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Monitoring current flow through a single nanopore has proved to be a powerful technique for the in situ detection of molecular structure, binding and reactivity. Transmembrane proteins, such as a-hemolysin, provide particularly attractive platforms for nanopore sensing applications due to their atomically precise structures. However, many nanopore applications require the introduction of functional groups to tune selectivity. To date, such modifications have required genetic modification of the protein prior to functionalization. Here we demonstrate the in situ synthetic modification of a wild-type a-hemolysin nanopore embedded in a membrane. We show that reversible dynamic covalent iminoboronate formation and the resulting changes in the ion current flowing through individual nanopores can be used to map the reactive behavior of lysine residues within the nanopore channel. Crucially, the modification of lysine residues located outside the nanopore channel were found not to affect the stability or utility of the nanopore. Finally, knowledge of the reactivity patterns enabled the irreversible functionalization of a single, assignable lysine residue within the nanopore channel. The approach constitutes a simple, generic tool for the rapid, in situ synthetic modification of protein nanopores that circumvents the need for prior genetic modification.