Formylglycine-generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site-specific oxidation of a cysteine residue to the aldehyde-containing amino acid C -formylglycine (FGly). This non-canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine-generating enzyme (FGE) and the iron-sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.

Strategies for site-specific modification of proteins have increased in number, complexity, and specificity over the last years. Such modifications hold the promise to broaden the use of existing biopharmaceuticals or to tailor novel proteins for therapeutic or diagnostic applications. The recent quest for next-generation antibody-drug conjugates (ADCs) sparked research into techniques with site selectivity. While purely chemical approaches often impede control of dosage or locus of derivatization, naturally occurring enzymes and proteins bear the ability of co-or post-translational protein modifications at particular residues, thus enabling unique coupling reactions or protein fusions. This review provides a general overview and focuses on chemo-enzymatic methods including enzymes such as formylglycine-generating enzyme, sortase, and transglutaminase. Applications for the conjugation of antibodies and antibody mimetics are reported.

Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody−drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglycine (FGly) residues are generated by enzymatic conversion of cysteine residues embedded in a certain amino acid sequence motif with a formylglycine-generating enzyme (FGE). The addition of copper ions increases FGE activity leading to the conversion of cysteines within less readily accepted sequences. With this tuned enzyme activity, it is possible to address two different recognition sequences using two aerobic formylglycine-generating enzymes. We demonstrate an improved and facile strategy for the functionalization of a DARPin (designed ankyrin repeat protein) and the single-chain antibody scFv425-Fc, both directed against the epidermal growth factor receptor (EGFR). The single-chain antibody was conjugated with monomethyl auristatin E (MMAE) and carboxyfluorescein (CF) and successfully tested for receptor binding, internalization, and cytotoxicity in cell culture, respectively.

Formylglycin-generierende Enzyme haben in den letzten Jahren breite Anwendung in der bioorthogonalen Chemie gefunden. Sie katalysieren die ortsspezifische Oxidation eines Cysteinrestes zur Aldehyd-haltigen Aminosäure C a -Formylglycin (FGly). FGly kann in jedem beliebigen Zielprotein generiert und anschließend fürb ioorthogonale Konjugationsreaktionen genutzt werden. Das prototypische Formylglycin-generierende Enzym (FGE) und das Eisen-Schwefel-Protein AtsB zeigen leichte Variationen in ihren Erkennungssequenzen. Spezifische Motive in Peptiden und Proteinen wurden entworfen, die selektiv von diesen Enzymen adressiert werden. Die Kombination dieser Motive innerhalb eines Peptids oder rekombinanten Proteins ermçglichte die unabhängige und sukzessive Einführung von zwei FGly-Resten und die Generierung hetero-difunktionalisierter Proteinkonjugate.

Formylglycine‐generating enzymes provide a convenient tool for site‐specific protein derivatization. Their ability to oxidize cysteine or serine residues within a defined consensus sequence to Cα‐formylglycine (FGly) allows for the targeted introduction of a unique chemical handle for various bioconjugation reactions. In recent years, oxygen‐dependent FGly‐generating enzyme saw broad use in protein functionalization and the generation of protein conjugates. Yet, the FGly‐generating system AtsB, along with its capability to convert unusual aldehyde tag sequences, remains mostly unused. Herein, the ability of AtsB from Methanosarcina mazei to convert nonclassical aldehyde tags of the SX(A/P)XR‐type and its potential use in bioconjugation chemistry are demonstrated.