New strategy for detection of hydrogen peroxide based on bi-nucleophilic reaction

Article abstract of DOI:10.1016/j.saa.2021.120131

Two novel fluorescent probes based on 7-hydroxy-4-methyl-coumarin, FAA-MC-OH (2-fluoro-4-nitro-phenylacetyl hydroxyl coumarin) and FBA-MC-OH (2-fluoro-4-nitro-benzoyl hydroxyl coumarin) are first synthesized, and spectral studies confirm that both the pro

Full text of DOI:10.1016/j.saa.2021.120131

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120131
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
New strategy for detection of hydrogen peroxide based on bi-
nucleophilic reaction
a,
Chenxin Mao a,1, Yafei Tian b,1, Shuoshuo Wang a, Bei Wang a, , Xiang Liu
⇑
⇑
a Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province, State Key Laboratory of Applied Organic Chemistry, and College of Chemistry
and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
b Department of Burns and Plastic Surgery & Wound Repair Surgery, Lanzhou University Second Hospital, Lanzhou 730000, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Two 7-hydroxy-4-methyl-coumarin based fluorescent probes FAA-MC-OH and FBA-MC-OH could detect
H2O2 in PBS buffer (pH= 7.40). Of these two fluorescent probes, FBA-MC-OH has a better selectivity and
sensitivity to H2O2 than the fluorescent probe FAA-MC-OH.
ꢀ
ꢀ
ꢀ
Two fluorescent probes FAA-MC-OH
and FBA-MC-OH were synthesized.
Both the fluorescent probes could
detect H2O2 in PBS buffer (pH = 7.40).
Fluorescent probe FBA-MC-OH was
more sensitive and selective to detect
H2O2 than the fluorescent probe FAA-
MC-OH.
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel fluorescent probes based on 7-hydroxy-4-methyl-coumarin, FAA-MC-OH (2-fluoro-4-nitro-
phenylacetyl hydroxyl coumarin) and FBA-MC-OH (2-fluoro-4-nitro-benzoyl hydroxyl coumarin) are
first synthesized, and spectral studies confirm that both the probes display highly selective and sensitive
to H2O2, especially FBA-MC-OH has a shorter response time. Moreover, it is worth noting that the reac-
tion mechanism is based on bi-nucleophilic substitution instead of oxidation or hydrolysis, which is dif-
ferent from previous reported probes’.
Received 30 March 2021
Received in revised form 23 June 2021
Accepted 25 June 2021
Available online 30 June 2021
Keywords:
Fluorescent probe
Coumarin
Ó 2021 Elsevier B.V. All rights reserved.
Detection
H2O2
Bi-nucleophilic substitution
1. Introduction
iology, but also in the pathology.[1–9] In the abnormal cells, it’s
recognized as a second messenger to transfer signal and indicate
As one of the reactive oxygen species (ROS) in the cells, hydro-
gen peroxide (H2O2) plays an important role not only in the phys-
the intracellular oxidant levels.[3,10,11]However, when the level
of H2O2 in cells is over normal capacity, it would lead to diseases,
such as cancers,[12] cardiovascular disease,[13] Alzheimer’s dis-
ease,[14] genetic instability and so on.[15] Therefore, it’s very
urgent for us to develop efficient methods for detection of H2O2
in the cells.
⇑
Corresponding authors.
E-mail addresses: wangbei@lzu.edu.cn (B. Wang), liuxiang@lzu.edu.cn (X. Liu).
1
These authors contributed equally.
https://doi.org/10.1016/j.saa.2021.120131
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

C. Mao, Y. Tian, S. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120131
Recently, there are many different tools that have been applied
into detection of H2O2 in the cells, such as mass probes,[16] pro-
teomics probes,[17]and fluorescent probes.[15] Especially, fluores-
cent probes have been widely used for the features, because of high
sensitivity and low destructivity. In the previous reported work,
fluorescent probes have been used to detect H2O2 base on these
reaction mechanisms, such as borate hydrolysis,[18,19] H2O2 oxi-
dation,[20–22] and sulfonate hydrolysis.[23] However, other spe-
cies in the cells may have some interference to the H2O2, such as
NO,[24–27] O–2,[28–31] and so on. So, it’s necessary to have a better
fluorescent probe to detect H2O2 in the cells which will also sup-
press interference of other species.
In this work, the compounds FAA-MC-OH and FBA-MC-OH
were synthesized as Scheme 1, their structures were confirmed
by [1]H NMR, [13]C NMR and Mass spectrum respectively
(Figure S1-S12), and both probes were used to detect H2O2 based
on the bi-nucleophilic substitution instead of oxidation or hydrol-
ysis. When in the basic system (pH = 7.40), H2O2 may knock off one
of two protons, and become a negative monovalent anion HO–2, a
very good nucleophilic group.[32–35] Then, a leaving group, such
as the fluorine atom of compound FAA-MC-OH or FBA-MC-OH, is
easy replaced by HO–2 when para-position of the fluorine is nitro,
which has a good character of electrical drawing. After the HO2–
concentration. Remarkably, 10 lM FAA-MC-OH and FBA-MC-OH
showed excellent linear correlation between fluorescence intensity
and the concentrations of H2O2 (0–100 eq) with R2 = 0.9946 and
0.9993 (Fig. S13).
In order to obtain the optimal time for probes FAA-MC-OH and
FBA-MC-OH to respond to hydrogen peroxide, time-dependent flu-
orescence emission experiments were performed on the solution of
probes in the presence of H2O2. As shown in Fig. 3, the fluorescence
emission intensity of FAA-MC-OH gradually increased with the
extension of time, reached stability around 40 min (Fig. 3a Insert).
While FBA-MC-OH had a shorter response time, reached stability
around 20 min (Fig. 3b Insert). Additionally, upon reaction with
H2O2, two probes display color change from colorless to blue-
fluorescence within 10 min, we saw that clearly with the naked
eye (Fig. 3 Insert).
To further understand the optical behavior between the probes
and H2O2, the response of various concentration of H2O2 to probes
were conducted. We can see from Fig. 4, as time went on, the cor-
responding florescence intensity increased with increasing concen-
tration of H2O2, which make sure that the probe can react with
H2O2. Meanwhile, the higher concentration of H2O2, the faster
reaction rate.
The results of comparison between two probes could tell us that
the performance of FBA-MC-OH was better than that of FAA-MC-
OH, because FBA-MC-OH could response to the equilibrium in a
short time. The reason could be speculated as follows. When
H2O2 reacted with these probes through bi-nucleophilic substitu-
tion route, the corresponding intermediates were shown in Fig. 5,
hold together with benzene ring through
a-oxygen, the other pro-
ton is also easy to be splited in the basic system, and
a’-oxygen will
replace the electrophilic group if it lies in the ortho-position of the
oxygen, and if the leaving group is a fluorescent molecule, it will
also accompany with the changes of the fluorescence intensity in
the system (Scheme 2). Meanwhile, the proposed mechanism
was evidenced by detection of the corresponding HR-ESI-MS as
well (Fig S14). Upon the addition of 3 equiv H2O2 in the solution
of FBA-MC-OH, the main peak was observed at m/z 199.0368,
which represented complex 5-nitro-3H-benzo[c][1,2]dioxol-3-one.
that is,
a
-O atom of H2O2 attacked FBA-MC-OH, and substituted
’-O, subsequently
F atom to form Fig. 5a, then another O atom,
a
acted on carbon labeled as C2 of carbonyl group, which linked ben-
zene ring directly, to split ester bond. Thus, the fluorescent probe
7-hydroxy-4-methyl-coumarin was set free. This finding could be
also found in the formation of Fig. 5b. But unlike Fig. 5a, a’-O atom
would connect to C4, which was apart from benzene ring by a –
2. Experimental section
CH2- spacer. It was a tiny structural distinction that led to the
results. In other words, when
C2 bonded benzene ring provided a exactly right position to meet
the ’-O atom, and a five-member conjugated ring was produced.
In this sense, the structure of FBA-MC-OH was benefit to the occur-
rence of recognition. On the other hand, if C4 contacted ’-O atom,
a
’-O atom attacked C2, sp2 hybrid
In order to prove the validity of hypothesis above, spectral
study experiments were carried out in PBS buffer (pH = 7.40). In
all the tests, the concentrations of the fluorescent probes are
a
10
l
M.
a
Firstly, the responses of probes FAA-MC-OH and FBA-MC-OH
it is necessarily for it to overturn to an appropriate position to meet
the requirement of the substitution. Further studies indicate that a
non-conjugated six member ring would be emerged. Both of the
inversion of configuration and the creation of non-conjugated ring
were disadvantageous and energy-consuming. So, the reaction rate
and recognition property of FBA-MC-OH were better than that of
FAA-MC-OH.
As we all known, there are many other species in the cells, so it
is very vital for us to know if other species have an effect on our
experiments. In order to prove that, the selective experiments were
completed, as shown in Fig. 6. The other relevant species were
Na2S2, Na2S, KO2, NaClO, t-BuOOH, ꢀOH, ONOO–, Na3PO4, Na2SO4,
KBrO3, KF, NaCl, KBr, Na2CO3, KNO3 and Na2SO4, the concentrations
toward H2O2 were evaluated by UV–vis absorption (Fig. 1). The free
probes FAA-MC-OH and FBA-MC-OH displayed a peak at 275 nm
with molar absorption coefficients of 16496 cm-1MÀ1 and
14148 cm-1MÀ1, respectively. Both bands were red shifted with
addition of H2O2 (200
lM) in the spectra, which was consistent
with the absorption of 7-hydroxy-4-methyl-coumarin, the results
indicated that the probes FAA-MC-OH and FBA-MC-OH could react
with H2O2 and may undergo the mechanism as described Scheme 2,
which was similar to other reported bi-nucleophilic reaction.[32]
We studied the fluorescent changes of 10
lM FAA-MC-OH
(Fig. 2a) and FBA-MC-OH (Fig. 2b) by titration with various con-
centrations of H2O2 in air. As shown in Fig. 2, the fluorescence
emission intensity at 440 nm gradually increased with increasing
Scheme 1. Synthesis of FAA-MC-OH and FBA-MC-OH.
2

C. Mao, Y. Tian, S. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120131
Scheme 2. Reaction process of H2O2 with fluorescent probes.
Fig. 1. UV–vis spectra changes of the probes FAA-MC-OH (a) and FBA-MC-OH (b) upon the addition of H2O2 in PBS buffer (pH = 7.40, 10 mM) in 2 h.
Fig. 2. The fluorescence increment (440 nm) of FAA-MC-OH (10
pH = 7.40). (kex = 325 nm).
lM, a) and FBA-MC-OH (10 lM, b) with various concentrations of H2O2 in PBS buffer after 30 min. (10 mM,
3

C. Mao, Y. Tian, S. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120131
Fig. 3. Time course of fluorescence changes of FAA-MC-OH (10
(kex = 325 nm).
l M, a) and FBA-MC-OH (10 lM, b) in the presence of H2O2 (1 mM) in PBS buffer (10 mM, pH = 7.40).
Fig. 4. Time course of the fold of fluorescence increment (440 nm) of FAA-MC-OH (10
(10 mM, pH = 7.40). (kex = 325 nm).
l M, a) and FBA-MC-OH (10 lM, b) with various concentrations of H2O2 in PBS buffer
CRediT authorship contribution statement
Chenxin Mao: Validation, Investigation, Resources, Data cura-
tion. Yafei Tian: Visualization, Investigation, Software. Shuoshuo
Wang: Formal analysis, Resources. Bei Wang: Data curation, Writ-
ing - original draft, Writing - review & editing. Xiang Liu: Concep-
tualization, Methodology, Supervision, Project administration.
Fig. 5. The structure of intermediates of FBA-MC-OH (a) and FAA-MC-OH (b) after
reacted with H2O2.
Declaration of Competing Interest
of all the species including H2O2 are 100 lM. We found that only
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
H2O2 could cause dramatic fluorescence intensity increasing for
both probes, while Na2S2 led to slight fluorescence changes as their
structures are similar. The other relevant species didn’t cause any
fluorescence response. Therefore, both of these probes showed
higher selectivity to the H2O2, especially the probe FBA-MC-OH.
In conclusion, we have successfully designed probes FAA-MC-
OH and FBA-MC-OH, and applied them to detect H2O2. It is worth
noting that the mechanism is based on bi-nucleophilic substitution
which is different from other reported probes. The spectral studies
confirmed that both the probes displayed highly selective and sen-
sitive to the H2O2, especially the FBA-MC-OH.
Acknowledgements
We thank the Natural Science Foundation of Gansu province
(1107RJZA086), the National Natural Science Foundation of China
(21401090).
The authors have no competing interests to declare.
4

C. Mao, Y. Tian, S. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 262 (2021) 120131
Fig. 6. Fluorescence response of FAA-MC-OH (10
pH = 7.40) in the 15 min. (kex = 325 nm).
l M, a) and FBA-MC-OH (10 lM, b) in the presence of H2O2 and other relevant agents (100 lM) in PBS buffer (10 mM,
[17] V.V. Belousov, A.F. Fradkov, K.A. Lukyanov, D.B. Staroverov, K.S. Shakhbazov, A.
V. Terskikh, S. Lukyanov, Nat. Methods 3 (2006) 281.
Appendix A. Supplementary data
[18] L.C. Lo, C.Y. Chu, Chem. Commun. (2003) 2728.
[19] D. Srikun, E.W. Miller, D.W. Domaille, C.J. Chang, J. Am. Chem. Soc. 130 (2008)
4596.
[20] N. Soh, O. Sakawaki, K. Makihara, Y. Odo, T. Fukaminato, T. Kawai, M. Irie, T.
Imato, Bioorg. Med. Chem. 13 (2005) 1131.
[21] Y.X. Liao, K. Li, M.Y. Wu, T. Wu, X.Q. Yu, Org. Biomol. Chem. 12 (2014) 3004.
[22] K.M. Zhang, W. Dou, P.X. Li, R. Shen, J.X. Ru, W. Liu, Y.M. Cui, C.Y. Chen, W.S.
Liu, D.C. Bai, Biosens. Bioelectron. 64 (2015) 542.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.120131.
References
[1] T. Finkel, Curr. Opin. Cell Biol. 15 (2003) 247.
[23] H. Maeda, Y. Fukuyasu, S. Yoshida, M. Fukuda, K. Saeki, H. Matsuno, Y.
Yamauchi, K. Yoshida, K. Hirata, K. Miyamoto, Angew. Chem. 43 (2004) 2389.
[24] E.W. Miller, C.J. Chang, Curr. Opin. Chem. Biol. 11 (2007) 620.
[25] H. Kojima, N. Nakatsubo, K. Kikuchi, S. Kawahara, Y. Kirino, H. Nagoshi, Y.
Hirata, T. Nagano, Anal. Chem. 70 (1998) 2446.
[26] Y. Gabe, Y. Urano, K. Kikuchi, H. Kojima, T. Nagano, J. Am. Chem. Soc. 126
(2004) 3357.
[27] E. Sasaki, H. Kojima, H. Nishimatsu, Y. Urano, K. Kikuchi, Y. Hirata, T. Nagano, J.
Am. Chem. Soc. 127 (2005) 3684.
[2] L. Yuan, W.Y. Lin, Y.N. Xie, B. Chen, S.S. Zhu, J. Am. Chem. Soc. 134 (2012) 1305.
[3] S.G. Rhee, Science 2006 (1882) 312.
[4] E.W. Miller, O. Tulyathan, E.Y. Isacoff, C.J. Chang, Nat. Chem. Biol. 3 (2007) 263.
[5] P. Niethammer, C. Grabher, A.T. Look, T.J. Mitchison, Nature 459 (2009) 996.
[6] R.E. Haskew-Layton, J.B. Payappilly, N.A. Smirnova, T.C. Ma, K.K. Chan, T.H.
Murphy, H. Guo, B. Langley, R. Sultana, D.A. Butterfield, S. Santagata, M.J.
Alldred, I.G. Gazaryan, G.W. Bell, S.D. Ginsberg, R.R. Ratan, Proc. Natl. Acad. Sci.
USA 107 (2010) 17385.
[7] B.C. Dickinson, C.J. Chang, Nat. Chem. Biol. 7 (2011) 504.
[28] H. Maeda, K. Yamamoto, Y. Nomura, I. Kohno, L. Hafsi, N. Ueda, S. Yoshida, M.
Fukuda, Y. Fukuyasu, Y. Yamauchi, N. Itoh, J. Am. Chem. Soc. 127 (2005) 68.
[29] K.M. Robinson, M.S. Janes, M. Pehar, J.S. Monette, M.F. Ross, T.M. Hagen, M.P.
Murphy, J.S. Beckman, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 15038.
[30] K. Xu, X. Liu, B. Tang, ChemBioChem 8 (2007) 453.
[31] K. Xu, X. Liu, B. Tang, G. Yang, Y. Yang, L. An, Chemistry 13 (2007) 1411.
[32] C. Liu, W. Chen, W. Shi, B. Peng, Y. Zhao, H. Ma, M. Xian, J. Am. Chem. Soc. 136
(2014) 7257.
[8] B.C. Dickinson, J. Peltier, D. Stone, D.V. Schaffer, C.J. Chang, Nat. Chem. Biol. 7
(2011) 106.
[9] M.P. Murphy, A. Holmgren, N.G. Larsson, B. Halliwell, C.J. Chang, B.
Kalyanaraman, S.G. Rhee, P.J. Thornalley, L. Partridge, D. Gems, T. Nystrom,
V. Belousov, P.T. Schumacker, C.C. Winterbourn, Cell Metab. 13 (2011) 361.
[10] B.C. Dickinson, C. Huynh, C.J. Chang, J. Am. Chem. Soc. 132 (2010) 5906.
[11] J.R. Stone, S. Yang, Antioxid. Redox Signaling 8 (2006) 243.
[12] H. Ohshima, M. Tatemichi, T. Sawa, Arch. Biochem. Biophys. 417 (2003) 3.
[13] A.M. Shah, K.M. Channon, Heart 90 (2004) 486.
[14] K.J. Barnham, C.L. Masters, A.I. Bush, Nat. Rev. Drug Discovery 3 (2004) 205.
[15] A.R. Lippert, G.C.V. De Bittner, C.J. Chang, Acc. Chem. Res. 44 (2011) 793.
[16] H.M. Cocheme, A. Logan, T.A. Prime, I. Abakumova, C. Quin, S.J. McQuaker, J.V.
Patel, I.M. Fearnley, A.M. James, C.M. Porteous, R.A.J. Smith, R.C. Hartley, L.
Partridge, M.P. Murphy, Nat. Protoc. 7 (2012) 946.
[33] M. Gao, R. Wang, F. Yu, J. You, L. Chen, Analyst 140 (2015) 3766.
[34] M. Gao, F. Yu, H. Chen, L. Chen, Anal. Chem. 87 (2015) 3631.
[35] L. Zeng, S. Chen, T. Xia, W. Hu, C. Li, Z. Liu, Anal. Chem. 87 (2015) 3004.
5