Method for providing a labeled single isomeric chemical entity targeting vector based on the use of an isomer-free dienophile

Method for providing a labeled single isomeric chemical entity targeting vector comprising:

• a) labeling a first chemical entity having inverse electron demand Diels-Alder cycloaddition reactivity and being conjugated to a pharmaceutic agent, an imaging agent, or a therapeutic agent, with a labeling agent;
• wherein the first chemical entity is selected from the group consisting of a symmetrical substituted diene wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, an unsymmetrical substituted diene, and an isomer-free dienophile;
• and
• b) ligating the labeled first chemical entity obtained in step a) with a second chemical entity having complementary inverse electron demand Diels-Alder cycloaddition reactivity and being conjugated to a targeting vector;
• wherein the second chemical entity is selected from the group consisting of a symmetrical substituted diene wherein at least one of the symmetry planes pass through the nitrogen-nitrogen bonds of at least one tetrazine ring, an unsymmetrical substituted diene, and an isomer-free dienophile,
• wherein the reaction kinetics for the inverse electron demand Diels-Alder cycloaddition between the first and second chemical entities has a minimum rate constant of 500 M−1 s−1 in phosphate-buffered saline (PBS) at 25° C., determined by stopped-flow spectrophotometry,
• with the proviso that when the labeling agent in step a) is 94Tc, 99mTc, 211At, 223Ra or 225Ac the labeling agent is conjugated to an unsymmetrical substituted diene;
• and
• c) oxidizing the ligated labeled targeting vector obtained from step b) at a temperature ranging from 15° C. to 50° C. for up to 60 minutes by adding from 1 to 100 equivalents of chloranil, fluoranil, DDQ or NaNO2.