Maleimide chemistry stands out in the bioconjugation toolbox by virtue of its synthetic accessibility, excellent reactivity, and practicability. The second‐generation of clinically approved antibody–drug conjugates (ADC) and much of the current ADC pipeline in clinical trials contain the maleimide linkage. However, thiosuccinimide linkages are now known to be less robust than once thought, and ergo, are correlated with suboptimal pharmacodynamics, pharmacokinetics, and safety profiles in some ADC constructs. Rational design of novel generations of maleimides and maleimide‐type reagents have been reported to address the shortcomings of classical maleimides, allowing for the formation of robust bioconjugate linkages. This review highlights the main strategies for rational reagent design that have allowed irreversible bioconjugations in cysteines, reversible labelling strategies and disulfide re‐bridging.

The antioxidant activity of the phenolic compounds present in industrial black liquors obtained from the two cooking processes (kraft and sulphite) used in Portugal to produce Eucalyptus globulus pulp was evaluated. The black liquors treated at several pH values were extracted with ethyl acetate. Phenolic fractions were further separated by liquid chromatography of the crude extracts of kraft liquor at pH = 6 and sulphite liquor at the original pH. Total phenolic content was determined in terms of gallic acid equivalents (Folin-Ciocalteu colorimetric method), and the antioxidant activity in the crude extracts at several pH values and in the separated fractions was measured using the DPPH test for radical scavenging capacity. The total phenolic content of crude extracts and separated fractions ranged from 92.7 to 181.6 and from 91.6 to 1,099.6 mg GAE/g, respectively, while the antioxidant activity index (AAI) ranged from 2.20 to 3.41 and from 2.21 to 11.47 respectively, showing very strong antioxidant activity in all studied cases. The fractions separated by column chromatography were submitted to mass spectrometry analysis and the results were compared to others in the literature of natural products, mainly from Eucalyptus, and the characteristic bands of functional groups were identified by ¹H-NMR and FTIR. These methods allowed the identification of 17 phenolic compounds.

The development of efficient metal‐promoted bioorthogonal ligations remains as a major scientific challenge. Demonstrated herein is that azides undergo efficient and regioselective room‐temperature annulations with thioalkynes in aqueous milieu when treated with catalytic amounts of a suitable ruthenium complex. The reaction is compatible with different biomolecules, and can be carried out in complex aqueous mixtures such as phosphate buffered saline, cell lysates, fetal bovine serum, and even living bacteria (E. coli). Importantly, the reaction is mutually compatible with the classical CuAAC.

We show that formyl benzeno boronic acids (2FBBA) selectively react with N-terminal cysteines to yield a boronated thiazolidine featuring a B–N bond. The reaction exhibits a very rapid constant rate (2.38 ± 0.23 × 10² M⁻¹ s⁻¹) under mild aqueous conditions (pH 7.4, 23 °C) and tolerates different amino acids at the position adjacent to the N-cysteine. DFT calculations highlighted the diastereoselective nature of this ligation reaction and support the involvement of the proximal boronic acid in the activation of the imine functionality and the stabilisation of the boronated thiazolidine through a chelate effect. The 2FBBA reagent allowed the effective functionalisation of model peptides (C-ovalbumin and a laminin fragment) and the boronated thiazolidine construct was shown to be stable over time, though the reaction was reversible in the presence of benzyl hydroxylamine. The reaction proved to be highly chemoselective, and 2FBBA was used to functionalize the N-terminal cysteine of calcitonin in the presence of a potentially competing in-chain thiol group. This exquisite selectivity profile enabled the dual functionalisation of calcitonin and the interactive orthogonal modification of this peptide when 2FBBA was combined with conventional maleimide chemistry. These results highlight the potential of this methodology to construct complex and well-defined bioconjugates.