I would add HOBt or HOAt (same concentration as HATU) to the activation
mixture, and make the deprotection mixture 0.1 M in HOBt. You have 3 Asps
in the first peptide, including a DG - very prone to aspartimide
formation. You also have a DP in that peptide, so I'd be careful during
cleavage and afterward, not to expose it to aqueous acid for a long time.
You may, however, still get a little cleavage at the DP bond, just to
complicate your purification situation. As I wrote to Lisa Bibbs, I've
used ammonium bicarbonate - 10 mM - for phosphopeptide purifications, but
ammonium acetate and triethylammonium acetate have also been suggested.
Lastly, I would extend the coupling time for the pTyr to 2 hours, and
double couple it.
Hope this helps.
Angela C. Murphy
On Thu, 31 Aug 2000, neil brew wrote:
>
> Dear All
> I have the following two Phospho-peptides to make;
>
> V S S D G H E Y* I Y* V D P M Q L P Y D S T
>
> K E P E E R P T F E Y* L Q A F L E D Y
>
> Y* = Tyr(PO(OH,OBzl))-OH
>
> Both will be made on a pioneer Fmoc synthesiser. I was going to use
> HATU .49M, DIPEA 1M activators, 20% piperidine deblock. 1hr couple for all
> A.A's, with Fmoc monitoring and auto extend deblock and couple.
> Anybody have anything to add/suggest that would aid in these
> syntheses?
>
*******************************************
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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