1. A method for preparing a cocarboxylase represented by formula I, comprising the steps of: eluting a mixture containing the co-carboxylase shown as the formula I by using weak base anion exchange resin through water and weak base aqueous solution with the pH value of 10-12 as eluent in sequence, and collecting eluate to obtain aqueous solution containing the co-carboxylase shown as the formula I; the mixture containing the cocarboxylase comprises thiamine monophosphate and the cocarboxylase shown in the formula I;

2. the method according to claim 1, wherein the reaction mixture,

the weak base anion exchange resin is macroporous weak base acrylic acid series anion exchange resin;

and/or the water is pure water;

and/or the pH value is 10-12, and the pH value is obtained by adjusting ammonia water;

and/or the mixture containing the cocarboxylase shown in the formula I also contains one or more of thiamine, thiamine triphosphate and higher phosphate;

and/or the elution by water is carried out until the content of thiamine monophosphate in eluate is less than one percent.

3. The process according to claim 1 or 2, wherein the weakly basic anion exchange resin is one of the following types: d315, D301, D335, or D345;

and/or collecting the eluate obtained by the step (a) of collecting the cocarboxylase shown in the formula I, wherein the purity of the cocarboxylase is more than 95%.

4. The method of claim 1, wherein the mixture comprising the cocarboxylase of formula I is prepared by the following steps: step (1), sequentially adding water and an alcohol solvent into a reaction liquid obtained after phosphorylation reaction of vitamin B1 and a phosphorylating agent, mixing, standing, and removing supernatant to obtain a crude product of a mixture containing the co-carboxylase shown in the formula I;

and (2) washing a solution formed by the crude product and water by using a water-insoluble tertiary amine or a mixed solvent of the water-insoluble tertiary amine and an organic solvent to obtain a mixture containing the cocarboxylase shown in the formula I.

5. The method according to claim 4,

in the step (1), the phosphating agent is one or more of pyrophosphoric acid, a mixture of pyrophosphoric acid and phosphorus pentoxide or phosphorus oxychloride, a mixture containing pyrophosphoric acid prepared by reacting phosphoric acid and phosphorus pentoxide, and high-concentration phosphoric acid;

and/or, in the step (1), the alcohol solvent is isopropanol and/or ethanol;

and/or in the step (1), the volume-to-mass ratio of the alcohol solvent to the vitamin B1 is 2:1-5: 1;

and/or in the step (1), the volume-to-mass ratio of the water to the vitamin B1 is 2:1-5: 1;

and/or, the temperature of the phosphorylation reaction is 100-170 ℃;

and/or in the step (2), the volume-to-mass ratio of the water to the vitamin B1 is 1:1-3: 1;

and/or, in step (2), the water-insoluble tertiary amine is a tertiary amine having 12 to 36 carbon atoms: one or more of an aliphatic tertiary amine, an aliphatic-cycloaliphatic tertiary amine, and an aliphatic-araliphatic tertiary amine;

in the step (2), the organic solvent is one or more of an alcohol solvent having 4 to 8 carbon atoms, an ether solvent having 4 to 8 carbon atoms, a ketone solvent having 5 to 8 carbon atoms and an aromatic hydrocarbon solvent;

in the step (2), the content of the water-insoluble tertiary amine is 10 to 80% by mass based on the mixed solvent.

6. The method according to claim 5,

in the step (1), when the phosphating agent is a mixture containing pyrophosphoric acid prepared from phosphoric acid and phosphorus pentoxide, the molar ratio of the phosphoric acid to the phosphorus pentoxide is 2:1-1: 1; e.g., 1.2:1 to 1.3: 1;

and/or, in the step (1), when the phosphating agent is a mixture containing pyrophosphoric acid prepared from phosphoric acid and phosphorus pentoxide, the molar ratio of the phosphorus pentoxide to the vitamin B1 is 4:1-2: 1; e.g., 3:1 to 3.5: 1;

and/or, when the phosphating agent is a mixture containing pyrophosphoric acid prepared from phosphoric acid and phosphorus pentoxide, the mixture is prepared by reacting phosphoric acid and phosphorus pentoxide at 100-180 ℃; preferably 150 ℃ to 160 ℃;

and/or in the step (1), the volume-to-mass ratio of the alcohol solvent to the vitamin B1 is 3:1-4: 1;

and/or in the step (1), the volume-to-mass ratio of the water to the vitamin B1 is 3:1-4: 1;

and/or, in the step (1), the temperature of the phosphorylation reaction is 130-140 ℃;

and/or in the step (2), the volume-to-mass ratio of the water to the vitamin B1 is 1.5:1-2: 1;

and/or, in the step (2), when the water-insoluble tertiary amine is an aliphatic tertiary amine with 12 to 36 carbon atoms, the aliphatic tertiary amine with 12 to 36 carbon atoms is one or more of trioctylamine, trihexylamine and tridecylamine;

and/or, in step (2), when the water-insoluble tertiary amine is an aliphatic-cycloaliphatic tertiary amine having 12 to 36 carbon atoms, the aliphatic-cycloaliphatic tertiary amine having 12 to 36 carbon atoms is N, N-dimethylcyclohexylamine;

and/or, in step (2), when said water-insoluble tertiary amine is an aliphatic-araliphatic tertiary amine of 12 to 36 carbon atoms, said aliphatic-araliphatic tertiary amine of 12 to 36 carbon atoms is N, N-dimethylbenzylamine;

and/or, in the step (2), when the organic solvent is an alcoholic solvent with 4 to 8 carbon atoms, the alcoholic solvent with 4 to 8 carbon atoms is one or more of methyl isobutyl carbinol, 3-ethyl pentanol, 1-hexanol and methyl cyclohexanol;

and/or, in the step (2), when the organic solvent is an ether solvent with 4 to 8 carbon atoms, the ether solvent with 4 to 8 carbon atoms is one or more of diethyl ether, isopropyl ether, methyl tert-butyl ether and di-n-butyl ether;

and/or, in the step (2), when the organic solvent is a ketone solvent with 5 to 8 carbon atoms, the ketone solvent with 5 to 8 carbon atoms is one or more of diethyl ketone, methyl isobutyl ketone, acetophenone and cyclohexanone;

and/or, when the organic solvent is an aromatic solvent, the aromatic solvent is toluene;

and/or, in the step (2), the mass percentage content of the water-insoluble tertiary amine based on the mixed solvent is 40 to 70 percent.

7. A preparation method of a cocarboxylase tetrahydrate is characterized by comprising the following steps: concentrating the water solution containing the cocarboxylase shown in the formula I, and filtering the precipitated solid to obtain a cocarboxylase tetrahydrate; the aqueous solution containing the co-carboxylase shown in the formula I is prepared by the preparation method of the co-carboxylase shown in the formula I according to any one of claims 1 to 6.

8. The method of claim 7, wherein the concentration is at a temperature of less than 35 ℃, preferably 30 ℃ to 35 ℃;

and/or when the aqueous solution containing the co-carboxylase shown as the formula I contains excessive alkali ions, neutralizing the excessive alkali ions by using acid, crystallizing, and filtering the separated solid to obtain a co-carboxylase tetrahydrate; the acid can be hydrochloric acid; the crystallization may comprise the steps of: adding an alcohol solvent into the aqueous solution containing the cocarboxylase shown in the formula I, and filtering the precipitated solid to obtain the hydrochloride of the cocarboxylase; wherein, the alcohol solvent can be isopropanol and/or ethanol; the volume ratio of the alcohol solvent to the solution containing the hydrochloride of the cocarboxylase shown in formula I can be 2:1-3: 1.

9. A method for preparing a hydrochloride salt of a cocarboxylase, comprising the steps of: concentrating the water solution containing the cocarboxylase shown in the formula I, and adding concentrated hydrochloric acid to adjust the pH to 2-3 to obtain a solution containing the hydrochloride of the cocarboxylase shown in the formula I; the aqueous solution containing the co-carboxylase shown in the formula I is prepared by the preparation method of the co-carboxylase shown in the formula I according to any one of claims 1 to 6.

10. The method according to claim 7,

the concentration temperature is below 35 ℃, preferably 30-35 ℃;

and/or, the preparation method of the cocarboxylase hydrochloride further comprises the following crystallization steps: adding an alcohol solvent into the solution containing the hydrochloride of the cocarboxylase shown in the formula I, and filtering the precipitated solid to obtain the hydrochloride of the cocarboxylase; the alcohol solvent can be isopropanol and/or ethanol; the volume ratio of the alcohol solvent to the solution containing the hydrochloride of the cocarboxylase shown in formula I can be 2:1-3: 1.

Background

Cocarboxylases (cocamidase, also known as Cocarboxylase, thiamine pyrophosphate or diphosphate) are the inner salts of vitamin B1 pyrophosphate, which are also usually present in the form of Tetrahydrate (cocamidase Tetrahydrate, also known as Cocarboxylase Tetrahydrate or thiamine pyrophosphate Tetrahydrate) or hydrochloride (also known as thiamine pyrophosphate or vitamin B1 pyrophosphate chloride).

Co-carboxylases can also form several enzymes with specific proteins, which catalyze many important reactions in the metabolism of human and animal organisms.

More methods for preparing thiamine pyrophosphate have been reported in the literature. In 1937, Stern and Hofer prepared thiamine pyrophosphate by reacting vitamin Bl with phosphorus oxychloride [ cf. science, Vol.85, p.483(1937) ]. Weijlard and h.tauber prepared thiamine pyrophosphate from vitamin B1 and a mixture of sodium pyrophosphate and phosphoric acid, and obtained thiamine pyrophosphate by treatment with calcium phosphate solution and a large amount of organic solvent in about 10% yield [ cf. In 1940, it was reported that VB1 analogue 5-bromovinyl thiazole reacts with silver phosphate to prepare VB1 pyrophosphate chloride, which is precipitated as silver salt, silver ions are removed by hydrogen sulfide, and then the silver salt is treated by hydrogen chloride to obtain thiamine pyrophosphate chloride [ cf. biochemical Journal, vol.34, p.980(1940) ], which requires toxic reagents or a large amount of organic solvents, or has high temperature and is not favorable for environmental protection.

US5047223, US2991284, CN1887891A and CN101787048B disclose that vitamin B1 is used as a raw material, and phosphorylation reaction is carried out on the raw material and a phosphorylation reagent (for example, pyrophosphoric acid generated by the reaction of phosphorus pentoxide and phosphoric acid), the raw material is firstly subjected to phosphorylation reaction by a weakly alkaline anion column (which mainly adsorbs by-products, such as vitamin B1 triphosphate, vitamin B1 tetraphosphate and the like, and phosphoric acid), the concentration is carried out, then the concentrated solution is eluted by weakly acidic cation exchange resin pure water (vitamin B1 phosphatase is firmly adsorbed during water elution, cocarboxylase can be washed out without reservation, when 10% hydrochloric acid is used for the resin column, vitamin B1 phosphate can be washed out completely), the product is concentrated at 35 ℃, ethanol is added for crystallization, the product can be obtained (the purity of the final product reaches 99%), the pretreatment and the regeneration process of an ion column is very complicated, 2 times of the ion column are adopted, and the water concentration time at 35 ℃ is longer.

JP03145495 mentions the isolation and purification of the product by passing the resulting phosphoric acid mixture through a strongly basic anion column ion exchange resin, washing with pure water by adding acetic acid (or formic acid, propionic acid), crystallizing the eluent at pH <5.5 with ethanol to obtain pyrophosphate, and crystallizing the eluent at pH > 5.5 with acetone to obtain monophosphate with a purity of up to 96%.

Example 2 of US2991284 mentions that 2kg of the phosphate mixture obtained by phosphorylation of thiamine hydrochloride is first passed over strongly basic ion exchange resin (permutit ES) at pH 6.6-6.0 to give a monophosphate; the pH value is 6.0-5.4, and a mixture of monophosphate and pyrophosphate is obtained, wherein the monophosphate accounts for 25% -30%; when the pH is 5.4-2, pure carboxylase tetrahydrate is obtained, the pure pyrophosphate content in the process is extremely low, about 3%, and the separation degree of thiamine phosphate and diphosphate is poor.

Disclosure of Invention

The invention aims to overcome the defects of complicated purification method and poor effect of the conventional cocarboxylase and provides a preparation method of the cocarboxylase and tetrahydrate or salt thereof. The purification method can obtain the high-purity (more than 99 percent) cocarboxylase through one-step ion exchange, and can further directly prepare and obtain the high-purity tetrahydrate or the thiamine pyrophosphate.

The present invention solves the above-mentioned problems by the following technical means.

The invention provides a preparation method of a cocarboxylase shown in formula I, which comprises the following steps: eluting a mixture containing the co-carboxylase shown as the formula I by using weak base anion exchange resin through water and weak base aqueous solution with the pH value of 10-12 as eluent in sequence, and collecting eluate to obtain aqueous solution containing the co-carboxylase shown as the formula I; the mixture containing the cocarboxylase shown in the formula I comprises thiamine monophosphate and the cocarboxylase (thiamine diphosphate) shown in the formula I;

the weak base anion exchange resin can be a weak base anion exchange resin which is conventional in the preparation method in the field, such as macroporous weak base acrylic anion exchange resin, and the macroporous weak base acrylic anion exchange resin is one of the following types: d315, D301, D335, D345; in the present invention, D301 is preferred.

The water may be water conventional in such preparation methods in the art, such as pure water.

The pH value is 10-12, and the weak alkaline aqueous solution can be obtained by adopting alkali regulation conventional in the field; in the present invention, it is preferably obtained by adjusting with ammonia water.

In one embodiment of the present invention, the mixture containing the cocarboxylase of formula I may further contain one or more of thiamine, thiamine triphosphate, and higher phosphate.

In one aspect of the invention, the elution with water may be a method of operation conventional in such reactions in the art, for example, elution to an eluate with thiamine monophosphate (monoester phosphate) content of less than one percent (liquid phase monitoring, specific conditions may be as shown in the examples).

In one aspect of the invention, the eluate is collected as an eluate having a purity of greater than 95% of the cocarboxylase of formula I (liquid phase monitoring, as the case may be, as shown in the examples).

In the present invention, the mixture containing the cocarboxylase of formula I may be obtained by conventional methods for preparing cocarboxylases in the art, for example, referring to the prior art: science, Vol.85, p.483 (1937); ② journal of the American Chemical Society, Vol.60, p.2263 (1938); ③ Biochemical Journal, Vol.34, p.980 (1940); US 5047223; US 2991284; sixthly, CN 1887891A; seventhly, CN 101787048B; or the method of JP03145495, or the selection of conventional conditions and operations can be carried out to prepare a mixture containing a cocarboxylase. In the invention, the mixture containing the cocarboxylase represented by the formula I is prepared by the following steps:

step (1), sequentially adding water and an alcohol solvent into a reaction liquid obtained after phosphorylation reaction of vitamin B1 and a phosphorylating agent, mixing, standing, and removing supernatant to obtain a crude product of a mixture containing the co-carboxylase shown in the formula I;

and (2) washing a solution formed by the crude product and water by using a water-insoluble tertiary amine or a mixed solvent of the water-insoluble tertiary amine and an organic solvent to obtain a mixture containing the cocarboxylase shown in the formula I.

In step (1), the phosphorylating agent may be one or more of phosphorylating agents conventional in this type of phosphorylation reaction in the art, such as pyrophosphoric acid, mixtures of pyrophosphoric acid with phosphorus pentoxide or phosphorus oxychloride, mixtures containing pyrophosphoric acid prepared by reacting phosphoric acid with phosphorus pentoxide, and high concentrations of phosphoric acid (e.g., pyrophosphoric acid containing 25% or more); preferred in the present invention are pyrophosphoric acid-containing mixtures prepared from phosphoric acid and phosphorus pentoxide (e.g., a molar ratio of phosphoric acid to phosphorus pentoxide of from 2:1 to 1:1, e.g., from 1.2:1 to 1.3: 1).

The molar ratio of said phosphorylating agent to said vitamin B1 may be a molar ratio as is conventional in such processes in the art; when the phosphorylating agent is a mixture containing pyrophosphoric acid prepared from phosphoric acid and phosphorus pentoxide, the molar ratio of phosphorus pentoxide to vitamin B1 in the present invention is preferably 4:1 to 2:1 (e.g., 3:1 to 3.5: 1).

When the phosphating agent is a mixture containing pyrophosphoric acid prepared from phosphoric acid and phosphorus pentoxide, the mixture can be prepared by reacting phosphoric acid and phosphorus pentoxide at 100-180 ℃, and the temperature is preferably 150-160 ℃.

In step (1), the alcohol solvent may be an alcohol solvent conventional in such reactions in the art, such as isopropanol and/or ethanol. The volume-to-mass ratio of the alcohol solvent to the vitamin B1 can be 2:1-5:1 (e.g., 3:1-4: 1).

In the step (1), the volume-to-mass ratio of the water to the vitamin B1 can be 2:1-5:1 (e.g., 3:1-4: 1).

In step (1), the temperature of the phosphorylation reaction may be a temperature conventional in this type of reaction in the art, for example, 100 ℃ to 170 ℃, and in the present invention, preferably 130 ℃ to 140 ℃.

In the step (2), the volume-to-mass ratio of the water to the vitamin B1 can be 1:1-3:1 (e.g., 1.5:1-2: 1).

In step (2), the water-insoluble tertiary amine may be a tertiary amine that is hardly soluble in water or only slightly soluble in water as is conventional in reactions of this type in the art, as will be understood by those skilled in the art; for example 8 to 40, preferably 12 to 36 carbon atoms: one or more of an aliphatic tertiary amine (especially one or more of trioctylamine, trihexylamine, and tridecylamine), an aliphatic-cycloaliphatic tertiary amine (e.g., N-dimethylcyclohexylamine), and an aliphatic-araliphatic tertiary amine (e.g., N-dimethylbenzylamine); trioctylamine and/or tridecylamine is preferred in the present invention.

In step (2), the organic solvent may be an organic solvent conventional in the reactions of this type in the art, as will be understood by those skilled in the art, such as a moderately polar water-immiscible organic solvent (see chr. Reichardt, solvent effect in organic chemistry, Verlag Chemie, 1979, especially page 242-245); for example, one or more of an alcoholic solvent having 4 to 8 carbon atoms (e.g., one or more of methyl isobutyl carbinol, 3-ethylpentanol, 1-hexanol, and methylcyclohexanol), an etheric solvent having 4 to 8 carbon atoms (e.g., one or more of diethyl ether, isopropyl ether, methyl tert-butyl ether, and di-n-butyl ether), a ketone solvent having 5 to 8 carbon atoms (e.g., one or more of diethyl ketone, methyl isobutyl ketone, acetophenone, and cyclohexanone), and an aromatic hydrocarbon solvent (e.g., toluene); isopropyl ether is preferred in the present invention.

In step (2), the amount of the water-insoluble tertiary amine may be an amount conventionally used in such reactions in the art, for example, a mixed solvent containing 10 to 80% by mass, preferably 40 to 70% by mass of the water-insoluble tertiary amine based on the mixed solvent. It will be appreciated by those skilled in the art that if mixtures containing only small amounts of amine are used, either large amounts of amine/solvent mixtures must be used or the extraction process must be repeated several times. If an amine/solvent mixture is used that contains a large amount of amine, a single extraction process may be sufficient. The amount of the mixed solvent may be an amount conventionally used in such reactions in the art, for example, when the pH of a solution formed by the mixture and water is 3 to 4, particularly 3.2 to 3.4.

The invention provides a preparation method of a cocarboxylase tetrahydrate, which comprises the following steps: concentrating the water solution containing the cocarboxylase shown in the formula I, and filtering the precipitated solid to obtain a cocarboxylase tetrahydrate; the aqueous solution containing the co-carboxylase shown in the formula I is prepared by the preparation method of the co-carboxylase shown in the formula I.

The concentration may be carried out at conditions and methods conventional in the art for concentrating such compounds, for example at a temperature below 35 ℃, preferably from 30 ℃ to 35 ℃.

When the aqueous solution containing the co-carboxylase shown in the formula I contains excessive alkali ions, preferably, acid is used for neutralizing the excessive alkali ions, crystallization is carried out, and the separated solid is filtered, so that the co-carboxylase tetrahydrate is obtained; the acid can be hydrochloric acid; the crystallization may comprise the steps of: adding an alcohol solvent into the aqueous solution containing the co-carboxylase shown in the formula I, and filtering the precipitated solid to obtain the hydrochloride of the co-carboxylase. The alcohol solvent may be an alcohol solvent conventional in the art for such post-treatment, such as isopropyl alcohol and/or ethanol. The alcoholic solvent may be used in amounts conventional in such post-treatments in the art, for example, the volume ratio of the alcoholic solvent to the solution containing the hydrochloride salt of the cocarboxylase of formula I may be from 2:1 to 3: 1.

The invention also provides a preparation method of the cocarboxylase hydrochloride, which comprises the following steps: concentrating the water solution containing the cocarboxylase shown in the formula I, and adding concentrated hydrochloric acid to adjust the pH to 2-3 to obtain a solution containing the hydrochloride of the cocarboxylase shown in the formula I (namely thiamine pyrophosphate); the aqueous solution containing the co-carboxylase shown in the formula I is prepared by the preparation method of the co-carboxylase shown in the formula I.

The concentration may be carried out at conditions and methods conventional in the art for concentrating such compounds, for example at a temperature below 35 ℃, preferably from 30 ℃ to 35 ℃.

The preparation method of the cocarboxylase hydrochloride may further comprise the following crystallization steps: adding an alcohol solvent into the solution containing the hydrochloride of the cocarboxylase shown in the formula I, and filtering the precipitated solid to obtain the hydrochloride of the cocarboxylase.

The alcohol solvent may be an alcohol solvent conventional in the art for such post-treatment, such as isopropyl alcohol and/or ethanol. The alcoholic solvent may be used in amounts conventional in such post-treatments in the art, for example, the volume ratio of the alcoholic solvent to the solution containing the hydrochloride salt of the cocarboxylase of formula I may be from 2:1 to 3: 1.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the purification method can obtain the high-purity (more than 99 percent) of the cocarboxylase through one-step ion exchange, and can further directly prepare the high-purity (more than 99 percent) tetrahydrate or the thiamine pyrophosphate.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1

Preparation of the Cocarboxylase

Phosphoric acid (85%, 24g, 0.20mol), phosphorus pentoxide (22.5g, 0.158mol) were weighed into a three-necked flask and reacted at 100 ℃ for 2 hours. VB1(16g, 0.048mol) was weighed in portions and reacted at 100 ℃ for 3.5h, TLC showed complete reaction, heating was stopped, 48ml of water was added, stirring was carried out to dissolve, stirring was carried out in an ice water bath, 60ml of isopropanol was added, stirring was carried out for 30min, and then the mixture was left in a refrigerator overnight.

Removing the supernatant to obtain a yellow oily substance, adding 24ml of water for dissolving, and adding trioctylamine after dissolving: isopropyl ether ═ 1: 1(20ml), washed twice. Putting a sample (after the sample is placed in a refrigerator overnight, a product is precipitated at the bottom in the form of yellow oily matter, after a supernatant is removed, the yellow oily matter is a mixture of partial phosphoric acid and phosphate, and the yellow oily matter is mainly washed by phosphoric acid) into a D301 anion exchange resin, eluting the mixture by pure water until the content of phosphate monoester in an eluate is monitored to be lower than one percent in a liquid phase, adjusting the pH value of the eluate to be 10-12 by ammonia water to be used as an eluent, collecting the eluate, allowing the obtained eluate to enter the liquid phase to monitor the purity, selecting a combined eluate with the purity of more than 95 percent, and obtaining the purity: 95.54 percent.

Preparation of cocarboxylase tetrahydrate

Concentrating the eluate at 30-35 deg.C to 2-3ml, adding hydrochloric acid (such as 1M hydrochloric acid) to neutralize excessive alkali ion, and precipitating with 2-3 times of isopropanol to obtain cocarboxylase tetrahydrate.

Preparation of thiamine pyrophosphate

The eluate was evaporated to 2-3ml at 40 ℃, concentrated hydrochloric acid was added to adjust the pH to 2-3, and the product (1.6g) was precipitated with 2-3 times the amount of isopropanol, melting point: 238-241 ℃ of temperature; purity: 96.55 percent.

(detection conditions: chromatographic column: C18, 4.6X 250mm,5mm, column temperature: 45 ℃, detection wavelength: 239nm, flow rate: 0.8ml/min, sample introduction: 20. mu.l, running time: 30min, mobile phase: mobile phase A: methanol, mobile phase B: sodium dihydrogen phosphate concentration of 23mmol/L, sodium octane sulfonate concentration of 5mmol/L, ion pair buffer adjusted to pH 2.5 with phosphoric acid, isocratic elution: A: B: 46: 54); m/z425[ M + H]⁺；¹H NMR(600MHz,D₂O)：δ9.66(s,1H),7.92(s,1H),5.56(s,2H),4.21-4.18(m,2H),3.34-3.32(t,2H),2.62(s,3H),2.56(s,3H)；¹³C NMR(600MHz,D₂O):δ163.16，162.96，154.93，144.12，143.40，135.82，106.43，64.89，49.88，27.59，20.90，11.08。

Example 2

Preparation of the Cocarboxylase

Phosphoric acid (85%, 24g, 0.20mol), phosphorus pentoxide (22.5g, 0.158mol) were weighed into a three-necked flask and reacted at 100 ℃ for 2 hours. VB1(16g, 0.048mol) was weighed in portions and reacted at 100 ℃ for 3.5h, TLC showed complete reaction, heating was stopped, 48ml of water was added, stirring was carried out to dissolve, stirring was carried out in an ice water bath, 60ml of isopropanol was added, stirring was carried out for 30min, and then the mixture was left in a refrigerator overnight.

Removing the supernatant to obtain a yellow oily substance, adding 24ml of water for dissolving, and adding trioctylamine after dissolving: isopropyl ether ═ 1: 1(20ml), washed twice. Loading a sample (phosphate mixture after washing phosphoric acid) into D301 anion exchange resin, eluting with pure water until the content of phosphate monoester in an eluate is less than one percent when the liquid phase is monitored, adjusting the pH value of the eluate to 10-12 by using ammonia water as an eluent, collecting the eluate, introducing the obtained eluate into the liquid phase, selecting a combined eluate with the purity of more than 95 percent, and mixing the combined eluate with the purity: 95.43 percent.

Preparation of thiamine pyrophosphate

The eluate was evaporated to 2-3ml at 30-35 ℃, concentrated hydrochloric acid was added to adjust pH 2-3, and 2-3 times the amount of isopropanol was used to precipitate the product (1.81g), purity: 99.03 percent.

EXAMPLE 3 preparation of thiamine pyrophosphate

Preparation of the Cocarboxylase

Phosphoric acid (85%, 24g, 0.20mol) and phosphorus pentoxide (22.5g, 0.158mol) were weighed into a three-necked flask and reacted at 150 ℃ and 160 ℃ for 2 h. VB1(16g, 0.048mol) is weighed and added in batches to react for 3.5h at the temperature of 140 ℃, after TLC shows that the reaction is complete, heating is stopped, 48ml of water is added to be stirred and dissolved, 60ml of isopropanol is added to be stirred in an ice-water bath and stirred for 30min, and then the mixture is placed in a refrigerator for overnight.

Removing the product supernatant to obtain a yellow oily substance, adding 24ml of water for dissolving, and adding trioctylamine: isopropyl ether ═ 1: 1(20ml extraction), extracted twice. Loading the sample into D301 anion exchange resin, eluting with pure water until the content of the monoester phosphate in the eluate is less than one percent, adjusting the pH value to 10-12 by using ammonia water as an eluent, collecting the eluate, introducing the eluate into a liquid phase, and selecting a combined eluate with the purity of more than 95%.

Preparation of thiamine pyrophosphate

The eluate was evaporated to 2-3ml at 30-35 ℃, concentrated hydrochloric acid was added to adjust pH 2-3, and 2-3 times the amount of isopropanol was used to precipitate a product (3.24g) having a purity: 99.21 percent.

Comparative example 1

Using the method of US2991284

Phosphoric acid (85%, 24g, 0.20mol), phosphorus pentoxide (22.5g, 0.158mol) were weighed into a three-necked flask and reacted at 100 ℃ for 2 hours. VB1(16g, 0.048mol) was weighed in portions and reacted at 100 ℃ for 3.5h, TLC showed complete reaction, heating was stopped, 48ml of water was added, stirring was carried out to dissolve, stirring was carried out in an ice water bath, 60ml of isopropanol was added, stirring was carried out for 30min, and then the mixture was left in a refrigerator overnight.

Removing the product supernatant to obtain a yellow oily substance, adding 24ml of water for dissolving, and adding trioctylamine: isopropyl ether ═ 1: 1(20ml extraction), extracted twice. Loading the sample into D301 anion exchange resin, adjusting pure water by using acetic acid, collecting eluent with pH less than 5.5, monitoring by HPLC to obtain monophosphate, continuously adjusting pH, trying to obtain monophosphate when pH is 6.5, obtaining eluent with a small amount of pyrophosphate when pH is 7.5 and pH is 8.5 by using ammonia water, continuously adjusting pH to 9-10, and collecting eluent with the purity of the thiamine pyrophosphate less than 10%.

Comparative example 2

Phosphoric acid (85%, 24g, 0.20mol), phosphorus pentoxide (22.5g, 0.158mol) were weighed into a three-necked flask and reacted at 100 ℃ for 2 hours. VB1(16g, 0.048mol) was weighed in portions and reacted at 100 ℃ for 3.5h, TLC showed complete reaction, heating was stopped, 48ml of water was added, stirring was carried out to dissolve, stirring was carried out in an ice water bath, 60ml of isopropanol was added, stirring was carried out for 30min, and then the mixture was left in a refrigerator overnight.

Removing the product supernatant to obtain a yellow oily substance, adding 24ml of water for dissolving, and adding trioctylamine: isopropyl ether ═ 1: 1(20ml extraction), extracted twice. Loading the sample into a D301 anion exchange resin, adjusting the pH value to 12-13 by ammonia water, and collecting the eluent with the thiamine pyrophosphate purity of less than 80%.

Comparative example 3

The method adopted in CN101787048

Phosphoric acid (85%, 5.4g, 0.0468mol), phosphorus pentoxide (4.19g, 0.0295mol) were weighed and added to a three-necked flask for reaction at 100 ℃ for 2 hours. VB1(4g, 0.0118mol) was weighed in portions and reacted at 100 ℃ for 3.5h, TLC showed the reaction was complete, heating was stopped, 12ml of water was added, the mixture was dissolved under stirring, 35ml of isopropanol was added under ice-water bath, and after stirring for 30min, the mixture was left in a refrigerator overnight.

Removing the product supernatant to obtain a yellow oily substance, adding 5ml of water for dissolving, and adding trioctylamine after dissolving: isopropyl ether ═ 1: 1(5ml extraction), extracted twice. And (3) loading the sample into D301 anion exchange resin, eluting with pure water, and monitoring the eluent by using a liquid phase to obtain the thiamine pyrophosphate of which the purity is less than 15% in the eluent.

Eluates with a purity of more than 95% could not be collected using the methods described above in comparative examples 1, 2, and 3.

In the art, in the purification of the cocarboxylase, the final purification is carried out under acidic conditions to obtain a product with high purity, such as the prior art of US5047223, US2991284, CN1887891A, CN101787048B, JP03145495, etc. The invention adopts different technical schemes in the prior art, and achieves the level of the prior art in terms of effect (particularly purity) under the condition of reducing the steps of the ion exchange resin column.