Preparation method of pyridoxal phosphate_industrial additives

Background and overview^[1-3]

Pyridoxal 5-phosphate monohydrate chemical name: (3-hydroxy-2-methyl-5-[(phosphoryloxy)methyl]-4-pyridinecarbaldehyde. Pyridoxal phosphate is pyridoxine An important substance in aldehydes, it is not only a coenzyme of transaminase and decarboxylase in amino acid metabolism, but also promotes the decarboxylation of glutamic acid and promotes the production of γ-aminobutyric acid. Pyridoxal phosphate is used in the synthesis of β-carbonyl amide drugs. It is also widely used in biological activity research and other fields. For example, it is mainly used in clinical treatment of Parkinson’s syndrome to promote the transamination of aminotransferase and increase the content of dopamine in the body.

Pyridoxal phosphate is the coenzyme form of vitamin B6. In the catalytic reaction, the aldehyde group in the pyridoxal phosphate molecule is combined with the amino group of α-amino acid to first form an aldimine, also known as Schiffbase, and then transaminate and decarboxylate the amino acid according to the characteristics of different enzyme proteins. Or racemization and other effects. As a coenzyme for amino acid transaminase, decarboxylase and racemase, it plays an extremely important role in amino acid metabolism.

Preparation^[1]

The preparation method of pyridoxal phosphate, the specific steps are as follows:

1. Synthesis of pyridoxal hydrochloride Schiff base:

1) Synthesis of pyridoxal hydrochloride: Add 5.0g (24.3mmo1) pyridoxine hydrochloride into 50.0mL of 5.0% dilute hydrochloric acid solution, stir until the solution is clear, control the temperature at 20.0°C, and add 2.1g (24.3mmol) manganese dioxide. After 0.5h, add 2.1g (24.3mmol) manganese dioxide and react for 6.0h to obtain pyridoxal hydrochloride. 2) Synthesis of pyridoxal hydrochloride Schiff base: Suction filter the pyridoxal hydrochloride synthesized in step 1) to remove unreacted manganese dioxide, add 12.3g (51.0mmo1) sodium sulfide nonahydrate to the filtrate, and stir 0.5h, suction filtration, adjust the pH of the solution to weak acidity, slowly add 6.3g (46.2mmol) p-ethoxyaniline dropwise at 17.0°C, continue stirring for 1.0h after the dripping is completed, an orange-yellow solid will be generated, suction filtration , wash the reaction product with pure water and n-hexane, and blow dry it at 80.0°C to obtain a yellow solid pyridoxal hydrochloride Schiff base 5.6 Japanese Mitsubishi g, yield = (5.6g/(24.3mmol*0.001*286.3))* 100% = 80.4%, compound characterization data are: ¹HNMR (MHz, DMSO-d6), δ: 14.20 (s, 1H), 9.23 (s, 1H), 8.06 (s, 1H) ,7.58(d,J＝8.7Hz,2H),7.13(d,J＝8.3Hz,2H),5.49(s,1H),4.85(s,2H),4.25–4.03(m,2H),2.51( s, 3H), 1.44 (d, J = 6.8Hz, 3H).

Use high-performance liquid chromatography (HPLC) to determine the conversion rate of pyridoxine hydrochloride into pyridoxal hydrochloride in step 1) (detection instrument: LC-20AT high-performance liquid chromatography (HPLC), Shimadzu Corporation, Japan ), the methods and results are as follows:

Put a certain amount of sample into a 100mL volumetric flask, add mobile phase, dissolve with ultrasound, and inject the sample after diluting the volume. Among them, liquid phase conditions: C18 (5μm, 250mm×4.6mm); mobile phase: phosphate buffer: methanol=95:5 (volume ratio); buffer solution: 20mmol/L Na₂HPO₄·12H₂O (use 10.0% phosphoric acid to adjust pH to 7.0); flow rate: 1.0mL/min; column temperature: 30.0℃; injection volume: 20.0μL; Detection wavelength: 254nm.

After detection by HPLC, the conversion rate of pyridoxine hydrochloride in step 1) is: 93.7%, and the selectivity is: 95.8%.

2. Synthesis of pyridoxal phosphate Schiff base

In a 250mL three-neck reaction bottle equipped with a stirrer and thermometer, add 5.0g (17.5mmol) pyridoxal hydrochloride Schiff base prepared in step 1 and 17.8g (52.5mmol) polyphosphoric acid, and react at 50.0°C After 8.0h, add 42.0g ice water. After stirring at 30.0°C for 0.5h, add 4.2g of concentrated hydrochloric acid, raise the temperature to 75.0°C, and stir for 1.0h. Cool the temperature to -5.0°C to 0.0°C and neutralize it with 50.0% sodium hydroxide solution. A large amount of orange-red solid will precipitate. Filter, wash with water and dry to obtain 3.8g of orange-red solid pyridoxal phosphate Schiff base. Yield =(3.8g/(17.5mmol*0.001*366.3))*100%=59.3%, melting point 202-203℃, EA(C16H19N2O6P), Calcd: C 52.46, H 5.23, N 7.65; Found: C 52.14, H 5.19 ,N7.38.

3. Synthesis of pyridoxal phosphate

In a 100mL three-necked reaction bottle equipped with a stirrer and thermometer, add 5.0g (13.6mmol) of the pyridoxal phosphate Schiff base prepared in step 2 to 27.2mL of 2mol/L sodium hydroxide solution. The temperature was raised to 35.0°C and reacted for 2.0h. After cooling to room temperature, the reaction solution was extracted twice with ethyl acetate and separated (25.0 ml × 2). Add 19.6g of strongly acidic cation exchange resin to the water phase, stir for 1.0h, filter with suction, and finally freeze-dry with a freeze dryer to obtain 2.3g of off-white solid pyridoxal phosphate, yield = (2.3g/(13.6mmol*0.001 *247.1))*100%=68.4%, melting point 141-142℃, ¹H NMR (MHz, DMSO-d6), &del hydroxybutyl acrylate ta;: 10.36 (s, 1H), 8.09 (s, 1H), 5.17 (d, J = 6.6Hz, 2H), 3.99 (dd, J = 13.0, 6.0Hz, 1H), 2.41 (s, 3H).

Use high-performance liquid chromatography (HPLC) to determine the purity of pyridoxal phosphate prepared in step 3) (using the LC-20AT high-performance liquid chromatograph (HPLC) produced by Shimadzu Corporation, Japan), methods and results As follows:

Put an appropriate amount of sample into a 100mL volumetric flask, add DMSO, dissolve with ultrasound, and then add mobile phase to adjust volume; where, liquid phase conditions: C18 (5 μm, 250 mm × 4.6 mm); mobile phase: phosphate buffer: acetonitrile = 95:5 (volume ratio);

The mobile phase is gradient eluted according to Table 1:

Table 1

Buffer solution: 20mmol/L KH₂PO₄ (Adjust the pH to 6.0 with a mass fraction of 10.0% potassium carbonate solution); flow rate: 1.0m L/ min; column temperature: 30.0℃; injection volume: 20.0μL; detection wavelength: 254nm.

After HPLC detection, the purity of pyridoxal phosphate obtained in step 3) is: 95.2%.

Figure 1 is a synthesis route diagram of pyridoxal phosphate provided by the present invention.

Measurement method^[3]

A rapid determination method for pyridoxal phosphate using gold nanoparticles as a chromogenic probe, which is characterized by utilizing the interaction between pyridoxal phosphate and gold nanoparticles to cause the gold nanoparticles to aggregate and exhibit solution color and ultraviolet-visible The concentration of pyridoxal phosphate is determined by changing the absorption spectrum characteristics; the concentration of pyridoxal phosphate is determined by changing the absorbance ratio (A700/A525) of gold nanoparticles at wavelengths of 700 nm and 525 nm; the gold nanoparticles used are prepared by the following steps Obtain: First, add μL of mercaptoethylamine hydrochloride with a concentration of 213 mmol/L and 2.23 mL of HAuCl₄ with a concentration of 10 mg/mL into a beaker containing 37.5 mL of water, and stir at room temperature. React for 20 minutes; after completion, add 10 μL of NaBH₄ with a concentration of 10 mmol/L to the above reaction solution, and then continue the stirring reaction at room temperature for 30 minutes to obtain crude mercaptoethylamine-nanogold; The crude mercaptoethylamine-nanogold product was purified using a dialysis bag with a molecular weight cutoff of 7000 to obtain pure mercaptoethylamine-nanogold product; the obtained pure mercaptoethylamine-nanogold product was sealed to avoid light and stored in a refrigerator at 4°C. ,spare.