We acknowledge financial support from the National Key R&#38;D Program of China (2021YFC2103900); the Natural Science Foundation of China grants (21778009, and 21977010); the Natural Science Foundation of Guangdong Province (2022A1515010996 and 2020A1515010521): the Shenzhen Science and Technology Innovation Committee, (RCJC20200714114433053, JCYJ201805081522131455, and JCYJ20200109140406047); and the Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions grant (2019SHIBS0004). The authors acknowledge journal support from Chemical Science, The Royal Society of Chemistry for reference 30 and The Journal of Organic Chemistry, American Chemical Society, for reference 31.

1. Equipment preparation
CAUTION: Morpholine, N,N-dimethylformamide (DMF), dichloromethane (DCM), N,N-diisopropylethylamine (DIPEA), TFA, morpholine, piperidine, diethyl ether, and methanol are toxic, volatile, and corrosive. These reagents can harm the human body through inhalation, ingestion, or skin contact. For all chemical experiments, use protective equipment, including disposable gloves, experimental coats, and protective eyeglasses.
<ol>
	<li>Construct all peptide substrates on Rink-amide 4-methylbenzhydrylamine (MBHA) resin by standard manual Fmoc-based solid-phase peptide synthesis (SPPS)29, as shown in Figure 1.</li>
	<li>Use either of the two options for constructing linear peptides: one, synthesize the peptide utilizing condensing agent 2-(7-azabenzotriazol-1-yl)-N,N, N&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HATU) and a reagent of DIPEA; or two, synthesize utilizing 1-hydroxybenzotriazole (HOBT) and N,N&#39;-diisopropylcarbodiimide (DIC) as the amide condensation reagent. Select an appropriate protocol for peptide synthesis according to the sequence of the peptide.</li>
	<li>To establish a manual peptide-synthesis device, set up modified vacuum solid phase extraction equipment in a fume hood and connect it with a three-way stopcock. Next, place a polypropylene filter cartridge or glass reactor on the equipment while connected to nitrogen (N2) gas. 
	NOTE: For modifying the vacuum solid phase extraction equipment, remove the extraction tube and keep the vacuum-sealed system.</li>
	<li>Load MBHA resin into the resin-filled columns and dissolve it in DMF. Adjust the switch of the three-way stopcocks for N2 bubbling, and then remove the solvent in the column using a working pump connected to a vacuum system. Calculate the amount of amino acid or condensing agent required using the following formula: 
	Amino acid (g) and condensing agent (g) = resin scale (g) &#215; resin loading capacity (mM/g) &#215; equivalent (5 eq) &#215; molecular weight (g/M) 
	&#8203;NOTE: Choose the amount of loaded resin according to the length of the coupling peptide. Multiple equivalents (eq) of amino acids are used to react more fully. Liquid solvents need to be converted to volume by density. The 5 eq shows that the input amount of the calculated compound is amplified by a factor of five.</li>
</ol>
2. Resin preparation
NOTE: Choose the amount of loaded resin according to the length of the coupling peptide.
<ol>
	<li>Weigh 302 mg of Rink-amide MBHA resin (0.331 mM/g loaded) into a column (20 mL reservoir). Add 5-10 mL of DCM or DMF into the column to swell the resin under N2 bubbling for 30 min.</li>
	<li>Next, close the N2 switch, and then turn on the vacuum pump suction switch to remove the solvent. Then, wash the resins sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.</li>
</ol>
3. N-terminal Fmoc deprotection
NOTE: Deprotection by morpholine requires 30 min, and deprotection by piperidine takes 5 min.
<ol>
	<li>Prepare the N-terminal 9-fluorenylmethyloxycarbonyl (Fmoc) deprotection solution: prepare a sufficient volume (500 mL) of 20% (v/v) piperidine or 50% (v/v) morpholine in DMF in a glass container for Fmoc group deprotection.</li>
	<li>Add 10 mL of the deprotection solution to the column, bubble N2 into the&#160;solution for 30 min or 5 min 2x, and repeat the deprotection procedures 2x.</li>
	<li>Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.</li>
	<li>Detect the resin as a dark yellow color solution by 5% ninhydrin (Kaiser test) after every deprotection to confirm the absence of the Fmoc group before the coupling step. In detail, add 1 mL of DMF to a small amount of resin and add 200 &#181;L of 5% ninhydrin to a glass tube heated at 130 &#176;C for 3 min to assess changes in the resin color.</li>
	<li>Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.</li>
</ol>
4. Coupling the linear peptide (Figure 2)
NOTE: When the synthetic peptide sequences contain two or more repeating units, the coupling procedure can be directly carried out by selecting the amino acid type, such as Fmoc-AA-OH or Fmoc-AAA-OH, and so on. Some special amino acids with steric hindrance and peptides with longer amino acid sequences are required to properly extend the reaction time for coupling.
<ol>
	<li>For a single coupling step, take the coupling of Cys residue as an example (synthesize 300 mg resin scales). Prepare a mixture solution including Fmoc-Cys(Trt)-OH (5 eq, 292 mg) and HATU (5 eq, 206 mg) or Fmoc-Cys(Trt)-OH (5 eq, 292 mg) and HOBT (5 eq, 106 mg) in a polypropylene tube and dissolve it in 3 mL of DMF.</li>
	<li>Add 173 &#181;L of DIPEA (10 eq) or 154 &#181;L of DIC (10 eq) to the solution of Cys to activate the amino acid. Let the mixture pre-activate for 1 min. Add the mixture to the column with resin, and bubble it with N2 for 2 h. 
	NOTE: The specific reaction time required should be standardized to detect 5% ninhydrin.</li>
	<li>After coupling, add 1 mL of DMF to a small amount of resin and add 200 &#181;L of 5% ninhydrin to a glass tube heated at 130 &#176;C for 3 min. Observe the resin change to colorless to confirm the absence of a free amino group before the deprotection step.</li>
	<li>Drain the solution using a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.</li>
	<li>Add 10 mL of the deprotection solution to the column, bubble with N2 for 30 min or 5 min 2x, add fresh solution, and repeat the deprotection procedures 2x.</li>
	<li>Repeat the following steps: deprotection of the Fmoc group; detecting resin deprotection; washing the resin; coupling the amino acid; detecting the coupling reaction. Repeat the coupling step until all the peptides are synthesized. 
	&#8203;NOTE: Use the Kaiser test to monitor whether every step of deprotection is complete or whether each step of the amino acid coupling is thorough. Alternatively, a small amount of peptide can be cleaved from the resin and checked by LC-MS for successful coupling.</li>
</ol>
5. Bisalkylation between Met and Cys (Figure 3)
<ol>
	<li>Construct a linear peptide with Met and Cys by repeating steps 4.1-4.6. Add 10 mL of anhydrous methanol to the column with resin for dehydrating and dry with N2 for the next use (repeat 2x) after linear peptide coupling.</li>
	<li>Weigh 100 mg of the resin obtained in the previous step into a column (20 mL reservoir) and wash the resin with DCM (5-10 mL) and DMF (5-10 mL) before the coupling step.</li>
	<li>Prepare a solution for removing the Cys trt(trityl) protection group. Prepare a sufficient volume (100 mL) of TFA/TIS/DCM (3:5:92) mixture in a glass container for removing the protectiove group.</li>
	<li>Add 5-10 mL of the solution of TFA/TIS/DCM (3:5:92) to the column to remove the protection group for 10 min. Repeat six times with N2 bubbling until the yellow color completely disappears.</li>
	<li>Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.</li>
	<li>Prepare a solution for reacting with deprotected Cys. Prepare a sufficient volume (50 mL) of mixture solution (DMF) including di-halogenated linker (2 eq) and DIPEA (4 eq) for reacting with deprotected Cys.</li>
	<li>Add 5-10 mL of the reaction solution to the column to react with deprotected Cys for at least 3 h with N2 bubbling. Dehydrate with anhydrous methanol and dry with N2 for the next use.</li>
	<li>Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.</li>
	<li>Prepare a solution for peptide cyclization: prepare a sufficient volume (20 mL) of TFA mixture (TFA:TIS:H2O = 95:2.5:2.5) in a glass container in the fume hood for peptide cyclization.</li>
	<li>Add 5-10 mL of the TFA mixture solution to the polypropylene tube and cleave the resin under the TFA cocktail for 3 h. 
	CAUTION: TFA is highly corrosive and irritating; the peptide cleavage process should be performed in a fume hood.</li>
	<li>Perform steps 7.1-7.5 to obtain a linear peptide solution by reverse-phase liquid phase purification (HPLC). Freeze-dry the solution for the next use.</li>
</ol>
6. Propargyl sulfonium salt cyclization (Figure 4)
<ol>
	<li>Construct a linear peptide with Met and Cys by repeating steps 4.1-4.6. Add 10 mL of anhydrous methanol to the column with the resin for dehydrating and dry with N2 for the next use (repeat twice) after linear peptide coupling.</li>
	<li>Weigh 100 mg of the resin obtained in the previous step into a column (20 mL reservoir) and wash the resin with DCM (5-10 mL) and DMF (5-10 mL) before the coupling step.</li>
	<li>Perform steps 7.1-7.5 to obtain a linear peptide solution by HPLC, and freeze-dry the sample, as mentioned, for next use.</li>
	<li>Prepare a 1% HCOOH aqueous solution (in volume) and 1.0 mM propargyl bromide (5 eq) and add to the Met peptide solution (0.2 mM, 1 eq; 0.2 mL of MeCN/H2O [1:1, v/v]).</li>
	<li>Next, shake the coupling reaction of Met and propargyl bromide at room temperature for 12 h.</li>
	<li>After the reaction, dissolve the product in a polypropylene tube in acetonitrile and filter it through a 0.22 &#181;mfilter membrane. Then, purify the solution by reverse-phase HPLC immediately and freeze-dry it into powder for the next use.</li>
	<li>In the last step, add the peptide with propargyl bromide to a polypropylene tube, maintain the reaction solution pH at 8.0 by adding (NH4)2CO3 solution, and shake the reaction mixture at 37 &#176;C for 12 h. This step obtains the cyclic peptide of propargyl sulfonium salt.</li>
	<li>Collect the last reaction mixture: dissolve it in a polypropylene tube in acetonitrile and filter it through a 0.22 &#181;m filter membrane. Then, purify the solution by reverse-phase HPLC immediately and freeze-dry it into powder for the next use.</li>
</ol>
7. Purification of cyclic peptides
<ol>
	<li>Construct linear peptide substrates on Rink-amide MBHA resin by repeating steps 4.1-4.6. Add 10 mL of anhydrous methanol to the column with resin for dehydrating and dry with N2 for the next use (repeat twice) after linear peptide coupling.</li>
	<li>Prepare a sufficient volume of cleavage cocktail (TFA/H2O/TIS, v/v/v, 95:2.5:2.5) in the fume hood. Add 1-5 mL of TFA mixture solution to the polypropylene tube and cleave the resin under the TFA cocktail for 3 h.</li>
	<li>Next, sequentially dry the resin under a steam of N2 in the column. Alternatively, use a filter device to filter the resin and collect the peptide solution. Then, add 20 mL of ether to the peptide solution to precipitate the peptide, centrifuge at 3,500 x g for 5 min, and repeat twice. Collect the crude peptide precipitate and dry it under a stream of N2 for the next step.</li>
	<li>Dissolve 200 mg of crude peptide in a polypropylene tube in 4 mL of acetonitrile-water solution and filter it through a 0.22 &#181;m filter. Transfer the peptide into an HPLC vial insert. Place the insert into the autosampler of a semi-preparative reverse-phase HPLC system equipped with a C18 5 &#181;m, 4.6 mm x 250 mm column, and a 1 mL injection loop.</li>
	<li>Purify and isolate the peptide using a gradient program of 5%-95% acetonitrile in water with 0.1% TFA over 30 min while monitoring the HPLC spectra using UV at 254 nm. Confirm the peptide molecular weight by LC-MS, collect the solution of the peptide, and freeze-dry it into powder for the next use.</li>
</ol>

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The authors have nothing to disclose.

The synthetic approach described in this paper provides a method for synthesizing cyclic peptides using Cys and Met in the peptide sequence, in which the basic linear peptides are constructed by common solid-phase peptide synthesis techniques. For the bisalkylation of cyclic peptides between Cys and Met, 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 cleavage solution. ...

Protein-protein interactions (PPIs)1 play a pivotal role in drug research and development. Constructing stabilized peptides with a fixed conformation by chemical means is one of the most important methods for developing mimetic motifs of PPIs2. To date, several cyclic peptides that target PPIs have been developed for clinical use3. Most peptides are constrained to an &#945;-helix conformation to decrease the conformational entropy and improve the metabolic stability, target-binding affinity, and cell permeability4,5. In the past 2 decades, the side chains of Cys6,7, lysine8,9, tryptophan10, arginine11, and Met12,13 have been inserted into unnatural amino acids to fix the peptide into a cyclic conformation. Such cyclic peptides can target a unique chemical space or special sites, thereby triggering a covalent reaction to form protein-peptide covalent binding14,15,16,17. In a recent report by Yu et al., a chloroacetamide was anchored onto the domain of peptide ligands, ensuring a covalent conjugation reaction with excellent protein specificity18. Moreover, electrophilic warheads, such as acrylamide and aryl sulfonyl fluoride (ArSO2F), were further incorporated into peptides by Walensky et al.19 to form stabilized peptide covalent inhibitors and improve the anti-tumor effect of peptide inhibitors. Therefore, it is very important to introduce an additional functional group in order to covalently modify protein-peptide ligands20. These groups not only react with proteins on the side chain but also stabilize the secondary structure of the peptide21. However, the application of covalently modified proteins induced by peptide ligands is limited due to the complicated synthetic route and the non-specific binding of the chemical groups22,23. Effective strategies for the synthesis of cyclic peptides are, therefore, urgently required.
Inspired by the multifarious strategies of cyclic peptides2,24,25,26, this protocol attempts to develop a simple and efficient method for stabilizing peptides. In addition, we noted that the side chain group of a stable peptide could react covalently with a target protein when it was spatially close to the peptide ligands. The lack of chemically modified Met was filled by the Deming group in 2013 by developing a novel method for producing selectively modified peptide methionine27. Based on this background, the Shi et al. focused on the development of the ring closure of side chains to form a sulfonium salt center. When the peptide ligand combines with the target protein, the sulfonium salt group reacts covalently with the spatially close Cys protein. In recent years, the Shi et al. have designed a new method for stabilizing cyclic peptide28. The sulfonium salt on the cyclic peptide was reduced by a reducing agent with a sulfhydryl group that was reversibly reduced to Met. However, the reaction had low efficiency, which was harmful to subsequent biological application studies. In the current study, a Met-Cys and propargyl bromide-Cys ring-closure reaction was designed, with a single sulfonium salt remaining on the side chain of the cyclic peptide. The sulfonium salt acted as a new warhead that reacted covalently with the protein Cys under spatial proximity. Briefly, a Cys and Met mutated peptide was cyclized by intramolecular alkylation, resulting in the generation of an on-tether sulfonium center. In this process, the formation of a side chain bridge was critical for cyclic peptides. Overall, this protocol describes a detailed sulfonium-based peptide cyclization that is achieved using simple reaction conditions and operations. The aim is to develop a potential method for further broad biological applications.

<table><tbody><tr><td>1,3-bis(bromomethyl)-benzen</td><td>Energy</td><td>D0215</td><td></td></tr><tr><td>1,3-Dimethylbarbituric acid</td><td>Energy</td><td>A46873</td><td></td></tr><tr><td>1H NMR and HSQC</td><td>Bruker</td><td>&amp;nbsp;AVANCE-III 400</td><td></td></tr><tr><td>1-Hydroxybenzotriazole hydrate</td><td>Energy</td><td>E020543</td><td></td></tr><tr><td>2-(7-azabenzotriazol-1-yl)-N,N,N&#039;,N&#039;-tetramethyluronium hexafluorophosphate (HATU)</td><td>Energy</td><td>A1797</td><td></td></tr><tr><td>2-mercaptopyridine</td><td>Energy</td><td>Y31130</td><td></td></tr><tr><td>6-Aminocaproic acid</td><td>Energy</td><td>A010678</td><td></td></tr><tr><td>Acetic anhydride</td><td>Energy</td><td>A01021454</td><td></td></tr><tr><td>Acetonitrile</td><td>Aldrich</td><td>9758</td><td></td></tr><tr><td>Ammonium carbonate</td><td>Energy</td><td>12980</td><td></td></tr><tr><td>Dichloromethane (DCM)</td><td>Energy</td><td>W330229</td><td></td></tr><tr><td>Digital Heating Cooling Drybath</td><td>&amp;nbsp;Thermo Scientific</td><td>88880029</td><td></td></tr><tr><td>Diisopropylethylamine (DIPEA)</td><td>Energy</td><td>W320014</td><td></td></tr><tr><td>Dimethyl formamide (DMF)</td><td>Energy</td><td>B020051</td><td></td></tr><tr><td>Dithiothreitol</td><td>Energy</td><td>A10027</td><td></td></tr><tr><td>Electrospray Ionization Mass</td><td>SHIMADZU2020</td><td>&amp;nbsp;LC-MS2020</td><td></td></tr><tr><td>Fmoc-Ala-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R30101</td><td></td></tr><tr><td>Fmoc-Arg(Pbf)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R30201</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R30501</td><td></td></tr><tr><td>Fmoc-Gln(Trt)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R30601</td><td></td></tr><tr><td>Fmoc-Glu(OtBu)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R30701</td><td></td></tr><tr><td>Fmoc-His(Boc)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R30902</td><td></td></tr><tr><td>Fmoc-Ile-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31001</td><td></td></tr><tr><td>Fmoc-Lys(Boc)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31201</td><td></td></tr><tr><td>Fmoc-Met-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31301</td><td></td></tr><tr><td>Fmoc-Pro-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31501</td><td></td></tr><tr><td>Fmoc-Ser(tBu)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31601</td><td></td></tr><tr><td>Fmoc-Thr(tBu)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31701</td><td></td></tr><tr><td>Fmoc-Trp(Boc)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31801</td><td></td></tr><tr><td>Fmoc-Tyr(tBu)-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R31901</td><td></td></tr><tr><td>Fmoc-Val-OH</td><td>Nanjing Peptide Biotech Ltd</td><td>R32001</td><td></td></tr><tr><td>Formic acid</td><td>Energy</td><td>W810042</td><td></td></tr><tr><td>High Performance Liquid&lt;br/&gt; Chromatography</td><td>SHIMADZU</td><td>LC-2030</td><td></td></tr><tr><td>Methanol</td><td>Aldrich</td><td>9758</td><td></td></tr><tr><td>Morpholine</td><td>Aldrich</td><td>M109062</td><td></td></tr><tr><td>N,N&#039;-Diisopropylcarbodiimide</td><td>Energy</td><td>B010023</td><td></td></tr><tr><td>Ninhydrin Reagent</td><td>Energy</td><td>N7285</td><td></td></tr><tr><td>Propargyl bromide</td><td>Energy</td><td>W320293</td><td></td></tr><tr><td>Rink Amide MBHA resin</td><td>Nanjing Peptide Biotech Ltd.</td><td></td><td></td></tr><tr><td>Solid Phase Extraction (SPE) Sample Collection Plates</td><td>&amp;nbsp;Thermo Scientific</td><td>60300-403</td><td></td></tr><tr><td>Tetrakis(triphenylphosphine) palladium</td><td>Energy</td><td>T1350</td><td></td></tr><tr><td>Three-way stopcocks</td><td>Bio-Rad</td><td>7328107</td><td></td></tr><tr><td>Triethylamine</td><td>Energy</td><td>B010737</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA)</td><td>J&amp;amp;K</td><td>101398</td><td></td></tr><tr><td>Triisopropylsilane (TIS)</td><td>Energy</td><td>T1533</td><td></td></tr></tbody></table>

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.

All the linear peptides were synthesized on Rink-amide MBHA resin by standard manual Fmoc solid-phase synthesis. A model cyclic hexapeptide (Ac (cyclo-I)-WMAAAC-NH2) was constructed as described in Figure 5A. Notably, a new on-tether chiral center was generated by Met alkylation, with the two epimers of cyclic peptide (Ia, Ib) confirmed by reverse-phase HPLC. Further, the conversion and ratio of epimers were determined using the integration of reverse-phase HPLC. Cyclic Ac-(cyclo-...

Watch this Scientific Journal Video about Constructing Cyclic Peptides Using an On-Tether Sulfonium Center at JoVE.com

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

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 ...

constructing-cyclic-peptides-using-an-on-tether-sulfonium-center

Research

JoVE Journal

Chemistry

2.6K Views.  Shenzhen Bay Laboratory. In dit protocol werd het sulfoniumzout geconstrueerd dat kon fungeren als een nieuwe hand die reageerde met het eiwit cysteïne op de speciale nabijheid. Het voordeel van deze techniek was dat deze reactie snel en efficiënt was, en Met-katalysator vrij en ontwikkelde een eenvoudige en efficiënte methode voor het stabiliseren van peptiden. Bereid om te beginnen de N-terminale 9-fluorinyl-methyloxycarbonyl-deprotectie-oplossing van 20% piperidine of 50% morfoline en DMF in een bekerglas of ko...

Video: Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

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

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.

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 ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

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 ...

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 ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

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