Note Cite This: J. Org. Chem. 2018, 83, 4882−4887
pubs.acs.org/joc
Supramolecular Encapsulation and Bioactivity Modulation of a Halonium Ion by Cucurbit[n]uril (n = 7, 8) Hang Yin,†,# Qiaoxian Huang,†,# Wenwen Zhao,† David Bardelang,‡ Didier Siri,‡ Xiuping Chen,† Simon M. Y. Lee,† and Ruibing Wang*,† †
State Key Laboratory of Quality Research in Chinese Medicine, and Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau China ‡ Aix Marseille University, CNRS, ICR, Marseille, France S Supporting Information *
ABSTRACT: This is the first time that cucurbit[7]uril and cucurbit[8]uril have been demonstrated to serve as synthetic receptors for a halonium guest species, diphenyleneiodonium, modulating its bioactivities and alleviating its cardiotoxicity, which further expands the onium family of guest molecules for the cucurbit[n]uril family and provides new insights for haloniumcucurbit[n]uril host−guest chemistry and its potential applications in pharmaceutical chemistry.
A
with both CB[7] and CB[8] (Figure 1), respectively, and the encapsulation of this halonium compound by CB[7] and CB[8] positively improved its inhibitory activity toward reactive oxygen species (ROS) generation and alleviated its cardiac toxicity. DPI, a widely used uncompetitive inhibitor of flavoenzymes, has been identified as a hypoglycemic agent that is able to block gluconeogenesis and mitochondrial respiration in rat liver.10 It was previously reported that DPI has the ability to reduce the activity of NADPH oxidase,11 nitric oxide synthase,12 and xanthine oxidase.11a In principle, these pharmacological properties of DPI could be leveraged to protect the heart, whereas a previous work demonstrated that DPI in fact induced cardiomyopathy on a rat model, suggesting its cardiac toxicity,13 which has limited the biomedical applications of DPI. Previously, we have demonstrated that supramolecular formulation of a variety of drug molecules may improve the biological activities of the guest drugs, e.g., reducing their nonspecific toxicities and maintaining or improving their therapeutic efficacies.4,5 Encouraged by these preliminary successes, we studied the supramolecular encapsulation of DPI by CB[7] and CB[8], respectively, by examining their binding behaviors as well as the influence of encapsulation on DPI’s bioactivities, such as its inhibitory activity against ROS generation in vitro and cardiac toxicity in vitro and in vivo. Binding interactions of DPI toward CB[7] and CB[8] were, respectively, studied by 1H NMR spectroscopy at neutral pH conditions (Figure 2 and Figure S1). As shown in Figure 2a, in the presence of increasing concentrations of CB[7] (up to 2.2 equiv), all protons of DPI (1 mM) experienced upfield shifts (complexation-induced shifts, Δδ < 0). Relatively large upfield
mong the families of synthetic and natural macrocycles, the cucurbit[n]uril (CB[n], n = 5−8 and 10) family, containing two oxygen-laced hydrophilic portals and one hydrophobic cavity, has attracted increasing attention due to their unique binding properties toward a variety of guest species.1 Compared to other members of the CB[n] family, due to its superior water-solubility property,1,2 good biocompatibility,3 and suitable cavity size to accommodate a variety of guest molecules of biological interest,4 CB[7] has attracted the most attention in pharmaceutical sciences.4,5 Meanwhile, with a relatively large cavity size that can simultaneously accommodate two guest molecules to form 2:1 or 1:1:1 ternary complexes,1 CB[8] has been extensively studied in various research areas including biomaterial sciences.6 The majority of guest species that were shown to bind strongly with CB[7] and/or CB[8] are cationic molecules as they may be drawn together with the carbonyl-laced portals of the host molecules via cation-dipole interactions.1 A variety of onium species such as ammonium,7 phosphonium,7a oxonium,8 sulfonium,7 and carbonium9 ions have been demonstrated to be suitable guest molecules for CB[n], whereas halonium ions have never been studied so far in the area of CB[n] chemistry. In this study, we demonstrated for the first time that a representative, bioactive halonium ion, diphenyleneiodonium (DPI, Figure 1), complexed strongly
Figure 1. Molecular structures of DPI (with the hydrogen atom numerically assigned) and of CB[n] (n = 7 or 8). © 2018 American Chemical Society
Received: February 27, 2018 Published: April 4, 2018 4882
DOI: 10.1021/acs.joc.8b00543 J. Org. Chem. 2018, 83, 4882−4887
Note
The Journal of Organic Chemistry
doubly charged peak at m/z 721.17, which is consistent with the calculated m/z values of 1441.31 (DPI-CB[7])1+ and 721.16 (DPI-CB[7] + H)2+, respectively (Figure S3a). Similarly, the binding stoichiometry of DPI for CB[8] was determined by the same continuous variation method affording a Job’s plot titration curve whose maximum is centered at 0.33 for the [CB[8]]/([CB[8]]+[DPI]) ratio, which is in agreement with 2:1 DPI/CB[8] complexation (Figure S2b). ESI-MS also supported this stoichiometry (Figure S3b), with a doubly charged peak observed at m/z 943.17, corresponding to the calculated m/z value of 943.16 (C72H64N32O16I2)2+, which is consistent with ((DPI)2-CB[8])2+ complexes. To determine the binding constant of DPI toward CB[7], the UV−visible absorption spectrum of DPI (0.025 mM) was monitored during titration with increasing concentrations of CB[7] (0−3.0 equiv). As shown in Figure S4, the absorbance of DPI at 263 nm gradually decreased with the addition of CB[7]. Nonlinear best fit of the absorbance at 263 nm against CB[7]’s concentration yielded a binding affinity, Ka, of (6.63 ± 0.42) × 104 M−1. In addition, we employed isothermal titration calorimetry (ITC) to confirm the complexation of DPI toward CB[7] and CB[8]. As shown in Figure 3 and Table S1, ITC of Figure 2. Excerpt of the aromatic region of 1H NMR spectra of DPI (1 mM) in the absence and in the presence of 0.5, 1.2, and 2.2 equiv of CB[7] (a) and DPI (0.01 mM) in the absence and in the presence of 0.5, 1.2, 2.4, and 3.6 equiv of CB[8] (b).
shifts of H1 (Δδ = −0.260 ppm), H2 (Δδ = −0.330 ppm), H3 (Δδ = −0.322 ppm), and H4 (Δδ = −0.514 ppm) suggest that the entire guest molecule was encapsulated in the cavity of CB[7], with H4 protons likely situated around the center of the cavity due to their largest complexation-induced shift of the resonances. Similarly, all DPI protons experienced significant upfield shifts in the presence of 1.2 equiv of CB[8], however, with opposite-direction movements when the CB[8] concentration was further increased (up to 3.6 equiv, Figure 2b). Larger complexation-induced δ changes for H1 (Δδ = −0.806 ppm), H2 (Δδ = −0.612 ppm), H3 (Δδ = −0.672 ppm), and H4 (Δδ = −0.817 ppm) in the presence of 1.2 equiv of CB[8] suggest that DPI was deeply encapsulated in the cavity of CB[8] (with H1 and H4 protons situated close to the center of the cavity due to their large complexation-induced resonances shifts) and experienced a stronger shielding effect than that in the case of CB[7], which could be a result of the presence of a second DPI in the cavity of CB[8] (likely in a binding ratio of 2:1 DPI/CB[8]). Less significant Δδ values and much broader peaks in the presence of higher concentrations of CB[8] suggest that 1:1 host−guest species may have formed when a large excess of CB[8] was present with possibly multiple binding equilibria. The stoichiometry of the DPI-CB[7] complex was determined by the continuous variation titration method as monitored by UV−visible spectroscopy. During the titration, the concentrations of DPI and CB[7] in water were continuously varied while keeping the total concentration of the system constant at 0.025 mM. The Job’s plot for DPI with CB[7] monitored by UV−visible changes at 265 nm, showing a clear maximum at 0.50 for the [CB[7]]/([CB[7]]+[DPI]) ratio, in line with 1:1 DPI/CB[7] complexation in this concentration range (Figure S2a). ESI mass spectrometry (MS) also supports a 1:1 stoichiometry for DPI toward CB[7], with the presence of a singly charged peak at m/z 1441.34 and a
Figure 3. ITC thermograms (top) and dependence of enthalpic changes against the guest−host molar ratios during the titrations. The solid line represents the best fit for a “one set of binding sites” isothermal model. CB[7] solution (2 mM) was injected into a DPI solution (0.2 mM) placed in the cell (a), and a DPI solution (1.2 mM) was injected into a CB[8] solution (0.04 mM) placed in the cell (b).
the DPI-CB[7] pair confirmed the previously found 1:1 binding ratio between the guest and the host and yielded a binding affinity of (3.13 ± 0.16) × 104 M−1, which is consistent with the Ka value determined by UV−visible spectroscopy. The ΔH and TΔS values obtained from ITC are −12.6 and 13.1 kJ/mol, respectively, indicating that the complexation of DPI with CB[7] is both enthalpy and entropy driven. Similarly, ITC of the DPI-CB[8] pair confirmed the 2:1 (DPI/CB[8]) stoichiometry and afforded binding constants of (1.50 ± 0.38) × 106 M−1 for each step of the 2:1 guest−host binding and 2.26 × 1012 M−2 for the final 2:1 complexation. Similar to the CB[7] binding, the ΔH and TΔS values of −23.2 and 12.1 kJ/mol, respectively, suggested that the formation of the DPI2CB[8] ternary complexes is favored by both enthalpic and entropic contributions. The enthalpic changes of both DPICB[7] and DPI-CB[8] complexations (ΔH < 0) suggested that electrostatic interactions play a positive role during the 4883
DOI: 10.1021/acs.joc.8b00543 J. Org. Chem. 2018, 83, 4882−4887
Note
The Journal of Organic Chemistry complexation process, while entropic changes (TΔS > 0) showed that hydrophobic interactions are also one of the main driving forces for the CB[n] complexation. ROS is involved in numerous physiological and pathophysiological processes. There is accumulating evidence that oxidative stress plays a crucial role in the pathogenesis and development of cardiovascular diseases especially atherosclerosis.14 In the same report, it has been shown that inhibition of macrophage oxidative stress represents a reasonable therapeutic strategy against atherosclerosis. Generally, there are several sources of ROS generation, including NADPH oxidases (Noxs), xanthine oxidase, mitochondrial electron transport chain, and uncoupled endothelial nitric oxide (NO) synthase.15 In macrophages, Noxs are major sources responsible for the production of ROS,16 and DPI has frequently been used to inhibit Noxsderived ROS production.17 In our study, LPS and t-BHP were used to trigger chronic and acute generation of ROS in a mouse macrophage cell line, RAW 264.7, respectively. First, we examined the respective effect of DPI, CB[7], and CB[8] on ROS generation in RAW 264.7. Our results showed that none of these individual species affected the ROS generation by the macrophages (Figure S5). As shown by Figure 4, LPS and t-
Figure 5. Cytotoxicity of DPI (0, 1.25, 2.50, 5.00 μM) in the presence of 400 μM CB[7]/30 μM CB[8] to H9c2 cells after 24 h incubation (mean ± SEM, n = 4, 570 nm). *P < 0.05, **P < 0.01, and ***P < 0.001 denote a significant difference compared to control group.
respectively, suggesting that CB[7] significantly reduced the toxicity of DPI, whereas the presence of CB[8] moderately alleviated the toxicity of DPI on the H9c2 cell line, likely attributed to the low concentration of CB[8] due to its limited water solubility. To further evaluate whether CB[7] or CB[8] would be able to reduce the cardiotoxicity of DPI, zebrafish were utilized as an in vivo model. As shown in Figure S6, the fish hearts are elongated with a thin atrium and a compact ventricle upon exposure to 50 μM DPI for 48 h, confirming the cardiac toxicity of DPI at a relatively low dose. Conversely, 100 μM CB[7] and 25 μM CB[8] solutions did not exhibit any obvious distortion of heart morphology, which is consistent with previous studies.3a,c However, DPI-CB[7] complexes (50 μM DPI and 100 μM CB[7]) resulted in similar but less compact ventricles as compared to those induced by DPI, and no significant difference was observed in the hearts of the fish treated by DPI and DPI-CB[8] (50 μM DPI and 25 μM CB[8]), respectively. As expected, no sign of heart distortion or deformation was found among the zebrafish hearts in the control group. Furthermore, the serious cardiac toxicity resulted in mortality in the DPI-treated groups of zebrafish. Thus, survival rates of these different treatment groups were monitored in order to further evaluate the cardiotoxicity of DPI in the absence and in the presence of CB[n]. As shown in Figure S7a, significant differences between the free DPI-treated group and the DPICB[7]/[8]-treated groups were observed. The group of zebrafish treated with DPI in the presence of CB[7] or CB[8] exhibited 100% survival rate, whereas the free DPItreated group only exhibited a survival rate of 46.7%. Meanwhile, the cardiac functions of zebrafish were closely monitored. As shown in Figure S7b−e, the encapsulation of DPI by CB[7] moderately improved the heart rate (HR) in comparison with that of the free DPI-treated group. Meanwhile, it is worth to note that 100 μM CB[7] or 25 μM CB[8] alone treated groups were similar to the control group regarding both the overall survival rate and the cardiac functionality, attesting their good biocompatibility at this concentration range toward the zebrafish hearts. In summary, we have discovered a new onium guest species for CB[n] (n = 7 or 8), a halonium compound (DPI), which may further extend the cucurbituril host−guest chemistry and associated scope of potential applications in pharmaceutical sciences. Through this investigation, we have demonstrated that
Figure 4. ROS generation by RAW 264.7 cells, which were pretreated with DPI (10 μM) in the absence and in the presence of CB[7] (100 μM) and CB[8] (10 μM), respectively, for 1 h, before being treated with 1 μg/mL of LPS (a) for 8 h or 100 μM t-BHP (b) for 1 h. *P < 0.05, **P < 0.01, and ***P < 0.001 denote a significant difference compared to the control group.
BHP induced a 5-fold (Figure 4a) and a 5.5-fold increase (Figure 4b), respectively, in ROS production when compared with the control group. The addition of DPI significantly reduced ROS generation in both the chronic and acute ROS models. The encapsulation of DPI by CB[7] and by CB[8], respectively, preserved and moderately enhanced the inhibitory activity of DPI against ROS generation, suggesting that CB[7] or CB[8] may provide a novel formulation strategy for potentially enhancing the therapeutic efficacy of haloniumbased drug molecules. It was previously reported that DPI was employed to establish an in vivo cardiomyopathy model due to its inherent cardiac toxicity.13 We have previously demonstrated that strong complexation of a toxic substance by CB[7] may alleviate its toxicity.18 Thus, herein we investigated the influence of the supramolecular encapsulation on the toxicity of DPI. The rat cardiac myoblast cell line H9c2 was utilized as a cellular model to examine the influence of CB[7] and CB[8] on cardiac toxicity of DPI in vitro. The cytotoxicity of DPI, DPI-CB[7], and DPI-CB[8] was respectively determined by MTT assays. As shown by Figure 5, the IC50 values (upon 24 h of incubation) of DPI, DPI-CB[7], and DPI-CB[8] are 1.286 ± 0.058 μM, 2.115 ± 0.144 μM, and 1.402 ± 0.104 μM, 4884
DOI: 10.1021/acs.joc.8b00543 J. Org. Chem. 2018, 83, 4882−4887
Note
The Journal of Organic Chemistry
ratio [CB[8]]/([CB[8]]+[DPI]) was adjusted from 0 to 1.0 by steps of 0.1. (An additional ratio of 0.33 was inserted.) To determine the binding constant of DPI-CB[7], the aqueous solution of DPI (0.025 mM) was titrated with various volumes of solution containing the same concentration of DPI (0.025 mM) and 3.0 equiv of CB[7] (0.075 mM). All of these samples were subjected to UV−visible measurements. ESI-MS Analysis. To estimate the possible binding stoichiometries of DPI-CB[7] and DPI-CB[8], solutions of DPI (0.01 mM) with CB[7] (0.03 mM) as well as DPI (0.01 mM) with CB[8] (0.01 mM) were prepared in Milli-Q water, respectively, and were subsequently filtered (0.25 μm) before being injected into the ESI-MS spectrometer for analysis. ITC Analysis. Aqueous solutions of DPI at 1 mM, CB[7] at 2 mM, and CB[8] at 0.03 mM were prepared in Milli-Q water, respectively. The solution volume in the titration syringe of ITC was 40 μL (0.4 μL for the first drop and 2 μL per drop for the rest (18 drops)), while the volume in the cell of ITC was 280 μL. The stirring rate was set at 750 rpm, while the reference power was at 10.0 μcal/s at 25 °C. The calorimetric results were autoanalyzed by the Malvern MicroCal PEAQ-ITC Analysis Software 1.1.0.1262. Evaluation of ROS Generation in Vitro. The effect of DPI, DPICB[7], DPI-CB[8], CB[7], and CB[8] on LPS- or t-BHP-induced ROS production was, respectively, measured by DCFH2-DA. Briefly, RAW 264.7 cells were pretreated with these compounds/complexes, respectively, for 1 h and was subsequently exposed to LPS for 8 h or tBHP for 1 h. After the treatment, the cells were washed with PBS three times and incubated with DCFH2-DA for 30 min in the dark at 37 °C. Subsequently, the cells were washed with PBS again, and the fluorescence density was measured by a flow cytometry using a FACSCantoTM system (BD Biosciences). Effect of CB[7]/CB[8] on Cardiac Toxicity of DPI in Vitro. To compare the cytotoxicity of DPI in the absence and in the presence of CB[7] or CB[8] on a rat cardiac myoblast cell line H9c2, the IC50 values of DPI, DPI-CB[7], and DPI-CB[8] in H9c2 cell lines were evaluated by MTT assays. The standard treatments for H9c2 cells were as follows: the cells were used until they reached 70−80% confluence and were subsequently seeded onto 96-well plates at a density of 4000−5000 cells/well containing 100 μL of culture medium and four duplicate wells in each group. After 24 h of treatment, 20 μL of 5 mg/mL MTT (final concentration 1 mg/mL) was added to each well, followed by incubation for 4 h at 37 °C. The culture medium was removed by aspiration, and the cells were washed twice with phosphate buffered saline (PBS). A total of 100 μL of DMSO was added to dissolve blue formazan in the living cells, and the absorbance was measured at 570 nm with a microplate reader. The cells incubated with the control medium were considered to be 100% viable. Cell viability percentage = the OD value of each treated group/OD value of the control group × 100%. Effect of CB[7]/CB[8] on Cardiac Toxicity of DPI in Vivo Using the Zebrafish Model. Tg (cmlc2:GFP) zebrafish embryos were used to evaluate the cardiotoxicity of DPI and DPI-CB[7]/[8]. All embryos were cultivated in E3 medium containing 0.003% (wt %) of PTU to block pigmentation. A total of 48 hpf zebrafish embryos were dechorionated, and healthy embryos were selected and placed into a 24-well microplate with 15 embryos per well. Embryos were treated with 1 mL solutions of 50 μM DPI, 100 μM CB[7], 25 μM CB[8], and 50 μM DPI in the presence of 100 μM CB[7] or 25 μM CB[8], respectively, for 48 h. The E3 medium-treated group served as the control group. Subsequently, zebrafish embryos were embedded into 1% (wt %) low-melting point agarose matrix (Gibco) to fix them in a dorsal orientation and limit their movement before a microscope was employed to observe the embryos. Ventricular functions were evaluated by various parameters, which were measured as described previously.3c,22 Videos (15 s in length for each) of beating hearts from individual zebrafish at the end of incubation were recorded using an Olympus Cell∧R imaging system comprising a IX71 microscope at room temperature. The heart rate (HR) was determined by counting the number of heartbeats in a 15 s interval. The ventricular volume at
supramolecular encapsulation of DPI by either CB[7] or CB[8] may preserve or moderately improve its inhibitory activities against ROS generation and, meanwhile, alleviate its cardiotoxicity in vitro and in vivo. This research discovery may provide novel insights on discovering new types of guest species for the CB[n] family and further exhibit the potential of CB[n] as emerging pharmaceutical excipients.
■
EXPERIMENTAL SECTION
Ethics Statement. The zebrafish study was approved by the ICMS Animal Ethics Committee, University of Macau. Materials. CB[7] and CB[8] were synthesized and purified by using a previously reported method.19 Diphenyleneiodonium chloride was purchased from Hanxiang Biotechnology (Shanghai, China) and used as received. LPS (Escherichia coli serotype 055:B5, lipopolysaccharide), t-BHP (tert-Butyl hydroperoxide), 5-(6)-carboxy2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA), and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) were purchased from Sigma−Aldrich (St. Louis, MO, USA). RPMI culture solution, penicillin-streptomycin (PS), trypsin, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). The E3 medium was composed of 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4 (pH 7.2− 7.3). Instrumentation. 1H NMR spectra were acquired on a Bruker Ultra Shield 600 PLUS NMR spectrometer. ESI-MS analyses were performed on a Thermo LTQ OrbiTrap XL instrument equipped with an ESI/APcI multiprobe. UV−visible spectroscopy was performed on a HACH DR6000 UV−visible spectrometer with a 1.0 cm path length quartz cell. Isothermal titration calorimetry (ITC) tests were conducted by using a Malvern MicroCal PEAQ-ITC. Milli-Q water was purified by the Milli-Q Integral System, Merck Millipore. MTT assay was monitored by a microplate reader (Spectra Max M5Microplate Reader, Molecular Devices, USA). The morphology of zebrafish was observed on an optical microscope (Olympus, SZ61). The heart morphology, heart rates, and quantitative assessment of cardiac functions were obtained from the analysis of video segments that were recorded on individual fish using an Olympus Cell∧R imaging system, consisting of an IX71 microscope at room temperature. Cell Culture. RAW 264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The rat cardiac myoblast cell line H9c2 was purchased from America Tissue Type Collection (Manassas, VA, USA). Two cell lines were maintained in DMEM media supplemented with 10% FBS and 1% penicillinstreptomycin in a humidified environment at 37 °C with 5% CO2. The medium was changed every 48 h. Zebrafish. Tg(cmcl2: GFP) zebrafish with GFP (green fluorescent protein) specifically expressed in the myocardial cells were used for cardiotoxicity assays.20 Zebrafish were maintained as described in the Zebrafish Handbook.21 Briefly, adult fish were raised in an aquaculture system with 12 h light/12 h dark cycles and fed twice daily with newly hatched brine shrimp. The culturing of the embryos was performed in E3 medium containing 0.003 wt % of 1-phenyl-2-thiourea (PTU) at 27 ± 1 °C. All zebrafish-related procedures were performed according to the ethical guideline of the Ethics Committee, University of Macau. 1 H NMR Sample Preparation. In order to prepare the solution for 1H NMR analysis, a 1 mM stock solution of DPI in D2O was prepared and was subsequently mixed with various amounts of CB[7] (up to 2.2 mM) or CB[8] (up to 0.04 mM). The solutions were sonicated for 10 min in NMR tubes before they were analyzed for 1H NMR spectroscopy. UV−Visible Absorption Measurement. Stock solutions of DPI at 1 mM, CB[7] at 2 mM, and CB[8] at 0.03 mM were prepared by using Milli-Q water, respectively. For continuous variation titrations (Job’s plot titration), a series of solutions with the total concentrations of DPI and CB[7] (0.025 mM) were prepared, and the ratio [CB[7]]/ ([CB[7]]+[DPI]) was adjusted from 0 to 1.0 by steps of 0.1. For the Job’s plot titration of DPI-CB[8], a series of solutions with the total concentrations of DPI and CB[8] (0.025 mM) were prepared, and the 4885
DOI: 10.1021/acs.joc.8b00543 J. Org. Chem. 2018, 83, 4882−4887
Note
The Journal of Organic Chemistry end-diastole (EDV) and end-systole (ESV) in the fish was calculated from the heart dimensions using the formula
V=
4 πab2 3
synthesis to high-affinity binding and catalysis. Chem. Soc. Rev. 2015, 44, 394−418. (3) (a) Uzunova, V. D.; Cullinane, C.; Brix, K.; Nau, W. M.; Day, A. I. Toxicity of cucurbit[7]uril and cucurbit[8]uril: an exploratory in vitro and in vivo study. Org. Biomol. Chem. 2010, 8, 2037−2042. (b) Oun, R.; Floriano, R. S.; Isaacs, L.; Rowan, E. G.; Wheate, N. J. The ex vivo neurotoxic, myotoxic and cardiotoxic activity of cucurbituril-based macrocyclic drug delivery vehicles. Toxicol. Res. 2014, 3, 447−455. (c) Chen, H.; Chan, J. Y. W.; Yang, X.; Wyman, I. W.; Bardelang, D.; Macartney, D. H.; Lee, S. M. Y.; Wang, R. Developmental and organ-specific toxicity of cucurbit[7]uril: in vivo study on zebrafish models. RSC Adv. 2015, 5, 30067−30074. (d) Li, F.; Gorle, A. K.; Ranson, M.; Vine, K. L.; Kinobe, R.; Feterl, M.; Warner, J. M.; Keene, F. R.; Collins, J. G.; Day, A. I. Probing the pharmacokinetics of cucurbit[7, 8 and 10]uril: and a dinuclear ruthenium antimicrobial complex encapsulated in cucurbit[10]uril. Org. Biomol. Chem. 2017, 15, 4172−4179. (4) Yin, H.; Wang, R. Applications of Cucurbit[n]urils (n = 7 or 8) in Pharmaceutical Sciences and Complexation of Biomolecules. Isr. J. Chem. 2017. (5) Kuok, K. I.; Li, S.; Wyman, I. W.; Wang, R. Cucurbit[7]uril: an emerging candidate for pharmaceutical excipients. Ann. N. Y. Acad. Sci. 2017, 1398, 108−119. (6) (a) Zhang, J.; Coulston, R. J.; Jones, S. T.; Geng, J.; Scherman, O. A.; Abell, C. One-Step Fabrication of Supramolecular Microcapsules from Microfluidic Droplets. Science 2012, 335, 690. (b) Li, S.; Jiang, N.; Zhao, W.; Ding, Y.-F.; Zheng, Y.; Wang, L.-H.; Zheng, J.; Wang, R. An eco-friendly in situ activatable antibiotic via cucurbit[8]urilmediated supramolecular crosslinking of branched polyethylenimine. Chem. Commun. 2017, 53, 5870−5873. (7) (a) St-Jacques, A. D.; Wyman, I. W.; Macartney, D. H. Encapsulation of charge-diffuse peralkylated onium cations in the cavity of cucurbit[7]uril. Chem. Commun. 2008, 4936−4938. (b) Liu, S.; Ruspic, C.; Mukhopadhyay, P.; Chakrabarti, S.; Zavalij, P. Y.; Isaacs, L. The Cucurbit[n]uril Family: Prime Components for Self-Sorting Systems. J. Am. Chem. Soc. 2005, 127, 15959−15967. (8) (a) Montes-Navajas, P.; Corma, A.; Garcia, H. Complexation and Fluorescence of Tricyclic Basic Dyes Encapsulated in Cucurbiturils. ChemPhysChem 2008, 9, 713−720. (b) Basílio, N.; Pischel, U. Drug Delivery by Controlling a Supramolecular Host−Guest Assembly with a Reversible Photoswitch. Chem. - Eur. J. 2016, 22, 15208−15211. (9) Wang, R.; Macartney, D. H. Cucurbit[7]uril stabilization of a diarylmethane carbocation in aqueous solution. Tetrahedron Lett. 2008, 49, 311−314. (10) Holland, P. C.; Clark, M. G.; Bloxham, D. P.; Lardy, H. A. Mechanism of action of the hypoglycemic agent diphenyleneiodonium. J. Biol. Chem. 1973, 248, 6050−6. (11) (a) Doussiere, J.; Vignais, P. V. Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. Eur. J. Biochem. 1992, 208, 61− 71. (b) Cross, A. R.; Jones, O. T. The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase. Biochem. J. 1986, 237, 111−6. (12) Stuehr, D. J.; Fasehun, O. A.; Kwon, N. S.; Gross, S. S.; Gonzalez, J. A.; Levi, R.; Nathan, C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 1991, 5, 98−103. (13) Brosnan, M. J.; Hayes, D. J.; Challiss, R. A. J.; Radda, G. K. Diphenyleneiodonium-induced cardiomyopathy. Biochem. Soc. Trans. 1986, 14, 1209. (14) Sugamura, K.; Keaney, J. F., Jr. Reactive oxygen species in cardiovascular disease. Free Radical Biol. Med. 2011, 51, 978−992. (15) (a) Santos, C. X.; Tanaka, L. Y.; Wosniak, J.; Laurindo, F. R. Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid. Redox Signal. 2009, 11, 2409−27. (b) Antonenkov,
(1)
where a refers to the longitudinal axis length and b refers to the lateral axis length between the myocardial borders of ventricles at enddiastole and end-systole, respectively. The stroke volume (SV), cardiac output (CO), and percent fractional shortening (% FS) were also calculated according to the following equations. The stroke volume (SV), cardiac output (CO), and percent fractional shortening (% FS) were calculated as follows: SV = EDV − ESV
(2)
CO = SV × HR
(3)
FS (%) =
diastolic diameter − dystolic diameter × 100% dystolic diameter
(4)
Statistical Analysis. All data are presented as mean ± SEM, from at least three independent experiments. Diagrams and statistical analysis using a one-way ANOVA test were performed with the GraphPad Prism Software (GraphPad Software, La Jolla CA, USA). The statistical significance was calculated using the t test.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.8b00543. 1 H NMR spectrum of DPI, Job’s plots of DPI-CB[7] and DPI-CB[8], ESI-MS spectra of DPI-CB[7] and DPICB[8], UV−vis spectra, ITC results, ROS generation of RAW 264.7 cells, representative fluorescent microscopic images, and cardiac function of zebrafish (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +853-8822-4689. ORCID
Hang Yin: 0000-0002-2898-844X David Bardelang: 0000-0002-0318-5958 Ruibing Wang: 0000-0001-9489-4241 Author Contributions #
H.Y. and Q.H. contributed to this work equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The work was supported by Macau Science and Technology Development Fund (FDCT020/2015/A1 and FDCT030/ 2017/A1), the University of Macau Research Committee (MYRG2016-00008- ICMS-QRCM and MYRG2017-00010ICMS), and the Open Fund of SKL of Supramolecular Structure and Materials, Jilin University, China (SKLSSM201820).
■
REFERENCES
(1) Barrow, S. J.; Kasera, S.; Rowland, M. J.; del Barrio, J.; Scherman, O. A. Cucurbituril-Based Molecular Recognition. Chem. Rev. 2015, 115, 12320−12406. (2) (a) Masson, E.; Ling, X.; Joseph, R.; Kyeremeh-Mensah, L.; Lu, X. Cucurbituril chemistry: a tale of supramolecular success. RSC Adv. 2012, 2, 1213−1247. (b) Assaf, K. I.; Nau, W. M. Cucurbiturils: from 4886
DOI: 10.1021/acs.joc.8b00543 J. Org. Chem. 2018, 83, 4882−4887
Note
The Journal of Organic Chemistry V. D.; Grunau, S.; Ohlmeier, S.; Hiltunen, J. K. Peroxisomes are oxidative organelles. Antioxid. Redox Signal. 2010, 13, 525−37. (16) Shin, M. H.; Moon, Y. J.; Seo, J. E.; Lee, Y.; Kim, K. H.; Chung, J. H. Reactive oxygen species produced by NADPH oxidase, xanthine oxidase, and mitochondrial electron transport system mediate heat shock-induced MMP-1 and MMP-9 expression. Free Radical Biol. Med. 2008, 44, 635−45. (17) Keith, K. E.; Hynes, D. W.; Sholdice, J. E.; Valvano, M. A. Delayed association of the NADPH oxidase complex with macrophage vacuoles containing the opportunistic pathogen Burkholderia cenocepacia. Microbiology 2009, 155, 1004−15. (18) (a) Li, S.; Chen, H.; Yang, X.; Bardelang, D.; Wyman, I. W.; Wan, J.; Lee, S. M. Y.; Wang, R. Supramolecular Inhibition of Neurodegeneration by a Synthetic Receptor. ACS Med. Chem. Lett. 2015, 6, 1174−1178. (b) Li, S.; Chan, J. Y.-W.; Li, Y.; Bardelang, D.; Zheng, J.; Yew, W. W.; Chan, D. P.-C.; Lee, S. M. Y.; Wang, R. Complexation of clofazimine by macrocyclic cucurbit[7]uril reduced its cardiotoxicity without affecting the antimycobacterial efficacy. Org. Biomol. Chem. 2016, 14, 7563−7569. (19) Bardelang, D.; Udachin, K. A.; Leek, D. M.; Margeson, J. C.; Chan, G.; Ratcliffe, C. I.; Ripmeester, J. A. Cucurbit[n]urils (n = 5−8): A Comprehensive Solid State Study. Cryst. Growth Des. 2011, 11, 5598−5614. (20) Huang, C.-J.; Tu, C.-T.; Hsiao, C.-D.; Hsieh, F.-J.; Tsai, H.-J. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn. 2003, 228, 30−40. (21) Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish; University of Oregon Press, 2000. (22) Chen, H.; Chan, J. Y. W.; Li, S.; Liu, J. J.; Wyman, I. W.; Lee, S. M. Y.; Macartney, D. H.; Wang, R. In vivo reversal of general anesthesia by cucurbit[7]uril with zebrafish models. RSC Adv. 2015, 5, 63745−63752.
4887
DOI: 10.1021/acs.joc.8b00543 J. Org. Chem. 2018, 83, 4882−4887
