Aspartimide formation is supressed except in the very worst cases of
asp-gly (where it is substantially reduced) by adding 0.1M HOBt to the
deprotection solution of 20% piperidine in DMF. I use this routinely
and never now see aspartimide more than 10% in any peptide. All of the
asp-asn containing peptides I have made have been fine. You do lose the
deprotection peak if you have UV monitoring but it is worth it !!!
Dear Colleagues,
I am planning the synthesis of a 52 amino acid polypeptide in which
residues 40 and 41 are Asp and Asn, respectively. Thanks to previous
discussions on this bulletin board, I am aware that with standard Fmoc
chemistry, an Asp preceding a Gly or Asn residue is particularly prone to
dehydration to yield an aspartimide, which can react with piperidine to
form a piperidide or hydrolyze to give, at least partly, the peptide having
a peptide bond at the beta, rather than the alpha, carboxyl group of Asp.
I also know that an excellent way to prevent this is to use a
2-hydroxy-4-methoxybenzyl protecting group on the alpha amino group of the
Asn residue. However, I have not found any company that sells the
necessary Fmoc(FmocHmb)Asn(Trt)OH. Is this compound commercially available
(and if so, what is the source), or does it need to be synthesized? If it
needs to be synthesized, what is a good reference on how to do so?
Alternatively, I gather that aspartimide formation can be suppressed by
including 0.1 M HOBt in the piperidine solution used for Fmoc deprotection
at the expense of being able to see the Fmoc deprotection peaks. I will be
doing the synthesis using a Milligen Biosearch 9050 peptide synthesizer,
and would be interested in knowing if anyone has used the HOBt method for
avoiding aspartimide formation with this synthesizer. Any general comments
about the wisdom of using HOBt in the deprotection solution vs. Hmb
protection are also welcome.
Finally, a general question. Most of the discussion of problems
with aspartimide formation has dealt with Asp residues in peptides. As a
result my experience with protein sequencing, I have read that the Asn-Gly
sequence is uniquely sensitive to cleavage by hydroxylamine under basic
conditions, a reaction that is thought to proceed via aspartimide
formation. Yet I have not read that Asn-Gly sequences are nearly as
sensitive to aspartimide formation during peptide synthesis. Is this true,
and if so, why are not Asn-Gly sequences more prone to aspartimide
formation during synthesis?
Thanks in advance for taking time to reply to these questions.
Sincerely,
Dan Brune
Dept. of Chemistry & Biochemistry
Arizona State University
Tempe, AZ 85287-1604
Graham Bloomberg
Dept Biochemistry
Medical School
University of Bristol
Bristol BS8 1TD
g.b.bloomberg@bristol.ac.uk
+44(0)1179-293205