Bisaryl hydrazones as exchangeable biocompatible linkers.

The selective enrichment of tagged molecules from complex biological mixtures is of primary importance for methods in chemical biology and proteomics. Affinity purification on (strept)avidin beads using biotin as an affinity tag is one of the most widely applied methods to achieve this, fully utilizing the high affinity of biotin for (strept)avidin (Ka ≈ 1.7 × 1015 M−1)[1]. However, the method is limited by the harsh, denaturing conditions required for elution, such as an SDS boil or treatment with 8 M Guanidine (pH 1.5). Protein structure and function are lost and target-proteins may be contaminated with proteins nonspecifically bound to the beads. Two strategies have been explored to elute under mild conditions: i) weakening of the biotin-(strept)avidin interaction by modulating the Ka[2] and ii) introduction of a proteolytically[3] or chemically[4] cleavable linker. While the first strategy does improve the release of biotinylated proteins from (strept)avidin beads, it adversely affects the stringency of the immobilization. The second strategy enables site-specific cleavage, but premature cleavage has been reported and cleavage conditions have no demonstrated compatibility with active biomolecules. In addition, the general applicability of the cleavable linkers is often limited by the need for multistep organic synthesis before implementation.