In both eukaryotes and prokaryotes, enzymes capable of introducing α-formylglycine (fGly) residues (Scheme 1) into proteins prior to folding are essential for making post-or co-translational modifications to type I sulfatases and some phosphonate monoester hydrolases / phosphodiesterases. A fGly residue is generated by the selective oxidation of a cysteine or serine residue embedded within a conserved consensus sequence (C/S)XPXRXXXLTG by either formylglycine generating enzymes (FGEs) or anaerobic sulfatase maturating enzymes (anSMEs) (Scheme 1). Oxidation of this residue is used to introduce an active-site aldehyde functionality into the sulfatase / phosphonate monoester hydrolase / phosphodiesterases, which when hydrated to a geminal diol serves as a nucleophile, allowing these enzymes to cleave sulfate/phosphonate esters. Incorporation of fGly consensus sequences into recombinant proteins can be used in conjugation with FGEs or anSMEs to produce fGly-labelled proteins, in which the fGly motif can serve as a unique bioconjugation handle. Scheme 1. The oxidation of cysteine or serine residues by either FGE or anSME enzymes to yield formylglycine residues. FGly-functionalized peptides are therefore useful in studies involving sulfatases, phosphonate monoester hydrolases/phosphodiesterases, FGEs and anSMEs, and would also prove useful in the development of bioconjugation techniques intended for selectively targeting fGly residues. Additionally, fGly can also be used as a handle through which pharmaceutically important rigid diaminoacrylamide motifs can be installed into peptides. Scheme 2. A) The preparation of protected fGly precursor 6 from vitamin C. B) The preparation of fGly functionalized peptides using protected fGly precursor 6.