The covalent chemistry of individual reactants bound within a protein nanopore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. An efficient method to incorporate unnatural amino acid is semisynthesis, which allows site-specific modification with a chemically-defined functional group. However, relatively little work has been done on engineered membrane proteins. This deficiency stems from attributes inherent to proteins that interact with lipid bilayer, notably the poor solubility in aqueous buffer. In the present work, four different derivatives α-hemolysin (αHL) monomer were obtained either by two- or three-way native chemical ligation. The semisynthetic αHL monomers were successfully refolded to heptameric pores and used as nanoreactors to study single-molecule chemistry. The semisynthetic pores show similar biophysical properties to native αHL pores obtained from an in vitro transcription and translation technique. Interestingly, when αHL pores with one semisynthetic subunit containing a terminal alkyne group were used to study Cu(I)-catalyzed azide-alkyne cycloaddition, a long-lived intermediate in the reaction was directly observed.