Whereas chemical approaches for fGly installation into peptides have been reported, these methods currently suffer from limitations associated with the intolerance of fGly to the acidic conditions and nucleophiles (e.g. silanes and thiol-based scavengers, or cleaved 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting groups) during the resin cleavage step of solid-phase peptide synthesis (SPPS) (see Figure ). Herein we describe the design and total synthesis of an SPPS-compatible fGly-precursor building block from vitamin C: a cheap, readily-available chiral feedstock (Scheme 2A). We demonstrate that this building block is compatible with conventional and microwave-assisted SPPS, and prepare the pharmaceuticallyrelevant peptide Callyaerin A, a potent anti-tuberculosis macrocyclic peptide which contains a (Z)-2,3-diaminoacrylamide moiety derived from an fGly residue inaccessible to FGE/anSME-based enzymatic efforts. Oxidative cleavage of 1,2-diols or 1,2-amino alcohols using periodate is a mild and commonly-used method for the installation of aldehydes into biomolecules. 1,2-diols presented on the side-chains of sialic acids are highly susceptible to periodate oxidation, yet are tolerant to the conditions required for resin cleavage after SPPS. We thus envisioned installing 1,2-diol functionalized 4-hydroxy-L-threonine (hThr) residues into peptides as formyl glycine precursors for the subsequent generation of fGly residues after resin cleavage via mild periodate oxidation (Scheme 2B). We hypothesized this approach would overcome issues associated with the intolerance of fGly residues to the conditions encountered during SPPS resin cleavage.