All initial radiolabelling studies were conducted with compound 5 as it was available in larger amounts than the other derivatives. Initial attempts at radiolabelling 5 with [ 201 Tl](I) in water were unsuccessful. Reactions were monitored by analytical radioHPLC utilizing a C18 column and a 0-100% water/acetonitrile gradient with 0.1% TFA. During the synthesis of the K222 derivatives, the last step involves purification on reverse phase columns and through trial and error it was found that neutralisation of the collected fractions via addition of 1 M NaOH was necessary, otherwise no chelation of TlPF6 could be observed by NMR. We hypothesised that this is due to protonation of the nitrogen atom in the macrocycle ring, as supported by DFT calculations. Visualisation of the HOMO for 5(Tl) revealed overlap of nitrogen and Tl-based orbitals, originating from the p and d orbitals, respectively (Supporting Information Figure , left image). This interaction was no longer observed in the HOMO upon protonation of the nitrogen (Supporting Information Figure , right image), and is reflected in the N-Tl distances where an elongation from 3.04 Å to 3.83 Å was observed upon protonation. In the absence of this stabilisation from N-Tl interactions, Tl(I) was found to reside further away from the aromatic oxygen donors, resulting in a noticeably less enveloped structure. Based on these findings we assume that concentration of collected fractions in the presence of small amounts of HPLC mobile phase acid additives (TFA or formic acid) resulted in the formation of a protonated cryptand which is unable to coordinate [ nat Tl]Tl(I).