A near-infrared fluorogenic probe for nuclear thiophenol detection

Article abstract of DOI:10.1039/d0cc07930h

Based on the thiolysis of dinitrophenyl ethers, a nucleus-targeted fluorescent probe,AQD, with bisalkylaminoanthraquinone as the fluorophore, was prepared for the near-infrared mapping of thiophenol in live cells, zebrafish, and mice.

Full text of DOI:10.1039/d0cc07930h

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A near-infrared fluorogenic probe for nuclear
thiophenol detection†
Cite this: Chem. Commun., 2021,
57, 2800
Jinye Niu,‡ab Yong Li,‡a Ran Zhang,a Jian Zhang,a Xu Wang,*a Xilei Xie,a
a
Xiaoyun Jiaoa and Bo Tang
*
Received 6th December 2020,
Accepted 27th January 2021
DOI: 10.1039/d0cc07930h
rsc.li/chemcomm
Based on the thiolysis of dinitrophenyl ethers, a nucleus-targeted efforts have been made to construct fluorescent probes for
fluorescent probe, AQD, with bisalkylaminoanthraquinone as the thiophenol monitoring with satisfactory response behaviors.7
fluorophore, was prepared for the near-infrared mapping of thio- However, challenges exist as none of the fluorescent probes
phenol in live cells, zebrafish, and mice.
reported so far were capable of accomplishing thiophenol specific
sensing in cellular nucleus.
Thiophenol is a crucial raw material and intermediate that has
Herein, based on the thiolysis of dinitrophenyl ethers,
been extensively employed in the manufacture of pharmaceuticals, we established a near-infrared bisalkylaminoanthraquinone–
agrochemicals, polymeric materials, and so on.1 However, in spite dinitrobenzene conjugate, AQD, for nuclear thiophenol detec-
of its broad synthetic utility, thiophenol is highly poisonous to tion with prominent sensitivity and specificity. By capitalizing
organisms,2 and has been evaluated as a No. P014 acute hazardous on outstanding response features of AQD, the nuclear thiophenol
pollutant by the united states environment protection agency.3 level fluctuations in HeLa cells, zebrafish, and mice were success-
Moreover, thiophenol can be easily absorbed in organisms via fully revealed. This work not only provided a potent probe for
inhalation and the skin, and the prolonged exposure to thiophenol thiophenol detection but also afforded information that may
tends to induce various human health issues, such as central nerve contribute to the intensive understanding of thiophenol cytotoxic
injury, nausea, muscle weakness, cough, and even death.4 The mechanism, especially that in cell nucleus.
cytotoxic mechanism study demonstrated that DNA damage has
The unique probe, AQD, was fabricated by integrating
been deeply involved in thiophenol-induced biotoxic events.5 dinitrophenyl groups into bisalkylaminoanthraquinone (AQ)
Therefore, considering that nucleus is the main subcellular loca- through an ether linker, in which the dinitrophenyl group
tion of the eukaryotic cell DNA, the fabrication of a superior was introduced both as a thiophenol trigger and a fluorescence
performance strategy for thiophenol tracing in cellular nucleus is quencher while AQ was employed as a near-infrared fluorescent
undoubtedly significant in the field of biotoxic evaluation of reporter and a nucleus-targeted unit.8 In the absence of thio-
thiophenol.
phenol, the fluorescence of AQD is supposed to be silenced by
Currently, fluorescence detection, especially, near-infrared the strong photoinduced electron transfer (PET) effect of the
imaging, is widely utilized to track various active molecules dinitrophenyl group. Upon thiophenol exposure, the ether
in vitro and in vivo by virtue of its unique spatiotemporal bond would be first cut off, and then the PET effect relieves,
resolution, noninvasive capacity, and so on.6 Accordingly, much thus switching on the fluorescence of AQD. Simultaneously, the
weakened intramolecular charge transfer (ICT) induced by an
electron-withdrawing dinitrophenyl group would eliminate
upon thiophenol recognition (Scheme 1). AQD was readily
a College of Chemistry, Chemical Engineering and Materials Science,
Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in
prepared and its structure was well characterized by high
Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry
1
resolution mass spectrometry (HRMS), H NMR, and 13C NMR
of Education, Shandong Provincial Key Laboratory of Clean Production of Fine
analysis (ESI†).
Chemicals, Shandong Normal University, Jinan 250014, P. R. China.
E-mail: tangb@sdnu.edu.cn, wangxu@sdnu.edu.cn
With AQD in hand, its spectral response towards thiophenol
was first investigated. After thiophenol incubation, an obvious
bathochromic-shift (60 nm) of the maximum absorption band
was obtained, thus exhibiting the effective ICT recovery caused
by the thiolysis of the dinitrophenyl group (Fig. S1, ESI†).
Concomitantly, the distinct colorimetric change (purple to blue)
b School of Chemical Engineering, Shandong University of Technology, Zibo 255049,
P. R. China
† Electronic supplementary information (ESI) available: Experimental details,
supplementary data, synthesis and characterization of compounds. See DOI:
10.1039/d0cc07930h
‡ These authors contributed equally to this work.
2800 | Chem. Commun., 2021, 57, 2800À2803
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Scheme 1 Chemical structure of AQD and the proposed response
mechanism for thiophenol.
Fig. 2 The specificity of AQD (10 mM) towards various bioanalytes: 1.
Blank; 2. Cys; 3. GSH; 4. Hcy; 5. HSÀ; 6. HSO3À; 7 Vc.; 8. ONOOÀ; 9. NO;
À
1
10. H2O2; 11. ClO ; 12. O2; 13. ꢀOH; 14. O2 À; 15. Na+; 16. K+; 17. Cd2+; 18.
ꢀ
in Fig. S1 (ESI†) hinted AQD’s naked-eye response capacity
toward thiophenol. Meanwhile, as shown in Fig. 1, the treatment
of AQD with an equivalent of thiophenol displayed a dramatic
fluorescence increment (39 folds) with the maximum emission
band at around 680 nm, presenting effective PET effect elimina-
tion. In addition, the fluorescence intensity of AQD showed
Pd2+; 19. Co2+; 20. Mg2+; 21. Hg2+; 22. Ca2+; 23. Zn2+; 24. Cu2+; 25. Fe2+
;
26. Al3+; 27. Fe3+; 28. Thiophenol (10 mM). The concentrations of GSH, Hcy,
and Cys were 1.0 mM, the concentrations of metal ions were 500 mM, and
the concentrations of the other substances were 100 mM unless otherwise
stated. The results were recorded after incubation of AQD with various
substances for 60 min in HEPES buffer (20 mM, pH 7.4, 30% DMSO) at
a
concentration-dependent augmentation towards thiophenol 37 1C. lex/lem = 600/680 nm. Slit width: 5 nm/5 nm.
with a linear relationship of y = 49.5221 + 427.5985x ([thiophenol]
= 0–3.0 mM, R2 = 0.993) and y = 1057.6457 + 67.7261x ([thiophenol]
= 3.0–10 mM, R2 = 0.991), respectively. And the limit of detection was
calculated as 15 nM based on 3s/k. Meanwhile, the time-dependent
fluorescence variation of AQD with reaction time was assessed
(Fig. S2, ESI†). After exposure to an equivalent of thiophenol, the
fluorescence intensity of AQD displayed a sharp enhancement
(35 folds) within 12 min, and then fully saturated (39 folds) after
60 min. Taken together, these results suggested that AQD was
capable of tracking thiophenol with high sensitivity and reaction
activity.
The proposed reaction mechanism between AQD and thio-
phenol was explored via HRMS. Upon thiophenol incubation,
the AQD solution showed a discriminable mass peak at m/z
413.2162 [M + H]+, which was in good agreement with com-
pound AQ (Fig. S3, ESI†). This observation presented high
consistence with the proposed thiophenol reaction pathway
as shown in Scheme 1.
was probably due to the fact that fluorophore AQ decreased the
electrophilicity of the 2,4-dinitrophenyl ether unit and thus
AQD could be only attacked by the stronger nucleophile
thiophenol.9 In addition, negligible fluorescence enhancement
was observed after treatment of AQD with other common active
substances, including reactive nitrogen and reactive oxygen
species (ONOOÀ, NO, H2O2, ClOÀ, 1O2, ꢀOH, and O2 À), and
ꢀ
metal ions (Na+, K+, Cd2+, Pd2+, Co2+, Mg2+, Hg2+, Ca2+, Zn2+,
Cu2+, Fe2+, Al3+, and Fe3+), thus implying the high specificity of
AQD towards thiophenol. After that, the effect of pH
on fluorescence response behavior of AQD was examined
(Fig. S4, ESI†). It can be found that AQD itself showed weak
and constant fluorescence emission at the wide pH range
3.0–10.0. In the presence of thiophenol, strong fluorescent
signals were detected in the pH range 5.0–8.0, demonstrating
that AQD can work effectively in biological specimen.
Encouraged by AQD’s outstanding thiophenol recognition
behaviors, the imaging profile of AQD in the living system was
exploited next. Prior to cell imaging, the high biocompatibility
(IC50 = 207 mM) of AQD was well verified through MTT assays
(Fig. S5, ESI†). Subsequently, the time-dependent thiophenol
imaging in living cells was performed. The HeLa cells were
pretreated with thiophenol followed by staining with AQD and
then imaged immediately. With the passage of incubation time,
the increased fluorescence intensity appeared gradually and
significantly, and reached the maximum at 40 min (Fig. 3 and
Fig. S6, ESI†), thus displaying that AQD can effectively capture
thiophenol in the living system within 40 min. After that, the
thiophenol dose-dependent fluorescence changes were
explored. As can be seen from Fig. 4, the HeLa cells loaded
with AQD alone emitted a negligible fluorescence signal, hinting
that there is no thiophenol in living cells. Contrastingly, a
remarkable increased fluorescence in cells appeared upon thio-
phenol administration. And the increment in fluorescence inten-
sity displayed a positive relation with the dose of thiophenol.
Furthermore, the cells co-stained with AQD and DAPI, a com-
mercially available nuclear marker, led to an excellent colocali-
zation with a high Pearson’s correlation of 0.91 (Fig. S7, ESI†),
The specificity of AQD towards thiophenol was estimated
(Fig. 2). Satisfactorily, no obvious fluorescence intensity change
was triggered when AQD was incubated with a panel of reactive
sulfur species, such as cysteine (Cys), glutathione (GSH), homo-
cysteine (Hcy), hydrogen sulfide (H2S), and bisulfite (HSO3À).
The specificity of AQD towards thiophenol over other biothiols
Fig. 1 (a) Fluorescence spectra variation of AQD (10 mM) with the addition
of increasing amount of thiophenol (0–10 mM). (b) Linear correlation
between the fluorescence intensity of AQD at 680 nm and thiophenol
concentration. The results were recorded after incubation of AQD with
thiophenol for 60 min in HEPES buffer (20 mM, pH 7.4, 30% DMSO) at
37 1C. lex = 600 nm. Slit width: 5 nm/5 nm.
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Fig. 5 Confocal fluorescence imaging of thiophenol in zebrafish. (a and d)
The zebrafish was incubated with AQD (10 mM) only for 60 min; (b and e)
the zebrafish was pretreated with thiophenol (5 mM) for 15 min, and then
incubated with AQD for 60 min; (c and f) the zebrafish was administrated
with thiophenol (10 mM) for 15 min and further stained with AQD for
60 min. (g) The relative fluorescence intensity of (a–c). The images were
recorded with 633 nm excitation and 640–730 nm collection. The values
are the mean Æ s.d. for n = 3 and ***p o 0.001. Scale bar = 250 mm.
Fig. 3 Time-dependent fluorescence imaging of AQD in HeLa cells.
(a) The cells were pretreated with thiophenol (10 mM) for 15 min and then
incubated with AQD (10 mM) followed by imaging at different time points
(0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 min); (b) the relative fluorescence
intensity of (a). The images were recorded with 633 nm excitation and
640–730 nm collection. Scale bar = 20 mm.
Fig. 6 Fluorescence imaging of thiophenol in nude mice. The mice were
injected intraperitoneally with various substances (200 mL): (a) AQD (50 mM,
60 min) only; (b) thiophenol (20 mM, 15 min) and then AQD (50 mM,
60 min); (c) thiophenol (40 mM, 15 min) and then AQD (50 mM, 60 min);
(d) fluorescence intensity readout of red circle in (a–c). The excitation filter
was 600 nm, and the emission filter was 670 nm.
Fig. 4 Confocal fluorescence imaging of thiophenol in HeLa cells. (a and d)
The cells were incubated with AQD (10 mM) only for 60 min; (b and e) the
cells were pretreated with thiophenol (5 mM) for 15 min, and then
incubated with AQD for 60 min; (c and f) the cells were pretreated with
thiophenol (10 mM) for 15 min and further stained with AQD for 60 min.
(g) The relative fluorescence intensity of (a–c). The images were recorded
with 633 nm excitation and 640–730 nm collection. The values are the
mean Æ s.d. for n = 3, **p o0.01, and ***p o 0.001. Scale bar = 20 mm.
observed in mice treated with AQD only. When pre-administrated
thiophenol, a notable fluorescent signal increase in a dose-
dependent manner was gained, demonstrating that AQD is
competent to differentiate variations of the thiophenol level in
thus establishing that AQD accumulated specifically in the mice. Overall, it is credible to draw the conclusion that AQD is
nucleus. Taken together, these solid results evidently indicated capable of monitoring thiophenol fluctuations in vivo.
that AQD can be employed as a robust tool for nuclear thiophenol
In conclusion, we rationally designed and synthesized a
fluctuation mapping.
specific fluorescent sensor, AQD, for thiophenol tracking in
Having well-established the thiophenol imaging capability nucleus via integrating the dinitrophenyl group into AQ. Based
of AQD in living cells, an attempt was made to reveal the on the effective thiolysis of dinitrophenyl ether, AQD was
feasibility of visualizing thiophenol fluctuation in vivo. Firstly, capable of capturing thiophenol with outstanding sensitivity
the imaging assays in zebrafish were performed. The zebrafish and specificity upon PET effect elimination and ICT process
were pretreated with various concentrations of thiophenol and recovery. Furthermore, the near-infrared activatable merit of
then imaged with AQD. As shown in Fig. 5, upon thiophenol AQD endowed its superior fluorescence imaging behaviors
administration, the zebrafish exhibited much brighter fluo- in vitro and in vivo. Therefore, we expect that AQD will be a
rescence than that imaged in the control group, which was potent candidate for thiophenol research in other physiopatho-
consistent with AQD’s fluorescence response behaviors logical contexts.
observed in the cells. Afterward, the imaging ability of AQD
This work was supported by the National Natural Science
in mice was investigated. The mice were successively given Foundation of China (91753111, 21927811, 21775093, 21877076,
intraperitoneal injection of thiophenol and AQD. As illustrated and 21907060) and the Key Research and Development Program
in Fig. 6, almost no distinguishable fluorescence signal was of Shandong Province (2018YFJH0502). All animal experiments
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2020, 11, 5127–5141; (c) J. Mei, N. L. Leung, R. T. Kwok, J. W. Lam and
B. Z. Tang, Chem. Rev., 2015, 115, 11718–11940; (d) Y. Tang, Y. Ma,
J. Yin and W. Lin, Chem. Soc. Rev., 2019, 48, 4036–4048; (e) J. Ohata,
K. J. Bruemmer and C. J. Chang, Acc. Chem. Res., 2019, 52, 2841–2848;
( f ) J. Zheng, R. Yang, M. Shi, C. Wu, X. Fang, Y. Li, J. Li and W. Tan,
Chem. Soc. Rev., 2015, 44, 3036–3055; (g) J. A. Robson, M. Kubankova,
T. Bond, R. A. Hendley, A. J. P. White, M. K. Kuimova and J. Wilton-
Ely, Angew. Chem., Int. Ed., 2020, 59, 21431–21435; (h) J. Niu, J. Fan,
X. Wang, Y. Xiao, X. Xie, X. Jiao, C. Sun and B. Tang, Anal. Chem.,
2017, 89, 7210–7215; (i) Y. Li, Y. Shu, M. Liang, X. Xie, X. Jiao,
X. Wang and B. Tang, Angew. Chem., Int. Ed., 2018, 57, 12415–12419.
7 (a) W. Jiang, Q. Fu, H. Fan, J. Ho and W. Wang, Angew. Chem., Int. Ed.,
2007, 46, 8445–8448; (b) J. Hong, Q. Xia, W. Feng and G. Feng, Dyes
Pigm., 2018, 159, 604–609; (c) Q. Zhai, S. Yang, Y. Fang, H. Zhang and
G. Feng, RSC Adv., 2015, 5, 94216–94221; (d) H. Ren, F. Huo,
Y. Huang, J. Chao, W. Wen, J. Fan and C. Yin, Dyes Pigm., 2020,
181, 108567; (e) Z. Li, C. Liu, C. Yu, R. Yuan, P. Jia, Z. Wang, Q. Duan,
H. Zhu, B. Zhu and W. Sheng, New J. Chem., 2019, 43, 6746–6752;
( f ) H. W. Liu, X. B. Zhang, J. Zhang, Q. Q. Wang, X. X. Hu, P. Wang
and W. Tan, Anal. Chem., 2015, 87, 8896–8903; (g) Q. Liu, A. Li, X. Li,
B. Li, Y. Zhang, J. Li and Y. Guo, Sens. Actuators, B, 2019, 283,
820–830; (h) W. Lin, L. Long and W. Tan, Chem. Commun., 2010,
46, 1503–1505; (i) W. Jiang, Y. Cao, Y. Liu and W. Wang, Chem.
Commun., 2010, 46, 1944–1946.
were approved by the Animal Care and Use Committee of
Shandong Normal University, and were performed in compliance
with the Animal Management Rules of the Ministry of Health of
the People’s Republic of China and the Guidelines for the Care
and Use of Laboratory Animals of Shandong Normal University.
Conflicts of interest
There are no conflicts to declare.
Notes and references
1 (a) J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo and
G. M. Whitesides, Chem. Rev., 2005, 105, 1103–1170; (b) X. Chen,
Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120–2135.
2 (a) F. Wu, H. Wang, J. Xu, H.-Q. Yuan, L. Zeng and G.-M. Bao, Sens.
Actuators, B, 2018, 254, 21–29; (b) R. Munday, Free Radical Biol. Med.,
1989, 7, 659–673.
3 G. J. Hathaway and N. H. Proctor, Proctor and Hughes’ chemical
hazards of the workplace, John Wiley & Sons, 2014.
4 (a) P. Amrolia, S. G. Sullivan, A. Stern and R. Munday, J. Appl. Toxicol.,
1989, 9, 113–118; (b) T. R. Juneja, R. L. Gupta and S. Samanta, Toxicol.
Lett., 1984, 21, 185–189; (c) R. Munday, J. Appl. Toxicol., 1985, 5,
409–413; (d) M. M. Villalba, V. J. Litchfield, R. B. Smith,
A. M. Franklin, N. S. Lawrence and J. Davis, J. Hazard. Mater., 2008,
154, 444–450.
5 R. P. Verma, S. Kapur, O. Barberena, A. Shusterman, C. H. Hansch
and C. D. Selassie, Chem. Res. Toxicol., 2003, 16, 276–284.
6 (a) X. Jiao, Y. Li, J. Niu, X. Xie, X. Wang and B. Tang, Anal. Chem.,
2017, 90, 533–555; (b) M. Yang, J. Fan, J. Du and X. Peng, Chem. Sci.,
8 (a) K. L. Njoh, L. H. Patterson, M. Zloh, M. Wiltshire, J. Fisher,
S. Chappell, S. Ameer-Beg, Y. Bai, D. Matthews, R. J. Errington and
P. J. Smith, Cytometry, Part A, 2006, 69, 805–814; (b) H. D. Ou, S. Phan,
T. J. Deerinck, A. Thor, M. H. Ellisman and C. C. O’Shea, Science,
2017, 357, 13; (c) V. J. Pansare, S. Hejazi, W. J. Faenza and
R. K. Prud’homme, Chem. Mater., 2012, 24, 812–827.
9 (a) Y. Hao, Q. Yin, Y. Zhang, M. Xu and S. Chen, Molecules, 2019,
24, 3716; (b) Z. Wang, D. M. Han, W. P. Jia, Q. Z. Zhou and
W. P. Deng, Anal. Chem., 2012, 84, 4915–4920.
This journal is The Royal Society of Chemistry 2021
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