Supporting Information for
A Mitochondria-Targeted Ratiometric Fluorescent Probe to
Monitor Intrinsically Generated Sulfur Dioxide Derivatives in
Living Cells
Wang Xu a, b, ‡, Chai Lean Teoh c, ‡, Juanjuan Peng c, Dongdong Su c, Lin Yuan a, d, * and Young-Tae
Chang a, c, *
a
Department of Chemistry and Medicinal Chemistry Programme, National University of
Singapore, 117543
b
Singapore Peking Oxford Research Enterprise (SPORE), Environmental Research
Institute (NERI), 5A Engineering Drive 1, #02-01, 117411, Singapore
c
Singapore Bioimaging Consortium, Agency for Science, Technology and Research
(A*STAR), 138667, Singapore
d
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, Hunan University, Changsha, Hunan 410082, P. R. China.
‡ These authors contributed to this work equally.
S1
Table of contents
Synthesis …………………………………………………………….………………………. S3-4
Quantum yield calculation ………… ……………………………….………………………. S5
Detection limit calculation ………… ……………………………….………………………. S5
Table S1…………………………………………………………….……………….………...S6
Figure S1…………………………………………………………….……………….………..S10
Figure S2…………………………………………………………….……………….………..S10
Figure S3…………………………………………………………….……………….………..S11
Figure S4…………………………………………………………….……………….………..S11
Figure S5…………………………………………………………….……………….………..S12
Figure S6…………………………………………………………….……………….………..S12
Figure S7…………………………………………………………….……………….………..S13
Figure S8…………………………………………………………….……………….………..S14
Figure S9…………………………………………………………….……………….………..S15
Figure S10…………………………………………………………….……………….….…. .S16
Figure S11…………………………………………………………….………….…….…..….S16
Table S2-S4…………………………………………………………….………….…….…S17-18
Figure S12…………………………………………………………….…….………….……. .S19
Figure S13…………………………………………………………….….…………….…..….S20
Figure S14…………………………………………………………….….…………….…..….S20
The copy of NMR ………………………………………………….….…………………. S21-22
S2
Synthesis
Compounds 1, 2, 3, 4 and 5 were prepared on the basis of a known procedure (Chem
Commun, 2010, 46, 7930; Org. Lett. 2007, 9, 33; Chem. Mater. 1996, 8, 541).
Compound 1 (Mito-Ratio-SO2)
1-methyl-2,3,3-trimethyl-3H-indolium iodide 3 (63.0 mg, 0.2 mmol) was treated with
coumarin aldehydes 3 (49.1 mg, 0.2 mmol) in anhydrous ethanol (10 mL). The reaction
mixture was then refluxed for 10 h, and the solvent was removed under reduced pressure.
The resulting residue was purified by column chromatography on silica gel (CH2Cl2 to
CH2Cl2 /acetone = 50: 1) to afford the compound 1 as a reddish brown powder (78.2 mg,
yield: 72.1%). 1H NMR (500 MHz, CDCl3) δ 10.02 (s, 1H), 8.57 (d, J = 15.9 Hz, 1H),
8.04 (s, 1H), 8.02 (d, J = 6.3 Hz, 1H), 7.51 (dd, J = 14.6, 5.9 Hz, 3H), 7.47 (t, J = 7.1 Hz,
2H), 6.67 (dd, J = 9.0, 2.0 Hz, 1H), 6.43 (d, J = 1.9 Hz, 1H), 3.50 (dd, J = 14.2, 7.0 Hz,
4H), 1.81 (s, 6H), 1.32 (s, 3H), 1.26 (t, J = 7.1 Hz, 6H).; 13C NMR (126 MHz, CDCl3) δ
181.41, 162.89, 161.14, 158.87, 154.65, 150.92, 142.82, 141.71, 134.47, 129.49, 129.12,
122.64, 113.56, 112.78, 111.11, 109.26, 96.90, 51.75, 45.71, 29.99, 27.54, 12.70. HRMS
(EI) m/z calcd for C26H29N2O2(M+): 401.2224. Found 401.2401.
Compound 5
Compound 5 was prepared according to literature (Chem Commun, 2010, 46, 7930–
7932.). Compound 1 (75.0 mg, 0.14 mmol) was dissolved in ethanol (2 mL), and then
NaBH4 (2.1 mg, 0.056 mmol in 0.5 mL ethanol) was added drop-wise over 10 min.
Subsequently, the reaction mixture was stirred at room temperature for 30 min, and the
solvent was removed under reduced pressure. The resulting residue was purified on a
S3
silica gel column (CH2Cl2/petroleum ether = 3: 7) to afford compound 5 as a yellow
powder (51.2 mg, yield: 87.9%). 1H NMR (400 MHz, CD3CN/CDCl3 = 4: 1) δ 1.04 (t, J
= 7.2 Hz, 3H), 1.06 (s, 3H), 1.90 (t, J = 7.4 Hz, 6H), 1.29 (s, 3H), 3.07-3.15 (m, 1H),
3.27-3.35 (m, 1H), 3.44 (q, J = 7.2 Hz, 4H), 3.69, 3.72 (1H), 6.49 (d, J = 8.0 Hz, 1H),
6.51 (s, 1H), 6.54 (d, J = 8.8 Hz, 1H), 6.59 (s, 1H), 6.63-6.68 (2H), 7.01 (d, J = 6.4 Hz,
1H), 7.04 (t, J = 7.6, 1.2 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H); 13C NMR (100
MHz, CD3CN/CDCl3 = 4: 1) δ 8.88, 11.09, 22.89, 24.57, 28.54, 38.60, 43.24, 43.62,
95.66, 106.37, 107.68, 108.40, 115.55, 120.83, 126.49, 127.25, 128.21, 128.28, 138.11,
138.27, 148.71, 149.84, 154.83, 160.39; HRMS (EI) m/z calcd for C 27H32N2O2 (M):
416.2458. Found 416.2435.
S4
Quantum yield calculation
Quantum yields were determined using rhodamine B (0.70 in MeOH) as a standard
according to a published method (Velapoldi, R. A.; Tønnesen, H. H. J. Fluoresc. 2004,
14, 465). The quantum yield was calculated according to the equation:
Φsample = Φstandard × (AstandardFsample/AsampleFstandard)
where Φsample and Φstandard are the fluorescence quantum yield of the sample and the
standard, respectively; Fsample and Fstandard are the integrated fluorescence intensities of the
sample and the standard spectra, respectively; Asample and Astandard are the optical densities
at the excitation wavelength, of the sample and the standard, respectively.
Quantum yield of probe Mito-Ratio-SO2: Φ = 0.11. After the complete reaction with
sulfite, the Quantum yield of adduct: Φ = 0.09.
Detection limit calculation
The detection limit was calculated based on the fluorescence titration (Joshi,B. P.; Park,
J.; Lee, W. I.; Lee, K. Talanta. 2009, 78, 903; Wu, M. Y.; Li, K.; Hou, J.T.; Huang, Z.;
Yu, X. Q. Org. Biomol. Chem. 2012, 10, 8342). Probe Mito-Ratio-SO2 was employed at
5 μM. To determine the S/N ratio, the emission intensity of Mito-Ratio-SO2 without
sulfite was measured by 20 times and the standard deviation of blank measurements was
determined. Under the present conditions, a good linear relationship between the
emission ratios (I480/I650) and the sulfite concentration could be obtained in the 0.3-50 μM
(R = 0.999), as shown in Figure S2. The detection limit is then calculated with the
equation: detection limit = 3σ/m, where σ is the standard deviation of blank
measurements, m is the slope between the emission ratios versus sample concentration.
The detection limit was measured to be 0.09 μM at S/N = 3 (signal-to-noise ratio of 3:1).
S5
Table S1. Summary of fluorescent probes for SO2 derivatives
ID
Probe Name
1
Anthracene
chemodosimeter
1[1]
2
Coumarin probe
3d[2]
ex/nma em/nmb
290
380
353/417
458
Detection
Limit/M
780
Detection Medium
Interaction
Mechanismc
SO32detection
Model
90% water/DMSO
solution (pH 7.2, 10
mM Tris -HCl
buffer)
FRET
---
ESIPT
HSO3detection in
granulated
sugar
0.37
pH 5.0
(NaH2PO4/Na2HPO4)
3
Resorufin probe
1[3]
487
588
49
HEPES buffered (pH
7.0, 10 mM) H2OCH3CN (98:2, v/v)
4
BODIPY-Le[4]
538
570/647
58
H2O/DMSO solution
(1:1)
5
Benzimidazol
probe 1[5]
325
368/498
0.4
6
Rhodamine B
probe 1[6]
510
580
0.89
7
Anthracene
probe[7]
370
420
0.01
8
Benzothiazol
probe 1[8]
310
373/468
5
9
DEACA[9]
390
483
0.187
446
480/578
34
410
465/592
0.2
10
11
Coumarin probe
1[10]
TSP[11]
Deprotection
of levulinate
moiety and
ESIPT
Deprotection
of levulinate
moiety and
ESIPT
---
---
20% v/v ethanol–
water solution
ICT
buffered at pH 4.6
NaAc-HAc
Water–ethanol
Ring
(90/10, v/v) at pH
opening of
4.8 Na2HPO4–citric
spiroacid buffer
rhodamine
pH 7.0 HEPES
aqueous solution
PET
containing DMSO
(2%, v/v)
HEPES buffered (pH Deprotection
7.4, 10 mM)
of levulinate
H2O/CH3CN (50%,
moiety and
v/v)
ESIPT
0.2 M Na2HPO4
citric acid (pH 5.0
ESIPT
aqueous buffer)
20% DMF buffer
ICT
solution
CTAB–20 mM PBS,
ICT
---
---
---
---
----SO32S6
pH 7.4, 25 ◦C
12
N1[12]
355
460
4
13
Coumarin
hemicyanine dye
1[13]
445
478/633
0.38
14
SP-2[14]
405
600
0.24
15
P-1[15]
330
395/515
2
350
460
0.39
216 phenylisothiazolo
probe 1[16]
17
Coumarin-TCF
probe 2[17]
18
Naphthalimide
probe 1[18]
19
Coumarin probe
1[19]
20
BIFS[20]
21
Benzopyrylium
probe 1[21]
22
466/580 520/660
430
448
530
585
322/470 460/595
420
0.00027
0.56
0.00083
0.0003
495/690
0.00104
Coumarin–
indolium probe
1[22]
450/550 485/667
0.0027
23
Coumarin–
benzopyrylium
probe 1[23]
430/605 485/640
0.0034
24
Mito-Ratio-SO2
(Current Work)
405
480/650
0.09
detection in
mineral
water, sugar
and white
wine
Buffered (HEPES 30
mm, pH 7.5)
ESIPT
suspensions
PBS buffer (pH 7.4,
10 mM, containing
ICT
30% DMF)
DMSO : HEPES
buffer (3 : 7, 10 mM,
MLCT
pH 7.5)
THF/H2O = 3/7, pH
ICT
5.0
Disruption
THF/water solution
(1/999, v/v) buffered
of 
by 20 mM HEPES at structure and
pH 7.2
AIE
20 mM pH 7.4
HEPES buffer
ICT
solution
10 mM PBS buffer
(pH 7.2–7.4): DMSO
ICT
= 9:1(v/v)
pH 7.4 HEPES
buffer (20.0 mM)
Glycerol/PBS
solution = 4/6, pH
7.40
HEPES buffer (20.0
mM, pH ¼ 7.4,
containing 2% DMF)
MeOH–PBS buffer
(20 mM, pH 7.4, 1:
1, v/v)
Phosphate buffer (pH
7.4, 20 mM,
containing 0.25%
ethanol as a
cosolvent)
pH 7.4 PBS
(containing 15%
--HeLa cells
HepG2 cells
HeLa cells
---
U-2OS cells
GES-1 cells
PET
A431 cells
and HepG2
cells
TICT
A549 cells
PET
HeLa cells
ICT
Paper strip
test and
HeLa cells
PET
HepG2 cells
ICT
Mitochondria
in HeLa cells
S7
EtOH as a cosolvent)
a
excitation wavelength (nm) of the probe. “/” indicates two excitation wavelengths for
one probe. b emission wavelength (nm) of the probe. “/” indicates ratiometric
fluorescence change excited by either excitation or two excitation wavelengths. c FRET:
Förster resonance energy transfer; ESIPT: Excited-state intramolecular proton transfer;
ICT: Intramolecular charge transfer; PET: Photo-induced electron transfer; MLCT: metalto-ligand charge transfer; AIE: Aggregation-induced emission.
References:
[1] Sun YM, Zhong C, Gong R, Mu HL, Fu EQ. A Ratiometric Fluorescent Chemodosimeter with Selective
Recognition for Sulfite in Aqueous Solution. J Org Chem. 2009;74:7943-6.
[2] Chen KY, Guo Y, Lu ZH, Yang BQ, Shi Z. Novel Coumarin-based Fluorescent Probe for Selective
Detection of Bisulfite Anion in Water. Chinese J Chem. 2010;28:55-60.
[3] Choi MG, Hwang J, Eor S, Chang SK. Chromogenic and Fluorogenic Signaling of Sulfite by Selective
Deprotection of Resorufin Levulinate. Org Lett. 2010;12:5624-7.
[4] Gu XF, Liu CH, Zhu YC, Zhu YZ. A Boron-dipyrromethene-Based Fluorescent Probe for Colorimetric
and Ratiometric Detection of Sulfite. J Agr Food Chem. 2011;59:11935-9.
[5] Wang G, Qi HP, Yang XF. A ratiometric fluorescent probe for bisulphite anion, employing
intramolecular charge transfer. Luminescence. 2013;28:97-101.
[6] Yang XF, Zhao ML, Wang G. A rhodamine-based fluorescent probe selective for bisulfite anion in
aqueous ethanol media. Sensor Actuat B-Chem. 2011;152:8-13.
[7] Yu CM, Luo M, Zeng F, Wu SZ. A fast-responding fluorescent turn-on sensor for sensitive and selective
detection of sulfite anions. Anal Methods-Uk. 2012;4:2638-40.
[8] Chen S, Hou P, Wang JX, Song XZ. A highly sulfite-selective ratiometric fluorescent probe based on
ESIPT. Rsc Adv. 2012;2:10869-73.
[9] Yang YT, Huo FJ, Zhang JJ, Xie ZH, Chao JB, Yin CX, et al. A novel coumarin-based fluorescent probe
for selective detection of bissulfite anions in water and sugar samples. Sensor Actuat B-Chem.
2012;166:665-70.
[10] Wu MY, He T, Li K, Wu MB, Huang Z, Yu XQ. A real-time colorimetric and ratiometric fluorescent
probe for sulfite. The Analyst. 2013;138:3018-25.
[11] Tian H, Qian J, Sun Q, Bai H, Zhang W. Colorimetric and ratiometric fluorescent detection of sulfite in
water via cationic surfactant-promoted addition of sulfite to alpha,beta-unsaturated ketone. Analytica
chimica acta. 2013;788:165-70.
[12] Santos-Figueroa LE, Gimenez C, Agostini A, Aznar E, Marcos MD, Sancenon F, et al. Selective and
Sensitive Chromofluorogenic Detection of the Sulfite Anion in Water Using Hydrophobic Hybrid OrganicInorganic Silica Nanoparticles. Angew Chem Int Edit. 2013;52:13712-6.
[13] Sun YQ, Liu J, Zhang J, Yang T, Guo W. Fluorescent probe for biological gas SO2 derivatives bisulfite
and sulfite. Chem Commun (Camb). 2013;49:2637-9.
[14] Li GY, Chen Y, Wang JQ, Lin Q, Zhao J, Ji LN, et al. A dinuclear iridium(III) complex as a visual
specific phosphorescent probe for endogenous sulphite and bisulphite in living cells. Chem Sci.
2013;4:4426-33.
[15] Cheng XH, Jia HZ, Feng J, Qin JG, Li Z. "Reactive" probe for hydrogen sulfite: Good ratiometric
response and bioimaging application. Sensor Actuat B-Chem. 2013;184:274-80.
[16] Yang BC, Niu XY, Huang ZX, Zhao CH, Liu Y, Ma C. A novel kind of dimmer (excimer)-induced-AIE
compound 2-phenylisothiazolo[5,4-b]pyridin-3(2H)-one as high selective bisulfite anion probe.
Tetrahedron. 2013;69:8250-4.
[17] Wu MY, Li K, Li CY, Hou JT, Yu XQ. A water-soluble near-infrared probe for colorimetric and
ratiometric sensing of SO2 derivatives in living cells. Chem Commun (Camb). 2014;50:183-5.
[18] Wang CC, Feng S, Wu LY, Yan SY, Zhong C, Guo P, et al. A new fluorescent turn-on probe for highly
sensitive and selective detection of sulfite and bisulfite. Sensor Actuat B-Chem. 2014;190:792-9.
[19] Chen W, Fang Q, Yang D, Zhang H, Song X, Foley J. Selective, highly sensitive fluorescent probe for
S8
the detection of sulfur dioxide derivatives in aqueous and biological environments. Analytical chemistry.
2015;87:609-16.
[20] Zhu L, Xu J, Sun Z, Fu B, Qin C, Zeng L, et al. A twisted intramolecular charge transfer probe for
rapid and specific detection of trace biological SO2 derivatives and bio-imaging applications. Chem
Commun. 2015.
[21] Chen W, Liu X, Chen S, Song X, Kang J. A real-time colorimetric and ratiometric fluorescent probe
for rapid detection of SO2 derivatives in living cells based on a near-infrared benzopyrylium dye. Rsc Adv.
2015;5:25409-15.
[22] Zhang Q, Zhang Y, Ding S, Zhang H, Feng G. A near-infrared fluorescent probe for rapid, colorimetric
and ratiometric detection of bisulfite in food, serum, and living cells. Sensor Actuat B-Chem.
2015;211:377-84.
[23] Chen Y, Wang X, Yang X-F, Zhong Y, Li Z, Li H. Development of a ratiometric fluorescent probe for
sulfite based on a coumarin–benzopyrylium platform. Sensor Actuat B-Chem. 2015;206:268-75.
S9
Fl. intensity (a.u.)
5000
4000
3000
2000
1000
0
600
700
800
Wavelength, nm
Figure S1. Fluorescence spectral changes of Mito-Ratio-SO2 (5 μM) upon addition of
sulfite (0-25 equiv.). The spectra were recorded after incubation of the probe with sulfite
for 30 min. λex = 572 nm.
Figure S2. Plot of fluorescent ratio (I480/I650) of the probe Mito-Ratio-SO2 as a function
of sulfite concentration.
S10
Figure S3. Emission spectral changes of Mito-Ratio-SO2 (5 μM) with time upon
addition of sulfite (100 equiv.) in pH 7.4 PBS (containing 15% EtOH as a co-solvent). λex
= 572 nm.
Absorption
0.45
0.30
0.15
0.00
400
500
600
700
Wavelength, nm
Figure S4. Absorption spectral changes of Mito-Ratio-SO2 (5 μM) with time upon
addition of sulfite (100 equiv.) in pH 7.4 PBS (containing 15% EtOH as co-solvent): 0
min (■), 0.5 min (★), 1 min (●), 1.5 min (▲), 2 min(★), 3 min (□).
S11
Figure S5. Pseudo first-order kinetic plot of Mito-Ratio-SO2 (5 μM) reaction with
sulfite (100 equiv.) in pH 7.4 PBS (containing 15% EtOH as co-solvent). Slope = -0.012
s-1.
Fl.intensity (a. u.)
120
80
40
0
500
600
700
Wavelength, nm
Figure S6. Time-dependent (0-40 min) emission spectra of Mito-Ratio-SO2 (5 μM) +
SO2 donor (200 μM) upon addition of 1 mM Cys in pH 7.4 PBS (containing 15% EtOH
as co-solvent).
S12
Figure S7. Fluorescence emission ratio (I480/I650) of probe Mito-Ratio-SO2 (5 μM) in the
absence (▲) and presence (■) of sulfite (25 equiv.) under different pH conditions.
S13
Figure S8. The ESI-MS of probe Mito-Ratio-SO2 (top) and probe with sulfite (bottom)
in pH 7.4 PBS (containing 15% EtOH as co-solvent). 401.2: Probe 1; 481.1: [1-SO3]-.
S14
Figure S9. Job plots for Mito-Ratio-SO2 and sulfite interaction with a total concentration
of [Mito-Ratio-SO2] + [sulfite] at 40 μM by fluorescence response. The fluorescence
emission at 480 nm was used.
S15
Figure S10. DFT optimized structure of Mito-Ratio-SO2, adduct c and adduct b. In the
ball-and-stick representation, carbon, nitrogen, oxygen, and sulphur atoms are colored in
gray, blue, red, and yellow, respectively. The purple color refers to sodium atom. The
calculation was performed using Gaussian 09 (Revision A.02).
Figure S11. Frontier molecular orbital plots of Mito-Ratio-SO2, adduct c and adduct b in
water (CPCM model). The calculation was performed using Gaussian 09 (Revision
A.02).
S16
Table S2. The frontier molecular orbitals (MOs) of Mito-Ratio-SO2.
Frontier Orbital Energy Level
Orbital Conformation
LUMO + 1
-1.98eV
LUMO
-3.28eV
HOMO
-5.86eV
HOMO - 1
-6.87eV
Table S3. The frontier molecular orbitals (MOs) of adduct b.
Frontier Orbital Energy Level
Orbital Conformation
LUMO + 1
-0.59eV
LUMO
-1.95eV
S17
HOMO
-5.15eV
HOMO - 1
-5.59eV
Table S4. The frontier molecular orbitals (MOs) of adduct c.
Frontier Orbital Energy Level
Orbital Conformation
LUMO + 1
-0.74eV
LUMO
-2.07eV
HOMO
-5.33eV
HOMO - 1
-5.51eV
S18
Figure S12. The absorbance and ESI-MS of probe Mito-Ratio-SO2 (top) and probe with
sulfite (bottom three rows) in pH 7.4 PBS (containing 15% EtOH as co-solvent). Adduct
a is least abundant with lowest absorbance intensity detected. Adduct b is non fluorescent
with shortest absorbance wavelength. Adduct c is the fluorescent form with high
abundance and long absorbance wavelength. 401.2: Probe 1; 481.1: [1-SO3]- / adduct a &
b & c.
S19
Figure S13. Cytotoxicity assay of probe Mito-Ratio-SO2 at different concentrations (a: 0
μΜ; b: 1 μΜ; c: 2 μΜ; d: 5 μΜ; e: 10 μΜ; f: 15 μΜ; g: 25 μΜ; h: 50 μΜ; i: 75 μΜ; j:
100 μΜ; k: 150 μΜ; l: 200 μΜ; m: 250 μΜ; n: 300 μΜ; o: 350 μΜ; p: 400 μΜ; q: 500
μΜ) for HeLa cells. LD50 for HeLa cells was calculated to be 93 μΜ.
Figure S14. Fluorescence images of HeLa cells. Cells were incubated with 1 μM probe
Mito-Ratio-SO2 and 200 nM LysoTracker Green or Hoechst 33258 at 37 °C for 20 min
in DMEM media supplemented with 10% FBS: (a) and (e) probe Mito-Ratio-SO2 with
excitation at 559 nm and a scan range of 600–700 nm; (b) LysoTracker Green with
excitation at 488 nm and a scan range of 520–570 nm; (c) overlay of a and b; (d) Colocalization coefficient (Pearson's coefficient) of Mito-Ratio-SO2 and LysoTracker green
is 0.72; (f) Hoechst 33258 with excitation at 460 nm and a scan range of 500–550 nm; (g)
overlay of d and e; (h) Co-localization coefficient (Pearson's coefficient) of Mito-RatioSO2 and Hoechst33258 is 0.68. Scale bar: 50 μm.
S20
S21
S22