Sulfur dioxide prodrugs: Triggered release of SO2 via a click reaction

Article abstract of DOI:10.1039/c6cc08844a

Sulfur dioxide (SO₂) is being recognized as a possible endogenous gasotransmitter with importance on par with that of NO, CO, and H₂S. Herein we describe a series of SO₂ prodrugs that are activated for SO₂ relea

Full text of DOI:10.1039/c6cc08844a

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DOI: 10.1039/C6CC08844A
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Sulfur Dioxide Prodrugs: Triggered Release of SO2 via a Click
Reaction
Wenyi Wang,a Xingyue Ji, a* Zhenming Du,a and Binghe Wanga*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
-
2-
Sulfur dioxide (SO2) is being recognized as a possible endogenous and HSO3 /SO3 ion pair in aqueous solution to release SO2.13,
-
gasotransmitter with importance on par with that of NO, CO, and 14 However, using the HSO3 /SO32- ion pair for SO2 release does
H2S. Herein we describe a series of SO2 prodrugs that are activated not allow easy control of its quantity and relies on conversion
for SO2 release via a bioorthogonal click reaction. The release rate kinetics, which can be very complex. Another drawback of this
can be tuned by adjusting the substituents on the prodrug.
method is that it is hard to de-convolute the biological effects
-
2-
of HSO3 /SO3 and molecular SO2, which have been
demonstrated to be different.15 Thus, there is a need to
develop prodrugs with controlled release of SO2 to imitate the
process of endogenous production of SO2. Several groups have
developed SO2 donor molecules with various triggering
mechanisms. Chakrapani and colleagues have reported clever
designs that utilize biological thiol (cysteine) and UV light to
trigger the release of SO2 gas from donors.16, 17 They also
developed elegant work on using benzosultines to generate
SO2 under physiological conditions.18 Recently, Xian’s group
reported benzothiazole sulfinate (BTS) as a pH dependent
water soluble SO2 donor.19 We are interested in developing
SO2 prodrug systems with controllable release rates under
physiological conditions. Such prodrugs would complement
what is already available in providing research tools to help
advance this area of research.
SO2 gas is well known as an air pollutant and may cause
respiratory and cardiovascular diseases upon chronic
exposure.1-3 SO2 has also long been recognized as an
antimicrobial agent, and widely used in food industry,
especially in the brewery industry. Recent years have seen
increasing evidence that SO2 can be produced endogenously
by oxidation of sulfur-containing amino acids (cysteine,
homocysteine).4-6 SO2 has also been shown to have possible
physiological functions. There has been an increasing level of
understanding of SO2’s mechanism of actions at the pathway
level,7-9 and fluorescent probes have been reported that can
11
detect endogenous production of SO2.10,
SO2 has been
reported to exacerbate ischemia-reperfusion injury, and
suppress hypertension, pulmonary hypertension, vascular
remodeling, and inflammation.12 Thus, interest in exploring
SO2’s functions as a gasotransmitter has increased in recent
years. Up until now, knowledge of SO2’s physiological and
pathological roles is still very limited, especially in comparison
with the other three gasotransmistters NO, CO, and H2S.
Our group has
a long-standing interest in studying
gasotransmitters.20-26 Previously, we have demonstrated the
feasibility of using an extrusion reaction as a way to deliver
carbon monoxide (CO).21 Herein we describe a strategy to cage
SO2 in thiophene dioxide and use a strained alkyne to trigger
the release of SO2 in a controllable fashion (Scheme 1). By
varying the substituents on the thiophene dioxide scaffold, the
release rates can also be tuned.
There are three main methods currently used to provide SO2
-
for research purposes: gaseous SO2, HSO3 /SO32- pair, and SO2
donors. Despite the broad application in research activities,
gaseous SO2 has obvious disadvantages including difficulties in
preparing solutions of precise concentrations. Besides, the
required equipment can be cumbersome with potential hazard
-
2-
for lab personnel. The HSO3 /SO3 ion pair method relies on
the equilibrium established between hydrated SO2 (SO2 H2O)
·
a.Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia
State University, Atlanta, Georgia 30303, USA
*Correspondence: xji@gsu.edu (X. Ji); wang@gsu.edu (B. Wang)
Scheme 1. Proposed SO2 donor system
Electronic Supplementary Information (ESI) available: Additional experimental
data. See DOI: 10.1039/x0xx00000x
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(A)
As early as 1976, Stille already examined the reaction of
thiophene dioxide and a terminal alkyne under reflux condition
in toluene. Though conditions were “harsh”, the cheletropic
reaction yielded a cyclized product with the release of SO2.27
We planned to take advantage of this reaction to construct
caged SO2 for release under near physiological conditions
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DOI: 10.1039/C6CC08844A
(room temperature to 37 C, pH 7.4, and aqueous solution). A
(B)
key issue in this project was how to lower the reaction
temperature to ambient temperature for applications under
near physiological conditions. 2,3,4,5-Tetrachlorothiophene
dioxide has been shown to possess high reactivity in
cycloaddition reactions with a variety of olefines. Specifically, it
was shown to react steadily with ethylene and to give a
Room temperature
37 ⁰C
0.5
0.4
0.3
0.2
0.1
0
o
cyclohexyldiene product and SO2 at 28 C.28 We reasoned that
a strained alkyne with increased HOMO energy would promote
this cycloaddition reaction. The formation of a stable phenyl
ring should also help to drive the subsequent cheletropic
reaction with the concomitant release of SO2. Therefore, we
synthesized 2,3,4,5-tetrachlorothiophene dioxide
commercially available perchlorothiophene 2. Then we first
examined the reaction of (20 mM in MeOH) with endo-BCN
, 4 equiv.) at room temperature as a proof-of-concept test.
Indeed, after 5 min, we saw complete conversion of into a
3 from
a
b
c
d
e
f
g
3
Figure 1. (A) DTNB test mechanism. (B) UV absorption at 412 nm of DTNB test after 45
min incubation at room temperature or 37 oC of (a) blank (5% DMSO/PBS), (b) 250 μM
of 3 only, (c) 2.5 mM of 1 only, (d) 250 μM of 4 only, (e) 250 μM of 3 +2.5 mM of 1, (f)
50 μM of Na2SO3, (g) 50 μM of NaHSO3.
(1
3
new product, which was spectroscopically characterized as
(Scheme 2).
4
confirming the formation of SO32-. Both experimental groups
We then examined the reaction kinetics. The UV absorption
of the product is significantly lower than that of the reactants
at 328 nm. Therefore, we used UV absorbance decrease to
monitor the reaction progress. The second order rate constant
2-
and positive control groups showed lower SO3 formation at
37 oC than at room temperature, presumably due to increased
SO2 escape at higher temperature (Figure 1B).
Encouraged by the initial success, we went on to synthesize
additional analogues to see whether we can tune the reaction
rates for various applications. Thus, we synthesized
was determined by first using a large excess of
the pseudo-first order reaction followed by plotting the
pseudo-first order reaction rate constants against different
1 to examine
1
compounds
5 and 6 with electron withdrawing groups at
concentrations. The second order rate constant was
determined to be 1.50 M-1 s-1 in MeOH at room temperature,
allowing the reaction to be finished in about 3 hours at mid-
μM levels (Figure S1B).
different positions (Figure 2A; for synthetic scheme see ESI).
We reasoned that by varying the electron density on the
thiophene ring, different LUMO energy will result in varied
reaction rate with BCN. We also tested reaction with a trans-
cyclooctene compound (8, equatorial isomer), which is known
to have high strain energy and high HOMO, to see if the
reaction rate can be further enhanced. In all these reactions at
room temperature, we successfully isolated the cheletropic
reaction products (see ESI). Interestingly, we had initial
difficulties in analyzing the room temperature NMR spectra of
the products containing the bicyclo[6.1.0]non-4-ene structure.
To further confirm the formation of SO2, we performed the
DTNB test, which has been applied in SO2 measurement in
atmosphere and food samples.29-31 DTNB would react with
2-
SO3 ion to give an reduced product, which can be
quantitatively measured by UV absorbance at 412 nm (Figure
1A).29 To allow hydration of SO2, we performed this test in 5%
DMSO/PBS. A reaction mixture was incubated at room
o
temperature or 37 C for 45 min (about 1 t1/2) and then the
1
This was due to peak overlap caused by line broadening in H
DTNB probe was added. Afterwards, the test solution was
incubated at room temperature for another 15 min and
subjected to UV absorbance reading at 412 nm. The
experimental group showed higher absorbance at both room
temperature and 37 oC than the negative control groups,
NMR. Structure elucidation by 13C NMR is equally difficult
because of missing peak(s) at high field. Given HRMS
confirmation, we reasoned that this phenomenon is caused by
the slow flipping of eight-member ring between the “chair”
and “boat” conformations. We successfully observed two
conformations by tracking the -CH2 group next to the -OH
group at low temperature (270 K). As temperature increases,
the exchange rate of the two populations increases, and the
two peaks observed for the –CH2 group coalesced and then
averaged to give a sharp peak. Unfortunately, within the
temperature range tested, the exchange rate is not fast
enough to average the –CH2 and –CH groups from the
Scheme 2. Proof-of-concept test of SO2 donor system
2 | J. Name., 2012, 00, 1-3
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(A)
(B)
Table 1. Kinetics study of SO2 donor pairs
View Article Online
DOI: 10.1039/C6CC08844A
SO2 donor
pairs
6 +
exo-BCN
0.04a
3 + 1
3 + 8
5 + 1
6 + 1
0.05a
b
7 + 1
k2 (M-1s-1)
1.50a
0.02a
0.33b
0.01c
a
Measured in MeOH under room temperature. Measured in ACN under room
temperature. c Measured in DMSO/PBS 4:1 at 37 oC.
SO2 generation due to delayed response.32 One alternative
strategy for real time monitoring of SO2 release is to devise
such SO2 prodrugs that would become fluorescent after SO2
release. Therefore, we were interested in designing SO2
prodrugs that would lead to the formation of a fluorescent
reporter, which allows for real-time monitoring of SO2 release.
We reasoned that attaching an aromatic substituent to the
thiophene ring would allow formation of an expanded
conjugation system after the cheletropic reaction and thus
result in pronounced changes in the spectroscopic behavior of
the aromatic system after the reaction. Therefore, we
synthesized thiophene dioxide with naphthalene conjugated at
(C)
-CH2OH
–CH2 and –CH groups
from the bicyclo[6.1.0]non-4-ene structure
270 K
278 K
288 K
298 K
308 K
318 K
the 3,4 positions (7, Figure 2A). The compound itself has weak
yellow fluorescence with an excitation wavelength of 299 nm,
emission wavelength of 556 nm, and a quantum yield of 0.006.
After reaction with BCN and SO2 release, the resulting product
shows strong cyan fluorescence with an emission wavelength
of 470 nm, excitation wavelength of 296 nm, and quantum
yield of 0.138 (Figure 2B). We also established the kinetics
profile of this reaction by monitoring the fluorescence increase
at 470 nm. A k2 of 0.01 M-1s-1 was obtained (Table 1; Figure 3;
Figure S2).
In summary, we have developed a SO2 donor system, which
releases SO2 through a Diels-Alder reaction with a strained
alkyne/alkene as a trigger. We explored the initial tunability of
the releasing rate by varying the electron density of thiophene
dioxide ring. With a small set of compounds, we were able to
tune the reaction rate in the range of 0.01-1.50 M-1s-1, giving a
150-fold range in reactivity.
Additional work is underway in further extending the
tunability of reaction rate and in testing the donor system in
triggering various biological responses.
Figure 2. (A) SO2 donor pairs. (B) Fluorescent SO2 donor and product after SO2 release
(100 μM in MeOH). (C) Coalescence of cheletropic product (6 + 1) observed in 13C NMR
(DEPT-135) with increasing temperature.
bicyclo[6.1.0]non-4-ene structure, which became broad above
278
phenomenon has been observed exclusively with endo-
product. As for product obtained from reaction between and
K and hard to track (Figure 2C). However, this
6
exo-BCN, sharp peaks at high field were observed at room
temperature (see ESI). By contrast, with chlorine substitutions,
we are able to track the carbons at room temperature,
presumably because of its fast flipping rate. Edited HSQC
experiment gave clear evidence of geminal protons splitting
from the eight-membered ring, confirming the structures of
cheletropic products (see ESI). We studied the reaction kinetics
for these reactions and obtained k2 values ranging from 0.02 to
300
Background
250 μM 7
250
2500 μM 1
200
5 min
18 min
150
30 min
100
50
0
1 h
2 h
3 h
4 h
0.33 M-1s-1 (Table 1; Figure S1). However, compound
5 was
found to be unstable in MeOH at room temperature, which
was presumably ascribed to its strong electrophlicity.
Therefore, compound
5 is not a suitable SO2 prodrug for
380
480
580
680
biological applications due to this stability issue.
Wavelength (nm)
Currently, several elegant fluorescent probes for SO2 have
-
been developed based on the nucleophilicity of SO32-/HSO3 .
Figure 3. Fluorescence response increases as cycloaddition reaction between 7 and 1
proceeds (λex=350 nm). Condition: 250 μM 7 + 2500 μM 1 in 4:1 DMSO/PBS at 37 oC.
However, few of them are suitable for real-time monitoring of
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30.
Z. Guo, Y. Li, X. Zhang, W. Chang and Y. Ci, VAienwaAl.rtiBclieoOannlainle.
Acknowledgement: Partial financial support from the National
Institutes of Health (CA180519) is gratefully acknowledged.
Chem., 2002, 374, 1141-1146.
DOI: 10.1039/C6CC08844A
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32.
Y. Li and M. Zhao, Food Control, 2006, 17, 975-980.
V. Lin, W. Chen, M. Xian and C. Chang, Chem. Soc. Rev.,
2015, 44, 4596-4618.
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