The Binding Behaviors between Cyclopentanocucurbit[6]uril and Three Amino Acids in the Solid and the Solution Phases

Binding behaviors between CyP 6 Q[6] and three amino acids have been investigated by means of X-ray crystallography, proton nuclear magnetic resonance ( 1 H NMR) spectroscopy, amino acids and isothermal titration calorimetry (ITC). The results showed that CyP 6 Q[6] forms a 1:2 inclusion complex with glycine, but 1:1 complexes with both leucine and lysine. Whereas the carboxyl group of glycine can enter the interior of the cavity of CyP 6 Q[6], only the alkyl chains of leucine and lysine can enter this cavity. Interestingly, leucine can adopt two different self-assembly modes upon its interaction with cucurbituril, depending on the external conditions, whereas glycine and lysine do not exhibit such behavior.

acid simultaneously. [24,25] Our research group reported the host-guest binding behavior of twisted cucurbit [14]uril and inverted cucurbit [7]uril with amino acids, [26,27] and for the rst time reported supramolecular complexes of cucurbituril and enantiomeric amino acids. [28] In recent years, studies on the binding behavior between macrocyclic compounds, such as cyclodextrin and crown ethers, and amino acid molecules have been extremely widespread. [29][30][31] However, the research on the host-guest chemistry of cyclopentyl cucurbiturils is still immature, so we are interested in the study on the host-guest properties of CyP6Q [6] and amino acids. For the present study, we took CyP 6 Q [6] as the host, and selected three different amino acids, namely glycine (Gly), L-lysine (L-Lys), and L-leucine (L-Leu), as guests. We examined the binding behavior between these components in the solid and liquid phases (Scheme 1).
Results And Discussion 2.1 Binding modes between CyP 6 Q [6] and amino acids in the solution phase shows that complex 1 belongs to the triclinic system with the centrosymmetric space group P-1. An oak ridge thermal-ellipsoid plot program (ORTEP) representation of the asymmetric unit is shown in the Supporting Information (Fig. S1). It contains two halves of CyP 6 Q [6], two protonated glycine molecules, and one free [CdCl 4 ] 2ion. In the single-crystal structure of complex 1, each CyP 6 Q [6] contains two glycine molecules. The carboxyl group of each glycine molecule is included in the cavity of the CyP 6 Q[6], but its amino and methylene groups remain outside. The nitrogen atoms (N26 and N13) of the respective glycine molecules form two hydrogen bonds with two portal oxygen atoms (O5, O6 and O15, O16) of CyP 6 Q [6], and the N-H···O distances are in the range 2.752-3.035 Å. It is interesting to note that a hydrogen bond is established between the nitrogen atom of a glycine molecule included by the cucurbituril and a chlorine atom (Cl1) of the counter ion [CdCl 4 ] 2-, with an N-H···Cl distance of 3.228 Å. This is not the case for the other amino acid molecule included by the cucurbituril. At the same time, there is also a dipolar interaction between this counter ion and a methylene proton on the outer wall of the other cucurbituril molecule. [CdCl 4 ] 2thus acts as a bridging unit to link the cucurbituril with the amino acid (shown by purple dotted lines in Fig. 1). Fig. 2 shows the crystal structure of L-Lys@CyP 6 Q[6] (complex 2). Analysis of the single-crystal structure shows that complex 2 belongs to the monoclinic system with centrosymmetric space group P21/c. An ORTEP representation of the asymmetric unit is shown in Fig. S2. It contains half of CyP 6 Q[6], a protonated lysine molecule (occupancy ratio 0.5), and a free [CdCl 4 ] 2ion. In the single-crystal structure of complex 2, each CyP 6 Q[6] contains a lysine molecule. The carboxyl and amino groups of this lysine molecule, and the carbon atom (C2) to which they are bound, lie outside of the portal of CyP 6 Q [6], while the rest of the molecule is within the cavity. The amino nitrogen atom (N1) and hydroxyl oxygen atom (O2) of the lysine molecule outside of the portal of CyP 6 Q[6] form four hydrogen bonds (N1-H1C···O4, N1-H1A···O6, N1-H1C···O7, and O2-H2···O7) with three oxygen atoms (O4, O6, and O7) of a portal of CyP 6 Q [6], with lengths in the range 2.334-3.062 Å. The nitrogen atom (N2) of the terminal amino group of lysine inside the cavity forms a hydrogen bond with a portal carbonyl oxygen atom of the cucurbituril with a distance of 2.952 Å. Unlike in complex 1, the counter ion does not interact with the amino acid in this complex, but only surrounds the cucurbituril through dipole interactions (Fig. S5). The counter ions of complex 3 are paired around the cucurbituril by ion-dipole interactions, and there is also an ion-dipole interaction between the two paired counter ions. Although the counter ions of complex 4 also surround the cucurbituril through ion-dipole interactions, there is no weak interaction between them. This difference results in very different stacking patterns of these two complexes. Fig. 4 shows stack views of complexes 3 and 4 along the c-axis. From Fig. 4a, b, it can clearly be seen that all of the cucurbituril units in complex 3 have the same orientation, whereas those in complex 4 have two orientations with an included angle of 67.9°. Due to the different orientations of the cucurbituril moieties, there is a signi cantly larger channel along the c-axis in complex 4 than in complex 3.

Interactions between CyP 6 Q[6] and amino acids in the solution phase
CyP 6 Q [6] shows good solubility in many solvents, most notably in water, in which it is 1 to 2 orders of magnitude more soluble than ordinary cucurbituril. Because the amino acids that make up the protein required for animal nutrition are mostly present in aqueous systems, the good water solubility of CyP 6 Q [6] facilitates the study of its interactions with amino acids. In the present work, the binding behavior of CyP 6 Q[6] with the above three amino acids was investigated in D 2 O. In 1 H NMR, the cavity of CyP 6 Q [6] has a shielding effect on proton signals, whereas outside of the portals, in the vicinity of the carbonyl oxygen atoms, the proton signals are subjected to a deshielding effect. According to this theory, analysis of the 1 H chemical shifts and splittings of the signals of protons of amino acids and the host provides insight into the binding mode between them. Fig. 5 shows the changes in the 1 H NMR spectrum of the guest Gly as it is dropped into a solution of the host CyP 6 Q [6]. The results show that the peak due to the α protons of Gly shifts up eld, indicating that this unit enters the cavity of the cucurbituril. Considering the ion-dipole and hydrophobic effects, it may be speculated that the carboxyl group and methylene unit of Gly enter the cavity, while the amino group is xed at the portal of CyP 6 Q [6]. This binding mode is basically similar to the crystal structure in the solid phase. However, a difference is that the methylene unit lies outside of the portal in the solid phase, but inside the cavity in the liquid phase. At up to two molar equivalents of Gly with respect to CyP 6 Q [6], the α protons show only one signal. Beyond two molar equivalents, two signals due to this unit appear, corresponding to bound and free Gly. This indicates that CyP 6 Q [6] and Gly form a 1:2 inclusion complex, and that the exchange frequency is slower than the operating frequency of the 1 H NMR spectrometer.
The changes in the 1 H NMR spectrum of the guest L-Leu upon its incremental addition to CyP 6 Q[6] are shown in Fig. 6. Two sets of proton resonances for L-Leu are observed, indicating that the frequency of binding and release of L-Leu in CyP 6 Q [6] is slower than the operating frequency of the 1 H NMR spectrometer, consistent with the observations for Gly. A difference is that the α proton signals of l-Leu shift down eld, whereas the signals of the remaining alkyl chain protons shift up eld, suggesting that only the alkyl chain of L-Leu enters cucurbituril, while the carboxyl group remains outside of the portal. At the same time, it can be further seen in Fig. 6 that the ε and λ proton signals of the two methyl groups are split into two groups of signals from the original overlapping signal, indicating that these methyl groups are in different positions in the cucurbituril. A similar splitting is seen for the β and γ proton signals. After L-Leu interacts with the cucurbituril, the environment of the two hydrogen protons of β protons will have a certain difference, causing it to split into two sets of signals, so three peaks due to the β and γ protons are seen. When a sub-stoichiometric amount of L-Leu is added to CyP 6 Q [6], it displays only one set of signals. Once it is in excess, another set of signals due to free L-Leu appears. This indicates that CyP 6 Q [6] forms a 1:1 complex with L-Leu, as in the solid-phase crystal structure.
As shown in Fig. 7, titration 1 H NMR spectroscopy was also used to investigate the binding behavior between CyP 6 Q[6] and L-Lys. Similarly to L-Leu, the α proton signals of L-Lys shift down eld, while the signals of the other alkyl chain protons shift up eld, indicating that its alkyl chain enters the cavity of the cucurbituril, while the two amino groups are xed at the portals, forming a structure similar to that of butanediamine@CyP 6 Q [6]. The methine unit at which the carboxyl α proton is linked to one of the amino groups remains outside of the portal, forming a 1:1 complex of L-Lys@CyP 6 Q [6]. The cavity of CyP 6 Q[6] is large enough to accommodate the L-Lys alkyl chain in its fully extended form, as corroborated by the crystal structure. It is worth noting that during the titration process, when the amount of L-Lys added reached 0.5 equivalents with respect to CyP 6 Q[6], its peaks suddenly broadened and even disappeared.
This phenomenon may feasibly be attributed to the exchange frequency of L-Lys in and out of the cavity of the cucurbituril exceeding the operating frequency of the 1 H NMR spectrometer, such that the detected 1 H NMR signals are averages of various intermediate states of the host-guest interaction. Averaging over a multitude of states will make the signals of the guest tend towards the baseline. Of course, it may also be enrichment of lysine that causes the disappearance of the signal of the guest proton. The speci c cause is still unclear, and further research is needed.

Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) experiments (Fig. S8) were performed to determine the thermodynamic parameters of the above three amino acids and CyP 6 Q [6] in water, providing insight into the thermal stability and driving force of the interactions. Table 1 shows that the enthalpies and entropies of the interactions of the three amino acids with CyP 6 Q[6] are both negative. From the contributions of these two thermodynamic parameters to the Gibbs free energy, it can be seen that the three systems are enthalpy-driven, and the driving force is determined by the ion-dipole interaction and the hydrophobic effect. The alkyl chain of the amino acid is more inclined to enter the cavity of the host due to the hydrophobic effect, allowing water molecules originally in the cavity of the CyP 6 Q[6] to enter the aqueous phase, thereby reducing the entropy of the system. Moreover, 2Gly@CyP 6 Q[6] evidently has the largest binding constant among the three studied systems, which may be due to the fact that there is some interaction between the two amino acids in addition to the interaction between glycine and cucurbituril.
Its crystal structure (Fig. 1) shows that the carboxyl groups of both glycine molecules are also involved in hydrogen bonds, forming a more stable structure, so their binding constants are an order of magnitude higher than those for the other two amino acids. For lysine and leucine with the same number of carbon atoms, the binding constants are relatively close, but that of lysine is slightly higher due to a dipolar interaction of the amino groups.

Conclusion
In summary, we have investigated the binding behavior between CyP 6 Q[6] and three amino acids in both the solid and liquid phases. For lysine and leucine, both X-ray crystallography and 1 H NMR spectroscopy indicate the formation of 1:1 host-guest complexes, with the respective alkyl chains within the cavity of CyP 6 Q [6]. Glycine is bound slightly differently in the two phases; although a 1:2 inclusion complex is formed in each case, the methylene units have different locations. In the crystal structure, the methylene units lie outside of the portals, whereas solution 1 H NMR shows that they lie within the cavity of the cucurbituril. ITC shows that binding of all three amino acids is enthalpy-driven. Leucine shows two selfassembly modes, but this is not seen for the other two amino acids. The results of these experiments not only add to the understanding of the molecular recognition of amino acids, but are also of value for the design and synthesis of new bioactive cucurbiturils for the purpose of biological recognition and simulation.

Materials and methods
All raw materials used in this study were purchased from Aladdin Industrial Corporation (AR, Shanghai, China). CyP 6 Q [6] was prepared according to a literature procedure. [14] 4.

Isothermal titration calorimetry
A 1.00×10 -4 mol/L solution of CyP 6 Q [6] in water (1.00 mL) was placed in the sample cell, and a 1.00×10 -3 mol/L Gly solution was drawn into a 250 mL syringe. The temperature was set at 25 °C, and the titration was conducted by adding 30 aliquots (6 μL) of the Gly solution at intervals of 300 s. A 1.00×10 -3 mol/L solution of CyP 6 Q [6] in water (1.00 mL) was placed in the sample cell, and a 1.00×10 -2 mol/L L-Leu (L-Lys) solution was drawn into a 250 mL syringe. The temperature was set at 25 °C, and the titration was conducted by adding 25 aliquots (10 μL) of the L-Leu (L-Lys) solution at intervals of 300 s.
The thermodynamic parameters of each system were determined on a Nano ITC isothermal calorimeter. After deleting the rst two unwanted data points, the data were analyzed with ORIGIN 8.0 software using an independent model.

Declarations
Ethics approval and consent to participate Not applicable.

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Availability of data and materials.
All data and materials are all provided.

Competing interests
The authors declare no con icts of interest.

Contributions
SYC performed all lab work related to obtaining of compound and chemical assay, and wrote the highest percentage of the manuscript. WWZ analyzed and re ned all crystal structures and wrote related content.
XNY revised the manuscript. LTW conducted the synthesis of the main substance. ZT guided the technical methods involved in the manuscript. PHM was the leader of the project, holds the original idea, designed the statistical experiments and coordinated the experimental activities among all engaged labs. All authors read and approved the nal manuscript.  Figure 1 Host-guest interaction of complex 1.

Figure 2
Host-guest interaction of complex 2.

Supplementary Files
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