A Facile, Versatile, and Highly Efficient Strategy for Peroxynitrite

Subscriber access provided by UNIV OF LOUISIANA

Article

A Facile, Versatile, and Highly Efficient Strategy for Peroxynitrite Bioimaging Enabled by Formamide Deformylation Xilei Xie, Guangzhao Liu, Xingxing Su, Yong Li, Yawen Liu, Xiaoyun Jiao, Xu Wang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01175 • Publication Date (Web): 19 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A Facile, Versatile, and Highly Efficient Strategy for Peroxynitrite Bioimaging Enabled by Formamide Deformylation Xilei Xie,*,† Guangzhao Liu,† Xingxing Su, Yong Li, Yawen Liu, Xiaoyun Jiao, Xu Wang,* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Shandong Normal University, Jinan 250014, P. R. China. E-mail: [email protected]; [email protected]; [email protected] ABSTRACT: Peroxynitrite (ONOO−) is attracting increasing attention due to its involvement in multiple facets of pathophysiological processes. However, ONOO− bioimaging are still challenging due to (1) the lack of highly specific reaction triggers, (2) the tedious and low-yielding synthesis of current sophisticated probes, and (3) the lack of availability of a versatile chemical strategy. To address these challenges, based on the amine formylation/deformylation chemistry, we have developed a novel strategy for ONOO− bioimaging. As proof of principle, we designed, synthesized, and evaluated four novel fluorescent probes equipped with the formamide functionality. Although featuring distinctly different fluorophore classes, all probes can be one-step synthesized in high yields and exhibit particularly specific, highly sensitive, and rapid responses to ONOO−. The bioimaging capability is well demonstrated by successfully visualizing ONOO− fluctuation in live cells and major organs of mice suffering from paraquat poisoning. The proposed strategy has proved to be a facile, versatile, and highly efficient methodology for ONOO− visualization, which will greatly facilitate ONOO− biochemistry and pathophysiology.

Peroxynitrite (ONOO−) is a short-lived and immensely powerful oxidant biogenerated from the nonenzymatic reaction of the free radicals superoxide and nitric oxide.1 In organisms, ONOO− is implicated in cellular signal transduction2 and serves as the microbicidal effector in host immune response.3 However, due to its extremely oxidizing and nitrating capabilities, aberrantly elevated ONOO− irreversibly damages a panel of biological targets, such as proteins, nucleic acids, and lipids, and thus contributes to the evolution and progression of inflammation, cancer, neurodegenerative disorders, and other severe diseases.1,4,5 Therefore, the elucidation of ONOO−-involved pathogenesis will provide crucial insights into early diagnosis and management of these related disorders. However, disentangling the complex contribution of ONOO− in biosystems urgently requires reliable and efficient strategies for its specific detection over other biological competing species. Currently, several reaction-based approaches for ONOO− bioimaging have been reported, including boronate oxidation,6−8 oxidative N-dearylation,9,10 C−C double bond cleavage,11−16 and other reaction strategies.17−26 However, these approaches still leave room for further improvement. First, to some extent, the selectivity of some reaction triggers for ONOO− is still unsatisfactory due to their cross-reactivity with hydrogen peroxide,27,28 29,30 hypochlorous acid, and other biogenic competitors.31 Second, due to their sophisticated chemical structures, the currently available probes often involve tedious multistep organic preparation and are obtained in extremely low yields. Therefore large-scale preparation remains unrealized, which severely limits their biomedical application. Third, the

development of new probes with desirable capabilities is timeand cost-consuming, which demands iterative optimization to tune reactivity and selectivity,32 since the versatile and general strategy for ONOO− bioimaging is still lacking. To address the aforementioned issues, we herein report a facile, versatile, and highly efficient strategy for ONOO− bioimaging based on ONOO− induced formamide deformylation chemistry. As proof of principle, we developed four ONOO− fluorescent probes (FPP-Blue, FPP-Green, FPPYellow, and FPP-Red) whose excitation and emission wavelengths span the visible spectral region. All four probes were easily prepared by treating the corresponding aminebearing fluorophore with formic acid and were obtained in excellent yields (> 80%). Additionally, the probes showed superior sensing performance in terms of high specificity, excellent sensitivity, and rapid response rate, which enabled direct visualization of ONOO− in live cells. In particular, with the aid of FPP-Yellow, overproduced ONOO− was visualized in situ in cultured cells and major organs (heart, liver, lung, and kidney) of mice suffering from paraquat poisoning. This well-demonstrated strategy will greatly simplify the future design and synthesis of ONOO− probes.

EXPERIMENTAL SECTION General Procedure for the Formylation Reaction. The one-step synthetic route was described in Scheme 1. Fluorophore 1 is commercially available. Fluorophores 2, 3, and 4 were synthesized according to the reported method.33−35 The corresponding fluorophore (1.0 mmol) was dissolved in formic acid (5 mL). The resulting solution was stirred at reflux temperature overnight. The solvent was evaporated, and the

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

residue was purified by column chromatography on silica gel using dichloromethane/methanol as eluent to obtain the corresponding probe. FPP-Blue. This probe was synthesized from fluorophore 1 according to the general procedure, and obtained in 83% yield. HRMS (ESI): calculated for C11H9NNaO3+ (M + Na)+ 226.0475, found 226.0424. 1H NMR (400 MHz, DMSO-d6): δ 10.63 (s, 0.75H), 10.49 (d, J = 10.2 Hz, 0.25H), 9.01 (d, J = 10.7 Hz, 0.25H), 8.38 (s, 0.75H), 7.82−7.63 (m, 1.75H), 7.47 (d, J = 8.6 Hz, 0.75H), 7.28 (s, 0.25H), 7.19 (d, J = 8.5 Hz, 0.25H), 6.28 (s, 0.75H), 6.26 (s, 0.25H), 2.40 (s, 3H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 162.72, 160.34, 159.91, 154.08, 153.60, 153.14, 153.00, 142.07, 141.34, 126.60, 126.08, 115.35, 115.18, 113.31, 112.47, 112.12, 105.86, 103.34, 17.97 ppm. FPP-Green. This probe was synthesized from fluorophore 2 according to the general procedure, and obtained in 80% yield. HRMS (ESI): calculated for C18H13N2OS+ (M + H)+ 305.0743, found 305.0749. 1H NMR (400 MHz, DMSO-d6): δ 10.56 (s, 0.7H), 10.49 (d, J = 10.9 Hz, 0.3H), 9.05 (d, J = 10.8 Hz, 0.3H), 8.63 (s, 1H), 8.41 (d, J = 9.2 Hz, 1.7H), 8.20−8.12 (m, 2.7H), 8.09 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 8.6 Hz, 0.7H), 7.97 (d, J = 8.5 Hz, 0.3H), 7.78 (s, 0.3H), 7.68 (d, J = 8.7 Hz, 0.7H), 7.60−7.52 (m, 1.3H), 7.48 (t, J = 7.4 Hz, 1H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 167.35, 162.73, 160.11, 153.66, 137.88, 137.49, 135.02, 134.85, 134.48, 130.53, 129.86, 129.55, 129.10, 128.93, 128.47, 128.06, 127.25, 127.08, 126.68, 125.49, 124.73, 124.52, 122.78, 122.35, 120.68, 119.38, 115.19, 112.36 ppm. FPP-Yellow. This probe was synthesized from fluorophore 3 according to the general procedure, and obtained in 87% yield. HRMS (ESI): calculated for C17H17N2O3+ (M + H)+ 297.1234, found 297.1219. 1H NMR (400 MHz, DMSO-d6): δ 10.98 (s, 1H), 8.68 (d, J = 8.5 Hz, 2H), 8.50 (d, J = 7.2 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.38 (br s, 1H), 7.89 (t, J = 7.8 Hz, 1H), 4.02 (t, J = 7.3 Hz, 2H), 1.67−1.55 (m, 2H), 1.42−1.28 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 169.55, 163.38, 162.81, 140.26, 131.54, 130.73, 129.10, 128.20, 126.25, 123.82, 122.17, 119.12, 117.30, 39.25, 29.65, 19.81, 13.71 ppm. FPP-Red. This probe was synthesized from fluorophore 4 according to the general procedure, and obtained in 81% yield. HRMS (ESI): calculated for C17H11N2O3+ (M + H)+ 291.0764, found 291.0758. 1H NMR (400 MHz, DMSO-d6): δ 10.80 (s, 0.6H), 10.66 (d, J = 10.8 Hz, 0.4H), 10.54 (s, 0.4H), 9.06 (d, J = 10.7 Hz, 0.4H), 8.62 (d, J = 7.8 Hz, 1H), 8.41 (s, 0.6H), 8.15 (d, J = 7.4 Hz, 1H), 7.92−7.80 (m, 3.6H), 7.50 (ddd, J = 12.9, 8.7, 2.1 Hz, 1H), 6.47−6.40 (m, 1H) ppm. 13C NMR spectrum is unavailable due to the very low solubility in common deuterated solvents. Synthesis of FPP-Ac. Fluorophore 3 (200 mg, 0.75 mmol) was dissolved in acetic acid (1.5 mL) and acetic anhydride (1.5 mL). The resulting solution was stirred at 100 °C overnight. The solvent was evaporated, and the residue was purified by column chromatography on silica gel using dichloromethane/methanol (100/1, V/V) as eluent to obtain a solid (198 mg, 85% yield). HRMS (ESI): calculated for C18H19N2O3+ (M + H)+ 311.1390, found 311.1404. 1H NMR (400 MHz, DMSO-d6): δ 10.38 (s, 1H), 8.69 (d, J = 8.5 Hz, 1H), 8.51 (d, J = 7.2 Hz, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 7.87 (t, J = 8.0 Hz, 1H), 4.03 (t, J = 7.2 Hz, 2H), 2.28 (s, 3H), 1.64−1.57 (m, 2H), 1.39−1.30 (m, 2H),

0.92 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 170.02, 163.85, 163.28, 140.73, 132.01, 131.20, 129.57, 128.67, 126.72, 124.29, 122.64, 119.58, 117.77, 39.72, 30.12, 24.56, 20.28, 14.18 ppm. Synthesis of FPP-TFA. Fluorophore 3 (200 mg, 0.75 mmol) was dissolved in trifluoroacetic anhydride (3 mL). The resulting solution was stirred at reflux temperature overnight. The solvent was evaporated, and the residue was purified by column chromatography on silica gel using ethyl acetate/petroleum ether (1/3, V/V) as eluent to obtain a solid (227 mg, 83% yield). HRMS (ESI): calculated for C18H16F3N2O3+ (M + H)+ 365.1108, found 365.1048. 1H NMR (400 MHz, DMSO-d6): δ 11.90 (s, 1H), 8.59−8.49 (m, 2H), 8.39 (d, J = 8.4 Hz, 1H), 8.00−7.90 (m, 7.8 Hz, 2H), 4.06 (t, J = 7.2 Hz, 2H), 1.67−1.60 (m, 2H), 1.41−1.32 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 163.29, 162.84, 156.04 (q, J = 37.4 Hz), 136.89, 131.19, 130.73, 129.47, 128.21, 127.45, 126.41, 124.51, 122.53, 121.09, 115.99 (q, J = 288.4 Hz), 39.48, 29.65, 19.83, 13.76 ppm. 19F NMR (376 MHz, DMSO-d6): δ −73.61 (s, 3F) ppm.

RESULTS AND DISCUSSION Design and Screening of Amide-Functionalized Fluorescent Probes. The Yang group and our lab demonstrated that the trifluoromethyl36 or aromatic nitro34,37 activated ketone group reacts smoothly with ONOO−, and designed fluorescent probes on the basis of this chemical transformation. However, this strategy demands complex molecular design, tedious organic synthesis and elaborate purification, and the feasibility of other fluorophores has not been well demonstrated.38 Additionally, the aromatic nitro group is documented to cross-react with nitroreductase,39 hydrogen sulfide,40 carbon monoxide,41 etc., which would reduce the selectivity and signal-to-noise ratio. To simplify and generalize the design and synthesis of ONOO− probes, we turned our attention to simple amides, including formamide, acetamide, and trifluoroacetamide, due to their facile synthesis and potential reactivity with ONOO−. We envisioned that the amide functionalized fluorescent probe might undergo a nucleophilic attack by ONOO− and release the amine-bearing fluorophore, which would result in fluorescence restoration through the strengthened intramolecular charge transfer process. With these considerations in mind, we respectively installed formamide, acetamide, and trifluoroacetamide functionalities onto the naphthalimide platform (fluorophore 3) to generate a series of novel fluorescent probes: FPP-Yellow, FPP-Ac, and FPP-TFA (Figure 1a). To the best of our knowledge, this is the first time that the chemical reaction between ONOO− and simple amides has been investigated. The fluorescence responses of these probes toward ONOO− were successively evaluated (Figures 1b and S1). As a result, FPP-Yellow presented ca. 104-fold fluorescence enhancement in the presence of 10 μM ONOO−. In sharp contrast, only 10- and 20-fold enhancements were observed for FPP-Ac and FPPTFA, respectively, in the same condition. This significant difference is ascribed to the reduced steric hindrance and higher electrophilic reactivity of formamide than its amide congeners, acetamide and trifluoroacetamide. The results indicate that formamide is a much more efficient reaction trigger for ONOO−, which provides a novel chemical strategy for ONOO− probing and manipulating.


Page 2 of 9

Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. (a) Chemical structures of FPP-Yellow, FPP-Ac, and FPPTFA. (b) Fluorescence responses of the probes (2 μM) toward 10 μM ONOO−. Data were acquired in 50 mM phosphate buffer (pH 7.4, 4‰ DMSO) after incubation at 37 °C for 10 min. λex/λem = 435/560 nm.

Developing a Series of ONOO− Fluorescent Probes with Formamide Functionality. To further exemplify this strategy, we developed another three fluorescent probes (FPP-Blue, FPP-Green, and FPP-Red) by caging the corresponding aminebearing fluorophores (coumarin 1, naphthalene derivative 2, and benzophenoxazine derivative 4) with the formamide functionality. Notably, all three probes, along with FPPYellow, were easily prepared in excellent isolated yields (>80%) by formylation of the amine-containing fluorophore in formic acid (Scheme 1). This one-step synthetic approach is facilely performed, timeand cost-saving, and environmentally friendly, which not only simplifies future ONOO− probe development but also allows for large-scale synthesis.

(Figure S2). The fluorescence responses were successively examined upon treatment with different concentrations of ONOO−. Dose-dependent fluorescence increases were observed (Figure 2), and the intensity at the emission maxima was linearly correlated with the ONOO− concentration (Figure S3). Specifically, FPP-Blue is blue emitting (Emmax = 447 nm) and shows a ca. 23-fold fluorescence turn-on response to 10 μM ONOO− upon excitation at 375 nm. FPP-Green is green emitting (Emmax = 506 nm) and exhibits a ca. 17-fold increase in fluorescence response to 10 μM ONOO− when excited at 385 nm. FPP-Yellow is yellow emitting (Emmax = 560 nm) and displays a ca. 104-fold fluorescence enhancement response to 10 μM ONOO− upon excitation at 435 nm. FPP-Red is red emitting (Emmax = 620 nm) and presents a ca. 20-fold increment in fluorescence response to 4 μM ONOO− when excited at 530 nm. All four probes gave high signal-to-noise ratios, and their detection limits were determined to be 46, 64, 28, and 102 nM, respectively, based on the formula of 3σ/k, which enabled real-time visualization of the traces of ONOO− in physiological environments.

Scheme 1. Synthesis and Response of Formamide Functionalized Probes for ONOO− Bioimaging. HCOOH, reflux yields > 80%

O

Peroxynitrite 37 oC, < 4 s

H2N

H

N H

Fluorophore

Fluorescent probe

O H2N

O

O

H

N H

1 Emmax = 447 nm

FPP-Blue

N

N S

H2N

S

O H

2 Emmax = 506 nm O

N H

FPP-Green

n-Bu N O

O

3 Emmax = 560 nm

O

O H

FPP-Yellow

N

4 Emmax = 620 nm

n-Bu N O

HN

NH2

H2N

Figure 2. Fluorescence spectra of (a) FPP-Blue, (b) FPP-Green, (c) FPP-Yellow, and (d) FPP-Red with ONOO− titration. The final concentration of the probes was 2 μM. Fluorescence spectra were acquired in 50 mM phosphate buffer (pH 7.4, 4‰ DMSO) immediately after solution preparation with excitation at (a) 375, (b) 385, (c) 435, and (d) 530 nm, respectively.

O

O

N

O O

H

N H

O

O

FPP-Red

The UV-vis spectra of the FPP series (FPP-Blue, FPPGreen, FPP-Yellow, and FPP-Red) were measured. Upon ONOO− addition, the absorption maxima were all red-shifted

HPLC and HRMS analyses were performed to confirm the resulting fluorescent products of the FPP series (Figures S4 and S5). For example, the HPLC chromatograms showed that FPP-Blue (tR = 18.6 min) was completely transformed into the coumarin fluorophore 1 (tR = 16.4 min) after being incubated with 5 equiv. of ONOO−, and the HRMS data also supported this conversion. Notably, it is rare for current ONOO− probes to have such high conversion efficiency. Additionally, the other three probes also experienced the same chemical transformation triggered by ONOO−. These outcomes evidently validated the ONOO− induced deformylation of the FPP series to generate the parent fluorophore described in Scheme 1. We proposed that the formyl carbon of the probe first underwent a nucleophilic attack by ONOO− to produce the peroxo intermediate, which homolyzed to a caged radial pair and then recombined to nitrite and the carbamic acid intermediate, which was unstable and quickly hydrolyzed to carbon dioxide and the corresponding fluorophore (Scheme 2).42



Page 4 of 9

Scheme 2. Proposed Reaction Mechanism of ONOO− and Formamide Functionalized Probes. O H ONOO

N H

H

homolysis

OH

H

N O O H N O

OH

+

N O H

NO2

cage HNO2

CO2 O H2N

HO

N H

ONOO−

The time-dependent fluorescence response to was recorded (Figure 3). After treatment with 10 μM ONOO−, the fluorescence intensities of FPP-Blue, FPP-Green, and FPPYellow rapidly increased and reached their maxima within 2 s. Similarly, the fluorescence intensity of FPP-Red also amplified rapidly and reached its plateau within 4 s upon the addition of 4 μM ONOO−. The rapid response rate enables the formamide functionalized probes to efficiently capture and visualize the transient ONOO− in real time.

Figure 4. Fluorescence responses of (a) FPP-Blue, (b) FPP-Green, (c) FPP-Yellow, and (d) FPP-Red toward 100 μM analytes unless otherwise noted: (1) ONOO−, (2) blank, (3) H2O2, (4) ClO−, (5) O2•−, (6) •OH, (7) 1O2, (8) t-BuOOH, (9) NO, (10) 5 mM GSH, (11) 5 mM Cys, (12) Hcy, and (13) H2S. ONOO− concentrations were 10 μM (a, b, c) and 4 μM (d), respectively. The final concentration of the probes was 2 μM. Fluorescence intensity was acquired in 50 mM phosphate buffer (pH 7.4, 4‰ DMSO) after incubation at 37 °C for 10 min, with excitation at (a) 375, (b) 385, (c) 435, and (d) 530 nm, respectively.

Figure 3. Time course of the fluorescence intensity at the emission maxima of (a) FPP-Blue, (b) FPP-Green, (c) FPP-Yellow, and (d) FPP-Red after addition of ONOO−. The final concentration of the probes was 2 μM, and ONOO− concentrations were 10 μM (a, b, c) and 4 μM (d), respectively. Fluorescence intensity was acquired in 50 mM phosphate buffer (pH 7.4, 4‰ DMSO) with excitation at (a) 375, (b) 385, (c) 435, and (d) 530 nm, respectively.

We next evaluated the ONOO− specificity of the FPP series (Figures 4 and S6). All probes achieved dramatic fluorescence enhancement only in the presence of ONOO−. In sharp contrast, the fluorescence intensities remained constant upon treatment with other biorelevant species, including reactive oxygen and nitrogen species (H2O2, HClO, O2•−, •OH, 1O2, tBuOOH, and NO), reactive sulfur species (GSH, Cys, Hcy, and H2S), and a panel of anions and metal ions (NO3−, NO2−, CH3COO−, SO42−, SO32−, CO32−, PO43−, Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, and Fe3+). The formamide functionality achieves superior performance in ONOO− specificity compared to the current reaction triggers. The pH effect on the fluorescence response was also examined (Figure S7). The probes maintained constant fluorescence emission across the pH range of 4.0−10.0 and showed higher fluorescence off/on ratios to ONOO− around pH 7.4. The high specificity and pHindependency enable ONOO− bioimaging in the complex biological milieu.

Figure 5. Fluorescence images of the FPP probes in response to ONOO− in HepG2 cells. Cells were treated with (a−d) PBS, (e−h) 500 μM SIN-1, or (i−l) 100 μM uric acid and then 500 μM SIN-1, followed by incubation with 5 μM probe. Images of FPP-Blue (a, e, i) were obtained by collecting the emissions at 420−500 nm upon twophoton excitation at 730 nm. Images of FPP-Green (b, f, j) were obtained by collecting the emissions at 480−560 nm upon two-photon excitation at 760 nm. Images of FPP-Yellow (c, g, k) were obtained by collecting the emissions at 520−600 nm upon two-photon excitation at 800 nm. Images of FPP-Red (d, h, l) were obtained by collecting the emissions at 600−680 nm upon one-photon excitation at 514 nm. Scale bar: 20 μm.


Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (Top) Fluorescence images of FPP-Yellow in response to ONOO− in (a−f) HepG2 and (g−l) A549 cells after paraquat treatment. Cells were treated with (a, g) 0 μM, (b, h) 50 μM, (c, i) 100 μM, (d, j) 150 μM, (e, k) 200 μM, or (f, l) 200 μM paraquat, followed by incubation with 5 μM FPP-Yellow. Cells in scavenging group (f, l) were pretreated with 100 μM uric acid in advance. Images were obtained by collecting the emissions at 520−600 nm upon two-photon excitation at 800 nm. Scale bar: 20 μm. (Bottom) Relative mean fluorescence intensities of cells in (a−l) were quantified. The values are the mean ± s.d. for n = 3, **p < 0.01, ***p < 0.001.

Visualizing ONOO− in Live Cells. Overall, the FPP series feature high signal-to-noise ratios, low detection limits, excellent specificity, and rapid response rates, demonstrating that the formamide functionality is a highly efficient and versatile reaction trigger for ONOO− detection. The cytotoxicities of the FPP series were then assessed in HepG2 cells by MTT assay, which confirmed that these four probes are nontoxic to cells at their working concentrations (Figure S8). We then utilized the FPP series to visualize ONOO− in live cells (Figures 5 and S9). As expected, the probe-loaded cells displayed faint fluorescence emission. By contrast, treatment with 3-morpholinosydnonimine hydrochloride (SIN1, a widely used ONOO− generator) resulted in significant blue, green, yellow, and red intracellular fluorescence for FPPBlue, FPP-Green, FPP-Yellow, and FPP-Red, respectively. Additionally, the fluorescence increases were significantly suppressed by the ONOO− scavenger uric acid. These positive results demonstrate that the formamide-based FPP series are capable of visualizing ONOO− in live cells. FPP-Yellow was selected for the further exploration due to its superior performance in terms of the higher signal-to-noise ratio and lower detection limit in the FPP series as well as the twophoton imaging capability. Visualizing ONOO− Fluctuation during Paraquat Poisoning. Paraquat is one of the most widely used herbicides and also highly toxic to human beings and animals. Therefore, paraquat has been banned in several countries. Paraquat poisoning is mainly attributed to overproduced ONOO−.43,44 However, there is no visible and convincing evidence to support this viewpoint. Herein, for the first time, we attempted to probe the ONOO− contribution during paraquat poisoning in cultured cells and major organs of mice.

Figure 7. Detection of intracellular ONOO− in flow cytometry stained by 5 μM FPP-Yellow. Cells were treated with 200 μM paraquat in the absence (c) or presence (d) of 100 μM uric acid. The unstained (a) and untreated (b) groups were taken as control. Fluorescence emissions were collected at 505−560 nm upon excitation at 405 nm. The left was the histograms of fluorescence intensity in cells, and the right was the relative mean fluorescence intensities calculated based on the left histograms. The values are the mean ± s.d. for n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

Specifically, HepG2 and A549 cells were treated with different doses of paraquat, and then the ONOO− levels were visualized by two-photon imaging with the aid of FPP-Yellow. As shown in Figure 6, both HepG2 and A549 cells displayed dose-dependent fluorescence increases, which were effectively blocked by the ONOO− scavenger uric acid. The generation of ONOO− requires the combination of O2•− and NO. To further confirm the ONOO− production, aminoguanidine and apocynin, as the selective nitric oxide synthase inhibitor and NADPH oxidase inhibitor, were introduced to abolish NO and O2•− formation, respectively. As expected, both inhibitors effectively attenuated ONOO− generation (Figure S10). Above



all, these observations clearly demonstrated that the cells suffered an ONOO− burst due to the simultaneous elevation of O2•− and NO after paraquat administration, and that upregulated ONOO− concentration was positively dependent on the paraquat dosage. Additionally, FPP-Yellow was also successfully applied to quantitatively monitor the intracellular ONOO− levels during paraquat poisoning in the flow cytometry platform (Figure 7).

appeared within 10 min, then presented a time-dependent increase, and finally reached a plateau ca. 25 min, indicating that ONOO− related stress emerged quickly after paraquat administration. FPP-Yellow can be systemically injected into live mice and distribute to the main organs to visualize the biogenetic ONOO− in situ. These solid data validate that the amine formylation/deformylation strategy enables ONOO− detection and visualization during a specific pathophysiological process in not only live cells but also animal models.

CONCLUSION

Figure 8. Visualization of ONOO− in the major organs of mice after paraquat administration. The mice were injected with PBS (200 μL) for the control group; paraquat (50 mg/kg, 200 μL) for the treated group, or uric acid (100 mg/kg, 200 μL) with paraquat (50 mg/kg, 200 μL) for the scavenging group. This treatment was followed by injection with FPP-Yellow (50 μM, 100 μL) via the tail vein. (a) The hearts, livers, lungs, and kidneys were isolated and cut into slices for imaging. (b) Real-time imaging of the liver tissues in live mice. Images were obtained by collecting the emissions at 520−600 nm upon two-photon excitation at 800 nm. Scale bar: 20 μm (a) and 50 μm (b).

Subsequently, FPP-Yellow was utilized to systematically explore ONOO− fluctuations in the major organs of mice suffering from paraquat poisoning. The mice were randomly divided into three groups. The treated and scavenging groups were challenged with paraquat through intraperitoneal injection. For the scavenging group, the mice were pretreated with uric acid to remove the overproduced ONOO−. All three groups were treated with FPP-Yellow by tail intravenous injection. After anesthesia, the hearts, livers, lungs, and kidneys were isolated and cut into slices for two-photon fluorescence imaging (Figures 8a and S11). Compared with the control and scavenging groups, the treated group showed significant fluorescence emission in all four organs, indicating the systematical upregulation of ONOO− levels during paraquat poisoning. Additionally, paraquat-induced ONOO− fluctuation was monitored in real-time in the livers of live mice (Figure 8b). The fluorescence intensity of liver tissues

In this work, for the first time, we investigated the chemical transformation of ONOO− induced simple amide (formamide, acetamide, and trifluoroacetamide) cleavage and promoted formamide as a novel reaction trigger for ONOO−. To demonstrate its feasibility and universality, four fluorescent probes (FPP-Blue, FPP-Green, FPP-Yellow, and FPP-Red) were designed by caging the corresponding amine-bearing fluorophore with the formamide functionality. The four novel probes feature quite different fluorophore classes whose excitation and emission profiles span the visible spectral region but can all be one-step synthesized in high isolated yields. The involved design and synthesis principle is simple and easily accessible to others with an interest in ONOO− probing and manipulation, even those with less knowledge of synthetic chemistry. Additionally, all probes present rapid fluorescence response with sufficient specificity and high sensitivity in the presence of ONOO− in the chemical context and live cells. Among them, FPP-Yellow shows the highest signal-to-noise ratio and the lowest detection limit, as well as two-photon imaging capability, which enables visualization of ONOO− fluctuation in live cells and major organs (heart, liver, lung, and kidney) of mice suffering from paraquat poisoning by confocal imaging and/or flow cytometry. Compared with the currently available strategies, amine formylation/deformylation chemistry has proved to be a facile, versatile, and highly efficient methodology for ONOO− bioimaging. The formamide functionality is potentially applicable to any amine-bearing functional template on demand by direct formylation. We envision this chemical strategy can be not only expanded to develop ONOO− fluorescent probes with long-wavelength emission, ratiometric signals, cellular and subcellular targeting abilities but also extended to other sensing platforms such as luminescence, photoacoustic imaging, and magnetic resonance imaging, as well as ONOO−responsive drug delivery. On the other hand, inspired by ONOO− induced deformylation chemistry, we plan to explore new formamide-bearing biological targets, such as proteins and nucleic acids, susceptible to ONOO− attack in biological pathways and disease progression. Overall, the seminal work presented here significantly simplifies ONOO− probe development and will greatly facilitate ONOO− biochemistry and pathophysiology.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.


Page 6 of 9

Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry Materials and instruments, experimental details, supplementary data, and NMR and HRMS spectra (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +86-531-86180017. *E-mail: [email protected]. *E-mail: [email protected].

Author Contributions †X.X

and G.L. contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21535004, 91753111, 21877076, and 21775093) and the Key Research and Development Program of Shandong Province (2018YFJH0502).

REFERENCES (1) Szabó, C.; Ischiropoulos, H.; Radi, R. Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat. Rev. Drug Discovery 2007, 6, 662−680. (2) Shacka, J. J.; Sahawneh, M. A.; Gonzalez, J. D.; Ye, Y. Z.; D'alessandro, T. L.; Estevez, A. G. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells. Cell Death Differ. 2006, 13, 1506−1514. (3) Fang, F. C. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2004, 2, 820−832. (4) Pacher, P.; Beckman, J. S.; Liaudet, L. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 2007, 87, 315−424. (5) Beal, M. F. Experimental models of Parkinson's disease. Nat. Rev. Neurosci. 2001, 2, 325−334. (6) Sikora, A.; Zielonka, J.; Lopez, M.; Joseph, J.; Kalyanaraman, B. Direct oxidation of boronates by peroxynitrite: mechanism and implications in fluorescence imaging of peroxynitrite. Free Radical Biol. Med. 2009, 47, 1401−1407. (7) Sun, X.; Xu, Q.; Kim, G.; Flower, S. E.; Lowe, J. P.; Yoon, J.; Fossey, J. S.; Qian, X.; Bull, S. D.; James, T. D. A water-soluble boronate-based fluorescent probe for the selective detection of peroxynitrite and imaging in living cells. Chem. Sci. 2014, 5, 3368−3373. (8) Zhang, J.; Zhen, X.; Upputuri, P. K.; Pramanik, M.; Chen, P.; Pu, K. Activatable photoacoustic nanoprobes for in vivo ratiometric imaging of peroxynitrite. Adv. Mater. 2017, 29, 1604764. (9) Peng, T.; Wong, N. K.; Chen, X.; Chan, Y. K.; Ho, D. H. H.; Sun, Z.; Hu, J. J.; Shen, J.; El-Nezami, H.; Yang, D. Molecular imaging of peroxynitrite with HKGreen-4 in live cells and tissues. J. Am. Chem. Soc. 2014, 136, 11728−11734. (10) Li, X.; Tao, R. R.; Hong, L. J.; Cheng, J.; Jiang, Q.; Lu, Y. M.; Liao, M. H.; Ye, W. F.; Lu, N. N.; Han, F.; Hu, Y. Z.; Hu, Y. H. Visualizing peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury. J. Am. Chem. Soc. 2015, 137, 12296−12303. (11) Jia, X.; Chen, Q.; Yang, Y.; Tang, Y.; Wang, R.; Xu, Y.; Zhu, W.; Qian, X. FRET-based mito-specific fluorescent probe for ratiometric detection and imaging of endogenous peroxynitrite: dyad of Cy3 and Cy5. J. Am. Chem. Soc. 2016, 138, 10778−10781. (12) Zhou, X.; Kwon, Y.; Kim, G.; Ryu, J. H.; Yoon, J. A ratiometric fluorescent probe based on a coumarin–hemicyanine scaffold for sensitive and selective detection of endogenous peroxynitrite. Biosens. Bioelectron. 2015, 64, 285−291. (13) Shuhendler, A. J.; Pu, K.; Cui, L.; Uetrecht, J. P.; Rao, J. Realtime imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing. Nat. Biotechnol, 2014, 32, 373−380.

(14) Qin, X.; Li, F.; Zhang, Y., Ma, G.; Feng, T.; Luo, Y.; Huang, P.; Lin, J. In vivo photoacoustic detection and imaging of peroxynitrite. Anal. Chem. 2018, 90, 9381−9385. (15) Zhou, D. Y.; Li, Y.; Jiang, W. L.; Tian, Y.; Fei, J.; Li, C. Y. A ratiometric fluorescent probe for peroxynitrite prepared by de novo synthesis and its application in assessing the mitochondrial oxidative stress status in cells and in vivo. Chem. Commun. 2018, 54, 11590−11593. (16) Zhou, D. Y.; Ou-Yang, J.; Li, Y.; Jiang, W. L.; Tian, Y.; Yi, Z. M.; Li, C. Y. A ratiometric fluorescent probe for the detection of peroxynitrite with simple synthesis and large emission shift and its application in cells image. Dyes Pigm. 2019, 161, 288−295. (17) Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. Mechanismbased molecular design of highly selective fluorescence probes for nitrative stress. J. Am. Chem. Soc. 2006, 128, 10640−10641. (18) Yu, F.; Li, P.; Li, G.; Zhao, G.; Chu, T.; Han, K. A near-IR reversible fluorescent probe modulated by selenium for monitoring peroxynitrite and imaging in living cells. J. Am. Chem. Soc. 2011, 133, 11030−11033. (19) Zhang, Q.; Zhu, Z.; Zheng, Y.; Cheng, J.; Zhang, N.; Long, Y. T.; Zheng, J.; Qian, X.; Yang, Y. A three-channel fluorescent probe that distinguishes peroxynitrite from hypochlorite. J. Am. Chem. Soc. 2012, 134, 18479−18482. (20) Lin, K. K.; Wu, S. C.; Hsu, K. M.; Hung, C. H.; Liaw, W. F.; Wang, Y. M. A N-(2-aminophenyl)-5-(dimethylamino)-1naphthalenesulfonic amide (Ds-DAB) based fluorescent chemosensor for peroxynitrite. Org. Lett. 2013, 15, 4242−4245. (21) Miao, J.; Huo, Y.; Liu, Q.; Li, Z.; Shi, H.; Shi, Y.; Guo, W. A new class of fast-response and highly selective fluorescent probes for visualizing peroxynitrite in live cells, subcellular organelles, and kidney tissue of diabetic rats. Biomaterials 2016, 107, 33−43. (22) Cheng, D.; Pan, Y.; Wang, L.; Zeng, Z.; Yuan, L.; Zhang, X.; Chang, Y. T. Selective visualization of the endogenous peroxynitrite in an inflamed mouse model by a mitochondria-targetable two-photon ratiometric fluorescent probe. J. Am. Chem. Soc. 2017, 139, 285−292. (23) Li, Z. H.; Liu, R.; Tan, Z. L.; He, L.; Lu, Z. L.; Gong, B. Aromatization of 9, 10-dihydroacridine derivatives: discovering a highly selective and rapid-responding fluorescent probe for peroxynitrite. ACS Sens. 2017, 2, 501−505. (24) Li, H.; Li, X.; Wu, X.; Shi, W.; Ma, H. Observation of the generation of ONOO− in mitochondria under various stimuli with a sensitive fluorescence probe. Anal. Chem. 2017, 89, 5519−5525. (25) Li, J. B.; Chen, L.; Wang, Q.; Liu, H. W.; Hu, X. X.; Yuan, L.; Zhang, X. B. A bioluminescent probe for imaging endogenous peroxynitrite in living cells and mice. Anal. Chem. 2018, 90, 4167−4173. (26) Cao, J.; An, W.; Reeves, A. G.; Lippert, A. R. A chemiluminescent probe for cellular peroxynitrite using a selfimmolative oxidative decarbonylation reaction. Chem. Sci. 2018, 9, 2552−2558. (27) Lippert, A. R.; Van De Bittner, G. C.; Chang, C. J. Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems. Acc. Chem. Res. 2011, 44, 793−804. (28) Zielonka, J.; Sikora, A.; Hardy, M.; Joseph, J.; Dranka, B. P.; Kalyanaraman, B. Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chem. Res. Toxicol. 2012, 25, 1793−1799. (29) Xu, Q.; Lee, K. A.; Lee, S.; Lee, K. M.; Lee, W. J.; Yoon, J. A highly specific fluorescent probe for hypochlorous acid and its application in imaging microbe-induced HOCl production. J. Am. Chem. Soc. 2013, 135, 9944−9949. (30) Sun, M.; Yu, H.; Zhu, H.; Ma, F.; Zhang, S.; Huang, D.; Wang, S. Oxidative cleavage-based near-infrared fluorescent probe for hypochlorous acid detection and myeloperoxidase activity evaluation. Anal. Chem. 2014, 86, 671−677. (31) Chen, Y.; Zhu, C.; Yang, Z.; Chen, J.; He, Y.; Jiao, Y.; He, W.; Qiu, L.; Cen, J.; Guo, Z. A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria. Angew. Chem., Int. Ed. 2013, 52, 1688−1691.



(32) Xie, X.; Li, M.; Tang, F.; Li, Y.; Zhang, L.; Jiao, X.; Wang, X.; Tang, B. Combinatorial strategy to identify fluorescent probes for biothiol and thiophenol based on diversified pyrimidine moieties and their biological applications. Anal. Chem. 2017, 89, 3015−3020. (33) Mao, G. J.; Wei, T. T.; Wang, X. X.; Huan, S. Y.; Lu, D. Q.; Zhang, J.; Zhang, X. B.; Tan, W.; Shen, G. L.; Yu, R. Q. Highsensitivity naphthalene-based two-photon fluorescent probe suitable for direct bioimaging of H2S in living cells. Anal. Chem. 2013, 85, 7875−7881. (34) Li, Y.; Xie, X.; Yang, X.; Li, M.; Jiao, X.; Sun, Y.; Wang, X.; Tang, B. Two-photon fluorescent probe for revealing drug-induced hepatotoxicity via mapping fluctuation of peroxynitrite. Chem. Sci. 2017, 8, 4006−4011. (35) Woods, C. M.; Fernandez, C.; Kunze, K. L.; Atkins, W. M. Allosteric activation of cytochrome P450 3A4 by α-naphthoflavone: branch point regulation revealed by isotope dilution analysis. Biochemistry 2011, 50, 10041−10051. (36) Yang, D.; Wang, H. L.; Sun, Z. N.; Chung, N. W.; Shen, J. G. A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells. J. Am. Chem. Soc. 2006, 128, 6004−6005. (37) Xie, X.; Tang, F.; Liu, G.; Li, Y.; Su, X.; Jiao, X.; Wang, X.; Tang, B. Mitochondrial peroxynitrite mediation of anthracyclineinduced cardiotoxicity as visualized by a two-photon near-infrared fluorescent probe. Anal. Chem. 2018, 90, 11629−11635. (38) Xie, X.; Yang, X.; Wu, T.; Li, Y.; Li, M.; Tan, Q.; Wang, X.; Tang, B. Rational design of an α-ketoamide-based near-infrared fluorescent probe specific for hydrogen peroxide in living systems. Anal. Chem. 2016, 88, 8019−8025. (39) Li, Y.; Sun, Y.; Li, J.; Su, Q.; Yuan, W.; Dai, Y.; Han, C.; Wang, q.; Feng, W.; Li, F. Ultrasensitive near-infrared fluorescenceenhanced probe for in vivo nitroreductase imaging. J. Am. Chem. Soc. 2015, 137, 6407−6416. (40) Wang, R.; Yu, F.; Chen, L.; Chen, H.; Wang, L.; Zhang, W. A highly selective turn-on near-infrared fluorescent probe for hydrogen sulfide detection and imaging in living cells. Chem. Commun. 2012, 48, 11757−11759. (41) Dhara, K.; Lohar, S.; Patra, A.; Roy, P.; Saha, S. K.; Sadhukhan, G. C.; Chattopadhyay, P. A new lysosome-targetable turn-on fluorogenic probe for carbon monoxide imaging in living cells. Anal. Chem. 2018, 90, 2933−2938. (42) Ferrer-Sueta, G.; Campolo, N.; Trujillo, M.; Bartesaghi, S.; Carballal, S.; Romero, N.; Alvarez, B.; Radi, R. Biochemistry of peroxynitrite and protein tyrosine nitration. Chem. Rev. 2018, 118, 1338−1408. (43) Denicola, A.; Radi, R. Peroxynitrite and drug-dependent toxicity. Toxicology 2005, 208, 273−288. (44) Dinis−Oliveira, R. J.; Duarte, J. A.; Sanchez−Navarro, A.; Remiao, F.; Bastos, M. L.; Carvalho, F. Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit. Rev. Toxicol. 2008, 38, 13−71.


Page 8 of 9

Page 9 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only


A Facile, Versatile, and Highly Efficient Strategy for Peroxynitrite

Recommend Documents