Peptide thioesters are indispensable for chemical synthesis of proteins by native chemical ligation (NCL)¹ . Despite the requirement of peptide thioesters for NCL, their preparation using Fmoc SPPS has some problems including decomposition of thioesters during peptide chain elongation. Previously, we reported that N-sulfanylethylanilide (SEAlide) peptides, which were synthesized from Fmoc amino acid-incorporated N-sulfanylethyl anilide linker, efficiently function as a peptide thioester equivalent² . Preparation of the N-sulfanylethyl anilide linkers was achieved by condensation of Fmoc amino acyl chlorides with aniline linker. However, a standard preparation protocol of Fmoc amino acyl chlorides using SOCl₂ in CH₂Cl₂ induces the loss of acid-labile protections such as tert-butyl group and fails to afford the anilide linker. Therefore, a practical method for introduction of Fmoc amino acid derivatives to the N-sulfanylethyl aniline linker has been required. Recently, we developed a practical method for condensation of Fmoc amino acid derivatives to the N-sulfanylethylaniline linker³ . The developed technique was successfully used for the preparation of Fmoc amino acid-incorporated linkerspossessing 20 naturally occurring amino acids without accompanying racemization and loss of side-chain protections. The use of various resulting linkers in Fmoc chemistry afforded SEAlide peptides which enabled the efficient formation of NCL products within 24-72 h. In this presentation, details of reaction conditions for introduction of Fmoc amino acids to the aniline linker and application of the resulting SEAlide peptides toNCL protocols will be discussed.