3. Formation Thermodynamics

Two different temperature regions can be observed on the van ’t Hoff plot (the logarithms of the stability constants were plotted against the reciprocal temperatures, Figure 4.) of the interaction where the thermodynamic parameters of the complex formation differ significantly.

Figure 4. van ’t Hoff plot of the complex formation of MTX and TAC.

These results imply different complex formation mechanisms in the different temperature regions. At lower temperatures, entropy gain is associated with enthalpy gain, but at higher temperatures, entropy loss is associated with enthalpy loss. Based on these observations, it is suggested that in the lower temperature region MTX interacts with the ethisterone moieties of TAC, i.e., with the chiral inlet of the host molecule. In this way, the entropy gain is caused by the removal of solvent molecules, but this process costs energy, this is the reason of the moderated positive enthalpy change. However, in the higher temperature region MTX interacts with the rigid inner cavity of TAC, i.e., with the lower lying aromatic ’basket’, which causes entropy loss associated with the decreased freedom of the cavitand skeleton during complex formation. The negative enthalpy change reflects attractive interactions between the MTX molecules and the ethisterone arms of the cavitand.

4. Modeling Studies

The total energies of the species interacted were calculated first at semi-empirical AM1 level using Hyper Chem 8 code. The host cavitand molecule shows two stable conformers according to the orientation of the large ethisterone moieties on the upper rim (see Figure 5.): the opened structure associated with more flexible skeleton compared to the closed structure where the ethisterone moieties possess reduced freedom due to the steric hindering of their motion. The differences between the calculated total Gibbs free energies of the parent ’opened’ or ’closed’ molecules in gas phase (partition of solvent motions is excluded) was found to be 21.7 kJ/mole at 298.16 K, which property support presence of the opened structure exclusively at around room temperature.

Figure 5. Side and top views of the equilibrium conformations of methotrexate–cavitand complexes associated with the opened (A1 and A2) and closed (B1 and B2) conformations of the cavitand (blue, red, white and black colors mean carbon, oxygen, hydrogen atoms and MTX molecule, respectively).

The thermodynamic parameters associated with the complex formation of the TAC with MTX were then calculated as described in the Materials and methods section. Results show positive enthalpy and entropy change during formation of the closed form complexes, while both the enthalpy and entropy term is negative when the complex show opened structure. Possible reason for these results is that the freedom of the flexible opened cavitand decreased considerably when the closed form complexes are stabilized. In contrast, the entropy gain can be obtained when the flexibility of the cavitand skeleton remains unchanged during formation of the opened complex structure. The good agreement between the experimental results derived in solution phase and that of the theoretical value calculated in gas phase reflects moderated entropy gain associated with the increased freedom of solvent molecules after leaving the solvation shell of the methotrexate guest.

5. Conclusions

Photoluminescence and molecular modeling studies of inclusion complexes formed by the antimetabolite drug, methotrexate and by the macrocyclic host molecule tetrakis(androst-4-en-3-one-17α-ethinyl)-cavitand highlight two temperature regions where the complexes possess different conformations: opened structure of the host cavitand with free androst-4-en-3-one-17α-ethinyl arms forms enthalpy stabilized structure while the stability of complexes formed by the closed form cavitand is stabilized by the entropy gain. This information can be applicable to design sensitive and selective molecular sensors for methotrexate drug.