Method for efficiently preparing chiral oxo-ionol by enzyme method

Abstract

The invention belongs to the technical field of perfume synthesis, and particularly relates to a method for efficiently preparing chiral oxo-ionol by an enzymic method, which comprises the following steps: putting a substrate containing oxo-ionone and an auxiliary substrate into a buffer solution, and stirring at room temperature until complete dissolution to obtain a substrate solution; the method comprises the following steps: dispersing carbonyl reductase ChKRED20 or a mutant thereof and coenzyme into a substrate solution, and carrying out heat preservation and stirring; and after the reaction is finished, adding water to dilute, extracting with ethyl acetate, washing with water, drying, and concentrating the ethyl acetate to obtain the chiral oxo-ionol. The enzyme catalysis method disclosed by the invention is mild in reaction condition and high in selectivity, and a target product can be obtained at high yield at room temperature and normal pressure. The coenzyme is regenerated by adopting a substrate coupling method, the use amount of the coenzyme is reduced, and a substrate coupling strategy that an auxiliary substrate is used as a substrate is adopted, so that a reaction system is simple, products are easy to separate, and the cost is reduced. According to the technical scheme, the conversion rate is greater than 99%, the ee value of the product reaches 99% or above, and the industrial application value is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of fragrance synthesis, and particularly relates to a method for efficiently preparing chiral oxyionol by an enzymatic method. Background Art

[0002] Megastigmatrienoside is an important aroma compound in tobacco. A small amount of addition can improve the taste of the smoke, cover up impurities, and make the aftertaste mellow. It is an indispensable flavor for the preparation of high-end cigarettes and has important applications in the flavor and fragrance industry and the tobacco industry. As a tobacco flavor precursor, oxyionol esters release aroma components such as megastigmatrienoside during the smoking process of cigarettes, which can give cigarettes a softer quality and improve the stability and storage resistance of the flavors. Oxyionol is not only an aroma precursor in tobacco, but also a key intermediate of megastigmatrienoside and oxyionol esters. Therefore, it is of great significance to develop an efficient synthesis method of oxyionol.

[0003] In the prior art, chemical synthesis is usually used to prepare oxyionol, which is generally divided into two approaches: one is to use ionone as a raw material, and obtain the product through carbonyl reduction, esterification, allylic oxidation, and hydrolysis. The synthetic route of this method affects the total yield of the product during the hydroxyl protection and deprotection process, and the steps are cumbersome. In addition, heavy metals are used as oxidants, which are harmful to the environment; the other is to use ionone as a raw material, and obtain the product through allylic oxidation and carbonyl reduction. This method has a short route, focuses on the chemical selectivity and regioselective reduction reaction of the carbonyl group, and requires strict control of the amount of reducing agent and the reaction temperature. The product needs to be separated and purified by column chromatography, and the separation is difficult. Both approaches have the problem of complex subsequent separation and purification, and the obtained oxyionols are all racemates.

[0004] Compared with chemical synthesis, the enzyme catalysis method has mild reaction conditions and high selectivity, and can obtain the target product in high yield at room temperature and normal pressure. At the same time, scientific research shows that the aroma characteristics of chiral spices are closely related to stereochemistry, so the efficient preparation of chiral ionols by biocatalysis will help the research of chiral spices. At present, there is no literature publicly reporting the method of synthesizing chiral oxyionols by enzyme method. Summary of the invention

[0005] The object of the present invention is to provide a method for efficiently preparing chiral oxyionol by an enzymatic method, using carbonyl reductase ChKRED20 or its mutant to catalyze the synthesis of chiral oxyionol, with a yield of more than 99% and an enantioselectivity of more than 99%, thereby solving the problems in the above-mentioned background technology.

[0006] To achieve the above objectives, this application is implemented through the following technical solutions:

[0007] A method for efficiently preparing chiral oxyionol by enzymatic method, comprising the following steps:

[0008] S1. Adding the substrate and the auxiliary substrate containing oxoionone into a buffer solution, stirring at room temperature until completely dissolved, to obtain a substrate solution;

[0009] S2, dispersing carbonyl reductase ChKRED20 or its mutant and coenzyme into the substrate solution of step S1, and stirring at a temperature of 100°;

[0010] S3. After the reaction in step S2 is completed, water is added to dilute, and then extracted with ethyl acetate, washed with water, dried, and the ethyl acetate is concentrated to obtain chiral oxyionol.

[0011] Furthermore, in step S1, the concentration of oxoionone in the substrate is 10-60 g / L.

[0012] Furthermore, in step S1, preferably, the concentration of oxoionone in the substrate is 20-25 g / L.

[0013] Further, in step S1, the auxiliary substrate is an alcohol substance, which can act as a cosolvent to promote the dissolution of the substrate oxoionone in the buffer solvent, and can also act as a hydrogen donor to better reduce the substrate to the corresponding product. The alcohol substance here is such as isopropanol, ethanol and n-propanol, etc. Preferably, the hydrogen donor is isopropanol, and the amount of isopropanol used is 10-60 times the weight of the substrate, preferably 20-40 times.

[0014] Furthermore, in step S1, the amount of the auxiliary substrate is 20-40 times the weight of the substrate.

[0015] Furthermore, in step S1, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, the pH range of the buffer solution is 7.0-7.5, and the amount of the buffer solution is 10-20 times the weight of the substrate (g / L).

[0016] Furthermore, in step S2, the amount of carbonyl reductase ChKRED20 used is 0.1-0.5 wt %, preferably 0.1-0.3 wt %, based on the weight of the substrate.

[0017] Further, in step S2, the mutant of carbonyl reductase ChKRED20 is a mutant in which the amino acid residues at at least two of positions 97, 153 or 188 are replaced. For example, mutant M2 and mutant M3C1, the amino acid sequence of carbonyl reductase ChKRED20 is shown in SEQ ID NO.1, the amino acid sequence of mutant M2 of carbonyl reductase ChKRED20 is shown in SEQ ID NO.2, and the amino acid sequence of mutant M3C1 of carbonyl reductase ChKRED20 is shown in SEQ ID NO.3.

[0018] The use of carbonyl reductase ChKRED20 or its mutants can specifically select non-cyclic carbonyl groups in substrates for asymmetric catalytic reduction, achieving good chemical selectivity, regioselectivity and enantioselectivity. At the same time, the carbonyl reductase-substrate coupling method can achieve the regeneration of coenzymes, reduce the amount of coenzymes, and keep the enzyme activity at a high level throughout the reaction. The final product has high selectivity and high yield, providing a new method for enzyme-catalyzed synthesis of chiral oxoionols using oxoionone as a raw material.

[0019] Further, in step S2, the coenzyme is selected from NAD + , which can promote the transfer of electrons in the catalytic reduction process. The amount of the coenzyme used here can be adjusted according to actual conditions and is not specifically limited. Preferably, the amount of the coenzyme used is 0.002-0.004 wt% of the amount of oxoionone in the substrate.

[0020] Furthermore, in step S2, the enzyme catalysis condition is 30-40° C. for 24-72 h. Preferably, the enzyme catalysis reaction condition is 37° C. for 24 h.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention provides a new preparation method of chiral oxyionol - enzyme catalysis method.

[0023] 2. The present invention adopts a substrate coupling method to regenerate the coenzyme, which reduces the amount of coenzyme used. The substrate coupling strategy using the auxiliary substrate as the substrate has a simple reaction system, is easy to separate the product, and reduces the cost.

[0024] 3. The technical solution of the present invention completes the preparation of chiral ionol with a conversion rate of more than 99%, and the product ee value reaches more than 99%, which has industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the H NMR spectrum of 3-oxo-α-ionol.

[0026] Figure 2 This is the carbon NMR spectrum of 3-oxo-α-ionol.

[0027] Figure 3 This is the H NMR spectrum of 4-oxo-β-ionol.

[0028] Figure 4 This is the carbon NMR spectrum of 4-oxo-β-ionol.

[0029] Figure 5 This is a graph showing the HPLC test results for the racemic form of 3-oxo-α-ionol.

[0030] Figure 6 This is the HPLC detection result of 3-oxo-α-ionol chiral body.

[0031] Figure 7 This is a graph showing the HPLC test results for the 4-oxo-β-ionol racemate.

[0032] Figure 8 This is the HPLC detection result of 4-oxo-β-ionol chiral body. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below in conjunction with embodiments. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.

[0034] The present application relates to a method for efficiently preparing chiral oxyionol by enzymatic method, and the synthetic reaction formula thereof is as follows:

[0035]

[0036] Unless otherwise specified, the raw materials or chemical reagents used in the embodiments of the present invention can be obtained through commercial channels.

[0037] In the present application, the amino acid sequence of carbonyl reductase ChKRED20 is shown in SEQ ID NO.1.

[0038] Example 1

[0039] S1. Preparation of substrate solution: Add substrate (3-oxo-α-ionone, 21 mg) and isopropanol (0.6 mL) into 0.25 mL of potassium dihydrogen phosphate-potassium hydrogen phosphate buffer solution (pH=7.05), stir at room temperature until completely dissolved, and obtain substrate solution.

[0040] S2, enzyme catalysis: 50uL carbonyl reductase ChKRED20, 100uL NAD + The mixture was added into the above substrate solution, kept warm at 37°C, stirred at 100 rpm, and reacted for 24 hours.

[0041] S3. Post-treatment: After the reaction is completed, add 1 mL of water to dilute, then extract with ethyl acetate three times, 1 mL each time, combine the organic phases, wash with saturated brine, dry, and concentrate the ethyl acetate to obtain 3-oxo-α-ionol.

[0042] Example 2

[0043] The synthesis method of 3-oxo-α-ionol in this embodiment is the same as that in Example 1, except that:

[0044] The carbonyl reductase ChKRED20 in step S2 is replaced by mutant M2 (i.e., the amino acids at positions 97 and 153 are mutated simultaneously, wherein the glutamine at position 97 is mutated to leucine, and the serine at position 153 is mutated to leucine), and the amino acid sequence of the mutant M2 of the carbonyl reductase ChKRED20 is shown in SEQ ID NO.2.

[0045] Example 3

[0046] The synthesis method of 3-oxo-α-ionol in this embodiment is the same as that in Example 1, except that:

[0047] The carbonyl reductase ChKRED20 in step S2 is replaced by the mutant M3C1 (i.e., the amino acids at positions 97, 153 and 188 are mutated, the glutamine at position 97 is mutated to leucine, the serine at position 153 is mutated to leucine, and the tyrosine at position 188 is mutated to serine). The amino acid sequence of the mutant M3C1 of the carbonyl reductase ChKRED20 is shown in SEQ ID NO.3.

[0048] Example 4

[0049] The synthesis method of 4-oxo-β-ionol in this embodiment is the same as that in Example 1, except that:

[0050] The 3-oxo-α-ionone in step S1 is replaced by 4-oxo-β-ionone.

[0051] Example 5

[0052] The synthesis method of 4-oxo-β-ionol in this embodiment is the same as that in Example 4, except that:

[0053] The carbonyl reductase ChKRED20 in step S2 is replaced by mutant M2 (i.e., the amino acids at positions 97 and 153 are mutated simultaneously, wherein the glutamine at position 97 is mutated to leucine, and the serine at position 153 is mutated to leucine), and the amino acid sequence of the mutant M2 of the carbonyl reductase ChKRED20 is shown in SEQ ID NO.2.

[0054] Example 6

[0055] The synthesis method of 4-oxo-β-ionol in this embodiment is the same as that in Example 4, except that:

[0056] The carbonyl reductase ChKRED20 in step S2 is replaced by the mutant M3C1 (i.e., the amino acids at positions 97, 153 and 188 are mutated, the glutamine at position 97 is mutated to leucine, the serine at position 153 is mutated to leucine, and the tyrosine at position 188 is mutated to serine). The amino acid sequence of the mutant M3C1 of the carbonyl reductase ChKRED20 is shown in SEQ ID NO.3.

[0057] Example 7

[0058] The synthesis method of 4-oxo-β-ionol in this embodiment is the same as that in Example 6, except that:

[0059] The enzymatic reaction time in step S2 was extended to 120 hours.

[0060] Test analysis:

[0061] 1. The products obtained in Examples 1-7 were subjected to hydrogen spectrum and carbon spectrum test analysis, wherein the test results of Example 1 are detailed in Figure 1 and Figure 2 ,in 1 H NMR (400 MHz, CDCl3) δ 5.90 (s, 1H), 5.72-5.64 (m, 1H), 5.60-5.50 (m, 1H), 4.40-4.31 (m, 1H), 2.55-2.50 (m, 1H), 2.34 (d, J = 16.7Hz, 1H), 2.09 (d, J = 16.8 Hz, 2H), 1.90 (d, J = 1.1 Hz, 3H), 1.30 (d, J =2.5 Hz, 3H), 1.03 (s, 3H), 0.97 (s, 3H), 13 C NMR (101 MHz, CDCl3) δ 199.38,162.13, 138.75, 126.74, 125.99, 68.47, 55.59, 47.62, 36.29, 28.02, 27.28,23.82, 23.67. Figure 1 and Figure 2 It can be seen that the present invention successfully synthesizes the target product 3-oxo-α-ionol.

[0062] The test results of Example 4 are shown in Figure 3 and Figure 4 ,in 1H NMR (400 MHz, CDCl3) δ 6.22 (d, J= 16.2 Hz, 1H), 5.70 (dd, J = 16.2, 5.8 Hz, 1H), 4.50-4.42 (m, 1H), 2.51-2.47(m, 2H), 1.86-1.81 (m, 2H), 1.79 (s, 3H), 1.35 (d, J = 6.4 Hz, 3H), 1.15 (d,J = 1.6 Hz, 6H), 13 C NMR (101 MHz, CDCl3) δ 199.55, 160.73, 140.45, 129.91,125.12, 68.60, 37.17, 35.45, 34.27, 27.28, 27.28, 23.55, 13.39. Figure 3 and Figure 4 It can be seen that the present invention successfully synthesizes the target product 4-oxo-β-ionol.

[0063] The chiral values ​​of the products obtained in Examples 1-7 were determined by high performance liquid chromatography (HPLC). Figure 5 and Figure 6 As shown, Figure 5 This is the liquid chromatogram of the racemic 3-oxo-α-ionol standard sample. Since 3-oxo-α-ionol contains two chiral carbon atoms, there are four optical isomers, corresponding to Figure 5 There are 4 peaks in the middle. Figure 6 This is the liquid chromatogram of 3-oxo-α-ionol prepared by enzyme catalysis. Since the alcohol prepared by enzyme catalysis is a chiral alcohol, the configuration of the chiral carbon atom on the ring remains unchanged, so a pair of diastereomers is obtained, corresponding to Figure 6 There are 2 peaks in the middle. Figure 5 and Figure 6 It can be seen that the 3-oxo-α-ionol prepared in this example is a chiral compound (1:1dr, >99%ee). Figure 7 and Figure 8 As shown, Figure 7 This is the liquid chromatogram of the racemic 4-oxo-β-ionol standard sample. Figure 8 Liquid chromatogram of 4-oxo-β-ionol prepared by enzyme catalysis, combined with Figure 7 and Figure 8 It can be seen that the 4-oxo-β-ionol prepared in this embodiment is a chiral compound (>99% ee).

[0064] 2. Chirality value and yield detection

[0065] The chiral values ​​and yields of the chiral oxoionols in Examples 1-7 were detected by high performance liquid chromatography (HPLC). The chiral analysis conditions were as follows: chiral analysis column = AD-H; mobile phase = n-hexane: isopropanol (95:5, v / v); flow rate = 1 ml / min (3-oxo-α-ionol); flow rate = 0.5 ml / min (4-oxo-β-ionol); temperature = 25°C; detection wavelength = 220 nm (3-oxo-α-ionol); detection wavelength = 254 nm (4-oxo-β-ionol). The results are shown in Table 1.

[0066] Table 1 Chirality value and yield test results

[0067]

[0068] As can be seen from Table 1 , mutant M2 has substrate universality similar to that of wild-type ChKRED20 and can efficiently synthesize chiral oxoionols with high enantioselectivity.

[0069] The specific embodiments described in the present invention are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the attached claims.

Claims

1. A method for efficiently preparing chiral oxyionol by enzymatic method, characterized in that: Use the following steps: S1. Add the substrate containing oxoionone and the auxiliary substrate into a buffer solution, and stir at room temperature until they are completely dissolved to obtain a substrate solution; S2, dispersing carbonyl reductase ChKRED20 or its mutant and coenzyme into the substrate solution of step S1, and stirring at a temperature of 100°; S3. After the reaction in step S2 is completed, water is added to dilute, and then extracted with ethyl acetate, washed with water, dried, and the ethyl acetate is concentrated to obtain chiral oxyionol.

2. The method for efficiently preparing chiral oxoionol by enzymatic method according to claim 1, characterized in that: In step S1, the concentration of oxoionone in the substrate is 10-60 g / L.

3. The method for efficiently preparing chiral oxoionol by enzymatic method according to claim 2, characterized in that: In step S1, the concentration of oxoionone in the substrate is 20-25 g / L.

4. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 1, characterized in that: In step S1, the auxiliary substrate is an alcohol substance, and the amount of the auxiliary substrate is 10-60 times the weight of the substrate.

5. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 4, characterized in that: In step S1, the amount of the auxiliary substrate is 20-40 times the weight of the substrate.

6. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 1, characterized in that: In step S1, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution, the pH range of the buffer solution is 7.0-7.5, and the amount of the buffer solution is 10-20 times the weight of the substrate.

7. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 1, characterized in that: In step S2, the amount of carbonyl reductase ChKRED20 used is 0.1-0.5 wt% of the weight of the substrate.

8. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 1, characterized in that: In step S2, the mutant of carbonyl reductase ChKRED20 is a mutant in which the amino acid residues at at least two of positions 97, 153 or 188 are replaced, the amino acid sequence of carbonyl reductase ChKRED20 is shown in SEQ ID NO.1, and the amino acid sequence of the mutant of carbonyl reductase ChKRED20 is shown in SEQ ID NO.2 and / or SEQ ID NO.

3.

9. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 1, characterized in that: In step S2, the coenzyme is selected from NAD+, and the amount of the coenzyme is 0.002-0.004 wt% of the amount of oxoionone in the substrate.

10. The method for efficiently preparing chiral oxyionol by enzymatic method according to claim 1, characterized in that: In step S2, the enzyme catalysis condition is 30-40°C for 24-72h.

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