The third approach to Tau(157-290) was based on the NCL ligation featuring a modified threonine (Scheme 3). We selected a surrogate ligation at the unhindered G204-T205 site using mercaptothreonine, which would generate 47-and 40-residue segements. We envisioned that the relevant fragments could be ligated in the C→N direction, and the thiol mutations would be removed through desulfurization. Initial attempts to generate T205-T245 on a thioester surrogate MeNbz resin were unsuccessful because δ-mercaptothreonine is incompatible with p-NO2PhOCOCl activation and we decided to switch to the hydrazide surrogate Dbz linker. This linker was protected with a BocDbz to help enable microwave-assisted SPPS and prevent over acylation. After coupling of methyldisulfide 22a onto 14, the resin was subject to global cleavage (TFA:H2O:TA:TIS), which produced reduced thiol 15. Further manipulations were challenging because mercaptothreonine 15 displayed self-splicing capabilities, likely through a retro-NCL producing 17. This time-dependent cleavage occurs readily under acidic conditions (~20% splicing after 1 h), but thiol intermediate 15 could be preserved by lyophilization of the HPLC fractions. To avoid further degradation, crude peptide thiol 15 was treated with MMTS to generate disulfide 16, 108 which remained stable towards HPLC purifications. The T205-K290 fragment was prepared by a one-pot-ligation with hydrazide 23, but the ligation product remained unstable in the reduced state, and to avoid degradation, MPAA was removed by desalting column followed by immediate treatment with MMTS to generate stable peptide 18 in 64% yield.