An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.


Introduction
Peptoids (or poly-N-substituted glycines) are a class of peptide-mimetics that offer similar properties to peptides and as such are increasingly being investigated for medicinal and materials applications. In peptides, the side chain of each amino acid is connected to the α-carbon of the amide backbone; in peptoids the side chains are shifted onto the nitrogen atom of the backbone. Crucially, this gives peptoids greater resistance to proteolysis.
Peptoids are commonly synthesized using the submonomer method pioneered by Zuckermann et al., where peptoid monomers can be built by sequential haloacetylation of an amine functionality attached to a solid support and subsequent displacement of the halogen using a primary amine. 1 Our group has recently developed an adaptation to this submonomer method to allow lysine-and arginine-type peptoid residues to be included within the same peptoid sequence for the first time. 2 This manual solid-phase approach to peptoid synthesis uses commercially available reagents and common laboratory equipment, making it accessible for the majority of laboratories. Peptoids have been shown to have promising activities against a wide range of Gram negative bacteria, Gram positive bacterial and fungal species that are comparable to many known antimicrobial peptides. [3][4][5][6][7][8][9] In our work, peptoids have been used as novel anti-infective compounds for treatment of the neglected tropical disease leishmaniasis. 5,10 Leishmaniasis is endemic in more than 80 countries worldwide and it is estimated that over 12 million people are infected globally. 11 The disease is caused by protozoan parasites that are transmitted by the bite of a sandfly. Leishmania species can cause cutaneous leishmaniasis, a condition that leads to scarring and damage to mucous membranes, or the life-threatening visceral leishmaniasis, which causes fatal organ damage. No vaccine is currently available for this disease and existing treatments rely on a small number of drugs that have severe side effects. In addition, resistance to existing drugs is an emerging and serious problem so new treatments are desperately needed to effectively treat leishmaniasis in the future. [12][13][14][15][16] In these antimicrobial applications, peptoids are often designed to be amphipathic with a mixture of cationic and hydrophobic monomers. 3,4 This can give peptoids a degree of selectivity towards bacterial cells, reduce toxicity to mammalian cells, and to improve their activity as molecular transporters. [17][18][19][20] The majority of the anti-infective peptoids in the literature contain cationic side chains that are exclusively comprised of either amino functionalized lysine-type monomers or arginine-type residues. Peptide-peptoid chimeras, where the cationic chains are comprised of the amino acids lysine or arginine, have also been synthesized to examine the effect of cationic groups on activity and toxicity.
Herein, we describe the synthesis, purification and characterization of two novel peptoids that contain both lysine-and arginine-type residues in the same sequence. The method uses orthogonal N-Boc and N-Dde protection on resin with pyrazole-1-carboxamidine as a guanidinylation reagent. The biological evaluation of these peptoids is also described in cytotoxicity assays against Leishmania mexicana, the causative agent of cutaneous leishmaniasis. This provides a practical method to access peptoids with dual cationic functionality and to assess their biological activity. It is expected that this method will aid the synthesis of amphipathic peptoids by the peptoid community in the future.

Solid-phase Synthesis of Peptoids
NOTE: Peptoids are synthesized manually using the submonomer procedure of solid-phase peptoid synthesis. This method allows high coupling efficiency and good final product yields. Synthesis on solid-phase also allows excess reactants to be removed easily at the end of each step and the method has been modified here to allow different functionalized cationic monomers (i.e., arginine-type and lysine-type residues) to be included within the same sequence.  To add an arginine-type monomer, follow step 1.7. Depending upon the desired peptoid sequence, different amines will be added.
6. Repeat steps 1.1.4 and 1.1.5. 7. To include a guanidine functionalized monomer (i.e., NArg), add 1 ml of unprotected diamine solution (1.5 M in DMF) to the resin and shake for 60 min at room temperature. 1. Drain the solution and wash the resin with 2 ml DMF three times. 2. Add 2-acetyldimedone (0.2 g, 1 mmol in 0.5 ml DMF, 10 equivalents) to add the Dde group to the free primary amine and shake for 60 min at room temperature. Drain the solution and wash the resin with 2 ml DMF three times.
8. Continue submonomer synthesis as in 1.1.4 to 1.1.7 until the desired sequence is made. Add 2 ml DMF to wash the resin and repeat three times. 9. To deprotect the Dde group on resin, add 4 ml 2% hydrazine solution (in DMF v/v) and shake for 3 min at room temperature. Drain the solution and repeat three times. 10. Drain the solution and wash the resin with 2 ml DMF three times. 11. Add pyrazole-1-carboxamidine (6 equivalents per free amine, i.e., per NArg monomers, in the minimum volume of DMF) and N,Ndiisopropylethylamine or DIPEA (6 equivalents per free amine) and shake at room temperature for 60 min. 12. Drain the solution and wash the resin with 2 ml dichloromethane three times. Leave the resin to dry in air for 10 min then the resin can be stored until cleavage (section 2). 13. To pause the synthesis, wash the resin with 2 ml DMF three times. Add 2 ml DMF, stopper the synthesis vessel and leave at room temperature in a fume hood. NOTE: The synthesis may be paused after any displacement step (except the second displacement step as diketopiperazines may be formed).
2. Side-chain deprotection and cleavage from resin 1. Undertake a test cleave to check the progress of synthesis (purity and mass) at any point during synthesis after the displacement steps, addition or removal of Dde-protecting group or after the final sequence has been made. 1. Transfer approximately 10 resin beads from the reaction vessel into a new 8 ml polypropylene fritted cartridge.
5.5 medium. 9. Remove 10 ml of cell culture and transfer to cell culture flask. Incubate at 32 °C for 5 days. 10. Day 14: Check appearance of parasites under the microscope (400X). They should be in the pathogenic amastigote stage, lacking the flagellum characteristic of promastigotes, and ready for assay.
3. Cytotoxicity assay on L. mexicana axenic amastigotes. NOTE: The biological testing of peptoids uses high-throughput assays carried out in 96 well plates. This protocol describes the testing of L. mexicana axenic amastigotes, but identical assays can also be carried out on the promastigote stage parasites in appropriate media. Compounds are incubated with parasites at concentrations from 3 -100 µM for 60 min and then incubated for 24 hr following a 10-fold dilution. Results are gained by measuring the fluorescence of wells after incubation with Resazurin-based cell viability solution (e.g., alamarBlue).

Representative Results
As a representative result, the synthesis and characterization of two 12 residue peptoids containing two lysine-type monomers and two-arginine type monomers each will be described. The subsequent results from cytotoxicity assay are also shown.
Two peptoids [(NnArgNspeNspe)(NaeNspeNspe)] 2 (a) and [(NhArgNspeNspe)(NLysNspeNspe)] 2 (b) were synthesized using 120 mg Rink Amide resin each (loading = 0.79 mmol/g). All acetylation and displacement steps were carried out as described above, with all reagents purchased commercially. For these residues, the following amines were used in the displacement step: Nspe (S)-(-)-α-methylbenzylamine, Nae N-(tert-butoxycarbonyl)-1,2-diaminoethane, NLys N-(tert-butoxycarbonyl)-1,4-diaminobutane. For the arginine derivative residues, the following unprotected diamines were coupled: NhArg 1,4-diaminobutane or NnArg 1,2-diaminoethane, followed by on-resin protection with 2-acetyldimedone (Dde-OH). After the entire sequence had been synthesized, hydrazine deprotection of the Dde yields free amines to guanidinylate. The purified peptoids (a) and (b) were tested in cytotoxicity assays against L. mexicana axenic amastigotes. Frozen stocks of L. mexicana were defrosted and transformed to the amastigote stage ready for the assay. 72 hours after defrosting, the parasites should be insect stage promastigotes in their procyclic form, in log phase with many dividing cells. At this stage the parasites can be transformed into the amastigote stage using the pH and temperature shift described. 27 At Day 9 of the transformation, the parasites will be in the non-replicating infectious metacyclic promastigote stage. Finally at Day 14, the parasites should be in the pathogenic amastigote stage where parasites lack the characteristic flagella of the promastigotes. 28 5 mM stock solutions of the compounds were made in cell culture grade DMSO and tested in triplicates on a minimum of two occasions to ensure a robust data set was collected. A representative 96-well plate plan is shown in Figure 6. At the end of the assay, the cell viability reagent was added to each well and the fluorescence was measured as described to calculate the viability of parasites at each concentration tested (see Figure 7). ED 50 values were calculated as peptoid (a) >100 µM and peptoid (b) 37 µM respectively. The error bars plotted, show the variation between wells as a standard deviation and it can be seen that these are reasonable for most bars.

Discussion
Peptoids are increasingly being studied within the chemical biology and medicinal chemistry fields in applications such as novel therapeutics [3][4][5] , cell delivery agents 18,20 and diagnostic tools. 29 Typically, these sequences are cationic to provide a degree of selectivity for the pathogen over mammalian cells, the ability to penetrate through cell membranes and also aid solubility in aqueous systems. There are numerous examples of cationic peptoids that contain solely lysine-or arginine-mimetic residues in the literature. However, to date, the synthesis of peptoids that contain both of these cationic residues in the same sequence has been hindered by lack of a suitable synthetic procedure. The protocol described here allows mixed cationic peptoids to be synthesized in an efficient manner and is highly desirable as it offers a route to modulate the biological and chemical properties of amphipathic peptoids.
Our method uses an adaptation to the commonly used submonomer peptoid synthesis and permits the addition of both lysine-and argininetype monomers within the same sequence. It uses room temperature couplings and established protecting group chemistry so it is anticipated that this method will be useful for the majority of research groups. To add orthogonal protection for the arginine-type residue, an unprotected diamine is added under standard displacement conditions and then protected in a 60-minute coupling with Dde-OH. A variety of diamines can be used which allows side chains from 2 carbons to 6 carbons long to be installed, i.e., 1,2-diaminoethane to 1,6-diaminohexane respectively. The protecting Dde-OH dissolves well in DMF and is a very efficient and selective protecting group for primary amines. The Dde-protection group leaves secondary amines unaffected, e.g., the unprotected N terminus of peptoid chain. 30 One limitation is that all of the synthesis was Copyright © 2016 Creative Commons Attribution 3.0 License November 2016 | 117 | e54750 | Page 11 of 12 undertaken manually; however, it is anticipated that the coupling conditions developed make the method amenable for use with automated peptide/peptoid synthesizers.
The on resin deprotection of the Dde-groups is undertaken using a 2% hydrazine solution in DMF to leave free amines that can be guanidinylated on resin using pyrazole-1-carboxamidine. Six equivalents of the pyrazole-1-carboxamidine and six equivalents of DIPEA are used per free amine on the resin (i.e., six equivalents for each NArg type monomer to be installed). Again, this reaction is also efficient and the pyrazole-1-carboxamidine reagent has good solubility in DMF. Completion of the reaction is typically seen via LC-MS after 60 minutes at room temperature.
Due to the versatility of the submonomer method, a wide variety of primary amines can be used in the displacement step so conditions may need to be optimized to increase coupling efficiency and overall product yields or purity. 31 For the sequences discussed above, no special conditions were necessary for successful couplings. However, longer displacement times or higher amine concentrations could be used for problematic displacements (i.e., for poorly nucleophilic or sterically bulky amines). Some amines may not be completely soluble in DMF, in which case it is recommended to dissolve these in N-methyl-2-pyrrolidone (NMP), or other appropriate solvents for solid-phase reactions instead as in a previous comprehensive method for submonomer synthesis of peptoids. 32 To incorporate monomers that contain unprotected heteroatoms in the side chains, acetylation using chloroacetic acid has been shown to be effective by other groups. 33 Additionally, other resins may be used with this method to yield peptoids with different C terminal functionalization. Wang resins and 2-chlorotrityl chloride resins are routinely used in the submonomer synthesis of peptoids. For example, this method has been successfully used with 2-chlorotrityl chloride resin in our group. 2 Different solid supports will require a different loading procedure to Rink Amide discussed here (dependent on the specific resin used) so this should be checked with the literature prior to synthesis.
Similar to peptide synthesis, the conditions for final cleavage of the peptoid off resin can also be optimized for the specific sequence. In this protocol, a TFA cleavage cocktail was used (with triisopropylsilane and water as scavengers). The peptoids presented here contained only Boc protection which is a reasonably acid labile group. To ensure complete deprotection of sequences with a greater proportion of protected residues or less acid labile protecting groups, longer cleavage times may be necessary (i.e., cleavage times in excess of 2 hours are recommended for sequences that contain Pbf or tertiary-butyl ester protected groups). Alternative scavengers can also be used for specialized side chains (for example, ethanedithiol or 2-mercaptoethanol are often used in peptides that have sulphur containing side chains like cysteine or methionine).
The biological assay presented is a standard cytotoxicity test which can be altered to suit different cell lines. It is important to note that each 96well plate should contain sufficient controls to allow confidence in the results obtained. In this case, amphotericin B is used as a positive control as it is a known drug used to treat the disease and DMSO is used as a negative control as this is the solvent used to make compound stocks for the assay. If other cell lines are being used, alternative, suitable controls should be obtained and validated before use. L. mexicana is incubated with the peptoid at concentrations between 100 and 2 µM for one hour, and then the parasite/peptoid solution is diluted by a factor of ten for overnight incubation (i.e., wells initially with 100 µM peptoid stock are diluted to 10 µM).
The cell viability reagent is added to each well (10% of total well volume) at the end of the assay. A visible color change is seen between wells with viable parasites (pink), control wells with no viable parasites (blue) and a spectrum between with intermediate numbers of viable parasites. The fluorescence is proportional to the number of living cells and corresponds to the metabolic activity of the cells; resazurin dye (nonfluorescent) is converted to the fluorescent resorufin by reduction reactions in metabolically active cells. 34 In this assay, the incubation time with the cell viability reagent has been optimized for L. mexicana. Incubation times with the viability reagent will vary for plates seeded at different cell concentrations or with different cell lines (for example this method can also be used with mammalian cells). Dependent on the exact plate reader used, considerations need to be made before fluorescence measurements are taken. Any air bubbles in wells should be removed to ensure accurate readings can be taken. Some plate readers read from the bottom of plates, in which case flat bottomed 96 well plates should be used. Other machines may read from the top of the plate, so the lid of the plate should be removed before measurement.
Finally, in the future, this protocol may be compatible with automated synthesizers that are capable of making many sequences in parallel. Additionally, the synthesis of cyclic peptoids is also possible using this method. This protocol should provide researchers with a practical synthetic procedure that can be used to access novel peptoid scaffolds with both lysine-and arginine-type monomers, which may be of use in many applications, including materials or medicinal fields.

Disclosures
The authors have nothing to disclose.