Peptide workup after Fmoc synthesis
Andrew, Tel. +39-6-91093434
WALLACE at IRBM.IT
Fri Sep 9 04:12:28 EST 1994
In reply to your message:
>Does anyone know the most recent protocol for extraction of peptides after
>Fmoc synthesis on a PEG-PS resin? We would like to know the cocktail
>composition. We are currently using a 9050 Millipore Peptide synthesizer.
The cocktail you need depends on the linker you have used to attach your
peptide and the side-chain protecting groups, but for the common mild acid-
labile linkers and groups we routinely use the Reagent B cocktail developed by
Barany.(reference: Sole, N.A. & Barany, G. (1992) J. Org. Chem. 57, 5399-5403.)
The composition is: Trifluoroacetic acid 88%
The nice feature of this is that it doesn't contain thiols so is relatively
odourless and more pleasant to handle than other mixtures. We have synthesised
many dozens of peptides with this reagent without side-chain deprotection
problems or other difficulties attributable to its use. After cleavage we
precipitate the peptide with cold (-80 C) tert-butyl methyl ether, wash with
the ether, recover the ppt. and dry it under vacuum then dissolve in a
volatile aqueous buffer (5% acetic acid for basic peptides, 1% ammonium
bicarbonate for acidic peptides) adding some organic solvent e.g. acetonitrile
if necessary, then put it on a suitable size-exclusion column (e.g. Fractogel
HW-50) to isolate the peptide from small MW species. After this we lyophilise
the peptide-contining fractions an purify further by HPLC if necessary. If
the peptide is less than 10 aa long and the purity is good then we often
lyophilise directly without gel filtration.
Hope this helps,
Andrew Wallace, IRBM P. Angeletti, Pomezia, Italia.
Email: wallace at irbm.it
"IRBM - The home of the Minibody"
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