A Dual-site Fluorescent Probe to Monitor Intracellular Nitroxyl and

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A Dual-site Fluorescent Probe to Monitor Intracellular Nitroxyl and GSH-GSSG Oscillations Longxue Nie, Congcong Gao, Tianjiao Shen, Jing Jing, Shaowen Zhang, and Xiaoling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05098 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Analytical Chemistry

A Dual-site Fluorescent Probe to Monitor Intracellular Nitroxyl and GSH-GSSG Oscillations Longxue Niea, Congcong Gaoa, Tianjiao Shena, Jing Jinga*,Shaowen Zhanga*, and Xiaoling Zhanga* aKey

Laboratory of Cluster Science of Ministry of Education, Beijing Key Laboratory of

Photo-electronic/Electro-photonic Conversion Materials, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, P. R. China. E-mail: [email protected], [email protected], [email protected] Abstract Nitroxyl (HNO), the one-electron-reduction product of NO has recently been revealed to have potentially beneficial pharmacological properties in cardiovascular health as a result of interactions with specific thiols such as glutathione (GSH). To disentangle the complicated inter-relationship between HNO and GSH in the signal transduction and oxidative pathways, we designed and synthesized a dual-site fluorescent probe NCF to indicate cellular HNO and GSH-GSSG balance. The sensitive and selective detection of HNO was achieved by incorporating an organophosphine group to naphthaldehyde-TCF. Then the resulted fluorescent product is able to monitor the conversion of GSH and GSSG reversibly. Additionally, outstanding biocompatibility make it capable of monitoring intracellular HNO and consequently GSH-GSSG oscillationsin living cells.

We anticipate that NCF

will be a unique molecular tool to investigate the interplaying roles of HNO and GSH. Introduction

important biological roles with therapeutic

To date, reactive nitrogen species (RNS) have gained increasing interests because of their multiple

biological

roles

in

living

organism.1-4Among all the RNS, nitric oxide (NO) is one of the best known signaling molecules

which

participates

in

many

physiological and pathological processes like anticanceractivity, neurotransmission, immune responses, blood pressure modulation, anticancer activity , and smooth muscle relaxation.5-8 Nitroxyl (HNO) is the one-electron reduced form of NO, which is generated directly from the oxidative degradation of L-arginine with the assistance of nitric oxide synthase under conditions.9-12

applications in a variety of diseases including treatments

for

heart

failure

and

alcohol

abuse.15-17

These unique pharmacological properties that oppose those of nitric oxide (NO), leads to the speculation that HNO may initiate unique biological responses by interacting with biotargets that are unable to reactive with NO. 18,19

The reaction of HNO with “soft” nucleophiles such as thiols is thermodynamically favorable. Thus, thiols and thiol containing proteins are considered to be primary targets associated with HNO biological activity, which will generate irreversible disulfide adducts. 20 Glutathione (GSH) is the smallest and also the

appropriate Unexpectedly, relevant investigations have show that HNO has

most

unique

effects ,which exhibits biological effects distinct

in all living aerobic cells.21,22 In general, glutathione exists in reduced state (GSH) as well

from NO.13,14 For instance, HNO displays

as oxidized state (GSSG).

and

potential

bio-pharmacological

abundant

intracellular

protein

thiol

molecule, prevalent in millimolar concentrations

23

Its function as 1

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

Page 2 of 12

sulfhydryl buffer is linked with the reduced state and oxidized state balance (GSSG/2GSH), as shown in Figure 1. In particular, reduced glutathione (GSH) is the primary reducing agent in tissue. Using the reducibility of GSH, glutathione peroxidase (GPx) catalyzes the reduction of hydrogen peroxide to water.24 In this catalyzed process, glutathione disulfide (GSSG) is generated accompanied by the disulfide bond

formation between two GSH

molecules. In the following, the glutathione reductase reduces glutathione disulfide (GSSG) to reduced glutathione (GSH) along with the oxidation of β-NADPH2. In the total glutathione pool, there is more than 90% glutathione in the form of reduced GSH under under normal physiological

conditions.

When

cells

are

exposed to highly oxidative conditions, the contant of GSSG increases as well as the ratio of GSSG to GSH. The increasing ratio between GSSG and GSH indicated the great increasement of oxidative stress.

Figure 1. Pathways for the reaction of HNO with GSH. Among

various

fluorescence

reported

microscopy

has

techniques, been

well

developed for indicating various cellular species owing to its high sensitivity, dynamic detection, noninvasiveness and good biocompatibility. 27-32 In recent years, numbers of well-designed fluorescent probes specific for HNO

33-38

or

39-50

GSH have been constructed, and great deal of progresses have been made to study HNO or GSH biology by using these fluorescent probes. However, dual-responsive fluorescent probe for HNO and GSH-GSSG remains in high demand.

Interestingly, under biological conditions, when

To fill the void, we designed and synthesized a

reduced GSH is exposed to HNO donors,

dual-site fluorescent probe NCF containing two

formation of GS(O)NH2 intermediate occurs,

individual

followed by the generation of oxidized GSSG

organophosphine is suitable for HNO detection

25

reactive

sites,

of

which

immediately. Thus, monitoring HNO and the ratio of GSSG to GSH in biological samples is

and the double bond between TCF and

of great importance. HPLC results reported by

(Scheme 1). In the presence of HNO, the

David A.Wink and coworkers indicate that free

triarylphosphine of NCF first produces the

HNO can be biosynthesized and thus may

corresponding phosphine oxide and aza-ylide,

function as an endogenous signaling agent that is

and then an amide was obtained via aza-ylide

content.26

naphthaline is employed for GSH addition

regulated by GSH However, the direct observing of HNO and GSH-GSSG are still not

linked to Staudinger, which ultimately produces

well elucidated because of the lack of efficacious

of resulted NCF-OH is switchable, reflecting the

analytical methods.

interconversion of GSH and GSSG, which can

a red-emissive NCF-OH. And the fluorescence

be regulated by H2O2 content either in tubes or in organisms with the assistant of GPx.

2


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Scheme 1. NCF releases emissive derivative NCF-OH when exposed to HNO, and the emission of resulted NCF-OH is switchable by GSH/GSSG conversion. Experimental methods

mM pH 7.4,). All spectra were obtained in a quartz cuvette (path length = 1 cm).

Materials and Instruments Unless stated otherwise, all the chemical and biological

reagents

were

procured

from

commercial sources and used without further purification.

Flash

chromatography

was

performed using Qingdao Ocean silica gel (200-300 mesh). Analytical NMR spectra were recorded using a Bruker Avance III spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) relative to internal Me4Si (1H and 13C)

and coupling constants (J) are given in hertz

Analyte stock solution (10 mM) was prepared in ultrapure water. PBS solution was prepared with Na2HPO4 and KH2PO4, and adjusted to pH 7.4. All the amino acids were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and were of the highest grade available. Synthesis of probe NCF Synthesis of NCF-OH 172.0

mg

(1.0

mmol)

of

219.0

mg

(Hz). Electrospray ionization (ESI) mass spectra

6-hydroxy-2-naphthaldehyde,

were measured with an Bruker Apex IV FTMS.

2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)

UV-vis

Purkinje

TU-1901

malonon-itrile ( TCF 51 )(1.1 mmol) and 85.0 mgammonium acetate (1.1 mmol) were

Fluorescence

emission

dissolved in 10 ml THF/EtOH = 4:1 mixture

spectra was measured on a Hitachi F-7000

solvents, and stired under dark for 24 h. After

fluorescence spectrometer with a 10mm quartz

distilling the solvent, the residues were extracted

cuvette. Fluorescence imaging was performed by

with 30 mL ethyl acetate three times. The

an Olympus IX81 confocal laser scanning

combined organic layers were dried over Na2SO4,

microscope (Japan). The pH was measured with

filtered,

a Mettler Toledo FE-30 pH meter.

pressure. The crude product was purified by

absorption

temperature

on

spectrophotometer.

were a

taken

at

room

General procedures for spectroscopic studies

and

concentrated

under

reduced

chromatography on a silica gel column using DCM/EA = 2:1 as the mobile phase, affording

Fluorescent probe NCF stock solution (1.0 mM)

NCF-OH as a red powder 185.1 mg (50% yield).

was prepared in dimethyl sulfoxide (DMSO).

1H

Test solutions were prepared by adding 50 μL of

8.32 (s, 1H), 8.09 (d, J = 16.1 Hz, 1H), 7.97 (d, J

NCF stock solutions into a test tube, appropriate

= 8.7 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.79 (d,

aliquot of each analyte stock solution into the

J = 8.6 Hz, 1H), 7.26 (d, J = 16.3 Hz, 1H), 7.17

above, then diluting the solution to 10 mL with

(d, J = 13.7 Hz, 1H), 1.82 (s, 3H).

NMR (400 MHz, DMSO) δ 10.31 (s, 1H),

the mixture of ethanol and water (v/v, 1:1) containing phosphate buffered saline (PBS, 10 3



Page 4 of 12

Scheme 2. Methodologies adopted for the synthesis of NCF.

Synthesis of probe NCF

Detection limit = 3/k

Methodologies adopted for the synthesis of NCF

where  is the standard deviation of blank

was shown in Scheme 2. A mixture of

measurements, k is the slope between the

(E)-2-(3-cyano-4-(2-(6-hydroxynaphthalen-2-yl)

fluorescence intensity vs AS (a commonly

vinyl)-5,5-dimethyl-furan-2(5H)-ylidene)malono

employed HNO donor) concentration.

nitrile (NCF-OH, 353.0 mg, 1.0 mmol), 2-(diphenylphosphino) benzoic acid (460.0 mg,

Cell culture and imaging

1.5 mmol), 4-dimethylaminopyridine (DMAP,

The HepG2 cells were grown in DMEM

122.0

and

supplemented with 10% FBS (fetal bovine

dicyclohexylcarbodiimide (DCC, 413.0 mg, 2.0

serum) and 50 µg mL−1 penicillin-streptomycin

mmol) in CH2Cl2 (40 mL) was stirred at 45 ◦C

at 37 °C and 5% CO2. Cell imaging was then

for 6 hours. Purification by silica gel column

carried out after the adherent cells were washed

chromatography afforded pure probe NCF after

with FBS-free DMEM (2 mL × 3 times) and

cooling to room temperature. (CH2Cl2 as the

then were incubated with 5 µM NCF in culture

mg,

1.0

mmol)

NMR (400 MHz, DMSO) δ 8.48 (s,

media for 5 min at 37◦C and then washed with

1H), 8.30 (s, 1H), 8.10 (dd, J = 17.3, 9.7 Hz, 2H),

PBS to remove the remaining probe (pH 7.4, 2

7.98 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 18.3 Hz,

mL × 2 times). The confocal fluorescence

2H), 7.38 (d, J = 25.5 Hz, 4H), 7.24 (s, 3H), 6.95

images of HepG2 cells were performed with an

eluent

)1H

13C

NMR (176 MHz, DMSO) δ 177.63, 175.51, 165.28, 150.29, 147.50, 140.67, 137.63, 137.56, 135.42, 134.37, 134.10, 133.98, 133.59, 132.53, 131.68, 131.35, 131.22, 129.53, 129.32, 129.28, 129.06, 125.19, 122.96, 119.35, 116.28, 113.17, 112.34, 111.42, 99.99, 99.88, 54.98, 25.55. ESI-HRMS calcd for + C41H28N3O3P[M+H] : 642.1940, found 642.1949.

(s, 1H), 1.84 (s, 3H).

60×oil

immersion

objective

lens.

The

fluorescence signal of cells incubated with NCF was

collected

at

580-630

nm,

using

a

semiconductor laser at 488 nm as excitation resource. Results and discussion Design and Synthesis of Fluorescent Probe NCF As shown in Scheme 2, NCF was designed by

Determination of the detection limit Refering to previous papers, the detection limit was calculated based on fluorescence titration. 52-53

A fluorescent titration operation was carried out in the mixture of ethanol and water (v/v, 1:1), containing PBS (10 mM,

Olympus IX81 confocal microscope with a

pH

7.4,)

incorporating anphosphine moiety to a GSH sensitive NCF-OH ， based on the documented selective reductive ligation to HNO. When exposed

to

HNO,

NCF

generates

high

fluorescence by releasing red emissive NCF-OH.

to obtained the detection limit that was then calculated with the following equation: 4


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Figure 2. Fluorescence spectra (a) and absorption spectra (b) of NCF (5 μM) in the absence and presence of AS (50 μM) in H2O/ethanol=1:1 (v/v), 10 mM PBS, pH=7.4. (c) The plot of fluorescence intensity at 610 nm vs AS concentrations (0-20 μM). (d)The dynamic fluorescence changes at 610 nm of NCF (5 μM) after the addition of AS (50 μM) in H2O/ethanol=1:1 (v/v), pH=7.4, 10 mM PBS, 37 °C. λex = 520 nm, slit widths: Wex = Wem = 10 nm. Each datum was acquired 3 min after AS was added at 37 °C. Then the emission of resulted NCF-OH fluorophore can be switched by the balance between GSH and GSSG. This probe exploits the use of phosphine derivative for specific recognition of HNO and the nucleophilic addition by reduced GSH instead of GSSG. When

NCF

incubated

containing PBS (10 mM, pH 7.4) by UV-vis absorption and fluorescence techniques. As shown in Figure 2 and S1, the addition of 50

triarylphosphine group of NCF is removed to

ca.6-fold fluorescence intensity enhancement at

afford a distinctly fluorescent enhancement,

610 nm and the absorption band centered at 445

which is attributed to compound NCF-OH.

nm barely changed. This can be attributed to the

Upon treatment of NCF-OH with GSH/GSSG,

dissociation of triphenylphosphine quenching

an

generate

and the formation of stronger ICT structures.

NCF-SG-OH (theirs tructures are shown in

The large spectral shift is due to the cleavage of

Scheme 1). The C=C double bond of NCF-OH

the electron-stretched benzoate derivative, and

is controlled by GSH/GSSG to obtain an

the

intermediate compound NCF-SG-OH, which

electron-promoting ability. As displayed in

exhibits the fluorescence signal pattern for NCF

Figure S4, NCF showed acceptable stable

in the presence of GSH/GSSH is dim - bright

spectral properties, and the absorption and

red.

fluorescence spectra of the probe did not change

occurs

to

AS,

in a mixture of H2O/ethanol solution (v/v, 1:1)

µM AS to the NCF solution (5 μM) triggered a

reaction

with

The recognition of NCF to HNO was measured

the

additive

is

Response of NCF to HNO

oxygen

anion

produces

a

stronger

5



significantly within the first 28 hours.

Page 6 of 12

over ROS, RNS, and high concentrations of

Quantification of HNO and Detection limit Calculation

biologically relevant species. These results confirm that the probe molecule NCF is specific toward HNO, which might be attributed to the

The fluorescence spectra of NCF with various

adoption of the phosphino recognition moiety.

concentrations of AS is shown in Figure 2c. The fluorescence band centered at around 610 nm increased gradually were induced by the increasing concentrations of AS content. In addition, a good linear relationship between the fluorescence

band

at

610

nm

and

the

concentrations of HNO in the range of 0 to 20 μM was observed. The detection limit was calculated

to

be

160

nM.These

results

demonstrated that NCF could detect AS quantitatively by fluorescence spectrometry method with an excellent sensitivity. The kinetic profile of NCF towards HNO Response rate is a crucial fundamental parameter for reaction-based probes and the kinetic profile of the reaction of NCF with HNO was investigated at room temperature. The response time of NCF towards AS was evaluated by fluorescence spectroscope. Upon addition of 50 μM AS into the solution of NCF (5 μM), the fluorescence intensity at 610 nm increased gradually, and finally levels off after 3 min ( Figure 2d ). The result shows that the reaction between NCF and HNO could complete with in 3 min

， which is favor for biological

applications and reported

is much faster than other

phosphine-based

fluorescent

probes54-56.

Figure 3. (a) The fluorescence spectra of NCF (5 μM) toward AS (30 μM), GSH and various analytes (30 μM) in H2O/ethanol=1:1 (v/v), pH=7.4, 10mM PBS at 37 °C. (b) Fluorescence intensity changes at 610 nm. (1) NCF (2) ClO- (3) H2O2 (4) TBHP (5) ∙OH (6) TBu (7) Cys (8) NO2- (9) NO3- (10) Na2S (11) GSH (12) NO (13) AS. λex = 520 nm, slit widths: Wex = Wem = 10 nm. Each datum was acquired 3 min after various analytes addition at 37 °C. Fluorescence NCF-OH

Reversibility Mediated

by

of

resulted

GSH-GSSG

transformation

The selectivity of NCF Except for HNO, which induces the expected bright red emission, no significant change in emission intensity was observed in the presence of any other RNS and ROS species, As shown in Figure 3. Only HNO gave 4 fold fluorescence enhancement. Thus, these results demonstrated

Next, we examined the emissive reversibility of freshly generated NCF-OH in GSH pool. In the organism, it is reported glutathione peroxidase (GPx) is able to oxidize reduced GSH into oxidized GSSG by catalytic reduction of H2O224. In vitro, GSH still can be oxidized into its

that NCF possesses high selectivity for HNO 6


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Figure 4. (a) The fluorescence spectra of NCF (5 μM) after the addition of 50 μM AS as followed by 10 μM of GSH at different times in H2O/ethanol=1:1 (v/v), pH=7.4, 10 mM PBS at room temperature. (b) The fluorescence spectra of NCF (5 μM) after the addition of 50 μM AS followed 10 μM GSH and then by 30 μM of H2O2 at different times in H2O/ethanol=1:1 (v/v), pH=7.4, 10 mM PBS at 37 °C. λex = 520 nm, slit widths: Wex = Wem = 10 nm. oxidized state at a relatively slow rate.57-58 In this

strong emissive NCF-OH is released from the

case, H2O2 was employed to control the

triarylphosphine

GSH-GSSG ratio in the total GSH pool. As

triggered by HNO. ESI-HRMS in Figure S11

displayed in Figure 4, when NCF-OH was

shows a main peak at m/z 352.1051[M-H]+

treated with 10 μM GSH, the fluorescence

which is corresponded to compound NCF-OH

intensity was dropped immediately to 25% and

(calculated at m/z 352.1164 [M-H]+). Previous

below. When the mixture was further treated

reports

with 30 μM H2O2, which will lead to GSSG

S12) indicate that a Michael addition reaction

accumulates and the increasement of GSSG to

occurs between NCF-OH and GSH. And the

GSH ratio. As displayed in Figure 4b, treatment

C=C conjugation of NCF-OH is broken by the

of H2O2 showed the recovery of fluorescence,

attacking of GSH to obtain a non-emissive

suggesting that the addition reaction-based

NCF-SG-OH, the ESI-HRMS spectrum of which

reaction site is efficient to monitor GSH-GSSG

is also presented in Figure S12, showing a main

conversion

different

peak at m/z 659.7986 [M-H]+(calculated at m/z

concentrations of GSH and H2O2 regulate the

659.8064 [M-H]+). Next, the GSH-GSSG ratio

transformation

was

in the total GSH pool is controlled by H2O2, and

showed in Figure S2. As displayed in Figure S3,

when the ratio of GSH-GSSG decreases, it

upon the decrease of GSH/GSSG, the absorption

recycles NCF-SG-OH to NCF-OH with the

peak at 450 nm decreased and the peak at 340

simultaneous elimination reaction.

reversibly. between

Next,

GSH-GSSG

45and

caged

parent

fluorophore,

mass spectrometry data (Figure

nm increased; the fluorescence at 610 nm gradually increases as the GSH/GSSG ratio decreases. Reaction Mechanism It has been well documented that reaction of HNO with triarylphosphine would generate the corresponding phosphine oxide and aza-ylide,

Scheme 3. The proposed reaction mechanism of

and then an amide was obtained via aza-ylide

probe NCF with HNO and GSH/GSSG

linked to Staudinger,

59

As shown in Scheme 3, 7



Page 8 of 12

Figure 5. (A) Real-time imaging of HNO and GSH-GSSG balance in living HepG2 cells. Cells were incubated with 50 µM AS at 37℃ for 30 min and then stained with NCF (5 μM, 5 min). (a) HNO was incubated for 1 min then incubated with NCF (5 μM) (b) 1 min (c) 3 min, (d) 6 min, (e) H2O2 (final concentration: 50 μM) was added at 10 min,(f) 15min. (B) Time course of the fluorescence intensity changes in HepG2 cells. The fluorescence intensity was measured at 580-630 nm with excitation at 488 nm. Scale bar 20 μm. Bioimaging applications and cytotoxicity

incubation (Figure 5c-d). These results imply

Furthermore, to evaluate the cytotoxicity of

that fresh generated NCF-OH is responsive to

NCF,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylt

GSH by nucleophilic addition to generate

etrazolium bromide (MTT) assays was first

non-emissive NCF-GS-OH. Fascinatingly, after

performed to evaluate the cytotoxicity of NCF;

adding H2O2 to convert GSH into GSSG as

0, 5, 10, 15, 20 µM of probe were incubated with

discribed in Figure 1, a significant fluorescence

HepG2 cells for 24 h, and cytotoxicity was

enhancement was observed in Figure 5e-f. More

measured. The viability results in Figure S5

specific calculated intracellular fluorescence

clearly indicated that NCF was low toxic to

intensity was given in Figure 5B. These results

cultured cells under the experimental conditions.

clearly

The obtained results showed that NCF is

cell-permeable and it behaves as an alternative

suitable for living cell imaging at its working

tool for imaging HNO, as well as the balance

concentration, which is 5 μM.

between GSH-GSSG in living cells.

Inspired by the in vitro experimental results and

Conclusion

encouraged by low cytotoxicity, we expected this highly sensitive HNO probe could have good performance inimaging cellular HNO. In order to verify our conjecture, HepG2 cells were incubated with AS (50 µM) for 30 min before washing off, and then incubated with NCF (5 µM). A obvious red fluorescence enhancement was observed straight away as shown in Figure 5b.

However,

intracellular

fluorescence

decreased after 3 minutes caused by the addiction of GSH. And the fluorescence became even lower to 50% after another 10 min’s

demonstrated

that

NCF

is

well

In this work, we have developed a dual site fluorescent NCF to monitor cellular nitroxyl and GSH-GSSG oscillations, and demonstarated its optical response and intracellular performance. Caged by a oganophosphine, NCF responds to HNO

and

generates

bright

red

emissive

NCF-OH. By reversible nucleophilic addition, the resulted NCF-OH exhibited swithable fluorescence along with the interconversion of GSH and GSSG in the total GSH pool. In conclusion, this work establishes a robust 8


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strategy for monitoring HNO, and GSH-GSSG

Proc. Natl. Acad. Sci. U. S. A. 1994, 91,

oscillations using a single fluorescent probe,

10992–10996.

which might become a unique optical tool to

(13) Fukuto, J. M.;Cisneros, C. J.; Kinkade, R. L.

investigate the interplaying roles of HNO and

J. Inorg. Biochem. 2013, 118, 201–208.

GSH-GSSG in complex signaling and redox

(14) Espey, M. G.; Miranda,. K. M.; Thomas, D.

pathways.

D.; Wink, D. A. Free Radical Biol. Med. 2002, 33, 827–834.

Supporting Information

(15) Sherman, M. P.; Grither, W. R.; McCulla, R.

The Supporting Information is available free of

D. J. Org. Chem. 2010, 75, 4014–4024.

charge on the ACS Publications website.

(16) Feelisch, M. Proc. Natl. Acad. Sci. U. S. A.

Experimental details, supplementary data, and

2003, 100, 4978–4980.

characterization of compounds (PDF)

(17) Paolocci, N.; Katori, T.; Champion, H.; St

Acknowledgements:

John, M.; Miranda, K.; Fukuto, J.; Wink, D.; Kass ,D. Proc. Natl. Acad. Sci. U. S. A. 2003,

We gratefully acknowledge financial support

100, 5537–5542.

from the National Natural Science Foundation of

(18) Wong, P. S.-Y.; Hyun,J.; Fukuto, J. M.;

China (No. 21575015 and 21505004).

Shirota,F. N.; DeMaster,E. G.; Shoeman, D. W.;

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