Re: pep syn/p-ser

Angela c. Murphy (acmurphy@helix.nih.gov)
Fri, 16 Jan 1998 20:42:45 -0500 (EST)

I've used the Novabiochem monobenzyl protected Fmoc-pSer and had
reasonably good results making a 16-mer with the pSer 6 residues in
from the N-terminus. This peptide has Trp in it as well as Met, and I
really didn't want to take a chance with post synthetic phosphitylation
and oxidation. Following a hint in the AnaSpec catalog about coupling
Fmoc-pTyr, I lengthened the coupling time (HBTU/HOBt mediated) on my ABI
430A synthesizer to 1 hour, and I double coupled it (expensive! when
you're using a 4-fold molar excess [1 mmole] each time). Next time I'll
do a quantitative ninhydrin test after the first coupling, and I may also
lengthen the coupling time further, as Gautam suggests. Also, since my
Peptide Companion indicated that this area of the peptide had a bunch of
"difficult" couplings, I used 2% DBU/2% Pip in NMP for deprotection up to
the residue immediately preceding pSer. After I'd coupled the pSer, I
switched to plain piperidine, because I didn't know what the DBU/Pip mix
would do to the phosphate. Wish I did, because I worry about coupling
*to* the pSer. It seemed to go all right in this synthesis, but some
folks have had trouble coupling to phosphoamino acids. Anyone have any
suggestions?
Angela C. Murphy

On Fri, 16 Jan 1998, Gautam Sarath wrote:
>
> Dear Jane: You need to have protected p-ser, unless this is the terminal
> residue, in which case use the boc derivative. Ther will be beta
> elimination when using base and losses and overall yields will be poor or
> non-existant. Anaspec also sells the fmoc-p-ser-benzyl derivatives and
> their material is good. I have used protected p-ser from various suppliers
> and novabiochem and Anaspec have worked very well. Also use a four-fold
> excess with HATU coupling for about 1 to 2 hours. This is really slick on
> the new Pioneer system, but should work well on all other automated
> systems. We have had much poorer yields when attempting to make
> phosphopeptides manually. good luck, gautam
>
> Gautam Sarath
> N-226, Beadle center
> Protein Core Facility
> Department of Biochemistry
> University of Nebraska-Lincoln
> Lincoln, NE 68588-0664
> Phone: 402-472-2928
> FAX: 402-472-7842
>
>

*******************************************
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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