Supporting Information
A novel colorimetric and ratiometric NIR fluorescent sensor for
glutathione based on dicyanomethylene-4H-pyran in living cells
WU XuMeng, SHAO AnDong, ZHU ShiQin, GUO ZhiQian & ZHU WeiHong*
Shanghai Key Laboratory of Functional Materials Chemistry, Key Laboratory for Advanced
Materials and Institute of Fine Chemicals, East China University of Science and Technology,
Shanghai 200237, P. R. China. Fax: (+86) 21-6425-2758. E-mail: whzhu@ecust.edu.cn.
S1
Materials and Methods
All reagents and solvents were purchased from commercial sources and are of analytical
grade. No further purification was operated. 1H and
13
C NMR in CDCl3 or DMSO-d6 were
recorded on a Bruker AvanceIII 400 MHz instruments with tetramethylsilane (TMS) as
internal standard. Data for 1H NMR spectra was reported as follows: chemical shift (ppm) and
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet). Data for 13C NMR
spectra is reported in ppm. HRMS was performed using a Waters LCT Premier XE
spectrometer. UV/Vis spectra were measured with a Varian Cary 100 spectrophotometer (1 cm
quartz cell). Emission spectra were measured with Varian Cary Eclipse (1 cm quartz cell). The
photofading experiment was induced by a continuous wavelength irradiation with Hg/Xe
lamp (Hamamatsu, LC8 Lightningcure, 300 W). The irradiation power was measured with a
photodiode from Ophir (PD300-UV). The time dependence of fluorescence of compounds
was induced in situ by a HORIBA FluoroMax-4 spectrofluorometer (1 cm quartz cell) at 37°C.
The relative quantum yield of GSH-treated DCM-S and free DCM-S was obtained by the
ratio of the integral area of their fluorescence spectra (on condition of the usage of the
isobestic point as the excitation wavelength). Flash chromatography was conducted by using
silica-gel column packages purchased from Qingdao Haiyang Chemical Co. (China).
Deionized water was used in the preparation of all samples.
S2
Scheme S1. Synthetic routes for DCM-S and DCM-C.
Experimental Section
The mediate compound 2-(2-methyl-4H-chromen-4-ylidene)malononitrile (DCM) was
synthesized by adapting published procedures.
Synthesis of DCM-NH2: DCM (1.0 g, 4.8 mmol) and N-(4-formylphenyl)acetamide (940 mg,
5.8 mmol) were dispersed in toluene (40 mL) with acetic acid (0.5 mL) and piperidine (1 mL).
The mixture was refluxed for 10 h under an argon atmosphere. The resulting suspension was
filtered and dried in vacuo to obtain crude DCM-NO as orange solid (1.2 g). Then the crude
DCM-NO was dispersed in ethanol/conc. Hydrochloric acid (20 mL/40 mL) solution. The
mixture was refluxed for 6 h and neutralized using anhydrous sodium carbonate. The aqueous
solution was extracted by CH2Cl2 which is dried by Na2SO4. The solvent was removed under
reduced pressure, and then the crude product was purified by silica gel chromatography using
dichloromethane as the eluent to afford DCM-NH2 as a deep red solid (520 mg): Yield 35%
for two steps. 1H NMR (400 MHz, DMSO-d6, ppm): δ = 8.72 (d, J = 8.3 Hz, 1H, Ph-H), 7.89
S3
(t, J = 7.8 Hz, 1H, Ph-H), 7.77 (d, J = 8.4 Hz, 1H, Ph-H), 7.64 (d, J = 15.8 Hz, 1H, alkene-H),
7.58 (t, J = 7.8 Hz, 1H, Ph-H), 7.49 (d, J = 8.3 Hz, 2H, Ph-H), 7.09 (d, J = 15.7 Hz, 1H,
alkene-H), 6.86 (s, 1H, Ph-H), 6.61 (d, J = 8.2 Hz, 2H, Ph-H), 6.03 (s, 2H, NH2). 13C NMR
(100 MHz, DMSO-d6, ppm): δ = 164.96, 157.76, 157.34, 157.25, 145.86, 140.21, 135.91,
131.09, 129.75, 127.49, 124.10, 123.01, 122.41, 121.65, 118.99, 117.60, 109.91, 62.55, 60.14.
Mass spectrometry (ESI-MS, m/z): [M + H]+ calcd for C20H14N3O, 312.1137; found,
312.1141.
Synthesis of DCM-S: To a mixture of DCM-NH2 (150 mg, 0.48 mmol), triphosgene (573 mg,
1.9 mmol) and dry toluene (30 mL) was added DIEA (1.0 g, 7.8 mmol) dropwise under an
argon atmosphere at room temperature. The resulting solution was refluxed under argon
protection for 3 h. After removal of unreacted phosgene gas by flushing argon gas, a solution
of 2,2’-dithiodiethanol (821 mg, 90%, 4.8 mmol) in CH2Cl2/THF (1:1, 10 mL) was added to
the mixture and the reaction mixture was stirred overnight at room temperature. After
removing the solvent under reduced pressure, the crude product was purified by silica gel
chromatography using ethyl acetate/PE (v/v, 1:1) as the eluent to afford DCM-S as a yellow
solid (80 mg): Yield 34%. 1H NMR (400 MHz, CDCl3, ppm): δ = 8.91 (d, J = 8.4 Hz, 1H,
Ph-H), 7.75 (t, 1H, Ph-H), 7.61-7.44 (m, 8H, Ph-H and alkene-H), 6.85 (s, 1H, Ph-H), 6.73 (d,
J = 16.0 Hz, 1H, alkene-H), 5.55 (s, 1H, NH), 4.48 (t, 2H, -O-CH2-), 3.93 (t, 2H, OH-CH2),
3.02 (t, 2H, -O-CH2-CH2), 2.95 (t, 2H, OH-CH2-CH2). 13C NMR (100 MHz, CDCl3, ppm): δ
= 157.62, 152.86, 152.33, 139.85, 138.24, 134.63, 129.85, 129.09, 125.96, 125.84, 118.75,
118.58, 117.86, 117.39, 116.87, 115.80, 106.63, 63.35, 60.41, 41.62, 37.52. Mass
spectrometry (ESI-MS, m/z): [M - H]- calcd for C25H20N3O4S2, 490.0895; found, 490.0906.
Synthesis of DCM-C: The compound DCM-C was synthesized using the same procedure
used in the case of DCM-S. DCM-C was afforded as a brown solid (80 mg): Yield 36.7%. 1H
NMR (400 MHz, CDCl3, ppm): δ = 8.92 (d, J = 8.4 Hz, 1H, Ph-H), 7.74 (t, 1H, Ph-H),
7.61-7.44 (m, 7H, Ph-H and alkene-H), 6.86 (d, J = 5.6 Hz, 2H, Ph-H and NH), 6.73 (d, J =
15.6 Hz, 1H, alkene-H), 4.20 (t, 2H, -O-CH2-), 4.08 (t, 2H, OH-CH2), 3.67 (q, 4H, -CH2-),
S4
1.31 (d, 4H, -CH2-).13C NMR (100 MHz, CDCl3, ppm): δ = 157.71, 152.88, 152.35, 140.23,
138.37, 134.61, 129.59, 125.94, 125.86, 118.58, 117.89, 117.22, 116.89, 115.83, 106.57,
64.79, 62.83, 29.09, 28.79, 25.78, 25.33. Mass spectrometry (ESI-MS, m/z): [M - H]- calcd
for C27H24N3O4, 454.1767; found, 454.1765.
0.25
Absorbance
0.15
400
0.10
200
Intensity (a.u.)
600
0.20
0.05
0.00
400
0
800
600
Wavelength (nm)
Figure S1. Absorption and fluorescence spectra of DCM-NH2 (10 μM) in DMSO/PBS (50/50,
v/v, pH =7.4, 10 mM), excitation wavelength at 492 nm.
A
1.2 B
DCM-NH2
DCM-S with GSH
1.0
1.0
Absorbance
Intensity (a.u.)
0.8
0.6
0.4
0.8
0.6
0.4
0.2
0.2
0.0
0.0
550
600
650
700
750
DCM-NH2
DCM-S with GSH
400
800
500
600
Wavelength (nm)
Wavelength (nm)
Figure S2. Normalized fluorescence (A) and absorption (B) spectra of DCM-NH2 (10 μM)
and DCM-S (10 μM with 250 equiv of GSH) in DMSO/PBS (50/50, v/v, pH =7.4, 10 mM),
excitation wavelength at 450 nm, each spectral curve was recorded 1 h after exposure at
37 °C.
S5
DCM-S
DCM-NH2
DCM-S + GSH
4
6
8
10
Retention time (min)
Figure S3. Reversed-phase HPLC spectra of DCM-S, DCM-NH2, DCM-S (treated with 20
equiv of GSH for 1 h at 37 °C), mobile phase : CH3OH/H2O = 9:1, flow rate of 0.6 mL/min,
detection wavelength at 450 nm.
DCM-S with GSH
DCM-S
500
I665 nm
400
300
200
100
0
3
4
5
6
7
8
9
pH
Figure S4. Fluorescence intensity at 665 nm of DCM-S (10 μM) in DMSO/H2O (50/50, v/v,
pH =7.4, 10 mM) with and without GSH (250 equiv) as a function of pH value. Each point
was recorded 1 h after exposure at 37 °C, excitation wavelength at 450 nm.
S6
(R-Rmin)/(Rmax-Rmin)
1.0
0.8
Equation
Weight
Residual Sum
of Squares
Pearson's r
Adj. R-Square
y = a + b*x
No Weighting
0.07001
0.96158
0.91522
Intercept
Slope
B
0.6
Value Standard Err
4.4821
0.39711
0.9704
0.09795
0.4
0.2
0.0
-4.8
-4.6
-4.4
-4.2
-4.0
-3.8
-3.6
Log([GSH])
Figure S5. Plot of (R - Rmin)/(Rmax - Rmin) vs. Log([GSH]). Note, the limit of detection of GSH
by DCM-S was calculated to be 2.4 × 10-5 M via linear fitting of the plot ((R - Rmin)/(Rmax Rmin) vs Log([GSH])), where the R is the fluorescence intensity ratio of 665 to 568 nm.
Figure S6. 1H NMR spectrum of DCM-NH2 in DMSO-d6.
S7
Figure S7. 13C NMR spectrum of DCM-NH2 in DMSO-d6.
Figure S8. HRMS spectrum of DCM-NH2.
S8
Figure S9. 1H NMR spectrum of DCM-S in CDCl3.
Figure S10. 13C HMR spectrum of DCM-S in CDCl3.
S9
Figure S11. HRMS spectrum of DCM-S.
Figure S12. 1H NMR spectrum of DCM-C in CDCl3.
S10
Figure S13. 13C NMR spectrum of DCM-C in CDCl3.
Figure S14. HRMS spectrum of DCM-C.
S11