Host-guest complexation of cucurbit[8]uril with two enantiomers

Host-guest complexation of cucurbit[8]uril (Q[8]) with two enantiomers, D-3-(2-naphthyl)-alanine (D-NA) and L-3-(2-naphthyl)-alanine (L-NA), has been fully investigated. Experimental data indicate that double guests reside within the cavity of Q[8] in both aqueous solution and solid state, generating highly stable homoternary complexes D-NA2@Q[8] and L-NA2@Q[8].

It is well known that all alpha amino acids but glycine usually exist in two enantiomers (L-or D-amino acid). To the best of our knowledge, however, the detection and recognition of specific enantiomeric amino acids by Q[n]s have never been reported. Previous investigation revealed that the host Q [8] is large enough to accommodate two phenyl, naphthyl or other aromatic groups simultaneously through host-stabilized charge-transfer interactions 14,[36][37][38] . This observation prompted us to explore the possibility of the formation of homoternary complexes between Q [8] and enantiomeric amino acid containing naphthyl residue, D-3-(2-naphthyl)-alanine and L-3-(2-naphthyl)-alanine (abbreviated as D-NA and L-NA, respectively, Fig. 1b). In the present work, we studied the host-guest complexation of Q [8] with D-NA and L-NA in aqueous solution by NMR, UV and fluorescence spectroscopy, MS and isothermal titration calorimetry (ITC), and in the solid state by X-ray crystallography.

Results and Discussion
Binding Behaviors in Aqueous Solution. The 1 H NMR spectroscopy measurements indicate that both D-NA and L-NA form host-guest inclusion complex with Q [8] host. Given that the changes induced by Q [8] host in the 1 H NMR spectra of guests D-NA (Fig. 2) and L-NA ( Figure S2) are similar, guest D-NA is taken as a representative to depict their binding interactions. In the presence of small amount of the Q[8] host (Fig. 2b), the signals of both free and complexed guests are simultaneously observed and are very broad, indicating slow exchange of free and complexed guests on the NMR time scale. All guest aromatic protons move upfield considerably, revealing deep insertion of the naphthyl group inside the cavity. On the other hand, the proton H 1 and one of the CH 2 protons of D-NA move downfield slightly, which indicates that they are located outside the cavity. At a 2:1 ratio of D-NA to Q [8], the aromatic peaks are completely shifted upfield. These observations suggest that the naphthyl moiety of the D-NA guest was encapsulated into the cavity of the Q [8] host.
To better understand the host-guest interaction between Q [8] and both enantiomers in aqueous solution, we carried out UV and fluorescence titration experiments. According to the UV absorption spectroscopic results, Fig. 3(A), upon the gradual addition of Q [8] into D-NA in H 2 O, the absorption underwent a slight bathochromic shift from 220 to 227 nm in addition to a significant decrease in its intensity due to the strong interaction between Q[8] and D-NA. This is actually also true for the case of Q [8] with L-NA ( Figure S3).
We also studied the fluorescence properties of both D-NA and L-NA in the presence of Q [8]. As can be seen in Fig. 3(B), the D-NA shows an emission peak at 334 nm in aqueous solution, when the excitation is λ = 274 nm. Successive addition of Q [8] caused decrease in the fluorescence intensity at 334 nm and appearance of a new emission peak at around λ = 410 nm. Moreover, we found that an isobestic point appears at 364 nm. These substantial changes in emission profiles further confirm the strong host-guest interaction between Q[8] and D-NA. When the D-NA is replaced by L-NA, similar fluorescence spectra are also observed ( Figure S3).
Their Job's plots (based on the continuous variation method) clearly show that both UV and fluorescence spectra data of both enantiomers fit well to 1:2 stoichiometry of the host-guest inclusion complexes (Fig. 3, inset). The formation of the homoternary complexes D-NA 2 @Q [8] and L-NA 2 @Q [8] was also established by the MS experiments. Their MALDI-TOF spectra ( Figure S4   It is should be noted that the homoternary complex D-NA 2 @Q [8] is completely different from the host-stabilized charge-transfer complexes, which Kim group perviously reported 14 . In the homoternary complex D-NA 2 @Q [8], the two encapsulated D-NA molecules are connected together through π ···π interactions. In the latter, the encapsulated guests were electron donor and acceptor pair, and the major driving force for the ternary complex formation appears to be strong charge-transfer interaction between the guests 14 . Description of ITC. ITC study (Fig. 5) on the complexation of Q [8] with both D-NA and L-NA affords the thermodynamic parameters (Table S1), and further confirms that the binding stoichiometry of Q [8] to both enantiomers is 1:2. From the Δ H and TΔ S values in the Table S1, it is clear that the formation of both homoternary complexes is enthalpically driven. The observed negative enthalpy change (Δ H 1 = −50.12 ± 2.59 kJ·mol −1 , Δ H 2 = −6.17 ± 2.56 kJ·mol −1 for D-NA 2 @Q [8]; Δ H 1 = −48.97 ± 4.42 kJ·mol −1 , Δ H 2 = −3.97 ± 4.77 for L-NA 2 @Q [8]) is probably due to the cooperativity of above mentioned four kinds of weak interactions. On the basis of the corresponding experimental results, we also obtained the association constants of Ka = (6.51 ± 0.19) × 10 11 M −2 and (3.17 ± 0.05) × 10 11 M −2 for Q [8] with D-NA and L-NA, which are much larger than that of Q [8] with tripeptides reported by Urbach 21 . Such a high binding constant suggests the relatively strong host-guest interaction between Q[8] and D-NA or L-NA, indicating the construction of stable homoternary complexes D-NA 2 @Q [8] and L-NA 2 @Q [8] in aqueous solution.

Conclusion.
In summary, we have investigated the host-guest complexation of Q [8] with two enantiomers D-NA and L-NA in both aqueous solution and solid state by using NMR, UV and fluorescence spectroscopy, MS, isothermal titration calorimetry (ITC), and X-ray crystallography. Driven by the cooperativity of electrostatic interactions, multiple C-H···π interactions, and hydrogen-bondings, both D-NA and L-NA can be encapsulated into the cavity of Q [8] to form stable homoternary complexes D-NA 2 @Q [8] and L-NA 2 @Q [8]. This study suggests that Q [8] host may be very useful in dimerisating specific amino acids, peptides and proteins with suitable binding groups.

Methods
Materials and methods. 3-(2-naphthyl)-D-alanine and 3-(2-naphthyl)-L-alanine were obtained from Aldrich and used as supplied without further purification. Q [8] was prepared according to a literature method 39,40 . All the 1 H NMR spectra were recorded on a Bruker DPX 400 spectrometer in D 2 O. Absorption spectra of the host-guest complexes were recorded on an Aglient 8453 spectrophotometer at room temperature. Fluorescence spectra of the host-guest complexes were performed with a Varian RF-540 fluorescence spectrophotometer. MALDI-TOF mass spectrometry was recorded on a Bruker BIFLEX III ultra-high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with a-cyano-4-hydroxycinnamic acid as matrix.
Single-crystal X-ray crystallography. Single crystals of D-NA 2 @Q [8] and for L-NA 2 @Q [8] were grown from hydrochloride acid solution by slow evaporation. Diffraction data of both complexes were collected at 273(2) K with a Bruker SMART Apex-II CCD diffractometer using graphite-monochromated Mo-K α radiation (λ = 0.71073 Å). Empirical absorption corrections were performed by using the multi-scan program SADABS. Structural solution and full-matrix least-squares refinement based on F 2 were performed with the SHELXS-97 and SHELXL-97 program packages, respectively 41,42 . Non-hydrogen atoms were treated anisotropically in all cases. All hydrogen atoms were introduced as riding atoms with an isotropic displacement parameter equal to 1.2 times that of the parent atom. Hydrogen atoms were given for all isolated water molecules.
Crystal data for D-NA 2 @Q [8]:   L). The heat of dilution was corrected by injecting the guest solution into deionized water and subtracting these data from those of the host-guest titration. All titrations were repeated three times. Computer simulations (curve fitting) were performed using the Nano ITC analyze software.