Method for non-aqueous phase in-vitro enzymatic synthesis of AMA

Abstract

The invention provides a method for non-aqueous phase synthesis of AMA by using AMA synthetase as a catalyst, and the method specifically comprises the following steps: adding substrates L-aspartic acid, O-phosphate-L-serine, AMA synthetase and a deep eutectic solvent into a buffer solution, reacting at 25-37 DEG C under the conditions of standing, oscillation or stirring to prepare AMA, and after the reaction is finished, carrying out anion-cation exchange chromatography and recrystallization separation and purification to obtain AMA. The non-aqueous-phase synthesis method provided by the invention can slow down adverse side reactions of degradation of a substrate O-phosphate-L-serine and AMA in an aqueous-phase synthesis method, reduces excessive input of the substrate, can improve catalytic efficiency and space time yield, and has a wide application prospect in the field of green and efficient synthesis of metal chelate antibiotic adjuvants.

Description

Technical Field

[0001] The present invention belongs to the field of novel biocatalysis technology and relates to a method for enzymatically synthesizing AMA in a non-aqueous phase in vitro.

[0002] Specifically, it relates to a process method for efficiently preparing AMA, which uses L-aspartic acid and O-phospho-L-serine as substrates, AMA synthase as a biocatalyst, and a low eutectic solvent-buffer as a reaction medium. Background Art

[0003] The main reason for the drug resistance of Gram-negative pathogens is the hydrolysis of β-lactam antibiotics by expressing metallo-β-lactamase (MBL). There is an urgent need to develop effective MBL inhibitor / β-lactam antibiotic combination drugs to treat infections caused by multidrug-resistant bacteria.

[0004] Aspergillomarasmine A, a fungal natural product, is a biotoxin that causes leaf spotting, wilt or necrosis in plants. As an aminopolycarboxylate natural metal chelator, AMA was found to have some important physiological activities in the 1980s and 1990s, such as an inhibitor of metal-dependent enzymes angiotensin-converting enzyme (ACE) and endothelin-converting enzyme (ECE). Recently, AMA has been re-identified as an MBL inhibitor, which can effectively inhibit a variety of B1-class MBL (including NDM, VIM and IMP), especially against the fast-spreading NDM-1. Therefore, AMA can restore the activity of carbapenems (such as meropenem) against a variety of clinically isolated resistant strains carrying the NDM-1 gene, and has the potential to be developed as an anti-infective drug for the treatment of carbapenem-resistant Gram-negative bacteria (such as CRE).

[0005] The synthesis of AMA mainly includes chemical synthesis, chemical-enzyme coupling synthesis and enzymatic synthesis (see: Angew. Chem., Int. Ed. 2016, 55, 4291-4295; Angew. Chem., Int. Ed. 2016, 55, 2210-2212; Angew. Chem., Int. Ed. 2016, 55, 13259-13262; Nat. Catal. 2018, 1, 186-191). The enzymatic synthesis of AMA was first reported in 2020. A natural AMA synthase isolated from Aspergillus oryzae 3.9544 catalyzed the synthesis of AMA via two-step continuous substitution reactions with L-aspartate and O-phospho-L-serine as substrates, with a yield of 85% and an isolated yield of 19% (see: ACS Catal. 2020, 10, 11, 6291–6298; CN 110982796). Due to the use of an enzyme catalyst with high site and stereo specificity, this method streamlines the AMA synthesis steps (from 4 to 19 steps to 1 step) and is more environmentally friendly. Recently, the applicant of this application identified and characterized a more active AMA synthase PteAMAS from the fungus Pyrenophora teres f.teres 0-1. By optimizing the reaction conditions, PteAMAS was used to achieve an AMA synthesis yield of >99% and a yield of 51% (see: Adv. Synth. Catal. 2024, 366, 24, 5160-5170; CN 116426491). In addition to the high yield, another major advantage of this method is that it can completely convert the intermediate toxin A, which has similar properties to AMA, thereby simplifying product purification and improving the separation yield.

[0006] However, the enzymatic AMA synthesis system requires a high input of O-phospho-L-serine, because AMA synthase also has lyase activity and can catalyze the hydrolysis of O-phospho-L-serine; when the concentration of O-phospho-L-serine in the system drops to a very low level, AMA synthase will further catalyze the decomposition of AMA back to L-aspartic acid. From the mechanism, it is speculated that the lyase activity mainly comes from the hydrolysis of the intermediate (as shown in the following reaction formula), which also competes with the product synthesis, resulting in a decrease in the efficiency of the synthesis reaction. These problems have greatly limited the application of enzymatic synthesis of AMA.

[0007]

[0008] The reaction equation of the synthesis of AMA catalyzed by AMA synthetase and the degradation of O-phospho-L-serine or AMA Summary of the invention

[0009] The purpose of the present invention is to provide a method for enzymatically synthesizing AMA in a non-aqueous phase, which reduces the adverse effects of water on the reaction by introducing an environmentally friendly non-aqueous solvent as a reaction medium, greatly improves the AMA yield, and overcomes the shortcomings of the prior art.

[0010] To achieve the above object, the technical solution adopted by the present invention is: a method for enzymatically synthesizing AMA in a non-aqueous phase, characterized in that the system contains at least one of the deep eutectic solvents, comprising the following steps:

[0011] (1) Adding substrates L-aspartic acid, O-phospho-L-serine and AMA synthetase and a low eutectic solvent into a buffer to prepare a reaction system.

[0012] The buffer solution is a buffer solution with a pH value of 7.0 to 9.0, preferably a potassium phosphate buffer solution with a pH value of 8.5.

[0013] The low eutectic solvent is a low eutectic mixture of a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is one of betaine or choline chloride, and the hydrogen bond donor is one of glycerol, ethylene glycol, and 1,2-propylene glycol; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2 to 1:9, and the concentration is 25%-90% (v / v). Preferably, the low eutectic solvent is a hydrogen bond acceptor of betaine, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2 to 1:9, and the concentration is 40%-60% (v / v); the most preferred is a betaine-glycerol (1:3) low eutectic solvent, with a concentration of 50% (v / v).

[0014] The concentration of the substrate L-aspartic acid is 5mM to 80mM, the concentration of O-phospho-L-serine is 10mM to 300mM, and the ratio of L-aspartic acid to O-phospho-L-serine is 1:1 to 1:15. Preferably, the concentration of O-phospho-L-serine is 20mM to 160mM, and the ratio of L-aspartic acid to O-phospho-L-serine is 1:2.5 to 1:4.

[0015] The AMA synthase is one of the AMA synthases from Pyrenophora teres, Aspergillus versicolor, Fusariumoxysporum, Colletotrichum gloeosporioides or other AMA natural producing bacteria, preferably the AMA synthase PteAMAS from Pyrenophora teres. The concentration of the AMA synthase is greater than or equal to 10 uM.

[0016] In the above technical solution, the order of adding the reagents is: first add the substrate into the buffer solution to dissolve, then add the low eutectic solvent and mix well, and finally add the AMA synthase PteAMAS.

[0017] (2) The reaction system obtained in step (1) is allowed to stand, shake or stir at 25-37° C. to react and prepare AMA. After the reaction is completed, the product and the low-solvent component are separated using anion and cation exchange resins and recrystallization, and AMA is obtained after freeze-drying.

[0018] In the above technical solution, the method of adding the substrate is selected from one of the following two methods: [a] preparing a high-concentration substrate stock solution, adjusting the pH value of the stock solution to the same as that of the reaction solution, and no longer adjusting the pH after adding the substrate to the reaction system; [b] directly adding the substrate to the buffer solution, and adjusting the pH to the required reaction value with hydrochloric acid, potassium hydroxide or sodium hydroxide after the substrate is completely dissolved.

[0019] In the above technical scheme, the inventor unexpectedly discovered that the low eutectic solvent with betaine as the hydrogen bond acceptor significantly slowed down the activity of the AMA synthetase PteAMAS lyase under medium concentration (40-60%) conditions, that is, the hydrolysis side reaction of O-phospho-L-serine and AMA was reduced, and the catalytic efficiency was improved. The yield of AMA in the low eutectic solvent formed by the choline chloride group as a hydrogen bond acceptor is lower than that in the aqueous phase system. It shows that only a specific low eutectic solvent can ensure the activity of the AMA synthetase PteAMAS lyase and enhance the synthesis efficiency. At the same time, the embodiment of the present invention has also been verified that the synthesis efficiency is related to the concentration of the low eutectic solvent, the molar ratio of betaine / glycerol, and the concentration of O-phospho-L-serine.

[0020] Finally, compared with the synthesis in an aqueous system, the betaine-based low eutectic solvent-buffer system can reduce the input of O-phospho-L-serine and obtain a higher space-time yield.

[0021] In the above technical scheme, the preparation of AMA synthase is described in Adv.Synth.Catal.2024,366(24),5160-5170 and ACS Catal.2020,10,11,6291-6298, and the reaction form is pure enzyme.

[0022] Beneficial Effects

[0023] Due to the use of the above technical solution, the present invention has the following advantages compared with the prior art:

[0024] (1) The present invention uses a non-aqueous phase to replace the aqueous phase for enzymatic synthesis of AMA. By introducing a non-aqueous solvent into the system, the adverse side reaction activity and reduced synthesis efficiency caused by hydrolysis of the intermediate are reduced, thereby improving the synthesis efficiency of the existing technology and obtaining a higher space-time yield. At the same time, while ensuring that the reaction can obtain a high conversion rate and high stereoselectivity, the input of the substrate O-phospho-L-serine is reduced, thereby achieving a more economical and efficient enzymatic synthesis of AMA.

[0025] (2) The non-aqueous solvent used in the present invention is a biodegradable low-melting solvent, which meets the requirements of green chemistry; compared with other commonly used non-aqueous solvents such as organic solvents, ionic liquids (ILs) and supercritical CO2 fluids, it has higher solubility for highly polar substrates; it is cheap and easy to obtain, and can be separated, recovered and recycled by ion exchange resins, thereby reducing costs.

[0026] (3) The present invention achieves a high space-time yield under high substrate concentration conditions, and the product purity is greater than 98%, which has good application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the effect of betaine / glycerol ratio on yield;

[0028] Figure 2 The effect of betaine-glycerol eutectic solvent content;

[0029] Figure 3 The effect of betaine-glycerol deep eutectic solvent on the hydrolysis of O-phospho-L-serine;

[0030] Figure 4 The figure shows the optimization of O-phospho-L-serine concentration; (A) the reaction progress curves at concentrations of 10 mM, 15 mM and 20 mM; (B) the highest yield under different substrate concentration conditions;

[0031] Figure 5 The reaction progress curve of PteAMAS catalyzing L-aspartic acid (64 mM) and O-phospho-L-serine (160 mM) in aqueous phase and DES-buffer system;

[0032] Figure 6 This is the H NMR spectrum of the isolated enzymatic synthesis product AMA. DETAILED DESCRIPTION

[0033] Example 1 Preparation of a low eutectic solvent formed by three hydrogen bond donors and choline chloride

[0034] As shown in Table 1, 10 g of choline chloride was weighed and mixed with corresponding volumes of glycerol, ethylene glycol and 1,2-propylene glycol, stirred at 80° C. until clear, and allowed to stand at room temperature for use.

[0035] Table 1 Formulas of three choline chloride based deep eutectic solvents

[0036]

[0037] Example 2 Enzymatic AMA Synthesis in Choline Chloride-Based DES-Buffer System

[0038] The preparation of pure PteAMAS enzyme was based on the method reported in the literature (Zhu et al, Adv. Synth. & Catal. 2024, 366, 5160-5170). The reaction process of PteAMAS catalyzing O-phospho-L-serine and L-aspartic acid is shown in Figure 1 .

[0039] 1ml reaction system includes: 10uM PteAMAS, 5mM L-aspartic acid, 15mM O-phospho-L-serine, 0%, 25%, 50%, 75% or 90% (v / v) of DES1, DES2 or DES3, and 100mM KH2PO4-KOH buffer (pH=8.5). The specific preparation method is: add 25μL L-aspartic acid solution (200mM) and 30μL O-phospho-L-serine solution (500mM) into the buffer, mix with the corresponding volume of low eutectic solvent, stir evenly with magnetic vortex, add PteAMAS, and react at 30℃ for 8 hours. Set three parallels for each sample. Detection method: Take 50uL of the reaction solution, add 50μl 100mM Fmoc-NHS solution, 50μl 0.2M boric acid (pH=10.0) and 50μl acetonitrile, react for 2h, and then quench the reaction with 50μl methanol for 1h. After filtering with a 0.22μm filter membrane, use the following liquid phase method for HPLC detection: chromatographic column YMC-Pack ODS-A (150mm x 4.6mm, 5μm), mobile phase A is water containing 0.1% TFA, phase B is acetonitrile containing 0.1% TFA, and the gradient elution is: 0-10 minutes, the concentration of phase B increases from 15% to 45%; 10-12 minutes, from 45% to 95%; 12-16 minutes, maintain 95%. Flow rate 1.2mL / min, column temperature 30℃, UV detection wavelength 260nm. The reaction percentage yield is calculated using the AMA standard curve.

[0040] result:

[0041] As shown in Table 2, under the same substrate concentration conditions, the yields of reactions containing DES1, DES2 and DES3 were all lower than those of pure water phase reactions. Choline chloride-based low melting solvents may inhibit the activity of PteAMAS; at the same time, as the DES content increased (25-50%), the AMA yield increased from low to high. The results of different hydrogen bond donors were different, but when the DES content was 50%, the AMA yield was similar, among which choline chloride / ethylene glycol formed a low eutectic solvent that was optimal. Continuing to increase the DES concentration to 75% and 90% did not further increase the yield, but instead led to a decrease in activity or even inactivation, reflecting that maintaining a certain water content in the reaction system may be necessary for the activity of PteAMAS.

[0042] Table 2 Effect of choline chloride-based deep eutectic solvent on the maximum yield of AMA

[0043]

[0044] Example 3 Preparation of three betaine-based deep eutectic solvents

[0045] As shown in Table 3, 10 g of betaine was weighed and mixed with corresponding volumes of glycerol, ethylene glycol and 1,2-propylene glycol, stirred at 80° C. until clear, and allowed to stand at room temperature for use.

[0046] Table 3 Preparation of deep eutectic solvents formed by three hydrogen bond donors and betaine

[0047]

[0048] Example 4 Enzymatic AMA Synthesis in Betaine-Based DES-Buffer System

[0049] The preparation of pure PteAMAS enzyme was based on the method reported in the literature (Zhu et al, Adv. Synth. & Catal. 2024, 366, 5160-5170). The reaction process of PteAMAS catalyzing O-phospho-L-serine and L-aspartic acid is shown in Figure 1 .

[0050] 1 ml reaction system includes: 10uM PteAMAS, 5mM L-aspartic acid, 15mM O-phospho-L-serine, 0%, 25%, 50%, 75% or 90% (v / v) DES4, DES5 or DES6, and 100mM KH2PO4-KOH buffer (pH=8.5). Other reaction conditions and detection methods are the same as those in Example 2.

[0051] result:

[0052] As shown in Table 4, under the same substrate concentration conditions, the reaction systems containing 25% and 50% DES4, DES5, and DES6 have significantly higher yields than the pure water phase reaction, among which the yield of the 50% DES4 system has increased most significantly; continuing to increase the DES concentration to 75% and 90% did not further increase the yield, but instead led to a decrease in activity or even inactivation, reflecting that maintaining a certain water content in the reaction system may be necessary for the activity of PteAMAS.

[0053] Table 4 Effect of betaine-based deep eutectic solvent on the maximum yield of AMA

[0054]

[0055] Example 5 Effect of Betaine-Glycerol DES Composition Ratio on Reaction

[0056] As shown in Table 5, low eutectic solvents DES7, DES8 and DES9 with betaine / glycerol molar ratios of 1:5, 1:7 and 1:9 were prepared, and the preparation method was the same as that in Example 3.

[0057] Table 5 Effect of betaine-glycerol DES composition ratio on reaction

[0058]

[0059] Effect of composition ratio on reaction yield: 1 ml reaction system includes: 10 uM PteAMAS, 5 mM L-aspartic acid, 15 mM O-phospho-L-serine, 50% (v / v) DES4, DES7, DES8, DES9 or glycerol, and 100 mM KH2PO4-KOH buffer (pH = 8.5). Other reaction conditions and detection methods are the same as those in Example 2.

[0060] result:

[0061] See Figure 1 Under the same content conditions, the highest yields of all betaine-glycerol low eutectic solvents and glycerol systems were higher than those of the aqueous phase system, and the yields decreased with the increase of glycerol concentration. DES4 (betaine: glycerol = 1:3) had the best effect.

[0062] Example 6 Effect of Betaine-Glycerol DES Content on Reaction

[0063] 1 ml reaction system includes: 10uM PteAMAS, 5mM L-aspartic acid, 15mM O-phospho-L-serine, 0%, 25%, 40%, 50%, 60% or 75% (v / v) DES4, and 100mM KH2PO4-KOH buffer (pH=8.5), react at 30°C for 1.5 hours, and other reaction conditions and detection methods are the same as Example 2.

[0064] result

[0065] See Figure 2 Under the same substrate concentration, as the DES4 content in the system increases, the reaction yield increases first and then decreases. The 50% DES4 system has the highest yield, reaching 90% after 45 minutes of reaction.

[0066] Example 7 Effect of Betaine-Glycerol DES on Hydrolysis of O-Phospho-L-Serine

[0067] The reaction process of PteAMAS-catalyzed O-phospho-L-serine hydrolysis is shown in Figure 1 .

[0068] 1 ml reaction system includes: 10 uM PteAMAS, 15 mM O-phospho-L-serine, 50% (v / v) DES4, 100 mM KH2PO4-KOH buffer (pH=8.5). Reaction at 30°C for 1.5 hours. Detection method is the same as in Example 2.

[0069] result:

[0070] See Figure 3 Compared with the pure water system, the PteAMAS-catalyzed hydrolysis of O-phospho-L-serine in the 50% betaine-glycerol (1:3) system was significantly slowed down.

[0071] Example 8 Optimization of O-phospho-L-serine concentration in DES-buffer system

[0072] 1ml reaction system includes: 10uM PteAMAS, 5mM L-aspartic acid, 10mM, 15mM, 20mM, 90mM, 160mM, 230mM or 300mM O-phospho-L-serine, 50vol% DES4, with 100mM KH2PO4-KOH buffer (pH=8.5). React at 30℃ for 1.5 hours. The specific preparation method and detection method are the same as Example 2.

[0073] result:

[0074] See Figure 4 , with the increase of O-phospho-L-serine input concentration, the maximum yield of the reaction first increased and then decreased. When it was increased to 20mM, the maximum yield reached 99%, which was significantly higher than the 50% of the aqueous phase reaction, and the reaction rate was accelerated; when the substrate concentration was further increased, the yield remained at 99%; but when the concentration exceeded 160mM, the reaction yield began to decrease significantly, indicating that there may be substrate inhibition.

[0075] Example 9 Optimization of L-aspartic acid concentration

[0076] DES-buffer system: 1 ml reaction system includes: 10uM PteAMAS, 40mM, 53mM, 64mM or 80mM L-aspartic acid, 160mM O-phospho-L-serine, 50%vol DES4, and KH2PO4-KOH buffer (100mM, pH=8.5). React at 30°C for 24 hours. The specific preparation method and detection method are the same as in Example 2.

[0077] Aqueous system: 1 ml reaction system includes: 10uM PteAMAS, 40mM, 53mM, 64mM or 80mM L-aspartic acid, 160mM O-phospho-L-serine, and KH2PO4-KOH buffer (100mM, pH=8.5). Reaction at 30°C for 24 hours. Detection method is the same as in Example 2.

[0078] result:

[0079] As shown in Table 6 and Figure 5 As shown in the figure, under the condition of 160mM O-phospho-L-serine, in the 50%vol betaine-glycerol (1:2) reaction system, when the concentration of L-aspartic acid is increased to 64mM, the yield can reach 99% in 3 hours; when it is increased to 80mM, the yield is reduced to 90% in 3 hours, and the highest yield is 96% in 6 hours. Under the same substrate concentration conditions, the 50%vol betaine-glycerol (1:2) reaction system has a higher yield and a shorter time than the aqueous phase system, and the time-space yield is increased by about 4 times.

[0080] Table 6 Optimization of L-aspartic acid concentration

[0081]

[0082] Example 10: Enzymatic in vitro preparation of AMA in large quantities using a DES-buffer system

[0083] Take 7.68mL of 500mM L-aspartic acid solution and 9.6mL of 1M O-phospho-L-serine solution into a 100mL round-bottom flask, add KH2PO4-KOH buffer (100mM, pH=8.5) and 30mL DES4, vortex mix, and let stand overnight for equilibrium; add PteAMAS (final concentration 10μM), and the total reaction volume is 60mL. After reacting at 30℃ for 3h, heat at 95℃ for 15min to terminate the reaction. The detection method is the same as in Example 2. The yield is greater than 99% after reacting for 3 hours.

[0084] Product separation and purification:

[0085] (1) Purification with anion exchange resin: Weigh 37.5 g of anion exchange resin (AG 1-X8, acetic acid type, 100-200 mesh), swell overnight, and load into the column. Rinse the column with 5 CV of deionized water, filter the reaction solution, and then load the sample. First, rinse the unbound compound with 5 CV of deionized water, and then elute the product with 3 CV of 1 M ammonium acetate solution. Monitor the purification process with the ninhydrin colorimetric method.

[0086] (2) Purification by cation exchange resin

[0087] Weigh 37.5g of cation exchange resin (Dowex 50W X8, ammonia type, 100-200 mesh), swell overnight, and load the column. First rinse the chromatographic column with 5CV of 2M ammonia water, then convert it to hydrogen type with 3CV of 2M hydrochloric acid, and then neutralize it with 5CV of deionized water. Take the 1M ammonium acetate eluate obtained by purification of the anion exchange resin, adjust the pH to 8.5 with ammonia water, and then load it on the cation exchange chromatographic column. First rinse the unbound compounds with 5CV deionized water, then elute the product with 3CV 2M ammonia water, and remove water by rotary evaporation at 50℃. Recrystallize with 75%vol methanol containing 1% glacial acetic acid to obtain 718mg of pure AMA, with an isolation yield of 61%. The product nuclear magnetic hydrogen spectrum data is: 1 H NMR(600MHz,0.25M NH4OH in D2O) δ3.91(dt,J=8.5,4.0Hz,2H),3.49(dd,J=9.6,4.4Hz,1H),3.39–3.32(m,2H),3.22(dd,J=13.0,9 .7Hz,1H),2.95(dd,J=13.4,3.9Hz,1H),2.89(dd,J=17.7,3.7Hz,1H),2.77(dd,J=17.7,9.2Hz,1H), see Figure 6 .

Claims

1. A method for enzymatically synthesizing AMA in a non-aqueous phase, characterized in that: Substrates L-aspartate, O-phospho-L-serine, AMA synthase and a deep eutectic solvent are added to a buffer to obtain AMA through an enzymatic reaction.

2. The method according to claim 1, characterized in that The low eutectic solvent is a low eutectic mixture of a hydrogen bond acceptor and a hydrogen bond donor, wherein the hydrogen bond acceptor is betaine or choline chloride; the hydrogen bond donor is any one of glycerol, ethylene glycol, and 1,2-propylene glycol; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2 to 1:9, and the concentration of the low eutectic solvent is 25%-75% v / v.

3. The method according to claim 1, characterized in that The concentration of the substrate L-aspartic acid is 5mM to 80mM, the concentration of O-phospho-L-serine is 10mM to 300mM, and the ratio of L-aspartic acid to O-phospho-L-serine is 1:1 to 1:

15.

4. The method according to claim 1, characterized in that: The buffer solution is a potassium phosphate buffer solution with a pH of 7.0 to 9.

0.

5. The method according to claim 1, characterized in that The concentration of the AMA synthase is greater than or equal to 10uM.

6. The method according to any one of claims 1 to 5, characterized in that: (1) First, the substrate is added to the buffer solution to dissolve, then the low eutectic solvent is added to mix thoroughly, and finally the AMA synthase is added; (2) The reaction system obtained in step (1) is allowed to stand, shake or stir at 25 to 37° C. to react and prepare AMA. After the reaction is completed, the product is separated using anion and cation exchange resins and recrystallized to obtain AMA.

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