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.

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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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Journal (Chemistry)

Journal (Chemistry)

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

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