A. Retrosynthetic Analysis of 2N4R Tau. In the pursuit of total synthesis of 2N4R Tau, we designed an approach where the protein was dissected into three major segments, each approximately 150 amino acids in length (Figure ). Our rationale for choosing the G156-A157 junction for ligation was its unhindered nature. In addition, we selected K290-C291 as the ultimate ligation site due to its status as one of the two natural cysteine residues in the protein. These segments -designated as Tau(1-156), Tau(157-290), and Tau(291-441) -would be subsequently assembled in the N-to-C direction. In order to retain the two natural cysteine residues, the final ligation was orchestrated between Tau(1-290) and Tau(291-441) at the K290-C291 junction. The Tau(1-290) construct would be achieved by ligation and followed by desulfurization at G156-A157C involving Tau(1-156) and Tau(157-290). These major segments were assembled from smaller synthetic peptides encompassing approximately 30-50 residues (Figure ). We note that our objective of this study is to develop chemical synthesis of the longest 2N4R isoform which is found in the adult human brain and relevant to neurodegenerative diseases. However, the presented chemical strategy is applicable to other five isoforms with minimal modifications in terms of disconnections and chemistries used for segment couplings.