Use of phosphorodithioate-based compounds as hydrogen sulfide donors

Article abstract of DOI:10.1016/bs.mie.2014.11.032

Synthetic hydrogen sulfide (H₂S) donors are useful research tools as well as potential therapeutic agents. In this chapter, we report the detailed protocols for the synthesis and evaluation of a series of phosphorodithioate-based H₂S donors. Fluorescence assays were used to determine H₂S release from the donors in both aqueous buffers and in cultured cells. These donors were found to be slow-release donors, much like the well-known GYY4137. These donors also showed some protective effects against hydrogen peroxide (H₂O₂)-induced oxidative damage in myocytes.

Full text of DOI:10.1016/bs.mie.2014.11.032

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SECTION II
Hydrogen Sulfide
Donors and Their
Pharmacological
Activity
125

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ARTICLE IN PRESS
Use of Phosphorodithioate-Based
Compounds as Hydrogen Sulfide
Donors
Chung-Min Park, Ming Xian1
Department of Chemistry, Washington State University, Pullman, Washington, USA
1Corresponding author: e-mail address: mxian@wsu.edu
Contents
1. Introduction
128
2. Synthesis of Phosphorodithioate-Based Donors
2.1 Materials
129
129
130
130
2.2 Synthetic route
2.3 Protocols and data
3. Measurements of H2S Generation from the Donors Using Fluorescence Methods 133
3.1 Materials and instrument
133
134
135
135
136
136
137
138
138
138
139
140
140
140
3.2 Calibration curve for fluorescence measurements
3.3 Determination of H2S release from donors
4. H2S Release from the Donors in Cultured Cells
4.1 Materials
4.2 Cell viability test protocol and results
4.3 Images of H2S release in cells
5. Donor's Activity Against H2O2-Induced Cell Damage
5.1 Materials
5.2 The optimal concentration of H2O2 for cell damage experiments
5.3 Evaluation of donor's protective effects against H2O2 damage
6. Summary
Acknowledgments
References
Abstract
Synthetic hydrogen sulfide (H2S) donors are useful research tools as well as potential
therapeutic agents. In this chapter, we report the detailed protocols for the synthesis
and evaluation of a series of phosphorodithioate-based H2S donors. Fluorescence assays
were used to determine H2S release from the donors in both aqueous buffers and in
cultured cells. These donors were found to be slow-release donors, much like the
well-known GYY4137. These donors also showed some protective effects against hydro-
gen peroxide (H2O2)-induced oxidative damage in myocytes.
#
Methods in Enzymology
ISSN 0076-6879
http://dx.doi.org/10.1016/bs.mie.2014.11.032
2015 Elsevier Inc.
All rights reserved.
127

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1. INTRODUCTION
Hydrogen sulfide (H2S) has been known as a colorless, flammable,
extremely hazardous gas with a rotten-egg smell for many years. However,
this gaseous molecule is recently classified as an important cell signaling
molecule with numerous physiological functions (Fukuto et al., 2012;
Lavu, Bhushan, & Lefer, 2011; Li, Rose, & Moore, 2011; Wang, 2012).
The production of H2S in mammalian systems has been involved in
transsulfuration pathways by at least three endogenous enzymes (Kimura,
2011; Kimura, Shibuya, & Kimura, 2012; Olson, 2012; Predmore,
Lefer, & Gojon, 2012): cystathionine β-synthase, cystathionine γ-lyase,
and 3-mercaptopyruvate sulfur-transferase. L-cysteine (Cys) or L-cysteine
derivatives are nature substrates of these enzymes. In addition, nonenzymatic
H2S production pathways are also known which utilize labile sulfane sulfurs
(compounds containing sulfur atoms bound only to other sulfur atoms) to
generate H2S under reducing conditions or simple degradation ( Jacob,
Anwar, & Burkholz, 2008; Nagy et al., 2014). Although the reported func-
tions of H2S are numerous, the chemical mechanisms behind its activities are
not well established. Nevertheless, several biochemical reactions of H2S are
suggested to be important. These include aerobic redox reactions, reactions
with endogenously generated oxidants like peroxynitrite, superoxide, and
hydrogen peroxide (Chang et al., 2008; Geng et al., 2004; Whiteman
et al., 2004), S-sulfhydration on protein cysteine residues (Krishnan, Fu,
Pappin, & Tonks, 2011; Mustafa et al., 2009; Sen et al., 2012), etc. Increas-
ing literature evidences have demonstrated that H2S, both endogenously
produced and exogenously administered, can have protective effects in
many pathologies such as vasodilation and anti-inflammation (Fukuto
et al., 2012; Lavu et al., 2011; Li et al., 2011; Wang, 2012; Whiteman
et al., 2004). These results strongly suggest that modulation of H2S levels
could have potential therapeutic values.
In H2S research, researchers often use sulfide salts (NaHS or Na2S) as H2S
sources. Sulfide salts are short-lasting H2S donors as they release H2S quickly
(within seconds). Noncontrollable and high-dose exposures may cause
insufficient cardiac output, irregular heartbeat, and conduction abnormali-
ties. In addition, sulfide concentrations in aqueous solution can rapidly
decrease due to volatilization (DeLeon, Stoy, & Olson, 2012), thus signif-
icantly limiting the utility of these chemical precursors.

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Scheme 1 Structures of several representative synthetic H2S donors.
Due to these considerations, researchers have started to use synthetic
H2S-releasing agents (i.e., H2S donors) to explore the biological functions
of H2S (Caliendo, Cirino, Santagada, & Wallace, 2010; Hughes,
Centelles, & Moore, 2009; Kashfi & Olson, 2013; Zhao, Tyler, & Xian,
2014). Several representative donors are shown in Scheme 1, including
(1) GYY4137 (Li et al., 2008), (2) the dithiolthione derivatives (Wallace,
Caliendo, Santagada, Cirino, & Fiorucci, 2007), (3) N-(benzoyl)-
thiobenzamide derivatives (Zhao, Wang, & Xian, 2011), (4) S-acylated per-
thiol derivatives (Zhao et al., 2013), and (5) amino acid-based thioacids (von
Wantoch Rekowski et al., 2010).
GYY4137 is perhaps the most often used donor among these synthetic
donors. The core structure of GYY4137 is the phosphorodithioate template,
which is believed to release H2S upon hydrolysis in aqueous buffers. Most
importantly H2S release from GYY4137 is very slow and only less than 10%
H2S can be generated even after 7 days (Lee et al., 2011). GYY4137 is thus
considered as a slow-release donor. Some studies have revealed that
GYY4137 can exhibit H2S-relevant biological activities such as vasodilation
and antihypertension. (Lee et al., 2011; Li et al., 2008; Yong et al., 2011). To
further expand the scope of GYY4137-like H2S donors, we have recently
developed a series of O-substituted phosphorodithioate-based donors
(PDDs) (Park et al., 2013). In this chapter, we provide a detailed description
of the preparation and evaluation of these donors.
2. SYNTHESIS OF PHOSPHORODITHIOATE-BASED
DONORS
2.1. Materials
Reagents and solvents employed were of the highest grade available.
Reagent grade solvents were used for either chromatography or extraction
without further purification before use. Carbon disulfide (CS2) and 1,8-
diazo[5.4.0]-bicycloundec-7-ene (DBU) were purchased from Aldrich

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and used as received. Elemental Sulfur (S8) was purchased from Alfar
Asar and used directly. All alcohols, amines, and 1,2-ethanedithiol were
purchased from Aldrich and used without further purification. Phospho-
rous trichloride (PCl3) was purchased from Aldrich and freshly distilled
before use. Redistilled diisopropyl ethylamine was purchased from Aldrich
and used without further purification. Biograde dimethyl sulfoxide
(DMSO) and hydrogen peroxide (H2O2, 3% solution) was purchased from
biomedical LLC and J.T. Baker, respectively, and used without further
purification.
2.2. Synthetic route
PDDs were prepared from PCl3 in three steps (Scheme 2). Briefly freshly
distilled PCl3 was treated with 1,2-dithioethane to provide
2-chloro-1,3,2-dithiaphospholane 1. Compound 1 was then treated with
aniline (PhNH2) and followed by sulfurization to give the key intermediate
2. Finally different phenol derivatives were reacted with 2 to furnish the
donors (PDD-1 to PDD-5).
2.3. Protocols and data
2-Chloro-1,3,2-dithiaphospholane 1: Commercially available 1,2-ethanedithiol
(3.56 mL, 42.5 mmol) was added to freshly distilled PCl3 (1.75 g, 127 mmol)
at room temperature under argon atmosphere. The mixture was stirred for 3 h
(HCl bubbles were generated and observed in this reaction). Then the excess
of PCl3 was removed by a simple distillation to yield the product,
2-chloro-1,3,2-dithiaphospholane, as pale yellow oil (6.4 g, 95%). This oil
was stored under argon atmosphere and used for the next step without
1
further purification. H NMR (300 MHz, CDCl3) δ 3.77–3.67 (m, 2H),
Scheme 2 Synthesis of phosphorodithioate-based H2S donors.

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131
3.62–3.52 (m, 2H); 31P NMR (122 MHz, CDCl3) δ 168.39; 13C NMR
(75 MHz, CDCl3) δ 42.99; MS (ESI) m/z calcd for C2H4ClPS2 [M]+
158.0, found 158.0.
2-(Phenylamino)-1,3,2-dithiaphospholane 2-sulfide 2: 2-Chloro-1,3,2-
dithiophospholane (1.1 g, 6.94 mmol) in deoxygenized MeCN (10 mL)
was added dropwise to a stirred solution of aniline (0.63 mL, 6.94 mmol)
and diisopropylethylamine (1.33 mL, 7.63 mmol) in dry, degassed MeCN
(4 mL) at ꢀ40 °C (dry-ice/MeCN bath). After stirring for 1 h under the
same temperature, the reaction mixture was warmed to room temperature
and stirred for additional 1 h (monitored by TLC). A solution of elemental
sulfur S8 (667 mg, 7.63 mmol) dissolved in CS2 (10 mL) was then added into
the reaction mixture. The resultant light yellow heterogeneous mixture was
stirred vigorously overnight. The reaction mixture was filtered and rinsed
with EtOAc. Precipitate formed in the filtrate was filtered again to remove
excess sulfur. Finally, the filtrate was concentrated under reduced pressure
and the crude product was purified by flash chromatography on
preneutralized (1% TEA in hexanes) silica gel to give the desired product
1
as a white solid (1.04 g, 61%). H NMR (300 MHz, CDCl3) δ 7.47–7.01
(m, 5H), 5.52 (d, J¼9.5 Hz, 1H), 3.75–3.28 (m, 4H); 31P NMR
(122 MHz, CDCl3) δ 96.91; 13C NMR (75 MHz, CDCl3) δ 139.11,
129.44, 124.91, 124.89, 123.22, 123.14, 42.09, 42.07; IR (thin film)
3247.0, 2963.6, 1596.5, 1494.6, 1374.5, 1273.0, 1219.8, 1029.6, 912.2,
747.0 cmꢀ1; MS (ESI) m/z calcd for C8H10NNaPS3 [M+Na]+ 270.0,
found 270.1.
General procedure of PDD donors: To a solution of intermediate 2
(1 equiv.) and the corresponding phenol (1 equiv.) in MeCN (total
c¼0.1–0.2 M) at room temperature was added DBU (1 equiv.). The mix-
ture was stirred for 1 h at room temperature. Upon completion, the solvent
was removed under reduced pressure. The crude product was frozen in
diethyl ether using a dry-ice acetone bath then the solid was crashed with
a specular. Solid product was formed under this condition. Diethyl ether
was decanted and the remaining solid was washed with cold diethyl ether
and dried to afford the desire product.
Compound PDD-1 was obtained as white solid (96% yield): mp 110 °C; 1H
NMR (300 MHz, CDCl3) δ 9.60 (s, 1H), 7.34–7.04 (m, 6H), 6.83
(t, J¼6.9 Hz, 1H), 6.73 (d, J¼9.0 Hz, 2H), 5.75 (d, J¼10.0 Hz, 1H),
3.70 (s, 3H), 3.34 (m, 6H), 2.78 (, J¼4.9 Hz, 2H), 1.87 (q, J¼5.9 Hz,
2H), 1.72–1.50 (m, 6H); 31P NMR (122 MHz, CDCl3) δ 94.95; 13C
NMR (75 MHz, CDCl3) δ 166.24, 155.99, 146.67, 142.45, 128.92,

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Chung-Min Park and Ming Xian
123.46, 123.40, 120.19, 118.20, 118.11, 113.99, 113.96, 55.75, 54.67,
48.88, 38.61, 33.03, 29.12, 26.88, 24.11, 19.64.; IR (thin film) 3224.7,
3100.8, 2924.8, 1637.5, 1601.6, 1575.5, 1503.8, 1438.6, 1389.8, 1282.2,
1190.9, 1102.9, 1034.5, 904.1 871.5, 832.4, 777.0, 679.3 cmꢀ1; MS
(ESI, negative mode) m/z calcd for C13H13NO2PS2 [MꢀDBU]ꢀ 310.0,
found 310.2.
1
Compound PDD-2 was obtained in 92% yield: H NMR (300 MHz,
CDCl3) δ 9.88 (br-s, 1H), 8.12 (d, J¼9.1 Hz, 1H), 8.04 (d, J¼9.1 Hz,
1H), 7.55–7.41 (m, 1H), 7.32–7.05 (m, 4H), 6.99–6.78 (m, 2H), 5.85
(d, J¼10.3 Hz, 1H), 3.41 (m, 6H), 2.90–2.70 (m, 2H), 1.94 (q, 2H),
1.80–1.49 (m, 6H); 31P NMR (122 MHz, CDCl3) δ 94.70; 13C NMR
(75 MHz, CDCl3) δ 166.42, 166.16, 129.09, 126.48, 125.05, 122.82,
122.75, 120.86, 118.39, 118.30, 116.54, 54.77, 48.92, 38.62, 33.10,
29.14, 26.90, 24.12, 19.65; IR (thin film) 3228.1, 3105.1, 3036.6,
2932.0, 2855.2, 1643.5, 1586.1, 1513.2, 1488.2, 1383.2, 1335.9, 1285.6,
1220.4, 1158.3, 1106.3, 906.3, 872.2, 753.0, 680.1 cmꢀ1; MS (ESI, nega-
tive mode) m/z calcd for C12H10N2O3PS2 [M–DBU]ꢀ 325.0, found
325.1.
Compound PDD-3 was obtained as white solid (95% yield): mp 108°C; 1H
NMR (300 MHz, CDCl3) δ 8.92 (br-s, 1H), 7.45–7.03 (m, 8H), 6.86
(t, J¼6.9 Hz, 1H), 5.77 (d, J¼10.1 Hz, 1H), 3.37 (m, 6H), 2.81
(m, 2H), 1.92 (m, 2H), 1.81–1.44 (m, 6H); 31P NMR (122 MHz, CDCl3)
δ 94.85, 13C NMR (75 MHz, CDCl3) δ 166.35, 142.12, 131.87, 128.99,
124.71, 124.65, 120.48, 118.29, 118.20, 54.71, 48.92, 38.61, 33.03,
29.17, 26.94, 24.12, 19.67; IR (thin film) 3269.9, 3222.6, 3106.5,
2939.2, 2857.7, 1640.4, 1597.7, 1578.0, 1479.0, 1388.2, 1319.8, 1288.9,
1200.7, 1158.7, 1066.3, 1009.2, 907.7, 866.3, 838.2, 767.7, 723.5, 699.8,
690.9 cmꢀ1; MS (ESI, negative mode) m/z calcd for C12H10BrNOPS2
[M–DBU]ꢀ 357.9, found 358.0.
Compound PDD-4 was obtained as sticky white solid (92% yield): 1H NMR
(300 MHz, CDCl3) δ 10.41 (br-s, 1H), 7.33–7.00 (m, 4H), 6.74 (m, 1H),
3.61–3.19 (m, 8H), 3.19–2.48 (m, 5H), 2.44–2.08 (m, 1H), 1.83 (m, 3H),
1.71–1.41 (m, 9H), 1.48–0.97 (m, 2H); 31P NMR (122 MHz, CDCl3) δ
91.02; 13C NMR (75 MHz, CDCl3) δ 166.14, 142.50, 128.79, 128.52,
119.99, 118.85, 118.07, 70.38, 54.63, 48.87, 42.20, 41.13, 38.49, 35.75,
32.84, 31.83, 29.15, 26.93, 25.68, 24.23, 19.68; IR (thin film) 3219.4,
3091.3, 3035.4, 2928.8, 2856.4, 1642.0, 1598.2, 1495.7, 1444.4, 1382.5,
1321.2, 1285.7, 1204.8, 1105.1, 895.4, 751.5, 692.2 cmꢀ1; MS (ESI, neg-
ative mode) m/z calcd for C12H17NOPS2 [M–DBU]ꢀ 286.4, found 286.3.

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Compound PDD-5 was obtained as sticky white solid (93% yield): 1H NMR
(300 MHz, CDCl3) δ 7.25 (m, 1H), 7.21–6.97 (m, 3H), 6.73 (m, 1H), 5.54
(d, J¼10.2 Hz, 1H), 3.86 (m, 1H), 3.35 (m, 6H), 2.82 (m, 3H), 2.80–2.66
(m, 2H), 1.85 (m, 2H), 1.58 (m, 6H), 1.36–0.98 (m, 6H), 0.98–0.62
(m, 4H); 31P NMR (122 MHz, CDCl3) δ 95.10; 13C NMR (75 MHz,
CDCl3) δ 166.14, 142.60, 128.76, 119.65, 117.67, 117.58, 65.63, 54.63,
48.88, 38.50, 32.84, 31.77, 30.59, 29.14, 26.93, 25.89, 24.21, 22.82,
19.68, 14.29; IR (thin film) 3224.6, 3095.9, 3034.0, 2928.2, 2856.7,
2799.2, 1642.1, 1598.4, 1495.9, 1381.8, 1321.1, 1285.4, 1228.2, 1204.8,
1105.3, 894.8, 750.7, 692.2 cmꢀ1; MS (ESI, negative mode) m/z calcd
for C12H19NOPS2 [M–DBU]ꢀ 288.1, found 288.2.
3. MEASUREMENTS OF H2S GENERATION FROM THE
DONORS USING FLUORESCENCE METHODS
In previous studies of phosphorodithioate-type donors like
GYY4137, the standard methylene blue assay (Fogo & Popowsky, 1949;
Lawrence, Davis, & Compton, 2000) was often used to determine H2S pro-
duction. In this assay, H2S released from donors is first rapidly trapped with
Zn(OAc)2 to form ZnS. The trapped H2S is then released under highly
acidic conditions and reacts with N,N-dimethyl-p-phenylenediamine in
the presence of FeCl3 to form the methylene blue dye which can be easily
detected by UV/vis at 670 nm. Although this method is convenient it
is not appropriate for the evaluation of phosphorodithioate-based donors.
It has been reported that the hydrolysis of phosphorodithioates is
pH-dependent. Acidic conditions can significantly enhance hydrolysis
(Ora, Jarvi, Oivanen, & Lo€nnberg, 2000) so methylene blue assay is likely
¨
to give false positive results for phosphorodithioate-based donors. Because
of this problem, we envisioned that fluorescence methods would be useful.
Recently, a number of H2S-specific fluorescent probes have been reported
(Lin & Chang, 2012; Lippert, 2014; Peng et al., 2012; Peng & Xian, 2014;
Pluth, Baisley, Hammer, & Montoya, 2013) and these detections usually
occur under neutral pH. In this study, we used two H2S fluorescent probes
(DNS-Az and WSP-1, Scheme 3) to measure H2S generation from the
donors.
3.1. Materials and instrument
DNS-Az was synthesized using the known method (Peng et al., 2011).
WSP-1 was synthesized as previously described (Liu et al., 2011). WSP-1

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Chung-Min Park and Ming Xian
Scheme 3 Fluorescent probes used in this study.
can also be purchased from Cayman Chemicals Co. Sodium sulfide non-
ahydrate (Na2Sꢁ9H2O) (>98.0%) was purchased from Acros and used with-
out further purification. Sodium phosphate buffer was freshly prepared and
used. The stock solution of DNS-Az in absolute ethanol (20 mM) was
freshly prepared and stored in a refrigerator. The stock solution of Na2S
(2 mM) in sodium phosphate buffer (20 mM, pH 7.4) was freshly prepared,
sealed, and kept in ice-bath. The stock solution of each donor (20 mM) in
biograde DMSO was freshly prepared, sealed, and kept in a refrigerator.
Cary Eclipse Fluorescence spectrophotometer (Agilent Technologies) was
used to measure fluorescence changes.
3.2. Calibration curve for fluorescence measurements
A 4-mL cuvette was charged with 3.94 mL mixture solution of sodium
phosphate buffer (20 mM, pH 7.4) and CH3CN (v/v, 1:1). To this solution
was then added 40 μL stock solution of DNS-Az (20 mM) and a requisite
volume of Na2S stock solution (4, 8, 12, 16, and 20 μL, respectively).
The final volume of the reaction solution was adjusted to 4 mL with sodium
phosphate buffer (20 mM, pH 7.4). After mixing thoroughly and then stand-
ing for 2 min at room temperature, the fluorescence intensity of reaction
solution was measured with λex ¼340 nm. In the meantime, a blank solution
containing no testing species sample was prepared and measured under the
same conditions for comparison (Fig. 1).

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Figure 1 Fluorescence emission spectra of DNS-Az (200 μM) with varied concentrations
of Na2S2 (0, 2, 4, 6, 8, 10 μM for curves 1–6, respectively). The reactions were carried out
for 2 min at room temperature in sodium phosphate buffer (20 mM, pH 7.4) with 50%
CH3CN. Data were acquired with excitation at 340 nm. The inset depicts the plot of rel-
ative fluorescence intensity (F/F0) of the reaction system at λex/em ¼340/535 nm against
the corresponding reagent blank (without Na2S), which is the calibration curve for
this study.
3.3. Determination of H2S release from donors
A 4-mL cuvette with a PTFE stopper was charged with 3.94 mL mixture of
sodium phosphate buffer (20 mM, pH 7.4) and CH3CN (v/v, 1:1) followed
by the addition of 40 μL the stock solution of DNS-Az. The solution was
gently shaken followed by the addition of 20 μL of each donor’s stock solu-
tion. After thoroughly mixing and standing for 2 min at room temperature,
the changes in fluorescence emission spectra at 535 nm (λex ¼340 nm) were
then monitored at 15 min intervals for 3 h at room temperature. Finally, the
fluorescence signals were converted to H2S concentrations based on the ref-
erence curve obtained with a series of Na2S standard solutions (Fig. 2).
4. H2S RELEASE FROM THE DONORS IN
CULTURED CELLS
These donors’ H2S release capabilities were evaluated in H9c2 cells. In
this study, we first examined cytotoxicity of three donors PDD-1, PDD-2,
and PDD-3. For comparison, NaHS and GYY4137 were also tested. Based
on cytotoxicity results, PDD-1 was used in cell imagine to prove its H2S
release in cells.

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A
B
Control
PDD-1
H S (µM)
1.0
2
Time (min)
PDD-2
PDD-1
PDD-2
PDD-3
GYY4137
PDD-3
0.8
0.6
0.4
0.2
0
5
0
0
0
0
GYY4137
0.09
0.09
0.05
0.01
30
60
90
120
150
180
0.31
0.51
0.56
0.53
0.55
0.62
0.31
0.51
0.56
0.53
0.55
0.62
0.32
0.25
0.38
0.41
0.62
0.73
0.24
0.23
0.26
0.41
0.51
0.64
0.0
0
30 60 90 120 150 180
Time (min)
Figure 2 (A) Time-dependent H2S releasing profiles of donors PDD-1, PDD-2, PDD-3,
and GYY4137. (B) Data of H2S concentration changes of each donor.
4.1. Materials
Cells: Rat embryonic cardiomyoblasts cell line H9c2 was purchased from the
American Tissue Culture Collection and cultured in Dulbecco’s modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum,
4 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin at
37 °C and with 5% CO2.
Cell viability Kit: Cell Counting Kit-8 (CCK-8) was purchased from
Dojindo Molecular Technologies, Inc. 10% CCK-8 solution in FBS-free
DMEM with 20 U/mL penicillin and 20 μg/mL streptomycin was directly
added to the cells. The cell viability was measured by a microplate reader
(Infinite M1000, TECAN Group Ltd.).
4.2. Cell viability test protocol and results
Cell viability was measured using CCK-8. H9c2 cells were cultured in
96-well plates, with four duplicate wells in each group. When 80–90% con-
fluence was reached, cells were treated with each donor (20 mM stock solu-
tions were prepared in DMSO) at different concentrations in 1% DMSO in
medium and incubated for 24 h. Then CCK-8 solution (10 μL) at a 1:10
dilution with FBS-free DMEM/F12 (100 μL) was added to each well
followed by a further 3 h incubation under 5% CO2 at 37 °C. Absorbance
was automatically measured at 450 nm with a microplate reader (Infinite
M1000, TECAN Group Ltd.). The mean optical density (OD, absorbance)
of four wells in the indicated groups was used to calculate the percentage of
cell viability as follows: percentage of cell viability¼(Atreatment ꢀAblank)/
(Acontrol ꢀAblank)ꢂ100% (where, A¼absorbance). Values were plotted
by averaging duplicate wells. Figure 3 shows the results of three donors

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137
120
110
100
90
80
70
60
50
40
30
20
10
0
50 µM 100 µM 200 µM
Control
VEH
PDD-1
PDD-2 PDD-3 GYY4137 NaHS
H2S donors
Figure 3 Effects of H2S donors on cell viability. H9c2 cells were treated with different
concentrations of PDD-1, PDD-2, PDD-3, GYY4137, and NaHS for 24 h. CCK-8 assay
was performed to measure cell viability. Reprinted with permission from Park et al.
(2013). Copyright 2013 Molecular BioSystems.
(PDD-1, PDD-2, and PDD-3). For comparison, GYY4137 and NaHS
were also tested and their results are included in Fig. 3.
4.3. Images of H2S release in cells
PDD-1 was selected as the example due to its low cytotoxicity. H9c2 cells
were incubated with PDD-1 at different concentrations (0, 100, 200 and
400 μM) for 24 h. Then a H2S-specific fluorescent probe, WSP-1 (Liu
et al., 2011), was introduced into the cells to monitor the production of
H2S. As expected, donor-treated cells (Fig. 4B–D) showed much enhanced
fluorescent signals compared to vehicle treated cells (Fig. 4A).
Method and instrument: H9c2 cells were incubated in 24-well plates in
medium for 3 days (> 90% confluence). After removing the fetal-serum
containing medium, the cells in each well were treated with different con-
centrations (0, 100, 200, and 400 μM) of PDD-1 (20 mM stock solution in
DMSO) in FBS-free DMEM (0.5 mL) medium and then incubated under
5% CO2 at 37 °C for 24 h. The old medium was removed and then the cells
were incubated for 1 h with 0.5 mL of a fresh medium containing a fluores-
cence probe WSP-1 (final c¼100 μM) and cetyltrimethylammonium bro-
mide (final c¼1 mM). After removing the medium, the cells were carefully
rinsed with PBS buffer (1 mLꢂ1). To each well was added 0.5 mL of PBS
and cell images were taken on EVOS fl microscope (Fig. 4). This experiment
was conducted in triplicate.

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Chung-Min Park and Ming Xian
Figure 4 H2S production of PDD-1 in H9c2 cells. Cells were incubated with vehicle (A),
100 μM (B), 200 μM (C), and 400 μM (D) of donor for 24 h. After removal of excess donor,
100 μM of WSP-1 was added. Images were taken afterwards. Reprinted with permission
from Park et al. (2013). Copyright 2013 Molecular BioSystems.
5. DONOR'S ACTIVITY AGAINST H2O2-INDUCED CELL
DAMAGE
This experiment was designed to investigate the protective effects of
PDD-1, PDD-2, GYY4137, and NaSH against H2O2-induced cell dam-
age in H9c2 cardiomyoblasts.
5.1. Materials
30% H2O2 was purchased from J.T. Baker and used without further puri-
fication. Biograde DMSO was purchased from biomedical LLC and directly
used. CCK-8 was purchased from Dojindo Molecular Technologies, Inc.
10% CCK-8 solution in FBS-free DMEM with 20 U/mL penicillin and
20 μg/mL streptomycin was prepared and directly added to the cell. The cell
viability was measured by a microplate reader (Infinite M1000, TECAN
Group Ltd.).
5.2. The optimal concentration of H2O2 for cell damage
experiments
To examine H2O2 induced injury, cells were exposed to different concen-
trations of H2O2 (50, 100, 150, 200, 300, 400, 500, 600, 700, 1000 μM) in

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A
B
100
75
50
25
0
0
50 100 150 200 300 400 500 600 700 1000
Concentration of H2O2 (mM)
Figure 5 Effects of H2O2 on H9c2 cell viability. The cells were treated with increasing
concentrations of H2O2 (0–1000 μM) for 5 h. (A) H9c2 cell viability measured by
CCK-8 assay. (B) Picture of H9c2 cell viability in a 96-well plate.
FBS-free DMEM medium and incubated with 5% CO2 at 37 °C for 5 h.
Cells exposed to highly concentrated H2O2 (>400 μM, see graph) were
not healthy, detached, and dead. This observation was similar to a literature
(Sun, Sun, Wang, Xiao, & Sun, 2011). Following the same procedure for
the CCK-8 kit as stated above, the mean OD (absorbance) was obtained
and used to calculate the cell viability (Fig. 5). The optimal concentration
of H2O2 was decided as 150 μM.
5.3. Evaluation of donor's protective effects against H2O2
damage
When cell confluence (cultured in 96-well plates, four duplicates) reached
80–90%, old media were removed and cells were washed with PBS buffer
(ꢂ1). The cells were treated with 100 μL of freshly prepared donor solutions
in medium which were prepared as follows: the stock solutions of PDD-1,
PDD-2, GYY4137 (20 mM in DMSO) were diluted with FBS-free
DMEM media to prepare solutions in three different concentrations (50,
100, and 200 μM). After incubation for 24 h under 5% CO2 at 37 °C,
3.75 μL of H2O2 (4 mM stock solution in PBS buffer) was directly added
to each well to afford 150 μM H2O2 final concentration. After incubation
for additional 5 h, CCK-8 solution (10 μL) at a 1:10 dilution with FBS-free
DMEM/F12 (100 μL) was added to each well and incubated for additional
3 h under 5% CO2 at 37 °C. Without further treatment, the absorbance
was recorded at 450 nm using a microplate reader. The percentage of
cell viability was calculated according to a following equation (p¼
(Atreatment ꢀAblank)/(Acontrol ꢀAblank)ꢂ100% (where, A¼absorbance)).
Values were plotted by averaging wells duplicated four times. As shown

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Chung-Min Park and Ming Xian
120
110
100
90
80
70
60
50
40
30
20
10
0
100 µM
200 µM
50 µM
Control
VEH
PDD-1
PDD-2 GYY4137
NaHS
Figure 6 H9c2 cell viability after oxidative injury by H2O2 in the presence of donors
PDD-1, PDD-2, GYY4137, and NaHS. Reprinted with permission from Park et al. (2013).
Copyright 2013 Molecular BioSystems.
in Fig. 6, cell viability was reduced by 35% when treated with H2O2 only. In
the presence of donors, however, cell viability increased significantly. These
results suggest that H2S donors indeed have some protective effects against
H2O2-induced injury.
6. SUMMARY
In this chapter, we provide detailed experimental protocols for the
synthesis and evaluation of a series of phosphorodithioate-based H2S donors.
A fluorescence method was used to determine their H2S release capability.
These donors were found to release H2S slowly in aqueous buffers, very sim-
ilar to the well-known GYY4137. In addition, these donors exhibited effec-
tive H2S production in cultured cells (as monitored by a fluorescent probe)
and protective effects against H2O2 induced oxidative damage in cells.
ACKNOWLEDGMENTS
This work was supported by NIH (R01HL116571) and the ACS-Teva USA Scholar Award.
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