DK157139B - PROCEDURE FOR PREPARING A MIXTURE OF 2-KETOGULONATE AND 2-KETOGLUCONATE - Google Patents

Description

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The present invention relates to a process for preparing a mixture of 2-ketogulonate and 2-ketogluconate. The mixture is useful for preparing ascorbic and erythorbic acids. Ascorbic acid or vitamin C is required in the human diet and widely used in tablet form as an additive to other nutrients to meet this need. Erythorbic acid or isoascorbic acid is useful as an antioxidant for use in nutrients.

2,5-Diketogluconic acid is readily prepared by the bacterial action of glucose, several species of Acetobacter and Pseudomonas being useful for this purpose. Japanese Patent No. 14493 (1964) i

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the name Shionogi and Co., Ltd. describes the use of Pseudomonas sesami for this preparation.

Previous work on the sodium borohydride reduction of 2,5-diketogluconic acid has been limited to complete reduction of both 2-keto and 5-keto groups to hydroxyl groups using a large excess of sodium borohydride and the preparation of 2-ketogulonic acid and 2- ketogluconic acid by stereoselective and regioselective non-catalytic reduction has so far not been reported. Wakisaka, Agr. Biol. Chem. 28,819 (1964), reduced the 2,5-diketogluconic acid in both 2- and the 5-keto positions by acting with an excess of sodium borohydride. The four isomers obtained were designated as D-gluconic acid, D-mannonic acid, L-idonic acid and L-gulonic acid. Ruff's oxidation of the resulting mixture of these isomers yielded D-arabinose and L-xylose. The yield of D-arabinose obtained was greater than the yield of L-xylose, which Wakisaka suggested could occur either by stereospecific reduction, by the presence of isomers or by transformations between the various structural isomers. The greater yield of D-arabinose suggests that reduction with hydride to form them. D-isomers were larger than the reduction to form the L-isomer, which is in contrast to the present process, which not only provides regioselective reduction at the 5-keto position, but also stereoselective reduction to produce larger amounts of the desired L-isomer of 2-ketogulonic acid. Complete reduction of 2,5-diketogluconic acid with an excess of sodium borohydride has also been reported by Katznelson, J. Biol. Chem., 204, 43 (1953), which obtained a "gluconic acid" meant to consist of four isomers which could not be separated in his experiment. Similarly, complete reduction of calcium 2,5-diketogluconate with sodium borohydride is reported by Bernaerts et al., Antonie van Leeuwenhoeck, 37,185 (1971).

Catalytic reduction of 2,5-diketogluconic acid using a Raney nickel catalyst and hydrogen gives, according to Wakisaka,

Agr. Biol. Chem. 28, 819 (1964), low yields of a mixture of 2-ketogulonic acid and 2-ketogluconic acid with 2-ketogluconic acid as the main product. This is undesirable if one seeks to use the mixture to prepare and isolate the more valuable ascorbic acid in high yields. For this purpose, a mixture containing a major portion of 2-ketogulonic acid is desirable as 2-ketogulonic acid is the precursor of ascorbic acid, while 2-ketogluconic acid is the precursor of erythorbic acid.

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The catalytic reduction of a 5-keto-D gluconate using noble metal catalysts to prepare a mixture of L-idonate and a D-gluconate is also known. The selectivity for L-idonate is forged using a metal boron catalyst prepared by treating a ash metaisai with sodium borohydride, Chen et al., Chem. Pharm. Bull., 18.1305 (1970). The sodium borohydride reduction of 5-keto-D-gluconic acid has also been described, J.A.C.S., 76 * 3543 (1954), but is non-stereoselective, yielding approximately equal amounts of D-gluconic acid and L-idonic acid.

The present invention relates to a process for the preparation of a mixture of 2-ketogulonate and a 2-ketogluconate, the process of which is characterized by the feature of claim 1. The resulting mixture of the 2-ketogulonate and 2-ketogluconate can be transferred into ascorbic and erythorbic acids.

The process of the invention has unexpectedly been found to enable the regioselective and stereoselective non-catalytic. . reduction of 2,5-diketogluconate at the 5-keto position in good total yield to a mixture of a 2-ketogulonate and a 2-ketogluconate. The ratio of the products in the resulting mixture can be varied from approx. 85:15 to approx. 45:55 depending on the conditions and reagents used, as will be described in greater detail below. Of particular interest is that the process of the present invention can yield a good yield of a mixture containing predominantly 2-keto-gulonic acid, which can be transferred in good yield to the more valuable ascorbic acid. However, mixtures containing approximately equal amounts of 2-ketogulonate and 2-ketogluconate are useful sources for the preparation of both ascorbic and erythorbic acids, and the process of the invention therefore offers advantages in the form of flexibility for producing varying amounts of ascorbic and erythorbic acids.

As mentioned, the 2,5-diketogluconate used in the present invention can e.g. be 2,5-diketogluconic acid or salts of the acid. Suitable salts include those salts which, as a counterion, have an alkali metal, an alkaline earth metal, ammonium or tetraalkylammonium, wherein the alkyl groups have 1-4 carbon atoms. Also useful as starting materials for the process of the invention are the novel normal alkyl esters of 2,5-diketogluconic acid, wherein the alkyl group has 1-4 carbon atoms. As used in the specification and claims, the terms 2,5-diketogluconate, 2-ketogulonate and 2-ketogluconate include the free acids and appropriate alkyl esters and salts thereof as described hereinbefore. 2.5-Diketoalucone Acid οσ salts thereof 4

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freins can be allowed in any manner known in the art. Generally, the 2,5-diketogluconate is prepared as the calcium salt in aqueous solution by fermentation using methods well known in the fermentation industry, see e.g. Japanese Patent No. 14493, and it can be used directly as the starting material in the process of the invention. The 2,5-diketogluconate may also be prepared by fermentation in the presence of other ions, such as sodium, and the resulting sodium 2,5-diketogluconate may also be used directly as a starting material. In an alternative method, the 2.5-diketogluconate is prepared in a conventional manner as the calcium-2,5-diketogluconate and it is transferred into the desired compound by the addition of a site capable of precipitating calcium to give 2,5-diketogluconate in solution with the Wanted counterion. Thus, e.g. sodium or ammonium 2,5-diketogluconate is prepared by adding sodium or ammonium carbonate, as appropriate, to a solution of calcium 2,5-diketogluconate prepared by fermentation. Calcium precipitates as calcium carbonate, leaving the 2,5-diketogluconate in solution with sodium or ammonium ions. The free acids can also be neutralized with an appropriate hydroxide or other site. If desired, the 2,5-diketogluconate can be isolated, purified and redissolved.

The normal alkyl esters of 2,5-diketogluconic acid, wherein the alkyl group has 1-4 carbon atoms, are novel compounds useful as starting materials in the process of the invention. The esters can be prepared by heating a solution of 2.5-diketogluconic acid or a suitable site thereof in the appropriate normal alkanol at 50 ° C to 100 ° C in the presence of a catalytic amount of a strong acid such as concentrated sulfuric acid, hydrochloric acid, p. -toluenesulfonic acid and the like, to give the corresponding alkyl-2,5-diketogluconate-5,5-dialkyl acetal. Suitable salts of 2,5-diketogluconic acid include alkali metal, alkaline earth metal, ammonium and tetraalkylammonium salts in which each alkyl group of the tetraalkylammonium ion has 1-4 carbon atoms. The acetal is then hydrolyzed with aqueous acid at a temperature of between ca. -10 ° C and 30 ° C to provide the desired alkyl ester of 2/5-diketogluconic acid. Suitable acids include aqueous hydrochloric acid, trifluoroacetic acid, sulfuric acid, sulfonic acid ion exchange resins and the like. The alkyl 2,5-diketogluconate-5,5-dialkyl acetal intermediates are also novel compounds. A preferred acetal and ester obtained by hydrolysis thereof is methyl 2,5-diketogluconate-5.5

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dimethyl acetal and methyl 2,5-diketogluconate, respectively.

When an alkali metal 2,5-diketogluconate is used as the starting material, the sodium salt is preferred. The sodium salt has been found to be a particularly desirable starting material for preparing mixtures of a 2-ketogulonate and a 2-ketogluconate containing the 2-ketogulonate as the main product, thereby facilitating the synthesis of ascorbic acid. A preferred alkaline earth metal 2,5-diketogluconate is the calcium salt. When tetraalkylammonium salts are used, tetramethylammonium is preferred because of its cost and availability. A preferred alkyl ester starting material is methyl 2,5-diketogluconate

A solution of the 2,5-diketogluconate is contacted with an alkali metal borohydride. The reaction is preferably carried out in aqueous solution, optionally containing organic co-solvents such as, but not limited to, alcohols of 1-4 carbon atoms, alkanediols of 2-4 carbon atoms, acetonitrile, dimethylsulfoxide and dimethylformamide. Methanol is a preferred co-solvent. The concentration of 2,5-diketogluconate is not critical, but is preferably between 5 and 20% by weight. The concentration of 2,5-diketogluconate formed by fermentation is generally located in this range thereby providing an appropriate aqueous solution of the starting material for use in the process of the invention. When an alkyl ester is used as the starting material, the reaction can be carried out in anhydrous solvents such as alkanols, especially methanol, dimethyl sulfoxide and dimethylformamide. In all cases, it is not necessary that the 2,5-diketogluconate be completely dissolved in the solvent, provided that a substantial portion of the starting material is in solution.

Q

The alkali metal borohydride can be used either in solution or in solid form. The preferred borohydride for use in the process of the invention is sodium borohydride. It has been found that the use of the sodium compound, especially when used with the sodium 2,5-diketogluconate as a substrate, leads to large proportions of 2-ketogulonate in the product mixture. The use of other alkali metal borohydrides has been found to yield somewhat lower amounts of 2-ketogulonate, and when selecting reagents, mixtures with ratios of 2-ketogulonate: 2-ketogluconate can be formed at 85:15 to 45:55.

This allows some flexibility in using these mixtures to obtain either ascorbic acid or erythorbic acid.

Good yields of a mixture of a 2-ketogulonate and a 2-ketogluconate can be obtained using between 0.8 approx.

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1.1 equivalents of the alkali metal borohydride per mole of 2,5-diketogluconate. By an equivalent alkali metal borohydride is meant the stoichiometric amount needed to transfer the 5-keto group in the 2,5-diketogluconate to hydroxyl. This can also be expressed as 0.8 to 1.1 equivalents of hydride ion. 1 mole of alkali metal borohydride contains four equivalents of hydride ion and the amount of reagent required can be similarly expressed as 0.200 to 0.275 mole of alkali metal borohydride. If the alkali metal borohydride is used in amounts less than approx. 0.8 equivalents per However, for the selective reduction of the 2,5-diketogluconate, the yield of the mixture of 2-keto acids will be correspondingly lower. The process of the invention aims to achieve the optimum total yields of the desired product mixture. Therefore, the disclosure and claims disclose a method for practicing this process in which only a portion of the 2,5-diketogluconate is reacted and unreacted starting material can then be recycled for further reaction.

During the reaction of the 2,5-diketogluconate with the alkali metal borohydride, the pH of the solution should be kept greater than 5, preferably between 6 and 10.5. When 2,5-diketogluconic acid is used as the starting material, the pH should preferably be adjusted to above 5 for the addition of the alkali metal borohydride. The pH of an aqueous solution of sodium or calcium 2,5-diketogluconate produced by fermentation is usually less than 5, and the pH should therefore preferably be adjusted correspondingly to a value greater than 5 before the addition of the borohydride. This can be done by the addition of any base, but it is preferred to use a sodium compound such as sodium carbonate or sodium hydroxide. Alternatively, the pH may be adjusted simultaneously with the borohydride addition by dissolving the borohydride in a basic solution, such as sodium hydroxide, so that when the basic borohydride solution is added, the aqueous solution pH is immediately adjusted to a value higher than 5. In this case, preferably by adding a small excess of the required stoichiometric amount allows a small part of the borohydride to decompose under the acidic conditions before the pH has been adjusted to above 5.

The borohydride may be added slowly portionwise over a period of time, e.g. by adding the basic solution of the borohydride dropwise while stirring the solution of the 2,5-diketogluconate. The boro hydride is preferably added at once to the reaction 7

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start at a temperature below 25 ° C.

The reduction may also be effected in a flow reaction system when a solution of the alkali metal borohydride is usually mixed with or injected into the 2,5-diketogluconate containing stream.

The time required to complete the reaction depends on the reaction temperature and the rate of addition of the borohydride to the 2,5-diketogluconate, but in general the reaction times will be relatively short and the reaction will be complete after a period of approx. 10 minutes to approx. 2 hours.

During the addition of the alkali metal borohydride, the temperature of the aqueous solution should preferably be maintained at between ca. -30 ° C and approx. 50 ° C and preferably between -25 ° C and 25 ° C. Above 50 ° C, decomposition of the reactants can be observed.

The reduction may advantageously be carried out in the presence of a boron complexing agent which is dissolved or dispersed in the reaction medium. Boric acid is formed during the reduction and it can complex with the 2,5-diketogluconate starting material. By a boron complexing agent is meant any compound or material which will inhibit or prevent the complex formation between boric acid and the 2,5-diketogluconate, e.g. by preferably reacting with or adsorbing the boric acid, but the agent does not damage the reaction. Suitable boron complexing agents include alkali metal fluorides, ammonium fluoride and boron absorbing ion exchange resins. A variety of such resins are commercially available. A particularly useful example of the latter is "Amberiite XE-243" (Rohm and Haas Company, Philadelphia, Pa.). A sufficient amount of boron complexing agent must be present to complex with the boric acid formed. Thus, approx. 4 moles of fluoride for each mole of sodium boron hydroxide used to effect reduction. The amount of ion exchange resin used will usually be from 0.5 to 1 volume of resin per volume 2.5-diketogluconate solution in a batch process, but the amount used will necessarily vary according to the particular resin used and the reaction conditions.

Upon completion of the selective reduction to form the mixture of a 2-ketogulonate and a 2-ketogluconate, unreacted 2.5-diketogluconate can be recycled for further reaction or it can be effectively removed by heating with acid or base.

If it is desired to subject the unreacted 2,5-diketogluconate further,

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like reduction reactions, the initial reduction is preferably carried out in the presence of a boron complexing agent as described above.

The mixture of 2-ketogulonic acid and 2-ketogluconic acid can be isolated by filtration of the reaction mixture and adjusting the pH of the filtrate to between 1.5 and 2 by the addition of acids such as concentrated sulfuric acid and filtration and disposal of formed precipitate. The 2-ketogulonic and 2-ketogluconic acids can be pooled, by removal of the water or water-organic co-solvent, e.g. by freeze drying. The ratio of 2-ketogulonic acid to 2-ketogluconic acid in the mixture can be determined by liquid chromatography on the methyl esters using a mixture of boronic acid (0.6 M) and ammonium formate (0.4 M) in water as the mobile phase with "Aminex Resin" type A-25 (TM. Bio-Rad Laboratories, Richmond, California), 50-100 mesh as the stationary phase or by thin layer chromatography using a cellulose carrier.

The mixture of 2-ketogulonic acid and 2-ketogluconic acid can be easily transferred into ascorbic and erythorbic acids. The mixture of 2-keto acids can be transferred into the methyl esters by boiling under reflux in methanol in the presence of an acidic catalyst such as hydrochloric acid or a sulfonic ion exchange resin for 3 to 24 hours. Other esters can be formed in this way using the appropriate alcohol. The esters are formed directly when an alkyl ester of 2,5-diketogluconic acid is the starting material for the selective reduction. The mixture of methyl esters can be separated and boiled there under reflux in methanol in the presence of a base, such as sodium bicarbonate, in an inert atmosphere. Upon cooling, sodium ascorbate and sodium erythorbate precipitate. The raw salts are collected by filtration, mixed with water and deionized with a cation exchange resin such as "Dowex 50" manufactured by Dow Chemical Co. The water is removed and the ascorbic acid and erythorbic acid are recrystallized from methanol / water to give a mixture of ascorbic and erythorbic acids. If desired, ascorbic acid can be obtained by recrystallization from a 4: 1 methanol / water solution. Other appropriate solvents or co-solvents may be used if desired. ascorbic acid and erythorbic acid, respectively, using the same conditions as described above for the ester mixture.

t

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9 ·:> *. r, * *

Ascorbic acid can be selectively prepared from a mixture of 2-ketogulonic acid and 2-ketogluconic acid. This is particularly advantageous when the mixture containing a shark proportion of 2-ketogulonic acid has been formed, for example, by sodium borohydride reduction of sodium 2,5-diketogluconate. The mixture of acids obtained by the borohydride reduction is heated in an appropriate organic solvent, such as xylene, to ca. 50-13 ° C, preferably 60-90 ° C, in the presence of an acid selected from hydrochloric, hydrobromic, sulfuric and sulfonic ion exchange resins. The preferred acid is hydrochloric acid. After heating for a period of 3-12 hours, depending on the temperature used, the lactonization of the 2-ketogulonic acid to ascorbic acid is substantially complete. In this process, erythorbic acid is not formed and it is therefore a simple method for selectively preparing ascorbic acid from mixtures of 2-ketogulonic acid and 2-ketogluconic acid prepared by borohydride reduction of a 2,5-diketogluconate. This acid-catalyzed lactonization can also be used to transfer mixtures of the alkyl esters of 2-ketogulonic acid and the 2-ketogluconic acid in ascorbic acid.

The following examples further explain the invention.

Example 1

To a rapidly stirred solution of 20 liters of filtered crude fermentation mixture containing 10% calcium-2,5-diketogluconate (CgH-yO 1,5, 1.5 H₂O, molar weight 238, 0.84 M) at 0 ° C (ice / water) -bath) 42.4 ml of 2.2 M NaBH 4 was added to 7 M NaOH (0.93 MH) at a rate of 1 ml per ml. mine. The pH of the solution increased rapidly from 3.65 to 10.2.

The resulting slurry was filtered, the filtrate adjusted to pH 1.6 with concentrated H 2 SO 4 and the resulting precipitate removed by filtration and discarded. Removal of water by freeze-drying gave 246 g of freeze-dried solid. Part of this remained esterified and analyzed by liquid chromatography with an internal standard to obtain a ratio of 78:22 between 2-ketogulonic acid and 2-ketogluconic acid in a total yield of 79%.

A solution of 10 g of freeze-dried 2-ketogulonic and 2-keto-gluconic acids from the above reduction in 50 ml of MeOH was treated with 1 g of "Dowex 50" (T. M. Dow Chemical Co.) resin and refluxed for 12 hours. Upon cooling, the resin was removed by filtration and the crude methyl esters isolated by solvent removal as an oil.

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Use of the product:

The crude mixture of methyl ester from before was placed in methanol with 1.5 equivalents of NaHCO 3 and refluxed under a nitrogen atmosphere for 6 hours. Upon decoction, the sodium salts of ascorbic and erythorbic acid precipitated from the solution. The crude salts were isolated by filtration, placed in water and deionized with "Dowex 50" cation exchange resin. Upon removal of water, the crude ascorbine / erythorbic acid residue crystallized from methanol / water to obtain a mixture of ascorbic and erythorbic acids. Recrystallization of 4: 1 methanol / water gave ascorbic acid.

Example 2

A 10% aqueous solution of sodium 2,5-diketogluconate was adjusted to a pH of 6.1 by the addition of sodium carbonate, methanol was added to 50% by volume and the solution was cooled to between -15 ° C and -25 ° C. An equivalent of sodium borohydride was added to the cooled solution, which was stirred for 6 hours at -15 ° C to -25 ° C and at room temperature overnight. A mixture of sodium 2-ketogulonate and -2-ketogluconate was isolated by precipitation with methanol and filtration. Analysis of the methyl esters by liquid chromatography showed a 78:22 ratio of 2-ketogulonic acid to 2-keto-gluconic acid in the product mixture.

Example 3

Using the method of Example 2, sodium borohydride reduction of calcium 2,5-diketogluconate was carried out at 0 ° C at various pH values. The mixture of 2-ketogulonate and 2-gluconate formed was analyzed by liquid chromatography to determine the ratio of 2-ketogulonic acid to 2-keto-gluconic acid. The results obtained were as follows:

Ratio 2-ketogulonic acid: pH Solvent 2-ketogluconic acid_ 6.37 Water: methanol (a) 69:31 8.23 Water: methanol 68:32 8.65 Water 45:55 (a) 1: 1 by volume

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Example 4

Using the method of Example 2, sodium borohydride reductone of sodium 2,5-diketogluconate in water: methanol solution (1: 1 by volume) was kept at between -15 ° C and -20 ° C at various pH. values. The mixtures of 2-keto-gulonate and 2-ketogluconate formed were analyzed by liquid chromatography to determine the ratio of 2-ketogulonic acid to 2-ketogluconic acid. The results obtained were as follows:

Ratio 2-ketogulonic acid: pH 2-ketogluconic acid_ 6.10 78:22 8.6o 77:23 8.80 77:23 10.20 71:29

Example 5

Using the method of Example 2, sodium borohydride reductlon of sodium 2,5-diketogluconate in water: methanol solution at pH between 7.9 and 8.6 was carried out at various temperatures. The ratio of target 2-ketogulonic acid to 2-ketogluconic acid in the resulting products was determined by liquid chromatography. The results obtained were as follows:

Ratio 2-ketogulonic acid:

Temp. ° C 2-Ketogluconic Acid 24 77:23 0 80:20 -15 to -20 77:23

Example 6

Using the method of Example 2, sodium borohydride reduction of sodium 2,5-üiketo-gluconic acid was carried out in water at 0 ° C at a pH of 8 with varying concentrations of sodium 2,5-diketogluconate. The ratio of 2-ketogulonic acid to 2-ketogluconic acid in the resulting products was determined by liquid chromatography. The results obtained were as follows:

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Concentration of Sodium 2,5- Ratio 2-ketogulonic acid: diketogluconate, weight% _ 2-ketogluconic acid_ 5 75:25 10 79:21 20 56:44

Example 7

The reduction was carried out using different alkali metal borohydrides and 2,5-diketogluconates of different counterions. The ratio of 2-ketogulonic acid to 2-ketogluconic acid in the resulting mixtures was determined by liquid chromatography. The reaction conditions and the results obtained were as follows:

Borohydride 2,5-diketo-Temp. Ratio of 2-ketoglucon gluconate ° C pH lonic acid: 2-ketogluconic acid

Ll Li -15 to -20 8.26 48:52

Na Li -15 to -20 8.06 63:37

Na Na -15 to -20 8.60 77:23

Na K -15 to -20 8.08 64:36 K K -15 to -20 7.97 67:33

Na Me4N -15 to -20 8.61 63:37

Na Ca 0 8.65 45:55

Ll Li '0 8.0 48:52

Ll Na 0 8.0 47:53

Reach

Li 0 8.0 63:37

Na Na 0 8.0 79:21

Example 8

The reduction of calcium 2,5-ketogluconate with 4.4 M sodium borohydride in 14 M sodium hydroxide was carried out at 0 ° C in water containing various co-solvents. The ratio of 2-ketogulonic acid to 2-kebogluconic acid in the resulting mixtures was determined by liquid chromatography. The results obtained were as follows: 13

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Ratio of water: Ratio of 2-ketogulonic acid:

Co-solvent with solvent 2-ketogluconic acid

Ethylene Glycol 6: 1 72:28

Acetonitrile 4: 1 76:24

Dimethylformamide 4: 1 72:28

Dimethylsulfoxide 6: 1 71:29

Nothing - 77:23

Example 9 15 g of isolated calcium 2,5-diketogluconate was dissolved in 150 ml of water and 6.61 g of sodium carbonate was added at 0 ° C with stirring of the solution. The pH of the solution increased to 9.57. 0.49 g of sodium borohydride was added to the solution at 0 ° C. After stirring for 15 minutes, the mixture was filtered and the filtrate was ion exchanged with an acidic ion exchange resin. After freeze-drying and formation of the methyl esters described in Example 1, analysis by liquid chromatography gave a ratio of 2-ketogulonic acid to 2-ketogluconic acid at 85:15:

Example 10

To 50 ml of 20% sodium 2,5-diketogulonate at 0 ° C was added 0.8 ml of 10% sodium hydroxide to adjust the pH from 5.15 to 9.70. Sodium borohydride powder (11.26 mmol, Alfa Products, Danvers, Ma. 01923) was added immediately. With a pH of 10.60 after 10 minutes, the mixture was adjusted to a pH of 7 with concentrated sulfuric acid. Analysis of the reduced mixture by HPLC (³minex A-25H resin using 0.5 M NH ^HCX ^ as eluent) showed pure reduction to a mixture of sodium 2-ketogulonate and sodium 2-ketogluconate in a yield. at 85%.

To determine the ratio of 2-ketogulonate to 2-ketogluconate, the freeze-dried solid from 5 ml of the reduced mixture was esterified with 15 ml of methanol and 0.275 ml of concentrated sulfuric acid. The methyl ester obtained was analyzed by gas chromatography as its persilvated dérivat (prepared by treatment with "Tri-Sil / TBT" [Pierce Chemical Company, Rockford, 111. 61105]). Separation of a 3% 1 s * OV-21O * column at 135 ° C (30 ml per min flow rate) showed a ratio of methyl 2-keto-gulonate acid to methyl-2-ketogluconate acid at 85:15.

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Example 11

A 250 ml round bottom flask with 5.0 g (19.8 mol) of methyl 2,5-diketogluconate-5,5-dimethyl acetal, 150 ml of water and 3 ml of 6N hydrochloric acid was stirred at 80 ° C for 45 minutes, cooled and the aqueous solution was passed through a beaker containing 40 ml of "Amberlyst A-21" ion exchange resin (Rohm and Haas Co., Philadelphia, Pa.).

The neutralized eluent was then freeze-dried to obtain 2.2 g (100%) of methyl 2,5-diketogluconate as a 10 pt. Unstable yellow powder homogeneous by HPLC analysis ("Aminex A-25" resin using of 0.5 M + + + 002 as eluent): IR (KBr) cm -1 3330 (s, OH), 1736 (s, methyl ester), NMR (D20) S, c 170.00 (s, ester carbon) ), 96.86 and 92.84 (singlets, anomers), 73.20 and 71.50 (doublets, -CH-OH), 65.99 (t, -CH2-0-), (53.95 (q , CH3-0).

The product was placed in 150 ml of water, cooled to 0 ° C and the pH adjusted to 7.5 with 1 N NaOH. To the rapidly stirred mixture was added 215 mg of sodium borohydride. After 1 minute, the mixture was passed through 40 ml of an ion exchanger in a column containing 50% "Dowex 50" and 50% "Amberlyst A-21" resin. The filtrate was concentrated in vacuo to a solid mixture of methyl 2-ketogulonate and methyl 2-ketogluconate comprising 3.7 g. The crude solid was placed in 50 ml of 95% ethanol with 5.99 g of sodium bicarbonate and boiled under reflux for 4 hours under nitrogen. After cooling, the reaction mixture was deionized with excess "Dowex 50" and concentrated in vacuo to a crude oil. GLPC analysis of the per-trimethylsilylated reaction mixture (δδ * 3.5 ft 0V-210 column) indicates a ratio of 78:22 between ascorbic acid and erythorbic acid in a total yield of 20% determined by iodine titration.

Example 12

To 50 ml of 12% aqueous sodium 2,5-diketogluconate (28.04 mmol) was added 45 ml of Amberlite XE-243 ion exchange resin (Rohm & Haas, Philadelphia, Pa. 19105). The mixture was stirred to 0 ° C in an ice-bath. After addition of 10% sodium hydroxide, the pH was adjusted to 10.8. Treatment with 0.265 g of sodium borohydride (7.01 mmol, Alfa Products, Danvers, Ma. 01923) for 10 minutes followed by adjustment to pH 7 with concentrated sulfuric acid. Stirring for 0.5 hours gave after filtration of the resin 15

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a reduced mixture from which approx. 50% of the initially present boron was removed. A 90% yield of an 85:15 mixture of 2-ketogulonic and 2-ketogluconic acids was obtained.

Example 13

To 50 ml of 12% aqueous sodium 2,5-diketogluconic acid (28.04 mmol) was added 35 ml of Amberlite EX-243 ion exchange resin (Rohm & Haas, Philadelphia, Pa. 19105). The mixture was stirred at 0 ° C in an ice / water bath. By dropwise addition of 10% sodium hydroxide, the pH was adjusted to 10.8. By adding 0.212 g of sodium borohydride (5.61 mmol, Alfa Products, Danvers, Ma. 01923), a pH increase to 11.55 was observed. After 10 minutes, the mixture was quenched with concentrated sulfuric acid to adjust the pH from 11.2 to 7. After stirring for 0.5 hours, the partially reduced mixture was filtered to remove the resin.

To the partially reduced solution was added an additional 10 ml of "Amberlite XE-243" resin. After cooling to 0 ° C, the pH of the solution was adjusted to 10.8 with 10% sodium hydroxide.

An additional 53 mg (1.40 mmol) of sodium borohydride was added.

After 10 minutes, concentrated sulfuric acid was added to adjust the pH to 7. After stirring for 0.25 hours, the mixture was filtered. HPLC analysis showed complete reduction of sodium 2,5-diketogluconate to sodium 2-ketogulonate and sodium 2-ketogluconate. Very little boric acid or possible over-reduction products were observed. Using HPLC analysis with 2-imidazolidone as the internal standard, a 96% yield of an 85: 15 mixture of 2-ketogulonic acid and 2-ketogluconic acid was determined.

Example 14

To a rapidly stirred 12% aqueous solution of sodium 2,5-diketogluconate (28.04 mmol), cooled to 0 ° C in an ice-water bath, was added 2.355 g (56.08 mmol) of sodium fluoride. By dropwise addition of 10% sodium hydroxide, the pH was adjusted from 4.3 to 10.8. Ten minutes after the addition of 0.530 g of sodium borohydride (14.02 mmol, Alfa Products, Danvers, Ma. 01923), the pH was adjusted to 7 with concentrated sulfuric acid. HPLC analysis ("Aminex A-25" resin using 0.5 M NH 2 H 2 as eluent) showed less boron present than without resin. After stirring the mixture overnight, filtration was removed

16 DK 157139 B

a little white solid precipitated. The yield of the 85:15 mixture of 2-ketogulonic and 2-ketogluconic acid was determined to be 90%.

Example 15

To a rapidly stirred solution of 55 mmol sodium 2,5-diketogluconic acid in 150 ml H2 O at 0 ° C and a pH of 9.5 was added 12.7 mmol sodium borohydride for 10bet of 15 minutes. After completion of the addition, the pH of the solution was adjusted to ca. 7 with 6 N hydrochloric acid and freeze-dried to give 16.3 g of solid. A portion of 15.0 g of the freeze-dried solid was dissolved in 250 ml of methanol: 95: 5 water with 100 ml of "Amberlyst 15" (Rohm and Haas, Philadelphia, Pa. 19105) ion exchange resin and boiled under reflux overnight. After decoction, the resin was removed by filtration, the filtrate passed through 40 ml of "Amberlyst A-21" (Rohm and Haas, Philadelphia, Pa. 19105) ion exchange resin and concentrated in vacuo. Crystals present on standing were removed by filtration and washed with acetone to give 2.21 g (21%) of methyl 2-ketogulonate (mp 150-154 ° C, literature 155-157 ° C), which HPLC analysis and C ^^ spekt spectroscopy were found to be 97% ischemic. The methyl 2-ketogulonate can be transferred into ascorbic acid by heating with sodium bicarbonate under nitrogen.

Claims (13)

17 DK 157139 B A process for preparing a mixture of 2-ketogluonate and 2-ketogluconate, characterized in that a 2,5-diketogluconate selected from 2,5-diketogluconic acid is a normal alkyl ester of this acid wherein the alkyl group has 1 4 carbon atoms, and a site of this acid having a counterion selected from alkali metals, alkaline earth metals, ammonium and tetraalkylammonium having 1-4 carbon atoms in each alkyl group is reduced by 0.8 to 1.1 equivalents of one alkali metal borohydride per liter. mole of 2,5-diketogluconate, in solution at a pH greater than 5 and a temperature between -30 ° C and 50 ° C. Process according to claim 1, characterized in that the reduction is carried out at a temperature of -25 ° C to 25 ° C. Method according to claim 1, characterized in that the reduction is carried out at a pH between 6 and 10.5. Process according to claim 1, characterized in that 2,5-diketogluconate constitutes between 5 and 20% by weight of the solution. Method according to claim 1, characterized in that the alkali metal counterion is sodium. Method according to claim 1, characterized in that the alkaline earth metal counterion is calcium. Process according to claim 1, characterized in that the alkyl ester of 2,5-diketogluconic acid is methyl-2,5-diketogluconate. Method according to claim 1, characterized in that the tetraalkylammonium counterion is tetramethyl ammonium. Process according to claim 1, characterized in that the alkali metal borohydride is sodium borohydride. 10. A process according to claim 1, characterized in that the reduction is carried out in aqueous solution. Process according to claim 10, characterized in that the aqueous solution contains a co-solvent selected from alkanols of 1-4 carbon atoms, alkanediols with 2-4 carbon atoms, acetonitrile, dimethylsulfoxide and dimethylformamide. Process according to claim 11, characterized in that the co-solvent is methanol. A process according to claim 1, characterized in that the reduction is carried out in the presence of a boron complexing agent selected from alkali metal fluorides, ammonium fluoride and boron absorbing ion exchange resins.