I recently synthesized a 60 mer containing several Asn-Gly,
Asp-Gly, and Asp-Asn bonds. I used
Fmoc-Asp(O-1-adamantyl)-OH (Novabiochem #04-12-1076) at all
Asp-Asn positions and Fmoc-(FmocHmb)Gly-OH (Novabiochem
#04-12-1135) at any Asp-Gly positions. I found that
Fmoc-Asn(Trt)-OH worked fine in all Asn positions. Peptide
was synthesized on a Milligen 9050 using HATU/DIPEA
chemistry. Mass analysis indicated no aspartamide
formation from the single peak observed from RP-HPLC.
HOBt is inexpensive so go ahead and add this to your 20%
PIP as well.
Good luck,
Jay Gambee
On Wed, 16 Dec 1998 03:05:49 +0000 (GMT)
mbgbb@seqnet.dl.ac.uk wrote:
>
> Dear Dan
>
> 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
>
----------------------
Jay Gambee
JEG@shcc.org