In the early days of Fmoc chemistry M. Bodanszky demonstrated that
proline ester deprotects Fmoc group in FmocAaONp at a detectable rate in
the course of acylation, which gives rise to some FmProOR
(N-fluorenylmethyl derivative, fulvene adduct).
FmocAaONp + H-ProOR --->FmocAaProOR + FmProOR + ....
Somewhat later Carpino introduced polymer-bound piperazine as a
Fmoc-cleaving reagent of choice (for solution synthesis), however I
can't recall any report dealing with this side reaction in SPPS, except
for one observation I noted while browsing ABRF archive one day. The
problem was in exclusive formation of FmProO-resin instead of expected
incorporation of second amino acid.
Since every peptide is unique and unpredictable (:-)), one could imagine
that at the Pro/Val-steps your peptides adopt a conformation with
partially buried N-terminal residue. Why not? Clearly, this will slow
down coupling reaction, but may have little or no effect on
Fmoc-deprotection and fulvene scavenging, which both are less sensitive
to steric hindrance at nitrogen. All this may result in amplification of
otherwise insignificant side reaction and high yield of deletion
products:
FmocLys(Boc)OX + H-ProVal-...-resin ------>
--->FmProVal-...-resin + FmocLys(Boc)ProVal-...-resin --->
--->H-ProVal-...-resin + H-Lys(Boc)ProVal-...-resin ->->->
My suggestions:
1. Hydrolyze peptide resins and analyze for Lys content.
2. Using BocLys(Boc) instead of Fmoc-derivative may fix your particular
problem.
3. Do not take conductivity tracings too seriously and check two last
couplings with ninhydrine(V)/isatine(P) tests.
4. If difficult couplings will be revealed, use appropriate protocol
enhancements (aggregation-related). Switching to Boc-chemistry + "in
situ neutralization" is recommended.
Good luck!
Igor
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Igor L. Rodionov
Laboratory of Peptide Chemistry
Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Science Avenue 6
Pushchino, Moscow Region
142292, Russian Federation
E-mail: <rodionov@fibkh.serpukhov.su>
------------------------------------------------
Original message:
>We have attempted syntheses of two peptides 19-mer and 9-mer with
identical N-term. residues K-P-V-... Both were synthesized with F-moc
chemistry. 19-mer on 432A 9-mer 433A. Conductivity tracings for both of
them do not indicate any synthetic problems. However, the first (19-mer)
synthesis yielded almost 100% HPLC pure product without N-terminal Lys.
The synthesis of the 9-mer gave 17% of the expected product, the main
product did not have Lys and there was 15% of peptide without Lys and
Pro.
It seems that Lys was coupled and then cleaved during deprotection or
workup.
Have anybody encounter similar problems. Any suggestions how to solve
the problem.
Thanks in advance, Krystyna>