Method for synthesizing erythritol ketose from formaldehyde and dihydroxy acetone
By spontaneously reacting formaldehyde and dihydroxyacetone in the buffer, the problem of difficulty in accumulation of erythrilose in microbial fermentation is successfully solved, and the method of producing erythrilose under normal temperature and pressure is realized, with wide application prospects.
Patent Information
- Application Number
- CN202510141581.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-08
- Publication Date
- 2025-05-30
AI Technical Summary
In the prior art, erythrilose is difficult to accumulate through microbial fermentation method, and faces the problem of strict control of bacterial growth environment and difficulty in isolating and purifying fermentation products.
Formaldehyde and dihydroxyacetone are used to react spontaneously in a buffer containing glycine ions and/or a buffer containing hydrogen phosphate ions to produce erythrilose. The reaction can be carried out at normal temperature and pressure and can be carried out within a wide pH range.
A new route for synthesizing erythrilose from a carbon compound formaldehyde has been achieved, overcoming the shortcomings of microbial fermentation methods and having good application prospects.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biochemistry, and more specifically, relates to a method for synthesizing erythrulose from formaldehyde and dihydroxyacetone. Background Art
[0002] Erythrulose is a rare tetrose sugar that can be used as a pharmaceutical precursor compound, a raw material for cosmetics, etc. It has a wide range of uses and a large consumption, and has high value in industrial applications. In the pharmaceutical industry, erythrulose is a precursor compound of pyruvaldehyde acetal, which can be further synthesized into a variety of biologically active compounds, such as the anticancer drug Bengamide E, the antifungal compound Tanikolide, the cholesterol-lowering drugs Crestor and Zetia, and the antiepileptic and antihypertensive drug γ-amino-8-hydroxybutyric acid. In the cosmetics industry, erythrulose can be used as a skin tanning agent. It produces a tea-colored effect through the Maillard reaction with skin keratin, which can ensure that the skin produces a lasting and uniform color, and at the same time has a moisturizing effect.
[0003] Currently, erythrulose is mainly obtained by microbial fermentation of erythritol and further oxidation to obtain erythrulose. However, the erythrulose produced by microbial conversion can be converted into phosphorylated erythrulose by erythrulokinase in the cells, and can further enter the metabolic pathway to generate downstream products to produce energy. Therefore, it is difficult to accumulate in the cells. At the same time, the microbial fermentation method also faces problems such as strict control of the growth environment of the bacteria and difficulties in separating and purifying the subsequent fermentation products.
[0004] In recent years, one-carbon compounds have received extensive attention as promising green energy substances. One-carbon compounds can be used to synthesize basic organic chemical raw materials, fuels, and other high-value chemicals. Due to their cheap and easily available characteristics, one-carbon compounds have become the most promising compounds for replacing petroleum to prepare high-value compounds, and have important scientific significance and development value in the fields of medicine, food, and chemical engineering. At the same time, the utilization and conversion of one-carbon compounds can also significantly reduce the negative impact on the ecological environment caused by people in the process of using fossil fuels and synthetic materials. Formaldehyde can be converted from other one-carbon compounds, and then converted into intermediate substances for biological utilization. At the same time, it also has the characteristics of wide source and low price. Synthesizing high-value compounds with formaldehyde as a precursor has important application prospects.
[0005] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure. Summary of the Invention
[0006] In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a method for synthesizing erythrulose from formaldehyde and dihydroxyacetone.
[0007] The object of the present invention is achieved by the following technical solutions: A method for synthesizing erythrulose from formaldehyde and dihydroxyacetone, the method comprising: using formaldehyde and dihydroxyacetone as substrates, and spontaneously reacting in a buffer solution containing glycine root ions and / or a buffer solution containing hydrogen phosphate ions to generate erythrulose.
[0008] Preferably, the spontaneous reaction is carried out at 30-50 °C for 24-48 h.
[0009] Preferably, the pH of the buffer solution containing glycine root ions is 7-12.
[0010] Preferably, the pH of the buffer solution containing hydrogen phosphate ions is 7-12.
[0011] Preferably, the concentration of the buffer solution containing glycine root ions is 50-900 mM.
[0012] Preferably, the concentration of the buffer solution containing hydrogen phosphate ions is 50-900 mM.
[0013] Preferably, the final concentration ratio of formaldehyde to dihydroxyacetone in the buffer solution containing glycine root ions is (1-9):(1-9).
[0014] Preferably, the final concentration of formaldehyde in the buffer solution containing glycine root ions is 100-1800 mM.
[0015] Preferably, the final concentration ratio of formaldehyde to dihydroxyacetone in the buffer solution containing hydrogen phosphate ions is (1-9):(1-9).
[0016] Preferably, the final concentration of formaldehyde in the buffer solution containing hydrogen phosphate ions is 100-1800 mM.
[0017] The present invention has the following advantages and effects compared with the prior art: The present invention provides a new route for synthesizing erythrulose from a one-carbon compound formaldehyde. Using formaldehyde and dihydroxyacetone as substrates, and spontaneously reacting in a buffer solution containing glycine root ions and / or a buffer solution containing hydrogen phosphate ions to generate erythrulose, this reaction can be carried out at normal temperature and pressure, and can be carried out within a wide pH range, having good application prospects.
[0018] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present disclosure. Description of the Drawings
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0020] Figure 1 It is the experimental result of the spontaneous reaction of formaldehyde and dihydroxyacetone in glycine - sodium hydroxide buffer in Example 1.
[0021] Figure 2 It is the experimental result of the spontaneous reaction of formaldehyde and dihydroxyacetone in potassium hydrogen phosphate - potassium dihydrogen phosphate buffer in Example 1.
[0022] Figure 3 It is the experimental result of the spontaneous reaction of formaldehyde and dihydroxyacetone in Tris - HCl - sodium hydroxide buffer in Example 1.
[0023] Figure 4 It is the experimental result of the spontaneous reaction of formaldehyde and dihydroxyacetone in ammonium chloride - ammonia buffer in Example 1.
[0024] Figure 5 It is the yield result of erythrulose generated by the reaction of formaldehyde and dihydroxyacetone with different concentrations in 300 mM potassium phosphate buffer at 30 °C and 40 °C for 24 h and 48 h in Example 7. Detailed implementation manners
[0025] The following will clearly and completely describe the implementation schemes of the present invention in combination with the embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The description of at least one exemplary embodiment is actually only illustrative and in no way limits the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0026] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0027] Example 1: Spontaneous reaction of formaldehyde and dihydroxyacetone in different types of buffers
[0028] Four different types of buffers with a pH of 10 and a concentration of 50 mM were prepared, namely glycine-sodium hydroxide buffer (50 mM glycine-sodium hydroxide, 5 mM magnesium sulfate), potassium phosphate buffer (50 mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate, 5 mM magnesium sulfate), Tris-HCl-sodium hydroxide buffer (50 mM Tris-HCl-sodium hydroxide, 5 mM magnesium sulfate), and ammonium chloride-ammonia buffer (50 mM ammonium chloride-ammonia, 5 mM magnesium sulfate). Then, formaldehyde with a final concentration of 100 mM and dihydroxyacetone with a final concentration of 100 mM were added to the above-mentioned buffer systems with different types at a pH of 10 and a concentration of 50 mM, and the reaction was carried out at 30 °C with 1000 rpm for 24 h. After the reaction was completed, the formation of erythrulose was detected by HPLC.
[0029] First, 30 μL of the reacted solution was taken and added to 30 μL of a 50 mM veratrol solution (as an internal standard) and 140 μL of a 2,3,4,5,6-pentafluorobenzylamine hydrochloride solution with a concentration of 20 mg / mL (prepared using a 100 mM citric acid and sodium citrate buffer with a pH of 4), and then HPLC detection was carried out after derivatization at 30 °C for 1 h.
[0030] HPLC detection conditions: Mobile phase A is water, and mobile phase B is 100% acetonitrile; the column is a C18 column, and detection is carried out using an ultraviolet (263 nm) detector. The flow rate is 1 mL / min, and the elution program is 30% of mobile phase B and 70% of mobile phase A from 0 - 3 min; from 3 - 18 min, the proportion of mobile phase B increases from 30% to 100%; from 18 - 18.3 min, the proportion of mobile phase B decreases from 100% to 30%; from 18.3 - 21 min, it is 30% of mobile phase B and 70% of mobile phase A. The total time is 21 min, the injection volume is 20 μL, and the column temperature is 30 °C.
[0031] The HPLC detection results are as Figures 1-4 shown. Through analysis, it can be seen that formaldehyde and dihydroxyacetone can spontaneously react to form erythrulose in glycine-sodium hydroxide buffer and potassium phosphate buffer, while they cannot spontaneously react to form erythrulose in Tris-HCl-sodium hydroxide buffer and ammonium chloride-ammonia buffer. It shows that glycine root and hydrogen phosphate ions in the solution help formaldehyde and dihydroxyacetone to spontaneously react to form erythrulose, and the peak area of erythrulose in the potassium phosphate buffer system is larger than that in the glycine-sodium hydroxide buffer system. Therefore, the hydrogen phosphate ion buffer was selected for subsequent experiments.
[0032] Example 2: Spontaneous reaction of formaldehyde and dihydroxyacetone to form erythrulose in potassium phosphate buffer with different pH values Prepare potassium phosphate buffers with different pH values (pH = 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12) (300 mM potassium dihydrogen phosphate - dipotassium hydrogen phosphate, 5 mM magnesium sulfate). Add formaldehyde with a final concentration of 100 mM and dihydroxyacetone with a final concentration of 100 mM to the potassium phosphate buffer systems with the above different pH values, and react at 30 °C with 1000 rpm for 24 h. After the reaction is completed, detect according to the derivatization method and HPLC detection method in Example 1, and the yield of erythrulose is obtained through calculation as shown in Table 1.
[0033] Table 1 Yield of erythrulose formed by the spontaneous reaction of formaldehyde and dihydroxyacetone in potassium phosphate buffers with different pH values
[0034] As can be seen from Table 1, formaldehyde and dihydroxyacetone can both spontaneously react to form erythrulose in the potassium phosphate buffer system with a pH of 7 - 12 and a concentration of 300 mM. Moreover, as the pH of the buffer in the reaction system increases, the yield of erythrulose first increases and then decreases, and reaches the highest yield of 34.90 mM at pH = 10.
[0035] Example 3: Spontaneous reaction of formaldehyde and dihydroxyacetone to form erythrulose in potassium phosphate buffers with different concentrations
[0036] Prepare potassium phosphate buffers with different concentrations (50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM) (potassium dihydrogen phosphate - dipotassium hydrogen phosphate with pH = 8, 5 mM magnesium sulfate). Add formaldehyde with a final concentration of 100 mM and dihydroxyacetone with a final concentration of 100 mM to the potassium phosphate buffer systems with the above different concentrations, and react at 30 °C with 1000 rpm for 24 h. After the reaction is completed, detect according to the derivatization method and HPLC detection method in Example 1, and the yield of erythrulose is obtained through calculation as shown in Table 2.
[0037] Table 2 Yield of erythrulose formed by the spontaneous reaction of formaldehyde and dihydroxyacetone in potassium phosphate buffers with different concentrations
[0038] As can be seen from Table 2, formaldehyde and dihydroxyacetone can both spontaneously react to form erythrulose in potassium phosphate buffer systems with different concentrations. Moreover, as the concentration of the buffer in the reaction system increases, the yield of erythrulose in the system further increases.
[0039] Example 4: Spontaneous reaction of different ratios of formaldehyde and dihydroxyacetone to form erythrulose in potassium phosphate buffer In potassium phosphate buffer (50 mM potassium dihydrogen phosphate - dipotassium hydrogen phosphate, 5 mM magnesium sulfate, pH = 8.0), formaldehyde and dihydroxyacetone solutions with final concentration ratios of 1:1, 1:1.5, and 1.5:1 were added (i.e., the final concentrations of formaldehyde and dihydroxyacetone were 100 mM:100 mM, 100 mM:150 mM, and 150 mM:100 mM respectively), and the reaction was carried out at 1000 rpm for 24 h at 30 °C. After the reaction was completed, detection was carried out according to the derivatization method and HPLC detection method in Example 1, and the yield of erythrulose obtained through calculation is shown in Table 3.
[0040] Table 3 Yields of erythrulose formed by the spontaneous reaction of formaldehyde and dihydroxyacetone in different ratios in potassium phosphate buffer
[0041] As can be seen from Table 3, formaldehyde and dihydroxyacetone in different ratios can all spontaneously react to form erythrulose in potassium phosphate buffer, that is, in the reaction system, whether the concentration of formaldehyde is higher than or equal to or lower than the concentration of dihydroxyacetone, they can all spontaneously react to form erythrulose.
[0042] Example 5: Spontaneous reaction of formaldehyde and dihydroxyacetone in different ratios to form erythrulose at different temperatures In potassium phosphate buffer (50 mM potassium dihydrogen phosphate - dipotassium hydrogen phosphate, 5 mM magnesium sulfate, pH = 8.0), formaldehyde and dihydroxyacetone solutions with final concentration ratios of 1:1, 1:1.5, and 1.5:1 were added (i.e., the final concentrations of formaldehyde and dihydroxyacetone were 100 mM:100 mM, 100 mM:150 mM, and 150 mM:100 mM respectively), and the reaction was carried out at 1000 rpm for 24 h at 30 °C, 40 °C, and 50 °C respectively. After the reaction was completed, detection was carried out according to the derivatization method and HPLC detection method in Example 1, and the yield of erythrulose obtained through calculation is shown in Table 4.
[0043] Table 4 Yields of erythrulose formed by the spontaneous reaction of formaldehyde and dihydroxyacetone in different ratios at different temperatures
[0044] As can be seen from Table 4, formaldehyde and dihydroxyacetone in different ratios can all spontaneously react to form erythrulose at different temperatures, and with the increase of the reaction temperature, the yield of erythrulose gradually increases.
[0045] Example 6: Reaction of dihydroxyacetone with formaldehyde at different concentrations in potassium phosphate buffer with different pH values Prepare potassium phosphate buffers with different pH values (pH = 8, 8.5, 9, 9.5, 10) (300 mM potassium dihydrogen phosphate - dipotassium hydrogen phosphate, 5 mM magnesium sulfate). Add dihydroxyacetone with a final concentration of 100 mM and formaldehyde with different final concentrations (final concentration of 200 - 900 mM) to the above potassium phosphate buffer systems with different pH values, and react at 1000 rpm at 30 °C for 24 h. After the reaction is completed, detect according to the derivatization method and HPLC detection method in Example 1, and the yield of erythrulose obtained through calculation is shown in Table 5.
[0046] Table 5 Yield of erythrulose produced by the reaction of dihydroxyacetone with formaldehyde at different concentrations in potassium phosphate buffer with different pH values
[0047] As can be seen from Table 5, under the condition of pH = 8, as the final concentration of formaldehyde increases, the yield of erythrulose in the reaction system further increases; as the pH of the buffer solution increases and the final concentration of formaldehyde in the reaction system increases, the yield of erythrulose first increases and then decreases.
[0048] Example 7: Erythrulose is produced by the reaction of formaldehyde and dihydroxyacetone with different concentrations in 300 mM potassium phosphate buffer at 30 °C and 40 °C for 24 h and 48 h
[0049] Add dihydroxyacetone with a final concentration of 200 mM and formaldehyde with different final concentrations (200 mM, 400 mM, 600 mM, 1000 mM, 1400 mM, 1800 mM) to 300 mM potassium phosphate buffer (300 mM potassium dihydrogen phosphate - dipotassium hydrogen phosphate, 5 mM magnesium sulfate), that is, the concentration ratios of dihydroxyacetone to formaldehyde are 1:1, 1:2, 1:3, 1:5, 1:7, 1:9 respectively. React at 1000 rpm at 30 °C and 40 °C for 24 h and 48 h respectively. After the reaction is completed, detect according to the derivatization method and HPLC detection method in Example 1, and the yield of erythrulose obtained through calculation is as Figure 5 shown.
[0050] From Figure 5 it can be seen that under the condition of 30 °C - 24 h, as the concentration of formaldehyde increases, the yield of erythrulose gradually increases; under the condition of 40 °C - 24 h, the highest yield is reached when the concentration ratio of dihydroxyacetone to formaldehyde is 1:2, and further increasing the concentration of formaldehyde has little effect on the yield of erythrulose. When the concentration of formaldehyde is low, the erythrulose produced by the reaction at 40 °C is higher than that at 30 °C; when the concentration of formaldehyde is high, the erythrulose produced by the reaction at 30 °C is higher than that at 40 °C. Comparing the reaction for 48 h with 24 h, the yield of erythrulose is helpful at low concentrations of formaldehyde and not helpful at high concentrations of formaldehyde. It may be that at high concentrations of formaldehyde, more erythrulose and formaldehyde are converted into other by-products.
[0051] Example 8: The spontaneous reaction of formaldehyde and dihydroxyacetone in potassium phosphate buffer containing different concentrations of magnesium sulfate to produce erythrulose
[0052] Prepare potassium phosphate buffers (300 mM potassium dihydrogen phosphate - dipotassium hydrogen phosphate, pH = 8) containing different concentrations of magnesium sulfate (0 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 200 mM, 300 mM) respectively. Add 100 mM dihydroxyacetone and 100 mM formaldehyde with a final concentration into the above potassium phosphate buffers containing different concentrations of magnesium sulfate, and react at 30 °C and 1000 rpm for 24 h. After the reaction is completed, detect according to the derivatization method and HPLC detection method in Example 1. The yield of erythrulose obtained through calculation is shown in Table 6.
[0053] Table 6 Yield of erythrulose produced by the spontaneous reaction of formaldehyde and dihydroxyacetone in potassium phosphate buffer containing different concentrations of magnesium sulfate
[0054] As can be seen from Table 6, the production of erythrulose was detected in the potassium phosphate buffer containing 0 - 300 mM magnesium sulfate, and the yield of erythrulose was the highest in the potassium phosphate buffer containing 0 mM, 5 mM and 10 mM magnesium sulfate.
[0055] In summary, the present invention provides a new route for synthesizing erythrulose from the one - carbon compound formaldehyde. Using formaldehyde and dihydroxyacetone as substrates, erythrulose is spontaneously generated in a buffer containing glycine root ions and / or a buffer containing hydrogen phosphate ions. This reaction can be carried out under normal temperature and pressure, and can be carried out within a wide pH range, having good application prospects.
[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.
Claims
1. A method for synthesizing erythrulose from formaldehyde and dihydroxyacetone, characterized in that: The method comprises: taking formaldehyde and dihydroxyacetone as substrates, and spontaneously reacting in a buffer containing glycine ions and / or a buffer containing hydrogen phosphate ions to generate erythrulose.
2. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The spontaneous reaction is carried out at 30-50°C for 24-48 hours.
3. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The pH of the buffer containing glycine ions is 7-12.
4. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The pH of the buffer solution containing hydrogen phosphate ions is 7-12.
5. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The concentration of the buffer containing glycine ions is 50-900 mM.
6. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The concentration of the buffer containing hydrogen phosphate ions is 50-900 mM.
7. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The final concentration ratio of formaldehyde and dihydroxyacetone in the buffer containing glycine ions is (1-9):(1-9).
8. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 7, characterized in that: The final concentration of formaldehyde in the buffer containing glycine ions is 100-1800 mM.
9. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 1, characterized in that: The final concentration ratio of formaldehyde and dihydroxyacetone in the buffer containing hydrogen phosphate ions is (1-9):(1-9).
10. The method for synthesizing erythrulose from formaldehyde and dihydroxyacetone according to claim 9, characterized in that: The final concentration of formaldehyde in the buffer containing hydrogen phosphate ions is 100-1800 mM.