IMHO:
This is reminiscent of a diketopiperazine loss of the first 2 amino acids
during the FMOC removal from dipeptide. (IJPPR 35 191 (1990). This is
usually highly sequence dependant, reliably when Pro is resin anchored (I
don't recall nor haven't experienced it with Lys). If your recoveries are
low or you C-terminus ragged, this is likely.
Try:
Lys(Boc)-cl-trityl resin: The extra steric hindrance can suppress DKP loss.
This has worked for Proline acids for me.
Making the amide: If the amide is successful, perhaps this is the reason
for poor yields.
Monitor your deprotections: The lack of FMOC removal at cycle 3 indicates
no more peptide on your resin, i.e. DKP loss of the growing chain.
Couple AA 2 and 3 as dipeptide: Prepare the dipeptide and couple it to
resin. Once you have 3 aa synthesized, you're past the problem.
I have successfully used Novabiochem FMOC-Lys(BOC)-PEG resin. One would
think that DKP is sequence, not linker dependant, but I could be mistaken?
Comments?
David H. Singleton
Scientist
Pfizer Central Research
PO Box 8118-101
Eastern Point Road
Groton, CT 06340
-----Original Message-----
From: Joe Gray [mailto:Joe.Gray@newcastle.ac.uk]
Sent: Tuesday, May 23, 2000 12:35 PM
To: Recipients of ABRF List
Subject: Re: Peptide Synthesis - Crude quality
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
**********************************
This archive was generated by hypermail 2b29 : Mon Jun 12 2000 - 13:09:41 EDT