N alpha-trifluoroacetylation of N-terminal hydroxyamino acids: a new side reaction in peptide synthesis.

In the synthesis of the double-chain bis-cystinyl hinge fragment 225-232/225'-232' of the human IgG1, which contains two N-terminal threonine residues, the final acidolytic deprotection step, with 99% aqueous trifluoroacetic acid, was accompanied by formation of remarkable amounts of an unknown side product. This has been identified as the N alpha-mono-trifluoroacetylated product; however, even bis-trifluoroacetylation was found to occur by prolonged exposure of the parallel dimer to the reaction medium. Similarly, the model compounds H-Thr(tBu)-Phe-OH, [Boc-Thr(tBu)-Cys-OH]2 and Boc-Thr(tBu)-Cys(StBu)-Ala-OH were acylated by treatment with trifluoroacetic acid at rates that suggest a rather pronounced sequence dependency. Since, on the other hand, the model compound [Boc-Ala-Cys-OH]2 was not trifluoroacetylated at all under identical conditions, the reaction has to proceed prevalently via intermediate formation of the trifluoroacetyl ester of N-terminal hydroxyamino acids, followed by O-->N shift according to the hydroxyoxazolidine mechanism. The experimental data indicate also that under favored conditions aminolysis of the trifluoroacetyl ester via an inter- or intramolecular pathway may contribute to the overall reaction rate.