An oxidative cyclization reaction based fluorescent “Turn-On” probe for highly selective and rapid detection of hypochlorous acid

Article abstract of DOI:10.1016/j.tetlet.2018.02.018

A novel fluorescent turn-on probe, PHC1, for the detection of hypochlorous acid has been developed based on hypochlorous acid-initiated oxidative intramolecular cyclization. Endowed by the fast reaction rate, PHC1 features rapid detection kinetics, as wel

Full text of DOI:10.1016/j.tetlet.2018.02.018

Accepted Manuscript
An Oxidative Cyclization Reaction Based Fluorescent “Turn-On” Probe for
Highly Selective and Rapid Detection of Hypochlorous Acid
Bo Chen, Hongyu Fu, Yaohong Lv, Xin Li, Yifeng Han
PII:
S0040-4039(18)30185-0
https://doi.org/10.1016/j.tetlet.2018.02.018
TETL 49703
DOI:
Reference:
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15 November 2017
6 February 2018
7 February 2018
Please cite this article as: Chen, B., Fu, H., Lv, Y., Li, X., Han, Y., An Oxidative Cyclization Reaction Based
Fluorescent “Turn-On” Probe for Highly Selective and Rapid Detection of Hypochlorous Acid, Tetrahedron
Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.02.018
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An Oxidative Cyclization Reaction Based
Fluorescent “Turn-On” Probe for Highly
Selective and Rapid Detection of
Hypochlorous Acid
Leave this area blank for abstract info.
Bo Chen, Hongyu Fu, Yaohong Lv, Xin Li,* and Yifeng Han*

1
Tetrahedron Letters
jo u r n a l h o m e p a g e : w w w . e ls e v ie r . c o m
An Oxidative Cyclization Reaction Based Fluorescent “Turn-On” Probe for Highly
Selective and Rapid Detection of Hypochlorous Acid
Bo Chen, Hongyu Fu, Yaohong Lv, Xin Li,* and Yifeng Han*
Department of Chemistry, Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University,
Hangzhou, 310018, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel fluorescent turn-on probe, PHC1, for the detection of hypochlorous acid has been
developed based on hypochlorous acid-initiated oxidative intramolecular cyclization. Endowed
by the fast reaction rate, PHC1 features rapid detection kinetics, as well as high degree of
selectivity and sensitivity for HClO.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probe
hypochlorous acid
Reaction-based
Oxidative Cyclization
BODIPY
1. Introduction
Hypochlorous acid (HClO),
a
well-known antiseptic
germicidal agent, is widely employed in our daily life, such as
disinfectant of public water supplies, household bleach, and as an
antimicrobial.1 In particular, it is also an important member of the
ROS (reactive oxygen species) family and plays crucial roles as a
potent microbicidal agent in the immune-defense system.2
Endogenous HClO is produced mainly from the peroxidation
reaction of hydrogen peroxide (H2O2) and chloride ions (Cl-)
catalyzed by the enzyme myeloperoxidase (MPO) mostly in
leukocytes
(including
neutrophils,
macrophages,
and
monocytes).3 However, the imbalanced homeostasis of HClO
was believed to cause oxidative injuries to DNA, proteins, and
lipids, which consequently led to many pathological states, such
as cardiovascular diseases, arthritis, atherosclerosis, nephropathy,
neuron degeneration and even cancer.4 Accordingly, it is strongly
desirable to develop new analytical methods for the sensitive and
accurate detection and monitoring of HClO.
Figure 1. Some reported HClO probes.
Many efforts have been focused on the studies of the
biological functions of HClO, and a number of analytical
methods such as potentiometry, electroanalysis, and
chemiluminescence have been developed.5 In comparison with
conventional methods for HClO, the non-invasive fluorescent
probe technique displays apparent advantages as exemplified by
their ease of application both in solution and in living organism
as well as their high sensitivity and selectivity for trace analytes
with spatial and temporal resolution.6
To date, a number of fluorescent probes have been synthesized
and applied to detect HClO, most of which were designed by
taking advantage of the strong oxidation properties of HClO.
Functional groups, such as p-methoxyphenol, p-alkoxyaniline,
oxime, hydroxamic acid, dibenzoylhydrazine, thiol, and selenide,
due to their tendency of being easily oxidized by HClO, have
been employed as reporters in fluorescent probe design.7
However, most of these probes are limited by pH dependency,
low sensitivity, or sluggish detection kinetics, compromising
their applicability for real-time detection of HClO. Thus, the
development of novel fluorescent probes with high sensitivity,

2
Tetrahedron
excellent selectivity, and real-time capability to measure HClO
Figure 2. UV-vis absorption spectra of PHC1 (5.0 μM) in
still arouses much attention.
HEPES buffer solution (10 mM, pH 7.4, containing 50% EtOH)
in the presence of different concentrations of HClO (0-15.0
equiv.). Inset: cuvette images of probe PHC1 before and after
addition of HClO.
Isomerization of the imine C=N bond is now known to be the
main non-radiative decay process of excited states in fluorogenic
compounds with an unbridged C=N structure, leading to the
Scheme 1. The strategy for the design of PHC1.
Figure 3. Fluorescence spectra of PHC1 (5.0 μM) in HEPES
buffer solution (10 mM, pH 7.4, containing 50% EtOH) in the
presence of different concentrations of HClO (0-80.0 equiv.) (λex
= 485 nm). Inset 1: the fluorescent intensity at 508 nm as a
function of HClO concentration. Inset 2: cuvette images of probe
PHC1 before and after addition of HClO taken under a hand-held
UV-lamp (λex = 365 nm).
knowledge, this is the first hypochlorous acid initiated oxidative
intramolecular cyclization based HClO fluorescent probe.
Scheme 2. Synthesis of PHC1: (a) 1) TFA, dry DCM, rt, 10 h; 2)
DDQ, rt, 4h; 3) Et3N, BF3.OEt2, rt, 2h, 20.1% over three steps; (b)
1) dry DMF, POCl3, 1, 2-dichloroethane, rt to 50 oC, 3h; 2) satd.
NaHCO3, 0.5 h, 82.3% over two steps; (c) diaminomaleonitrile,
MeOH, conc. H2SO4, rt, 2 h, 97.1%; (d) EtOH, HClO, 0oC, 5
min, 75.2%.
compounds weak-fluorescence or even non-fluorescence.8
However, the fluorescence intensity could be enhanced by
removal of the unbridged imine C=N bond or blockage of its
bond rotation by the formation of bridged C=N structure.8
Inspired by these works, we report here a novel boron-
dipyrromethene (BODIPY) based fluorescence turn-on probe,
2. Results and discussions
As shown in Scheme 2, PHC1 can be readily prepared in
three convenient steps under facile conditions with high yield
starting with commercially available benzaldehyde and 2, 4-
1
dimethylpyrrole. The product (PHC1) was characterized by H,
13C NMR, and HR-MS (ESI†).
With PHC1 in hand, we firstly assessed its UV-vis
spectroscopic properties in HEPES buffer solution (10 mM, pH
7.4, containing 50% EtOH). PHC1 (5.0 μM) displayed two
moderate UV-vis absorption bands around 375 and 530 nm,
respectively. Upon incremental addition of HClO (0-15.0 equiv.),
both the peaks at 375and 560 nm dramatically decreased, and a
new band at 503 nm, which is characteristic for the BODIPY
fluorophore emerged simultaneously with clear isosbestic points
at 445 and 523nm respectively, which is in concert with the
observation by naked eyes that the solution color changed from
pink to red, indicating that compound 4 was formed in the
presence of HClO (Fig. 2 and Scheme 1). Furthermore, a good
linear relationship (R2 = 0.99) was observed between the ratios of
absorbance at 560 and 503 nm (Fig. S1, ESI†).
PHC1, for the detection of HClO. The probe is
a
diaminomaleonitrile derivative of BODIPY (see Scheme 1).
We envisioned that the rapid isomerization of the imine moiety
in the excited state would repress the fluorescence emission of
PHC1. However, HClO promoted intramolecular oxidative
cyclization of γ-aminoimine would block its bond rotation and in
turn restore the strong fluorescence of PHC1. To the best of our
The emission spectra of PHC1 and its fluorescent titration
with HClO were recorded in HEPES buffer solution (10 mM, pH
7.4, containing 50% EtOH) (Fig. 3 and S2, ESI†). As expected,
PHC1 alone is almost non-fluorescence (λex = 485 nm, Φ =
0.029, Table S1, ESI†) due to the rapid isomerization of the
imine moiety in the excited state (Scheme 1). However, upon
progressive addition of HClO (0-80.0 equiv.), the emission band
at 508 nm appeared instantly with an obvious yellowish-green
color emission (Fig. 3),which is attributed to the formation of
substituted imidazole ring by the HClO-induced intramolecular
oxidative cyclization of γ-aminoimine to compound 4 (Fig. 3 and

3
Scheme 1). Moreover, the fluorescence titration curve revealed
that the fluorescence intensity at 508 nm increased linearly with
increasing concentrations of HClO in the range of 0-50.0 equiv.
(R2 = 0.9936) (Fig. S3, ESI†) and further smoothly increased
until a maximum was reached up to 250 μM HClO (λex = 485 nm,
λem = 508 nm, Φ = 0.383, Table S1, ESI†). Based on these
results, the detection limit of PHC1 for HClO was calculated to
be 1.18
Figure 5. Fluorescence responses of PHC1 (5.0 μM) to various
reactive oxygen species, reactive nitrogen species (RNS), and
common coexisting ions (including ·OH, 1O2, Fe3+, H2O2,
ONOO-, BuOOH, NO, and HClO) at 250.0 μM. Bars represent
t
the emission intensity of PHC1 at 508 nm after addition of each
analyte in HEPES buffer solution (10 mM, pH 7.4, containing
50% EtOH) (λex = 485 nm).
Scheme 3. The proposed mechanism of PHC1-HClO
interactions.
Moreover, the influence of pH on the detection assay was
investigated. As depicted in Fig. S7-S8, ESI†, the fluorescence of
PHC1 alone was inert to pH in the range of 6.5-9.5. On the other
hand, it still responded to HClO sensitively within this pH range
(6.5-9.5). These results indicate that PHC1 could be used in
neutral natural systems, or a mildly acidic or basic environment.
Figure 4. Time-dependent fluorescence intensity changes of
PHC1 (5.0 μM) upon addition of various concentration of HClO
(0, 20, 35, and 50 equiv., respectively) in HEPES buffer solution
(10 mM, pH 7.4, containing 50% EtOH) (λex = 485 nm).
 10-7 M, which reveals the high sensitivity for the analysis of
HClO.
The plausible mechanism of the HClO-induced fluorescence
response was shown in Scheme 3. Chlorination at α-nitrogen of
imine group by HClO leads to the formation of active
intermediate 5,7w which is then followed by the base induced 1,
Subsequently, the time-dependence of PHC1 fluorescence was
evaluated in the presence of HClO in HEPES buffer solution (10
mM, pH 7.4, containing 50% EtOH) (Fig. 4 and S4, ESI†).The
results showed that no change in fluorescence were detected in
the absence of HClO. On the other hand, the fluorescence of
tested solutions (in the presence of 20, 35, and 50 equiv. of
HClO, respectively) remarkably increased to their maximum
value within 15 seconds (Fig. S4, ESI†), which indicated that
PHC1 possesses a real-time capability to measure HClO.
2-elimination reaction. The intermediate
6 undergoes a
nucleophilic intramolecular cyclization reaction to form
substituted imidazole structure 7, which, after deprotonation by
water transformed to the final product 4.
To make further confirmation on this proposed mechanism, 1H
NMR titration experiment was conducted to clarify the actual
PHC1-HClO interaction. As shown in Fig. 6, upon incremental
addition of HClO to compound 3 in d6-DMSO, the aromatic
proton at δ 6.36 ppm attributed to H-6 and the imine proton at δ
8.21 ppm attributed to H-9 were all decreased significantly, while
the proton at δ 6.16 ppm assigned to H-6' of compound 4 was
increased gradually. This result indicated that the substituted
imidazole structure 4 was generated in the PHC1-HClO system.
Continuing on, the fluorescence titration of PHC1 towards
1
t
various reactive oxygen species (·OH, O2, H2O2, and BuOOH),
reactive nitrogen species (ONOO-, and NO), and cations
commonly found in the biological systems was conducted to
examine the selectivity (Fig. 5 and S5-S6, ESI†). Much to our
delight, the tested analytes caused little change to the
fluorescence profile of PHC1. It is noteworthy that PHC1 still
responds to HClO sensitively even in the presence of other
relevant competing species. Therefore, these results suggest that
PHC1 displays high selectivity toward HClO.
1
Moreover, the H, 13C NMR spectra and HR-MS of the isolated
product of the PHC1-HClO solution were also measured to
support the HClO-induced intramolecular oxidative cyclization
of PHC1 (see ESI†).

4
Tetrahedron
Y.; Tang, B. Z. Chem. Soc. Rev., 2015, 44, 4228-4238; (f) Yin, J.;
Hu, Y.; Yoon, J. Chem. Soc. Rev., 2015, 44, 4619-4644. (g) Kaur,
M.; Choi, D. H. Chem. Soc. Rev., 2015, 44, 58-77; (h) Guo, Z.;
Park, S.; Yoon, J.; Shin, I. Chem. Soc. Rev., 2014, 43, 16-29. (i)
Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev.,
2013, 42, 622-661. (j) Fan, J.; Hu, M.; Zhan, P.; Peng, X. Chem.
Soc. Rev., 2013, 42, 29-43.
1
Figure 6. H NMR titration experiment of PHC1 (compound 3)
in the presence of different concentrations of HClO (0-6.0
equiv.), and compound 4 (control) in d6-DMSO.
7. (a) Cheng, X.; Jia, H.; Long, T.; Feng, J.; Qin, J.; Li, Z. Chem.
Commun., 2011, 47, 11978-11980. (b) Wang, B.; Li, P.; Yu, F.;
Song, P.; Sun, X.; Yang, S.; Lou, Z.; Han, K. Chem. Commun.,
2013, 49, 1014-1016. (c) Liu, S.; Wu, S. Org. Lett., 2013, 15, 878-
881. (d) Liu, S.; Vedamalai, M.; Wu, S. Anal. Chim. Acta, 2013,
800, 71-76. (e) Xu, Q.; Lee, K. -A.; Lee, S.; Lee, K. M.; Lee, W.-
J.; Yoon, J. J. Am. Chem. Soc., 2013, 135, 9944-9949. (f)
Emrullahoglu, M.; Ucuncu, M.; Karakus, E. Chem. Commun.,
2013, 49, 7836-7838. (g) Cheng, G.; Fan, J.; Sun, W.; Cao, J.; Hu,
C.; Peng, X. Chem. Commun., 2014, 50, 1018-1020. (h) Hu, J. J.;
Wong, N. -K.; Gu, Q.; Bai, X.; Ye, S.; Yang, D. Org. Lett., 2014,
16, 3544-3547. (i) Yu, S.; Hsu, C.; Chen, W.; Wei, L.; Wu, S.
Sens. Actuators B, 2014, 196, 203-207. (j) Li, J.; Huo, F.; Yin, C.
RSC Adv., 2014, 4, 44610-44613. (k) Liu, F.; Gao, Y.; Wang, J.;
Sun, S. Analyst, 2014, 139, 3324-3329. (l) Kim, J.; Kim, Y.
Analyst, 2014, 139, 2986-2989. (m) Zhu, H.; Fan, J.; Wang, J.;
Mu, H.; Peng, X. J. Am. Chem. Soc., 2014, 136, 12820-12823. (n)
Goswami, S.; Das, A. K.; Manna, A.; Maity, A. K.; Saha, P.;
Quah, C. K.; Fun, H.; Abdel-Aziz, H. A. Anal. Chem., 2014, 86,
6315-6322. (o) Hou, J.; Wu, M.; Li, K.; Yang, J.; Yu, K.; Xie, Y.;
Yu, X. Chem. Commun., 2014, 50, 8640-8643. (p) Reja, S. I.;
Bhalla, V.; Sharma, A.; Kaur, G.; Kumar, M. Chem. Commun.,
2014, 50, 11911-11914. (q) Zhang, Y.; Chen, X.; Shao, J.; Zhang,
J.; Yuan, Q.; Miao, J.; Zhao, B. Chem. Commun., 2014, 50,
14241-14244. (r) Zhao, J.; Li, H.; Yang, K.; Sun, S.; Lu, A.; Xu,
Y. New J. Chem., 2014, 38, 3371-3374. (s) Venkatesan, P.; Wu, S.
Analyst, 2015, 140, 1349-1355. (t) Manna, A.; Goswami, S. New
J. Chem., 2015, 39, 4424-4429. (u) Xiao, H.; Xin, K.; Dou, H.;
Yin, G.; Quan, Y.; Wang, R. Chem. Commun., 2015, 51, 1442-
1445. (v) Ding, S.; Zhang, Q.; Xue, S.; Feng, G. Analyst, 2015,
140, 4687-4693. (w) Chen, W.; Venkatesan, P.; Wu, S. Anal.
Chim. Acta, 2015, 882, 68-75. (x) Chen, W.; Venkatesan, P.; Wu,
S. New J.Chem., 2015, 39, 6892-6898. (y) Tian, F.; Jia, Y.; Zhang,
Y.; Song, W.; Zhao, G.; Qu, Z.; Li, C.; Chen, Y.; Li, P. Biosensors
and Bioelectronics, 2016, 86; 68-74. (z) Xing, P.; Gao, K.; Wang,
B.; Gao, J.; Yan, H.; Wen, J.; Li, W.; Xu, Y.; Li, H.; Chen, J.;
Wang, W.; Sun, S. Chem. Commun., 2016, 52, 5064-5066. (aa)
Cui, K.; Zhang, D.; Zhang, G.; Zhu, D. Tetrahedron Lett., 2010,
51, 6052-6055.
3. Conclusions
In conclusion, we have rationally developed a novel BODIPY-
based sensitive fluorescence probe, PHC1, for the selective
detection of HClO via HClO-induced intramolecular oxidative
cyclization reaction. Based on this specific reaction, the probe
displayed a specific fluorescence response towards HClO under
mild conditions with a low detection limit (1.18  10-7 M). We
anticipate that the experimental results of this study will inspire
the future design of ROS sensors for a variety of chemical and
biological applications.
Acknowledgments
This work was supported by Zhejiang Provincial Natural
Science Foundation of China (Grant No. LY17B070008),
Zhejiang Provincial Top Key Academic Discipline of Chemical
Engineering and Technology, Zhejiang Sci-Tech University
(YR2015006), the Graduate Innovative Research Program of
Zhejiang Sci-Tech University (YCX15001), and the Project
Grants 521 Talents Cultivation of Zhejiang Sci-Tech University.
References and notes
1. (a) Aokl, T.; Munemorl, M. Anal. Chem., 1983, 55, 209-212. (b)
Hidalgo, E.; Bartolome, R.; Dominguez, C. Chem.-Biol. Interact.,
2002, 139, 265-282. (c) Pattison, D. I.; Davies, M. J.
Biochemistry, 2005, 44, 7378-7387. (d) Rutala, W. A.; Weber, D.
J. Clin. Microbiol. Rev., 1997, 10, 597-610.
2. (a) Steinbeck, M. J.; Nesti, L. J.; Sharkey, P. F.; Parvizi, J. J.
Orthop. Res., 2007, 25, 1128-1135. (b) Pattison, D. I.; Davies, M.
J. Chem. Res. Toxicol., 2001, 14, 1453-1464. (c) Weitzman, S. A.;
Gordon, L. I. Blood, 1990, 76, 655-663.
8. (a) Su, X.; Aprahamian, I. Chem. Soc. Rev., 2014, 43, 1963-1981.
(b) Zugazagoitia, J. S.; Maya, M.; Damián-Zea, C.; Navarro, P.;
Beltrán, H. I.; Peon, J. J. Phys. Chem. A, 2010, 114, 704-714. (c)
Wang, P.; Liu, J.; Lv, X.; Liu, Y.; Zhao, Y.; Guo, W. Org. Lett.,
2012, 14, 520-523. (d) Liu, Z.; Peng, C.; Wang, Y.; Pei, M.;
Zhang, G. Org. Biomol. Chem., 2016, 14, 4260-4266.
3. (a) Harrison, J. E.; Schultz, J. J. Biol. Chem., 1976, 251, 1371-
1374. (b) Yap, Y. W.; Whiteman, M.; Cheung, N. S. Cell.
Signalling, 2007, 19, 219-228.
4. (a) Prokopowicz, Z. M.; Arce, F.; Biedron, R.; Chiang, C. L. L.;
Ciszek, M. Katz, D. R.; Nowakowska, M.; Zapotoczny, S.;
Marcinkiewicz, J.; Chain, B. M. J. Immunol., 2010, 184, 824-835.
(b) Daugherty, A.; Dunn, J. L.; Rateri, D. L.; Heinecke, J. W. J.
Clin. Invest., 1994, 94, 437-444.
5. Zhan, X. Q.; Yan, J. H.; Su, J. H.; Wang, Y. C.; He, J.; Wang, S.
Y.; Zheng, H.; Xu, J. G. Sens. Actuators, B, 2010, 150, 774-780.
6. (a) Carter, K. P.; Young, A. M.; Palmer, A. E. Chem. Rev., 2014,
114, 4564-4601. (b) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.;
Yoon, J. Chem. Rev., 2012, 112, 1910-1956. (c) Lin, V. S.; Chen,
W.; Xian, M.; Chang, C. J. Chem. Soc. Rev., 2015, 44, 4596-4618.
(d) Zhu, H.; Fan, J.; Wang, B.; Peng, X. Chem. Soc. Rev., 2015,
44, 4337-4366. (e) Kwok, R. T. K.; Leung, C. W. T.; Lam, J. W.
Supplementary Material
Supplementary data (additional spectroscopic of PHC1)
associated with this article - copies of H and 13C NMR Spectra
1
can be found, in the online version, at http://dx.doi.org.
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5
Highlights
1.
A
novel hypochlorous acid-initiated oxidative
intramolecular cyclization based fluorescent probe has
been developed.
2. The probe features rapid detection kinetics (< 15 sec.).
3. The probe shows high degree of selectivity and
sensitivity for HClO.

6
Tetrahedron