The development and application of a novel endo furan-protected maleimide building block is reported. The endo isomer undergoes deprotection at temperatures ~50 °C below the exo derivative. This enables a simple and powerful approach to quantitatively and selectively introduce functional maleimide groups via temperature modulation.

The thiol-maleimide linkage is widely used for antibody-drug conjugate (ADC) production; however, conjugation of maleimide-drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels-Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol-maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.

Herein, we report the development of a scalable and synthetically robust building block based on norbornadiene (NBD) that can be broadly incorporated into a variety of macromolecular architectures using traditional living polymerization techniques. By taking advantage of a selective and rapid deprotection with tetrazine, highly reactive "masked" cyclopentadiene (Cp) functionalities can be introduced into synthetic polymers as chain-end groups in a quantitative and efficient manner. The orthogonality of this platform further enables a cascade "click" process where the "unmasked" Cp can rapidly react with dienophiles, such as maleimides, through a conventional Diels−Alder reaction. Coupling proceeds with quantitative conversions allowing high molecular weight star and dendritic block copolymers to be prepared in a single step under ambient conditions.

The synthesis of nucleoside analogues incorporating 4-(5-pyrimidinyl)-1,2,3-triazole aglycons as expanded purine nucleobase mimics were accessed using the copper-catalyzed azide-alkyne Huisgen cycloaddition between a ribosyl azide and 5-alkynylpyrimidines. Depending on the nature of the alkyne employed, other nucleoside analogues that possess fluorescence or potential metal-binding properties were prepared. Computational studies were undertaken on the purine analogues and indicate that the heterocycles of the unfused nucleobase prefer a coplanar arrangement and the anti-glycosidic conformer is favoured in most instances.

Here, we describe a diene‐containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron‐demand Diels–Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self‐reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug‐linker AZ1508 at a rate of ≈79 m−1 s−1 to produce functional antibody–drug conjugates (ADCs) in a one‐step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity‐based stapling through a DA dimerization reaction.