Self-Immolative Hydroxybenzylamine Linkers for Traceless Protein Modification

Traceless self-immolative linkers are widely used for the reversible modification of proteins and peptides. This article describes a new class of traceless linkers based on ortho- or para-hydroxybenzylamines. The introduction of electron-donating substituents on the aromatic core stabilizes the quinone methide intermediate, thus providing a platform for payload release that can be modulated. To determine the extent to which the electronics affect the rate of release, we prepared a small library of hydroxybenzylamine linkers with varied electronics in the aromatic core, resulting in half-lives ranging from 20 to 144 h. Optimization of the linker design was carried out with mechanistic insights from density functional theory (DFT) and the in silico design of an intramolecular trapping agent through the use of DFT and intramolecular distortion energy calculations. This resulted in the development of a faster self-immolative linker with a half-life of 4.6 h. To demonstrate their effectiveness as traceless linkers for bioconjugation, reversible protein-polyethylene glycol conjugates with a model protein lysozyme were prepared, which had reduced protein activity but recovered ≥94% activity upon traceless release of the polymer. This new class of linkers with tunable release rates expands the traceless linkers toolbox for a variety of bioconjugation applications.