Suppression of Deletion Sequences

During reaction of these blocking agents an acidic function (3-nitro-benzoyl-2-carbox-ylate or benzoyl-2-sulfonate) from the intramolecular anhydride bond is Uberated in its salt form. All blocked defective sequences therefore additionally are marked by this acidic moiety. They can be separated from the end product by ion exchange chromatography, since the acidity of the marker function is much more pronounced than any peptide car-boxylate on terminal or side [168] positions (Fig. 51). Though acetylation likewise suppresses the formation of failure sequences, it effects no facilitated separability of synthetic peptide by-products. 

Today we routinely use the 3-nitrophthalic anhydride blocker It is applicated in a single dose of a ten- to twenty fold excess on estimated amounts of remaining amino functions and added as a 0.1 niolar solution in pure pyridine with ten minutes reaction time at the end of each peptide synthesis stage, in which all chemical operations are monitored by photometric control and forced to approach completion. We have no indications to date that the acidically marked, blocked sequences on polymer cause undesired side reactions in subsequent stages of the synthesis, particularly in further peptide coupUngs, probably because of the acidity of the blocker functions (pK 2), which in their anionic salt form possess only a very diminished nucleophilicity. 

Under the acidic conditions of the Edman degradation (trifluoroacetic acid/40 °C/ 

45 minutes), the blocker was found to be completely cleavable from peptides on polymer containing sequences terminated by 3-nitro-2-carboxy-benzoyl. Therefore, we are in the position to determine directly on polymer the contamination of the target sequence with defective ones, though the latter originally were blocked at their N-terminus. This is done by quantitatively exploitable Edman degradation in gel phase, a technique which was already mentioned in Sect. 3.3.6. 

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