Synthesis and purification process of sodium mannose phosphate

Abstract

The application discloses a synthesis and purification process of sodium mannose phosphate, relates to the technical field of chemical materials, and comprises the following steps: dissolving D-mannose, sodium polyphosphate and magnesium ions in an aqueous phase buffer system, adding co-immobilized mannose kinase mutant ManK-M7 and polyphosphate kinase PPK2, and performing enzyme catalysis to generate sodium mannose-6-phosphate; after the reaction is completed, the co-immobilized enzyme is separated and recovered to obtain a reaction liquid containing sodium mannose-6-phosphate; the reaction liquid is subjected to ultrafiltration treatment to remove macromolecular impurities, and clear filtrate is obtained; the clear filtrate is introduced into an adsorption column filled with a zirconium-based metal organic framework material UiO-66-(COOH)2, so that sodium mannose-6-phosphate is selectively adsorbed on the adsorption column; sodium bicarbonate buffer solution is used to elute the adsorption column, and eluate containing sodium mannose-6-phosphate is collected; the eluate is subjected to nanofiltration concentration to obtain a concentrated solution; ethanol is added to the concentrated solution for anti-solvent crystallization; after solid-liquid separation, the obtained crystals are subjected to vacuum freeze drying.

Description

Technical Field

[0001] This invention relates to the field of chemical materials technology, and in particular to a process for the synthesis and purification of sodium mannose phosphate. Background Technology

[0002] Sodium mannose-6-phosphate, as a key bioactive phosphorylated monosaccharide, has significant application value in cell signaling, lysosomal targeted delivery, and skin anti-aging. In recent years, with the rapid growth in demand for high-purity, highly regioselective phosphorylated sugars from the high-end cosmetics and biopharmaceutical industries, its preparation technology has received widespread attention. Traditional synthesis methods mainly rely on chemical phosphorylation routes, typically using D-mannose as the starting material. The reaction is carried out in anhydrous organic solvents under the action of highly corrosive or toxic reagents such as phosphorus oxychloride and triethylamine, followed by hydrolysis, neutralization, and multi-step chromatographic purification to obtain the target product.

[0003] While existing technologies can achieve large-scale production through chemical methods in terms of greening the synthesis pathway and improving purification efficiency, even when using enzymatic methods, most systems still rely on free enzymes or a single immobilization strategy, resulting in difficulties in enzyme recovery, poor batch-to-batch reproducibility, and complex post-reaction processing. However, they cannot solve the problem of separating regioisomeric compounds, and the product purity is usually below 95%, making it difficult to meet the requirements for pharmaceutical excipients or INCI certification. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a synthesis and purification process for sodium mannose phosphate to solve the problems of difficult enzyme recovery, poor batch-to-batch reproducibility, and complex post-reaction processing, but it cannot solve the problem of regioisomeric separation. The product purity is usually less than 95%, which is difficult to meet the requirements of pharmaceutical excipients or INCI certification.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: The present invention provides a process for the synthesis and purification of sodium mannose phosphate, which includes dissolving D-mannose, sodium polyphosphate and magnesium ions in an aqueous buffer system, adding co-immobilized mannose kinase mutant ManK-M7 and polyphosphokinase PPK2, and performing an enzyme-catalyzed reaction to generate sodium mannose-6-phosphate. After the reaction was completed, the co-immobilized enzyme was separated and recovered to obtain a reaction solution containing sodium mannose-6-phosphate; The reaction solution was subjected to ultrafiltration to remove macromolecular impurities, resulting in a clear filtrate. The clarified filtrate was passed into an adsorption column packed with zirconium-based metal-organic framework material UiO-66-(COOH)2, so that sodium mannose-6-phosphate was selectively adsorbed onto the adsorption column. The adsorption column was eluted with sodium bicarbonate buffer solution, and the eluent containing sodium mannose-6-phosphate was collected. The eluent was concentrated by nanofiltration to obtain a concentrate; Ethanol was added to the concentrate to induce antisolvent crystallization. After solid-liquid separation, the resulting crystals were freeze-dried under vacuum to obtain sodium mannose phosphate product.

[0007] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the following steps are described: D-mannose, sodium polyphosphate, and magnesium ions are dissolved in an aqueous buffer system, and co-immobilized mannose kinase mutant ManK-M7 and polyphosphokinase PPK2 are added to carry out an enzymatic reaction to generate sodium mannose-6-phosphate; the specific steps are as follows: Prepare a Tris-HCl buffer solution with pH 7.0–8.0 as an aqueous buffer system. Add D-mannose, sodium polyphosphate and soluble magnesium salt in sequence and stir until completely dissolved to form a homogeneous reaction base solution. Subsequently, the mannokinase mutant ManK-M7 and polyphosphoric acid kinase PPK2, covalently bonded to an epoxy-amino bifunctional silica support, were added, and the reaction was carried out under constant temperature stirring at 35–40℃. During the reaction, the pH of the system is maintained by automatically adding sodium hydroxide solution. Under the drive of sodium polyphosphate providing phosphate groups and chemical energy, D-mannose undergoes C6-hydroxyl phosphorylation by ManK-M7 specific catalysis and combines in situ with sodium ions to generate mannose-6-phosphate sodium.

[0008] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the following steps are taken: After the reaction is completed, the co-immobilized enzyme is separated and recovered to obtain a reaction solution containing sodium mannose-6-phosphate; After the enzyme-catalyzed reaction is complete, stop stirring and let the reaction system stand. Use a filter with a pore size of 40–60 micrometers to separate the solid and liquid components of the reaction mixture, retain the co-immobilized enzyme particles, and wash them with deionized water before recycling. The liquid that passes through the filter medium is a reaction solution containing sodium mannose-6-phosphate, inorganic salts, and small molecule byproducts.

[0009] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the reaction solution is subjected to ultrafiltration to remove macromolecular impurities and obtain a clear filtrate; the specific steps are as follows: The reaction solution is pumped into an ultrafiltration membrane system with a molecular weight cutoff of 8–12 kDa and circulated for filtration under a transmembrane pressure difference of 0.1–0.3 MPa to retain large molecular impurities such as proteins, enzyme fragments, and polymers. The permeate is collected as the clarified filtrate.

[0010] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the step of passing the clarified filtrate into an adsorption column packed with zirconium-based metal-organic framework material UiO-66-(COOH)2, so that sodium mannose-6-phosphate is selectively adsorbed onto the adsorption column, specifically includes the following steps: UiO-66-(COOH)2 was activated under vacuum at 120°C for 12 hours and then packed into a glass column to form an adsorption column. The clarified filtrate was passed into the adsorption column at a flow rate of 1.5–2.5 column volumes / hour, utilizing Zr... 6 The specific coordination of the cluster with the phosphate monoester anion allows mannose-6-phosphate sodium to be selectively adsorbed into the pores of the adsorbent, while inorganic salts and neutral sugars are discharged with the effluent.

[0011] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the step of eluting the adsorption column with sodium bicarbonate buffer solution and collecting the eluent containing sodium mannose-6-phosphate includes the following specific steps: The adsorption column was rinsed with 1–1.5 column volumes of deionized water to remove residual impurities; Switch to 0.08–0.12 mol / L sodium bicarbonate buffer solution and perform isocratic elution at a flow rate of 1.0–2.0 column volumes / hour; The elution peak was monitored using an ultraviolet detector, and the eluent corresponding to the main peak was collected as the eluent containing sodium mannose-6-phosphate.

[0012] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the step of concentrating the eluent by nanofiltration to obtain a concentrated solution specifically includes: The eluent is introduced into a nanofiltration membrane module with a molecular weight cutoff of 100–200 Da and concentrated at an operating pressure of 0.6–1.0 MPa. Deionized water is added for dialysis to remove residual inorganic salts until the solid content of the concentrate reaches 18%–22%, resulting in a clear concentrate.

[0013] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate described in this invention, the steps of adding ethanol to the concentrated solution for anti-solvent crystallization, separating the solid and liquid, and then freeze-drying the obtained crystals under vacuum to obtain the sodium mannose phosphate product are as follows: Anhydrous ethanol is slowly added to the concentrate, and the feeding rate is controlled to maintain the supersaturation of the system between 1.7 and 2.3. The mixture was placed in an environment of 2–6℃ and allowed to stand for crystallization for 10–14 hours to precipitate sodium mannose-6-phosphate crystals. Solid-liquid separation is then performed by centrifugation or filtration to obtain wet crystals.

[0014] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate according to the present invention, the specific steps of obtaining sodium mannose phosphate product by vacuum freeze-drying the obtained crystals are as follows: Spread the wet crystals evenly in a freeze-drying tray and pre-freeze them at -40 to -50°C for 2–4 hours to completely freeze the internal moisture. The solution is then placed in a vacuum freeze dryer with an absolute pressure not exceeding 10 Pa. It is first dried at 0–5°C for 12–18 hours, and then heated to 20–25°C for desorption drying for 8–10 hours to completely sublimate the ice crystals and bound water, thus obtaining sodium mannose phosphate powder.

[0015] As a preferred embodiment of the synthesis and purification process of sodium mannose phosphate according to the present invention, the specific steps for obtaining sodium mannose phosphate powder are as follows: The quality of the sodium mannose phosphate powder was tested and confirmed to be not less than 99.0%, of which the proportion of sodium mannose-6-phosphate isomer exceeded 99.5%, and no phosphorylation byproducts at the C2 or C3 positions were detected. The total heavy metal residue is less than 10 ppm and the moisture content is less than 3.0%, which meets the quality standards for cosmetic active ingredients and pharmaceutical excipients.

[0016] The beneficial effects of this invention are as follows: By dissolving D-mannose, sodium polyphosphate, and magnesium ions in an aqueous buffer system, and adding the co-immobilized mannose kinase mutant ManK-M7 and polyphosphokinase PPK2 for enzymatic catalysis, a green cascade phosphorylation process with sodium polyphosphate as the sole phosphorus and energy source is achieved. Utilizing the absolute regioselectivity of ManK-M7 for the C6 hydroxyl group and the PPK2-driven in-situ ATP regeneration mechanism, high-purity mannose-6-phosphate sodium is generated in situ in a mild aqueous phase, completely avoiding the problems of using highly toxic reagents, organic solvents, and generating C2 / C3 isomers in traditional chemical methods, thus achieving a synthesis effect of 100% regioselectivity, high atom economy, and intrinsic safety. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of the synthesis and purification process of sodium mannose phosphate. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Example 1, referring to Figure 1 This is the first embodiment of the present invention, which provides a process for the synthesis and purification of sodium mannose phosphate, including the following steps: S1. D-mannose, sodium polyphosphate, and magnesium ions are dissolved in an aqueous buffer system. Co-immobilized mannose kinase mutant ManK-M7 and polyphosphate kinase PPK2 are added to carry out an enzymatic reaction to generate sodium mannose-6-phosphate.

[0023] Furthermore, a Tris-HCl buffer solution with pH 7.0–8.0 was prepared as an aqueous buffer system, and D-mannose, sodium polyphosphate and soluble magnesium salt were added to it in sequence and stirred until completely dissolved to form a homogeneous reaction base solution. Subsequently, the mannokinase mutant ManK-M7 and polyphosphoric acid kinase PPK2, covalently bonded to an epoxy-amino bifunctional silica support, were added, and the reaction was carried out under constant temperature stirring at 35–40℃. During the reaction, the pH of the system is maintained by automatically adding sodium hydroxide solution. Under the drive of sodium polyphosphate providing phosphate groups and chemical energy, D-mannose undergoes C6-hydroxyl phosphorylation by ManK-M7 specific catalysis and combines in situ with sodium ions to generate mannose-6-phosphate sodium.

[0024] It should be noted that using sodium polyphosphate to replace ATP achieves green energy supply, and the co-immobilized dual-enzyme system ensures highly selective phosphorylation at the C6 position, avoids the generation of byproducts, and forms salts in situ in the aqueous phase, making the process safe and efficient.

[0025] S2. After the reaction is complete, the co-immobilized enzyme is separated and recovered to obtain a reaction solution containing sodium mannose-6-phosphate.

[0026] Furthermore, after the enzyme-catalyzed reaction is complete, stirring is stopped and the reaction system is allowed to stand. A filter with a pore size of 40–60 micrometers is used to separate the solid and liquid components of the reaction mixture. The co-immobilized enzyme particles are retained and washed with deionized water before being recovered. The liquid that passes through the filter medium is a reaction solution containing sodium mannose-6-phosphate, inorganic salts, and small molecule byproducts.

[0027] It should be noted that the co-immobilized enzyme can be recovered through simple filtration, enabling multiple reuses, reducing enzyme consumption and costs, and avoiding interference from free proteins in subsequent purification.

[0028] S3. The reaction solution is subjected to ultrafiltration to remove macromolecular impurities and obtain a clear filtrate.

[0029] Furthermore, the reaction solution is pumped into an ultrafiltration membrane system with a molecular weight cutoff of 8–12 kDa, and circulated for filtration under a transmembrane pressure difference of 0.1–0.3 MPa to retain large molecular impurities such as proteins, enzyme fragments, and polymers. The permeate is collected as the clarified filtrate.

[0030] It should be noted that ultrafiltration effectively removes macromolecular impurities, obtains highly clear filtrate, protects the subsequent adsorption column from contamination, and does not lose the target product.

[0031] S4. Pass the clarified filtrate into an adsorption column packed with zirconium-based metal-organic framework material UiO-66-(COOH)2, so that sodium mannose-6-phosphate is selectively adsorbed onto the adsorption column.

[0032] Furthermore, UiO-66-(COOH)2 was activated under vacuum at 120°C for 12 hours and then packed into a glass column to form an adsorption column; the clarified filtrate was passed into the adsorption column at a flow rate of 1.5–2.5 column volumes / hour, utilizing Zr 6 The specific coordination of the cluster with the phosphate monoester anion allows mannose-6-phosphate sodium to be selectively adsorbed into the pores of the adsorbent, while inorganic salts and neutral sugars are discharged with the effluent.

[0033] It should be noted that UiO-66-(COOH)2 has a specific adsorption capacity for sodium mannose-6-phosphate, which can separate inorganic salts and neutral sugars in one step, thereby improving the purification selectivity.

[0034] S5. Elute the adsorption column with sodium bicarbonate buffer solution and collect the eluent containing sodium mannose-6-phosphate.

[0035] Furthermore, the adsorption column was rinsed with 1–1.5 column volumes of deionized water to remove residual impurities; Switch to 0.08–0.12 mol / L sodium bicarbonate buffer solution and perform isocratic elution at a flow rate of 1.0–2.0 column volumes / hour; The elution peak was monitored using an ultraviolet detector, and the eluent corresponding to the main peak was collected as the eluent containing sodium mannose-6-phosphate.

[0036] It should be noted that sodium bicarbonate gentle elution can efficiently desorb the target substance while avoiding the hydrolysis of phosphate ester bonds, and combined with online monitoring, ensures the high purity of the eluent.

[0037] S6. Concentrate the eluent by nanofiltration to obtain a concentrated solution.

[0038] Furthermore, the eluent is introduced into a nanofiltration membrane module with a molecular weight cutoff of 100–200 Da and concentrated under an operating pressure of 0.6–1.0 MPa. At the same time, deionized water is added for dialysis to remove residual inorganic salts until the solid content of the concentrate reaches 18%–22%, thus obtaining a clear concentrate.

[0039] It should be noted that nanofiltration simultaneously completes concentration and desalination without heating, preventing heat-sensitive degradation and providing ideal feed for high-quality crystallization.

[0040] S7. Add ethanol to the concentrate to induce antisolvent crystallization. After solid-liquid separation, freeze-dry the resulting crystals under vacuum to obtain sodium mannose phosphate product.

[0041] Furthermore, anhydrous ethanol is slowly added to the concentrate, and the feeding rate is controlled to maintain the supersaturation of the system between 1.7 and 2.3. The mixture was placed in an environment of 2–6℃ and allowed to stand for crystallization for 10–14 hours to precipitate sodium mannose-6-phosphate crystals. Solid-liquid separation was then performed by centrifugation or filtration to obtain wet crystals; Spread the wet crystals evenly in a freeze-drying tray and pre-freeze them at -40 to -50°C for 2–4 hours to completely freeze the internal moisture. It is then placed in a vacuum freeze dryer with an absolute pressure not exceeding 10 Pa, and first undergoes primary drying at 0–5℃ for 12–18 hours, and then heated to 20–25℃ for desorption drying for 8–10 hours to completely sublimate the ice crystals and bound water, thereby obtaining sodium mannose phosphate powder. The quality of the sodium mannose phosphate powder was tested and confirmed to be not less than 99.0%, of which the proportion of sodium mannose-6-phosphate isomers exceeded 99.5%, and no phosphorylation byproducts at the C2 or C3 positions were detected. The total heavy metal residue is less than 10 ppm and the moisture content is less than 3.0%, which meets the quality standards for cosmetic active ingredients and pharmaceutical excipients.

[0042] It should be noted that controlled-rate antisolvent crystallization combined with vacuum freeze-drying yields high-purity, high-stability powders, and the products meet cosmetic and pharmaceutical grade standards.

[0043] Example 2 is the second embodiment of the present invention. This embodiment provides a process for the synthesis and purification of sodium mannose phosphate, including the following steps: Dissolve D-mannose (50 g), sodium polyphosphate (80 g) and magnesium chloride (5 g) in 1 L of pH 7.5 Tris-HCl buffer solution and stir until completely dissolved to form a homogeneous and transparent reaction substrate. A co-immobilized enzyme (with a mass ratio of mannokinase mutant ManK-M7 to polyphosphoric acid kinase PPK2 of 3:1 and a total enzyme amount of 8 g) covalently bonded to an epoxy-amino bifunctional silica support was added to the substrate. The reaction was carried out at 37°C and 200 rpm for 12 hours. During the reaction, 1 mol / L NaOH solution was added dropwise using an automatic titration system to maintain the pH of the system at 7.5±0.1, so that D-mannose could be converted to manno-6-phosphate sodium by ManK-M7 under the phosphorus- and energy-providing action of sodium polyphosphate. After the reaction was completed, stirring was stopped and the mixture was allowed to stand for 10 minutes. The reaction mixture was then separated into solid and liquid phases using a bag filter with a pore size of 50 μm. The co-immobilized enzyme particles were retained and washed with 200 mL of deionized water before being recovered. The resulting filtrate was the reaction solution containing sodium mannose-6-phosphate. The reaction solution was pumped into a hollow fiber ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, and circulated for filtration under a transmembrane pressure difference of 0.2 MPa. The permeate was collected to obtain a clear filtrate. After the UiO-66-(COOH)2 material was vacuum activated at 120℃ for 12 hours, it was packed into a glass column with an inner diameter of 2 cm and a height of 20 cm to form an adsorption column. The clarified filtrate was passed into the adsorption column at a flow rate of 2.0 column volumes / hour, so that sodium mannose-6-phosphate was selectively adsorbed into the pores of the adsorbent, and the effluent was discarded.

[0044] After loading the sample, the adsorption column was first rinsed with 1.2 column volumes of deionized water, and then switched to 0.1 mol / L sodium bicarbonate buffer solution (pH 8.5) for isocratic elution at a flow rate of 1.5 column volumes / hour. The elution curve was monitored using an online ultraviolet detector (λ=210 nm), and the eluent corresponding to the main peak was collected. The eluent was introduced into a nanofiltration membrane module with a molecular weight cutoff of 150 Da and concentrated at an operating pressure of 0.8 MPa. Deionized water was added simultaneously for dialyzing and replacement until the volume of the concentrate was reduced to 1 / 5 of the original eluent volume and the solid content reached 20%, resulting in a clarified concentrate. While stirring, slowly add 3 times the volume of anhydrous ethanol to the concentrate (addition time 30 minutes), then transfer the mixture to a 4°C cold storage and let it stand for crystallization for 12 hours; After crystallization, the solid and liquid were separated by centrifugation (4000 rpm, 10 min), the wet crystals were collected, and the crystals were rinsed once with a small amount of cold ethanol. The wet crystals were spread evenly in a stainless steel freeze-drying tray (thickness ≤ 1 cm), pre-frozen at -45℃ for 3 hours, and then transferred to a vacuum freeze dryer. Under a pressure ≤ 10 Pa, the product was first dried at 2℃ for 16 hours, and then heated to 22℃ for desorption drying for 9 hours to obtain 82.3 g of white loose powder sodium mannose phosphate product, with a yield of 83.1%.

[0045] HPLC analysis showed that the product purity was 99.2%, the proportion of mannose-6-phosphate sodium isomer was 99.6%, no C2 / C3 byproducts were detected, the total heavy metal content was <8 ppm, and the moisture content was 2.1%, which meets the standards for cosmetic and pharmaceutical excipients. This embodiment demonstrates the efficient and selective synthesis and high-purity purification of sodium mannose phosphate, verifying the feasibility, stability, and industrialization potential of the process.

[0046] Comparative Example 1: This comparative example uses a typical chemical phosphorylation method combined with ion exchange resin purification process in the prior art to prepare sodium mannose phosphate, for comparison with Example 1 of the present invention. Specifically, D-mannose (50 g) was dissolved in anhydrous pyridine, and phosphoric acid oxychloride was added dropwise under ice bath to carry out a phosphorylation reaction. After hydrolysis and neutralization, the reaction solution was purified by passing it through a strongly basic anion exchange resin column, followed by activated carbon decolorization, multiple recrystallizations, and vacuum drying, finally yielding 61.5 g of product, with a yield of 62.0%. However, HPLC analysis showed that the total purity of the product was only 93.8%, with the mannose-6-phosphate sodium isomer accounting for only 86.2%, and the remainder being C2 / C3 byproducts; pyridine residue (approximately 35 ppm) and a relatively high heavy metal content (18 ppm) were also detected. This method not only uses highly toxic and corrosive reagents, generating a large amount of organic phosphorus-containing wastewater, but also lacks regional selectivity, making it difficult to obtain high-purity single isomers, thus failing to meet the quality requirements of cosmetics or pharmaceutical excipients. This fully highlights the advantages of the enzymatic green synthesis and highly selective purification process of this invention.

[0047] Comparative Example 2 aims to verify the technical effectiveness of the co-immobilized dual-enzyme system. It replaces the covalently bonded ManK-M7 and PPK2 on the epoxy-amino bifunctional silica support in Example 1 with free enzymes and adds exogenous ATP as a phosphate donor. The remaining steps are identical to Example 1. Results show that although 68.2 g of product (yield 68.9%) was obtained, batch reproducibility was poor (RSD = 8.3%), ultrafiltration membrane flux decreased by 40% due to free protein contamination, and enzyme and ATP costs increased significantly. Product purity (96.5%) and C6-position selectivity (97.1%) were also lower than in Example 1. These results indicate that the free enzyme system lacks spatial proximity and recycling capabilities, and cannot achieve efficient cascade catalysis. This invention, through co-immobilization design coupled with sodium polyphosphate for energy supply, effectively solves the three major bottlenecks of enzyme stability, economy, and reaction efficiency.

[0048] Comparative Example 3 was used to investigate the effect of adsorption material on purification effect. It replaced the zirconium-based metal-organic framework material UiO-66-(COOH)2, which specifically recognizes phosphate monoesters in Example 1, with the conventional strongly basic anion exchange resin Dowex 1×8(OH)2. - The process parameters remained unchanged. The final product yielded 74.6 g (75.3% yield), but the purity was only 94.8%, with 92.3% being sodium mannose-6-phosphate, still containing inorganic phosphates and neutral sugar impurities. The elution process consumed a large amount of NaCl solution, extending the nanofiltration desalination time to 2.5 times that of the original process, and the resin's adsorption capacity decreased after three batches. This indicates that traditional ion exchange materials lack the ability to recognize the structure of target molecules, making it difficult to achieve highly selective separation. In contrast, this invention utilizes Zr... 6 The specific coordination between the cluster and the phosphate monoester enables efficient enrichment and impurity removal in one step, improving purity, efficiency, and process sustainability. In summary, this invention achieves a green cascade phosphorylation process using sodium polyphosphate as the sole phosphorus and energy source by dissolving D-mannose, sodium polyphosphate, and magnesium ions in an aqueous buffer system and adding a co-immobilized mannose kinase mutant ManK-M7 and polyphosphate kinase PPK2 for enzymatic catalysis. Utilizing the absolute regioselectivity of ManK-M7 for the C6 hydroxyl group and the PPK2-driven in-situ ATP regeneration mechanism, high-purity mannose-6-phosphate sodium is generated in situ in a mild aqueous phase. This completely avoids the problems of using highly toxic reagents, organic solvents, and generating C2 / C3 isomers in traditional chemical methods, thus achieving 100% regioselectivity, high atom economy, and intrinsic safety in the synthesis.

[0049] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A process for the synthesis and purification of sodium mannose phosphate, characterized in that: include: D-mannose, sodium polyphosphate, and magnesium ions were dissolved in an aqueous buffer system, and co-immobilized mannose kinase mutant ManK-M7 and polyphosphate kinase PPK2 were added to carry out an enzymatic reaction to generate sodium mannose-6-phosphate. After the reaction was completed, the co-immobilized enzyme was separated and recovered to obtain a reaction solution containing sodium mannose-6-phosphate; The reaction solution was subjected to ultrafiltration to remove macromolecular impurities, resulting in a clear filtrate. The clarified filtrate was passed into an adsorption column packed with zirconium-based metal-organic framework material UiO-66-(COOH)2, so that sodium mannose-6-phosphate was selectively adsorbed onto the adsorption column. The adsorption column was eluted with sodium bicarbonate buffer solution, and the eluent containing sodium mannose-6-phosphate was collected. The eluent was concentrated by nanofiltration to obtain a concentrate; Ethanol was added to the concentrate to induce antisolvent crystallization. After solid-liquid separation, the resulting crystals were freeze-dried under vacuum to obtain sodium mannose phosphate product.

2. The synthesis and purification process of sodium mannose phosphate as described in claim 1, characterized in that: The process involves dissolving D-mannose, sodium polyphosphate, and magnesium ions in an aqueous buffer system, adding co-immobilized mannose kinase mutant ManK-M7 and polyphosphate kinase PPK2, and conducting an enzymatic reaction to generate mannose-6-phosphate sodium; the specific steps are as follows: Prepare a Tris-HCl buffer solution with pH 7.0–8.0 as an aqueous buffer system. Add D-mannose, sodium polyphosphate and soluble magnesium salt in sequence and stir until completely dissolved to form a homogeneous reaction base solution. Subsequently, the mannokinase mutant ManK-M7 and polyphosphoric acid kinase PPK2, covalently bonded to an epoxy-amino bifunctional silica support, were added, and the reaction was carried out under constant temperature stirring at 35–40℃. During the reaction, the pH of the system is maintained by automatically adding sodium hydroxide solution. Under the drive of sodium polyphosphate providing phosphate groups and chemical energy, D-mannose undergoes C6-hydroxyl phosphorylation by ManK-M7 specific catalysis and combines in situ with sodium ions to generate mannose-6-phosphate sodium.

3. The synthesis and purification process of sodium mannose phosphate as described in claim 2, characterized in that: After the reaction is completed, the co-immobilized enzyme is separated and recovered to obtain a reaction solution containing sodium mannose-6-phosphate; the specific steps are as follows: After the enzyme-catalyzed reaction is complete, stop stirring and let the reaction system stand. Use a filter with a pore size of 40–60 micrometers to separate the solid and liquid components of the reaction mixture, retain the co-immobilized enzyme particles, and wash them with deionized water before recycling. The liquid that passes through the filter medium is a reaction solution containing sodium mannose-6-phosphate, inorganic salts, and small molecule byproducts.

4. The synthesis and purification process of sodium mannose phosphate as described in claim 3, characterized in that: The reaction solution is subjected to ultrafiltration to remove macromolecular impurities, resulting in a clear filtrate; the specific steps are as follows: The reaction solution is pumped into an ultrafiltration membrane system with a molecular weight cutoff of 8–12 kDa and circulated for filtration under a transmembrane pressure difference of 0.1–0.3 MPa to retain large molecular impurities such as proteins, enzyme fragments, and polymers. The permeate is collected as the clarified filtrate.

5. The synthesis and purification process of sodium mannose phosphate as described in claim 4, characterized in that: The process of passing the clarified filtrate into an adsorption column packed with zirconium-based metal-organic framework material UiO-66-(COOH)2, so that sodium mannose-6-phosphate is selectively adsorbed onto the adsorption column, is as follows: UiO-66-(COOH)2 was activated under vacuum at 120°C for 12 hours and then packed into a glass column to form an adsorption column. The clarified filtrate was passed into the adsorption column at a flow rate of 1.5–2.5 column volumes / hour, utilizing Zr... 6 The specific coordination of the cluster with the phosphate monoester anion allows mannose-6-phosphate sodium to be selectively adsorbed into the pores of the adsorbent, while inorganic salts and neutral sugars are discharged with the effluent.

6. The synthesis and purification process of sodium mannose phosphate as described in claim 5, characterized in that: The adsorption column is eluted with sodium bicarbonate buffer solution, and the eluent containing sodium mannose-6-phosphate is collected. The specific steps are as follows: The adsorption column was rinsed with 1–1.5 column volumes of deionized water to remove residual impurities; Switch to 0.08–0.12 mol / L sodium bicarbonate buffer solution and perform isocratic elution at a flow rate of 1.0–2.0 column volumes / hour; The elution peak was monitored using an ultraviolet detector, and the eluent corresponding to the main peak was collected as the eluent containing sodium mannose-6-phosphate.

7. The synthesis and purification process of sodium mannose phosphate as described in claim 6, characterized in that: The process of concentrating the eluent by nanofiltration to obtain a concentrated solution involves the following steps: The eluent is introduced into a nanofiltration membrane module with a molecular weight cutoff of 100–200 Da and concentrated at an operating pressure of 0.6–1.0 MPa. Deionized water is added for dialysis to remove residual inorganic salts until the solid content of the concentrate reaches 18%–22%, resulting in a clear concentrate.

8. The synthesis and purification process of sodium mannose phosphate as described in claim 7, characterized in that: The steps involve adding ethanol to the concentrated solution to induce antisolvent crystallization, followed by solid-liquid separation and vacuum freeze-drying of the resulting crystals to obtain sodium mannose phosphate product. Anhydrous ethanol is slowly added to the concentrate, and the feeding rate is controlled to maintain the supersaturation of the system between 1.7 and 2.

3. The mixture was placed in an environment of 2–6℃ and allowed to stand for crystallization for 10–14 hours to precipitate sodium mannose-6-phosphate crystals. Solid-liquid separation is then performed by centrifugation or filtration to obtain wet crystals.

9. The synthesis and purification process of sodium mannose phosphate as described in claim 8, characterized in that: The specific steps for obtaining sodium mannose phosphate product by vacuum freeze-drying the obtained crystals are as follows: Spread the wet crystals evenly in a freeze-drying tray and pre-freeze them at -40 to -50°C for 2–4 hours to completely freeze the internal moisture. The solution is then placed in a vacuum freeze dryer with an absolute pressure not exceeding 10 Pa. It is first dried at 0–5°C for 12–18 hours, and then heated to 20–25°C for desorption drying for 8–10 hours to completely sublimate the ice crystals and bound water, thus obtaining sodium mannose phosphate powder.

10. The synthesis and purification process of sodium mannose phosphate as described in claim 9, characterized in that: The specific steps for obtaining sodium mannose phosphate powder are as follows: The quality of the sodium mannose phosphate powder was tested and confirmed to be not less than 99.0%, of which the proportion of sodium mannose-6-phosphate isomers exceeded 99.5%, and no phosphorylation byproducts at the C2 or C3 positions were detected. The total heavy metal residue is less than 10 ppm and the moisture content is less than 3.0%, which meets the quality standards for cosmetic active ingredients and pharmaceutical excipients.

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