Selective Modification of Streptozotocin at the C3 Position to Improve Its Bioactivity as Antibiotic and Reduce Its Cytotoxicity towards Insulin-Producing β Cells.

With the increasing resistance of bacteria to current antibiotics, novel compounds are urgently needed to treat bacterial infections. Streptozotocin (STZ) is a natural product that has broad-spectrum antibiotic activity, albeit with limited use because of its toxicity to pancreatic β cells. In an attempt to derivatize STZ through structural modification at the C3 position, we performed the synthesis of three novel STZ analogues by making use of our recently developed regioselective oxidation protocol. Keto-STZ (2) shows the highest inhibition of bacterial growth (minimum inhibitory concentration (MIC) and viability assays), but is also the most cytotoxic compound. Pre-sensitizing the bacteria with GlcNAc increased the antimicrobial effect, but did not result in complete killing. Interestingly, allo-STZ (3) revealed moderate concentration-dependent antimicrobial activity and no cytotoxicity towards β cells, and deoxy-STZ (4) showed no activity at all.


S5
Lividosamine hydrochloride (154 mg, 0.771 mmol) and 4-nitrophenyl Nnitroso-N-methylcarbamate (191 mg, 0.848 mmol) were dissolved in DMF (5 mL) at r.t., then the reaction mixture was cooled to 0 °C under N2 atmosphere, followed by the addition of N,N-diisopropylethylamine (161 μL, 0.925 mmol), The reaction mixture was stirred at 0 °C for 2 h, the DMF was evaporated and the residue purified by flash chromatography on a 15 g silica cartridge with 100% EtOAc to afford the product (135 mg, 70%) as colorless oil. NMR showed the major form is α-pyranose, in approximately 60%.

-nitrophenyl N-nitroso-N-methylcarbamate (6)
To a solution of N-methyl 4-nitrophenyl carbamate (1 g, 5.1 mmol) in 20 mL DCM in a 100 mL flask, was added a solution of NaNO2 (2.5 g, 36.2 mmol) in 20 mL of water. The mixture was chilled to 0 °C, and 5.2 mL conc. HCl aq was added dropwise and slowly (one drop per 3 seconds), upon which the color of the solution changed from yellow to green. The reaction was monitored by TLC, and after 3 h, a very small amount of starting material remained.

Minimal Inhibitory Concentration (MIC) Assays
MIC determination in rich medium. MIC determination was carried out in 96-well plates following the CLSI guidelines [3]. The pre-culture of E. coli TOP10 (K12 origin) was prepared from the glycerol stock in LB medium and incubated overnight at 37 °C and shaking. The next day this pre-culture was used to inoculate fresh LB medium and E. coli cells were grown to OD600 of 0.08-0.1 (turbidity of the 0.5 McFarland standard). The culture was subsequently diluted with LB medium to reach the density of approximately 5 × 10 6 CFU/mL and then 10 μL of the resulting suspension were transferred to the exposure wells (total volume 100 μL), yielding the final culture density of 5 × 10 5 CFU/mL. The serial dilutions of the compounds were prepared in 10× concentrated form, and 10 μL of each respective series were transferred to the exposure well (100 μL final volume) resulting in 1× concentrated series. Within 15min of adding the inoculum the 96-well plate was incubated at 37 °C for 16-22h in an plate-reader (Synergy TM 2 Multi-Mode Microplate Reader, BioTek Instruments) under ambient air with OD600 measurements every 20 min preceded by 30 s of shaking.
MIC determination in minimal medium with pre-sensitizing. The pre-culture of E. coli TOP10 (K12 origin) was prepared from glycerol stock in minimal medium (50mM Na2HPO4, 25mM KH2PO4, 10mM NaCl, 5mM MgSO4, 0.2mM CaCl2, 50uM FeCl3, 0.1% NH4Cl, 1% casein hydrolysate) in the presence of 4% (w/v) of the pre-sensitizer (N-acetylglucosamine or ribose) and incubated overnight at 37 °C with shaking. The next day cells were pelleted by centrifugation and washed with PBS (phosphate buffer saline) three times to remove the traces of presensitizer. After the final washing step pellet was resuspended in 5 milliliters of PBS and used to inoculate minimal medium containing 1% glycerol. The resulting culture was allowed to grow until OD600 of 0.08-0.1 (turbidity of the 0.5 McFarland standard). The culture was subsequently diluted with minimal medium to reach the density of approximately 5 × 10 6 CFU/mL and then 10 μL of the resulting suspension were transferred to the exposure wells (total volume 100 μL), yielding the final culture density of 5 × 10 5 CFU/mL. The serial dilutions of the compounds were prepared in 10x concentrated form and 10 μL of each respective series were transferred to the exposure well (100 μL final volume) resulting in 1× concentrated series. Within 15min of adding the inoculum the 96-well plate was incubated at 37 °C for 16-22h in an ambient air incubator with OD600 measurements every 20 min preceded by 30 s of shaking.

Viability Assays
Growth-based viability assay. In order to determine the inhibitory effect of streptozotocin and its derivatives, the growth-based viability assay developed by C.L. Haynes et al. [4] was used. The underlying principle of the assay is to estimate the viability of the cells after exposure to an inhibitory compound by the delay in the subsequent outgrowth in fresh medium. This effect stems from the fact that the fewer the remaining cells, the longer it takes to reach a certain density threshold, indicating the bactericidal activity of the compound used. In the assay, bacterial cells were first exposed to the streptozotocin derivatives (either in rich or minimal medium) at room temperature and constant shaking to ensure good mixing. Afterwards, a small fraction of the exposure mixture was transferred into the fresh medium (yielding a 40× dilution and therefore alleviating the effect of the antibiotic) and the cells were incubated in a Biotek plate reader for 16 hours at 37 °C with optical density measurements at 600 nm taking place every 20 min preceded by 30 s of shaking.
Plate layout. As recommended in the paper of Haynes 4 the original layout with water evaporation control was used in this work. For all experiments the following plate sections were included: calibration series with outgrowth in duplicates, 10× dilution series of the compound, exposure wells and after-exposure outgrowth wells in duplicates.
Viability assay in rich medium. Bacterial culture of E. coli TOP10 was grown in LB medium (37 °C, shaking) until cells entered the exponential growth phase (OD600 0.3-0.5) and was diluted to working density of 0.05 (calibration curve series) and 0.1 (exposure). For exposure, 20 μL of the 10× compound preparation in an appropriate solvent was transferred to 180 μL of the bacterial culture, yielding 1× concentration of the compound for exposure. The plate was then incubated in the plate reader at room temperature and constant shaking for 2 hours. Afterwards, 5 μL of the exposure mixture were added to 195 μL of fresh LB to dilute the antibiotic and allow remaining bacterial cells to outgrow. The same outgrowth procedure was performed for the calibration series. As a last step, the plate was incubated for 16 hours in the plate reader at 37 °C with OD600 measurements every 20 min preceded by 30 s of shaking.
Viability assay in minimal medium on pre-sensitized E. coli cells. Bacterial culture of E. coli TOP10 was pre-grown overnight in minimal medium (50mM Na2HPO4, 25mM KH2PO4, 10mM NaCl, 5mM MgSO4, 0.2mM CaCl2, 50uM FeCl3, 0.1% NH4Cl, 1% casein hydrolysate) containing 0.4% Nacetylglucosamine or ribose at 37 °C with shaking. Next day, cells were harvested and washed three times with Dulbecco's phosphate saline buffer (DPBS) and resuspended in the DPBS to the original volume of the overnight culture. The resulting bacterial suspension was then used to inoculate minimal medium containing 1% glycerol which was further incubated at 37 °C and shaking. The bacterial culture was allowed to reach exponential phase densities of 0.3-0.5 and then was diluted to working densities of 0.05 (calibration curve) and 0.1 (exposure). Exposure and outgrowth procedures were performed in the same way as described in the section "Viability assay in rich medium".
Calculation of viability. The procedure to calculate viability was taken from the original article by Haynes et al [4]. In short, the viability of the bacterial culture after exposure to the STZ analogues is defined as the percentage of cells remaining. This percentage is calculated from a calibration curve of defined optical densities (OD at 600 nm, related to percentage of cells), and their associated delays in time for outgrowth above a threshold OD (defined as Ct).
To construct the calibration curve, a serial dilution was performed to obtain 100%, 50%, 25%, 12.5%, 6.25% of total viable cells. Outgrowth was performed as described above and monitored over time with 20 minute intervals (defined as cycles). A plot was generated of the cycle number

Cell Viability
The effect of streptozotocin (STZ) and the derivatives on β-cell viability was determined by the cell proliferation reagent WST-1 (Roche, Indianapolis, USA). Briefly, MIN6 cells (1 × 10 5 cells/well) were seeded in 96-well plates. Cells were cultured overnight and the following day incubated with or without STZ or analog (Sigma-Aldrich) at 5 mM for 48 and 72 hours followed by WST-1 assay. After 30 min incubation with WST-1 (10 μL/well) at 37 °C, the absorbance was measured at 450 nm using a Bio-Rad Benchmark Plus microplate spectrophotometer reader (Bio-Rad Laboratories B.V, Veenendaal, the Netherlands).