Optimizing the Diels–Alder (DA) reaction for aqueous
coupling
has resulted in practical methods to link molecules such as drugs
and diagnostic agents to proteins. Both normal electron demand (NED)
and inverse electron demand (IED) DA coupling schemes have been employed,
but neither mechanism entails a common multipurpose reactive group.
This report focuses on expanding the bioconjugation toolbox for cyclopentadiene
through the identification of reactive groups that couple through
NED or IED mechanisms in aqueous solution. Dienophiles and tetrazine
derivatives were screened for reactivity and selectivity toward antibodies
bearing cyclopentadiene amino acids to yield bioconjugates. Twelve
NED dienophiles and four tetrazine-based IED substrates were identified
as capable of practical biocoupling. Furthermore, tetrazine ligation
to cyclopentadiene occurred at a rate of 3.3 ± 0.5 M–1 s–1 and was capable of bioorthogonal transformations,
as evidenced by the selective protein labeling in serum. Finally,
an antibody–drug conjugate (ADC)-bearing monomethyl auristatin
E was prepared via tetrazine conjugation to cyclopentadiene. The resulting
ADC was stable and demonstrated potent activity in vitro. These findings expand the utility of cyclopentadiene as a tool
to couple entities to proteins via dual DA addition mechanisms.