Our next goal was to prepare the 133-mer Tau(157-290). Native cysteines are absent in Tau(157-290), but alanine residues in this segment are suitable NCL-desulfurization (Figure ). We initially selected several potential disconnection sites such as Q165-A166, N167-A168, P172-A173, P177-A178, V226-A227, S238-A239, T245-A246. The G156-A157 junction that would be used for a large segment coupling (vide infra) is unhindered and has a low likelihood of epimerization. Chemical ligations at C-terminal glutamine and asparagine are typically avoided because of a potential formation of cyclic imides and β-branched products. However, subsequent studies that used MPAA to facilitate the NCL showed that the amount of side products at the glutamine and, to a lesser extent, asparagine sites could be reduced. With this considerations in mind, we selected the Q165-A166 over the N167-A168 site. The Tau(157-290) region also contains two proline-alanine junctions, but these ligations were considered less favorable because they show slow reaction kinetics together with Xaa-Pro deletions due to diketopiperazine formation. The following three disconnections V226-A227, S238-A239, and T245-A426 were expected to proceed uneventfully. Finally, the T245-A246 site was also deemed achievable because it uses a 45-mer peptide, which is sufficiently short to be produced in a homogeneous form, and this site was used in a previous synthesis. With these considerations in mind, we initially focused on four peptide segments that would be assembled through ligations at the Q165-A166, V226-C227, and T245-C246 sites. We were able to synthesize A227C-K290 in acceptable yields from two smaller segments C246-K290 and C227-T245 but attempts to produce longer segment C166-K290 by NCL with C166-V226 were unsuccessful, and the peptide could not be purified to homogeneity. These problematic disconnections led us to consider an alternative approach using a threonine ligation. Our plan was to dissect Tau(157-290) into three segments of lengths no more than fifty amino acids. After dissected the region evenly, we decided to use the unhindered G204-T205 junction and serine-threonine ligation (STL). The three required segments A157-G204, T205-T245, and A246-K290 were prepared by SPPS, and the key ligation between C-terminal salicylaldehyde ester C157-G204 and N-terminal threonine of T205-T245 was unsuccessful under basic, neutral, or acidic conditions.