Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
Experimental
Ninhydrin Test of TLQ-C. TLQ-C (40 μmol) in DMSO
(100 μL) and a reducing agent or a control such as NaBH4,
NADPH, NADH, NAD+, or NADP+ (400 μmol, 10 equiv)
in 1X PBS (900 μL) were mixed and stirred for 1 h. Ninhy-
drin in EtOH (100 μL, 0.08 g/mL) was then added to the
reaction mixture, which was heated at 80ꢀC for 5 min using
heat blocks. After cooling to room temperature, the absor-
bance at 570 nm was measured using a UV spectrometer.
Fluorescence Response of Q-TLQ-F.
Q-TLQ-F
(0.03 mg, 24.7 nmol) was dissolved in 30 μL DMSO.
NaBH4 (123 nmol), NADPH (123 nmol), NADH (123 nmol),
NADP+ (123 nmol), glucose-6-phosphate (G6P) (0.03 mg,
123 nmol), and glucose-6-phosphate dehydrogenase (G6PD)
(1.02 mU/well) were dissolved in 20 μL of PBS. Q-TLQ-F
and the reagents were mixed in a well plate. The well plate
was stirred for 30 min, and the fluorescence intensity of each
solution was measured using a fluorescence microplate reader.
The excitation wavelength was 496 nm, and the emission
wavelength was 515–645 nm. The fluorescence imaging of
each sample was visualized using an IVIS® imaging system
(Caliper Life Sciences Lumina II, Hopkinton, MA, USA).
Fluorescent Cell Imaging. MDA-MB-231 cells were cul-
tured in RPMI 1640 supplemented with 10% FBS, penicil-
lin (100 units/mL), and streptomycin (100 μg/mL). The
cells were plated in a 96-well plate and incubated for 24 h.
The medium was replaced with 48 μL of HBSS and 2 μL
of fluorescein solution or Q-TLQ-F solution (10 μM in
DMSO). The cells were then incubated at 37ꢀC and washed
twice with HBSS. The incubation time was 30 min for fluo-
rescein and 30 min or 1 h for Q-TLQ-F. The fluorescence
images of the cells were taken using an inverted fluores-
cence microscope (Carl Zeiss, Oberkochen, Germany) with
an EGFP filter.
Figure 1. (a) Synthetic route and proposed detection mechanism
of TLQ C. (i) N-hydroxysuccinimide, DCC, DCM, 30 min;
(ii) CPB, DIEA, DMF, 5 h. (b) UV absorption spectra of 4 mM
TLQ-C with reducing agents after the ninhydrin test. left: UV
absorption spectra of TLQ-C upon addition of NADPH from
4 mM to 80 mM. Right: UV absorption spectra of TLQ-C upon
addition of NaBH4 (20 mM), NADPH (80 mM), NADH
(80 mM), NAD+ (80 mM), and NADP+ (80 mM).
δ-lactonization reaction, and the reporting group (CPB) was
subsequently released. CPB was used to detect the color
change through the ninhydrin test after δ-lactonization. The
most intense color change was observed with NaBH4, which
was the strongest reducing agent screened (Figure 1(b)).
The TLQ-C containing a trimethyl lock derivative with an
amide bond, along with CPB, produced ninhydrin-active
signals with NaBH4 and NAD(P)H, but no color change
was observed with NAD+ and NADP+.
Next, we designed a fluorescent sensing system, designated
as the quencher-TLQ-fluorophore-type probe (Q-TLQ-F), to
sense NADPH. Q-TLQ-F was comprised of a TLQ and
a fluorescein linked by an amide bond (Figure 2). Moreover,
Q-TLQ-F contained a quencher moiety to efficiently absorb
the fluorescence of a fluorescein moiety. For detailed synthetic
schemes and experimental procedures for TLQ-C and Q-
TLQ-F, see Supporting Information. The fluorescence
response of Q-TLQ-F with reducing agents was investigated.
The concentrations of the reducing agents were well below
1 mM, similar to what is observed under normal biological
conditions. As shown in Figure 2(b), the fluorescence inten-
sity of Q-TLQ-F (0.5 mM) gradually increased and became
saturated after incubation with up to 5 equiv of NADPH for
150 min at 519 nm, whereas it took less than 10 min to
observe the quick saturation of fluorescence with NaBH4. The
higher (nearly five times) green fluorescence emission
indicated that Q-TLQ-F was reduced by the reducing agents
(NaBH4 and NADPH) followed by the removal of a fluores-
cein from the quencher moiety.
Inhibitor-Dependent Fluorescence Cell Imaging. MDA-
MB-231 cells were plated in a 96-well plate and incubated
for 24 h. The medium was removed, and the cells were fur-
ther incubated with 50 μL of DHEA (50, 100 μM in serum-
free media) for 24 h at 37ꢀC. The cell medium was then
replaced with 48 μL of HHBSS and 2 μL of FQD solution
(10 μM in DMSO). After 2 h at 37ꢀC, the HHBSS and Q-
TLQ-F solutions were removed, the cells washed twice
using HHBSS, and the fluorescent cell images taken.
Results and Discussion
For the proof-of-concept study, we synthesized a TLQ-
chloramphenicol base (TLQ C), which is comprised of a
TLQ moiety and a reporting group (chloramphenicol base
and CPB) connected by an amide bond (Figure 1). The syn-
thesis of TLQ-C was accomplished by the reported method,
as shown in Figure 1(a).26 Briefly, Compound (1) was acti-
vated with N-hydroxysuccinimide and coupled to a chlor-
amphenicol base to produce TLQ C. When TLQ-C was
applied to a reducing agent, δ-lactone was formed by a
Prior to the examination of Q-TLQ-F as a turn-on fluores-
cent probe for the in-cell detection of NADPH, its sensing
Bull. Korean Chem. Soc. 2019, Vol. 40, 807–811
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
808
Yu, Geunhyeok
