To implement the abovementioned strategy, we first aimed to prepare individual oligopeptide fragments. Because the Tau(1-156) region lacks any native cysteine residues, we decided to use alanine ligation sites for the first Tau(1-156) fragment (Figure ). A four-segment approach was deemed as practical since this region contains numerous alanine residues suitable for segment coupling. For the first ligation site we selected D40-A41 disconnection because the alternative disconnections at H14 and D65 would require peptide fragments over 50 amino acid long. The next alanine ligation site marked the halfway point after 35 amino acids at the T76-A77 junction, and the final ligation site was selected at A118-A119 junction which requires 41-and 37-mer fragments. We decided to merge the peptides Tau(1-76) and Tau (77-156) first and then couple these to obtain Tau(1-156) (Scheme 2B). Furthermore, all cysteine mutations within this sequence could be converted back into alanine residues using a global metal free desulfurization. Before moving forward with the synthesis, we also considered the limitation of the proposed ligation at D40-A41 that could be potentially problematic due to the presence of a C-terminal aspartic acid residue in the Tau(1-40) segment. NCL reactions at C-terminal aspartic acid may result in a cyclic anhydride which is hydrolysis prone, and problematic bbranched ligation products are formed even with aryl thiols (MPAA, PhSH/BnSH) used as a catalyst. Previous studies have shown that protection of aspartic acid inhibits cyclization of the side-chain carboxylate with the thioester, and this prompted us to the allyl ester for the side chain of D40.