A strategy for last-step 18F fluorination of bioconjugated peptides is reported that exploits an “Achilles heel” in the substrate specificity of the fluorinase enzyme. An acetylene functionality at the C-2 position of the adenosine substrate projects from the active site into the solvent. The fluorinase catalyzes a transhalogenation of 5′-chlorodeoxy-2-ethynyladenosine (ClDEA) to 5′-fluorodeoxy-2-ethynyladenosine (FDEA). Extending a polyethylene glycol linker from the terminus of the acetylene allows the presentation of bioconjugation cargo to the enzyme for 18F labelling. The method uses an aqueous solution (H218O) of [18F]fluoride generated by the cyclotron and has the capacity to isotopically label peptides of choice for positron emission tomography (PET).