JPH049796B2 - - Google Patents

Description

[Detailed Description of the Invention] [Field of Industrial Application] This invention is directed to the production of solid acetyl phosphoric acid (meaning monoacetyl phosphoric acid) using orthophosphoric acid (hereinafter also simply referred to as phosphoric acid) as a raw material. It concerns a method by which lithium salts can be obtained.

Certain salts of monoacetyl phosphate, such as dilithium salt, diammonium salt, disodium salt, lithium potassium salt, etc., are substrates for acetate kinase, and adenosine triphosphate (ATP) is involved.
It is known that it can be used in enzymatic reactions. The present invention relates precisely to a method for obtaining monoacetyl phosphate that can be used in such fields.

Acetyl phosphate, which can be used in enzyme reactions, has so far been obtained by relatively complicated methods, but according to the present invention, acetic anhydride as an acetylating agent and lithium ions as a precipitant are added to phosphoric acid. By simply allowing it to act, a highly pure target product can be obtained without the need for conventional complicated separation processes.

[Conventional technology and problems]

It is known that acetylation of phosphoric acid using an acetylating agent such as acetic anhydride or ketene yields mixtures of mono- and diacetyl phosphoric acids, and optionally triacetyl phosphoric acids. A typical example of a method for obtaining crystalline salts of monoacetyl phosphate, which can be used in the enzymatic reactions described above, from these reaction products is 100%
% phosphoric acid with acetic anhydride using an ethyl acetate solvent is added to cold ammonia-saturated methanol (G.
M. Whitesides et al.; J.Org.Chem. 44 , 864). However, this method involves operation at low temperatures (-30 to -10°C), and monoacetyl phosphate diammonium salt is easily decomposed by excess ammonia.
Strict control of reaction conditions is required for smooth over-separation. Acetyl phosphate can also be obtained as an aqueous solution from a similar acetylation reaction solution (DCCrans, GM
Whitesides; J.Org.Chem., 48 , 3130-32), the fact that the product is an aqueous solution is more inconvenient to store and use than a solid product.

On the other hand, it is also known to obtain acetyl phosphate by acetylating a salt of phosphoric acid with acetic anhydride. West German Patent No. 2831831 and East German Patent No.
No. 213902 is a prior art related to this, and uses acetic anhydride itself or acetic acid added thereto as a medium, but the examples in these patents use sodium or potassium salts of phosphoric acid as a medium. This shows that when acetylation is performed using acetic anhydride or acetic anhydride/acetic acid, which does not have the ability to dissolve, the reaction progresses to the diacetyl phosphate stage. Therefore, in order to obtain monoacetyl phosphate, another solvolysis treatment with alcohol (diacetyl→monoacetyl) was required, as shown in the latter part of the East German patent.

In the present invention, the lithium salt of monoacetyl phosphoric acid selected as suitable for obtaining acetyl phosphoric acid in the form of a solid salt has been conventionally used as a means for purifying and isolating monoacetyl phosphoric acid. Known (ERStadtman, F.
Lipmann, J.Biol.Chem., 185 , 549−551:
Methods in Enzymology, vol, p.228−
231, ed. SPColowick, NO Kaplan (1957); D.
E. Koshland, Jr., J. Amer. Chem. Soc., 73 ,
4103; AWDavison, J.Chem.Soc., 1955, 736
etc.). These separation methods utilize the property that dilithium monoacetyl phosphate is sparingly soluble in an ethanol-water mixed solvent.

According to RW Porter et al. (see J. Biol. Chem., 244 , 1847), lithium acetate was added to the monoacetyl phosphoric acid salt obtained by carrying out the reaction in an acetonitrile solvent and distilling off the volatile components such as the solvent. By adding the containing methanol, the dilithium salt of acetyl phosphate is precipitated.

As described above, the medium conventionally used to precipitate the lithium salt of acetyl phosphate is an alcohol-based solvent that is reactive with acetic anhydride, and therefore the precipitation method using lithium salt is similar to the acetylation method using acetic anhydride. It could not be directly linked to the reaction.

Thus, until now, there has been no known method for precipitating easy-to-handle monoacetyl phosphate crystals from an acetylated product of phosphoric acid by a simple method that does not use low temperatures. In addition, the acetylation of phosphates resulted in excessive diacetylation, and in order to precipitate the lithium salt, it was necessary to replace the solvent with a solvent different from that used in the acetylation reaction.

[Means for solving problems]

The present invention solves these problems seen in the prior art and provides a new simplified method for producing monoacetyl phosphate in which an acetylation reaction and precipitation separation are directly connected.

That is, the present invention provides a reaction mixture obtained by acetylating orthophosphoric acid with acetic anhydride in an inert solvent, in which the molar ratio of lithium ions to phosphate groups is
This is a method for producing acetyl phosphate, which is characterized by precipitating a lithium salt of acetyl phosphate in solid form by applying lithium ions in an amount of 1.5 to 2.3. In the present invention, the term molar ratio means the ratio of not only molecules but also atoms, atomic groups, and ions.

[Effect]

As a typical embodiment of the present invention, a case where the inert solvent is acetic acid will be described.

That is, orthophosphoric acid is first monoacetylated with a mixture of acetic acid and acetic anhydride, the reaction is stopped by adding approximately 2 moles of lithium ion to orthophosphoric acid to the reaction mixture, and monoacetyl phosphoric acid is obtained by continuing stirring and mixing. A lithium salt of the acid is formed and precipitated in the system and separated from the mother liquor. The same applies when an inert solvent other than acetic acid is used.

By setting the Li/PO ₄ molar ratio to approximately 2 (1.5 to 2.3), the lithium salt of acetyl phosphate precipitates from the reaction mixture containing an inert solvent as a solid that is easily separated from the mother liquor at around room temperature. It was discovered that
As a result, a simple process that directly connects the acetylation reaction and precipitation separation was realized.

The present invention does not require substitution of the alcoholic medium conventionally used to precipitate lithium salts, nor does it require the low temperatures of ammonium salt precipitation methods. According to the present invention, acetyl phosphate, which is suitable as a substrate for acetate kinase, can be easily obtained by using readily available starting materials and performing reaction operations at around room temperature.

The orthophosphoric acid used in the present invention may be anhydrous or hydrated. For example, 100% phosphoric acid (see e.g. J.Org.Chem., 40 , 2518 for preparation),
98% phosphoric acid (crystalline), 89% phosphoric acid, 85% phosphoric acid, or 75% phosphoric acid can be used.

The acetylation reaction of orthophosphoric acid is carried out in an inert solvent. Here, the inert solvent is a solvent that can dissolve a mixture of acetic anhydride and phosphoric acid and does not irreversibly react with them. Examples include acetate esters such as methyl acetate, isopropyl acetate, and 2-ethoxyethyl acetate, and ethers such as ethyl ether, isopropyl ether, tetrahydrofuran, dioxane, and methyl-t-butyl ether.

A suitable molar ratio of inert solvent to phosphoric acid is generally in the range of 15 to 2, more preferably in the range of 13 to 3, at the start of the acetylation reaction.

The inert solvent has the function of lowering the concentration of phosphoric acid and acetic anhydride in the acetylation reaction, thereby allowing the reaction to proceed slowly, and ensuring good mixing conditions during acetylation and precipitate formation. In the absence of an inert solvent, the condensation reaction of phosphoric acid becomes an important side reaction. Low temperature (e.g. -5 to +5℃) to suppress this side reaction
If this is done, the solvent-free acetylation reaction system becomes viscous, making it difficult to stir or control the reaction temperature.

Acetic anhydride, in addition to serving as an acetylating agent and medium, reacts with water in the reaction mixture to form acetic acid,
Since it also has the role of keeping the reaction system in a substantially anhydrous state, its usage amount is determined in consideration of the influence of the molar ratio of water and PO ₄ groups contained in the raw phosphate. The value obtained by subtracting the molar ratio of water to PO ₄ from the molar ratio of acetic anhydride to phosphate groups (PO ₄ ) used here is called the effective acetic anhydride molar ratio, and it is usually a value of 1.0 or more. The amount of acetic anhydride to be used is determined within the range. The preferred range of effective acetic anhydride molar ratio in the present invention is
It is 1.5 to 10, more preferably 2.0 to 8.0.

After adding the ionic lithium compound, acetic anhydride may be added as necessary.

The acetylation reaction temperature needs to be sufficiently controlled to suppress rapid generation of reaction heat and to suppress side reactions such as condensation of phosphoric acid. The reaction temperature must also be set to maintain the viscosity of the reaction mixture and thus good stirring and mixing conditions.

Although the preferred reaction temperature varies depending on the type and amount of the inert solvent, the reaction is usually carried out at a temperature of 30° C. or lower where side reactions and exothermic peaks are not noticeable. When using acetic acid, the temperature is usually 10 to 30°C.

The reaction time can be determined experimentally using reaction results (yield and purity) as indicators for each reaction temperature. In the case of acetic acid solvent, the time is usually 30 minutes to 2 hours, preferably 30 minutes to 1 hour.

Following the acetylation reaction or in parallel with the acetylation reaction, lithium ions are applied to form a precipitate.

Preferred ionic lithium compounds that supply lithium ions include lithium acetate (anhydrous), lithium acetate (dihydrate), lithium hydroxide (anhydrous), lithium hydroxide (monohydrate), and lithium carbonate. These may be added in powder form or in the form of a dispersion in an inert solvent such as acetic acid or acetic anhydride. After the addition, the reaction mixture is further stirred until it becomes a uniform slurry. The amount of ionic lithium compound used is such that the molar ratio of lithium ions to orthophosphoric acid is 1.5 to 2.3, preferably 1.8 to 2.3.
It is determined to be 2.3, more preferably in the range of 1.9 to 2.2. It is desirable that the addition is normally carried out at a temperature of 30°C or lower, more preferably 20°C or lower.

The lithium salt of acetyl phosphate forms as a white crystalline solid and can be easily isolated from the reaction mixture by solid-liquid separation means such as filtration. The powder obtained by washing with an organic solvent such as methanol acetate and drying under reduced pressure can be used for enzyme assay (GM Whitesides et al., J.Org.Chem., 40 ).
2516-9) and liquid chromatography (reference example).
It can be confirmed that it is a lithium salt of monoacetyl phosphoric acid containing 5.8 mmol/g.

〔Effect of the invention〕

In the present invention, a specific amount of lithium ions with a Li/PO ₄ molar ratio close to 2 is applied to a reaction mixture using an inert solvent such as acetic acid as a medium for the acetylation reaction of phosphoric acid with acetic anhydride. By doing so, we have made it possible to directly obtain acetyl phosphate in the form of a crystalline solid that has high purity and is easy to handle using an enzyme assay, through a separation process that is directly connected to the acetylation reaction.

〔Example〕

The present invention will be explained in more detail below using specific examples.

Reference example Liquid chromatography analysis of acetyl phosphate Samples of acetyl phosphate can be separated and analyzed using the following requirements. Acetate, diacetyl phosphate and monoacetyl phosphate are detectable.

[Liquid chromatography conditions]

Column: Nagel Nucleosil 10-( _CH3 ) _2N 4.6mmφ×5cmL+4.6mmφ×25cmL Column temperature: 0°C ₍ ice water bath) Mobile phase: 1.6wt _{% NH4H2PO4} aqueous solution (PH4.60) Flow rate: 1.5 ml/min Detector: JASCO UVIDEC-, 210nm Data processor: SIC model 7000AS [Retention time of each component] Component Retention time (min) Acetate 4.3 Diacetyl phosphate 4.8 Monoacetyl phosphate 6.7 [Analysis method ] Dissolve approximately 50 mg of acetyl phosphate sample in 1 ml of distilled water (cooled overnight in an ice-water bath), take a 10μ aliquot using a microsyringe, and obtain a chromatogram.
Relative purity is determined by comparing the peak area of monoacetyl phosphate with that of a standard sample (lithium potassium acetyl phosphate, manufactured by Boehringer Mannheim). The purity of the standard sample was determined by enzyme assay (method was provided by GM).
Whitesides et al., J.Org.Chem., 40 , 2516-9.
(1975)). Purity is the concentration of active phosphoric acid (abbreviated as ~P) expressed in units of mmol/g.

Example 1 Acetic anhydride (183.5 g. , 1.80 mol) and continued stirring at 30°C for 60 minutes. Lithium carbonate (26.7 g, 0.361
mol) was added over a period of 7 minutes and stirring was continued for a further 4 hours at a temperature between 20 and 25°C. The resulting colorless precipitate was suctioned off using a glass filter (G-2), washed with 200 ml of acetic acid and 350 ml of methanol,
After drying under reduced pressure, 51.1 g of the lithium salt of monoacetyl phosphate was obtained as a crystalline powder. The purity according to the liquid chromatography method described in the reference example is ~P
= 5.49 mmol/g, and the purity by enzyme assay was ~P = 5.41 mmol/g.

Example 2 Acetic anhydride (105.3 g, 1.03 mol) was added dropwise to a mixture of acetic acid (110.2 g, 1.84 mol) and 100% phosphoric acid (40.2 g, 0.410 mol) with stirring for 60 minutes at 10°C. , and lithium carbonate (29.2g,
0.395 mol) was added at the same temperature. The resulting slurry was stirred at 20°C for 3 hours and then at 35°C for 5 hours, and the precipitate was suctioned off using a glass filter. Thereafter, 50.3 g of acetyl phosphate was obtained in the same manner as in Example 1. The purity determined by liquid chromatography was ~P=5.79 mmol/g.

Example 3 Acetic anhydride (190.2 g, 1.86 mol) was added to a mixture of acetic acid (149.9 g, 2.50 mol) and 100% phosphoric acid (36.5 g, 0.372 mol) and stirring was continued for 60 minutes at 20°C. . Lithium carbonate (27.3 g, 0.369 mol) was added to the resulting homogeneous reaction mixture and the mixture was heated at 20°C for 3 hours.
Stirring was continued for an hour. After the reaction mixture was left at the same temperature overnight, the resulting precipitate was suctioned off using a glass filter. Thereafter, the same treatment as in Example 1 was carried out to obtain 55.6 g of acetyl phosphate. Purity by liquid chromatography is ~P=5.26m
It was mol/g.

Example 4 Acetic anhydride (214.4 g, 2.10 mol) was added dropwise to a mixture of commercially available 85% phosphoric acid (40.1 g, 0.348 mol) and acetic acid (110.2 g, 1.84 mol) at 10° C. over 40 minutes with stirring. In addition, lithium carbonate (27.5g,
0.372 mol) was added at the same temperature. The resulting slurry was stirred at 20° C. for 3 hours and then at 30° C. for 17 hours, and then treated in the same manner as in Example 1 to obtain 55.1 g of acetyl phosphate. The purity determined by liquid chromatography was ~P=5.70 mmol/g.

Example 5 Acetic anhydride (213.1 g, 2.09 mol) was added dropwise to a mixture of commercially available 85% phosphoric acid (40.2 g, 0.349 mol) and acetic acid (120.4 g, 2.00 mol) with stirring for 40 minutes at 10°C. And then lithium hydroxide (anhydrous)
(17.0, 0.710 mol) and stirred the resulting slurry at about 25°C for 3 hours and further at 35°C for 5 hours.
When the same treatment as in Example 1 was carried out, 53.6 g of acetyl phosphate was obtained. The purity determined by liquid chromatography was ~P=5.35 mmol/g.

Example 6 Acetic anhydride (176.6 g, 1.73 mol) was added dropwise to a mixture of commercially available 85% phosphoric acid (40.2 g, 0.349 mol) and acetic acid (164.9 g, 2.75 mol) with stirring, and the mixture was heated at 20°C.
Stirring was continued for 2 hours. Add lithium carbonate (25.7 g, 0.348 mol) to the resulting homogeneous reaction product,
After stirring at the same temperature for 3 hours, the mixture was further left overnight. Thereafter, the same treatment as in Example 1 was carried out to obtain 44.9 g of acetyl phosphate. The purity determined by liquid chromatography was ~P=4.82 mmol/g.

Example 7 Acetic anhydride (109.6 g, 1.07 mol) was added dropwise to a mixture of commercially available 85% phosphoric acid (40.2 g, 0.349 mol) and acetic acid (140.2 g, 2.33 mol) with stirring for 60 minutes at 15°C. Then, lithium carbonate (26.7g,
0.361 mol) was added at the same temperature. After stirring for 1 hour, acetic anhydride (33.4 g, 0.327 mol) was added and 35
Stirring was continued for an additional 5 hours at °C. Thereafter, the same treatment as in Example 1 was carried out, and 53.3 g of acetyl phosphate was obtained. The purity determined by liquid chromatography was ~P=5.36 mmol/g.

Example 8 Acetic anhydride (124.0 g, 1.21 mol) was added dropwise to a mixture of commercially available 85% phosphoric acid (40.0 g, 0.347 mol) and acetic acid (66.8 g, 1.11 mol) at 10° C. for 40 minutes while stirring. Then, at the same temperature, lithium acetate (anhydrous) (46.7 g, 0.708 mol) was added over a period of 10 minutes, and stirring was continued for a further 5 hours at a temperature between 20 and 25°C. After that, when the same process as in Example 1 is performed, the result is 50.6
g of acetyl phosphate were obtained. The purity determined by liquid chromatography was ~P=5.31 mmol/g.

Example 9 A mixture of 100.0 g of 100% phosphoric acid, 150.2 g of ethyl acetate, and 129.5 g (1.24 mole times) of acetic anhydride was heated at 0°C.
The mixture was kept for 3.0 hours to obtain an acetylated mixture.

20 g of the above reaction solution was taken out and added to each of the following mixtures, followed by stirring at about 20°C.

○B Acetic acid 10ml + Lithium acetate○B Acetic acid 10ml + Sodium acetate○C Acetic acid 10ml + Potassium acetate Approximately 2 times the mole of phosphoric acid○D Acetic acid 10ml + Magnesium acetate tetrahydrate
Almost equimolar to phosphoric acid ○I produced a crystalline precipitate and confirmed acetyl phosphate by HPLC method.

No formation of precipitate was observed in ○B and ○C, which remained almost clear.

○D has solidified into a gel-like consistency.

Example 10 100% phosphoric acid 22g, methyl acetate 29g, acetic anhydride
30g of the mixture was kept at 0-3℃ for 3.5 hours, and the reaction mixture was diluted by first adding 50g of acetic acid.
16 g of Li ₂ CO ₃ was added. When the cooling bath was removed and stirring was continued, a powdery precipitate formed instead of dissolving the Li ₂ CO ₃ . Separate a portion of the precipitate and perform HPLC
Acetyl phosphate was confirmed by method.

Example 11 Acetyl was substituted in the same manner as in Example 10, except that the following solvent system was used instead of methyl acetate. Precipitation of acetyl phosphate was similarly confirmed in these cases as well.

() Isopropyl ether + ethyl acetate 3:2
mixture () methyl-t-butyl ether + ethyl acetate 3:2 mixture () tetrahydrofuran () isopropyl acetate

Claims (1)

[Claims] 1. A reaction mixture obtained by acetylating orthophosphoric acid with acetic anhydride in an inert solvent is treated with lithium ions in an amount such that the molar ratio of lithium ions to phosphoric acid groups is 1.5 to 2.3 to form acetyl phosphoric acid. A method for producing acetyl phosphate, characterized by precipitating lithium salt in solid form.