A RuAAC-Based Fluorescent Labeling Strategy for Efficient Detection of Alkyne-Containing Natural Products

Natural products (NPs) represent an indispensable reservoir for drug discovery, with alkyne-containing natural products (ANPs) exhibiting diverse biological activities. However, the efficient discovery of ANPs is severely hindered by low natural abundance, inherent chemical instability, and the constraints of conventional resource screening strategies, highlighting an urgent need for efficient discovery strategies to unlock the hidden potential of ANPs. Current fluorescent labeling methodologies, predominantly relying on copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, offer selective detection of terminal alkyne-containing natural products (TANPs) but are inherently incapable of targeting internal alkyne-containing natural products (IANPs). This limitation has long remained an unresolved bottleneck in comprehensive ANP screening. To address this limitation, we developed a fluorescent labeling strategy employing ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) reaction with 3-azido-7-hydroxycoumarin as a probe. This method enables efficient and selective labeling of both IANPs and TANPs under mild reaction conditions (20 °C, 5 h). Under the optimized conditions, the assay exhibits detection limits of 3.95 μM for internal alkyne and 1.55 μM for terminal alkyne, respectively, alongside broad substrate tolerance and excellent selectivity. To demonstrate its practical utility, we applied this method to screen 22 medicinal plants. Remarkably, seven samples showed significant fluorescence enhancement, indicating the presence of ANPs. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS) analysis observed the existence of labeled products in target plants, validating the accuracy and reliability of the method. Overall, this work provides a rapid, efficient, and high-throughput screening strategy for the comprehensive discovery of ANPs and holds great promise for accelerating the discovery of lead compounds from complex NP mixtures.