Q. Zhang et al.
Dyes and Pigments 196 (2021) 109753
were more attractive in the wavelength range of 650–900 nm, in which
biomolecules had almost no interference, the depth of sample penetra-
tion was large and it has minimal harm to biological system [41].
Therefore, it is urgent to develop NIR fluorescent dyes that can sensi-
2.4. Synthesis of B1
3-aminophenol (2.05 g, 18.785 mmol), NaHCO
3
(6.3125 g, 75.14
mmol) and 1-bromo-3-chloropropane (11.83 g, 75.14 mmol) were
added to a 100 mL round bottom flask and were evenly dissolved in DMF
ꢀ
2ꢀ
tively detect HSO
3
/SO
3
.
◦
Keeping these considerations in mind, herein, using 3-aminophenol
as raw material, a new NIR ratiometric fluorescent probe BAOA (11-
(30 mL). Then the reaction mixture was stirred at 70 C for 12 h until the
reaction was completed. After cooling to room temperature, the reaction
solution was poured into ice water. The solution was extracted with
ethyl acetate, and then washed with water. The remaining solution was
′
′
′
′
′
′
′
′
oxo-2,2 ,3,3 ,6,7,7 ,8 -octahydro -1H,1 H,5H,6 H,11H- [10,12 -bipyrano
[
2,3-f] pyrido [3,2,1-ij] quinolin]-13 -ium perchlorate) was designed
and developed (ba, figures S1, S2 and S3), in which the oxygen positive
dried by Na
2 4
SO and further purified by silica gel column chromatog-
ion on the benzopyran ring was a strongly electron-absorbing group,
raphy ((PE: EA = 20 : 1, v/v) to get white solid B1 (900 mg, yield:
–
1
reducing the electron cloud density and making the C
–
C double bonds
25.3%). H NMR (600 MHz, DMSO‑d
6
) δ 8.63 (s, 1H), 6.48 (d, J = 8.0
ꢀ
strongly electrophilic. The strong nucleophilic HSO
carbon double bond to form BAOA-HSO
3
can attack carbon-
Hz, 1H), 6.00 (d, J = 8.0 Hz, 1H), 3.03–2.97 (m, 4H), 2.57 (t, J = 6.5 Hz,
1
3
3
by Michael addition reaction.
2H), 2.51 (d, J = 6.3 Hz, 2H), 1.86–1.80 (m, 4H). C NMR (151 MHz,
DMSO‑d
The sensing mechanism had been verified by nuclear magnetic titration
6
) δ 153.0, 143.6, 126.0, 112.0, 107.7, 103.0, 49.6, 49.1, 26.7,
experiments and high resolution mass spectrometry. Probe BAOA can
22.1, 21.4, 21.0.
ꢀ
quantitatively detect HSO
3
in the concentration range of 1.25–8.75
μ
mol/L and the LOD was calculated to be 63.0 nmol/L. Cell experiments
2.5. Synthesis of B2
showed that BAOA could not only locate mitochondria, lysosomes, ER
ꢀ
and Golgi but also detect exogenous HSO
3
in living cells. Further
2 mL of dried DMF was placed in a round neck flask and stirred in an
studies showed that probe BAOA had good selectivity and anti-
ice-water bath for 15 min. Then 0.5 mL of POCl was added dropwise to
3
ꢀ
interference ability toward HSO
3
against common anions, cations
the flask and stirring for 30 min. The product B1 (189.2 mg, 1 mmol)
and small biomolecules. Moreover, BAOA not only had good localization
from the previous step dissolved in DMF was added dropwise to the
reaction system and stirred at room temperature for 1 h. Then, the
ability in mitochondria, lysosomes, ER and Golgi but also could detect
ꢀ
◦
exogenous HSO
3
in living cells.
temperature was raised to 100 C for 1 h and the heating was stopped.
After cooling to room temperature, 10 mL of water was added to the
reaction flask while the pH of the reaction system was adjusted to 6–8
with saturated potassium carbonate solution, and stirring was continued
2
. Experimental
.1. Materials
-Aminophenol, 2-bromo-3-chloropropane, acetoacetic ester, N,N-
2
2 2
for 1 h. After the reaction was completed, it was extracted with CH Cl ,
and then the organic phase was washed with saturated brine, and dried
overnight with anhydrous sodium sulfate. After silica gel column chro-
3
dimethylformamide, acetic ether, dichloromethane, anhydrous
ethanol, anhydrous methanol, petroleum ether, dimethyl sulfoxide,
matography, a light yellow solid product was obtained. (154 mg, yield:
1
70.9%). H NMR (600 MHz, DMSO‑d
6
) δ 11.85 (s, 1H), 9.36 (s, 1H), 6.98
methyl sulfonic acid, NaHCO
3
, POCl
, AlCl
, AgCl, Ni(NO
, NaH PO , Na
, Na , NaNO , Ala, Arg, Asp, Cys, Gln, Glu,
Gly, GSH, Hcy, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Val, H . All
3
, K
2
CO
3
, Na
2
SO
4
, NaHSO
, FeCl , SnCl
, Co(NO
, NaBF , NaI, NaN
3
, NaOH,
, Pb
, Cd
(s, 1H), 3.28 (dd, J = 12.2, 7.3 Hz, 4H), 2.62 (t, J = 6.2 Hz, 2H), 2.54 (t,
13
NaF, NaCl, NaBr, NaI, KCl, CaCl
2
3
, ZnCl
2
, FeCl
2
3
2
J = 6.4 Hz, 2H), 1.83 (dd, J = 12.1, 6.1 Hz, 4H). C NMR (151 MHz,
DMSO‑d
(
NO
NO
3
)
)
2
, CuCl, CuCl
, Na PO , Na
, NaHSO
2
, MgCl
2
3
)
2
, MnCl
2
3
)
2
6
) δ 191.6, 158.4, 149.2, 130.9, 113.4, 110.0, 104.2, 49.5, 49.0,
(
3
2
3
4
2
HPO
4
2
4
2
CO
3
4
3
,
26.5, 21.0, 19.9, 19.2.
NaSCN, NaNO
3
4
C
2 2
O
4
2
2
O
2
2.6. Synthesis of B3
reagents and drugs were analytical grade and used without any further
purification. Twice-distilled water was used throughout all the
experiments.
B2 (217 mg, 1 mmol) was dissolved in 5 mL of absolute ethanol, and
ethyl acetate (260 mg, 2 mmol) and piperidine (80 L, 0.81 mmol) were
μ
added to the reaction solution. Under the protection of argon, it was
heated to reflux for 5 h and then cooled to room temperature. Filtered
with suction and washed with n-hexane to obtain an orange solid (205.6
2
.2. Laboratory apparatus
Dual-beam UV–vis Spectrophotometer (TU-1901), Fluorospec-
trophotometer (F-4600), pH meter (PHS–2F), 400 M NMR spectrometer
mg, yield: 72.6%). 1H NMR (600 MHz, CDCl
1H), 3.34 (dd, J = 12.0, 6.5 Hz, 4H), 2.88 (t, J = 6.4 Hz, 2H), 2.76 (t, J =
3
) δ 8.33 (s, 1H), 6.96 (s,
1
3
(
AVIII HD 400), 600 M NMR spectrometer (AVIII HD 600), High-
resolution mass spectrometer (IonSpec4.7), Rotary evaporator (RE-
000B), Electronic analytical balance (FA2004), Vacuum drying oven
DZF-6020), Constant temperature magnetic stirrer (85-2), Uultrasonic
6.2 Hz, 2H), 2.67 (s, 3H), 2.01–1.94 (m, 4H). C NMR (151 MHz, CDCl
3
)
δ 196.0, 161.2, 153.8, 148.8, 147.8, 127.8, 114.9, 108.1, 105.6, 50.4,
2
50.0, 30.6, 27.4, 21.2, 20.2, 20.1.
(
cleaner (SB-100D), Circulating water vacuum pump (SHB-3), Vacuum
oil pump (2XZ-4), Digital camera (D3300), Portable UV analyzer (ZF-5).
2.7. Synthesis of BAOA
B3 (283.4 mg, 1 mmol) and B2 (260.4 mg, 1.2 mmol) were dissolved
◦
2
.3. The parameters of the solution in spectra
in methyl sulfonic acid and stirred at 90 C for 6 h. After the reaction was
cooled to room temperature, the reaction droplets were added to the ice
salt water to form precipitate, the precipitate was filtered and washed
with water, and the crude product was separated by silica gel column
In Fig. S4, the solution containing 2100
μ
L PBS and 900 L DMSO
μ
was chosen because it has the most excellent fluorescence change before
ꢀ
ꢀ 3
and after adding HSO
3
. The concentration of BAOA was 1 × 10 mol/
chromatography to obtain blue and black solid (220 mg, yield: 47.3%).
1
L (dissolved in DMSO solution). The BAOA solution of 30
μ
L was
H NMR (400 MHz, CDCl
3
) δ 8.54 (s, 1H), 8.25 (d, J = 8.3 Hz, 1H), 8.17
removed with a liquid transfer gun and added to 3000
containing 2100 L PBS and 900
μ
L solution
(d, J = 8.4 Hz, 1H), 7.39 (d, J = 16.9 Hz, 2H), 3.72–3.61 (m, 4H), 3.47
μ
μ
L DMSO (VPBS:VDMSO = 7:3), the final
(dd, J = 10.1, 5.3 Hz, 4H), 3.16 (t, J = 5.8 Hz, 2H), 2.93–2.81 (m, 6H),
ꢀ 5
13
concentration of BAOA in the test system was 1 × 10 mol/L.
2.16 (d, J = 27.5 Hz, 8H). C NMR (101 MHz, CDCl ) δ 162.0, 158.7,
3
The concentrations of anion ions, metal ions and small biomolecules
153.4, 153.0, 152.2, 150.8, 145.1, 143.8, 129.0, 127.4, 127.3, 121.4,
117.9, 110.3, 109.9, 105.8, 105.4, 104.9, 51.3, 51.0, 50.4, 27.7, 27.3,
27.2, 20.9, 20.3, 19.9, 19.8, 19.4.
used in the detection were 0.01 mol/L initially. The concentration of
ꢀ
HSO
3
was 0.0025 mol/L.
2