The overall goal of this procedure is to introduce both lysine and arginine-type monomers within the same peptoid sequence and to evaluate the activity of these compounds against Leishmania mexicana, the causative parasites of the neglected tropical disease cutaneous leishmaniasis. Novel therapeutics are urgently needed for cutaneous leishmaniasis, and this procedure can help to identify and develop peptoids as a new class of potential anti-infectives for this neglected tropical disease. The main advantage of this protocol is that peptoids can be synthesized that contain mixed cationic functionality.

This is highly desirable as it can help to modulate the biological activities of the sequences. Although this tests against the mammalian form of the parasite, it can also be used against the insect stage of Leishmania mexicana or applied to mammalian cell lines. Visual demonstration of this method is really useful because it shows the stepwise nature of synthesis and also the parasite culture at various points in the life cycle of Leishmania mexicana.

To begin this procedure prepare solutions in dimethylformamide as outlined in the text protocol. Then, add 0.1 millimoles of Fmoc-protected rink amide resin to a capped 20-milliliter polypropylene reaction vessel with two frits. Add five milliliters of dimethylformamide and let the vessel stand for 60 minutes at room temperature.

Then, drain the DMF using a solid phase extraction vacuum platform. Next, add two milliliters of prepared piperidine solution. Place the vessel on a shaker platform at 450 RPM at room temperature and shake for five minutes.

Drain the solution using the vacuum station. Then, add two milliliters of piperidine solution and shake at the same conditions for 15 minutes. After shaking is complete, drain the solution via a vacuum station.

Add two milliliters of DMF and mix for 30 seconds to wash the resin. Drain the DMF and repeat the wash three times. To begin submonomer synthesis, add one milliliter of prepared bromoacetic acid solution and 0.2 milliliters of prepared DIC solution.

Shake for 20 minutes at room temperature. Then, drain the solution and wash three times using two milliliters of DMF each time. Add one milliliter of prepared amine solution.

Then, shake the resin for 60 minutes at room temperature. Drain the solution and wash the resin three times using two milliliters of DMF each time. Next, add one milliliter of bromoacetic acid solution and 0.2 milliliters of DIC solution.

Shake for 20 minutes at room temperature. Then, drain the solution and wash three times using two milliliters of DMF each time. To introduce a guanidine-functionalized monomer, add one milliliter of unprotected diamine solution to the resin.

Shake for 60 minutes at room temperature. Then, drain the solution and wash the resin with two milliliters of DMF three times. Add 0.5 milliliters of prepared 2-acetyl dimedone solution.

Shake for 60 minutes at room temperature. Then, drain the solution and wash the resin with two milliliters of DMF three times. Continue the submonomer synthesis until the desired sequence is made.

Then, add four milliliters of prepared hydrazine solution and shake for three minutes at room temperature. Drain the solution and repeat three times. Then, wash the resin three times using two milliliters of DMF for each wash.

Add DIPEA and six equivalents of pyrazole-1-carboxamidine per free amine in the peptoid sequence. Shake for 60 minutes at room temperature. After shaking is complete, drain the solution.

Wash the resin three times using two milliliters of dichloromethane for each wash. Air dry the resin for 10 minutes and store for future use. A test cleave may be performed after amine additions to check the progress of synthesis.

To begin the final cleavage, add four milliliters of cleavage cocktail to the same fritted reaction cartridge used for synthesis and cover the vessel. Shake for 90 minutes at room temperature. Then using the fritted reaction vessel, filter the cleavage cocktail from the resin into a round-bottomed flask.

Evaporate the cleavage cocktail using a rotary evaporator. Add two milliliters of anhydrous diethyl ether to precipitate the peptoid. Using a pipette, remove the diethyl ether and discard.

Then, repeat the diethyl ether precipitation three times. Once the precipitation is complete, dissolve the crude peptoid in 10 milliliters of prepared acetonitrile and acidified water solution. Transfer to a pre-weighed container.

Then, freeze at minus 20 degrees Celsius and lyophilize to a dry powder ready for purification by reverse phase HPLC. After culturing the Leishmania mexicana parasites and transformation to the axenic amastigote stage, begin the cytotoxicity assay by preparing compound stock solutions as outlined in the text protocol. Add two microliters of five millimolar peptoid stock solution, amphotericin B control and DMSO control to the top row of a 96-well plate as outlined in the text protocol.

Using a multi-channel pipette, add 48 microlitres of fresh medium to the top row. Add 25 microlitres of fresh medium to all other rows. Next, carry out a serial dilution by pipetting 25 microlitres of solution from the top row, adding it to the row below and mixing.

Repeat dilutions for the entire plate discarding the last 25 microlitres pipetted from the bottom row. Then, transfer a parasite culture to a 50-milliliter centrifuge tube. Centrifuge for five minutes at 447 times g.

Pour off the old medium and add 10 milliliters of fresh medium. Next using a pipette, gently resuspend the pellet of parasites in the new medium. Count using a Neubauer improved hemocytometer and dilute the culture to eight million parasites per milliliter.

After the culture has been diluted, add 25 microliters of parasites to each well. Incubate the plate for 60 minutes at 32 degrees Celsius. Once incubation is complete, remove 40 microlitres of solution from each well.

Add 90 microlitres of fresh medium to each well and incubate at 32 degrees Celsius for 24 hours. Then, add 10 microlitres of resazurin-based cell viability solution to each well. Incubate at 32 degrees Celsius for four hours.

After incubation is complete, use a plate reader to measure the fluorescence. Analyze the data by removing the average background and comparing the fluorescence of wells after normalization with resect to the DMSO controls. In this study, two peptoids are synthesized using 120 milligrams of rink amide resin each.

Products are purified via reverse-phase high-pressure liquid chromatography, and fractions corresponding to the target mass are combined and obtained as white powders. The amount gathered for the two peptoids is shown here with yields of approximately 30%for fractions over 90%pure. Product purity is assessed using an analytical reverse-phase HPLC and visualized at 220 nanometers, the absorbance of the amide backbone.

The peptoids are both seen to be homogeneous. The representative results of the cytotoxicity assays against L Mexicana are shown here. Five-millimolar stock solutions of the compounds are made in cell culture grade DMSO and tested in triplicates at least twice to ensure robust data.

It can be seen that peptoid B is more effective than peptoid A at reducing the percentage of viable parasites with both having a dose-dependent effect. After watching this method, you should have a good understanding of how to synthesize, characterize and purify peptoids that contain both lysine type and arginine type monomers and how to use these in cytotoxicity assays against Leishmania mexicana. Our method to add dual cationic functionality into peptoids adds only one extra simple step to the commonly used submonomer method of peptoid synthesis.

Following this method, lysine or arginine-type submonomers of different lengths can be added to help increase library diversity, for example, with side chains of two to six carbons in length. While attempting the peptoid synthesis, it's really important to remember to wash the resin well between steps to prevent unwanted addition or deletion products. This method paves the way for researchers to explore the biological activity of mixed cationically-functionalized peptoids and to compare this activity to those that contain lysine or arginine-type monomers exclusively.

Finally, don't forget that the parasites and several of the reagents used in this protocol can be extremely hazardous. Therefore, it's important to always use adequate personal protective equipment and to work inside a fume hood or microbiological safety cabinet where appropriate.

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