Having accomplished the SPPS of the Tau(1-156) fragments, we began with their assembly (Scheme 2B). Ligation between Tau(1-40) 6 and Tau(41-76) 7 in the 4-mercaptiophenylacetic acid (MPAA) 55 buffer was complete within 2 h. We noted that products 10 were unstable and over longer reaction times (6 h), cyclization to aspartimide occurred at the allyl ester side chain (41%; 15% aspartimide after 6 h, but complete conversion and hydrolysis of the aspartimide occurs after 16 h resulting in a mixture of inseparable branched and epimerized isomers). Therefore, it was critical to use efficient NCL and de-allylation conditions to preserve the ligation product. A survey of the literature for allyl ester removal identified a Ru(II) catalyst that can deprotect allyl esters in aqueous Gnd•HCl buffers and uses residual MPAA to scavenge the allyl cations. We were pleased to find that adding Cp*Ru(COD)Cl (30 mM) at the end of the We next investigated desulfurization of the A41C mutant of Tau (1-76). We found that t-BuSH (5% v/v) was the most effective H-atom transfer regent, and the product was generated in 77% yield. Furthermore, using t-BuSH as opposed to other Hatom donors (e.g., GSH) caused only minimal aspartimide formation (~10%) that could be separated at this stage by preparative HPLC. The resultant Tau(1-76) peptide was then subjected to Dbz activation with NaNO2 and converted into MES thioester 10e. To prepare Tau(77-156) 11, coupling of Tau(77-118) 8 and Tau(119-156) 9 proceeded smoothly in MPAA buffer (200 mM) and was followed by a one-pot thiazolidine removal with MeONH2. This step required additional optimizations of the thiol catalyst -replacing MPAA with methyl thioglycolate (MTG, 200 mM) or trifluoroethanethiol (TFET, 5% v/v) resulted only in diminished yields (48% and 27%, respectively). Having accomplished the synthesis of Tau(77-156), we proceeded towards the assembly of Tau(1-156). To this end, ligation between Tau(1-76) 10e and Tau(77-156) 11 proceeded uneventfully and provided the ligation product in 97%. Subsequent double desulfurization of A77®C77 and A119®C119 regenerated the native sequence in good yield (89%). Finally, activation of Dbz and thiolysis afforded thioester intermediate Tau(1-156) 12 in excellent yield and purity. A dehydrated benzimidazole impurity was formed during this step which is inseparable from the thioester but does not couple in subsequent ligations (benzimidazole formation has been observed previously at glycine; for details, see the SI).