Method for modifying medicine by utilizing microalgae whole-cell cluster

Abstract

The invention discloses a method for modifying drugs by using microalgae whole-cell clusters. According to the invention, an anti-tumor drug is taken as a substrate, and in the presence of microalgae whole cells, a cosolvent and a buffer solution, the drug is subjected to cluster modification through various enzyme systems in microalgae cells, so that a compound with a modified structure is obtained. Microalgae used in the method belong to photosynthetic organisms, biological catalysis conditions are mild, and compounds which cannot be obtained through chemical modification can be obtained through cluster modification. The invention provides a new technical method for drug structure modification, can enrich compound types, lays a foundation for new drug screening, is a new thought for drug development, and has a very wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis / pharmaceutical technology, specifically to a method for modifying drugs using whole-cell clusters of microalgae. Background Technology

[0002] Currently, many drugs suffer from poor water solubility, low bioavailability, or strong toxic side effects, limiting their clinical application. Therefore, structural modification of natural drug molecules to optimize their pharmacokinetic properties and uncover new biological activities has become a crucial step in drug development. While traditional chemical catalytic modification methods are highly efficient, they often face significant challenges, such as harsh reaction conditions (e.g., high temperature, high pressure, strong acids and bases), difficulty in controlling regio and stereoselectivity, and the environmental damage caused by toxic reagents and solvents. Therefore, developing green and pollution-free biocatalytic methods has become a research hotspot.

[0003] Whole-cell catalysis utilizes intact microbial cells as "miniature factories" for enzymatic reactions, employing intracellular enzymes without the need for enzyme extraction and purification. Whole-cell catalysis occurs within the cell, providing a stable supporting structure and protection for intracellular enzymes. It boasts advantages such as high cascade reaction efficiency, strong adaptability to reaction conditions, and tolerance to high substrate concentrations. Compared to enzyme-based catalysis alone, whole-cell catalysis is simpler to operate, reduces enzyme purification, and lowers reaction costs. Compared to microbial fermentation, whole-cell catalysis offers shorter production cycles, higher product purity, and higher substrate conversion rates.

[0004] Microalgae are a type of photosynthetic autotrophic organism with a rapid growth cycle. They can provide energy and reducing power for whole-cell catalysis through photosynthesis without the need for additional NADP+, NADPH, or hydrogen donors. Furthermore, microalgae possess a rich and diverse array of unique enzyme systems (cytochrome P450, monooxygenases, glycosyltransferases, methyltransferases, etc.) that can perform unique cluster modifications of drugs without any toxic side effects, making them ideal materials for whole-cell catalysis.

[0005] Despite the numerous advantages of whole-cell catalysis by microalgae, research on whole-cell cluster modification of drugs using microalgae has not yet been conducted. Current research mainly focuses on whole-cell catalysis by fungi (yeast) and bacteria (Escherichia coli). For example, a patented method for whole-cell catalysis to prepare eligliflozin intermediates (CN112941124 B) uses E. coli as a whole-cell catalytic carrier to prepare eligliflozin intermediates, and requires the addition of hydrogen donors, NADP+, and NDAPH, resulting in high costs. Another patented method for transcription factors, recombinant cells, and their application in the preparation of tumor therapeutic drugs (CN115558021 B) also uses recombinant E. coli for drug modification, and the operation is relatively complex. Currently, common drug modification methods often utilize chemical methods. The patented method for modifying calix [3]carbazole derivatives with bridged methylene modification and its synthesis method and application in drug molecule recognition (CN 116102568 B) uses the oxidant 2,3-dichloro-5,6-dicyanobenzoquinone and heating at 90-120℃ to modify drugs. The reaction conditions are harsh, and the oxidant is prone to polluting the environment. This invention utilizes whole-cell microalgae to perform cluster modification of drugs. Not only is it unnecessary to add additional hydrogen donors, NADP+ and NDAPH, reducing the cost of cluster modification, but microalgae also contain enzymes that are not found in bacteria and fungi, which helps to modify drugs in new ways and obtain new compounds. This invention provides a new technical method for whole-cell microalgae cluster modification of drugs, which has good development prospects and advantages. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned problems by providing a method for drug modification using whole-cell microalgae. This method utilizes whole-cell microalgae for drug cluster modification, optimizes catalytic conditions to obtain structurally modified compounds, and then purifies them through concentration and drying, organic solvent extraction, column chromatography, and preparative liquid chromatography. The microalgae used in this method are photosynthetic organisms, exhibiting mild biocatalytic conditions, and cluster modification can yield compounds that cannot be obtained through chemical modification. This invention provides a novel technical method for drug structure modification, enriching the types of compounds that can be obtained, laying the foundation for new drug screening, and representing a new approach to drug development with very broad application prospects.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for modifying drugs using whole-cell clusters of microalgae, the specific steps of which are as follows: Using antitumor drugs as substrates, and in the presence of a certain proportion of whole microalgal cells, a solubilizer, and culture medium, the drugs are subjected to cluster modification by various enzyme systems within the microalgal cells to obtain structurally modified compounds. The specific method is as follows: the drug is dissolved in a solubilizer and added to the microalgal solution in a certain proportion. Under specific conditions, the drug is subjected to whole-cell cluster modification of the microalgae. The microalgae are then separated from the culture medium and subjected to concentration and drying, organic solvent extraction, column chromatography, and preparative liquid chromatography purification to obtain the whole-cell biocatalytic product of the microalgae.

[0008] The invention is described below: The microalgae mentioned include, but are not limited to, *Phaeodactylum tricornutum* (… Phaeodactylum tricornutum Synechocystis ( Synechocystis sp.), Spirulina ( Spirulina sp.), Chlamydomonas laevigata (sp.), Chlamydomonas lanensis ( Chlamydomonas reinhardi Chlorella ( Chlorella sp.), Dunaliella salina ( Dunaliella salina Microalgae species such as ).

[0009] The anti-tumor drugs mentioned include, but are not limited to, gefitinib, olaparib, methotrexate, tamoxifen, etc.

[0010] The co-solvents include, but are not limited to, DMSO, 0.1% formic acid aqueous solution, and 0.1% acetic acid aqueous solution. The co-solvents account for 0-0.5% of the algal solution.

[0011] The microalgae in the microalgae solution should be in a viable division and maturation phase, and the OD value of the microalgae cell concentration should be between 0.1 and 0.8.

[0012] The concentration of the antitumor drug is 0.5 mg / L-500 mg / L.

[0013] The culture medium is suitable for different microalgae, including but not limited to seawater culture medium, BG-11 medium, SOT medium, etc.

[0014] The catalytic conditions are as follows: catalytic time of 0.5-10 days; catalytic temperature of 15-40℃; catalytic light intensity of 2000-8000 lux; and catalytic light-dark ratio of 10-14 h:10-14 h.

[0015] The organic solvents used in the organic solvent extraction include, but are not limited to, methanol, chloroform, ethyl acetate, and petroleum ether.

[0016] The column chromatography method includes, but is not limited to, silica gel columns, octadecylsilane-bonded silica gel columns, ion exchange columns, etc.

[0017] Compared with the prior art, the beneficial technical effects of this invention are as follows: 1. Microalgae can provide energy and reducing power for whole-cell catalysis through photosynthesis, without the need for additional hydrogen donors, NADP+, and NDAPH, and without the need for recombination technology, thus reducing costs and making the operation simple, fast, and universal.

[0018] 2. Microalgae possess a rich variety of unique enzyme systems (cytochrome P450, monooxygenase, glycosyltransferase, methyltransferase, etc.) that differ from those of bacteria and fungi, enabling them to perform unique cluster modifications on drugs, resulting in more diverse compound structures.

[0019] 3. The whole-cell catalytic reaction of microalgae is a mild method that does not require the addition of strong acids, strong bases, or strong oxidants, making it an environmentally friendly approach. Attached Figure Description

[0020] Figure 1 This is the structure of the main compound obtained by modifying gefitinib with whole-cell clusters of *Phaeodactylum tricornutum*. Detailed Implementation

[0021] To better understand the essence of the present invention, the following will use embodiments of the present invention to illustrate a method for modifying drugs using whole-cell clusters of microalgae, but these embodiments are not intended to limit the present invention.

[0022] Example 1: A method for modifying drugs using whole-cell clusters of microalgae The specific preparation method is as follows: Whole-cell cluster modification of *Phaeodactylum tricornutum* with gefitinib *Phaeodactylum tricornutum* was selected as the microalgal strain for whole-cell cluster modification, and gefitinib was used as the substrate. A 0.1% acetic acid aqueous solution was used as a solubilizer to dissolve gefitinib. *Phaeodactylum tricornutum* strains with good activity in the logarithmic growth phase were selected, with OD values... 730 The *Phaeodactylum tricornutum* was cultured in artificial seawater medium at a concentration of approximately 0.2 mg / L. Dissolved gefitinib was added to the *Phaeodactylum tricornutum* culture to achieve a gefitinib concentration of 4 mg / L. The catalytic temperature was 15℃; the catalytic light intensity was 2000 lux; the catalytic light-dark ratio was 12 h:12 h; and the catalytic time was 10 days. After catalysis, the *Phaeodactylum tricornutum* and its supernatant were collected by centrifugation, concentrated, and dried. Methanol was used to extract substances from the *Phaeodactylum tricornutum* and its culture medium, followed by purification by silica gel column chromatography. The catalytic efficiency of the *Phaeodactylum tricornutum* was determined to be 86.7% using liquid chromatography. High-resolution mass spectrometry was used to detect the structurally modified compounds in the *Phaeodactylum tricornutum* and its culture medium. The structurally modified compounds 1-3 are shown below. Figure 1 .

[0023] Example 2: A method for modifying drugs using whole-cell clusters of microalgae The specific preparation method is as follows: Synechocystis PCC6803 whole-cell cluster modified olapaline Synechocystis PCC6803 was selected as the microalgal strain for whole-cell cluster modification, and olaparib was used as the substrate. DMSO solution was selected as a solubilizer to dissolve olaparib. Synechocystis PCC6803 with good activity in the logarithmic growth phase was selected, and its OD... 730 The catalytic concentration was approximately 0.2 mg / L, and the cells were grown in BG-11 medium. Dissolved olaparib was added to the *Syneocarpus cytogenes* PCC6803 algal culture to achieve a concentration of 8 mg / L. The catalytic temperature was 30℃, the catalytic light intensity was 4000 lux, the light-dark ratio was 12 h:12 h, and the catalytic time was 10 days. After catalysis, the *Syneocarpus cytogenes* PCC6803 cells and the culture supernatant were collected by centrifugation, concentrated, and dried. Methanol was used to extract substances from the *Syneocarpus cytogenes* PCC6803 cells and the culture medium, followed by purification by silica gel column and reversed-phase column chromatography. Liquid chromatography showed a catalytic efficiency of 75.3% for the *Syneocarpus cytogenes* PCC6803 cells and the culture medium. High-resolution mass spectrometry was used to detect structurally modified compounds in the *Syneocarpus cytogenes* PCC6803 cells and the culture medium.

[0024] Example 3: A method for modifying drugs using whole-cell clusters of microalgae The specific preparation method is as follows: Spirulina was selected as the microalgal strain for whole-cell cluster modification, and tamoxifen was used as the substrate. No solubilizer was required; Spirulina strains with good activity in the logarithmic growth phase were chosen, as their OD... 730 The spirulina was cultured in SOT medium at a concentration of approximately 0.2 mg / L. Dissolved piracetam was added to the spirulina culture to achieve a concentration of 50 mg / L. The catalytic temperature was 30°C; the catalytic light intensity was 3500 lux; the catalytic light-dark ratio was 14 h:10 h; and the catalytic time was 10 days. After catalysis, the spirulina and culture supernatant were collected by centrifugation, concentrated, and dried. Chloroform was used to extract substances from the spirulina and culture medium, followed by silica gel column purification. Liquid chromatography determined the catalytic efficiency of the spirulina to be 88%. The structurally modified compounds in the spirulina and culture medium were then detected.

[0025] Comparative Example 1: Whole-cell cluster modification of *Phaeodactylum tricornutum* with gefitinib (1) The steps are the same as in Example 1, except that only the early stage of *Phaeodactylum tricornutum* (*Phaeodactylum tricornutum* without light exposure) is used, with an OD value of 0.05.

[0026] The experimental results show that the early growth rate of *Phaeodactylum tricornutum* is slow, the catalytic rate is low, and the conversion rate is low, at 51.8%.

[0027] Comparative Example 2: Synechocystis PCC6803 whole-cell cluster modified olapaline (1) The steps are the same as in Example 2, except that the catalytic conditions are changed to a catalytic temperature of 35°C, a catalytic light intensity of 10,000 lux, a catalytic light-dark ratio of 12 h:12 h, and a catalytic time of 10 days. Under these catalytic conditions, all Synechocystis PCC6803 cells died and could not be modified into whole-cell clusters.

[0028] Comparative Example 3: Synechocystis PCC6803 whole-cell cluster modified olapaline (1) The steps are the same as in Example 2, except that the catalytic conditions are changed to a catalytic temperature of 30°C, a catalytic light intensity of 1000 lux, a catalytic light-dark ratio of 12 h:12 h, and a catalytic time of 10 days. Under these catalytic conditions, Synechocystis PCC6803 grows slowly, has a low catalytic rate, and a poor conversion efficiency of 61.69%.

[0029] Comparative Example 4: Spirulina whole-cell cluster modification olapaline (1) The steps are the same as in Example 3, except that the catalytic conditions are changed to a catalytic temperature of 45°C, a catalytic light intensity of 5000 lux, a catalytic light-dark ratio of 12 h:12 h, and a catalytic time of 10 days. Under these catalytic conditions, all Spirulina died and whole-cell cluster modification could not be performed.

Claims

1. A method for modifying drugs using whole-cell clusters of microalgae, characterized in that: Using drugs as substrates, in the presence of whole microalgae cells, solubilizers, and culture medium, the drugs are clustered and modified by enzyme systems in the microalgae cells to obtain structurally modified compounds.

2. The method as described in claim 1, characterized in that: The specific process is as follows: The drug is dissolved in a solubilizer and added to the microalgae solution in a certain proportion. The drug is subjected to whole-cell cluster modification of microalgae under the conditions of light intensity of 2000~8000 lux and temperature of 15-40℃. The microalgae are separated from the culture medium and concentrated and dried, extracted with organic solvents, column chromatography and preparative liquid phase separation and purification to obtain whole-cell biocatalytic products of microalgae.

3. The method as described in claim 1, characterized in that: The microalgae mentioned include, but are not limited to, *Phaeodactylum tricornutum* (… Phaeodactylum tricornutum Synechocystis ( Synechocystis sp.), Spirulina ( Spirulina sp.), Chlamydomonas laevigata (sp.), Chlamydomonas lanensis ( Chlamydomonas reinhardi Chlorella ( Chlorella sp.), Dunaliella salina ( Dunaliella salina ), Microcystis thuringiensis ( Nannochloropsis salina Haematococcus pluvialis ( Haematococcus Pluvialis Microalgae species such as ).

4. The method as described in claim 1, characterized in that: The drugs mentioned include, but are not limited to, gefitinib, olaparib, methotrexate, tamoxifen, amoxicillin, cefixime, etc.

5. The method as described in claim 1, characterized in that: The co-solvents include, but are not limited to, DMSO, 0.1% formic acid aqueous solution, 0.1% acetic acid aqueous solution, etc., and the co-solvents account for 0-0.5% of the algal solution.

6. The method as described in claim 1, characterized in that: The microalgae in the microalgae solution should be in a viable division and maturation phase, and the OD value of the microalgae cell concentration should be between 0.1 and 0.

8.

7. The method as described in claim 1, characterized in that: The drug concentration is 0.5 mg / L-500 mg / L.

8. The method as described in claim 1, characterized in that: The culture medium is suitable for different microalgae, including but not limited to seawater culture medium, BG-11 medium, SOT medium, etc.

9. The method as described in claim 1, characterized in that: The modification time is 0.5-10 days; the light-to-dark ratio is 10-14 h:10-14 h.

10. The method as described in claim 1, characterized in that: The organic solvents used in the organic solvent extraction include, but are not limited to, methanol, chloroform, ethyl acetate, petroleum ether, etc.; the column chromatography includes, but is not limited to, silica gel columns, octadecylsilane-bonded silica gel columns, ion exchange columns, etc.

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