Chemical Synthesis of Microtubule-Associated Protein Tau 2N4R

Deposits of the microtubule-associated protein tau (MAPT) serve as a hallmark of neurodegenerative diseases known as tauopathies. Numerous studies have demonstrated that in diseases such as Alzheimer’s disease (AD), tau undergoes extensive remodeling. The attachment of post-translational modifications distributed throughout the entire sequence of the protein cor-relates with the clinical presentation. A systematic examination of these protein alterations can shed light on their roles in both healthy and diseased states. However, the ability to access these modifications in the entire protein chain is limited as tau can only be produced recombinantly or through semi-synthesis. In this paper, we describe the first chemical synthesis of the longest 2N4R isoform of tau, consisting of 441 amino acids. The 2N4R tau was divided into three major segments and a total of eleven fragments, all of which were prepared via solid-phase peptide synthesis. The successful chemical strategy re-lied on the strategic use of two cysteine sites (C291 and C322) for native chemical ligations (NCL). This was combined with modern preparative protein chemistries, such as threonine ligation (T205), diselenide-selenoester ligation (D358), and mutations of mercaptoamino acids into native residues via homogeneous radical desulfurization (A40, A77, A119, A157, A246, A390). The successful completion of the synthesis established a robust and scalable route to the native protein in multimilligram quantities and high purity. In broader terms, the presented strategy can be applied to the preparation of other shorter isoforms of tau, as well as to introduce all post-translational modifications that are characteristic of tauopathies such as AD, progressive supranuclear palsy, and frontotemporal dementia.