Investigating Ugi/Passerini Multicomponent Reactions for the Site‐Selective Conjugation of Native Trastuzumab**

Abstract

Site-selective modificationo fp roteins hasb een the object of intense studies over the past decades, especially in the therapeutic field. Prominent resultsh ave been obtained with recombinant proteins,f or which site-specific conjugation is made possible by the incorporation of particular aminoa cid residues or peptides equences. In parallel, methods for the site-selective and site-specific conjugation of native andn atural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues.P ursuingt he efforts in this field, we planned to develop an ew typeo fs ite-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasonedt hat this should give higher chances of developing as ite-selective strategy compared to the great majority of existing methods that solely target as ingle residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the sidechain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein,w es how that this strategyc an give access to valuable antibody conjugates bearings everald ifferent payloads;f urthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.

Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins

“…Suitable bioconjugation techniques for antibody-conjugates development are still being extensively explored and are required for vectorized immunotherapies or cancer diagnostics. [49][50][51] We finally evaluated eY-click with 10 for antibody labelling. Trastuzumab (Tras, ~150kDa) was selected as a model monoclonal antibody referenced for breast cancer therapies due to its high affinity for the overexpressed Human Epidermal Growth Factor Receptor-2 (HER2).…”

Luminol Anchors Improve the Electrochemical-Tyrosine-Click Labelling of Proteins

“…Convenient bioconjugation techniques for antibodyconjugates development are still extensively explored and required in vectorised immunotherapies or cancer diagnostics. [48][49][50] We finally evaluated eY-click with 10 for antibody labelling. Trastuzumab (Tras, ~150kDa) was selected as a model monoclonal antibody referenced for breast cancer therapies due to its high affinity for the overexpressed Human Epidermal Growth Factor Receptor-2 (HER2).…”

Site-selective modification strategies in antibody–drug conjugates

“…14D). 227 The Ugi reaction was used to conjugate the side-chain amine and carboxylate groups of two neighbouring lysine and aspartic acid/glutamic acid residues via reaction with an aldehyde and isocyanidemodified payload. It was reasoned that the requirement for two amino acids to be sufficiently spatially close to undergo reaction would increase the site-selectivity of the reaction.…”