"'Jennifer Woodman'"@
<Jennifer_Woodman@dfci.harvard.edu>
Subject: RE: Peptides
Date: Wed, 23 Feb 2000 13:07:12 -0600
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Jen,
I would agree with others that a good approach would be to biotinylate the
N-terminus of the peptide on the
resin, then to cleave and deprotect the peptide. We have had good success
in using Ekathiox on a 8-mer which had both C- and N-terminal Cys. Other on
resin methods (Tl(III) trifluroacetate and iodine) were not effective. The
Ekathiox reagent is expensive, but could be tested on a small scale first to
be sure it works.
The protocol we used, which resulted in complete oxidation to the disulfide
for the peptide mentioned above in 1.5 hours, is listed below:
-Use peptide after work up and lyophilization at conc. of 1 mM (in 2:3
acetonitrile:0.5% aq. acetic acid)
-Add Ekathiox resin so that have 10 eq per free thiol (may get by with less
if willing to wait longer)
-Agitate to suspend resin for 1-3 hours (depends on sequence)
-Filter resin off with glass wool
-Test for remaining free thiols (Ellman's or other method)
-If rxn complete proceed to A, if not repeat steps 2-5 above (have never had
to repeat)
A) Purify peptide as usual (crude was quite clean in above case)
Best of luck.
Randy Wilhelm
Sr. Research Chemist
Mallinckrodt, Inc
Discovery Research Peptide Team
Phone (314)654-3965
Fax (314)654-8911
rrwilhe@mkg.com
> ----------
> From: Jennifer Woodman[SMTP:Jennifer_Woodman@dfci.harvard.edu]
> Reply To: Jennifer Woodman
> Sent: Tuesday, February 22, 2000 11:45 AM
> To: Recipients of ABRF List
> Subject: Peptides
>
> Hello,
> I was wondering if anyone out there could offer some advice on cyclic
> peptides. We are currently working on a project that involves making a
> cyclic peptide in which either the C or N terminus would be biotinylated.
> My concerns are:
>
> I believe I will need the free amine group to cyclize the peptide and
> therefore will not have a free amine group to biotinlate.
>
> In addition, both the C and N terminus have a Cysteine. We were thinking
> that we might be able to cyclize the peptide by forming a disulfide bond
> between the two ends. That way, we might be able to use the free amine to
> biotinylate. If this is a possibility, how do we selectively have the
> single peptide form a disulfide bond between its two ends rather than
> having a chain formation.
>
> I thank you for any advice,
>
> Jen
>
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