DE2923268A1 - Riboflavin prodn. from D-glucose - via gluconic acid, arabinose, ribose, ribityl-xylidine and ribityl:amino-di:methyl-phenyl:azo-benzene - Google Patents

Abstract

Prodn. of riboflavin (I) is carried out by (a) oxidising D-glucose to D-gluconic acid or an alkali metal gluconate, (b) reacting with hypochloride to form D-arabinose, (c) treating with a hexavalent Mo cpd. to form D-ribose, (d) hydrogenating in the presence of nitroxylene or xylidine, (e) reacting the resulting N-D-ribityl-3,4-xylidine (II) with the corresp. diazonium salt to form 1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene (III),and (f) reacting (III) with barbituric acid (IV). Step (a) is pref. effected by fermentative oxidn., e.g. with Acetobacter suboxydans. Step (b) is pref. effective by rapidly adding a 10-20% Nacl soln. to a 10-40% soln. of D-glutamic acid at 30-90 deg.C and pH 4-6. Step (c) can be effected with molybdic acid, followed by crystallisation to remove most of the unconverted arabinose. Step (d) is pref. effected at 50-80 deg.C and 3 bar in the presence of a Pd/C catalyst.

Description

Process for the production of

Riboflavin The invention relates to a new method of production of riboflavin, starting from D-glucose.

Processes for the production of riboflavin are known.

The process usually carried out on an industrial scale starts with D-glucose which is oxidized to D-arabonic acid, which is epimerized to D-ribonic acid and then is converted to D-ribonolactone, from which D-ribose is formed by amalgam reduction which is hydrogenated via the xylidine riboside to the N-D-ribityl-3,4-xylidine.

The N-D-ribityl-3,4-xylidine can then be reacted with diazotized Aniline and barbituric acid riboflavin are obtained.

The yield from this process is for the. Levels of D-glucose to N-D-ribityl-3,4-xylidine at about 20-23%. This gives an overall yield of riboflavin, starting from D-glucose, from 15-16% of theory. Downside besides The relatively poor overall yield in this process is also the epimerization step from D-arabonic acid to D-ribonic acid, in the case of a number of resinous By-products are formed which require a complicated purification do. In particular, however, the amalgam reduction causes very great problems because Considerable effort must be made when working with large quantities of mercury need to keep both the products and the waste materials free of mercury keep.

It has therefore already been proposed methods that work avoid with mercury.

So can D-ribonic acid or D-ribonolactone in the presence of 3,4-xylidine or 4-nitro-1,2-xylene can be hydrogenated in one step to give N-D-ribityl-3,4-xylidine.

However, this requires a very investment-intensive high-pressure hydrogenation system, because the hydrogenation takes place at a pressure of about 250-300 bar. Despite the savings of intermediate stages and in spite of the high costs involved in this process Yield of N-D-ribitylxylidine starting from D-glucose only at a maximum of 35 %, resulting in a riboflavin yield of less than 25%.

It has also been suggested to ferment D-ribose directly in a microbiological process with the help of suitable microorganisms D-glucose to produce and then in the usual way N-D-ribityl-xylidine and To gain riboflavin. Although this approach has advantages, the large-scale one Carrying out the complicated and, above all, failure-prone biochemical process Considerable difficulties are to be expected, affecting the economics of the process affect.

In addition, the yield of N-D-ribityl-xylidine is also in this process, starting from D-glucose, only at about 34%, resulting in a riboflavin yield of about 24%.

There was therefore the object of a process for the production of Finding riboflavin that can be done in simple fashion without large investments Reaction steps using inexpensive starting materials and avoiding them environmentally harmful, highly toxic reaction components in good yield to one pure product leads. This object has been achieved by the present invention.

It has been found that, surprisingly, a completely new Path that also starts from D-glucose, but not via arabonic acid and ribonic acid leads, but via gluconic acid and arabinose D-ribose and N-D-ribityl-3,4-xylidine delivers, avoids the disadvantages of the known processes and even with higher yields than the known processes leading to riboflavin. This is surprising in that known as such as the individual steps of the method according to the invention was. However, it could not be foreseen that this particular combination of individual reactions would lead to such an advantageous overall result, especially since Numerous attempts have been made to make this important product even larger Scale advantageous to manufacture.

The invention accordingly provides a method for production of riboflavin, which is characterized in that one is known per se consecutively a) D-glucose to D-gluconic acid or an alkali gluconate oxidized, b) the gluconate thus obtained with the aid of hypochlorite in D-arabinose transferred, c) D-arabinose with a molybdenum (VI) compound converts into D-ribose, d) D-ribose hydrogenated in the presence of nitroxylene or xylidine, e) the obtained N-D-ribityl-3,4-xylidine by treatment with the corresponding diazonium salt L-D-Ribitylamino-3, 4-dimethyl-6-phenylazobenzene converts and f) this azo compound converted into riboflavin by reaction with barbituric acid.

It is surprisingly advantageous in this process that the individual Steps without difficulty with inexpensive and non-toxic reaction components can be carried out and that in part on the isolation of intermediate products can be dispensed with without loss of yield, so that one can without great labor and Investment outlay reaches the end product.

It is particularly surprising that very good yields are nevertheless achieved that are above those achieved with the previously known methods will.

Thus, N-D-ribitylxylidine is used as a starting point according to the process according to the invention of D-glucose, obtained in a yield of about 40%, equivalent to a riboflavin yield of almost 30%, based on D-glucose.

To carry out the method, as in the case of the known methods can be assumed from the easily accessible, inexpensive D-glucose. D-gluconic acid is obtained from it in very high yields of over 90% in a known manner by fermentative, to obtain chemical or electrochemical oxidation. An overview above such procedures can be found e.g. in Ind.Eng.Chem.

11, pp. 370-372 (1972). Fermentative oxidation is preferred carried out. For this purpose, D-glucose is used in an aqueous solution with the addition of nutrients in a fermenter under sterile conditions with a suitable bacterial strain such as Acetobacter suboxydans with aeration and neutralization of the resulting Acid treated with a base, preferably caustic soda, until the test on sugar is negative, i.e. all of the glucose is converted. Depending on the batch size and bacterial strain, the oxidation is complete in about 10 to 40 hours.

The approximately 20% that is obtained in practically quantitative yield Gluconate solution can be used directly for further oxidation to arabinose. Surprisingly, it has been found that the per se known oxidation of gluconate (preferably alkali gluconate) to arabinose with hypochlorite significantly improved can be, if the oxidation with high gluconate concentration and at high Temperature. In the case of the J.Amer. Chem. Soc., Volume 81, page 5190 ff (1959), known reaction produces an approximately 2% gluconate solution within approximately 20-30 hours at room temperature with an approximately 2.5-fold molar excess of hypochlorite reacted to provide arabinose in about 40% yield.

In contrast, in the method of the present invention, about 10-40% by weight aqueous gluconate solution is used, preferably about 20% by weight Fermentation solution. This takes only 10 - 60 minutes at one temperature from about 30-90 ° C., preferably about 50-70 ° C., with 1.0-1.5 equivalents, preferably about 1.1-1.2 equivalents of hypochlorite set, which is slowly added during the reaction.

The pH value of the solution is determined by the simultaneous addition of acid, preferably concentrated hydrochloric acid, kept constant at about 4-6. It will not only because of the high concentration of the reactants and the short reaction time a very good space / time yield is achieved, but also the absolute yield about twice as much as compared to the known reaction.

Even when using the fermentation solution obtained from glucose a yield of arabinose of about 70-75%, based on glucose, is achieved. The arabinose can be released in the usual way after the addition of hypochlorite the reaction solution can be isolated.

For this purpose, the reaction solution of common salt and unreacted gluconate e.g. freed by electrodialysis or by ion exchange and the arabinose solution evaporated to crystallization. By stirring with an alcohol, e.g. B. methanol, very pure D-arabinose is obtained in high yield. The one that is already very high Overall yield can be increased if the recovered gluconate is renewed is used.

The D-arabinose thus obtained can by a method known per se under catalysis by molybdic acid or another molybdenum (VI) compound to form a Epimer mixture are implemented, the D-arabinose and D-ribose in the ratio of contains about 3: 1, but also pentoses such as D-LyY.ose and D-xylose as well other by-products: e. Although left for the further implementation to riboflavin alone desired D-ribose makes up only the minor part of the epimer mixture In general, this route to ribose turns out to be very advantageous in the overall process proven. On the one hand, that can be present as the main component of the mixture of epimers D-Ara- binose very simply, almost completely from the mixture of epimers be separated; on the other hand it is not necessary to remove the D-ribose from the epimer mixture isolate at all, but the mixture can go directly into the per se as well known hydrogenation in the presence of nitroxylene or xylidine can be used. From the mixture obtained in the hydrogenation of ribityl, arabityl, xylityl and lyxityl-xylidine surprisingly crystallizes only that which is desired N-D-Ribityl-3,4-xylidine, so that time-consuming cleaning steps are saved can.

The process runs in detail so that the arabinose in water is dissolved and the catalyst is added at an elevated temperature. The concentration the arabinose is not critical, but it is in the interests of good utilization The highest possible concentrations of the existing equipment are used, e.g. about 10-20% by weight solutions. It can also be the result of gluconate oxidation Solution can be used without isolation of the arabinose. This solution is based on a Temperature of about 80-1000 C and heated with about 1 wt .-% catalyst, z. B.

Molybdic acid, based on arabinose, added, whereby it is advantageous is when a pH is adjusted around 3. With increasing amount of catalyst and as the temperature increases, there will be a faster adjustment of the epimeric equilibrium achieved. At a temperature of about 90 - 950 C and 1 wt .-% molybdic acid is equilibrium is reached after about 2 hours, i.e. there is a mixture of the 4 pentoses arabinose, ribose, lyxose and xylose in addition to a certain proportion of decomposition and oxidation products. To avoid an excessive proportion The apparatus can remove oxidation products when reacting with an inert gas, e.g. nitrogen.

After the reaction has ended, the catalyst is removed from the solution, z. B. with the help of ion exchangers. Then, preferably under reduced Pressure, concentrated. By adding a lower alcohol, e.g. methanol or, preferably, Ethanol, most of the arabinose present in the reaction mixture can crystallize, separated in pure form and used again in a further batch in the reaction will.

From the mother liquor, which contains about 10-20% solids, the to about 75% from D-ribose, about 10% from D-arabinose, about 5% from D-xylose and D-lyxose and consists of about 10% by-products, the ribose lets through Chromatography over a cation exchanger loaded with calcium or barium ions to win.

According to the method according to the invention, however, this can be expensive The D-ribose is not shown in pure form and the mother liquor can be used instead used for catalytic hydrogenation in the presence of 4-nitro-o-xylene or 3,4-xylidine will. To do this, the mother liquor is diluted with aqueous alcohol and mixed with the equivalent amount (based on the total amount of pentoses) of 4-nitro-1,2-xylene offset.

The hydrogenation itself can, as in the Japanese patent application No. 6665 (1964) described, with Raney nickel as a catalyst at a hydrogen pressure from about 50 - 100 bar. At a hydrogenation temperature of about At 60-80 ° C., the reaction is complete after about 30-60 minutes and after removal of the catalyst and concentration of the solution, the pure crystallizes on cooling N-D-ribityl-3,4-xylidine. While with the previously known method nickel-catalyzed hydrogenation was used almost exclusively now found that the hydrogenation is very advantageous even with palladium on charcoal can be carried out at only a low hydrogen pressure of about 3 bar. Again, in a mixture of water with a lower alcohol at increased Temperature of about 50 - 80 ° C worked, with the hydrogen uptake after about 2 - 3 hours is finished. The yield is comparable to that of high pressure hydrogenation; However, there are advantages in the fact that the investment in the hydrogenation plant are significantly lower and that, on the other hand, the catalyst is recovered quantitatively and can be used again. Post-purification of the wastewater for removal of nickel ions can therefore be omitted. After separating the catalyst by filtration When the solution cools, the pure N-D-ribityl-3,4-xylidine crystallizes to a high degree Yield and purity.

The N-D-ribityl-3,4-xylidine is a key product in riboflavin synthesis view, which is also run through in the other known riboflavin syntheses. As is known, this intermediate product is converted to riboflavin Azo coupling of ribitylxylidine with a benzene diazonium salt and then Implementation of the azo compound with barbituric acid. These conversions are known. One A review of the literature on these processes can be found e.g.

in Kirk-Othmer, Encyclopedia of Chemical Technology, Volume 17 (1968), P. 451.

Usually it is acidic, aqueous or aqueous / alcoholic Solution or suspension of the N-D- Ribitylxylidins at low temperature mixed with phenyldiazoium chloride and isolated the azo compound formed. the subsequent reaction with barbituric acid in acetic acid solution yields with cleavage of aniline riboflavin, which is optionally dissolved in aqueous hydrochloric acid, Treating with hydrogen peroxide and precipitating with water can still be purified can.

The overall yield in the process according to the invention, starting out of D-glucose, is above the yields achieved with the known processes, so that with the method according to the invention a very valuable new method of manufacture of riboflavin is available.

Example 1 a) Conversion of D-glucose to sodium D-gluconate.

In a fermenter, 10 l of a sterile aqueous solution, the 1.5 kg D-GlucoseH2O, 10 g Cornsteep dry matter, 10 g ammonium dihydrogen phosphate, Contains 10 g of potassium dihydrogen phosphate, 5 g of magnesium sulfate and a pH of 6.5, at a temperature of 30 OC with a shaking culture of Acetobacter suboxydans ATCC 621 inoculated and aerated with stirring. By adding baking soda lye a pH of 5.5-6.0 is maintained. After about 40 hours when the Glucose is completely converted to gluconate, is cooled and centrifuged. Sodium gluconate can be obtained by evaporating the centrifuged solution in crystalline form with over 90 % Yield can be obtained.

b) Conversion of sodium D-gluconate to D-arabinose.

A solution of 218 g of sodium D-gluconate in 872 ml of water or the The corresponding amount of the fermentation solution from example la) is heated to 60.degree and at that temperature with 532 ml of a sodium hypochlorite solution containing of 16% (w / v) of active chlorine added, with the simultaneous addition of About 80 ml of concentrated hydrochloric acid kept the pH constant at 4.5-5.0 will. Thereafter, the solution is made strong by electrodialysis or by ion exchange ( acidic and weakly basic exchanger) freed from electrolytes. By evaporation 108 g (72%) of crystalline D-arbinose can be obtained by stirring the residue with methanol be won.

c) Conversion of D-arabinose to D-ribose.

A solution of 100 g of D-arabinose in 500 ml of water or the equivalent The amount of arabinose solution from Example 1b) is increased to 92 while flushing with nitrogen OC heated and 1 g of molybdic acid (commercial quality, mostly from Consisting of ammonium molybdate) for 1 - 2 hours. After that, will the Electrodialysis or ion exchange catalyst (strongly acidic and weakly basic Exchanger) removed. The solution becomes one which still contains about 10% water Concentrated syrup and extracted with 200 ml of ethanol. 70 g of D-arabinose crystallize that are separated and reused.

The aqueous / alcoholic mother liquor, which contains about 21 g of D-ribose, 3 g of D-arabinose, 3 g of a mixture of D-lyxose and D-xylose and 3 g of further product contains, can be used to obtain D-ribose via a calcium or barium ion loaded cation exchanger are chromatographed d) conversion of D-ribose to N-D-ribityl-3,4-xylidine The alcoholic mother liquor from Example lc) is with Dilute the same volume of water with 5 g of sodium acetate and acetic acid to pH 5.8 adjusted, mixed with 30 g of 4-nitro-o-xylene and 15 g of moist Raney nickel, heated to 80 ° C. and hydrogenated at a hydrogen pressure of 50 bar for 1/2 hour. After filtration from the catalyst and evaporation of part of the ethanol crystallize 30 g of N-D-ribityl-3,4-xylidine on cooling.

Additional N-D-ribityl-3,4-xylidine can be obtained by evaporation the mother liquor, stirring the residue with 10% hydrochloric acid, with only that easily soluble hydrochloride of Ribitylxylidins goes into solution, and neutralize the solution separated from the residue, with N-D-ribityl-3,4-xylidine crystallizing out.

e) Conversion of N-D-ribityl-3,4-xylidine to l-D-ribitylamino-3,4-dimethyl-6-phenylazoben A suspension, cooled to -5 ° C., of 616 g of aniline, 1320 ml of water and 1732 ml of 37% strength Hydrochloric acid is treated within one hour with a solution of 456 g of sodium nitrite in 1140 ml of water are added. The diazonium salt solution thus obtained is left with a Temperature of max. 5 ° C within 1 hour to a suspension, the 1532 g of N-D-ribityl-3,4-xylidine, 2200 ml of water, 1800 ml of 100% acetic acid and 470 ml Contains 37% hydrochloric acid, the pH value by simultaneous addition of 32% Sodium hydroxide solution is kept constant at 1.5. After stirring for a while, it is set with Sodium hydroxide solution pH 3t5, stir the precipitated crystals for a few hours Room temperature and sucks off. 2564 g of crude product are obtained, which is obtained by recrystallization can be cleaned from ethanol.

f) conversion of l-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene to Riboflavin A solution of 35.9 g of crude 1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene (obtained according to Example le) and 21.6 g of barbituric acid in 135 ml of dioxane and 25 ml Glacial acetic acid is boiled for 16 hours. After cooling, the precipitated riboflavin becomes filtered, washed with 100 ml of water at 50 ° C. and dried, 32.7 g Rohriboflavin received.

Purification can be carried out in such a way that 100 g of Rohriboflavin at 5Q OC in 130 ml of 37% hydrochloric acid, 29 ml of water and 7.1 ml of 35% hydrogen peroxide be dissolved and the filtered solution with 1144 ml Water one Hour to 90 - 100 OC is heated.

After cooling, it is filtered, with 380 ml of water and 160 ml of methanol washed and dried to give 88.1 g of pure riboflavin.

Total yield of stages a - f, based on D-glucose: 28% of theory.

Example 2 The procedure is analogous to Example 1, instead of the under d) described hydrogenation is proceeded as follows: A solution of 5.243 kg epimer mixture from example lc), which contains about 60% ribose, and 5.442 kg 4-Nitro-oxylene in 72 liters of 50% aqueous methanol (v / v) is combined with 0.6 kg of palladium / carbon catalyst hydrogenated at a temperature of about 62 ° C. and a hydrogen pressure of 3 bar. After about 2.5 hours, when the uptake of hydrogen has ceased, it is filtered and the filtrate was cooled to 0 ° C. for crystallization. After centrifuging, washing and drying gives a total of 4.2 kg of N-D-ribityl-3,4-xylidine.

The catalyst can be reused after washing with glacial acetic acid and water.

Claims (5)

Claims 1. Process for the production of riboflavin from D-glucose via N-D-ribityl-3,4-xylidine, characterized in that one is known per se Way in succession a) D-glucose to D-gluconic acid or an alkali gluconate oxidized, b) the gluconate thus obtained with the aid of hypochlorite in D-arabinose transferred, c) D-arabinose with a molybdenum (VI) compound converts into D-ribose, d) D-ribose hydrogenated in the presence of nitroxylene or xylidine, e) the obtained N-D-ribityl-3,4-xylidine by treatment with the corresponding diazonium salt L-D-Ribitylamino-3, 4-dimethyl-6-phenylazobenzene converts and f) this azo compound converted into riboflavin by reaction with barbituric acid. 2. The method according to claim 1, characterized in that the D-gluconic acid at a pH of 4 - 6, a temperature of 30 - 90 OC and a gluconic acid concentration from 10 to 40% by weight by quickly adding an approximately 10 to 20% by weight hypochlorite solution is oxidized. 3. The method according to claim 1, characterized in that the D-arabinose with a molybdenum (VI) compound to one also containing D-arabinose and D-ribose Epimer mixture is implemented and the main amount of D-arabinose from the epimer mixture is separated. 4. The method according to claim 1, characterized in that for formation of N, D-ribityl-3,4-xylidine that obtained in the epimerization of D-arabinose Ribose-containing epimer mixture after coarse separation of D-arabinose in the presence of Nitroxylene or xylidine is hydrogenated and the N-D-ribityl-3, 4-xylidine by crystallization is won. 5. The method according to claim 4, characterized in that the hydrogenation at a temperature of 50 - 80 OC and a hydrogen pressure of about 3 bar a palladium / carbon catalyst is carried out.