Abstract:

Antibody-drug conjugates (ADCs) have garnered significant research interest due to their highly selective treatment of cancers. While considerable efforts have focused on improving ADC stability in serum and enhancing therapeutic indices, these compounds still face challenges in treating heterogeneous tumors. ADCs carrying multiple, different drugs have shown promise in effectively treating resistant tumors. However, traditional bioconjugation techniques, such as thiol-maleimide click reactions or NHS-ester reactions, often have drawbacks including reversibility in serum or uncontrolled protein modification. Disubstituted maleimides, are proposed to form less reversible protein-maleimide bonds and offer greater selectivity than NHS-ester reactions. This dissertation explores the use of disubstituted maleimides, specifically amidino- and azidomaleimides, as a method to synthesize multifunctional bioconjugates.

Since the imide present on these maleimides is replaced during imide transfer, attempts to functionalize these disubstituted maleimides instead focused on the C-C π bond. We successfully functionalized 3-methyl N-phenyl maleimide with an azide group, as confirmed by NMR. While this vinyl azide was not compatible with copper-catalyzed azide-alkyne click reactions, it could react with an aldehyde and a secondary amine to perform a three-component amidine synthesis. We then suggested that these amidinomaleimides could potentially be utilized for the synthesis of multifunctional bioconjugates through the imide transfer mechanism associated with disubstituted maleimides.

However, when these amidinomaleimides were reacted with the antibody Herceptin, no conjugate was observed. As the imide transfer reaction failed, we synthesized 3-azido-4-methyl-N-2-azidoethylmaleimide with an overall yield of 7%, proposing to utilize this maleimide for further functionalization after bioconjugation. Alternative bioconjugation strategies using the thiol-maleimide click reaction or utilizing the protein as the aldehyde component in the amidine-forming reaction were unsuccessful.
Notably, we discovered a novel N-terminal proline-azidomaleimide conjugation that yields greater than 55% modification of protein. While the details of this reaction remain to be fully elucidated, this discovery opens new avenues for bioconjugation research and applications.