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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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+reaction+toolbox+for+peptide+macrocyclization+and+stapling.">Multicomponent reaction toolbox for peptide macrocyclization and stapling.</a> Chemical Reviews. 119 (17), 9836-9860 (2019).</li></ol>

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. Notably, the removal of the protective group of Cys was found to be a critical step for the subsequent ring-closure reaction. Therefore, trt-Cys was deprotected, and this was done until the solution had no apparent yellow color. Further studies showed that cyclic peptides developed by the bisalkylation between Cys and Met methodology could be extended to loop closure in a variety of peptides, with the condition being that the peptide contains the amino acids Cys and Met. In addition, the peptide sequence and linker could also be further adjusted according to the experimental design (Figure 5). It was, therefore, necessary to monitor the synthesis by LC-MS.
For cyclic peptides in the thiol-yne type reaction, firstly, linear peptides were also constructed by common solid-phase peptide synthesis techniques on resin, with the following reactions carried out in the liquid phase solution. Crude peptides were then cleaved from the resin and purified by reverse-phase HPLC. A solution of peptides and propargyl bromide was added to the reaction for 12 h, along with 0.2 mL of MeCN/H2O (1:1, v/v) and 1% HCOOH aqueous solution, and the products were then purified immediately by reverse-phase HPLC. Surprisingly, the ring-closure reaction occurred when the pH of the reaction solution was increased to 8.0. This study has developed a method that constrains the peptide to a cyclic conformation structure with good stability via a thiol-yne type reaction. Moreover, it was necessary to adjust the solvent for the peptide loop closure due to the hydrophilicity and hydrophobicity of the peptides being different. For example, the pH of the hydrophilic peptide was adjusted by (NH4)2CO3, and the hydrophobic peptide was then adjusted by a mixed solvent (1% Et3N solution in 50% MeCN/H2O; Figure 6).
In recent years, various ligation technologies have been developed for synthesizing cyclic peptides, with these technologies generating natural peptide bonds and unnatural backbone linkages32. However, challenges remain in the synthetic cyclic peptide field. Firstly, the amino acid sequence of the peptide has a steric effect, and the efficiency of cyclization may be affected by the residues close to the cyclization site. Secondly, the spatial structure of peptides is diverse, and it may be necessary to carefully choose the connection site during ring closure at the same site. Finally, peptide sequences contain hydrophilic or hydrophobic amino acids, and, therefore, the solubility of the reaction solvent needs to be considered. Therefore, more effort should be put into identifying cyclization at the site, solubility, and isomers to further develop cyclic peptide applications in the pharmaceutical industry.
In this research, a series of facile macrocyclization protocols using bisalkylation between Cys and Met or via a thiol-yne type reaction were designed to resolve the challenges of synthesizing cyclic peptides. Fortunately, these reactions were facile, highly efficient, and metal catalyst-free. The methods developed have been demonstrated to perform both intermolecularly and intramolecularly and have satisfying functional group tolerance. Furthermore, these methods were developed to introduce constraint cyclization, ensuring the peptide chain is more conformationally stabilized, thereby improving target protein binding affinity and reducing nonspecific protein binding. In addition, by using reasonable designs, the reaction site selectivity could also be enhanced by forming a conformationally stabilized cyclization peptide. Overall, peptide chain cyclization generated biologically active compounds, indicating that cyclic peptides are promising drug candidates.

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.

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			<td>2-(7-azabenzotriazol-1-yl)-N,N,N&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HATU)</td>
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			<td>&#160;Thermo Scientific</td>
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			<td>Nanjing Peptide Biotech Ltd</td>
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			<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>
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			<td>Fmoc-Trp(Boc)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
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			<td>Fmoc-Tyr(tBu)-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
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			<td>Fmoc-Val-OH</td>
			<td>Nanjing Peptide Biotech Ltd</td>
			<td>R32001</td>
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			<td>Formic acid</td>
			<td>Energy</td>
			<td>W810042</td>
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			<td>High Performance Liquid 
			Chromatography</td>
			<td>SHIMADZU</td>
			<td>LC-2030</td>
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			<td>Methanol</td>
			<td>Aldrich</td>
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			<td>Morpholine</td>
			<td>Aldrich</td>
			<td>M109062</td>
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			<td>N,N&#39;-Diisopropylcarbodiimide</td>
			<td>Energy</td>
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			<td>Ninhydrin Reagent</td>
			<td>Energy</td>
			<td>N7285</td>
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			<td>Propargyl bromide</td>
			<td>Energy</td>
			<td>W320293</td>
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			<td>Rink Amide MBHA resin</td>
			<td>Nanjing Peptide Biotech Ltd.</td>
			<td></td>
			<td></td>
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			<td>Solid Phase Extraction (SPE) Sample Collection Plates</td>
			<td>&#160;Thermo Scientific</td>
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			<td>Tetrakis(triphenylphosphine) palladium</td>
			<td>Energy</td>
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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-I)-WMAAAC-NH2 peptides 1-Ia and 1-Ib, generated from hexapeptide Ac-WMAAAC-NH2, exhibited distinct retention times and identical molecular weights (Figure 5B). Next, the tolerance of the cyclic peptide to different functional groups was further tested, as shown in Figure 5C. The loop closure efficiency of the 10 linear peptides was evaluated using a di-halogenated linker, with the results showing that all the peptides efficiently generated the corresponding cyclic peptides. Compared with the other cyclic peptides, a model hexacyclic cyclic peptide with high conversion rates produced a differential ratio of 1:1 of the epimers and could be separated by HPLC. However, in some cases, the peptide epimers could not be separated under HPLC conditions, likely due to the sulfonium chiral center of the epimer not being very stable and being slowly racemized into epimer mixtures. The reaction efficiency of the epimers (1-Ia) with pyridinrthiols (PyS) (10 mM) was then examined by HPLC. Figure 5D shows the HPLC traces of the time-dependent conversion between the cyclic peptide (1 mM) and its conjugated product. The traces clearly show the time-dependent reduction of peptide 1-Ia with PyS in PBS (pH 7.4).
Another strategy for peptide ring closure was that, firstly, a propargyl group was attached to Met, and then the formed propargyl sulfonium center triggered a thiol-yne reaction to generate a cyclic peptide. In this strategy, the ring size and peptide sequence did not affect the cyclization reaction, with the peptide containing two or three amino acids just used as a model to close the loop. The synthetic route of intramolecular peptide cyclization is described in Figure 6A. In addition, a simplified propargylated Met model peptide was constructed as described in Figure 6B. The results showed that the model peptide MC&#39;s yield could be up to 80% when the reaction solution pH was set to 8.0 (MC refers to a model cyclic peptide synthesized by this route; Figure 6A). Moreover, the model peptide MC was isolated and purified by HPLC (Figure 6D), and its molecular weight was confirmed by LC-MS (Figure 6C). As shown in Figure 6E, the chemical shift was further characterized by 1H NMR and heteronuclear single quantum coherence (HSQC). In addition, the dithiothreitol (DTT) was added to attempt to open the sulfonium ring to explore the stability of MC. The results showed no addition or ring-opening products after 24 h&#160;(Figure 6F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64289/64289fig01.jpg" /> 
Figure 1: Diagram of the experimental setup for a Fmoc-based solid phase peptide for synthesizing peptides. The peptide column was placed on the solid phase reactor via three-way stopcocks with nitrogen or argon bubbling through the column during peptide synthesis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64289/64289fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64289/64289fig02.jpg" /> 
Figure 2: On-resin intermolecular synthetic linear CM peptides. All the linear peptides were synthesized on Rink-amide MBHA resin by standard manual Fmoc solid-phase synthesis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64289/64289fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64289/64289fig03.jpg" /> 
Figure 3: Synthetic routes for peptide cyclization via Cys and Met bisalkylation. The linear peptides containing Met and Cys were constructed as stabilized cyclic peptides. First, the trt-protected Cys was deprotected with 3% TFA in DCM. Then, a di-halogenated linker (2 eq) and DIPEA (4 eq) were added to react with Cys for 3 h. Finally, cyclization between Cys and Met was completed when the resin was cleaved in a TFA cleavage solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64289/64289fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64289/64289fig04.jpg" /> 
Figure 4: Synthetic routes for peptide cyclization via Cys and Met thiol-yne. First, the linear peptides were purified and characterized by HPLC and LC-MS. The reaction occurred under the following conditions: a solution including the compound (0.2 mM, 1.0 eq) in 0.2 mL of MeCN/H2O (1:1, v/v), 1% HCOOH aqueous solution (in volume), and propargyl bromide (1.0 mM, 5.0 eq) was shaken at room temperature for 12 h at a pH of 8.0. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64289/64289fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64289/64289fig05.jpg" /> 
Figure 5: Synthesis scheme of cyclic peptides using bisalkylation between Cys and Met. (A) Schematic illustration of peptide cyclization by Cys and Met. (B) The HPLC and&#160;LC-MS spectrum of the epimers (1-Ia and 1-Ib). (C) The functional residue tolerance of the different peptides was tested. (D)&#160;HPLC traces of the time-dependent conversion between epimers (1-Ia; 1 mM) and their conjugated products. This figure is a modification of that reported by Wang et al.30. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64289/64289fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64289/64289fig06.jpg" /> 
Figure 6: Synthesis scheme of cyclic peptides using a thiol-yne type reaction. (A) Facile construction of peptide cyclization by the thiol-yne reaction. MC refers to a model cyclic peptide synthesized by the route. (B)&#160;Intramolecular peptide cyclization of different linear peptides. a = 1 mL of 1 M (NH4)2CO3 aqueous solution. b = 1% Et3N in 1 mL of MeCN/H2O (1:1). Abbreviations: Ahx = 6-aminocaproic acid. The yield was calculated as the percentage of the product determined by weighing. Conversion represented the amount of starting material that reacted by HPLC. (C) The LC-MS spectrum of the MC cyclization peptide. (D) The HPLC spectrum of the MC cyclization peptide. (E)&#160;1H NMR and HSQC spectra characterization of the MC cyclic peptides. (F) 1H NMR spectra of the time-dependent conversion between MC cyclic peptide and DTT in D2O for 3 h, 6 h, 12 h, and 24 h. This figure is a modification of that reported by Hou et al.31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64289/64289fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Chemistry

2.6K Views.  Shenzhen Bay Laboratory. 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.

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

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 ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

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 ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

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 ...

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.