One- And Two-Photon-Activated Cysteine Persulfide Donors for Biological Targeting

Article abstract of DOI:10.1021/acs.joc.9b01224

Persulfides have been considered as potential signaling compounds similar to the H₂S in "S-persulfidation", a sulfur-mediated redox cycle. The research of this sulfur-mediated species is hindered because of the lack of efficient persulfide donors. In this current study, we have developed one- and two-photon-activated persulfide donors based on an o-nitrobenzyl (ONB) phototrigger, which releases the biologically active persulfide (N-acetyl l-cysteine persulfide, NAC-SSH) in a spatiotemporal manner. Next, we have demonstrated the detection of persulfide release both qualitatively and quantitatively using the well-known "turn on" fluorescence probe, that is, monobromobimane, and the trapping agent, that is, 2,4-dinitrofluorobenzene, respectively. Furthermore, we examined the cytotoxicity of synthesized persulfide donors on HeLa cells and the cytoprotective ability in the highly oxidizing cellular environment.

Full text of DOI:10.1021/acs.joc.9b01224

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Article
One- and Two-Photon Activated Cysteine
Persulfide Donors for Biological Targeting
Amrita Chaudhuri, Yarra Venkatesh, Joyjyoti Das, Moumita
Gangopadhyay, Tapas K Maiti, and N. D. Pradeep Singh
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01224 • Publication Date (Web): 21 Aug 2019
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One- and Two-Photon Activated Cysteine Persulfide
Donors for Biological Targeting
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Amrita Chaudhuri,†§ Yarra Venkatesh,†§ Joyjyoti Das,‡ Moumita Gangopadhyay,†
Tapas K. Maiti‡ and N. D. Pradeep Singh*†
‡
†Department of Chemistry, Department of Biotechnology, Indian Institute of Technology
Kharagpur, 721302, West Bengal, India.
ABSTRACT: Persulfides (RSSH) have been considered as potential signaling compounds
similar to the H2S in “S-persulfidation”, a sulfur-mediated redox cycle. The research of this
sulfur-mediated species is hindered due to the lack of efficient persulfide donors. In this current
study, we have developed a one- and two-photon activated persulfide donors based on o-
nitrobenzyl (ONB) phototrigger, which releases the biologically active persulfide (N-acetyl L-
cysteine persulfide, NAC–SSH) in a spatiotemporal manner. Next, we have demonstrated the
detection of persulfide release both qualitatively and quantitatively using the well-known “turn
on” fluorescence probe i.e., monobromobimane and the trapping agent i.e., 2,4-
dinitrofluorobenzene, respectively. Furthermore, we examined the cytotoxicity of synthesized
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persulfide donors on HeLa cells and the cytoprotective ability in the highly oxidizing cellular
environment.
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INTRODUCTION:
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Hydrogen sulfide (H2S) is an important small gaseous signaling molecule that has been
recently explored as a novel therapeutic agent for the treatment of cancer, inflammation,
metabolic syndrome, obesity, cardiovascular disease, and neurodegenerative disorders.1 Looking
into the therapeutic prospect and potential of H2S, several research groups started understanding
mechanisms of action that underlie the effects of H2S.2 Since H2S is endogenously produced by
various mammalian tissues at nanomolar concentrations, it is difficult to fully elucidate the
sulfide-based signaling mechanism.3 In order to advance the field of sulfur biology, it is
important to devise chemical tools that facilitate precise exogenous production of various
specific sulfur species to understand the role of these sulfur species at the molecular level.
To enable the exogenous production of various specific sulfur species, several research groups
developed different types of H2S-releasing compounds based on different triggering mechanisms
(e.g., water, pH, thiols, enzymes, and light).4-13 There are three main ways in which H2S exerts
its biological effects: (i) metal center interactions, (ii) reactive oxygen and nitrogen species, and
(iii) persulfidation. Among these, persulfidation has been shown to be an important signaling
pathway involving H2S.14 Protein persulfidation (sometimes referred to as S-sulfhydration) is an
oxidative process, which involves the post-translational modification of cysteine to cysteine
persulfide (thiol -SH is converted to a perthiol -SSH). Recent investigation has shown that much
of the biological activity associated with H2S can be attributed to persulfides and the closely
related polysulfides.15 It has even been suggested that H2S could only be a degradation product
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of persulfides.16,17 Furthermore, persulfidation modulates the biological activity of proteins due
to the decrease in pKa and increase in nucleophilicity of perthiols relative to thiols. In addition,
other findings also support that reactive sulfur species (RSS) such as sulfane sulfurs or
polysulfides are more effective in S-sulfhydration than H2S.18 Hence, there is a clear need for the
investigation of persulfide chemistry in the sulfide-based signaling mechanisms. A major
obstacle in the study of the biological roles of persulfides is a lack of chemical tools capable of
generating well-defined persulfide species in response to specific biologically relevant triggers.
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a) An Esterase-Sensitive Persulfide Donor (Previous work)
O
Enzyme Promoted
SH
Hydrolysis
R2
R1
O
R1
S
S
R1
S
Persulfide
b) Reactive Oxygen Species Responsive Persulfide Donor (Previous work)
S
S
R
H2O2
RSSH
O
B
O
Persulfide
self-immolation
c) Light Activated Persulfide Donor (Present work)
R1
MeO
S
R2SSH
h
S
R2
Persulfide
MeO
NO2
R1 = H, CH3
Figure 1. (a) An esterase-sensitive persulfide donor (b) H2O2 responsive persulfide donor and
(c) Designed light activated persulfide donor based on o-nitrobenzyl phototrigger.
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To date, very few donors have been developed for the release of persulfide.19 Galardon and
co-workers developed a pH-triggered persulfide analog of the nitrosothiol (SNAP).20 Of late,
Wang and co-workers developed esterase-triggered persulfide prodrugs capable of releasing
either a persulfide or hydrogen persulfide (H2S2) (Figure 1a).21-23 Recently, Xian group also
reported a class of pH- and fluoride-dependent persulfide donors.24 In addition, Matson and co-
workers also developed ROS-responsive (ROS: reactive oxygen species), self-immolative
persulfide donor (Figure 1b).25 The main limitation of the abovementioned persulfide donors is
their inability to provide temporal control over the persulfide release.
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Light-triggered persulfide generation from a photochemically active chromophore will be a
suitable activation strategy because it will provide precise spatial and temporal control over the
persulfide release. However, to the best of our knowledge, there is no report in the literature for
the release of persulfide using an external stimulus light. Herein, we designed for the first time
one- and two-photon activated persulfide donors based on o-nitrobenzyl (ONB) phototrigger. To
design photochemically active persulfide donor, o-nitrobenzyl (ONB) phototrigger was chosen
for the following reasons (i) ONB is more widely reported as a phototrigger for caging several
biologically active compounds for its fast and clean photorelease ability. (ii) It can be photolyzed
upon both one- and two-photon excitation (1PE and 2PE) (Figure 1c).26,27 Two-photon uncaging
provides finer spatial resolution and less photo-toxicity with a deeper tissue penetration.28
RESULTS AND DISCUSSION:
Here, we have synthesized two persulfide donors based on o-nitrobenzyl phototrigger, namely
(i) N-acetyl-S-((4,5-dimethoxy-2-nitrobenzyl)thio)cysteine (5a) and (ii) N-acetyl-S-((1-(4,5-
dimethoxy-2-nitrophenyl)ethyl)thio)cysteine (5b). Next, we also synthesized N-acetyl-L-
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(benzylthio)cysteine (7) lacking the ONB group (Scheme 1). The compound 5a is well known
for its two-photon uncaging efficiency and the significance of developing 5b is that it undergoes
rapid and clean photolysis with high quantum efficiency, which will be suitable for the
biomedical applications, compared to 5a under one photon excitation.26 In the present study, we
choose N-acetyl L-cysteine (NAC) due to its biocompatibility and antioxidant properties to
protect cells in highly oxidative environments.25
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Scheme 1. Synthesis of persulfide donors 5a-b and 7
R
R
R
MeO
MeO
MeO
MeO
MeO
MeO
SH
NO2
2 M HCl
MeOH
i. PBr3
SAc
OH
NO2
NO2
ii. KSAc
3a-b
S
1a-b
2a-b
R
COOH
MeO
MeO
COOH
NHAc
MeOH
S
NHAc
3a-b
S
R = H (a)
R = CH3 (b)
N
S
NO2
4
5a-b
COOH
NHAc
MeOH
S
S
COOH
NHAc
SH
S
4
N
S
7
6
In order to synthesize light activated persulfide donors 5a-b, first, compounds 1a-b were
prepared according to literature procedures.27,29 Then, compounds 1a-b were treated with PBr3,
followed by reaction with potassium thioacetate afforded thioacetates 2a-b. Deprotection of
acetyl group was then carried out by refluxing compounds 2a-b in the presence of 2N HCl to
give thiols 3a-b. Next, the treatment of 3a-b with N-Acetylcysteine pyridine disulfide (NAC-
pyDS, 4) gave our target persulfide donors 5a-b. Finally, the compound 7 was obtained by the
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treatment of benzyl mercaptan (6) with NAC-pyDS (4). The product obtained in each step of the
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synthesis was characterized by H NMR, C NMR, and mass spectrometry (Fig. S1–S17 in the
Supporting Information (SI)) and we also provided the purity information of final compounds
(5a, 5b and 7) by RP-HPLC (Fig. S18–S20).
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The photophysical properties of persulfide donors 5a-b (10 µM) were investigated in
ACN/PBS buffer (3:7 v/v) (Figure 2). The absorption spectrum of 5a-b showed a broad
absorption band in the wavelength range of 300-430 nm.
Figure 2. The absorption spectrum of (a) 5a and (b) 5b in ACN/PBS buffer (3:7 v/v) system.
Next, the hydrolytic stability of 5a-b and 7 (100 µM) were tested in ACN/PBS (3:7 v/v, pH =
7.4) containing 10% fetal bovine serum and also in the presence of cellular thiols (1 mM) (e.g;
NAC and GSH) separately for 10 days under dark conditions. The aliquots collected after 10
days were analyzed by HPLC and the results showed that the decomposition of the donors was
less than 20% (average of three runs) (Figure S21 in SI). Therefore, our designed persulfide
donors are sufficiently stable and suitable for biological applications.
To analyze the photouncaging of persufide from 5a-b, a solution of 5a and 5b (100 µM in
ACN: PBS buffer (3:7 v/v); pH=7.4) was irradiated by one-photon excitation (1PE) at λ ≥ 365
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nm, individually. The progress of photodecomposition was monitored by reverse phase (RP)-
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HPLC (Figure S22 in SI). As a proof of concept, we have represented the photolysis of 5b by 1H
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NMR study in CD3OD/D2O (1:1 v/v) solvent system (Figure 3). At 0 min, the H NMR
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spectrum of 5b showed two characteristic signals of aromatic protons at 7.55 ppm (doublet) and
7.19 ppm (doublet). With the gradual increase in an irradiation time, a decrease in the intensity
of the signals at 7.55 and 7.19 ppm was observed, indicating the photodecomposition of 5b. On
the other hand, four new aromatic signals were appeared, corresponding to the photoproduct,
4,5-dimethoxy-2-nitrosoacetophenone (at 7.68 and 7.22 ppm) and might be an adduct, formed
between photoproduct (reactive nitroso acetophenone, 13b)30 and released NAC-SSH (at 7.06
and 6.36 ppm). With the progress of irradiation time, a new signal at 2.12 ppm appeared with an
increase in the intensity, represents the –CH3 proton of free NAC-SSH or NAC (as persulfide
1
species are not enough stable) (see full H NMR spectra Figure S23 in SI). In addition, the 1H
NMR spectra indicated the formation of few minor by-products, possibly due to the further
decomposition of 4,5-dimethoxy-2-nitrosoacetophenone. The major photoproducts obtained
(characterized by NMR study) from 5a and 5b were also characterized using LC-MS analysis by
immediate injection of the photolysis mixture (Figures S24-S27 in SI).
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Figure 3. H NMR study of 5b (CD3OD/D2O 1:1 v/v) during photolysis (0-10 min). Irradiation
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wavelength of λ ≥365 nm.
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From the HPLC data, the photochemical quantum yields (Фp) for the decomposition of 5a and
5b (ACN: PBS buffer; 3:7 v/v) were calculated using potassium ferrioxalate as an actinometer31
and they found to be 0.07 and 0.36, respectively.
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To explore the two-photon uncaging ability of our persulfide donor 5a, we have measured the
two-photon uncaging cross-section (δu) of 5a by comparing the photolysis rate of 5a with that of
DMNB-OAc (4,5-dimethoxy-2-nitrobenzyl acetate) as a reference (δu = 0.035 GM at 730 nm).32
The individual solutions of 5a and DMNB-OAc (100 µM in ACN/PBS buffer, 3:7 v/v, 100 μL),
were irradiated by varying the time (0–30 min) with a 730 nm Ti: sapphire laser (pulse width 100
fs, 80 MHz) emitting at an average of 300 mW. The progress of photolysis was monitored by
HPLC and the first-order decay constants were calculated. Then, the value of δu (where δu=
δa×Φu) was calculated using the formula δaΦu(5a) = δaΦu(reference) × kobs(5a)/kobs(reference) and
the two-photon uncaging cross-section for 5a was found to be ~ 0.033 GM (See Table S1 in SI).
Now, our main focus was to detect and quantify the release of NAC-SSH from 5a and 5b. For
this purpose, we used monobromobimane (mBB) and 2, 4-dinitrofluorobenzene (DNFB) to
detect the released persulfide by qualitative and quantitative fashion, respectively, as shown in
Scheme 2.
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Scheme 2. Quantitative and Qualitative Detection of released NAC-SSH
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O
O
O
O
ACN/PBS (3:7 v/v)
pH = 7.4
N
N
N
N
HOOC
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S
S
AcHN
Br
mBB
Bimane-SS-NAC
COOH
NHAc
S
NO2
HS
ACN/PBS (3:7 v/v)
pH = 7.4
COOH
F
S
AcHN
S
O2N
NO2
NO2
DNFB
DNB-SS-NAC
For the qualitative detection of persulfide, the solution of 5a and 5b (100 µM in ACN/PBS
buffer (3:7 v/v); pH = 7.4) was irradiated, individually. The aliquots collected from the
photolysis mixture at different time intervals were incubated with mBB (100 μM, ACN/PBS
buffer (3:7 v/v); pH = 7.4) for 30 min.16c,33 Then, the emission spectrum of the aliquots was
recorded and we observed a gradual increase in fluorescence intensity at λ= 455 nm with an
increase in irradiation time, indicating the formation of Bimane-SS-NAC (Figure 4 and Figure
S28a in SI), which was further confirmed by mass spectral (MS) analysis (Figure S29, S30 in
SI). Next, compound 7, which lacks the nitro group, was subjected to mBB experiment under
light irradiation for 30 min and the results showed that the compound 7 was unable to produce
Bimane-SS-NAC, indicating that the presence of nitro group is responsible for the generation of
cysteine persulfide (Figure S28b in the SI). Furthermore, we also subjected the persulfide
donors 5a and 5b to the mBB experiment against dark conditions (absence of light). However,
the donors 5a and 5b showed no response towards mBB experiment, suggesting that the
generation of cysteine persulfide is possible only under light irradiation (Figure S28c & S28d in
the SI).
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Figure 4. (a) Qualitative detection of persulfide release by mBB. Emission spectra recorded at a
different interval of irradiation time (0-10 min) after incubation of photolysis mixture of 5b (100
µM) with mBB and (b) % of fluorescence enhancement for 5a (blue dots) and 5b (red dots) by
1PE at different time intervals after incubation with mBB. Excitation wavelength: 380 nm.
Values are presented as mean ± standard deviation (SD).
Next, to detect the persulfide release quantitatively, we carried out the photolysis of 5a and 5b
(100 µM, ACN/PBS buffer (3:7 v/v) at pH= 7.4) in the presence of different concentration of
DNFB [100 μM, 200 μM, 500 μM, 1 mM and 5 mM, ACN/PBS buffer (3:7 v/v, at pH= 7.4)] for
55 min and 20 min, respectively. The photolysis mixture was further incubated for 30 min at 37
°C and it was then analyzed by RP-HPLC to detect NAC-SSH as in the form of DNB-SS-NAC
(Figure S32 in SI). To quantify the amount of persulfide release, the modal compound DNB-SS-
NAC (9) was synthesized by reacting DNFB with NaSH, followed by the treatment of NAC-
pyDS (4) (Scheme 3, Figure S31 in SI).
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Scheme 3: Synthetic Scheme of modal compound DNB-SS-NAC.
COOH
6
7
8
9
NHAc
F
i. NaSH
EtOH/water
S
S
NO2
NO2
ii. MeOH
NHAc
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NO2
NO2
DNB-SS-NAC (9)
S
HOOC
S
N
8
From the HPLC data, the generation of DNB-SS-NAC was quantified as 57 ± 3 μM and 59 ± 4
μM from 5a and 5b, respectively at 5 mM DNFB (Figure 5 and Figure S33). The formation of
DNB-SS-NAC after photolysis was further confirmed by mass spectroscopy (Figure S34 in SI).
To confirm the persulfide release was due to photocleavage of the ONB protecting group, control
experiments were performed using compound 7. As shown in Figure 5 & S33c, the irradiation (λ
≥ 365 nm) of compound 7 (100 µM) in ACN/PBS buffer (3:7 v/v, at pH= 7.4) solution in the
presence of DNFB was incapable to generate DNB-SS-NAC. Henceforth, the compound 7,
which lacks the ONB group does not release persulfide upon photolysis, confirmed that the
carbon-disulfide linkage is not photodegraded under light irradiation.
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Figure 5. Time-dependent quantification of persulfide release from 5a and 5b (100 µM each)
was determined in the form of DNB-SS-NAC. The DNFB concentration is 5 mM. Values are
presented as mean ± SD.
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Furthermore, to show the precise control over the persulfide release from 5a and 5b (100 µM)
by external stimuli light, we monitored the formation of DNB-SS-NAC (in presence of 100 µM
DNFB) under light and dark conditions periodically, respectively. The HPLC data clearly
indicated that only external stimulus light induces the release of persulfide (Figure 6).
Figure 6: Progress of the formation of DNB-SS-NAC from persulfide donors 5a and 5b under
light and dark conditions (ON indicates the start of light irradiation and OFF indicates the end of
light irradiation).
Based on the literature,26 we proposed a possible photorelease mechanism for NAC-SSH from
5a-b (Scheme 4) and it is known to proceeds through the Norrish-type II reaction pathway.
After absorption of a photon by o-nitro benzyl group, it gets excited to singlet state (S1) and then
undergoes rapid intersystem crossing (ISC) to its triplet excited state. In the triplet excited state,
the oxygen atom of the nitro group abstracts a proton from the methylene carbon in the γ-H
position, resulting in intramolecular electron redistribution and formation of the aci-nitro
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tautomer 11. The aci-nitro intermediate then cyclises to form benzisoxazoline intermediate 12.
Thereafter, subsequent decomposition of the resonance stabilized five-membered ring 12 rapidly
generates NAC-SSH and photoproducts (13a-b).
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It has been found from the literature studies that in the case of the ONB-type uncaging
process, the released photoproduct 13 is known to undergo a "self-interaction" with the released
thiol-type species and form a substantial amount of the adduct. This type of undesirable product
formed between 13 and released NAC-SSH can reduce the yield of released persulfide, but not
the rate of release 30c
Scheme 4. Proposed photorelease mechanism for persulfide from ONB phototrigger
3*
R1
R1
MeO
MeO
S
MeO
MeO
S
S
R2
ISC
h

S
R2
S1
OH
NO2
N
O
5a-b
O
R1 = H (a), CH3 (b)
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R1
N
R1
R2
S
MeO
MeO
MeO
MeO
R1
S
MeO
MeO
S
S
O H
R2
O
NO
13a-b
N
O
OH
12
11
R2SSH
Further, to examine the rate and efficiency of persulfide release under biological environment,
we carried out the photolysis experiment in PBS buffer solution containing 5% DMSO and no
significant difference in persulfide release profile was observed compared to ACN/PBS buffer
(3:7 v/v) solution (Table S2).
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To investigate the cytotoxicity of persulfide donors 5a, 5b and compound 7, we carried out the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay using two set of
experiments: i) persulfide donors incubated in HeLa cells without irradiation; ii) persulfide
donors incubated in HeLa cells and subjected to the irradiation (Figure 7). From the MTT assay,
we have found that there is no evidence for inhibition of cell proliferation by 5a, 5b and 7 up to a
concentration of 200 µM before and after photolysis. Therefore, in vitro cytotoxicity studies
showed that persulfide donors 5a-b did not generate substantial toxicity at concentrations up to
200 uM under the studied experimental conditions.
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Figure 7: Cell viability assay of 5a, 5b and 7 on the HeLa cell line: (a) before and (b) after
photolysis. Values are presented as mean ± SD from three independent experiments with
triplicates per experiment.
The persulfide was found to be persuasive to exhibit the cytoprotective effects in a highly
oxidative cellular environment. In this regard, we used persulfide donors 5a-b to rescue the cells
from the highly oxidizing cellular environment. First, we treated the HeLa cells with 200 µM of
H2O2 and found that cell viability was reduced by ~ 50% compared to the cell line without H2O2
treatment. Next, the cells were incubated with persulfide donors 5a-b (200 µM) in the presence
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of 200 µM of H2O2 and then photolysis was carried out. From the MTT assay, we observed that
the cell viability increased with increasing irradiation time and ~ 68% and ~ 71% cell viability
was found for 5a and 5b after photolysis, respectively (Figure 8). Thus, we envisioned that the
persulfide donors 5a and 5b are effective in rescuing cells under oxidative stress.20 In addition,
HeLa cells were also treated with different thiols for e.g; GSH, NAC, HCys, and Na2S prior to
the addition of H2O2 (Figure S35). The cell viability assay demonstrated that NAC-SSH was
comparatively effective in defending cells from the highly oxidative environment than the
studied thiols and H2S. On the other hand, we also carried out the cell viability assay of
compound 7 under the above experimental conditions, and the results showed that compound 7
was incapable to defend cells from the oxidising environment because compound 7 (lacks the
nitro group) does not release NAC-SSH under light irradiation (see Figure S36 in the SI).
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Additionally, to validate our results, a DCFDA staining experiment was also performed using
HeLa cells to realize intracellular ROS generation (oxidative stress) by H2O2 and reduction of
ROS by NAC-SSH. Initially, DCFDA is nonfluorescent in nature. Addition of H2O2 to the HeLa
cells in the presence of DCFDA produces an intense green color fluorescence, which can be
attributed to the formation dichloro fluorescein (DCF) due to the ROS generation. On the other
hand, irradiation of the persulfide donor 5b in the presence of H2O2 for 10 min and subsequent
addition of DCFDA resulted in relatively less intense green color fluorescence, clearly indicating
that the cysteine persulfide, released from donor 5b has the cytoprotective effects in the highly
oxidative environment (Figure S37 in the SI).
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Figure 8. Cell viability assay of (a) 5a and (b) 5b (200 µM) before and after irradiation in
presence of H2O2 (200 µM; to generate highly oxidative environment on HeLa cell line). Cell
viability was evaluated by MTT assay. Data represent the mean (± standard deviation, SD) of
three independent experiments; each performed in triplicates, and is presented relative to control.
Error bars indicate SDs.* significant differences between groups at the level of p<0.05.
In summary, we have synthesized photoactivated persulfide donors 5a and 5b, which release
persulfide NAC-SSH spatiotemporally under one- and two-photon excitation. Next, the
photochemical properties of the persulfide donors under physiological conditions were followed
1
by RP-HPLC, H NMR, and MS analysis. Furthermore, we detected the persulfide release both
qualitatively and quantitatively using the well-known “turn on” fluorescence probe i.e.,
monobromobimane and the trapping agent 2,4-dinitrofluorobenzene, respectively. Finally, we
demonstrated the cytotoxic effect and the cytoprotective ability of persulfide donors in the highly
oxidative cellular environment within the HeLa cells. We intend that the current light-activated
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persulfide donors will help to understand the persulfide biology to a greater extent by providing
insight into sulfur redox cycles and sulfur-mediated cell signaling.
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EXPERIMENTAL SECTION:
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1. General Information: All commercially available anhydrous solvents dimethylformamide
(DMF), dichloromethane (DCM), petroleum ether (PE) and ethyl acetate (EtOAc) and other
chemicals were used without further purification. Acetonitrile and dichloromethane were
distilled from CaH2 before use. NMR spectra were recorded on a 600 and 400 MHz instrument.
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1H NMR chemical shifts were referenced to the tetramethylsilane signal (0 ppm), C NMR
chemical shifts were referenced to the solvent resonance (77.23 ppm, Chloroform-d (CDCl3) and
39.97 ppm, DMSO-d6). Chemical shifts (δ) are reported in ppm, and spin-spin coupling constants
(J) are given in Hz. The following abbreviations were used to explain multiplicities: s = singlet, d
= doublet, t = triplet, q = quadruplet, m = multiplet. UV/vis absorption spectra were recorded on
a Shimadzu UV-2450 UV/vis spectrophotometer and fluorescence spectra were recorded on a
Hitachi F-7000 fluorescence spectrophotometer. High-resolution mass spectra (HRMS) were
recorded on ESI-TOF (electrospray ionization-time-of-flight). Photolysis was carried out using a
125 W medium pressure mercury lamp. RP-HPLC was taken using mobile phase
acetonitrile/water (1:1), at a flow rate of 1 mL /min (detection: UV 310 nm). Chromatographic
purification was done with 60–120 mesh silica gel. For reaction monitoring, precoated silica gel
60 F254 TLC sheets were used. HRMS spectra were recorded by electron spray ionization (ESI)
method on a Q-TOF Micro with lock spray source (High Resolution Q-Tof Mass).
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EXPERIMENTAL PROCEDURE AND SPECTROSCOPIC DATA:
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(4,5-dimethoxy-2-nitrophenyl)methanol (1a)27: Light yellow solid (0.28 g, 93%). H NMR
(400 MHz, Chloroform-d) δ 7.71 (s, 1H), 7.18 (s, 1H), 4.96 (d, J = 5.3 Hz, 2H), 4.01 (s, 3H),
3.96 (s, 3H), 2.63 (t, J = 6.0 Hz, 1H). 13C {1H} NMR (151 MHz, CDCl3) δ 153.9, 147.9, 139.7,
132.3, 111.0, 108.1, 62.8, 56.5 (purity > 98% from HPLC).
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1-(4,5-dimethoxy-2-nitrophenyl)ethan-1-ol (1b) 29: Yellow solid (0.23 g, 94%). H NMR (600
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MHz, Chloroform-d) δ 7.58 (d, J = 2.1 Hz, 1H), 7.33 – 7.31 (m, 1H), 5.58 (qd, J = 6.2, 1.9 Hz,
1H), 4.02 (d, J = 1.6 Hz, 3H), 3.96 (d, J = 1.6 Hz, 3H), 1.57 (dd, J = 6.3, 2.0 Hz, 3H). 13C {1H}
NMR (151 MHz, CDCl3) δ 153.7, 147.7, 139.6, 136.8, 108.5, 107.6, 65.7, 56.4, 24.3 (purity >
98% from HPLC).
S-4,5-dimethoxy-2-nitrobenzyl ethanethioate (2a): PBr3 (1.39 g, 0.48 mL 5.15 mmol)
in DCM was added dropwise to the solution of compound 1a (1 g, 4.7 mmol) in DCM (30
mL) under an N2 atmosphere with external cooling. After stirring 1 h at room temperature
the reaction mixture was poured into H2O (50 mL) and was neutralized with a 2 N
aqueous NaOH solution (100 mL). The organic phase was then separated, and the
aqueous phase was rinsed twice with DCM (50 mL). The solution was then dried over
Na2SO4 and the solvent was removed under vacuum. The residue was purified over silica gel
column (10% EtOAc: hexane) (1.03 g, 80%) as a yellow solid. To the solution of brominated
compound (1 g, 3.62 mmol) in ACN (40 mL), was added potassium thioacetate (0.496 g, 4.34
mmol) and the reaction mixture was stirred for 3 h. White solid formed during the reaction was
filtered away and the filtrate was concentrated by removing the ACN under reduced pressure.
The residue was purified over silica gel column (10 % EtOAc: hexane) to afford compound 2a
(0.90 g, 92%) as a yellow solid. 1H NMR (600 MHz, CDCl3) δ 7.69 (s, 1H), 7.09 (s, 1H), 4.44 (s,
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2H), 4.01 (s, 3H), 3.96 (s, 3H), 2.35 (s, 2H). C{1H} NMR (150 MHz, CDCl3) δ 195.9, 153.3,
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148.2, 140.1, 128.9, 114.0, 108.24, 56.5, 56.4, 31.8, 30.2. HRMS (ESI+) calcd for C11H13NO5S
[M + H]+, 272.0587; found: 272.0594.
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S-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl) ethanethioate (2b): PBr3 (1.31 g, 0.45 mL,
5.15 mmol) in DCM was added dropwise to the solution of compound 1b (1 g, 4.4 mmol)
in DCM (30 mL) under a N2 atmosphere with external cooling. After stirring 1 h at room
temperature the reaction mixture was poured into H2O (50 mL) and was neutralized with
a 2 N aqueous NaOH solution (100 mL). The organic phase was then separated, and the
aqueous phase was rinsed twice with DCM (50 mL). The solution was then dried over
Na2SO4 and the solvent was removed under vacuum. The residue was purified over silica gel
column (5% EtOAc: hexane) to afford compound (0.99 g, 78%) as a dirty yellow solid. To
this solution of brominated compound (0.9 g, 3.44 mmol) in ACN (40 mL), was added potassium
thioacetate (0.43 g, 4.12 mmol) and the reaction mixture was stirred for 3 h. White solid formed
during the reaction was filtered away and the filtrate was concentrated by removing the ACN
under reduced pressure. The residue was purified over silica gel column (10 % EtOAc: hexane)
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to afford compound 3b (0.81 g, 92%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 7.46 (s,
1H), 6.95 (s, 1H), 5.35 (q, J= 6.8 Hz, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 2.25 (s, 2H), 1.68 (d, J= 8
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Hz, 3H). C{1H} NMR (101 MHz, CDCl3) δ 194.4, 152.9, 147.7, 141.3, 132.7, 110.8, 107.62,
56.4, 56.3, 38.8, 30.3, 21.7. HRMS (ESI+) calcd for C12H19N2O5S [M + NH4]+, 303.1009;
found:303.1009.
(4,5-dimethoxy-2-nitrophenyl)methanethiol (3a): The methanolic solution of
compound 3a (0.9 g, 3.32 mmol) was refluxed in presence of 2 N HCl. After 3 h the
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solvent was evaporated under reduced pressure and neutralized by NaHCO3. Then the
aqueous layer was extracted by EtOAc (3 × 15 mL). The combined organic layer was
then dried over Na2SO4 and the solvent was removed under vacuum. The residue was
purified over silica gel column (10% EtOAc: hexane) to afford compound 5a as a yellow solid
(0.67 g, 88%). 1H NMR (400 MHz, CDCl3) δ 7.65 (s, 1H), 6.86 (s, 1H), 4.02 (d, J = 8.5 Hz, 2H),
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3.99 (s, 3H), 3.94 (s, 3H), 2.21 (t, J = 8.4 Hz, 1H). C{1H} NMR (101 MHz, CDCl3) δ 153.5,
148.1, 140.1, 131.9, 112.8, 108.6, 56.4, 26.9. HRMS (ESI+) calcd for C9H15N2O4S [M + NH4]+,
247.0747; found: 247.0741.
1-(4,5-dimethoxy-2-nitrophenyl)ethanethiol (3b): The methanolic solution of
compound 3b (0.8 g, 2.80 mmol) was refluxed in presence of 2 N HCl. After 3 h the
solvent was evaporated under reduced pressure and neutralized by NaHCO3. Then the
aqueous layer was extracted by EtOAc (3 × 15 mL). The combined organic layer was
then dried over Na2SO4 and the solvent was removed under vacuum. The residue was
purified over silica gel column (10% EtOAc: hexane) to afford compound 5b as a yellow solid
(0.60 g, 88%). 1H NMR (400 MHz, CDCl3) δ 7.43 (s, 1H), 7.18 (s, 1H), 5.02 (p, J = 6.4 Hz, 1H),
3.98 (s, 3H), 3.91 (s, 3H), 2.23 (d, J = 5.1 Hz, 1H), 1.66 (d, J = 6.9 Hz, 3H). 13C{1H} NMR (101
MHz, CDCl3) δ 153.3, 147.6, 140.1, 135.7, 109.5, 107.5, 56.4, 32.9, 25.4. HRMS (ESI+) calcd
for C10H17N2O4S [M + NH4]+, 261.0904; found: 261.0902.
2-acetamido-3-(pyridin-2-yldisulfanyl)propanoic acid (4): According to a previously reported
procedure,25 a round bottom flask was charged with N-acetyl-L-cysteine (2.0 g, 12.3 mmol), H2O
(17 mL), and a stirbar to give a clear solution. A solution of 2,2’-dipyridyl disulfide (5.40 g, 24.5
mmol) in MeOH (17 mL) was added in one portion resulting in a clear, yellow solution. The
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reaction mixture was stirred at rt (16 h). Reaction progress was monitored by TLC (EtOAc),
showing complete consumption of starting material. The resulting yellow solution was
concentrated via rotary evaporation. The crude product, obtained as a yellow solid, was then
purified by silica gel chromatography eluting with 5 % to 15 % MeOH in DCM, yielding a light
yellow powder (2.30 g, 69% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.41 (dd, J = 22.5, 5.9 Hz,
2H), 7.83 – 7.72 (m, 2H), 7.26 – 7.21 (m, 1H), 4.46 (d, J = 8.7 Hz, 1H), 3.19 (dd, J = 13.7, 3.9
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Hz, 1H), 3.06 (dd, J = 13.5, 9.1 Hz, 1H), 1.84 (s, 3H). C{1H} NMR (101 MHz, DMSO) δ
172.3, 169.9, 159.2, 150.1, 138.27, 121.7, 119.8, 51.8, 22.8. NMR spectral data are in
accordance with those previously reported.25
3-(2-(4,5-dimethoxy-2-nitrobenzyl)disulfanyl)-2-acetamidopropanoic acid (5a): To a
solution of compound 4a (0.2 g, 0.87 mmol) in methanol NAC-pyDS (4) (0.213 g, 0.78 mmol)
was added. The reaction mixture was stirred for 2 h at rt, monitoring reaction progress with TLC
(50:50; hexane: EtOAc). Once complete, the solvent of the reaction mixture was evaporated in
rotary evaporator to get a crude yellow solid which was further purified by silica gel
chromatography (5% methanol in DCM), yielding a yellow solid (0.26 g, 78 % yield) (purity >
99% from HPLC chromatogram). 1H NMR (600 MHz, CDCl3) δ 7.72 (s, 1H), 6.90 (s, 1H), 6.66
(s, 1H), 4.78 – 4.72 (m, 1H), 4.34 – 4.27 (m, 2H), 4.02 (s, 3H), 3.98 (s, 3H), 3.07 (d, J = 13.6 Hz,
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1H), 2.95 (d, J = 13.4 Hz, 1H), 2.08 (d, J = 8.8 Hz, 3H). C{1H} NMR (151 MHz, CDCl3) δ
172.9, 171.3, 153.2, 148.4, 140.1, 128.0, 113.9, 108.7, 56.7, 56.4, 52.3, 41.8, 39.8, 22.9. HRMS
(ESI+) calcd for C14H19N2O7S2 [M+H]+, 391.0628, found 391.0628.
2-acetamido-3-(2-(1-(4,5-dimethoxy-2-nitrophenyl)ethyl)disulfanyl)propanoic acid
(5b): To a solution of compound 4a (0.2 g, 0.82 mmol) in methanol NAC-pyDS (4) (0.201 g,
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0.73 mmol) was added. The reaction mixture was stirred for 2 h at rt, monitoring reaction
progress with TLC (50:50; hexane: EtOAc). Once complete, the solvent of the reaction mixture
was evaporated in rotary evaporator to get a crude yellow solid which was further purified by
silica gel chromatography (5% methanol in DCM), yielding regioselectively diastereomeric pair
(1:1) (yellow solid, 0.25 g, 78 % yield) (purity > 99% as diastereomeric pair from HPLC
chromatogram). 1H NMR (600 MHz, CDCl3) δ 7.52 (d, J = 2.2 Hz, 1H), 7.06 (d, J = 6.6
Hz, 1H), 6.51 – 6.43 (m, 1H), 5.14 – 5.08 (m, 1H), 4.75 – 4.70 (m, 1H), 4.01 (d, J = 5.1
Hz, 3H), 3.97 (s, 3H), 3.04 – 2.96 (m, 2H), 2.84 (dd, J = 14.0, 6.3 Hz, 1H), 2.08 (dt, J =
6.1, 3.0 Hz, 3H), 1.71 (d, J = 6.9 Hz, 3H). 13C{1H} NMR (151 MHz, CDCl3) δ 171.9, 171.2,
153.2, 153.0, 148.5, 148.3, 140.2, 140.1, 128.29, 127.8, 114.0, 113.9, 108.7, 108.5, 56.7, 56.6,
56.4, 56.3, 52.1, 42.1, 41.8, 39.5, 23.0. HRMS (ESI+) calcd for C15H21N2O7S2 [M+H]+ 405.0785,
found 405.0775.
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N-acetyl-S-(benzylthio)cysteine (7): To a solution of benzyl marcaptan (0.1 g, 0.80 mmol)
in methanol NAC-pyDS (4) (0.196 g, 0.72 mmol) was added . The reaction mixture was stirred
for 2 h at rt, monitoring reaction progress with TLC (50:50; hexane: EtOAc). Once complete, the
solvent of the reaction mixture was evaporated in rotary evaporator to get a grey solid which was
further purified by silica gel chromatography (2% methanol in DCM), yielding a white solid,
0.25 g, 78 % yield) (purity > 99% from HPLC chromatogram). 1H NMR (400 MHz, DMSO-d6)
δ 12.89 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.30–7.32 (m, 4H), 7.26 (m, 1H), 4.46 (dt, J = 8.1, 4.5
Hz, 1H), 3.99 – 3.91 (m, 2H), 2.97 – 2.89 (m, 1H), 2.75 (dd, J = 13.6, 9.3 Hz, 1H), 1.84 (s, 3H).
13C{1H} NMR (101 MHz, CDCl3) δ 172.5, 169.8, 137.8, 129.7, 128.9, 127.7, 51.6, 42.4, 40.1,
22.9. HRMS (ESI+) calcd for C12H16NO3S2 [M+H]+ 286.0572, found 286.0574.
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N-acetyl-S-((2,4-dinitrophenyl)thio)cysteine (DNB-SS-NAC, 9): NaSH (0.15g, 2.68 mmol) in
water was added dropwise to an ethanolic solution of 2,4 dinitrofluorobenzene (0.5g, 2.68
mmol). The resulting solution was stirred 1 h at room temperature. After 1 h, the ethanol was
evaporated in the rotary evaporator and acidified with 1 N HCl to make the pH of the solution to
6. The aqueous fraction was extracted with EtOAc (3 × 30 mL). The organic layers were dried
over Na2SO4, evaporated and purified to get the pure compound (Yield: 0.318g, 60%). To the
resulting yellow solid compound (0.1 g, 0.5 mmol) the methanolic solution of linker 4 (0.12 g,
0.44 mmol) was added and heated to 60°C for 1 h. After 1 h, the methanol was evaporated and
purified by column chromatography to get the yellow solid (Yield: 0.16 g, 90%). 1H NMR (400
MHz, DMSO-d6) δ 8.88 (s, 1H), 8.57 (d, J = 8.8 Hz, 1H), 8.49 (d, J = 8.9 Hz, 1H), 8.27
(d, J = 6.9 Hz, 1H), 4.41 – 4.35 (m, 1H), 3.22 (s, 1H), 3.14 (s, 1H), 1.84 (s, 3H). 13C{1H}
NMR (101 MHz) δ 171.9, 169.9, 145.8, 145.2, 145.0, 129.4, 128.6, 121.9, 52.2, 40.1,
22.9. HRMS (ESI+) calcd for C11H12N3O7S2 [M+H]+ 362.0117, found 362.0111.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
NMR and HRMS spectra of all synthesized compounds, stability studied of donors, All
experimental details of photolysis, qualitative and quantitative analysis, Related emission, HPLC
and LC-MS spectra, Details information of biological studies, Cell viability assay (PDF)
AUTHOR INFORMATION
Corresponding Author
* ndpradeep@chem.iitkgp.ac.in
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Author Contributions
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§These authors contributed equally.
ACKNOWLEDGMENT
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We thank DST SERB (Grant No. EMR/2016/005885) for financial support. Amrita Chaudhuri is
thankful to UGC-New Delhi for fellowship and Yarra Venkatesh is thankful to the Indian
Institute of Technology Kharagpur for the fellowship. We are grateful to S. Bhattacharya
(Physics Department, IIT Kharagpur) for helping us to perform the two-photon uncaging
experiments.
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