We thank Lauren Van Wassenhove, Sunhee Hwang, and Daria Mochly-Rosen for helpful discussions. The work was supported by the National Institutes of Health Grant NIH RC4 TW008781-01 C-IDEA (SPARK) to N.Q. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Equipment and Reagents Preparation
<ol>
	<li>Preparing equipment
	<ol>
		<li>Perform all steps inside a fume hood using proper personal protective equipment.</li>
		<li>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).</li>
		<li>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.</li>
		<li>To drain reaction mixtures or washes, connect to the house vacuum via a waste trap.</li>
		<li>Place the fiber-optic probe into the reaction vessel.</li>
	</ol>
	</li>
	<li>Preparing reagents
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Prepare 0.45 M activator solution mix by dissolving 18.96 g O-(benzotriazol-1-yl)-N,N,N&#8242;,N&#8242;-tetramethyluronium hexafluorophosphate (HBTU) in 100 ml DMF and vortexing the mixture until the solid is dissolved (Table 1).</li>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
</ol>
2. Fmoc-protected Amino Acid Coupling
<ol>
	<li>Amino acid coupling
	<ol>
		<li>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 &#176;C, Table 2). Drain the solution.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Fmoc deprotection
	<ol>
		<li>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 &#176;C, Table 2).</li>
		<li>Drain the reaction mixture.</li>
		<li>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 &#176;C, Table 2).</li>
		<li>Drain the reaction mixture.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>After amino acid coupling step, wash the resin with DCM and store for at least several days at 4 &#176;C (for a longer period store the resin at - 20 &#176;C).
	<ol>
		<li>Move the resin from the reaction vessel to a polypropylene cartridge.</li>
		<li>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.</li>
		<li>Seal the polypropylene cartridge tightly with a top cap and stopcock.</li>
		<li>Before starting a new synthesis, swell the resin for 3-4 hr in DMF (7 ml).</li>
	</ol>
	</li>
	<li>Monitoring the synthesis
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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).</li>
</ol>
3. Anhydride/Acid Coupling
<ol>
	<li>Anhydride coupling
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Add a 10:1:10 mixture of anhydride/DMAP/DIEA to the resin and incubate for 300 sec (25 W, 75 &#176;C, Table 2). Drain the solution.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Acid coupling
	<ol>
		<li>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.</li>
		<li>Dissolve 10 equivalents of the corresponding dicarboxylic acid in DMF (5 ml). Add 1 equivalent DMAP and 10 equivalents N,N&#39;-Diisopropylcarbodiimide (DIC) to the solution (Table 1).</li>
		<li>Pre-activate the mixture by mixing for 30 min.</li>
		<li>Add the mixture to the resin and incubate for 300 sec (25 W, 75 &#176;C, Table 2) and drain the solution.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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 conditions82,83. Deprotect Mtt protecting group manually on a shaker without microwave energy.
<ol>
	<li>Transfer the resin to a polypropylene cartridge equipped with cap plug and stopcock.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Keep concentrated solutions of TFA in the hood at all times.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Place the polypropylene cartridge on a shaker and shake for 5 min at RT.</li>
	<li>Drain the solution from the polypropylene cartridge by applying vacuum.</li>
	<li>Repeat steps 4.3-4.5, three times.</li>
	<li>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.</li>
</ol>
5. Cyclization of the Linear Peptide
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>Add the mixture to the resin and incubate for 300 sec (25 W, 75 &#176;C, Table 2). Drain the solution.</li>
	<li>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.</li>
</ol>
6. Cleavage and Deprotection of Side-chain Groups
<ol>
	<li>Wash the resin with DCM and diethyl ether.
	<ol>
		<li>Add DCM to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat two times.</li>
		<li>Add diethyl ether to the resin for 120 sec (7 ml, 0 W, rt) and drain the solution. Repeat two times.</li>
	</ol>
	</li>
	<li>Dry the resin in vacuum desiccator at RT for at least 3 hr over potassium hydroxide (KOH, 1-10 g).</li>
	<li>Weigh the dried resin and transfer it to a polypropylene cartridge.</li>
	<li>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.</li>
	<li>Shake for 3 hr at RT.</li>
	<li>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.</li>
	<li>Add cold diethyl ether (35 ml) to the tube.</li>
	<li>Centrifuge for 5 min at 1,207 x g at 4 &#176;C.</li>
	<li>Decant the ether layer.</li>
	<li>Repeat Step 6.7-6.9, five times.</li>
</ol>
7. Drying the Backbone Cyclic Peptide
<ol>
	<li>Keep the precipitated peptide in the same tube and dry it in a hood for 30 min.</li>
	<li>Dissolve the peptide in a 1:1 mixture of water and acetonitrile (ACN).</li>
	<li>Freeze the final product solution in liquid nitrogen.</li>
	<li>Lyophilize the final product.</li>
</ol>
8. Characterizing the Backbone Cyclic Peptide
<ol>
	<li>Dissolve a small sample (1 mg) of the product in water (400 &#181;l).</li>
	<li>Inject the dissolved peptide (10-200 &#181;l) to a reverse phase high-performance liquid chromatography (RP-HPLC) system to test the peptide purity 34.</li>
	<li>Check the mass of the peptide using mass spectrometry matrix-assisted laser desorption ionization (MS-MALDI) 84.
	<ol>
		<li>Mix 1 &#181;l (100 &#181;M) peptide in a 1:1 (v/v) mixture of acetonitrile:water with 1 &#181;l of matrix (5 mg/ml, &#945;-cyano-4-hydroxycinnamic acid) in a 1:1 (v/v) mixture of acetonitrile:water with TFA (0.1%).</li>
		<li>Spot 1 &#181;l on the MS-MALDI plate.</li>
		<li>Dry the sample and place it in the mass spectrometer.</li>
	</ol>
	</li>
	<li>Weigh the peptide and calculate the percent yield.</li>
	<li>Store at -20 &#176;C.</li>
</ol>
9. Monitoring the Synthesis
<ol>
	<li>Kaiser (ninhydrin) test 85
	<ol>
		<li>Prepare the reagent solutions.
		<ol>
			<li>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.</li>
			<li>Prepare Solution B by dissolving 1 g of ninhydrin in 20 ml of ethanol.</li>
			<li>Prepare Solution C by dissolving 40 g phenol in 20 ml ethanol.</li>
		</ol>
		</li>
		<li>Use the Kaiser test to check the completion of amino acid coupling or the deprotection of the protecting group.
		<ol>
			<li>Transfer a few beads from the resin to a test tube.</li>
			<li>Add three drops (~ 100 &#181;l) of each solution (A, B and C) and mix.</li>
			<li>Heat the test tube on a heating block at 110 &#176;C for 5 min. 
			Note: Blue colored beads (positive result) indicate incomplete coupling reaction or deprotection of Fmoc protecting group.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Chloranil test 86
	<ol>
		<li>Prepare the following reagents fresh for each test.
		<ol>
			<li>Prepare a solution of 2% Chloranil in DMF, solution A.</li>
			<li>Prepare a solution of 2% acetaldehyde in DMF, solution B.</li>
		</ol>
		</li>
		<li>Perform the Chloranil test to check the completion of amino acid coupling or the deprotection of the protecting group.
		<ol>
			<li>Mix 100 &#181;l of solution A with 100 &#181;l of solution B in a 1.5 ml tube.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Small scale cleavage reaction
	<ol>
		<li>Remove a small amount of resin to a 3 ml polypropylene cartridge equipped with cap plug and stopcock.</li>
		<li>Treat with a 2 ml mixture of 95% TFA, 2.5% water and 2.5% TIS.</li>
		<li>Shake the mixture for 30 min at RT.</li>
		<li>Remove the resin by filtration using the frit of the polypropylene cartridge and evaporate the solvents by a stream of nitrogen.</li>
		<li>Dissolve the residue in water and analyze the product using HPLC and/or MS.</li>
	</ol>
	</li>
</ol>
10. Leishmania donovani Promastigote Viability in Culture Assay
<ol>
	<li>Leishmania donovani (L. donovani) growth and treatment conditions
	<ol>
		<li>Culture L. donovani promastigotes in Dulbecco&#39;s Modification of Eagle&#39;s Medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate at 26 &#176;C .</li>
		<li>Treat the L. donovani promastigotes with cyclic peptides (100 &#181;M) for 24 hr at 26 &#176;C.</li>
	</ol>
	</li>
	<li>Leishmania donovani (L. donovani) viability assay
	<ol>
		<li>Assess parasite viability with 20 &#181;l alamarBlue according to manufacturer&#39;s protocol.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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....

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 interfaces2,3. 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 PPIs4,5. 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 flexibility8,9. One alternative to surmount these drawbacks is to apply different modifications such as local (D-amino acid and N-alkylation) and global (cyclization) constraints8,10-12. These modifications also occur naturally. For example, cyclosporin A, an immunosuppressant cyclic natural peptide, contains a single D-amino acid and undergoes N-alkylation modifications13,14.
Modification of natural amino acids to induce local constraints, such as D- and N-alkylation, often affects the peptide&#39;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 counterparts15-17.
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 conformation9,18.
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-&#945;-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 &#34;gold standard&#34; 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 sequences21,22, fluorescent modifications23, and peptide libraries24,25. SPPS is also very useful for other poly-chain assemblies such as oligonucleotides26,27, oligosaccharides28,29, and peptide nucleic acids30,31. Interestingly, in some cases, SPPS was shown to be advantageous for synthesizing small molecules that are traditionally made in solution32,33. SPPS is used both in small scale for research and teaching34,35 as well as large scale in industry36-38.
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-&#945;-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-&#945;-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).
<img alt="Figure 1" src="/files/ftp_upload/53589/53589fig1.jpg" /> 
Figure 1. General scheme of solid phase peptide synthesis. The N-&#945;-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-&#945; (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). <a href="https://www.jove.com/files/ftp_upload/53589/53589fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After assembly of the complete peptide chain, cyclization can be achieved by several alternatives: (A) head-to-tail cyclization &#8212; 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&#160;&#8212; 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).
<img alt="Figure 2" src="/files/ftp_upload/53589/53589fig2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/53589/53589fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Microwave-assisted synthesis uses microwave irradiation to heat reactions, thus accelerating organic chemical transformations40,41. 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 controls43,44. 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 sequences46-53.
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 glycopeptides55-58 or phosphopeptides59-61, 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(&#945;) of an amino acid residue with a nitrogen atom62, or peptoids, whose side chain is connected to the amide nitrogen rather than the C&#945; atom63,64; (D) synthesis of cyclic peptides65-71; and (E) synthesis of combinatorial libraries51,72. In numerous cases, the authors reported higher efficiency and reduced synthesis time using microwave irradiation as compared to the conventional protocol.
Using a rational design73-75, we developed anti-parasitic peptides that were derived from the scaffold Leishmania&#39;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 TAT47-57 (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.

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

development of a backbone cyclic peptide library as potential antiparasitic therapeutics using microwave irradiation

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&#39; 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.

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 ...

Watch this Scientific Journal Video about Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation at JoVE.com

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

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.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

development-backbone-cyclic-peptide-library-as-potential

Research

JoVE Journal

Chemistry

11.8K Views.  Stanford University School of Medicine. 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.

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

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.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Biochemistry

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Bioengineering

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

Versatile Home-Built Microwave-Assisted System for Peptide Synthesis

An Inexpensive Adaptation of a Commercial Microwave Reactor for Solid Phase Peptide Synthesis

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to conventional SPPS reaction vessels, which drain solvent and byproducts via a frit located at the bottom of the vessel, the presented apparatus employs a gas dispersion tube under vacuum to remove solvent, byproducts, and excess reagents. The same gas dispersion tube supplies nitrogen gas agitation of the SPPS beads during the reaction steps of coupling and deprotection. Microwave heating is beneficial for SPPS couplings of sterically hindered residues, such as alpha-aminoisobutyric acid (Aib), an alpha,alpha-dialkylated amino acid residue. This home-built apparatus has been used to prepare, via manual Fmoc SPPS methods, heptameric and octameric peptides dominated by the Aib residue, which is notoriously difficult to couple under standard room temperature conditions and reagents. Further, typical commercial microwave SPPS reactors are dedicated exclusively to SPPS synthesis rendering them inaccessible to non-SPPS users. In contrast, the presented apparatus preserves the versatility of the microwave reactor for conventional microwave acceleration of chemical reactions, as the apparatus is trivially removed from the commercial microwave reactor.

A simple, inexpensive, and novel apparatus for performing solid phase peptide syntheses in a commercial microwave reactor is presented.

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to ...

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Immunology and Infection

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (RCH3), dimethylated arginine (R(CH3)2), and/or methylated tryptophan (WCH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-RCH3, WCH3-RCH3, and W-R(CH3)2, were tested using acellular assays and were found to have IC50 values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 &#181;M, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]5-[W-R]9) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 &#181;M) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Presented here is a protocol for designing and testing dipeptides that can inhibit Src kinase enzyme activity using acellular and cellular assays for anti-cancer applications. The peptides formulated here (W-RCH3, WCH3-RCH3, and W-R(CH3)2)&#160;inhibited Src kinase with IC50 values of 510 nM, 916 nM, and 1 &#181;M, respectively.

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated ...

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

Summary

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