![]() ![]() 4Īmong all bioorthogonal reactions, the Diels–Alder cycloaddition with inverse electron demand (DA inv) between alkenes or alkynes (dienophiles) and N-heteroaromatic compounds (dienes) has become the most promising chemoselective bond-forming chemistry in terms of catalyst-free reaction conditions and reaction rates. ![]() To fully unleash the potential of bioorthogonal chemistry for the production of advanced biologicals, the method of conjugation needs to be highly efficient, quantitative and free of undesired side products to obtain homogeneous products-essential criteria to meet the high requirements of pharmaceutical manufacturing. ![]() Protein fusion and conjugate architectures accessible through expression (only left), enzymatic ligation strategies or site-specific bioorthogonal chemistry. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DA inv). Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Bioorthogonal chemistry holds great potential to generate difficult-to-access protein–protein conjugate architectures. ![]()
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