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.

Effects of ligands on the migratory insertion of alkenes into rhodium-oxygen bonds.

Migratory insertions of olefins into metal-oxygen bonds are elementary steps of important catalytic processes, but well characterised complexes that undergo this reaction are rare, and little information on the effects of ancillary ligands on such reactions has been gained. We report a series of alkoxo alkene complexes of rhodium(i) that contain a range of bidentate ligands and that undergo insertion of the alkene. Our results show that complexes containing less electron-donating ancillary ligands react faster than their counterparts containing more electron-donating ancillary ligands, and that complexes possessing ligands with larger bite angles react faster than those with smaller bite angles. External added ligands had several effects on the reactions, including an inhibition of olefin isomerisation in the product and acceleration of the displacement of the product from complexes of ancillary ligands with small bite angles. Complementary computational studies help elucidate the details of these insertion processes.

Gold-Catalyzed Hydroamination of Propargylic Alcohols: Controlling Divergent Catalytic Reaction Pathways To Access 1,3-Amino Alcohols, 3-Hydroxyketones, or 3-Aminoketones.

A versatile approach to the valorization of propargylic alcohols is reported, enabling controlled access to three different products from the same starting materials. First, a general method for the hydroamination of propargylic alcohols with anilines is described using gold catalysis to give 3-hydroxyimines with complete regioselectivity. These 3-hydroxyimines can be reduced to give 1,3-amino alcohols with high syn selectivity. Alternatively, by using a catalytic quantity of aniline, 3-hydroxyketones can be obtained in high yield directly from propargylic alcohols. Further manipulation of the reaction conditions enables the selective formation of 3-aminoketones via a rearrangement/hydroamination pathway. The utility of the new chemistry was exemplified by the one-pot synthesis of a selection of N-arylpyrrolidines and N-arylpiperidines. A mechanism for the hydroamination has been proposed on the basis of experimental studies and density functional theory calculations.

Gold-Catalyzed Hydrophenoxylation of Propargylic Alcohols and Amines: Synthesis of Phenyl Enol Ethers.

A practical method for the synthesis of phenyl enol ethers is reported. The combination of a gold(I) catalyst and potassium carbonate selectively mediates the addition of phenols to propargylic alcohols/amines in a chemo-, regio-, and stereoselective fashion in high yield. The resulting enol ethers are formed exclusively with a Z-configuration and can be obtained from a wide array of phenols and propargylic alcohols or amines with the reaction showing excellent functional group tolerance.

Catalytic direct amidations in tert-butyl acetate using B(OCH2CF3)3.

Catalytic direct amidation reactions have been the focus of considerable recent research effort, due to the widespread use of amide formation processes in pharmaceutical synthesis. However, the vast majority of catalytic amidations are performed in non-polar solvents (aromatic hydrocarbons, ethers) which are typically undesirable from a sustainability perspective, and are often poor at solubilising polar carboxylic acid and amine substrates. As a consequence, most catalytic amidation protocols are unsuccessful when applied to polar and/or functionalised substrates of the kind commonly used in medicinal chemistry. In this paper we report a practical and useful catalytic direct amidation reaction using tert-butyl acetate as the reaction solvent. The use of an ester solvent offers improvements in terms of safety and sustainability, but also leads to an improved reaction scope with regard to polar substrates and less nucleophilic anilines, both of which are important components of amides used in medicinal chemistry. An amidation reaction was scaled up to 100 mmol and proceeded with excellent yield and efficiency, with a measured process mass intensity of 8.

Dihalohydration of Alkynols: A Versatile Approach to Diverse Halogenated Molecules.

In this paper we outline how dihalohydration reactions of propargylic alcohols can be used to access a wide variety of useful halogenated building blocks. A novel procedure for dibromohydration of alkynes has been developed, and a selection of dichloro and dibromo diols and cyclic ethers were synthesized. The dihalohydration of homo-propargylic alcohols provides a useful route to 3-halofurans, which were shown to readily undergo cycloaddition reactions under mild conditions. Finally, a novel ring expansion of propargylic alcohols containing a cyclopropylalkyne provides access to halogenated alkenylcyclobutanes.

Stereodivergent Carbamate Synthesis by Selective in Situ Trapping of Organic Carbonate Intermediates.

Trans carbamates have been prepared in a diastereoselective approach by a judicious one-pot combination of organic carbonates, prepared in situ, and suitable amine reagents under appropriate reaction conditions. This unprecedented approach allows for stereodivergence from a single oxirane substrate with easy access to both cis and trans carbamate isomers with high stereoselectivity (>19:1 d.r.). Key to the control of the diastereoselective nature of the conversions that lead to the trans carbamates is the in situ formation of trans-configured oligo/polycarbonates through Al catalysis, which provides the targeted products after aminolysis. The present results demonstrate the valorization of a renewable carbon-based reagent (CO2) into new valuable scaffolds and an unusual stereocontrol exerted through carbonate intermediates. A series of control experiments support the proposed mechanistic rationale towards the trans carbamate products, which is based on the trapping of an in situ formed trans-configured oligo/polycarbonate.

Carbon dioxide as a protecting group: highly efficient and selective catalytic access to cyclic cis-diol scaffolds.

The efficient and highly selective formation of a wide range of (hetero)cyclic cis-diol scaffolds using aminotriphenolate-based metal catalysts is reported. The key intermediates are cyclic carbonates, which are obtained in high yield and with high levels of diastereo- and chemoselectivity from the parent oxirane precursors and carbon dioxide. Deprotection of the carbonate structures affords synthetically useful cis-diol scaffolds with different ring sizes that incorporate various functional groups. This atom-efficient method allows the simple construction of diol synthons using inexpensive and accessible precursors and green metal catalysts and showcases the use of CO2 as a temporary protecting group.

Highly active aluminium catalysts for the formation of organic carbonates from CO2 and oxiranes.

Al(III) complexes of amino-tris(phenolate) ligand scaffolds have been prepared to attain highly Lewis acidic catalysts. Combination of the aforementioned systems with ammonium halides provides highly active catalysts for the synthesis of organic carbonates through addition of carbon dioxide to oxiranes with initial turnover frequencies among the highest reported to date within the context of cyclic carbonate formation. Density functional theory (DFT) studies combined with kinetic data provides a rational for the relative high activity found for these Al(III) complexes, and the data are consistent with a monometallic mechanism. The activity and versatility of these Al(III) complexes has also been evaluated against some state-of-the-art catalysts and the combined results compare favorably in terms of catalyst construction, stability, activity, and applicability.

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.