Uncatalyzed Click Reaction between Phenyl Azides and Acrolein: 4

Apr 12, 2016 - Consequently, developing new analytical tools for acrolein detection that are straightforward, cost-effective, selective, and preferabl...
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Uncatalyzed Click Reaction between Phenyl Azides and Acrolein: 4‑Formyl-1,2,3-Triazolines as “Clicked” Markers for Visualizations of Extracellular Acrolein Released from Oxidatively Stressed Cells Ambara R. Pradipta,†,# Misako Taichi,†,# Ikuhiko Nakase,*,‡ Elena Saigitbatalova,§ Almira Kurbangalieva,§ Shinobu Kitazume,∥ Naoyuki Taniguchi,∥ and Katsunori Tanaka*,†,§,⊥ †

Biofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan § Biofuctional Chemistry Laboratory, A. Butlerov Institute of Chemistry, Kazan Federal University, 18 Kremlyovskaya street, Kazan 420008, Russia ∥ Disease Glycomics Team, Global Research Cluster, RIKEN-Max Planck Joint Research Center for System Chemical Biology, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ⊥ JST-PRESTO, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ‡

S Supporting Information *

ABSTRACT: Acrolein, a highly toxic α,β-unsaturated aldehyde, has been a longstanding key biomarker associated with a range of disorders related to oxidative stresses. Currently available analytical methods rely on the indirect protocols, e.g., derivatization/HPLC or mAb detection of the lysine adducts. Consequently, developing new analytical tools for acrolein detection that are straightforward, cost-effective, selective, and preferably feasible using live cells remains a highly essential pursuit in the diagnosis and therapeutic treatment of oxidative stress-related diseases. We demonstrated that for the first time aryl azides can rapidly and selectively react with acrolein in a “click” manner to provide 4-formyl-1,2,3-triazolines and 4-formyl-1,2,3-triazoles, which represents an unexplored reactivity of aryl azides. When treating a fluorescently labeled phenyl azide with oxidatively stressed or smoking-associated cell models, these heterocyclic compounds could be selectively taken up by the cells and preferably localized at the endoplasmic reticulum (ER) and lysosome, leading to a new tool for both effectively detecting acrolein level and directly imaging live cells that are under stress. The detection method developed here is convenient: target cells may be treated with fluorescently labeled azides to enable the direct and efficient detection of acrolein in live system. The simple stuctures of the azide probes allows for functional groups other than fluorescent groups to be readily linked to aryl azides to image, examine, or target cells associated with oxidative stress processes. We developed a new method for detecting and imaging acrolein extracellularly released by cells in the context of oxidative stress processes or introduced via environmental exposure. KEYWORDS: acrolein, aryl azide, oxidative stress, cell imaging, click chemistry

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hydrazines,10,11 although such methods require quite harsh conditions or offer poor selectivity in the presence of other aldehydes. More sensitive and efficient methods that do not rely on HPLC, including a solid-supported two-reaction system, were recently developed for detecting acrolein in mouse serum.12 Antibodies that recognize the acrolein−lysine conjugate, 3-formyl-3,4-dehydropiperidine (FDP)-substituted 2-aminohexanoic acid,13,14 are currently available for the immunochemical detection of oxidatively stressed cells and tissues in the context of

crolein is a highly toxic unsaturated aldehyde1 produced during smoking or generated by cells under oxidative stress conditions through the enzymatic oxidation of threonine or polyamines.2−4 Acrolein may also be generated during reactive oxygen species (ROS)-mediated oxidation of highly unsaturated lipids.5 Acrolein, which is sometimes generated on the millimolar scale in oxidatively stressed cells,6 is more toxic to cells than ROS, such as hydrogen peroxide (H2O2) or hydroxyl radicals (·OH), the major oxidative stress factors that lead to a variety of disorders.7 The detection of acrolein level is important for understanding oxidative stress and, hence, diseases at the molecular level. Acrolein has traditionally been analyzed by forming derivatives with nucleophiles, e.g., 3-aminophenol8,9 or fluorescently labeled © XXXX American Chemical Society

Received: February 23, 2016 Accepted: March 31, 2016

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various disease states.15−17 In addition to incurring high costs, and complicated and time-consuming procedures, the formation of FDP conjugates is very slow (over a few days).18,19 This method does not sensitively detect the presence of acrolein directly in live systems, which would be highly desirable. The 1,3-dipolar cycloaddition between an azide and an acetylene is extensively applied in chemical biology studies and the production of organic functional materials.20,21 The reaction may be accelerated in the presence of a Cu(I) catalyst22 or by placing the acetylene group within a strained ring system.23 In the chemical biology fields, the reaction has especially been used to achieve the bioorthogonal labeling or bioconjugation by introducing an azide genetically, metabolically, or enzymatically at a desired position within a protein or on a cell surface. Aside from these “click reactions”, terminal azides can participate in similar 1,3-dipolar cycloaddition reactions with the α,β-unsaturated carbonyl compounds to produce 1,2,3-triazoline derivatives. Several examples of this type of reaction exist. Reactions with unsaturated ketones, esters, or amides have been reported.24−27 The synthetic utility of these reactions with biologically active compounds or bioconjugation applications is unfortunately not well understood. The reactions required elevated temperatures, and under such conditions, the intermediary 1,2,3-triazolines readily decompose into the reactive azomethine intermediates, from which a variety of byproducts is produced.27 During our study on exploring the new reactivity of the unsaturated aldehyde,28 we noticed the high reactivity of the phenyl azide toward acrolein: after mixing the two reagents at room temperature, the solution turned red, and the 4-formyl1,2,3-triazoline, as a 1,3-dipolar cycloaddition product, was obtained (see Figure 2). Yao and co-workers recently reported that the triazole derivative is obtained in 75% yield from the benzyl azide with acrolein under oxidation conditions involving heating in the presence of a Cu(I) catalyst and with exposure to oxygen.29 The inherent reactivity of the aryl azide to acrolein, however, has not yet been reported in the literature. In this paper, we would like to report the first uncatalyzed “click” reaction between phenyl azides and acrolein. The reaction proceeded in both organic and aqueous solution under physiological conditions. The unexplored reactivity of azide to acrolein highlighted the importance of detecting acrolein under oxidative stress conditions (Figure 1). Fluorescently labeled

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EXPERIMENTAL SECTION

1,3-Dipolar Cycloaddition between Phenyl Azide 1 and Acrolein. Bromobenzene (3.0 mL, 4.5 g, 29 mmol), sodium azide (3.7 g, 57 mmol), sodium L-ascorbate (289 mg, 1.43 mmol), CuI (544 mg, 2.86 mmol), and trans-N,N′-dimethylcyclohexane-1,2-diamine (690 μL, 622 mg, 4.29 mmol) were stirred in ethanol/water (7:3) (60 mL, [bromobenzene] = 0.5 M). The reaction mixture was refluxed under N2 atmosphere for 2 h. After removal of ethanol by rotary evaporation, the mixture was extracted with CHCl3. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by short silica gel column chromatography using hexane−EtOAc (10:1) as the eluent to give phenyl azide 1 as a yellow oil (2.6 g, 77%). Phenyl azide 1 (117 mg, 982 μmol) and acrolein (728 μL, 9.82 mmol) were stirred in THF (stabilizer free, 655 μL, [phenyl azide 1] = 1.5 M) at ambient temperature. After being stirred for 30 min, the reaction mixture was concentrated in vacuo. The crude product was purified by silica gel chromatography using a gradient of eluents [hexane−EtOAc (4:1 to 2:1)] to give 4-formyl-1,2,3-triazoline 2 as yellow-brown oil (55 mg, 32%), 4-formyl-1,2,3-triazole 3 as yellow-brown oil (26 mg, 15%), and together with recovery of the starting material 1 (7 mg, 6%). Synthesis of Fluorescently Labeled Phenyl Azides 4. The 4-bromobenzylamine (748 μL, 1.10 g, 5.90 mmol), Boc2O (1.9 g, 8.9 mmol), and NaOH (355 mg, 8.90 mmol) were stirred in dioxane/ water (1.5:1) (25 mL, [4-bromobenzylamine] = 0.2 M) at ambient temperature. After being stirred for 30 min, the solvents were removed in vacuo. The residual crude was taken up with chloroform, and washed with sat. aq. NH4Cl. The organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give compound 6 as colorless crystals (1.4 g, 82%). Compound 6 obtained above (227 mg, 790 μmol), sodium azide (155 mg, 2.38 mmol), sodium L-ascorbate (8.0 mg, 40 μmol), CuI (15 mg, 80 μmol), and trans-N,N′-dimethylcyclohexane-1,2-diamine (26.0 μL, 23.0 mg, 160 μmol) were stirred in ethanol/water (7:3) (8.0 mL, [compound 6] = 0.1 M). The reaction mixture was refluxed under N2 atmosphere for 3.5 h. After removal of ethanol by rotary evaporation, the mixture was extracted with chloroform. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography using hexane−EtOAc (4:1) as the eluent to give the product compound 7 as pale yellow crystal (160 mg, 81%). Compound 7 obtained above (16 mg, 65 μmol) in TFA (600 μL, [compound 7] = 0.1 M) was stirred vigorously at 0 °C. After being stirred for 3 h, the pH was adjusted to 8 by adding aqueous NaOH (10 M) solution and then extracted with CHCl3. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product compound 8 as a dark yellow oil (8.3 mg, 87%). Compound 8 prepared above (5.3 mg, 36 μmol) and 5-(and 6-)TAMRA-succinimidyl ester (18.9 mg, 35.8 μmol) were stirred in anhydrous DMF (720 μL, [compound 8] = 0.05 M) under N2 atmosphere at ambient temperature. After being stirred for 24 h, DMF

Figure 1. Detection of acrolein extracellularly released by cells, based on the azide/acrolein cycloaddition.

azide readily and selectively reacted with acrolein generated from the smoke or the oxidatively stressed cells within 30 min to provide the 4-formyl-1,2,3-triazoline and 4-formyl-1,2,3-triazole derivatives. These products were efficiently taken up by the cells and were selectively localized in the endoplasmic reticulum (ER) and lysosome; hence, acrolein extracellularly released by the oxidatively stressed cells was directly and conveniently visualized simply by treating the live cells with the fluorescent azide.

Figure 2. Reaction of azides with acrolein: reactivity and product profiles. B

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Figure 6. Detection of acrolein introduced into or extracellularly released by HUVECs, upon treatment with TAMRA-labeled azide 4 (10 μM at room temperature for 30 min). The cells were fixed by paraformaldehyde and analyzed by microscopy. The scale bar indicate 20 μm. (a) Control cells. Cells were treated with (b) only 4; (c) 4 preincubated with acrolein; 4 in media containing (d) 50% or (e) 100% tobacco smoke; (f) 4 after treating the cells with 500 μM H2O2 for 2 h; or (g) in the presence of 10 mM Ac-Cys. (h) Comparison of fluorescence intensities.

Figure 3. Reaction of phenyl azide with acrolein in aqueous solution, e.g., buffer or medium, in the presence of various metals or interferences.

Figure 4. Stability of phenyl azide 1 in the presence of interference in cells. In part (b), most of the azide was recovered, but the unidentified products could also be produced.

Figure 7. HPLC profiles of 1 mM H2O2-treated cell culture medium in the presence of phenyl azide 1 (upper) and authentic “clicked” product 2 (lower). Conditions of reversed-phase HPLC: Column, Cosmosil 5C18-AR300 (Nacalai Tesque, Inc.) 4.6 × 250 mm; Mobile phase A, 0.1% TFA in H2O; B, 0.1% TFA in CH3CN; Gradient elution, 0−4 min at 50% B, 4−14 min at 50−80% B, 14−15 min at 80% B; Flow rate at 1 mL/min; UV detection at 254 nm. labeled phenyl azides 4 as dark purple solid (12 mg, 60%). Conditions of reversed-phase HPLC: Column, Cosmosil 5C18-AR300 (Nacalai Tesque, Inc.) 10 × 250 mm; Mobile phase A, 0.1% TFA in H2O; B, 0.1% TFA in CH3CN; Gradient elution, 0−4 min at 50% B, 4−14 min at 50−80% B, 14−15 min at 80% B; Flow rate at 4 mL/min; UV detection at 254 nm. Cell Culture. Human umbilical vein endothelial cells (HUVECs) (Takara Bio. Inc. Shiga, Japan) were cultured in EBM-2 (Lonza, Walkersville, MD, USA) supplemented with 2% fetal bovine serum (FBS) and EGM-2 SingleQuots (Lonza) and used within four passages.

Figure 5. Structures of TAMRA-labeled phenyl azide 4 and “clicked” product 9, and their fluorescent properties in water. Absorption spectra (dashed line) and fluorescence spectra (solid line) of phenyl azide 4 (2 μM, black line) and “clicked” product 9 (2 μM, gray line). was removed azeotropically with toluene in vacuo. The crude product was purified by reversed-phase HPLC to give the product fluorescently C

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Figure 8. Detection sensitivity of acrolein present in cell culture medium by use of (a,b) 3-aminophenol and (c) TAMRA-labeled phenyl azide 4. (a,b) 3-Aminophenol (17 mM) was reacted with the culture medium containing various concentrations of acrolein (0, 2, 5, 10, and 15 μM) in the presence of hydroxylamine hydrochloride and 3 N HCl for 10 min under reflux conditions, according to the reported method.8,9 X-axis denoted the concentration of acrolein in medium. Y-axis denoted (a) fluorescent intensity of 7-hydroxyquinoline (ex: 350 nm, em: 516 nm) and (b) HPLC peak area of 7-hydroxyquinoline. Detection limit for both analyses was evaluated at 2 μM. (c) Azide 4 (10 μM) was reacted with culture medium containing various concentrations of acrolein (0, 1, 10, and 100 nM) at 37 °C for 10 min, and treated with the cells at 37 °C for 30 min. X-axis denoted concentration of acrolein in medium. Y-axis denoted increase of TAMRA fluorescence imaged on the cells. Detection limit was at 100 nM. ***p < 0.001. Confocal Microscopy. HUVECs were seeded on 8-well chamber slides coated with type I collagen (4 × 104 cells/well), and left for attachment for 12 h before treatment. As a control experiment, the cells were treated with a solution of 4 (10 μM, 200 μL) in the culture medium, and incubated for 30 min at room temperature. In order to prepare the azide-acrolein conjugates, to a solution of 4 (700 μg, 1.30 μmol) in THF (25 μL) was added acrolein (1.3 μL, 20 μmol for 4). The reaction mixture was stirred for 12 h at room temperature, and the solvent and acrolein were removed in vacuo. The cells were incubated with the resulting mixtures (10 μM, 200 μL) in the culture medium for 30 min at room temperature. For the tobacco smoke experiment,30 a tobacco solution was prepared by bubbling smoke from one cigarette (Marlboro, Philip Morris International, NY, USA) into 10 mL of culture medium, and this medium was referred to as a 100% tobacco solution. The azide 4 (1.0 μL, 10 mM in DMSO) were incubated with the 100% or 50% tobacco solution (1.0 mL) for 1 h at room temperature, and the cells were incubated with the azide−tobacco mixture for 30 min at room temperature.

As for the hydrogen peroxide experiment, the cells were incubated with various concentrations of hydrogen peroxide in the culture medium (200 μL) in the presence or absence of 10 mM acetyl cysteine for 2 h at room temperature. Then, a medium solution of 4 (10 μL, 200 μM) was added to the cells, and the resulting cells were incubated for 30 min at room temperature (final concentration of 4 = 10 μM). The cells were observed using an FV-1000D laser scanning confocal microscope (Olympus, Tokyo, Japan), without fixation, or after fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min, and mounted in CC/Mount (Diagnostic BioSystems). In the experiments of live cell imaging, HUVECs (3 × 104 cells, 200 μL/well) were plated on 1 μ-Slide 8 well ibiTreat microscopy chamber (ibidi, Martinsried, Germany) and incubated for 24 h at 37 °C under 5% CO2. After complete adhesion, the cell culture medium was removed and treated with the azide−acrolein conjugates (10 μM, 200 μL/well) for 30 min at 37 °C under 5% CO2 in the presence of ER-Tracker Green (500 nM; Molecular Probes, Eugene, OR, USA), LysoTracker Green DND-26 (500 nM; Molecular Probes), or MitoTracker Green FM (1 μM; Molecular Probes). The cells were D

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Figure 9. Live imaging of acrolein generated by HUVECs, upon treatment with TAMRA-labeled azide 4 (10 μM at 37 °C for 30 min). The scale bar indicates 20 μm. (a) Control cells; HUVECs were treated with 4 after treating the cells with (b) 0 μM; (c) 50 μM; (d) 100 μM; (e) 500 μM; and (f) 1000 μM of H2O2 for 2 h. (g) Comparison of fluorescence intensities. Red: TAMRA; Blue: nucleus. Based on the standard fluorescence in Figure 8c, approximately 100 nM of acrolein could be generated by treatment of HUVECs with 50 μM H2O2. then washed with PBS (three times, 200 μL/well) and analyzed using a FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 40× or 60× objective without cell fixation. For experiments of low temperature conditions,31 the cells were pretreated for 30 min at 4 °C before treatment with the azide−acrolein conjugates (10 μM)-containing cell culture medium (200 μL/well) for 30 min at 4 °C. For experiments using the endocytosis inhibitor, dynasore hydrate32 (Sigma-Aldrich, St. Louis, MO, USA), the cells were pretreated with dynasore hydrate (300 μM) for 30 min at 37 °C before treatment with the azide−acrolein conjugates (10 μM)-containing cell culture medium (200 μL/well) for 30 min at 37 °C. Statistic Analyses. All statistical analyses were performed using GraphPad Prism software (v 5.00; GraphPad, San Diego, CA, USA). For comparison of two groups, the Welch’s correction was used after verification of the equal variances by F-test. For multiple comparison analyses, one-way analysis of variance (ANOVA) followed by Tukey’s posthoc test was used.

Interestingly, the product 2 was the triazoline, but the double bond isomerized at the conjugation position of the C4-aldehyde. Decomposition was not observed, even over an incubation period of several days; hence, the 1,3-dipolar cycloadduct was stabilized by this isomerization.27 We thus recognized the high reactivity of the phenyl azide with acrolein, i.e., within 30 min at room temperature in the absence of a catalyst. It is worth noting that the uncatalyzed azide cycloaddition reaction was selective to the unsubstituted acrolein. No products formed through the reaction with the β- or α-substituted acrolein, e.g., crotonaldehyde, trans-2-octenal as the model of the lipid metabolites, or methacrolein, as well as the cis-double bond and styrene, even after an incubation period of 1 day at room temperature (Figure 2). The higher reactivity of the azide toward acrolein compared to the substituted acroleins is likely due to the same steric and electronic reasons that explain its higher reactivity toward various nucleophiles. This process may be applied to the selective and highly sensitive detection of acrolein extracellularly released by cells (see below). Upon a finding of high reactivity and selectivity of the aryl azide with acrolein in millimolar concentration under mild conditions, we envisioned that alkyl azides with functional groups, e.g., fluorescent groups, could readily react with the environmentally generated acrolein, which damages cell functions, or

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RESULTS AND DISCUSSION The reaction of phenyl azide 1 with 10 equiv of acrolein present in the THF at the millimolar level (which mimicked the acrolein concentration produced by oxidatively stressed cells, see below), smoothly gave the new products within 30 min at room temperature (Figure 2). After silica gel column chromatography, the products were identified to be the 4-formyl-1,2,3-triazoline 2 (32% yield) and 4-formyl-1,2,3-triazole 3 (15% yield).29 E

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Figure 10. Trials on detecting acrolein generated from H2O2-treated cells by the conventional 3-aminophenol method. 3-Aminophenol (17 mM) was reacted with the cell culture medium in the presence of hydroxylamine hydrochloride and 3 N HCl for 10 min under reflux conditions, according to the reported method.8,9 X-axis denoted concentration of H2O2. Y-axis denoted (a) fluorescent intensity of 7-hydroxyquinoline (ex: 350 nm, em: 516 nm), and (b) HPLC peak area of 7-hydroxyquinoline.

even with biogenic acrolein produced by the oxidatively stressed cells (see Figure 1). If the reaction were efficient and the triazoline or triazole products internalized into the cells, the acrolein-exposed or -producing cells could be selectively detected simply by treating the cells with the functionalized azides. In order to check the applicability of the azide/acrolein click reaction toward the biologically relevant conditions, we performed the reaction in aqueous solution, e.g., buffer or medium, in the presence of various metals or interferences (Figure 3). While the presence of the oxidants converted the initially produced triazoline 2 to triazole 3, all conditions performed in Figure 3 gave the “clicked” products in about 30−40% yields. We also found that our phenyl azide was inert to hydrogen peroxide and glutathione under the experimental conditions investigated in this research (Figure 4). When bubbling H2S gas in the solution, most of the azide was recovered, but at the same time, the mixture of unidentified products could also be obtained (Figure 4). Sodium hydrogen sulfide, which is the main cell constituent, on the other hand, rapidly reduced the azide to aniline derivative, but the aniline could not be internalized into the cells, so that it does not disturb the acrolein detection when excess azide was applied (see Supporting Information, Figure S3). Then, the reaction-based cell imaging approach was put into practice using the tetramethylrhodamine (TAMRA)-labeled phenyl azide 4 (Figure 5, TAMRA-fluorescence properties of azide 4 and “clicked” product 9 are identical). We investigated the three systems using this acrolein labeling approach (Figure 6). Human umbilical vein endothelial cells (HUVECs) were treated with a 10 μM solution of TAMRA-labeled azides at room temperature for 30 min, accompanied by [1] pretreatment with excess acrolein; [2] exposure to tobacco smoke; and [3] the presence of hydrogen peroxide, which induced cellular oxidative stress. We observed that under the cell labeling conditions applied to Figure 6, the conversion reached close to maximum between 30 min and 1 h; hence, as early as “30 min” was evaluated as an appopriate incubation time for convenient and rapid detection of acrolein in live systems, without affecting significant cell viability

Figure 11. Detection of early formation of ROS and late formation of acrolein. HUVECs were treated with (a,b) menadione (25 μM) and (c,d) PMA (phorbol 12-myristate 13-acetate, 25 μM) as nonperoxide sources for 10, 30, and 60 min at 37 °C. (a,c) Real-time ROS production was monitored by Total ROS Detection Kit (cell-permeable total ROS detection dye, Enzo Life Sciences), while (b,d) acrolein production was detected by the present protocol using 10 μM of TAMRA-labeled azide 4. See Supporting Information, Figures S6 and S7, for the live cells images.

(see Figure S1). The cell fluorescence corresponding to the TAMRA-azide 4 was monitored by microscopy with or without fixing the cells using paraformaldehyde. Figure 6 shows microscopy images of the fixed HUVECs (the live image data are also provided in the Figure S2). Gratifyingly, the fluorescence intensity across the whole cells increased significantly (Figure 6c) upon pretreatment with TAMRA-azide 4 (Figure 6b). Thus, the azide−acrolein conjugates, i.e., the fluorescently labeled 4-formyltriazole or 4-formyltriazoline derivatives, interacted with the cells and could be clearly visualized. We next examined the fluorescence images of cells exposed to tobacco smoke30 (Figure 6d and e). This cell model is widely used to investigate the effects of the toxic acrolein in tobacco smoke on the cells,33,34 especially on the lung cells,35 which can lead to chronic obstructive pulmonary disease (COPD).36−38 The fluorescence uptake by the cells was notably enhanced upon exposure to tobacco smoke in a dose-dependent manner (Figure 6b,d,e). Acrolein generated by the cells treated with 500 μM hydrogen peroxide was also detected using our TAMRA-labeled azides (Figure 6b and f). In order to prove that the acrolein is F

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Figure 12. Confocal microscopy images of HUVECs treated with the azide−acrolein conjugate (derived from 4, 10 μM) for 30 min at 37 °C in the presence of (a) ER-Tracker, (b) LysoTracker, or (c) MitoTracker; or (d) costained with Hoechst. The scale bars indicate 10 μm. The white dotted squares show the regions of the enlarged images presented in each lower-right panel.

extracellularly released by the hydrogen peroxide-treated cells, we have also identified the 4-formyl-1,2,3-triazoline product from the medium (Figure 7; also see Figure S4). It should be noted that the fluorescence intensity on the cells decreased in the presence of acetyl cysteine (Figure 6g), an inhibitor of oxidative stress processes;39 hence, our method sensitively dictated the quality of acrolein extracellularly released under oxidative stress conditions. Detection of acrolein in situ generated from the live cells was still possible under exposure of 50 μM of the hydrogen peroxide, and the fluorescence intensity increased with the increasing concentration of hydrogen peroxide (see H2O2 dosedependent detection of acrolein in Figure 9). The formation of the triazoline/triazole products does not significantly change the intensity or wavelength of the fluorescence in comparison with the azide (Figure 5). Thus, the TAMRA-labeled phenyl azide is not a fluorescence “on/off” probe upon the reaction with acrolein. When acrolein is generated by the cells by exposure of the hydrogen peroxide; however, the azide react with acrolein and triazoline/triazole products endocytosed into the cells more efficiently than the starting azide (mechanism described below), and hence the cells are fluorescently visualized. To evaluate the sensitivity for acrolein detection, we then compared our protocol with the conventional HPLC- and fluorescence-based detection using 3-aminophenol,8,9 which produces the fluorescence 7-hydroxyquinoline by the reaction with acrolein (Figures 8 and S5). Our method is superior in detection sensitivity; while the traditional method only detected 2 μM of acrolein in cell medium, our new protocol can detect even 100 nM of acrolein. We could then evaluate the acrolein produced during the treatment of the cells with a certain concentration of hydrogen peroxide (Figure 9), which could never be estimated by the traditional methods (Figure 10). We additionally investigated whether our azide/acrolein click reaction can sensitively detect the late formation of acrolein during the oxidative stress, which was induced by nonperoxide

source, i.e., menadione and PMA (Figures 11, S6, and S7). Using both ROS probes and our fluorescent azide, the early formation of ROS and late formation of acrolein were independently imaged on the live cells. The mechanism by which the cells took up the azide−acrolein conjugates were examined by assessing the intracellular localization of the azide−acrolein conjugates after accumulation in cells (Figure 12). Organelle staining with Hoechst 33342 (nucleic acid staining),40 ER-Tracker Green (ER staining),41 MitoTracker Green FM (mitochondrial staining),42 or LysoTracker Green DND-26 (lysosome) were applied to the detection of TAMRA-labeled azide−acrolein conjugate accumulation in the cellular organelles without cell fixation. Confocal microscopy images revealed a high colocalization of the azide− acrolein conjugates using ER-Tracker Green and Lysotracker Green DND-26 and weak colocalization with MitoTracker Green FM (Figure 12a−c). The azide−acrolein conjugates did not flow into the nucleus (Figure 12d). Next, we visualized the conjugate internalization process using time-course confocal microscopy measurements. Figure 13 shows time-course fluorescence images of HUVECs treated with the TAMRAlabeled azide−acrolein conjugate (10 μM) at 37 °C without washing the cells. The cellular uptake of the TAMRA-labeled azide−acrolein conjugate rapidly increased, and fluorescence accumulation inside the cells began 30 s after treatment. At low temperatures (4 °C),31 which inhibited energydependent cellular uptake pathways, including clathrin-mediated endocytosis, and reduced membrane fluidity, the cellular uptake efficacy of the TAMRA-labeled azide−acrolein conjugates decreased significantly (Figure 14a). Treatment of dynasore,32 which is an inhibitor of dynamin for clathrin- or caveolaemediated endocytosis, reduced cellular uptake of the azide− acrolein conjugates (Figure 14b). These results suggested that endocytosis was an important pathway for the cellular uptake of the azide−acrolein conjugates. The conjugates penetrated the G

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delivery of future diagnostic and therapeutic molecules at the organelle level under oxidative stress conditions. The azide− acrolein conjugates were found not to be cytotoxic under the experimental conditions, based on the cellular morphologies.

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CONCLUSIONS In conclusion, we developed a new method for detecting the acrolein exogenously generated by cells in the context of oxidative stress processes or introduced via environmental exposure, e.g., exposure to tobacco smoke. The detection method described here is convenient: target cells may be treated with fluorescently labeled azides to enable the direct and efficient detection of acrolein in live system. When applying the azide/acrolein click reaction to in vivo research, further optimization of the azide structures and reactivity should be considered, i.e., in vivo toxicity, in vivo kinetics, in vivo specificity and selectivity to acrolein in the presence of other biologically relevant molecules, when intravenously administered. Nevertheless, the simple structures of the azide probes allows for functional groups other than fluorescent groups to be readily linked to aryl azides to image, examine, or target cells associated with oxidative stress processes. Azides can be reduced under certain conditions, such as hypoxia.43 The results described here indicate that azide should also be used with caution under oxidative stress conditions.

Figure 13. Live cell images by time-course confocal microscopic observations at 37 °C. HUVECs were treated with 10 μM of azide 4 preincubated with acrolein. The scale bars indicate 30 μm. TAMRA: fluorescent signals of 4+acrolein in HUVECs. Intensity: colored indicator of fluorescent intensity of TAMRA. ***p < 0.001.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00122. Additional experiments and cell imaging data, synthetic schemes, compound and characterization data, NMR and ESI mass spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

Ambara R. Pradipta and Misako Taichi contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 22651081, 23681047, and 25560410); by a MEXT Grantin-Aid for Scientific Research on Innovative Areas (Nos. 26102743 and 15H05843); by Suntory Foundation for Life Sciences, Kyoto (Japan); and by a subsidy of the Russian Government “Program of Competitive Growth of Kazan Federal University among World’s Leading Academic Centers”. We are grateful to Prof. R. Hsung (University of Wisconsin-Madison) for carefully reading the manuscript and for giving valuable comments.

Figure 14. (a) Live cell images on cellular uptake of azide−acrolein conjugates at 37 or 4 °C. HUVECs were treated with 10 μM of azide 4 preincubated with acrolein. (b) Cellular uptake of the azide−acrolein conjugates (10 μM) for 30 min at 37 °C in the presence or absence of endocytosis inhibitor, dynasore. The scale bars indicate 50 μm. Intensity: colored indicator of fluorescent intensity of TAMRA. ***p < 0.0001.

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membrane and accumulated intracellularly at the ER and lysosome during the very early stages of endocytosis. Membrane fluidity may play a crucial role in membrane penetration by the azide−acrolein conjugates. Although further studies are needed to explore the mechanism in detail, enhanced organelle accumulation among the azide− acrolein conjugates may be applicable to intracellular controlled

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