Dear Krystyna,
It looks like you have lost the chains at the dipeptide stage due to the
diketopiperazine formation, which is strongly favored by any N-alkyl
amino acid in the last two C-terminal positions (like Pro does).
Use trityl resin (any substitution) and couple the 3rd amino acid as
symmetrical anhydride or, at least, exclude acidic HOBt & HOAt at this
stage.
Try also to couple N-Me amino acids using the technique best suited for
these derivatives. As I remember, they are unusually prone to
racemization even when urethane-protected, with addition of HOBt being
ineffective, if not deleterious. Presence of tertiary amine salt was
shown to be particularly harmful and this is the case when you use TBTU
or the other uroniums. 15 years ago best results were achieved using
DCC-HOSu, mixed anhydrides with pivalic acid, Ph2P(O)Cl and BOP-Cl.
I hope that Leo Benoiton, the top expert in the chemistry of N-Me amino
acids, will comment on the current status of this problem.
Good luck.
Igor Rodionov
Laboratory of Peptide Chemistry
Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
6 Science Avenue, Pushchino, Moscow Region
142292, Russian Federation
rodionov@fibkh.serpukhov.su
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