Have you checked the resins for completeness of loading? If any free ben=
zyl
alcohol remains exposed from the linker, typical HBTU/HOBt active esters
don't couple well, but do couple to form an ester linkage. I've seen thi=
s
phenomenon with resins I've loaded myself and reactions didn't go to
completion.=20
Either:
1.) End-cap your existing resin with a symmetric anhydride (Ac2O or bette=
r
yet Benzoic anhydride). The manufacturer should have, but may not have d=
one
this already.
2.) Try the amide, if the terminus charge isn't critical to your intended
experiments. HBTU/HOBt will react ~100% leaving no terminus ambiguity.
-----Original Message-----
From: Marcus Macht [mailto:Marcus.Macht@uni-koeln.de]
Sent: Thursday, May 06, 1999 5:03 AM
To: Recipients of ABRF List
Subject: PepSyn: Degradation during cleavage?
Dear colleagues:=20
During the last time I observed a strange problem during several peptide
synthesis. Amongst others, I tried to synthesize the following peptide
sequences:
A H2N-MARGSVSDEEMMELREAF-COOH
B H2N-RLRPGGKKK-COOH
The conductivity monitoring on our 433A looked fine in both cases, but
after cleavage from the Wang-resin, both peptides showed a missing
C-terminus. In the case of peptide A, the -F-product was the most dominan=
t
signal in MALDI-MS (approximately 70-80%) with smaller amounts of -REAF,
-EAF and -AF as well. In the case of peptide B, -KKK and -GKKK were the
most prominent signals in MS. Both peptides showed nearly a quantitative
loss of the C-terminal AA. The cleavage reagent in both cases was 95% TFA=
,
2.5% TES and 2.5% water.=20
Since both peptides have been synthesized twice and the mass spectra of t=
he
cleaved peptides looked almost identical, I don=B4t think, that it is a
problem related to the synthesis but more likely related to the cleavage
procedure. For peptide B I tried a time course monitoring of the cleavage.
After 5 minutes I already observed a complete loss of the C-terminal K
while Pmc protection group is still present (most abundant peak is
-KKK+Pmc). After 30 minutes of cleavage, the Pmc is completely removed bu=
t
the most abundant peptide species remaining are -KKK and -GKKK. I have al=
so
tried different cleavage reagents like Reagent K, 90%TFA/5%TES/5%water,
80%TFA/7.5%TES/7.5%thioanisol/5%water, but the loss of amino acid remaine=
d
identical.
Is this a problem observed in other labs as well? At the moment I think,
that maybe some rective tBu-species from the protection groups (Boc, tBu,
OtBu) could be the reason. Do you think that this could be possible? If s=
o,
is anybody aware of a method to circumvent this problem (e.g. special
cleavage solutions)? The next thing I would try is to use a different
resin for the synthesis (like amide- or Sasrin-resin). Do you think that
this would make sense?
I appreciate your comments and suggestions!
Yours sincerely,
Marcus
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Dr. Marcus Macht
Centre for molecular medicine - Service laboratory
Joseph-Stelzmann-Str. 52
50931 Cologne, Germany
Tel.: +49 221 478-6995
Fax: +49 221 478-6977
e-mail: Marcus.Macht@uni-koeln.de
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