Self-Immolative Hydroxybenzylamine Linkers for Traceless ProteinModification

Traceless self-immolative linkers are widely used forthe reversible modification of proteins and peptides. This articledescribes a new class of traceless linkers based onortho-orpara-hydroxybenzylamines. The introduction of electron-donatingsubstituents on the aromatic core stabilizes the quinone methideintermediate, thus providing a platform for payload release that canbe modulated. To determine the extent to which the electronicsaffect the rate of release, we prepared a small library ofhydroxybenzylamine linkers with varied electronics in the aromaticcore, resulting in half-lives ranging from 20 to 144 h. Optimizationof the linker design was carried out with mechanistic insights fromdensity functional theory (DFT) and thein silicodesign of anintramolecular trapping agent through the use of DFT and intramolecular distortion energy calculations. This resulted in thedevelopment of a faster self-immolative linker with a half-life of 4.6 h. To demonstrate their effectiveness as traceless linkers forbioconjugation, reversible protein-polyethylene glycol conjugates with a model protein lysozyme were prepared, which had reducedprotein activity but recovered >= 94% activity upon traceless release of the polymer. This new class of linkers with tunable release ratesexpands the traceless linkers toolbox for a variety of bioconjugation applications.