OBJECTIVES: This study presents a versatile, AI-guided workflow for the targeted isolation and characterization of prenylated flavonoids from Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae). METHODS: The approach integrates established extraction and chromatography-based fractionation protocols with LC-UV-HRMS/MS analysis and supervised machine-learning (ML) custom-trained classification models, which predict prenylated flavonoids from LC-HRMS/MS spectra based on the recently developed Python package AnnoMe (v1.0). RESULTS: The workflow effectively reduced the chemical complexity of plant extracts and enabled efficient prioritization of fractions and compounds for targeted isolation. From the pre-fractionated plant extracts, 2687 features were detected, 42 were identified using reference standards, and 214 were annotated via spectra library matching (public and in-house). Furthermore, ML-trained classifiers predicted 1805 MS/MS spectra as derived from prenylated flavonoids. LC-UV-HRMS/MS data of the most abundant presumed prenyl-flavonoid candidates were manually inspected for coelution and annotated to provide dereplication. Based on this, one putative prenylated (C5) dihydroflavonol (1) and four geranylated (C10) flavanones (2-5) were selected and successfully isolated. Structural elucidation employed UV spectroscopy, HRMS, and 1D as well as 2D NMR spectroscopy. Compounds 1 and 5 were isolated from a natural source for the first time and were named 6-prenyl-4'-O-methyltaxifolin and 3',4'-O-dimethylpaulodiplacone A, respectively. CONCLUSIONS: This study highlights the combination of machine learning with analytical techniques to streamline natural product discovery via MS/MS and AI-guided pre-selection, efficient prioritization, and characterization of prenylated flavonoids, paving the way for a broader application in metabolomics and further exploration of prenylated constituents across diverse plant species.

Core Facility Bioactive Molecules Screening and Analysis BOKU University Konrad Lorenz Str 20 3430 Tulln Austria

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 CZ 613 00 Brno Czech Republic

Department of Natural Drugs Masaryk University Palackeho 1946 1 CZ 612 00 Brno Czech Republic

Institute of Bioanalytics and Agro Metabolomics Department of Agricultural Sciences BOKU University Konrad Lorenz Str 20 3430 Tulln Austria

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