SPECIFICITY AND FORMATION OF UNUSUAL AMINO-ACIDS OF AN AMIDE LIGATIONSTRATEGY 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 aldehyd e and the N-terminal amino acid bearing a beta-thiol or beta-hydroxide . A new method was developed to define the specificity of this reactio n using a dye-labeled alanyl ester aldehyde to react with libraries of 400 dipeptides which contained all dipeptide combinations of the 20 g enetically coded amino acids. Three different ester aldehydes of the d ye-labeled alanine: alpha-formylmethyl (FM), beta-formylethyl (FE), an d beta,beta,beta-dimethyl and formylethyl esters (DFE), were examined. The DFE ester was overly hindered and reacted with N-terminal Cys dip eptides (Cys-X). Interestingly, it also reacted slowly with the sequen ces of X-Gly where Gly was the second amino acid and the X-Gly amide b ond participated in the ring formation. Although the FE ester reacted similarly as the FM ester in the ring formation, the subsequent O,N-ac yl transfer was at least 30-fold slower than those of the FM-ester. Th e FM a-formyl methyl ester was the most suitable ester and was reactiv e with dipeptides of six N-terminal amino acids: Cys, Thr, Trp, Ser, H is and Asn. The order and extent of their reactivity were highly depen dent on pH, solvent and neighboring participation by the adjacent amin o 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 o xazolidine slowly in aqueous buffer (t(1/2)>300 h) but rapidly and com pletely within 20 h in organic-water solvents. (2) N-Terminal Trp, His and Ser were comparatively much less reactive than Cys or Thr. Trp re acted slowly and completely in aqueous buffer but significantly more s lowly and incompletely in water-organic solvents. Both His and Ser rea cted very slowly and incompletely in both solvent systems. (3) Finally , Asn reacted nearly insignificantly in both solvent systems. The sign ificant rate enhancement by the water-miscible organic solvent on Thr was particularly important to allow the synthesis of disulfide-rich pr otein domains. Furthermore, the ring formation with N-terminal Trp, Hi s and Asn provided a convenient route to prepare their bicyclic and un usual heterocyclic derivatives for structure-activity study. (C) Munks gaard 1995.