Bisaryl Hydrazones as Exchangeable Biocompatible Linkers

摘要

The selective enrichment of tagged molecules from complex biological mixtures is of primary importance in chemical biology and proteomics. One of the most widely applied enrichment methods is affinity purification on (strept)avidin beads with biotin as an affinity tag. This strategy takes full advantage of the high affinity of biotin for (strept)avidin (K_a ≈1.7x10~(15)M~(-1)). However, the method is limited by the harsh, denaturing conditions required for elution, such as boiling in a buffer containing sodium dodecyl sulfate (SDS) or treatment with an 8 m solution of guanidine (pH 1.5). Under these conditions, protein structure and function are lost, and target proteins may be contaminated with proteins bound nonspecifically to the beads. Two strategies have been explored for elution under mild conditions: 1) weakening of the biotin-(strept)avidin interaction by modulation of the K_a value and 2) introduction of a proteolytically or chemically cleavable linker. Although the first strategy does improve the release of biotinylated proteins from (strept)a-vidin beads, it adversely affects the stringency of the immobilization. The second strategy enables site-specific cleavage; however, premature cleavage has been reported, and cleavage conditions have no demonstrated compatibility with active biomolecules. Furthermore, the general applicability of the cleavable linkers is often limited by the need for multistep organic synthesis before implementation.