A modular template for the design of thiol-triggered sensors and prodrugs

Article abstract of DOI:10.1016/j.saa.2020.119072

A unique reaction between thiols (RSH) and alkyl sulfonylbenzothiazole was discovered. This reaction was specific for thiols and produced a sulfinic acid (RSO₂H) as the intermediate, which further triggered an intramolecular cyclization to release a -OH containing payload. This reaction was used to develop thiol-triggered fluorescent sensors and prodrugs. The modular design of this template provides tunability of the release profiles of the payloads.

Full text of DOI:10.1016/j.saa.2020.119072

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A modular template for the design of thiol-triggered sensors and
prodrugs
Jessica Renee Knight, Yingying Wang, Shi Xu, Wei Chen,
Clifford E. Berkman, Ming Xian
PII:
S1386-1425(20)31051-9
https://doi.org/10.1016/j.saa.2020.119072
SAA 119072
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 24 September 2020
Revised date:
6 October 2020
October 2020
Accepted
date:
7
Please cite this article as: J.R. Knight, Y. Wang, S. Xu, et al., A modular template for the
design of thiol-triggered sensors and prodrugs, Spectrochimica Acta Part A: Molecular
and Biomolecular Spectroscopy (2020), https://doi.org/10.1016/j.saa.2020.119072
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A modular template for the design of thiol-triggered sensors and
prodrugs
a
a
b
a
Jessica Renee Knight , Yingying Wang , Shi Xu , Wei Chen , Clifford E.
a
b,
Berkman , Ming Xian *
a
Department of Chemistry, Washington State University, Pullman, WA 99164, USA
b
Department of Chemistry, Brown University, Providence, RI 02912, USA
Abstract
A unique reaction between thiols (RSH) and alkyl sulfonylbenzothiazole was
discovered. This reaction was specific for thiols and produced a sulfinic acid (RSO H)
2
as the intermediate, which further triggered an intramolecular cyclization to release a
-OH containing payload. This reaction was used to develop thiol-triggered fluorescent
sensors and prodrugs. The modular design of this template provides tunability of the
release profiles of the payloads.
Keywords: Fluorescent probe; Prodrug; Biothiols; Sulfinic acid
*
Corresponding author
E-mail addresses: ming_xian@brown.edu

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Introduction
Biothiols include both small molecule compounds (such as cysteine-Cys,
homocysteine-Hcy, and glutathione-GSH) and protein bound cysteine residues. These
–
SH containing species play crucial roles in biological systems, especially in
maintaining cellular redox homeostasis via –SH based redox reactions to form a
variety of thio-derivatives (such as disulfides, S-nitrosothiols, and sulfenic acids) [1-3].
Abnormal levels of biothiols are known to be associated with many diseases such as
AIDS, cardiovascular diseases, and neurodegenerative diseases [4-6]. In addition,
various types of cancer have elevated levels of biothiols, especially GSH. This
elevated level of GSH impacts apoptotic resistance and drug resistance in cancer cells
[
7-11].
Due to the importance of thiols in biology, many reactions and reagents have
been developed to specifically label or sense thiol residues [12-17]. These
reactions/reagents normally take the advantage of much stronger nucleophilicity of
thiols (-SH) than other functional groups (such as amines or hydroxyls) under
physiological conditions. Among these reactions/reagents, iodoacetamide (IAM),
N-ethylmaleimide (NEM), and their derivatives are perhaps most well-known
(Scheme 1a). These reagents react with thiols very rapidly and in high yields and have
been used as standard protocols for blocking cellular thiols. However, these reagents
are not without limitations. For example, recent studies have revealed cross reactivity
of IAM/NEM with other cellular thiol-adducts such as sulfenic acids (-SOH) [18-19].
In the search for more specific thiol-blocking reagents, we have discovered several
heterocyclic thiosulfonate reagents which showed high reactivity and specificity for
thiols. Most importantly they do not show cross reactivity with sulfenic acids [19,20].
These compounds, for instance, methylsulfonylbenzothiazole (MSBT, shown in
Scheme 1b), have been used in the labeling of cellular thiol (-SH) and the
development of thiol-specific sensors [21]. A unique feature of MSBT is that its
reaction with thiols not only produces the desired labeling product (e.g.
thio-benzothiazole) but also produces a sulfinic acid (RSO H) as the byproduct. This
2
is different from the reactions of IAM or NEM, which just produce the corresponding

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thiol-adducts. We envisioned the reaction of MSBT could be advantageous as RSO H
2
is a potentially reactive functional group under physiological conditions. The
formation of RSO H could allow us to develop thiol-triggered tandem reactions or
2
modular templates for the design of thiol-sensitive sensors or prodrugs. Herein we
would like to report our progress in this attempt.
Please, insert Scheme 1 here.
Experimental Section
Materials and instruments
All solvents were reagent grade. Reactions were magnetically stirred and
monitored by thin layer chromatography (TLC) with 0.25 mm pre-coated silica gel
plates. Flash chromatography was performed with silica gel 60 (particle size
0
.040-0.062 mm). Yields refer to chromatographically and spectroscopically pure
compounds, unless otherwise stated. Proton and carbon-13 NMR spectra were
recorded on a 400 MHz spectrometer. The solvent for NMR measurements were
CDCl or DMSO-d . Fluorescence measurements were taken using a Fluostar Omega
3
6
Microplate Reader running Omega software version 1.02 and Mars Data Analysis
Software Program version 1.10 or on Cary Eclipse fluorescence spectrophotometer
Synthesis of SBTP-1, SBTP-2, and SBTP-NO
Compound 1 was prepared from methylsulfonylbenzothiazole (MSBT) and
thiosalicylic acid following the known procedure in 80% yield.
Compound 2 was obtained as an off-white solid in quantitative yield. Compound 1
was dissolved in acetonitrile in a round bottom flask. In a separate flask Oxone was
dissolved in PBS buffer (50 mM, pH 7.4). The oxone solution was slowly added to
the solution of compound 1. The reaction stirred at rt for 5 h. The product was
extracted with ethyl acetate and dried over MgSO and concentrated. It was used for
4
next step without further purification.
SBTP-1 was obtained as a white solid in 76% yield (38 mg). Compound 2 (0.043 g,

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0
.125 mmol) was dissolved in 1.0 mL of acetonitrile in an oven dried 25 mL round
bottom flask. Then DCC (0.044 g, 0.213 mmol) and DMAP (0.001 g, 0.008 mmol)
was added to the flask. The flask was placed under argon and allowed to stir for 10
min. Fluorescein (0.018 g, 0.05 mmol) was then added to the reaction flask. The
reaction was stirred under argon for 18 h. The reaction was then worked up with water
1
and ethyl acetate. The product was purified column chromatography. H NMR (400
MHz, CDCl ) δ 8.55(m, 2H) , 8.07 (t, J=7.2 , 2H), 7.98 (d, J=8.9 , 1H), 7.95-7.83(m,
3
8
2
1
1
H), 7.71(dt, J=26.3,7.6 3H), 7.48-7.58 (m, 4H) 7.24 (s, 2H), 6.98 (dd, J=8.7, 2.6,
1
3
H), 6.88 (dd, J=8.7, 2.8, 2H); C (100 MHz, CDCl ) δ167.23, 164.42, 152.28,
3
51.70, 151.67, 136.87,136.58, 135.35, 134.65, 132.34, 132.11, 130.31, 129.13,
28.08, 127.87, 127.69, 127.40, 126.15, 125.40, 125.30, 122.37, 122.31, 117.70,
+
1
17.06, 110.54, 42.40; MS(ESI) [M+H] calculated C H N O S 935.05, found
4
8
27
2
11 4
+
[
M+H] 935.0.
Compound 3 was obtained in quantitative yield as an off-white solid.
-Marcaptobenzothiazol (670 mg) was dissolved in THF (10 mL), potassium
2
carbonate (1.1 g) was added, and the reaction was stirred for 10 min under argon.
Then 3-bromoproponic acid (910 mg) was added. The reaction was allowed to stir
overnight at room temperature under argon. The reaction was neutralized with 2 M
HCl and the product was extracted with DCM.
Compound 4 was obtained in quantitative yield following the same procedure as
1
compound 2. H NMR (400 MHz, CDCl ) δ 8.22 (d, J= 8.0, 1H), 8.03 (d, J= 8.5, 1H),
3
1
3
7
1
.57-7.68 (m, 2H), 3.83 (t, J=7.5, 2H), 3.01 (t, J=7.5, 2H); C (100 MHz, CDCl ) δ
3
64.93, 152.56, 136.76, 128.28, 127.82, 125.57, 122.39, 49.95, 27.45; MS (ESI)
+
+
[
M+Na] calculated C H NNaO S 293.99, found [M+Na] 294.0.
10 9 4 2
SBTP-2 was obtained in 67% yield (67 mg). Compound 4 (54 mg) was added to a dry
5 mL round bottom flask and placed under argon. 5 mL of dry CH Cl was added
2
2
2
followed by oxalyl chloride (25 μL). Then DMF (0.5 mL) was added dropwise. When
the reaction was completed the mixture was concentrated under vacuum. The product
was dissolved in dry CH Cl (5 mL) under argon. Then fluorescein (20 mg) was
2
2
added followed by TEA (20 μL). After the reaction was completed the mixture was

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1
purified by flash column chromatography to afford 67 mg SBTP-2. H NMR (400
MHz, CDCl ) δ 8.19 – 8.12 (m, 2H), 8.13 – 8.06 (m, 2H), 8.05 – 7.99 (m, 2H), 7.65 –
3
7
.57 (m, 4H), 7.40 – 7.34 (m, 2H), 7.27 (t, J = 0.8 Hz, 2H), 7.27 (s, 2H), 7.26 – 7.23
1
3
(
m, 2H), 4.06 (t, J = 7.0 Hz, 4H), 3.78 (t, J = 6.9 Hz, 4H). C (100 MHz, CDCl ) δ
3
1
1
82.63, 174.49, 169.20, 161.87, 155.86, 149.64, 128.16, 127.71, 127.21, 125.91,
25.55, 122.36, 122.07, 121.14, 117.81, 114.44, 106.25, 77.21, 49.83, 29.70. MS (ESI)
+
+
[
M+H] Calculated C H N O S 839.05, found [M+H] 839.0.
40 27 2 11 4
Compound 10 was prepared in 76% yield as a white solid. Compound 9 (50 mg) and
carbon tetrabromide (60 mg) were added to an oven dried round bottom flask and
placed under argon. 1 mL of dry CH Cl was then added and the flack was placed in
2
2
an ice bath. In a separate oven dried flask triphenylphosphine (68 mg) was dissolved
in 1 mL of dry CH Cl then slowly added to the reaction. After stirring overnight, the
2
2
solvent was removed, and the product was purified by flash column chromatography
to yield 38 mg product.
SBTP-NO was obtained as yellow solid in 65% yield. Compound 10 (38 mg) and
compound 11 (11 mg) were added to an oven dried round bottom flask. The flask was
sealed and backfilled with argon. Then dry DMF (1mL) was added and the reaction
was placed on an ice bath. Then potassium iodide was added. After stirring overnight
under argon, the reaction was quenched with water and the product was extracted with
ethyl acetate. After purification by flash column chromatography the reaction yielded
1
3
7
2
2 mg product. H NMR (400 MHz, CDCl ) δ 8.55 (dd, 1H), 8.08 (d, J = 7.5 Hz, 1H),
3
.90 (d, J = 6.4 Hz, 2H), 7.87 – 7.80 (m, 2H), 7.58 – 7.47 (m, 2H), 7.44 (d, J = 8.5 Hz,
H), 7.23 (d, J = 8.5 Hz, 2H), 5.18 (s, 2H), 3.53 (p, J = 3.5 Hz, 4H), 1.94 (p, J = 3.5
1
3
Hz, 4H). C NMR (100 MHz, CDCl ) δ 167.44, 164.81, 152.27, 150.39, 136.80,
3
1
1
36.61, 134.64, 134.25, 132.77, 132.32, 131.95, 130.36, 129.97, 127.77, 127.31,
+
25.30, 122.26, 121.57, 74.45, 50.91, 22.81. MS (ESI) [M+Na] Calculated
+
C H N NaO S 561.09, found [M+Na] 561.0.
25
22
4
6 2
Fluorescence measurements in PBS buffer
All of the measurements were carried out at 37 C for 60 min in 10 mM PBS buffer
o

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(pH 7.4) containing 50% DMSO according to the following procedure: in a test tube,
1
.8 mL of 10 mM PBS buffer (pH 7.4) and 1.8 mL DMSO were mixed. A 200 μL of
the probe stock solution (0.2 mM) was then added to the mixture. The resulting
solution was well-mixed, followed by the addition of the requisite volume of testing
species solution. The final volume of the solution was adjusted to 4 mL with 10 mM
o
PBS buffer (pH 7.4). After mixing and standing for 60 min at 37 C, a 100 μL portion
of the reaction solution was loaded onto a 96 well plate to measure fluorescence with
λex/em = 485/520 nm. Error bars represent the standard deviation from triplicate
experiments.
Fluorescence detection in Plasma
3
6
mL of commercially available fetal bovine serum (FBS) was deproteined by adding
mL cold pure ethanol and then centrifuging for 30 min. The supernatant was then
decanted and used for testing. 1 mL samples were prepared in a test tube. 400 μL
deproteinized FBS was added followed by 400 μL DMSO. Then 20 μL of 0.5 mM
stock solution of SBTP-1 and 80 μL DMSO was added, followed by the addition of
the requisite volume of testing species solution. The final volume of the solution was
o
adjusted to 1 mL with deproteinized FBS. After the samples were incubated at 37 C
for 60 min. 100 μL of each sample were loaded on a 96 well plate and the
fluorescence emission were recorded (λex/em = 485/520 nm). Error bars represent the
standard deviation from triplicate experiments.
Results and Discussion
Probe synthesis and design
Our idea of the modular template is shown in Scheme 1b. We expected that a
hydroxyl containing payload could be tethered with the thiol-reacting site
(sulfonylbenzothiazole) via an ester linker. Upon the reaction with thiols the sulfinic
acid group would be formed, which should undergo an intramolecular cyclization
with the ester group to release the payload. This modular design allows easy
modifying all three components: the heterocycle part, the linker, and the payload.

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To test this idea, we first employed benzoic acid as the linker and fluorescein as
the payload. Fluorescein was chosen because of its excellent fluorescent property and
stability in esters. More importantly the hydroxyl group of fluorescein or its analogue
is known as the photo-switch as its acylation would quench the fluorescence while
acyl de-protection would restore fluorescence [22-27]. With the target molecule, e.g.
SBTP-1, we could evaluate this template’s sensitivity and specificity to thiols using
readily available and sensitive fluorescence spectroscopy. The preparation of SBTP-1
is shown in Scheme 2. Briefly, conjugation between MSBT and thiosalicyclic acid
under basic conditions produced compound 1, which was then subjected to oxidation
and esterification with fluorescein to furnish SBTP-1. To study the effects of linker on
the sensitivity of the template, we also prepared another fluorescein-based compound
SBTP-2 using a more flexible and linear linker propionic acid. SBTP-2 was similarly
synthesized from thiol-alkylation between 2-marcaptobenzothiazole and
3
-bromoproponic acid followed by oxidation and esterification.
Please, insert Scheme 2 here.
Fluorescence measurement
With SBTP-1 and SBTP-2 in hand, we tested their fluorescence responses to
thiols. As shown in Fig 1, these two compounds appeared to be stable in buffers. They
did not show obvious fluorescence changes if thiols were absent. When they were
treated with thiols (GSH, Cys, and Hcy), time- and concentration-dependent
fluorescence increases were observed. We also noticed that SBTP-1 (10 µM) reacted
more quickly, with the fluorescence emission reaching a maximum steady state in 30
min with 1 mM GSH. In comparison at 60 min SBTP-2 showed very little fluorescent
enhancement. These results suggested that benzoic acid was a better linker to facilitate
the intramolecular cyclization and the release of the payload.
Please, insert Fig.1 here.
Because SBTP-1 showed better reactivity to thiols than SBTP-2, we then

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carefully examined the selectivity of SBTP-1. It was found that SBTP-1 did not
respond to other reactive sulfur species such as disulfide and sulfite. It also did not
respond to hydrogen peroxide and hypochlorite (Fig. 2a). We further tested SBTP-1 in
the presence of representative amino acids (Fig 2b). These species (alanine, lysine,
histidine, tryptophan, arginine, serine, leucine, and tyrosine) did not turn on the
fluorescence and they did not affect the effects of thiols on SBTP-1 neither.
Please, insert Fig.2 here.
To further demonstrate thiol concentration dependence for the release of the
payload SBTP-1 was tested with varying GSH concentrations from 0 μM to 100 μM
(Fig. 3) The fluorescent intensity was shown to increase with increasing
concentrations of GSH demonstrating that the release of the payload was dependent
on the thiol concentration.
Please, insert Fig.3 here.
We further validated the reaction mechanism of the SBTP sensor by using a
model compound SBTP-phenol. The reaction between SBTP-phenol and a cysteine
derivative 5 was carried out and the desired product, compound 6, was obtained in 83%
yield, together with phenol and compound 7. These results confirmed our proposed
mechanism.
Please, insert Scheme 3 here.
Detection of thiol in plasma
Diseases such as Motor neuron disease, Parkinson’s disease, and Alzheimer’s
disease, show deregulation of plasma thiols, Cys in particular [28]. To demonstrate the
practical applicability SBTP-1 was also evaluated in deproteinized fetal bovine serum.
As shown in Fig 4, the reaction of SBTP-1 with thiols can still cause remarkable
fluorescence enhancement in deproteinized fetal bovine serum. And a stronger
fluorescence was observed after treatment with increasing concentration of Cys. It
indicated that SBTP-1 could be used for sensitive detection of biothiols in biological

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samples.
Please, insert Fig.4 here.
Application in thiol-triggered NO release
All the results with SBTP-1 confirmed that the template was selective and
sensitive to thiol-triggering. We next applied this template to prepare a nitric oxide
prodrug (e.g. SBTP-NO shown in Scheme 4). N-Hydroxy-N-nitroso pyrrolidinamine
(
pyrrolidine-NONOate) was used as the drug payload. It has been previously found
that the release NO from another template-furoxans could be mediated by thiol
cofactor. Particularly in phenylsulfonyl-substituted furoxans and
furoxancarboxamides. Phenylsulfonyl-substituted furoxan has been coupled with
anti-cancer drugs for a synergistic effect [29-31]. Our new template provides a
versatile tool to expand on thiol-triggered NO release. The synthesis of SBTP-NO
started from the esterification of compounds 2 and 8. The product 9 was then
subjected
to
TBS
deprotection,
bromination,
and
alkylation
with
pyrrolidine-NONOate 11 to afford the prodrug SBTP-NO.
Please, insert Scheme 4 here.
Thiol-triggered NO release from SBTP-NO was then evaluated by using a
well-known NO fluorescent probe DAF-2. As shown in Fig 5, DAF-2 alone, DAF-2
with SBTP-NO, and DAF-2 with GSH (columns 1-3) did not show any fluorescence
increase. These indicated that SBTP-NO was stable in testing conditions and not
releasing NO on its own. With the authentic NO donor pyrrolidine-NONOate (column
4
) we observed strong fluorescence increase. This signal was significantly decreased
but still very obvious when GSH was presented (column 5). This could be explained
by the competing reaction between NO and GSH that diminished the reaction between
NO and DAF-2. Columns 6-8 showed the behavior of SBTP-NO (100 μM) in the
presence of thiols. Column 6 contained 1 mM GSH. Similar to the results shown in
column 5, while NO release in this sample was obvious, the competing GSH/NO

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reaction decreased the fluorescence signal. In columns 7 and 8 Cys and Hcy were
used at a concentration of 200 μM. This lower concentration of thiol did not have as
much of the competing reaction with NO, therefore, fluorescence signal was much
stronger. These results demonstrated that thiol indeed could trigger NO release from
this template.
Please, insert Fig.5 here.
Conclusion
In summary, we reported herein the discovery of a unique reaction between thiols
(RSH) and alkyl sulfonylbenzothiazole. This reaction was found to be specific for
thiols. Unlike other thiol-specific reactions that normally do not produce reactive
intermediates, this reaction produces a sulfinic acid (RSO H) as the intermediate,
2
which can further trigger an intramolecular cyclization to release -OH containing
payloads. Moreover, the reaction template allows easy modifying all three
components: the heterocycle part, the linker, and the payload. As such, it could serve
as a modular template for the development of thiol-triggered payload release. This
was demonstrated by SBTP-1 (a fluorescent sensor) and SBTP-NO (a NO prodrug).
Further optimization of this template and its applications are still undergoing in our
lab and will be reported in due course.
Acknowledgement
This work was supported by NSF (CHE1954826) and NIH (GM125968).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version.
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Credit Author Statement
Jessica Knight: Conceptualization, Methodology, Visualization, Investigation
Yingying Wang: Methodology, Visualization, Investigation
Shi Xu: Methodology, Investigation
Wei Chen: Methodology, Writing-review&editing
Clifford E. Berkman: supervision, writing review & editing
Ming Xian: Conceptualization, writing review & editing, supervision, funding
acquisition.

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conflict of interests
There are no conflicts to declare.

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Schemes and Figures:
Scheme 1. The design strategy of thiol-labeling reagents.
Scheme 2. Synthesis of SBTP-1 and SBTP-2.
Scheme 3. Model reaction of the sensor with thiols.
Scheme 4. Synthesis of SBTP-NO.
Fig. 1. (A) Time-dependent fluorescence enhancement (F/F ) of 10 μM SBTP-1 to
0
different biothiols: 100 μM Cys; 100 μM Hcy; 100 μM GSH; and 1 mM GSH. (B)
Fluorescence enhancement (F/F ) of 10 μM probes to different biothiols in 60 min: (1)
0
1
00 μM Cys; (2) 100 μM Hcy; (3) 100 μM GSH; (4) 1 mM GSH. The reactions were
carried out at 37 °C in PBS buffer (10 mM, pH 7.4) with 50% DMSO.
Fig. 2. (A) Fluorescence measurement of SBTP-1 (10 μM) to common oxidants and
various RSS: (1) probe alone; (2) 100 μM NaClO; (3) 100 μM H O ; (4) 100 μM
2
2
NaNO ; (5) 100 μM Na SO ; (6) 100 μM Na S O ; (7) 100 μM GSSG; (8) 100 μM
2
2
3
2
2
3
Cys; (9) 100 μM Hcy; (10) 100 μM GSH. (B) Competitive fluorescence intensity
changes of SBTP-1 (10 μM) with and without GSH (100 μM) in the presence of
various amino acids (100 μM). All the reactions were carried out for 60 min in PBS
o
buffer (10 mM, pH = 7.4) with 50% DMSO at 37 C.
Fig. 3. Fluorescence emission spectra of SBTP-1 (10 μM) with GSH at varied
concentrations (0, 1, 5, 10, 15, 20, 30, 40, 50, 100 μM for curves 1-10 respectively).
o
The reactions were carried out for 60 min at 37 C in PBS buffer (10 mM, pH = 7.4)
with 50% DMSO.
Fig. 4. Fluorescence response of 10 μM SBTP-1 to varied concentrations of Cys in
o
deproteinized fetal bovine serum. The reactions were carried out for 60 min at 37 C
in deproteinized fetal bovine serum with 50% DMSO.
Fig. 5 Fluorescence intensity at of 10 μM DAF-2 in the presence of various reactive
species: (1) DAF-2 alone ; (2) SBTP-NO (100 μM); (3) GSH (1 mM); (4)
pyrrolidine-NONOate (100 μM); (5) GSH (1 mM) and pyrrolidine-NONOate (100
μM); (6) SBTP-NO (100 μM) and GSH (1 mM); (7) SBTP-NO (100 μM) and Cys
(200 μM); (8) SBTP-NO (100 μM) and Hcy (200 μM). All the reactions were carried
o
out for 60 min in PBS buffer (10 mM, pH = 7.4) with 50% DMSO at 37 C.

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Graphical abstract
Highlights


A reaction template based on thiol/alkyl sulfonylbenzothiazole reaction was
designed.
This reaction template was specific for biolthiols and produced a sulfinic acid
(
RSO H) as the intermediate, which further triggered an intramolecular
2
cyclization to release a -OH containing payload.
This template could be used to design thiol-specific fluorescent sensors and
thiol-triggered prodrugs.


Figure 1

Figure 2

Figure 3

Figure 4

Figure 5