A triphenylamine as a fluorophore and maleimide as a bonding group selective turn-on fluorescent imaging probe for thiols

Article abstract of DOI:10.1016/j.dyepig.2015.12.031

With the biological importance of biothiols, the development of probes for thiols has been an active research area in recent years. Here, we report a novel thiol-reactive fluorescent probe based on Michael addition reaction for selectively detecting thiol

Full text of DOI:10.1016/j.dyepig.2015.12.031

Accepted Manuscript
A triphenylamine as a fluorophore and maleimide as a bonding group selective turn-
on fluorescent imaging probe for thiols
Tao Liu, Fangjun Huo, Caixia Yin, Jianfang Li, Jianbin Chao, Yongbin Zhang
PII:
S0143-7208(16)00037-1
10.1016/j.dyepig.2015.12.031
DYPI 5079
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 4 November 2015
Revised Date: 15 December 2015
Accepted Date: 22 December 2015
Please cite this article as: Liu T, Huo F, Yin C, Li J, Chao J, Zhang Y, A triphenylamine as a fluorophore
and maleimide as a bonding group selective turn-on fluorescent imaging probe for thiols, Dyes and
Pigments (2016), doi: 10.1016/j.dyepig.2015.12.031.
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ACCEPTED MANUSCRIPT
Abstract Graphic
The Title:
A triphenylamine as a fluorophore and maleimide as a bonding group selective turn-on
fluorescent imaging probe for thiols
List of Authors:
Tao Liu a,1, Fangjun Huo b ,*, Caixia Yin a,**, Jianfang Li a,1, Jianbin Chao b, Yongbin Zhang b
The statement:～～
Based on the Michael addition reaction of thiol with the C=C bond under mild conditions,
we herein report a new fluorescent probe which features a rapid signal response time, a
good linearity range and a low detection limit. The potential application of this new
fluorescent probe was demonstrated by fluorescent imaging of thiol in living cells.
～
1

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A triphenylamine as a fluorophore and maleimide as a bonding group selective
turn-on fluorescent imaging probe for thiols
～
Tao Liu a,1, Fangjun Huo b ,*, Caixia Yin a,**, Jianfang Li a,1, Jianbin Chao b, Yongbin Zhang b
a Institute of Molecular Science, Shanxi University, Taiyuan 030006, China.
b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China.
*Corresponding author: F. J. Huo, E-mail: huofj@sxu.edu.cn; C. X. Yin, E-mail:
yincx@sxu.edu.cn, Tel/Fax: +86-351-7011022ꢀ
Abstract: With the biological importance of biothiols, the development of probes for
thiols has been an active research area in recent years. Here, we report a novel
thiol-reactive fluorescent probe based on Michael addition reaction for selectively
detecting thiols over other relevant biological species. The thiol adduct was
characterized using NMR and mass spectroscopy and detection mechanism was further
confirmed. This sensor with excellent selectivity for biothiols over other amino acids
features a rapid signal response time, a good linearity range and a low detection limit.
For the practical application of the sensor, it can be used to monitor thiol in live cells
with turn-on fluorescence imaging.
Keywords: Thiols; Detection; Fluorescent probe; Bioimaging
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1. Introduction
Biological thiols including cysteine (Cys), homocysteine (Hcy), and glutathione
(GSH) are components of many peptides, which play crucial roles in maintaining the
biological redox homeostasis through the equilibrium of free thiols and oxidized
disulfides in biological systems [1-3]. It is known that intracellular concentration of
GSH is much higher than Cys (Cys: 30-200 µM; GSH: 1-10 mM) [4,5]. In contrast, in
healthy human plasma, Cys concentration is typically 10 times that of GSH, 20-30 times
that of Hcy, which normally presents below 12-15 µM [4]. However, the alterations in
the level of thiols in biological fluids are implicated in a variety of diseases [6-8]. For
example, Cys deficiency is involved in many syndromes such as slow growth in
children, hair depigmentation, edema, lethargy, liver damage, loss of muscle and fat,
skin lesions, and weakness [9]. At elevated levels in plasma, Hcy is a risk factor for
Alzheimer’s disease [10], folate and cobalamin (vitamin B12) deficiencies [11], and
cardiovascular diseases [12]. GSH is the most abundant intracellular non-protein thiol
[13], which serves many cellular functions, including maintenance of intracellular redox
activities, xenobiotic metabolism, intracellular signal transduction, and gene regulation
[14, 15]. Owing to their important roles, sensitive and selective detection of thiols has
received growing attention in recent years.
In recent times, fluorescent molecular probes have emerged as an attractive tool for
selective detection of various chemical and biological components [16–19], including
thiols [20–27]. Compared to traditional techniques, chemical probes based on
absorption or fluorescence changes are more feasible for detecting analyses owing to
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their many appealing advantages such as low detection limit, high selectivity, its
real-time monitoring and its potential for in vivo imaging of living cells. However,
fluorescence quenching may be caused by a number of factors other than the target
analyte, and thus the sensing behavior may be nonspecific [28]. Probes that rely on
fluorescence quenching suffer from inherent drawbacks including low signal-to-noise
ratio and non-specific quenching [29], so that “turn-on” type fluorescence probes are
preferred [30-33].
Maleimide groups are known to react fairly selectively with thiols via addition
reactions involving their C=C double bond. They are also known to quench
fluorescence in their conjugated form, but not as their thiol adduct products [34]. These
properties were demonstrated in the characterization of fluorophores bearing a
maleimide group whose fluorescence increased dramatically upon reaction with thiols
[35-37]. Based on these issues, we synthesized a new thiol-reactive fluorescent probe
containing triphenylamine and maleimide (Scheme 1). When the concentration of probe
is low, Hcy/GSH induced a significant enhancement in fluorescence intensity whereas
Cys induced almost no change, while increasing amount of probe, fluorescence
enhancement induced by Cys was enough to make clear the recognition. Furthermore,
this probe was successfully applied in fluorescent imaging in living cells.
<Inserted Scheme 1>
2. Materials and Methods
2.1. Materials
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from
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Sigma-Aldrich (St. Louis, MO). Sodium hydroxide solution (0.1 mol/L) was added to
aqueous HEPES (10 mmol/L) to adjust the pH to 7.4. Amino acids were purchased from
Shanghai Experiment Reagent Co., Ltd (Shanghai, China). All other chemicals used
were of analytical grade.
2.2. Instruments
A pH meter (Mettler Toledo, Switzerland) was used to determine the pH.
Ultraviolet–visible (UV–vis) spectra were recorded on an Agilent 8453 UV-Visible
spectrophotometer. Fluorescence spectra were measured on F-7000 FL
Spectrophotometer. A PO-120 quartz cuvette (10 mm) was purchased from Shanghai
1
Huamei Experiment Instrrument Plants, China. H NMR, 13C NMR spectra were
recorded on a Bruker AVANCE-300 MHz (and 600MHz) and 75 MHz NMR
spectrometer, respectively (Bruker, Billerica, MA). ESI was measured with an LTQ-MS
(Thermo) instrument. LC-MS was measured with Bruker solari X FTMS. The ability of
probe reacting to thiols in the living cells was also evaluated by laser confocal
fluorescence imaging using an Olympus FV1000 laser scanning microscope.
2.3. Preparation and characterization of probes
2.3.1. Preparation and characterization of B
The synthesis route is summarized in Scheme 1. POCl3 (11 mL) was added to a
DMF solution (250 mL) with stirring for 2 h in ice-water bath. After the color change to
nacarat, added triphenylamine (100 mmol) to the solution with stirring at 40ꢁ. After
the reaction was complete, the reaction mixture was poured into ice water (1000 mL)
and adjusted pH to 9 with NaOH to separate faint yellow crystals out. The solid
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obtained by filtered, washed with water and recrystallized in ethanol to give compound
1
B in 86% as a faint yellow powder. H NMR (DMSO-d6, 300 MHz): δ (ppm): 9.77 (s,
1H), 7.72 (d, 2H, J = 8.7 Hz), 7.43 (t, 4H, J = 15.6 Hz), 7.23 (m, 6H), 6.88 (d, 2H, J =
13
8.7 Hz); C NMR (DMSO-d6, 75 MHz): δ (ppm): 117.7, 125.0, 126.0, 128.1, 129.6,
130.9, 145.1, 152.3, 190.1; Elemental analysis (calcd. %) for C19H15NO: C, 83.49, H,
5.53, N, 5.12, Found: C, 83.46, H, 5.54, N, 5.14; ESI–MS m/z: [B + H]+ Calcd for
C19H16NO 274.12, Found 273.92 (Fig. S1).
2.3.2. Preparation and characterization of C
A mixture of C (triphenylphosphine, 0.16 mol) and 4-nitrobenzyl chlorine (0.15
mol) in paraxylene (200 mL) was stirred and refluxed at 150ꢁ for 2 h. The mixture
was then cooled to 0ꢁ and filtered. The taupe solid thus obtained was dried under
vacuum to give compound D in 92%. 1H NMR (DMSO-d6, 300 MHz): δ (ppm): 8.11 (d,
2H, J = 8.1 Hz), 7.93 (s, 3H), 7.75 (d, 12H, J = 9.9 Hz), 7.30 (d, 2H, J = 8.1 Hz), 5.60 (s,
1H), 5.54 (s, 1H); 13C NMR (DMSO-d6, 75 MHz): δ (ppm): 116.0, 117.1, 122.9, 129.3,
129.5, 131.4, 133.2, 133.4, 134.5, 135.4, 135.6, 146.5; Elemental analysis (calcd. %) for
C25H21ClNO2P: C, 69.21, H, 4.88, N, 3.23, Found: C, 69.20, H, 4.90; N, 3.20 (Fig. S2).
2.3.3. Preparation and characterization of D
In a 250 mL 3 mouth flask, added potassium tert-butoxide to a THF solution (100
mL) of D (12.5 mmol) in ice-water bath with stirring for 1h. Afterwards 8.5 mmol B
was added to the solution with stirring and refluxing at 70ꢁ. After the reaction was
complete, parts of the solvents were then evaporated in vacuo and the residue was
dissolved in dichloromethane. 30 mL of water was then added to the dichloromethane
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solution, and the organic layer was purified by chromatography on a silica gel column
to give a crimson product in 82% yield. A CH3Cl solution containing the product was
allowed to evaporate slowly at room temperature for several days, and the crystals that
subsequently formed were suitable for X-ray crystallography. 1H NMR (DMSO-d6, 300
MHz): δ (ppm): 8.21 (d, 2H, J = 8.7 Hz), 7.81 (d, 2H, J = 8.7 Hz), 7.55 (t, 2H, J = 15.6
13
Hz), 7.30 (m, 6H), 7.09 (m, 6H), 6.95 (d, 2H, J = 8.4 Hz); C NMR (DMSO-d6, 75
MHz): δ (ppm): 121.7, 123.2, 124.1, 126.4, 127.9, 129.1, 129.6, 132.4, 144.0, 145.3,
146.2, 147.3; Elemental analysis (calcd. %) for C26H20N2O2: C, 79.57, H, 5.14, N, 7.14,
Found: C, 79.59, H, 5.14, N, 7.12; ESI–MS m/z: [E + H]+ Calcd for C26H21N2O2 393.15,
Found 393.08; Crystal data for C26H20N2O2: crystal size: 0.20 × 0.16 × 0.06, monoclinic,
space group P 1 21/n 1. a = 8.4057(13) Å, b = 8.8548(13) Å, c = 27.015(4) Å, β =
96.654(3)°, V =1997.2(5) Å3, Z = 4, T = 173.1500 K, max = 27.483°, 13867 reflections
measured, 4562 unique (Rint = 0.0355). Final residual for 271 parameters and 4562
reflections with I > 2(I): R1 = 0.0596, wR2 = 0.1248 and GOF = 1.104 (Fig. S3).
2.3.4. Preparation and characterization of F
A solution of E (10 mmol) dissolved in 150 mL ethanol was added into a
round-bottom flask equipped with a magnetic stirrer and heated at 80ꢁ. Then 0.3 g of
Pd/C catalyst was added into the preceding reaction system and a solution of 4.9 mL of
85% hydrazine hydrate was added dropwise for about 0.5 h. The reaction was
monitored by TLC. After the completion of the reaction, the reaction mixture was
filtered immediately and the solution was poured into saturated sodium chloride
solution to give a faint yellow solid. The solid obtained by filtered was purified by
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chromatography on a silica gel column to give compound F in 65% yield. 1H NMR
(DMSO-d6, 300 MHz): δ (ppm): 7.40 (d, 2H, J = 8.1 Hz), 7.27 (m, 6H), 6.85 (m, 10H),
6.53 (d, 2H, J = 8.1 Hz), 5.26 (s, 2H); 13C NMR (DMSO-d6, 75 MHz): δ (ppm): 112.2,
112.7, 121.0, 121.7, 122.5, 12.7, 125.6, 126.2, 126.7, 128.3, 131.5, 14.4, 145.9, 147.3;
Elemental analysis (calcd. %) for C26H22N2: C, 86.15, H, 6.12, N, 7.73, Found: C, 86.14,
H, 6.12, N, 7.74; ESI–MS m/z: [F + H]+ Calcd for C26H23N2 363.18, Found 362.92 (Fig.
S4).
2.3.5. Preparation and characterization of probe 1
The synthesis of probe 1 is that a mixture of F (2 mmol), maleic anhydride (2
mmol) in glacial acetic acid (10 mL) was heated under reflux with monitoring by TLC.
The mixture solution was poured into saturated sodium carbonate solution to separate
crystals out. The solid obtained by filtered was purified by chromatography on a silica
gel column to give compound F in 58% yield. 1H NMR (DMSO-d6, 600 MHz): δ (ppm):
7.67 (d, 2H, J = 8.5 Hz), 7.53 (d, 2H, J = 8.6 Hz), 7.35 – 7.32 (m, 4H), 7.31 (d, 2H, J =
2.0 Hz), 7.25 (d, 2H, J = 16.4 Hz), 7.19 (s, 2H), 7.15 (d, 2H, J = 16.4 Hz), 7.07 (t, 2H, J
= 7.4 Hz), 7.05 (d, 4H, J = 7.6 Hz), 6.97 (d, 2H, J = 8.6 Hz). 13C NMR (DMSO-d6, 75
MHz): δ (ppm): 122.5, 122.9, 123.8, 125.5, 126.1, 126.5, 127.4, 128.4, 129.2, 130.0,
130.7, 134.3, 136.4, 146.5, 169.5; Elemental analysis (calcd. %) for C30H22N2O2: C,
81.43, H, 5.01, N, 6.33, Found: C, 81.44, H, 5.02, N, 6.31. LC–MS m/z: [probe +
CH3OH] Calcd for C31H26N2O3 474.19434, Found 474.19364 (Fig. S5).
2.4. General UV-Vis and fluorescence spectra measurements
Probe stock solutions were prepared in DMSO. Aqueous amino acid solutions
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were also prepared using deionized water. Fluorescence measurements were carried out
with a slit width of 5 nm (λex = 370 nm). Fluorescence spectra were obtained in HEPES
aqueous buffer (10 mmol/L, pH 7.4) solutions.
2.5. Detection range
Fluorescence spectra were measured from 385 nm to 620 nm with excitation at 370
nm. The detection threshold for Cys, Hcy and GSH was 10−8 to 10−7 mol/L, and at this
level the fluorescence color change was very obvious.
3. Results and discussion
3.1. The Selectivity over thiols
The spectral responses of probe 1 towards various amino acids, including thiols
with mercapto groups (GSH, Hcy and Cys), and those without mercapto groups
(L-alanine (Ala), L-arginine (Arg), L-asparagine(Asp), L-glutamine (Gln), L-glutamic
acid (Glu), L-glycine (gly), L-histidine (His), L-isoleucine (Ile), L-leucine (Leu),
L-lysine (Lys), L-methionine (Met), L-phenylalanine (Phe), L-proline (Pro), L-serine
(Ser), L-threonine (Thr), L-tryptophan (Trp), L-tyrosine (Tyr), L-valine (Val) and SH–)
were examined. The probe 1 is essentially non-fluorescent in the absence of thiol. Only
in the presence of thiols there was a significant enhancement in fluorescence intensity
observed for probe 1. In the buffer containing 0.5 µmol/L probe, addition of Hcy/GSH
will induce a significant fluorescence enhancement, whereas Cys induce a little (Fig.1).
Interestingly, only using 5 µmol/L probe 1, fluorescence enhancement induced by Cys
was obvious (Fig. S6). The other amino acid samples exhibited no noticeable increase
of the fluorescence signal. For ultraviolet spectrum, there is no significant change even
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in the presence of 20 eq. of thiols (Fig. S7). For other thiols, such as ME
(2-mercaptoethanol), 1 showed similar fluorescence responses (Fig. S6).
<Inserted Figure 1>
3.2. The fluorescence spectra for thiol
As a typical biological thiol, GSH was used to further examine the fluorescence
response of probe 1. The changes in the fluorescence spectra of probe 1 (0.5 µmol/L) in
the absence or presence of GSH (0–3.5 µmol/L) in HEPES buffer are displayed in Fig.
2. The probe 1 is essentially non-fluorescent in the absence of GSH; however, the
addition of GSH caused a dramatic change in the fluorescence spectra. A strong new
emission peak at 480 nm appeared, and an enhancement of the fluorescence intensity by
up to 24-fold was observed. Upon addition of Hcy (3.0 µmol/L) to probe, a similar
phenomenon on fluorescence spectra could be observed (Fig. S6). Cys induced a
negligible response for 0.5 µmol/L probe 1. However, Cys (35 µmol/L) caused 12-fold
fluorescence enhancement in the presence of 5 µmol/L probe 1.
<Inserted Figure 2>
To investigate the detection limit of probe for GSH, probe 1 (0.5 µmol/L) was
treated with various concentrations of GSH (0–3.5 µmol/L) and the relative emission
intensity at 480 nm was plotted as a function of the GSH concentration (Fig. 3). The
emission intensity of probe 1 was linearly proportional to GSH concentrations of 0–3.5
µmol/L. The detection limit, based on the definition by IUPAC (CDL = 3 Sb/m) [38],
was found to be 0.085 µmol/L. Similarly, the detection limits were estimated to be 0.12
µmol/L for Hcy and 0.13 µmol/L for Cys, respectively (Fig. S8). The probe shows a
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high sensitivity towards thiols which is comparable to that of other reported thiol
chemosensors (Table 1) [39-41].
<Inserted Figure 3>
<Inserted Table 1>
3.3. Time-dependence in the detection process of thiols
Time-dependent modulations in the fluorescence spectra of probe 1 were
monitored in the presence of 10 eq. of thiol. The kinetic study (Fig. 4) showed that the
reaction was complete within 75 s for Hcy and GSH, indicating that probe 1 reacts
rapidly with Hcy/GSH under the experimental conditions. Slightly inferior to Hcy/GSH,
the reaction time for Cys is 150 s which is also faster than that of reported probes
[26,42].
<Inserted Figure 4>
3.4. pH dependence
To investigate the effect of pH on the fluorescence response of 1 to GSH, the
fluorescence intensity changes of 1 induced by GSH were measured at various pHs (Fig.
S9). When the solution pH was between 2.0 and 4.0, the probe has slight fluorescence.
When pH value exceeds 4.0, the probe 1 almost has no fluorescence. However, when
the solution pH exceeds 5.0, fluorescence increase induced by GSH is obvious with
intensifying pH value. Therefore, physiological pH 7.4 was chosen for further studies
3.5. Michael addition mechanism
Maleimide groups are known to react fairly selectively with thiols via addition
reactions involving their C=C double bond. Michael addition of thiols to the
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electron-deficient alkene groups in probe 1 gave rise to emission enhancement (λex =
370 nm, λem = 480 nm). Mass spectrometry analysis of a product obtained from the
reaction of probe with 2-mercaptoethanol also supports the formation of probe-thiol
derivatives. A peak at 521.16 corresponding to [probe-ME + H]+ was clearly observed
(Fig. S10). Further NMR spectroscopic analysis also provided the evidence for the
1,2-addition of the thiol to the maleimide ring in probe 1. With addition of 2 eq. of
2-mercaptoethanol (ME) to probe in DMSO-d6, the resonance of the original proton
(CHmaleimide ring) at 7.19 ppm disappeared and new peaks at 4.90, 4.19, 3.62, 2.7–3.0 ppm
appeared (Fig. S11). Thus, the sensing mechanism of probe 1 towards thiol, which is in
keeping with reported literature [43], was based on the Michael addition as shown in
Scheme 2.
<Inserted Scheme 2>
3.6. Cellular Imaging
In order to evaluate the cell permeability and capability of probe 1 to selectively
detect intracellular thiols, live-cell imaging studies were carried out. As shown in Fig.
5a, HepG2 cells that were pre-treated with 10 µmol/L N-ethylmaleimide (NEM, a thiol
blocking reagent) and then incubated with 0.5 µmol/L probe 1 for 30 min at 37 ꢁ
showed no fluorescence. In a further experiment it was found that HepG2 cells
displayed cyan fluorescence when the cells were first incubated with 0.5 µmol/L of
probe 1 for 30 min at 37ꢁ and then incubated with 3.5 µmol/L GSH (Fig. 5b). These
cell experiments show the good cell-membrane permeability of probe, and it can thus be
used to mark thiols within living cells.
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<Inserted Figure 5>
4. Conclusions
In summary, here we report an easy-prepared probe based on triphenylamine for
the detection of thiol-containing molecules with high selectivity and sensitivity. The
observed fluorescence enhancement is ascribed to a selective thiol-induced addition
reactions to C=C double bond of maleimide group. The results of the investigation show
that thiol induce an enhancement of the fluorescence intensity of 1 in pH 7.4 solutions.
Moreover, because of its good water solubility, cell-penetration ability and
biocompatibility, 1 serves as a fluorescent sensor to visualize thiol in live cells at
micromolar concentrations. However, one shortcoming is that the excitation and
emission wavelengths are in the ultraviolet region, which might be avoided in the future
directions.
Acknowledgments
The work was supported by the National Natural Science Foundation of China (No. 21472118),
the Shanxi Province Science Foundation for Youths (No. 2013011011-1), the Shanxi Province
Foundation for Returnee (No. 2012-007), the Taiyuan Technology star special (No. 12024703), the
Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of
Shanxi (TYMIT, No. 2013802), talents Support Program of Shanxi Province (No. 2014401) and
Shanxi Province Outstanding Youth Fund (No. 2014021002).
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Figure captions
Scheme 1. The synthesis of the probe 1.
Fig. 1. (a) Fluorescence emission spectra of the probe 1 (0.5 µmol/L) in
CH3OH–HEPES buffer (10 mmol/L, pH=7.4, 1 : 1, v/v) in the presence of 50 µmol/L
Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Val and
3.5 µmol/L Cys, GSH, Hcy. (b) Optical density two-dimensional graph of the probe at
480 nm upon the addition of amino acids.
Fig. 2. Fluorescence spectra of the probe (0.5 µmol/L) in the presence of various
concentrations of GSH (0–3.5 µmol/L) in CH3OH–HEPES buffer (10 mmol/L, pH 7.4,
1 : 1, v/v) (λex = 370 nm, slit: 5 nm/5 nm).
Fig. 3. The linearity of the relative fluorescence intensity versus GSH concentration.
Table 1. A comparison table of the detection limits for Hcy.
Fig. 4. Reaction time profiles of probe (0.5 µmol/L) with GSH, Hcy and probe (5
µmol/L) for Cys.
Scheme 2. Proposed detection mechanism of the probe to thiols.
Fig. 5. Confocal fluorescence images of HepG2 cells. (a) Fluorescence image of HepG2
cells pre-treated with 10 µmol/L NEM and then incubated with 0.5 µmol/L probe for 30
min at 37ꢁ and its bright field image (c); (b) fluorescence image of HepG2 cells first
incubated with 0.5 µmol/L of probe for 30 min at 37ꢁ and then incubated with 3.5
µmol/L GSH for 30 min at 37ꢁ and its bright field image (d).
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Scheme 1
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Figure 1(a)
(a)
Figure 1 (b)
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Figure 2
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Figure 3
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Table 1
Method
Ref.[39]
Ref.[40]
Ref.[41]
This work
Analyte
Hcy
Signal output
Fluorescence
Fluorescence
Fluorescence
Fluorescence
Solvent
Detection limit
1.19 µM
DMSO/H2O
DMSO/H2O
CH3CN/H2O
DMSO/H2O
Hcy
1.96µM
Hcy
0.13µM
Hcy
0.12µM
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Figure 4
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Scheme 2
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Figure 5
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Supporting Information for
A triphenylamine as a fluorophore and maleimide as a bonding group
selective turn-on fluorescent imaging probe for thiols
b
Tao Liu a,1, Fangjun Huo b ,*, Caixia Yin a,**, Jianfang Li a,1, Jianbin Chao ,
Yongbin Zhang b
a Molecular Science, Shanxi University, Taiyuan 030006, China; bInstitute of Research Institute of Applied
Chemistry (RIAC), Shanxi University, Taiyuan, 030006, China *Corresponding author: F. J. Huo, E-mail:
huofj@sxu.edu.cn; Caixia Yin, E-mail: yincx@sxu.edu.cn, Tel/Fax: +86-351-7011022.
Figure S1: The characterization data of the compound B
Figure S2: The characterization data of the compound D
Figure S3: The characterization data of the compound E
Figure S4: The characterization data of the compound F
Figure S5: The characterization data of the probe 1
Figure S6: The fluorescence titration spectra of Cys, Hcy and ME
Figure S7: The absorption titration spectra of thiols
Figure S8: The detection limits of Hcy and Cys
Figure S9: Choice of pH range for the measurements
Figure S10: LC-MS spectra of the probe-ME adduct
Figure S11: NMR spectra of probe and probe-ME
29