fGly building block 6 was used to prepare a test peptide NH2-Gly-Leu-Tyr-Arg-hThr-Ala-Gly-COOH 7 (Figure ) using Fmoc SPPS in a 95% yield and with excellent purity. No acid-catalyzed breakdown products were observed during the cleavage of 9 from the peptide resin, and no side-products derived from the reaction of fGly with the cleaved Pbf group of the Arg residue were observed. Conversion of the hThr residue of 7 into fGly was then readily achieved via treatment of a 2 mM aqueous solution of 7 with 1.2 equivalents of sodium metaperiodate (NaIO4), yielding peptide 8 (Figure ). Peptides 7 and 8 were found to have the same retention time in LC-MS (most likely due to the similar polarities of hydrated fGly and hThr motifs), but mass spectrometry peaks attributable to 7 were fully lost within 20 minutes of treatment with NaIO4, leaving only signals attributable to 8 (Figure ). Additionally, while oxidation of serine/threonine residues to fGly using RuO4 is known to lead to side-products via oxidative scission/retroaminal fragmentation, no such byproducts were detected after the treatment of 7 with NaIO4 (see Figure ). Having verified the compatibility of 6 with SPPS, and with conditions established for fGly installation via periodate treatment, we applied the methodology to the synthesis of Callyaerin A 10, which contains a rare (Z)-2,3-diaminoacrylamide motif derived from an fGly residue (Figure ). The only previous synthesis of 10 used an alternative Fmoc-protected building block, but reported problems associated with the epimerization of this building block in SPPS, in addition to acid-catalyzed fragmentation of the fGly-functionalized linear Callyaerin A peptide upon resin cleavage. By contrast, we hypothesized that the protected hThr building blocks would be no-more prone to epimerization than threonine, and would not undergo peptide fragmentation under acidic conditions via the fGly-dependent pathway that has previously been postulated. Microwave-assisted fully-automated Fmoc SPPS, was used to prepare linear Callyaerin A precursor 9 (Figure ), verifying that 6 is suitable for use in microwave-assisted peptide synthesis. A 2 mM solution of 9 was then treated with 1.2 equivalents of NaIO4, and the hThr residue of 9 was cleanly converted to fGly in a one-pot reaction within 20 minutes, yielding a mixture of linear Callyaerin A 9′ and Callyaerin A 10 (see Figure ). Complete cyclisation of 9′ into 10 was facilitated via the extraction of the peptide mixture into MeCN and the addition of 0.1% formic acid and MgSO4. Due to the profound change in the shape accompanying cyclisation, 9 and 10 have distinct retention times as well as distant mass spectrometry peaks (Figure ).