Chemistry

Sulfones 2, 3, and 4 were synthesized according to the scheme shown in Fig. 7A and Supplementary data. The starting compound in the synthesis of sulfones was commercially available sodium p-toluene sulfinate, which was subjected to the reaction with chloroform and potassium hydroxide solution. Obtained dichloromethyl-p-toluene sulfone 1 was then oxidized to the 4-dichloromethylbenzoic acid by potassium dichromate 2. Compound 3 was then brominated with sodium hypobromite to give bromodichloromethylsulfonyl benzoic acid 4.

Fig. 7

Synthesis of sulfone derivatives A 1, 2 and 3, B dichloro- and (bromodichloromethylsulfonyl)phenylpropanoic acids 7 and 8, C 11 and 12 and D, E. attachment of carboxylic acids to ZnO quantum dots. Conjugate synthesis L-1, L-2, L-3

Sulfones 7 and 8 were prepared according to Fig. 7B and Supplementary data. The substrate for this synthesis was commercial 3-phenylpropanoic acid, which in reaction with chlorosulfonic acid gave 3-(4-chlorsulfonylphenyl) propanoic acid 5, which after reduction with sodium sulfite gave sodium 4- (2-carboxyethyl phenyl) methanesulfinate 6. This salt was used for the reaction with chloroform in the presence of potassium hydroxide to give 3- [4-(dichloromethylsulfonyl) phenyl] propanoic acid 7. This compound was converted into 3-[4-(bromodichloromethylsulfonyl)phenyl] propanoic acid 8 by reaction with an aqueous solution of sodium hypobromite.

The syntheses of compounds 11 and 12 are shown in Fig. 7C and Supplementary data. The starting material was commercially available 4-methylbenzenethiol 9, which was reacted with difluorochloromethane in a water-dioxane sodium hydroxide solution. The obtained difluoromethyl-4-toluene sulfide 10 was oxidized to difluoromethyl 4-methylphenyl sulfone 11 by hydrogen peroxide in acetic acid. Then the methyl group in sulfone 11 was oxidized to the carboxyl moiety using potassium dichromate in sulfuric acid to give sulfone 12.

Sulfones 7, 12, and 8 were attached to ZnO quantum dots that could act as fluorescent markers. Surrounding reactions ZnO quantum dots by sulfone ligands were carried out according to the unique procedure developed by [17], the so-called OSSOM method (one-pot self-supporting organometallic approach), shown in Fig. 7D and Supplementary data. Stable ZnO nanoparticles were not obtained as a result of the L-3 and Et2Zn reaction. In this case, an alternative method of conjugate synthesis was used. In the first step, non-functionalized ZnO nanoparticles were obtained as a result of a direct reaction of diethyl zinc with water. In the second step, an equilibrium amount of ligand was added to the ZnO nanoparticles (Fig. 7E).

BiologyStrains and media

Candida albicans strains used in the study are listed in Table 3. The quality control wild-type strain C. albicans SC5314 was obtained from the American Type Culture Collection (ATCC). All the strains were stored on ceramic beads in a Microblank tube (Prolab Diagnostics, Canada) at −70 °C. Before the respective examinations, routine cultures were performed in yeast extract-peptone-dextrose medium (YEPD) at 30 °C for 18 h [4].

Table 3 Candida albicans strainsAntifungal activity assays against the C. albicans planktonic growth

A susceptibility of SC5314 and CNB1 mutant planktonic cells to novel sulfone derivatives was determined using the M27-A3 method [5]. The final inoculum of 2.5 × 102 cells/mL was prepared in the synthetic RPMI 1640 medium (ThermoFisher). Compound concentrations ranging from 0.5 to 256 µg/mL were prepared with a stock solution (12,800 µg/mL) dissolved in DMSO. The cell growth was measured using a microtiter plate reader Synergy H4 Hybrid Reader (BioTek Instruments, USA) after incubation at 35 °C without agitation for 23, 28, and 48 h. Minimal inhibitory concentration (MIC) was defined as the lowest concentration of the compounds yielding cell growth (visual assessment) [18]. The percentage of cell growth reduction was calculated according to the following formula: % inhibition = 100% x [1 - (OD405 CTW – OD405 SCW)/(OD405 GCW – OD405 SCW)]. CTW was prepared with compound, medium, and C. albicans inoculum; SCW sterility control wells contained compound, RPMI 1640 medium, and sterile water replacing inoculum; GCW growth control wells were prepared with inoculum, RPMI 1640 medium, and the same amount of DMSO used in CTW. The minimal fungicidal concentration (MFC) was determined as described previously [6]. Briefly, 100 µL of aliquots from the selected wells of plates were removed after incubation at 35 °C for 48 h. Next, 100 µL of aliquots (1–10,000 diluted) were placed onto YEPD and incubated at 35 °C for 48 h. The MFC results for each sulfone derivative concentration tested were defined as the reduction viability record (R) and calculated using the formula: R = lg A – lg B, where A means CFU/mL of growth control wells (GCW), B means CFU/mL of sulfone-treated wells (CTW), and A or B were calculated according to the following quotation: CFU/mL= (mean number of colonies from 3 plates) x (inverse dilution coefficient plated) x 10 (counted per 1 mL). The lowest sulfone concentration that killed ≥ 99.9% of viable cells, compared with the GCW (lgR ≥ 3) was defined as MFC [18] and the concentration of a compound is considered to be fungistatic when 0 ≤ lgR < 3.

Metabolic activity assay

Antifungal activity of sulfone derivatives was assessed using the MTS test (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS, Promega, USA) [5, 6]. Briefly, 10 µL of MTS was added to each well after incubation of cells with the compounds for 48 h. The optical density was measured at 490 nm and 660 nm (reference wavelength) using a microtiter plate reader after 3h-incubation of MTS with the treated cell suspension. Specific absorbance (SA) was calculated using the following formula: SA = A490 – A660. The reduction of viability was calculated using the following formula: % inhibition = 100% x [1 - (SA CTW – SA SCW)/(SA GCW – SA SCW)] [5, 6].

Antifungal activity of sulfone derivatives against the C. albicans biofilm

Biofilms of C. albicans were formed using a 96-well microtiter plate as described previously [6, 19]. The antifungal effect of sulfone derivatives against the C. albicans mutants’ and wild-type strains’ biofilm was monitored using the metabolic (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS, Promega, USA) assay. After 48-h incubation at 35 °C, the microplates were read as described above. The antifungal effect was measured by comparing the reduction in the mean absorbance of the compound-treated well to that of the untreated growth (control). The lowest concentration showing a distinct reduction in the metabolic activity compared with the growth control was determined and the results were plotted as % of metabolic activity. Three replicates of biofilm were included for each experiment [6, 19].

In vitro cytotoxicity

The sulfone cytotoxicity against the kidney epithelial cell line Vero (ATCC CCL-81, LGC Poland) was assessed by MTS (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay) (MTS, Promega, USA) [6]. Vero cells were maintained in EMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco) at 37 °C and 5% CO2. Vero cells (1 × 106 cells/mL of EMEM) were plated in 96-well microtiter plates for 24 h, following which the cell monolayer was treated with sulfone derivatives at different concentrations and incubated under the same conditions for 20 h. Subsequently, 10 µL of MTS was added to each well, and incubation for another 3 h in darkness proceeded. The concentration reducing cell viability by 50% compared with the untreated control was recorded as the cytotoxic concentration (CC50) [7]. Additionally, the cytotoxicity of 4 and 12 was evaluated against the lung fibroblast cell line MRC-5 (ATCC CCL-171) maintained and collected as described above, using MTS as an assessment of cytotoxicity.

Rhodamine 123 (RH-123) dye efflux property

The final inoculum of 1.2 × 108 SC5314 cells/mL was prepared in phosphate-buffered saline (PBS) pH 7.4 with the addition of glucose and RH-123 at 5 mM or 15 µM [20, 21]. After 30 min of incubation at 35 °C, the cells were pelleted at 6000 x g for 2 min and resuspended in PBS containing 1 mM glucose and sulfones (4, 7, 8, 12) at 16 µg/mL or 128 µg/mL. Incubation at 35 °C was carried out for 15, 30, 45 min, 1 h, and 18 h, after which the cells were centrifuged (6000 × g, 2 min), and RH-123 fluorescence in the supernatants was assessed spectrophotometrically (Synergy H4 Hybrid Reader BioTek Instruments, USA) using excitation at 521 nm and emission at 627 nm. Additionally, CNB1/cnb1∆ and cnb1∆/cnb1∆ treated with 4 or 12 were analyzed. The fluorescence of RH-123 released from cells treated with sulfones compared with the untreated control was recorded as % efflux. The experiment was conducted in three independent replications. The antifungal sulfone concentrations were selected based on the MIC/MFC studies [20, 21].

Flow cytometry analyses

Flow cytometry was applied to evaluate the mechanism of the C. albicans cell death caused by the tested sulfone derivatives at concentrations of 16 µg/mL and 128 µg/mL [5]. The final inoculum of 6.9 × 1011 cells/mL was prepared in sterile Mili-Q water, harvested (1500 × g, 4 °C, 10 min), and cells were incubated in 2.5 mL of buffer I (1 M sorbitol; 1 mM EDTA (pH 8.0); 50 mM 2-mercaptoethanol) at 28 °C for 30 min (180 rpm). The cell pellet collected by centrifugation under the same condition as before was washed twice in 3 mL 1 M sorbitol. Then, the cells were harvested in buffer II (1 M sorbitol; 1 mM EDTA, pH 5.8; 10 mM sodium citrate) and digested with lyticase (12.5 µg/mL) for 1 h at 37 °C (200 rpm). The pellet was centrifuged (3000 rpm, 4 °C, 10 min) and washed twice with 2.5 mL 1 M sorbitol. The protoplasts were resuspended in 1 mL of sterile Mili-Q water. The suspensions of cells or protoplasts were prepared in an MMD medium with the addition of tested compounds and incubated at 28 °C for 24 h (180 rpm). After centrifugation, the pellet was resuspended in Mili-Q water, and cells and protoplasts were stained with annexin V (Invitrogen), and incubated for 15 min at room temperature. The cell/protoplast suspensions were prepared in an annexin binding buffer (BD Pharmingen) and propidium iodide (PI) (Invitrogen) was added. 50 µL of the suspension was mixed with 450 µL PBS before the cytometric analysis was undertaken [5].

Ergosterol assay (HPLC)

The effect of sulfone derivatives on ergosterol synthesis in Candida SC5314 wild-type cells was determined using the previously described method [22]. Briefly, yeast cells at a density of 7 × 106 cells/mL in MMD with compounds (2, 3, 11, and 12) at two concentrations of 0.5 µg/mL or 16 µg/mL were incubated for 24 h with shaking (100 rpm) at 30 °C. The cells were harvested by centrifugation at 1500 × g for 5 min, washed, and weighed. Each pellet was then treated with 3 mL of freshly prepared alcoholic potassium hydroxide solution (25%) and vortexed for 1 min. Cell suspensions were then incubated for 1 h at 80 °C in a water bath and cooled. Sterols were extracted by adding 3 mL of petroleum ether followed by shaking for 3 min. The organic phase was then transferred to a clean glass tube to evaporate petroleum ether on a rotary evaporator (Bunchi) at 60 °C under a vacuum. The extracted sterols were redissolved in 1 mL of methanol (Chempur) before HPLC-UV analysis. The sample containing ergosterol was analyzed at 281 nm with a C18 reverse-phase column. The mobile phase was a solution of acetonitrile/ methanol (97/3 v/v).

Antifungal activity of sulfone derivatives against exogenous ergosterol

The tested sulfone derivatives’ interaction with exogenous ergosterol (Sigma) was determined using the method M27-A3 [18] with a few modifications. The compounds (2, 3, 7, and 11) were tested at the conc. range from 0.5 to 16 µg/mL. Briefly, the final inoculum of yeast suspension (103 CFU/mL) was prepared in MMD supplemented with ergosterol and distributed to the wells, giving ergosterol a final concentration of 400 µg/mL in the tested wells [22]. The ergosterol was prepared directly before the experiment. It was dissolved in 10% DMSO (v/v), and 1% Triton X-100 (v/v), heated to augment the solubility, and then diluted with the MMD medium. The tested compounds were added to the microtiter plate wells. Yeast growth control and sterility control were also analyzed. The plates were incubated at 30 °C for 4 days. Absorbance measurements were taken at 405 nm with an automatic plate reader every 24 h [22].

Antifungal activity of sulfone derivatives and amphotericin B (AmB) against exogenous ergosterol - assessment of the fractional inhibitory concentration (FIC)

The activity of sulfones (2, 3, 11, and 12) at final conc.: 0.5 µg/mL, 1 µg/mL, 2 µg/mL, 4 µg/mL, 8 µg/mL and 16 µg/mL against C. albicans SC5314 wild type strain (1 × 103 CFU/mL) in the presence of ergosterol (400 µg/mL), was tested with addition of AmB at final cons.: 0.04 µg/mL, 0.09 µg/mL, 0.16 µg/mL, 0.31 µg/m, 0.63 µg/mL and 1.25 µg/mL, respectively. The antifungal effect was measured by comparing the mean absorbance reduction of the compound-treated well to that of untreated growth control at 405 nm after 3 days using an automatic plate reader Synergy H4 Hybrid Reader (BioTek Instruments, USA). To characterize the interaction of each combination tested, the fractional inhibitory concentrations (FICs) of each agent tested and their sums are used to calculate the FIC index for the cell metabolic reduction, according to the following formula: index FIC = A/MICA + B/MICB, where A/B denotes the concentrations of tested compound (A) or amphotericin B (B), MICA and MICB mean the concentrations of a tested compound or AmB, respectively, which were defined as MIC of compound against C. albicans. The FIC index of <1 is the expression of the agents’ synergism, whereas the FIC index of >1 represents an antagonism [23].

In addition, the minimal fungicidal concentration (MFC) was determined as described previously. Briefly, to verify if 0.5 µg/mL of 2, 3, 11, or 12 used with 0.09 µg/mL of AmB could be considered as a fungicidal cons., the cell suspension treated for 48 h with the compounds and exogenous ergosterol (400 µg/mL) was plated on agar medium. The fungicidal conc. induced reduction of cells in 99.9%. The decimal logarithm of R was calculated.

Confocal laser scanning microscopy

The suspension of SC5314 (1 × 105 CFU/mL) in YEPD was inoculated on sterile glass disks in the 24-well culture plates to obtain biofilms by incubation at 37 °C for 20 h. The biofilm was washed with PBS and fresh YEPD medium with 15 µM rhodamine 123 and 5 mM glucose. After 5-h incubation, the cells were rinsed and treated with fluorescently labeled sulfones (L1, L2, and L3) at 128 µg/mL (selected based on previous studies), and incubated for 18 h at 37 °C. Subsequently, the observations were made using a confocal scanning microscope (CLSM, Fluoroview FV1000, Olympus) at excitation and emission wavelengths: λex = 553 nm and λam = 627 nm (rhodamine 123), λex = 400 nm and λam = 655 nm (fluorescently labeled compounds) [23].