A method for synthesizing doxorubicin by using enzyme

Abstract

This invention provides a method for the enzymatic synthesis of doxorubicin, specifically a method using the cytochrome P450 enzyme DoxA and hydrogen peroxide catalysis to convert daunorubicin into doxorubicin. This invention achieves a one-step enzymatic method to replace the complex and polluting multi-step chemical resolution process in current doxorubicin synthesis, and through optimized conditions, achieves a yield of 23.5%, providing a new and simple route for the industrial synthesis of doxorubicin. The method features mild and easily controllable reaction conditions; strong enzyme catalysis specificity; no byproduct generation; and easy removal of the catalyst from the reaction system, significantly improving the process's green index and demonstrating good economic efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biological agent preparation technology, specifically relating to a method for synthesizing doxorubicin using an enzymatic method, namely a method for synthesizing doxorubicin (DXR) using an H2O2 enzymatic method. Background Technology

[0002] Doxorubicin (DXR, chemical structure see below) Figure 1 DXR is a glycoside antibiotic extracted from the fermentation broth of *Streptomyces peucetius* var. *caesius*. It is a broad-spectrum antitumor drug currently used clinically, effective against various tumors. Its mechanism of action involves inhibiting RNA and DNA replication, thereby suppressing tumor cell proliferation. Despite its cardiotoxic side effects, DXR remains one of the preferred treatment options worldwide due to its outstanding anticancer advantages.

[0003] Currently, the main industrial production method of doxorubicin is semi-chemical synthesis, which uses daunorubicin, an intermediate product obtained by microbial fermentation, as raw material. Then, it needs to go through seven chemical reactions to achieve the introduction of the hydroxyl group at the C-14 position. The yield is only 33%. The whole process is difficult, complicated, has a low yield, and causes serious environmental pollution, resulting in high production costs for doxorubicin. Summary of the Invention

[0004] This invention provides a method for synthesizing doxorubicin using an enzymatic method, specifically a method for converting daunorubicin (DNR) to doxorubicin (DXR) using the cytochrome P450 enzyme DoxA and catalyzed by hydrogen peroxide (H2O2). Figure 2 ).

[0005] This invention provides a DoxA enzyme derived from *Streptomyces coeruleorubidus*, the amino acid sequence of which is as follows (SEQ ID NO:1):

[0006] VAVDPFACPMMTMQRKPEVHDAFREAGPVVEVNAPAGGPAWVITDDALAREVLADPRFVKDPDLAPAAWRGVDDGLDIPVPELRPFTLIAVDGEAHRRLRRIHAPAFNPRRLAERTDRIAAIAGRLLTELADTSGRSGKPAELIGGFAYHFPLLVICELLGVPVTDPAMAREAVSVLKALGLGGPQSGGGDGTDPAGGVPDTSALESLLLEAVHSARRNDTPTMTRVLYERAQAEFGSVSDDQLVYMITGLIFAGHDTTGSFLGFLLAEVLAGRLAADADEDAVSRFVEEALRYHPPVPYTLWRFAATEVTIGGVRLPRGAPVLVDIEGTNTDGRHHDDPHAFHPDRPSWRRLTFGDGPHYCIGEQLAQLESRTMIGVLRSRFPEARLAVPYDELRWSRKGAQTARLTELPVWLR;

[0007] The nucleotide sequence of its coding gene is as follows (SEQ ID NO: 2):

[0008]

[0009] In another aspect, the present invention provides an use of the DoxA enzyme in the catalytic production of doxorubicin from daunorubicin;

[0010] The present invention also provides a method for synthesizing doxorubicin, which uses the aforementioned DoxA enzyme and utilizes H2O2 as the sole oxygen and electron donor to catalyze the production of doxorubicin from daunorubicin;

[0011] Furthermore, in the reaction, the concentration of H2O2 was 100mM, the concentration of DoxA was 6μM, and the concentration of daunorubicin was 100μM. The reaction was carried out at 30℃ for 30min to obtain the highest doxorubicin yield.

[0012] This invention realizes a one-step enzymatic method to replace the complex and polluting multi-step chemical resolution in the current synthesis of doxorubicin, and achieves a yield of 23.5% through optimized conditions, providing a new and simple route for the industrial synthesis of doxorubicin. The method has mild and easy-to-control reaction conditions; the enzyme catalysis is highly specific and produces no by-products; the catalyst is easy to remove from the reaction system, significantly improving the green index of the process and having good economic benefits. Attached Figure Description

[0013] Figure 1 Chemical structure diagram of doxorubicin

[0014] Figure 2 Synthetic pathway diagram of DoxA enzyme catalyzing the oxidation of daunorubicin to doxorubicin using H2O2.

[0015] Figure 3 : SDS-PAGE electrophoresis image of DoxA enzyme

[0016] Figure 4 HPLC chromatograms of doxorubicin synthesis catalyzed by DoxA enzyme using different concentrations of H2O2.

[0017] Figure 5 Bar chart showing the optimization of doxorubicin DXR production. Detailed Implementation

[0018] This invention uses daunorubicin as a raw material, P450 DoxA enzyme as a catalytic enzyme, and H2O2 as the sole oxygen and electron donor to synthesize doxorubicin with higher application value.

[0019] Example 1: DoxA protein catalyzes the conversion of daunorubicin DNR to doxorubicin DXR using H2O2.

[0020] 1) Inducible expression of DoxA protein

[0021] The *E. coli* strain BL21(DE3) expressing the DoxA protein was streaked onto LB agar plates (containing 50 μg / mL Kan) and incubated at 37°C for 24 h. Single colonies were picked and cultured overnight at 37°C and 220 rpm in 50 mL LB agar (containing 50 μg / mL Kan). A 1% inoculum was then added to 2 L Erlenmeyer flasks containing 500 mL TB or LB agar (containing 50 μg / mL Kan) and incubated at 37°C and 200 rpm until the OD600 reached 0.8–1. IPTG (200 μM), 500 μM 5-aminolevulinic acid (5-ALA), and 500 μM vitamin B1 (VB1) were added to final concentrations. The mixture was then incubated at 18°C ​​and 150 rpm for 18–20 h to induce protein expression.

[0022] (2) Purification of DoxA protein

[0023] Collect bacterial cells by centrifugation, add 30-40 mL of Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0) and vortex to resuspend the bacterial cells. After sonicating to disrupt the cells, centrifuge at 10,000 rpm at 4°C for 60 min. Incubate the supernatant with Ni-NTA at 4°C for 60 min. Then, contaminating proteins were eluted with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0); the target protein was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0), and the protein solution was concentrated using a 50 kDa ultrafiltration tube; finally, imidazole was removed using a PD-10 desalting column, with desalting buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0); SDS-PAGE detected significant amounts of soluble DoxA protein (…). Figure 3 ).

[0024] (3) DoxA utilizes H2O2 to catalyze the production of doxorubicin from daunorubicin.

[0025] Using 100 μM daunorubicin as a substrate, 2 μM DoxA and different concentrations of H2O2 (500 μM, 1 mM, 5 mM, 10 mM, 20 mM, 40 mM) were added, and the reaction was carried out at 30 °C for 2 h. The reaction was terminated by adding twice the volume of methanol to the reaction system and mixing thoroughly. After high-speed centrifugation, the supernatant was collected for high-performance liquid chromatography (HPLC) detection. The results showed that when the H2O2 concentration was 10 mM, DXR was detected, but the yield was only 5%. With increasing H2O2 concentration, the doxorubicin yield significantly increased. Figure 4).

[0026] Example 2: Optimizing the reaction system to improve DXR yield

[0027] H2O2, as a strong oxidant, can affect the activity of P450 DoxA enzyme to some extent. In order to maximize the DoxA protease activity and thus increase the yield of doxorubicin, an orthogonal experimental table was designed using the online software SPSSAU (Table 1).

[0028] Table 1: SPSSAU Orthogonal Experiment Design Table

[0029]

[0030] The optimal reaction system and reaction time were determined to be: 100 mM H2O2, 6 μM DoxA, 100 μM daunorubicin, reacted at 30℃ for 30 min, increasing the doxorubicin yield from 5% to 23.5%. Figure 5 ).

Claims

1. A method for synthesizing doxorubicin, characterized in that, The method utilizes H2O2 as the sole oxygen and electron donor and uses the DoxA enzyme with the amino acid sequence SEQ ID NO:1 to catalyze the production of doxorubicin from daunorubicin.

2. The method as described in claim 1, characterized in that, The method described herein uses a reaction system with H2O2 concentration of 100 mM, DoxA concentration of 6 μM, and daunorubicin concentration of 100 μM, and reacts at 30°C for 30 min.

3. The method as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the DoxA enzyme is SEQ ID NO:2.

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