I have been attempting to couple BBA(p-benzoyl benzoic acid) to the
N-terminal of a resin bound side-chain protected peptide. The BBA was
activated with HBTU/HOBt in DIEA/NMP and reacted overnight. Ninhydrin
monitoring indicated the primary amine had been covered. Cleavage
conditions for the peptide were 95% TFA/2.5%water and 2.5%DTT. After HPLC
purification FAB-MS analysis showed a parent ion mass with a net increase
of 43 instead of the expected 209 for the BB moiety. The peptide should
have a M+H mass of 1822, the BB-peptide should be 209 higher i.e. 2031 but
MS identifies a major portion of the synthesis at mass 1864. This net mass
difference has been observed on several BB containing peptides. The
amino-acid analysis of the 1864 molecular weight material appears normal
and sequencing indicated a blocked N-terminal. My question is how stable
is the BB group to TFA cleavage? I know it can be alkylated by thiol
scavengers but DTT is supposed to be less reactive. Has anyone with
experience using BB incorporation for photocoupling seen this problem? It
looks like an acetylation but any suggestions of how this could occur would
be appreciated.
Thanks in advance.
Darryl Hardie