Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.

This journal is © The Royal Society of Chemistry 2022

Supplementary Information

for

Thioredoxin reductase-triggered fluorogenic donor of hydrogen sulfide: a

model study with a symmetrical organopolysulfide probe with turn-on near-
infrared fluorescent emission

Sulendar K. Mahato,a Debojit Bhattacherjee,a,b Pallavi Barmana and Krishna P. Bhabaka,b,*

aDepartment of Chemistry, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India.

bCentre for the Environment, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India.
Email: [email protected]

-S1-
Sl. No. CONTENT Page No.
1 Abbreviations S3
2 Experimental section S3-S8
3 Effect of DMSO on the absorption and emission spectral S8-S9
pattern of DCI-OH
4 UV-Vis and Fluorescence Spectroscopic Studies with DCI- S9-S10
DS
5 Summary of the variation of composition of polysulfide S10-S11
alcohol 6 and its effect on H2S release profile.
6 Time-dependent H2S release profile overlay of DCI-PS and S11-S12
DCI-DS in the presence of DTT and GSH.
7 HPLC chromatogram for the reaction of DCI-DS with PhSH S12-S13
and DTT
8 ESI-MS spectra for the reaction mixture of DCI-DS and S13
PhSH
9 HPLC chromatogram for the composition of DCI-PS S14
10 ESI-MS spectra for the reaction mixture of DCI-PS and PhSH S14-S15
11 HPLC chromatogram for the reaction of DCI-PS with DTT S16
12 Dose-dependent anti-proliferative activities of DCI-DS and S16
DCI-PS in MDA-MB-231 cells
13 Fluorescent microscopic images of DCI-DS and DCI-PS in S17
MDA-MB-231 cells
14 Fluorescent microscopic images for WSP2 mediated detection S18
of H2S from DCI-DS and DCI-PS in MDA-MB-231 cells
15 Summary docking parameters for the interactions of DCI-DS S18
and DCI-PS with TrxR1
16 NMR (1H and 13C) and ESI-MS spectra of the intermediates S19-S30
and final compounds
17 References S30

-S2-
Abbreviations
Glu, L-Glutamic acid; Asp, L-Aspartic acid; Ser, L-Serine; Gly, L-Glycine; Met, L-Methionine;
Arg, L-Arginine; Val, L-Valine; Ala, L-Alanine, NaCl, Sodium chloride; H2O2, Hydrogen
peroxide; tBuOOH, tert-butyl hydroperoxide; BSA, Bovine serum albumin; GR, Glutathione
disulfide reductase; GPx, Glutathione peroxidase; Na2S.9H2O, Sodium sulfide nonahydrate;
DNCB, 2,4-Dinitrochlorobenzene, Ph3PAuCl, Chlorotriphenyl phosphinegold(I); NADPH, β-
Nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt; PhSH, Thiophenol; 2-
ME, 2-Mercaptoethanol; Cys, L-Cysteine; GSH; Glutathione reduced; DTT, Dithiothreitol;
TCEP, Tris(2-carboxyl)phosphine.

Experimental section

Compound 2:1 To a solution of 3, 5, 5-trimethyl-2-cyclohexen-1-one (0.20 g, 1.47 mmol) and

malononitrile (0.11 g, 1.72 mmol) in absolute ethanol was added piperidine (13.00 mg, 0.15
mmol) and acetic acid (9.00 mg, 0.15 mmol) under argon atmosphere. The reaction mixture was
refluxed at 90 °C for 12 h upon which the reaction mixture turned dark black in colour. The
progress of the reaction was monitored by TLC analysis. After completion, the mixture was
cooled to room temperature and the solvent was evaporated under reduced pressure and the
residue obtained was dissolved in CH2Cl2. The organic layer was washed with water, brine and
dried over anhy sodium sulfate. The solvent was evaporated to afford the crude product, which
was subjected to silica gel column chromatography using pet ether and dichloromethane as
eluents to afford the pure product 2 as a white solid. Rf = 0.5 (20% dichloromethane in pet ether).
Yield: 0.20 g (74%). 1H NMR (600 MHz, CDCl3) δ (ppm): 6.62 (s, 1H), 2.52 (s, 2H), 2.18 (s,
2H), 2.03 (s, 3H), 1.01 (s, 6H). 13C NMR (150 MHz, CDCl3) δ (ppm): 170.4, 159.9, 120.6, 113.2,
112.4, 78.2, 45.6, 42.6, 32.4, 27.8, 25.3.

Compound DCI-OH:1 To a solution of 4-hydroxybenzaldehyde (0.20 g, 1.07 mmol) in

acetonitrile (20 mL) was added compound 2 (0.14 g, 1.18 mmol) and piperidine (10 μl, 0.10
mmol). The mixture was heated to reflux at 90 °C for 5 h under inert atmosphere and the reaction
mixture turned red in colour. Progress of the reaction was monitored by TLC analysis. Upon
completion, the solvent was removed under reduced pressure. The resulting residue was
dissolved in dichloromethane, washed with water and dried over anhy sodium sulfate. The crude
product was purified by silica gel column chromatography using methanol and dichloromethane
as eluents to afford the pure DCI-OH as an orange solid. Rf = 0.5 (30% ethyl acetate in pet ether).
Yield: 0.20 g (41%). 1H NMR (600 MHz, CDCl3) δ (ppm): 7.42 (d, J = 8.4 Hz, 2H), 7.01 (d, J
-S3-
= 16.2 Hz, 1H), 6.87-6.84 (m, 3H), 6.80 (s, 1H), 2.59 (s, 1H), 2.46 (s, 1H), 1.08 (s, 3H). 13C
NMR (150 MHz, CDCl3) δ (ppm): 169.4, 157.2, 154.4, 136.8, 129.4, 128.6, 127.0, 122.7, 116.1,
113.7, 112.9, 77.7, 43.0, 39.2, 32.0, 28.0. ESI-MS: m/z calcd. for C19H17N2O2 [M−H]−: 289.1346;
obs. [M−H]−: 289.1448.

Compound 3:2 To a solution of 2-mercaptoethanol (0.50 g, 6.40 mmol) in ethyl acetate (5.0 mL)
was added a catalytic amount of NaI (5.00 mg, 0.03 mmol), followed by the dropwise addition
of hydrogen peroxide (30% solution in water, 0.70 mL, 6.4 mmol), upon which the reaction
mixture turned brown in colour. The reaction mixture was stirred at room temperature for
additional 30 min and the colour of the solution turned brownish. The reaction was quenched by
addition of saturated Na2S2O3 solution (10.0 mL). Upon completion of the reaction, the organic
layer was washed with saturated Na2CO3 solution (10.0 mL) followed by brine solution (10.0
mL). The organic layer was collected and dried over anhy. sodium sulfate. The solvent was
evaporated under vacuum to afford compound 3 as colourless oil. Rf = 0.5 (60% ethyl acetate in
pet ether). Yield: 0.20 g (48%). 1H NMR (400 MHz, CDCl3) δ (ppm): 3.92 (t, J = 6.0 Hz, 4H),
2.89 (t, J = 5.6 Hz, 4H), 2.33 (d, J = 4.0 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ (ppm): 60.4,
41.3. ESI-MS: m/z calcd. for C4H10NaO2S2 [M+Na]+: 177.0020; Obs. [M+Na]+: 177.0036.

Compound 4:3 To a solution of compound 3 (0.20 g, 1.30 mmol) in anhy dichloromethane (6.0
mL) was added pyridine (0.50 mL, 6.40 mmol) and the mixture was cooled to 0 °C. To the above
mixture, a solution of 4-nitrophenyl chloroformate (0.60 g, 3.20 mmol) in anhy dichloromethane
was added dropwise at 0 °C. The reaction mixture was then allowed to attain room temperature
and stirred for 8 h. The progress of the reaction was monitored by TLC analysis. Upon
completion, the mixture was diluted with dichloromethane and the mixture was sequentially
washed with saturated NaHCO3 (10.0 mL), water (10.0 mL), 10% citric acid (10.0 mL), water
(10.0 mL) and brine (10.0 mL). The organic layer was dried over anhy sodium sulfate and the
solvent was evaporated under reduced pressure to afford compound 4. The crude residue was
purified by silica gel column chromatography using ethyl acetate and pet ether as eluents to
obtain pure compound 4 as white solid. Rf = 0.5 (30% ethyl acetate in pet ether) Yield: 0.40 g
(62%). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.28 (d, J = 7.2 Hz, 4H), 7.39 (d, J = 9.2 Hz, 4H),
4.58 (t, J = 6.4 Hz, 4H), 3.09 (t, J = 6.4 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ (ppm): 155.3,
152.3, 145.5, 125.4, 121.8, 66.7 and 36.8. ESI-MS: m/z calcd. for C14H16N2O10S2 [M+K]+:
522.9883; obs. [M+K]+: 522.9885.

Compound 6:4, 5 To a solution of sodium sulfide nonahydrate (0.70 g, 3.10 mmol) in deionized
water (12.0 mL) was added sulfur powder (0.20 g, 0.80 mmol) and the reaction mixture was

-S4-
stirred for 2.5 h at room temperature during which the solution turned reddish brown indicating
the formation of Na2Sn (n = 2, 3, 4, 5) with Na2S3 as the major component. To the above solution,
2-chloroethanol (0.40 mL, 6.20 mmol) was added and the reaction mixture was stirred for 6 h at
room temperature. Progress of the reaction was monitored by TLC analysis. Upon completion,
the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with
brine solution and dried over anhy sodium sulfate. The solvent was evaporated under reduced
pressure to afford polysulfide 6 as faint yellow oil with almost quantitative yield. The crude
compound was purified to remove the major amount of the disulfide from the mixture and the
remaining inseparable polysulfide was used directly for the next step without any further
purification. Rf = 0.5 (45% ethyl acetate in pet ether). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.01
– 3.97 (m, overlap of two triplets for –OCH2- group from trisulfide, tetrasulfide and pentasulfide,
4H), 3.18 (t, J = 6.0 Hz -SCH2- group from pentasulfide), 3.14 (t, J = 6.0 Hz, -SCH2- group from
tetrasulfide) and 3.09 (t, J = 6.0 Hz, -SCH2- group from trisulfide). 13C NMR (150 MHz, CDCl3)
δ (ppm): 60.32, 59.97, 59.62, 42.44, 41.78, 41.67. ESI-MS: m/z calcd. for C4H10O2S3 (trisulfide)
[M+Na]+: 208.9741; Obs. [M+Na]+: 208.9749, ESI-MS: m/z calcd. for C4H10O2S4 (tetrasulfide)
[M+Na]+: 240.9461; Obs. [M+Na]+: 240.9468, ESI-MS: m/z calcd. for C4H10O2S5 (pentasulfide)
[M+Na]+: 272.9182; Obs. [M+Na]+: 272.9196.

Preparation of polysulfide alcohol 6 with variation of the ratio of sulfur powder and
Na2S.9H2O

General Procedure: To a solution of sodium sulfide nonahydrate (3-10 equiv) in deionized

water (10 mL) was added sulfur powder (0.20 g, 0.78 mmol, 1 equiv) under argon atmosphere.
The reaction was continued for 2.5 hours at room temperature and the reaction mixture turned
yellow in colour that indicates the formation of polysulfide (Na2Sn) species in the reaction
mixture. After that 2-chloroethanol (0.50 g, 6.24 mmol) was added dropwise to the reaction
mixture and the reaction was continued for 6 hours at room temperature. The progress of the
reaction was monitored by thin layer chromatography. Upon completion of the reaction, the
reaction mixture was extracted with ethyl acetate (3 x 25 mL). The organic layer was then washed
with brine (30 mL) and dried over anhydrous sodium sulfate. The organic layer was evaporated
under reduced pressure to afford the crude mixture as light yellow oil. The crude mixture was
purified by silica gel column chromatography that mostly removed the disulfide component and
afforded the polysulfide mixture as a colourless oil.

Synthesis of polysulfide alcohol 6 with 1:3 ratio of Sulfur powder to Sodium sulfide
nonahydrate: Sulfur powder (0.20 g, 0.78 mmol) and sodium sulfide nonahydrate (0.56 g, 2.34

-S5-
mmol). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.00-3.98 (m, overlap of three triplets for –OCH2-
group from trisulfide, tetrasulfide and pentasulfide), 3.18 (t, J = 6.0 Hz -SCH2- group from
pentasulfide), 3.14 (t, J = 6.0 Hz, -SCH2- group from tetrasulfide) and 3.09 (t, J = 6.0 Hz, -SCH2-
group from trisulfide).

Synthesis of polysulfide alcohol 6 with 1:4 ratio of Sulfur powder to Sodium sulfide
nonahydrate: Sulfur powder (0.20 g, 0.78 mmol) and sodium sulfide nonahydrate (0.75 g, 3.12
mmol). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.00-3.98 (m, overlap of three triplets for –OCH2-
group from trisulfide, tetrasulfide and pentasulfide), 3.18 (t, J = 6.0 Hz -SCH2- group from
pentasulfide), 3.14 (t, J = 6.0 Hz, -SCH2- group from tetrasulfide) and 3.09 (t, J = 6.0 Hz, -SCH2-
group from trisulfide).

Synthesis of polysulfide alcohol 6 with 1:5 ratio of Sulfur powder to Sodium sulfide
nonahydrate: Sulfur powder (0.20 g, 0.78 mmol) and sodium sulfide nonahydrate (0.94 g, 3.90
mmol). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.00-3.98 (m, overlap of two triplets for –OCH2-
group from trisulfide and tetrasulfide), 3.14 (t, J = 6.0 Hz, -SCH2- group from tetrasulfide) and
3.09 (t, J = 6.0 Hz, -SCH2- group from trisulfide).

Synthesis of polysulfide alcohol 6 with 1:6 ratio of Sulfur powder to Sodium sulfide
nonahydrate: Sulfur powder (0.20 g, 0.78 mmol) and sodium sulfide nonahydrate (1.12 g, 4.68
mmol). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.00-3.98 (m, overlap of two triplets for –OCH2-
group from trisulfide, tetrasulfide and pentasulfide), 3.18 (t, J = 6.0 Hz -SCH2- group from
pentasulfide), 3.14 (t, J = 6.0 Hz, -SCH2- group from tetrasulfide) and 3.09 (t, J = 6.0 Hz, -SCH2-
group from trisulfide).

Synthesis of polysulfide alcohol 6 with 1:10 ratio of Sulfur powder to Sodium sulfide
nonahydrate: Sulfur powder (0.20 g, 0.78 mmol) and sodium sulfide nonahydrate (1.87g, 7.80
mmol). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.00-3.98 (m, overlap of two triplets for –OCH2-
group from trisulfide and tetrasulfide), 3.15 (t, J = 6.0 Hz, -SCH2- group from tetrasulfide) and
3.09 (t, J = 6.0 Hz, -SCH2- group from trisulfide).

Compound 7:6 To a solution of polysulfide 6 (0.20 g, 1.0 mmol) in anhy. dichloromethane (6.0
mL) under inert atmosphere was added pyridine (0.45 mL, 5.30 mmol) and the mixture was
cooled to 0 °C with external ice bath. A solution of 4-nitrophenyl chloroformate (0.54 g, 2.60
mmol) in anhy dichloromethane (6.0 mL) was added to the above reaction mixture in a dropwise
manner at 0 °C. The resulting solution was allowed to attain room temperature and was stirred
for another 8 h. The progress of the reaction was monitored by TLC analysis. Upon completion,

-S6-
the mixture was diluted with dichloromethane and sequentially washed with saturated NaHCO3
(15.0 mL), water (15.0 mL), 10% citric acid (15.0 mL) and brine solution (15.0 mL). The organic
layer was dried over anhy. sodium sulfate and the solvent was evaporated under reduced pressure
to afford compound 7 as white solid with almost quantitative yield, which was used for the next
step without further purification. Rf = 0.5 (20% ethyl acetate in pet ether). 1H NMR (600 MHz,
CDCl3) δ (ppm): 8.29 (d, J = 9.6 Hz, 4H), 7.39 (d, J = 9.6 Hz, 4H), 4.64-4.62 (m, overlap of two
triplets for –OCH2- group from trisulfide and tetrasulfide, 4H), [3.35-3.30 (m, overlap of two
triplets for –OCH2- group from tetrasulfide and pentasulfide) and 3.26 (t, J = 6.4 Hz, –OCH2-
group from trisulfide), 4H]. 13C NMR (150 MHz, CDCl3) δ (ppm): 155.3, 152.3, 145.5, 125.4,
121.7, 66.6, 66.4, 36.9, 36.4. ESI-MS: m/z calcd. for C18H16N2O10S3 (trisulfide) [M+Na]+:
538.9865; Obs. [M+ Na] + : 538.9914, ESI-MS: m/z calcd. for C18H16N2O10S4 (tetrasulfide)
[M+Na]+: 570.9585; Obs. [M+Na]+: 570.9563, ESI-MS: m/z calcd. for C4H10O2S5 (pentasulfide)
[M+Na]+: 602.9306; Obs. [M+Na]+: 602.9212.

Protein-ligand docking study

Protein Preparation: For the docking studies, the recently published crystal structure of TrxR1
(PDB: 1H6V) was extracted from RCSB (Protein Data Bank).7 The protein structure was
prepared using AutoDock 4.2 Tools (MGL Tools 1.5.6) and Pymol (Schrodinger LLC) software
package.8 Initially, the protein structure was assembled to the original form having all the
important subunits depicted by the crystallographic study. During the protein preparation,
addition of missing hydrogens and Gasteiger charges were applied.

Receptor grid generation: The protein TrxR1 was co-crystallized with FAD+ and NADPH and
the crystal structure contained Cysteine (Cys 498A) residue in its native form. A modified protein
was prepared and used in the present study upon the point mutation at the cysteine residue (Cys
498A) with selenocysteine (Sec 498A) using Chimera 1.14 software package.9, 10 In order to
locate the binding site, the grid around the selenocysteine sites without altering the entire protein
structure was calculated and the grid box was generated by performing the Auto grid. The grid
size was set to 40 × 40 × 40 points with grid spacing of 1.000 Å and the grid was centred at Se
atom (x = 0.938, y = 8.852, z = 156.484).

Ligand preparation: The 2D structures of DCI-DS and DCI-PS were drawn carefully and
converted to 3D structure using Chem3D Pro (Version: 19.0.1.28). The 3D structures of ligands
were subjected to MM2 energy minimization calculations and the correct bond orders were
assigned. Finally, for the docking studies, extended PDB format, termed PDBQT, was used for

-S7-
coordinate files, which includes atomic partial charges and atom types. Torsion angles were
calculated to assign the fixable and non-bonded rotation of molecules.11

Docking Study: Docking simulations were performed using AutoDock Vina software package
by using Perl programming for handling multiple ligands at a time.8 The number of runs is set
by exhaustiveness parameter. Since the individual runs were executed in parallel, proper
exhaustiveness parameter is essential. The exhaustiveness parameter of 8 was considered
throughout the study.10 Additionally, we have mentioned 20 binding modes and energy
difference of 5 in the conformation file.

0.9
5% DMSO
0.8 10% DMSO
20% DMSO
0.7
30% DMSO
0.6 40% DMSO
Absorbance

50% DMSO
0.5

0.4

0.3

0.2

0.1

0
320 400 480 560 640
Wavelength (nm)

Figure S1. Absorption spectra of DCI-OH (5.0 µM) in phosphate buffered saline (20 mM) of pH
7.4 in the presence of varying percentage of DMSO (5% to 50%).

-S8-
 160

140

Fluorescence Intensity (a.u.) x 103

5% DMSO
120 10% DMSO
20% DMSO
100 30% DMSO
40% DMSO
50% DMSO
80

60

40

20

0
570 620 670 720 770
Wavelength (nm)

Figure S2. Emission spectra of DCI-OH (5.0 µM) in phosphate buffered saline (20 mM) of pH
7.4 in the presence of varying percentage of DMSO (5% to 50%).

0.4

0.35
DCI-DS
0.3 DCI-DS + DTT
DCI-DS + GSH
0.25 DCI-OH
Absorbance

0.2

0.15

0.1

0.05

0
330 380 430 480 530 580 630
Wavelength (nm)

Figure S3. Absorption spectra of DCI-DS (5.0 µM) in the presence and absence of different
thiols (200 µM) in phosphate buffered saline (20 mM) of pH 7.4 in the presence of 50% DMSO
after incubating for 60 min. An absorption spectrum of DCI-OH was included for comparison.

-S9-
 14

Fluorescence Intensity (a.u.) X 103

DCI-DS + DTT
12
DCI-DS +GSH
DCI-DS
10
DCI-OH

0
580 630 680 730 780
Wavelength (nm)

Figure S4. Emission spectra of DCI-DS (5.0 µM) in the presence and absence of different thiols
(200 µM) in phosphate buffered saline (20 mM) of pH 7.4 in the presence of 50% DMSO (after
incubating for 60 min) along with the emission spectrum of DCI-OH. λex = 550 nm; λem = 570 -
800 nm; slit width 5/5 nm.

Table S1. Summary of the relative distribution of tetrasulfide and pentasulfide with respect to
trisulfide as observed in 1H NMR spectra (integration). The integration of the peak of –(S)n-CH2-
group corresponding to the trisulfide was considered as 2.00.

S8 : Na2S.9H2O Trisulfide : Tetrasulfide : Pentasulfide

1:3 2.00 : 1.22 : 0.76
1:4 2.00 : 0.32 : 0.21
1:5 2.00 : 0.33
1:6 2.00 : 0.32 : 0.23
1 : 10 2.00 : 0.41

-S10-
 3.0 equiv Na2S 4.0 equiv Na2S
250
5.0 equiv Na2S 6.0 equiv Na2S
10.0 equiv Na2S

200

H2S (μM) 150

100

50

0
0 20 40 60 80 100 120
Time (min)

Figure S5. Comparative H2S release profile of the different batches of polysulfide alcohols 6 (25
µg/mL) in the presence of DTT (1 mM) over 120 min. The batches were obtained upon the
variation of the relative ratio of sulfur powder to Na2S.9H2O during their synthesis.

(A) 0.9 (B) 1

0.9
0.8
0.8
0.7
0.7
0.6
Absorbance
Absorbance

0.6
0.5 MB pattern
0.5
0.4
0.4
0.3
120 min 0.3 MB pattern
0.2 0.2

0.1 0.1 120 min

0 0
400 500 600 700 800 400 500 600 700 800
Wavelength (nm) Wavelength (nm)

Figure S6. Time-dependent H2S release profile of DCI-PS (25 µg/mL) in the presence of (A)
DTT (1 mM) and (B) GSH (1 mM) over 120 min using MB assay. The representative methylene
blue formation pattern was observed at 620-800 nm region.

-S11-
 (A) 0.45 (B) 1

0.8
0.35

Absorbance
0.6
0.25
Absorbance

0.4
0.15

No MB pattern 0.2
No MB pattern
0.05 120 min 120 min

0
400 500 600 700 800 400 500 600 700 800
-0.05 Wavelength (nm)
Wavelength (nm)

Figure S7. Time-dependent H2S release profile of DCI-DS (25 µM) in the presence of (A) DTT
(1 mM) and (B) GSH (1 mM) over 120 min using MB assay. The formation of methylene blue
was not observed in the presence of both the thiols.

2000
1800
1600 (h)
Absorbance (mAU)

1400 (g)

1200 (f)

1000 (e)

800 (d)

600 (c)

400 (b)

200 (a)

0
0 2 4 6 8 10 12 14 16 18 20
Retention Time (min)

Figure S8. HPLC chromatogram (0 - 20 min) for the reaction of DCI-DS with PhSH. Lines: (a)
PhSH; (b) PhSSPh; (c) DCI-OH; (d) DCI-DS; (e) DCI-DS + PhSH 1:1@254 nm; (f) DCI-DS +
PhSH 1:1@433 nm; (g) DCI-DS + PhSH 1:5@ 433 nm; (h) DCI-DS + PhSH 1:5@ 254 nm. The
chromatograms for the reaction mixtures were recorded after 60 min of incubation.

-S12-
 800
700

Absorbance (mAU)
600
(e)
500
(d)
400
(c)
300
(b)
200
(a)
100
0
0 2 4 6 8 10 12 14 16 18 20
Retention Time (min)

Figure S9. HPLC chromatogram (0-20 min) for the reaction of DCI-DS with DTT. Lines: (a) DTT
@230 nm; (b) DCI-OH; (c) DCI-DS; (d) DCI-DS + DTT 1:1@ 433 nm; (e) DCI-DS + DTT 1:1@
230 nm. The chromatograms for the reaction mixtures were recorded after 60 min of incubation.

[M + NH4]+ NC CN

O
S
S O O
M

Figure S10. ESI-MS (+ve) spectrum of the reaction mixture of DCI-DS and PhSH (1:5) in
acetonitrile, representing the formation of intermediate 8. ESI-MS calcd. for [M + NH4]+ =
520.1729, observed [M + NH4]+ = 520.1850.

-S13-
Figure S11. HPLC chromatogram of DCI-PS showing the presence of disulfide (4.1%), trisulfide
(73.8%), tetrasulfide (19.5%) and pentasulfide (2.4%).

[A + NH4 ] +

[B + NH4 ] +

[C + NH4 ] +

NC CN NC CN NC CN

O O O
S S S
S O O S S
S O O S S O O
A
B C

-S14-
Figure S12. ESI-MS (+ve) spectrum of the reaction mixture of DCI-PS and PhSH (1:5) in
acetonitrile. The formation of unsymmetrical polysulfide intermediates was detected in mass
spectrometric condition. The structures and the detailed mass data of the intermediates A, B
and C: ESI-MS calcd. for C28H26N2O3S2 [A + NH4]+ = 520.1729, obs. [ A + NH4]+ = 520.1737,
ESI-MS calcd. for C28H26N2O3S3 [B + NH4]+ = 552.1449, obs. [B + NH4]+ = 552.1536, calcd. for
C28H26N2O3S4 [C + NH4]+ = 584.1170, obs. [C + NH4]+ = 584.1246.

NC CN
[M-H]-

O
M

Figure S13. ESI-MS (-ve) spectrum of the reaction mixture of DCI-PS and PhSH (1:5) in
acetonitrile. Formation of the released fluorophore DCI-OH was detected under the mass
spectrometric condition. ESI-MS (-ve) calcd. for C19H18N2O [M - H]- : 289.1346, obs. [M - H]-:
289.1341.

-S15-
 (e)

(d)

(c)

(b)

(a)

Figure S14. HPLC chromatogram (0-25 min) for the reaction of DCI-PS with DTT. Lines: (a)
DTT @ 230 nm; (b) DCI-OH; (c) DCI-PS; (d) DCI-PS + DTT 1:1 @ 230 nm; (e) DCI-PS + DTT
1:1 @ 433 nm The chromatograms for the reaction mixtures were recorded after 60 min of
incubation.

120
DCI-DS DCI-PS
100

80
%Proliferation

60

40

20

0
Control 5 10 25
Concentration (µM or µg/mL)

Figure S15. Dose-dependent anti-proliferative activities of DCI-DS (µM) and DCI-PS (µg/mL) in
MDA-MB-231 cell line over an incubation over 48 h.

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 DCI-DS DCI-PS DNCB + DCI-PS Ph3PAuCl + DCI-PS NEM + DCI-PS
(a) (d) (g) (j) (m)
Bright Field

(b) (e) (h) (k) (n)

Red Channel

(c) (f) (i) (l) (o)

Merged

Figure S16. Fluorescence microscopic images (bright field, red channel and merged) of MDA-
MB-231 cells for the bio-analytes-triggered release of DCI-OH from DCI-DS (a-c) and DCI-PS
(d-f) and the inhibition of TrxR-triggered release of DCI-OH from DCI-PS using DNCB (10 µM, g-
i) and PPh3AuCl (2 µM, j-l). The endogenous thiol/selenol was quenched upon the pre-treatment
of cells with NEM (2 mM, m-o). The scale bar represents 50 µm.

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 Bright Field Blue Channel Red Channel Merged

WSP2
A

B
DCI-PS + WSP2

C
DCI-DS + WSP2
PAG + NEM + DCI-PS + WSP2

Figure S17. Fluorescence microscopic images (bright field, blue, red channels and merged) of
MDA-MB-231 cells for the WSP2-mediated detection of (A) endogenous level of H2S; (B)
released H2S from DCI-PS in the presence of bio-analytes; (C) released H2S (if any) from DCI-
DS in the presence of bio-analytes and (D) released H2S from DCI-PS in the presence of bio-
analytes upon the pre-incubation of cells with NEM (2 mM) and PAG (20 µM). The scale bar
represents 50 µm.

Table S2. Summary of distances of various sulfur atoms of the ligands with the selenium center
of TrxR1 at the active site and the list of proximal amino acid residues with which the ligands
have non-bonding interactions.

Probes Binding Energy Distance of S atoms of Amino acid residues involved

(∆Gbind) (kcal/mol) ligands from the Se atom in non-bonding interactions
of Sec498 of TrxR (Å)
DCI-DS -8.3 4.2, 4.5 K29, Y116, S404, W407, H472,
G496
DCI-PS (n = 1, -9.2 7.8, 8.1, 9.3 K33, K123, S404, F406, W407,
trisulfide) L409, H472, V474, S483
DCI-PS (n =2, -8.7 6.5, 7.6, 8.1, 8.1 K33, Y116, R351, W407, N419,
tetrasulfide) H472, G499
DCI-PS (n = 3, -9.4 7.2, 7.4, 8.0, 8.4, 8.7 S22, K29, Y116, W407, L409,
pentasulfide) N418, V474, E477, Q494, G496

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 NMR (1H, 13C and Mass Spectrometric Data)

Figure S18. 1H NMR (600 MHz) spectrum of compound 2 in CDCl3.

Figure S19. 13C NMR (150 MHz) spectrum of compound 2 in CDCl3.

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Figure S20. 1H NMR (600 MHz) spectrum of DCI-OH in CDCl3.

Figure S21. 13C NMR (150 MHz) spectrum of DCI-OH in CDCl3.

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Figure S22. 1H NMR (400 MHz) spectrum of compound 3 in CDCl3.

Figure S23. 13C NMR (100 MHz) spectrum of compound 3 in CDCl3.

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Figure S24. 1H NMR (400 MHz) spectrum of compound 4 in CDCl3.

Figure S25. 13C NMR (100 MHz) spectrum of compound 4 in CDCl3.

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Figure S26. 1H NMR (400 MHz) spectrum of DCI-DS in CDCl3.

Figure S27. 13C NMR (100 MHz) spectrum of DCI-DS in CDCl3.

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Figure S28. ESI-MS (+ ve) spectrum of DCI-DS. ESI-MS :m/z calcd. for C44H46N5O6S2 [M + Na]+
809.2443 obs. [M + Na]+:809.2449.

Figure S29. 1H NMR (600 MHz) spectrum of compound 6 in CDCl3.

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Figure S30. 13C NMR (150 MHz) spectrum of compound 6 in CDCl3

Figure S31. 1H NMR (600 MHz) spectrum of compound 7 in CDCl3.

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Figure S32. 13 C NMR (150 MHz) spectrum of compound 7 in CDCl3.

Figure S33. 1H NMR (400 MHz) spectrum of DCI-PS in CDCl3.

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 Figure S34. 13C NMR (100 MHz) spectrum of DCI-PS in CDCl3.

Figure S35. Zoomed ESI-MS (+ ve) of DCI-PS showing disulfide, trisulfide, tetrasulfide and
pentasulfide forms of DCI-PS. ESI-MS m/z calcd. for C44H46N5O6S3 [M + NH4]+ 836.2610 ,obs.
836.2535, m/z calcd. for C44H46N5O6S4 [M + NH4]+: 868.2331, obs. 868.2245, m/z calcd. for
C44H46N5O6S5 [M + NH4]+: 900.2052 , obs. 900.1947.

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Figure S36. 1H NMR (600 MHz) spectrum of compound 6 in CDCl3.The compound was prepared
using sulfur powder and Na2S.9H2O in 1:3 ratio.

Figure S37. 1H NMR (600 MHz) spectrum of compound 6 in CDCl3.The compound was prepared
using sulfur powder and Na2S.9H2O in 1:4 ratio.

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Figure S38. 1H NMR (600 MHz) spectrum of compound 6 in CDCl3.The compound was prepared
using sulfur powder and Na2S.9H2O in 1:5 ratio.

Figure S39. 1H NMR (600 MHz) spectrum of compound 6 in CDCl3.The compound was prepared
using sulfur powder and Na2S.9H2O in 1:6 ratio.

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Figure S40. 1H NMR (600 MHz) spectrum of compound 6 in CDCl3.The compound was prepared
using sulfur powder and Na2S.9H2O in 1:10 ratio.

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