A light-gated regulation of the reaction site by a cucurbit[7]uril macrocycle

Competitive inhibition can be overcome by increasing the amount of catalyst in the reaction mixture. Here we present a pseudorotaxane system that circumvents this rule. A merocyanine inhibitor linked with the substrate obstructs the binding of the macrocyclic catalyst at the electrophilic reaction site preventing catalysis. Under UV light merocyanine is converted to the spiropyran form, losing its inhibition properties, thereby allowing the catalyst to bind the reaction center and promote the reaction. Moreover, when more than one nucleophile is present in the reaction mixture, the pseudorotaxane can scavenge a selected nucleophile and change the final product ratio. This work is a step forward in the development of new types of regulation in catalytic systems with remote control.

Synthesis of 6. Compound 5 was synthesized following the procedure described previously [N. Rad et al. Angew. Chem. Int. Ed. 2019, 58, 11340]. To a solution of 5 (2.91 g, 14 mmol) S4 in 85 mL of MeOH, 33% aq. solution of HNMe 2 (5 mL, 28 mmol) was added. The mixture was stirred overnight, then cooled to 0 o C, and NaBH 4 (0.80 g, 21 mmol) was added portionwise for 15 min. The cooling bath was removed, and the reaction mixture was stirred for 5 h. The mixture was quenched with 50 mL of water and extracted with EtOAc (3x30 mL). The extracts were dried with Na 2 SO 4 , filtered, and concentrated under reduced pressure.

Isomerization
Isomerization was studied by NMR spectroscopy. The proton NMR spectra made it possible to observe the isomerization of the less stable SP form into the more stable MCH form during thermal relaxation in the dark ( Figure S23). Importantly, each photostationary state contained only one form, which promised high operation efficiency of the devised pseudorotaxane.

S21
Isomerization was also studied by UV-Vis spectroscopy. The formation of the MCH form was accompanied by the appearance of a new band at 420 nm ( Figure S28). The use of light with a wavelength close to this value (430 nm) induced a rapid back isomerization (few seconds) to the SP form. To slow this process, we used a shorter wavelength (365 nm) of very low intensity (5% of maximum power), see Figure S29.

S22
UV-Vis spectroscopy has been also used to determine isomerization kinetics:

Solvent
Solvent was prepared by mixing 30 mL of H 2 O with 30 mL of MeCN and adjusting to pH ≈ 3.0 with 2M HCl (15.0 µL).

Axle solution
1.38 mg of the axle was dissolved in 2.69 mL of solvent. A 2.4 mL portion was diluted with 36 mL of the solvent to give a 50 µM solution.

Macrocycle solution
9.54 mg of the macrocycle was dissolved in 848 µL of solvent to give a 7.5 mM solution.

Isomerization from SP to MCH form
The UV-Vis cuvette was filled with 1.5 mL of axle solution. Afterward, either 50 µL of solvent or 50 µL of macrocycle solution was added. The mixture was then irradiated for 1 min using a 365 nm LED at 50% maximum power to convert the axle into the SP form. Next, the solution was left in the dark and UV-Vis spectra were recorded at 1 min intervals ( Figure   S30). The experiment was halted when no changes in the intensity of the MCH form were observed.

Isomerization from MCH to SP form
The UV-Vis cuvette was filled with 1.5 mL of axle solution and left in the dark for 2 h to convert the SP form into the MCH form. Afterward, either 50 µL of solvent or 50 µL of macrocycle solution was added. The mixture was then irradiated using a 365 nm LED at 10% maximum power to prevent the back isomerization observed at 5% light intensity. The absorbance at 420 nm (maximum of MCH band) was recorded at 3 s intervals ( Figure S31).
The experiment was stopped when no changes in the intensity were observed. The degree of isomerization was calculated as: where A is the absorbance at time t, A 0 is the absorbance at the beginning of the reaction, and A ∞ is the absorbance at 100% conversion.
Since the isomerization obeys first order kinetics, the isomerization rate was determined from: The half-live of each species under these conditions can be obtained from:

Complexation
Initially, we thought to determine the binding constant directly from NMR titration. To this end, the axle (5 mg With this, we could find the binding strengths of SP and MCH by their displacement in a complex with benzaldehyde ( Figures S36-37). Briefly, the axle (6.44 mg) and the macrocycle  is the dissociation constant.
Equilibrium concentrations can be also written as: where and are the initial concentrations of MCH and CB7, respectively.

7
Inserting them into the chemical equilibrium equation we obtain a quadratic equation with one unknown : The solution of this equation gives: After substituting the association constant for the dissociation constant = 1 the equation takes the following form: Having an equilibrium concentration, we can get a molar fraction of the assembled pseudorotaxane in solution: In the same manner, we can obtain a molar fraction of the pseudorotaxane in the SP form (under irradiation).
For NMR experiments carried out at and , 7 = 5.25 = = 5 and , respectively. The molar fractions of • 7 = 0.972 • 7 = 0.921 pseudorotaxane in UV-Vis experiments ( ) at different amounts of the = = 50 macrocycle are summarized in Table S2.  The relative time that the macrocycle spends on the aromatic station in the OFF state, i.e., its molar fraction, can be calculated as: = -where is the hypothetical shift of H a , when the macrocycle is entirely located on the aliphatic station; is the hypothetical shift of H a , when the macrocycle is entirely located on the aromatic station; is the average (observed) shift of H a , i.e., when the macrocycle is located on both stations.
In the same way we can express the molar fraction of the macrocycle on the aromatic station in the ON state:

Hydrazonation
To determine at which pH the hydrazide would have the greatest buffering capacity, we determined its acidity by titration with aq. hydrochloric acid (see below). It was found that the pK a ≈ 3.2, so all experiments were performed at a pH close to this value. Figure S39. Titration of methyl hydrazinocarboxylate with hydrochloric acid.

Solvent
Solvent was prepared by mixing 30 mL of H 2 O with 30 mL of MeCN and adjusting to pH ≈ 3.2 with 2M HCl (9.5 µL).

Hydrazide solution
11.25 mg of methyl hydrazinocarboxylate was dissolved in 4.16 mL of solvent, and adjusted to pH ≈ 3.0 with 30 μL of 2M HCl to give a 30 mM solution.

Axle solution
1.63 mg of SP was first dissolved in 3.18 mL of solvent. A 2.4 mL portion was diluted next with 36 mL of solvent to give a 50 µM solution.

Macrocycle solution
13.76 mg of CB7 was dissolved in 1.33 mL of solvent to give a 7.5 mM solution.

Hydrazonation of the pseudorotaxane (MCH form)
A UV-Vis cuvette was filled with 1.5 mL of axle solution and, if necessary, with the appropriate amount of macrocycle solution and left in the dark for 2 h to convert the SP form S34 into the MCH form. Next, 50 μL of hydrazide solution was added, and the absorbance at 282 nm was recorded every 3 s.

Hydrazonation of the pseudorotaxane( SP form)
A UV-Vis cuvette was filled with 1.5 mL of axle solution and, if necessary, with the appropriate amount of macrocycle solution. The solution was irradiated for 1 min using a 430 nm LED at 20% maximum power to convert the entire axle to the SP form. Immediately after that, the UV-Vis spectrum was recorded to determine A 0 . Then, 50 μL of hydrazide solution was added, and absorbance at 282 nm (maximum of hydrazone band) was recorded at ~ 4 s intervals. Within these intervals, the sample was irradiated with a 430 LED at 20% maximum power.
The reaction rate was calculated in respect to the product (hydrazone), on which the reaction shows a pseudo-first-order dependence due to a large excess of hydrazide. The product yield was calculated as follows: where A is the absorbance at a time t, A 0 is the absorbance at the beginning of the reaction, and A ∞ is the absorbance at 100% conversion.
The rate constant was calculated as the slope of the function:   Figure S42. Kinetic traces for the hydrolysis of SP-hydrazone in the presence of CB7 at pH=2.

Controlling the outcome of concurrent reactions
Axle ( Figure S45. Titration of 4-nitrobenzaldehyde with increasing amounts of CB7 (0.00 0.14, 0.27, 0.39, 0.50, 0.69, 0.86, 1.00, 1.24, 1.50, 1.75 equiv.) in MeCN-d 3 /D 2 O at 298 K. Figure S46. 1 H NMR spectra of Ald compound (1 equiv.), which emulates the benzaldehyde station in the pseudorotaxane, with methyl hydrazinocarboxylate (0.75 equiv.) and in a mixture with semicarbazide (0.75 equiv.) in the absence and presence of CB7 (1 equiv.) in MeCN-d 3 /D 2 O at 298 K. Based on more pronounced upfield shifts of a", b" and c" compared to a', b' and c', it can be concluded that CB7 ring in the complex with Ald-N product is closer to hydrazone residue than in the complex with Ald-O product, indicating some additional interaction between them.