Molecular Design Strategy of Protein Isoform-Specific Fluorescent Probes by Considering Molecule in Its Entirety

Generally, different isoforms of proteins exert separate biological functions. However, due to similar structures and identical catalysis functions, distinguishing isoforms is challenging. Summarizing a molecular design strategy has great significance in developing a protein-specific fluorescent probe. Usually, recognition of a group was deemed to be the key to a protein isoform-specific response. However, some novel literature reported that fluorophore could play a vital role in the protein isoform-specific response. It means that any part of the fluorescent probe could affect the detected properties. In this work, we report the generation of the first probe to specifically recognize HexA­(β-N-acetylhexosaminidase A), Hex-C4, by adjusting the length of the linker. Hex-C4 exhibits specific recognition of HexA both in vitro and in living cells. The integration of the fluorescent spectrum and the MD (molecular dynamics) results provide two factors for the molecular design of isoform-specific fluorescent probes. One is the interaction between tetraphenyl ethylene (AIE fluorogen) and amino acid residues, and the other is the interaction between amino acid residues and the binding group. In this work, a powerful tool to detect HexA in living cells is reported for the first time. Further, a workable molecular design strategy for protein isoform-specific fluorescent probes is summarized.