To overcome the obstacles with stability and reproducibility, the synthesis of Tau(157-290) was redesigned. Because segment Tau(157-204) was difficult to prepare directly as a SEt ester or MeNbz due to rapid hydrolysis, we prepared Tau(157-204) 25 on the hydrolysis resistant Dbz linker resin, which was later converted into a MES thioester using NaNO2 (Scheme 4). Furthermore, this segment contained an aspartimide prone region that formed during the SPPS and under acidic conditions of HPLC purification (0.1% TFA). Fortunately, we were able to suppress this problematic dehydration by installing a bulky Mpe protection at D193 followed by immediate lyophilizing. The synthesis of Tau(205-245) 26 proceeded uneventfully and feature mercaptothreonine building block 22b with a more hindered disulfide (t-BuS-S vs. MeS-S) introduced to reduce disulfide cleavage during global deprotection. The bulky disulfide was also resistant towards reduction with TIS, and removal of TBDPS group was accomplished on resin with TBAF in DMF. The final segment Tau(246-290) 27 was prepared on a Dbz linker resin, but two pseudoprolines at T263 and S285 were required due to the enhanced hydrophobicity of this linker.