- UDP-glucose: furaneol glucosyltransferase (VvFGT)：Furaneol is the specific substrate of this enzyme, which catalyzes the glucosylation of Furaneol to form furaneol β -D-glucoside. [1]

1. Enzymatic glucosylation of Furaneol by recombinant VvFGT:
- Recombinant VvFGT (expressed in E. coli) catalyzed the conversion of Furaneol to furaneol-β -D-glucoside in the presence of UDP-glucose (cofactor) [1]
- Optimal reaction conditions for Furaneol glucosylation: pH 7.5, temperature 30°C; under these conditions, the enzyme showed maximum activity toward Furaneol, with a reaction efficiency 3.2-fold higher than that toward other structurally similar substrates (e.g., 4-hydroxy-2,5-dimethyl-3(2H)-furanone) [1]
- Substrate specificity: VvFGT exhibited high selectivity for Furaneol; no detectable glucosylation activity was observed when using other aroma compounds (e.g., vanillin, hexanol) as substrates [1]
- Product identification: The reaction product was confirmed as furaneol-β -D-glucoside via high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), with a retention time matching the authentic standard [1]

1. VvFGT enzyme activity assay (using Furaneol as substrate):
- Reaction system composition: Total volume 100 μl, containing 50 mM Tris-HCl buffer (pH 7.5), 5 mM UDP-glucose (cofactor), 1 mM Furaneol (substrate), and 10 μg recombinant VvFGT protein [1]
- Incubation conditions: The reaction mixture was incubated at 30°C for 30 minutes; the reaction was terminated by adding 10 μl of 10% trichloroacetic acid (TCA) [1]
- Product detection: The mixture was centrifuged at 12,000×g for 10 minutes; the supernatant was filtered through a 0.22 μm membrane and analyzed by HPLC [1]
- HPLC conditions: C18 column (4.6 × 250 mm), mobile phase (acetonitrile: water = 10:90, v/v), flow rate 1 ml/min, detection wavelength 280 nm; furaneol-β -D-glucoside was quantified using a standard curve [1]
2. Recombinant VvFGT expression and purification:
- The VvFGT gene was cloned into a prokaryotic expression vector and transformed into E. coli BL21 (DE3) cells [1]
- Cells were cultured in LB medium containing antibiotics at 37°C until OD₆₀₀ reached 0.6; isopropyl β -D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce protein expression, followed by incubation at 18°C for 16 hours [1]
- Cells were harvested by centrifugation (6,000×g for 10 minutes), resuspended in lysis buffer, and sonicated on ice; the lysate was centrifuged at 15,000×g for 20 minutes to collect the supernatant [1]
- The recombinant VvFGT protein was purified from the supernatant using affinity chromatography (based on the His-tag fused to the protein) and desalted using a desalting column; protein purity was verified by SDS-PAGE [1]

Absorption, Distribution and Excretion
2,5-Dimethyl-4-hydroxy-3[2H]furanone (furanol, DMHF) [3658-77-3] is an important flavor component of strawberry fruit. In this study, fresh strawberries were used as the natural source of DMHF, and four male and two female volunteers were given DMHF. Excretion was determined by measuring the levels of DMHF and its glucuronide in urine. The synthesis and structure of DMHF glucuronide were analyzed using 1H NMR (¹³C), 1C NMR (¹³C), and 2D NMR and mass spectrometry data. DMHF glucuronide in human urine was identified and quantified using an XAD-2 solid-phase extraction column combined with reversed-phase high-performance liquid chromatography (HPLC) and online UV/Vis or electrospray tandem mass spectrometry. Male and female volunteers excreted 59-69% and 81-94% of the total DMHF dose (free and glycoside-bound DMHF in strawberries) in their urine as DMHF glucuronide, respectively, within 24 hours. The amount of DMHF excreted was independent of the dose and the ratio of free DMHF to glycoside-bound DMHF in strawberry fruit. Naturally occurring DMHF, DMHF glucoside, and its 6'-O-malonyl derivatives in strawberries were not detected in human urine. Aromatic hydroxyfuranones and dihydroxypyranone derivatives generated from sugars and amino acids in the Maillard reaction are present in various processed foods, and in vitro studies have shown that they possess DNA single-strand disrupting activity. This study investigated the plasma absorption of two common soy sauce compounds—2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF)—after intraperitoneal or oral administration in mice at doses of 0.5–1.0 g/kg. Following intraperitoneal injection, both compounds appeared in plasma within 15 minutes and disappeared after 2 hours. After oral administration, both DMHF and HEMF appeared in plasma within 5 minutes, peaked after 15–45 minutes, and then gradually disappeared after 2 hours, indicating their absorption by the digestive tract. Oral administration of DMHF and HEMF resulted in dose-dependent production of micronucleated reticulocytes (MNRETs) in mouse peripheral blood. These results suggest that oral administration of DMHF and HEMF can lead to genetic damage.
4-Hydroxy-2,5-dimethyl-3(2H)-furanone is expected to have the same metabolic pathway as the main substance, namely, conjugation with glucuronic acid and excretion in urine.

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Metabolism/Metabolite>
4-Hydroxy-2,5-dimethyl-3(2H)-furanone is expected to have the same metabolic pathway as the main substance, namely, conjugation with glucuronic acid and excretion in urine.

Toxicity Summary
Identification and Uses: Dimethylhydroxyfuranone is a beige powder. It is used as a flavoring agent and experimental drug. Human Exposure and Toxicity: 2,5-Dimethyl-4-hydroxy-3(2H)-furanone (2,5-DMHF) is a caramel-like aromatic compound found in many processed foods and has been reported to be mutagenic. 2,5-DMHF produces superoxide, which subsequently generates hydrogen peroxide, thereby inducing metal-dependent DNA damage. Animal Studies: Sixty male and sixty female rats were fed diets containing 2,5-DMHF at doses of 0 (control group), 100, 200, or 400 mg/kg body weight/day for 24 months. At doses of 100 and 200 mg/kg body weight/day, no significant adverse reactions related to this compound were reported in any of the animals. In the highest dose group (400 mg/kg body weight/day), the mean body weight and weight gain of both males and females were significantly lower than those in the control group at 24 months. The mean survival rate of males in the highest dose group was also significantly lower than that in the control group at 24 months (approximately 20%, p<0.05). The authors attribute this result to the increased incidence of distal pituitary adenomas in males in the highest dose group, leading to compression of the hypothalamic region. The study concludes that these adenomas are common, spontaneous tumors unrelated to administration of 2,5-dimethylhydrofluoric acid (2,5-DMHF). 2,5-DMHF is mutagenic to Salmonella Typhimurium TA100 strain, regardless of metabolic activation, and induces micronucleation of peripheral blood reticulocytes in mice. Oral administration of 2,5-dimethylfuranone (DMHF) (at doses of 0.5–1.0 g/kg) resulted in the appearance of micronucleated reticulocytes in the peripheral blood of mice in a dose-dependent manner. The results showed that oral administration of DMHF could lead to genetic damage.

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Interaction
2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is a product of the Maillard reaction of sugars/amino acids and is found in various foods. This compound is mutagenic to Salmonella Typhimurium TA100 strain, inducing micronucleation in mouse peripheral blood reticulocytes regardless of the addition of the S9 mixture. At pH 7.4, the DNA strand breakage activity of this compound increased with increasing dosage and incubation time. The presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin traps, thiols, and metal chelators all inhibited the DNA strand breakage activity, as did the removal of dissolved oxygen from the incubation mixture. The addition of Fe(III) ions to the incubation mixture enhanced the DNA strand breakage activity. Incubation of DMHF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced the characteristic electron spin resonance signal of the DMPO-OH adduct, indicating the generation of hydroxyl radicals. The study found that DMHF generates hydroxyl radicals with the assistance of trace metal ions and induces DNA strand breaks. The mutagenicity of DMHF and its induction of micronucleated reticulocytes may be due to the modification of DNA by hydroxyl radicals. We analyzed the pro-oxidative properties of furanone compounds (including 2,5-furanone (furanol, 4-hydroxy-2,5-dimethyl-furan-3-one), 4,5-furanone (4,5-dimethyl-3-hydroxy-2(5H)-furanone) (sotropone), and cyclopentene (2-hydroxy-3-methyl-2-cyclopenten-1-one)) and their relationship with metal reducing activity. Only 2,5-furanone, known as "strawberry or pineapple furanone," inactivates aconitase, the enzyme most sensitive to reactive oxygen species (ROS), in the presence of ferrous sulfate, indicating that furanol/iron mediates ROS generation. 2,5-furanone causes single-strand breaks in pBR322 DNA in the presence of copper. Treatment of calf thymus DNA with 2,5-furanone and copper generates 8-hydroxy-2'-deoxyguanosine (8-DG). 2,5-furanone exhibits potent copper-reducing activity; therefore, it can induce DNA strand breaks and 8-DG formation by reducing copper ions to cuprous ions to generate superoxide radicals, which are then converted into hydrogen peroxide and hydroxyl radicals. However, isomers and analogs of 2,5-furanone, such as 4,5-furanone and cyclopentene, did not exhibit inhibition of aconitase activity, DNA damage (including strand breaks and 8-DG formation), or copper-reducing activity. 2,5-Furfural possesses a hydroxyketone structure, and its cytotoxicity can be attributed to its pro-oxidative properties: furanol/transition metal complexes generate reactive oxygen species, leading to aconitase inactivation and the formation of hydroxyl radicals, causing DNA base damage.

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Non-human toxicity values
Oral LD50 in mice: 1608 mg/kg

[1]. Molecular cloning and characterization of UDP-glucose: furaneol glucosyltransferase gene from grapevine cultivar Muscat Bailey A (Vitis labrusca × V. vinifera. J Exp Bot. 2015 Oct;66(20):6167-74.

4-Hydroxy-2,5-dimethylfuran-3-one is a furan compound with the structure 2,5-dimethylfuran, with additional oxo groups at the 3 and 4 positions, and a hydroxyl group at the 4 position. It is mainly found in strawberries and other similar fruits. 4-Hydroxy-2,5-dimethylfuran-3-one can be used as a flavoring agent, spice, and plant metabolite. It belongs to the furan class, enol class, and cyclic ketone class. It is the conjugate acid of 4-hydroxy-2,5-dimethylfuran-3-ol. Furan alcohols have been reported to be found in durian (Durio zibethinus), chili peppers (Capsicum annuum), and several other organisms with relevant data. Furan alcohols are metabolites found or produced in Saccharomyces cerevisiae.

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Therapeutic Use
/EXPL THER/ ... 4-hydroxy-5-methyl-3(2H)-furanone (HMF) and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) were added separately to semi-purified diets and fed to female ICR mice previously treated with benzo[a]pyrene (1.5 mg/week, orally, for 4 weeks) to induce forestomach tumors. Mice were sacrificed at 30 weeks of age. Both furans reduced forestomach tumors, with HDMF being more potent. Data suggest that HDMF and HMF inhibit carcinogenicity in this system by acting in the post-initiation phase.

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1. Chemical background of furanol:
- Furanol (4-hydroxy-2,5-dimethyl-3(2H)-furanone) is a naturally occurring volatile compound with a caramel-like aroma; it is widely found in fruits such as grapes, strawberries and pineapples, and imparts a unique flavor to these fruits[1]
2. Metabolism of furanol in grapes:
- In grape berries, furanol is synthesized during ripening and is stored mainly in the form of its glycoside (furanol-β-D-glucoside) to reduce volatility and increase stability; this glycosylation reaction is catalyzed by the enzyme VvFGT (UDP-glucose:furanol glycosyltransferase)[1]
3. Significance of VvFGT research on furanol:
- VvFGT The cloning and characterization of furanols provide important information for a deeper understanding of the molecular mechanisms of furanol glycosylation in grapes, which is crucial for regulating the aroma quality of grape products (e.g., wines) by controlling the content of free furanols and their glycosides [1].

C6H8O3

128.12592

128.047

3658-77-3

19309

White to off-white solid powder

1.3±0.1 g/cm3

215.5±40.0 °C at 760 mmHg

73-77 °C(lit.)

90.5±20.8 °C

0.0±0.9 mmHg at 25°C

1.513

0.34

1

3

0

9

181

0

INAXVXBDKKUCGI-UHFFFAOYSA-N

InChI=1S/C6H8O3/c1-3-5(7)6(8)4(2)9-3/h3,8H,1-2H3

4-hydroxy-2,5-dimethylfuran-3-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~780.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (19.51 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (19.51 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (19.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)