Name: constructing cyclic peptides using an on-tether sulfonium center
Uploaded: 2022-09-28T13:30:00
Description: Watch this Scientific Journal Video about Constructing Cyclic Peptides Using an On-Tether Sulfonium Center at JoVE.com

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 su...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysine+and+arginine+side+chains+in+glycosaminoglycan-protein+complexes+investigated+by+NMR,+cross-Linking,+and+mass+spectrometry:+a+case+study+of+the+factor+h-heparin+Interaction.">Lysine and arginine side chains in glycosaminoglycan-protein complexes investigated by NMR, cross-Linking, and mass spectrometry: a case study of the factor h-heparin Interaction.</a> Journal of the American Chemical Society. 132 (18), 6374-6381 (2010).</li><li>Petitdemange, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+tuning+of+elastin-like+polypeptide+properties+via+methionine+oxidation.">Selective tuning of elastin-like polypeptide properties via methionine oxidation.</a> Biomacromolecules. 18 (2), 544-550 (2016).</li><li>Kadlcik, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+modification+of+a+methionine+residue+in+the+amyloid-beta+peptide.">Reductive modification of a methionine residue in the amyloid-beta peptide.</a> Angewandte Chemie International Edition. 45 (16), 259 (2006).</li><li>Reguera, L., Rivera, D. 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M., Schoffelen, S., Kofoed, C., Meldal, M., Diness, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+tuning+of+fluorobenzene+probes+for+cysteine-selective+protein+modification.">Rational tuning of fluorobenzene probes for cysteine-selective protein modification.</a> Angewandte Chemie International Edition. 57 (27), 8022-8026 (2018).</li><li>Jiang, H. F., Chen, W. J., Wang, J., Zhang, R. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+covalent+targeting+of+anti-apoptotic+BFL-1+by+cysteine-reactive+stapled+peptide+inhibitors.">Selective covalent targeting of anti-apoptotic BFL-1 by cysteine-reactive stapled peptide inhibitors.</a> Cell Chemical Biology. 23 (9), 1123-1134 (2016).</li><li>Chow, H. Y., Zhang, Y., Matheson, E., Li, X. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation+technologies+for+the+synthesis+of+cyclic+peptides.">Ligation technologies for the synthesis of cyclic peptides.</a> Chemical Reviews. 119 (17), 9971-10001 (2019).</li><li>Zhang, H. Y., Chen, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+peptide+drugs+approved+in+the+last+two+decades+(2001-2021).">Cyclic peptide drugs approved in the last two decades (2001-2021).</a> RSC Chemical Biology. 3 (1), 18-31 (2021).</li><li>Lee, Y. J., Han, S. H., Lim, Y. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+stabilization+and+multimerization+of+a+peptide+alpha-helix+by+stapling+polymerization.">Simultaneous stabilization and multimerization of a peptide alpha-helix by stapling polymerization.</a> Macromolecular Rapid Communications. 37 (13), 1021-1026 (2016).</li><li>Karthikeyan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-viral+activity+of+methyl+1-chloro-7-methyl-2-propyl-1h-benzo[d]+imidazole-5-carboxylate+against+white+spot+syndrome+virus+in+freshwater+crab+(Paratelphusa+hydrodromous).">Anti-viral activity of methyl 1-chloro-7-methyl-2-propyl-1h-benzo[d] imidazole-5-carboxylate against white spot syndrome virus in freshwater crab (Paratelphusa hydrodromous).</a> Aquaculture International. 30, 989-998 (2022).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinked+aspartic+acids+as+helix-nucleating+templates.">Crosslinked aspartic acids as helix-nucleating templates.</a> Angewandte Chemie International Edition. 55 (39), 12088-12093 (2016).</li><li>Hu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in-tether+chiral+center+modulates+the+helicity,+cell+permeability,+and+target+binding+affinity+of+a+peptide.">An in-tether chiral center modulates the helicity, cell permeability, and target binding affinity of a peptide.</a> Angewandte Chemie International Edition. 55 (28), 8013-8017 (2016).</li><li>Hu, K., Sun, C., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+and+versatile+on-tether+modification+of+chiral-center-induced+helical+peptides.">Reversible and versatile on-tether modification of chiral-center-induced helical peptides.</a> Bioconjugate Chemistry. 28 (7), 2001-2007 (2017).</li><li>Kramer, J. R., Deming, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+chemoselective+tagging+and+functionalization+of+methionine+containing+peptides.">Reversible chemoselective tagging and functionalization of methionine containing peptides.</a> Chemical Communications. 49 (45), 5144-5146 (2013).</li><li>Shi, X. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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&#39;,N&#39;-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>&#160;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>&#160;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 
			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&#39;-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>&#160;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&#38;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 ...

In this protocol, the sulfonium salt was constructed that could act as a new hand that reacted with the protein cysteine on the special proximity. The advantage of this technique was that this reaction was fast and efficient, and Met-catalyst free and developed a simple and efficient method for stabilizing peptides. To begin, prepare the N-terminal 9-fluorinyl-methyloxycarbonyl deprotection solution of 20%piperidine or 50%morpholine and DMF in a 500-milliliter beaker or flask.Then, add 10 milliliters of the deprotection solution to the column and bubbled nitrogen into the solution for 30 minutes or twice for five minutes. Drain the solution by a vacuum pump and wash the resin sequentially for five times with dichloromethane and N, N-dimethylformamide. To detect the resin as a darky yellow color solution, add one milliliter of N, N-dimethylformamide to a small amount of resin and add 200 microliters of 5%ninhydrin to a glass tube.Heat it at 130 degrees Celsius for three minutes to assess changes in the resin color. For the coupling of peptides, prepare a mixture solution as described in the manuscript in a polypropylene tube and dissolve it in three milliliters of N, N-dimethylformamide. Then, add 173 microliters of N, N-diisopropylethylamine or 154 microliters of N, N'diisopropylcarbodiimide to the solution to inactivate the amino acid.Next, add the mixture to the column with resin and bubble it with nitrogen for two hours. After coupling, add one milliliter of N, N-dimethylformamide to a small amount of resin and add 200 microliters of 5%ninhydrin to a glass tube. Heat it at 130 degrees Celsius for three minutes while the resin changed to colorless which confirms the absence of a free amino group before the deprotection step.After draining the solution, wash the resin as demonstrated previously and add 10 milliliters of the deprotection solution to the column. Then, bubble with nitrogen for 30 minutes or twice for five minutes. And again, add fresh solution.Add 10 milliliters of anhydrous methanol to the column with resin for dehydrating and dry with nitrogen for the next use. Weigh 100 milligrams of the resin into a column and wash the resin with dichloromethane and N, N-dimethylformamide before the coupling step. Prepare a solution for removing the trityl-L-cysteine protection group by adding trifluoroacetic acid, triisopropylsilane, and dichloromethane mixture in a 100 milliliter beaker or flask for removing the protective group.Add five to 10 milliliters of the prepared solution to the column to remove the protection group for 10 minutes. Repeat six times with nitrogen bubbling until the yellow color completely disappears. After draining the solution by a vacuum pump, wash the resin as demonstrated previously.Then, prepare a solution for reacting with deprotected cysteine by adding dihalogenated linker and N, N-diisopropylethylamine in a 50 milliliter beaker or flask with N, N-dimethylformamide. Add five to 10 milliliters of the reaction solution to the column to react with protected cysteine for at least three hours with nitrogen bubbling. Again, drain the solution by a vacuum pump and wash the resin sequentially as demonstrated previously.To prepare the peptide cyclization solution, add trifluoroacetic acid mixture in a 20 milliliter beaker or flask in the fume hood for peptide cyclization. Then, add five to 10 milliliters of the mixture solution to the polypropylene tube and cleave the resin under the trifluoroacetic acid cocktail for three hours. After dehydrating and washing the resin before the coupling step is demonstrated previously, prepare a 1%formic acid aqueous solution and one millimolar propargyl bromide and add to the methionine peptide solution.Next, shake the coupling reaction of methionine and propargyl bromide at room temperature for 12 hours. After the reaction, dissolve the product in a polypropylene tube and acetonitrile and filter it through a 0.22 micrometer filter membrane. Then, purify the solution by reverse phase HPLC immediately and freeze dry it into powder for the next use.The synthesized cyclic peptides using bis-alkylation between cysteine and methionine were characterized by HPLC and LCMS spectrum, which shows that the epimers exhibited distinct retention times and identical molecular weights. The HPLC traces of the epimers and its conjugated product demonstrated the time-dependent conversion between the cyclic peptide and its product. The molecular weight of cyclic peptides synthesized using a thiol-yne type reaction was determined by by LCMS and the peptide was isolated and purified by HPLC.The peptides were further characterized by proton NMR and heteronuclear single quantum coherent spectra, revealing the chemical shift. Proton NMR spectra of the time-dependent conversion between cyclic peptide and dithiothreitol and deuterium oxide was obtained to explore the stability of peptide due to opening of the sulfonium ring. The results showed that no addition or ring opening products were formed after 24 hours.This procedure established an effective strategy for synthesizing cyclic peptides. The reaction says selectivity also enhanced by forming a confirmation that stabilize the cyclization peptide, indicating that cyclic peptides were promising drug catalyst.

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