Alternative Pathway for the Formation of 4,5-dihydroxy-2,3-pentanedione, the Proposed Precursor of 4-hydroxy-5-methyl-3(2H)-furanone As Well As Autoinducer-2, and Its Detection As Natural Constituent of Tomato Fruit

The formation of 4-hydroxy-5-methyl-3(2H)-furanone (HMF, norfuraneol) by spinach ribosephosphate isomerase was reinvestigated. Incubation experiments using D-ribose-5-phosphate and D-ribulose-5-phosphate clearly revealed a spontaneous nonenzymatic formation of the hydroxy-furanone from the ketose-phosphate under physiological conditions at 35 degrees C and pH 7.5, whereupon up to 1.3% of D-ribulose-5-phosphate was transformed to HMF within 15 h. 4,5-Dihydroxy-2,3-pentanedione was deduced as ultimate precursor of HMF, since addition of o-phenylenediamine to the incubation mixture led to lower amounts of HMF and to the formation of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline, which was identified by means of high pressure liquid chromatography with diode array detection (HPLC-DAD), HPLC-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) and NMR spectroscopy. Additionally, the spontaneous formation of 4,5-dihydroxy-2,3-pentanedione was demontrated by its conversion to the respective alditol acetate using either NaBH(4) or NaBD(4) for the reduction. Comparative gas chromatography-mass spectrometry (GC-MS) analysis revealed the incorporation of two deuterium atoms and confirmed the dicarbonyl structure. Application of 1-13C-D-ribulose-5-phosphate as well as 5-13C-D-ribulose-5-phosphate and analysis of the derived quinoxaline derivatives by HPLC-ESI-MS/MS demonstrated the formation of the methyl-group at C-5 of the carbohydrate phosphate in consequence of a nonenzymatic phosphate elimination. Application of o-phenylenediamine into ripe tomatoes led to the detection of 3-(1,2-dihydroxyethyl)-2-methylquinoxaline by means of HPLC-MS/MS analysis implying the genuine occurrence of 4,5-dihydroxy-2,3-pentanedione in this fruit.