1 A Thiol Specific and Mitochondria Selective Fluorogenic

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A Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Non-protein Thiol Imaging and Quantification in Mitochondria Shenggang Wang, Huihui Yin, Yue Huang, and Xiangming Guan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01469 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Analytical Chemistry

A Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Non-protein Thiol Imaging and Quantification in Mitochondria Shenggang Wang, Huihui Yin, Yue Huang, and Xiangming Guan* Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions South Dakota State University, Brookings, SD 57007

*Corresponding Author: Xiangming Guan, Ph.D. Department of Pharmaceutical Sciences, College of Pharmacy & Allied Health Professions, Box 2202C, South Dakota State University, Brookings, SD 57007. Phone: (605) 688-5314; Fax: (605) 688-5993; E-mail: [email protected]

Current address for Huihui Yin: Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning, P.R. China

Abstract Cellular thiols are divided into two major categories: non-protein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'thiobis(N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence microscopy. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λex = 550 nm, λem = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two co-localization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

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INTRODUCTION Mitochondria are double-membrane subcellular organelles. They are one of the most important subcellular organelles in the cell. In mammalian cells, mitochondria provide most of the cellular energy in the form of ATP by means of the oxidative phosphorylation accompanied by the reduction of molecular oxygen. They are also the main producer of reactive oxygen species (ROS). Although ROS are oxidants and harmful, a physiological amount of ROS are needed in cellular signaling. However, excessive ROS generated in mitochondria can cause oxidation of lipids, proteins, and other biomolecules resulting in apoptosis and necrosis. Mitochondrial thiols serve as the major antioxidants and play a critical role in the removal of ROS to maintain a delicate balance of ROS to meet the physiological need. In addition, mitochondrial thiols, mainly mitochondrial glutathione (mGSH), is essential in removing toxic electrophiles in mitochondria.1,2 A decrease in the level of mitochondrial thiol reduces the antioxidant defense system and has been linked to aging, cardiovascular and autoimmune disorders, and neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. 1,2 Monitoring thiol status in mitochondria provides valuable information in understanding the roles of thiols in the functions and dysfunctions of mitochondria. Cellular thiols can be divided into two major categories: protein thiols (PSH) and non-protein thiols (NPSH). NPSH include glutathione (GSH), cysteine, homocysteine, and other thiol-containing short peptides. Although GSH is the major NPSH in mitochondria, cysteine, γ-glutamylcysteine (precursor of GSH), and likely other short peptide thiols are also present in mitochondria.2-5 Most conventional analytical methods for the quantification of mitochondrial thiols, mostly mGSH, are through isolation and homogenization of mitochondria followed by an analytical thiol assay method such as HPLC,6,7 LC/MS,8 or enzyme assay.9 Our understanding of the physiological/pathological roles of thiols in mitochondria is mostly based on the determination of thiol concentration from homogenized mitochondria. Analytical methods to monitor thiol density and distribution in mitochondria in live cells are limited though it is known that thiols are not evenly distributed in cells.10,11 Monitoring an analyte in live cells through fluorescence microscopy has been one of the most common approaches for live cell analyte analysis. Compared with conventional analytical methods that require tissue/cell/subcellular organelle homogenization, live cell analyte fluorescence microscopy has the advantage of allowing us to visualize the analyte in its native environment and revealing information that cannot be revealed by cell homogenates, such as intracellular distribution and dynamitic movement of the analyte.12 The major challenge for detecting an analyte through fluorescence microscopy in live cells is to turn the analyte to a fluorescent derivative that can be readily detected by fluorescence microscopy in a non-cytotoxic manner. Quantification of the analyte is even more challenging. It requires the conversion of the analyte into a fluorescent derivative in a quantitative and measurable manner. Lim and colleagues reported the first mitochondria selective fluorescent thiol imaging agent (SSH-Mito) based on a thiol specific reaction.13 This is the only reagent reported to be able to react with GSH, cysteine and other small molecule thiols and to image thiols in mitochondria in live cells. Followed after that, various mitochondria-targeting fluorescent/fluorogenic thiol imaging reagents have been reported for selective imaging of GSH13-16 or cysteine2-5 based on their preferred reactions with GSH or cysteine. Here we would like to report 7,7'-thiobis(N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS) as a thiol-specific mitochondria-selective fluorogenic reagent. TBROS successfully imaged and quantified NPSH in mitochondria in live cells through fluorescence microscopy using 550 nm and 580 nm as the excitation and emission wavelengths respectively. To our knowledge, this is the first reagent that is able not only to image mitochondrial NPSH and but also to provide quantitative information in live cells through fluorescence microscopy. EXPERIMENTAL SECTION Unless otherwise stated, all reagents and solvents were obtained from commercial sources and used without further purification. Information on materials, solutions, instruments, experimental procedures of 2


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chemical stability determination of TBROS, quantum yield determination, reactivity of TBROS towards PSH, pH effects on the reactivity of TBROS towards thiols and live cell mitochondrial NPSH imaging, determination of cell viability and optimal cell culture condition for live cell mitochondria NPSH imaging, and photostability of TBROS’ thiol adducts in live cells are provided in the Support Information. Synthesis of TBROS. To a stirred solution of benzofurazan sulfonamide (1) (20 mg, 0.046 mM) in 10 mL acetonitrile was added with (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) (97.2 mg, 0.187 mM), 1-hydroxybenzotriazole (HOBT) (28.6 mg, 0.187 mM), Rhodamine B (44.07 mg, 0.092 mM), N,N-diisopropylethylamine (DIPEA) (50 µl), and a small amount of 4-dimethylaminopyridine (4-DMAP) as a catalyst. The mixture was stirred at room temperature for 3 h. Solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethanol/ethyl acetate) to yield TBROS as a red solid (49 mg, 83.3%). TBROS was fully characterized by 1HNMR and HRMS. The purity (>95%) was confirmed by HPLC (254 nm as detection wavelength). 1H NMR [400 MHz, (CD3)2SO)] δ = 8.06-7.99 (m, 2H), 7.64 (d, J = 7.1Hz, 2H), 7.60-7.54 (m, 4H), 7.40 (d, J = 7.2 Hz, 2H), 7.08-6.99 (m, 2H), 6.48 (d, J = 9.5 Hz, 8H), 6.41 (d, J = 9.1 Hz, 4H), 3.37 (q, J = 6.8 Hz, 16H), 1.05 (t, J = 6.9 Hz, 24H). HRMS: calculated for C68H66N10O10S32+: 639.2057, found: 639.2054. Cell Culture. NCI-H226 cells (human lung cancer cells) were obtained from the National Cancer Institute and maintained in RPMI 1640 growth medium supplemented with 10% FBS, 100 units/mL penicillin (Mediatech, Inc., Herndon, VA) and 100 µg/mL streptomycin (Mediatech, Inc., Herndon,VA) in a humidified atmosphere containing 5% CO2 at 37 °C. Reaction of TBROS with NPSH and non-thiol containing amino acids. The reaction between TBROS with NPSH or non-thiol containing amino acids was conducted in a Tris buffer (pH 7 or 8.0, 0.1 M) with 2 mM EDTA and 50% acetonitrile at 37 °C. Briefly, TBROS (50 µM) was mixed with NPSH or a non-thiol containing amino acid at different ratios (1:0, 1:1, 1:10, 1:100, 1:500). Aliquots were withdrawn for HPLC/UV (20 µL) analysis or fluorescence monitoring (100 µL). For HPLC analysis, the HPLC condition is presented in the Supporting Information. For fluorescence monitoring, the aliquots were transferred to a 96 well plate for measuring fluorescence on a SpectraMax M2 microplate reader using 550 and 580 nm as λex and λem respectively with a cutoff wavelength of 570 nm. Confirmation of imaging in mitochondria Co-localization with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. NCI-H226 cells were seeded on a 15 mm diameter microscope cover glass in a 12-well plate in the RPMI 1640 growth medium with 10% FBS. After 24 h attachment, the cells were washed three time with (Dulbecco's phosphate-buffered saline) DPBS, treated with TBROS (100 nM) and DiOC6(3) (Thermo Fisher Scientific, Waltham, MA) (50 nM) in a RPMI 1640 medium without FBS for 180 min in a 37 °C incubator with 5% CO2. The medium was removed and the cells were washed 3 times with DPBS. The microscope cover glass was transferred to a microscope slide with DPBS as the mounting medium. The fluorescence images were taken at a 100x oil objective on an upright fluorescence microscope (Zeiss AXIO Imager A1) connected to a camera (AxioCam MRc5). Rhodamine channel filter was set for thiol imaging by TBROS while FITC channel filter was set for DiOC6(3). The merged images were processed by ImageJ, a software obtained from the Web site of the National Institutes of Health (http://rsbweb.nih.gov/ij/). Co-localization with GFP (green fluorescence protein)-labeled mitochondria. Co-localization with GFP-labeled mitochondria followed the procedure provided by the manufacture. Briefly, NCI-H226 cells were seeded in 12well plates. After 24 h attachment, 30 µl of CellLight® Mitochondria-GFP (Thermo Fisher Scientific, Waltham, MA) was added to the cells. The cells were incubated in a 37 °C incubator with 5% CO2 for 16 h. The cells were washed with DPBS and then treated with TBROS (10 µM) for 2.5 h before images taken as described above. FITC filter was used for the green fluorescence of GFP. Confirmation of thiol imaging and quantification. The procedure is the same as presented in the section of [co-localization with DiOC6(3)] except no DiOC6(3) was used and the cells were pretreated with different concentration of N-ethylmaleimide (NEM) (Sigma-Aldrich, St. louis, MO) for 3 h to establish cells with 3



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different levels of thiols. For quantification, fluorescence intensity of TBROS thiol adducts derived from cells treated with NEM was compared with the control in which cells were treated with no NEM. RESULTS AND DISCUSSION Design and Synthesis TBROS is a symmetric benzofurazan sulfide (Figure 1). Its design as a thiol-specific mitochondriaselective fluorogenic reagent was based on a benzofurazan sulfide skeleton structure shown in Figure 1. We found previously that the symmetric nature of the benzofurazan sulfide skeleton structure made the molecule non-fluorescent. The structure reacted with a thiol in a thiol specific manner rapidly and quantitatively to form a fluorescent thiol adduct and a non-fluorescent thiophenol (Figure 1).17 Our plan to design a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide was to link a mitochondria-targeting moiety to the basic skeleton structure. TBROS was evolved from two earlier designed thiol-specific mitochondria-selective fluorogenic agents TBOP and RhOD-2 (Figure 1). TBOP employed an alkyltriphenylphosphonium cation (TPP) as the brain-targeting structure. Alkyltriphenylphosphonium cations (TPPs) are the most well-established mitochondria-targeting structures.18-20 We gave up TBOP due to the reason that the fluorescence intensity derived from TBROP thiol adducts was not strong enough for quantification when thiols in mitochondria were lower than normal. Nevertheless, TBOP was able to image and quantify total NPSH in cells when given enough time.21 RhOD-2 used a rhodamine structure which is a mitochondria-targeting structure also.22 RhOD-2 was abandoned due to its slow hydrolysis of the sulfonamide bond resulting in a release of strong fluorescent Rhodamine. The released Rhodamine interfered with the quantification of thiols in mitochondria. To overcome this problem, we removed the N-methyl group in the sulfonamide structure so that the sulfonamide would become an ionizable acidic functional group. The anion resulted from the ionization should make the structure less electron deficient and less likely for hydrolysis. The resulting structure is TBROS which was later confirmed to be resistant to hydrolysis and able to image and quantify NPSH in mitochondria in live cells by fluorescence microscopy. The synthesis of TBROS was completed by reacting a benzofurazan sulfonamide (1) with Rhodamine in the presence of PyBOP, HOBT, 4-DMAP, and DIPEA (Scheme 1). Column separation of the reaction mixture produced TBROS in 83% yield. TBROS was fully characterized by 1H NMR and HRMS. The purity (>95%) was confirmed by HPLC. The starting material benzofurazan sulfonamide (1) was prepared according to a reported procedure.17 Once TBROS was synthesized, the chemical stability of TBROS was checked in a Tris buffer (pH 7, 0.1 M) with 2% SDS and 2 mM EDTA. When TBROS was dissolved in the buffer at 37 °C for 3 h, no hydrolysis was observed confirming that removal of the N-methyl group in the sulfonamide of RhOD-2 make the molecule much less susceptible to hydrolysis (Figure S-1). Thiol specificity, reaction rate, and pH effects Like those previously reported benzofurazan sulfide derivatives,17,21 TBROS was shown to be thiols specific. When TBROS reacted with a thiol, the reaction yielded a thiol adduct and a released thiophenol (Figure 2). Figure 3 shows representative HPLC chromatograms obtained from TBROS’ reaction with a thiol molecule N-acetylcysteine methyl ester (NAC methyl ester) using a UV/vis detector (a, b) or fluorescence detector (c, d). Figures 3a and 3b demonstrate that disappearance of the TBROS peak was followed by appearance of two product peaks. These two products were confirmed by mass spectrometry to be the thiol adduct and corresponding thiophenol (Figure 3b). When TBROS reacts with serine - a non-thiol amino acid containing -OH, -NH2, and -COOH groups, no reaction was observed suggesting the inability of TBROS to react with -OH, -NH2, and -COOH groups. The functional groups of -OH, -NH2, and -COOH are the most commonly seen nucleophilic groups in biological systems. The thiol specificity was further confirmed by the inability of TBROS to react with other non-thiol amino acids. These non-thiol amino acids include one acidic amino acid (glutamic acid), two basic amino acids (lysine, arginine), one amino acid with a phenolic group (tyrosine), one aromatic amino acid (tryptophan), and two neutral amino acids (valine, glycine). When TBROS was mixed with 500 equiv of these amino acids at 37 ºC for 4 h, no reaction was observed (Figure 4). 4


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R

R

R'

S

N O N

Thiol adduct (fluorescent)

SH

N O N

+

N

Thiophenol (non-fluorecent) R'-SH (Thiol specific reaction)

R

O

N

N O N

O

N Cl-

O O S

N H

R S

N

ClS S

N O N

N N O

O O

O

O

N H

N N O

TBROS

Basic skeleton structure of thiol-specific fluorogenic benzofurazan sulfides

N

O

N

N

O

O

P Br

S

N

O

O

O S

S

N ON

O O

Br- P

N

N Cl-

Cl-

N

N NO

S

O

O

S

N O N

TBOP

O O

S

RhOD-2

N

N N O

Figure 1. Design of thiol-specific and mitochondria-selective fluorogenic agent TBROS. O H2N

S

O

O

S S

N O N

N

O a

NH2

N

O

N

O

N Cl-

ClO O

N N O

N H

S

S S

N O N

1 a. Rhodamine B, DIPEA, PyBOP, HOBT, DIPEA , and 4-DMAP in acetonitrile at room temperature.

O O

O

O

N H

N N O

TBROS (83%)

Scheme 1. Synthesis of TBROS N

N

O

O

ClO O

O S

R1S

HO

N H

Serine or other non-thiol amino acids

N Thiol Adduct (Fluorescent) N O (R1SH = NAC methyl ester; MW=799.2)

λex = 550 nm, λem = 580 nm

N

O

N

N

O

O O N

O

N H

R1SH (NPSH)

ClO O N H

S

O

N O N

N Cl-

Cl-

+ N

OH NH2

O

TBROS

N O N

S

S

O O

O S

N H

N N O

PSH

SH

Thiophenol (Fluorescent) λex = 550 nm, λem = 570 nm; (R1SH = NAC methyl ester; MW=656.2)

Figure 2. TBROS’ reaction with NPSH, PSH, and serine (a non-thiol amino acid). The non-florescent TBROS reacted with NPSH (R1SH) to form a fluorescent thiols adduct and a fluorescent thiophenol. No reaction was observed when TBROS was mixed with serine and PSH. 5



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Figure 3. HPLC chromatograms derived from the reaction of TBROS (90 µM) with NAC methyl ester (45 mM) in a Tris buffer (pH 8.0, 0.1 M) containing 50% acetonitrile at 37 ºC for 0 min (a and c) and 30 min (b and d). UV/Vis (a, b) or fluorescence (c, d) (λex = 550 nm, λem = 580 nm) was employed for detection. Compared with GUALY’s reagent, one of the first thiol-specific fluorogenic benzofurazan sulfides reported from this group,17,21 TBROS appeared to react at a much slower rate with thiols. GUALY’s reagent completed the reaction with NAC methyl ester in five minutes at 1:1 reactant ratio in a Tris buffer (pH 7.0, 0.1 M) at 37 ºC. Under the same condition, no significant reaction was observed in 1 h for TBROS. When the ratio of NAC methyl ester to TBROS was increased to 100:1 (5 mM:50 µM), noticeable reaction was observed (Figure S-2). Since mitochondria are more alkaline than other parts of the cell and have a pH value of 8,2 the reaction rate of TBROS with NAC methyl ester at pH 8 was evaluated. TBROS was found to react at a much faster rate at pH 8 than at pH 7 (Figure S-2). With a ratio of 100:1 of NAC methyl ester vs TBROS, TBROS completed the reaction in 40 min. The reaction of GSH with TBROS under the same condition appeared to be slower (Figure S-2). Similarly, homocysteine and cysteine reacted much faster with TBROS at pH 8 than that at pH 7 (Figure S-2). To determine which thiol reacts faster, TBROS (25 µM) was mixed with 125 equiv of NAC, NAC-methyl ester, cysteine, homocysteine or GSH in a Tris buffer (pH 8.0, 0.1 M, 2 mM EDTA) with 50% acetonitrile at 37 °C for 2 h. Our data show that the reaction order was NAC methyl ester > homocysteine > cysteine >GSH > NAC with unreacted remaining TBROS as 3%±1 for NAC methyl ester, 46%±1 for homocysteine, 50%±2 for cysteine, 71%±1 for GSH, and 88%±4 for NAC (Figure S-2d). The slow reaction rate of TBROS at pH 7 is an advantage for mitochondria selectivity. If a reagent reacts too rapidly with a thiol, it will start to react with thiols before reaching mitochondria and interfere with mitochondria thiol imaging and quantification. Therefore, it was expected that the slow reaction rate of TBROS at pH 7 could reduce the possibility of TBROS to react with thiols on its path to mitochondria. Once it enters mitochondria, the alkaline pH together with higher TBROS concentration in mitochondria would facilitate TBROS’ reaction with thiols. This expected mitochondrial thiol selectivity was indeed observed when TBROS was used to image thiol in live cells. The fluorescence produced by TBROS was strong in mitochondria but 6


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barely visible in other parts of the cell. TBROS not only imaged but was also able to quantify thiols in mitochondria even when mitochondrial thiols were lower than normal (data presented below). To further confirm the impact of pH on TBROS’ reaction rate with mitochondrial thiol in live cells, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was employed. CCCP is a reagent that can increase acidity in mitochondria in live cells through a reduction of mitochondria cross-membrane potentials.23,24 At the concentration of 20 µM, CCCP has been shown to decrease mitochondrial pH by 0.7.23,24 When NCI-H226 cells, a cell line employed previously by this group for thiol imaging in live cells,17,21 were treated with TBROS, the fluorescence in mitochondria was much weaker in the presence of CCCP (20 µM) than that in the absence of CCCP (Figure S-3). The data provide an additional piece of evidence that alkaline pH in mitochondria facilitated the reaction of TBROS with mitochondrial thiols though it is recognized that a reduction of crossmembrane potential by CCCP could lead to less accumulation of TBROS in mitochondria which would also lead to a slower reaction with mitochondrial thiols. Reactivity toward PSH TBROS’ reactivity with PSH was first checked with bovine serum albumin (BSA). BSA contains 36 thiol groups/molecule. When TBROS was mixed with BSA in a Tris buffer (pH 8, 0.1 M) at 37 ºC for 60 min, no fluorescence change was observed suggesting no fluorescent thiol adducts formed (please refer to the Supporting Information for detailed experimental conditions). When the supernatant of the reaction was checked by HPLC, it was found that the peak area of TBROS remained the same for the sample obtained at 0 min vs that at 60 min even at a ratio of 1: 2024 for TBROS vs thiol in BSA confirming that no reaction had occurred between TBROS and thiols of BSA during the time (Table S-1). The same results were obtained with proteins from cell homogenates. When protein precipitates were mixed with TBROS (450 µM), no change on fluorescence intensity as well as no change on the HPLC peak area of TBROS were observed from aliquots of the reaction mixtures at 0 min and 60 min (Table S-1). The inability of TBROS to react with PSH was similar to that of TBOP21 and RhOD-2. TBOP and RhOD-2 were unable to react with PSH either due to likely a steric hindrance of the large molecular size since GUALY’s reagent, a much smaller molecule reported by us earlier, was able to react with both PSH and NPSH.17 TBROS’ inability to react with PSH was likely caused by the same reason. 3.50E+05

Control GSH

3.00E+05 Fluorescence Intensity (a.u.））

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


NAC methyl ester Serine Cysteine

2.50E+05

Homocysteine NAC Tyrosine

2.00E+05

Tryptophan Lysine

1.50E+05

Valine Glycine Glutamic acid

1.00E+05

Arginine

5.00E+04 0.00E+00 560

590 620 Wavelength(nm)

650

7



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Figure 4. Emission spectra of TBROS (25 µM) in the absence and presence of various amino acids (12.5 mM) at 37 ºC for 4 h in 50% acetonitrile in a Tris buffer (pH 8.0, 0.1 M). Fluorescence property After confirming that TBROS is a thiol-specific reagent and reacts with only NPSH, we then moved to confirm if TBROS was a fluorogenic reagent like other previously reported benzofurazan sulfides.17,21 In Figure 4, TBROS was shown to exhibit minimum fluorescence when excited at 550 nm (control). However, when TBROS was mixed with a thiol molecule, strong fluorescence was observed (Figure 4) confirming that TBROS was a fluorogenic agent for thiols. On the other hand, no fluorescence change was observed when TBROS was mixed with non-thiol amino acids providing an additional piece of evidence that no reaction occurred between TBROS and non-thiol amino acids (Figure 4). As shown in Figure 4, the maximum emission wavelength derived from the reaction of TBROS with different thiols was the same (580 nm) though the fluorescence intensities were different with the intensity order of homocysteine > cysteine > NAC methyl ester > GSH > NAC suggesting that the wavelength 580 nm can be used to detect various different thiols. However, due to different fluorescence intensity from different thiols, quantification of an absolute amount of thiols would not be possible. The quantum yield (Φ) of the NAC methyl ester thiol adduct of TBROS was determined by following a literature procedure.25 The Φ value of the thiol adduct was determined to be 0.4 while at the same condition the Φ value for Rhodamine B is 0.7.25 The fluorogenicity of TBROS was further confirmed by HLPC with a fluorescence detector. As shown in Figures 3a and 3b, HPLC/UV chromatograms demonstrate that TBROS reacted with a thiol to form a thiol adduct and a thiophenol. However, when the reaction was monitored by HPLC/fluorescence, no peak was observed for TBROS (Figure 3c) confirming that TBROS was not fluorescent while both thiol adduct and thiophenol were fluorescent (Figures 3d). Mitochondria thiol imaging in live cells Optimal TBROS concentration, incubation length, cell viability, and photostability of thiol adducts Once confirmed to be thiol-specific and fluorogenic, TBROS was evaluated for its ability to image NPSH in mitochondria in live cells using NCI-H226 cells. Before imaging, the optimal concentration and incubation length required for live cell NPSH imaging were determined. When cells were incubated with different concentrations of TBROS for 2 h 30 min, fluorescence intensity increased with an increase in TBROS’ concentration until it reached 10 µM. No further increase in fluorescence was observed when TBROS’ concentration was higher than10 µM suggesting 10 µM TBROS was needed in order to turn all NPSH to thiol adducts for quantification (Figure S-4). When cells were incubated with 10 µM of TBROS, fluorescence intensity continued to increase for 2 h 30 min before a plateau was reached suggesting that 2 h 30 min was needed for NPSH quantification (Figure S-5). To ensure it was live cell imaging, the viability of cells treated with 10 µM of TBROS was checked by the Trypan blue assay. It was found that the cells remained alive (>95%) when treated with 10 µM TBROS for 12 h. The same viability was observed with CV-1 cells (a normal monkey kidney cell line). CV-1 cells remained viable (94%) even when treated with 50 µM of TBROS for 12 h. Finally, the decay property of the fluorescence derived from thiol imaging in live cells by TBROS was evaluated with NCI-H226 cells. The decay rate was comparable to that of FITC, a commonly used fluorescence reagent for fluorescence microscopy (Figure S-6). Imaging of mitochondrial NPSH The ability of TBROS to image mitochondrial NPSH in live cells was determined by confirming first that the imaging was occurring in mitochondria followed by confirmation that the observed fluorescence was derived from thiols. Determination of imaging in mitochondria was achieved by two co-localization methods: one with a mitochondria imaging reagent DiOC6(3), and the other using cells with GFP labeled mitochondria. 8


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Confirmation of the observed fluorescence derived from thiols was accomplished by the use of NEM. NEM is a reagent employed to deplete thiols in cells by forming a covalent bond with a thiol group.17 Mitochondria would not be expected to show any fluorescence by TBROS after thiol depletion by NEM if the fluorescence was derived from thiol imaging. Co-localization with DiOC6(3) DiOC6(3) is a commercially available green fluorescent lipophilic dye that is selective for imaging mitochondria in live cells. Cells were first treated with TBROS (100 nM) and DiOC6(3) (50 nM) at 37 °C for 3 h. Figure 5a provides representative fluorescence images obtained from TBROS (image a), DiOC6(3) (image b), and the merged image (image c) of images a and b. The red image (image a) obtained from TBROS matches well with the green image (image b) obtained from DiOC6(3) suggesting that the red fluorescence was located in mitochondria. a.

b.

Images from cells treated with TBROS followed by DiROC6(3)

Image a (TBROS) Image b [DiOC6(3)] Image c (merge of images a and b) Images from cells with GFP-labeled mitochondria and treated with TBROS

Image d (TBROS) Image e (GFP labeled Mitochondria) Image f (merge of images d and e) Figure 5. Representative fluorescence images obtained from TBROS-treated cells; a: NCI-H226 cells; b: NCIH226 cells with GFP-labeled mitochondria. The fluorescence was recorded from different fluorescence channels. The red fluorescence channel (images a, d) recorded the fluorescence derived from TBROS and the green fluorescence channel (images b, e) recorded the fluorescence derived from DiOC6(3) or cells with GFPlabeled mitochondria. Image c was obtained by merging image a with image b while image f was derived from merging image d with image e. Co-localization with GFP-labeled mitochondria To further confirm the imaged subcellular structure by TBROS was mitochondria, cells with GFPlabeled mitochondria were employed. Rhodamine channel filter was set to take images for thiols imaged by TBROS (red), FITC channel filter was set for taking images which represent the fluorescence of GFP (green). The results are presented in Figure 5b. As shown in the figure, the image from TBROS (image d) matches well 9



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with the image from GFP-labeled mitochondria (image e). The data further confirm that the imaged subcellular structure by TBROS was mitochondria. Confirmation of thiol imaging To confirm that the observed fluorescence was from thiol imaging, NEM was employed. When cells were pretreated with NEM before treated with TBROS, fluorescence intensity was decreased with an increase in NEM concentration (Figure 6a). When NEM concentration reached 100 µM, no fluorescence was observed confirming unambiguously that the red fluorescence was derived from thiol imaging by TBROS (Figure 6a). Quantification of mitochondrial NPSH Since fluorescence intensity of TBROS’ thiol adducts derived from different thiols varies (Figure 4), it would not be possible to provide an absolute quantity of NPSH in mitochondria by fluorescence microscopy. Therefore, the presented quantification will be a relative quantification - relative to control. The relative NPSH quantification in mitochondria of cells with different levels of NPSH was achieved by comparing the fluorescence intensity from cells pre-treated with different concentration of NEM with that of the control where no pre-treatment was conducted. The results are presented in Figure 6b. As shown in the figure, about 90% of NPSH was depleted by 20 µM NEM. Figure 6a

Control

10 µM NEM

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Figure 6b 120%

RFUs(% of Control)

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100%

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y = -0.0423x + 0.9696 R² = 0.965

0% 0

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NEM Concentration (µM)

16%

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Figure 6. Detection and quantification of mitochondrial NPSH by TBROS. Figure 6a shows that TBROS effectively detected the change of thiol concentration in mitochondria caused by pre-treatment with different concentration of NEM while Figure 6b reflects the relative quantity of thiols in the presence of various NEM concentrations determined on a microplate reader. The data are presented as percentage of control where no NEM treatment was conducted. The data are from one representative of triplicate experiments.

Although no data on mitochondrial NPSH are available, the relative percentage of mitochondrial NPSH obtained by TBROS are close to total relative percentage cellular NPSH reported previously for cells treated with similar concentration of NEM using other analytical methods (Table 1). Table 1. Thiol determination by different analytical methods Methods NEM (µM) 0 3 5 TBROS (mitochondrial NPSH) 100%±3% 86%±63% 79%±3% TBOP (total cellular NPSH)21 100%±4% 80%±5% 70%±8% 21 HPLC (total cellular NPSH) 100% 81% 63%

8 53%±1% 49%±5% 52%

In summary, we have demonstrated that TBROS is a thiol-specific mitochondria-selective fluorogenic agent. TBROS was able to image NPSH in mitochondria in live cells and provide quantitative information on the change of NPSH in mitochondria. This is the first reagent capable of providing quantitative measurement of NPSH change in mitochondria in live cells through fluorescence microscopy. The reagent will be useful in exploring the roles of mitochondrial NPSH in cell function/dysfunction through fluorescence microscopy in live cells. Supporting Information. Information on materials, solutions, instruments, experimental procedures of chemical stability determination of TBROS, quantum yield determination, reactivity of TBROS towards protein thiols, pH effects on the reactivity of TBROS towards thiols and live cell mitochondrial NPSH imaging, determination of cell viability and optimal cell culture condition for live cell mitochondrial NPSH imaging, and photostability of TBROS’ thiol adducts in live cells is provided in the Support Information. REFERENCES (1) Ribas, V.; Garcia-Ruiz, C.; Fernandez-Checa, J. C. Front Pharmacol 2014, 5, 151. (2) Quintana-Cabrera, R.; Bolanos, J. P. Biochem Soc Trans 2013, 41, 106-110. (3) Niu, W.; Guo, L.; Li, Y.; Shuang, S.; Dong, C.; Wong, M. S. Anal Chem 2016, 88, 1908-1914. (4) Han, C.; Yang, H.; Chen, M.; Su, Q.; Feng, W.; Li, F. ACS Appl Mater Interfaces 2015, 7, 27968-27975. (5) Kim, C. Y.; Kang, H. J.; Chung, S. J.; Kim, H. K.; Na, S. Y.; Kim, H. J. Anal Chem 2016, 88, 7178-7182.

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(6) Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis, W. W.; Potter, D. W. Anal Biochem 1980, 106, 55-62. (7) Newton, G. L.; Dorian, R.; Fahey, R. C. Anal Biochem 1981, 114, 383-387. (8) Guan, X.; Hoffman, B.; Dwivedi, C.; Matthees, D. P. J Pharm Biomed Anal 2003, 31, 251-261. (9) Tietze, F. Anal Biochem 1969, 27, 502-522. (10) Hansen, J. M.; Go, Y. M.; Jones, D. P. Annu Rev Pharmacol Toxicol 2006, 46, 215-234. (11) Go, Y. M.; Jones, D. P. Biochim Biophys Acta 2008, 1780, 1273-1290. (12) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Curr Opin Biotechnol 2007, 18, 17-25. (13) Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J Am Chem Soc 2011, 133, 1113211135. (14) Zhang, H.; Wang, C.; Wang, K.; Xuan, X.; Lv, Q.; Jiang, K. Biosens Bioelectron 2016, 85, 96-102. (15) Zhang, J.; Bao, X.; Zhou, J.; Peng, F.; Ren, H.; Dong, X.; Zhao, W. Biosens Bioelectron 2016, 85, 164-170. (16) Liu, X. L.; Niu, L. Y.; Chen, Y. Z.; Zheng, M. L.; Yang, Y.; Yang, Q. Z. Org Biomol Chem 2017, 15, 10721075. (17) Li, Y.; Yang, Y.; Guan, X. Anal Chem 2012, 84, 6877-6883. (18) Porteous, C. M.; Logan, A.; Evans, C.; Ledgerwood, E. C.; Menon, D. K.; Aigbirhio, F.; Smith, R. A.; Murphy, M. P. Biochim Biophys Acta 2010, 1800, 1009-1017. (19) Abu-Gosh, S. E.; Kolvazon, N.; Tirosh, B.; Ringel, I.; Yavin, E. Mol Pharm 2009, 6, 1138-1144. (20) Ross, M. F.; Prime, T. A.; Abakumova, I.; James, A. M.; Porteous, C. M.; Smith, R. A.; Murphy, M. P. Biochem J 2008, 411, 633-645. (21) Yang, Y.; Guan, X. Anal Chem 2015, 87, 649-655. (22) Walsh, D. W. M.; Siebenwirth, C.; Greubel, C.; Ilicic, K.; Reindl, J.; Girst, S.; Muggiolu, G.; Simon, M.; Barberet, P.; Seznec, H.; Zischka, H.; Multhoff, G.; Schmid, T. E.; Dollinger, G. Sci Rep 2017, 7, 46684. (23) Takahashi, A.; Zhang, Y.; Centonze, E.; Herman, B. Biotechniques 2001, 30, 804-808, 810, 812 passim. (24) Leung, C. W.; Hong, Y.; Chen, S.; Zhao, E.; Lam, J. W.; Tang, B. Z. J Am Chem Soc 2013, 135, 62-65. (25) Divya, K. P.; Savithri, S.; Ajayaghosh, A. Chem Commun (Camb) 2014, 50, 6020-6022. ACKNOWLEDGMENTS This work was supported by a grant from the National Institutes of Health (1R15GM107197-01A1). Conflict of Interest Disclosure The authors declare no competing financial interest. TOC N Cl-

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Live cell mitochondrial non-protein thiol imaging and quantification λex = 550 nm, λem = 580 nm

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1 A Thiol Specific and Mitochondria Selective Fluorogenic

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