Steric Control in Activator‐Induced Nucleophilic Quencher Detachment‐Based Probes: High‐Contrast Imaging of Aldehyde Dehydrogenase 1A1 in Cancer Stem Cells

Abstract

Turn‐on fluorescence probes can visualize the enzyme activity with high contrast. We have established a new turn‐on mechanism, activator‐induced nucleophilic quencher detachment (AiQd), and developed AiQd‐based turn‐on fluorescence probes for the detection of enzymes. Herein, we demonstrate that the precise steric control efficiently quenches the fluorescence of AiQd‐based turn‐on probes before the enzymatic transformation. Theoretical calculation appropriately predicted the ratio of the fluorescence‐quenched closed‐ring form of probes. βC5S‐A, which has a sterically demanding methyl group at the β‐position of a fluorescence‐quenching nucleophilic mercapto group, showed a low background signal. βC5S‐A responded to aldehyde dehydrogenase 1A1 (ALDH1A1) with high selectivity, thereby enabling high‐contrast live imaging of cancer stem cells (signal‐to‐noise ratio >10). The ALDH1A1‐responsiveness of βC5S‐A was not significantly affected by amino acids and biological thiols, such as cysteine and glutathione.

Real-time fluorescent monitoring of phase I xenobiotic-metabolizing enzymes

“…Probe 23 represents an innovative approach to detecting ALDH1A1 activity with improved specificity and reduced background noise, positioning it as a promising tool for the identification and study of cancer stem cells. 75…”

“…Probe 23 represents an innovative approach to detecting ALDH1A1 activity with improved speci-city and reduced background noise, positioning it as a promising tool for the identication and study of cancer stem cells. 75 Pereira and colleagues devised a radioactive aromatic aldehyde probe, denoted as 24, designed for ALDH1A1 imaging through the implementation of a prodrug strategy. The probe, initially inert, undergoes activation by esterase enzymes within cells.…”

Dual-responsive near-infrared turn-on fluorescent probe for cancer stem cell-specific visualization

“…11 The always-on fluorescent probe ALDEFLUOR 15 and analogous derivatives [16][17][18] are frequently used for CSC isolation based on fluorescence-activated cell sorting. Recently, turn-on fluorescent probes, [19][20][21][22][23][24] which are non-emissive before ALDH1A1-mediated transformation, have been developed for both visualization and isolation of CSCs in cancer cells. Some of the near-infrared (NIR) turn-on fluorescent probes were successfully applied to CSC-specific visualization in vivo and ex vivo.…”