H2S donors with optical responses

Article abstract of DOI:10.1016/bs.mie.2020.04.039

Reactive sulfur species, including hydrogen sulfide (H₂S), are important biological mediators and play key roles in different pathophysiological conditions. Small molecules that release H₂S on demand, often referred to as “H₂S donors,” constitute a key investigative tool for H₂S-related research. A significant challenge, however, is correlating the rate of H₂S release from such donors in complex systems with biological outcomes, because release rates are commonly perturbed by different biological environments. In this chapter, we outline an approach to use H₂S donors that provide a fluorescent response upon H₂S release to address this problem. These compounds leverage the intermediate release of carbonyl sulfide (COS), which is quickly converted to H₂S by the endogenous enzyme carbonic anhydrase (CA), to provide activatable donors with an optical response. The described donors are activated by biological thiols and provide a fluorescence response that correlates directly with H₂S delivery, which allows for delivered H₂S levels to be measured in real time by fluorescence techniques.

Full text of DOI:10.1016/bs.mie.2020.04.039

ARTICLE IN PRESS
H S donors with optical responses
2
Michael D. Pluth*, Yu Zhao, Matthew M. Cerda
Department of Chemistry and Biochemistry, Materials Science Institute, Knight Campus for Accelerating
Scientific Impact, Institute of Molecular Biology, University of Oregon, Eugene, OR, United States
*
Corresponding author: e-mail address: pluth@uoregon.edu
Contents
1
2
. Introduction
. Preparation and properties of fluorescent H S donors
2
2
3
2
2
2
2
2
2
2
2
2
.1 General design principles
.2 Materials, equipment, and reagents
.3 Preparation procedures
.4 Donor usage in vitro
.5 Correlating fluorescence signal to H S release
3
4
5
6
8
9
9
9
10
10
12
12
13
13
14
14
2
.6 Materials, equipment, and reagents
.7 Measurement of fluorescence intensity of FLD donors
.8 Measurement of H
2
S release from FLD donors
.9 Prepare an H S calibration curve
2
3
4
. Probe usage and application in cell models
3
3
3
.1 Materials
.2 Procedure
.3 Anti-inflammatory activities of FLD-1 and control compounds
. Concluding remarks
Acknowledgments
References
Abstract
Reactive sulfur species, including hydrogen sulfide (H
2
S), are important biological medi-
ators and play key roles in different pathophysiological conditions. Small molecules that
release H S on demand, often referred to as “H S donors,” constitute a key investigative
tool for H S-related research. A significant challenge, however, is correlating the rate of
S release from such donors in complex systems with biological outcomes, because
2
2
2
H
2
release rates are commonly perturbed by different biological environments. In this
chapter, we outline an approach to use H S donors that provide a fluorescent response
2
upon H S release to address this problem. These compounds leverage the intermediate
2
release of carbonyl sulfide (COS), which is quickly converted to H S by the endogenous
2
enzyme carbonic anhydrase (CA), to provide activatable donors with an optical
response. The described donors are activated by biological thiols and provide a fluores-
cence response that correlates directly with H
2 2
S delivery, which allows for delivered H S
levels to be measured in real time by fluorescence techniques.
Methods in Enzymology
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.039
# 2020 Elsevier Inc.
All rights reserved.
1

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Michael D. Pluth et al.
1. Introduction
Reactive sulfur species (RSS) have gained significant interest in the
last decade due to their roles in different biological processes and systems.
Of such species, hydrogen sulfide (H S) has garnered significant interest
2
because it is produced endogenously from both enzymatic and non-
enzymatic pathways and functions as an important signaling molecule akin
to nitric oxide (NO) and carbon monoxide (CO) (Wang, 2002, 2012). As
brief examples of this activity, misregulation of endogenous H S has been
2
implicated in different (patho)physiological processes, including angiogen-
esis, wound healing, carcinogenesis, protection against ischemia/reperfusion
injury, as well as other activities (Cortese-Krott, Fernandez, Kelm, Butler, &
Feelisch, 2015; Filipovic, Zivanovic, Alvarez, & Banerjee, 2018; Kolluru,
Shen, Bir, & Kevil, 2013; Szabo et al., 2014; Yuan, Shen, & Kevil,
2
017). Complementing the expansion of biological activities associated with
H S and related RSS, small molecule chemical tools for H S detection and
2
2
delivery have emerged as key tools for investigate complex RSS in diverse
environments (Levinn, Cerda, & Pluth, 2020; Li, Yin, & Huo, 2015; Lin,
Chen, Xian, & Chang, 2015; Powell, Dillon, & Matson, 2018; Szabo &
Papapetropoulos, 2017).
Of such tools, small molecules that release H S on demand form a cor-
2
nerstone of our ability to manipulate biological levels of H S (Levinn et al.,
2
2
020; Powell et al., 2018; Szabo & Papapetropoulos, 2017). Simple sources
of H S, such as Na S or NaSH, are commonly used in biological investiga-
2
2
tions, yet lack physiological relevance due to the rapid release and subse-
quent oxidation of H S from these salts. Building from the need for
2
slow-releasing H S donors, researchers have made significant advances in
2
the development of passive releasing donors, which are often activated by
hydrolysis or reaction with common biological nucleophiles, as well as
activatable donors, which are often activated by specific biological or bio-
orthogonal stimuli. One approach that has enabled different donors to be
tuned for activation by different stimuli is the development of systems that
release carbonyl sulfide (COS) as a precursor for H S delivery. Under bio-
2
logical conditions, COS is quickly converted to H S by the endogenous
2
enzyme carbonic anhydrase (CA).
One significant challenge in using small molecule donors for H S or
2
other biologically-relevant species, is relating measured release rates in buffer
with H S-related activities in more complex systems. Release in buffer,
2
however, typically does not match release rates in more complex systems,

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H
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S donors with optical responses
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such as cell culture or animal models. For example, even the presence of
simple proteins, such as bovine serum albumin (BSA), has been demon-
strated to significantly change the rate of H S release from certain donors
2
(Zhao, Steiger, & Pluth, 2018a). H S release rates from donors can be readily
2
measured in buffer or in vitro using H S-responsive electrodes, or analytical
2
methods including the methylene blue (MB) or monobromobimane (mBB)
methods (Hartle & Pluth, 2016). All of these methods, however, are destruc-
tive and consume H S and also often require significant sample preparation.
2
One approach toward addressing this challenge is to use H S donors that
2
couple an optical signal with H S releasing, such that H S delivery can be
2
2
monitored in real time by common spectroscopic methods. In this chapter,
we describe the procedure for preparing and using the fluorescent H2S
donors FLD-1 and FLD-3, which enable direct real-time monitoring
of H S release using fluorescence spectroscopy and microscopy (Zhao,
2
Cerda, & Pluth, 2019).
2
. Preparation and properties of fluorescent H S donors
2
2
To access H S donors that release H S slowly under physiological condi-
.1 General design principles
2
2
tions with a concomitant fluorescence readout, we developed a system that
could be readily activated by biological thiols to release both H S and fluo-
2
rescein. By using the thiol-mediated reduction of sulfenyl thiocarbonates,
reaction with biological thiols including reduced glutathione (GSH) or cys-
teine (Cys) results in disulfide exchange and the intermediate release COS
(Scheme 1). The released COS is quickly converted to H S by CA. This
2
strategy for leveraging intermediate COS release to enable H S delivery is
2
now a broadly-used approach to develop H S donor compounds (Cerda,
2
Mancuso, Mullen, Hendon, & Pluth, 2020; Levinn, Cerda, & Pluth,
2
019), and has been used to develop donors activated by cellular nucleophiles
Scheme 1 Activation and response mechanism for the sulfenyl thiocarbonate donors.
Reaction with cellular thiols results in disulfide reduction and COS release, which is
quickly converted to H S by carbonic anhydrase (CA).
2

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Michael D. Pluth et al.
(Powell, Foster, Okyere, Theus, & Matson, 2016; Zhao, Steiger, & Pluth,
2
018b, 2019), reactive oxygen species (Chauhan, Jos, & Chakrapani, 2018;
Hu et al., 2019; Zhao, Henthorn, & Pluth, 2017; Zhao & Pluth, 2016),
enzymes (Chauhan, Bora, Ravikumar, Jos, & Chakrapani, 2017; Levinn,
Steiger, & Pluth, 2019; Steiger, Marcatti, Szabo, Szczesny, & Pluth, 2017),
light (Sharma et al., 2017; Stacko, Muchova, Vitek, & Klan, 2018; Zhao,
Bolton, & Pluth, 2017), and other stimuli (Gilbert, Zhao, Otteson, &
Pluth, 2019; Steiger, Yang, Royzen, & Pluth, 2017).
On the basis of these design principles, two different fluorescein-based
H S donors (FLD-1 and FLD-3) can be readily prepared. Treatment with
2
either fluorescein or 3-O-methylfluorescein with ((benzyl)dithio)carbonyl
chloride yields the fluorescent donors FLD-1 and FLD-3, respectively, in
moderate yield (Scheme 2). Both compounds are readily purified by SiO2
column chromatography and are stable at room temperature, but should
be stored in a freezer over extended periods of time. These donors pro-
vide systems with a turn on fluorescence response that corresponds to
H S donation. The chromophores used for FLD-1 (λ ¼490nm; λ
¼
2
ex
em
5
00–650nm) and FLD-3 (λ ¼454nm; λ ¼500–650nm) correspond to
ex
em
commonly used FITC/GFP green channels and filter cubes used for fluo-
rescence microscopy. Fluorescein is commercially available from many
sources, and ((benzyl)dithio)carbonyl chloride and 3-O-methylfluorescein
are readily available through simple synthetic procedures (Mugherli,
Burchak, Chatelain, & Balakirev, 2006).
2
.2 Materials, equipment, and reagents
2
Fluorescein (CAS# 2321-07-5) or 3-O-methylfluorescein (CAS#
0672-05-8)
(Benzyl(dithio))carbonyl chloride (CAS# 31331-36-9)
7
2
Scheme 2 Synthesis of FLD-1 and FLD-2.

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H
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S donors with optical responses
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2
2
2
2
2
2
2
2
2
2
2
2
Chloroform
Ethyl acetate
Hexanes
Silica gel
Diisopropyl ethylamine (DIPEA)
Magnesium sulfate (MgSO4)
Brine
Round bottom flask
Separatory funnel
Column for chromatography
NMR instrument to verify compound identity and purity
HPLC to verify compound purity
2.3 Preparation procedures
2
1
.3.1 General procedure for preparation of FLD-1 and FLD-3
. Add 1.0 equiv. of fluorescein and 3.0 equiv. of ((benzyl)dithio)carbonyl
chloride to CHCl (ꢀ5–10mL) under nitrogen.
3
2
3
. Cool the reaction mixture to 0°C for 5min in an ice bath, and then
slowly add 3.0 equiv. of diisopropyl ethylamine (DIPEA).
. Remove the ice bath and stir the reaction mixture for 2h, or until the
reaction is complete as indicated by thin layer chromatography.
. Quench the reaction mixture by adding 25mL of brine.
4
5
6
7
8
. Extract the aqueous solution with ethyl acetate (3ꢁ15mL).
. Combine the organic layers and dry over MgSO .
4
. Filter off the MgSO and remove the solvent under vacuum.
4
. Purify the crude product using SiO column chromatography using
2
ethyl acetate/hexanes as the eluent.
9. The product can be isolated as a yellow solid in approximately
40–55% yield.
2.3.2 Reaction scales and product identification for FLD-1 and FLD-3
Reaction scale for FLD-1: fluorescein (332mg, 1.00mmol), ((benzyl)dithio)
carbonyl chloride (654mg, 3.00mmol), DIPEA (390mg, 3.00mmol);
SiO2 purification with 1:3 ethyl acetate: hexanes (v/v), R ¼0.31.
f
1
Characterization data for FLD-1: H NMR (500MHz, DMSO-d ) δ
6
(
ppm): 8.09 (d, J ¼5.0Hz, 1H), 7.85 (t, J ¼5.0Hz, 1H), 7.79 (t, J ¼
5
1
.0Hz, 1H), 7.38 (m, 13H), 7.01 (d, J ¼10.0Hz, 2H), 6.95 (d, J ¼
13
1
0.0Hz, 2H), 4.18 (s, 4H). C{ H} NMR (125MHz, DMSO-d ) δ
6
(
ppm): 168.8, 167.8, 152.6, 151.2, 136.5, 131.1, 130.1, 130.0, 129.0,
ꢂ1
1
28.2, 125.7, 125.6, 124.6, 118.4, 117.5, 110.6, 81.1, 42.4. IR (cm ):

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Michael D. Pluth et al.
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981, 1744, 1608, 1408, 1420, 1237, 1143, 1107, 1060, 988, 881, 751.
+
HRMS m/z [M+H] calcd. For [C H O S ] 697.0483; found
+
36
25 7 4
6
97.0474.
Reaction scale for FLD-3: 3-O-methylfluorescein (69.0mg,
0.207mmol), ((benzyl)dithio)carbonyl chloride (136mg, 0.623mmol),
DIPEA (81.0mg, 0.623mmol); SiO purification with 1:1 ethyl acetate:
2
1
hexanes (v/v) R ¼0.64. Characterization data for FLD-3: H NMR
f
(
500MHz, DMSO-d ) δ (ppm): 8.06 (d, J ¼10.0Hz, 1H), 7.83
6
(
t, J ¼5.0Hz, 1H), 7.77 (t, J ¼5.0Hz, 1H), 7.36 (m, 7H), 6.98
d, J ¼10.0Hz, 2H), 6.91 (d, J ¼10.0Hz, 2H), 6.77 (d, J ¼10.0Hz, 1H),
(
13
1
6
.73 (d, J ¼10.0Hz, 1H), 4.18 (s, 2H), 3.84 (s, 3H). C{ H} NMR
(
125MHz, DMSO-d ) δ (ppm): 168.9, 167.8, 161.7, 152.7, 152.4, 151.9,
6
1
1
51.5, 136.5, 136.4, 130.9, 130.1, 130.0, 129.5, 129.0, 128.2, 126.1,
25.4, 124.5, 118.0, 117.8, 113.0, 110.9, 110.4, 101.3, 81.9, 56.2, 42.4.
ꢂ1
IR (cm ): 2981, 1747, 1607, 1491, 1420, 1241, 1220, 1144, 1103,
+
+
1
060, 986, 874. HRMS m/z [M+H] calcd. For [C H O S ]
29 21 6 2
5
29.0780; found 529.0779.
2.4 Donor usage in vitro
Both FLD donors react with thiols to release COS, which is subsequently
converted to H S by CA. For simplicity, Cys can be used to verify donor
2
activity due to the higher solubility of Cys in buffer than GSH. As an exam-
ple of the reactivity of FLD-1, incubation of 10μM of FLD-1 with 100μM
Cys in PBS buffer (pH 7.4, 10mM) containing physiologically-relevant
concentrations of CA (25μg/mL) results in a significant fluorescent
response as shown in Fig. 1A. Over the course of 2h under these reaction
conditions, a ꢀ500-fold increase in fluorescence intensity is observed for
FLD-1. The release rate from the FLD compounds is dependent on both
the donor and thiol concentration. As an example of this dependence,
Fig. 1B shows the Cys concentration dependence of the fluorescence
response from FLD-1. Using conditions of 200μM Cys and 10μM
FLD-1, the activation of the donor is complete within 20min.
Both FLD-1 and FLD-3 become fluorescent when activated by thiols
to release COS/H S, but the magnitude of this fluorescence response is dif-
2
ferent. FLD-1, which releases 2 equivalents of COS/H S, shows a more
2
gradual fluorescence response because the intermediate in which 1 equiva-
lent of COS/H S has been released is moderately fluorescent. By contrast,
2
FLD-3 shows a faster fluorescence response, although a smaller dynamic
range (Fig. 2).

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S donors with optical responses
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Fig. 1 (A) Fluorescence response of FLD-1 (10μM) in PBS (pH 7.4, 10mM) containing Cys
100μM) and CA (25μg/mL). (B) Cys-dependent (0–200μM) fluorescence turn on of
FLD-1 (10μM) in PBS. General fluorescence acquisition parameters: λex ¼490nm,
em ¼500–650nm. The data shown is the average of three replicates and errors are
shown as meanꢃSD (n ¼3). Modified from Zhao, Y., Cerda, M. M., & Pluth, M. D.
2019). Fluorogenic hydrogen sulfide (H S) donors based on sulfenyl thiocarbonates enable
(
λ
(
2
H2S tracking and quantification. Chemical Science, 10(6), 1873–1878 with permission from
the R.S.C.
Fig. 2 Fluorescence turn on of FLD-1 (gray) and FLD-3 (blue). General conditions: 10μM
FLD in PBS (pH 7.4, 10mM) with Cys (100μM). λex ¼490nm for FLD-1, λ ¼454nm for
ex
FLD-3, λem ¼500–650nm. The data shown is the average of three replicates and errors
are shown as meanꢃSD (n¼3). Modified from Zhao, Y., Cerda, M. M., & Pluth, M. D. (2019).
2 2
Fluorogenic hydrogen sulfide (H S) donors based on sulfenyl thiocarbonates enable H S
tracking and quantification. Chemical Science, 10(6), 1873–1878 with permission from
the R.S.C.

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Michael D. Pluth et al.
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The H S release from the fluorescent FLD compounds can be readily mea-
.5 Correlating fluorescence signal to H S release
2
2
sured and quantified using the methylene blue (MB) assay. This method
allows for trapping of released H S to form the methylene blue dye, which
2
can then be quantified by UV–vis spectroscopy. Incubation of FLD-3
(
10μM) with Cys (100μM) resulted in a rapid fluorescence response with
9
6% of the expected H S release measured by the MB assay. In the absence
2
of CA, negligible H S was detected, which is consistent with the inter-
2
mediate formation of COS, which requires CA to be converted to H2S
(Fig. 3A). Using FLD-3, the fluorescence response can be monitored and
plotted against H S measurements from the MB assay under the same con-
2
ditions (Fig. 3B). These experiments show a linear correlation between the
fluorescence response and quantified H S, which allows for the fluorescence
2
response to be correlated directly with H S concentrations. Note that
2
because all fluorimeters will have different sensitivities, this calibration curve
should be repeated for each instrument system used to ensure accurate
correlations from fluorescence to H S concentrations.
2
Fig. 3 Fluorescence response (red) and H
FLD-3 (10μM) in PBS (pH 7.4, 10mM) with Cys (100μM) and CA (25μg/mL). No H
was detected in the absence of CA (black). λex ¼454nm, λ ¼500–650nm.
2
S release (blue) after treatment of
2
S
em
(
2
B) Correlation between quantified H S released and fluorescence response. The data
shown is the average of three replicates and errors are shown as meanꢃSD (n ¼3).
Modified from Zhao, Y., Cerda, M. M., & Pluth, M. D. (2019). Fluorogenic hydrogen sulfide
(
H S) donors based on sulfenyl thiocarbonates enable H S tracking and quantification.
2 2
Chemical Science, 10(6), 1873–1878 with permission from the R.S.C.

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S donors with optical responses
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2.6 Materials, equipment, and reagents
2
Carbonic anhydrase (from bovine erythrocytes, CAS# 9001-03-0;
EC# 4.2.1.1)
2
2
2
2
2
2
2
2
2
2
2
2
Quartz fluorescent cuvette
Disposable plastic cuvettes
Fluorimeter
UV–vis spectrophotometer
Cysteine
Zinc acetate (Zn(OAc) dihydrate; CAS# 5970-45-6)
2
Iron trichloride (FeCl hexahydrate; CAS# 10025-77-1)
3
Hydrochloric acid (HCl)
N,N-dimethyl-p-phenylene diamine (HCl salt, CAS# 536-46-9)
Sodium hydrosulfide (NaSH, CAS# 16721-80-5)
Dimethyl sulfoxide (DMSO)
PBS buffer (pH 7.4, 10mM PBS)
2.7 Measurement of fluorescence intensity of FLD donors
1
2
. Prepare a 10.0mM stock solution of the desired FLD donor in DMSO.
. Add 3.00μL of the stock solution to 3.00mL of PBS (pH 7.4, 10mM)
containing carbonic anhydrase (25.0μg/mL) in a quartz cuvette.
. Prepare a 10.0mM stock solution of cysteine in PBS buffer.
3
4
. Measure the background fluorescence spectrum of the FLD donor
(
λ ¼454 or 490nm depending on the donor; λ ¼500–650nm).
ex
scan
5
6
. Add 30μL of the cysteine stock solution to the cuvette.
. Repeat fluorescence measurements of the sample after the addition of
cysteine until the reaction is complete (ꢀ120min).
. Integrate the fluorescence signal from 500 to 650nm and plot as a
function of time.
7
2
.8 Measurement of H S release from FLD donors
2
1. Prepare a 10mM stock solution of the FLD donor in DMSO and add
20.0μL of the stock solution to 20.0mL of PBS (pH 7.40, 10.0mM)
containing CA (25.0μg/mL) in a 20mL scintillation vial.
. Prepare a 100mM cysteine stock solution in PBS buffer.
2
3
. Prepare stock solutions for the methylene blue (MB) analysis. These stock
solutions include 1.00% (w/v) Zn(OAc) , 30.0mM FeCl in 1.20M
2
3
HCl, and 20.0mM N,N-dimethyl-p-phenylene diamine in 7.20M HCl.

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Michael D. Pluth et al.
4
. Remove a 300μL aliquot from the FLD solution as a time¼0 point, and
add this to a disposable plastic UV–vis cuvette containing 300μL of MB
cocktail (60.0μL Zn(OAc) (1.00% w/v), 120μL FeCl (30.0mM in
2
3
1
.20M HCl), and 120μL N,N-dimethyl-p-phenylene diamine
(20.0mM in 7.20M HCl)).
5
6
. Add 20.0μL of the cysteine stock solution to the FLD donor solution in
the scintillation vial.
. Remove a 300μL aliquot from the reaction mixture and add it to a dis-
posable plastic UV–vis cuvette containing 300μL of MB cocktail
(
60.0μL Zn(OAc) (1.00% w/v), 120μL FeCl (30.0mM in 1.20M
2
3
HCl), and 120μL N,N-dimethyl-p-phenylene diamine (20.0mM in
.20M HCl)) at different time points.
7
7
8
. Allow each aliquot to equilibrate for 1h, and then measure the absor-
bance value at 670nm.
. Plot the absorbance values at 670nm as a function of time. To convert
this data to quantitative H S levels, prepare an H S calibration curve
2
2
(described below).
2
.9 Prepare an H S calibration curve
2
1
. Add 500μL of the MB cocktail (100μL Zn(OAc) (1.00% w/v), 200μL
2
FeCl3 (30.0mM in 1.20M HCl), and 200μL N,N-dimethyl-p-
phenylene diamine (20.0mM in 7.20M HCl)) and 500μL of PBS
(pH 7.4, 10mM) to a 1.5mL disposable cuvettes.
2
3
. Prepare a 1.00mM NaSH stock solution in PBS buffer.
. Add aliquots of the 1.00mM NaSH stock solution to the cuvettes con-
taining the MB cocktail to result in final concentration of 1, 3, 5, 10, 15,
and 20μM sulfide.
4
5
. Incubate the cuvettes for 1h prior to measuring the absorbance
at 670nm.
. Plot the absorbance as a function of sulfide concentration. The resultant
plot should produce a straight line, which can be used as a calibration
curve for quantitation experiments.
3. Probe usage and application in cell models
The FLD donor compounds can also be used to deliver H S in live
2
cell environments after activation by endogenous thiols. As an example of
this activity, treatment of HeLa cells with FLD-1 results in donor internal-
ization and release of COS/H S by endogenous thiols and CA. As shown in
2

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S donors with optical responses
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Fig. 4 H
responsive probe C7-Az (50μM) in DMEM only (top row) or DMEM containing FLD-1
50μM) (bottom row) for 30min. Cells were then washed with PBS and cell images were
taken in PBS using a fluorescent microscope. Bar scale: 50μm. Modified from Zhao, Y.,
Cerda, M. M., & Pluth, M. D. (2019). Fluorogenic hydrogen sulfide (H S) donors based on
sulfenyl thiocarbonates enable H S tracking and quantification. Chemical Science,
0(6), 1873–1878 with permission from the R.S.C.
2 2
S delivery from FLD-1 in HeLa cells. HeLa cells were treated with the H S-
(
2
2
1
Fig. 4, donor activation results in an increase in fluorescence in the green
channel due to liberation of fluorescein after donor activation. The released
H S can be visualized by using a reaction-based H S responsive probe that
2
2
does not overlap with the FLD-1 emission spectrum. Co-incubation with
the commercially available, H S-responsive 7-azido-4-methylcoumarin
2
(
C7-Az) fluorescent probe results in C7-Az activation, confirming H2S
release from FLD-1 (Fig. 4). In the absence of FLD-1, minimal background
fluorescence from C7-Az is observed.
A second example of cell-based applications of the FLD system is
blocking NO formation in Raw 264.7 macrophage cells treated with lipo-
polysaccharide (LPS). Prior reports have demonstrated that H S can reduce
2
inflammatory responses and decrease NO levels in LPS-stimulated macro-
phage cells, and this assay has been used previously to investigate anti-
inflammatory response of H S-releasing compounds (Whiteman et al.,
2
2010). To measure the impacts of FLD-1 on NO formation, cultured
Raw 264.7 macrophage cells were treated with FLD-1 (0–25μM) for 2h
followed by LPS (0.5μg/mL) administration and further incubation 24h.
After this incubation period, accumulated nitrate (NO2 ), which is the
ꢂ
downstream oxidation product of NO, was measured using a commercially
available Griess assay kit. From these experiments, a dose-dependent de-
ꢂ
crease in NO2 formation was observed, which is consistent with both
H S release from FLD-1 under these experimental conditions and also
2
anti-inflammatory activity of the FLD donor (Fig. 5).

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Michael D. Pluth et al.
ꢂ
Fig. 5 Effects of FLD-1 on LPS-induced NO2 accumulation. Raw 264.7 cells were
pretreated with FLD-1 (0–25μM) for 2h, followed by a 24-h treatment of LPS
(
0.5μg/mL). Results are expressed as meanꢃSD (n ¼4). *** P <0.001 vs the control
group; ## P <0.01 vs vehicle-treated group; ### P <0.001 vs vehicle-treated group.
Modified from Zhao, Y., Cerda, M. M., & Pluth, M. D. (2019). Fluorogenic hydrogen sulfide
(
H
2
S) donors based on sulfenyl thiocarbonates enable H
2
S tracking and quantification.
Chemical Science, 10(6), 1873–1878 with permission from the R.S.C.
3
.1 Materials
2
2
2
2
2
2
2
2
2
HeLa cells (ATCC: CCL-2).
Raw 264.7 cells (ATCC: TIB-71).
Dulbecco’s modified Eagle’s medium.
Fetal bovine serum (FBS).
Penicillin.
Streptomycin.
Cell incubator with 5% CO2.
Poly-D-lysine coated plates (MatTek).
7-Azido-4-methylcoumarin (C7-Az); commercially available from
Santa Cruz Biotech. or readily prepared by previously published
methods (Thorson, Majtan, Kraus, & Barrios, 2013).
Inverted fluorescence microscope with filter cubes for blue (DAPI) and
green (GPF, FITC) channels.
2
2
2
Lipopolysaccharide (LPS).
Griess nitrite assay kit for nitrite quantification (ThermoFisher, G7921).
3
.2 Procedure
3.2.1 Cell culture
1
. Culture HeLa cells (ATCC) in high glucose Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin/streptomycin at 37°C under 5% CO2.

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H
2
S donors with optical responses
13
2
3
. Plate HeLa cells in poly-D-lysine coated plates (MatTek) containing
.00mL of DMEM and incubated at 37°C under 5% CO for 24h.
2
2
. Wash the cells with PBS and then add C7-Az (50.0μM) and FLD
50.0μM) and incubate for 30min.
. Wash the cells with PBS and bathe in 2.0mL of PBS for imaging.
(
4
5
. Collect fluorescence images for using an inverted fluorescence micro-
scope. The excitation and emission profiles for C7-Az (λ ¼365nm;
ex
λ
em
match common filter sets used for blue/DAPI and green/GFP,
¼400–550nm) and FLD-1 (λ ¼490nm; λ ¼500–650nm)
ex
em
respectively.
3
.3 Anti-inflammatory activities of FLD-1 and control
compounds
. Seed Raw 264.7 macrophage cells (ATTC) in a 24-well plate
1
5
(
5ꢁ10 cells/well) containing 500μL of DMEM and incubate at
3
7°C under 5% CO for 24h.
2
2
. Wash the cells with PBS and incubate with the FLD donor (0–25.0μM)
at 37°C for 2h.
3
4
. Wash the cells with PBS to remove excess FLD donor.
. Incubate the FLD-treated cells with FBS-free DMEM containing LPS
(
0.500μg/mL) for 24h.
ꢂ
5
. Measure NO levels were measured by using a Griess Reagent Kit.
2
4. Concluding remarks
The delivery of small molecules from donor platforms provides a
key tool for perturbing levels of biological analytes in complex systems.
By being able to monitor the release in real time using non-invasive spec-
troscopic techniques, researchers now have an additional methods moni-
tor experiments under different conditions. In addition to the FLD-1 and
FLD-3 donors described in this chapter, additional optically-responsive
H S-releasing donors have recently been reported. Select examples include
2
colorimetric donors (Zhao et al., 2018a), photoactivatable fluorescent
donors (Venkatesh et al., 2018), and other fluorescent donor motifs
(Hu et al., 2019; Kim et al., 2019). In addition, fluorescent donors are
also available for NO donors (Hibbard & Reynolds, 2019; Ravikumar,
Bagheri, Saini, & Chakrapani, 2017) and CO donors (De La Cruz
et al., 2018; Popova, Soboleva, Ayad, Benninghoff, & Berreau, 2018),
which provide a diverse palette for monitoring the release of these impor-
tant small gaseous signaling molecules in complex environments.

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Michael D. Pluth et al.
Acknowledgments
This work was supported by the NIH (R01GM113030) and Dreyfus Foundation. NMR,
fluorescence microscopy, and MS instrumentation in the UO CAMCOR facility that
enabled this work is supported by the NSF (CHE-1427987, CHE-1531189, and CHE-
1
625529).
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