Synthesis method of florfenicol and derivative thereof

Abstract

The invention discloses a brand-new synthesis method of florfenicol and a derivative thereof. According to the method, an enzyme chemical grade combined synthesis route is adopted, three steps are needed in total, and florfenicol and the derivative thereof are directly synthesized from p-methylsulfonyl benzaldehyde, so that the cost is reduced, the process is simplified, and industrial amplification is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of chemical synthesis, and particularly relates to a method for synthesizing florfenicol. Background Art

[0002] Florfenicol is a widely used new generation of chloramphenicol veterinary antibiotics, which has the advantages of rapid absorption, wide distribution in the body, long half-life, no side effects of aplastic anemia, not easy to develop drug resistance, no residue, no cross-resistance, etc. It is mainly used to treat bacterial diseases and mycoplasma infections in cattle, pigs, chickens, ducks, fish and other animals. In recent years, the global demand for florfenicol has increased, and its current market size has exceeded 3 billion yuan.

[0003] The structural formula of florfenicol is shown below:

[0004]

[0005] At present, the most mature process routes for synthesizing florfenicol in China adopt the p-methylsulfonylbenzaldehyde route (Tobibiki Hisao; Ger Offen, DE2349496), which uses chloramphenicol, thiamphenicol and other raw materials or their related intermediates as starting materials, and is prepared by reduction, protection, fluorination, hydrolysis and ring opening, and acylation. It involves the reaction of p-methylsulfonylbenzaldehyde with glycine and copper sulfate to prepare copper salt, and then esterification to obtain racemic D and L-type serine ethyl ester, which is obtained by tartaric acid separation, and one of the isomers, the L-type, is discarded, which increases the cost of the entire route, and a large amount of copper sulfate wastewater is generated when preparing copper salt, and the wastewater treatment cost is very high, so that the enterprise is temporarily closed due to production costs and environmental pressure.

[0006] With the development of asymmetric chemistry, various chiral catalysts have been applied to the synthesis of florfenicol. Jon E. Clark et al. in the United States used enzyme catalysis to study this type of reaction. Later, Feng Li et al. used an asymmetric synthesis method to synthesize the intermediate oxazoline, which was then hydrolyzed and dichloroacetylated to obtain the product. This process route has the disadvantages of low yield and long process, and is difficult to apply to industrial production (Tetrahedron: Asymmetry 22 (2011) 1337-1341).

[0007] At present, the production process routes of florfenicol of various domestic manufacturers are relatively consistent. They all use (2S,3R)-p-methylsulfonylphenylserine ethyl ester (D-ethyl ester) as the raw material, and prepare oxazoline by reduction, reaction with benzonitrile, fluorination under the action of Ishikawa reagent, hydrolysis and dichloroacetylation to obtain florfenicol raw materials.

[0008]

[0009] An equivalent amount of Ishikawa reagent is required in the fluorination reaction step. This type of reagent has a relatively low fluorine atom utilization rate and a relatively high cost. Due to the stability of the Ishikawa reagent, factories usually adopt a make-as-you-go method. The production process of this reagent requires ultra-low temperature, high pressure, high temperature and other conditions, which increases energy consumption and safety risks. In addition, a large amount of N,N-diethyl-2,3,3,3-tetrafluoropropionamide will be formed after the reaction, which is difficult to recover and reuse, and has great pressure on environmental protection and production costs.

[0010] At present, D-ethyl ester is a key intermediate in the production of florfenicol, and its use cost is relatively high. The main methods for large-scale production of D-ethyl ester at home and abroad are chemical synthesis and bio-enzyme catalysis. The chemical synthesis method uses p-methylsulfonylbenzaldehyde and glycine as raw materials to obtain (2S, 3R)-p-methylsulfonylphenylserine through condensation, esterification and splitting. There are two main routes for the preparation of (2S, 3R)-p-methylsulfonylphenylserine by bio-enzyme catalysis. One route is to use L-threonine aldolase to catalyze the condensation reaction of p-methylsulfonylbenzaldehyde and glycine. The other route is to use p-methylsulfonylbenzaldehyde and L-threonine as raw materials to synthesize (2S, 3R)-p-methylsulfonylphenylserine through whole cell or bio-enzyme catalysis.

[0011] In summary, the existing florfenicol production route has high production costs and produces a large amount of wastewater that is difficult to treat. Therefore, it is very valuable to find an industrial production route with high yield and less other waste. In the prior art, the method for preparing florfenicol often has insufficient overall yield, or although the yield is high, the operation process is too cumbersome, which is destined to be unable to be used for large-scale production.

[0012] With the regulation of food safety in recent years, the use of florfenicol in the international animal health market has continued to expand. Therefore, developing a new synthesis process for florfenicol to reduce production costs has very important economic and social significance for environmental friendliness and green safety production. Summary of the invention

[0013] In view of the shortcomings of the prior art, the present invention provides a new method for synthesizing florfenicol and its derivatives, which adopts an enzyme chemical cascade synthesis route and requires a total of three steps to directly synthesize florfenicol and its derivatives from p-methylsulfonylbenzaldehyde, thereby reducing costs, simplifying the process, and facilitating industrial scale-up.

[0014] The structural formula of florfenicol and its derivatives described in the present invention is shown in Formula I:

[0015]

[0016] In formula I, X is selected from CH 3 , OH, F, Cl, Br or I.

[0017] When X is F, the compound represented by formula I is florfenicol.

[0018] The technical solution adopted by the present invention is as follows:

[0019] A method for synthesizing florfenicol and its derivatives comprises the following steps:

[0020] 1) reacting compound 1 (p-methylsulfonylbenzaldehyde) and compound 2 under the action of pyruvate decarboxylase or whole Escherichia coli cells expressing pyruvate decarboxylase to obtain compound 3;

[0021]

[0022] Wherein, the definition of X in compound 2 is the same as that of formula I; the definition of X in compound 3 is the same as that of formula I;

[0023] 2) reacting compound 3 with an amine substance under the action of aminotransferase or whole Escherichia coli cells expressing aminotransferase to obtain compound 4;

[0024]

[0025] Wherein, the definition of X in compound 4 is the same as that of formula I;

[0026] 3) subjecting compound 4 to an amidation reaction with methyl dichloroacetate to obtain a compound of formula I.

[0027] In step 1) of the above method, the pyruvate decarboxylase is pyruvate decarboxylase E1; and the whole Escherichia coli cells expressing pyruvate decarboxylase are whole Escherichia coli cells E1.

[0028] Furthermore, the pyruvate decarboxylase is from Bacillus megateriu.

[0029] In an embodiment of the present invention, the amino acid sequence of the pyruvate decarboxylase E1 is SEQ ID NO:1.

[0030] Furthermore, the pyruvate decarboxylase is added in the form of a crude enzyme solution, and the dosage ratio of the compound 1, the compound 2 and the pyruvate decarboxylase crude enzyme solution is 10 mmol: (40-60) mmol: (60-90) mL; specifically, 10 mmol: 50 mmol: 80 mL.

[0031] Furthermore, the crude pyruvate decarboxylase enzyme solution is prepared according to the following steps: after inducing the recombinant bacteria expressing pyruvate decarboxylase with an OD value of 12-16 with isopropyl-β-D-thiogalactopyranoside (IPTG) for 18-22 hours, the bacteria are collected, and then the PBS buffer is added to the bacteria at a ratio of 1 g: 8-12 mL of the PBS buffer, and the bacteria are crushed, and the supernatant of the crushed product is collected to obtain a crude enzyme solution;

[0032] The recombinant bacteria expressing pyruvate decarboxylase are recombinant bacteria obtained by introducing the coding gene of pyruvate decarboxylase into Escherichia coli.

[0033] According to one embodiment of the present invention, a crude pyruvate decarboxylase enzyme solution is prepared according to the following steps: a recombinant bacterium expressing pyruvate decarboxylase with an OD value of 15 is induced by IPTG for 20 hours, and then the cells are collected, and then the buffer is added to the cells in an amount of 1 g:10 mL of the buffer solution, and the cells are crushed, and the supernatant of the crushed product is collected to obtain a crude pyruvate decarboxylase E1 enzyme solution;

[0034] The recombinant bacteria expressing pyruvate decarboxylase are recombinant bacteria obtained by introducing the coding gene of pyruvate decarboxylase into Escherichia coli via plasmid pET28a-E1;

[0035] The temperature of IPTG induction is 30°C;

[0036] The concentration of IPTG induction is 0.5 mM;

[0037] The collected bacteria were centrifuged at 6000 rpm for 15 min to obtain a precipitate;

[0038] The buffer was 50 mM PBS buffer at pH 8.0.

[0039] The crushing is high-pressure homogenization crushing.

[0040] The supernatant of the crushed product is collected by centrifuging the crushed product at 13000 rpm for 60 min, taking the supernatant, and filtering it through a 0.45 μm membrane to obtain a crude enzyme solution.

[0041] Furthermore, in the step 1), the dosage ratio of the compound 1, the compound 2 and the whole Escherichia coli cells expressing pyruvate decarboxylase is 10 mmol: (40-60) mmol: 20-35 g;

[0042] The whole Escherichia coli cells expressing pyruvate decarboxylase are the cells collected after the recombinant bacteria expressing pyruvate decarboxylase are induced by IPTG for 18-22 hours.

[0043] According to one embodiment of the present invention, the whole Escherichia coli cells expressing pyruvate decarboxylase are prepared according to the following steps: the recombinant bacteria expressing pyruvate decarboxylase with an OD value of 15 are induced by IPTG for 20 hours and then the bacteria are collected to obtain whole Escherichia coli E1 cells expressing pyruvate decarboxylase.

[0044] The temperature for IPTG induction was 30°C.

[0045] The concentration of IPTG induction was 0.5 mM.

[0046] The collected bacteria were centrifuged at 6000 rpm for 15 min to obtain a precipitate.

[0047] In step 1) of the above method, during the reaction, MgCl 2 , ThDP (thiamine pyrophosphate), FAD (flavin adenine dinucleotide), DMSO.

[0048] According to an embodiment of the present invention, compound 1, MgCl 2 , ThDP (thiamine pyrophosphate), FAD (flavin adenine dinucleotide), and DMSO were fed in a ratio of 10 mmol: 5 mmol: 0.1 mmol: 0.05 mmol: 125 mL, respectively.

[0049] In step 1) of the above method, the reaction is carried out in a HEPES buffer, which can be a 100 mM HEPES buffer with a pH of 7.5.

[0050] In step 1) of the above method, the reaction conditions are: 25-35° C., stirring reaction for 18-22 hours, and the stirring speed can be 220-260 rpm.

[0051] After the reaction of step 1) is completed, the obtained compound 3 can be directly subjected to the reaction of step 2) in the same reactor without separation and purification. That is, the reaction of step 1) and step 2) is prepared by a one-pot method.

[0052] In step 2) of the above method, the aminotransferase is aminotransferase E2; and the whole Escherichia coli cells expressing the aminotransferase are whole Escherichia coli cells E2.

[0053] Furthermore, the aminotransferase is derived from Paludibacterium sp.

[0054] In an embodiment of the present invention, the amino acid sequence of the aminotransferase E2 is SEQ ID NO:2.

[0055] Furthermore, the aminotransferase is added in the form of a crude enzyme solution, and the dosage ratio of the compound 3, the amine substance and the aminotransferase crude enzyme solution is 10mmol: (100-200)mmol: (60-90)mL; specifically, 10mmol: 100mmol: 50mL.

[0056] Furthermore, the aminotransferase crude enzyme solution is prepared according to the following steps: after inducing the recombinant bacteria expressing aminotransferase with an OD value of 12-16 with isopropyl-β-D-thiogalactopyranoside (IPTG) for 18-22 hours, the bacteria are collected, and then the PBS buffer is added to the bacteria at a ratio of 1 g: 8-12 mL of the bacteria: PBS buffer, and the bacteria are crushed, and the supernatant of the crushed product is collected to obtain a crude enzyme solution;

[0057] The recombinant bacteria expressing the aminotransferase are recombinant bacteria obtained by introducing the aminotransferase encoding gene into Escherichia coli.

[0058] According to one embodiment of the present invention, the aminotransferase crude enzyme solution is prepared according to the following steps: after the recombinant bacteria expressing aminotransferase with an OD value of 15 are induced by IPTG for 20 hours, the bacteria are collected, and then the buffer is added to the bacteria at a ratio of 1 g: 10 mL of the bacteria: buffer, and the bacteria are crushed, and the supernatant of the crushed product is collected to obtain the aminotransferase E2 crude enzyme solution;

[0059] The recombinant bacteria expressing the aminotransferase are obtained by introducing the aminotransferase coding gene into Escherichia coli via plasmid pET28a-E2;

[0060] The temperature of IPTG induction is 30°C;

[0061] The concentration of IPTG induction is 0.5 mM;

[0062] The collected bacteria were centrifuged at 6000 rpm for 15 min to obtain a precipitate;

[0063] The buffer was 50 mM PBS buffer at pH 8.0.

[0064] The crushing is high-pressure homogenization crushing.

[0065] The supernatant of the crushed product is collected by centrifuging the crushed product at 13000 rpm for 60 min, taking the supernatant, and filtering it through a 0.4 μm membrane to obtain a crude enzyme solution.

[0066] Furthermore, in the step 2), the dosage ratio of the compound 3, the amine substance and the whole Escherichia coli cells expressing the aminotransferase is 10 mmol: (100-200) mmol: 15-25 g;

[0067] The whole Escherichia coli cells expressing aminotransferase are the cells collected after the recombinant bacteria expressing aminotransferase are induced by IPTG for 18-22 hours.

[0068] According to one embodiment of the present invention, the whole Escherichia coli cells expressing aminotransferase are prepared according to the following steps: the recombinant bacteria expressing aminotransferase with an OD value of 15 are induced by IPTG for 20 hours and then the bacteria are collected to obtain whole Escherichia coli E2 cells expressing aminotransferase.

[0069] The temperature for IPTG induction was 30°C.

[0070] The concentration of IPTG induction was 0.5 mM.

[0071] The collected bacteria were centrifuged at 6000 rpm for 15 min to obtain a precipitate.

[0072] In step 2) of the above method, the amine substance as the amino donor is selected from at least one of the following: isopropylamine, α-methylpropylamine, 1-methyl-3-phenylpropylamine, 1-aminoindane, benzylamine, 2-heptylamine, 1,5-dimethylhexylamine, 2-aminobutane, aminobutyric acid, L-alanine, etc.

[0073] In step 2) of the above method, pyridoxal phosphate (PLP) is also added as a coenzyme during the reaction.

[0074] The dosage ratio of the p-methylsulfonylbenzaldehyde to pyridoxal phosphate (PLP) is 10mmol:0.1-0.3mmol.

[0075] In step 2) of the above method, the reaction conditions are: 25-35° C., stirring reaction for 20-35 hours, and the stirring speed can be 150-250 rpm.

[0076] In step 3) of the above method, the initial molar ratio of compound 4 to methyl dichloroacetate is 1:(3-6), specifically 1:5.

[0077] In step 3) of the above method, the amidation reaction is carried out in the presence of a base, and the base can be selected from triethylamine (TEA) or diisopropylethylamine.

[0078] Further, the molar ratio of the compound 4 to the base is 1:(1-1.5), specifically 1:1

[0079] In step 3) of the above method, the amidation reaction is carried out in a solvent, and the solvent can be selected from methanol or THF.

[0080] In step 3) of the above method, the reaction conditions of the amidation reaction are: stirring the reaction at room temperature (25-33° C.) for 22-26 hours, and the specific reaction conditions may be: stirring the reaction at room temperature for 24 hours.

[0081] Compared with the prior art, the present invention has the following beneficial effects:

[0082] The present invention provides a novel enzymatic chemistry cascade synthesis route for synthesizing florfenicol and its derivatives, and the entire route only requires three steps. Compared with the currently used route of synthesizing florfenicol and its derivatives from D-ethyl ester, the production cost of florfenicol is significantly reduced, the synthesis process is simplified, and it also makes it more in line with the needs of today's green industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The present invention provides a route map for producing florfenicol and its derivatives using p-phenylsulfone benzaldehyde as a raw material. DETAILED DESCRIPTION

[0084] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0085] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0086] Terms and Definitions

[0087] In the present invention, amino acids are represented by single-letter or three-letter codes and have the following meanings: A: Ala (alanine); I: Ile (isoleucine); G: Gly (glycine); N: Asn (asparagine); W: Trp (tryptophan); L: Leu (leucine); T: Thr (threonine); K: Lys (lysine); R: Arg (arginine); H: His (histidine); F: Phe (phenylalanine); V: Val (valine); S: Ser (serine); Q: Gln (glutamine) Example 1, strain construction and expression.

[0088] Step 1: Construction of recombinant bacteria

[0089] In the present invention, pyruvate decarboxylase E1 is derived from Bacillus megateriu (SEQ ID NO: 1), and aminotransferase E2 is derived from Paludibacterium sp. (SEQ ID NO: 2). Both genes are obtained by gene synthesis, and each gene is connected to a vector pET28a to obtain plasmid pET28a-E1 and plasmid pET28a-E2.

[0090] The above plasmids pET28a-E1 and pET28a-E2 were transformed into Escherichia coli BL21 (DE3) strain, cultured on plates, and finally single clones were picked to obtain recombinant bacteria BL21 / pET28a-E1 and recombinant bacteria BL21 / pET28a-E2.

[0091] Step 2: Preparation of whole cells

[0092] The above recombinant bacteria were cultured in liquid step by step as follows:

[0093] Seed solution: Respectively transfer the recombinant bacteria BL21 / pET28a-E1 and the recombinant bacteria BL21 / pET28a-E2 monoclonal into 3 mL of LB culture medium containing 50 μM kanamycin (37°C) and culture overnight to obtain BL21 / pET28a-E1 seed solution and BL21 / pET28a-E2 seed solution;

[0094] Shake flask culture: 1% of the above seed solution was transferred to 250 mL of LB culture medium containing 50 μM kanamycin and cultured for 18 h to obtain BL21 / pET28a-E1 culture medium and BL21 / pET28a-E2 culture medium;

[0095] Fermentation culture: 5% of the BL21 / pET28a-E1 culture solution and BL21 / pET28a-E2 culture solution were transferred to a 10L culture fermenter containing 5L LB medium for fermentation culture for 2-4h until the cell OD in the fermentation culture product reached 15;

[0096] Induce expression: add 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the above fermentation culture tank with OD~15 to induce protein expression at 30°C for 20 hours, and finally collect the cells by high-speed centrifugation (6000 rpm, 15 min) to obtain 150-180 g of wet cells (i.e., Escherichia coli E1 whole cells or Escherichia coli E2 whole cells), and store them at -20°C for use.

[0097] Step 3: Preparation of crude enzyme solution

[0098] Take 10 g of the wet cells of BL21 / pET28a-E1 and BL21 / pET28a-E2 obtained in step 2 above, resuspend them in 100 mL of (PBS) buffer (50 mM, pH 8.0), and mix them evenly.

[0099] The two cell resuspensions were then broken by high pressure homogenization (500-700 bar, 3-5 cycles), centrifuged at 13000 rpm for 60 min, and the supernatant was filtered through a 0.45 μm membrane to obtain a crude enzyme solution. The supernatant was collected to obtain two crude enzyme solutions, which were recorded as E1 crude enzyme solution and E2 crude enzyme solution.

[0100] Example 2: Preparation of (1R,2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol by 1-pot 2-step cascade reaction

[0101] In a 1L reaction system, add 5mM MgCl 2 , 0.1mM ThDP, 0.05mM FAD, 10mM p-methylsulfonylbenzaldehyde, 12.5% ​​DMSO, 50mM pyruvic acid, 80mL of the above E1 crude enzyme solution (16.2U / mL), dilute to 1L with 100mM pH 7.5 HEPES buffer, react at 30°C, 250rpm for 4h, then add 0.2mM PLP, 50mL of the above E2 crude enzyme solution (26.6U / mL) and 100mM isopropylamine into the reactor, stir and react at 30°C, 250rpm for 20 hours, and detect the product ((1R,2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol) by HPLC, with a conversion rate of 73%.

[0102] Raw material conversion rate = 1-(detected substrate concentration c*reaction solution volume v / molecular weight of p-methylsulfonylbenzaldehyde M) / (p-methylsulfonylbenzaldehyde feed amount m / molecular weight of p-methylsulfonylbenzaldehyde M)*100%

[0103] Detected substrate concentration = c (obtained by HPLC detection)

[0104] Reaction liquid volume v = 1L

[0105] The molecular weight of p-methylsulfonylbenzaldehyde is M=184.21

[0106] The amount of p-methylsulfonylbenzaldehyde added was m=1.84 g.

[0107] Example 3: Preparation of (1R,2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol by one-pot two-step cascade reaction

[0108] In a 1L reaction system, add 5mM MgCl 2, 0.1mM ThDP, 0.05mM FAD, 10mM p-methylsulfonylbenzaldehyde, 12.5% ​​DMSO, 50mM pyruvic acid, 30g / L Escherichia coli E1 whole cells, dilute to 1L with 100mM pH 7.5 HEPES buffer, react for 3 hours, add 0.2mM PLP, 20g / L Escherichia coli E2 whole cells, 100mM isopropylamine to the reactor, 30℃, 250rpm, stir and react for 30 hours, HPLC detection of the product ((1R,2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol), the conversion rate is 65%.

[0109] Example 4: Preparation of Florfenicol

[0110] Methyl dichloroacetate (7.22 g, 0.0505 mol) and TEA (1.02 g, 0.0101 mol) were added to a solution of (1R, 2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol (2.5 g, 0.0101 mol) in methanol (25 mL). The mixture was stirred at room temperature for 24 h. Methanol was evaporated under reduced pressure, and toluene (10 mL) was added to the residue. The mixture was cooled to 0°C and filtered after 6 h. The filter cake was washed with a small amount of isopropanol to obtain light yellow crystals 1 (3.24 g, 90%). The light yellow crystals were confirmed to be the target product, florfenicol, by structural identification.

[0111] Example 5: Preparation of Florfenicol

[0112] Methyl dichloroacetate (7.22 g, 0.0505 mol) and diisopropylethylamine (1.31 g, 0.0101 mol) were added to a solution of (1R, 2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol (2.50 g, 0.0101 mol) in methanol (25 mL). The mixture was stirred at room temperature for 24 h. Methanol was evaporated under reduced pressure, and toluene (10 mL) was added to the residue. The mixture was cooled to 0°C and filtered after 6 h. The filter cake was washed with a small amount of isopropanol to obtain light yellow crystals 1 (3.12 g, 86.7%). The light yellow crystals were confirmed to be the target product, florfenicol, by structural identification.

[0113] Example 6: Preparation of Florfenicol

[0114] Methyl dichloroacetate (7.22 g, 0.0505 mol) and diisopropylethylamine (1.31 g, 0.0101 mol) were added to a solution of (1R, 2S)-1-[4-(methylsulfonyl)phenyl]-2-amino-3-fluoro-1-propanol (2.50 g, 0.0101 mol) in THF (25 mL). Stir at room temperature for 24 h. THF was evaporated under reduced pressure, toluene (10 mL) was added to the residue, cooled to 0°C, and filtered after 6 h. The filter cake was washed with a small amount of isopropanol to obtain light yellow crystals 1 (3.23 g, 89.7%). The light yellow crystals were confirmed to be the target product florfenicol by structural identification.

[0115] The amino acid sequence of pyruvate decarboxylase E1 is SEQ ID NO: 1, as follows:

[0116] MASSGTTTSTRKRFTGAEFIVHFLERQGIQIVTGIPGGSILPVYDALSQSTQIRHILARHEQGA

[0117] GFIAQGMARTDGKPAVCMACSGPGATNLVTAIADARLDSIPLVCITGQVPASMIGTDAFQEV

[0118] DTYGISIPITKHNYLVRHIDELPQVMSDAFRLAQSGRPGPVWIDIPKDVQTAVFEIDAMPAPA

[0119] EKMAAPAFSEQSVRDAAAMINAAKRPVLYLGGGVINAPERVRQLAEKAQLPTTMTLMALG

[0120] MLPKAHPLSLGMLGMHGARSTNYILQEADLLVVLGARFDDRAIGKTEQFCPNARIIHVDID

[0121] RAELGKIKQPHVAIQADVDEVLSQLIPQIEAQPRTEWHQLVRDLQREFPCTIPQESDPLSHYG

[0122] LINAVAACVDDNAIITTDVGQHQMWAAQAYPLNRPRQWLTSGGLGTMGFGLPAAIGAALA

[0123] NPDKKVLCFSGDGSLMMNIQEMATASENQLDVKIILMNNEALGLVHQQQSLFYKQGVFAA

[0124] TYPGMINFMQIAAGFGLETCDLNNEADPQAALQAIISRPGPALIHVRIDAEEKVYPMVPPGA

[0125] ANTEMVGE

[0126] The amino acid sequence of aminotransferase E2 is SEQ ID NO:2, as follows:

[0127] MKQHNTEVLRQQDAAHHLHPFSDMQSLNRQGSRVITHADGIYLWDSEGNKIIDGMAGLW

[0128] CVNIGYGRKDLPEVAKKQMEELAYYNTFFKTTHPAVVELSALLNEVSPEGFSHVFYTNSGS

[0129] ESVDTMMRMVRRFWDVQGKPQKKTLIGRWNGYHGSTIGGASLGGMTYMHEQGDLPIPGI

[0130] AHVEQPWWYKHGKDMTPDEFGLQAARWVEEKILEIGADKVAAFVGEPIQGAGGVIIPPAT

[0131] YWPEVERICRKYDVLLVADEVICGFGRTGEWFGHQTLGFRPDIFTTAKGLSSGYLPIGAVFV

[0132] GDRVAEGLLAGGDFNHGFTYSGHPVAAAVAQANVRALRDEGIVERVKNETGPYMQRRWH

[0133] ETFGQFAHVDDIRGVGLIQAFTLVKDRAKRELFPNWGDMGMKCRDIFFRNNLIMRACGDH

[0134] IVSAPPLIISKEEIDKMLGLAARCIETFEKEVLF。

Claims

1. A method for synthesizing florfenicol and its derivatives shown in formula I, comprising the following steps: In formula I, X is selected from H, OH, F, Cl, Br or I; 1) allowing compound 1 and compound 2 to react under the action of pyruvate decarboxylase or whole Escherichia coli cells expressing the pyruvate decarboxylase to obtain compound 3; in, The definition of X in compound 2 is the same as that in formula I; the definition of X in compound 3 is the same as that in formula I; 2) reacting compound 3 with an amine substance under the action of an aminotransferase or whole Escherichia coli cells expressing the aminotransferase to obtain compound 4; 3) subjecting compound 4 to an amidation reaction with methyl dichloroacetate to obtain a compound of formula I.

2. The method according to claim 1, characterized in that: In the step 1), the pyruvate decarboxylase is added in the form of a crude enzyme solution, and the dosage ratio of the compound 1, the compound 2 and the pyruvate decarboxylase crude enzyme solution is 10 mmol: (40-60) mmol: (60-90) mL; Further, the crude pyruvate decarboxylase enzyme solution is prepared according to the following steps: after the recombinant bacteria expressing pyruvate decarboxylase with an OD value of 12-16 are induced by IPTG for 18-22 hours, the PBS buffer is added to the bacteria in an amount of 1g:8-12mL of the ratio of bacteria:PBS buffer, and the PBS buffer is crushed, and the supernatant of the crushed product is collected to obtain a crude enzyme solution; the recombinant bacteria expressing pyruvate decarboxylase are recombinant bacteria obtained by introducing the coding gene of the pyruvate decarboxylase into Escherichia coli; And / or, in the step 1), the dosage ratio of the compound 1, the compound 2 and the whole Escherichia coli cells expressing pyruvate decarboxylase is 10 mmol: (40-60) mmol: 20-35 g; The whole Escherichia coli cells expressing pyruvate decarboxylase are the cells collected after the recombinant bacteria expressing pyruvate decarboxylase are induced by IPTG for 18-22 hours.

3. The method according to claim 1 or 2, characterized in that: In the step 1), the pyruvate decarboxylase is pyruvate decarboxylase E1, and the amino acid sequence of the pyruvate decarboxylase E1 is SEQ ID NO:

1.

4. The method according to any one of claims 1 to 3, characterized in that: In the step 1), MgCl2, ThDP, FAD and DMSO are also added during the reaction.

5. The method according to any one of claims 1 to 4, characterized in that: In the step 1), the reaction is carried out in a HEPES buffer; And / or, in the step 1), the reaction conditions of the reaction are: 25-35° C., stirring reaction for 18-22 hours, and stirring speed of 220-260 rpm.

6. The method according to any one of claims 1 to 5, characterized in that: In the step 1), after the reaction is completed, the obtained compound 3 does not need to be separated and purified, and the reaction of step 2) is directly carried out in the same reactor, that is, the reaction of step 1) and step 2) is prepared by a one-pot method.

7. The method according to any one of claims 1 to 6, characterized in that: In the step 2), the amine substance as the amino donor is selected from at least one of the following: isopropylamine, α-methylpropylamine, 1-methyl-3-phenylpropylamine, 1-aminoindane, benzylamine, 2-heptylamine, 1,5-dimethylhexylamine, 2-aminobutane, aminobutyric acid, and L-alanine.

8. The method according to any one of claims 1 to 7, characterized in that: In the step 2), the aminotransferase is added in the form of a crude enzyme solution, and the dosage ratio of the compound 3, the amine substance and the aminotransferase crude enzyme solution is 10 mmol: (100-200) mmol: (60-90) mL; Furthermore, the crude enzyme solution of aminotransferase is prepared according to the following steps: after inducing the recombinant bacteria expressing aminotransferase with an OD value of 12-16 with isopropyl-β-D-thiogalactopyranoside (IPTG) for 18-22 hours, the bacteria are collected, and then the PBS buffer is added to the bacteria at a ratio of 1g:8-12mL of bacteria:PBS buffer, and the bacteria are crushed, and the supernatant of the crushed product is collected to obtain a crude enzyme solution; the recombinant bacteria expressing aminotransferase is a recombinant bacteria obtained by introducing the coding gene of the aminotransferase into Escherichia coli; And / or, in the step 2), the dosage ratio of the compound 3, the amine substance and the whole Escherichia coli cells expressing the aminotransferase is 10 mmol: (100-200) mmol: 15-25 g; The whole Escherichia coli cells expressing aminotransferase are the cells collected after the recombinant bacteria expressing aminotransferase are induced by IPTG for 18-22 hours.

9. The method according to any one of claims 1 to 8, characterized in that: In the step 2), the aminotransferase is aminotransferase E2, and the amino acid sequence of the aminotransferase E2 is SEQ ID NO:

2.

10. The method according to any one of claims 1 to 9, characterized in that: In the step 2), pyridoxal phosphate is added as a coenzyme during the reaction; the dosage ratio of the compound 1 to pyridoxal phosphate is 10 mmol: 0.1-0.3 mmol.

11. The method according to any one of claims 1 to 10, characterized in that: In the step 2), the reaction conditions are: 25-35° C., stirring reaction for 20-35 hours, and stirring speed of 150-250 rpm.

12. The method according to any one of claims 1 to 11, characterized in that: In the step 3), the amidation reaction is carried out in the presence of a base, and the base is selected from triethylamine or diisopropylethylamine; the amidation reaction is carried out in a solvent, and the solvent is selected from methanol or THF.

13. The method according to any one of claims 1 to 12, characterized in that: In the step 3), the molar ratio of compound 4 to methyl dichloroacetate is 1:(3-6); And / or, the molar ratio of the compound 4 to the base is 1:(1-1.5); And / or, the reaction conditions of the amidation reaction are: stirring the reaction at 25-35° C. for 18-22 hours.

Images