Synthesis and evaluation of cyclic sulfite diesters as sulfur dioxide (SO2) donors
Although sulfur dioxide (SO2) finds widespread use in the food industry as its hydrated form, sulfite, a number of aspects of SO2 biology remain to be completely understood. Among the tools available for intracellular enhancement of SO2, most suffer from poor cell permeability and a lack of control over SO2 release. We report 1,2-cyclic sulfite diesters as a new class of reliable SO2 donors that dissociate in buffer through a nucleophilic displacement to produce SO2 with tuneable release profiles. We provide data in support of the suitability of these SO2 donors to enhance intracellular levels of SO2 at an efficiency superior to sodium bisulfite, the most commonly used SO2 donor for cellular studies.
Introduction<!>Results and Discussion<!>Scheme 3. Reaction of probe 23 with sulfites<!>Conclusions
<p>Sulfur dioxide (SO2) is an environmental pollutant that is also produced during metabolism of sulfur containing amino acids [1] as well as hydrogen sulfide (H2S), which is known to mediate a number of cellular processes. [2] The known vasodilatory effects of SO2 in animal models suggest possible signaling roles for this gas as well. [3] SO2 is also used in the food industry as a preservative and an anti-bacterial agent. [4] At elevated levels SO2 is known to cause biomacromolecular damage and cell death; [5] these damaging effects are perhaps responsible for the anti-bacterial properties of this gas. However, due to the limited understanding of molecular mechanisms of action of this gas, reliably producing [6] and detecting SO2 within cells are necessary. While there are numerous probes for SO2, biological studies have thus far relied on gaseous SO2 or a complex formulation of inorganic sulfites. [7] Both methods may not be well suited for enhancing SO2 within cells and offer no temporal control over SO2 release. Furthermore, they are useful for studying effects of SO2 as a single dose, which is unsuitable for study of prolonged exposure. Thus, the chemical biology of SO2 remains largely uncharacterized. 15 Our laboratory has developed several strategies having different triggers for generating SO2 under physiological conditions using small organic molecules. [8] First, 2,4-dinitrophenylsulfonamides with tunable rates of generation of SO2 when triggered by biological thiols were reported (Scheme 1). The use of thiol is used as a trigger may complicate biological studies as targets of SO2 include biologically relevant disulfides and thiols. [8a, 8b, 9] In a second strategy, a series of benzosultines as SO2 donors with controlled rate of generation of SO2 under physiological condition, having heat as a trigger was reported (Scheme 1). [8c] Although enzosultines are stable as solids, they are not highly suited for prolonged storage at room temperature as stock solutions. In third strategy, benzosulfones were reported as photochemically activated SO2 donors by our group and others (Scheme 1). [8d] These compounds might have limitations associated with the inconvenience associated with using a light source in cellular studies and possibly by intensity of light that was required for SO2 generation. [10] Xian et al. reported sodium benzothiazole sulfonate as water-soluble SO2 donor, having limitation of prolonged half-life (t1/2 = 13 days) at physiological pH 7.4. [11] Recently, SO2 donors, using esterase as a trigger, [8f, 12] and click reaction as a mode of SO2 donation, have been reported by our, and Wang's group (Scheme 1). These strategies rely on the use of cellular enzyme, and bio-orthogonal reaction.</p><p>Scheme 1. Reported strategies for generation of SO2 under physiological conditions.</p><p>Therefore, new self-emulative SO2 donors which are stable at room temperature and permeate cells to enhance intracellular SO2 could help better understand cellular responses to this important gaseous molecule.</p><!><p>1,2-Cyclic sulfite diesters were considered as SO2 donors (Scheme 2). Upon attack by a nucleophile (such as water), a sulfite monoester would be formed, which could spontaneously decompose to produce SO2. Modulating substituents "R" or perhaps the pKa of the leaving group would help tune the rate of substitution by a nucleophile and possibly SO2 release. Here, we report results of synthesis and evaluation of a series of 1,2-cyclic sulfite diesters as SO2 donors. 1,2-diols used for synthesis of 1,2-cyclic sulfite diesters were either commercially available (1 and 2; Figure 1) or prepared by Upjohn dihydroxylation from corresponding olefin by OsO4 mediated dihydroxylation [13] (diols 3-5 and 11; Figure 1) or by nucleophilic ring opening of 4-substituted styrene oxide by 10% aq. K2CO3 solution. The diol 6 and 10 were synthesized from Ltartaric acid, and NaBH4 mediated reduction [14] of (±)-benzoin respectively. The diols 7 and 8-9 were prepared by ring opening of corresponding (±)-phenyloxirane and substituted (±)phenyloxirane, in 10% K2CO3 reflux condition respectively. Various 1,2-cyclic sulfite diesters (12-22) were prepared by the reaction of 1,2-diols with thionyl chloride, triethylamine and imidazole in DCM at 0 °C (Table 1). [15] The cis-1,2-diols 3 and 4, gave an isomeric mixture (depending on sulfoxide orientation) of 14 (1:0.68), 15 (1:0.64) by 1 H NMR. [16] The diols 5, 6 and 11 afforded mixture of diastereomers of 16, 17 and 18 in the ratios (depending on chirality of sulfoxide), 16 (1:0.95), 17 (~1:1) and 22 (1:0.86) by 1 H NMR. The racemic 1,2-diols 7-10 afforded mixture of diastereomers for 18-21.</p><p>The aforementioned derivatives 12-22 were evaluated for SO2 release in pH 7.4 phosphate buffer. First, ethylene glycol derivative 12 was incubated at 37 °C in pH 7.4 buffer for 30 min. The reaction was monitored for SO2 generation by ion chromatography equipped with an ion conductivity detector; [8b] SO2 was quantified as sulfite, SO3 2-. After 30 min, 12 gave 45% of SO2 (Table 2, entry 1). The pinacol derivative 13 produced negligible amounts of SO2 and a 2% yield was recorded (Table 2, entry 2). These results suggest that increasing sterics on the carbon bearing the sulfite functional group reduced the propensity for decomposition of the compound supporting direct displacement at the carbon, which involves formation of a sulfite monoester, which in turn rapidly rearranges to produce SO2 and an alcohol (Scheme 2) Scheme 1. Sulfite diesters can decompose in pH 7.4 buffer to produce SO2. In the cases of 12-15, nearly similar pKa values for the alcohols implies that any difference in SO2 yields must be due to increased steric hinderance at the carbon bearing the sulfite ester. The diesters 12, 14 and 15 gave SO2 yields > 20% after 30 min, whereas pinacol derivative 13 gave 2% of SO2. These results suggest that when leaving group was similar the important determinant of observed reaction rates was sterics supporting the proposed mechanism (Scheme 2). These results are also consistent with previous reports [17] of 1,2-cyclic sulfite diesters undergoing nucleophilic substitution with various nucleophiles such as chloride, azide and (CH3OOC)2HC − at one of the activated carbon atoms (Scheme 2). [17][18] The hydrolysis of cyclic sulfite esters of normal, or cis or trans diols under acidic (cat. H2SO4/HClO4) or basic (2 eq. NaOH) reflux condition, results in sulfur-oxygen bond fission to give corresponding diol without change in stereo-configuration. [17,19] In order to study the electronic effect on decomposition, diesters 16 and 17 were incubated in pH 7.4 buffer and 93% and 98% SO2, respectively were recorded (Table 2, entry 5 and 6). These compounds contain an electron withdrawing substituent as compared to ethylene glycol diester 12. The electron withdrawing nature of the ester enhances electrophilicity of the carbon bearing the sulfite functional group contributing to an increased rate of displacement.</p><p>Next, decomposition of derivatives with phenyl substituent 18-21 was carried out. After 30 min, the phenyl derivative 18 gave 73% of SO2 (Table 2, entry 7). The 4-NO2-phenyl derivative 19 on the other hand produced higher amount of SO2 and a 96% yield was obtained (Table 2, entry 8). Incubation of the 4-F-phenyl derivative 20 (Table 2, entry 9) resulted in a nearly similar SO2 yield as that of phenyl derivative 18. The diphenyl derivative 21 gave 83% SO2 after 30 min (Table 2, entry 10). These results suggest that electron withdrawing group on phenyl substituent increases the rate of decomposition, again consistent with a nucleophilic displacement mechanism. Values are for the corresponding 1,2-diol (most acidic proton) calculated using ChemBioDraw Ultra 16.0.</p><p>A number of SO2 probes that typically use the distinct nucleophilic properties of sulfite/bisulfite have been developed by various groups. [20] . For example, Sun et al. have reported probe 23 for selective detection of SO2 derivatives HSO3 -/SO3 2in pH 7.4 buffer (Scheme 3). [20c] The probe 23 absorbs at 545 nm and upon reaction with HSO3 -/SO3 2-, a distinct and ratiometric shift to an absorbance at 410 nm (for 24) was observed (see Supporting Information, Figures S1 and S2). A similar UV profile was observed when 23 was reacted with the SO2 donor 17 supporting the intermediacy of sulfite (Figure 2a). The probe 23 fluoresces in the red region and upon reaction with SO3 2-/HSO3it forms green fluorescing adduct 24 (Figure 2b). When 23 was treated with sulfite diester 17, we find a similar shift in emission (Figure 2b). Together, these data independently confirm the ability of 17 to generate SO2 in pH 7.4 buffer.</p><!><p>The expected product formed during hydrolysis in buffer is the diol (Scheme 2). In order to confirm this, we incubated 19 in pH 7.4 buffer and monitored the decomposition and formation of 1-(4-nitrophenyl)ethane-1,2-diol 8 (Figure 3). We find nearly complete disappearance of 19 during 30 min with the formation of 8 as the exclusive product (Figure 3) and a nearly quantitative yield of SO2 (Table 2, entry 8). Thus, the decomposition of the sulfite monoester was and SO2 is likely to be generated as soon as the intermediate is produced (Scheme 2). Having established the suitability of cyclic sulfite esters for molecular biology studies, cell permeability as well as the suitability of these compounds for enhancing intracellular levels of SO2 was examined. The ratiometric probe 23 has been previously reported to be suitable for detection of intracellular sulfite/bisulfite. When DLD-1 cells treated with 23 (10 μM), we found a distinct fluorescence signal only in the red channel but not in the green channel (Figure 4A-C). [20c] When cells pre-treated with 23 were exposed to 17 (20 μM), a decrease in fluorescence signal in the red channel with concomitant increase in fluorescence increase in green channel was observed (Figure 4D-F). Under similar conditions, when a similar experiment was conducted with authentic bisulfite, we found a similar profile. [20c] However, an increased concentration of 200 M was necessary to elicit this response whereas with the donor developed in this study, a significantly lower concentration could achieve enhancement of intracellular SO2. Above results suggest the compatibility of cyclic sulfite diesters with cellular nucleophiles. Lastly, a cell viability assay conducted with human cervical cancer cells (HeLa) revealed SO2 donors 17 and 13 were not significant inhibitors of proliferation at 100 μM (see Supporting Information, Figures S3 and S4). Thus, the SO2 donor 17 might find convenient use for studying cellular responses to enhanced reactive sulfur species.</p><!><p>In summary, we report a series of 1,2-cyclic sulfite diesters that: can be easily synthesized; are stable at room temperature; have tunable SO2 release profiles; and are well suited to study effects of enhanced intracellular levels of SO2 and duration of exposure to this reactive species. Together, we present superior alternatives to inorganic sulfites, the most commonly used SO2 donors. Due to the fundamental importance of redox regulation in cellular growth and survival, perturbation of redox homeostasis has emerged as a possible mechanism for the development of new therapeutics. [21] Thus, reliably generating reactive oxygen, [22] nitrogen [23] and sulfur species [8a, 8b, 8e] may have a range of applications including developing small molecule-based inhibitors of against bacteria such as Staphylococcus aureus, [8e, 22e] Mycobacterium tuberculosis [8a, 8b, 22a, 22b] as well as cancer. [24] Experimental Section Synthesis and characterization data for all new compounds and assay protocols.</p>