A highly selective ratiometric fluorescent probe for 1,4-dithiothreitol (DTT) detection

Article abstract of DOI:10.1039/b923754b

A highly selective ratiometric fluorescent probe, which contains an aminonaphthalimide fluorophore and a self-immolative spacer for 1,4-dithiothreitol (DTT) detection was designed and synthesized. The probe displays a 66 nm red-shift of fluorescence emiss

Full text of DOI:10.1039/b923754b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A highly selective ratiometric fluorescent probe for 1,4-dithiothreitol (DTT)
detection†
Baocun Zhu,a Xiaoling Zhang,*a Hongying Jia,b Yamin Li,a Haipeng Liuc and Weihong Tan*c
Received 13th November 2009, Accepted 18th January 2010
First published as an Advance Article on the web 3rd February 2010
DOI: 10.1039/b923754b
A highly selective ratiometric fluorescent probe, which contains an aminonaphthalimide fluorophore
and a self-immolative spacer for 1,4-dithiothreitol (DTT) detection was designed and synthesized. The
probe displays a 66 nm red-shift of fluorescence emission and the color changes from colorless to
jade-green upon reaction with DTT. These properties are mechanistically ascribed to the strong
reducing capability of DTT.
Introduction
Results and discussion
1,4-Dithiothreitol (DTT),1 a synthetic thiol, has been widely used
in cell biology, biochemistry, and biomedical application. For
example, DTT has been used as a powerful reducing agent,2 an
antidote protecting cells and tissues,3 and a radioprotector,4 to
name a few. However, it is also noted that DTT is a highly toxic
substance.5 It can cause oxidative damage of biomolecules in the
presence of transition metals6 and enhance the toxic action of
some arsenic- and mercury-containing compounds.7 Therefore,
developing an appropriate analytical probe for selective detection
of DTT is central for the safe use of this important substance.
Among the various methods used to detect thiols, fluores-
cent probes are commonly developed due to their operational
simplicity and high sensitivity.8 However, current probes show
that most of them have poor selectivity between biothiols and
DTT.8d,e,g,j,k Accordingly, fluorescent indicators that can effectively
discriminate DTT from biothiols are needed. Although some
probes9 for the detection and discrimination of specific thiols
have been reported, to the best of our knowledge, there has been
no specific fluorescent indicator reported for DTT. Additionally,
most of current probes detect thiols only by intensity-responsive
fluorescence signal, which is known to be disturbed in quantitative
detection by many factors, such as variabilities in excitation and
emission efficiency, sample environments, and probe distribution.
Ratiometric fluorescent probes can eliminate most or all ambi-
guities by self-calibration of two emission bands.10 In this paper,
we describe a novel, highly selective, colorimetric and ratiometric
fluorescent DTT probe 1 containing self-immolative spacer based
on the internal charge transfer (ICT) mechanism.
Very recently, Chang et al.11 designed a novel ratiometric fluores-
cent probe PL1 for H2O2 and PL1 features a chemospecific H2O2
switch to modulate ICT with a marked blue-to-green emission
color change. This strategy inspired us to design ratiometric
fluorescent probes for other analytes. As illustrated in Scheme 1,
our probe can be divided into three distinct parts. The first
part is a fluorophore, 4-aminonaphthalimide12 was chosen due
to its desirable spectroscopic properties and outstanding ICT
structure. The second part consisted of an organoselenium8j,k,13
as DTT receptor. Finally, the third part, 3,4-diaminophenyl
methanol, a self-immolatvie spacer was chosen to connect the
fluorophore and receptor. We hypothesize that reaction of 1
with DTT triggers the cleavage of piazselenole-based carbamate
protecting group, as a result, restores the green fluorescence of 4-
aminonaphthalimide 2. The reaction mechanism of 1 with DTT is
shown in Scheme 1. Scheme 2 outlines the synthesis of probe 1. The
detailed synthetic procedures and characterizations are described
in the Experimental Section.
aDepartment of Chemistry, School of Science, Beijing Institute of Technology,
Beijing 100081, P. R. China. E-mail: Zhangxl@bit.edu.cn
bInstitute of Chemistry, Chinese Academy of Sciences, Beijing 100190,
P. R. China
Scheme 1 Reaction mechanism of 1 with DTT.
cCenter for Research at Bio/nano Interface, Department of Chemistry and
Department of Physiology and Functional Genomics, Shands Cancer Center,
UF Genetics Institute and McKnight Brain Institute, University of Florida,
Gainesville, Florida 32611-7200, USA. E-mail: tan@chem.ufl.edu
The spectral properties of 1 were measured under a mixture
of ethanol and water (1 : 1, v/v) solution containing phosphate
buffered saline (PBS) (20 mM, pH 7.4). 1 exhibits hunchbacked ab-
sorption band (absorption peak around 340 nm from piazselenole
and around 370 nm from carbamate-derived naphthalimide)
† Electronic supplementary information (ESI) available: The determina-
tion of quantum yield, additional spectroscopic data, 1H-NMR, 13C-NMR
and MS spectra of probe 1. See DOI: 10.1039/b923754b
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Scheme 2 The synthesis of probe 1.
(Fig. 1). When DTT was added to the solution of 1, the maximum
absorption peak showed a 60 nm red shift (Fig. 1) and the color of
the solution turned from colorless to jade-green (Fig. S1, ESI†). In
the fluorescence emission spectrum, 1 displays the maximum emis-
sion peak at 461 nm with a quantum yield of 0.69 (ESI†). Upon
addition of DTT (final concentration: 100 mM), the maximum
emission peak undergoes a red shift to 527 nm with a quantum
Fig. 2 Fluorescence response of 1 (5 mM) toward different concentration
of DTT (final concentration: 0, 5, 10, 20, 30, 40, 50, 60 mM) in
PBS (20 mM) solution (ethanol/water = 1 : 1, v/v, pH 7.4). Excitation
wavelength was 410 nm, excitation and emission slit widths were 2.5 nm
and 5.0 nm. (a) Fluorescence spectra of 1 in the presence of increasing
concentrations of DTT; (b) Fluorescence intensity ratio F461/F527 of 1
versus increasing concentrations of DTT. Each spectrum was acquired
10 h after DTT addition at 25 ◦C.
yield of 0.48, and the ratio of fluorescence intensities (F527/F461
)
changes from 0.2 to 19 (R = 95-fold). Moreover, a ratiometric
response was observed when adding different concentrations of
DTT to the solution of 1. A well-defined isoemission point at
507 nm can be clearly seen (Fig. 2a). Also, there was a good
linearity between the ratio of fluorescence intensities (F461/F527
)
and concentrations of DTT in the range of 5 to 60 mM with
a linear coefficient of 0.9916 (Fig. 2b). This data demonstrated
that probe 1 could detect DTT qualitatively and quantitatively by
ratiometric fluorescence method. Therefore, 1 is a very promising
probe for the detection of DTT in environmental and life science
because DTT become toxic at a higher concentration (10 mM).14
To confirm this hypothesis, 1,2-dimercaptoethane (200 mM), a
reducing compound similar to DTT, was added to the solution of
1, and a relative large change of the intensity ratio (R = 26-fold)
was observed (Fig. 4). According to the previous reports,15 this
result favors the reducing ability of thiols as a key element in the
initiation of the fluorescence response. Therefore, in this regard, we
believe that 1 will have great potential for studying the redox state
in living systems because the normal reducing environment in the
cytosol equalled to a solution containing 10 mM DTT.16 We next
studied the effect of interference of above-mentioned analytes on
monitoring DTT. The results show that the probe 1 possesses high
selectivity toward DTT when present with other analytes (Fig. 3b).
To confirm the reaction mechanism of 1 with DTT, the reaction
of 1 with DTT was conducted under the same conditions as
described above. The unique fluorescent reaction product was
obtained and characterized by 1H NMR, 13C NMR and HRMS17
to be compound 2. Additionally, to further demonstrate the
reaction of 1 with DTT by thiols, we added N-ethylmaleimide
(NEM, a known thiol-blocking agent) to the system. The addition
of thiol-blocking agent, however, showed no obvious fluorescence
emission peak at 527 nm (Fig. 5). The above data indicate that the
probe most likely undergoes a designed mechanism as showed in
Scheme 1.
Next, to further demonstrate the practical application of the
probe, we carried out experiments in living cells. HeLa cells
incubated with the probe 1 (10 mM) for 1 h showed an intense
intracellular fluorescence in the cytosol (Fig. 6a, b). The results
reveal that the probe 1 can penetrate the cell membrane and labeled
designedly the cytosol. Furthermore, HeLa cells were pretreated
with 50 mM N-ethylmaleimide (as a thiol-blocking reagent, NEM)
for 2 h to decrease the reducing capability because reduced thiols
maintain the reducing environment in living cells, and then they
were incubated with the probe 1 (10 mM) for another 1 h. Distinct
Fig. 1 Absorption response of 1 (5 mM) toward DTT (final concentration:
100 mM) in PBS (20 mM) solution (ethanol/water = 1 : 1, v/v, pH 7.4).
Each spectrum was acquired 10 h after DTT addition at 25 ◦C.
The selectivity of probe 1 toward DTT was evaluated by testing
the response of the assay to physiologically important reducing
agent ascorbic acid (Vc) and other environmentally relevant thiols,
including L-cysteine (L-Cys), b-mercaptoethylamine (MEA), thio-
glycerol (TG), thioglycolic acid (TA), and glutathione (GSH) at a
concentration of 200 mM. As showed in Fig. 3a, compared with
DTT, other thiols and Vc showed very little in the change of the
fluorescence intensities ratio (F527/F461). This result is inconsistent
with the previously reported conclusion by Tang et al.8j,k and
Zhang et al.13 We suspect that the reaction of 1 with DTT
is mainly triggered by the strong reducing capability of thiols
rather than the reported nucleophilic substitution of sulfhydryl.
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Fig. 5 Fluorescence response of 1 (5 mM) toward NEM, a mixture of
NEM and DTT, DTT in PBS (20 mM) solution (ethanol/water = 1 : 1, v/v,
pH 7.4). Excitation wavelength was 430 nm, and excitation and emission
slit widths were 2.5 nm. Each spectrum was acquired 10 h after various
analytes addition at 25 ◦C.
Fig. 3 (a) Fluorescence response of 1 (5 mM) to different thiols and Vc
(200 mM). Thiols: L-Cys (200 mM), MEA (200 mM), TG (200 mM),
TA (200 mM), GSH (200 mM) and dithiothreitol (DTT) (100 mM). Bars
represent the change of fluorescence intensities ratio (F527/F461). Excitation
wavelength was 410 nm. (b) Fluorescence response of 1 (5 mM) to DTT
(100 mM) in the absence and presence of other analytes (200 mM),
including L-Cys, MEA, TG, TA, GSH, and Vc. Bars represent the
fluorescence intensity at 527 nm. Excitation wavelength was 430 nm. Each
spectrum was acquired 10 h after various analytes addition at 25 ◦C.
Fig. 6 Confocal fluorescence images of living HeLa cells: HeLa cells
incubated with the probe 1 (10 mM) for 1 h (a) blue channel, (b) green
channel, and (c) bright-field image; HeLa cells incubated with the probe
1 (10 mM) for 1 h after preincubation with 50 mM NEM for 2 h (d)
blue channel, (e) green channel, and (f) bright-field image; HeLa cells
incubated with the probe 1 (10 mM) for 1 h after preincubation with
100 mM DTT for 30 min (g) blue channel, (h) green channel, and (i)
bright-field image. Incubation was performed at 37 ◦C under a humidified
atmosphere containing 5% CO2. Scale bar = 20 mm.
were incubated with the probe 1 (10 mM) for another 1 h. The
enhancement of green fluorescence was observed (Fig. 6h), which
demonstrates the probe 1 can be used for imaging of DTT in living
cells. These results also suggest that the fluorescence changes were
really due to the changes in the intracellular redox state.
Fig. 4 Fluorescence response of 1 (5 mM) to 1,2-dimercaptoethane
(DME) and DTT in PBS (20 mM) solution (ethanol/water = 1 : 1,
v/v, pH 7.4). Bars represent the change of fluorescence intensities ratio
(F527/F461). Excitation wavelength was 410 nm and excitation and emission
slit widths were 2.5 nm. Each spectrum was acquired 10 h after various
analytes addition at 25 ◦C.
Conclusions
In conclusion, we have presented the synthesis and properties
of a new, ICT and naphthalimide fluorescent probe with 3,4-
diaminophenyl methanol as self-immolative spacer and organose-
lenium as receptor. The probe exhibits high DTT-selectivity over
biothiols, including cysteine and glutathione, which was ascribed
decrease of green fluorescence in HeLa cells was observed (Fig. 6e).
In addition, HeLa cells pretreated with DTT (100 mM) for 30 min
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to the strong reducing capability of DTT. Additionally, the probe
displays a 66 nm red-shift of fluorescence emission and the color
changes from colorless to jade-green upon addition of DTT, and
thus can serve as a “naked-eye” probe for DTT. Importantly,
probe 1 can detect DTT quantitatively by ratiometric fluorescence
method. The living cell image experiments further demonstrate
its value in detecting DTT and studying the changes of redox
environment in living systems.
14 mmol) and dibenzoylperoxide (as a catalyzer) were dissolved
in 15 mL CCl4 and 7 mL CHCl3. The resulting solution was
heated to reflux for 5 h. The hot reaction mixture was filtrated
and the portion of solvent was removed under reduced pressure.
After cooling to room temperature, the crystal product (1.0487 g,
3.8 mmol, 54% yield) was obtained after filtration. 1H-NMR
(400 MHz, CDCl3) d (*10-6): 4.58 (s, 2H), 7.51 (d, J = 9.2 Hz,
1H), 7.81–7.84 (m, 2H).
2,1,3-Benzoselenadiazol-5-methanol. 5-Bromomethyl-2,1,3-
benzoselenadiazol (552.0 mg, 2 mmol) was suspended in 100 mL
H2O containing K2CO3 (621.9 mg, 4.5 mmol). The resulting solu-
tion was heated to reflux for 30 min under nitrogen atmosphere.
After cooling to room temperature, the crystal product (277.6 mg,
1.3 mmol, 65% yield) was obtained after filtration. 1H-NMR
(400 MHz, CDCl3) d (*10-6): 2.37 (s, 1H), 4.81 (s, 2H), 7.43 (d, J =
9.2 Hz, 1H), 7.76–7.78 (m, 2H).
Experimental section
Materials and general methods
All chemicals used in this paper were commercial products of
analytical grade. 1H-NMR and 13C-NMR were recorded on
a Bruker AV-400 spectrometer with chemical shifts reported
as ppm (in CDCl3 or DMSO-d6, TMS as internal standard).
Mass spectral analyses were carried out on a MALDI-TOF
spectrometer. IR spectrum was recorded on Nicolet AVATAR FT-
IR 360 infrared spectrophotometer using KBr pellet sample. High-
resolution mass data were measured with Fourier transform ion
cyclotron resonance mass spectrometer (APEX IV). Absorption
spectra were recorded on TU-1901 UV-vis spectrophotometer.
Fluorescence emission and excitation spectra were measured on
Hitachi F-7000. All pH measurements were made with a Sartorius
basic pH-meter PB-10.
Probe 1. To a mixture of 218 (268.3 mg, 1 mmol) and DIPEA
(439.4 mg, 3.4 mmol) in 20 mL toluene was added a solution of
triphosgene (305.9 mg, 1 mmol) in 5 mL toluene dropwise. The
resulting solution was heated to reflux for 3 h. After cooling to
room temperature, the reaction mixture was diluted with 20 mL
THF and filtered. After removal of the solvents, to the residues
was added the 2,1,3-benzoselenadiazol-5-methanol (213.1 mg,
1 mmol) in 20 mL THF. The solution was stirred at room
temperature for an additional 12 h. After removal of THF, the
residues were purified by silica gel column chromatography using
chloroform as eluent to afford 1 (264.9 mg, 0.52 mmol, 52% yield).
1H-NMR (400 MHz, DMSO-d6) d (*10-6): 0.93 (t, J = 7.4 Hz, 3H),
1.32–1.38 (m, 2H), 1.60–1.63 (m, 2H), 4.04 (t, J = 7.4 Hz, 2H),
5.43 (s, 2H), 7.64 (d, J = 8.6 Hz, 1H), 7.84–7.91 (m, 2H), 7.98 (s,
1H), 8.23 (d, J = 8.0 Hz, 1H), 8.48–8.52 (m, 2H), 8.75 (d, J =
8.8 Hz, 1H), 10.47 (s, 1H). 13C-NMR (100 MHz, DMSO-d6) d
(*10-6): 14.1, 20.2, 30.1, 66.2, 117.7, 118.7, 121.4, 122.7, 123.7,
124.4, 126.9, 128.8, 129.7, 129.8, 131.4, 132.1, 138.4, 141.0, 154.3,
159.8, 160.1, 163.4, 163.9. IR (KBr, cm-1): 3267, 2956, 2865, 1697,
1652, 1544, 1358, 1236, 1062, 783, 658. MALDI-TOF calcd for
C24H21N4O4Se [M+H]+ 509.1, found 509.1. HRMS (ESI positive)
calcd for C24H21N4O4Se [M+H]+ 509.07239, found 509.07176.
Cell culture
HeLa cells (gifted from the center of cells, Peking Union Medical
College) were cultured in culture media (DMEM/F12 supple-
mented with 10% FBS, 50 unit/mL penicillin, and 50 mg mL-1 of
streptomycin) at 37 ◦C under a humidified atmosphere containing
5% CO2. HeLa cells were seeded in a 6-well plate at a density
of 104 cells per well in culture media. After 24 h, the cells were
incubated with 10 mM 1 in culture media for 1 h at 37 ◦C. HeLa
cells were pretreated with 50 mM NEM for 2 h to decrease the
reducing capability, and then they were incubated with the probe
1 (10 mM) for another 1 h. In addition, HeLa cells were pretreated
with 100 mM DTT for 30 min, and then they were incubated
with the probe 1 (10 mM) for another 1 h. After the medium was
removed and the cells were carefully washed with PBS for twice,
fluorescence imaging of living HeLa cells was observed under
confocal fluorescence microscope (excitation light source: Blue;
Olympus IX 71 S 8F-2).
Acknowledgements
This work was supported by the National Nature Science Foun-
dation of China (No. 20975012) and the 111 Project (B07012).
Notes and references
Synthesis of probe 1
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1
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17 1H NMR, 13C NMR and HRMS data of compound 2: 1H-NMR
(400 MHz, DMSO-d6) d (*10-6): 0.92(t, J = 7.4 Hz, 3H), 1.27-1.36(m,
2H), 1.55-1.60(m, 2H), 4.01(t, J = 7.4 Hz, 2H), 6.85(d, J = 8.4 Hz,
1H), 7.43(s, 2H), 7.65(t, J = 7.8 Hz, 1H), 8.19(d, J = 8.4 Hz, 1H),
8.42(d, J = 7.2 Hz, 1H), 8.61(d, J = 7.6 Hz, 1H). 13C-NMR (100 MHz,
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162.87. HRMS (ESI positive) calcd for C16H17N2O2 [M+H]+ 269.12845,
found 269.12817; calcd for C16H16N2NaO2 [M+Na]+ 291.11040, found
291.11020.
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