Message-Id: <l03010d01af14baa3fe45@[131.111.47.52]>
In-Reply-To: <9701281541.ZM6275@prion.ucsf.edu>
Date: Wed, 29 Jan 1997 08:48:45 +0000
From: Len Packman <lcp2@mole.bio.cam.ac.uk>
Subject: Re: Asp dehydration
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Haydn,
Asp-Gly and Asp-Asn sequences are the most prone to dehydration in
synthetic peptide sequences. There is nothing you can do to the peptide you
already have except purify out the target material....BUT this assumes that
you have kept the peptide in mild acid conditions since cleavage. If the
peptide has been into basic conditions, then the aspartimide can open up to
give you a peptide of the correct mass but containing a mixture of alpha
and beta peptide, D and L forms. The best way to avoid aspartimnide is to
inspect a sequence for likely problem areas and then take steps to prevent
aspartimide formation during the synthesis; your cleavage cocktail will
have had little effect on aspartimide formation. The dehydration occurs
generally a few percent per deprotection cycle in Fmoc chemistry, so
sequences nearer to the C-terminus are more prone to being a problem as
they experience the largest number of deprotection cycles with piperidine.
Piperidine can add into the imide to form a piperidide product too. The
literature gives a number of ways around aspartimide formation, but my
choice is Hmb protection of the Gly residue in your peptide. Fmoc2-Gly-OH
can be purchased from Novabiochem. It should give you close to 100%
prevention of aspartimide.
Len
TI: N-2 HYDROXY-4-METHOXYBENZYL (HMB) BACKBONE PROTECTION STRATEGY
PREVENTS DOUBLE ASPARTIMIDE FORMATION IN A DIFFICULT PEPTIDE SEQUENCE
AU: PACKMAN_LC
JN: TETRAHEDRON LETTERS, 1995, Vol.36, No.41, pp.7523-7526
TI: THE N-(2-HYDROXYBENZYL) PROTECTING GROUP FOR AMIDE BOND PROTECTION IN
SOLID-PHASE PEPTIDE-SYNTHESIS
AU: JOHNSON_T, QUIBELL_M
JN: TETRAHEDRON LETTERS, 1994, Vol.35, No.3, pp.463-466
TI: A REVERSIBLE PROTECTING GROUP FOR THE AMIDE BOND IN PEPTIDES - USE IN
THE SYNTHESIS OF DIFFICULT SEQUENCES
AU: JOHNSON_T, QUIBELL_M, OWEN_D, SHEPPARD_RC
JN: JOURNAL OF THE CHEMICAL SOCIETY-CHEMICAL COMMUNICATIONS, 1993, No.4,
pp.369-372
TI: SUPPRESSION OF PIPERIDINE-MEDIATED SIDE PRODUCT FORMATION FOR
ASP(OBU(T))-CONTAINING PEPTIDES BY THE USE OF N-(2-HYDROXY-4-
METHOXYBENZYL) (HMB) BACKBONE AMIDE PROTECTION
AU: QUIBELL_M, OWEN_D, PACKMAN_LC, JOHNSON_T
JN: JOURNAL OF THE CHEMICAL SOCIETY-CHEMICAL COMMUNICATIONS, 1994, No.20,
pp.2343-2344
TI: REVERSIBLE MODIFICATION OF THE ACID-LABILE 2-HYDROXY-4-METHOXYBENZYL
(HMB) AMIDE PROTECTING GROUP - A SIMPLE SCHEME YIELDING BACKBONE
SUBSTITUTED FREE PEPTIDES
AU: QUIBELL_M, TURNELL_WG, JOHNSON_T
JN: TETRAHEDRON LETTERS, 1994, Vol.35, No.14, pp.2237-2238
>--------------------------------------------------------------------------
>We have recently synthesized the following sequence using Fastmoc chemistry on
>an ABI433A - ESQAYYDGRRSS. Our problem is that the final product contains a
>very high proportion (~45%) of the dehydrated Asp impurity. We are using EDT,
>thioanisole, water and TFA (1:2:2:35) for cleaving the peptide from the resin.
>Any suggestions as to how we can minimize this problem, which seems to be
>sequence related since other Asp-containing peptides we have made do not
>contain the impurity, would be greatly appreciated.
>
>Many thanks
>
>Haydn Ball, PhD
>Dept of Neurology,
>UCSF
>San Francisco
>CA 94143-0518
>
>hlb@prion.ucsf.edu
>Tel (415) 476 6485
******************************************************
Dr Len C. Packman
Assistant Director of Research
Protein and Nucleic Acid Chemistry Facility
Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK
Tel: +44 (1223) 333639 (including answerphone)
FAX: +44 (1223) 333345
e-mail: lcp2@mole.bio.cam.ac.uk
Visit my WWW page at http://www.bio.cam.ac.uk/proj/adr/PNAC/pnac.html