Application of hydantoinase in hydrolysis or preparation of chiral monomeric compound

Abstract

The invention discloses an application of hydantoinase in hydrolysis or preparation of a chiral monomeric compound. Specifically, the invention discloses application of hydantoinase or an enzyme preparation thereof in hydrolysis of a chiral monomeric compound as shown in a formula (iii) and / or preparation of a chiral monomeric compound as shown in a formula (ii). The hydantoinase is used for preparing the chiral monomeric compound shown in the formula (ii), and compared with a traditional chemical synthesis method, the method has the advantages of being high in reaction efficiency, good in stereoselectivity, mild in reaction condition, low in energy consumption, environmentally friendly and the like, and can be used for large-scale application and popularization.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to the application of hydantoin in the hydrolysis or preparation of chiral monomeric compounds. Background Technology

[0002] Hydantoinase, also known as hydantoinase, is a class of amide hydrolases that catalyze the cleavage of cyclic amide bonds in hydantoin, 5'-monosubstituted hydantoin, and their derivatives. They are widely found in various organisms, including microorganisms, plants, and animals. However, hydantoinase with industrial application potential primarily originates from microorganisms. Hydantoinase is classified under cycloamidinases (EC 3.5.2), and based on its substrate, it is further divided into four categories: dihydropyrimidineases (EC 3.5.2.2); allantoinases (EC 3.5.2.5); carboxymethylhydantoinases (EC 3.5.2.4); and N-methylhydantoinases (EC 3.5.2.14). In addition, there are some unnamed hydantoinases, including amidases with known metabolic pathways, carboxyethylhydantoinases, and some hydantoinases with unclear metabolic mechanisms. Based on the specificity of their substrate or the optical activity of their products, hydantoinase can also be classified into L-type, D-type, or non-selective hydantoinases. Currently, the main industrial application of hydantoin is to produce D-type and L-type amino acids.

[0003] (S)-4-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione can be used as a synthetic intermediate for compounds with medicinal value. Currently, it is mainly prepared via chemical methods, requiring complex and cumbersome processes such as layering, filtration, drying, and fractional distillation. From an economic and environmental perspective, it is unsuitable for large-scale industrial production. Summary of the Invention

[0004] To address the technical problem of the lack of a method for preparing (S)-4-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione and its structural analogs using bio-enzymatic catalysis, this invention takes a different approach. It uses hydantoin to selectively hydrolyze one configuration (R configuration) to obtain another configuration (S configuration) of chiral monomeric compounds. Based on this, this invention provides the application of hydantoin in the hydrolysis or preparation of chiral monomeric compounds.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] The first aspect of the present invention provides the use of hydantoin or an enzyme preparation thereof in the hydrolysis of a chiral monomer compound of formula (iii) and / or in the preparation of a chiral monomer compound of formula (ii);

[0007]

[0008] Among them, R1 and R2 are each independently -H, -OH, -OTBS, -NO2, -F or -NH2.

[0009] In one embodiment of the present invention, the hydantoin is derived from Geobacillus skaustophilus.

[0010] In one embodiment of the present invention, the chiral monomer compound represented by formula (ii) is selected from one or more of the following: (S)-4-amino-2-(2,6-dioxadipin-3-yl)isoindole-1,3-dione, (S)-2-(2,6-dioxadipin-3-yl)-5-nitroisoindole-1,3-dione, (S)-5-amino-2-(2,6-dioxadipin-3-yl)isoindole-1,3-dione, and (S)-2-(2,6-dioxadipin-3-yl)-5-hydroxyisoindole-1,3-dione; their structural formulas are as follows:

[0011]

[0012] In one embodiment of the present invention, the hydantoin is selected from the group consisting of:

[0013] (1) The amino acid sequence of the hydantoin is shown in SEQ ID NO:1.

[0014] (2) The amino acid sequence of the hydantoin has at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO:1, and has the ability to hydrolyze the chiral monomer compound shown in formula (iii).

[0015] A second aspect of the present invention provides a method for hydrolyzing a chiral monomer compound of formula (iii) and / or preparing a chiral monomer compound of formula (ii), the method comprising:

[0016] The hydantoin or its enzyme preparation is contacted with and reacted with a reaction substrate; said reaction substrate is a chiral monomeric compound as shown in formula (iii), or a mixture thereof with a chiral monomeric compound as shown in formula (ii); and

[0017] Hydrolyzing the chiral monomer compound as shown in formula (iii) and optionally, preparing the chiral monomer compound as shown in formula (ii);

[0018]

[0019] Among them, R1 and R2 are each independently -H, -OH, -OTBS, -NO2, -F or -NH2.

[0020] In one embodiment of the present invention, the hydantoin is derived from *Geobacillus skaustophilus*.

[0021] In one embodiment of the present invention, the chiral monomer compound represented by formula (ii) is selected from one or more of the following:

[0022]

[0023] In one embodiment of the present invention, the hydantoin is selected from the group consisting of:

[0024] (1) The amino acid sequence of the hydantoin is shown in SEQ ID NO:1.

[0025] (2) The amino acid sequence of the hydantoin has at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO:1, and has the ability to hydrolyze the chiral monomer compound shown in formula (iii).

[0026] In one embodiment of the invention, the method employs a co-solvent to dissolve the reaction substrate. The co-solvent used should be miscible with water to further increase the solubility of the substrate. Typically, the co-solvent is an organic solvent, such as selected from dimethyl sulfoxide, alcohol solvents, toluene, or combinations thereof. The alcohol solvents include, but are not limited to, isopropanol. In a preferred embodiment of the invention, the co-solvent is dimethyl sulfoxide or isopropanol.

[0027] In one embodiment of the present invention, the reaction system contains metal ions to activate hydantoinase, wherein the metal ions are zinc ions, manganese ions or cobalt ions, preferably zinc ions.

[0028] In one specific embodiment of the present invention, the reaction system contains zinc chloride.

[0029] In one embodiment of the present invention, the reaction substrate is a chiral monomer compound represented by formula (iii) and a racemic mixture of the chiral monomer compound represented by formula (ii).

[0030] In one embodiment of the present invention, the added mass of the hydantoin is 0.5-100 times the added mass of the reaction substrate, preferably 1-4 times.

[0031] In one embodiment of the present invention, the added mass of zinc chloride is 0.01-0.5% of the added mass of the substrate, preferably 0.05-0.2%.

[0032] In one embodiment of the present invention, the reaction parameters of the method are selected from one or more of the following:

[0033] (1) The reaction is carried out in Tris-HCl buffer; preferably, the concentration of Tris-HCl buffer is 10-200 mM, more preferably 50-100 mM;

[0034] (2) The pH value of the reaction is 6 to 10, preferably 6.5 to 7.5;

[0035] (3) The reaction temperature is 10℃~50℃, preferably 20℃~45℃, more preferably 25℃~32℃;

[0036] (4) The reaction time is 0.1 to 96 hours, preferably 12 to 25 hours.

[0037] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0038] The reagents and raw materials used in this invention are all commercially available.

[0039] The positive and progressive effects of this invention are as follows:

[0040] The present invention provides a method for preparing (S)-4-amino-2-(2,6-dioxanidin-3-yl)isoindole-1,3-di Compared with traditional chemical synthesis methods, the methods for synthesizing ketones and their structural analogs have advantages such as high reaction efficiency, good stereoselectivity, mild reaction conditions, low energy consumption, and environmental friendliness, and can be used for large-scale application. Attached Figure Description

[0041] Figure 1 SFC analysis revealed the (S)-4-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione obtained from the reaction.

[0042] Figure 2 The SFC assay results showed that the reaction yielded (S)-2-(2,6-dioxadiazine-3-yl)-5-nitroisoindole-1,3-dione.

[0043] Figure 3 The SFC assay results showed that the reaction yielded (S)-5-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione.

[0044] Figure 4 The SFC assay results showed that the reaction yielded (S)-2-(2,6-dioxadiazine-3-yl)-5-hydroxyisoindole-1,3-dione. Detailed Implementation

[0045] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0046] Preparation Example

[0047] (1) Construction and transformation of recombinant vectors

[0048] Activate E. coli DH5α / pET-30a(+) strain [E. coli DH5α purchased from Novizan Biotechnology Co., Ltd.], add 3-5 μL of pET-30a-DPYS (50 ng / μL) to 50-100 μL of E. coli BL21(DE3) competent cells (purchased from Novizan Biotechnology Co., Ltd.), place on ice for 20 min, heat shock at 42℃ for 90 sec, quickly return to ice for 5 min, add 800 μL of antibiotic-free LB medium, incubate at 37℃, 200 rpm for 1 h, then spread on LB agar plates containing kanamycin resistance, and incubate overnight at 37℃ for 12 h to obtain recombinant E. coli containing pET-30a-DPYS. BL21(DE3).

[0049] The nucleotide sequence of DPYS is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:1.

[0050] The formula for antibiotic-free LB medium is as follows: yeast extract 5g / L, sodium chloride 10g / L, peptone 10g / L;

[0051] The formulation of LB agar plate medium containing cannabinoid resistance is as follows: yeast extract 5 g / L, sodium chloride 10 g / L, peptone 10 g / L, agar powder 20 g / L, cannabinoid 25 μg / mL.

[0052] (2) Construction and expression of expression carriers

[0053] The transformed recombinant E. coli BL21(DE3) containing pET30a-DPYS was directly plated onto solid LB agar plates containing 25 μg / mL kanamycin resistance and cultured at 37°C for 12-14 h to obtain single colonies. Single colonies of E. coli BL21(DE3) containing the DPYS recombinant vector were picked from the kanamycin plates and inoculated into 1 mL of liquid LB medium containing 25 μg / mL kanamycin resistance, and cultured at 37°C with shaking for 12 h. Then, at a 2% (v / v) inoculation rate, the inoculum was added to 1 L of fresh liquid LB medium containing 25 μg / mL kanamycin resistance and cultured at 37°C until OD (out of control). 600When the concentration was approximately 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L. Expression was induced at 200 rpm and 25℃ for 20 h. After centrifugation (4℃, 4000 rpm, 30 min), the supernatant was removed, and the resulting bacterial sludge was washed and resuspended in 30 ml of 0.9% NaCl solution for later use.

[0054] (3) Preparation of lyophilized hydantoin powder

[0055] The collected bacterial suspension was washed twice with 50 mmol / L Tris-HCl, and then resuspended in 50 mmol / L Tris-HCl (pH 8.0) buffer. The cells were then sonicated in an ice bath (amplitude bar 6, power 500W, on for 2 seconds, off for 5 seconds, 30 minutes). The sonicated sample was centrifuged at 12000 rpm at 4°C for 30 minutes, and the supernatant was placed in a -80°C freeze dryer for 24 hours. The resulting freeze-dried sample was then ground to prepare lyophilized hydantoin enzyme powder.

[0056] SEQ ID NO:1, Heinz amino acid sequence

[0057] MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDMPFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVIDYGFHLMISEITDDVLEELPKVIEEEGITSFKVF MAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALED GHTDPIYHALTRPPELEGEEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKI PNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0058] SEQ ID NO:2, the DNA sequence encoding hydantoin.

[0059]

[0060] Example 1

[0061] (1) Prepare a 50 mM Tris-HCl buffer solution: Weigh 6.057 g Tris into a 1 L glass bottle, stir at room temperature until the solid dissolves, and make up to 1 L with ddH2O. Adjust the pH to 7.0 with HCl / NaOH.

[0062] (2) Weigh 0.01 mg ZnCl2 and 20 mg hydantoin powder (amino acid sequence as shown in SEQ ID NO:1) and add them to a 4 mL glass tube containing 2 mL of 50 mM Tris-HCl buffer solution. Vortex and mix well.

[0063] (3) Weigh 20 mg of substrate ((R / S)-4-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione) and add it to 100 μL of dimethyl sulfoxide (DMSO), and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube, vortex and mix well.

[0064] (4) Tightly cap the glass tube and place it in a shaker at 30℃ and 1000rpm for 20 hours. Take 100μL of the reaction solution into a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex to mix, and then centrifuge at 12000rpm for 2 minutes. Take 300μL of the supernatant for SFC detection; the results are as follows. Figure 1 As shown in Table 1, the peak at t = 2.743 min is SM1, which is the target compound (S)-4-amino-2-(2,6-dioxadiazin-3-yl)isoindole-1,3-dione, and the peak at t = 3.175 min is SM2, which is the isomer (R)-4-amino-2-(2,6-dioxadiazin-3-yl)isoindole-1,3-dione with an ee value of 95.82% and a residual S / DMSO of 49.58% for the target configuration.

[0065] Table 1. Chromatographic data of (S)-4-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione

[0066] Peak Name RT Peak area area(%) Peak height 1 DMSO 0.750 3712850 84.69 1300110 2 SM1 2.743 657285 14.99 94339 3 SM2 3.175 13871 0.32 1517

[0067] Example 2

[0068] (1) Weigh 0.01 mg ZnCl2 and 25 mg hydantoin powder (amino acid sequence as shown in SEQ ID NO:1) and add them to a 4 mL glass tube containing 2 mL of 50 mM Tris-HCl buffer solution. Vortex and mix well.

[0069] (2) Weigh 20 mg of substrate ((R / S)-2-(2,6-dioxadiazine-3-yl)-5-nitroisoindole-1,3-dione) and add it to 100 μL of dimethyl sulfoxide (DMSO), and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube, vortex and mix well.

[0070] (3) Tightly cap the glass tube and place it in a shaker at 30℃ and 1000rpm for 20 hours. Take 100μL of the reaction solution into a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex to mix, and then centrifuge at 12000rpm for 2 minutes. Take 300μL of the supernatant for SFC detection; the results are as follows. Figure 2 As shown in Table 2, the peak at t = 1.402 min is SM-1, which is the target compound (S)-2-(2,6-dioxadipinidin-3-yl)-5-nitroisoindole-1,3-dione, and the peak at t = 1.647 min is SM-2, which is the isomer (R)-2-(2,6-dioxadipinidin-3-yl)-5-nitroisoindole-1,3-dione with an ee value of 85.43% and a residual S / DMSO of 20.09% for the target configuration.

[0071] Table 2. Chromatographic data of (S)-2-(2,6-dioxadiazine-3-yl)-5-nitroisoindole-1,3-dione

[0072] Peak Name RT Peak area area(%) Peak height 1 DMSO 0.778 3820858 76.12 875718 2 SM-1 1.402 1111270 22.14 233573 3 SM-2 1.647 87231 1.74 19882

[0073] Example 3

[0074] (1) Weigh 0.01 mg ZnCl2 and 25 mg hydantoin powder (amino acid sequence as shown in SEQ ID NO:1) and add them to a 4 mL glass tube containing 2 mL of 50 mM Tris-HCl buffer solution. Vortex and mix well.

[0075] (2) Weigh 20 mg of substrate ((R / S)-5-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione) and add it to 100 μL of dimethyl sulfoxide (DMSO), and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube, vortex and mix well.

[0076] (3) Tightly cap the glass tube and place it in a shaker at 30℃ and 1000rpm for 20 hours. Take 100μL of the reaction solution into a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex to mix, and then centrifuge at 12000rpm for 2 minutes. Take 300μL of the supernatant for SFC detection; the results are as follows. Figure 3As shown in Table 3, the peak at t = 3.532 min is SM1, which is the target compound (S)-5-amino-2-(2,6-dioxadiazin-3-yl)isoindole-1,3-dione, and the peak at t = 4.733 min is SM2, which is the isomer (R)-5-amino-2-(2,6-dioxadiazin-3-yl)isoindole-1,3-dione with an ee value of 84.15% and a residual S / DMSO of 26.31% for the target configuration.

[0077] Table 3. Chromatographic data of (S)-5-amino-2-(2,6-dioxadiazine-3-yl)isoindole-1,3-dione

[0078] Peak Name RT Peak area area(%) Peak height 1 DMSO 0.850 2967021 77.77 534875 2 SM1 3.532 780727 20.46 71137 3 SM2 4.733 67261 1.76 6233

[0079] Example 4

[0080] (1) Weigh 0.01 mg ZnCl2 and 100 mg hydantoin powder (amino acid sequence as shown in SEQ ID NO:1) and add them to a 4 mL glass tube containing 2 mL of 50 mM Tris-HCl buffer solution. Vortex and mix well.

[0081] (2) Weigh 20 mg of substrate ((R / S)-2-(2,6-dioxadiazine-3-yl)-5-hydroxyisoindole-1,3-dione) and add it to 100 μL of dimethyl sulfoxide (DMSO), and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube, vortex and mix well.

[0082] (3) Tightly cap the glass tube and place it in a shaker at 30℃ and 1000rpm for 20 hours. Take 100μL of the reaction solution into a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex to mix, and then centrifuge at 12000rpm for 2 minutes. Take 300μL of the supernatant for SFC detection; the results are as follows. Figure 4 As shown in Table 4, the peak at t = 1.508 min is SM-S, which is the target compound (S)-2-(2,6-dioxadiazin-3-yl)-5-hydroxyisoindole-1,3-dione, and the peak at t = 1.741 min is SM-R, which is the isomer (R)-2-(2,6-dioxadiazin-3-yl)-5-hydroxyisoindole-1,3-dione with an ee value of 92.23% and a residual S / DMSO of 27.71% for the target configuration.

[0083] Table 4. Chromatographic data of (S)-2-(2,6-dioxadiazine-3-yl)-5-hydroxyisoindole-1,3-dione

[0084] Peak Name RT Peak area area(%) Peak height 1 DMSO 0.766 489077 77.62 267413 2 SM-S 1.508 135526 21.51 30437 3 SM-R 1.741 5497 0.87 1230

[0085] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. The use of hydantoin or its enzyme preparations in the hydrolysis of the chiral monomer compound represented by formula (iii) and / or in the preparation of the chiral monomer compound represented by formula (ii); wherein R1 and R2 can each be independently -H, -OH, -OTBS, -NO2, -F or -NH2; The hydantoin is preferably derived from *Geobacillus kaustophilus*.

2. Use according to claim 1, wherein The chiral monomer compound represented by formula (ii) is selected from one or more of the following:

3. Use according to claim 1 or 2, characterized in that, The hydantoin is selected from the following group: (1) The amino acid sequence of the hydantoin is shown in SEQ ID NO:

1. (2) The amino acid sequence of the hydantoin has at least 50% sequence identity with SEQ ID NO:1 and has the properties of the chiral monomer compound shown in formula (iii).

4. A method for hydrolyzing a chiral monomer compound of formula (iii) and / or preparing a chiral monomer compound of formula (ii), characterized in that, The method includes: The hydantoin or its enzyme preparation is contacted with and reacted with a reaction substrate; said reaction substrate is a chiral monomeric compound as shown in formula (iii), or a mixture thereof with a chiral monomeric compound as shown in formula (ii); and Hydrolyzing the chiral monomer compound as shown in formula (iii) and optionally, preparing the chiral monomer compound as shown in formula (ii); Among them, R1 and R2 are each independently -H, -OH, -OTBS, -NO2, -F or -NH2; The hydantoin is preferably derived from *Geobacillus kaustophilus*.

5. The method of claim 4, wherein, The chiral monomer compound represented by formula (ii) is selected from one or more of the following:

6. The method of claim 4 or 5, wherein, The hydantoin is selected from the following group: (1) The amino acid sequence of the hydantoin is shown in SEQ ID NO:

1. (2) The amino acid sequence of the hydantoin has at least 50% sequence identity with SEQ ID NO:1 and has the properties of the chiral monomer compound shown in formula (iii).

7. The method according to any one of claims 4 to 6, wherein, The method uses a co-solvent to dissolve the reaction substrate; the co-solvent is selected from dimethyl sulfoxide, alcohol solvents, toluene or combinations thereof, preferably dimethyl sulfoxide.

8. The method according to any one of claims 4 to 7, wherein, The reaction system contains metal ions, which are zinc ions, manganese ions, or cobalt ions, preferably zinc ions; Preferably, the reaction system contains zinc chloride.

9. The method according to any one of claims 4 to 8, wherein, The reaction substrates are the chiral monomeric compound shown in formula (iii) and the racemic mixture of the chiral monomeric compound shown in formula (ii); Preferably, the mass of the hydantoin added is 0.5-100 times the mass of the reaction substrate added, more preferably 1-4 times; and / or The amount of zinc chloride added is 0.01-0.5% of the mass of the substrate, preferably 0.05-0.2%.

10. The method according to any one of claims 4 to 9, characterized in that, The reaction parameters of the method are selected from one or more of the following: (1) The reaction is carried out in Tris-HCl buffer; preferably, the concentration of Tris-HCl buffer is 10-200 mM, more preferably 50-100 mM; (2) The pH value of the reaction is 6 to 10, preferably 6.5 to 7.5; (3) The reaction temperature is 10℃~50℃, preferably 20℃~45℃, more preferably 25℃~32℃; (4) The reaction time is 0.1 to 96 hours, preferably 12 to 25 hours.

Images