Cleavage Cocktail for Methionine Containing Peptides (Reagent H)

Cleavage Cocktail for Methionine Containing Peptides (Reagent H) Peptides prepared by Fmoc protocols on acid labile resin can be cleaved from the support and sidechain deprotected under mild acid conditions. Methionine residues are subject to oxidation under these mild cleavage conditions, however, so many popular cleavage mixtures contain a reducing agent such as thioanisole. Even with thioanisole added, significant oxidation methionine residues may still occur. A cleavage cocktail containing trifluoroacetic acid, phenol, thioanisol, 1,2-ethanedithiol, dimethylsulfide, ammonium iodide and water has been shown to prevent methionine oxidation during cleavage.1 Dimethylsulfoxide and iodine are generated from the reduction of methionine sulfoxide, so peptides containing Cys(Trt) residues can be isolated as linear peptides or as cyclized products upon extended treatment with the cleavage cocktail.

Preparation of Cleavage Cocktail (Reagent H)

Mix trifluoroacetic acid (81% w/w), phenol (5% w/w), thioanisole, (5% w/w), 1,2-ethanedithiol (2.5% w/w), water (3% w/w), dimethylsulfide (2% w/w) and ammonium iodide (1.5% w/w).

Peptide Cleavage (Without Disulfide Formation)

1. Suspend the peptide resin in the cleavage cocktail (30 mL/g resin).

 2. Allow the mixture to stand three hours at room temperature under inert gas. 

3. Filter and wash the resin with trifluoroacetic acid. 

4. Combine the filtrates and add methyl tert-butyl ether to precipitate the crude product. 

Peptide Cleavage with Disulfide Formation

1. Suspend the peptide resin in the cleavage cocktail (30 mL/g resin). 

2. Allow the mixture to stand ten hours at room temperature. 

3. Filter and wash the resin with trifluoroacetic acid. 

4. Combine the filtrates and add methyl tert-butyl ether to precipitate the crude product.