Encapsulation of L-Valine, D-Leucine and D-Methionine by Cucurbit[8]uril

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Introduction
As the basic building blocks of peptides and proteins, the twenty natural amino acids play fundamental and significant roles in sustaining life on earth.The deletion of any amino acid may lead to some diseases.0][11][12][13][14] Each Q[n] molecule possesses two identical portals and a rigid hydrophobic cavity, which can selectively accommodate and bind guest molecules.][17][18][19][20][21][22][23][24][25][26][27] It is well-known that the larger Q[n] homologue Q[8] has a spacious hydrophobic cavity, which can accommodate two aromatic groups simultaneously. 28In theory, the Q[8] cavity also has the capability of encapsulating a pair of amino acids.With this idea in mind, we have started to address two important issues.Firstly, how does the shape and size of the     The binding behaviour of Q[8] with D-Leucine and D-Methionine is illustrated in Figures 3 and 4, and these are similar to our observations for Q[8] with L-Valine.In the other words, both D-Leucine and D-Methionine can also be encapsulated into the Q[8] cavity in aqueous solution.There is no doubt that the major driving forces for the formation of the Q[8]-based inclusion complexes will be hydrophobic effects.Meanwhile, the van der Waals interaction between the surfaces of the amino acids and the inner wall of the Q[8], the ion-dipole interactions between the ammonium group of the amino acids and the carbonyl groups on the Q[8] portal as well as hydrogen bonds between the amino acids, may all also contribute to the formation of the Q[8]-based inclusion complexes.To better understand the host-guest interactions between Q[8] and these 3 amino acids, we carried out isothermal titration calorimetry (ITC) experiments at 298.15 K. Table S1 and Figure S1-S3 show the equilibrium association constants (Ka) and thermodynamic parameters for the Q[8]-amino acid systems for L-Valine, D-Leucine, and D-Methionine.The experimental results revealed the association constant (Ka) for the complexes of the amino acids with the Q[8] to be 5.847×10 7 , 7.648×10 7 and 2.825×10 7 M -2 , respectively, which indicates strong binding between the Q[8] and the three amino acids.

Structural Analysis of Inclusion Complexes in the Solid State.
We also examined the binding behaviour of the Q[8] host and with the L-Valine, D-Leucine, and D-Methionine guests in the solid state by X-ray crystallography (see experimental section).Slow vapour evaporation of aqueous HCl solutions containing the Q[8] host and the corresponding amino acid guest in the presence of CdCl2 (see experimental section) successfully afforded single crystals of the inclusion complexes (2), and (C5H12NO2S)

Please do not adjust margins
Please do not adjust margins    The X-ray crystallography confirms that compound 3 crystallizes in the triclinic space group P-1.There are two crystallographically independent Q[8] hosts in the asymmetric unit of compound 3.One of them accommodates two D-Methionine molecules and generates a homoternary inclusion complex D-Met2@Q[8], the other accommodates some disorder water molecules or is empty.As can be seen in Figure 7

Materials and methods:
The amino acid was commercially available and used as received.The Q[8] host was prepared according to a literature method. 32All the 1 H NMR data were recorded on a JNM-ECZ400s MHz nuclear magnetic resonance ( 1 H NMR) spectrometer in D2O at 293.15K.Elemental analyses (C, H, and N) were carried out on a PE 240C elemental analyzer.ITC measurements.Microcalorimetric experiments were performed using an isothermal titration calorimeter Nano ITC (TA, USA).The heat evolved was recorded at 298.15 K.All solutions were degassed prior to titration experiments by sonication.1.0×10 -3 mol/L stock solution of amino acids and 1.0×10 -4 mol/L stock solution of Q[8] were prepared in deionized water.A typical ITC titration was carried out by titrating the amino acid solution (1.0×10 -3 mol/L) into a Q[8] solution.The concentration of Q[8] in the sample cell (1.3 mL) was 1.0×10 -4 mol/L at pH=7.Computer simulations (curve fitting) were performed using the Nano ITC analyze software.First points in the ITC data are excluded when fitting the model to acquire the binding constant, enthalpy change, and entropy change.

Single-crystal X-ray crystallography:
Single-crystal data for the compounds 1 , 2 and 3 were collected on a Bruker D8 VENTURE diffractometer (for compounds 1 and 3) and a Bruker Smart Apex CCD diffractometer (for compound 2) with a graphite-monochromated Mo-Kα radiation source (λ = 0.71073 Å) respectively.Empirical absorption corrections were applied by using the multiscan program SADABS.Structural solution and full matrix least-squares refinement based on F 2 were performed with the SHELXS-97 and SHELXL-97 program package, 33 respectively.Anisotropical thermal parameters were applied to all the non-hydrogen atoms.All hydrogen atoms were treated as riding atoms with an isotropic displacement parameter equal to 1.2 times that of the parent atom.The SQUEEZE routine of Platon was employed for all compounds because of the disordered solvent water molecules. 34A summary of crystal data, intensity measurements, structure solution, and refinement for all the three compounds are given in Table 1.CCDC 2122314, 2122315 and 2122316 contain the supplementary crystallographic data for this paper.These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif.

Conclusions
In conclusion, by using1 H NMR spectroscopy and X-ray crystallography, we have demonstrated the capacity of the Q[8] host to recognize L-Valine, D-Leucine, and D-Methionine molecules, both in aqueous solution and in the solid state.NMR spectroscopic titration experiments revealed that the Q[8] host can encapsulate the L-Valine, D-Leucine, and D-Methionine molecules to form host-guest inclusion complexes in aqueous solution.We also successfully obtained single crystals of these three amino acids complexed with the Q[8] host.Their crystal structures unambiguously confirmed that the Q[8] host can accommodate two amino acid molecules with suitable size and shape.To accommodate different guests, the Q[8] host may experience a certain degree of deformation, which depends on the molecular size of the encapsulated guests.
amino acid affect the binding of the Q[8] to the amino acid?Secondly, does the chirality of the amino acid affect the binding of the Q[8] to the amino acid?Here, three specific amino acids, L-Valine (L-Val), D-Leucine (D-Leu), and D-Methionine (D-Met) were chosen as the guests.In the present work, the binding interactions of the Q[8] host with these three amino acids in aqueous solution is investigated by 1 H NMR spectroscopy.In particular, single crystals of these three amino acids complexed with the Q[8] host have been prepared.The X-ray crystallography clearly shows how the Q[8] host can encapsulate the amino acids to form homoternary inclusion complexes through host-guest interactions.Significant ellipsoidal deformation of the Q[8] host was also observed in these inclusion complexes.Structural comparison of the homoternary inclusion complexes reveals that the deformation degree of the Q[8] host depends on the molecular size of the encapsulated amino acids.The purpose of this article is to probe the recognition properties of the Q[8] host toward amino acids.
Single crystal X-ray diffraction analysis reveals that compound 1 crystallizes in the monoclinic crystal system, space group P21/c.The asymmetric unit of the compound 1 contains one half of the Q[8] host, one protonated Val molecule, one tetrahedral [CdCl4] 2-anion, and seven lattice water molecules.As shown in Figure5, two protonated L-Val molecules were encapsulated into the Q[8] host, forming a homoternary inclusion complex L-Val2@Q[8], in agreement with what we observed in aqueous solution.The ammonium group of the L-Val molecule points toward two carbonyl oxygen atoms of the Q[8] host.One proton of the ammonium group is involved in hydrogen bonding with one carbonyl oxygen atom, with an N-H•••O distance of 2.219 Å.The other two protons of the ammonium group form hydrogen bonds with solvent water molecules.At the same time, the carboxyl group on the other side of the L-Val molecule forms a hydrogen bond with a water molecule (O2W), which links with two carbonyl oxygen atoms (O3 and O8) at the portal of the Q[8]host through hydrogen bonds.Given that the encapsulated L-Val molecule is protonated, there must be electrostatic interactions and van der Waals contacts between the guest molecules and the Q[8] host, as we previously reported.29

Figure 6 .
Figure 6.The distance of the nitrogen atom out of the mean plane of the Q[8] portal.

Figure 7 .
Figure 7. Crystal structure of the inclusion complex D-Met2@Q[8] (up) and 2D planar structure of compound 3 viewed down the a axis (down).
, the two D-Methionine guests are located inside the Q[8] cavity.Both the ammonium group and the carboxyl group point toward the Q[8] portal.They form hydrogen bonds with the carbonyl oxygens of the Q[8] host or the solvent water molecules (N•••O and O•••O distances in the ranges of 2.833(5)-This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins3.097(6)Å).It is worth mentioning that in compound 3, each Q[8] host of the inclusion complex D-Met2@Q[8] forms numerous C-H•••O hydrogen bonds with four neighbouring empty Q[8] hosts.At the same time, the empty Q[8] hosts also connect with four inclusion complexes through C-H•••O hydrogen bonds.As a result, they form an interesting grid-like planar structure, parallel to the bc-plane of the unit cell, as shown in Figure 7.The stacking of the planar structure further generates numerous 1-D channels along the a-axis, which are filled with [CdCl4] 2-anions and water molecules.The solvent water accessible volume of the channels is 1903.1 Å 3 , about 38.3% of the total unit cell volume (4970.0Å 3 , estimated with Platon). 31It is interesting to compare the deformation degree of these three inclusion complexes.Normally, the Q[8] host is a relatively rigid macrocycle with centrosymmetric symmetry.However, all the Q[8] hosts in the inclusion complexes L-Val2@Q[8], D-Leu2@Q[8], and D-Met2@Q[8] experience a significant deformation.As can be seen in Figure 8, for these three inclusion complexes, the largest and smallest O•••O diameters of the Q[8] portals are in the ranges 10.721-11.064Å and 8.879-9.850Å, respectively.The deformation degree of the Q[8] host is related to the molecular size of the encapsulated guests.The deformation degree of the Q[8] host for these three inclusion complexes is in the order L-Val2@Q[8] < D-Leu2@Q[8] < D-Met2@Q[8], because the molecular size of the encapsulated guests is in the order L-Val < D-Leu < D-Met.