With these segments in hand, we proceeded towards the assembly of Tau(157-290) 28 as shown in Scheme 4. Threonine ligation between Tau(157-204) 25 and Tau(205-245) 26 was rapid (2 h) with MPAA, but purification was challenging because of the previously discovered instability d-mercaptothreonine. These results prompted us to investigate a one pot ligationdesulfurizaiton approach. Free radical desulfurization is not compatible with aromatic thiols (MPAA, PhSH), and aliphatic thiols such as t-BuSH, GSH, 113 DTT, and MESNa 112 are used as H-atom donors in the desulfurization step. To thie end, the ligation between 25 and 26 was complete within 2 h using TFET as the catalyst, and efficient one-pot desulfurization was accomplished upon adding desulfurization buffer with GSH (28). The Dbz group was then activated with NaNO2 and treated with MPAA/27, followed by monodesulfurization of A246→C246 with GSH and Acm removal with PdCl2 and MgCl2 furnishing 29. 114 D. Synthesis of Tau(291-441). Our next goal was to develop a synthesis of Tau(291-441). Since this region contains two natural cysteine residues (C291 and C322), the first ligation would use the K321-C322 junction. We also selected the G389-A390 site, which would require a 51-mer peptide segment Tau(390-441). Therefore, the immediate goal was to prepare Tau(322-389) using a surrogate ligation approach, and we selected the L357-D358 site for the mercaptoaspartic acid ligation. The first approach for assembling Tau(322-389) was based on the NCL at the L357-D358 junction with mercaptoaspartic acid 31 and Tau(322-357) 30, which afforded product 32 in high yield (Scheme 5). The subsequent elaboration of 32 involved a NCL with Tau(390-441) 37 and homogeneous desulfurization of 33, which produced a complex mixture of products. Alternatively, metal-free desulfurization of mercaptoaspartic acid 32 was also problematic and resulted in a mixture of 34a, 34c, and 34d (entries 1 and 2). Heterogeneous reduction with H2 and Pd/Al2O3 was clean but generated a mixture of 34a and 34e (entry 3). Other reducing conditions including phosphine-borane desulfurization (entry 4) 116 and increased reaction temperature with low pH (entry 5) were equally difficult. As an alternative, we investigated diselenide-selenoester ligation (DSL). The reaction between diselenides and selenoesters proceeds under non-reducing conditions without an additive 118 and can be accompanied by a rapid one-pot deselenization. In order to preserve the natural cysteines, we first prepared Tau(322-389) using DSL followed by ligations the C to N direction. Tau(322-357) 35 features N-terminal Acm protective group and C-terminal selenoester that was installed with DPDS/PBu3 after protected cleavage from HMPB resin (Scheme 6). Tau(358-389) 36 was prepared with the Dbz linker at the C-terminus and PMB protected selenoaspartic acid at the N-terminus, which was converted into the reactive diselenide 36 using oxidation with DMSO/TFA. The 51-mer segment Tau(390-441) 37 was then prepared on a Wang resin. The Tau(291-321) fragment 38 featured N-terminal thiazoline and was prepared on HMPB resin followed by a solution phase esterification with EtSH/PyBOP. To complete the synthesis of Tau(291-441), the DSL/deselenization reactions between the 36 and 37 proceeded well producing 39 cleanly, and the subsequent steps afforded Tau(291-441) 41 as a homogenous material.