Article Cite This: ACS Appl. Polym. Mater. 2019, 1, 1532−1539
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Small Organic Catalase Mimic Encapsulated in Micellar Artificial Organelles as Reactive Oxygen Species Scavengers Carina Ade, Edit Brodszkij, Bo Thingholm, Noga Gal, Fabian Itel, Essi Taipaleenmäki, Martin Juul Hviid, Philipp S. Schattling, and Brigitte Städler* Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus, Denmark
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S Supporting Information *
ABSTRACT: Biocatalytic intracellular active nanoreactors (artificial organelles) aim to support their host cells. Here, we report the first successful micelle-based artificial organelles containing a salen−manganese complex (EUK) as catalase mimic with intracellular activity in HepG2 cells to act as reactive oxygen species (ROS) scavengers. Four different EUKs were synthesized and compared in their ability to convert hydrogen peroxide to water and oxygen as free compounds and when encapsulated into micelles assembled from the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate). An EUK candidate with an asymmetric substitution of chemical groups at the ortho and the meta position (EUK-B) was identified as lead candidate. HepG2 cells continued proliferating when preincubated with low concentrations of EUK-B-containing micelles (MB). Importantly, HepG2 cells equipped with MB showed improved viability compared to the controls when stressed with paraquat, a compound that induces ROS generation. The intracellular activity of MB was supported by lower amounts of intracellular detectable ROS. This first report on the combination of artificial enzymes and artificial organelles further extends the opportunities in therapeutic cell mimicry. KEYWORDS: artificial organelle, EUK, micelles, HepG2 cells, reactive oxygen species
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INTRODUCTION Artificial organelles are nanoreactors with intracellular biocatalytic activity aimed to replace missing or lost cellular function. While diverse concepts for the assembly of nanoreactors were proposed as recently discussed in several comprehensive reviews,1−6 approaches with reported intracellular function remain scarce. Nonetheless, a first report on the subject dates back around 10 years,7 and substantial developments have been witnessed since then.8−11 Recent interesting examples include the use of subcompartmentalized reactors exhibiting intracellular activity to convert glucose into cytotoxic hydrogen peroxide (H2O2)12 or to interact with model substrates.13 In contrast to immortalized cell lines, the van Hest group illustrated the ability of intracellularly placed catalase-loaded polymersomes to protect patient-derived human-complex-I-deficient primary fibroblasts against the toxicity of exogenous H2O2.14 In another report, polymersomes with engineered protein gates to control the material flow were found functional in cell culture and in vivo in zebrafish embryos.15 In an attempt to circumvent challenges involved with artificial organelles internalized via endocytosis, microinjection was successfully employed to introduce exogenous organelles.16 In addition, artificial organelles based on GC-rich double-stranded oligonucleotides bound to nanogold showed a reduction in hepatotoxicity in cancer treatment due to the doxorubicin-scavenging ability of the nucleic acid strands both in cell culture and in mice.17 In another report, reactive oxygen species (ROS)-mediated apoptosis in HT-29 cells was demonstrated using copper© 2019 American Chemical Society
impregnated mesoporous silica nanoparticles containing a catalase inhibitor.18 Apart from the latter two cases, artificial organelles employ enzymes as their active entity, inherently limiting their long-term performance since enzymes often lose their catalytic function within a few days. Metallosalenes, first reported over 80 years ago,19 have fascinated researchers due to the vast structure−function correlation opportunities they offer.20 Eukarion (EUK) compounds are salen−manganese complexes, which possess superoxide dismutase and catalase activities and are an excellent candidate to be considered in the context of artificial organelles. These compounds offer broad opportunities to manipulate their structure and coordination chemistry for envisioned applications as recently outlined in detail by Erxleben.21 Unlike enzymes, EUK has reduced cost, higher stability, and it exhibits less environmental sensitivity. These molecules have been considered in reactive oxygen speciesrelated diseases; i.e., EUK compounds were associated with a positive effect in models for Alzheimer’s disease,22 Parkinson’s disease,23 multiple sclerosis,24 and a rodent stroke model25 as well as their ability to mitigate injury to normal tissues caused by ionizing radiation.26 However, biomedically relevant modifications such as the addition of targeting units or the encapsulation of EUK into nanoparticulate formulations remain largely unexplored. In this context, mitochondria Received: April 2, 2019 Accepted: May 3, 2019 Published: May 3, 2019 1532
DOI: 10.1021/acsapm.9b00308 ACS Appl. Polym. Mater. 2019, 1, 1532−1539
Article
ACS Applied Polymer Materials targeting of EUK-13427 and the loading of EUK-134 into antiPECAM (platelet endothelial cell adhesion molecule)modified liposomes for targeting the endothelium28 were illustrated. Herein, we report the first use of small organic enzyme mimics, imitating catalase, in micellar artificial organelles with intracellular activity in HepG2 cells as ROS scavengers (Scheme 1). Specifically, we (i) synthesized a selection of
peroxide (30%), LysoTracker Red DND-99, and CellMask Deep Red Plasma Membrane Stain were obtained from Thermo Fisher Scientific. Dimethyl-d6 sulfoxide, chloroform-d1, chloroform anhydrous (≥99%), ethanol, methanol, toluene, hexane, diethyl ether, chloroform, acetone, and hydrochloric acid (HCl) were purchased from VWR. Fetal bovine serum (FBS), penicillin (10000 U mL−1), and streptomycin (10000 μg mL−1 Pen Strep) were purchased from Gibco Life Technologies. Uncoated μ-Slide VI 0.4 was purchased from iBidi. Poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate) (pCMA-b-pDMAEMA, P1) and fluorescein-labeled P1 (P1f) were synthesized by RAFT polymerization following a prior published protocol29 (P1: Mn_pCMA = 5.2 kDa, Mn_PDMAEMA = 21 kDa; P1f: Mn_pCMA = 5.2 kDa, Mn_pDMAEMA = 28 kDa, Mn_pflMA = 2.8 kDa). The NMR spectra can be found in the Supporting Information (Figure S1b). All experiments were performed using a HEPES buffer solution consisting of 10 mM HEPES and 150 mM NaCl at pH 7.4. Ultrapure water (18.2 MΩ cm−1 resistance) was provided by an ELGA Purelab Ultra system (ELGA LabWater, Lane End). EUK Synthesis. An overview reaction scheme (Scheme S1) and the NMR spectra (Figure S1a) can be found in the Supporting Information. EUK-A. o-Vanillin (3 g, 19.7 mmol) was dissolved in methanol (20 mL). 3,4-Diaminobenzoic acid (1.5 g, 9.86 mmol) was added, and the mixture was stirred and refluxed for 2 h. The product was then filtered off and washed with cold methanol four times, yielding an orange powder. Yield: 90%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.82 (6, CH3−O−), 6.92, 7.15 (tr, 1−1H), 7.2−7.33 (m, 2−2H, Ar2), 7.53 (d, 1H, Ar1), 7.94−7.97 (s, d, 1−1 H, Ar1) (1−1 H), 8.96 and 9.02 (s−s, 1−1H, CHN−)), 10.27, 12.62 and 12.84 (s, −OH intermol H-bond). To activate the EUK-A ligand, the compound (1 g, 2.19 mmol) was suspended in ethanol (10 mL); manganese(II) acetate (650 mg, 2.20 mmol) was added at room temperature and stirred overnight. Ethanol was removed, and the product was washed four times with acetone, yielding a dark brown powder. Yield: 95%. EUK-B. 2,4-Dihydroxybenzaldehyde (0.85 g, 6.16 mmol) and 4allyl-2-hydroxybenzaldehyde (1.00 g, 6.16 mmol) were suspended in methanol (30 mL). o-Phenylenediamine (0.670 g, 6.16 mmol) was added, and the mixture was stirred and refluxed for 2 h. The product was then filtered off and washed with cold methanol four times, yielding a bright orange powder. We note that EUK-B might contain EUK-C and EUK-D. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.39 (d, 2−2 H, −CH2−), 5.05 (m, 2−2 H, H2C=), 6.04 (br, −CH=), 6.36, 6.98 (br, Ar), 7.00−7.60 (br, Ar), 8.75−8.95 (br, CHN−), 13.11−13.51 (br, −OH intermol H-bond). To activate the EUK-B ligand, the compound (30 mg) was suspended in ethanol (1 mL), and manganese(II) acetate (19.6 mg, 0.080 mmol) was added at room temperature and stirred for 2 h. Ethanol was removed, and the product was washed four times with acetone, yielding a black powder. EUK-C. 4-Allyl-2-hydroxybenzaldehyde (517 mg, 3.08 mmol) was suspended in methanol (10 mL). o-Phenylenediamine (173 mg, 1.54 mmol) was added, and the mixture was stirred for 4 h at 40 °C and further 24 h at RT. The product was separated and washed with cold methanol three times, yielding a bright orange, viscous liquid. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.39 (d, 2−2 H, −CH2−), 5.05 (m, 4H, H2C=), 6.03 (m, 1−1H, −CH=), 6.94 (tr, 1−1H, Ar2), 7.30 (d, 1−1H, Ar2), 7.4−7.5 (d and m, 1−1 H and 4H, Ar2 and Ar1), 8.95 (s, 1H, CHN−), 13.51 (s, −OH intermol H-bond). To activate the EUK-C ligand, the compound (600 mg, 1.51 mmol) was suspended in ethanol (10 mL), and manganese(II) acetate (367 mg, 150 mmol) was added at room temperature and stirred overnight. Ethanol was removed, and the product was washed four times with acetone, yielding a brown powder. Yield: 40%. EUK-D . 2,4-Dihydroxybenzaldehyde (7.88 g, 5.71 mmol) was dissolved in methanol (15 mL), and o-phenylenediamine (3.09 g, 2.85 mmol) dissolved in methanol (25 mL) was added slowly and stirred under reflux. After 2 h the solution was allowed to cool to room temperature and was stirred for another 16 h. The product was filtered off and was washed three times with cold methanol, yielding a
Scheme 1. (a) Schematic Illustration of the Internalization of the Micellar Artificial Organelles Including Their Lysosomal Escape and Intracellular Biocatalytic Activity; (b) Chemical Structure of Amphiphilic Block Copolymer Poly(cholesteryl methacrylate)-block-Poly(2(dimethylamino)ethyl methacrylate) (P1, PCMA-bPDMAEMA) Used To Assemble the Artificial Organelles and the Salen−Manganese Complex EUK That Serves as Catalase Mimic
EUK complexes and compared their catalytic activity, (ii) loaded the different EUKs into the core of micellar artificial organelles made from the amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate) and assessed their properties including the catalytic activity, and (iii) biologically evaluated the micellar artificial organelles with the highest catalytic activity in HepG2 cells considering viability, uptake, lysosomal escape, and intracellular catalytic activity.
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EXPERIMENTAL SECTION
Materials. o-Vanilin, 3,4-diaminobenzoic acid, 2,4-dihydroxybenzaldehyde, 4-allyl-2-hydroxybenzaldehyde, o-phenylenediamine, 2-(dimethylamino)methacrylate (DMAEMA), fluorescein O-methacrylate (flMA), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester (CTA-NHS), 2,2′-azobis(2-methylpropionitrile) (AIBN), manganese(II) acetate, catalase, Cell Counting Kit-8 (CCK-8), EUK-134, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), L-glutamine (200 mM), MEM non-essential amino acid solution 100×, sodium chloride (NaCl), paraquat dichloride hydrate (PQ), phosphate-buffered saline (PBS), and trypsin-EDTA 0.25% were purchased from Sigma-Aldrich. Amplex UltraRed Reagent, CellROX Green Reagent, dimethyl sulfoxide (DMSO), horseradish peroxidase (HRP), stabilized hydrogen 1533
DOI: 10.1021/acsapm.9b00308 ACS Appl. Polym. Mater. 2019, 1, 1532−1539
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ACS Applied Polymer Materials light orange powder. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 6.30 (d, 1−1H, Ar2), 6.40 (d, 1−1H, Ar2), 7.30−7.38 (m, 4 H, Ar1), 7.43 (d, 1−1H, Ar2), 8.75 (s, 1H, CHN−), 10.25 (s, −OH), 13.38 (s, −OH intermol H-bond). To activate the EUK-D ligand, the compound (349 mg, 1.427 mmol) was suspended in ethanol (20 mL), and manganese(II) acetate (500 mg, 1.327 mmol) was added at room temperature and stirred for 2 h. Ethanol was removed, and the product was washed four times with acetone, yielding a brown powder. Micelle Assembly and Characterization. 1 mL (2 mg mL−1 in ethanol) of P1 was added to a 25 mL round-bottom flask and dried while slowly turning the flask under a steady stream of N2 until a visible dry film was left. The flask was put on a vacuum line for at least 1 h followed by rehydration with 1 mL of HEPES buffer to obtain a 2 mg mL−1 solution of the polymer. Then, the solution was dialyzed against HEPES buffer for 35 h. The buffer was changed 3−4 times, and M0 were obtained. For EUK-loaded micelles, 1 mL (0.5 mg mL−1 in ethanol) of EUK and 1 mL (2 mg mL−1 in ethanol) of P1 were mixed before drying. Micelles loaded with EUK will be named MX, where X refers to the type of used EUK. To obtain MB, 10% ethanol was added to the buffer during rehydration. All micelle solutions were dialyzed against HEPES buffer for at least 24 h. For fluorescently labeled micelles, P1 and P1f were mixed in 1:1 (w/w) yielding in M0f. The hydrodynamic radius of the micelles (Z-average size) was determined by using dynamic light scattering (DLS, Malvern Zeta sizer Nano-590) at 25 °C. In addition, the size of the micelles was assessed from transmission electron microscopy (TEM) by measuring the major axis (D1) and the minor axis (D2) of 120 individual micelles in each sample using Inkscape software. TEM grids were prepared by glow discharge treatment (30 s, PELCO easiGlow) followed by adding 5 μL of the micelle stock solution, washing the grids twice with 5 μL of ultrapure water after 1 min, and staining twice with 5 μL of 2% uranyl acetate. TEM images were taken using a Tecnai G2 Spirit instrument (TWIN/BioTWIN, FEI Co.). The critical micelle concentration (cmc) was determined by fluorescent measurement of encapsulated pyrene.30 10 μL of a 200 μM pyrene solution (25% THF/HEPES) was added to a 90 μL micelle solution with concentrations between 0 and 2 mg mL−1. The fluorescence signal was read out with a multimode plate reader (EnSight, PerkinElmer) (λex = 337 nm) at 374 nm (pyrene 1) and 383 nm (pyrene 3). The concentration of the micelle solution was estimated. First, the buffer was exchanged to DI water by spin columns (Vivaspin 500, MWCO 3000, 10K rpm for 10 min, followed by refilling with water and vortexing for 20 s, repeated eight times) or dialyzed against DI water. Empty Eppendorfs were measured with a sensitive balance (Mettler Toledo, Excellence Plus XP6), filled with a known amount of micelle solution in water (between 150 and 400 μL), freeze-dried, and remeasured. The measured concentrations were between 0.6 and 1.2 mg mL−1. Catalytic Activity. The activity of EUK to convert H2O2 into water and oxygen was assessed using the Amplex UltraRed assay. 25 μL of 10 μM H2O2 and 25 μL of EUK in water/DMSO (0−2 mM EUK), whereas DMSO never exceeded 2.5 vol %, were added to the wells of a black 96-well plate and allowed to incubate for 30 min at room temperature. 50 μL of the working solution (0.2 U mL−1 HRP and 0.1 mM Amplex UltraRed Reagent in water) was added to each well, which was then protected from light. The fluorescence signal was read out with a multimode plate reader (EnSight, PerkinElmer) (λex = 568 nm and λem = 590 nm) 30 min after the addition of the working solution. The background signal was subtracted from all values using the read-out from a sample consisting of 0 μM H2O2 in water. In addition, the fluorescent signals were normalized to the value obtained for 5 μM H2O2. Two independent repeats were performed. The decrease in fluorescence intensity due to the dark-brown color of the EUKs during Amplex UltraRed assay measurement was assessed. To this end, 25 μL of 10 μM H2O2 was incubated with the working solution for 30 min. 25 μL of EUK-B with increasing concentrations (0−1 mM) was added, and the fluorescence intensity
was recorded and compared to the sample without EUK-B (Figure S3b). The activity of the micelles was assessed by mixing 25 μL of micelle stock solution with 25 μL of 10 μM H2O2. The remaining H2O2 after a 30 min incubation time was determined to be outlined for the EUK activity. Three independent repeats were performed. For the long-term catalytic activity assessment, the samples were prepared as described above and incubated at 37 °C in Eppendorf vials. Samples were taken from the top layer to avoid precipitates. Two independent repeats were performed. Loading Efficiency of EUK into Micelles. The absorptions of 50 μL solutions of EUK-A/B/D in water/DMSO (no more than 2.5%) and EUK-C in 100% ethanol (0−2 mM concentration) were recorded with a multiplate reader (EnSight, PerkinElmer) in a range of λ = 280−780 nm (Figure S3a, example of 1 mM solutions). Read-outs at the absorption maximum (λ = 354 nm for EUK-B/C/D and λ = 310 nm for EUK-D) were used to make a calibration curve (not shown). 50 mL of EUK loaded micelles was also measured. Shifts of the recorded curves (read-outs at λ = 310/338/371/354 nm for EUKA/ B/C/D, respectively) were used to determine the loading efficiency. Biological Evaluation. The immortalized human hepatocellular carcinoma HepG2 cell line was purchased from European Collection of Cell Cultures. HepG2 cells were cultured in 75 cm2 culture flasks in Minimum Essential Medium Eagle with Earle’s Salts and sodium bicarbonate (from Sigma-Aldrich) supplemented with 10% FBS, 2 mM L-glutamine and 1% MEM Non-essential Amino Acid Solution, 100 μg mL−1 streptomycin, and 100 U mL−1 penicillin at 37 °C and 5% CO2. All cell experiments were conducted in at least three independent repeats. Data are displayed as mean ± standard deviation (SD), indicating the number n of independent repeats. The statistical significance used to compare the distribution was determined using a two-way ANOVA followed by a Tukey’s multiple comparison posthoc test (*p < 0.05). Cell Viability. 50000 HepG2 cells per well were seeded in a 96-well plate (100 μL media per well) and allowed to adhere overnight at 37 °C in 5% CO2. Different volumes of MB stock solution were added to the wells and incubated for 6 or 24 h. In the former case, the MBcontaining medium was exchanged with fresh medium, and the cells were allowed to recover for 18 or 42 h at 37 °C in 5% CO2. Following on, the cells were washed twice with PBS buffer, and 110 μL of medium containing 10 μL of Cell Counting Kit-8 solution (CCK-8) was added to each well followed by 2 h incubation at 37 °C in 5% CO2. Then, 100 μL of the solution from each well was transferred to a new 96-well plate and analyzed using a multimode plate reader (EnSight, PerkinElmer) by measuring the absorbance (λ = 450 nm). The obtained values were normalized to untreated cells after 24 h incubation. Uptake Efficacy. 50000 HepG2 cells per well were seeded in a 96well plate (100 μL media per well) and allowed to adhere overnight at 37 °C in 5% CO2. A 0.5 μL aliquot of MBf stock solution in HEPES buffer was added per well followed by 3, 6, or 24 h incubation at 37 °C in 5% CO2. Then, the cells were washed twice with PBS buffer, and 35 μL of trypsin-EDTA (5 min at 37 °C) was used to detach the cells. Trypsin was neutralized with 65 μL of cell medium before analysis by flow cytometry (Guava easyCyte Single Sample Flow Cytometer, Merck) using an excitation wavelength of 488 nm. At least 2000 cells were analyzed. The autofluorescence of untreated cells was subtracted. Additionally, the cell mean fluorescence values were normalized to the fluorescence intensity of MBf, which was measured in the multiplate reader to minimize MBf batch-to-batch variations. Lysosomal Escape of M0f. 100000 HepG2 cells per channel were seeded in an ibidi VI 0.4 uncoated slide (120 μL media per channel) and allowed to adhere overnight at 37 °C in 5% CO2. 20 μg mL−1 M0f was added to each channel and incubated for 6 or 24 h. The LysoTracker dye and corresponding cell media were heated to 37 °C prior to being mixed for a final Lysotracker concentration of 100 nM, which was added to each channel (120 μL) and incubated for 1.5 h. In addition, the CellMask Deep Red Plasma Membrane Stain was diluted to a final concentration of 5 μg mL−1 in cell media or PBS, 1534
DOI: 10.1021/acsapm.9b00308 ACS Appl. Polym. Mater. 2019, 1, 1532−1539
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ACS Applied Polymer Materials which was also heated to 37 °C before. After addition of 120 μL of the CellMask membrane stain solution to each channel and an incubation of 10 min at 37 °C, the cells were washed once with PBS and 120 μL of PBS was added to each channel for storage. The cells were immediately visualized using a Zeiss LSM700 confocal laser scanning microscope (CLSM) (Carl Zeiss, Germany). Two images with the same settings were taken by using either an emission cutoff at 572 or 644 nm to ensure the absence of crosstalk. Three independent experiments were performed. The colocalization of the micelles with the lysosomes was determined via the total Pearson correlation coefficient (PCC) using the Coloc 2 plug-in in ImageJ. Subtraction of the background (50 pixel ball pen size) and adjustments of the contrast was performed before the analysis for all of the images. Intracellular Activity of MB. 50000 HepG2 cells per well were seeded in a 96-well plate and allowed to adhere overnight at 37 °C in 5% CO2. 0−2 μL of MB and MD stock solution was added to the wells and incubated for 6 h. The cells were washed twice with PBS buffer and left to recover for 16−18 h in media. Next, PQ (dissolved in HEPES buffer) was added to the cells to a final concentration of 75 μg mL−1 and incubated for 24 h before measuring the cell viability as described above. The obtained absorption values were normalized to untreated cells after 24 h incubation. Intracellular ROS. 50000 HepG2 cells per well were seeded in a 96-well plate and allowed to adhere overnight at 37 °C in 5% CO2. Then, the cells were incubated with 0−2 μL of MB or MD for 6 h, washed twice with PBS buffer, and left to recover for 16−18 h in media before adding PQ (75 μg mL−1 final concentration) for 3 h to stimulate the intracellular ROS. Then, CellROX Green Reagent (5 μM final concentration) was added to the wells and allowed to incubate for 30 min. Following detachment via trypsin, the cell mean fluorescence (CMF) was measured by flow cytometry. Autofluorescence from the cells incubated with CellROX in the absence of PQ was subtracted from the CMF. The obtained values were normalized to the CMF of the cells not exposed to PQ, resulting in the nCMF.
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RESULTS AND DISCUSSION EUK Synthesis. As a first step, we synthesized four alternatives to prior reported EUK versions with the aim to obtain catalase mimics with higher activity toward hydrogen peroxide (H2O2). It was early on shown that EUK compounds with 3-methoxy substituents and aromatic bridges exhibited improved catalase activity, e.g., EUK-178.31 The catalytic performance of these molecules strongly depends on their electron donation ability since the catalase activity of the salen−manganese complexes rely on the conversion of MnIII to MnV. Therefore, the EUK compounds were designed to possess enhanced electron-donor abilities compared to EUK134 (Figure 1a). In addition, pendant groups were chosen to introduce options for future further chemical modification, i.e., the addition of the carboxylic acid group in EUK-A. This modification should also improve the solubility in aqueous environment. EUK-B, EUK-C, and EUK-D were synthesized to compare the symmetric and asymmetric substitution of chemical groups at the ortho and the meta position. The hydroxy group was expected to enhance water solubility while the allyl group contributes to the stability. (For details on the synthesis see Supporting Information Scheme S1 and Figure S1a and the related text.) The catalytic ability of these five different EUKs was elucidated by comparing their ability to convert 5 μM H2O2 to water and oxygen within 30 min.
