Aldehyde group assisted thiolysis of dinitrophenyl ether: A new promising approach for efficient hydrogen sulfide probes

Article abstract of DOI:10.1039/c4cc03818e

We report herein a new approach, which combines fast nucleophilic addition of H₂S to an aldehyde group and the subsequent intramolecular thiolysis of dinitrophenyl ether, and can be used to develop efficient and effective H₂S probes.

Full text of DOI:10.1039/c4cc03818e

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Aldehyde group assisted thiolysis of dinitrophenyl
ether: a new promising approach for efficient
hydrogen sulfide probes†
Cite this: Chem. Commun., 2014,
50, 9185
Received 19th May 2014,
Accepted 24th June 2014
Zijun Huang, Shuangshuang Ding, Dehuan Yu, Feihu Huang and Guoqiang Feng*
DOI: 10.1039/c4cc03818e
www.rsc.org/chemcomm
We report herein a new approach, which combines fast nucleophilic
We are interested in the thiolysis of dinitrophenyl ether for H2S
addition of H2S to an aldehyde group and the subsequent intra- probes because the dinitrophenyl ether is very easy to be introduced to
molecular thiolysis of dinitrophenyl ether, and can be used to develop a fluorophore, and such probes have very low background fluorescence
efficient and effective H2S probes.
due to the strong quenching effect of the nitro group. However, the
reported probes for H2S based on this approach by several groups7a–d
Much attention has been focussed in the last few years on the including us7e used intermolecular thiolysis of dinitrophenyl ether (A in
development of small molecular probes for hydrogen sulfide (H2S) Scheme 1), and they generally suffered from low reaction rates and thus
since the recognition of the biological significance of H2S recently.1 long response times are also needed. Although addition of a surfactant
Among them, the fluorescent detection method is highly attractive such as cetyltrimethylammonium bromide (CTAB)7a,e or Tween-207c
due to its simplicity, high sensitivity, low cost and ability of real-time can enhance the reaction rates significantly, the use of surfactant limits
detection. Accordingly, highly selective and sensitive fluorescent the applications of these probes. Since an intramolecular reaction can
probes with rapid response for H2S under mild conditions have be performed more efficiently than an intermolecular one, we thought
become high in demand, as they have huge potential benefits for a that the thiolysis reaction could proceed much faster if it was switched
better and deeper understanding of the biological functions of H2S. into an intramolecular fashion. Using the unique dual nucleophilic
Since 2011, a number of fluorescent probes for H2S have been characters of H2S, we envisaged that combining the fast nucleophilic
developed based on several significant chemical properties of H2S, addition of H2S to an aldehyde group and the subsequent spontaneous
such as good reducing property,2,3 high binding affinity towards intramolecular instead of intermolecular thiolysis of dinitrophenyl ether
copper ions,4 specific nucleophilic addition to unsaturated double could achieve this purpose (B in Scheme 1). Motivated by this concept,
bonds,5 dual nucleophilicity6 as well as efficient thiolysis of dinitro- we herein report that this new approach can be very promising to
phenyl ether.7 Among them, using the dual nucleophilicity of H2S is develop efficient H2S probes.
especially attractive for H2S probes because a high selectivity for H2S
To investigate the feasibility of our design concept, we chose
can be well guaranteed by this strategy.6 Although the advantage of a known excited state intramolecular proton transfer (ESIPT)
this strategy has been utilized in several innovative H2S probes,6
most of them showed a delayed response time (typically 420 min)
to detect H2S. Actually, this is a common problem for most reaction-
based H2S probes reported to date, which is a disadvantage for
efficient capturing of the transient H2S.1 Besides, some other draw-
backs of these reported probes can also be found such as either they
need a relatively high probe loading due to the probe consumption
by other thiols,6a or need complicated synthetic procedures,6b–d or
need the use of surfactants to promote the reactions.6f,h,i Therefore,
new methods are highly expected to overcome these shortcomings.
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education,
College of Chemistry, Central China Normal University, 152 Luoyu Road,
Wuhan 430079, P. R. China. E-mail: gf256@mail.ccnu.edu.cn
† Electronic supplementary information (ESI) available: Synthesis and structure
Scheme 1 Sensing of hydrogen sulfide by thiolysis of dinitrophenyl ether,
characterizations of probe 1 and 2, and additional optical spectra. See DOI:
the ESIPT dyes and probes in this work.
10.1039/c4cc03818e
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dye, compound 3 in Scheme 1, as the fluorophore, because this type pseudo-first-order rate constant kobs at 25 1C was determined to be
of dye has been widely used to construct fluorescent probes due to about 2.077 minÀ1 (t1/2 E 0.334 min) and the emission intensity was
their large Stokes shift and good photostability.6f,8 In addition, it can found to increase about 120-fold at 545 nm. Since the reaction between
be easily prepared, and an aldehyde group can also be easily probe 1 and NaHS cannot complete even after 18 hours under the
introduced to the ortho position of the hydroxyl group in this dye same conditions (Fig. S2, ESI†), probe 2 responds at least more than
to afford an aldehyde containing fluorophore 4. Thus, two new hundreds of times faster than probe 1 for H2S, indicating that our
probes (probe 1 and 2) for the purpose of this study can be readily design concept works very well. In fact, probe 2 is able to detect
obtained by protection of the hydroxyl group in 3 and 4 with a H2S more rapidly than most of the reported reaction-based probes for
dinitrophenyl group, respectively. The synthesis of these two probes H2S.1–3,5,6 In addition, it was also found that probe 2 can work with less
is illustrated in Scheme S1 (ESI†), and their structures are confirmed amount of CH3CN and can work over a wide range of pH with the
by NMR, IR and HR-MS analysis. Detailed synthetic procedures and optimal pH around 7.0 (Fig. S3, ESI†). All these results indicate that this
structure characterization are given in the ESI.†
new combined approach is effective and attractive.
Since 3 and 4 are the expected thiolysis products of probe 1 and
To confirm the thiolysis of dinitrophenyl ether and release of 4, the
2 with H2S, respectively, we first checked the differences in their reaction product of probe 2 with NaHS was isolated and subjected to
optical spectra. As shown in Fig. S1 (ESI†), 3 absorbs light in the UV thin layer chromatography (TLC) and 1H NMR analysis, and the
range (lmax 335 nm), however, lmax of 4 red-shifted to the visible results proved that the product is 4 (Fig. S4 and S5, ESI†). Considering
light region (lmax 445 nm), which indicates that 4 is visible light that the aldehyde group in probe 2 provides a larger steric hindrance
excitable. Fluorescent spectra showed that both probe 1 and 2 are to the dinitrophenyl ether moiety for a direct thiolysis by H2S and the
non-fluorescent, while 3 and 4 show much stronger fluorescence. low reactivity of probe 1 for H2S, the sensing process of probe 2 for
Besides, 4 shows not only longer emission wavelength, but also HSÀ most likely happened through route B as shown in Scheme 1.
higher fluorescence quantum yield than 3 (4: lem 545 nm, F 0.18 vs.
The above results show that probe 2 can be used as a rapid and
3: lem 480 nm, F 0.04), indicating that 4 is a better fluorophore sensitive dual colorimetric and fluorescent turn-on sensor for HSÀ in
(Fig. S1b, ESI†). Anyway, if the thiolysis reactions of both probes aqueous solution. To shed further light on the sensitivity of probe 2
with H2S are fast, then both of them should give rapid and for H2S, the changes in its fluorescence were investigated by addition
significant optical signal changes.
of various concentrations of NaHS. As shown in Fig. S6 (ESI†), probe 2
The sensing ability of probe 1 and 2 for H2S was then investigated in is reasonably stable under the test conditions, and upon addition of
20 mM PBS buffer (pH 7.4) with 25% CH3CN (v/v) at 25 1C. Under these 0.6–10 equiv. HSÀ, the fluorescence intensity of the probe 2 solutions
conditions, we observed that addition of 10 equiv. of NaHS (a standard showed significant enhancement within 5 min. When the reaction
source for H2S) to the probe 1 and 2 (20 mM of each) solutions resulted was monitored after 5 min of HSÀ addition, a saturation of fluores-
in significant differences. As shown in Fig. 1, the probe 1 solution cence could be observed after the addition of more than 2 equiv. HSÀ
showed very small changes upon addition of NaHS and even the (Fig. S7, ESI†). In addition, we also observed a good linearity between
reaction time was prolonged to 18 h. In contrast, the probe 2 solution the fluorescent intensity at 545 nm and the concentration of HSÀ in
showed fast and significant optical changes within a few minutes upon the range of 0–12 mM (Fig. 2), thus, the detection limit (S/N = 3) of
addition of NaHS, and meanwhile, the colorless solution of 2 changed probe 2 for HSÀ is calculated to be about 48 nM under the test
into yellow, and gave out strong green fluorescence as compound 4. conditions. This indicates that probe 2 has high sensitivity for H2S.
Kinetic studies showed that the reaction (20 mM probe 2 + 100 mM
NaHS) was completed at about 2 min (Fig. S2, ESI†). The observed competitors such as FÀ, ClÀ, BrÀ, IÀ, NO3À, NO2À, AcOÀ, SCNÀ,
We then investigated the selectivity of probe 2 for HSÀ over various
CO32À, HCO3À, H2PO4À, CNÀ, SO42À, HSO4À, SO32À, HSO3À, S2O32À
,
S2O72À, cysteine (Cys), homocysteine (Hcy), glutathione (GSH),
N-acetyl-cysteine (NAC), C6H5NH2, C6H5CH2NH2, H2NCH2CH2NH2
and HOCH2CH2NH2, and the results showed that probe 2 is highly
Fig. 2 (a) Fluorescence spectral changes of probe 2 (20 mM) upon addi-
tion of different concentrations of HSÀ in PBS buffer (20 mM, pH 7.4)
containing 25% CH3CN (v:v). The final concentrations of HSÀ are 0, 1, 2, 4,
6, 8, 10, 12 mM, respectively. (b) A linear calibration curve between the
fluorescent intensity changes of probe 2 at 545 nm and the concentration
of HSÀ in the range of 0 to 12 mM. Each spectrum was collected at 5 min
after HSÀ addition. lex = 440 nm, slit width: dex = dem = 5 nm.
Fig. 1 UV-vis and fluorescent spectral changes of probe 1 (a and b,
respectively) and probe 2 (c and d, respectively) against time in the presence
of HSÀ (10 equiv.). All spectra were measured in PBS buffer (20 mM, pH 7.4)
with 25% CH3CN (v/v) at 25 1C with [probe] = 20 mM.
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ether, and can be used to develop more efficient probes for H2S.
Using this approach, we showed that a new dual colorimetric
and fluorescent probe (probe 2) was able to sense H2S with
rapid response (o5 min), high selectivity and sensitivity, and
was also able to detect H2S in living cells, indicating that this
approach is applicable and very promising. We believe that this
approach could aid the design of future H2S probes for efficient
and effective detection of H2S.
Fig. 3 (a) Fluorescent spectral changes of probe
2 (20 mM) in the
presence of various analytes (10 eq.). (b) Fluorescent intensity changes of
probe 2 (20 mM) at 545 nm for various analytes (200 mM). lex = 440 nm,
dem = dex = 5 nm. The numbers (1–29) represent probe 2 with analytes:
We thank the National Natural Science Foundation of China
(Grant No. 21172086 and 21032001) for financial support.
(1) none, (2) HSÀ, (3) FÀ, (4) ClÀ, (5) BrÀ, (6) IÀ, (7) SCNÀ, (8) CO32À, (9) AcOÀ,
(10) NO3À, (11) NO2À, (12) PO43À, (13) HPO42À, (14) H2PO4À, (15) S2O3
,
2À
Notes and references
(16) S2O72À, (17) SO42À, (18) SO32À, (19) HSO3À, (20) HSO4À, (21) CNÀ, (22)
GSH, (23) Hcy, (24) Cys, (25) NAC, (26) C6H5NH2, (27) C6H5CH2NH2, (28)
H2NCH2CH2NH2, (29) HOCH2CH2NH2 respectively.
1 Useful reviews, see: (a) W. Xuan, C. Sheng, Y. Cao, W. He and W. Wang,
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selective for HSÀ. As shown in Fig. 3 and Fig. S8 and S9 (ESI†), only
addition of HSÀ resulted in significant optical changes to the probe 2
solution. In contrast, addition of other analytes showed almost no
effect. Notably, detection of HSÀ using probe 2 in the presence of
these analytes is still effective (Fig. S10, ESI†), even biothiols Cys, Hcy,
GSH and NAC are present in a millimolar range (Fig. S11, ESI†).
Despite the fact that aryl-aldehydes have been reported to react with
biothiols such as Cys to form stable thiazolidines,9 our experiments
showed that compound 4, the product of probe 2 with HSÀ, is not
fluorescent responsive to Cys even upon incubation for more than
10 hours (Fig. S12, ESI†). Therefore, all these results indicate that
probe 2 is highly selective for HSÀ.
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The ability of probe 2 to visualize H2S in living cells was also
tested. As shown in Fig. 4, no fluorescence was observed when
HeLa cells were incubated with probe 2 only. In contrast, addition
of H2S to the cells pre-incubated with probe 2 produced strong
green fluorescence. This result indicates that probe 1 can be used
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Fig. 4 Fluorescence imaging of H2S in HeLa cells incubated with 20 mM
probe 2. (A) and (B) show that cells were incubated with 2 only for 20 min.
(C) and (D) show that cells were pre-incubated with 2 for 20 min, and then
incubated with 200 mM H2S for 30 min. (A) and (C) are bright field images,
(B) and (D) are fluorescence images of (A) and (C), respectively.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 9185--9187 | 9187