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Radioisotopes of Cu, such as 64 Cu and 67 Cu, are alluring targets for imaging (e.g., positron emission tomography, PET) and radiotherapeutic applications. Cyclen-based macrocyclic polyaminocarboxylates are one of the most frequently examined bifunctional chelators in vitro and in vivo, including the FDA-approved 64 Cu radiopharmaceutical, Cu(DOTATATE) (Detectnet); however, connections between the structure of plausible reactive intermediates and their stability under physiologically relevant conditions remain to be established. In this study, we share the synthesis of a cyclen-based, N , N -alkylated spirocyclic chelate, H 2 DO3A C4H8 , which serves as a model for N -protonation. Our combined experimental (in vitro and in vivo) and computational studies unravel complex pH-dependent speciation and enable side-by-side comparison of N - and O -protonated species of relevant 64 Cu radiopharmaceuticals. Our studies suggest that N- protonated species are not inherently unstable species under physiological conditions and demonstrate the potential of N , N -alkylation as a tool for the rational design of future radiopharmaceuticals.