Highly homogeneous ADCs are generated by the efficient bridging of interchain disulfide bonds in trastuzumab, using next generation maleimides.

Herein we report the use of next generation maleimides (NGMs) for the construction of a potent antibody-drug conjugate (ADC) via functional disulfide bridging. The linker has excellent stability in blood serum and the ADC, armed with monomethyl auristatin E (MMAE), shows excellent potency and cancer cell selectivity in vitro.

The cyclopentenone unit is a very powerful synthon for the synthesis of a variety of bioactive target molecules. This is due to the broad diversity of chemical modifications available for the enone structural motif. In particular, chiral cyclopentenones are important precursors in the asymmetric synthesis of target chiral molecules. This Review provides an overview of reported methods for enantioselective and asymmetric syntheses of cyclopentenones, including chemical and enzymatic resolution, asymmetric synthesis via Pauson-Khand reaction, Nazarov cyclization and organocatalyzed reactions, asymmetric functionalization of the existing cyclopentenone unit, and functionalization of chiral building blocks.

Disulfide bridging offers a convenient approach to generate site-selective antibody conjugates from native antibodies. To optimise the reagents available to achieve this strategy, we describe here the use of dibromomaleimides designed to undergo accelerated post-conjugation hydrolysis. Conjugation and hydrolysis, which serve to 'lock' the conjugates as robustly stable maleamic acids, is achieved in just over 1 h. This dramatic acceleration is also shown to infer significant improvements in homogeneity, as demonstrated by mass spectrometry analysis.

Delivering potent, stable, targeted and in vivo efficacious antibody–drug conjugates (ADCs) using pyridazinedione functional disulfide re-bridging reagents.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Lightactivated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues. P 2X receptors and acid-sensing ion channels (ASICs) are trimeric membrane ion channels gated by binding extracellular ligands. P2X receptors are gated by extracellular ATP, and their physiological roles include neuroeffector transmission, primary afferent transmission (e.g., taste, hearing, chemoreception), central control of respiration, and neuroinflammation (1-3). ASICs are gated by protons and are involved in pain sensation (4, 5). The experimental study of ligand-gated channels in intact tissues is often hampered by difficulties in application of the appropriate ligand while recording ion channel activity in the millisecond time domain, and there are advantages to controlling channel activation by surrogate optical methods. The increase in our knowledge of molecular and atomic structure of ligand-gated channels over the past 10 years has allowed one such approach (photoswitchable tethered ligands) to become much more sophisticated, because cysteines can be introduced into the channel protein exactly where required to form an attachment point. The method has been applied to pentameric nicotinic receptors (6) and tetrameric glutamate receptors (7,8). Although attaching ligands through photoswitchable tethers is proving extremely valuable, an intimate structural knowledge of a closed and open state of a channel also allows for optical control of conformation at parts of the protein that are remote from the agonist binding site (9-11).High-resolution structures are available for P2X receptors (closed: ref. 12; open: ref. 13) and ASICs (closed: refs. 14 and 15; open: ref. 16). In both these trimeric channels the second of the...

Graphical abstract

Thiol-stable albumin biologics are enabled by controlled, quantitative hydrolysis of maleimide–albumin conjugates, i.e. with no retro-Michael.