Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Summary

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.

Abstract

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.

Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.

We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites' viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

Introduction

Protein-protein interactions (PPIs) play a pivotal role in most biological processes, from intracellular signal transduction to cell death¹. Hence, targeting PPIs is of fundamental importance to basic research and therapeutic applications. PPIs can be regulated by specific and stable antibodies, but antibodies are expensive and difficult to manufacture and have poor bioavailability. Alternatively, PPIs can be targeted by small molecules. Small molecules are easier to synthesize and inexpensive compared to antibodies; however, they are relatively less flexible and fit better to small cavities than to large protein-protein interfaces^2,3. Diverse studies have demonstrated that peptides, which are simpler and cheaper than antibodies and more flexible than small molecules, can bind protein interfaces and regulate PPIs^4,5. The global therapeutic peptide market was valued around fifteen billion dollars in 2013 and is growing 10.5% annually⁶. Furthermore, there are more than 50 marketed peptides, around 270 peptides in different phases of clinical testing, and about 400 peptides in advanced preclinical phases⁷. Although numerous peptides are being used as drugs, peptides still pose several challenges that limit their widespread application including poor bioavailability and stability, inefficiency in crossing cell membranes, and conformational flexibility^8,9. One alternative to surmount these drawbacks is to apply different modifications such as local (D-amino acid and N-alkylation) and global (cyclization) constraints^8,10-12. These modifications also occur naturally. For example, cyclosporin A, an immunosuppressant cyclic natural peptide, contains a single D-amino acid and undergoes N-alkylation modifications^13,14.

Modification of natural amino acids to induce local constraints, such as D- and N-alkylation, often affects the peptide's biological activity. However, cyclization, in which the sequence of interest can remain the same, is more likely to preserve biological activity. Cyclization is a highly attractive way to restrict peptide conformational space by reducing the equilibrium between different conformations. It usually increases biological activity and selectivity by restricting the peptide to the active conformation that mediates only one function. Cyclization also improves peptide stability by keeping the peptide in a conformation that is less recognized by degrading enzymes. Indeed, cyclic peptides were shown to have improved metabolic stability, bioavailability, and selectivity compared to their linear counterparts^15-17.

However, cyclization can be a double-edged sword since in some cases the restriction may prevent the peptides from achieving a bioactive conformation. To overcome this hurdle, a focused library in which all peptides have the same primary sequence and consequently constant pharmacophores can be synthesized. Peptides in the library differ in parameters that influence their structure, such as ring size and position, in order to subsequently screen for the most bioactive conformation^9,18.

Peptides can be synthesized both in solution and by a solid-phase peptide synthesis (SPPS) approach, which is now the more prevalent peptide synthesis approach and will be discussed further. SPPS is a process by which chemical transformations are performed on a solid support via a linker to prepare a wide range of synthetic compounds¹⁹. SPPS enables assembling peptides by consecutive coupling of amino acids in a stepwise manner from the C-terminus, which is attached to a solid support, to the N-terminus. The N-α-amino acid side-chains must be masked with protecting groups that are stable in the reaction conditions used during peptide elongation to ensure the addition of one amino acid per step. In the final step, the peptide is released from the resin and the side-chain protecting groups are concomitantly removed. While the peptide is being synthesized, all soluble reagents can be removed from the peptide-solid support matrix by filtration and washed away at the end of each coupling step. With such a system, a large excess of reagents at high concentration can drive coupling reactions to completion and all the synthesis steps can be performed in the same vessel without any transfer of material²⁰ .

Although SPPS has some limitations such as the production of incomplete reactions, side reactions, impure reagents, as well as difficulties monitoring the reaction²¹, the advantages of SPPS have made it the "gold standard" for peptide synthesis. These advantages include the option to incorporate non-natural amino acids, automation, easy purification, minimized physical losses, and the use of excess reagents, resulting in high yields. SPPS has been shown to be extremely useful in the synthesis of difficult sequences^21,22, fluorescent modifications²³, and peptide libraries^24,25. SPPS is also very useful for other poly-chain assemblies such as oligonucleotides^26,27, oligosaccharides^28,29, and peptide nucleic acids^30,31. Interestingly, in some cases, SPPS was shown to be advantageous for synthesizing small molecules that are traditionally made in solution^32,33. SPPS is used both in small scale for research and teaching^34,35 as well as large scale in industry^36-38.

Two synthesis strategies that are mainly used in SPPS methodology for the synthesis of peptides are butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). The original strategy introduced for SPPS was Boc, which requires strong acid conditions to remove side-chain protecting groups and cleave the peptide from the resin. Fmoc-based peptide synthesis, however, utilizes moderate base conditions and is a milder alternative to the acid-labile Boc protocol³⁹. The Fmoc strategy utilizes orthogonal t-butyl (tBu) side-chain protection that is removed in the last step of the synthesis while cleaving the peptide from the resin under acid conditions.

The general principle for peptide synthesis on solid support is presented in Figure 1. The initial amino acid, masked by a temporary protecting group on the N-α-terminus, is loaded onto the resin from the C-terminus. A semi-permanent protecting group to mask the side chain is also used if necessary (Figure 1, Step 1). The synthesis of the target peptide is assembled from the C-terminus to the N-terminus by repetitive cycles of deprotection of the N-α-temporary protecting group (Figure 1, Step 2) and coupling of the next protected amino acid (Figure 1, Step 3). After the last amino acid is loaded (Figure 1, Step 4), the peptide is cleaved from the resin support and the semi-permanent protecting groups are removed (Figure 1, Step 5).

Figure 1. General scheme of solid phase peptide synthesis. The N-α-protected amino acid is anchored using the carboxyl group via a linker to the resin (Step 1). The desired peptide is assembled in a linear fashion from the C-terminus to the N-terminus by repetitive cycles of deprotection of the temporary protecting group (TPG) from the N-α (Step 2) and amino acid coupling (Step 3). After accomplishing the synthesis (Step 4), the semi-permanent protecting groups (SPG) are deprotected during peptide cleavage (Step 5). Please click here to view a larger version of this figure.

After assembly of the complete peptide chain, cyclization can be achieved by several alternatives: (A) head-to-tail cyclization — this is a convenient way but limited since it provides only one option for cyclization (Figure 2A), (B) cyclization using the amino acids from the sequence of interest that contain bioactive functional groups — however, the use of these amino acids may influence the biological activity (Figure 2B), and (C) cyclization by adding amino acids (or other building blocks) without disturbing the bioactive sequence. Introducing these molecules is widespread as it allows production of focused libraries without modifying the sequence of interest (Figure 2C).

Figure 2. Alternative peptide cyclization strategies. (A) head to tail cyclization, through a peptide bond between the C-terminus and N-terminus; (B) cyclization between functional groups such as a disulfide bond between cysteine residues (1), or an amide bond between the side chains of lysine to aspartic/glutamic acid (2), or side chain to N- or C-terminus (3-4); (C) cyclization by adding extra amino acids or amino acid derivatives or small molecules, for example before (R0) and after (R7) the bioactive sequence. Please click here to view a larger version of this figure.

Microwave-assisted synthesis uses microwave irradiation to heat reactions, thus accelerating organic chemical transformations^40,41. Microwave chemistry is based on the ability of the reagent/solvent to absorb the microwave energy and convert it to heat⁴². Before the technology became widespread, major drawbacks had to be overcome, including the controllability and reproducibility of synthesis protocols and lack of available systems for adequate temperature and pressure controls^43,44. The first report of microwave-assisted peptide synthesis was done using a kitchen microwave to synthesize several short peptides (7-10 amino acids) with significant improvement of the coupling efficiency and purity ⁴⁵. Moreover, microwave energy was shown to decrease chain aggregation, reduce side reactions, limit racemization, and improve coupling rates, which are all critical for difficult and long sequences^46-53.

Currently the use of microwave irradiation for the synthesis of peptides or related compounds on a solid support is extensive, including (A) synthesis in water instead of organic solvent⁵⁴; (B) synthesis of peptides with common post-translational modifications, such as glycopeptides^55-58 or phosphopeptides^59-61, whose synthesis is typically difficult due to the low coupling efficiency of sterically hindered amino acid derivatives; (C) synthesis of peptides with modification in the backbone, such as azapeptides, which can be formed by the replacement of the C(α) of an amino acid residue with a nitrogen atom⁶², or peptoids, whose side chain is connected to the amide nitrogen rather than the Cα atom^63,64; (D) synthesis of cyclic peptides^65-71; and (E) synthesis of combinatorial libraries^51,72. In numerous cases, the authors reported higher efficiency and reduced synthesis time using microwave irradiation as compared to the conventional protocol.

Using a rational design^73-75, we developed anti-parasitic peptides that were derived from the scaffold Leishmania's receptor for activated C-kinase (LACK). LACK plays an important role in the early phase of Leishmania infection⁷⁶. Parasites expressing lower levels of LACK fail to parasitize even immune-compromised mice⁷⁷ as LACK is involved in essential parasite signaling processes and protein synthesis⁷⁸. Therefore, LACK is a key scaffold protein⁷⁹ and a valuable drug target. Focusing on sequences in LACK that are conserved in the parasites, but not in the host mammalian homolog RACK, we identified an 8 amino acid peptide (RNGQCQRK) that decreased Leishmania sp. viability in culture.

Here, we describe a protocol for the synthesis of backbone cyclic peptides derived from the LACK protein sequence described above. The peptides were synthesized on a solid support using microwave heating by SPPS methodology with Fmoc/tBu protocol. Peptides were conjugated to a TAT_47-57 (YGRKKRRQRRR) carrier peptide through an amide bond as part of the SPPS. TAT-based transport of a variety of cargoes into cells has been used for over 15 years and delivery of the cargo into subcellular organelles has been confirmed⁸⁰. Four different linkers, succinic and glutaric anhydride as well as adipic and pimelic acid, were used to perform the cyclization to generate carboxylic acid linkers of two to five carbons. Cyclization was done using microwave energy, and the final cleavage and side-chain deprotection steps were done manually without microwave energy. The use of an automated microwave synthesizer improved the product purity, increased the product yield, and reduced the duration of the synthesis. This general protocol can be applied to other studies that utilize peptides to understand important molecular mechanism in vitro and in vivo and further develop potential drugs for human diseases.

Protocol

1. Equipment and Reagents Preparation

1. Preparing equipment
   1. Perform all steps inside a fume hood using proper personal protective equipment.
   2. Chemically synthesize peptides on solid support using a Microwave Peptide Synthesizer with an additional module of Discover equipped with a fiber-optic temperature probe for controlling the microwave power delivery in a Teflon reaction vessel (30 ml, with a glass frit) or in a disposable polypropylene cartridge (12 ml, with a coarse frit).
   3. For proper mixing, connect nitrogen supply to the reaction vessel, or alternatively seal both ends of the polypropylene cartridge, and place on a rotary shaker.
   4. To drain reaction mixtures or washes, connect to the house vacuum via a waste trap.
   5. Place the fiber-optic probe into the reaction vessel.
2. Preparing reagents
   1. Prepare the resin by weighing Rink Amide AM resin 100-200 mesh (0.204 mg), add 5 ml of 1:1 mixture of N,N-dimethylformamide (DMF)/dichloromethane (DCM) to the reaction vessel/polypropylene cartridge to wash the resin down, shake for 2-4 hr to swell properly, and drain.
   2. Prepare 0.2 M 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid solutions by dissolving the corresponding Fmoc-amino acid in DMF and vortex the mixture until the amino acids are dissolved (Table 1).
   3. Prepare 0.45 M activator solution mix by dissolving 18.96 g O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) in 100 ml DMF and vortexing the mixture until the solid is dissolved (Table 1).
   4. Prepare 2 M activator base solution mix by combining 34.8 ml N,N-diisopropylethylamine (DIEA) with 65.2 ml 1-methyl-2-pyrrolidinone (NMP) (Table 1).
   5. Prepare 0.1 M deprotection solution mix by dissolving 3.37 g 1-hydroxybenzotriazole hydrate (HOBt) in 250 ml of a 20% v/v solution of piperidine in DMF and vortexing the mixture until the solid is dissolved (Table 1).

2. Fmoc-protected Amino Acid Coupling

1. Amino acid coupling
   1. Add amino acid (2.5 ml)/activator (1 ml)/activator base (0.5 ml) to reaction vessel/polypropylene cartridge and let the reaction proceed for 300 sec (25 W, 75 °C, Table 2). Drain the solution.
   2. Wash the resin with DMF. Add DMF to the resin for 120 sec (7 ml, 0 W, RT) and drain the solution. Repeat five times.
2. Fmoc deprotection
   1. Add 7 ml of 20% piperidine in DMF with 0.1 M HOBt to reaction vessel/polypropylene cartridge and incubate for 30 sec (45 W, 75 °C, Table 2).
   2. Drain the reaction mixture.
   3. Add 7 ml of 20% piperidine in DMF with 0.1 M HOBt to reaction vessel/polypropylene cartridge and incubate for 180 sec (45 W, 75 °C, Table 2).
   4. Drain the reaction mixture.
   5. Wash the resin with DMF. Add DMF to the resin for 120 sec (7 ml 0 W, rt) and drain the solution. Repeat five times.
      Note: Optionally, pause the procedure here and resume at a later date.
3. After amino acid coupling step, wash the resin with DCM and store for at least several days at 4 °C (for a longer period store the resin at - 20 °C).
   1. Move the resin from the reaction vessel to a polypropylene cartridge.
   2. Wash the resin with DCM. Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat three times.
   3. Seal the polypropylene cartridge tightly with a top cap and stopcock.
   4. Before starting a new synthesis, swell the resin for 3-4 hr in DMF (7 ml).
4. Monitoring the synthesis
   1. Use the Kaiser (ninhydrin) test or Chloranil test to quickly determine the progress of the synthesis. Optionally, perform a small-scale cleavage reaction to determine the purity and mass of the synthesized peptide. See section 9.
      Note: For further troubleshooting see Table 3.
5. Repeat steps 2.1 and 2.2 as desired to synthesize targeted peptide: Arg(Pbf)-Asn(Trt)-Gly-Gln(Trt)-Cys(Trt)-Gln(Trt)-Arg(Pbf)-Lys(Boc)-Lys(Mtt)-Gly-Gly-Tyr(But)-Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf).

3. Anhydride/Acid Coupling

1. Anhydride coupling
   1. Wash the resin with NMP. Add NMP to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat three times.
   2. Dissolve 10 equivalents of the corresponding anhydride in NMP (5 ml), add 1 equivalent 4-Dimethylaminopyridine (DMAP) and 10 equivalents DIEA to the solution (Table 1).
   3. Add a 10:1:10 mixture of anhydride/DMAP/DIEA to the resin and incubate for 300 sec (25 W, 75 °C, Table 2). Drain the solution.
   4. Wash the resin with NMP. Add NMP to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat three times.
2. Acid coupling
   1. Wash the resin with DMF. Add DMF to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat three times.
   2. Dissolve 10 equivalents of the corresponding dicarboxylic acid in DMF (5 ml). Add 1 equivalent DMAP and 10 equivalents N,N'-Diisopropylcarbodiimide (DIC) to the solution (Table 1).
   3. Pre-activate the mixture by mixing for 30 min.
   4. Add the mixture to the resin and incubate for 300 sec (25 W, 75 °C, Table 2) and drain the solution.
   5. Wash the resin with DMF. Add DMF to the resin for 120 sec (7 ml, 0 W, rt) and drain solution. Repeat step three times.

4. N-methyltrityl (Mtt) Protecting Group Deprotection

Note: The lysine side chain was protected with N-methyltrityl (Mtt)⁸¹, a protecting group that can be deprotected selectively under acid labile conditions^82,83. Deprotect Mtt protecting group manually on a shaker without microwave energy.

1. Transfer the resin to a polypropylene cartridge equipped with cap plug and stopcock.
2. Wash the resin with DCM. Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat three times.
3. Add 15-25 ml of a mixture of 1% Trifluoroacetic acid (TFA), 5% Triisopropylsilane (TIS), and 94% DCM to the polypropylene cartridge per one gram of resin.
   Note: TFA is a strong acid and corrosive and is extremely irritating to the skin, eyes, and lung tissue.
   1. Keep concentrated solutions of TFA in the hood at all times.
   2. Use proper personal protective equipment (eye protection, a lab coat and gloves) and work in a well-ventilated hood. Change gloves promptly if they come in contact with TFA and immediately clean-up any spills. If skin or eyes come in contact with the acid, flush the affected area immediately with water and wash for additional 15 min.
4. Place the polypropylene cartridge on a shaker and shake for 5 min at RT.
5. Drain the solution from the polypropylene cartridge by applying vacuum.
6. Repeat steps 4.3-4.5, three times.
7. Wash the resin with DCM. Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat five times.

5. Cyclization of the Linear Peptide

1. Wash the resin with DCM. Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat five times.
2. In a 50 ml polypropylene tube, dissolve 5 equivalents Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate (PyBOP) in Dibromomethane (DBM, 5 ml) and add 10 equivalents DIEA to the solution (Table 1).
3. Add the mixture to the resin and incubate for 300 sec (25 W, 75 °C, Table 2). Drain the solution.
4. Wash the resin with DCM. Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat three times.

6. Cleavage and Deprotection of Side-chain Groups

1. Wash the resin with DCM and diethyl ether.
   1. Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat two times.
   2. Add diethyl ether to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat two times.
2. Dry the resin in vacuum desiccator at RT for at least 3 hr over potassium hydroxide (KOH, 1-10 g).
3. Weigh the dried resin and transfer it to a polypropylene cartridge.
4. Add 10 ml of a pre-cooled trifluoroacetic acid (TFA) cleavage cocktail (e.g., 90% TFA, 2.5% water, 2.5% TIS and 5% phenol) to every one gram of resin.
5. Shake for 3 hr at RT.
6. Collect the TFA cleavage solution by filtering the resin into a 50 ml polypropylene tube. For the filtration, use the frit that is in the 12 ml polypropylene cartridge.
7. Add cold diethyl ether (35 ml) to the tube.
8. Centrifuge for 5 min at 1,207 x g at 4 °C.
9. Decant the ether layer.
10. Repeat Step 6.7-6.9, five times.

7. Drying the Backbone Cyclic Peptide

1. Keep the precipitated peptide in the same tube and dry it in a hood for 30 min.
2. Dissolve the peptide in a 1:1 mixture of water and acetonitrile (ACN).
3. Freeze the final product solution in liquid nitrogen.
4. Lyophilize the final product.

8. Characterizing the Backbone Cyclic Peptide

1. Dissolve a small sample (1 mg) of the product in water (400 µl).
2. Inject the dissolved peptide (10-200 µl) to a reverse phase high-performance liquid chromatography (RP-HPLC) system to test the peptide purity ³⁴.
3. Check the mass of the peptide using mass spectrometry matrix-assisted laser desorption ionization (MS-MALDI) ⁸⁴.
   1. Mix 1 µl (100 µM) peptide in a 1:1 (v/v) mixture of acetonitrile:water with 1 µl of matrix (5 mg/ml, α-cyano-4-hydroxycinnamic acid) in a 1:1 (v/v) mixture of acetonitrile:water with TFA (0.1%).
   2. Spot 1 µl on the MS-MALDI plate.
   3. Dry the sample and place it in the mass spectrometer.
4. Weigh the peptide and calculate the percent yield.
5. Store at -20 °C.

9. Monitoring the Synthesis

1. Kaiser (ninhydrin) test ⁸⁵
   1. Prepare the reagent solutions.
      1. Prepare Solution A by dissolving 16.5 mg of Potassium cyanide (KCN) in 25 ml of distilled water. Dilute 1 ml of the above solution with 49 ml of pyridine.
      2. Prepare Solution B by dissolving 1 g of ninhydrin in 20 ml of ethanol.
      3. Prepare Solution C by dissolving 40 g phenol in 20 ml ethanol.
   2. Use the Kaiser test to check the completion of amino acid coupling or the deprotection of the protecting group.
      1. Transfer a few beads from the resin to a test tube.
      2. Add three drops (~ 100 µl) of each solution (A, B and C) and mix.
      3. Heat the test tube on a heating block at 110 °C for 5 min.
         Note: Blue colored beads (positive result) indicate incomplete coupling reaction or deprotection of Fmoc protecting group.
2. Chloranil test ⁸⁶
   1. Prepare the following reagents fresh for each test.
      1. Prepare a solution of 2% Chloranil in DMF, solution A.
      2. Prepare a solution of 2% acetaldehyde in DMF, solution B.
   2. Perform the Chloranil test to check the completion of amino acid coupling or the deprotection of the protecting group.
      1. Mix 100 µl of solution A with 100 µl of solution B in a 1.5 ml tube.
      2. Drop the beads in and gently shake for 5 min.
         Note: Dark brown colored beads (positive result) indicate deprotection of the Fmoc protecting group or an incomplete coupling reaction.
3. Small scale cleavage reaction
   1. Remove a small amount of resin to a 3 ml polypropylene cartridge equipped with cap plug and stopcock.
   2. Treat with a 2 ml mixture of 95% TFA, 2.5% water and 2.5% TIS.
   3. Shake the mixture for 30 min at RT.
   4. Remove the resin by filtration using the frit of the polypropylene cartridge and evaporate the solvents by a stream of nitrogen.
   5. Dissolve the residue in water and analyze the product using HPLC and/or MS.

10. Leishmania donovani Promastigote Viability in Culture Assay

1. Leishmania donovani (L. donovani) growth and treatment conditions
   1. Culture L. donovani promastigotes in Dulbecco's Modification of Eagle's Medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate at 26 °C .
   2. Treat the L. donovani promastigotes with cyclic peptides (100 µM) for 24 hr at 26 °C.
2. Leishmania donovani (L. donovani) viability assay
   1. Assess parasite viability with 20 µl alamarBlue according to manufacturer's protocol.
   2. Determine alamarBlue reduction by measuring fluorescence (at 570 nm excitation and 590 nm emission). Higher fluorescence values indicate greater metabolic activity and increased parasite viability.

Results

Here we describe the development of a focused small library of backbone cyclic peptides that specifically target vital PPIs of the Leishmania parasite and act as antiparasitic agents (for review about peptides that target PPIs as antiparasitic agents⁸⁷). Through the synthesis of novel backbone cyclic peptides, pharmacophores are conserved in a scaffold of extendable size. The strength of the focused library proposed here is the ability to vary peptide scaffold sizes while allowing a restricted degree ...

Discussion

The synthesis of a focused library of backbone cyclic peptides derived from the LACK protein of the Leishmania parasite using a fully automated microwave synthesizer is described. A focused library of cyclic peptides was developed with conserved pharmacophores and various linkers. Addition of various linkers such as glutaric anhydride, succinic anhydride, adipic acid, pimelic acid, lysine, ornithine, and other building blocks can be used to increase the variety of the conformational space of the cyclic peptides....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Solid support, Rink Amide AM resin ML	CBL	BR-1330	loading: 0.49 mmol/g
Fmoc-Ala-OH	Advanced Chemtech	FA2100
Fmoc-Arg(Pbf)-OH	Advanced Chemtech	FR2136
Fmoc-Asn(Trt)-OH	Advanced Chemtech	FN2152
Fmoc-Asp(OBut)-OH	Advanced Chemtech	FD2192
Fmoc-Cys(Trt)-OH	Advanced Chemtech	FC2214
Fmoc-Gln(Trt)-OH	Advanced Chemtech	FQ2251
Fmoc-Glu(OtBu)-OH	Advanced Chemtech	FE2237
Fmoc-Gly-OH	Advanced Chemtech	FG2275
Fmoc-His(Trt)-OH	Advanced Chemtech	FH2316
Fmoc-Ile-OH	Advanced Chemtech	FI2326
Fmoc-Leu-OH	Advanced Chemtech	FL2350
Fmoc-Lys(Boc)-OH	Advanced Chemtech	FK2390
Fmoc-Met-OH	Advanced Chemtech	FM2400
Fmoc-Phe-OH	Advanced Chemtech	FF2425
Fmoc-Pro-OH	Advanced Chemtech	FP2450
Fmoc-Ser-(tBu)-OH	Advanced Chemtech	FS2476
Fmoc-Thr(tBu)-OH	Advanced Chemtech	FT2518
Fmoc-Trp(Boc)-OH	Advanced Chemtech	FW2527
Fmoc-Tyr(But)-OH	Advanced Chemtech	FY2563
Fmoc-Val-OH	Advanced Chemtech	FV2575
1-Methyl-2-pyrrolidinone (NMP)	Sigma	328634	Caution Toxic/Highly flammable/Irritant.
N,N-Dimethylformamide (DMF)	Alfa Aesar	43465	Caution Toxic. Use high quality DMF to eliminate side reactions such as Fmoc removal as a result of the dimethylamine traces from DMF decomposition.
Dichloromethane (DCM)	Sigma	D65100	Caution Harmful
Dibromomethane (DBM)	Sigma	D41868	Caution Harmful
Trifluoroacetic acid (TFA)	Sigma	T62200	Caution Corrosive/Toxic
Trifluoroacetic acid, HPLC grade (TFA)	Sigma	91707	Caution Corrosive/Toxic
Diethylether	Sigma	31690	Caution Highly flammable/Harmful
Triisopropylsilane (TIS)	Sigma	233781	Caution Irritant/Flammable
Water, HPLC grade	Sigma	270733
Acetonitroile, HPLC grade (ACN)	Fisher Scientific	A998-4	Caution Flammable/Irritant/Harmful
N,N-Diisopropylethylamine (DIEA)	Sigma	3440	Caution Corrosive/Highly flammable
Piperidine	Sigma	W290807	Caution Toxic/Highly flammable
Pyridine	Sigma	270970	Caution Highly flammable/Harmful
Ethanol (EtOH)	Sigma	459844	Caution Highly flammable/Irritant
1-Hydroxybenzotriazole hydrate (HOBt)	Sigma	157260	Caution Highly flammable/Irritant/Harmful
O-(Benzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate (HBTU)	Sigma	12804	Caution Irritant/Harmful
Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate (PyBOP)	Advanced Chemtech	RC8602	Caution Irritant
Ninhydrin	Sigma	454044	Caution Harmful
Phenol	Sigma	P3653	Caution Corrosive/Toxic
Potassium cyanide (KCN)	Sigma	11813	Caution Very Toxic
Potassium hydroxide (KOH)	Sigma	221473	Caution Toxic
N,N’-Diisopropylcarbodiimide (DIC)	Sigma	38370	Caution Flammable/ Toxic
4-Dimethylaminopyridine (DMAP)	Sigma	522805	Caution Toxic/Irritant
Glutaric anhydride	Sigma	G3806	Caution Flammable/Irritant/Harmful
Succinic anhydride	Sigma	239690	Caution Irritant/Harmful
Adipic acid	Sigma	A26357	Caution Toxic/Irritant
Pimelic acid	Sigma	P45001	Caution Toxic/Irritant
Chloranil	Sigma	23290	Caution Toxic/Irritant
Acetaldehyde	Sigma	402788	Caution Flammable/ Toxic
EQUIPMENT
Centrifuge	Beckman Coulter	Allegra 6R centrifuge
Lyophilizer	Labconco	freezone 4.5
Vacuum pump	Franklin Electric	model 1101101416 with 3/4 HP	Alcatel pump with Franklin Motor
Polypropylene cartridge 12 ml	Applied Separation	2419
Cap plug for 12 ml polypropylene cartridge	Applied Separation	8157
Polypropylene cartridge 3 ml	Applied Separation	2413
Cap plug for 3 ml polypropylene cartridge	Applied Separation	8054
Stop cocks PTFE	Applied Separation	2406
Tubes flat, 50 ml	VWR	21008-240
Extraction manifold, 20 pos, 16 x 100 mm tubes	Waters	WAT200609
Shaker, BD adams nutator mixer	Fisher scientific	22363152
Nalgene HDPE narrow mouth IP2 bottles, 125 ml	Fisher scientific	03-312-8
Erlenmeyer flask	Fisher Scientific	FB-501, 500 ml
Heating block	Thermolyne	1760 dri bath
Disposable borosilicate glass tubes with plain end	Fisher Scientific	14-961-25
Micropipettes and tips Finnpipette	Thermo	20–200 and 100–1,000 μl
HPLC vials - micro vl pp 400 µl PK100	VWR	69400-124
HPLC vial- Blue Snap-It Cap	VWR	66030-600
Analytical HPLC column	Peeke Scientific	U1-5C18Q-JJ	ultro 120 5 µm C18Q, 4.6 mm ID 150 mm
Prep HPLC column, XBridge	Waters	OBD C18 5 µm column	19 mm × 150 mm
Mass spectrometer	Applied Biosystems	Voyager DE-RP
Nitrogen cylinder
Desiccator
Analytical RP-HPLC system Shimadzu LC-20			equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC-20AD solvent delivery unit, SIL-20AC autosampler, DGU-20A5 degasser (Shimadzu, MD, USA).
Preparative RP-HPLC system Shimadzu LC-20			equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC-6AD solvent delivery unit and FRC-10A fraction collector (Shimadzu, MD, USA).