**1. Introduction**

Formation of host-guest type inclusion complexes typically occurs when the host molecule uses its cavity to encapsulate a guest through noncovalent interactions. According to the significant practical utility of macrocyclic molecules, such as calixarenes [1,2], cavitands [3,4], and cyclodextrins (CDs) [5,6] in host-guest complex formations, chemists, biologists, and material scientists got interested the physical properties, chemical nature, and related biological activity of these molecules. However, utilization of these noncovalent interactions (hydrogen-bonding, π-stacking, electrostatic interaction, van der Waals force, and hydrophobic/hydrophilic attraction) are still a great challenge [7–10]. CDs, a fascinating class of macrocycles, are composed of six, seven, or eight glucose units, called α-, β-, and γ-CDs, respectively. CDs are used as host components for the construction of various interesting supramolecular structures [11,12]. Sulfonamide antibiotics are widely used in both human medicine and livestock production to treat some bacterial infections of the urinary tract, ears, lungs, skin, and soft

tissues [13,14]. Furthermore, sulfonamides can appear as contaminants in various foods, which may cause adverse health effects [15–17]. The host-guest type complex formation of these antibiotics with CDs is an extensively studied field [18–24]. Zoppi et al. focus on the increased water solubility of sulfonamide drugs in the presence of native and methylated β-CD [23,24]. In the case of sulfamethazine (SMT), their nuclear magnetic resonance (NMR) and molecular modeling results demonstrate that SMT included the substituted pyrimidine ring into the β-CD cavity. Contradictory, NMR and quantum chemical results of Bani-Yaseen and Mo'ala revealed that complex formation is favorable with inclusion of the aniline moiety through the β-CD cavity [18]. various foods, which may cause adverse health effects [15–17]. The host-guest type complex formation of these antibiotics with CDs is an extensively studied field [18–24]. Zoppi et al. focus on the increased water solubility of sulfonamide drugs in the presence of native and methylated β-CD [23,24]. In the case of sulfamethazine (SMT), their nuclear magnetic resonance (NMR) and molecular modeling results demonstrate that SMT included the substituted pyrimidine ring into the β-CD cavity. Contradictory, NMR and quantum chemical results of Bani-Yaseen and Mo'ala revealed that complex formation is favorable with inclusion of the aniline moiety through the β-CD cavity [18]. Several studies have been performed to get an insight into the factors which affect the

ears, lungs, skin, and soft tissues [13,14]. Furthermore, sulfonamides can appear as contaminants in

Several studies have been performed to get an insight into the factors which affect the thermodynamic and kinetic stability or selectivity of host-guest complexes [25,26], because the deeper understanding of these interactions has high importance. The pH-responsive host-guest encapsulation is also a highly studied field in material sciences [27] and in pharmacology [28,29]. Therefore, besides the complex stability and stoichiometry of SMT – β-CD complex and along the contradictory description of the related structures [18,23,24], the investigation of the pH dependence interaction of SMT with CDs is also reasonable. thermodynamic and kinetic stability or selectivity of host-guest complexes [25,26], because the deeper understanding of these interactions has high importance. The pH-responsive host-guest encapsulation is also a highly studied field in material sciences [27] and in pharmacology [28,29]. Therefore, besides the complex stability and stoichiometry of SMT – β-CD complex and along the contradictory description of the related structures [18,23,24], the investigation of the pH dependence interaction of SMT with CDs is also reasonable. In our recent study [30], we demonstrated the importance of pH-dependent dipole moment of

In our recent study [30], we demonstrated the importance of pH-dependent dipole moment of SMT molecule, which phenomenon can affect the complex geometry formed with β-CD (BCD) and randomly methylated β-CD (RAMEB) (Figure 1). Now we focus on the thermodynamic properties of the formation of inclusion complexes at different pH values. Our aim is to analyze the weak interactions between the pH dependent ionic and neutral forms of SMT and native or methylated CDs at molecular level to clarify the previous contradictory results. In this way, the involvement of weak molecular interactions (electrostatic forces and hydrogen bonds) have been tested by the temperature-dependent measurements and molecular modeling studies. SMT molecule, which phenomenon can affect the complex geometry formed with β-CD (BCD) and randomly methylated β-CD (RAMEB) (Figure 1). Now we focus on the thermodynamic properties of the formation of inclusion complexes at different pH values. Our aim is to analyze the weak interactions between the pH dependent ionic and neutral forms of SMT and native or methylated CDs at molecular level to clarify the previous contradictory results. In this way, the involvement of weak molecular interactions (electrostatic forces and hydrogen bonds) have been tested by the temperature-dependent measurements and molecular modeling studies.

**Figure 1.** Chemical structures of sulfamethazine (SMT), native β-cyclodextrin (BCD), and randomly **Figure 1.** Chemical structures of sulfamethazine (SMT), native β-cyclodextrin (BCD), and randomly methylated β-cyclodextrin (RAMEB).

#### methylated β-cyclodextrin (RAMEB). **2. Results and Discussion**

determined to analyze further the related processes.

#### **2. Results and Discussion**  *2.1. Temperature Dependence of the Association Constants of Sulfamethazine-CD Complexes at Di*ff*erent pH*

*2.1. Temperature Dependence of the Association Constants of Sulfamethazine-CD Complexes at Different pH*  Figure 2 shows the van't Hoff plot of SMT-CD complexes, based on association constants determined at different temperatures. In accordance with our earlier findings [30], significant difference between the association constants at elevation pH (pH = 5 and pH = 7) and at strong acidic environment (pH = 2) has been found. The slight dependence of complex stabilities on the temperature reflects low enthalpy changes, i.e., weak interactions between the molecules. At pH 7, where the nonionic and anionic guest molecules are dominant, higher stability is associated to the complexes at decreased temperatures. In contrast, in the presence of considerable amount of cationic guest at pH 2, the complex stability increases with the elevation of the temperature. Although, only one form of the guest molecule (nonionic SMT) is available at pH 5, the substitution of the β-CD affects the change of the association constants with the temperature. The association constants of CD complexes generally decrease with the elevation of the temperature [19,31]. However, one of our Figure 2 shows the van't Hoff plot of SMT-CD complexes, based on association constants determined at different temperatures. In accordance with our earlier findings [30], significant difference between the association constants at elevation pH (pH = 5 and pH = 7) and at strong acidic environment (pH = 2) has been found. The slight dependence of complex stabilities on the temperature reflects low enthalpy changes, i.e., weak interactions between the molecules. At pH 7, where the nonionic and anionic guest molecules are dominant, higher stability is associated to the complexes at decreased temperatures. In contrast, in the presence of considerable amount of cationic guest at pH 2, the complex stability increases with the elevation of the temperature. Although, only one form of the guest molecule (nonionic SMT) is available at pH 5, the substitution of the β-CD affects the change of the association constants with the temperature. The association constants of CD complexes generally decrease with the elevation of the temperature [19,31]. However, one of our earlier work showed an opposite example [32]. The thermodynamic parameters have been also determined to analyze further the related processes.

earlier work showed an opposite example [32]. The thermodynamic parameters have been also

**Figure 2.** The van't Hoff plots of SMT-BCD and SMT-RAMEB complex formations at different pH **Figure 2.** The van't Hoff plots of SMT-BCD and SMT-RAMEB complex formations at different pH values.

values. Thermodynamic parameters (Table 1) were calculated from the slopes and the intercepts of the lines fitted to the experimental data based on the van't Hoff plot (Equation (1), see Figure 2). The negative ΔG values yield spontaneous complex formation between SMT and CDs. Results showed exothermic association at high pH (pH = 7), while an endothermic molecular association was obtained at low pH (pH = 2). At pH 5, the endothermic character of the complex formation was just changed to exothermic as a result of the methyl substitution of BCD. In each interaction, an entropy gain was observed; however, the entropy increase during the complex formation correlates with the enthalpy change. The entropy increase during the association reaction was probably due to the process when SMT enters the CD cavity (it releases its solvation shell). Furthermore, higher entropy gain associated with positive or less negative enthalpy, which property reflects to the removal of more or less water molecules from the solvation shell regarding the molecules interacted during formation of complexes. Decreased ΔS at higher pH values suggest the release of less water molecules from the solvation shell of SMT molecules, because the stabilization is also supported by the attractive coulomb forces between the negatively charged SMT and the dipole moments of the solvent molecules. The correlation between the enthalpy and the entropy changes can be described by the changes of the solvation shell of guests, since the removal of less water molecules from the solvation shell costs less energy. This description agrees with the enthalpy-entropy compensation and Thermodynamic parameters (Table 1) were calculated from the slopes and the intercepts of the lines fitted to the experimental data based on the van't Hoff plot (Equation (1), see Figure 2). The negative ∆G values yield spontaneous complex formation between SMT and CDs. Results showed exothermic association at high pH (pH = 7), while an endothermic molecular association was obtained at low pH (pH = 2). At pH 5, the endothermic character of the complex formation was just changed to exothermic as a result of the methyl substitution of BCD. In each interaction, an entropy gain was observed; however, the entropy increase during the complex formation correlates with the enthalpy change. The entropy increase during the association reaction was probably due to the process when SMT enters the CD cavity (it releases its solvation shell). Furthermore, higher entropy gain associated with positive or less negative enthalpy, which property reflects to the removal of more or less water molecules from the solvation shell regarding the molecules interacted during formation of complexes. Decreased ∆S at higher pH values suggest the release of less water molecules from the solvation shell of SMT molecules, because the stabilization is also supported by the attractive coulomb forces between the negatively charged SMT and the dipole moments of the solvent molecules. The correlation between the enthalpy and the entropy changes can be described by the changes of the solvation shell of guests, since the removal of less water molecules from the solvation shell costs less energy. This description agrees with the enthalpy-entropy compensation and highlights that the exothermicity of molecular association usually restricts the movement of the constituents, thereby causing growing entropy loss.

highlights that the exothermicity of molecular association usually restricts the movement of the constituents, thereby causing growing entropy loss. **Table 1.** Thermodynamic parameters associated to the formation of SMT-CD complexes. Data are determinate based on temperature-dependent fluorescence spectroscopic measurements. (∆H [kJ mol−<sup>1</sup> ], ∆S [J K−<sup>1</sup> mol−<sup>1</sup> ] ∆G298K [kJ mol−<sup>1</sup> ]).


#### BCD 15.4 ± 0.8 90.0 ± 2.5 −11.4 ± 1.5 2.2 ± 0.5 63.3 ± 1.7 −16.7 ± 1.0 −2.2 ± 0.5 51.0 ± 1.7 −17.3 ± 1.0 *2.2. Modeling Studies*

RAMEB 18.9 ± 0.8 102.7 ± 2.6 −11.7 ± 1.6 −6.4 ± 1.0 37.2 ± 3.3 −17.5 ± 2.0 −8.5 ± 1.2 28.8 ± 3.8 −17.1 ± 2.3 *2.2. Modeling Studies*  To get a deeper insight into the complex formation processes, molecular modeling studies were performed at semi-empirical level. During these calculations, the energetically favorable To get a deeper insight into the complex formation processes, molecular modeling studies were performed at semi-empirical level. During these calculations, the energetically favorable deprotonation route of SMT molecule was determined first in aqueous solutions considering the presence of other ions as described in the Materials and Methods section. Sulfamethazine exists as cationic (SMT+), anionic (SMT−), nonionic (SMT<sup>0</sup> ) and zwitterionic (SMT+/−) forms in aqueous solutions. Figure 3 shows that the aromatic amine moiety, which is protonated at low pH loses first the proton while the second

deprotonation route of SMT molecule was determined first in aqueous solutions considering the

deprotonation occurs at the sulfonamide nitrogen. The associated experimental pKa<sup>1</sup> and pKa<sup>2</sup> values at room temperature were 2.07 and 7.49, respectively. We should mention here that the Gibbs free energy difference between the nonionic and zwitterionic forms of SMT was found to be 12.3 kJ mol−<sup>1</sup> in this environment. This result suggests presence of SMT in nonionic rather than zwitterionic form in the solutions, however, it is known that zwitterionic form can stabilized e.g., in adsorbed state [33]. Then the interactions of these three forms of SMT (cationic, nonionic and anionic) were examined with BCD and RAMEB host molecules in the aqueous buffer. Due to the huge computation time of the large systems, in the case of RAMEB the electron releasing property of the methyl groups was considered as negatively charged specie of the native BCD molecules. Thus, the repulsive Coulomb interaction between the negatively charged RAMEB cavity (simulated by −1 BCD) and the deprotonated SMT species will reduce the secondary interactions between the host and guest molecules. In contrast, the charged SMT species showed stronger interactions with the negatively charged cavity of RAMEB. Furthermore, the host molecules formed even more stable complexes with the anionic form of the guest. From the point of view the enthalpy (Table 2), the following process is responsible for these unexpected results: at low pH the cationic SMT molecule enters into the host cavity with its aromatic amine moiety. However, at higher pH, SMT molecule enters with its methyl substituents. In the former cases, hydrogen bridges between the (guest amine) N-H ··· O (host hydroxyl), while in the latter cases, the hydrogen bridges between the (guest methyl) C-H ··· O (host hydroxyl) are moderate the weak interactions between the host and guest (Figure 4). Noted here, that this pH dependent orientation of guest molecule in the complexes support the earlier described structures based on the inclusion of the aniline moiety [18] as well as the pyrimidine ring [24] through the CD cavity. cationic (SMT+), anionic (SMT−), nonionic (SMT0) and zwitterionic (SMT+/−) forms in aqueous solutions. Figure 3 shows that the aromatic amine moiety, which is protonated at low pH loses first the proton while the second deprotonation occurs at the sulfonamide nitrogen. The associated experimental pKa1 and pKa2 values at room temperature were 2.07 and 7.49, respectively. We should mention here that the Gibbs free energy difference between the nonionic and zwitterionic forms of SMT was found to be 12.3 kJ mol−1 in this environment. This result suggests presence of SMT in nonionic rather than zwitterionic form in the solutions, however, it is known that zwitterionic form can stabilized e.g., in adsorbed state [33]. Then the interactions of these three forms of SMT (cationic, nonionic and anionic) were examined with BCD and RAMEB host molecules in the aqueous buffer. Due to the huge computation time of the large systems, in the case of RAMEB the electron releasing property of the methyl groups was considered as negatively charged specie of the native BCD molecules. Thus, the repulsive Coulomb interaction between the negatively charged RAMEB cavity (simulated by −1 BCD) and the deprotonated SMT species will reduce the secondary interactions between the host and guest molecules. In contrast, the charged SMT species showed stronger interactions with the negatively charged cavity of RAMEB. Furthermore, the host molecules formed even more stable complexes with the anionic form of the guest. From the point of view the enthalpy (Table 2), the following process is responsible for these unexpected results: at low pH the cationic SMT molecule enters into the host cavity with its aromatic amine moiety. However, at higher pH, SMT molecule enters with its methyl substituents. In the former cases, hydrogen bridges between the (guest amine) N-H ··· O (host hydroxyl), while in the latter cases, the hydrogen bridges between the (guest methyl) C-H ··· O (host hydroxyl) are moderate the weak interactions between the host and guest (Figure 4). Noted here, that this pH dependent orientation of guest molecule in the complexes support the earlier described structures based on the inclusion of the aniline moiety [18] as well as the pyrimidine ring [24] through the CD cavity.

presence of other ions as described in the Materials and Methods section. Sulfamethazine exists as

**Figure 3.** The energetically most favorable deprotonation routes of SMT (cationic: left, nonionic: middle, anionic: right, zwitterionic: bottom) determined by MINDO/3 approximation using the TIP3P solvation model for the buffer [34]. Gibbs free energy between the nonionic and zwitterionic forms suggest presence preferably of nonionic form in the solution. **Figure 3.** The energetically most favorable deprotonation routes of SMT (cationic: left, nonionic: middle, anionic: right, zwitterionic: bottom) determined by MINDO/3 approximation using the TIP3P solvation model for the buffer [34]. Gibbs free energy between the nonionic and zwitterionic forms suggest presence preferably of nonionic form in the solution.

Furthermore, the inclusion of SMT by its aromatic amine moiety in case of RAMEB host enhances formation of zwitterionic form of SMT in the cavity. This is due to the tautomerization of **Table 2.** Thermodynamic parameters associated to the formation of SMT-CD complexes. Semiempirical MINDO/3 method with TIP3P solvation model is applied. (∆H [kJ mol−<sup>1</sup> ], ∆S [J K−<sup>1</sup> mol−<sup>1</sup> ]).


of the BCD host.

of the SMT's zwitterion formation in the RMAEB's cavity, simultaneous analysis of complexation behavior has been done using infrared (IR) spectroscopy. In general, our results are in agreement with the IR analyses of SMT-BCD complexes prepared by a freeze-drying method [23], the characteristic bands of SMT shifted and are more or less intense in the presence of CD. Moreover, IR spectra of the SMT-RAMEB complexes and the species interacted support our idea described above (Figure 5): significant changes of two characteristic vibrations of SMT molecules were observed upon complexation by the RAMEB host as follows. Quantum chemical analysis revealed that belting vibration of SNH bond angle at sulfonamide moiety (1103 cm−1) disappeared while the bond stretching associated to the aromatic NH3 is appeared at 2812 cm−1 in the experimental IR spectra of the complexes. These changes in the experimental IR spectra indicate the stabilization of the

**Figure 4.** Equilibrium conformation of SMT-BCD complexes. (**a**) SMT molecules with their aromatic **Figure 4.** Equilibrium conformation of SMT-BCD complexes. (**a**) SMT molecules with their aromatic amine moiety and (**b**) with their methyl groups enter into the cavities of hosts.

amine moiety and (**b**) with their methyl groups enter into the cavities of hosts. **Table 2.** Thermodynamic parameters associated to the formation of SMT-CD complexes. Semiempirical MINDO/3 method with TIP3P solvation model is applied. (ΔH [kJ mol<sup>−</sup>1], ΔS [J K<sup>−</sup><sup>1</sup> mol<sup>−</sup>1]). **Host Specie Host Simulated as Guest's Charges +1 (Cationic) 0 (Nonionic) 0 (Zwitterionic) −1 (Anionic)**  ΔH ΔS ΔH ΔS ΔH ΔS ΔH ΔS BCD 0 BCD 16.3 93.0 9.3 78.2 5.4 68.4 −3.7 47.5 RAMEB −1 BCD 19.1 105.4 14.3 99.7 −8.7 35.2 −9.4 26.4 In all eight situations, the interactions show an increased entropy term (Table 2). This property is associated with two facts: the solvent water molecules leave the host's cavity prior to the complex formation and the guest molecules (at least partly) lose their hydration shell. Both processes increase the entropy. In particular, the entropy gain decreases by the second deprotonation step. This is probably due to the increase in the stability of the hydration shell regarding the anionic SMT molecules. Considering that the formation of hydrogen bridges between the host and guest always assumes Furthermore, the inclusion of SMT by its aromatic amine moiety in case of RAMEB host enhances formation of zwitterionic form of SMT in the cavity. This is due to the tautomerization of the proton from the sulfonamide to the aromatic amine moiety enhanced by the Coulomb interaction of the proton with the negatively charged cavity of RAMEB. With the aim to justify this conception of the SMT's zwitterion formation in the RMAEB's cavity, simultaneous analysis of complexation behavior has been done using infrared (IR) spectroscopy. In general, our results are in agreement with the IR analyses of SMT-BCD complexes prepared by a freeze-drying method [23], the characteristic bands of SMT shifted and are more or less intense in the presence of CD. Moreover, IR spectra of the SMT-RAMEB complexes and the species interacted support our idea described above (Figure 5): significant changes of two characteristic vibrations of SMT molecules were observed upon complexation by the RAMEB host as follows. Quantum chemical analysis revealed that belting vibration of SNH bond angle at sulfonamide moiety (1103 cm−<sup>1</sup> ) disappeared while the bond stretching associated to the aromatic NH<sup>3</sup> is appeared at 2812 cm−<sup>1</sup> in the experimental IR spectra of the complexes. These changes in the experimental IR spectra indicate the stabilization of the zwitterionic form of SMT in the RAMEB cavity. This phenomenon has not been observed in the case of the BCD host. *Molecules* **2019**, *24*, x FOR PEER REVIEW 6 of 12

**Figure 5.** Infrared spectra of SMT – RAMEB complexes. **Figure 5.** Infrared spectra of SMT – RAMEB complexes.

*2.3. Driving Forces of the SMT-CD Complex Formations*  Taking into account the binding conformations suggested by theoretical modeling, we can discuss in detail the thermodynamic parameters of complex formation between CDs and SMT. However, it should be noted here, that thermodynamic parameters derived from temperaturedependent spectroscopic measurements assume that these parameters are constants within the In all eight situations, the interactions show an increased entropy term (Table 2). This property is associated with two facts: the solvent water molecules leave the host's cavity prior to the complex formation and the guest molecules (at least partly) lose their hydration shell. Both processes increase the entropy. In particular, the entropy gain decreases by the second deprotonation step. This is probably due to the increase in the stability of the hydration shell regarding the anionic SMT molecules.

temperature range of investigation. Furthermore, these data reflect not only for the temperaturedependent change of the association constants, but also for the way how the association constants have been determined. Spectroscopic identification of association constant based on changes of the Considering that the formation of hydrogen bridges between the host and guest always assumes dehydration of the appropriate part of the host and guest molecules, the energy cost of dehydration compensated by the entropy gain associated to the increased freedom of the water molecules after

environment around the guest when the molecule enters from the polar aqueous media the

determined based on fluorescence spectroscopic measurements, using the van't Hoff equation. Relevant experiments showed [19] that the results of calorimetric studies are similar to the spectroscopic findings regarding host-guest type CD complexes, and the data of thermodynamic parameters only slightly differs between the two methods. This property supports our conclusions

The possible driving forces which stabilize the host-guest complexes of CDs are electrostatic interaction, van der Waals interaction, hydrophobic interaction, hydrogen bonding, relief of conformational strain, charge transfer interaction, and release of water molecules from the hydrophobic cavity of the host to the bulk phase [35]. The values of thermodynamic parameters consist the contribution of the species' desolvation and the different kind of noncovalent interactions listed above. In general, the combination of both negative or positive enthalpy and entropy changes indicate that van der Waals forces and hydrogen bonding or hydrophobic interaction take places in complex formation, respectively. While higher negative values of ΔH combined with positive ΔS have found for the electrostatic driving forces combined with hydrogen bonds of ionized groups [36]. However, the given values can be strongly affected by intensive dehydration and solvent reorganization. In the discussion of the present experimental data (Table 1) we focus on two tendencies observed in the thermodynamic parameters: both the enthalpy and entropy changes associated to the complex formation decrease while the charge of the guest SMT molecules varies from +1, 0 to −1. On this base, considering the attractive forces between the anionic cavity of the host and the cationic guest at pH = 2, highly negative enthalpy changes should be observed in vacuo. However, the desolvation of the guest costs more energy than it is causes during the association of

made on the spectroscopic data.

the dehydration. This assumption is supported by the good agreement between the measured and calculated thermodynamic parameters.
