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BULLETIN OF THE
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KOREAN CHEMICAL SOCIETY
hexane/ethyl acetate (1:2, v/v) to yield the desired product
1
(
15 mg, 0.014 mmol, yield: 37%). H NMR (300 MHz,
CDCl ): δ 8.17 (s,1H), 8.07 (d, J = 2.8 Hz, 1H), 7.93
3
(
(
d, J = 7.9 Hz, 1H), 7.80 (d, J = 2.8 Hz, 1H), 7.74
d, J = 8.2 Hz, 2H), 7.51 (app br, 2H), 7.38–7.36 (over-
lapped, 1H), 7.33–7.35 (app d, J = 8.2 Hz, 2H), 7.25–7.23
app t, 1H), 7.04 (d, J = 7.5 Hz, 1H), 6.91 (s, 2H), 6.86 (s,
(
2
3
H), 6.38 (app br, 1H), 6.35 34 (app s, 1H), 5.56 (s, 2H),
.75–3.68 (m, 2H), 3.58–3.55 (m, 1H), 3.19 (s, 3H), 2.98
13
(t, J = 7.14 Hz, 2H), 2.25 (s, 6H), 1.76–1.08 (m, 10H);
C
NMR (75 MHz, CDCl ): δ 168.2, 167.4, 163.0, 150.6, 149.2,
3
Scheme 1. Schematic representation of ER-targeting fluorogenic
ROS chemosensor (ER-Flu) in cells. ER-Flu has two states: Non-
fluorescent, reduced form (left) and fluorescent, oxidized form
1
1
1
3
48.7, 146.1, 145.8, 145.1, 143.2, 138.0, 133.5, 132.1, 131.5,
30.9, 130.6, 130.4, 129.5, 129.2, 128.8, 128.5, 127.4, 127.3,
25.8, 122.8, 121.3, 111.9, 62.3, 58.3, 49.0, 40.6, 37.8, 35.2,
2.8, 25.3, 24.4, 20.5; HR-MS (FAB+): calculated 1058.188
(right). Two stepwise reactions for ER-Flu: (a) deacetylation by
esterases. (b) Oxidation by ROS. Ac = acetyl.
35
(100.0%) for C H Cl N O S, 1060.185 (95.9%) for
50 45 3 6 12
35
37
C H Cl2 ClN O S, observed 1059.194 (93.4%),
50
45
6 12
+
3
-propargyl ester of DCFH DA and N -glibenclamide. The
1061.202 (100%) for [M + H] .
3
ROS sensing capability of ER-Flu in the live cells and its ER
localization were confirmed by confocal experiments in the
cellular ER stress models using thapsigargin, brefeldin A, and
tunicamycin.
Deacetylation of Dihydrofluorescein Derivatives
Deacetylation. Each probe stock solution was diluted in
methanol (90 μL) to give the final concentration (1 mM).
Each probe solution (100 μL, 1 mM) was added to a deox-
ygenated NaOH solution (400 μL, 10 mM) and the reaction
ꢀ
mixture was incubated in Eppendorf ThermoMixer (37 C,
Materials and Experiments
1
000 rpm for 30 min). The reaction was checked by TLC
(
dichloromethane: methanol = 10:1, v/v). To quench the
0 0
2 ,7 -Dichlorodihydrofluorescein diacetate
Materials.
D6883), fluorescein (46955), hematin (porcine, H3281),
reaction, 1X PBS (2 mL) was added to the reaction.
(
RP HPLC analysis: Column condition: flow
rate = 1 mL/min; solvent A = acetonitrile, solvent
B = 0.1% trifluoroacetic acid in water; a linear gradient of
solvent B, 0–90% over 30 min; DAD detection at 280 nm.
ROS Experiment of Flu-Acid and Flu-Ester. The prepa-
ration of ROS was summarized at Supporting Information S3.
Dulbecco’s modified Eagle’s medium (DMEM) (D6429,
high glucose), menadione (M5625), brefeldin A (B6542),
thapsigargin (T9033), tunicamycin (T7765), and protease
inhibitor cocktail (P8340 for use with mammalian cell and
tissue extracts) were purchased from Sigma-Aldrich. HeLa
(
species: human, tissue: cervix, cell type: epithelial, disease:
Potassium superoxide (KO , 14 058), hydrogen peroxide
®
2
adenocarcinoma, ATCC CCL-2™). Fetal bovine serum
FBS, Gibco™, 12483-020), ER-Tracker™ Red (E34250),
(H O , 33 323), sodium nitroferricyanide(III) dehydrate
2 2
(
(A15656), sodium hypochlorite (NaOCl, L14709) and tert-
LysoTracker™ Deep Red (L12492), and MitoTracker™
Deep Red FM (M22426) were purchased from Thermo-
Fisher Scientific.
butyl hydroperoxide (A13926) were purchased from Alfa
Aesar. Sodium nitrate (NaNO , 71 752) was purchased from
Fluka; Angeli’s salt (Na N O , 82 23O) from Cayman Chem-
3
2
2 3
Synthesis and Characterization. The synthesis and char-
acterization of dihydrofluorescein diacetate (Flu-acid DA),
dihydrofluorescein methyl ester (Flu-ester), and DCFH-
propargyl ester DA were summarized in Supporting
ical; sodium hydroxide pellets (SO620) from Samchun Chem-
ical; isoamyl nitrite (I0089) and diethylenetriamine-pentaacetic
acid (D0504) from TCI.
Ester Stability of ER-Flu in Cell Lysate
Information S1. N -glibenclamide was prepared by our pre-
vious report. ER-Flu DA was prepared following the pro-
cedure below.
DCFH-propargyl ester DA (20.0 mg, 0.038 mmol) was
dissolved in 3 mL of THF/water (2:1, v/v), and
Cell lysate preparation. HeLa cells were rinsed with 1X
PBS two times and harvested (10 000 rpm, 1 min, 4 C).
3
12
ꢀ
The cells were lysed with cell homogenate buffer (250 mM
sucrose, 10 mM HEPES, 1 mM EDTA, protease inhibitor
cocktail [1:100; Sigma-Aldrich] [pH 7.4]) using a Dounce
homogenizer. The homogenate was centrifuged (10 000 rpm,
N -glibenclamide (20.4 mg, 0.038 mmol) was added to the
3
ꢀ
solution. A 1:1 (v/v) mixture of CuSO (60 mM in water)
15 min, 4 C). The supernatant containing total cell number
4
5
and TBTA (60 mM in THF/ethanol (4:1, v/v)) were pre-
15 × 10 in 700 mL was collected for the ester stability
2
+
pared. Then, the Cu /TBTA cocktail (63 μL) and sodium
ascorbate (36 μL, 50 mM) were added to the reaction mix-
ture. After stirring at room temperature at 2 h, the solvent
was removed by rotary evaporation. The residual was
purified by silica gel column chromatography using
analysis.
Sample preparation. The 100 μL of a solution of probe
(1 mM in methanol) was added to 400 μL of NaOH
ꢀ
(10 mM) and the mixture was incubated at 37 C for
30 min. The reaction was quenched by adding 2 mL of 1X
Bull. Korean Chem. Soc. 2021, Vol. 42, 279–285
© 2020 Korean Chemical Society, Seoul & Wiley-VCH GmbH
www.bkcs.wiley-vch.de
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