Turning Cucurbit[8]uril into a Supramolecular Nanoreactor for Asymmetric Catalysis

Chiral macromolecules have been widely used as synthetic pockets to mimic natural enzymes and promote asymmetric reactions. An achiral host, cucurbit[8]uril (CB[8]), was used for an asymmetric Lewis acid catalyzed Diels–Alder reaction. We achieved a remarkable increase in enantioselectivity and a large rate acceleration in the presence of the nanoreactor by using an amino acid as the chiral source. Mechanistic and computational studies revealed that both the amino acid–Cu2+ complex and the dienophile substrate are included inside the macrocyclic host cavity, suggesting that contiguity and conformational constraints are fundamental to the catalytic process and rate enhancement. These results pave the way towards new studies on asymmetric reactions catalyzed in confined achiral cavities.

enantioselectivity (40% ee). Finally, a decrease in the catalyst loading from 3 to 0.5 mol% led to only a slight drop of the conversion and ee value (entry 9). Hence, we retain high enantioselectivity at extremely low catalyst loading, which is especially important in the case of CB [8] with its limited solubility in water.

Kinetic and thermodynamic measurements
Scheme S1. Proposed catalytic cycle of D-A reaction.
All kinetic and thermodynamic measurements were performed as reported by Otto and Engberts. [3] A typical procedure of measuring k app is as follows: the samples contained 112 μM CB[8], 84 μM amino acid, 56 μM Cu(NO 3 ) 2 , 22 μM azachalcone (1a), and 3.67 mM (2.5 mM in the case of CB [8]•e•Cu 2+ ) of cyclopentadiene (2) with a total volume of around 2.1 mL in 1mM PBS buffer at pH=7.4. The apparent second-order rate constant (k app ) was determined according to the following expression: In which d [A 1a ]/dt is the slope of the absorption of 1a at a certain wavelength vs. time for the first 10% decrease of substrate conversion. d is the path length of the cuvette and ε 1a and ε 3a represent the extinction coefficients of 1a and 3a, respectively.

Determination of the molar extinction coefficients:
Since the maximum absorption wavelength of 1a was significantly influenced by the presence of CB[8], the molar extinction coefficient of 1a and 3a were determined at various wavelengths which were related to the different catalyst combinations. The absorbance of 1a and 3a at a certain wavelength was measured with the fixed concentration of 10, 20, 30, 40, and 50 μM. The molar extinction coefficient was estimated from the slope of the fitting curve.

Determination of the equilibrium constants K eq :
At 25 o C, equilibrium constants were obtained by measuring the extinction coefficient of partially complexed dienophile (ε obs ) at a proper wavelength where the extinction coefficient of uncomplexed and complexed 1a show maximal differences. ε obs as a function of the concentration of copper ion can be derived as following: ε 1a and ε complex represent the extinction coefficients of the uncomplexed and complexed dienophile, respectively. When [Cu 2+ ]/(ε 1aε obs ) was plotted vs.[Cu 2+ ], a linear relation was observed, which was used to determine the equilibrium constant K eq which equals the ratio of slope and intercept.

Determination of k cat :
The k cat values were calculated according to the following equation: Herein, the concentration of unbound copper complex can be calculated from the measured K eq .

Computational studies
The geometries of the catalytic complexes both with and without CB[8] were optimized using DFT B3LYP with Grimme D3 dispersion correction [4] and 6-31G* basis set.Additionally, a polarizable continuum solvent model (PCM) [5] was used throughout the calculations to represent the standard water dielectric medium.

Geometric isomers of azachalcone in complex with Cu 2+ and Trp
Optimized geometries for isomers of the complex tryptophan-Cu 2+ -azachalcone were calculated using DFT B3LYP/6-31G* level of theory with Grimme D3 dispersion corrections [4] and the PCM continuum solvent model as implemented in Gaussian09 [5] .
Additional calculations were also run with a single water molecule explicitly,to take into account possible solvent interactions with the copper ion. Subsequently, single point energies were determined with the aug-ccpVTZ-PP basis set at the same level of theory. The trans-cisoid conformation was found as the most stable isomer. These relative energies were determined with aug-ccpVTZ-PP basis set.
b This structure is represented in Figure S4a.
Once the complex was inserted in CB[8], in the presence of a single explicit water molecule, the energy was minimized again to assure that the copper is indeed coordinated to the water molecule and not to the macrocyle rim(see Figure S4b).
Therefore, optimized geometries for the complex with and without CB[8] in presence of cyclopentadiene (Cp) approaching from different directions were alsocalculated as described above. Since the Cp approach was significantly hindered from the Trp-side attack and the geometry optimizations resulted in a non-reactive conformation, a cis -transoid 9.7 --distance constraint was added in all the calculations to obtain models closely related to the transition state. In all of the approaches studied, two constraints were set at 3.0 Å each between the two pairs of reacting carbon atoms. Oncewe obtained the optimized geometries, the constraints were released and the unconstrained optimum geometries calculated as well. Note that only the 6-31G* basis set was used in the calculations with CB[8] on account of the large number of atoms to analyze when the macrocycle is included. As a general trend, we observed similar but slightly lower energy differences between the various approach conformations when using the larger basis set (aug-ccpVTZ-PP), both in the calculations of the complex on its own and in presence of Cp (Tables S1 and S2). Therefore,it is reasonable to assume that the relative energies obtained for the complex with CB[8] would have only been lower if calculated with the larger basis set. The obtained relative energies (Table S2) suggested that the endo complex is slightly favored as compared to the exo complex.
The Trp-side approach was much less favorable both with and without CB[8], in addition, the complex seemed totwist and open slightly to allow the Cp to interact with the azachalcone (Figure 2b and S6). Importantly, the energies obtained are in good agreement with the product ratios obtained experimentally.

Synthesis
Standard procedure for Diels-Alder reactions employing cucurbituril nanoreactor: An aqueous solution composed of 75µL of 1mM amino acid, 50μL of 1 mM Cu(NO 3 ) 2 and 50µL of PBS buffer(10mM, pH 7.4) was shaken for 30minutes.To this components a solution of CB[8] (666µL, 150µM in water) was added, and the mixture was shaken at room temperature for another 30 minutes. Then an aliquot of a stock solution of azachalcone in CH 3 CN (10 μL of 150 mM solution) was added and the mixture was cooled to 6 °C. The reaction was started by addition of the freshly distilled cyclopentadiene (4 μL, 48 μmol) and proceeded for 24 h at 6 °C, followed by extraction with diethyl ether (3×1 ml). After removal of the solvent, the ee of crude products were directly determined by HPLC on a chiral stationary phase.

Preparation of 2-Acetyl-3-methylpyridine
Under an argon atmosphere, n-BuLi (7.3 mL, 11.6mmol, 1.6 M inn-hexane) was added to a mixture of 2-bromo-3-methyl-pyridine (1.0 g, 5.8mmol) and anhydrous Et 2 O (30 mL) at -78 0 C. The reaction mixture was stirred for 30 minutes. Then, N,N-dimethylacetamide (2.0 mL, 23.2 mmol) was added, and the mixture was allowed to reach ambient temperature within 1 h. The reaction mixture was quenched by adding saturated NH 4 Cl, and was then extracted with Et 2 O three times.
The combined organic layers were washed with brine, dried, and concentrated. The crude product was purified on silica gel column using n-hexane/AcOEt (10:1) to yield the desired product. 1