Structure-reactivity relationship of probes based on the H2S-mediated reductive cleavage of the CC bond

Article abstract of DOI:10.1039/d0nj02307h

Recently, we discovered the H2S-mediated reductive cleavage of the CC bond, and applied this reaction to design probes for the detection of H2S. To extensively elucidate the structure-reactivity relationship, our current work further investigated the effect of substitutes that connected to the CC bond on the H2S-mediated reductive cleavage, wherein different kinds of electron-withdrawing groups (such as pyridine, bipyridine, terpyridine) and electron donating groups (such as carbazole, N,N-dimethylaniline and phenothiazine) were conjugated to the CC bond of interest. Experimental results and DFT calculations showed that the strength of the electron-donating and withdrawing substitutes could significantly affect the reductive cleavage of the CC bond. Unexpectedly, the reductive cleavage was not influenced by the change of the C(2) and C(3) positions in phenothiazine. On this basis, two probes (NPTZ-P1 and NPTZ-P2) with a C(3)-substituted phenothiazine were thus developed and successfully applied for sensing exogenous H2S in living HeLa cells, which implied their potential in bioimaging. This study provides further understanding on the structure-reactivity relationship of the H2S-mediated reductive cleavage of the CC bond, and is also valuable for exploring new reactions of the CC bond in organic synthesis. This journal is

Full text of DOI:10.1039/d0nj02307h

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Structure-reactivity relationship of probes based on H2S-mediated
reductive cleavage of C=C bond
Chunfei Wang, Jingyun Tan and Xuanjun Zhang *
Received 00th January 20xx,
Accepted 00th January 20xx
Recently, we discovered H2S-mediated reductive cleavage of C=C bond and applied this reaction for probe design in detecting
H2S. To extensively elucidate the structure-reactivity relationship, current work further investigated the effect of substitutes
that connected to C=C bond on H2S-mediated reductive cleavage, wherein different kinds of electron withdrawing groups
such as pyridine, bipyridine, terpyridine, as well as electron donating groups such as carbazole, N,N-dimethylaniline and
phenothiazine, were conjugated to the C=C bond of interest. Experimental results and DFT calculation showed that the
strength of electron donating and withdrawing substitutes could significantly affect the reductive cleavage of C=C bond.
Unexpectedly, the reductive cleavage was not influenced by the change of C(2) and C(3) position in phenothiazine. On this
basis, two probes (NPTZ-P1 and NPTZ-P2) with C(3)-substituted phenothiazine were thus developed and successfully applied
for sensing exogenous H2S in living HeLa cells which implied their potential bioimaging. This study provides a further
understanding about structure-reactivity relationship of H2S-mediated reductive cleavage of C=C bond and is also valuable
for exploring new reactions of C=C bond in organic synthesis.
DOI: 10.1039/x0xx00000x
reductive cleavage of C=C bond, this new reaction is a very
promising strategy for the design of fluorescent probes with
unprecedented selectivity of H2S.
Introduction
Carbon-carbon double bond (C=C) is of great importance in π-
conjugation, because carbon atoms cannot rotate freely around
the axis of the double bond, resulting in the rigid planarity with
C=C bond 1. Especially, different substituents connected to the
C=C bond have significant effects on its reactivity, as well as the
optical properties of the whole molecules. As well known,
addition reaction is one of the characteristic reactions of C=C
In donor-acceptor structures, the photophysical properties
of fluorescent probes are obviously impacted by different
substitutes with electron donating and accepting ability.
Generally, medium-strength intramolecular charge transfer
(ICT) is conducive to enhance fluorescence quantum yield,
whereas too strong ICT can quench fluorescence, especially in
polar solvents 21-24. Therefore, rational design and proper
selection of substituents are crucial for probe design.
Malononitrile is extensively used as acceptor and anchor,
presumably related to an increase in spectral response through
ICT and good electron injection properties 25-27. Pyridine has a
moderate electron withdrawing ability. The condensation
between ethylidene malononitrile and pyridine by Knoevenagel
reaction, not only lengthened the π-conjugation, but also
increased the electron withdrawing ability. As depicted in
Scheme 1A, PTZ-P3 has been reported in our previous study,
wherein phenothiazine acts as a strong electron donor, and
dicyano-pyridine group is a strong electron acceptor. The very
strong ICT resulted in fluorescence quenching of PTZ-P3. Upon
detecting H2S, the Ca=Cb bond could be reductively cleaved
together with the formation of amine-functionalized thiophene
ring by consumption of a cyano group. Consequently, the
medium-strength ICT in the product leads to turn on
fluorescence.
bond. Meanwhile, oxidative cleavage of C=C bond is also
2-5
extensively reported in organic synthesis
. Recently
developed methods used for confirming the position of C=C
bond in fatty acids and lipids, are still based on oxidative
cleavage of C=C bond 1, 6, 7. Of particular note is that only limited
studies about reductive cleavage of alkenes have been reported
so far 8-11, due to the particularity of C=C bond. We recently
reported the first H2S-mediated reductive cleavage of C=C bond
(Ca=Cb) under mild condition 12. As we know, H2S is an important
gas signalling molecule in physiology, whereas the biological
activities relevant to H2S are also affected in the absence of
hydrogen polysulfides (H2Sn, n>1) 13-15. H2S can regulate
intracellular redox status, thereby large numbers of fluorescent
probes have been developed based on nucleophilicity and/or
reduction ability of H2S and H2Sn 16-20. In view of the anomaly of
Cancer Centre and Centre of Reproduction, Development and Aging, Faculty of
Health Sciences, University of Macau, Taipa, Macau SAR, 999078, China.
E-mail: xuanjunzhang@um.edu.mo
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Attracted by this interesting reaction, current study was
thus performed to reveal the structure-reactivity relationship of
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H2S-mediated reductive cleavage of C=C bond. Pyridine is a in living HeLa cells, which provide an evidence for their potential
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DOI: 10.1039/D0NJ02307H
moderate electron acceptor, different kinds of electron pulling applications for bioimaging. This study provides a deep
and pushing groups were firstly implemented to replace understanding about H2S-mediated reductive cleavage of C=C
pyridine for exploring their impact on reactivities with H2S. bond as an effective strategy for probe design.
Hence, a series of phenothiazine ethylidene malononitrile
derivatives were reasonably synthesized by condensation with
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4-(dimethylamino)benzaldehyde, 4-hydroxybenzaldehyde, 2,2'-
bipyridine-4-carbaldehyde and [2,2':6',2''-terpyridine]-4'-
Results and Discussions
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carbaldehyde, respectively (Scheme 1B). Furthermore,
carbazole, N,N-dimethylaniline and C(3)-substituted
phenothiazine with different electron donating abilities, were
subsequently employed to replace C(2)-substituted
phenothiazine for illustrating their influence on H2S-mediated
reductive cleavage of Ca=Cb bond (Scheme 1C).
Design and synthesis
In heteroaromatic phenothiazine, the two phenyl rings are
arranged with a small torsion angle between the N(10) and S(9)
atoms, so that π-delocalization can be extended over the entire
chromophore 28-30. This nonplanar butterfly conformation of
phenothiazine can sufficiently inhibit molecular aggregation
and the formation of intermolecular excimers 31. Thanks mainly
to the electron-rich nitrogen and sulfur heteroatoms,
phenothiazine displays high electron-donating ability 32-34. Here,
phenothiazine acts as a strong electron donor in PTZ-P3, while
the dicyano-pyridine group is a strong electron acceptor.
Considering the influence of pyridine moiety in this donor-
acceptor structures, different electron donors/acceptors were
introduced to replace pyridine moiety, yielding PTZ-P1, PTZ-P2,
PTZ-P4, PTZ-P5 and PTZ-P6.
(A) Previous Study
Very Strong ICT
Medium-strength ICT
S
CN
CN
2
S
CN
S
CN
S
N
H
CN
R2
Ca
H2
S
H2S
R2
CN
Cb
R1
NH2
N
H
NH2
PTZ-P3
N
(B) Change of R1 group
(C) Change of R2 group
S
CN
CN
S
CN
CN
N
H
CN
N
CN
H
N
N
KZ-P
N
N
CN
CN
N
PTZ-P1
PTZ-P4
Generally speaking, electrophilic substitutions easily occur
in the C(3) or C(7) position of phenothiazine because the para-
position is occupied by a nitrogen atom with strong electron-
donating ability. However, the acetyl group was substituted at
the position of C(2) in phenothiazine from PTZ-P1 to PTZ-P6;
thereby, we moved the acetyl group to the C(3) position in
NPTZ-P1 and NPTZ-P2 to explore the positional impact on the
reactivity of the H2S-mediated reductive cleavage of Ca=Cb
bond.
S
CN
N
NNP-P
S
N
N
H
CN
CN
N
H
CN
CN
CN
N
S
N
N
NPTZ-P1
NPTZ-P2
N
OH
CN
PTZ-P2
PTZ-P5
N
S
CN
CN
CN
CN
N
H
S
S
N
N
H
CN
O
-N
B
- O
N
+
N+
B
N
PTZ-P3
PTZ-P6
N
In addition, carbazole has only one less sulfur atom than
phenothiazine. Carbazole has drawn dramatic attentions for the
versatility in functionalization, high fluorescence quantum yield
and excellent photostability 35-37. Similar with phenothiazine,
carbazole is also a strong electron donor due to π-conjugated
Scheme 1 (A) Our previous study of H2S-mediated reductive
cleavage of C=C bond. (B) The chemical structures of probes
with differing electron-withdrawing group R1. (C) The chemical
structures of probes with differing electron-donating group R2.
system and electron-rich tertiary amine moiety. As
a
Isolation of reaction, spectrophotometric results and DFT
calculation demonstrated that the ability of electron donating
and withdrawing appears to be concerned with the reactivity.
Among the short series from PTZ-P1 to PTZ-P6, PTZ-P3, PTZ-P4
and PTZ-P6 exist moderate electron accepting moieties.
Additionally, PTZ-P3, NPTZ-P1 and NPTZ-P2 have strong
electron donating phenothiazine in comparison to KZ-P and
NNP-P. Therefore, PTZ-P3, PTZ-P4, PTZ-P6, NPTZ-P1 and NPTZ-
P2 showed effective reductive cleavage of Ca=Cb bond,
producing thiophene-based products. More importantly, the
reductive cleavage of Ca=Cb bond was identified to be not
influenced by the change of C(2) and C(3) position in
phenothiazine. The newly developed probes, NPTZ-P1 and
NPTZ-P2, were successfully applied for sensing exogenous H2S
consequence, carbazole and N,N-dimethylaniline were further
utilized as donors to replace phenothiazine in the donor-
acceptor structure namely KZ-P and NNP-P for investigating
their influence on the reductive cleavage of Ca=Cb bond
mediated by H2S.
The synthesis of PTZ-P3, PTZ-P4 and PTZ-P6 were reported
in our previous study 12. The detailed synthetic procedures of
above-mentioned other probes were described in experimental
section. All intermediates and final probes were fully
1
characterized by HNMR, 13CNMR and HRMS spectra (Fig. S1-
S30 and S39-S46, ESI†).
Reactivity of Probes toward H2S and reaction mechanism study
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In order to clearly validate the reactivity of H2S-mediated different substitute in C(2) and C(3) position of phenothiazine
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DOI: 10.1039/D0NJ02307H
reductive cleavage of C=C bond, PTZ-P1, PTZ-P2, PTZ-P3, PTZ- but both exhibited effective reaction.
P4, PTZ-P5, KZ-P, NNP-P and NPTZ-P1 were used to react with
NaHS in DMSO containing 50% distilled water respectively
(details were described in experimental section). The reactions
of PTZ-P3 and PTZ-P4 to H2S were thoroughly confirmed in our
previous study and thus not discussed too much here. After the
reactions were completed, the products were isolated and
characterized. Fortunately, the green fluorescent product, SF4,
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1
was successfully isolated. Unlike NPTZ-P1, the H NMR data of
SF4 showcased the disappearance of pyridine moiety (Fig. 1)
and this product was further confirmed by HRMS result at the
same time (Fig. S47, ESI†). Although KZ-P and NNP-P could react
with H2S, the thiophene-based products induced by reductive
cleavage of Ca=Cb bond were not isolated, as well as the failing
characterization of the reaction mixture by HRMS data. We
inferred that addition reaction occurred in the reaction mixture
of NNP-P and H2S, because the product of addition reaction was
confirmed by HRMS data (Fig. S48, ESI†), but other compounds
were not clearly confirmed. Additionally, there were too many
products in the reaction between KZ-P and H2S to confirm these
products from HRMS data. In the rationally designed series of
Fig. 1 Comparison of 1H-NMR spectra between NPTZ-P1 and
SF4.
probes, the results indicated that PTZ-P3, PTZ-P4 and NPTZ-P1
were successfully proved to be effectively cleaved Ca=Cb bond
mediated by H2S. Unexpectedly, PTZ-P3 and NPTZ-P1 displayed
Fig. 2 (A) Absorption and fluorescent spectra of thiophene-based products (10 μM) in PBS buffer (pH=7.4)/DMSO (1/2 v/v, 2% PEG
400), Dotted lines and full lines represent absorption and fluorescent spectra respectively. (B-I) Absorption and fluorescent spectra
of probes (10 μM, PTZ-P1, PTZ-P2, PTZ-P3, PTZ-P4, PTZ-P5, KZ-P, NNP-P and NPTZ-P1) in PBS buffer (pH=7.4)/DMSO (1/2 v/v, 2%
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PEG 400) in the absence and presence of H2S (100 μM), λex=330 nm. Black lines and red lines represent reaction without and with
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DOI: 10.1039/D0NJ02307H
H2S respectively. Dotted lines and full lines represent absorption and fluorescent spectra respectively.
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In our previous study, we found that PTZ-P3 effectively well photophysical properties in photovoltaic performance due
reacted with H2S producing thiophene-based products. Herein, to nitrogen atom in para-position 28, 30, 44, 45. However, making a
different thiophene-based products (SF1, SF2, SF3 and SF4) comparison between NPTZ-P1 and PTZ-P3, the change of
involved in this study were elaborately synthesized with other substituted position in phenothiazine almost has no impact on
method as references in the following detection of absorption the reductive cleavage of Ca=Cb bond mediated by H2S in current
and fluorescent spectra. The detailed synthetic procedures study. As a result, this was further employed for the rational
were illustrated in experimental section and they all were well design of a dual-color fluorescent probe which is similar with
characterized (Fig. S31-S38, ESI†). As illustrated in Fig. 2B, 2C PTZ-P6. According to previous study about PTZ-P6, NPTZ-P2
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and 2F, the absorption and fluorescent spectra of PTZ-P1, PTZ- was synthesized by condensation of a red emitting part, namely
46
P2 and PTZ-P5 were almost no changes in the absence and BOBPY, which was first reported by Jiao and co-workers
.
presence of H2S, which presumably indicated the poor reactivity Then, upon detecting H2S, both green and red emitting
of PTZ-P1, PTZ-P2 and PTZ-P5 with H2S. This is consistent with fluorophores will be released after reductive cleavage of Ca=Cb
the above-mentioned reaction results. Moreover, the bond, which could be utilized for effective sensing H2S.
absorption and fluorescent spectra of KZ-P and NNP-P
Optical properties
significantly changed after the addition of H2S, which revealed
their active reaction with H2S (Fig. 2G-2H). However, the
fluorescent spectra of KZ-P upon adding H2S are totally different
with SF2, which demonstrated the reaction is not concerned
with reductive cleavage of Ca=Cb bond. The similar results are
also observed for NNP-P. Therefore, we infer that Ca=Cb bond in
KZ-P and NNP-P could not be reductively cleaved by H2S but
could react with H2S through other reactions. For PTZ-P3, PTZ-
P4 and NPTZ-P1, their absorption and fluorescent spectra after
reacting with H2S not only obviously changed comparing with
their absorption and fluorescent spectra before reaction
respectively, but also are similar with that of SF1 and SF4 (Fig.
2A, 2D, 2E and 2I). Their yielding of SF1 and SF4 were
successfully supported by isolation and MS data.
The absorption and fluorescent spectra of probes (PTZ-P1, PTZ-
P2, PTZ-P3, PTZ-P4, PTZ-P5, KZ-P, NNP-P and NPTZ-P1) were
measured in different solvents (Methanol, DMF and THF). As
shown in Fig. S49A-49E (ESI†), the ability of electron accepting
group increased from aminobenzene to terpyridine in the short
series of PTZ-P1 to PTZ-P5, which weakened the strength of ICT
showing reduced absorption peaks at around 500 nm.
Compared with those possessing strong electron donor (KZ-P
and NNP-P), these probes substituted in different position of
phenothiazine (PTZ-P3 and NPTZ-P1) possessed weaker
strength of ICT (Fig. S49C and S49F-S49H, ESI†). In addition, all
absorption spectra displayed no significant shifts in different
solvents, as well as their fluorescent spectra.
N
N
N
N
N
NH
S
S
NH2
S
N
S
N
SH
S
N
S
N
After the study of structure-reactivity relationship, NPTZ-
P1 and NPTZ-P2 were developed as new fluorescent probes,
their absorption and fluorescence spectra toward H2S were
tested in a mixture of PBS and DMSO (pH=7.4, v/v=1:2,
containing 2% PEG 400), using aqueous NaHS as H2S source. As
described in Fig. 4A, NPTZ-P1 showed a main absorption peak
at 325 nm along with an ICT band at around 500 nm. The
absorption intensity at 325 nm decreased upon the gradual
addition of H2S (0–700 μM). However, NPTZ-P1 showed turn-on
green fluorescence at 455 nm with a 15-fold enhancement (Fig.
4B). As shown in Fig. 4E, NPTZ-P2 displayed primary absorption
peaks both at 390 nm and 630 nm. The representative
absorption peaks at 390 nm decreased with the addition of
NaHS (0–600 μM), as the main peaks at 630 nm increased,
which was attributed to reductive cleavage of C=C bond for
releasing BOBPY-related moiety. Because the red fluorescence
was so strong that the weak green fluorescence was covered
with the insignificant ratiometric change (Fig. S50, ESI†). NPTZ-
P2 exhibited very weak fluorescence peak at 635 nm, which
increased with a 20-fold intensity enhancement after gradually
adding NaHS (0–600 μM) (Fig. 4F)
CN
N
N
N
H2
NPTZ-P1
S
MP1
MP2
SF4
Fig. 3 Proposed possible reaction mechanism.
Based on the above results, the possible reaction
mechanism was proposed. As described in Fig. 3, unsaturated
C=C acts as a well-known Michael acceptor, whereas dicyano
group is a strong electron-withdrawing group functioned as an
electrophile. Initially, the Michael addition intermediate MP1
was constructed by H2S. Afterwards, the free thiol-group
attacked cyano group via intramolecular nucleophilic addition
producing a cyclized adduct MP2 38-40. Subsequently, a more
stable product SF4 yielded by rearrangement. This is possible
reaction pathway, while the exact one is still need further
investigation.
In pharmacology, C(2)-substituted phenothiazine was
reported with better activity than that of C(1), C(3) and C(4)-
substituted one, while this was decided by the stronger electron
accepting ability in C(2) position 41-43. Nevertheless, the
substituents in C(3) position of phenothiazine were proved with
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Fig. 4 (A,B) Absorption and fluorescent spectra of NPTZ-P1 (10 μM) in PBS buffer (pH=7.4)/DMSO (1/2 v/v, 2% PEG 400) with
addition of H2S (0–700 μM), λex=330 nm. (C) The corresponding linear relationship between the fluorescent intensity of NPTZ-P1
(10 μM) and different concentrations of H2S (0–700 μM). (D) The fluorescence spectra of NPTZ-P1 (10 μM) in the presence of H2S
(100 μM) and various analytes (100 μM) in PBS buffer (pH=7.4)/DMSO (1/2 v/v, 2% PEG 400), excitation: 330 nm. (E,F) Absorption
and fluorescent spectra of NPTZ-P2 (10 μM) in PBS buffer (pH=7.4)/DMSO (1/2 v/v, 2% PEG 400) with addition of H2S (0–600 μM),
λex=550 nm. (G) The corresponding linear relationship between the fluorescent intensity of NPTZ-P2 (10 μM) and different
concentrations of H2S (0–600 μM). (H) The fluorescence spectra of NPTZ-P2 (10 μM) in the presence of H2S (100 μM) and various
analytes (100 μM) in PBS buffer (pH=7.4)/DMSO (1/2 v/v, 2% PEG 400), excitation: 550 nm.
In comparison to PTZ-P3, NPTZ-P1 with corresponding for H2S versus biological relevant thiols, reactive oxygen species
-
substitute in C(3) position of phenothiazine also displayed (ROS) such as H2O2 and ClO-, ions including K+, Na+, Ca2+, HSO3 ,
2-
effective reductive cleavage of Ca=Cb bond by H2S. The main SO32-, SO4 and S2O32-, and so on. Even if cysteine, cystine or
difference is that NPTZ-P1 exhibits a slower response to H2S for glutathione just induced negligible changes of fluorescent
about 3 h (Fig. S51A and S51C, ESI†). And the similar results intensity. Therefore, both NPTZ-P1 and NPTZ-P2 showed high
were also shown in comparing NPTZ-P2 with PTZ-P6 (Fig. S51B selectivity to H2S.
and S51D, ESI†). Therefore, the differing substitute position in
phenothiazine does not affect the reaction mechanism.
DFT calculation
To better understand the novel reaction, DFT calculation was
provided for the eight selected probes and the optimized
Selectivity study
As an effective strategy for probe design, the selectivity of the molecular structures and frontier molecular orbitals were
two probes will attract more attentions. Subsequently, the shown in Fig. 5 and Table S1 (ESI†). Both the HOMO and LUMO
selectivity of the NPTZ-P1 and NPTZ-P2 towards H2S was strictly of PTZ-P1 arise on dicyano-dimethylaniline group with energy
identified. NPTZ-P1 and NPTZ-P2 (10 μM) was both treated with gap of 2.2110 eV. The HOMO of PTZ-P2, PTZ-P3, PTZ-P4 and
various biological relevant analytes in the mixture of PBS and PTZ-P5 mainly located on phenothiazine moiety, but PTZ-P3
DMSO (pH=7.4, v/v=1:2, containing 2% PEG 400) for 30 mins, and PTZ-P4 are the exception with the LUMO most locating on
respectively. As shown in Fig. 4D and 4H, the turn-on dicyano-pyridine and dicyano-bipyridine group.
fluorescent response of NPTZ-P1 and NPTZ-P2 is highly selective
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Fig. 5 The frontier molecular orbitals energy diagram of PTZ-P1, PTZ-P2, PTZ-P3, PTZ-P4, PTZ-P5, KZ-P, NNP-P and NPTZ-P1 in
vacuum. Transition energies were calculated using the TD-B3LYP method with 6-31G basis sets.
The HOMO of KZ-P mainly located on carbazole and As reported that the level of H2S fluctuated in physiological
dicyano moiety, while the LUMO located on the most part of system as low as 30 μM while as high as 200 μM 47-50. However,
the structure. The HOMO of NNP-P mainly located on N,N- the limit of detection of NPTZ-P1 and NPTZ-P2 for H2S were
48
dimethylaniline and dicyano moiety, and the LUMO located on respectively calculated as 5 μM and 4 μM (ESI†), which are
almost the whole structure. Differently, the HOMO of PTZ-P3 lower than that of physiological levels. Prior to cell study, the
entirely located on phenothiazine moiety, and the LUMO cytotoxicity of NPTZ-P1 and NPTZ-P2 was evaluated in Hela cells
located mainly on dicyano and pyridine group. This indicated by MTT assay. As described in Fig. S52, NPTZ-P1 and NPTZ-P2
that charge-transfer transition from phenothiazine to dicyano exhibited relatively low toxicity from 10 to 60 μM towards Hela
and pyridine group in PTZ-P3. Analogously, the HOMO of NPTZ- cells with excellent viability (>90%). Moreover, NPTZ-P1 and
P1 mainly located on phenothiazine and dicyano moiety, and NPTZ-P2 maintained satisfactory stability across a pH range of
the LUMO located mainly on dicyano-pyridine group. Compared 2.79–8.37 (Fig. S53).
with KZ-P and NNP-P, PTZ-P3 and NPTZ-P1 also exhibited lower
We applied NPTZ-P1 and NPTZ-P2 for monitoring
exogenous H2S in living HeLa cells. Firstly, both groups of HeLa
cells were exposed to 20 μM NPTZ-P1 for 2 h, then NaHS
solution was used as exogenous H2S source at 100 μM,
incubating with only one group of cells for another 2 h.
Compared with cells treated with sole NPTZ-P1, there
presented obvious green fluorescence in cells treatment with
both H2S and NPTZ-P1 (Fig. 6A). We hypothesized that both
green and red fluorophores could be released in NPTZ-P2 after
reductive cleavage of Ca=Cb bond by H2S. The results indicated
that both green and red fluorescence were observed in cells
energy gap as 1.8044 eV and 2.1048 eV. We inferred that the
reductive cleavage of Ca=Cb bond was related to the strength of
electron donating and withdrawing substitutes. In PTZ-P3, PTZ-
P4 and NPTZ-P1, medium electron withdrawing pyridine and
bipyridine as acceptor and strong electron donating
phenothiazine as donor that show different abilities of charge
transfer from donor to acceptor, are suitable for reductive
cleavage of Ca=Cb bond.
Cell imaging
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Journal Name
treatment with both H2S and NPTZ-P2 (Fig. 6B). This is an
ARTICLE
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S
Ammonium acetate,
Acetic acid,C3H2N2
Aldehyde
Piperidine
S
S
CN
CN
View Article OCnNline
N CN
O
H
DOI: 10.1039/D0NJ02307H
N
H
N
H
implication of NPTZ-P2 for dual color bioimaging in the future.
Toluene, reflux
Overnight
Dichloromethane
RT, overnight
R
PTZCN
5
6
7
8
Scheme 2 Synthetic route of intermediates and PTZCN, PTZ-P1,
PTZ-P2, PTZ-P3, PTZ-P4 and PTZ-P5.
9
Synthesis of PTZCN, PTZ-P1, PTZ-P2, PTZ-P3, PTZ-P4 and PTZ-P5
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PTZCN, PTZ-P1, PTZ-P2, PTZ-P3, PTZ-P4 and PTZ-P5 were
synthesized by method reported previously 12, 51-53. A solution
of PTZCN (1.1 mmol) and aldehydes (1 mmol) in
dichloromethane was added with piperidine as catalyst (100
µL). The mixture was stirred at room temperature overnight.
Evaporation of dichloromethane gave the crude product and
purified by silica gel column to get products.
PTZ-P1. Yield 58.05%. 1H NMR (400 MHz, DMSO) δ=8.81 (s, 0H),
7.58 (d, J=9.0, 0H), 7.21 (d, J=15.1, 0H), 7.09 (d, J=7.9, 0H), 7.05–
6.97 (m, 0H), 6.95 (d, J=7.6, 0H), 6.84–6.78 (m, 0H), 6.74 (d,
J=9.0, 0H), 6.70–6.64 (m, 0H), 3.04 (s, 6H). 13C NMR (101 MHz,
DMSO) δ=170.98, 153.40, 150.63, 142.55, 142.55, 141.76,
133.35, 132.22, 132.22, 128.40, 127.03, 127.03, 126.83, 122.91,
122.91, 122.77, 122.07, 120.57, 117.87, 116.01, 115.51, 115.14,
115.14, 114.78, 114.73, 112.50, 74.47. HRMS (ESI): calcd for
C26H20N4S [M+H]+, 421.1408; found, 421.1511.
Fig. 6 Confocal images of exogenous H2S in HeLa cells in the
presence and absence of 100 μM H2S with 20 μM NPTZ-P1 (A)
and NPTZ-P2 (B). Scale bar: 20 μm. Green channel: λex=405 nm,
λem=420-550 nm; Red channel: λex=543 nm, λem=600-750 nm.
PTZ-P2. Yield 45.03%. 1H NMR (400 MHz, DMSO) δ=9.59 (s, 1H),
8.90 (s, 1H), 8.72 (s, 1H), 7.04 (d, J=8.1, 1H), 7.02–6.96 (m, 2H),
6.93 (d, J=8.1, 1H), 6.91–6.86 (m, 2H), 6.79 (d, J=1.7, 1H), 6.74
(ddd, J=8.1, 5.6, 3.4, 2H), 6.67 (dd, J=15.3, 7.9, 2H), 6.53 (d,
J=1.7, 1H). 13C NMR (101 MHz, DMSO) δ=168.20, 158.34,
142.42, 142.38, 141.37, 134.97, 128.52, 128.13, 127.13, 127.04,
126.75, 123.10, 122.20, 119.14, 116.49, 115.80, 115.57, 115.11,
114.96, 113.68, 111.43, 57.02. HRMS (ESI): calcd for C24H15N3OS
[M+H]+, 394.0936; found, 394.1011.
Experimental Section
General materials and methods
Chemicals, such as 2,4-dimethyl-3-ethylpyrrole, pyridine-4-
aldehylde and MgSO4 were provided by Dieckman (Hong Kong)
chemical industry company Ltd (Hong Kong, China). Fetal bovine
serum (FBS) was purchased from Gibco/BRL (Grand Island,
USA). Dulbecco’s modified eagle medium (DMEM) and other
chemicals were obtained from Sigma-Aldrich (St. Louis, USA).
Besides, all other reagents were AR grade and used without
further purification. Distilled water was purified with a Milli-Q
water purification system provided by Millipore (Bedford, USA).
PTZ-P3. Yield 65.13%. 1H NMR (400 MHz, DMSO) δ=8.86 (s, 1H),
8.65 (dd, J=4.5, 1.5, 2H), 7.73–7.63 (m, 3H), 7.17–7.09 (m, 2H),
7.01 (td, J=7.7, 1.4, 1H), 6.93 (ddd, J=12.5, 7.8, 1.6, 2H), 6.79 (td,
J=7.5, 1.2, 1H), 6.74-6.65 (m, 2H). 13C NMR (101 MHz, DMSO)
δ=169.74, 151.06, 145.60, 142.66, 141.72, 141.62, 132.30,
128.70, 128.47, 127.17, 126.83, 123.17, 122.85, 122.67, 121.85,
115.84, 115.18, 114.79, 114.13, 113.33, 83.61. HRMS (ESI):
calcd for C23H14N4S [M+H]+, 379.0939; found, 379.1132.
NMR spectra detection were recorded by a Bruker AV-400
instrument (Bruker, Germany) and reported in ppm downfield.
High resolution mass spectra (HRMS) were obtained by a Xevo
G2-XS QTof spectrometer (Waters, UK). All tested compounds
were determined through a reverse phase C18 column by peak
area integration (Agilent HPLC 1260, USA). UV/Vis absorption
and fluorescent spectroscopy were measured by UV
spectrophotometer (UV-1800, Shimadzu, Japan) and
fluorescence spectrophotometer (FluoroMax-4, Horiba, Japan).
Cell imaging was evaluated with confocal fluorescence
microscopy (LMS 718, Carl Zeiss, Germany).
PTZ-P4. Yield 62.31%. 1H NMR (400 MHz, DMSO) δ=8.87 (s, 1H),
8.76 (d, J=5.1, 1H), 8.71 (dd, J=3.2, 2.4, 1H), 8.62 (s, 1H), 8.40 (d,
J=7.9, 1H), 7.97 (td, J=7.8, 1.8, 1H), 7.78 (dd, J=5.1, 1.6, 1H), 7.72
(d, J=15.7, 1H), 7.49 (ddd, J=7.5, 4.8, 1.1, 1H), 7.29 (d, J=15.7,
1H), 7.14 (d, J=7.9, 1H), 7.01 (td, J=7.7, 1.4, 1H), 6.95 (d, J=1.8,
2H), 6.79 (s, 1H), 6.75 (d, J=1.8, 1H), 6.69 (d, J=1.0, 1H). 13C NMR
(101 MHz, DMSO) δ=169.81, 156.71, 155.07, 150.83, 149.83,
145.78, 142.99, 142.64, 141.64, 137.96, 132.26, 128.81, 128.46,
127.14, 126.82, 125.08, 123.21, 122.93, 122.82, 121.82, 121.15,
This journal is © The Royal Society of Chemistry 20xx
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119.21, 115.86, 115.17, 114.86, 114.16, 113.45, 83.64. HRMS J=8.7, 1.7, 1H), 7.62 (dd, J=8.5, 2.2, 2H), 7.54 (ddd, J=8.2, 7.1,
View Article Online
13
(ESI): calcd for C28H17N5S [M+H]+, 456.1204, found, 456.1383.
1.2, 1H), 7.30 (td, J=7.6, 1.0, 1H), 3.98 (s, 3H), 2.67 (s, 3H).
C
DOI: 10.1039/D0NJ02307H
NMR (101 MHz, Acetone) δ=205.63, 205.43, 205.23, 205.04,
196.17, 143.61, 141.87, 129.03, 126.42, 126.08, 123.00, 122.32,
121.62, 120.42, 119.82, 109.37, 108.50, 29.51, 29.32, 29.13,
28.94, 28.74, 28.66, 28.55, 28.36, 25.79.
5
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8
PTZ-P5. Yield 60.18%. 1H NMR (400 MHz, DMSO) δ=8.88 (s, 1H),
8.74 (dd, J=4.7, 0.8, 2H), 8.69–8.62 (m, 4H), 8.03 (d, J=1.7, 2H),
7.78 (d, J=15.8, 1H), 7.54 (s, 2H), 7.48 (s, 1H), 7.15 (d, J=7.9, 1H),
7.05-6.92 (m, 3H), 6.76 (d, J=1.8, 2H), 6.68 (dd, J=7.9, 1.0, 1H).
13C NMR (101 MHz, DMSO) δ = 169.93, 156.42, 154.96, 149.87,
9
Synthesis of KZ-3 55. KZ-2 (10 mmol, 2.23 g) and
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146.00, 144.30, 142.63, 141.67, 138.04, 132.25, 128.89, 128.44, malononitrile (12 mmol, 0.79 g) dissolved in the mixture
127.12, 126.81, 125.25, 123.25, 122.79, 121.79, 121.46, 119.42, solution of dichloromethane (20 mL) and methanol (10 mL), and
115.91, 115.17, 114.93, 114.17, 113.57, 83.65, 55.39. HRMS then added ammonium acetate (12 mmol, 0.92 g). The reaction
(ESI): calcd for C33H20N6S [M+H]+, 534.1470, found, 533.1627.
mixture was refluxed for about 10 h. The reaction solution was
removed under reduced pressure. The crude product was
purified with chromatography (dichloromethane/hexane=1/1)
N
H
N
Acetic anhydride
AlCl3, DCM, RT
KOH, CH3
DMF, RT
I
N
1
to give the desired product as yellow solid (yield 58.1%). H
O
NMR (400 MHz, Acetone) δ=8.66 (d, J=1.6, 1H), 8.25 (d, J=7.8,
1H), 7.93 (dd, J=8.7, 1.9, 1H), 7.72 (d, J=8.7, 1H), 7.65 (d, J=8.2,
1H), 7.60–7.54 (m, 1H), 7.35–7.29 (m, 1H), 4.00 (s, 3H), 2.82 (s,
3H). 13C NMR (101 MHz, Acetone) δ=205.43, 205.24, 205.16,
204.98, 176.22, 143.18, 141.87, 126.86, 126.59, 125.78, 122.71,
122.49, 121.05, 120.50, 120.10, 114.36, 113.90, 109.58, 109.14,
80.66, 29.51, 29.32, 29.13, 28.94, 28.75, 28.55, 28.36, 23.61.
KZ-1
KZ-2
N
Malononitrile,
O
CN
CN
N
Ammonium acetate
Piperidine
DCM, RT

CN
CN
DCM/EtOH=2:1, 45C
N
KZ-3
KZ-P
N
Scheme 3 Synthetic route of carbazole intermediates and KZ-P.
Synthesis of KZ-P 53. a solution of KZ-3 (2.1 mmol, 0.57 g)
and pyridine-4-aldehylde (2 mmol, 0.21 g) in dichloromethane
was added of piperidine as catalyst (100 µL). The mixture was
stirred at room temperature overnight. Evaporation of
dichloromethane gave the crude product and purified by silica
gel column (dichloromethane/ethyl acetate=10/1) to get KZ-P
Synthesis of KZ-P
Synthesis of KZ-1 36. The mixture of potassium hydroxide (90
mmol, 5.05 g) and carbazole (30 mmol, 5.02 g) in 50 mL N,N-
dimethylformamide was stirred at room temperature for 40
min. Subsequently, a solution of methyl iodide (40 mmol, 5.68
g) was added dropwise with stirring. The resulting reaction
mixture was then stirred at room temperature for 12 h. After
the reaction was completed, the mixture was poured into cold
water. White precipitate was obtained and recrystallized from
ethanol to give KZ-1 (yield 92.5%). 1H NMR (400 MHz, Acetone)
δ=8.16-8.11 (m, 2H), 7.53 (d, J=8.2, 2H), 7.46 (ddd, J=8.2, 7.1,
1.2, 2H), 7.20 (ddd, J=7.9, 7.1, 1.0, 2H), 3.90 (s, 3H). 13C NMR
(101 MHz, Acetone) δ=205.33, 205.25, 205.08, 141.08, 125.63,
122.62, 120.01, 118.71, 108.72, 29.53, 29.34, 29.15, 28.95,
28.76, 28.57, 28.37, 28.33.
1
(yield 62.3%). H NMR (400 MHz, Acetone) δ=8.66 (dd, J=4.5,
1.6, 2H), 8.45 (d, J=1.3, 1H), 8.24 (d, J=7.8, 1H), 7.92 (d, J=15.7,
1H), 7.78 (d, J=8.5, 0H), 7.73–7.61 (m, 1H), 7.58 (ddd, J=8.3, 7.2,
1.1, 0H), 7.33–7.27 (m, 0H), 7.15 (d, J=15.7, 0H), 4.04 (s, 3H). 13
C
NMR (101 MHz, Acetone) δ = 205.43, 205.24, 205.14, 204.98,
171.06, 150.75, 145.80, 142.77, 141.78, 141.71, 129.28, 127.20,
126.81, 123.35, 122.84, 122.38, 121.94, 120.65, 119.92, 114.08,
113.15, 109.46, 109.36, 82.80, 29.52, 29.32, 29.13, 28.94, 28.75,
28.56, 28.36. HRMS (ESI): calcd for C24H16N4 [M+H]+, 361.1409;
found, 361.1487.
Synthesis of KZ-2 54. To a stirred mixture of KZ-1 (20 mmol,
3.62 g) in 30 mL dichloromethane, anhydrous AlCl3 (40 mmol,
5.33 g) was added. Then acetic anhydride (60 mmol, 6.12 g) in
dichloromethane (10 mL) was added at 0 °C with an ice bath.
After that, the reaction was left at room temperature for
overnight. After the reaction was finished, the reaction mixture
was quenched with 1 N HCl and cold water. The mixture was
extracted with dichloromethane for three times. The combined
organic extracts were washed with 1N Na2CO3 aqueous
solution. The organic phase was dried over anhydrous Na2SO4,
filtered, and concentrated in vacuum. The crude product
obtained was purified over silica gel column chromatography
using hexane/dichloromethane (1/1, v/v) to obtain the pure
product KZ-2 as a white solid (yield 68.3%). 1H NMR (400 MHz,
Acetone) δ 8.86 (dd, J=1.7, 0.4, 1H), 8.30–8.26 (m, 1H), 8.15 (dd,
Malononitrile
NC
CN
NC
CN
O
O
Ammonium acetate
Piperidine
DCM, RT

Acetic acid, toluene, 140C
N
N
N
N
N
NNP-1
NNP-P
Scheme 4 Synthetic route of intermediates and NNP-P.
Synthesis of NNP-P
Synthesis of NNP-1 51. To a 100 mL round-bottom was added
malononitrile (50 mmol, 3.30 g), ammonium acetate (10 mmol,
0.77 g), 1-[4-(Dimethylamino)phenyl]ethanone (50 mmol, 8.16
g), acetic acid (55 mmol, 3.30 g), and toluene (50 ml). The round-
bottom was heated to reflux overnight. The reaction was
removed from heat, cooled to room temperature, and then
washed with water. The organic layer was dried with MgSO4 and
8 | J. Name., 2012, 00, 1-3
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concentrated in vacuo. The resulting solid was recrystallized in saturated aqueous solution of NaCl, dried with VNiewa2ASrtOicl4e,Oanlninde
1
DOI: 10.1039/D0NJ02307H
ethanol to get NNP-1 (yield 95.8%). H NMR (400 MHz, CDCl3) concentrated. The residue was purified by flash column
δ=7.68 (d, J=9.1, 2H), 6.69 (d, J=9.1, 1H), 3.09 (s, 3H), 2.58 (s, chromatography on silica gel (dichloromethane/hexane=1/1) to
2H). 13C NMR (101 MHz, CDCl3) δ=172.20, 153.24, 130.32, give PTZ-2 (yield 54.3%). 1H NMR (400 MHz, CDCl3) δ=7.77 (dd,
122.14, 115.17, 114.78, 111.12, 39.99, 22.96.
J=8.5, 2.0, 1H), 7.70 (d, J=2.0, 1H), 7.21–7.15 (m, 1H), 7.13 (dd,
J=7.6, 1.3, 1H), 6.97 (t, J=7.3, 1H), 6.81 (dd, J=14.4, 8.3, 2H), 3.40
(s, 3H), 2.52 (s, 3H). 13C NMR (101 MHz, CDCl3) δ=196.14,
149.90, 144.41, 131.62, 128.61, 127.71, 127.36, 127.28, 123.39,
123.23, 122.67, 114.58, 113.36, 35.66, 26.29.
Synthesis of NNP-P 53. To a solution of pyridine-4-aldehylde
(2 mmol, 0.21 g) and NNP-1 (2.1 mmol, 0.44 g) in
dichloromethane was gradually added several drops of
piperidine. The mixture was stirred at room temperature
overnight. After evaporation of the solvent, the residue was
purified on a silica gel column (ethyl acetate/dichloromethane,
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Synthesis of PTZ-3. The synthesis of PTZ-3 (yield 95.8%) is
similar with NNP-1. 1H NMR (400 MHz, CDCl3) δ=7.53 (dd, J=8.6,
2.3, 1H), 7.32 (d, J=2.3, 1H), 7.23–7.17 (m, 1H), 7.11 (dd, J=7.6,
1.5, 1H), 6.99 (td, J=7.5, 1.1, 1H), 6.84 (dd, J=11.0, 4.7, 2H), 3.41
(s, 3H), 2.56 (d, J=3.2, 3H). 13C NMR (101 MHz, CDCl3) δ=172.58,
149.61, 144.03, 129.30, 128.07, 127.97, 127.35, 126.22, 124.21,
123.69, 122.11, 114.77, 113.70, 113.63, 113.45, 81.52, 35.64,
23.55.
1
1/50, v/v) to afford NNP-P (yield 65.2%). H NMR (400 MHz,
CDCl3) δ=8.67 (dd, J=4.6, 1.4, 2H), 7.64 (d, J=15.7, 1H), 7.45–7.40
(m, 2H), 7.37 (d, J=6.1, 2H), 6.95 (d, J=15.7, 1H), 6.77 (t, J=5.9,
2H), 3.11 (s, 6H). 13C NMR (101 MHz, CDCl3) δ=169.24, 153.04,
150.80, 144.37, 141.68, 131.79, 129.40, 121.75, 119.30, 114.65,
113.84, 111.43, 79.27, 40.06. HRMS (ESI): calcd for C19H16N4
[M+H]+, 301.1409; found, 301.1520.
General procedure for NPTZ-P1 and NPTZ-P2
O
Malononitrile
Piperidine
S
N
S
Acetic anhydride
H3PO4, RT
t-BuOK, CH3
I
S
BOBPY-CHO was synthesized according to our previous study 12
.
N
H
DMF, RT
Acetic acid, EtOH, RT
N
A solution of PTZ-3 (2.1 mmol) and aldehydes (2 mmol) in
dichloromethane was added of acetic acid (100 µL) and
piperidine (100 µL). The mixture was stirred at room
temperature overnight. Evaporation of dichloromethane gave
the crude product and purified by silica gel column to get
products.
PTZ-1
PTZ-2
NC
CN
NC
CN
O
Piperidine
Acetic acid, EtOH, RT
S
S
N

N
N
N
PTZ-3
NPTZ-P1
O
BOBPY-CHO
NC
CN
- O
N
N+
B
S
N
NPTZ-P2
O
- N
Piperidine, acetic acid, EtOH, RT
B
N+
1
NPTZ-P1 (yield 65.8%): H NMR (400 MHz, DMSO) δ=8.64 (dd,
J=4.5, 1.5, 2H), 7.73–7.62 (m, 3H), 7.46–7.37 (m, 2H), 7.32–7.23
(m, 1H), 7.20 (dd, J=7.6, 1.4, 1H), 7.15 (d, J=8.5, 1H), 7.10–6.99
(m, 3H), 3.41 (s, 3H). 13C NMR (101 MHz, DMSO) δ=151.02,
148.78, 145.64, 144.66, 141.80, 130.40, 128.94, 128.63, 128.14,
127.50, 127.02, 123.85, 122.96, 122.67, 121.72, 115.76, 115.16,
114.53, 113.60, 82.65, 40.62, 40.41, 40.20, 39.99, 39.78, 39.58,
39.37, 35.94. HRMS (ESI): calcd for C24H16N4S [M+H]+, 339.1129;
found, 339.1302.
Scheme 5 Synthetic route of phenothiazine intermediates and
NPTZ-P1 and NPTZ-P2.
Synthesis of NPTZ-P1 and NPTZ-P2
Synthesis of PTZ-1 56. To a solution of phenothiazine (40 mmol,
7.96 g) in anhydrous DMF (30 mL) was added 60 mmol of t-BuOK
(6.73 g) in portions, and the mixture was stirred for 3 h at room
temperature. Then, 120 mmol of Iodomethane (17.03 g) was
added dropwise, and the mixture was stirred overnight. After
the addition of water, the mixture was extracted with ethyl
acetate, and the organic phase was washed with a saturated
aqueous solution of NaCl, dried with Na2SO4, and concentrated.
The residue was purified by flash column chromatography on
silica gel (dichloromethane/hexane=1/1) to give PTZ-1 (yield
65.5%). 1H NMR (400 MHz, CDCl3) δ=7.22–7.12 (m, 4H), 6.94 (t,
J=7.5, 2H), 6.82 (d, J=8.0, 2H), 3.38 (s, 1H). 13C NMR (101 MHz,
CDCl3) δ=145.85, 127.45, 127.18, 123.40, 122.47, 114.10, 35.33.
1
NPTZ-P2 (yield 55.7%): H NMR (400 MHz, DMSO) δ=8.33 (d,
J=8.3, 1H), 8.23 (d, J=8.1, 1H), 8.17–8.12 (m, 2H), 7.61 (t, J=7.5,
1H), 7.51–7.41 (m, 2H), 7.32 (dd, J=11.4, 5.1, 3H), 7.30–7.21 (m,
4H), 7.14 (t, J=6.5, 3H), 7.08–6.97 (m, 4H), 6.83 (d, J=15.5, 1H),
3.36 (d, J=6.8, 3H), 2.42 (s, 3H), 2.35 (q, J=7.4, 2H), 2.28 (s, 3H),
0.99 (t, J=7.5, 3H). 13C NMR (101 MHz, DMSO) δ=170.12, 156.47,
150.70, 149.52, 148.48, 144.69, 142.70, 135.34, 134.76, 134.01,
133.11, 132.22, 130.46, 130.18, 129.69, 128.58, 128.40, 128.09,
128.03, 127.44, 127.34, 125.89, 123.75, 122.84, 121.69, 118.59,
115.69, 115.02, 79.40, 56.49, 40.62, 40.42, 40.21, 40.00, 39.79,
39.58, 39.37, 35.86, 31.42, 22.53, 19.03, 17.29, 15.19, 14.44,
13.27, 9.75. HRMS (ESI): calcd for C48H36BN5OS [M], 741.2734;
found, 741.2742.
Synthesis of PTZ-2 57. The reaction mixture (PTZ-1 and
acetic anhydride ratio 20 mmol:200 mmol and phosphoric acid
in catalytic amounts) was heated at 65 °C overnight. After the
addition of water, the mixture was extracted with
dichloromethane, and the organic phase was washed with a
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CN
S
CN
CN
THF, NaHCO3 (aq)
elemental sulfur
added and extracted with ethyl acetate. The orgaVnieicw AlartyicelerOwnlianes
DOI: 10.1039/D0NJ02307H
separated and dried with Na2SO4. After concentrating in vacuo,
the crude product was purified by silica gel column.
R
R
NH2
S
S
N
R=
N
H
N
Computational studies
N
8
9
Scheme 6 Synthetic route of thiophene-based products.
Density functional theory (DFT) calculations of PTZ-P1, PTZ-P2,
PTZ-P3, PTZ-P4, PTZ-P5, KZ-P, NNP-P and NPTZ-P1 were
performed in vacuum. Transition energies were calculated using
the TD-B3LYP method with 6-31G basis sets. DFT calculation of
the lowest 25 singlet-singlet excitation energies was calculated
with a basis set composed of 6-31G (d, p) for C, N, H, O, S atoms.
General procedure for synthesizing thiophene-based products
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The synthesis of thiophene-based products were based on
previous study 58. A mixture of ethylidene malononitrile
derivatives including PTZCN, KZ-3, NNP-1 and PTZ-3 (5 mmol),
elemental sulfur (5 mmol), 20 mL tetrahydrofuran and
saturated solution of NaHCO3 in water (5 mL) was stirred at
room temperature overnight. After completion of reaction, the
solvent was evaporated in vacuo and the crude products were
purified by column chromatography to obtain corresponding
thiophene-based products.
Preparation of the test solution
Sodium hydrosulfide (NaHS) was dissolved in distilled water to
10 mM. A stock solution (1 mM) of NPTZ-P1 and NPTZ-P2 were
both prepared in DMSO. Take NPTZ-P1 for example, test
solutions of NPTZ-P1 were diluted to 2 mL with PBS buffer
(pH=7.4)/DMSO (1/2 v/v, 2% PEG 400), while small aliquots of
NaHS solution was added for detecting UV-vis absorption and
fluorescence property.
SF1 (yield 65.3%): 1H NMR (400 MHz, DMSO) δ=8.73 (s, 1H), 7.25
(s, 2H), 6.95 (s, 2H), 6.91 (d, J=1.8, 2H), 6.83 (d, J=1.7, 1H), 6.77
(t, J=7.5, 1H), 6.71 (d, J=7.3, 1H), 6.45 (s, 1H); 13C NMR (101 MHz,
DMSO) δ=166.88, 142.67, 142.22, 138.39, 134.34, 128.11,
126.83, 126.71, 122.38, 120.91, 116.92, 116.66, 116.54, 115.01,
113.10, 105.23, 83.40.
The solutions of various analytes were prepared from H2O2,
HClO, NaCl, KCl, CaCl2, NaHSO3, Na2S2O3, Na2SO3, Na2SO4, K3PO4,
Phenylalanine, Arginine, Glutamic, Cysteine and Glutathione
respectively. Small aliquots of each testing species solution
were added. The resulting solution was shaken well and
incubated for 30 min at room temperature before recording the
fluorescent spectra.
1
SF2 (yield 85.8%): H NMR (400 MHz, DMSO) δ=8.31 (t, J=1.2,
1H), 8.16 (d, J=7.6, 1H), 7.63 (dd, J=14.4, 4.7, 3H), 7.49 (ddd,
J=8.3, 7.2, 1.2, 1H), 7.27–7.18 (m, 3H), 6.51 (s, 1H), 3.90 (s, 3H).
13C NMR (101 MHz, DMSO) δ=166.69, 141.52, 140.65, 140.03,
126.50, 126.04, 125.47, 122.54, 122.38, 120.78, 119.43, 119.17,
117.41, 109.80, 109.68, 103.99, 84.37, 29.56.
Analysis of Cell Viability by MTT
SF3 (yield 80.8%): 1H NMR (400 MHz, DMSO) δ=7.42–7.35 (m,
2H), 7.13 (s, 2H), 6.79–6.71 (m, 2H), 6.30 (s, 1H), 2.92 (s, 6H). 13
NMR (101 MHz, DMSO) δ=166.61, 150.40, 139.27, 127.97,
122.87, 117.43, 112.62, 102.50, 83.83.
HeLa cells were cultured in complete DMEM medium,
containing 10% fetal bovine serum (FBS) and 1% penicillin-
streptomycin at 37 °C in atmosphere containing 5% CO2. After
90% confluence, the cells were cultured into 96-well plates
(5000 cells/well, 100 μL). Cells were treated with different
concentrations of NPTZ-P1 or NPTZ-P2 (10, 20, 30, 40, 50 and
60 μM) for 24 h. Then, MTT stock solution (5.0 mg/mL, 100 μL)
was added to each well for incubating 4 h to form purple crystal
formazan. At the end of incubation, DMSO (100 μL) was added
for 10 min microvibration after removing medium. The
absorbance was measured at 570 nm on a microplate reader
(Thermo, USA).
C
SF4 (yield 70.5%): 1H NMR (400 MHz, DMSO) δ=7.40 (dd, J=8.4,
2.1, 1H), 7.32 (d, J=2.1, 1H), 7.26–7.20 (m, 3H), 7.18 (dd, J=7.9,
1.5, 1H), 6.99 (dd, J=15.2, 7.6, 3H), 6.50 (s, 1H), 3.34 (s, 3H). 13
NMR (101 MHz, DMSO) δ=166.84, 145.45, 145.33, 137.57,
129.35, 128.37, 127.34, 126.70, 125.40, 123.13, 122.84, 122.08,
117.13, 115.19, 115.07, 104.63, 83.38, 35.66.
C
Reaction confirmation of probes by reacting with NaHS
Cell treatment and imaging exogenous H2S
PTZ-P1, PTZ-P2, PTZ-P3, PTZ-P4, PTZ-P5, KZ-P, NNP-P and NPTZ-
P1 were all used to react with NaHS in DMSO (containing 50%
distilled water) respectively. Here, NPTZ-P1 was taken as an
example. The general procedure is as follows: NPTZ-P1 (0.5
mmol, 0.196 g) was dissolved in 10 mL DMSO and added
dropwise with NaHS (1 mmol, 0.056 g) dissolved in 10 mL
distilled water. Then the mixture was stirred for 4 h at room
temperature. After reaction completed, distilled water was
After 90% confluence, cells were seeded in confocal culture
dishes for 24 h. Aqueous NaHS was used as exogenous H2S
source. Cells were preincubated with 20 μM NPTZ-P1 or NPTZ-
P2 for 2 h. Subsequently, cells were washed with PBS (pH=7.4)
for three times and incubated with H2S (100 μM) for 2 h.
Residual medium was removed by washing with PBS (pH=7.4)
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for three times and fixed. Images were collected by 6. N. Cetraro, R. B. Cody and J. Y. Yew, Analyst, 2019, 144, 5848-
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5855.
DOI: 10.1039/D0NJ02307H
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49, 3214-3216.
We have studied that the structure-reactivity relationship about
H2S mediated reductively cleavage of C=C bond. Through
rational design, different kinds of electron pulling or pushing
groups were used to replace pyridine group, meanwhile
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carbazole,
N,N-dimethylaniline
and
C(3)-substituted
phenothiazine with different electron donating abilities were
employed to replace C(2)-substituted phenothiazine part. The
impact of substitutes on the reductive cleavage of C=C were
investigated for these probes. PTZ-P3 and PTZ-P4 with
moderate electron accepting pyridine and bipyridine, were
proved with effective reductive cleavage of Ca=Cb bond
mediated by H2S, as well as NPTZ-P1 with the C(3)-substituted
phenothiazine. It is worth noting that the reductively cleavage
of Ca=Cb bond significantly affected by the strength of electron
donating and withdrawing substitutes, but not influenced by
the changing of C(2) and C(3) position in phenothiazine. Based
on the study of structure-reactivity relationship, two new
fluorescent probe, NPTZ-P1 and NPTZ-P2, were further
developed and successfully applied for imaging exogenous H2S
in living HeLa cells. As a new strategy for designing fluorescent
H2S probes, this study provides a further understanding of
reaction mechanism and structure-reactivity relationship. This
is also anticipated to promote the development of C=C cleavage
in organic synthesis.
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Conflicts of interest
There are no conflicts to declare
Acknowledgements
This work was funded by the Science and Technology
Development Fund, Macau SAR (File No.: 082/2016/A2,
0114/2019/A2, and 019/2017/AMJ) and the Research Grant of
University of Macau under grant no.: MYRG2019-00051-FHS
and MYRG2017-00066-FHS.
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