Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.     

2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF); antimicrobial compound with cell cycle arrest in nosocomial pathogens

2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF), an aroma compound found in a number of fruits and foods, has shown various biological properties in animal models, but its antimicrobial effect remains poorly understood. The current study investigated the antimicrobial effect of DMHF using human pathogenic microorganisms including clinically isolated antibiotics-resistant strains. The results indicated that DMHF exhibited broad spectrum antimicrobial activities in an energy-dependent manner without hemolytic effect on human erythrocytes. To confirm antifungal effect of DMHF, we investigated the effect on dimorphism of Candida albicans induced by FBS, which plays a key role for pathogenesis in host invasion. The result showed that DMHF exerted a potent antifungal activity on the serum-induced mycelia of C. albicans. To elucidate the physiological changes of the fungal cells induced by DMHF, cell cycle analysis was performed, and the results showed that DMHF arrested the cell cycle at the S and G2/M phase in yeast. Therefore, it could be expected that DMHF may have potential as an anti-infective agent in human microbial infections.     

Formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone by Zygosaccharomyces rouxii: identification of an intermediate

The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from D-fructose-1,6-bisphosphate by the yeast Zygosaccharomyces rouxii was studied with regard to the identification of intermediates present in the culture medium. Addition of o-phenylenediamine, a trapping reagent for alpha-dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from D-fructose-1,6-bisphosphate under the applied growth conditions (30 degrees C; pH 4 to 5). Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3). Q1 and Q2 were formed independently of Z. rouxii cells, whereas Q3 was detected only in incubation systems containing the yeast. Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF. Since HDMF was detected only in the presence of Z. rouxii cells, additional enzymatic steps were presumed. Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with D-fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts.     

Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from D-fructose 1,6-diphosphate

The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from D-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC-DAD and HPLC-UV-MS/MS. The temperature optimum for HDF formation was 30 degrees C, whereas the pH value (pH 3-10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and D-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-(2)H(2)]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-(13)C]-D-fructose 1,6-diphosphate with [4R,S-(2)H(2)]-NADH revealed that the hydride is transferred to C-5 or C-6 of the D-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from D-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated.     

FaQR, required for the biosynthesis of the strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone, encodes an enone oxidoreductase

The flavor of strawberry (Fragaria x ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of approximately 37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripening-induced, auxin-dependent putative quinone oxidoreductase, Fragaria x ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.     

Nonenzymatic formation of 4,5-dimethyl-3-hydroxy-2(5H)-furanone in foodstuffs

The mechanism of formation of 4,5-dimethyl-3-hydroxy-2/5H/-furanone in some food-stuffs was investigated. The compound was found to result from condensation of acetaldehyde under oxidative conditions. The intermediate product of the condensation is 2-oxobutanoic acid.     

Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development.     

The formation mechanism by yeast of 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in Miso

The mechanism of the formation of 4-hydroxy-2(or 5)-ethyl-5-(or 2)-methyl-3(2H)-furanone (HEMF) with yeast under caltivation in a medium containing amino-carbonyl reactants of ribose and glycine was investigated using stable isotopes of the corresponding compounds. It was confirmed that the skeleton of the five-membered ring and the methyl group of the side chain of HEMF was formed from ribose, and that the ethyl group was derived from the glucose metabolite by yeast. The formation of HEMF was confirmed when acetaldehyde as the glucose metabolite and a cell-free extract from yeast were added to the medium containing amino-carbonyl reactants. These results suggest that the role of yeast in HEMF formation is not only to provide the glucose metabolite, but also in combining the amino-carbonyl reactants with the glucose metabolite.     

Genotoxic effects of the chlorinated hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-dichloro-5-hydroxy-2[5H]-furanone in Tradescantia micronucleus assays

This is the first report of clastogenic effects of chlorinated hydroxyfuranones (CHFs) in plants. Two byproducts of water chlorination, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-dichloro 5-hydroxy-2[5H]-furanone (MA) induced a dose dependent increase of micronuclei (MN) in pollen mother cells of Tradescantia when doses up to 100 μg MX and 500 μg MA were applied directly to the inflorescences. In contrast, exposure of the stems in aqueous solutions containing up to 1 mg/I MX and 10 mg/I MA did not cause a positive response.     

Developmental toxicity evaluation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in Wistar rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a genotoxic chlorination by-product in drinking water. There is some evidence that it has developmental toxic effects in vitro but its potential to cause developmental effects in vivo is not known. The developmental effects were evaluated in Wistar rats. Rats (22-26 dams per dose group) were administered MX by gavage at the dose levels of 3, 30, or 60 mg/kg in water on gestation days 6-19. Control animals received plain water. Clinical signs, body weight, and food and water consumption were recorded for the dams. On gestation day 20, a cesarean section was performed and the ovaries anduterine contents of the dams were examined and the liver, kidneys, spleen, and thyroid glands weighed. The fetuses of all dose groups were weighed, sexed, and observed for external and skeletal malformations and the fetuses of the two highest dose groups were evaluated for visceral malformations. The highest dose, 60 mg/kg of MX, was slightly toxic to the dams. It decreased the corrected body weight gain of dams by 32% and the water consumption by 16-17%. Kidney and liver weights were slightly increased. MX did not affect the number of implantations nor did it cause any resorptions. The body weights of fetuses were not significantly affected. MX did not cause external malformations or skeletal anomalies. Two fetuses at 60 mg/kg and one fetus at 30 mg/kg had major visceral malformations (persistent truncus arteriosus, diaphragmatic hernia, dilated aorta with a stenosis of pulmonary arteries) and two minor artery abnormalities were observed in those animals. The frequency of unilateral displaced testis was slightly higher (9.2%) in the 60-mg/kg dose group than in controls (1.6%). Since the abnormalities did not form a consistent pattern and occurred most at maternally toxic dose, we conclude that MX can be regarded as non-teratogenic.     

2,3-Dimethylpyrazine (3DP) and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) generated by the Maillard reaction in foods affect autonomic nervous activity and central nervous activity in human

ABSTRACT

This study investigated the effect of the odors generated by the glycine/glucose Maillard reaction and the potent odorants 2,3-dimethylpyrazine (3DP) and 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) on the human mood and integrative physiological activity. The score of certain subjective moods, especially anger-hostility, and tension-anxiety were decreased significantly after inhalation of the Maillard reaction sample and DMHF, and fatigue-inertia mood was also significantly decreased by DMHF, suggesting a sedative effect of these odors on mood, while 3DP had no effect. Miosis rate and fingertip temperature increased significantly following inhalation of the odor from the Maillard reaction sample and both potent odorants, suggesting that the parasympathetic nervous system dominates through suppression of the sympathetic activity. The physiological relaxing effect of these odors was also confirmed by decreased flicker frequency value and decreased oxyhemoglobin in the prefrontal cortex.     

Structural Basis for the Enzymatic Formation of the Key Strawberry Flavor Compound 4-Hydroxy-2,5-dimethyl-3(2H)-furanone

The last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H-dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in six different states or complexes at resolutions of ≤1.6 Å, including those with HDMF as well as three distinct substrate analogs. Our crystallographic analysis revealed a monomeric enzyme whose active site is largely determined by the bound NAD(P)H cofactor, which is embedded in a Rossmann-fold. Considering that the quasi-symmetric enolic reaction product HDMF is prone to extensive tautomerization, whereas its precursor HMMF is chemically labile in aqueous solution, we used the asymmetric and more stable surrogate product 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (EHMF) and the corresponding substrate (2E)-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone (EDHMF) to study their enzyme complexes as well. Together with deuterium-labeling experiments of EDHMF reduction by [4R-²H]NADH and chiral-phase analysis of the reaction product EHMF, our data show that the 4R-hydride of NAD(P)H is transferred to the unsaturated exocyclic C6 carbon of HMMF, resulting in a cyclic achiral enolate intermediate that subsequently becomes protonated, eventually leading to HDMF. Apart from elucidating this important reaction of the plant secondary metabolism our study provides a foundation for protein engineering of enone oxidoreductases and their application in biocatalytic processes.     

Identification of 4,5-Dimethyl-3-hydroxy-2(5H)-furanone (Sotolon) and Ethyl 9-Hydroxynonanoate in Botrytised Wine and Evaluation of the Roles of Compounds Characteristic of It

Two new components of botrytised wine were identified: 4,5-dimethyl-3-hydroxy-2(5H)-furanone (Sotolon) and ethyl 9-hydroxynonanoate. Sotolon, the key substance of cane sugar aroma, was identified as the sugary flavor substance of botrytised wine by means of gas chromatography-mass spectrometry after column chromatographic separation on DEAE-Sephadex and silica gel. Ethyl 9-hydroxynonanoate was identified by chemical ionization and electron impact mass spectrometry. To evaluate the role of 17 volatile and 5 nonvolatile compounds characteristic of botrytised wine, these compounds were added to a normal wine. This produced a sweet, honey-like flavor similar to that of botrytised wine. The importance of Sotolon and the role of each group of flavor substances in producting this flavor was clarified by omission tests.     

Formation by Yeast of the HEMF (4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone) Aroma Component in Miso with Aging

Two kinds of miso, one with added precultured yeast and the other without, were compared with respect to the changes in the concentration of HEMF formed and the number of yeast cells in the process of aging. In miso without added yeast, the HEMF concentration increased with the increasing number of existing yeast cells. In miso without yeast aged for 21 days after the miso mash, 0.06 ppm HEMF was detected when the cell number was 2.2 × 10³ cell/g. In yeast-added miso aged for 7 days after the miso mash, no HEMF was detected, although the number of yeast cells was 1.6 × 10⁶ cell/g. In yeast-added miso aged for 14 days after the miso mash, HEMF was first detected. The pH levels of miso without yeast and with added yeast when HEMF was first detected were 5.59 and 5.57, respectively. It is suggested that the formation of HEMF in miso containing a high concentration of reducing sugar and salt was related to the growth of yeast and started when the pH level fell to less than 5.6.     

Evaluation of multiwavelength culture fluorescence for monitoring the aroma compound 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) production

Fluorescence spectra of a 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) fermentation culture broth were combined with measurable process variables for off-line and on-line process monitoring. Culture broth fluorescence in UV and visible ranges was acquired by a fiber optic LCD array spectrometer. Process dynamics was followed on-line using a fiber optic probe attached to an external recirculation loop of the bioreactor. Partial least squares and stepwise regression methods were used to correlate measurable process parameters with the components of the fluorescence spectra. Both methods provided adequate approximation of yeast density, HEMF, glucose, and ethanol concentrations from fluorescence spectra. HEMF production was observed during the oxido-reductive growth phase when there was a lack of measurable oxygen in the culture broth and an excess of glucose. The addition of glucose resulted in the rapid production of HEMF and other metabolite intermediates such as ethanol, acetate, and glycerol.     

2,2-Dimethyl-4,5-diaryl-3(2H)furanone derivatives as selective cyclo-oxygenase-2 inhibitors

A series of 2,2-dimethyl-5-[4-(methylsulfonyl)phenyl]-4-phenyl-3(2H)furanones was prepared and evaluated for their ability to inhibit cyclo-oxygenase-2 (COX-2).