Cleavage Cocktails HEADING_TITLE

“Odorless” Cleavage Cocktail (Reagent B)[1]

In place of highly odorous ethane dithiol and thioanisole, triisopropylsilane is used in Reagent B to scavenge cationic species.  Reagent B is especially useful when the resin-product contains trityl-based protecting groups.  Reagent B will not prevent oxidation of methionine residues during cleavage. 

Composition of Reagent B

            trifluoroacetic acid (88% v/v)

            phenol (5% v/v)

            water (5% v/v)

            triisopropylsilane (2% v/v)

Use immediately after preparation

Cleavage with Reagent B

  1. If necessary, remove any Fmoc-protecting groups using the standard Fmoc deprotection procedure.
  2. Wash the resin with dichloromethane.
  3. Suspend the resin in Reagent B (100 mL/mg of peptide resin)
  4. Stir the mixture for 1 hour at room temperature.
  5. Filter and wash the resin with a little additional Reagent B.
  6. Cool the combined filtrates to 4°C and add cold methyl t-butyl ether (3 times the volume of the combined filtrates) to precipitate the crude peptide.

Cleavage Cocktail for Methionine Containing Peptides (Reagent H)

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.[2]  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.

Composition of Reagent H

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)

ammonium iodide (1.5% w/w)

Peptide Cleavage (Without Disulfide Formation)

  1. If necessary, remove Fmoc-protecting groups using the standard Fmoc removal procedure.
  2. Suspend the peptide resin in the cleavage cocktail (30 mL/g resin).
  3. Allow the mixture to stand three hours at room temperature under inert gas.
  4. Filter and wash the resin with trifluoroacetic acid.
  5. 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.

Cleavage Cocktail for Peptides Containing Cys, Met, Trp and Tyr Residues (Reagent K)[3]

This cleavage cocktail is commonly used to cleave peptides containing combinations of sensitive residues such as cysteine, methionine, tryptophan and tyrosine.  Since this reagent is suitable to most of the sensitive amino acid residues, it is often used as a general cleavage reagent.  This reagent is recommended when peptides prepared on PAL or BAL resins contain tryptophan residues.

Composition of Reagent K

trifluoroacetic acid (82.5% v/v)

phenol (5% v/v)

water (5% v/v)

thioanisole (5% v/v)

1,2-ethanedithiol (2.5% v/v)

Cleavage and Deprotection with Reagent K

  1. If necessary, remove and Fmoc protecting groups using the general Fmoc removal procedure.
  2. Suspend the resin in Reagent K (10 mL to 40 mL per gram of peptide resin)
  3. Stir at room temperature for 1 to 2.5 hours.  Peptides containing multiple arginine residues may require longer for complete deprotection.
  4. Filter and rinse the resin with trifluoroacetic acid.
  5. Concentrate the combined filtrates to a syrup under reduced pressure.
  6. Dissolve the syrup in a minimum volume of trifluoroacetic acid.
  7. Add the solution to methyl t-butyl ether (approximately 300:1 v/v methyl t-butyl ether: peptide solution).
  8. Filter or centrifuge to collect the crude peptide.

Low Odor Cleavage Cocktail (Reagent L)[4]

In this cleavage cocktail, dithiothreitol (DTT) replaces the pungent scavengers ethanedithiol (EDT) and thioanisole.  Additionally, unlike EDT, DTT does not react readily with the benzophenone moiety of 4-benzoylphenylalanine (Bpa), making Reagent L a preferred cleavage cocktail for Bpa containing peptides.

Composition of Reagent L

trifluoroacetic acid (TFA, 88% v/v)

triisopropylsilane (TIS, 2% v/v)

dithiothreitol (5% w/v)

water (5% w/w)

Use immediately after preparation

Cleavage and Deprotection with Reagent L

  1. If necessary, remove Fmoc-protecting groups using the standard Fmoc removal procedure.
  2. Suspend the resin in Reagent L (5 ml to 10 mL/g resin).
  3. Allow the mixture to stand at room temperature for 90 minutes.
  4. Filter the resin.
  5. Wash the resin with TFA (5 mL to 10 mL/g resin).
  6. Combine the filtrates and add cold ether to precipitate the crude peptide.

Cleavage Cocktail for Peptides Containing Arg Residues (Reagent R)[5]

Reagent R is especially suited for cleaving and deprotecting peptides that contain arginine residues protected with sulfonyl protecting groups.  This reagent also is recommended for tryptophan-containing peptides prepared on PAL or BAL resin, for it minimizes reattachment of the peptide to the linker at the tryptophan residue.

Composition of Reagent R

            trifluoroacetic acid (90% v/v)

            thioanisole (5% v/v)

            1,2-ethanedithiol (3% v/v)

            anisole (2% v/v)

 Use immediately after preparation

Cleavage and Deprotection with Reagent R

  1. If necessary, remove Fmoc-protecting groups using the standard Fmoc removal procedure.
  2. Suspend the resin in Reagent R (10 mL/g of peptide resin).
  3. Allow the mixture to stand at room temperature for 2 hours.
  4. Filter the resin.
  5. Wash the resin with Reagent R (2 x 5 mL /g resin).
  6. Combine the filtrates and add cold ether to precipitate the crude peptide.

 


[1] Solé, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403.

[2] Huang, H.; Rabenstein, D. L. J. Peptide Res. 1999, 53, 548-553.

[3] King, D.; Fields, C. G.; Fields, G. B. Int. J. Peptide Protein Res. 1990, 36, 255-266.

[4] Bonner, A. G.; Udell, L. M.; Creasey, W. A.; Duly, S. R.; Laursen, R. A. J. Peptide Res.  2001, 57, 48-58.

[5] Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R.I.; Hudson, D.; Barany, G. J.Org.Chem. 1990, 55, 3730-3743.