Esterase Sensitive Self-Immolative Sulfur Dioxide Donors

Article abstract of DOI:10.1021/acs.orglett.7b02544

A series of cell-permeable esterase-sensitive sulfonates that undergo self-immolation to produce sulfur dioxide (SO₂), a gaseous pollutant with new and emerging biological roles, is reported. These compounds should facilitate the study SO₂ biology and will lay the platform for newer stimuli-responsive donors of this gas.

Full text of DOI:10.1021/acs.orglett.7b02544

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Esterase Sensitive Self-Immolative Sulfur Dioxide Donors
Kundansingh A. Pardeshi, Govindan Ravikumar, and Harinath Chakrapani*
Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pune 411 008,
Maharashtra, India
S
* Supporting Information
ABSTRACT: A series of cell-permeable esterase-sensitive sulfonates that
undergo self-immolation to produce sulfur dioxide (SO2), a gaseous
pollutant with new and emerging biological roles, is reported. These
compounds should facilitate the study SO2 biology and will lay the platform
for newer stimuli-responsive donors of this gas.
SO2 and again, incorporation of a trigger will help in controlling
delivery of this gas. Xian and co-workers have recently reported a
sulfinate which undergoes hydrolysis to produce SO2.14 They
have also shown the vasodilatory effects of this molecule
underscoring the importance of controlled generation of SO2.
However, this donor does not contain a physiological trigger and
this may be a limitation for further exploitation of therapeutic
potential of this gas.
Photolabile SO2 donors have also been reported. Benzosul-
fones were found to be photolabile under ultraviolet irradiation
conditions to produce SO2.15 A similar class of phototriggerable
SO2 donors were reported by Uchida and co-workers.16 The use
ofultravioletlight, however, haslimitedutilityandmaybeharmful
to cells due to phototoxicity. Our laboratory has reported 2,4-
dinitrophenylsulfonamides (DNs-Amine) as thiol-activated
sulfur dioxide donors (Figure 1).17−19 While these donors have
triggerable SO2 donation, the use of thiols, which are a major
antioxidant withincells, astriggersmaynotbeusefultostudySO2,
a redox-active gaseous species. It is thus desirable to have a
relatively innocuous stimulus such as esterase (ES) to trigger SO2
generation within cells.
In order to design a new class of esterase-sensitive SO2 donors,
we considered the chemistry associated with decomposition of
carbonates, which have been extensively used for drug delivery
and they operate by generation of carbon dioxide (CO2), which
presumably is an irreversible reaction. An appropriately placed
trigger and subsequent self-immolation can produce CO2 from a
carbonate (Scheme 1a). The key bond that breaks is the C−O
bond shown by an arrow, whose estimated bond dissociation
energy (BDE) is 68 kcal·mol−1. Similarly, it was envisaged that
cleavage of a C−S bond in sulfonates (shown by an arrow) will
similarly trigger generation of SO2 and an alcohol. The BDE of
this bond is estimated to be 57 kcal·mol−1, which is comparable
with the C−O bond.20 If successful, such a method may have
broad relevance as a probe for SO2 biology as well as a
methodology for codelivery of an alcohol-based drug. Further-
aseous signaling molecules derived from nitrogen, carbon,
Gand sulfur are ubiquitous in nature and are emerging as
major modulators of diverse physiological processes.1 For
example, hydrogen sulfide (H2S) has numerous roles in
neuromodulation, pathogen response to antibiotics,2 and blood
vessel relaxation. Sulfur dioxide (SO2),3 a potential product of
oxidation of H2S, similarly has recently assumed importance as a
mediator of cellular processes.4 SO2 is widely used in the food
industry as a preservative and as an antibacterial agent.5 At
diminished concentrations, SO2 can mediate signaling such as
vasodilation.6 However, at elevated concentrations, SO2 can
damage biomacromolecules such as DNA, especially in the
presence of metal ions.7−9 Thus, controlled generation of this
gaseous molecule has enormous potential but is challenging.
Biochemical and cellular experiments carried out with gaseous
SO2 are somewhatunreliable, associated withpotential variations,
and have limited therapeutic potential. The most commonly used
inorganicdonorofsulfite, whichisamixture ofsulfiteandbisulfite
has limited permeability and is thus used in high millimolar
concentrations.5,6 These elevated concentrations may compro-
mise appropriate interpretations regarding the effects of SO2.
Thus, small molecule donors of this gas assume importance.10
Our laboratory reported benzosultines as donors of SO2 under
ambient physiological pH conditions (Figure 1).11 These donors
spontaneously undergo retro Diels−Alder to generate SO2 with
moderately tunable rates. Binghe Wang and co-workers have
recently shown a “click and release” strategy for generation of
sulfur dioxide (Figure 1).12,13 These donors have the unique
advantage of having a wide range of rates of SO2 generation.
However, these donors are more akin to spontaneous donors of
Received: August 17, 2017
Revised: December 2, 2017
Figure 1. Representative examples of sulfur dioxide donors.
© XXXX American Chemical Society
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Scheme 1. (a) Esterase-Cleavable Carbonate Should Generate
CO2 and an Alcohol (1a, R = Umbelliferone (Figure 2)); (b)
Proposed EsteraseCleavableSulfonatesThat AreExpected To
Generate SO2 and an Alcohol
Table 1. Synthesis of Sulfonates 2a−k
a
R
pKa of ROH
product
yield (%)
umbelliferone
7.7
9.9
2a
2b
2c
2d
2e
2f
50
75
65
70
65
72
23
27
24
53
44
Ph
4-OMePh
10.2
7.1
4-NO2Ph
(2-tert-b
10.2
9.4
(3,5-dimethyl-4-Cl)Ph
Bn
15.4
15.5
13.6
16.0
17.0
2g
2h
2i
allyl
propargyl
n-butyl
iPr
2j
2k
more, the nature of the leaving group i.e. the alcohol may
determine the SO2 generation capability.
In order to test this hypothesis, first, the fluorophore
umbelliferone (Umb, Figure 2) was derivatized. The carbonate
a
Values are from literature and are either for the compound itself or
analogues with similar structures. References 27−31.
Figure 2. Structures of umbelliferone and 8.
Figure 3. (a) HPLC analysis of 2a. Compound 2a (50 μM) in PBS (pH
7.4, 10 mM) at 37 °C was incubated in the presence of esterase showed
the disappearance of 2a. Rate constant for disappearance was found as
0.13 0.01 min−1. (b) HPLC analysis of the reaction mixture for Umb.
Rate constant for appearance of Umb was found as 0.18 0.006 min−1
(for a detailed protocol, see the SI).
1a (Scheme 1a) was synthesized (see the SI). Due to the
improved hydrolytic stability of cyclopropyl esters when
compared with other aliphatic esters, this ester was chosen.21,22
The pivaloyloxymethyl group was not considered as sulfite is
known to react with formaldehyde, a byproduct of decom-
position.23 In order to synthesize the sulfonate 2a, first, the
aldehyde 3 was synthesized from 4-hydroxybenzaldehyde
(Scheme 2) Reduction with sodium borohydride gave 4, which
of Umb was 80% suggesting an efficient conversion of 2a to Umb
in the prescence of esterase.
Acoumarin−hemicyaninedye7(Scheme3),26 forcolorimetric
as well as fluorescence-based detection of sulfite (SO32−), the
Scheme 2. Synthesis of the Sulfonyl Chloride 6
Scheme 3. Dye 7 and the Adduct It Forms upon Reaction with
Sulfite
hydratedformofSO2,wassynthesizedusingreportedprocedures.
This probehas beenfoundtobeuseful ininvitro aswellas cellular
studies to detect SO2. The dye 7 reacts with sulfite to produce a
covalent adduct (Scheme 3). This results in a characteristic
decrease in absorbance at 545 nm with concomitant increase in
the absorbance signal at 410 nm (Figure 4a). In the presence of 2a
and esterase, a decrease in the absorbance at 545 nm was seen
(Figure 4b). Due to the significant absorbance of Umb at 410 nm,
the use of 7 to accurately determine sulfite is not possible.
However, based on the diminution of the absorbance at 545 nm,
the generation of sulfite during decomposition of 2a in the
presence of esterase was inferred (see Figure S6).
was then converted to the thioacetate 5 in two steps. The
thioacetate was treated with N-chlorosuccinamide to afford the
sulfonyl chloride 6.24 Treatment of 6 with umbelliferone (Figure
2) gave 2a (Table 1). The structure of 2a was confirmed by X-ray
diffraction analysis of crystalline material (see Figure S1).
The carbonate 1a and the sulfonate 2a were independently
treated with esterase.22,25 HPLC analysis of the reaction mixture
after 10 min revealed complete disappearance of the 1a (see
Figure S2). The disappearance of 2a (Figure 3a) was somewhat
slower when compared with 1a (0.54 0.08 min−1). The rate
constant was found to be 0.13 min−1 and the half-life was 5 min.
This rate is comparable with the formation of umbelliferone, 0.18
min−1 monitored under similar conditions (Figure 3b). The yield
Intheabsenceofesterase, nosignificantdecompositionof1aor
2a was observed (see Figure S3), suggesting that the cyclo-
propylester is not susceptible to hydrolysis. When 2a was
B
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absorbance profile for 2e is shown in Figure 4c. The ratio of
absorbance at 410 nm (A410) to the absorbance at 545 nm (A545
)
provides an estimate of the ability of the compound to produce
SO2. Colorimetric analysis revealed that all the aforementioned
compounds were capable of generating sulfite. The dye 7 is also
used as a sulfite-sensitive fluorescence probe: the free probe
displayed a red emission with the maximum at 633 nm (excitation
at 545 nm). In the presence of sulfite, the fluorescence signal at
this wavelength is diminished with a concomitant increase in a
newblueemissionpeakat478nm(excitationat410nm). I478/I633
is hence a measure of SO2 donating capability. The fluorescence
data corroborated the absorbance data that was obtained with the
donors. Together, our data supports a mechanism of ester
hydrolysis leading to formation of intermediate I, which
undergoes self-immolation to produce an alkoxide and a quinone
methide (Scheme 4).
a
Scheme 4. Mechanism of Generation of SO2 from Sulfonates
Figure 4. (a) Incubation of 7 (10 μM) in the presence of NaHSO3 (50
μM) for 15 min shows a decrease in absorbance at 545 nm and a
corresponding increase in absorbance at 410 nm.; (b) Incubation of 2a +
ES inthe presence of 7 (10 μM) for 15 min. Disappearance of the 545 nm
signal was observed and is indicative of sulfite formation. Concentration
of2awas 50 μM. Thisexperiment was carried outin the presenceof Umb
(see Figure S6). (c) Incubation of 7 (10 μM) in the presence of 2e + ES
for 15 min shows an absorbance profile comparable with NaHSO3. (d)
Ratio of absorbance at 410 nm (A410) to the absorbance at 545 nm (A545
)
during incubation of 7 with various compounds (Ctrl) and in the
presenceofES(seeFigureS7). Concentrationof2ewas50μM. Allofthe
experiments were conducted in PBS (pH 7.4, 10 mM) at 37 °C (for a
detailed protocol, see the SI).
a
For sulfonates derived from aromatic alcohols, SO2 generation is
pretreated with an esterase inhibitor (phenylmethanesulfonyl
fluoride, PMSF)32 and subsequent exposure to ES, a diminished
signal for Umb was observed (see Figure S4). Preincubation of ES
with PMSF and subsequent addition of 2a showed a dose-
dependent decrease in fluorescence, again supporting catalysis by
esterase is necessary for activation of 2a (see Figure S4, inset). In
the presence of common biological nucleophiles, 2a was found to
be stable (see Figure S4). In basic pH (9.2), significant formation
of Umb from the hydrolysis of the carbonate was observed; while
the sulfonate remained stable. This enhanced stability of the
sulfonate may present opportunities for delivering SO2-based
hybrid drugs.
In order tostudythereactivity ofthe sulfonatefunctional group
toward esterase, compound 8 (Figure 2) was synthesized. When
treated with esterase, no significant fluorescence signal
corresponding to Umb formation was recorded (see Figure S4).
A similar result was observed in human cervical cancer HeLa cell
lysate during 1 h suggesting that the sulfonate group is stable
under cellular conditions (see Figure S5). Incubation of 2a, as
expected, produced a fluorescence signal confirming the
production of Umb. Taken together, our data supports the
selectivity of cleavage by esterase and the broad-range stability of
the sulfonate group to cellular nucleophiles.
dominant. Certain sulfonates derived from aliphatic alcohols were
susceptible to hydrolysis (through II) and were poor SO2 donors or
are possibly trapped as intermediate I.
Next, sulfonates 2g−k, which are derived from aliphatic
alcohols were synthesized. When estimated for SO2 in the
presence of esterase, 2g, 2h, and 2k did not show a significant shift
in the absorbance profile, and accordingly, the ratio of A410/A545
was diminished (Figure 4d). This data suggests that these
compounds are poor donors of SO2 when compared with their
counterparts. Being aliphatic alcohols (Scheme 4), their leaving
group ability is significantly lower than aromatic alcohols and this
may contribute to diminished yield (see Table 1 for pKa values).
Prolonged incubation in buffer of these reaction mixtures also did
notproducesignificantlevelsofsulfite.Sincethecyclopropylester
is stable toward hydrolysis in pH 7.4 buffer, the propensity for the
sulfonate group to undergo hydrolysis to produce II was next
examined. Compounds 2g−k were independently incubated in
pH 7.4 buffer, and we found that all compounds except for the
benzyl and allyl compounds were stable toward hydrolysis (see
Figure S12). When the benzyl derivative was incubated in buffer,
nearly complete disappearance in 30 min was seen. While partial
hydrolysis was observed in the case of the allyl derivative, this data
suggested that certain sulfonates were susceptible to hydrolysis.
Thus, amongaliphatic alcohol-derived sulfonates, SO2 generation
afteractivation byesteraseispossibleprovidedthesulfonateisnot
susceptible to hydrolysis and if the alcohol was a good leaving
group (such as propargyl alcohol).
Having established that 2a was capable of undergoing self-
immolation in the presence of esterase to generate SO2, we next
proceeded to study the effect of the leaving group on this process.
Aseriesofaromaticalcoholswereindependentlyreactedwith6to
produce the corresponding sulfonates 2b−f (Table 1). Again, the
SO2-sensitive dye 7 was used to assess the capability of these
compounds to generate SO2. These compounds were next
exposed to esterase in the presence of the dye 7. A representative
Next, we investigated the capability of the SO2 donors to
permeate cells and generate SO2. Accordingly, the dye 7 was used
C
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in A549 lung carcinoma cells. In the presence of sulfite (200 μM),
a distinct increase in the signal in the blue channel with
concomitant decrease in signal in the red channel was observed
(Figure5andFigureS13). TheSO2 donors2cwassimilarlyfound
ACKNOWLEDGMENTS
■
We thank the Department of Science and Technology (DST,
Grant No. EMR/2015/000668) and the Council for Scientific
and Industrial Research (CSIR) for financial support.
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ASSOCIATED CONTENT
* Supporting Information
■
S
TheSupportingInformationisavailablefreeofchargeontheACS
Publications website at DOI: 10.1021/acs.orglett.7b02544.
Synthesis, characterization data, protocols for assays, and
X-ray data for 2a (PDF)
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2013, 49, 2637.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: harinath@iiserpune.ac.in.
ORCID
(33) Wang, W.; Wang, B. Chem. Commun. 2017, 53, 10124.
Harinath Chakrapani: 0000-0002-7267-0906
Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acs.orglett.7b02544
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