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Total Chemical Synthesis of a Functionalized GFP Nanobody

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb‐drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino‐acid Nb against GFP. A native chemical ligation‐ desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on‐demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb‐PA was correctly folded after internal disulfide bond formation. The synthetic Nb‐PA was functionalized with a biotin or a sulfo‐cyanine5 dye by copper(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull‐down and confocal microscopy settings.