Activated thioesters cannot be anchored at the C-terminus during Fmoc-SPPS due to their sensitivity to base treatment which is routinely used during deprotection. Consequently, inert precursors including the refined Dawson linker have been developed (Table , Entry 5). Following SPPS, the base-stable 3,4-diaminobenzoic acid (Dbz) derivative is activated and subjected to thiolysis to yield the thioester for native chemical ligation. More recently, a second generation Dawson linker recruits an N-methylated amino group, minimizing unwanted acylation when using an excess of glycine reagent. Assembly of peptides directly onto the linker amine is a simple and useful feature of the Dawson linker. Alternatively, the C-terminal peptide hydrazide can be used to create a C-terminal thioester (Table , Entry 4). Activated by the addition of sodium nitrite, the hydrazide can be oxidized into an acyl azide which can be subjected to thiolysis with an acidic thiol in situ. Hydrazine resins are prepared fresh before use, but it has recently been shown that resins can be prepared with Fmoc-hydrazine facilitating long term storage and facile loading quantification. In the convergence of multiple peptide fragments to prepare larger synthetic proteins, some central fragments will be ligated at both N-and C-termini. To prevent intramolecular side-reactions (cyclization), the peptide can be prepared as a thioester surrogate, whereby a C-terminal functional group remains inert in NCL and can be subsequently activated for the next ligation step. The peptide hydrazide is the most common choice in this instance due to the facile in situ activation and has been used to prepare numerous D-proteins, including the mirror-image TIGIT domain discussed earlier.