JPH0699417B2 - D-Glucosaccharoascorbic acid and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to D-glucosaccharoascorbic acid or a salt thereof, and a method for producing the same.

More specifically, the present invention relates to D-glucosaccharoascorbic acid in which the 6-hydroxymethyl group of D-erythorbic acid has become a carboxyl group (“D-erythro-hexo-2-
Enaro-1,4-lactone ") or a salt thereof and a process for producing the same, and the present compound has an antioxidant effect.

Prior art L-ascorbic acid, D-erythorbic acid, L-grosaccharoascorbic acid [US Pat. No. 2,428,438,
No. 2,483,251; Carbo Hydrate Research (Carbo
hydrate Research), 60 (1978) 251-258] and the like are known as compounds having an antioxidant effect.

Problems to be Solved by the Invention The present inventor has conducted various studies for the purpose of synthesizing L-ascorbic acid, D-erythorbic acid, and related compounds thereof, and found that 2-keto-D-glucaric acid or its 2 ,
The present invention has been completed by discovering that a novel compound, D-glucosaccharoascorbic acid, can be obtained by treating a 3-O-acetal or a ketal with an acid, and that this compound has an antioxidant action.

That is, the present invention includes (1) D-glucosaccharoascorbic acid or a salt thereof, and (2) 2-keto-D.
A method for producing D-glucosaccharoascorbic acid, which comprises treating glucanic acid or its 2,3-O-acetal or ketal with an acid.

The D-glucosaccharoascorbic acid of the present invention is 2-keto-D-glucaric acid (“D-Arabino-2-hexulosaric a
cid ") or its 2,3-O-acetal or ketal by treatment with an acid. As the acetal or ketal body, the constituent part of the acetal or ketal is represented by the general formula (In the formula, R ₁ and R ₂ are the same or different and are hydrogen, an alkyl group, an optionally substituted phenyl group, or R ₁ and R ₂
Which may together form a cycloalkyl group) can be used.

The alkyl group represented by R ₁ and R ₂ has 1 carbon atom.
To 5 alkyl groups, and specific examples thereof include methyl, ethyl, propyl, i-propyl, butyl, i-butyl, sec-butyl, t-butyl, amyl, i-amyl and the like. .

As the substituent of the phenyl group which may be substituted, halogen such as fluorine, chlorine, bromine and iodine, hydroxyl group, alkoxy having 1 to 3 carbon atoms, alkyl having 1 to 3 carbon atoms, amino group, nitro group, cyano Group, carboxyl group, ester group, carbamoyl group, trifluoromethyl group and the like.

When R ₁ and R ₂ together form a cycloalkyl group,
The carbon number thereof is usually 2 to 6, and specific examples thereof include ethylene, trimethylene, tetramethylene, pentamethylene and hexamethylene.

Further, as a salt of D-glucosaccharoascorbic acid,
Alkali metal salts (eg, lithium salt, sodium salt, potassium salt), alkaline earth metal salts (eg, magnesium salt,
Examples thereof include calcium salt, barium salt) and ammonium salt.

2 used as a raw material compound in the production method of the present invention
The keto-D-glucaric acid or its 2,3-O-acetal or ketal may be used alone or as a mixture. In addition, these raw material compounds may be used as salts, and examples thereof include alkali metal salts (eg, lithium salt, sodium salt, potassium salt), alkaline earth metal salts (eg, magnesium salt, calcium salt, barium salt). ) Or an ammonium salt.

The acid used in the method for producing D-glucosaccharoascorbic acid of the present invention is not particularly limited, but examples thereof include hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, fluorosulfuric acid, and perfluorosulfuric acid. Mineral acids such as chloric acid, trifluoromethanesulphonic acid, methanesulphonic acid, paratoluenesulphonic acid, acetic acid, monochloroacetic acid, trichloroacetic acid, trifluoroacetic acid and organic acids such as H ⁺ type ion exchange resins can be mentioned by themselves. Alternatively, it may be dissolved or suspended in water as needed. The acid used may be a single acid or a mixture of two or more of the above-mentioned acids.

The amount of the acid used is not particularly limited, but a large excess amount of about 0.1 times to about 100 times the molar amount of the starting material is usually used, and more preferably about 0.2 times to about 50 times the molar amount.

2-keto-D-glucanic acid salt or 2 as a starting material
When a salt of 2,3-O-acetal or 2,3-O-ketal of keto-D-glucaric acid is used, it is neutralized by adding a theoretical amount or a slight excess of acid for neutralizing these salts, At this time, the by-produced salt may be previously removed from the reaction system by a known technique such as a filtration method, an ion exchange membrane method, an ion exchange resin method, an extraction method with an appropriate solvent, column chromatography or electrodialysis method. .

Water is usually used as the reaction solvent used in the production method of the present invention, but any solvent that does not inhibit the reaction can be used. For example, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene,
Examples thereof include nitromethane, nitroethane, t-butanol, ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, acetone, methyl ethyl ketone, cyclopentanone, cyclohexanone, acetonitrile, propionitrile and benzonitrile. The reaction can be carried out in a mixed solvent composed of two or more of these solvents. When an organic solvent is used as the reaction solvent, the reaction rate may be improved by adding a surfactant to the reaction system.

Examples of the surfactant include nonionic surfactants such as 18-crown-6, dibenzo-18-crown-6, [2,2,2] -cryptate and polyoxyethylene alkylallyl ether, such as tetrapropylammonium. Various ammonium salts such as chloride, benzyltrimethylammonium bromide, phenyltriethylammonium chloride, cetyltrimethylammonium chloride, stearyltrimethylammonium bromide, 1-phenacylpyridinium chloride, stearylpropylenediamine dihydrochloride, and for example benzyltriphenylphosphonium chloride , Vinyl triphenylphosphonium bromide and other phosphonium salts and other cationic surfactants, such as higher fatty acids and alkylallyl sulfonates and other anionic interfaces Include sexual agent, it may safely any be used alone or alone or in combination. The amount of surfactant added is about 0.05 to 5% by weight, preferably 0.1 to 3% by weight, based on the substrate.

The reaction temperature is not particularly limited, but is usually about 0 ° C to about 150 ° C.
The temperature is within a range of about 10 ° C to about 10 ° C.
It is in the range of 100 ° C.

The reaction time varies depending on the type of starting material, the type of salt, the type of acid, the amount used, and the reaction conditions, but it is usually about 10 minutes to 24 hours, preferably about 20 minutes to 12 hours. Is.

The isolation of D-glucosaccharoascorbic acid thus produced from the reaction system is slightly different depending on whether the starting material used is a free acid or a salt thereof. That is, the raw material used is a salt of 2-keto-D-glucaric acid or 2-keto-D-
Removal of a salt of glucanic acid, which is a salt of 2,3-O-acetal or 2,3-O-ketal, and which has not been removed in advance during the reaction by a neutralization reaction with an acid. Is required. For the desalting operation, the reaction solution as it is or after treatment such as solvent removal, a known technique such as filtration, ion exchange membrane method, ion exchange resin method, extraction method with a suitable solvent, column chromatography, or electrodialysis method is used. This is done alone or in combination as appropriate.

The starting material is a salt of 2-keto-D-glucanic acid or a 2,3-O-acetal of 2-keto-D-glucanic acid or 2,3
Even if the salt of -O-ketal is desalted during the reaction, or 2-keto-D-glucanic acid or 2-
2,3-O-acetal of keto-D-glucanic acid or
In the case of 2,3-O-ketal, the solvent and the low-boiling substance are distilled off from the reaction product by a conventional method to extract the residue,
The target D-glucosaccharoascorbic acid can be easily isolated and purified by known means such as column chromatography and recrystallization. D-Glucosaccharoascorbic acid is isolated as an anhydrate or a monohydrate.

When isolated as a salt of D-glucosaccharo-ascorbic acid, D-glucosaccharo-ascorbic acid obtained by the above method and a suitable base such as alkali metal oxide, alkali metal hydroxide or alkali metal carbonate. Or reacted with bicarbonate, alkaline earth metal oxides, alkaline earth metal hydroxides or alkaline earth metal carbonates or suitable amines, or suitable alkali metal or alkaline earth metal or suitable ammonium It can be converted by contact with a cation exchange resin substituted with. Further, the reaction product is not isolated once as D-glucosaccharoascorbic acid, and the reaction product is treated with a suitable base such as an alkali metal oxide, an alkali metal hydroxide, an alkali metal carbonate or bicarbonate, an alkaline earth metal oxide. , Neutralized with alkaline earth metal hydroxides or carbonates or suitable amines, or contacted with a cation exchange resin substituted with a suitable alkali metal or alkaline earth metal or suitable ammonium The desired salt of D-glucosaccharoascorbic acid is isolated by a known means or technique such as direct conversion to a salt of D-glucosaccharoascorbic acid by distilling the solvent, and then distilling off the solvent to recrystallize or reprecipitate. ，
It may be purified.

In the production method of the present invention, 2-keto-D-glucanic acid or a salt thereof used as a raw material compound is obtained by the following method. Among the raw material compounds, the calcium salt of 2-keto-D-glucaric acid is a known compound synthesized by subjecting D-glucaric acid to an oxidation reaction using a microorganism belonging to the genus Pseudomonas [Czechoslovak Patent No.
222, 577 (1984) and Chemical Abstracts, Vol. 101, 22036x]. In addition, 2-keto-D-glucaric acid is subjected to general fermentation oxidation reaction of D-glucose using Pseudoglyconobacter spp. [Eg, Pseudogluconobacter Saccharoketgenes K591s (FERM P-8481, IFO14464)]. It can also be synthesized by the method described above, and can be easily isolated as a sparingly water-soluble calcium salt. By reacting a calcium salt of 2-keto-D-glucaric acid with an acid or a cation exchange resin (H ⁺ type), it is possible to obtain free 2-keto-D-glucaric acid, and from this, a conventional method can be used. By 2-keto-D-
It can lead to the salt of glucaric acid.

Then, 2-keto-D-glucanic acid is acetalized or ketal is obtained. By converting this acetalization or ketalization to 2,3-O of 2-keto-D-glucaric acid.
-Acetalization or ketalization is carried out by reacting 2-keto-D-glucaric acid or a salt thereof with an aldehyde, a ketone or a reactive derivative thereof in the presence of an appropriate acetalization or ketalization catalyst. The above-mentioned aldehyde or ketone has the general formula R ₁ CO
A compound of R ₂ (R ₁ and R ₂ are as defined above) is used.

Further, as the reactive derivative of aldehyde or ketone, (In the formula, R ₁ and R ₂ have the same meanings as described above, and R ₃ and R ₄
Are the same or different and have 1 to 5 carbon atoms, or
R ₃ and R ₄ together show that may form a cycloalkyl group of 2-6 carbon atoms). Specific examples of R ₃ and R ₄ include, for example, methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t
-Butyl, amyl, i-amyl and R ₃ and R ₄ together form ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene and the like.

The acetalization or ketalization reaction can be carried out using a known acid catalyst (Japanese Patent Laid-Open No. 60-72895), for example, by the method shown in the reference example described later.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples along with Reference Examples.

Reference Example 1 Peptone 0.5%, yeast extract 0.5%, glucose 1.0%, K
30 ml of a medium containing ₂ % HPO ₄ (PYG medium) was dispensed into a 200 ml Erlenmeyer flask, and the above sterilization was performed at 120 ° C. for 20 minutes. To this flask, 1 platinum loop of Pseudomonas aeruginosa IFO3448, which had been grown for 2 days at 28 ° C. on a slant medium prepared by adding 2.0% agar to PYG medium, was inoculated. Incubate at 30 ° C with shaking (200 rpm) for one day,
It was used as a seed culture.

D-glucaric acid / monopotassium salt (manufactured by Sigma) in advance
The pH was adjusted to 7.0 with NaOH, and the bacteria were slowly filtered using a 0.45 micron filter and added to PYG medium so as to be 1%. 1 ml of the above-mentioned seed culture solution was transferred to a 200 ml Erlenmeyer flask containing 20 ml of this medium, and cultured by shaking at 30 ° C. for 24 hours.
The thus obtained culture solution contained 9.02 mg / ml of 2-keto-D.
-Glucaric acid was measured by high performance liquid chromatography [High performance liquid chromatography using a sulfonated polystyrene gel packed column (Shimadzu SCR-101H column, 7.9 mm x 30 cm) (mobile phase: dilute sulfuric acid with pH 2.2, Flow rate;
0.5 ml / min, detector; differential diffractometer))]. The bacteria were removed from 590 ml of the culture broth by centrifugation to obtain 580 ml of supernatant. The supernatant is Amberlite cation exchange resin IR120
(H ⁺ type, 200 ml) flow down the column, wash the column with 150 ml of deionized water to remove cations, then activate carbon (70
ml) column, and the column was washed with 50 ml of deionized water for decolorization. The pH of the 780 ml of the passing liquid was adjusted to 6.5 with Ca (OH) ₂ , the white turbidity was removed by filtration, and the filtrate was concentrated under reduced pressure to about 20 ml. White amorphous crystals are formed in the concentrate. The crystals were collected on a glass filter, washed with a small amount of cold water, methanol and ethyl ether, dried under reduced pressure, and 5.
04 g of 2-keto-D-dicalcium glucarate 3.5 hydrate was obtained. The analytical values of this crystal were as follows.

Mp: S152 to S157 ° C. (decomposition) Elemental analysis _{(%) C 6 H 6 O} 8 · Ca · 3.5H 2 O as a theory: C; 23.30, H; 4.24 , Ca; 12.96 Found: C; 23.15, H; 4.18, Ca; 14.00 Specific light intensity: ▲ [α] ²⁵ _D ▼ ＝ -11.0 ° (c = 1.075%, 0.1
N HCl, immediately after dissolution) ▲ [α] ²⁵ _D ▼ ＝ ＋ 9.0 ° (c = 1.075%, 0.1N HCl, after stabilization) Infrared absorption spectrum (IR) Wavenumber of major absorption maximum measured by KBr method ( cm ^-1 ) is as follows.

3590,3500,3400 ~ 2700 (br), 1650,1600,1430,1380,136
0,1300, 1250,1240,1220,1125,1095,1065,1040,1005,99
5,955,900,840,800,765,725 Note that the water content of 2-keto-D-glucaric acid dicalcium salt varies depending on the drying conditions, and the elemental analysis results show that C ₆ H ₆ O ₈ · Ca.
・ 1H ₂ O to C ₆ H ₆ O ₈・ Ca ・ 4H ₂ O were obtained.

Reference Example 2 D-glucose 2.0%, peptone 1.0%, dry yeast 1.0%
20 ml of a seed medium consisting of 2.0% CaCO ₃ and CaCO ₃ was dispensed into a 200 ml Erlenmeyer flask and steam sterilized in advance at 120 ° C. for 20 minutes. In this flask, 2.5% D-sorbitol, 1.0% peptone, 1.0% yeast extract, 0.2% CaCO ₃ and agar 2.
They were grown at 28 ° C for 4 days on a 0% slope. Pseudogluconobacter saccharoketgenes K591S strain (F
ERM P-8481, IFO 14464) inoculated with 1 platinum loop
Culturing was carried out at 200C for 2 days with shaking (200 rpm) to obtain a seed culture solution. 200 ml of medium with the same composition as the seed medium was dispensed into a 1-volume Erlenmeyer flask and sterilized in the same manner as above.
After transplanting ml, shaking culture was carried out at 30 ° C. for 3 days.

In the obtained culture solution, 19.4 mg / ml of 2-keto-D-glucaric acid was produced.

1600 ml of this culture solution is centrifuged (7,000 rpm, 10 minutes),
The precipitate containing the cells was removed to obtain 1520 ml of supernatant. 4 supernatants
Amorphous 2-keto-D after cooling to ℃ for 3 days
-Glucaric acid dicalcium salt crystals form. The crystals formed were collected on a glass filter (No. 3), washed with a small amount of cold water, methanol and ethyl ether, and dried over phosphorus pentoxide under reduced pressure. Thus 18g of 2-keto-
D-glucaric acid dicalcium trihydrate was obtained. The analytical values of the obtained crystals were as follows.

Mp: S152 to S157 ° C. (decomposition) Elemental analysis _{(%) C 6 H 6 O} 8 · Ca · 3H 2 O and a theoretical: C; 24.00, H; 4.03 , Ca; 13.35 Found: C; 23.96, H ; 4.16, Ca; 13.00 Specific light intensity: ▲ [α] ²⁵ _D ▼ ＝ -12.3 ° (c = 1.065%, 0.1
N HCl: Immediately after dissolution) = + 7.9 ° (c = 1.065%, 0.1N HCl: After stabilization) This crystal showed the same infrared spectrum as the crystal obtained in Reference Example 1. The retention time (9.4 minutes) by the high performance liquid chromatography method and the ratio of the ultraviolet absorption at 214 nm to the differential diffraction intensity (about 1.0) were the same as those of the crystal obtained in Reference Example 1. Furthermore, regarding each of the standards obtained in Reference Example and Reference Example 1, phenol: formic acid: water (75: 5: 25)
Using the solvent and the cellulose plate (Merck)
The Rf values by thin layer chromatography when developed at room temperature for 3 hours were all 0.20. Both of them showed the same color of black brown with the silver nitrate reagent, yellow with the O-phenylenediamine reagent, and yellow with the anilinephthalic acid reagent.

From the above analysis results, the glucose metabolite of Pseudogluconobacter saccharoketgenes K591S strain was identified as 2-keto-D-glucaric acid.

Reference Example 3 Crude 2-keto-D obtained by the same method as Reference Example 2
- by dicalcium salt [High Performance Liquid Chromatography of glucaric acid as a quantitative ^(※ Note) in 2-keto-D-3 hydrate of dicalcium salt of glucaric acid _{(C 6 H 6 O 8 Ca} · 3H 2 O) 5.0 g (containing 85.1%) was put into a 500 ml Meyer, 300 ml of distilled water was added to this, and the mixture was suspended. To this suspension, 60 ml of cation exchange resin IR-120B (manufactured by Rohm and Haas Company, USA) (H ⁺ type) was added, and the mixture was stirred at room temperature for 1 hour. Filter the reaction through a glass filter to remove solids.
It was washed with 100 ml of distilled water. Combine the filtrate and wash solution,
Concentrate to about 50 ml under reduced pressure, finely filter with Millipore filter paper (GSWPO 4700, Millipore US, pore size 0.22 μm) covered with about 10 g of Hyflo Supercell (Johnmanville, USA Celite), and further distill 20 ml. It was washed with water and combined with the filtrate. The filtrate was quantified by high performance liquid chromatography and contained 2.66 g of 2-keto-D-glucaric acid (recovery rate 90.1%). This filtrate was passed through a column packed with 10 g of activated carbon (White Sagi A for chromatography, manufactured by Takeda Pharmaceutical Co., Ltd.) and washed with 100 ml of distilled water. 500 ml of two effluents
The mixture was placed in an eggplant-shaped flask for 2 minutes, and water was distilled off by a freeze-drying method. After 7 hours, the obtained colorless oily matter was dried under reduced pressure for 3 hours in the presence of phosphorus pentoxide to obtain a sample for elemental analysis and IR spectrum.

Elemental analysis value (%) Calculated as C ₆ H ₈ O ₈・ 1.4H ₂ O C 30.88; H 4.66 Measured value C 31.12; H 4.57 High performance liquid chromatography ^* Retention time 7.30 min IR (film) cm ^-1 3700- 2700,1730,1700 sh, 1680 sh, 1640 ^* Note High-performance liquid chromatography measurement conditions HPLC: Shimadzu LC-3A type column: Aminex ion exclusion HPX-87H 300 × 7.8 mm (Bi
Flow rate: 0.6ml / min Column temperature: 25 ℃ Mobile phase: 0.008NH ₂ SO ₄ Detector: UV (210nm) and RI (Showa Denko SE-31)
Type) Reference Example 4 Crude 2-keto-D obtained by the same method as Reference Example 2
-Dicalcium salt of glucaric acid [determined by high performance liquid chromatography under the same conditions as in Reference Example 2-
The containing 85.1% by trihydrate di calcium salts of keto -D- glucaric acid _{(C 6 H 6 O 8 Ca} · 3H 2 O)] 100g placed in 2l beaker, stirred with distilled water 500ml thereto Then, it was suspended (at this time, pH was 8.27). Cation-exchange resin IR-120B (Rohm and Haas, USA) (H ⁺ type) (about 1) was gradually added to this suspension while stirring,
After stirring for 1 hour after addition, the pH of the reaction solution will be
It was 1.36 and became constant. The reaction product was filtered through a glass filter, and the solid product was washed with distilled water (about 400 ml × 4).
The filtrate (730 ml) and the washing liquid (1740 ml) were quantified by liquid chromatography, respectively.
In addition, 9.1 g of 2-keto-D-glucaric acid was contained in the cleaning solution of g, and the total amount was 57.8 g, and the recovery rate of free acid was 97.9%. The filtrate and washings were combined and concentrated under reduced pressure to about 300 ml. 1 g of activated carbon was added to this concentrated solution and stirred, and then finely filtered with Millipore filter paper (US Millipore GSWPO 4700, pore size 0.22 μm) covered with about 20 g of Hyflo Supercell (US John Marville Co. Celite), and further distilled. It was washed with water (50 ml × 2) and combined with the filtrate. The filtrate was determined by high performance liquid chromatography to contain 52.3 g of 2-keto-D-glucaric acid, and the recovery rate was 88.6%. This was concentrated under reduced pressure to obtain 62.5 g of a pale yellow syrup. The content of 2-keto-D-glucaric acid was 83.7%. Yield 88.7%.

Reference Example 5 10 ml of an aqueous solution containing 2.71 g (0.013 mol) of 2-keto-D-glucaric acid was diluted to 30 ml with distilled water, and 0.96 g (0.013 mol) of calcium hydroxide was added with stirring. After stirring at room temperature for 2 hours, insoluble matter was collected by filtration, washed with about 200 ml of water, and dried under reduced pressure in a desiccator to obtain 3.84 g of 2
A dicalcium salt of keto-D-glucaric acid was obtained. yield
98.4%.

Melting point: approx. 155 ° C (decomposition) Elemental analysis value (%) Calculated as C ₆ H ₆ O ₈ Ca ・ 3H ₂ O C24.00; H4.03 Measured value C23.94; H3.99 It varies easily depending on the drying conditions. For example, if it is dried at room temperature for a long time, it becomes a 2.5 hydrate, if it is dried at 40 ° C for 6 hours it is a dihydrate, and if it is dried at 60 ° C for 6 hours it is
It became a hydrate.

IR (KBr) cm ^-1 3650-2900,1650,1600,1420 ¹³ CNMR (D ₂ O + DCl) δ 182.7 (s), 182.4 (s), 174.
6 (s), 174.3 (s), 172.1 (s), 105.8 (s), 100.6
(S), 82.8 (d), 81.7 (s), 79.4 (d), 79.1
(D), 78.5 (d), 77.4 (d) Reference Example 6 5.0 g of crude 2-keto-D-glucaric acid dicalcium salt (3
_{Hydrate, C 6 H 6 O 8 Ca} · 3H 2 O as a 85.1% content) from 10ml prepared according to the method of Reference Example 4 2-keto -D- glucaric acid solution (2 in quantitative by HPLC.
It contains 71 g (0.013 mol) of 2-keto-D-glucaric acid.
The yield (92.0%) was passed through a column packed with 30 ml of Dowex 50W-X8 (cation exchange resin name of Dow Chemical Co., USA) (Na ⁺ type). The column was eluted with distilled water to obtain 400 ml of an eluate (showing pH 3.4). The eluate was concentrated to dryness under reduced pressure, 400 ml of methanol was added to the residue, and the mixture was stirred to give a powder. The crystals were collected by filtration, washed with a small amount of acetone, and dried under reduced pressure in a desiccator to obtain 2.44 g of monosodium salt of 2-keto-D-glucaric acid. Yield 75.6
%.

Mp 155-160 ° C. (decomposition) Elemental analysis _{(%) C 6 H 7 O} 8 Na · H 2 O Calculated C29.04; H3.66 Found C28.72; H3.80 IR (KBr) cm - ¹ 3600-2800,1720,1410 Reference Example 7 From 5.0 g of crude 2-keto-D-glucaric acid dicalcium salt (trihydrate, containing 85.1% as C ₆ H ₆ O ₈ Ca · 3H ₂ O) 10 ml of 2-keto-D-glucaric acid solution (containing 2.71 g (0.013 mol) of 2-keto-D-glucaric acid as determined by high performance liquid chromatography) prepared according to the method of Reference Example 4 was Dowex 50W-X8. It was passed through a column packed with 30 ml (K ⁺ type). Elution was performed with distilled water to obtain 400 ml of an eluate (showing pH 3.42). The eluate was concentrated under reduced pressure and the residue
When 0 ml of methanol was added and stirred, it became powdery.
The crystals were collected by filtration, washed with a small amount of acetone, and dried under reduced pressure in a desiccator to obtain 2.12 g of monopotassium salt of 2-keto-D-glucaric acid. Yield 61.7%.

Mp 135 to 150 ° C. (decomposition) Elemental analysis _{(%) C 6 H 7 O} 8 K · H 2 O Calculated C27.27; H3.43 Found C26.83; H3.66 IR (KBr) cm - ¹ 3600-2800,1720,1610,1410 Reference example 8 5.0 g (0.0167 mol) of 2-keto-D-glucaric acid dicalcium salt trihydrate and 250 ml of a acetone suspension while stirring 2.5 ml concentrated sulfuric acid was added dropwise little by little.
Stir for an additional 3 hours after dripping and then add about 20g of reaction solution
Hi-Flo Supercell (made by John Marville, USA)
Was used as a filter aid to remove insoluble matter by filtration. About 1000 ml
The insoluble matter was washed with acetone and combined with the previous filtrate. After concentrating the washing solution under reduced pressure, the precipitated crystals were collected by filtration, washed with a small amount of ethyl acetate and dried in a desiccator.
3.80 g (colorless needles) of 2,3-O-isopropylidene-2
-Keto-D-glucaric acid was obtained. Yield 91.7%.

Melting point 180-210 ° C (decomposition) (recrystallized from acetone-ether) Elemental analysis value (%) Calculated as C ₉ H ₁₂ O ₈ C43.56; H4.87 Found C43.57; H4.99 IR (KBr ) Cm ^-1 3400,3300-2800,1750,1690 ¹ H-NMR (d ₆ -DMSO) δ 1.30 (s, 3H), 1.39 (s, 3H),
4.35-4.7 (m, 3H), 8-10 (br, 3H) ¹³ C-NMR (D ₂ O) δ 25.3 (q), 25.5 (q), 76.7
(D), 86.7 (d), 111.8 (s), 116.7 (s), 170.4
(S), 173.0 (s) Reference Example 9 7.10 g of syrupy crude 2-keto-D-glucaric acid (content 83.7% as determined by high performance liquid chromatography,
0.0286 mol), 15 ml of benzaldehyde, 50 ml of acetonitrile and 2 drops of concentrated sulfuric acid were stirred under reflux for 4.5 hours. During this period, 20 g of Molecular Sieves 3A was incorporated between the reactor and the cooling tube, and the reflux solvent was dried. Analysis of reactants by high performance liquid chromatography (Analysis conditions-column; Aminex HPX-87H (manufactured by Bio-rad); mobile phase; 0.008NH ₂ SO ₄ ; flow rate: 0.6 ml / min; detector: SE-31 (Showa (Denko Co., Ltd.), the isomer of 2,3-O-benzylidene compound is about 6
It was confirmed that they were produced at a ratio of 0:40. The reaction mixture was concentrated under reduced pressure, and ethyl ether was added to the paste-like residue, whereby mainly isomers in a large amount were powdered. This was collected by filtration, washed with a small amount of ether, dried, and recrystallized from acetonitrile to obtain 3.81 g of 2,3-O-benzylidene-2-keto-D-glucaric acid. When this product was analyzed by high performance liquid chromatography, most of the isomers were mostly found, and only a small amount of the isomers was observed. yield
45.0%.

Melting point 198-199 ° C (decomposition) Elemental analysis value (%) Calculated as C ₁₃ H ₁₂ O ₈ C52.71; H4.08 Found C52.73; H4.02 IR (KBr) cm ^-1 3500-3000, 1740,1710 ^1- NMR (d ₆ -DMSO) δ 4.50 (s, 2H), 4.75 (d, 1H), 6.
15 (s, 1H), 7.43 (s, 5H), OH, COOH are very broad and difficult to quantify.

Example 1 24.8 g (0.1 mol) of 2,3-O-isopropylidene-2
A mixture of keto-D-glucaric acid and 50 ml of concentrated hydrochloric acid was stirred for 20 minutes at 50 ° C. The reaction solution is concentrated to dryness under reduced pressure,
10 ml of distilled water was added to the residue, and this solution was passed through a column filled with 50 ml of a characteristic white heron for chromatography (manufactured by Takeda Pharmaceutical Co., Ltd.) using distilled water. After elution with distilled water, the eluate was concentrated again, a small amount of dichloromethane was added to the residue, and the insoluble material was collected by filtration and dried to give 19.0 g of crude D-glucosaccharoascorbic acid (D-erythro-hexo- A hydrate of 2-enalo-1,4-lactone) was obtained (purity 98.5).
%, Yield 99.0%). By recrystallizing this from dry acetonitrile, pure D-glucosaccharoascorbic acid was obtained.

Melting point 188-189 ° C (decomposition) (recrystallized from acetonitrile) Elemental analysis value (%) Calculated as C ₆ H ₆ O ₇ C37.91; H3.16 Found C37.80; H3.21 IR (KBr) cm ^-1 3580,3500,3400 ~ 3000,1770,1720,1690,
1590 ¹ H-NMR (d ₆ -DMSO) δ 4.42 (d, 1H), 4.95 (d, 1H),
6.5-9.5 (br, 2H), 9.5-13 (br, 2H) ¹³ C-NMR (d ₆ -DMSO) δ 69.5 (d, c-5 position), 77.2
(D, C-4th place), 118.9 (s, C-2nd place), 152.3 (s, C-3rd place)
Position), 170.5 (s, C-1 position), 171.7 (s, C-6 position) Example 2 2.48 g (0.01 mol) of 2,3-O-isopropylidene-2-
A mixture consisting of keto-D-glucaric acid and an aqueous solution of the acid shown in Table 1 was stirred at 65 ° C. for 2 hours. After the reaction was completed, the total volume was adjusted to 100 ml and high performance liquid chromatography (column: Zorb
ax BP-NH ₂ 4 mm × 250 mm; mobile phase: 25% 0.01 NKH ₂ PO ₄ −75%
Acetonitrile; Flow rate: 1.5 ml / min; Detector: SE-31 (manufactured by Showa Denko KK) to determine D-glucosaccharoascorbic acid in the yield shown in Table 1.

Example 3 3.06 g (0.01 mol) of 2,3-O-cyclohexylidene-2
A mixture of -keto-D-glucaric acid and 6 ml of concentrated hydrochloric acid was stirred at 70 ° C for 30 minutes. The reaction solution was concentrated to dryness under reduced pressure, a small amount of activated carbon and 50 ml of distilled water were added to the residue,
Heated for about 5 minutes. After hot filtration, the filtrate was concentrated to dryness under reduced pressure, a small amount of dichloromethane was added to the residue, the insoluble matter was collected by filtration, and dried to obtain 1.69 g of D-glucosaccharoascorbic acid monohydrate (purity 98.0%) yield obtained
79.6%. This product has the same melting point as that obtained in Example 1, 1R, ¹
The retention times in 1 H-NMR, ¹³ C-NMR and high performance liquid chromatography were exactly the same.

Example 4 1.48 g (0.005 mol) of 2,3-O-benzylidene-2-keto-D-glucaric acid was dissolved in 10 ml of 20% hydrochloric acid, and this solution was heated with stirring at 70 ° C. for 1 hour. After the reaction is complete,
When the amount was set to 0 ml and quantified by high performance liquid chromatography, 0.82 g of D-glucosaccharoascorbic acid was formed. Yield 85.8%.

Example 5 24.9 g of crude 2-keto-D-glucaric acid in the form of a syrup (content rate 83.7% as determined by high performance liquid chromatography,
0.10 mol), 15 ml of concentrated hydrochloric acid and 150 ml of toluene were stirred at an internal temperature of 65 ° C. for 4 hours. After the reaction, the mixture was concentrated to dryness under reduced pressure, 1.0 g of activated carbon and 200 ml of water were added,
Overheated for about 5 minutes. After filtration using a glass filter, the filtrate was quantified by high performance liquid chromatography according to a conventional method to find that 17.7 g of D-glucosaccharoascorbic acid was produced. (Yield 93.3%).

The filtrate was concentrated to dryness again under reduced pressure, a small amount of ether was added to the residue, the insoluble matter was collected by filtration, washed with a small amount of dichloromethane, dried and recrystallized from an acetone-benzene mixture to give 15.8 g of the mixture. D-glucosaccharoascorbic acid was obtained.
Yield 83.1%.

Example 6 50.0 g of crude 2-keto-D-glucaric acid dicalcium salt [2-keto-determined by high performance liquid chromatography]
Trihydrate of dicalcium salt of D-glucaric acid (C ₆ H ₆ O ₈ Ca
・ 3H ₂ O) containing 85.1%, 0.1417 mol] was suspended in 800 ml of acetone, 17.19 g of 97% sulfuric acid (0.17 mol) was gradually added dropwise at room temperature with stirring, and stirring was continued for 10 hours after the addition was completed. . The insoluble matter was filtered off, washed with about 300 ml of acetone and combined with the filtrate. The mixture was concentrated to dryness under reduced pressure, 100 ml of concentrated hydrochloric acid was added to the residue, and the mixture was heated at 70 ° C for 20 minutes. The reaction mixture was concentrated to dryness under reduced pressure, 100 ml of water and 0.5 g of activated carbon were added to the residue, the mixture was heated at 70 ° C. for about 5 minutes, the activated carbon was removed by filtration, and the mixture was concentrated to dryness under reduced pressure. Ether-acetonitrile was added to the residue, the insoluble matter was collected by filtration, washed with a small amount of dichloromethane, and dried in a desiccator under reduced pressure to obtain 22.2 g of D.
-Glucosaccharoascorbic acid monohydrate (purity 98.8
%) Was obtained. Yield 74.4%.

Melting point 188-189 ° C (decomposition) (recrystallized from acetone-benzene).

Example 7 From 5 mmol of 2-keto-D-glucaric acid salt of 2,3-O-ketal or 2-keto-D-glucaric acid salt, or a mixture thereof, from a total of 5 mmol and 10 ml of 15% hydrochloric acid. The resulting mixture was stirred at 65 ° C. for 1 hour. After completion of the reaction, the total amount was adjusted to 50 ml and the quantitative determination was carried out by high performance liquid chromatography in the same manner as in Example 2 to produce D-glucosaccharoascorbic acid in the yield shown in Table 2.

Example 8 500 g of crude 2-keto-D-glucaric acid dicalcium salt [2-keto-by quantitative determination by high performance liquid chromatography]
Trihydrate of dicalcium salt of D-glucaric acid (C ₆ H ₆ O ₈ Ca
・ 3H ₂ O) containing 85.1%, 1.417 mol] is suspended in 1500 ml of distilled water and 214.9 g of 97% sulfuric acid (2.1255 mol) is gradually added dropwise at room temperature with stirring. Stir it. The insoluble matter was filtered off, washed with about 1000 ml of distilled water and combined with the filtrate. The filtrate was concentrated under reduced pressure using a water bath having a bath temperature of 55 ° C. to about 300 ml. After cooling, the insoluble matter was removed by filtration, the mixture was again heated in a water bath having a bath temperature of 55 ° C. for 8 hours, and then concentrated under reduced pressure. After adding about 200 ml of distilled water to the concentrate and filtering off the insoluble matter, the solution was passed through a column filled with 300 ml of Kuroto's characteristic Shirasagi (manufactured by Takeda Pharmaceutical Co., Ltd.) using distilled water to remove distilled water. Elute with. The eluate was concentrated under reduced pressure, and the precipitated crystals were collected by filtration and dried to obtain first crude crystals. The filtrate was passed through activated carbon chromatography again, the eluate was concentrated under reduced pressure, and the precipitated crystals were collected by filtration and dried to obtain second crude crystals. The filtrate was subjected to the same treatment again to obtain a third crude crystal. The first to third crude crystals thus obtained were mixed well and a total of 247 g of crude D-glucosaccharoascorbic acid monohydrate (purity 9
6.8%, yield 81.1%) was obtained.

When the obtained crude D-glucosaccharoascorbic acid was recrystallized from distilled water, pure D-glucosaccharoascorbic acid monohydrate was obtained.

Mp 134-138 ° C. Elemental analysis (recrystallized from water) (%) calcd _{C 6 H 8 O 8 C34.63;} H3.87 Found C34.52; H3.89 IR (KBr) cm -1 3580 , 3500,3400−3000,1770,1720,1690,
To remove water of crystallization from 1590 D-glucosaccharoascorbic acid monohydrate, recrystallization from an anhydrous organic solvent or drying under reduced pressure can be carried out. The method is easy.

That is, 1 g of 10.0 g of D-glucosaccharoascorbic acid
100 ml of acetonitrile is added to the hydrate, and the acetonitrile is distilled off using an atmospheric distillation device. During this period, a reduced amount of acetonitrile and the same amount of aetonitrile were continuously injected to distill off a total of about 250 ml of acetonitrile. By distilling off the acetonitrile, the anhydride of D-glucosaccharoascorbic acid begins to precipitate. After completion of the reaction, the mixture was cooled and the precipitate was collected by filtration and dried to obtain 8.8 g of D-glucosaccharoascorbic acid. Yield 96.3%. This product had exactly the same melting point, IR, ¹ H-NMR, ¹³ C-NMR and retention time as those obtained in Example 1 in high performance liquid chromatography.

Example 9 1.0 g (5.26 mmol) of D-glucosaccharoascorbic acid was dissolved in 10 ml of distilled water, and 30 ml of Dowex 50W-X8 (cation exchange resin name of Dow Chemical Company, USA) (Na ⁺ type) was charged. Passed through the column. Elute the column with distilled water
400 ml of eluate was obtained. The eluate was concentrated under reduced pressure, 10 ml of methanol was added to the residue, the mixture was stirred overnight, the precipitate was collected by filtration, and dried under reduced pressure in a desiccator to give 0.57 g.
A monosodium salt of D-glucosaccharoascorbic acid was obtained. Yield 45.3%.

Melting point approx. 170 ° C (decomposition) Elemental analysis (%) Calculated as C ₆ H ₅ O ₇ Na ・ 1.5H ₂ O C30.14; H3.37 Found C30.38; H3.21 IR (KBr) cm ^-1 3600-2800,1750,1680,1620,1420 Example 10 1.90 g (0.01 mol) of D-crucosaccharoascorbic acid was added to 30 ml of distilled water and stirred at room temperature. In this aqueous solution
1.06 g (0.01 mol) of sodium carbonate was added, and the carbonic acid was stirred until the generation of soot stopped. 300m of the obtained reaction solution
It was added dropwise to l of methanol. The precipitate was collected by filtration and dried under reduced pressure to obtain 2.50 g of a disodium salt of D-glucosaccharoascorbic acid. Yield 99.2%.

Melting point 185 ° C (decomposition) Elemental analysis value (%) Calculated value as C ₆ H ₄ O ₇ Na ₂ · H ₂ O C 28.59; H 2.40 Measured value C 28.67; H 2.61 Example 11 1.0 g in 10 ml of distilled water ( 5.26 mmol of D-glucosaccharoascorbic acid was dissolved in IR-120B (US ROHM
Ander's cation exchange resin name) (K ⁺ type) 100m
It was passed through a column packed with l. The column was eluted with distilled water to obtain 400 ml (pH 5.17) of eluate. The eluate was concentrated under reduced pressure, 400 ml of methanol was added to the residue, and the precipitate was collected by filtration and dried under reduced pressure in a desiccator 1.
12 g of monopotassium salt of D-glucosaccharoascorbin was obtained. Yield 83.5%.

Melting point approx. 250 ° C (decomposition) Elemental analysis value (%) Calculated as C ₆ H ₅ O ₇ K ・ 1.5H ₂ O C 28.24; H 3.16 Measured value C 28.10; H 2.83 IR (KBr) cm ^-1 3600-2800 , 1760, 1660, 1600 Example 12 1.90 g (0.01 mmol) of D-glucosaccharoascorbic acid was added to 30 ml of distilled water and stirred at room temperature. To this aqueous solution, 1.38 g (0.01 mol) of potassium carbonate was added and stirred until the generation of carbon dioxide gas stopped. The reaction solution obtained is 30
It was added dropwise to 0 ml of methanol. The precipitate was collected by filtration and dried under reduced pressure to obtain 2.70 g of D-glucosaccharoascorbic acid dipotassium salt. Yield 95.0%.

Melting point 175 ° C (decomposition) Elemental analysis value (%) Calculated as C ₆ H ₄ O ₇ K ₂ · H ₂ O C 25.35; H 2.13 Found C 25.23; H 2.31 IR (KBr) cm ^-1 3600-2900, 1720, 1590, 1400 Example 13 1.0 g (5.26 mmol) of D-glucosaccharoascorbic acid was dissolved in 10 ml of distilled water to dissolve Dowex 50W-X8 (cation exchange resin name of Dow Chemical Company, USA) (Ca ⁺⁺ Type) through a column packed with 30 ml. Elute the column with distilled water
400 ml of eluate (representing pH 3.5) was obtained. The eluate was concentrated under reduced pressure, 20 ml of acetone was added to the concentrated solution, and the resulting precipitate was collected by filtration and dried under reduced pressure in a desiccator to obtain 0.96 g of monocalcium D-glucosaccharoascorbic acid. I got salt. Yield 81.6%.

Melting point 190-205 ℃ (decomposition) Elemental analysis value (%) Calculated as C ₆ H ₅ O ₇ 1 / 2Ca ・ 0.8H ₂ O C 32.27; H 2.98 Found C 32.13; H 3.19 IR (KBr) cm ^-1 3600-2800,1760,1680,1600 Example 14 1.90 g (0.01 mmol) of D-glucosaccharoascorbic acid was added to 30 ml of distilled water and stirred at room temperature. To this aqueous solution, add 0.741 g (0.01 mol) of calcium hydroxide,
The precipitate was collected after 30 minutes and dried under reduced pressure in a desiccator to obtain 2.2 g of dicalcium salt of D-glucosaccharoascorbic acid. Yield 78.0%.

Melting point 175 ° C. (decomposition) Elemental analysis value (%) Calculated value as C ₆ H ₄ O ₇ Ca · 3H ₂ O C 25.54; H 3.57 measured value C 25.23; H 3.84 Effect of the invention The present invention provides D-glucosaccharoascorbin. An acid and a salt thereof are provided, and the present compound is a novel compound which exhibits an antioxidant effect and is useful. That is, since this compound has an enediol group showing a reducing property in the molecule and exerts an antioxidant action based on this, various fields (eg,
It can be used as an antioxidant in foods, chemical products such as fats and oils). For example, when added to fruit juice, fruits, meat products, seafood, etc., it is effective in preventing browning and maintaining flavor.

Claims (2)

[Claims] 1. D-Glucosaccharoascorbic acid or a salt thereof. 2. 2-Keto-D-glucaric acid or its 2,3.
A method for producing D-glucosaccharoascorbic acid, which comprises treating -O-acetal or ketal with an acid.