Study on the Binding Interaction of the α,α′,δ,δ′-Tetramethylcucurbit[6]uril With Biogenic Amines in Solution and the Solid State

1H NMR spectroscopy and MALDI-TOF mass spectrometry were utilized to examine the binding interaction of α,α′,δ,δ′-tetramethylcucurbit[6]uril (TMeQ[6]) and six biogenic amines (spermine, spermidine, 2-phenethylamine, tyramine, histamine, and tryptamine). Their 1H NMR spectra both at pD = 7 and pD = 3 revealed that four biogenic amines (spermine, spermidine, 2-phenethylamine, and histamine) can fit in the TMeQ[6] cavity, respectively, and other biogenic amines were located outside of the TMeQ[6] portal. In addition, a solid-state evaluation with single-crystal X-ray diffraction analysis showed the binding interaction of spermine, spermidine, 2-phenethylamine, and tyramine with TMeQ[6].


INTRODUCTION
Biogenic amines (BAs) belong to a group of low-molecular weight nitrogen-containing organic compounds with remarkable biological activity. BAs are categorized by structure into three groups: aliphatic amines (e.g., spermine and spermidine), aromatic amines (e.g., tyramine and phenylethylamine), and heterocyclic amines (e.g., histamine and tryptamine) (Sentellas et al., 2016). These amines can develop via the thermal or enzymatic decarboxylation of amino acids due to the storage and handling of food, and increased levels of BAs suggest spoilage of food. Thus, BAs are pivotal markers for food poisoning (Santos, 1996;Shalaby, 1996). Since biogenic amines also take part in critical physiological functions, the determination of the BA content in food is important.

Nature of Binding in Aqueous Solution
The interactions between the host TMeQ[6] and the biogenic amine guests in aqueous solution were examined by NMR spectroscopy. Then, 1 H NMR spectroscopy was conducted on each biogenic amine guest with TMeQ[6], and the NMR assignments were made through 1 H-1 H COSY ( Figures S1-S6). The ambiguously binding behavior will be discussed.
As shown in Figure 2, upon continuous addition of additional hosts, the signals of free guest 1 gradually disappeared, and new signals of the supramolecular complexes appeared as a result of the binding behavior. The resonances of α, γ, and β of guest 1 moved downfield by 0.70, 0.37, and 0.60 ppm, respectively, whereas the resonances of δ and ε of guest 1 shifted upfield by 0.40 and 0.96 ppm, respectively. This observation indicates that only a portion of guest 1 is buried deeply inside the host, forming a stable inclusion complex TMeQ[6]@ 1. The α, γ, and β protons are located outside of TMeQ[6], whereas the δ and ε protons of guest 1 are located in TMeQ [6].
The 1 H NMR spectra in Figure 3A reveal the presence of 2 in neutral D 2 O solution in the absence and presence of 1.1 equiv. of TMeQ [6]. The α, γ, and β proton resonances of guest 2 moved downfield by 0.67, 0.35, and 0.40 ppm, respectively, while the resonances for ϕ, δ, and ε protons of guest 2 moved upfield by 0.30, 0.81, and 1.04 ppm, respectively. The outcomes suggest that nearly identical to guest 1, guest 2 is somewhat concealed within the TMeQ[6] cavity and establishes the stable inclusion complex TMeQ[6]@ 2.
With 1.1 equiv of TMeQ[6] ( Figure 3B) the α, β, and γ proton resonances of guest 3 shifted upfield by 0.86, 0.92, and 0.86 ppm, respectively, while those of the ε and δ protons of guest 3 shifted downfield by 0.21 and 0.54 ppm, respectively. The NMR results indicated that the phenyl group of guest 3 is buried in TMeQ[6], whereas the alkyl chain of guest 3 is located outside the TMeQ[6], forming an inclusion complex TMeQ[6]@ 3.
The 1 H NMR spectra of guest 4 with 1.1 equiv. of TMeQ[6] are revealed in Figure 3C, and the resonance for the α, β, γ, and δ protons of guest 4 shifted downfield by 0.07, 0.06, 0.06, and 0.04 ppm, respectively. The results show that guest 4 is located outside of TMeQ [6]. Given the presence of the hydroxyl group, guest 4 is not encapsulated in TMeQ [6].
The binding conduct of guest 5 with TMeQ[6] is altered from that of the other guests in this evaluation and is revealed in Figure 3D. The resonances for the α, β, δ, and γ protons of guest 5 shifted upfield by 0.09, 1.50, 1.05, and 0.81 ppm, respectively. This discovery suggested that guest 5 was fully concealed in the cavity of TMeQ[6], verifying the creation of the inclusion complex.
When guest 6 was combined with TMeQ[6], the resonance for its ϕ and η protons moved downfield by 0.22 and 0.13 ppm, respectively, and the resonance for the protons of the indolyl group did not notably move ( Figure 3E). Based on this outcome, due to the large size of guest 6, it is found outside the portal of TMeQ[6]. All of 1 H NMR spectroscopy indicate the guest 1 and 2 formed host-guest inclusion complexes that display slow kinetics of exchange on the 1 H NMR time scale, while guests 3 and 5 show fast kinetics of exchange, and the 1 H NMR complexationinduced shifts (CIS) of the protons of guests 1-6 are listed in Table 1.
The host-guest interactions of TMeQ[6] with charged BAs were also investigated at pD = 3 by 1 H NMR spectroscopy ( Figures S8-S13). An NMR titration experiment was conducted at pD = 3 and revealed that guests 1, 2, 3, and 5 created an inclusion complex with the TMeQ[6] host, while guests 4 and 6 were found outside of the portal of the TMeQ[6]. This reaction phenomenon is in accord with the titration experiments conducted in D 2 O. The results indicate that BAs can form complexes with TMeQ[6] both in aqueous and acidic solutions.  The interaction of TMeQ[6] with guest 6 was also evaluated by UV absorbance spectrophotometry and fluorescence spectroscopy. Based on the UV absorption spectroscopy outcomes ( Figure S14), the addition of TMeQ[6] to guest 6 in buffered solution (pH 7) was joined by a minor rise in the intensity at 218 nm, suggesting a weak interaction of TMeQ[6] and guest 6. As revealed in Figure S15, guest 6 showed an 358-nm emission peak at an excitation wavelength of 218 nm. The addition of TMeQ[6] with 1:1 stoichiometry induced a minor reduction and bathochromic shift from 358 to 360 nm in fluorescence intensity at 358 nm. These alterations in the emission profile additionally verify the weak host-guest interaction of TMeQ[6] and guest 6 (Mallick et al., 2016). The results of the spectral analysis are consistent with those from the NMR experiments.

Isothermal Titration Calorimetry
To improve our comprehension of host-guest interactions of TMeQ[6] and BAs, we performed isothermal titration calorimetry (ITC) experiments at 25 • C in 10 mM sodium phosphate (pH 7.0). Table 2 and Figures S16-S20 reveal the equilibrium association constants (K a ) and thermodynamic parameters, respectively, for TMeQ[6] and for the BA guests, with the exception of guest 3. The experimental outcomes showed K a values ranging from 10 3 to 10 7 M −1 . Among which TMeQ[6] binds with spermine with the highest binding affinity, which similar to parent cucurbit[6]uril or cucurbit[6]uril derivatives (Mock and Shih, 1986;Kim et al., 2009;Lucas et al., 2011;Wen et al., 2017). In addition, the K a constants of guest 1 and 2 with TMeQ[6] are lower than the corresponding K a of guest 1 and 2 with cyclohexyl substituted cucurbit[6]uril(CyH 6 Q[6]), suggesting that the substitution impacts the shape of the cavity (Kim et al., 2009;Wen et al., 2017) (Table S1). From the H • and T S • values revealed in Table 2, the intermolecular complexation interactions of the TMeQ[6] host and all guests seem to be compelled by negative enthalpy alterations, while the interactions of TMeQ[6] host and guests 2 and 4 were compelled by negative enthalpy alterations, along with small negative (unfavorable) entropy alterations. The ITC experiment for guest 3 was not successful because of the weak interaction of TMeQ[6] and guest 3.
The binding conduct of TMeQ [6] with BAs in aqueous solution is detailed above. Guests 1 and 2 are favorably encapsulated into TMeQ [6]. Given their long chain, parts of guests 1 and 2 are located outside TMeQ [6]. The phenyl group of guest 3 is completely encapsulated into TMeQ[6], and the alkyl chain of guest 3 cannot be accommodated due to a lack of available space. Guest 4 was not favorably encapsulated into  1, 2, 3, and 4. The selected hydrogen bonding (Å) of the complexes is listed in Table S2. The TMeQ[6]@1 complex crystallized in the monoclinic systems in space group C 2/c. As shown in Figure 4, guest 1 is encapsulated in TMeQ[6], exiting the back-folded geometry. Moreover, the atoms (C14G, C16I, and C15H) of guest 1 were found in the portal of TMeQ[6], while the atoms C6 and C25A were concealed within the TMeQ[6] cavity, and these structural observations are in accord with the evaluation of the 1 H NMR spectroscopic data. Guest 1 shows back-folded geometry due to the tight hydrogen bonds between the guest 1 nitrogen atoms and the oxygen atoms of TMeQ [6], which is in accord with the literature (Shan et al., 2017).
It was noted that the TMeQ[6]@2 complex crystallized in the triclinic systems with space group P-1. As revealed in Figure 5, the TMeQ[6]@2 complex is made up of the TMeQ[6] host, guest 2, tetrachloride zincate anions, and some water molecules. The asymmetry of the TMeQ[6]@2 complex results from the asymmetrical structure of guest 2. Guest 2 is encapsulated in the TMeQ[6] host, the C82, C83, C84, and N50 atoms were buried in the TMeQ[6] host, and the C81, C85, C86, C87, N49, and N51 atoms were located outside the TMeQ[6] host. The information obtained from the structure is in agreement with the 1 H NMR analysis. As observed in the crystal structure, there is a N-H· · ·O hydrogen bond between N52 of guest 2 and O21 of the TMeQ[6] host, and a N-H· · ·O hydrogen bond between N53 and O16 of the portal O atoms of TMeQ[6], as well as a N-H· · ·O hydrogen bond between N53 and O18 of the portal O atoms of TMeQ[6]. The nearby molecules create a 1D chain via the hydrogen bonds of the N atoms of guest 2 and the O atoms of water. Thus, guest 2 exhibited a straight chain, whereas guest 1 exhibited a back-folded geometry.
It was observed that the TMeQ[6]@3 crystallized in a triclinic system with the P-1 space group. As shown in Figure 6, two guest 3 molecules exist on the portal of the TMeQ [6]. The N-H· · ·O hydrogen bonds exist between the portal O atoms of TMeQ [6] and the N atoms of guest 3. The structure suggests that guest 3 was found outside the TMeQ[6]; this discovery runs counter to the outcome of the evaluation of 1 H NMR. The experimental outcomes indicate that the TMeQ[6]-guest inclusion states could be changed by altering the synthetic conditions. The cavity of TMeQ[6] is big enough to accommodate the guest 3 molecule in neutral aqueous solution, and guest 3 was found outside TMeQ[6], in an acidic environment and with [CdCl 4 ] 2− anions. The hydrogen bond interaction and outer surface interaction of Q[n] has a pivotal part in producing the inclusion complex [20].
As revealed in Figure 7, the TMeQ[6]@4 complex is made up of guest 4 found outside of the TMeQ[6] host. There are strong hydrogen bonds between the guest 4 N atoms and the O atoms of the TMeQ[6] host. Moreover, several fascinating hydrogen bonding interactions are present between guest 4 and the TMeQ[6] host, such as the C-H···O hydrogen bond between

CONCLUSION
The binding interactions of biogenic amines and TMeQ [6] in aqueous solution were examined by NMR spectroscopy, ITC, and MALDI-TOF mass spectrometry. The binding interactions of the biogenic amine guest and the TMeQ[6] host were examined in the solid state by X-ray crystallography, with the exception of guests 5 and 6. As far as we know, the current evaluation is the initial one to structurally detail examples of cucurbit[n]uril and biogenic amine host-guest complexes. The results show that guests 1, 2, 3, and 5 can be encapsulated into the TMeQ[6] host, whereas guests 4 and 6 are located outside of the TMeQ [6] host. The forces that facilitate the connection between the host TMeQ[6] and the guest biogenic amines include balanced  hydrophobic, hydrogen bonding, and ion-dipole interactions. These outcomes will not just improve our understanding of the molecular identification of biogenic amines, but they may also be substantial for planning and generating novel macrocyclic complexes for biological recognition and replication.

Materials and Methods
Spermine, spermidine, 2-phenethylamine, tyramine, histamine, and tryptamine were purchased. TMeQ[6] was prepared according to a literature method (Zhao et al., 2004). All NMR data were received in D 2 O at pD = 7 and pD = 3, respectively, the temperature is 298K, the concentration of the solution with guest is 1.00 × 10 −3 mol/L, then TmeQ[6] was added into the solution gradually. MALDI-TOF mass spectra were taken on an ultrahigh-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with α-cyano-4-hydroxycinnamic acid as matrix.

X-ray Crystallography
All the crystals of complexes were grown from water. The crystal data was collected through the Bruker Apex II WITH CCD diffractometer. The crystal data was solved by the software SHELXS-204/7 (Sheldrick, 1997 ,35.34;H,3.58;N,19.14. Found: C,39.98;H,3.62;N,19.

AUTHOR CONTRIBUTIONS
LY, XX, and ZT write the manuscript. JK, FW, and QL analyse the data of the experiment. XW did the measurement. YZ synthesis the complex.

ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China (Grant No. 21561007), the Innovation Program for High-level Talents of Guizhou Province (No. 2016-5657), the Science and Technology Fund of Guizhou Province (No. -1030, the Major Program for Creative Research Groups of Guizhou Provincial Education Department (2017-028) and the Henan science and technology project (Nos.172102210161) for financial support.