A fast response and red emission probe for mammalian thioredoxin reductase

Article abstract of DOI:10.1039/c6cc04984b

The first red emission off-on probe, TRFS-red, for thioredoxin reductase was reported. Compared to the previous green emission probe TRFS-green, TRFS-red maintains a high selectivity to the redox enzyme yet with improved response rate and sensitivity.

Full text of DOI:10.1039/c6cc04984b

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Fast response and red emission probe for mammalian thioredoxin
reductase
Received 00th January 20xx,
Accepted 00th January 20xx
Huilong Ma, Junmin Zhang, Zhenzhe Zhang, Yaping Liu, and Jianguo Fang *
DOI: 10.1039/x0xx00000x
www.rsc.org/
The first red emission off-on probe, TRFS-red, of thioredoxin published procedures. The fluorophore 5 reacted with triphosgene
reductase was reported. Compared to the previous green emission in the presence of N, N-diisopropylethylamine (DIPEA) in
probe TRFS-green, TRFS-red maintains high selectivity to the redox dichloromethane (DCM), followed by the addition of 2 furnishing
enzyme yet with improved response rate and sensitivity.
the final product TRFS-red in 28% yield. In step c, two
methoxycarbonylalkyl groups (R group) were introduced and
maintained in the probe. This would be expected to facilitate the
probe to penetrate cell membranes. Once inside cells, the ester
bond is hydrolyzed by cellular esterases to yield the charged dye
that is entrapped inside cells. This strategy has been well
Fluorescence sensing is one of the most powerful and popular
tools to explore the complicated biological processes. Remarkable
progress has been achieved in design of fluorogenic probes for
various biomolecules, including small molecules, nuclear acids and
1
-13
3
1
various enzymes.
Development of red or near-infrared
established to construct live cell imaging probes such as DCFH-DA.
The chemical structures of TRFS-red and the synthetic
fluorescent probes with emission wavelength >600 nm is ideal for
interrogating biological systems, as longer wavelengths provide less
background autofluorescence and higher tissue penetration. We
recently reported the first probe (TRFS-green) for sensing the redox
1
13
intermediates were characterized by H NMR, C NMR and MS. The
purity of TRFS-red is >95% determined by HPLC analysis (Figure S4B).
The original NMR and MS spectra were included in the Supporting
Information (Figures S5-17).
1
4
enzyme thioredoxin reductase (TrxR).
strategies, Bu et al. also reported several inhibitor-oriented TrxR
Based on different
1
5-16
probes.
TRFS-green has high selectivity to TrxR with green
OH
emission, and is readily applied to image the enzyme activity in
OH
a
OH
b
1
7-25
Cl
Cl
HS
SH
various types of live cells.
In this work, we reported the
S
S
2
synthesis and evaluation of a red emission probe of TrxR, TRFS-red.
The probe maintains the high specificity to TrxR, and displays an off-
on response with the maxima emission wavelength at ~660 nm. In
addition, TRFS-red gives faster response to TrxR with improved
sensitivity compared to the parent probe TRFS-green.
1
c
d
N
N
NO
2
R
R
R
HO
NH2
HO
N
R
N
OH
R
3
4
We replaced the naphthalimide fluorophore in the parent
molecule TRFS-green with the nile blue scaffold to generate TRFS-
red, whose synthetic routes were outlined in Scheme 1 according to
e
f
N
N
O
O
OH
1
4, 26-30
R
the published procedures.
The red fluorophore nile blue
N
O
NH2
N
R
N
O
H
derivative (5) was synthesized in 3 steps (steps c-e) with the overall
yield of 44% from the readily available starting material m-
aminophenol by adopting the published procedures. The TrxR-
recognizing part, 1,2-dithiolan-4-ol (2), was synthesized in two steps
R
ClO4
-
5
ClO4
S
S
2
S S
R= -CH
2
CH
2
COOCH
3
TRFS-red
Scheme 1 Synthesis of TRFS-red. Reagents and conditions: (a)
CS , 60 C, 59%; (b) I /NaHCO /CH Cl , rt, 89%; (c) methyl
2 3 2 3 2 2
acrylate/NaBr/AcOH, 95 C, 86%; (d) p-nitroaniline/NaNO /HCl/H O,
o
Na
(
steps a & b) with overall yield of 52% from the commercially
o
2
2
o
available 1,3-dichloropropan-2-ol by following our previously
7
5%; (e) 1-naphthylamine/HClO4/DMF, 165 C, 68%; (f)
triphosgene/DIPEA/DMAP/DCM/THF, 28%.
Initially, we examined the absorption and emission spectra of
TRFS-red in responding to the purified TrxR (Figure.1) in vitro. TRFS-
red (10 μM) has a maximal absorption at ~530 nm in TE buffer (50
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mM Tris-HCl and 1 mM EDTA, pH 7.4). After addition of the Figure 1 Response of TRFS-red to TrxR. Time course of absorbance
recombinant enzyme (75 nM) and NADPH (200 μM), its maximum (A) and emission (B) changes of TRFS-red (10 μM) in the presence of
absorbance is red-shifted to ~615 nm (Figure. 1A) in a time- TrxR (75 nM). Arrows show the change of absorbance at 530 nm
dependent manner, and the absorbance reaches a plateau in ~120 and 615 nm (A), and the change of emission at 661 nm excited at
min. TRFS-red (10 μM) itself is almost non-fluorescent when excited 615 nm. (C) Time course of the fold of fluorescence increment of
at 530 nm (φ<0.001). However, the emission centered at ∼660 nm TRFS-red (10 μM, λex=615 nm; λem=661 nm) and TRFS-green (10 μM,
(
λex=615 nm) increases remarkably after addition of TrxR (75 nM)
λex=438 nm; λem=538 nm) with various concentrations of TrxR and
and NADPH (φ =0.12, Figure. 1B). The increment of the fluorescence U498C TrxR. (D) Influence of pH on TRFS-red. The fold of
intensity is dependent on the incubation time, and the maxima fluorescence increase at 661 nm was determined after mixing TRFS-
emission intensity reaches after 120 min, consistent with the red with 1 mM TCEP for 120 min in 0.1 M PBS with pH ranges from
observation from the absorbance spectra. It should be noted that 5.5 to 8.5. NADPH (0.2 mM) was included in all enzymatic reactions.
Nile blue derivatives showed a maximum of fluorescence in water
3
2
Since the fluorescence signal of TRFS-red could be switched on by
TrxR, we next determined the selectivity of TRFS-red to various
related species. As shown in Figure. 2A, neither reduced glutathione
at ~670 nm. Our Nile blue fluorophore (compound 5) has a
maximal emission at ~660 nm (Figure S1D), which is close to the
reported value (658 nm) of the same molecule. This difference is
likely due to the different substituted groups of the dye. Next, we
compared the response rate and fold of fluorescence increment of
TRFS-red to those of the parent probe TRFS-green. As shown in
Figure. 1C, TRFS-red displays faster response to TrxR compared to
its parent molecule TRFS-green: The fluorescence increment of
TRFS-red triggered by TrxR reaches a plateau in ~120 min, while
that of TRFS-green appears a sluggish reaction and keeps increasing
within 180 min. Furthermore, TRFS-red exhibits ~90 fold of
fluorescence increment, while TRFS-green only gives ~35 fold of
elevation of the emission intensity after 120 min. The strong
electron-withdrawing iminium cation moiety in nile blue moiety
endues the nile blue fluorophore good leaving ability. In addition,
the iminium cation enables TRFS-red to bear a positive charge,
which facilitates it binding to the negatively-charged C-terminal
active site of TrxR via electrostatic interaction. Both of them might
contribute to the improved response rate of TRFS-red. The pH-
dependent response of TRFS-red was shown in Figure. 1D. Under
(
GSH, 1 mM) nor Cys (1 mM) causes significant increment of the
signal. Glutathione reductase (GR), whose structure is closely
related to TrxR, has little effect on the reduction of TRFS-red in the
presence of NADPH. One of the most important hallmarks of
mammalian TrxRs is they all contain a selenocysteine (Sec) residue
at their C-terminal redox center, and the Sec residue is critical for
3
3
their function. Thus, we generated U498C TrxR, where the Sec498
was replaced by Cys, and compared the efficiency of the mutant
enzyme with the wild type TrxR in reducing the probe. We observed
that the U498c TrxR (0.2 μM) also displays negligible interference of
the emission signal, supporting the indispensable role of the Sec
residue of the enzyme to turn on the fluorescence. The probe also
gives marginal response to amidehydrolase (AHL), Sec, and NADPH.
We further determined the reduction of TRFS-red by cell extract. As
shown in Figure. 2B, incubation of the probe with HeLa cell lysate
elicits a time-dependent increase of the fluorescence intensity.
However, pretreatment of the lysate with the specific TrxR inhibitor
auranofin (AF) or less specific inhibitor 2,4-dinitro-1-chlorobenzene
m
TrxR catalysis, the K /kcat values for TRFS-red and TRFS-green were
-1
-1
(
DNCB) drastically suppresses the fluorescence. Taken together,
determined to be 51.8 ꢀM/0.17 s and 63.2 ꢀM/0.11 s ,
respectively (Figure S2), supporting the faster response of TRFS-red
than TRFS-green. To exclude the potential interference of TrxR
activity by pH, we employed tris(2-carboxyethyl)phosphine (TCEP)
to reduce the probe in this experiment as the response of TRFS-red
to TCEP is similar to its response to TrxR (Figure. S1). In the pH
range of 5.5-8.5, TRFS-red displays reliable fluorescence signal
TRFS-red maintains the high selectivity towards TrxR. This is not
surprising as the recognition part of TRFS-red is the same as that of
the specific TrxR probe TRFS-green.
(
F/F >75), indicating the probe is suitable for application in living
0
systems.
Figure 2 Selective recognition of TRFS-red by TrxR. (A) Response of
TRFS-red (10 μM) to various related species. The fold of
fluorescence increase at 661 nm was determined (λex=615 nm) after
the probe was incubated at 37 °C for 120 min. Sec (10 μM) was
generated in situ by mixing Cys (1 mM) and selenocystine (5 μM). (B)
Inhibition of cell lysate-mediated TRFS-red reduction by TrxR
inhibitor DNCB or AF. The NADPH-prereduced HeLa cell lysate (25
o
μL) was incubated with DNCB (20 μM) or AF (2 μM) at 37 C for 30
min, and further incubation with TRFS-red (10 μM) and NADPH (0.2
mM) for additional 2 h. The fold of fluorescence increment (λ =615
ex
nm; λem=661 nm) was determined. Total protein concentration of
the lysate is 10 mg/mL determined by the Bradford procedure using
BSA as a standard.
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We then applied TRFS-red to image the enzyme activity in live
COMMUNICATION
Based on the proposed mechanism of selective recognition of
1
4
cells. Figure 3A shows imaging of TrxR activity in live HeLa cells. The TRFS-green by TrxR, we reasoned that TRFS-red follows the same
cells emit red signal after incubation with TRFS-red for 30 min. pathway: Firstly TrxR cleaved the disulfide bond in TRFS-red to
Extending incubation time gives stronger fluorescence. After 60 min, generate free thiols, and then the nascent thiolate attacks the
fluorescence signal appears reaching maximum. We further co- amide carbonyl carbon, yielding
a
five-membered cyclic
stained the cell nuclei with the Hoechst dye, and found that the red thiocarbonate and the fluorophore 5 (Scheme 2). To confirm this
fluorescence is predominantly distributed in the cytosol (Figure S3), process, we quantified the probe and fluorophore from the reaction
indicating TRFS-red is triggered on by the cytosolic TrxR (TrxR1). mixture by HPLC. Since TCEP has similar effect to react with the
Pretreatment of the cells with TrxR inhibitor AF or DNCB severely probe (Figure. S3), we incubated the probe with TCEP instead of the
suppresses the fluorescence (Figure. 3B), supporting selective enzyme to facilitate HPLC analysis. When incubation of TRFS-red (10
recognition of TrxR by TRFS-red in live cells. The relative μM) with TCEP (1 mM) in TE buffer for 3 h at 37 °C, the desiring
fluorescence intensity (RFI) in individual cells was quantified by the product 5 is the only one in the reaction mixture with ∼85%
ImageJ, and the data were listed under each picture. We next conversion based on TRFS-red (Table S1 and Figure. S5). At the time
compared the effects of TRFS-red with TRFS-green in imaging TrxR point of analysis, the remaining TRFS-red (1.44 μM) plus the
activity in live cells. Consistent with the in vitro observation, TRFS- product 5 (8.51μM) almost equals to the amount of the starting
red displays faster response and higher sensitivity than TRFS-green TRFS-red (10 μM), demonstrating that there is no side reactions and
in imaging cellular TrxR activity (Figure. 4). Treatment of the cells TRFS-red was exclusively converted to the fluorophore 5.
with TRFS-red (1 μM) for 1 h gives bright red fluorescence and the
fluorescence is stable for at least 4 h (Figure. 4B). Increasing the
probe concentration to 10 μM does not cause significant alteration
of the cellular fluorescence intensity. In contrast, it takes longer
time (4 h) for TRFS-green (10 μM) to give visible green fluorescence
(
Figure. 4A).
Figure 4 Imaging TrxR activity by TRFS-green (A) and TRFS-red (B).
Figure 3 Imaging TrxR activity in live cells. (A) HeLa cells treated HeLa cells were treated with varying concentrations of dyes for
with TRFS-red (1 μM) for the indicated times, and bright field (top different times, and the fluorescence images were acquired under
panel) and fluorescence (bottom panel) images were shown. (B) the same conditions. Scale bar: 20 μm.
HeLa ells were pretreated with TrxR inhibitors DNCB (20 μM) or AF
(
2 μM) for 3h, and then were further stained with TRFS-red (1μM)
for 60 min. Bright field (top panel) and fluorescence (bottom panel)
images were shown. Relative fluorescence intensity (RFI) in
individual cells was quantified by the ImageJ, and the values were
given under each fluorescence images. Scale bar: 20 μm.
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1
1
0. H. S. Jung, J. Han, J. H. Lee, J. H. Lee, J. M. Choi, H. S. Kweon, J.
H. Han, J. H. Kim, K. M. Byun, J. H. Jung, C. Kang and J. S. Kim,
J Am Chem Soc, 2015, 137, 3017-3023.
1. M. H. Lee, N. Park, C. Yi, J. H. Han, J. H. Hong, K. P. Kim, D. H.
Kang, J. L. Sessler, C. Kang and J. S. Kim, J Am Chem Soc, 2014,
1
36, 14136-14142.
2. P. Zhou, J. Yao, G. Hu and J. Fang, ACS Chem Biol, 2016, 11,
098-1105.
3. B. Zhang, C. Ge, J. Yao, Y. Liu, H. Xie and J. Fang, J Am Chem
Soc, 2015, 137, 757-769.
1
1
1
1
1
1
1
1
1
4. L. Zhang, D. Duan, Y. Liu, C. Ge, X. Cui, J. Sun and J. Fang, J
Am Chem Soc, 2014, 136, 226-233.
5. B. Liang, W. Shao, C. Zhu, G. Wen, X. Yue, R. Wang, J. Quan, J.
Du and X. Bu, ACS Chem Biol, 2016, 11, 425-434.
6. L. Huang, Y. Chen, B. Liang, B. Xing, G. Wen, S. Wang, X. Yue,
C. Zhu, J. Du and X. Bu, Chem Commun, 2014, 50, 6987-6990.
7. J. Zhang, Y. Li, D. Duan, J. Yao, K. Gao and J. Fang, Biochem
Pharmacol, 2016, 102, 34-44.
Scheme 2 Proposed mechanism for the activation of TRFS-red by
TrxR/NADPH.
8. D. Duan, J. Zhang, J. Yao, Y. Liu and J. Fang, J Biol Chem, 2016,
2
91, 10021-10031.
9. B. Zhang, D. Duan, C. Ge, J. Yao, Y. Liu, X. Li and J. Fang, J
Med Chem, 2015, 58, 1795-1805.
In summary, TRFS-red, the first red emission probe for TrxR, 20. J. Yao, B. Zhang, C. Ge, S. Peng and J. Fang, J Agric Food
Chem, 2015, 63, 1521-1531.
was prepared and evaluated. Compared to the parent probe TRFS-
2
1. S. Peng, B. Zhang, J. Yao, D. Duan and J. Fang, Food Funct,
015, 6, 2091-2100.
improved response rate and sensitivity. The discovery of TRFS-red 22. S. Peng, B. Zhang, X. Meng, J. Yao and J. Fang, J Med Chem,
green, TRFS-red maintains the high selectivity to TrxR but exhibits
2
2
015, 58, 5242-5255.
enriches toolbox for exploring TrxR function in living systems.
2
2
2
2
2
3. S. Peng, J. Yao, Y. Liu, D. Duan, X. Zhang and J. Fang, Food
Funct, 2015, 6, 2813-2823.
4. J. Yao, C. Ge, D. Duan, B. Zhang, X. Cui, S. Peng, Y. Liu and J.
Fang, J Agric Food Chem, 2014, 62, 5507-5518.
5. Y. Liu, D. Duan, J. Yao, B. Zhang, S. Peng, H. Ma, Y. Song and J.
Fang, J Med Chem, 2014, 57, 5203-5211.
6. Y. Liu, H. Ma, L. Zhang, Y. Cui, X. Liu and J. Fang, Chem
Commun, 2016, 52, 2296-2299.
7. E. J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti, K.
S. Hong and J. S. Kim, J Am Chem Soc, 2014, 136, 13888-
ACKNOWLEDGEMENTS
The financial support from the Natural Science Foundation of
China (21572093), the Natural Science Foundation of Gansu
Province (145RJZA225), and the Fundamental Research Funds for
the Central Universities (lzujbky-2016-49) is acknowledged. The
13894.
8. S. Maiti, N. Park, J. H. Han, H. M. Jeon, J. H. Lee, S. Bhuniya, C.
Kang and J. S. Kim, J Am Chem Soc, 2013, 135, 4567-4572.
2
authors also express appreciation to Prof. Arne Holmgren 29. T. Guo, L. Cui, J. Shen, W. Zhu, Y. Xu and X. Qian, Chem
(Karolinska Institute, Sweden) for the recombinant rat TrxR1. There
Commun, 2013, 49, 10820-10822.
3
3
0. J. Jose and K. Burgess, J Org Chem, 2006, 71, 7835-7839.
1. A. Gomes, E. Fernandes and J. L. Lima, J Biochem Biophys
Methods, 2005, 65, 45-80.
are no conflicts of interest.
3
3
2. J. Jose, Y. Ueno and K. burgess, Chem Eur J, 2009, 15, 418-
423.
3. W. Cai, L. Zhang, Y. Song, B. Wang, B. Zhang, X. Cui, G. Hu, Y.
Liu, J. Wu and J. Fang, Free Radic Biol Med, 2012, 52, 257-
Notes and references
1
2
3
4
5
6
7
8
9
. Y. Tang, X. Kong, A. Xu, B. Dong and W. Lin, Angew Chem Int
Ed Engl, 2016, 55, 3356-3359.
. X. Liu, B. Hu, R. Cheng, F. Kong, X. Pan, K. Xu and B. Tang,
Chem Commun, 2016, 52, 6693-6696.
. P. Li, L. Liu, H. Xiao, W. Zhang, L. Wang and B. Tang, J Am
Chem Soc, 2016, 138, 2893-2896.
. R. Hata, H. Nonaka, Y. Takakusagi, K. Ichikawa and S. Sando,
Angew Chem Int Ed Engl, 2016, 55, 1765-1768.
2
65.
. K. Gu, Y. Xu, H. Li, Z. Guo, S. Zhu, S. Zhu, P. Shi, T. D. James, H.
Tian and W. H. Zhu, J Am Chem Soc, 2016, 138, 5334-5340.
. Q. Cai, T. Yu, W. Zhu, Y. Xu and X. Qian, Chem Commun, 2015,
5
1, 14739-14741.
.
J. Miao, Y. Huo, X. Lv, Z. Li, H. Cao, H. Shi, Y. Shi and W. Guo,
Biomaterials, 2016, 78, 11-19.
. H. Zhang, J. Liu, Y. Q. Sun, Y. Huo, Y. Li, W. Liu, X. Wu, N. Zhu,
Y. Shi and W. Guo, Chem Commun, 2015, 51, 2721-2724.
. Y. H. Lee, W. X. Ren, J. Han, K. Sunwoo, J. Y. Lim, J. H. Kim
and J. S. Kim, Chem Commun, 2015, 51, 14401-14404.
4
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The first red emission probe, TRFS-red, of thioredoxin reductase
was reported with improved response rate and sensitivity.
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