Radioisotopes
of Cu, such as 64Cu and 67Cu,
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 64Cu 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, H2DO3AC4H8
, 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 64Cu 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.