A method for the synthesis of salicylic acid derivatives catalyzed by enzymes

Abstract

The application provides a method for synthesizing a salicylic acid derivative by using an enzyme as a catalyst, and the enzyme is a mutant of a lipase with an amino acid sequence shown in SEQ ID NO. 1, and the mutant catalyzes an esterification reaction of salicylic acid and an acid; and the mutation points of the mutant are V194A, L212A, P256A, R258A and I284A. The method of the application realizes green and efficient preparation of an ester, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of synthesizing compounds using enzymatic methods, specifically, it relates to an enzyme-catalyzed green synthesis technology, and more specifically, it relates to the use of enzymes derived from *Sphingosine monocytogenes* (…). Sphingorhabdus sp. A method for synthesizing salicylic acid ester derivatives catalyzed by lipase mutants. Background Technology

[0002] Salicylic acid possesses anti-inflammatory and keratin-regulating properties, but its lipid solubility is relatively low. Esterification with long-chain fatty acids can significantly improve its lipid solubility and sustained-release properties. Traditional chemical esterification methods suffer from problems such as high temperatures, strong acid catalysis, and environmental pollution. Lipases can catalyze esterification reactions in non-aqueous systems, exhibiting high selectivity and mild reaction conditions. Developing new sources of lipases can avoid the homogenization problem of commercial enzymes and enhance their potential for industrial applications. Summary of the Invention

[0003] This invention aims to overcome the aforementioned shortcomings and solve the problems of high temperature, high energy consumption, strong acid corrosion, numerous byproducts, and environmental pollution associated with traditional chemical esterification methods. It provides a mild, environmentally friendly, highly selective, efficient, and industrially scalable biosynthetic method for salicylic acid derivatives via an enzyme-catalyzed synthetic route. This invention utilizes screening of *Sphingosine monocytogenes* (… Sphingorhabdus sp. A lipase mutant of [a specific enzyme] was developed to catalyze the esterification reaction of salicylic acid with branched fatty acids in a non-aqueous system, thereby achieving the efficient preparation of isostearyl salicylic acid and isopalmitoyl salicylic acid.

[0004] This invention provides a novel lipase for catalyzing salicylation reactions.

[0005] This lipase originates from *Sphingosine monocytogenes* (…). Sphingorhabdus sp. The amino acid sequence of the compound is shown in SEQ ID NO.1. A mutant was obtained through molecular modification. This mutant preferentially catalyzes the esterification reaction between the phenolic hydroxyl group of salicylic acid and the carboxyl group of fatty acids in a low water activity system without affecting the carboxyl structure of the salicylic acid molecule, thus achieving highly selective conversion.

[0006] The preparation method of this lipase is as follows: the enzyme sequence was obtained from the NCBI database, the target gene was inserted into the expression vector pET-28a(+) using whole-genome synthesis, and then the recombinant plasmid was transformed into... E.coli In BL21(DE3). Cells were cultured at 37°C until OD. 600 IPTG was added at a final concentration of 0.2 mM between 0.6 and 0.8, and expression was induced at 16°C for 20 hours. Finally, the cells were collected by centrifugation and sonicated to obtain crude enzyme solution, which can be used for the synthesis of salicylic acid derivatives.

[0007] Furthermore, the present invention also provides the application of the above-mentioned lipase as an enzyme catalyst in the synthesis of salicylic acid derivatives.

[0008] In addition, the present invention provides a specific method for the biosynthesis of salicylic acid derivatives using the above-mentioned lipase. The method is as follows: salicylic acid and fatty acid are mixed in a molar ratio of 1:1 to 1:3, a solvent and a lipase mutant are added, and a molecular sieve is added to regulate the water activity. The mixture is shaken and reacted at a temperature below 60°C for 12 to 36 hours to finally obtain the product.

[0009] Branched-chain fatty acids are preferably derived from isostearic acid or isopalmitic acid, etc.

[0010] This invention further provides a liquid chromatography method for the detection of salicylic acid, isostearic acid, and isopalmitic acid. Specifically, an appropriate amount of reaction solution is centrifuged at 12000 rpm for 5 min, and the supernatant is collected. After filtration through a 0.22 μm organic phase filter membrane, liquid chromatography analysis is performed using a DAD detector and a SinoChrom ODS-BP C18 (5 μm, 4.6 × 150 mm) column. The detection conditions are: mobile phase A is isopropanol, mobile phase B is acetonitrile, and the ratio of mobile phase A to B is 60:40. The flow rate is 1 mL / min, and the column temperature is 40 °C.

[0011] The effects and benefits of this invention are: (1) High selectivity: This invention uses a novel lipase mutant for catalysis, which avoids the side reactions that may occur under strong acid conditions, resulting in high selectivity.

[0012] (2) Mild reaction conditions: The reaction temperature is below 60℃ and no high pressure is required.

[0013] (3) Green and environmentally friendly: No need to use corrosive catalysts such as concentrated sulfuric acid.

[0014] (4) Great industrialization potential: the substrate concentration can reach more than 100 mM and the product yield is more than 85%. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the materials and reagents used in the following examples are all available through conventional commercial channels.

[0017] Example 1. Preparation of lipase The specific steps are as follows: (1) Gene synthesis and cloning: The whole gene from the enzyme sequence is synthesized, and plasmid pET-28a(+) is selected as the expression vector. After obtaining the recombinant plasmid, it is transformed into... E.coli Recombinant strains were obtained from BL21(DE3) competent cells.

[0018] (2) Induction and expression of the target protein: After successful transformation, a single colony was picked from the plate and inoculated into 4 mL of LB liquid medium containing 50 μg / mL Kan. The culture was incubated at 37℃ and 200 rpm for 12 h. Then, 1 mL of the bacterial culture was inoculated into a shake flask containing 100 mL of LB liquid medium (containing 50 μg / mL Kan) at a 1% inoculation rate. The culture was incubated at 37℃ and 200 rpm for 3 h until OD was reached. 600 When the bacterial count reaches 0.6-0.8, IPTG with a final concentration of 0.2 mM is added for induction, and the shake flask is transferred to a shaker at 16℃ and 200 rpm for 20 h. After 20 h of induction, the fermentation broth is collected and centrifuged at 10,000 rpm for 5 min in a floor centrifuge at 4℃ to collect the bacterial cells.

[0019] (3) Cell disruption and enzyme extraction: Cells were disrupted by ultrasonic disruption, and the supernatant was collected by centrifugation to obtain a crude extract containing enzymes. The crude enzyme activity was found to be above 800 U / mg.

[0020] Lipase amino acid sequence (SEQ ID No. 1) MTETTPFVRPDVALFLQFLNAVPGPKFWEVSADEARAMTSAMRDVADAPVGELAVIRDINIPGPAGTIPARIYDSRADRGAGPVMVFYHGGGFVIGSLYSYEPYCAEVARLLDLPVISIDYRLSPEHPFPAPAEDCEAATRWIASSPTELGLTVTGLITS GDSAGGNLTIVTSMALRDKPAAVPVLVQFPIYPVVTLNSDWPSMRDYANGYLLTEAMTYFGEGHAAVPGDYRSEPLNFPQEGMPPSLVTTASLDPLRDQGLAYVEKLKQADVRVQHVSADGNIHGHINIRQAIPSVQTDIEGYIRALKIILAEVTPAA.

[0021] Example 2. Synthesis of salicylic acid derivatives Using salicylic acid and an acid as substrates, and employing lipase as a catalyst, an esterification reaction is carried out in an organic solvent system at a temperature below 60°C for 12-36 hours to generate salicylic acid derivatives. The reaction system is an anhydrous organic solvent system, preferably alkanes, alcohols, and esters, such as n-hexane, isooctane, tert-butanol, or ethyl acetate. The acid can be any organic acid, such as fatty acids, olefinic acids (e.g., acrylic acid, cinnamic acid), aromatic acids, and heterocyclic acids (e.g., furanyl acid, thiophene acid).

[0022] The preferred reaction temperature is 40℃-50℃.

[0023] The molar ratio of salicylic acid to fatty acids is 1:1 to 1:3.

[0024] In this reaction system, a surfactant is preferably added, with a mass percentage of 0.1-5%, and can be selected from one of tetrabutylammonium bromide, tetrabutylammonium iodide, Tween 20, Tween 60 or Tween 80, preferably a nonionic surfactant.

[0025] In this reaction system, it is also preferable to add water-removing materials, such as 4Å molecular sieves, to remove the water generated in the reaction.

[0026] The following esterification reaction was used as an example to conduct the following condition screening experiment: Example 2.1. Synthesis of isostearyl salicylic acid 1.38 g (10 mmol) of salicylic acid and 5.68 g (20 mmol) of isostearic acid were weighed and placed in a 100 mL three-necked round-bottom flask. 50 mL of anhydrous n-hexane was added as the reaction solvent, followed by 5 g of 4 Å molecular sieve (pre-dried at 120 °C for 12 h). Finally, 1 mL of the crude enzyme solution from Example 1 was added. The reaction was carried out at 50 °C for 24 h. HPLC analysis showed that the product yield was 15% (purity 95%).

[0027] Example 2.1.1. Screening of lipases Since the catalytic efficiency of the lipase shown in SEQ ID NO.1 is obviously insufficient, molecular modification of the lipase was attempted in this group of experiments. Mutants N167A, Y192A, P193A, V194A, L212A, P256A, R258A and I284A were constructed using classical site-directed mutagenesis. Alternatively, mutations can be performed using methods similar to those in Biocatalytic Heteroaromatic Amide Formation in Water Enabled by a Catalytic Tetrad and Two Access Tunnels, Erna Zukic, etc. ACS Catal. 2024, 14, 8913−8921.

[0028] When the enzyme in Example 2.1 was replaced with the mutant described above, the yields were 17% (95% purity), 26% (95% purity), 34% (95% purity), 55% (96% purity), 48% (97% purity), 63% (98% purity), 43% (97% purity) and 51% (97% purity).

[0029] The results show that mutants V194A, L212A, P256A, R258A, and I284A all doubled the yield compared to the conventional mutants, indicating a significant improvement in catalytic efficiency. Among them, P256A is the optimal mutant and will be the subject of further research.

[0030] Example 2.1.2. Screening of Surfactants Our study found that surfactants may influence enzyme activity and stability by regulating the enzyme's microenvironment, thereby improving conversion rates. In this experiment, tetrabutylammonium bromide, tetrabutylammonium iodide, Tween 20, Tween 60, and Tween 80 were selected for the study, with an addition amount of 1%. After adding surfactants, the product yields were 53% (97% purity), 56% (97% purity), 78% (98% purity), 81% (98% purity), and 86% (98% purity), respectively, using the mutant P256A as a catalyst. Increasing the amount of Tween 80 to 3% did not change the product yield or purity.

[0031] The results above show that Tween 80 is the optimal surfactant. Therefore, in subsequent studies, 1% Tween 80 was added to carry out the reaction.

[0032] Example 2.1.3. Screening of reaction temperature In this group of reactions, the reaction temperature was screened, and the reaction was carried out at 30°C, 40°C, 50°C, and 60°C. The conversion rate was monitored at each temperature. The product yields at the four temperatures were 38% (96% purity), 69% (98% purity), 86% (98% purity), and 52% (97% purity), respectively. The results showed that the product yield was highest at 50°C, reaching 86%, and 50°C was subsequently selected as the reaction temperature for isostearyl salicylic acid.

[0033] Example 2.1.4. Screening of substrate molar ratio and enzyme based on isostearyl salicylic acid This group screened the molar ratio of salicylic acid to isostearic acid, setting it to 1:1, 1:1.5, 1:2, and 1:3. The reactions were carried out at 50℃, and the product yields were 68% (98% purity), 85% (98% purity), 76% (98% purity), and 77% (98% purity), respectively. The results show that the highest product yield was achieved when the molar ratio of salicylic acid to isostearic acid was 1:1.5; therefore, a molar ratio of 1:1.5 was selected for further investigation.

[0034] In this group of experiments, the eight mutants in Example 2.1.1 were used for the reaction. The product yields of N167A, Y192A, P193A, V194A, L212A, P256A, R258A and I284A were 18% (purity 89%), 23% (purity 90%), 31% (purity 90%), 36% (purity 89%), 52% (purity 95%), 66% (purity 98%), 45% (purity 97%) and 58% (purity 98%), respectively. Among them, mutant P256A showed the best results and was selected as the subject of further research.

[0035] The optimal reaction conditions were thus obtained as follows: 1.38 g (10 mmol) of salicylic acid and the corresponding fatty acid or aromatic acid (15 mmol) were weighed into a 100 mL three-necked round-bottom flask, 50 mL of anhydrous n-hexane was added as the reaction solvent, 0.5 g of Tween 80 was added, followed by 5 g of 4Å molecular sieve (pre-dried at 120 °C for 12 h), and finally 1 mL of crude enzyme solution was added. The reaction was carried out at 50 °C for 24 h, and the yield and purity of the product were obtained by HPLC detection or column chromatography purification.

[0036] The product data is as follows: Yield 92%, purity 95% 1 H NMR (400 MHz, DMSO- d 6) δ 12.41 (s, 1H), 7.99 (dd, J = 7.4, 1.6 Hz, 1H), 7.64 (td, J = 7.3, 1.5 Hz, 1H), 7.37 (td, J = 7.6, 1.6 Hz, 1H), 7.21(dd, J = 7.6, 1.7 Hz, 1H), 2.51 (q, J = 7.1 Hz, 1H), 2.21 (hept, J= 6.8 Hz,1H), 1.81 – 1.51 (m, 4H), 1.55 – 1.47 (m, 1H), 1.43 – 1.31 (m, 3H), 1.26 (dd, J = 12.3, 7.0 Hz, 1H), 0.97 (d, J = 11.2 Hz, 19H), 0.97 – 0.88 (m, 4H).

[0037] 13 C NMR (100 MHz, DMSO- d 6) δ 173.32, 168.70, 151.14, 133.42, 131.13,125.02, 120.68, 118.64, 50.56, 49.68, 47.17, 35.12, 33.20, 32.27, 31.64,30.35, 29.48, 29.41, 25.78, 20.94, 18.59.

[0038] M / Z=404.28.

[0039] The product data is as follows: Yield 89%, purity 98%.

[0040] 1 H NMR (400 MHz, DMSO- d 6) δ 12.49 (s, 1H), 7.97 (dd, J = 7.4, 1.6 Hz, 1H), 7.64 (td, J = 7.4, 1.5 Hz, 1H), 7.37 (td, J = 7.6, 1.6 Hz, 1H), 7.21(dd, J = 7.4, 1.6 Hz, 1H), 2.45 (p, J = 7.0 Hz, 1H), 1.68 – 1.49 (m, 3H), 1.52 – 1.31 (m, 3H), 1.35 – 1.18 (m, 8H), 0.95 – 0.83 (m, 4H).

[0041] 13 C NMR (100 MHz, DMSO- d6) δ 172.94, 168.70, 151.23, 133.39, 130.99,125.00, 120.68, 119.46, 45.27, 31.93, 31.70, 31.13, 31.11, 29.19, 29.09, 28.24, 27.31, 26.97, 22.66, 22.58, 14.07.

[0042] M / Z=376.27.

[0043] Example 2.2 Salicylic acid and benzoic acid (too much steric hindrance, no reaction) No product was obtained from salicylic acid and benzoic acid under optimal reaction conditions.

[0044] Example 2.3 Salicylic acid and tertivalic acid (too much steric hindrance, no reaction) No product was obtained from salicylic acid and terpentine under optimal reaction conditions.

[0045] Example 2.4 Salicylic acid and trans-cinnamic acid Salicylic acid and trans-cinnamic acid react under optimal reaction conditions to yield the product. CAS: 1338444-04-4, yield 50%, purity 90%.

[0046] Example 2.5 Salicylic acid and phenylacetic acid Salicylic acid and trans-cinnamic acid react under optimal reaction conditions to yield the product. CAS: 643747-30-2, yield 72%, purity 95%.

[0047] Example 2.6 Salicylic acid and phenylpropionic acid Salicylic acid and phenylpropionic acid react under optimal reaction conditions to yield the product. CAS: 1554285-83-4, Yield 82%, Purity 96%.

[0048] Example 2.7 Salicylic acid and isobutyric acid Salicylic acid and isobutyric acid react under optimal reaction conditions to yield the product. CAS: 77836-44-3, yield 25%, purity 91%.

[0049] Example 2.8 Salicylic acid and propionic acid Salicylic acid and propionic acid react under optimal reaction conditions to yield the product. CAS: 6328-44-5, yield 88%, purity 96%.

[0050] Example 2.9 Salicylic acid and butyric acid Salicylic acid and butyric acid react under optimal reaction conditions to yield the product. CAS: 71974-02-2, yield 86%, purity 96%.

[0051] Example 2.10 Salicylic acid and furan acid (some aromatic acids can also react) Salicylic acid and furan acid react under optimal reaction conditions to yield the product. CAS: 363590-43-6, yield 32%, purity 88%.

[0052] Example 2.11 Salicylic acid and 2-thiophenecarboxylic acid Salicylic acid and 2-thiophenecarboxylic acid react under optimal reaction conditions to yield the product. CAS: 95232-68-1, Yield 22%, Purity 87%.

Claims

1. A method for synthesizing an enzyme-catalyzed salicylic acid derivative, characterized in that: A mutant lipase with an amino acid sequence as shown in SEQ ID NO.1 was used as an enzyme catalyst to catalyze the esterification reaction of salicylic acid and acid. The mutation sites of the mutant are V194A, L212A, P256A, R258A, and I284A.

2. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: The branched fatty acid is isostearic acid or isopalmitic acid.

3. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: The reaction is carried out under solvent conditions; The solvent is selected from anhydrous organic solvents.

4. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: Surfactants are also added to the reaction system; The amount of surfactant added is 1-5% of the total weight.

5. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: The molar ratio of salicylic acid to fatty acid is 1:1 to 1:

3.

6. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: The reaction temperature is below 60℃.

7. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: The reaction time is 12 to 36 hours.

8. The method for synthesizing an enzyme-catalyzed salicylic acid derivative as described in claim 1, characterized in that: A dehydrating material was also added to the reaction system.

9. A lipase, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

10. The application of the lipase as described in claim 9 in catalyzing esterification reactions.