Specificity and formation of unusual amino acids of an amide ligation strategy for unprotected peptides

An important step in the recently developed ligation strategy known as domain ligation strategy to link unprotected peptide segments without activation is the ring formation between the C‐terminal ester aldehyde and the N‐terminal amino acid bearing a 8‐thiol or 8‐hydroxide. A new method was developed to define the specificity of this reaction using a dye‐labeled alanyl ester aldehyde to react with libraries of 400 dipeptides which contained all dipeptide combinations of the 20 genetically coded amino acids. Three different ester aldehydes of the dye‐labeled alanine: α‐formylmethyl (FM), β‐formylethyl (FE), and β,β,β‐dimethyl and formylethyl esters (DFE), were examined. The DFE ester was overly hindered and reacted with N‐terminal Cys dipeptides (Cys‐X). Interestingly, it also reacted slowly with the sequences of X‐Gly where Gly was the second amino acid and the X‐Gly amide bond participated in the ring formation. Although the FE ester reacted similarly as the FM ester in the ring formation, the subsequent O,N‐acyl transfer was at least 30‐fold slower than those of the FM‐ester. The FM α‐formyl methyl ester was the most suitable ester and was reactive with dipeptides of six N‐terminal amino acids: Cys, Thr, Trp, Ser, His and Asn. The order and extent of their reactivity were highly dependent on pH, solvent and neighboring participation by the adjacent amino acid. In general, they could be divided into three categories. (1) N‐Terminal Cys and Thr were the most reactive. Cys reacted very rapidly and completely within 0.5 h to form thiazolidine in both aqueous and high content of water‐miscible organic solvents. Thr reacted to form oxazolidine slowly in aqueous buffer (t1/2 > 300 h) but rapidly and completely within 20 h in organic‐water solvents. (2) N‐Terminal Trp, His and Ser were comparatively much less reactive than Cys or Thr. Trp reacted slowly and completely in aqueous buffer but significantly more slowly and incompletely in water‐organic solvents. Both His and Ser reacted very slowly and incompletely in both solvent systems. (3) Finally, Asn reacted nearly insignificantly in both solvent systems. The significant rate enhancement by the water‐miscible organic solvent on Thr was particularly important to allow the synthesis of disulfide‐rich protein domains. Furthermore, the ring formation with N‐terminal Trp, His and Asn provided a convenient route to prepare their bicyclic and unusual heterocyclic derivatives for structure‐activity study.