A Study of the Inclusion Complex Formed Between Cucurbit[8]uril and N,4-Di(pyridinyl)benzamide Derivative

Abstract

The interaction between cucurbit[8]uril (Q[8]) and the guest 1-methyl-4-(4-(1-methylpyridin-1-ium-4-yl)benzamido)pyridin-1-ium (PB²⁺) has been thoroughly investigated. Multiple techniques were employed, including ¹H NMR spectroscopy, mass spectrometry, isothermal titration calorimetry (ITC), UV–vis absorption spectrophotometry, and quantum chemistry calculations. The experimental results and calculation analysis have clearly shown that in aqueous solution, the host Q[8] preferentially encapsulates the phenylpyridinium salt moiety of the PB²⁺ guest within its hydrophobic cavity, forming a 1:2 inclusion complex.

1. Introduction

Cucurbit[n]urils (Q[n]s) are a class of macrocyclic compounds composed of glycoluril units connected by methylene bridges [1,2,3]. Their unique hydrophobic cavities and polar carbonyl ports endow them with excellent host–guest recognition abilities, which have attracted extensive attention in supramolecular chemistry, drug delivery, biomedicine, and functional materials [4,5,6,7]. Based on the cavity size of Q[n], cucurbit[8]uril (Q[8]) has a relatively large cavity size (~8.8 Å). This large cavity enables Q[8] to exhibit a unique molecular recognition performance and allows specific guest molecules to dimerize in its cavity in a controlled manner [8,9,10,11,12]. Two guest molecules or large-volume functional groups, such as phenyl, naphthyl, indole, quinoline, and larger aromatic rings, can be included in the cavity of Q[8] to form homoternary inclusion complexes. These complexes have become an important platform for constructing multi-component complex supramolecular systems [13,14,15,16]. In recent years, research on the host–guest interaction based on Q[8] has been extended to ionic aromatic compounds, especially derivatives containing pyridine or benzene rings [17,18,19,20]. These molecules can form stable inclusion complexes with Q[8] through hydrophobic interactions, π-π stacking, and electrostatic effects. However, their binding modes and structural regulation mechanisms still need further exploration [21,22,23,24]. For instance, Zhang’s group constructed a variety of supramolecular self-assembly systems for photocatalytic applications through the self-assembly of linear molecules and Q[8] [25,26]. Liu’s group constructed a variety of supramolecular room-temperature phosphorescence systems based on 4-(4-bromophenyl)-pyridinium derivatives and Q[8] [27,28,29], which have shown good results in many fields such as biological imaging, molecular detection, drug delivery, and so on. Li’s group synthesized a tetrahedral molecule featuring tetraphenylmethane as its core and combined it with Q[8] to create a novel water-soluble three-dimensional supramolecular organic framework. This framework was utilized as a broad-spectrum reversal agent for heparin anticoagulation [30]. Moreover, a self-assembly process utilizing hexa-armed [Ru(bpy)₃]²⁺ derivatives and Q[8] constructs another three-dimensional cubic framework. This framework can serve as a heterogeneous catalyst for hydrogen production and organic reactions [31]. Xiao’s group has constructed a series of Q[8]-based ternary self-assemblies and achieved a series of excellent results in the fields of fluorescence sensing and molecular recognition [32,33]. Overall, non-covalent interactions-based supramolecular functional materials have shown great potential in many fields [34,35].

The N,4-dipyridylbenzamide derivative, as a bi-cationic aromatic molecule containing phenylpyridinium salt structural units, is an ideal object for studying the ternary supramolecular self-assembly of Q[8] [11,29]. Phenylpyridinium salt is one of the building blocks for supramolecular network structures. In the field of supramolecular self-assembly, we have conducted some related research and achieved certain results. A series of supramolecular polymer systems were constructed by using the antiparallel arrangement of naphthyl and styrene pyridine dimers in the Q[8] cavity [36,37,38,39]. For instance, an irreversible covalent component was created through the photocatalytic photodimerization of carbon–carbon double bonds, which was utilized to construct the first highly water-soluble 3D supramolecular covalent organic framework. When the framework material supported a POM catalyst and a Ru²⁺ complex photosensitizer to form a composite catalytic system, the electron transfer of protons to H₂ was significantly enhanced [40]. Consequently, we believe that cucurbit[n]uril is one of the ideal macrocyclic host molecules for constructing a supramolecular network system.

Herein, in this study, we selected Q[8] and the guest 1-methyl-4-(4-(1-methylpyridin-1-ium-4-yl)benzamido)pyridin-1-ium (PB²⁺) (Figure 1) as the research objects. We systematically examined the host–guest interaction between Q[8] and PB²⁺ in an aqueous solution, employing techniques such as ¹H NMR, ITC, UV–vis, ESI–MS, and quantum chemical calculations. The experiments revealed that Q[8] selectively encapsulated the phenylpyridine salt of PB²⁺ within its hydrophobic cavity, forming a stable inclusion compound with a stoichiometric ratio of 1:2. Additionally, it engaged in polar interactions with the pyridine group through the carbonyl port. This research not only reveals the supramolecular binding mechanism between Q[8] and PB²⁺ molecules for the first time but also offers a theoretical and experimental foundation for the development of multi-component functional materials based on Q[n]s and phenylpyridine salts.

2. Materials and Methods

Cucurbit[8]uril was prepared and purified following our previously published procedure [41,42]. Add 500 g of semicarbazide and 245 g of paraformaldehyde (maintaining a weight ratio of 2:1) to a 3000 mL three-necked flask. Subsequently, introduce 600 mL of ice-cold concentrated hydrochloric acid. Using a constant temperature magnetic stirrer, maintain the temperature at 90~110 °C for 4 to 6 h. Following the reaction, add approximately 1 L of distilled water to the mixture. Upon stirring with a rod, a large amount of white solid (crude product Q[8]) is observed to form. The mixture is then left to stand overnight. The solid is subsequently purified through recrystallization (3M hydrochloric acid solution).

• Synthesis of compound PB²⁺

Compound PB²⁺. Compound 1 (4-Aminopyridine, 0.52 g, 5.1 mmol), 2 (1.0 g, 5.0 mmol), EDCI (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) (0.96 g, 5.0 mmol), and HOBT (1-Hydroxybenzotriazole) (0.68 g, 5.0 mmol) were dissolved in DMF (10 mL) and stirred at 60 °C for 24 h. After the reaction was completed, it was allowed to cool to room temperature. The resulting precipitate was filtered, washed with ethanol three times, and dried in vacuum to obtain compound 3 as a white solid.

Compound 3 (0.58 g, 2.1 mmol) and methyl iodide (0.71 g, 4.0 mmol) were added to MeCN (15 mL), and the mixture was stirred at 80 °C for 24 h. After the reaction was completed, it was allowed to cool to room temperature. The precipitate was filtered, washed with a small amount of MeCN, and vacuum-dried to obtain a crude product. The solid was then dissolved in a small amount of water, and a saturated aqueous solution of ammonium hexafluorophosphate was added dropwise to the solution. The compound was heated until it was completely dissolved. The precipitate was filtered, washed with cold water, and vacuum-dried to obtain a pale white solid product. Finally, the product was dissolved in a small amount of acetonitrile. A saturated aqueous solution of tetrabutylammonium chloride (TBA-Cl) was added dropwise to this solution, and the resulting precipitate was filtered, washed with cold acetonitrile, and further dried under vacuum to obtain the compound. PB²⁺ (0.95 g, 62%). M.p. > 280 °C (decomp). ¹H NMR (400 MHz, DMSO-d₆): δ 11.10 (s, 1H), 9.14 (d, J = 4.0 Hz, 2H), 8.62 (d, J = 2.0 Hz, 2H), 8.50 (d, J = 2.0 Hz, 2H), 8.30 (dd, J = 8 Hz, 2H. J = 8 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 4.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ = 166.8, 153.3, 152.2, 146.5, 146.3, 138.0, 136.0, 129.9, 128.9, 125.2, 116.1, 47.8, 47.0. HRMS (ESI): Calcd for C₁₉H₁₉N₃O²⁺ (305.1528) [M-2Cl]²⁺/2 = 305.1528/2 = 152.5764. Found: 152.5741. We have only analyzed the double charge mode of the molecule (Figure S6).

• Nuclear magnetic resonance measurements.

Deuterated water was used as the solvent to prepare a 2.0 mM deuterated aqueous solution of PB²⁺ and a supersaturated deuterated aqueous solution of Q[8]. Different proportions of Q[8] were then added to PB²⁺ to create nuclear magnetic resonance tubes with varying host–guest molar ratios (Q[8]/PB²⁺). All NMR spectra were measured by VARIAN INOVA-400 spectrometer at 25 °C, and the resulting nuclear magnetic resonance data for the different host–guest ratios were collected and analyzed.

• UV–vis absorption.

The UV–vis absorption spectrum of the host–guest complex was tested using Agilent 8453 spectrophotometer (1 cm quartz cells) at 25 °C. Accurately prepare a 1.0 × 10⁻³ mol/L aqueous solution of PB²⁺, transfer 0.2 mL into a 10 mL volumetric flask, and add varying amounts of Q[8] aqueous solution with a concentration of 1.0 × 10⁻⁴ mol/L to prepare a series of host–guest interaction test solutions with different molar ratios (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5). Adjust the volume to 10 mL, allow it to stand for half an hour, and measure its electronic absorption spectrum and fluorescence emission spectrum at room temperature. The absorption spectra were obtained at a concentration of 2.0 × 10⁻⁵ mol L⁻¹ PB²⁺ and at varying Q[8] concentrations for the PB²⁺@Q[8] system at 25 °C.

• Isothermal titration calorimetry (ITC) experiments.

The association constants and thermodynamic parameters for the 1:2 complexation of PB²⁺ with Q[8] were determined using isothermal titration calorimetry with a Malvern MicroCal VP-ITC instrument. We did not conduct repetitive experiments in the ITC lab; we only performed the experiment once. In a typical procedure, an aqueous solution (0.1 mM) of Q[8] was placed in the sample cell (170 μL), and a solution (1.0 mM) of PB²⁺ was added in a series of 20 injections (2 μL) at 150 s intervals, with the heat evolved recorded at 298.15 K. The data is fitted by MicroCal ITC analysis software (Origin 7 SR4 v7.0552). We utilized the Multiple Sites mode for fitting. The results of the Multiple Sites mode fitting are detailed in the supporting materials, based on experimental data (Table S1).

• High-resolution electrospray mass spectrometer (HR ESI–MS).

HR ESI–MS was tested on a Bruker 5 MicrOTOF II. The ESI–MS experiments were conducted by adding a PB²⁺ solution (1.0 mM, 1.0 mL) to a Q[8] solution (0.1 mM, 10.0 mL). The resulting solution concentration was approximately 0.1 mM (Q[8]/PB²⁺ = 1:2).

• Quantum chemistry calculations.

Utilize the Gaussian 09 software package to perform all calculations. With the assistance of the HyperChem Release 7.52 software package, the initial geometries of all structures are constructed. Becke’s three-parameter mixed functional, combined with the correlation functional of Lee, Yang, and Parr (B3LYP), is employed to optimize the elements C, H, N, and O at the theoretical level of 6-311G(d, p).

3. Results and Discussion

Binding behaviour in aqueous solution: ¹H NMR spectroscopy. The interaction between PB²⁺ and Q[8] was explored through ¹H NMR. It has been previously reported that the ¹H NMR peak of the guest proton within the low polarizability cavity of Q[8] has experienced a field shift. Simultaneously, due to proton unmasking, its interaction with the carbonyl oxygen of Q[8] results in a low field displacement. The ¹H NMR spectroscopy measurements clearly indicate that PB²⁺ forms a host–guest inclusion complex with the Q[8] host. Figure 2 presents the titration ¹H NMR spectra, where distinct changes in the proton signals were observed compared with those of the free guest PB²⁺. Specifically, the H5 and H6 proton signals gradually shifted upfield by approximately 1.6 and 0.1 ppm, respectively. Additionally, the H3 and H4 proton signals shifted upfield by approximately 0.7 and 1.3 ppm, respectively. The chemical shift in protons to a higher field is due to the shielding effect of electron-rich regions in Q[8]. It strongly suggests that the N-dimethyl and benzene ring moiety of the guest PB²⁺ are deeply included in the cavity of Q[8] due to the formation of the complex PB²⁺@Q[8]. In contrast, under a 1:1 host–guest ratio in D₂O, the signal for the H1 and H2 protons showed an obvious downfield shift of approximately 0.2 ppm. Moreover, the H7 and H8 proton signals for the N-methyl also exhibited a downfield shift. These experimental results indicate that the phenylpyridinium salt is included in the low-polarizability cavity of Q[8]. At the same time, the amidopyridinium salt and the N-methyl on the pyridine ring are mainly located in a deshielding environment, resulting in the formation of a 1:1 host–guest inclusion complex in aqueous solution. The formation of the inclusion complex between PB²⁺ and Q[8] was further confirmed by ¹H-¹H COSY NMR spectra (Figure S1).

UV–vis absorbance spectrophotometry. First, the changes in absorption intensity during the complexation of Q[8] with PB²⁺ were investigated. As shown in Figure 3a, in an aqueous solution, PB²⁺ exhibits an absorption maximum centred at 298 nm. When Q[8] is added, the complexation of PB²⁺ leads to a decrease in absorption from 0.99 to 0.80, along with a bathochromic shift from 298 to 301 nm. As the amount of Q[8] increases, the intensity of the absorption maximum peak gradually decreases, which is a clear indication of an interaction between PB²⁺ and Q[8]. Moreover, the shape of the absorption spectrum of Q[8] remains unchanged, suggesting a non-covalent interaction between PB²⁺ and Q[8]. With a further increase in Q[8], the UV–vis absorption intensity of PB²⁺ no longer shows obvious changes. These experimental results imply the formation of supramolecular complexes PB²⁺@Q[8]. Based on the continuous variation method, the Job plots clearly demonstrate that the UV–−vis spectra data of PB²⁺ fit well with a 1:2 stoichiometry for the host–guest inclusion complexes (Figure 3b). Furthermore, the binding stoichiometry was determined through Job plot analysis at a constant total concentration of the host and guest molecules. By plotting the changes in absorption intensity (ΔA) against the molar fraction of PB²⁺, a peak was observed at a molar fraction of 0.66, indicating a 1:2 stoichiometry for the Q[8]:PB²⁺ inclusion complex. (Figure S2).

Isothermal titration calorimetry (ITC). ITC is one of the most effective techniques for studying supramolecular interactions. It can provide not only thermodynamic parameters (ΔS, ΔH, ΔG) but also the binding constant (Ka). To gain a deeper understanding of the nature of the host–guest complexations between Q[8] and PB²⁺, we conducted ITC experiments, as shown in Figure 4. We only conducted one ITC experiment, and did not conduct many repeated experiments. First, from the ΔH and TΔS values presented in Table S1, it was evident that the formation of homoternary complexes was enthalpically driven. The observed negative enthalpy change (ΔH₁ = −60.70 kJ∙mol⁻¹, ΔH₂ = −80.89 kJ∙mol⁻¹ for PB²⁺@Q[8]) was likely due to the cooperativity of weak interactions. Secondly, the intermolecular complexation between Q[8] and PB²⁺ were driven by favorable enthalpy changes. Based on the corresponding experimental results, we also obtained the association constant of Ka = 4.413 × 10⁹ M⁻² for the interaction with PB²⁺. The high binding constant indicates a relatively strong host–guest interaction between Q[8] and PB²⁺, suggesting the formation of a stable homoternary complex PB²⁺@Q[8] in aqueous solution.

Electrospray ionization–mass spectrometry (ESI–MS). In addition, the 2:1 complex PB²⁺@Q[8] was further characterized by ESI–MS. The presence of a peak at m/z = 484.6730, corresponding to PB²⁺@Q[8] (calculated for [2(C₁₉H₁₉ON₃Cl₂)@(C₄₈H₄₈N₃₂O₁₆)-4Cl⁻]⁴⁺/4, 484.6746) (Figure 5). This observation confirmed that the binding ratio between Q[8] and PB²⁺ is 1:2. This result is consistent with the findings from ¹H NMR spectroscopy, ITC, and UV–vis absorption.

Quantum chemistry calculations. To gain a more in-depth proof of the host–guest complexation between Q[8] and PB²⁺, molecular modeling was utilized. Density functional theory (DFT) calculations were performed at the B3LYP/6-31G(d, p) level to obtain structural information on supramolecular self-assembly. Figure 6 depicts the low energy conformations of the host–guest complexes. These conformations clearly indicate that PB²⁺ forms stable 1:2 inclusion complexes. Specifically, the phenylpyridinium salt of PB²⁺ is situated within the cavity of Q[8]. Meanwhile, the amidopyridinium salt and the N-methyl group on the pyridine ring are located just outside the host’s portal. This finding is in excellent agreement with the NMR results.

4. Conclusions

In summary, we have conducted an investigation into a novel supramolecular host–guest complex formed between the N,4-di(pyridinyl)benzamide derivative (PB²⁺) and Q[8] in aqueous solutions. Multiple analytical techniques, including ¹H NMR spectroscopy, ESI–mass spectrometry, UV–vis absorbance spectrophotometry, and ITC, were employed for this study. The results obtained from these analyses revealed that a PB²⁺@Q[8] (2:1) inclusion complex was formed. Specifically, the phenylpyridinium salt of the PB²⁺ was found to be located within the cavity of Q[8], while the N-methyl on the pyridine ring was situated just outside the host’s portal. To the best of our knowledge, the present result represents the first example of structurally characterized Q[n]s-isoniazid host–guest complexes. Based on this study, we conclude that the Q[8] host and the molecules containing phenylpyridinium units can serve as building blocks to construct a diverse range of supramolecular network structures.