>Dear Joe
I found your analysis very interesting. One thing that I am
wondering is whether there are also any Prolines in the C-terminal region.
Pro-Lys is notorious for diketopiperazine formation
Best regards
Denis Scanlon
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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