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Benzocoumarin hydrazine: A large Stokes shift fluorogenic sensor for detecting carbonyls in isolated biomolecules and in live cells. Kamalika Mukherjee, Tak Ian Chio, Han Gu, Abhijit Banerjee, Anthony M. Sorrentino, Dan L. Sackett, and Susan L. Bane ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00616 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 9, 2017
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Benzocoumarin hydrazine: A large Stokes shift fluorogenic sensor for detecting carbonyls in isolated biomolecules and in live cells. Kamalika Mukherjee,†⊥ Tak Ian Chio,† Han Gu,† Abhijit Banerjee,† ⱡ Anthony M. Sorrentino,† Dan L. Sackett ‡ and Susan L. Bane † * †
Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York 13902, USA. Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. ⊥Current address: Massachusetts General Hospital, Harvard Medical School, 149 13th Street, ⱡ Charlestown, MA 02129, USA. Current address: Chemical Division, Thermax Ltd., 97-E, General Block, Bhosari, Pune 411026. India. KEYWORDS: Fluorophore, hydrazone, coumarin, bioconjugation, carbonylation, oxidative stress, live cells, microscopy. ‡
ABSTRACT: Detection and quantification of biomolecule carbonylation, a critical manifestation of oxidative stress, allows better understanding of associated disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomolecules in cells. Live cell detection of these species requires sensors that are non-toxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomolecule carbonyl detection: a hydrazine derivative of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is especially well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission maximum of BzCH undergoes a substantial red shift upon hydrazone formation (from ~430 to ~550 nm), which is the result of fluorophore disaggregation. Additionally, the hydrazone exhibits an exceptionally large Stokes shift (~195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomolecule carbonylation can be detected and quantified in cells and in cell extracts in a one-step procedure using this probe.
Oxidative stress is a feature common to a wide range of disease states, including diabetes, cancer and neurodegenerative conditions. 1 One major outcome of oxidative stress is the carbonylation of lipids, proteins and nucleic acids.2 Carbonylated biomolecules such as 4-hydroxy-2-nonenal are known to alter cellular function and survival by regulating gene expression.3 Thus, observation, detection and quantitation of biomolecule carbonylation in cells can add value to our understanding of disease states. The common methods for detecting biomolecule carbonylation involve chemical labeling followed by immunochemistry, which cannot be performed in living systems. Fluorescence-based reporters of carbonylation are therefore an attractive alternative that can allow direct visualization of the modified biomolecules in live cells. Commercially available reagents that become fluorescent in living cells upon oxidation by reactive oxygen species (ROS) such as superoxide, nitrous oxide and hydrogen peroxide have been widely used to detect oxidative stress in cells and in isolated systems.4 But comparable reagents for carbonylation detection are sparse.5 Additionally, a drawback of using commercially available carbonylation detection reagents is that a separation or washing step is employed5b, 6 and therefore the reagent must be removed from the products sometime during analysis. A more useful fluorescent sensor for measuring biomolecule carbonylation would be one that undergoes a
change in its optical properties upon reaction with an aldehyde or ketone.7 Recently, we showed that a coumarin-based probe (7-hydrazinyl 4-methyl coumarin, CH) undergoes a shift in emission maximum and an increase in fluorescence intensity upon hydrazone formation with carbonylated biomolecules in live cells.5a The neutral CH probe is easily taken up and washed out of the cells. A drawback of this probe is that its absorption band is mainly in the ultraviolet region of the electromagnetic spectrum. In this work, we report the synthesis, spectroscopic characterization and biological application of a hydrazinyl derivative of a benzocoumarin, 7-hydrazinyl-4-methyl-2Hbenzo[h]chromen-2-one (BzCH, 1). The Stokes shift of the fluorophore is substantially greater than that of CH (BzCH ~9,000 cm-1 vs. ~4500 cm-1 for CH in DMSO). BzCH displays an unexpectedly robust propensity to self-associate into Haggregates, which do not absorb light at 405 nm, a common excitation wavelength used in fluorescence microscopy. Gratifyingly, the product hydrazone remains monomeric in most solvents and can be excited at 405 nm, which yields an increase in fluorescence intensity that can readily be observed in the presence of unreacted reagent. BzCH can therefore be used as a sensor for biomolecule carbonylation in isolated systems and is particularly useful for visualizing and quantifying carbonylation in live cells using fluorescence microscopy.
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The latter method may be used to observe the effect of exogenous anti-oxidants on the carbonylation state of cells.
EXPERIMENTAL SECTION Materials Synthetic reagents and materials were obtained from Acros. All solvents for synthesis and fluorescence experiments were analytical grade or spectroscopy grade and purchased from Fisher Scientific and used without further purification. Hydrogen peroxide (30%) was purchased from Thermo Scientific. Fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Atlanta Biologicals. Bovine serum albuminfraction V (BSA) was purchased from Rockland. Sephadex G25 fine resin was from GE Healthcare. F12K medium and RPMI 1640 medium were purchased from the American Type Culture Collection (ATCC) and Gibco (Thermo Scientific), respectively. Prostate cancer (PC3) cells were purchased from ATCC. Lung carcinoma (A549) cells (originally purchased from ATCC) were a gift from Professor Ming An. Synthesis of sensing molecule and model hydrazone All NMR spectra were collected and processed on a Bruker Avance III 600 NMR Spectrometer at 298 K. Mass spectra were collected and processed using a Waters (Waltham, MA USA) Time-of-Flight mass spectrometer model LCT Premier. 7-Hydrazinyl-4-methyl-2H-benzo[h]chromen-2one (BzCH, 1) was synthesized in four steps from 5-amino-1napthol as described in Supporting Information. 1H NMR (DMSO d6): δ 10.48 (s, 3H), 9.12 (s, 1H), 7.99 (d, J = 8.49 Hz, 1H), 7.97 (d, J = 9.25 Hz, 1H), 7.84 (d, J = 9.06 Hz, 1H), 7.65 (t, J = 8.01 Hz, 1H), 7.17 (d, J = 7.70 Hz, 1H), 6.52 (s, 1H), 2.54 (s, 3H). 13C NMR (DMSO d6): δ 159.53, 154.00, 149.53, 140.63, 127.20, 124.10, 122.82, 120.74, 117.95, 115.47, 114.76, 114.26, 109.95, 18.59. MS: 241.0 (M+1) and 224.0 (-CH3). HRMS (ESI-TOF) m/z: Calcd for C14H12N2O2 241.0977; Found 241.0973. 4-Methyl-7-(2-(2-methylpropylidene)hydrazinyl)-2Hbenzo[h]chromen-2-one (BzCZ, 2) was prepared from BzCH by reaction of the free base with isobutyraldehyde in methanol as described inSupporting Information. 1H NMR (600MHz, DMSO d6): δ 10.10 (s, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.73 (t, J=8.24Hz, 2H), 7.59 (d, J = 4.86 Hz, 1H), 7.54(t, J = 8.11 Hz, 1H), 7.49 (d, J = 7.75 Hz, 1H), 6.50 (s, 1H), 2.58 (m ,1H), 2.55 (s, 3H), 1.14 (d, J = 6.83 Hz, 6H). 13C NMR (600MHz, DMSO d6): δ 159.78, 154.17, 149.78, 149.26, 141.65, 128.15, 123.26, 121.96, 119.17, 117.83, 114.94, 113.81, 110.82, 108.61, 30.94, 20.08, 18.62. HRMS (ESITOF) m/z: Calcd for C18H19N2O2 295.1447; Found 295.1448. Optical spectroscopy of the fluorophores Absorption spectra were obtained at room temperature using a Hewlett Packard 8453 or 8452A diode array spectrophotometer. Excitation and emission spectra were recorded at room temperature using a Jobin Yvon Horiba FluoroMax-3 spectrofluorometer. Fluorescence spectra were collected using a 2 x 10 mm fluorescence cuvette, oriented such that the light passes through the shorter path. The effective path length, measured empirically, is 1 mm. Corrected excitation spectra are reported. Emission spectra are uncorrected. Spectra not shown in Results and Discussion are in Supporting Information.
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Detection of carbonyls in oxidized BSA BSA was oxidized as described previously.5a Briefly, BSA (10 mg/ml (w/v)) solubilized in oxidizing buffer (25 mM HEPES, 25 mM ascorbic acid, 100 µM FeCl3, pH 7.2) was incubated for 5 h at 37 °C. The protein solution was then applied to a gel filtration chromatography column containing G25 resin in HE buffer (50 mM HEPES, 1 mM EDTA, pH 7.2). The resultant oxidized BSA was stored at -50 °C until further use. Protein samples (oxidized BSA or unmodified BSA) were allowed to react with BzCH in HE buffer containing 0.5% DMSO (v/v) for 4.5 h at room temperature. The final concentration of BzCH was 300 µM and that of protein was 2 µg/µL. SDS PAGE analysis of the fluorophore ligated protein samples was then performed. The gel was first imaged under long wavelength UV to observe the fluorophore conjugated protein and then Coomassie stained for protein. Cell culture Cells were maintained in a humidified incubator containing 5% CO2 at 37°C. F12K medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin (F12K standard medium) was used as cell culture medium. Based on experimental requirements, cells were seeded either on 96-well plates, 6 well plates or Lab-Tek II chambered coverglass. The effect of BzCH on cell viability was assessed by a resazurin assay,5a described in Supporting Information. Detecting and quantification of carbonylation in cell lysate A549 cells originally grown in standard F12K medium were incubated in RPMI-1640 medium with or without FBS for 24 h. The cells were then treated with BzCH (50 µM; 1% DMSO in RPMI 1640 medium (with or without FBS)) for 30-60 min. Cells were then washed with PBS twice and lysed. The cell lysates with equal protein concentration were excited at 405 nm using the Synergy Mx microplate reader (BioTek). Emission intensity at 550 nm was recorded. To quantify the amount of hydrazone in the lysate samples, the fluorescence values were compared to a standard curve generated to measure hydrazone in cell lysate. The standard curve was obtained by plotting the fluorescence against increasing concentrations of BzCZ in lysates of cells grown in medium with or without FBS. The percentage of DMSO in all lysate samples was 5% (v/v). Limit of detection (LOD) of BzCZ in cell lysate was obtained by plotting the fluorescence of BzCZ at 550 nm (excitation: 405 nm) as a function of concentration and calculated as described elsewhere8. DMSO (5%, v/v) was used as a cosolvent in A549 cell lysate. A Cytation 5 imaging reader (BioTek) was used to measure the fluorescence signal. Microscopic visualization of cellular carbonylation For serum starvation-induced cellular carbonylation, PC3 or A549 cells (30,000 cells per chamber) in standard F12K medium (F12K medium containing FBS and penicillinstreptomycin) were allowed to attach to an 8-well chambered coverglass. For the next 24 h, the cells were grown in fresh standard medium or medium without FBS. At the end of the incubation period, the cells were treated with 20 µM BzCH for 30 min at 37°C. DMSO (0.5%, v/v) was used as a co-solvent for BzCH in medium. Cells were imaged using Zeiss LSM 510 Meta confocal microscope at room temperature within 20
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min after fluorophore incubation. The samples were excited by a diode 405 nm laser and the emission was collected using long pass (LP) 420. Photomicrographs were processed identically in each cell line for visual clarity. Inhibition of hydrogen peroxide-induced carbonylation by pyruvate was measured in A549 cells. Cells grown in standard RPMI medium were incubated with or without 2 mM sodium pyruvate for 1 h. The cells were then treated with 200 µM hydrogen peroxide for 3.5 h before incubating with 20 µM BzCH for 30 min. Photomicrographs were obtained as described above. Quantitative image analysis Quantitative analysis of the images was done with ImageJ (NIH). Briefly, the mean fluorescence of each cell and background in photomicrographs (3 sets) were measured using ImageJ software. Mean background signal was subtracted from the mean fluorescence of each cell and the intensity was normalized to the control set. Data are represented as mean ± SEM of experimental replicates. A two-tailed unpaired t-test was used for statistical analysis. P < 0.05 was considered significant; **** = P < 0.0001.
ples of fluorescent H-type dimer aggregates have been documented,11 including H-aggregates of coumarins.12 To test if the observed spectroscopic characteristics were indeed contributed by H-type-aggregates, experiments were performed in DMSO containing 10% water (v/v) as solvent. Although the absorption spectrum was unchanged (shown in Supporting Information, Figure S1), the intensity ratio of the two emission bands shifts in favor of the higher energy peak (Figure 2D). The excitation spectra (Figure 2E) reflect the greater contribution of the H-aggregate to the emission spectrum in Figure 2D. These data support the hypothesis that the hydrazine forms H-aggregates in DMSO solution, which is exacerbated with water.
RESULTS and DISCUSSION Synthesis and photochemical characterization of the sensor BzCH and its model hydrazone The probe 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2one (benzocoumarin hydrazine, BzCH, 1, Figure 1) was synthesized in 4 steps as described in Supporting Information. A model compound for the hydrazone of BzCH with an aliphatic aldehyde was prepared using isobutyraldehyde (2). O
O 1 O 10
3
O 9
4 5
8 7 NHNH2
6 1
H HN N 2
Figure 1. Structures of benzocoumarin hydrazine (BzCH, 1) and the hydrazone of isobutyraldehyde and BzCH (BzCZ, 2). The conventional numbering system for benzo[h]coumarin is shown.
Absorption and emission spectra of the benzocoumarin molecules were investigated. Figure 2A shows an absorption spectrum of BzCH in DMSO. The emission spectrum of BzCH was collected using the excitation wavelength corresponding to the low energy band, 365 nm. Surprisingly, two emission peaks at ~430 nm and ~540 nm were observed (Figure 2B). This observation indicated that there was more than one fluorescent species in solution. The origin of the two species was ascertained from the excitation spectra. Excitation spectra collected from excitation at each apparent emission maximum revealed two different excitation spectra for the two emission bands (Figure 2C). The excitation spectrum corresponding to the higher energy emission is blue shifted and more structured than that for the lower energy emission. These spectral features are characteristic of fluorophores in H-aggregates.9 Another property of BzCH is that the aggregates are fluorescent. H-aggregates are often non-emissive;10 however, exam-
Figure 2. Absorption, emission, and excitation spectra of BzCH. A: Absorption spectrum of BzCH in DMSO. B: Emission spectrum of BzCH in DMSO, excited at 365 nm. Note the appearance of two peaks, indicating that more than one fluorescent species is excited at this wavelength. C: Excitation spectra of BzCH in DMSO collected at the two emission maxima observed for BzCH in DMSO, illustrating the presence of two fluorescent species in this solution. D: Emission spectrum of BzCH in DMSO/water (9:1 v/v), excited at 360 nm. Note that the low energy emission band observed in panel B is greatly decreased. E: Excitation spectra of BzCH in DMSO/water (9:1 v/v) collected at the two emission maxima observed for BzCH in DMSO. The addition of water increases the relative intensity of the higher energy excitation and emission bands, indicating increased Haggregates in these solutions (panels D and E).
Absorption and fluorescence spectra of the hydrazone in DMSO, however, are quite different from those of the hydrazine. Figure 3A shows the absorption spectrum of BzCZ, which resembles the excitation spectrum of the BzCH monomer rather than the absorption spectrum of the mixture of monomers and aggregates (Figure 2A). Figure 3B shows that the emission spectrum of BzCZ consists of a single band, the shape of which is not affected by the excitation wavelength
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(Figure S2). No fluorescence is observed when the solution is excited at the emission maximum for the H-aggregate (Figure 3C). A small solvent study revealed that the BzCZ did not aggregate in acetonitrile or DMSO, and aggregated to a small extent in methanol, whereas significant aggregation of BzCH was observed in all three solvents. (Figures S2-S4). It may be that facial stacking of the molecule is less favorable for BzCZ than BzCH owing to the sp3-hybridized carbon in the hydrazone. Similar behavior has been observed in merocyanines.13 The presence of H-aggregates in solutions containing the hydrazine probe is beneficial in the detection of biomolecular carbonyls in aqueous environments. Since aggregation of the reagent shifts its absorption to higher energy, fluorescence from any unreacted BzCH is unlikely to be observed when samples are excited at 405 nm, a common laser used in fluorescence microscopes.
Figure 3. Absorption, emission, and excitation spectra of BzCZ. A: Absorption spectrum of BzCZ in DMSO. B: Emission spectrum of BzCZ in DMSO, excited at 365 nm. C: Excitation spectra of BzCZ collected at the two emission maxima observed for BzCH in DMSO. No evidence of H-aggregates is observed in these solutions.
Reaction of BzCH with oxidized protein Having demonstrated that a model compound possesses suitable fluorescence properties for aliphatic carbonyl detection, the next task was to confirm that BzCH reacts with biomolecule-associated aldehydes in aqueous solution. This was accomplished using a carbonylated model protein. Commercially available BSA was oxidized to introduce carbonyls. It was then allowed to react with BzCH at pH 7.2 prior to SDS PAGE analysis. Figure 4 shows that aldehyde-functionalized BSA forms covalent bonds with BzCH but very little reaction is observed with unmodified BSA. We conclude that BzCH can be successfully employed to detect carbonylated proteins, thereby suggesting that this fluorophore may be useful for detecting cellular carbonyls.
Figure 4. BzCH is a fluorescent reporter molecule for biomolecule carbonyls (oxidized BSA) in an isolated system. Oxidized BSA (1) or unmodified BSA (2) (final concentration 2 µg/µL) was allowed to react with 300 µM BzCH at room temperature. The protein samples were then subjected to SDS PAGE analysis. Left panel: Gel imaged under long wavelength UV. Right panel: Coomassie stained gel.
BzCH as a probe for carbonylation in cells Using BzCH as an optical sensor for cellular carbonyls requires that the hydrazone reaction takes place within living cells. Additionally, the ability to quantitate the amount of hydrazone in the cells is desirable. In this series of experiments, A549 cells subjected to serum starvation, which is a conventional model to induce oxidative stress and therefore biomolecule carbonylation, were prepared in parallel with control cells. The cells were allowed to react with BzCH prior to the terminal wash and lysing step. Fluorescence of the lysate was measured for control and for serum starved cells. Quantitation of the amount of cellular carbonylation was performed by comparing the fluorescence of the sample to that of a standard curve. The amount of hydrazone in the lysate of cells treated with BzCH, representing the carbonyl content, was quantified by comparing the emission intensity with that of the standard curve (Figure S5). These values were normalized for cell number by protein concentration and are presented in Table 1. Serum starvation increased the amount of carbonylation in these cells by about a factor of five. Additionally, the LOD of BzCZ in cell lysate was determined to be 0.98 ± 0.16 µM (Figure S6). These data therefore suggest that BzCH can be used as a sensor for biomolecule carbonylation in cells. Table 1: Quantitation of serum starvation induced cellular carbonylation Lysate
Hydrazone (nmol/mg protein)a
Control
5.2 ± 0.6
Serum Starved
25 ± 6.1
a Values denote mean ± standard deviation from three separate determinations.
BzCH as a sensor for visualization of carbonylation in live cells We have shown that BzCH reacts with protein carbonyls, that the hydrazone-forming reaction occurs inside live cells, and that the extent of carbonylation in total cellular lysates can be determined quantitatively. We also determined that BzCH is not toxic to the cells of interest (Supporting Information,
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Figure S7). We therefore turned our attention to visualizing biomolecule carbonylation in live cells. Two cancer cell lines, A549 and PC3, were subjected to serum starvation-induced oxidative stress. Cells grown in medium devoid of serum were incubated with BzCH for 30 min before microscopic analysis. Cells grown in standard medium were similarly treated with BzCH and used as control. The cell samples were excited by a 405 nm diode laser and the increase in emission due to hydrazone species that resulted from the reaction of BzCH and serum starvation induced cellular carbonyls were visualized using fluorescence confocal microscopy. It is seen in Figure 5 that BzCH detects basal level of carbonyls in both cell lines. Additionally, serum starvation induced an increase in carbonylation, resulting in more fluorescent serum starved cells in comparison to the control cells. Quantitative analysis of the photomicrographs show that serum starvation increases carbonylation in both A549 and PC3 cells (by a factor of 1.9 and 1.6, respectively, p < 0.0001). The lower contrast between the control and serum starved PC3 cells is in agreement with our previous results.5a
Figure 5. BzCH is a tool for microscopic visualization of carbonylation due to oxidative stress in live cells. A: A549 or PC3 cells were grown in F12K medium with (control) or without serum (serum starved) for 24 h. Cells were then incubated with 20 µM BzCH for 30 min before imaging. A 405 nm diode laser was used for excitation and the emission was monitored using long pass (LP) filter: LP 420, Scale bar = 20 µm. B: Bar graph comparing the means ± SEM for cellular fluorescence. Mean of A549 control (n=28) is compared to that of A549 serum starved cells (n=26) in the upper graph. Mean of PC3 control (n=59) is compared to that of PC3 serum starved cells (n=41) in the lower graph. ****= P
