Self-Immolative Hydroxybenzylamine Linkers for Traceless Protein Modification

Abstract

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.

Synthesis and Application of Trehalose Materials

“…Additionally, it is likely that a more conscious selection of conjugation sites could further reduce the loss in bioactivity, as thus far conjugation sites have been chosen for their accessibility as site-selective points of modification without considering the effects on the biomolecule activity. Alternatively, the use of self-immolative linkers to form polymer/protein conjugates, that release the native protein following certain stimuli, could also mitigate the loss of activity. , …”

“…Alternatively, the use of self-immolative linkers to form polymer/protein conjugates, that release the native protein following certain stimuli, could also mitigate the loss of activity. 103 , 104 …”

“…Alternatively, the use of self-immolative linkers to form polymer/protein conjugates, that release the native protein following certain stimuli, could also mitigate the loss of activity. 103,104 Interestingly, polymers with trehalose in the backbone have never been tested as an excipient or conjugate for protein stabilization but only for delivery of genetic material as discussed below. In the conjugate case, this might be due to a lack of general conjugation strategies that can be applied to a variety of backbone polymers because of the difficulty in obtaining mono-end-functionalized (i.e., semitelechelic) polymers.…”

Recent advances in self-immolative linkers and their applications in polymeric reporting systems

“…Related to these studies have been the application of self-immolative linkers in protein activity assays that take advantage of the highly reactive (aza) quinone methide 78 and quinone methide species. 79 Building upon these observations, Phillips and co-workers have reported protected 4-aminobenzyalcohol linkers such as 24, substituted by electron donating groups, that allow tunable release of phenols with pK a values ≤11.5, directly connected to the benzylic carbon and under neutral conditions (Fig. 2).…”

“…Related to these studies have been the application of self-immolative linkers in protein activity assays that take advantage of the highly reactive (aza)quinone methide 78 and quinone methide species. 79…”