Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

Homo- and hetero-bifunctional linkers play vital roles in constructing a variety of functional systems, ranging from protein bioconjugates with drugs and functional agents, to surface modification of nanoparticles and living cells, and to the cyclization/dimerization of synthetic polymers and biomolecules. Conventional approaches for assaying conjugation extents typically rely on ex situ techniques, such as mass spectrometry, gel electrophoresis, and size-exclusion chromatography. If the conjugation process involving bifunctional linkers was rendered fluorogenic, then in situ monitoring, quantification, and optical tracking/visualization of relevant processes would be achieved. In this review, conventional non-fluorogenic linkers are first discussed. Then the focus is on the evolution and emerging applications of fluorogenic bifunctional linkers, which are categorized into hetero-bifunctional single-caging fluorogenic linkers, homo-bifunctional double-caging fluorogenic linkers, and hetero-bifunctional double-caging fluorogenic linkers. In addition, stimuli-cleavable bifunctional linkers designed for both conjugation and subsequent site-specific triggered release are also summarized.