Insights into the Mechanism of Thiol-Triggered COS/H2S Release from N-Dithiasuccinoyl Amines

Article abstract of DOI:10.1021/acs.joc.0c00559

The hydrolysis of carbonyl sulfide (COS) to form H2S by carbonic anhydrase has been demonstrated to be a viable strategy to deliver H2S in a biological system. Herein, we describe N-dithiasuccinoyl amines as thiol-triggered COS/H2S donors. Notably, thiol species especially GSH and homocysteine can trigger the release of both COS and H2S directly from several specific analogues via an unexpected mechanism. Importantly, two representative analogues Dts-1 and Dts-5 show intracellular H2S release, and Dts-1 imparts potent anti-inflammatory effects in LPS-challenged microglia cells. In conclusion, N-dithiasuccinoyl amine could serve as promising COS/H2S donors for either H2S biological studies or H2S-based therapeutics development.

Full text of DOI:10.1021/acs.joc.0c00559

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Article
Insights into the Mechanism of Thiol Triggered
COS/H2S Release from N-Dithiasuccinoyl Amines
Shengchao Zhou, Yujie Mou, Miao Liu, Qian Du, Basharat Ali,
Ramprasad Jurupula, Chunhua Qiao, Li-Fang Hu, and Xingyue Ji
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00559 • Publication Date (Web): 04 Jun 2020
Downloaded from pubs.acs.org on June 4, 2020
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Insights into the Mechanism of Thiol Triggered
COS/H2S Release from N-Dithiasuccinoyl Amines
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Shengchao Zhou,a‡ Yujie Mou, b‡ Miao Liu, a Qian Du, b Basharat Ali,a Ramprasad Jurupula,a
Chunhua Qiao,a Li-Fang Hu bc* and Xingyue Ji a*
aCollege of Pharmaceutical Science, Soochow University, Suzhou, Jiangsu 215021,
China.
bJiangsu Key Laboratory of Neuropsychiatric Diseases and Institute of Neuroscience,
Soochow University, Suzhou, Jiangsu 215123, China.
c
Department of Neurology and Suzhou Clinical Research Center of Neurological
Disease, the Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
KEYWORDS: COS donor; H2S prodrug; anti-inflammatory effects; N-dithiasuccinoyl
amines
ABSTRACT: The hydrolysis of COS to form H2S by carbonic anhydrase has been
demonstrated to be a viable strategy to deliver H2S in biological system. Herein, we
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describe N-dithiasuccinoyl amines as thiol-triggered COS/H2S donors. Notably, thiol
species especially GSH and homocysteine can trigger the release of both COS and H2S
directly from several specific analogues via an unexpected mechanism. Importantly, two
representative analogues Dts-1 and Dts-5 show intracellular H2S release, and Dts-5
imparts potent anti-inflammatory effects in LPS challenged microglia cells. In conclusion,
N-dithiasuccinoyl amine could serve as promising COS/H2S donors for either H2S biology
studies or H2S-based therapeutics development.
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R = H, alkyl
O
R
Only COS
No H2S
S
GSH
S
N
O
GSH
R1 = EDG, H2S Yield
R1 = EWG, H2S Yield
R1
COS + H2S
R =
1. Introduction
H2S has now been well-recognized as a gasotransmitter with importance on par with that
of NO and CO.1-3 It is mainly generated endogenously by the catabolism of cysteine and
homocysteine in the presence of cystathionine β-synthase (CBS), cystathionine γ-lyase
(CSE), and cysteine aminotransferase (CAT)/3-mercaptopyruvate sulfur transferase (3-
MST).4 In addition to its important physiological functions, H2S has also been found to
show therapeutic effects against a wide range of human diseases, including CNS
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disorders,5-7 ischemia-reperfusion injury8 and other inflammation related diseases.9-11
However, the translation of H2S to clinical applications has lagged far behind due to the
controversy on the therapeutic potential of H2S and the lack of a pharmaceutical acceptable
delivery form. In some cases, contradictory pharmacological phenotypes were even
reported for H2S, and the underlying reason for this is at least partially associated with the
usage of different H2S donors/prodrugs with varied release rate. For example, mixed results
were reported with the fast releasing donors NaHS or Na2S,12 whereas the phenotype
observed with other H2S donors was primarily anti-inflammatory effects.13 In another
example, ample studies indicated that H2S promoted the proliferation of cancer cells.14,15
However, GYY4137, a H2S donor with sluggish H2S release rate, was reported to induce
apoptosis in some cancer cell lines.16,17 It has been argued that the apoptotic effects
observed with GYY4137 may not be associated with H2S but GYY4137 itself, in that the
majority of GYY4137 stays in its original form due to a sluggish H2S release profile.
Altogether, these results showcased the importance of developing reliable H2S
donors/prodrugs for H2S related biology studies.
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Figure 1. A) A selected subset of H2S donors/prodrugs with direct formation of H2S; B) The
general strategy used for most of the COS donors reported previously; C) New COS/H2S donors
reported in this work with well-defined but yet unexpected mechanism.
A series of H2S donors/prodrugs have been elegantly devised during the past decades with
several of them being investigated in clinical trials.18-20 These donors can be generally
classified into two categories: the ones with direct formation of H2S and the others with
indirect generation of H2S. The former includes H2S donors/prodrugs with a variety of
release mechanisms as exemplified by JK1,21 BW-HP-101,13 N-benzoylthiobenzamides,22
acylpersulfides,23 S-aroylthiooximes24 and gem-dithiols,25,26 among many others (Figure
1A).27-30 These donors/prodrugs have made tremendous contributions to elucidate H2S
biological effects. In comparison, the latter employed a totally different strategy, and it
delivered H2S indirectly by the release of carbonyl sulfide (COS), which can be hydrolyzed
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into H2S in the presence of an ubiquitously distributed enzyme carbonic anhydrase (CA).
The proof-of-concept studies were established by capping the COS in the form of
thiocarbamate by the Pluth group,31,32 and a number of COS donors in response to different
triggers have been elegantly developed following such a strategy. As shown in Figure 1B,
the release mechanism for most of the reported COS donors are quite similar, and they all
relied on either endogenous or exogenous triggers (i.e. Light or chemical) to activate the
masked self-immolative linker for COS release. 33, 34-42 Normally, the release of COS from
these donors is accompanied with the formation of an electrophilic byproduct (i.e. quinone
methide), which may form adduct with biomacromolecules or metabolites in its proximity,
and thereby induce some unwanted biological effects to complicate the deconvolution of
observed biological phenotypes. Very few of COS donors/prodrugs are precedented with
no electrophilic byproducts formed. 43-45 In order to mitigate this limitation, we hereby
describe a class of thiol activated COS/H2S donors with a brand-new release mechanism,
which release two equivalences of COS/H2S without the formation of any electrophilic
byproduct (Figure 1C), and display tuneable release rate with well-defined H2S-spent by-
product. Interestingly, it was unexpectedly found that specific thiols can directly trigger
considerable amount of H2S release from some specific analogues in the absence of
carbonic anhydrase (Figure 1C, path A). Such an observation is well-explained by detailed
mechanism studies, and some conclusive structure and H2S release relationships were
revealed, which provide significant insights into the COS/H2S release mechanism.
Importantly, two representative donors were demonstrated to be able to deliver H2S
intracellularly and recapitalize H2S-associated potent anti-inflammatory effects.
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R1SH
O
O
S
S
R
S
R
N
RNH2 + R1SSR1
N
S
R1
H
COS
O
COS
R1SH
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Figure 2. The mechanism for thiol triggered deprotecion of N-dithiasuccinoyl amines
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N-Dithiasuccinoyl group has been widely used in peptide synthesis for the protection of
amino group, which can be smoothly removed in the presence of thiol and a base with the
formation of two equivalence of COS (Figure 2). Normally, this deprotection is performed
in organic solvent with the utilization of an organic base (i.e. Et3N).46 We postulate that N-
dithiasuccinoyl amines could serve as promising COS donors, provided that the
deprotection and thus COS release can occur under physiological conditions. Actually,
during the preparation of this manuscript, the Pluth group just reported that N-
dithiasuccinoyl amines could serve as thiol activated COS donor via a mechanism shown
in Figure 2.47 However, in this work, our results show that thiol-triggered COS/H2S release
from N-dithiasuccinoyl amines can go through an alternative pathway, by which direct H2S
formation in the absence of CA is observed. The mechanism studies reveal that the direct
formation of H2S from N-dithiasuccinoyl amines is case sensitive, and is dependent on the
thiol used and the substituent on the nitrogen (R group in Figure 1C): In the absence of CA,
both GSH and homocysteine (Hcy) but not cysteine (Cys) can trigger substantialH2S
release directly from N-dithiasuccinoyl amines with a substituted phenyl ring on N.
However, no H2S release is detected from the analogues with alkyl or proton substitution.
Additionally, two representative COS/H2S donors Dts-1 and Dts-5 showed intracellular
H2S release. Importantly, Dts-5 also recapitulated H2S’s strong anti-inflammatory effects
in BV2 microglia cells.
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2. Results and Discussions
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A)
1) NaH, N,N-dimethylethanolamine, rt;
O
R
S
2) CHCl , formyl thiohypochlorite, rt.
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3
NCS
R
S
N
1a-d
O
Dts-1-4: R = Ph, 4-CF -Ph, 4-OMe-Ph, Bn
3
B)
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O
O
S
Toluene, formyl thiohypochlorite, rt
S
H
N
H
N
1e
O
Dts-5
C)
1) Et O, Et N, formyl thiohypochlorite, rt;
2
3
O
S
S
2) HCl (aq.), reflux.
S
H N
O
HN
2
O
1f
Dts-6
Scheme 1. The synthesis of N-dithiasuccinoyl amines with different substituents
Figure 3. The H2S release profiles of Dts-1 (25 M) in response to different thiol species in the
presence of CA (25 g/mL) at 25 oC in 1% of DMSO/PBS.
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Figure 4. The H2S release profiles of Dts-1 (25 M) in response to different thiol species in the
absence of CA at 25 oC in 1% of DMSO/PBS. ME: mercaptoethanol.
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Several N-dithiasuccinoyl amines Dts-1-6 with different R groups were synthesized
according to previously described methods (Schemes 1A-C).48-50 With those compounds in
hand, we moved to test their COS/H2S release profiles in response to different thiols.
Initially, compound Dts-1 was employed to probe its H2S release profiles under a variety
of conditions, and the methylene blue assay was employed to quantify H2S released. As
expected, a fast H2S release was observed from Dts-1 in response to different thiols
including GSH, cysteine (Cys) and homocysteine (Hcy) in the presence of CA (Figure 3).
The peak H2S concentration can reach around 30 M, indicating more than one equivalence
of COS was released, which is in consistence with the release mechanism (Figure 2). In the
absence of any thiol, a slow H2S release from Dts-1 was also observed, while only
negligible H2S (< 1 M) was generated in the absence of both thiol and CA. Therefore, the
H2S formation in the presence of CA is likely attributed to the chemical hydrolysis of Dts-1,
which is expected to afford COS along with CO2, amine and elemental sulfur. A hydrolysis
mechanism for Dts-1 in PBS is thus proposed (Figure S2).51. Unexpectedly, in the absence
of CA, GSH and Hcy also triggered considerable amount of H2S release with a peak H2S
concentration of around 10 M (Figure 4). Although Cys can also activate direct H2S
release from Dts-1, the peak H2S concentration (4 M) is much lower as compared to that
of GSH or Hcy, and the underlying reason for such a discrepancy remains unclarified at
this stage. Similarly, 2-mercaptoethanol also induced direct H2S release from Dts-1 with a
peak H2S concentration of 5.4 M. Altogether, these results indicated the existence of an
alternative pathway for thiol-induced H2S formation from Dts-1. Noteworthy, the Pluth
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group also reported a different chemotype of COS/H2S donor with independent COS and
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H2S release.43
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R1SH
H
R1SH
N
S
R1
R
S
RNH2 + R1SSR1
A
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O
2
COS
Path
Path
O
R
H
S
S
N
S
R
SR1
R1SH
N
R1SH
COS
O
O
Dts-1
H
H
B
2
N
SR1
+ R1SSH
N
S
R1
R1SSR1
R
R
S
H2S
O
O
2
3a
3b
: R1SH = GSH
: R1
=
HO
Figure 5. Proposed thiol-induced COS/H2S release from compound Dts-1.
To elucidate the COS/H2S release mechanism from Dts-1, a LC-MS experiment was
performed. Compound Dts-1 was incubated with 500 M GSH in 5% CH3CN/PBS, and an
aliquot from this mixture was taken for LC-MS analysis. As shown in Figure S3, an
additional peak (TR = 2.16 min) with a molecular weight of 427.4 was observed in addition
to aniline (TR = 4.59 min), and this new unexpected peak must be closely associated with
the direct H2S formation from Dts-1. Based on these results, a COS/H2S release mechanism
was proposed as illustrated in Figure 5. Initially, R1SH attacks the disulfide bond to yield
intermediate compound 2, which will react further with another molecule of R1SH to
release either COS and aniline (Path A) or R1SSH and compound 3a (Path B). R1SSH from
path B with react further with R1SH to afford H2S directly and R1SSR1. The calculated
molecule weight for compound 3a (427.4 for [M+H]+) matched well with the one observed
in the LC-MS experiment, supporting the proposed H2S release mechanism. In addition,
the recovery rate for aniline is also only 60% because some portion of aniline is trapped in
the form of compound 3a. Since the direct H2S formation induced by cysteine is much
lower than that of GSH, so the recovery rate of aniline increased to 80% accordingly.
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Moreover, when Dts-1 (25 M) was treated with only 1 equivalence of GSH (25 M), a
peak H2S concentration of around 15 M was observed in the presence of CA, while only
negligible (< 1 M) H2S was detected in the absence of CA under the same conditions
(Figure 6). Such results provided additional layer of evidence on step 1 (Figure 5).
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Figure 6. The GSH (25 M) triggered H2S release profiles of Dts-1 (25 M) with or
without CA (25 g/mL) in 1% DMSO/PBS at 25 oC.
To further confirm such a H2S release mechanism, the reaction between 2-
mercaptoethanol and Dts-1 was also studied. Since 2-mercaptoethanol induced very similar
COS/H2S release profiles from Dts-1 as compared to GSH, so the reaction between 2-
mercaptoethanol and Dts-1 should go through the same pathway to that of the reaction
between GSH and Dts-1. As expected, aside from aniline, compound 3b as well as R1SSR1
were also isolated from the reaction mixture, of which the structures were thoroughly
elucidated by NMR and MS. However, we were unable to trap the reactive intermediate
R1SSH with dinitrofluorobenzene due to the coexistence of thiol in the reaction mixture,
which is expected to react with both R1SSH and the trapping reagent. Nevertheless, these
results consolidated the proposed thiol-triggered COS/H2S release mechanism from Dts-1.
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Figure 7. The H2S release profiles of Dts-2-6 (25 ) in response to 500 M of GSH in the
presence of CA (25 g/mL) at 25 oC in 1% of DMSO/PBS.
Figure 8. The H2S release profiles of Dts-2-6 (25 ) in response to 500 M of GSH in the
absence of CA at 25 oC in 1% of DMSO/PBS.
We next turned to probe the COS/H2S release profiles from other analogues. As expected,
compound Dts-2 and Dts-3 showed very similar H2S/COS release profiles in response to
GSH either in the presence or absence of CA (Figure 7 and 8). For Dts-3 with an electron
donating group, the peak H2S concentration for GSH-induced direct H2S formation is only
around 6 M, which is lower as compared to Dts-1, whereas the direct H2S release yield
from Dts-2 with an electron withdrawing group is the highest with a peak concentration of
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around 14 M among all analogues tested. Such a discrepancy in the H2S release yield is
probably attributed to the different substitution on the phenyl ring, which will affect the
electrophilicity of the carbonyl center of intermediate 2. Take Dts-3 as an example, the
carbonyl center of its intermediate 2 is more electrophilic as compare to its counterparts
formed with Dts-1 and Dts-2, and thereby the possibility for GSH to attack the carbonyl
center of compound 2 is increased (Path B), leading to elevated yield in direct H2S
formation. Such an explanation is further supported by the results with analogues Dts-4-6.
Compounds Dts-4-6 showed much slower COS/H2S release as compared to Dts-1-2 under
the same conditions, and very interestingly, no H2S release was detected in the presence of
GSH without CA, indicating that the reactions between GSH and Dts-4-6 go through path
A only (Figure 5). The reaction mixture between GSH and Dts-4 was further taken for
HPLC analysis, and the results showed that benzylamine came as the sole product, and no
GSH adduct product (3) was detected (Figure S5). The reaction between 2-mercaptoethanol
and Dts-4 also gave the same results (Figure S6). As compared to Dts-1, the carbonyl center
of intermediate 2 with alkyl amino substitution (Dts-4-6) is less electrophilic, and thus GSH
will preferably attack the disulfide bond instead of the carbonyl center, resulting in COS
release only (Path A in Figure 5).
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Figure 9. The confocal cell imaging (a) and fluorescence intensity (b) of H2S release from Dts-1
and Dts-5 in BV2 cells (a mouse microglia cell line). Con: Probe only (5 M); NaHS (100 M),
Dts-1 (2.5 M) and Dts-5 (2.5 M). The images were taken under the LSM700 confocal
microscope with the excitation at 488 nm, and the fluorescence intensity was calculated using
ImageJ software. A.U., arbitrary unit. **P<0.01, ***P<0.001, compared to control group. Results
are expressed as mean ± SEM (n = 3 independent experiments for control group, n = 5 independent
experiments for H2S donor treatment). One-way ANOVA followed by Tukey’s post hoc analysis.
Having confirmed the COS/H2S release from compounds Dts-1-6, we next moved to
probe whether these compounds can deliver H2S in a real biological milieu. To this end,
compound Dts-1 and Dts-5 were picked to test their intracellular H2S release and H2S-
associated pharmacological effects. Initially, BV2 microglia cells were cotreated with Dts-
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1/Dts-5 and HSip-1 (a specific H2S fluorescent probe) for 30 min, briefly washed with
PBS, and then subjected to different treatments with H2S donors for 10 min. The images
were taken with the excitation at 488 nm using confocal microscope. As shown in Figure
9, the cells treated with Dts-1/Dts-5 and HSip-1 showed much enhanced fluorescence, as
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compared to the control treated with the probe only. Since the incubation of Dts-1 with
HSip-1 in the absence of thiol species did not yield turn-on fluorescence in a cuvette (data
not shown), precluding the possibility of fluorescence turn-on by Dts-1 itself, these results
indicated intracellular H2S release from compound Dts-1/Dts-5. Noteworthy, 2.5 M Dts-
1/Dts-5 treatment achieved comparable magnitude of enhancement in fluorescent intensity
to that of 100 M NaHS treatment, indicating the advantages of COS/H2S donors with
triggered release mechanism.
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Compound Dts-1 were then tested for H2S-associated anti-inflammatory effects, as
indicated by the suppression of LPS-induced TNF-α production. As shown in Figure 10,
treatment with Dts-1, at the concentration as low as 1 M and 2.5 M, was sufficient to
dose-dependently inhibit LPS-induced TNF-α secretion in BV2 microglia cells, and no
decrease in the cell viability was observed throughout the experiment.
Figure 10. The effect of Dts-1 on LPS-stimulated TNF-α generation (a) and cell viability (b) in
BV2 microglia cells. Cells were pretreated with Dts-1 at different concentrations for 30 min and
then stimulated with 100 ng/ml LPS for 24 h. (a) The TNF-α level in the culture supernatant was
determined using an ELISA kit (n = 6 independent experiments for each group). (b) Cell viability
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was measured using the MTT method (n = 3 independent experiments for each treatment group
with 8 replicates per experiment). **P<0.01, ***P<0.001, compared to LPS-stimulated group.
Results are expressed as mean ± SEM. One-way ANOVA followed by Tukey post hoc analysis,
n.s., not significant.
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3. Conclusion
In conclusion, our work provided significant insights into the mechanism of thiol-triggered
COS/H2S release from N-dithiasuccinoyl amines, and some conclusive structure and H2S
release relationships have been established (Figure 11), which could serve as invaluable
guidelines for the design of new COS/H2S donors. Importantly, these donors are also able
to deliver H2S in a real biological milieu to recapitulate H2S associated anti-inflammatory
effects. Taking together, N-dithiasuccinoyl amines can serve as either a research tool for
H2S related biology or H2S-based therapeutics.
R = H, alkyl
O
R
Only COS
No H2S
S
GSH
S
N
O
GSH
R1 = EDG, H2S Yield
R1 = EWG, H2S Yield
R1
COS + H2S
R =
Figure 11. Concluded structure and H2S release relationships (EDG: electron donating group,
EWG: electron withdrawing group).
4. Experimental
4.1 Chemistry
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General: All reagents and chemicals were reagent grade and were used without further purification.
NMR spectra were recorded on a INOVA NMR spectrometer at 400 MHz for 1H, Agilent NMR
spectrometer at 600 MHz and Bruker AVANCE NEO 400 MHz for 13C at room temperature unless
otherwise specified. UV-Vis absorption spectra were required on a Shimadzu PharmaSpec UV-
2600 UV-Visible spectrophotometer and Infinite M1000 PRO full wavelength microplate reader.
The mechanism studies and copies of NMR spectra can be found in supporting information.
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4.1.1 The synthesis of Dts-1-4
Dts-1-4 were synthesized according to previously reported method.48
Dts-1: white solid (198 mg). Yield: 47%. 1H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3)
δ 7.52 (p, J = 6.4 Hz, 3H), 7.28 (d, J = 7.6 Hz, 2H); MS m/z 211.8 [M+H]+.
Dts-2: White solid (168 mg). Yield: 35%. 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.1 Hz, 2H),
7.45 (d, J = 8.1 Hz, 2H); MS m/z 279.8 [M+H]+.
Dts-3: White solid (145 mg). Yield: 50%. 1H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 7.9 Hz, 2H),
7.03 (d, J = 7.9 Hz, 2H), 3.84 (s, 3H); MS m/z 241.8 [M+H]+.
Dts-4: White solid (158 mg), yield: 52%. 1H NMR (400 MHz, CDCl3) δ 7.41 (s, 2H), 7.34 (d, J =
2.1 Hz, 3H), 4.91 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 167.5, 134.1, 129.0, 128.8, 128.7, 49.3.
MS m/z 225.8 [M+H]+ .
4.1.2 The synthesis of Dts-5 50
To a stirring solution of N-methyl-formamide (300 mg, 1 equiv.) in dry toluene (5 mL),
chlorocarbonylsulfenyl chloride (1.46 g, 2.2 equiv) in dry toluene (5 mL) was slowly added. The
resulting solution was stirred at room temperature under nitrogen atmosphere until the completion
of the reaction, which was monitored by TLC (PE : EA = 20:1). The resulting solution was
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evaporated under vacuum. The crude product was purified by column chromatography (silica gel,
Hexane/EA=40:1) to yield Dts-5 as a yellowish solid (250 mg, yield: 33%). 1H NMR (400 MHz,
CDCl3) δ 3.29 (s, 3H). MS m/z 149.9 [M+H]+.
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4.1.3 The synthesis of Dts-6
Dts-6 was synthesized according to previously reported procedure (120 mg).49 13C NMR (125MHz,
D2O) δ 172.2. MS m/z 135.9 [M+H]+ .
4.1.4 The reaction between 2-mercaptoethanol and Dts-1
Dts-1 (200 mg, 1 equiv.) in 9 mL of CH3CN was added to a stirred solution of 2-mercaptoethanol
(222 mg, 3 equiv) in 2 mL of PBS buffer (100mM, pH = 7.4). After completion of the reaction as
indicated by TLC (PE : EA = 5:1, Figure S4), the reaction mixture was concentrated under vacuum,
and the residue was further extracted with ethyl acetate (20 mL × 3). The obtained organic layer
was dried over anhydrous Na2SO4. After filtration and concentration, the residue was purified on
a silica gel column to get the product B 53 (colorless oil, 130 mg, yield, 90%) and C 54 (3b, colorless
oil, 28 mg, yield 15%) (For TLC, see Figure S4).
1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.50 (d, J = 7.9 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.04 (t, J =
7.2 Hz, 1H), 4.97 (t, J = 5.3 Hz, 1H), 3.53 (dd, J = 12.2, 6.2 Hz, 2H), 2.95 (t, J = 6.6 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ 164.7, 139.0, 128.8, 123.3, 118.9, 60.7, 31.6. MS m/z 197.9 [M+H]+
1H NMR (400 MHz, CDCl3) δ 3.92 (t, J = 5.5 Hz, 4H), 2.89 (t, J = 5.7 Hz, 4H), 2.55 (s, 2H).13C NMR (125
MHz, CDCl3) δ 60.5, 41.4. MS m/z 176.9 [M+Na]+
4.2 H2S Release profiles from Dts-1-6 in the presence/absence of CA
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A solution of GSH/Cys/Hyc/ME (1mM, 1.98 mL in PBS) and CA (50 µg/mL, 1.98 mL in PBS,
CA was replaced with PBS only for H2S release profiles in the absence of CA) was degassed for
10 min at room temperature. Then Dts-1/2/3/4/5/6 (40.0 µL, 2.5 mM in DMSO) was added, and
the resulting mixture was incubated at 25 ℃. For every five minutes, 100 µL of the mixture was
taken and transferred to a 96 well plate containing 100 µL of MB cocktail (zinc acetate (20 µL
1.00% w/v), FeCl3 (40 µL, 30 mM in 1.20 N HCl), and N,N-dimethyl-p-phenylene diamine (40
µL, 20 mM in 7.20 N HCl)). After 30 minutes at room temperature, the absorbance at 676 nm of
each well was recorded using a plate reader. The concentration of H2S released was determined
using the calibration curve as shown in Figure S1.
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4.3 Intracellular H2S Release profiles from Dts-1/Dts-5-treated BV2 microglial cells
BV2 microglia cells were seeded in black 96-well plates with transparent bottoms. After 24 h, cells
were incubated with a highly selective fluorescent probe for H2S HSip-1 DA (5 M, Dojino,
Shanghai, China) for 30 min and briefly washed with PBS twice. Next, cells were subjected to 2.5
M Dts-1/Dts-5 treatment for 10 min. Treatment with 100 M NaHS served as a positive control.
The images were taken under the LSM700 confocal microscope (Zeiss, German) with the
excitation at 488 nm, and the fluorescence intensity was calculated using ImageJ software.
4.4 Anti-inflammatory effects of Dts-1
To test the biological function of Dts-1, BV2 cells were pretreated with Dts-1 at various
concentrations (0.5, 1, 2.5 M) for 30 min and then challenged with lipopolysaccharide (LPS, 100
ng/mL) for 24 hours. The content of the pro-inflammatory cytokine TNF-α in the culture
supernatant was measured using the enzyme-linked immunosorbent assay (ELISA, R&D,
Shanghai, China). To evaluate the impact of cell survival on TNF generation, the cell viability was
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determined using the MTT method. In brief, after removing the culture supernatant for TNF-α
assay at the end of treatment, 100 L fresh medium containing 0.5 mg/ml 3-[4,5-dimethylthiazol]-
2,5-diphenylterazolium bromide assay (MTT) was added and incubated for 4 h. Next, the medium
was replaced with 150 L DMSO, and the absorbance was read at 490 nm using the microplate
reader. The cell viability was calculated and expressed as the percentage of the mean value in the
control group.
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4.5 Statistical analysis
All results are expressed as mean ± SEM. The statistical significance was analyzed using GraphPad
Prism 8 software. Multiple groups comparisons were performed using one-way of variance
(ANOVA) followed by Tukey post hoc analysis. The significance level was defined as P < 0.05.
Supporting Information
The following files are available free of charge.
The mechanism studies along with copies of NMR spectra (PDF).
Corresponding Author
*hulifang@suda.edu.cn (L.F. Hu); jixy@suda.edu.cn (X. Ji).
Author Contributions
‡These authors contributed equally.
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ACKNOWLEDGMENT
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This work was financially supported by National Natural Science Foundation of China
(81773608, 81870997), Suzhou Clinical Research Center of Neurological Disease
(Szzx201503), the Priority Academic Program Development of the Jiangsu Higher
Education Institutes (PAPD) and the Six Major Talents Peak in Jiangsu Province in China
(YY-050, YY-053).
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