Interplay between structural parameters and reactivity of Zr6-based MOFs as artificial proteases

Structural parameters influencing the reactivity of metal–organic frameworks (MOF) are challenging to establish. However, understanding their effect is crucial to further develop their catalytic potential. Here, we uncovered a correlation between reaction kinetics and the morphological structure of MOF-nanozymes using the hydrolysis of a dipeptide under physiological pH as model reaction. Comparison of the activation parameters in the presence of NU-1000 with those observed with MOF-808 revealed the reaction outcome is largely governed by the Zr6 cluster. Additionally, its structural environment completely changes the energy profile of the hydrolysis step, resulting in a higher energy barrier ΔG‡ for NU-1000 due to a much larger ΔS‡ term. The reactivity of NU-1000 towards a hen egg white lysozyme protein under physiological pH was also evaluated, and the results pointed to a selective cleavage at only 3 peptide bonds. This showcases the potential of Zr-MOFs to be developed into heterogeneous catalysts for non-enzymatic but selective transformation of biomolecules, which are crucial for many modern applications in biotechnology and proteomics.


Experimental Procedures
Synthesis procedures.H 4 TBAPy linker was synthesized according to published methods via standard Suzuki-Miyaura reaction between 1,3,6,8-tetrabromopyrene and 4-ethoxycarbonylphenylboronic acid with some modifications.The ester is saponified by excess of base to get full conversion of the ester and after acidification the carboxylate linker is obtained with an overall yield of 47 % (Figure S1).The Zr-MOF NU-1000 was synthesized according to literature. 1 NU-1000 is prepared via an upscaled solvothermal method in which zirconyl chloride octahydrate is mixed with the H 4 TBAPy linker in N,N-dimethylformamide with benzoic acid as modulator. 1The modulator is removed postsynthetically by washing with a 37 % HCl solution.After thermal activation, the desired product is characterized with powder X-ray diffraction, fourier-transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis and N 2 physisorption. 1 H NMR spectra are obtained by acidic digestion of 1 mg NU-1000 with 5 drops of D 2 SO 4 in 1 mL of DMSO-d 6 .
Before hydrolysis experiments, NU-1000 was activated at 120 °C for 20 h to evacuate adsorbed molecules from the pores.
Hydrolysis studies of dipeptides.800 µL of D 2 O was mixed with 4.32 mg NU-1000, corresponding to 2 µmol of Zr 6 clusters.200 µL of a 10 mM Gly-Gly (GG) solution was added and pD was adjusted to 7.4 with NaOD.Of note, pD was measured in a conventional pH meter observing the relationship pD = pH read + 0.41 to account for the isotopic composition of the solvent. 2 Reactions were done in individual vials at 60 °C and at certain time points, the solution of a vial was centrifuged at 14000 rpm for 2 x 10 min, after which the supernatant solution was sampled for analysis.Temperature dependence studies followed the same procedure with reactions performed at 37, 50, 60, 70 and 80 °C over time.For pD dependence, reactions were carried out in a pD range between 3.4 and 9.4 with intervals of one, each after 3 days of reaction at 60 °C.All the reactions were followed with 1 H NMR spectroscopy and 3-(trimethylsilyl)propionic-2,2,3,3-d 4 acid sodium salt (TMSPA-d 4 ) was added to the supernatant before measurements.Reaction rates were determined by fitting a first-order decay function to the data.
Hydrolysis of proteins.800 µL of H 2 O was added to 2 µmol NU-1000 and mixed with 200 µL of hen egg white lysozyme (HEWL) solution (0.1 mM); pH was adjusted to 7.0.Reactions were done at 60 °C and 15 µL samples, collected at different time points, each from a separate reaction vial, were mixed with 5 µL sample buffer.Samples were run on 18 % Laemmli gels in a OmniPAGE electrophoretic cell at 200 V for 1.5 h.Page Ruler unstained low range protein ladder was used as a standard.Silver staining was used for imaging the gel and analysis was done with ImageLab. 3ution studies of HEWL.800 µL of H 2 O was added to 2 µmol NU-1000 and mixed with 200 µL of hen egg white lysozyme (HEWL) solution (0.1 mM); pH was adjusted to 7.0.Reactions were done at 60 °C for 3 days and centrifuged.1 mL of elution buffer was added to the precipitate and stirred for 3 days at room temperature.15 µL samples were mixed with 5 µL sample buffer.Samples were run on 18 % Laemmli gels in a OmniPAGE electrophoretic cell at 200 V for 1.5 h.Page Ruler unstained low range protein ladder was used as a standard.Coomassie staining was used for imaging the gel and analysis was done with ImageLab. 3cycling experiment.Reactions were conducted the same way as described above for the hydrolysis of dipeptides.After 24 h, the catalyst was recovered by centrifugation, and was regenerated by stirring overnight in D 2 O, followed by centrifugation at 14000 rpm for 10 min.This cycle was repeated in total five times, each for 24 h with 2 mM GG and after each cycle, centrifugation and stirring overnight in D 2 O was performed before starting the next cycle.Conversion rates were calculated from 1 H-NMR analysis of the supernatant whereas stability was determined with powder X-ray diffraction and inductively coupled plasma optical emission spectrometry.
Adsorption studies of HEWL.800 µL of H 2 O was mixed with 2 µmol NU-1000.200 µL of a 0.1 mM hen egg white lysozyme (HEWL) solution was added and pH was adjusted to 7.0 with NaOH.Reactions were done in individual vials at room temperature and at certain time points, the solution of a vial was centrifuged at 14000 rpm for 2 x 10 min, after which the supernatant solution was sampled for analysis with SDS-PAGE, UV-Vis and Tryptophan fluorescence spectroscopy.
Adsorption studies of GG.A stock solution of 40 mM GG was prepared and dilutions were made by adding the appropriate amount of GG to 2 µmol NU-1000 in D 2 O resulting in samples of 1, 2, 4, 10, 20, 30 and 40 mM GG. Concentration series were measured at pD 7.4, 5.4 and 3.4 by adjusting pD with NaOD and DCl.After 6 hours of stirring at room temperature, samples were centrifuged and 1 H NMR analysis of the supernatant was performed to determine the substrate uptake.
Instrumentation. 1 H NMR spectra were recorded with a Bruker Avance 400 and 600 spectrometer in deuterated solvents and with 0.1 M TMSPA-d 4 as an internal reference.Spectra were analyzed using Topspin software. 4Powder X-ray diffraction (PXRD) patterns were collected on a Malvern PANalytical Empyrean diffractometer (in transmission mode) over a 1.3 -45° 2θ range, using a PIXcel3D solid state detector and Cu anode (Cu K α1 : 1.5406 Å; Cu K α2 : 1.5444 Å).Fourier-transform infrared spectra (FTIR) were recorded on a Bruker Vertex 70 spectrometer and analyzed with the Bruker OPUS software (version 7.5). 5The solid samples were measured directly, without sample preparation, using the attenuated total reflectance module (Platinum ATR).Scanning electron microscopy (SEM) micrographs were recorded using a JEOL-6010LV SEM after depositing a palladium/gold layer on the samples with a JEOL JFC-1300 autofine coater under Ar plasma.N 2 physisorption isotherms were measured on a Micromeritics 3Flex surface analyzer at -196 °C.Prior to measurements, samples were evacuated at 120 °C under vacuum for 12 h.Surface areas were calculated using the multi-point BET method applied to the isotherm adsorption branch taking into account the Rouquerol consistency criteria and the micropore volume was calculated at P/P 0 = 0.5. 6Thermal gravimetric analyses (TGA) were performed under air atmosphere on a NETZSCH STA 449 F3 Jupiter® thermal analyser with a heating rate of 4 °C per minute.Inductively coupled plasma optical emission spectrometry (ICP-OES) was measured on a PerkinElmer optical emission spectrometry Optima 8300 instrument.UV-Vis measurements were performed on a Varian Cary 5000 UV-VIS-NIR spectrometer.Tryptophan fluorescence was measured on a Edinburgh Instruments FLS980 Spectrofluorimeter.
Synthesis of tetraethyl-4,4′,4″,4″′-(pyrene-1,3,6,8-tetrayl)tetrabenzoate (1) 4 g of 1,3,6,8-tetrabromopyrene (7.72 mmol), 6.6 g of 4-ethoxycarbonylphenylboronic acid (34.02 mmol), 13.2 g of K 3 PO 4 (62.18mmol) and 0.6 g of Pd(PPh 3 ) 4 (0.52 mmol) are added together in 216 mL dioxane under N 2 atmosphere under continuous stirring.The suspension is stirred for 48-72 h at 90 °C under N 2 atmosphere.The solution becomes more and more yellow and turns black when the reaction is complete.160 mL of H 2 O is added and the mixture is cooled down.The solution is filtered and the yellow precipitate is washed twice with 80 mL of H 2 O and twice with 160 mL of acetone.The precipitate is then dissolved in 240 mL of boiling chloroform.The volume is reduced under vacuum by half and subsequently 240 mL of methanol is added.After 30 minutes a yellow precipitate is recovered by centrifugation and dried at 70 °C under vacuum for 12 h (yield: 3.17 g; 52 %).

Figure S2. 1 H
Figure S2.1  H NMR spectra recorded at various time increments for the reaction of 2 mM GG with 2 µmol NU-1000 at 60 °C and pD 7.4.

Figure S3 .Figure S4 .
Figure S3.First order decay fit of ln[GG] as a function of time for the reaction of 2 mM GG with 2 µmol NU-1000 at 60 °C and pD 7.4.a) b)

Figure S6 .
Figure S6.Influence of temperature on reaction rate of hydrolysis of 2 mM GG with 2 µmol NU-1000, pH 7.0, as a function of reciprocal temperature.

Figure S7 .
Figure S7.Arrhenius plot of reaction rate as a function of reciprocal temperature for reaction of 2 mM GG and 2 µmol NU-1000, pH 7.0.

Figure S8 .
Figure S8.ln(k/T) plotted as a function of reciprocal temperature for reaction of 2 mM GG with 2 µmol NU-1000, pH 7.0.

Figure S9 .
Figure S9.Coomassie stained SDS-PAGE gel of sample of HEWL incubated at 60 °C and pH 7.0, in the presence of 2 µmol NU-1000 under different elution techniques.

Figure S11 .
Figure S11.PXRD patterns of NU-1000 as synthesized and after incubation with 2 mM GG at 60 °C for 3 days at different pD values.

Figure S14 .
Figure S14.% Conversion of GG in function of time before (black) and after (red) removal of NU-1000.

Figure S16 .
Figure S16.PXRD patterns of NU-1000 as synthesized (black) and after three (red) and five (blue) reaction cycles with 2 mM GG, 60 °C, pH 7.0, 24 h and solvent exchange with D 2 O overnight.

Figure S17 .
Figure S17.Influence of amount of MOF on reaction rate for hydrolysis of 2 mM GG by NU-1000, 60 °C, pH 7.0.

Figure S20 .
Figure S20.Silver stained SDS-PAGE gel of supernatant after centrifugation of sample of HEWL incubated at 60 °C and pH 7.0, in the absence and presence of 2 µmol NU-1000 at different time increments.