It depends on whether you are using Boc or Fmoc chemistry. If you are
using Boc chemistry, the low/high HF cleavage method of Jimmy Tam (J.P Tam
and others, various references in JACS, PNAS, Int. J. Pept. Prot. Res.,
etc.) may work for you. If you are using Fmoc chemistry, cleavage with
Reagent K - 82.5% TFA/5% thioanisole/5% water/5% phenol/2.5% EDT or DTT
may go a long way toward reducing your methionine without harming your Trp
or Cys. You can modify Reagent K a bit and cut the phenol to 2.5% and add
2.5% p-thiocresol. After cleavage if you still have a lot of Met(O), then
N-methyl mercaptoacetamide in HOAc at 37 degrees C. is the standard
reduction method. If you really have Met sulfone, it may be next to
impossible to reduce it, and your only recourse may be to purify it away
from the correct sequence after reduction. I hope you don't have much
Met sulfone.
Hope this helps.
Angela C. Murphy
On Thu, 11 May 2000, Auspepstaff wrote:
> Dear abrf
> I am currently manually synthesising a 62AA peptide which
> has 4 methionines and I am 50 cycles into this beast. I have checked by
> cleaving a small resin sample to see how it is going at 10 cycle intervals.
> I have seen a gradual increase in Met[O] until it is now the major product
> ( plus diMet{o})in the crude mass spec at 50 cycles. My question is does
> anyone have a bulletproof method that will convert the Met[O] back to Met. I
> have found in the past that if the oxidation occurs readily in a peptide,
> then DTT at 37C will convert the Met[O] back to Met but other times it is
> quite intractable . The peptide has also got Cys and Trp which make it
> sensitive to some of the methods I have seen in the literature. Please does
> anyone have any suggestions.
>
> Denis Scanlon
>
> AUSPEP PTY.LTD.
> P.O.Box 806 Parkville
> Vic. 3052 AUSTRALIA
> Phn: + 613 9328 1211
> Fax: +613 9326 8810
>
*******************************************
Angela C. Murphy, Chemist
Lab. of Cell Biology, NHLBI, NIH
3 Center Drive, MSC 0301, Rm. B1-22
9000 Rockville Pike
Bethesda, MD 20892-0301 USA
tel.: (301) 496-2324
fax: (301) 402-1519
email: acmurphy@helix.nih.gov
*******************************************
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