E. Assembly of Tau(1-441). With all the segments in hand, we were able to proceed to the completion of the synthesis of 2N4R Tau (Scheme 7). The ligation of Tau(1-156) 12 and Tau(157-290) 29 segments proceeded smoothly, and Tau(1-290) 43 was isolated in 64%. Next, we removed the A157C mutation using metal free desulfurization, and this step required HEPES (200 mM) and lysine (20 mM) in the desulfurization buffer to inhibit decomposition. We then converted Tau(1-290) 43 into active MES thioester using hydrazide activation chemistry. The final ligation step between Tau(1-290) 43 and Tau(291-441) 41 was carried out with lysine and MPAA buffer, and Tau(1-441) 1 was isolated in 41% yield (4.9 mg) after preparative reversed-phase sample displacement chromatography. To further corroborate the identity and purity of the synthetic material, we compared the synthetic 1 with recombinant Tau(2-441). Although the material produced in E. coli lacked the first methionine residue, both samples showed comparable HRMS spectra and purity on SDS-PAGE (Scheme 7C).