Single Mutation on Trastuzumab Modulates the Stability of Antibody-Drug Conjugates Built Using Acetal-Based Linkers and Thiol-Maleimide Chemistry.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

Dichloro Butenediamides as Irreversible Site-Selective Protein Conjugation Reagent.

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.

Protein Conjugation by Electrophilic Alkynylation Using 5-(Alkynyl)dibenzothiophenium Triflates.

5-(Alkynyl)dibenzothiophenium triflates are introduced as new reagents to prepare different protein conjugates through site-selective cysteine alkynylation. The protocol developed allows a highly efficient label of free cysteine-containing proteins with relevant biological roles, such as ubiquitin, the C2A domain of Synaptotagmin-I, or HER2 targeting nanobodies. An electrophilic bis-alkynylating reagent was also designed. The second alkynylating handle thus introduced in the desired protein enables access to protein-thiol, protein-peptide, and protein-protein conjugates, and even diubiquitin dimers can be prepared through this approach. The low excess of reagent needed, mild reaction conditions used, short reaction times, and stability of the S-C(alkyne) bonds at physiological conditions make this approach an interesting addition to the toolbox of classical, site-selective cysteine-conjugation methods.

A powerful aluminum catalyst for the synthesis of highly functional organic carbonates.

An aluminum complex based on an amino triphenolate ligand scaffold shows unprecedented high activity (initial TOFs up to 36,000 h(-1)), broad substrate scope, and functional group tolerance in the formation of highly functional organic carbonates prepared from epoxides and CO(2). The developed catalytic protocol is further characterized by low catalyst loadings and relative mild reaction conditions using a cheap, abundant, and nontoxic metal.