Chemistry
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Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation
Chapters
Summary January 26th, 2016
A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.
Transcript
The overall goal of this procedure is to develop a focused backbone cyclic peptidomimetic library with conformational diversity using microwave irradiation for novel anti-parasitic therapeutics. This method can help develop novel tools to specifically target protein-protein interactions. To begin this procedure, add 2.5 milliliters of the desired amino acid, one milliliter of activator, and 0.5 milliliters of activator base to a polypropylene cartridge.
Place the cartridge in an automated microwave synthesizer and allow the coupling reaction to proceed for 300 seconds at 25 watts and 75 degrees Celsius. Once the reaction is complete, wash the resin with seven milliliters of N, N-dimethylformamide, or DMF, for 120 seconds at room temperature. After repeating the previous step five times, add seven milliliters of 20%piperidine in DMF with 0.1 molar 1-hydroxybenzotriazole, or HOBt, to the polypropylene cartridge and incubate for 30 seconds at 45 watts and 75 degrees Celsius.
When finished, drain the reaction mixture. Next, add seven milliliters of 20%piperidine in DMF with 0.1 molar HOBt to the polypropylene cartridge and incubate for 180 seconds at 45 watts and 75 degrees Celsius. After draining the reaction mixture, wash the resin with seven milliliters of DMF for 120 seconds at room temperature.
Then, wash the resin with seven milliliters of dichloromethane, or DCM, for 120 seconds at room temperature. For anhydride coupling, wash the resin with seven milliliters of 1-methyl-2-pyrrolidinone, or NMP, for 120 seconds at room temperature. When finished, drain the reaction mixture.
After repeating the previous step three times, dissolve 10 equivalents of the corresponding anhydride in five milliliters of NMP in a 50 milliliter polypropylene tube. Then, add one equivalent of 4-Dimethylaminopyridine, or DMAP, and 10 equivalents of N, N-diisopropylethylamine, or DIEA, to the solution. Add this mixture to the resin, and incubate for 300 seconds at 25 watts and 75 degrees Celsius.
After washing the resin with NMP, wash it with seven milliliters of DMF for 120 seconds at room temperature. At this point, transfer the resin to a polypropylene cartridge equipped with a cap plug and stopcock. Once the resin has been washed with DCM, add 15 to 25 milliliters of a mixture of 1%trifluoroacetic acid, 5%triisopropylsilane, and 94%DCM to the polypropylene cartridge per 1 gram of resin.
Place the polypropylene cartridge on a shaker, and shake for five minutes at room temperature. Following this, drain the solution from the polypropylene cartridge by applying a vacuum. After repeating the previous steps three times, wash the resin with seven milliliters of DCM for 120 seconds at room temperature.
These steps are highly important, as the trityl protecting groups in several resins were found to be partially cleaved in the DCM-trifluoroacetic acid solutions. Removing methyltrityl groups is a slow process that takes multiple washes. In a 50 milliliter polypropylene tube, dissolve five equivalents of Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate in five milliliters of dibromomethane.
Then, add 10 equivalents of DIEA to the solution. Add the mixture to the DCM-rinsed resin and incubate for 300 seconds at 25 watts and 75 degrees Celsius. Following this, wash the resin with seven milliliters of DCM for 120 seconds at room temperature.
After two washes with DCM, transfer the resin to a polypropylene cartridge and wash twice with diethyl ether. Dry the resin in a vacuum desiccator at room temperature for at least three hours over potassium hydroxide. Next, add 10 milliliters of a pre-cooled trifluoroacetic acid cleavage cocktail to every one gram of resin.
Place the polypropylene cartridge on a shaker, and shake for 3 hours at room temperature. Following this, collect the cleavage solution by filtering the resin into a 50 milliliter polypropylene tube. Add 35 milliliters of cold diethyl ether to the tube.
Centrifuge for five minutes at 1, 207 times g at four degrees Celsius. Then, decant the ether layer. For the Kaiser test, prepare Solution A by dissolving 16.5 milligrams of potassium cyanide in 25 milliliters of distilled water.
Dilute one milliliter of the solution with 49 milliliters of peridine. Next, prepare Solution B by dissolving one gram of ninhydrin in 20 milliliters of ethanol. Prepare Solution C by dissolving 40 grams of phenol in 20 milliliters of ethanol.
After the solutions have been prepared, transfer a few beads from the resin to a test tube. Add three drops of each solution to the tube, and mix. Finally, heat the test tube on a heating block at 110 degrees Celsius for five minutes.
Synthesis of the backbone cyclic peptides was performed using an automated microwave synthesizer on solid support following the FMOC, t-butyl protocol. The product was analyzed by mass spectrometry, and its degree of purity was determined using HPLC. Biological screening showed that peptide pL1 was active against Leishmania donovani, a parasite causing visceral leishmaniasis, the most severe leishmaniasis in humans.
Peptide pL1 reduced parasite viability by 75%as compared with the control treatment. Once mastered, this technique can be done in three to five days if it is performed properly. After it's development, this technique paved the way for researchers in a wide array of fields to explore peptides as pharmacological regulators in basic research and therapeutics.
After watching this video, you should have a good understanding of how to develop a focused library of peptidomimetics with conformational diversity to specifically target protein-protein interactions. Don't forget that working with trifluoroacetic acid can be extremely dangerous, and precautions, such as wearing eye protection, a lab coat, and gloves while working in a well ventilated hood should always be taken while performing this procedure.
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