Coupling two enzymes into a tandem nanoreactor utilizing a hierarchically structured MOF† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01438k Click here for additional data file.

A hierarchically structured MOF is utilized to couple two enzymes in a tandem manner. A stepwise encapsulation with a specific order is the only way to achieve this goal.

. Rietveld refinement patterns of PCN-888 using synchrotron PXRD data (λ = 0.72768Å): observed (blue), calculated (red), and difference (grey) profiles are shown; the tick marks below the curves indicate Bragg positions. The X-ray diffraction pattern between 1.1° and 4.2° is magnified in the inset. The full pattern shows a precise match between the experimental PXRD data and those simulated from the proposed structure.

Materials and Instruments
Aluminum chloride hexahydrate (AlCl3 . 6H2O), anhydrous aluminum chloride, chromium(VI) oxide (CrO3), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and N,N-diethylformamide (DEF) is purchased from Alfa Aesar. Phosphorous pentachloride (PCl5), phosphorous oxochloride (POCl3), glucose and horseradish peroxidase (HRP) is purchased from Sigma Aldrich. Glucose oxidase and trypsin is purchased from MP biomedicals. Synchrotron powder X-ray diffraction (PXRD) was carried out on a Bruker D8-Discover diffractometer equipped with a Mo sealed tube (λ = 0.72768Å) on the beamline 17-BM at the Advanced Photon Source, Argonne National Laboratory. Nuclear magnetic resonance (NMR) data were collected on a Mercury 300 spectrometer. Low pressure gas adsorption measurements were performed on an ASAP 2020 with the extra-pure quality gases. The UV-Vis absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer. Scheme S1. Isoreticular extension strategy and synthetic routes of HTB ligand. In a 100 mL Schlenk flask was charged 8 g anhydrous AlCl3 and 20 mL anhydrous toluene. The mixture was heated to 60 o C under stirring. 3.2 g compound 2 was added to the flask in small portions over 20 minutes. The reaction mixture changed from yellow-green to dark red once compound 2 was added in and white gas was generated. The reaction was kept at 60 o C for 5 hours and it was cooled down to room temperature. The mixture was poured into ice water with rigorous stirring. The resulting yellow solid was filtered and washed with water and cold THF. Yield: 1. 1 g compound 3, 20.5 g HOAc, 1.23 mL H2SO4 was added in a 100 mL flask in ice bath. 2.03 g CrO3 was added slowly to the flask. Then 1.35 g Ac2O was added in. The flask was kept at 0 o C for 10 minutes and it was allowed to stay at room temperature overnight. The reaction mixture was poured into 300 mL deionized water and stirred for 1 hour. The solid was collected by centrifuge and washed with water for three times. The product was dissolved with 2.5 M KOH aqueous solution and the residue solid was removed by centrifuge. To the clear solution was added 6 M HCl until pH=1. The product is collected by filtration. Yield: 0.5 g, yellow solid. 1 H NMR (300MHz, DMSO): 8.14 (d, 2H), 8.50 (d, 2H).

Synthesis of PCN-888
AlCl3•6H2O (20 mg), HTB (10 mg) and TFA (0.1 mL) was dissolved in 2 mL DEF in a 4 mL pyrex vial. The mixture was kept in a 135 o C oven for 10 hours. The solid was collected by centrifuge. Yield: 4 mg. Enzyme immobilization of PCN-888 12 mg glucose oxidase (GOx) was dissolved in 2 mL water. 20 mg horseradish peroxidase (HRP) was dissolved in 2 mL water. 4 mg as-synthesized PCN-888 was washed with water twice and dispersed in 1 mL water. 1 mL GOx solution was added to the MOF slurry and incubated at room temperature for 50 minutes. The solid was collected by centrifuge and washed with DI water twice. The MOF was dispersed in 1 mL water. 1mL HRP solution was added to the MOF slurry and incubated at room temperature for another 50 minutes. The solid was collected by centrifuge and washed with water twice. The supernatants were collected for the determination of the amount of immobilized enzymes in PCN-888.

Activation of PCN-888
Freshly prepared PCN-888 was washed with DMF for three times. The sample was evacuated with supercritical CO2 in a Tousimis Samdri PVT-3D critical point dryer. Briefly, the DMF-containing sample was placed in the chamber and DMF was completely exchanged with liquid CO2. After that the chamber containing the sample and liquid CO2 was heated up around 40 °C and kept under the supercritical condition (typically 1300 psi) for 30 minutes. The CO2 was slowly vented from the chamber at around 40 °C, yielding porous material. The yellow solid was further activated by heating at 150°C for two hours.

The measurement of kinetic parameters of PCN-888-en/PCN-888-enR
ABTS and glucose are dissolved in 1mL 50 mM pH=7.4 tris-HCl buffer solution to yield desired concentration of ABTS (10 mM) and glucose (0.6-9 mM) solution. MOF powder is added in the solution and the system is monitored by UV-vis spectrometer at 403 nm spontaneously. The measurement is stopped when the adsorption reaches plateau.

The measurement of kinetic parameters of free enzymes
The procedure is similar with that of MOF nanoreactors instead of the glucose concentration range (1-9 mM), GOx concentration (0.0141 μM,) and HRP concentration (0.05057 μM).

Trypsin digestion conditions of PCN-888-en
PCN-888-en (10 mg) was suspended in 2 mL 50 mM pH=7.4 tris-HCl buffer. 2 mg trypsin was added and the media was incubated at 37 o C for 60 minutes. The powder was obtained by centrifugation and washed with fresh buffer for 3 times.