Process for the preparation of leucomycin based on a biosynthetic process

Abstract

The application relates to the technical field of medicines, and particularly provides a preparation method of lecomycin based on a biosynthesis process. The application first provides a 4''-ketone erythromycin A compound and a recombinant host cell capable of efficiently fermenting the compound. A brand-new semi-chemical synthesis route of lecomycin is designed by taking the key intermediate as a raw material, and the route comprises, in sequence, epoxidation, amination, 6-position oximation, configuration inversion, rearrangement, reduction and ring expansion. The application completely avoids the cumbersome hydroxyl and amino protection and deprotection steps in the prior art, avoids the use of noble metal catalysts, thereby significantly simplifies the process, improves the atomic economy, and the total yield can reach about 32.1%. The application provides a brand-new green synthesis route of lecomycin, which is simple in steps, high in yield, low in cost and easy to industrialize, and meanwhile protects a plurality of novel intermediates in the route.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, specifically providing a method for preparing lycomycin based on a biosynthetic process. Background Technology

[0002] Respiratory infections are common infectious diseases in animal husbandry, and their outbreaks and epidemics can cause huge economic losses to livestock production. The factors leading to respiratory infections are numerous and complex, which is the direct reason why there are no particularly effective preventive measures for this infectious disease. Currently, the prevention and treatment of animal respiratory infections mainly rely on drugs. Among them, macrolide antibiotics, due to their good tissue penetration, can reach concentrations in lung tissue several times higher than blood drug concentrations, and their tissue half-life is much longer than their serum half-life, playing a crucial role in clinical treatment.

[0003] Lekemycin is a novel macrolide antibiotic with the chemical name (2R,3S,4R,5S,8R,10R,11R,12S,13S,14R)-13-{[2,6-dideoxy-3-C-methyl-3-O-methyl-4-C-[(cyclopropylamino)methyl]-α-L-ribopyranosyl]oxy}-2-ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-{[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy}-1-oxa-7-azacyclopentadecan-15-one. Its core structure is derived by modifying the 6-carbonyl and 4''-hydroxyl groups of erythromycin A. Currently, the preparation of this compound mainly relies on total chemical synthesis, but existing technical routes all have significant drawbacks.

[0004] US patent 9765105B2 describes a novel macrocyclic lactone structure with the general formula:

[0005] , and its preparation method, when R is cyclopropylamine, it is lycomycin.

[0006] The preparation route is as follows:

[0007]

[0008] This route uses commercially available gamimycin intermediates as starting material. After protecting the 2'-hydroxyl group with benzooxycarbonyl chloride (Cbz-Cl), the core structure is obtained through oxidation, epoxidation, and deprotection. The target structure is then obtained through reaction with an amine. The drawbacks of this method are: the protection and deprotection steps are cumbersome, and the oxidation process involves numerous side reactions, resulting in low overall yield and unstable product quality, which is detrimental to industrialization.

[0009] To address the aforementioned issues, patent CN113666978A discloses a method for preparing lycomycin, as shown in the figure below:

[0010]

[0011] The method disclosed in this patent uses erythromycin A(E) oxime as the starting material. After oxime hydroxyl isomerization, rearrangement, reduction, and ring expansion, both the 7-NH and 2'-hydroxyl groups are protected to reduce the prevalence of single-protected products and their adverse effects on subsequent control. Following oxidation, epoxidation, and deprotection, the parent ring structure is obtained, which then reacts with cyclopropylamine to yield lycomycin. This method, by increasing the protecting group, improves the purity and reaction stability of key intermediates to some extent and simplifies process control. However, this method still requires protection of the 7-amino and 2'-hydroxyl groups and subsequent deprotection operations. The lengthy protection and deprotection steps not only reduce synthesis efficiency (poor atom economy) but also may involve the use of noble metal catalysts (such as palladium) during deprotection, increasing production costs and environmental burden.

[0012] In summary, the existing synthetic routes for lycomycin involve cumbersome protection and deprotection processes, resulting in long synthetic routes, low overall yields, difficult purification, and high costs, which limits its large-scale industrial production.

[0013] Therefore, there is an urgent need in this field to develop a new method for preparing lycomycin that is simplified, easy to purify, has a high yield, and is environmentally friendly. Summary of the Invention

[0014] One of the objectives of this application is to provide a method for preparing lycomycin based on a biosynthetic process, thereby providing a new route for the preparation of lycomycin that is simpler in steps, has a higher yield, lower cost, is more environmentally friendly, and is easier to industrialize.

[0015] To achieve the above objectives, this application adopts the following technical solution:

[0016] A 4''-ketoerythromycin A compound having the structure of Formula 2:

[0017] Equation 2.

[0018] A recombinant host cell for producing the above-mentioned 4''-ketoerythromycin A compound, wherein the recombinant host cell is introduced into and expresses an exogenous erythromycin biosynthesis gene cluster, the gene cluster comprising at least the eryF gene, eryBII gene, eryBIII gene, eryBV gene, eryBVI gene, eryBVII gene, ermE gene, eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryK, and eryG gene; and the recombinant host cell is capable of fermenting to produce the 4''-ketoerythromycin A compound;

[0019] Preferably, the recombinant host cell is *Escherichia coli*;

[0020] Preferably, the recombinant Escherichia coli further contains an exogenous molecular chaperone gene;

[0021] Preferably, the molecular chaperone gene is the GroES-GroEL operon.

[0022] A method for preparing lecomycin, using the above-mentioned 4''-ketoerythromycin A compound as a starting material, and without chemically protecting its 2'-hydroxyl group throughout the synthetic route.

[0023] Furthermore, the method includes the following steps:

[0024] (1) Epoxidation reaction of 4''-ketoerythromycin A shown in Formula 2 is carried out to obtain compound of Formula 3;

[0025] (2) The compound of formula 3 is subjected to an amination and ring-opening reaction with cyclopropylamine to obtain the compound of formula 4;

[0026] (3) The compound of formula 4 is subjected to an oxime reaction with hydroxylamine or its salt at the 6-position carbonyl group to obtain the compound of formula 5;

[0027] (4) The compound of formula 5 is subjected to configuration inversion under alkaline conditions to obtain the compound of formula 6;

[0028] (5) The compound of formula 6 is subjected to a rearrangement reduction and ring expansion reaction to obtain lycomycin as shown in formula 1;

[0029] Among them, Equations 1, 2, 3, 4, 5, and 6 have the following structures:

[0030] Formula 2; Formula 3; Equation 4; Formula 5; Formula 6; Formula 1.

[0031] Furthermore, the 4''-ketoerythromycin A represented by Formula 2 in step (1) is obtained by fermenting the above-mentioned recombinant host cells and is isolated and purified from the fermentation products.

[0032] Furthermore, the epoxidation reaction described in step (1) is carried out in the presence of a ylide reagent;

[0033] Preferably, the ylide reagent is generated by reacting trimethylsulfonium bromide, trimethyl sulfoxide, or a combination thereof with a strong base;

[0034] Preferably, the strong base is selected from KHMDS, potassium tert-butoxide, and sodium hydride;

[0035] Preferably, in the amination ring-opening reaction described in step (2), cyclopropylamine serves as both a reactant and a solvent;

[0036] Preferably, the amination ring-opening reaction in step (2) is carried out in a solvent selected from isopropanol, ethanol, and tert-butanol;

[0037] Preferably, the oxime reaction in step (3) is carried out in a pyridine solvent by reacting with hydroxylamine hydrochloride;

[0038] Preferably, the configuration inversion reaction in step (4) is carried out in a solvent selected from isopropanol and ethanol in the presence of lithium hydroxide or sodium hydroxide;

[0039] Preferably, the rearrangement-reduction-ring-expansion reaction described in step (5) is a one-pot reaction in which the Beckmann rearrangement reaction and the reduction reaction are carried out in series.

[0040] Preferably, the one-pot reaction is carried out in a pyridine solvent;

[0041] Preferably, the Beckmann rearrangement reaction is carried out in the presence of a sulfonating agent;

[0042] Preferably, the sulfonating agent is selected from p-methylbenzenesulfonyl chloride and p-nitrobenzenesulfonyl chloride;

[0043] Preferably, sodium borohydride is used as a reducing agent in the reduction reaction.

[0044] A compound as shown in Formula 3:

[0045] Formula 3.

[0046] A method for preparing the above-mentioned compound of formula 3 involves subjecting the above-mentioned compound of formula 2 to an epoxidation reaction with a sulfur ylide reagent without protecting the 2'-hydroxyl group.

[0047] A compound as shown in Formula 4:

[0048] Formula 4.

[0049] A compound as shown in Formula 5:

[0050] Formula 5.

[0051] A compound as shown in Formula 6:

[0052] Formula 6.

[0053] The use of the above compounds in the preparation of lycomycin.

[0054] The technical effects of this application are as follows:

[0055] This application successfully combines bio-fermentation with chemical synthesis, providing a new route for the preparation of lycomycin that is simpler in steps, has a higher yield, lower cost, is more environmentally friendly, and is easier to industrialize.

[0056] The synthetic route is significantly simplified and efficiency is greatly improved: This application innovatively uses 4''-ketoerythromycin A obtained through fermentation by genetically engineered bacteria as a key intermediate, and designs a novel semi-chemical synthetic route based on this. This route completely avoids the cumbersome protection and deprotection steps of hydroxyl and amino groups in the prior art, greatly simplifying the traditional multi-step synthesis, thereby significantly shortening the process cycle and improving the synthetic efficiency.

[0057] High product yield and good atom economy: By avoiding material loss and side reactions caused by the protection / deprotection steps, the overall yield of this application can reach approximately 32.1%. At the same time, the use of precious metal catalysts (such as palladium on carbon) in the deprotection step is avoided, which not only reduces costs but also improves atom economy, in line with the concept of green and environmentally friendly production.

[0058] The process is simple to operate, easy to purify, and produces stable quality: The intermediates involved in this application route have good stability, and the purification process is simple (such as extraction and crystallization), avoiding complex and time-consuming column chromatography purification, making it more suitable for industrial-scale production. The entire process conditions are mild, and the parameters are easy to control, ensuring the high purity and excellent quality stability of the final product, lycomycin.

[0059] This application not only protects a novel preparation method but also discloses and protects for the first time several structurally novel key intermediates (Formulas 3, 4, 5, and 6) generated in the synthetic route. These intermediates provide more synthetic route options for the preparation of lycomycin, enriching the technological toolbox in this field. Detailed Implementation

[0060] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0061] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0063] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0064] "Lycomycin" refers to the final target product with the structure of Formula 1 as specified in the specification, and its chemical name is (2R,3S,4R,5S,8R,10R,11R,12S,13S,14R)-13-{[2,6-dideoxy-3-C-methyl-3-O-methyl-4-C-[(cyclopropylamino)methyl]-α-L-ribopyranosyl]oxy}-2-ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-{[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy}-1-oxa-7-azacyclopentadecan-15-one.

[0065] "4''-ketoerythromycin A" (Formula 2) refers to the key starting material of this application, which is a compound with a keto group (-C=O) at the 4'' position of the cladinose in erythromycin A.

[0066] "Recombinant host cell" refers to a cell into which a foreign DNA sequence has been introduced through genetic engineering. This includes, but is not limited to, prokaryotic cells (such as *Escherichia coli*) and eukaryotic cells (such as yeast and filamentous fungi). Preferably, the host cell is *Escherichia coli*.

[0067] "Yellide reagents" refer to key reagents used in epoxidation reactions to form epoxides. These include, but are not limited to, sulfur ylides generated on-site by reacting trimethylsulfonium bromide or trimethyl sulfoxide with strong bases (such as KHMDS, potassium tert-butoxide, and sodium hydride).

[0068] "One-pot reaction" refers to a synthetic method in which multiple chemical reaction steps are carried out continuously in the same reaction vessel without the need to separate intermediate products. In this application, it specifically refers to step (5), in which the Beckmann rearrangement reaction of intermediate 6 and the subsequent reduction reaction are carried out continuously in the same reaction system.

[0069] This application provides a 4''-ketoerythromycin A compound having the structure of Formula 2:

[0070] Equation 2.

[0071] Compound 2 (4''-ketoerythromycin A) is a novel compound with high application value as a key intermediate in the synthesis of lycomycin.

[0072] This application also provides a recombinant host cell for producing the 4''-ketoerythromycin A compound, comprising exogenous eryF, eryBII, eryBIII, eryBV, eryBVI, eryBVII, and ermE genes, as well as exogenous eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryK, and eryG genes; and the recombinant host cell is capable of fermenting to produce the 4''-ketoerythromycin A compound.

[0073] This recombinant host cell contains key genes from an exogenous erythromycin biosynthesis gene cluster, which work synergistically to achieve the fermentation production of 4''-ketoerythromycin A. The specific functions of each gene are as follows:

[0074] 1. Macrolide nucleus synthesis and early modification genes

[0075] The eryF gene encodes deoxyerythronolide B hydroxylase. This enzyme catalyzes the hydroxylation reaction at the C-9 position of the macrolide parent nucleus, a key step in the synthesis of erythromycin B.

[0076] 2. Genes involved in carbamolybdenum synthesis, modification, and linkage.

[0077] This group of genes is responsible for synthesizing L-mycarose and linking it to the C-12 position of the macrolide core. The key structural feature of 4''-ketoerythromycin A described in this application—the keto group at the C-4'' position—is precisely achieved through metabolic engineering on this glycosyl group.

[0078] The eryBII gene encodes TDP-4-keto-6-deoxyhexose-2,3-reductase, which catalyzes the production of dTDP-4-oxo-2,6-dideoxy-D-allose.

[0079] The eryBIII gene encodes NDP-4-keto-2,6-dideoxyhexose-3-methyltransferase, which is responsible for the methylation modification at position 3 in carbamoyl synthesis.

[0080] The eryBV gene encodes TDP-carbamoyl glycosyltransferase, which catalyzes the glycosylation of activated TDP-carbamoyl with the C-12 hydroxyl group of the erythromycin lactone core, linking the carbamoyl unit to the macrolide backbone.

[0081] The eryBVI gene encodes NDP-4-keto-6-deoxy-glucose-2,3-dehydratase, which catalyzes the production of dTDP-3,4-dioxo-2,6-dideoxy-D-glucose, forming a key intermediate in the carbamolybdenum synthesis pathway.

[0082] eryBVII gene: dTDP-3-methyl-4-oxo-2,6-dideoxy-D-glucose 5-isomerase.

[0083] 3. Deoxyglucose synthesis and linker genes

[0084] eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI genes: This group of genes encodes a series of enzymes that work together to synthesize TDP-D-deoxyamino sugars from primary metabolites and attach them to the C-10 hydroxyl group of the macrolide nucleus.

[0085] 4. Post-modification genes

[0086] The eryK gene encodes C-3 hydroxylase, which is responsible for introducing a hydroxyl group at the C-3 position of the macrolide nucleus.

[0087] The eryG gene encodes a 3''-O-methyltransferase, which is responsible for methylation modification of the hydroxyl group at the C-3'' position of deoxyamino sugar.

[0088] 5. Resistance genes

[0089] The ermE gene encodes a 23S rRNA methyltransferase. This enzyme confers resistance to macrocyclic lactones synthesized by the host cell by methylating the host ribosome, which is crucial for ensuring the survival of engineered strains under high-concentration product conditions and achieving efficient fermentation.

[0090] In some embodiments, the host cell is *Escherichia coli*. To further enhance the soluble expression and activity of the exogenous protein, the recombinant *E. coli* may also contain a molecular chaperone system, such as, but not limited to, the GroES-GroEL operon.

[0091] In some embodiments, the recombinant *E. coli* introduces exogenous genes via multiple compatible vectors. For example, a first vector (e.g., pFGB62735E) carries the eryF, eryBII, eryBIII, eryBV, eryBVI, eryBVII, and ermE genes; a second vector (e.g., pC451623KG) carries the eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, eryK, and eryG genes; a third vector (e.g., pBP144) carries the gene that synthesizes the precursor (PCC) and initiates polyketide chain synthesis (DEBS1); and a fourth vector (e.g., pBP130) carries the genes that ultimately complete the synthesis and cyclization of the polyketide chain (DEBS2 and DEBS3).

[0092] In a preferred embodiment, the four vectors described above are co-transformed into Escherichia coli strain BAP1, and the resulting recombinant engineered strain is named OEA. This strain is capable of efficiently fermenting and producing 4''-ketoerythromycin A (Formula 2).

[0093] This application provides a method for preparing lycomycin, comprising the following steps:

[0094] (1) Epoxidation reaction of 4''-ketoerythromycin A shown in Formula 2 is carried out to obtain compound of Formula 3;

[0095] (2) The compound of formula 3 is subjected to an amination and ring-opening reaction with cyclopropylamine to obtain the compound of formula 4;

[0096] (3) The compound of formula 4 is subjected to an oxime reaction with hydroxylamine or its salt at the 6-position carbonyl group to obtain the compound of formula 5;

[0097] (4) The compound of formula 5 is subjected to configuration inversion under alkaline conditions to obtain the compound of formula 6;

[0098] (5) The compound of formula 6 is subjected to a rearrangement, reduction, and ring-expansion reaction to obtain lycomycin as shown in formula 1. The reaction procedure is as follows:

[0099] .

[0100] The preparation method described in this application is an innovative process integrating bio-fermentation and chemical synthesis. The core advantage of this method is that it eliminates the need for protection and deprotection of numerous reactive functional groups (such as 2'-OH, 7-NH2) in the erythromycin A route, thereby greatly simplifying the process.

[0101] In some embodiments, the epoxidation reaction in step (1) of this application preferably employs the Corey-Chaykovsky reaction mechanism. Under these reaction conditions, the thioyl ylide generated in-situ acts as a nucleophile, attacking the carbonyl group in the molecule. In the specific starting material of this application—4''-ketoerythromycin A represented by Formula 2—despite the presence of multiple potential reaction sites within the molecule (such as the 14-ester group and the 6-carbonyl group), the epoxidation reaction exhibits excellent regioselectivity under the aforementioned ultra-low temperature reaction conditions (e.g., -90°C to -100°C).

[0102] 14-Ester group: Due to the low electronegativity of its carbonyl carbon, it does not participate in the competing reaction under these reaction conditions.

[0103] 6-carbonyl group: Due to the significant steric hindrance effect caused by the complex spatial structure around it, its reactivity is effectively suppressed under these low temperature (e.g. -90℃ to -100℃) reaction conditions.

[0104] Therefore, thioyl ylides can react with the 4''-keto group of compound formula 2 with high selectivity and specificity to efficiently generate compound formula 3. This unique selectivity allows the '2'-hydroxyl protection' step, which is necessary in the prior art to avoid side reactions, to be completely omitted in this application, which is one of the core reasons for the simplification of the route in this application.

[0105] In some embodiments, the epoxidation reaction in step (1) is carried out in the presence of a ylide reagent. The reaction temperature range is -100°C to -70°C, preferably -90°C to -100°C, and the reaction time is 1.5 to 3 hours.

[0106] In some embodiments, the ylide reagent is generated by reacting trimethylsulfonium bromide, trimethyl sulfoxide, or a combination thereof with a strong base; preferably, the strong base is selected from KHMDS, potassium tert-butoxide, and sodium hydride; preferably, the reaction temperature is -20°C to -30°C.

[0107] In some embodiments, for the amination ring-opening reaction in step (2), cyclopropylamine may be used as both a reactant and a solvent, in amounts far exceeding the stoichiometric ratio. Alternatively, the reaction may be carried out in an inert organic solvent, including but not limited to isopropanol, ethanol, tert-butanol, and mixtures thereof.

[0108] In some embodiments, for the oxime reaction in step (3), the "salt of hydroxylamine" is preferably hydroxylamine hydrochloride. The reaction can be carried out in pyridine, which acts as both a solvent and a base to neutralize the acid produced in the reaction.

[0109] In some embodiments, the "basic condition" for the configuration inversion in step (4) can be achieved by adding an inorganic base (such as lithium hydroxide, sodium hydroxide) or an organic base (such as triethylamine). The reaction solvent can be selected from isopropanol, ethanol, methanol, etc.

[0110] The term "configuration inversion" as used in this article specifically refers to the transformation of the 6-oxime group in Formula 5 from the E-configuration to the Z-configuration, resulting in Formula 6.

[0111] In some embodiments, the preferred approach for the rearrangement reduction and ring-expansion reaction in step (5) is a one-pot process. The Beckmann rearrangement reaction is carried out in the presence of a sulfonating agent, including but not limited to p-toluenesulfonyl chloride, p-nitrobenzenesulfonyl chloride, and methanesulfonyl chloride. In the subsequent reduction reaction, the reducing agent includes, but is not limited to, sodium borohydride and sodium cyanoborohydride. This one-pot reaction is preferably carried out in a pyridine solvent.

[0112] In the above-mentioned synthetic route, this application, for the first time, discovered and isolated a series of intermediate compounds represented by Formulas 3, 4, 5, and 6. These compounds have novel structures and are important precursors for the synthesis of lycomycin.

[0113] The use of these intermediate compounds in the preparation of lycomycin is an integral part of this application. Any act of using these intermediates to produce lycomycin falls within the scope of protection of this application.

[0114] The present application is further described below with reference to specific embodiments. The advantages and features of the present application will become clear from the description. The embodiments described are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0115] Example 1: Construction of the metabolic engineered strain OEA of Formula 2

[0116] The method for constructing the metabolically engineered strain OEA in this embodiment includes the following steps:

[0117] Step 1: Construction of the pFGB62735E vector

[0118] (1) The 6-deoxyerythromycin lactone B hydroxylase gene eryF (nucleotide sequence as shown in SEQ ID NO: 1), the 4''-ketocarboxylic acid synthase encoding genes eryBII, eryBIII, eryBV, eryBVI, eryBVII (nucleotide sequences as shown in SEQ ID NO: 2-6), and the erythromycin resistance gene ermE (nucleotide sequence as shown in SEQ ID NO: 7) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. after optimization using the GenSmart™ codon optimization tool.

[0119] (2) Using the commercially available plasmid pCDFDuet-1 as a vector, the gene eryF, the E. coli expression molecular chaperone genes GroES-GroEL, eryBVI, eryBII, eryBVII, eryBIII, eryBV and ermE were sequentially linked with restriction endonucleases Nco I, EcoR I, Xba I, Spe I or Sac I to construct the vector pFGB62735E.

[0120] Step 2: Construction of the pC451623KG vector

[0121] (1) The dTDP-D-erythromycin deoxyglycosamine synthesis genes eryCI, eryCII, eryCIII, eryCIV, eryCV, eryCVI, 3"-O-methyltransferase gene eryG, and C-12 hydroxylase gene eryK (nucleotide sequences as shown in SEQ ID NO: 8-15) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. after optimization using the GenSmart™ codon optimization tool.

[0122] (2) Using the commercially available plasmid pACYCDuet-1 as a vector, the genes eryCIV, eryCV, eryCI, eryCVI, eryCII, eryCIII, eryK and eryG were sequentially linked using restriction endonucleases Nco I, EcoRI, Xba I, Spe I or Sac I to construct the vector pC451623KG.

[0123] Step 3: Construction of engineered strain OEA

[0124] (1) BAP1 strain was purchased from Kerafast, catalog number ESU002.

[0125] (2) pBP130 plasmid was purchased from Kerafast, catalog number ESU003.

[0126] (3) pBP144 plasmid was purchased from Kerafast, catalog number ESU004.

[0127] (4) The pBP130, pBP144, pFGB62735E and pC451623KG vectors were transformed into Escherichia coli BAP1 by chemical transformation. After revival culture, they were spread on LB solid medium plates containing 50 μg / mL ampicillin resistance, 50 μg / mL kanamycin resistance, 50 μg / mL streptomycin resistance and 50 μg / mL chloramphenicol resistance, and incubated at 37℃ for about 24 h.

[0128] (5) The single colony that grows on the LB solid medium plate is the recombinant Escherichia coli BAP1 (pBP130, pBP144, pFGB62735E, pC451623KG), named OEA.

[0129] Example 2: Preparation of compound 2

[0130] The OEA strain was inoculated into LB medium containing ampicillin, kanamycin, streptomycin, and chloramphenicol resistance, and cultured at 37°C with shaking until OD. 600 =0.6; Add 0.5 mM IPTG, induce at 28℃ for 72 hours; centrifuge the fermentation broth, extract the fermentation broth with methanol, and purify by silica gel column chromatography (eluent: CH2Cl2 / MeOH 20:1); m / z 731.4, 1H NMR (500 MHz, CDCl3): δ5.21 (ddd, J = 4.8, 3.7, 2.0 Hz, 1H), 4.97 (t, J = 5.4 Hz, 1H), 4.66 – 4.57(m, 2H), 4.54 (dt, J = 5.1, 2.5 Hz, 1H), 4.40 (d, J = 6.0 Hz, 1H), 4.11 (dd,J = 9.4, 8.5 Hz, 1H), 3.78 (dd, J = 9.6, 6.5 Hz, 1H), 3.64 – 3.51 (m, 2H), 3.42 (s, 1H), 3.31 (d, J = 14.5 Hz, 5H), 2.92 (dq, J = 9.7, 6.5 Hz, 1H), 2.87– 2.68 (m, 2H), 2.61 (dddd, J = 8.9, 7.7, 6.3, 3.2 Hz, 1H), 2.36 – 2.27 (m,7H), 2.18 – 2.07 (m, 2H), 2.03 (dd, J = 13.7, 7.6 Hz, 1H), 1.82 – 1.70 (m,1H), 1.70 – 1.58 (m, 3H), 1.54 (ddd, J = 12.4, 6.2, 3.8 Hz, 1H), 1.41 (s,2H), 1.38 – 1.32 (m, 9H), 1.21 (d, J = 7.3 Hz, 3H), 1.18 – 1.06 (m, 12H), 0.86 (t, J = 7.1 Hz, 3H).

[0131] Example 3: Preparation of compound 3

[0132] (2R,3S,4R,5R,7R,9R,10R,11R,12S,13R)-10-{[3-(dimethylamino)-3,4,6-trideoxy-β-D-xylo-hexopyranosyl]oxy}-12-{[8-methoxy-4,8-dimethyl-1,5-dioxaspiro[2.5]oct-6-yl]oxy}-2-ethyl-3,4,9-trihydroxy-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-6,14-dione

[0133] Method 3.1: Under nitrogen protection, 25.73 g (163.8 mmol) of pre-dried trimethylsulfonium bromide was added to 180 ml of anhydrous tetrahydrofuran. The mixture was stirred and cooled to -20 °C. 136.5 ml (136.5 mmol) of a tetrahydrofuran solution of potassium di(trimethylsilyl)amino (KHMDS) was added, and the reaction was carried out for 2 h. The temperature was lowered again to -90 to -100 °C, and 20 g (27.3 mmol) of anhydrous tetrahydrofuran solution of compound 2 was added dropwise. The reaction was maintained at low temperature for 2 h. The low temperature environment was removed, and 150 ml of water was added to quench the reaction. The mixture was stirred, allowed to stand and separate into layers, and the organic phase was evaporated to dryness under reduced pressure to obtain 17.6 g of compound 3, with a yield of 86.3% and m / z 745.5. 1 H NMR (500 MHz, CDCl3): δ 5.18 (ddd, J = 4.6, 3.7, 1.8 Hz, 1H), 4.97 (t,J = 5.4 Hz, 1H), 4.84 (qd, J = 5.3, 3.7 Hz, 1H), 4.60 (d, J = 6.4 Hz, 1H), 4.54 (dt, J = 5.1, 2.5 Hz, 1H), 4.40 (d, J = 6.0 Hz, 1H), 4.12 (dd, J = 9.3,8.4 Hz, 1H), 3.78 (dd, J = 9.6, 6.5 Hz, 1H), 3.64 – 3.51 (m, 2H), 3.42 (s,1H), 3.35 – 3.26 (m, 5H), 3.09 – 2.98 (m, 2H), 2.92 (dq, J = 9.7, 6.5 Hz,1H), 2.87 – 2.68 (m, 2H), 2.62 (dddd, J = 10.2, 7.5, 5.0, 3.6 Hz, 1H), 2.48(dd, J = 12.5, 4.6 Hz, 1H), 2.32 (d, J = 1.6 Hz, 6H), 2.22 (dd, J = 12.3, 1.8Hz, 1H), 2.17 – 1.98 (m, 2H), 1.82 – 1.70 (m, 1H), 1.70 – 1.58 (m, 3H), 1.54(ddd, J = 12.4, 6.2, 3.8 Hz, 1H), 1.41 (s, 2H), 1.35 (d, J = 6.4 Hz, 3H), 1.24 – 1.06 (m, 21H), 0.86 (t, J = 7.1 Hz, 3H).

[0134] Method 3.2: Under nitrogen protection, 21.04 g (95.60 mmol) of pre-dried trimethyl sulfoxide was added to 200 ml of anhydrous tetrahydrofuran. The mixture was stirred and cooled to -20 °C. 9.20 g (82.0 mmol) of potassium tert-butoxide was added, and the reaction was allowed to proceed for 2 h. The mixture was then cooled again to -90 to -100 °C. A solution of 10.03 g (13.7 mmol) of compound 2 in anhydrous tetrahydrofuran (60 ml) was added dropwise. The reaction was maintained at low temperature for 2 h. The low-temperature environment was removed, and 100 ml of water was added to quench the reaction. The mixture was stirred, allowed to stand and separate into layers, and the organic phase was evaporated to dryness under reduced pressure to obtain 7.2 g of compound 3, with a yield of 70.6%.

[0135] Method 3.3: Under nitrogen protection, 128.8 g (820.0 mmol) of pre-dried trimethylsulfonium bromide was added to 800 ml of anhydrous tetrahydrofuran. The mixture was stirred and cooled to -20 °C. 685.0 ml (685.0 mmol) of a tetrahydrofuran solution of potassium di(trimethylsilyl)amino (KHMDS) was added, and the reaction was carried out for 2 h. The temperature was lowered again to -90 to -100 °C, and 100 g (136.6 mmol) of anhydrous tetrahydrofuran solution of formula 2 (500 ml) was added dropwise. The reaction was maintained at low temperature for 2 h. The low temperature environment was removed, and 700 ml of water was added to quench the reaction. The mixture was stirred, allowed to stand and separate into layers, and the organic phase was evaporated to dryness under reduced pressure to obtain 86.3 g of formula 3, with a yield of 84.7%.

[0136] Example 4: Preparation of compound 4

[0137] (2R,3S,4R,5R,7R,9R,10R,11R,12S,13R)-10-{[3-(dimethylamino)-3,4,6-trideoxy-β-D-xylo-hexopyranosyl]oxy}-12-{[2,6-dideoxy-3-C-methyl-3-O-methyl-4-C-[(cyclopropylamino)methyl]-α-L-ribo-hexopyranosyl]oxy}-2-ethyl-3,4,9-trihydroxy-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-6,14-dione

[0138] Method 4.1: 50 g (67.03 mmol) of compound 3 was added to 500 ml of cyclopropylamine (7.2 mol), stirred and heated, and refluxed for 24 h. The solvent was removed by vacuum evaporation and the mixture was treated to obtain 49.3 g of compound 4, with a yield of 91.6% and m / z 802.5.

[0139] 1H NMR (500 MHz, CDCl3) δ 5.06 (ddd, J = 4.8, 3.8, 2.0 Hz, 1H), 4.97 (t, J = 5.4 Hz, 1H), 4.60 (d, J = 6.4 Hz, 1H), 4.54 (dt, J = 5.1, 2.5 Hz,1H), 4.40 (d, J = 6.0 Hz, 1H), 4.16 – 4.03 (m, 2H), 3.78 (dd, J = 9.6, 6.5Hz, 1H), 3.64 – 3.51 (m, 2H), 3.45 (d, J = 23.8 Hz, 2H), 3.31 (d, J = 6.0 Hz, 5H), 3.08 (dt, J = 5.9, 4.2 Hz, 1H), 3.00 – 2.68 (m, 5H), 2.67 – 2.56 (m,1H), 2.52 (dp, J = 5.7, 4.0 Hz, 1H), 2.32 (d, J = 1.6 Hz, 6H), 2.25 (dd, J =12.5, 4.8 Hz, 1H), 2.17 – 1.98 (m, 3H), 1.82 – 1.70 (m, 1H), 1.70 – 1.58 (m,3H), 1.54 (ddd, J = 12.4, 6.2, 3.8 Hz, 1H), 1.41 (s, 2H), 1.35 (d, J = 6.4Hz, 3H), 1.27 – 1.06 (m, 21H), 0.86 (t, J = 7.1 Hz, 3H), 0.63 (dp, J = 4.1,1.4 Hz, 4H).

[0140] Method 4.2: 5 g (6.70 mmol) of compound 3 was dissolved in 25 ml of isopropanol, 5 ml of cyclopropylamine (72.2 mmol) and 1 g of potassium iodide were added, the mixture was stirred and heated, and the reaction was maintained at 50 °C for 240 h. After adding 100 ml of water, 3.2 g of compound 4 was obtained, with a yield of 59.5%.

[0141] Example 5: Preparation of Compound Formula 5

[0142] (2R,3S,4R,5S,7R,9R,10R,11R,12S,13R)-10-{[3-(dimethylamino)-3,4,6-trideoxy-β-D-xylo-hexopyranosyl]oxy}-12-{[2,6-dideoxy-3-C-methyl-3-O-methyl-4-C-[(cyclopropylamino)methyl]-α-L-ribo-hexopyranosyl]oxy}-2-ethyl-3,4,9-trihydroxy-6-E-hydroxyamine imide-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-14-one

[0143] Method 5.1: 50 g (62.3 mmol) of compound 4 was added to 500 ml of pyridine, and 108.4 g (1.56 mol) of hydroxylamine hydrochloride was added with stirring. The resulting mixture was stirred at room temperature for 32 h to terminate the reaction. The mixture was concentrated under vacuum at about 40°C until no fraction remained. 500 ml of ethanol was added and stirred. The mixture was filtered, washed, and the filtrates were combined. The mixture was concentrated under reduced pressure to dryness. The mixture was stirred again with a mixture of sodium bicarbonate solution and dichloromethane (the system was kept slightly alkaline). The layers were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, concentrated again, and evaporated to dryness. The mixture was recrystallized with ethyl acetate / n-heptane and dried under vacuum to obtain 45.4 g of compound 5, with a yield of 89.1% and m / z 817.5.

[0144] 1H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 5.06 (ddd, J = 4.8, 3.8, 2.0Hz, 1H), 4.91 (t, J = 5.4 Hz, 1H), 4.54 (dt, J = 5.1, 2.5 Hz, 1H), 4.40 (d, J= 6.0 Hz, 1H), 4.16 – 4.03 (m, 2H), 3.98 (d, J = 6.1 Hz, 1H), 3.87 (dd, J =9.3, 6.2 Hz, 1H), 3.65 – 3.51 (m, 2H), 3.47 (d, J = 2.9 Hz, 2H), 3.40 – 3.30(m, 1H), 3.31 (d, J = 2.0 Hz, 5H), 3.08 (dt, J = 5.9, 4.2 Hz, 1H), 2.96 (dd,J = 14.5, 4.2 Hz, 1H), 2.91 – 2.72 (m, 3H), 2.66 – 2.56 (m, 1H), 2.52 (dp, J= 5.7, 4.0 Hz, 1H), 2.32 (d, J = 1.6 Hz, 6H), 2.25 (dd, J = 12.5, 4.8 Hz,1H), 2.17 – 2.04 (m, 2H), 1.97 (dd, J = 12.8, 7.1 Hz, 1H), 1.82 – 1.70 (m,1H), 1.70 – 1.58 (m, 3H), 1.54 (ddd, J = 12.4, 6.2, 3.8 Hz, 1H), 1.41 – 1.32(m, 5H), 1.27 – 1.17 (m, 16H), 1.10 (dd, J = 16.8, 6.3 Hz, 6H), 0.86 (t, J =7.1 Hz, 3H), 0.63 (dq, J = 3.8, 1.2 Hz, 4H)。

[0145] Method 5.2: 10 g (12.5 mmol) of compound 4 was added to 100 ml of pyridine, and 108.4 g (296.5 mmol) of hydroxylamine hydrochloride was added with stirring. The resulting mixture was stirred at room temperature for 38 h to terminate the reaction. The mixture was concentrated under vacuum at about 40°C until no fraction remained. 120 ml of ethanol was added and stirred. The mixture was filtered, washed, and the filtrates were combined. The mixture was concentrated to dryness under reduced pressure and stirred again with a mixture of sodium bicarbonate solution and dichloromethane (the system was kept slightly alkaline). The layers were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, concentrated again, and evaporated to dryness. The mixture was recrystallized from ethyl acetate / n-heptane and dried under vacuum to give compound 5, 8.82 g, with a yield of 86.6%.

[0146] Example 6: Preparation of compound 6

[0147] (2R,3S,4R,5S,7R,9R,10R,11R,12S,13R)-10-{[3-(dimethylamino)-3,4,6-trideoxy-β-D-xylo-hexopyranosyl]oxy}-12-{[2,6-dideoxy-3-C-methyl-3-O-methyl-4-C-[(cyclopropylamino)methyl]-α-L-ribo-hexopyranosyl]oxy}-2-ethyl-3,4,9-trihydroxy-6-Z-hydroxyamine imido-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-14-one

[0148] Method 6.1: 50 g (61.2 mmol) of compound 5 was added to 500 ml of isopropanol, and 7.2 g (144.1 mmol) of lithium hydroxide monohydrate was added. The mixture was stirred at room temperature for 36 h, concentrated under reduced pressure, and extracted with ethyl acetate / water. After concentration, the mixture was crystallized with nitromethane, filtered, and dried to obtain 35.2 g of compound 6, with a yield of 70.4% and m / z 817.5.

[0149] Method 6.2: 20 g (24.5 mmol) of compound 5 was added to 200 ml of ethanol, 2.5 g (63.4 mmol) of sodium hydroxide was added, and the mixture was stirred at room temperature for 36 h. The mixture was concentrated under reduced pressure, dissolved and extracted with ethyl acetate / water, concentrated, purified with dichloromethane, and dried to obtain 13.2 g of compound 6, with a yield of 66.0%.

[0150] Example 7: Preparation of compound of formula 1 (Lycomycin)

[0151] (2R,3S,4R,5S,8R,10R,11R,12S,13S,14R)-13-{[2,6-dideoxy-3-C-methyl-3-O-methyl-4-C-[(cyclopropylamino)methyl]-α-L-ribopyranosyl]oxy}-2-ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-{[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy}-1-oxa-7-azacyclopentadecan-15-one

[0152] Method 7.1: Dissolve 50 g of compound 6 (61.2 mmol) in 500 mL of pyridine. Maintain the reaction temperature at 0 ± 2 °C. Slowly add 23.3 g (122.4 mmol) of p-toluenesulfonyl chloride in 150 mL of isopropyl ether solution. After the addition is complete, maintain the reaction temperature at 0 °C and stir for 4 h. Add 500 mL of n-heptane to separate the layers, discarding the n-heptane layer. Add the lower layer to a mixture of isopropanol / water, maintaining the temperature below 5 °C. Add 23.5 g (621 mmol) of sodium borohydride and stir for 18 h. Quench the reaction with dilute hydrochloric acid, process, concentrate, recrystallize from acetone, filter, and dry to obtain 31.9 g of compound 1, with a yield of 64.8%.

[0153] Method 7.2: Dissolve 10 g of compound 6 (12.2 mmol) in 100 mL of pyridine. Maintain the reaction temperature at 0 ± 2 °C. Slowly add 4.9 g (22.1 mmol) of p-nitrobenzenesulfonyl chloride in 50 mL of tert-butyl methyl ether solution. After the addition is complete, maintain the reaction temperature at 0 °C and stir for 4 h. Add 200 mL of n-heptane to separate the layers (100 mL × 2 times), discarding the n-heptane layer. Add the lower layer to a mixture of isopropanol / water, maintaining the temperature below 5 °C. Add 6.2 g (98.7 mmol) of sodium cyanoborohydride and stir for 24 h. Quench the reaction with dilute hydrochloric acid, process, concentrate, recrystallize from acetone, filter, and dry to obtain 6.21 g of compound 1, with a yield of 63.3%.

[0154] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

[0155] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. A 4''-ketoerythromycin A compound having the structure of Formula 2: Equation 2.

2. A recombinant host cell for producing the 4''-ketoerythromycin A compound of claim 1, characterized in that, The recombinant host cell introduced and expressed an exogenous erythromycin biosynthesis gene cluster, which includes the following gene clusters: eryF gene (nucleotide sequence as shown in SEQ ID NO: 1), eryBII gene (nucleotide sequence as shown in SEQ ID NO: 2), eryBIII gene (nucleotide sequence as shown in SEQ ID NO: 3), eryBV gene (nucleotide sequence as shown in SEQ ID NO: 4), eryBVI gene (nucleotide sequence as shown in SEQ ID NO: 5), eryBVII gene (nucleotide sequence as shown in SEQ ID NO: 6), ermE gene (nucleotide sequence as shown in SEQ ID NO: 7), eryCI gene (nucleotide sequence as shown in SEQ ID NO: 8), eryCII gene (nucleotide sequence as shown in SEQ ID NO: 9), eryCIII gene (nucleotide sequence as shown in SEQ ID NO: 10), eryCIV gene (nucleotide sequence as shown in SEQ ID NO: 11), eryCV gene (nucleotide sequence as shown in SEQ ID NO: 12), eryCVI gene (nucleotide sequence as shown in SEQ ID NO: 13), eryK gene (nucleotide sequence as shown in SEQ ID NO: 15), and eryCVI gene (nucleotide sequence as shown in SEQ ID NO: 15). NO: 14 shows the eryG gene; and the recombinant host cell is able to ferment and produce the 4''-ketoerythromycin A compound; The recombinant host cell is Escherichia coli; The recombinant Escherichia coli also contains exogenous molecular chaperone genes; The molecular chaperone gene is the GroES-GroEL operon.

3. A method for preparing lycomycin, characterized in that, The 4''-ketoerythromycin A compound described in claim 1 was used as the starting material, and its 2'-hydroxyl group was not chemically protected throughout the synthetic route.

4. The preparation method according to claim 3, characterized in that, The method includes the following steps: (1) Epoxidation reaction of 4''-ketoerythromycin A shown in Formula 2 is carried out to obtain compound of Formula 3; (2) The compound of formula 3 is subjected to an amination and ring-opening reaction with cyclopropylamine to obtain the compound of formula 4; (3) The compound of formula 4 is subjected to an oxime reaction with hydroxylamine or its salt at the 6-position carbonyl group to obtain the compound of formula 5; (4) The compound of formula 5 is subjected to configuration inversion under alkaline conditions to obtain the compound of formula 6; (5) The compound of formula 6 is subjected to a rearrangement reduction and ring expansion reaction to obtain lycomycin as shown in formula 1; Among them, Equations 1, 2, 3, 4, 5, and 6 have the following structures: Formula 2; Formula 3; Equation 4; Formula 5; Formula 6; Formula 1.

5. The preparation method according to claim 4, characterized in that, The 4''-ketoerythromycin A represented by Formula 2 in step (1) is obtained by fermenting the recombinant host cell of claim 2 and isolating and purifying it from the fermentation product.

6. The preparation method according to claim 4, characterized in that, The epoxidation reaction in step (1) is carried out in the presence of ylide reagent.

7. The preparation method according to claim 6, characterized in that, The ylide reagent is generated by reacting trimethylsulfonium bromide, trimethyl sulfoxide, or a combination thereof with a strong base.

8. The preparation method according to claim 7, characterized in that, The strong base is selected from KHMDS, potassium tert-butoxide, and sodium hydride.

9. The preparation method according to claim 4, characterized in that, In the amination ring-opening reaction described in step (2), cyclopropylamine serves as both a reactant and a solvent.

10. The preparation method according to claim 4, characterized in that, The amination ring-opening reaction in step (2) is carried out in a solvent selected from isopropanol, ethanol, and tert-butanol.

11. The preparation method according to claim 4, characterized in that, The oxime reaction described in step (3) is carried out in a pyridine solvent by reacting with hydroxylamine hydrochloride.

12. The preparation method according to claim 4, characterized in that, The configuration inversion reaction described in step (4) is carried out in a solvent selected from isopropanol and ethanol in the presence of lithium hydroxide or sodium hydroxide.

13. The preparation method according to claim 4, characterized in that, The rearrangement-reduction-ring-expansion reaction described in step (5) is a one-pot reaction in which the Beckmann rearrangement reaction and the reduction reaction are carried out in series.

14. The preparation method according to claim 13, characterized in that, The one-pot reaction is carried out in a pyridine solvent.

15. The preparation method according to claim 13, characterized in that, The Beckmann rearrangement reaction was carried out in the presence of a sulfonating agent.

16. The preparation method according to claim 15, characterized in that, The sulfonating agent is selected from p-methylbenzenesulfonyl chloride and p-nitrobenzenesulfonyl chloride.

17. The preparation method according to claim 13, characterized in that, The reduction reaction uses sodium borohydride as a reducing agent.

18. A compound represented by Formula 3: Formula 3.

19. A compound represented by Formula 4: Formula 4.

20. A compound represented by Formula 5: Formula 5.

21. A compound as shown in Formula 6: Formula 6.

22. Use of the compound of claim 1 or any one of claims 18 to 21 in the preparation of lycomycin.

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