title: Fluorescent labeling of cranberry proanthocyanidins with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF).
abstract: A novel methodology was developed to elucidate proanthocyanidins (PAC) interaction with extra-intestinal pathogenic Escherichia coli (ExPEC). PAC inhibit ExPEC invasion of epithelial cells and, therefore, may prevent transient gut colonization, conferring protection against subsequent extra-intestinal infections, such as urinary tract infections. Until now PAC have not been chemically labeled with fluorophores. In this work, cranberry PAC were labeled with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (DTAF), detected by high-performance liquid chromatography with diode-array detection and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We report single and double fluorescent-labeled PAC with one or two chlorine atoms displaced from DTAF in alkaline pH via nucleophilic substitution. Fluorescent labeling was confirmed by fragmentation experiments using MALDI-TOF/TOF MS. Fluorescent labeled PAC were able to promote ExPEC agglutination when observed with fluorescence microscopy. DTAF tagged PAC may be used to trace the fate of PAC after they agglutinate ExPEC and follow PAC-ExPEC complexes in cell culture assays.
authors: University of Wisconsin-Madison, Dept. of Food Science, 1605 Linden Drive, Madison, WI 53706, USA; University of Wisconsin-Madison, Reed Research Group, Dept. Animal Sciences, 1675 Observatory Drive, Madison, WI 53706, USA.
authors: Rodrigo P
authors: RP
authors: Feliciano
authors: Biological & Biomaterials Preparation, Imaging & Characterization Facility, Dept. Animal Sciences, 1675 Observatory Drive, Madison, WI 53706, USA.
authors: Joseph A
authors: JA
authors: Heintz
authors: University of Wisconsin-Madison, Reed Research Group, Dept. Animal Sciences, 1675 Observatory Drive, Madison, WI 53706, USA; Complete Phytochemical Solutions, LLC, 317 South Street, Cambridge, WI 53523, USA.
authors: Christian G
authors: CG
authors: Krueger
authors: University of Wisconsin-Madison, Dept. of Chemistry, 1101 University Ave., Madison, WI 53706, USA.
authors: Martha M
authors: MM
authors: Vestling
authors: University of Wisconsin-Madison, Reed Research Group, Dept. Animal Sciences, 1675 Observatory Drive, Madison, WI 53706, USA; Complete Phytochemical Solutions, LLC, 317 South Street, Cambridge, WI 53523, USA. Electronic address: jdreed@wisc.edu.
authors: Jess D
authors: JD
authors: Reed
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry*; D005638:Fruit / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D010936:Plant Extracts / Q000737:chemistry*; D044945:Proanthocyanidins / Q000737:chemistry*; D029799:Vaccinium macrocarpon / Q000737:chemistry*
chemical_list: D010936:Plant Extracts; D044945:Proanthocyanidins; D019793:Fluorescein
keywords: 5-([4,6-Dichlorotriazin-2-yl]amino)fluorescein; Cranberry; DTAF; Fluorescence; MALDI-TOF MS; MTAF; Microscopy; Proanthocyanidins; TAF