Dear Joe
I can't answer your question directly but I can offer some comments.
I have used Lys-Wang resin (ACT) for MPS synthesis for many years and have
never really experienced any problems. I have also used Calbiochem Wang
resin but not lys, and this was also fine. Asp-X is prone to aspartimide
but not all X does it under standard conditions of synthesis in Fmoc
chemistry. From my experience DG is worst closely followed by DN and DD.
these can show unacceptable levels of aspartimide formation.
The rest, show small amounts of aspartimide if any. I have seen on the odd
occasion 20-30% aspartimide with DR and DK but rarely. It was in a peptide
that went xxxxxxDRDKxxxxx. It is very sequence sequence dependent.
These observations are based on about 4000 single column syntheses with
PEG resin.
You can reduce aspartimide formation by deblocking with HOBt/piperidine.
This reduces it to acceptable levels except sometimes with DG close to the
C-term followed by a long sequence. The worst case I have seen is 90%
aspartimide down to 30%. Peptide was then purifiable by HPLC.
I am happy to let you try some of my Wang resin
Dr Graham Bloomberg
Peptide Synthesis Facility
Dept. Biochemistry
Medical School
University of Bristol
Bristol BS8 1TD
UK
Tel. +44(0)1179-293205
On Tue, 23 May 2000, Joe Gray wrote:
> Dear ABRFers
>
> I think I've stumbled upon something rather interesting that
> affects the quality of crude peptides, and I'd like to hear
> comments / input from other users of this group.
>
> I normally run standard peptide synthesis on a 431A using the
> stated PE/ABI HBTU ssFmoc procedures (with capping).
>
> My problem first came to light last August when I purchased
> (among other things) a batch of Fmoc-Lys(Boc)-Wang resin from
> NovaBiochem (04-12-2057), lot number A23012, in order to generate
> a series of overlapping (by 10) 20er peptides.
>
> All went well until I came to a sequence with a C-terminal Lys.
> Suddenly the crude yield and quality deteriorated rapidly.
> Repeated attempts to make this peptide all failed, so I moved on.
>
> Earlier this year I while doing the same thing with another
> protein, I came upon another C-terminal Lys peptide, and again
> the same thing happened! I began to suspect the resin.
>
> Last week while working on yet another protein, I synthesized 3
> peptides, again part of a study of overlapping 20ers. The
> 3 C-terminal residues of these peptides were: Asn (ACT), Lys (Nova)
> and Leu (Nova) respectively. The crude yields (0.05 mmol) were
> 92 mg, 14mg and 105 mg respectively!! The HPLC purities were ca.
> 85%, no major peak, and ca. 85% respectively. All peptides were
> made with the same reagents on sequential days.
>
> Why does the peptide with C-terminal Lys fail so badly? I asked
> NovaBiochem UK and they said that the batch has now sold out, but
> that they have no reported problems.
>
> I've been looking back at my synthesis records for the last 18
> months, and this 'junking' of sequences as it turns out, has
> happened with previous Fmoc-Lys(Boc)Wang resin batches.
>
> Furthermore, I think I've spotted a pattern, which I'd like users
> of this group to help me confirm. Here are my observations based
> on what I've noticed over the last 200 or so syntheses :
>
> By coincidence / luck I've noticed that all the peptides
> involved (I've made a couple without lys) are aspartimide rich,
> containing Asp - X sequences close to the C-terminal.
>
> - The presence of an Asp (but not Asn) residue in the C-terminal
> decapeptide has a markedly deleterious effect.
>
> - If the Asp is within 5 of the C-terminal Lys, the peptides are
> invariably 'junk' and of low yield.
>
> - Within 6 - 10 and a major peak can usually be identified.
>
> - Peptides containing Lys in the penultimate position with Asp
> nearby are not affected.
>
> - The reverse does not hold true - Peptides with C-terminal Asp
> and a nearby Lys are OK.
>
> The one recorded synthesis I do have with C-terminal Lys & no
> Asp, produced a very pure crude, but with only ca. 50% expected
> crude yield ??
>
> Has anyone else been using this particular batch for synthesis?
>
> Does anyone out there have a explanation for this phenomenon?
>
> Is this just a Wang resin thing? Can anyone else confirm this
> phenomenon for other supports?
>
> Is there a database of known difficult / problem syntheses to
> compare against?
>
> Apologies for such a long e-mail, but it's not often a lurker has
> something interesting to report!
>
> Regards, JG.
>
>
>
>
> **********************************
> Dr J. Gray
> University of Newcastle Upon Tyne
> Molecular Biology Unit
> 3rd Floor Cookson Building
> Framlington Place
> Newcastle NE2 4HH
> United Kingdom.
>
> Tel. / FAX : +44 (0) 191 222 8612
> Email : joe.gray@newcastle.ac.uk
> **********************************
>
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