I have synthesized few peptides containing cysteine, but in this case the peptide have sulfur in oxidized form (sulfoxide not disulfide).I am using solid phase peptide synthesizer.
Which scavenger did you use for the cleavage? Try using thioanisole/1,2-ethanedithiol. If this does not help you can reduce the sulfoxide using TMSBr in TFA/EDT.
According to Jan-Patrick u can use Reagent K. It's a solution of : TFA/thioanisole/water/phenol/EDT (82.5:5:5:5:2.5 v/v).
I recommend "reagent B" Trifluoroacetic acid (TFA)/phenol/water/triisopropylsilane (TIPS) (at a volume to volume ratio of 88/5/5/2) for Cys containing peptides. I found that increasing the amount of TIPS (at the expense of TFA, ie., volume ratio of up to 80/5/5/10) helped keep Cys residues in their reduced form. One added benefit is that this reagent doesn't have a pungent sulfurous odor. We used a high purity grade of TFA (suitable for peptide sequencing). We also did the cleavage reactions in the dark or under low light conditions.
Additionally to Bruce suggestion you can do cleavage in Argon environment, to cut oxygen out.
While all of the above may work, simple soft reducing agents such as 2-mercaptoehanol or dithiothreitol (DTT) always worked for me.
I usually use TFA/TA/EDT (90/7/3; v/v/v) for the cleavage of Cys-, Met- or Trp-containing peptides.
In Fmoc-based solid phase peptide synthesis, the thiol function of Cys residues is protected with trityl group. Peptide is cleaved from the resin using trifluoroacetic acid/H20/ethanedithiol/ phenol/thioanisole (80/5/2.5/7.5/5 by vol). Peptide solution is bubbled with argon prior to HPLC purification of the linear (unfolded) peptide.
As Marco said, we have used Reagent K many times and it works great. Although the smell is terrible. Make sure you have a bottle with bleach to drop all the waste and use double gloves.
the simple answer is that you have to use a 'sacrificial scavenger' in your cleavage cocktail mixture. Compounds like DTT, Thioanisole or even TCEP would be effective.
Hope this helps.
Even we use Reagent K many times and it works great for most of the free thiol peptides.
if your sequence contains have more than 6 free thiols then may be have to optimize reagent K with more concentration of thiol scavenger.
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