Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation

Established methodology for the preparation of peptide thioesters requires the use of t-butoxycarbonyl chemistry owing to the lability of thioester li nkers to the nucleophilic reagents used in Fmoc solid phase peptide synthes is. Both the greater ease of use and the broad applicability of the method has led to the development of an Fmoc-based methodology for direct peptide thioester synthesis. It was found that successful preparation of a peptide thioester could be achieved when the non-nucleophilic base, 1,8-diazabicycl o[5.4.0]undec-7-ene, together with 1-hydroxybenzotriazole in dimethylformam ide, were used as the N-alpha-Fmoc deprotection reagent. Native chemical li gation of the resulting thioester product to an N-terminal cysteine-contain ing peptide was successfully performed in aqueous solution to produce a fra gment peptide of human alpha-synuclein. The formation of aspartimide (cycli c imide) in a base-sensitive hexapeptide fragment of scorpion toxin II was found to be significant under the deprotection conditions used. However, th is could be controlled by the judicious protection of sensitive residues us ing the 2-hydroxy-4-methoxybenzyl group. Copyright (C) 2000 European Peptid e Society and John Wiley gr Sons, Ltd.