Biochemistry
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Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth
Chapters
Summary January 7th, 2022
A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.
Transcript
Masarimycin is one of the few inhibitors of bacterial autolysins that halts bacterial cell growth. It can be used to investigate the differences between chemical and genetic inactivation of autolysins, and provides an orthogonal approach to genetics to study bacterial physiology. Synthesis of masarimycin may seem daunting.
Closely follow the steps shown. When in doubt if a reaction step worked, confirm by thin-layer chromatography and the changes in RF values. To begin, prepare a 0.1-molar solution of cyclohexylamine, cyclohexyl carboxaldehyde, ortho-iodobenzoic acid, and cyclohexyl isocyanide in methanol.
Add magnetic stir bar, then, mix five milliliters of cyclohexylamine and five milliliters of cyclohexyl carboxaldehyde in a capped round-bottom flask and place the flask in a sand bath on a hot plate stirrer for 30 minutes at 40 degrees Celsius. Monitor the temperature using a thermometer placed approximately one centimeter below the sand surface. After 30 minutes, add five milliliters of cyclohexyl isocyanide to the solution and stir for an additional 20 minutes at 50 degrees Celsius.
Then, add five milliliters of ortho-iodobenzoic acid to the reaction mixture and continue stirring at 55 degrees Celsius for three to five hours. After three hours of stirring, monitor the progress of the reaction periodically by thin-layer chromatography, or TLC, at approximately one-hour intervals. Consider the reaction complete when only one spot with a retention factor equal to 0.3 is visible on the TLC plate.
Remove the solvent in rotatory evaporator under reduced pressure. Once all methanol is evaporated, dissolve the dried crude product in 30 milliliters of ethyl acetate and transfer it to a separatory funnel. Extract ethyl acetate sequentially with one-molar hydrochloric acid, water, saturated sodium bicarbonate solution, water again, and saturated sodium chloride solution, discarding the aqueous layers at each extraction.
Then, remove the ethyl acetate layer from the separatory funnel and collect it in an Erlenmeyer flask. Add a spatula full of anhydrous sodium sulfate to remove residual water from ethyl acetate. Filter the dried ethyl acetate solution through a number one filter paper to remove the sodium sulfate.
Then, wash the filter paper with a small amount of ethyl acetate. The filtered ethyl acetate solution was evaporated to dryness on a rotatory evaporator under reduced pressure to obtain masarimycin as oil once all the ethyl acetate is removed. Dissolve the masarimycin oil in one to two milliliters of a 9:1 hexane to isopropanol solution and stir on a magnetic stir plate until the entire compound is dissolved.
To purify the dissolved masarimycin, perform flash chromatography with a 12-gram, normal-phase, silica flash column. Equilibrate the flash column with 10 column volumes of mobile phase with the instruments set at a flow rate of 15 milliliters per minute. After the equilibration is complete, draw the dissolved masarimycin using a five-milliliter syringe.
Connect the syringe directly to the top of the equilibrated flash column and inject the solution into the column. Reconnect the loaded column to the flash chromatography system and initiate the gradient elution. Elute masarimycin from the column using gradient elution to a final mobile phase concentration of 10:90 hexane to isopropanol over 12 column volumes.
Monitor the elution of masarimycin via absorption at 230 and 254 nanometers. For the morphology study, grow bacillus subtilis 11774 on LB agar plates at 37 degrees Celsius. In all subsequent experiments, use second-passage cells grown in five milliliters of LB broth until the optical density at 600 nanometers reaches 1.
Next, using a pipette, add masarimycin to the culture tubes labeled treated to a final concentration of 3.8 micromoles. For culture tubes labeled control, add an equivalent volume of DMSO. Place the samples in an incubator at 37 degrees Celsius for 90 minutes with shaking at 150 RPM.
After 90 minutes, chemically fix the cultures in a 1:10 mixture of culture media and fixing buffer at four degrees Celsius overnight. The following day, use a pipette to apply 10 to 20 microliters of the chemically-fixed samples to glass microscope slides and allow them to air dry. Then, heat-fix the air dried samples by heating the glass slides using a Bunsen burner.
After heat-fixing, stain the samples with 100 microliters of 0.1%methylene blue. After a 10 minute incubation with the stain, wash away the excess dye with distilled water. Then, gently heat the stained slides to 60 degrees Celsius in an oven for 15 to 20 minutes to bring the cells to a common focal plane.
Seal the stained samples by placing a microscope cover slip over the stained cells. Then, seal the edges using microscope slide cement. Place the sealed microscope slide on the microscope stage and bring the image into focus at 100X magnification using bright-field microscopy.
Place a drop of immersion oil on the microscope slide and bring the field-of-view to focus using 1000X magnification. Acquire micrographs using a camera attached to the microscope and its associated software. Acquire images using the software's auto-white balance and aperture settings.
Flash chromatography allows for rapid purification of masarimycin with high purity. Evaluation of synergy or antagonism with the ATPase inhibitor optochin in S.pneumoniae is presented here. The lowest concentration of the compound to inhibit bacterial growth, indicated by the blue color, is considered the minimum inhibitory concentration value in the presence of a co-drug.
In contrast, wells with pink color indicate bacterial growth. Phenotypic assays using masarimycin at 0.75 times the minimum inhibitory concentrations demonstrated a sausage-like phenotype for B.subtilis and a clumping phenotype for S.pneumoniae. Pay attention to the temperature of the reaction.
Ensure that the reaction is complete by monitoring with TLC. This method can be used in conjunction with fluorescently-labeled antibiotics to investigate changes to the cell wall by microscopy.
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