6-methyl-3,5-dioxooctanoyl coenzyme a, and a preparation method and application thereof

Abstract

The present application relates to a kind of preparation method and application of 6-methyl-3,5-dioxoctanoyl coenzyme A, the preparation method with 2-methyl butyric acid as starting material, through activation, condensation, coenzyme A coupling such as step, 6-methyl-3,5-dioxoctanoyl coenzyme A is chemically synthesized, by LC-MS / MS, 1 HNMR etc. Identify its structure and purity. The present application successfully prepares and identifies nine carbon coenzyme A by chemical synthesis method for the first time, solves the core problem that the intermediate is unstable and cannot be obtained in vitro, provides stable substrate for in vitro acyl transfer; For the first time, QS-21 in vitro acyl transfer reaction is realized, and an in vitro catalytic system of "chemically synthesized intermediate + insect cell expressed enzyme" is established, which lays a foundation for QS-21 total synthesis process development.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, specifically to a 6-methyl-3,5-dioxooctyl coenzyme A, its preparation method, and its applications. Background Technology

[0002] QS-21 (Quillajasaponaria saponin) is a high-value triterpenoid saponin vaccine adjuvant derived from the soap tree (Quillajasaponaria). The 18-carbon acyl chain in its structure is crucial for its immunostimulatory activity, and the formation of this acyl chain depends on the stepwise transfer and linkage of two nine-carbon acyl units (6-methyl-3,5-dioxooctyl-CoA). The acyl transfer reaction is the core step in the biosynthesis of QS-21, directly determining the product's immunomodulatory activity and structural integrity, and is a key step in the large-scale in vitro synthesis of QS-21.

[0003] The existing QS-21 acyl transfer relies on an in vivo enzyme catalytic system, which uses polyketide synthase (PKSIII) to catalyze the reaction of 2-methylbutyryl-CoA with malonyl-CoA to generate a nine-carbon CoA intermediate, followed by acyl transfer mediated by BAHD family acyltransferases ACT2 and ACT3. However, this nine-carbon CoA intermediate is extremely unstable and cannot be prepared in vitro. Even in vivo, the intact molecule is difficult to detect, as it readily degrades spontaneously into inactive products such as C9-δ-lactone. This results in low efficiency of the acyl transfer reaction, becoming the core technical bottleneck for the in vitro synthesis of QS-21. Summary of the Invention

[0004] Based on the above description, the present invention provides a 6-methyl-3,5-dioxooctyl-CoA, its preparation method and application, aiming to provide an in vitro synthesis method for 6-methyl-3,5-dioxooctyl-CoA.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for preparing 6-methyl-3,5-dioxooctyl-CoA, comprising: S10. Chiral boron reagent, Z-crotonyllithium, 3-(tert-butyldimethylsiloxy)propionaldehyde and solvent were mixed and reacted under alkaline conditions. After neutralization with acidic solution, the mixture was extracted, washed, dried, filtered, and purified to obtain compound 2. S20. Compound 2, benzyl bromide, sodium di(trimethylsilyl)aminomethylene and solvent were mixed and reacted. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted, washed, dried, filtered and concentrated, and purified to obtain compound 3. S30. Compound 3, tetrabutylammonium fluoride and solvent are mixed and reacted, the solvent is concentrated and removed, the mixture is redissolved in dichloromethane, washed, dried, filtered and concentrated, and purified to obtain compound 4. S40. Compound 4, oxalyl chloride, triethylamine and solvent were mixed and reacted. The reaction was quenched with water, extracted, washed, dried, filtered and concentrated, and purified to obtain compound 5. S50. Compound 5, (R)-2-acetoxy-1,1,2-triphenylethanol, diisopropylaminolithium, magnesium bromide and solvent were heated and mixed under an inert atmosphere, and then cooled and mixed. The reaction was quenched by adding acetic acid and saturated sodium bicarbonate solution. The mixture was extracted, washed, dried, filtered and concentrated, and purified to obtain compound 6. S60. Compound 6, methanol, sodium methoxide and solvent are mixed and reacted. Ammonium chloride is added to quench the reaction. The mixture is then extracted, washed, dried, filtered, concentrated and purified to obtain compound 7. S70. Compound 7, imidazole, tert-butyldimethylchlorosilane and solvent were mixed and reacted, and sodium bicarbonate solution was added. The mixture was then extracted, dried, filtered, concentrated and purified to obtain compound 8. S80. Compound 8, methanol, and lithium hydroxide solution were mixed and reacted, purified, and dried to obtain compound 9; S90. Compound 9, coenzyme A, BOP, potassium carbonate and solvent were mixed and reacted, purified and dried to obtain compound 10; S100. Compound 10 is mixed with an aqueous solution of formic acid, reacted, purified, and dried to obtain compound 11; S110. Compound 11, palladium on carbon catalyst and solvent were mixed and reacted in a hydrogen atmosphere. The mixture was then filtered, concentrated and dried to obtain 6-methyl-3,5-dioxooctanoyl-CoA.

[0006] Furthermore, step S10 includes: S11. Mix potassium tert-butoxide with tetrahydrofuran, cool to below -78°C, add cis-2-butene and 1.6 M / L n-butyllithium, mix, and then mix at -50°C to -40°C for 20 min to 30 min. S12. Add (+)-B-diisomenthyl methoxyborane dropwise to the solution after the mixed reaction within 20 min, mix at -80℃ to -70℃ for 40 min to 50 min, add 20 mmol of tetrahydrofuran solution of 3-(tert-butyldimethylsiloxy)propionaldehyde, and mix at -80℃ to -70℃ for 6 h to 10 h. S13. Add 3M NaOH aqueous solution, mix at 20℃~30℃ for 12h~16h, add hydrochloric acid and mix, extract, wash, dry, filter, purify, and obtain compound 2; The molar ratio of potassium tert-butoxide, cis-2-butene, and n-butyllithium is 1:(6~8):(1~2). The molar ratio of potassium tert-butoxide, (+)-B-diisomenthyl methoxyborane and 3-(tert-butyldimethylsilyloxy)propionaldehyde is 1:(1~1.5):(1~2).

[0007] Furthermore, step S20 includes: S21. Mix compound 2, benzyl bromide, and tetrahydrofuran, cool to below 0°C, add sodium di(trimethylsilyl)aminodiaminodimethyl ... S22. Extract, wash, dry, filter, and purify to obtain compound 3; The molar ratio of compound 2, benzyl bromide and sodium di(trimethylsilyl)amino is 1:(6~7):(1~2).

[0008] Furthermore, step S30 includes: S31. Mix compound 3 with tetrahydrofuran, add tetrabutylammonium fluoride, mix at 20℃~30℃ for 30min~60min, concentrate to remove tetrahydrofuran, and obtain concentrated residue; S32. Dissolve the concentrated residue in dichloromethane, wash with ammonium chloride aqueous solution and water respectively, dry, filter, concentrate and purify to obtain compound 4; The molar ratio of compound 3 to tetrabutylammonium fluoride is 1:(1~2).

[0009] Furthermore, step S40 includes: S41. After mixing oxalyl chloride and dichloromethane, cool to below -78°C, add dimethyl sulfoxide and mix, add compound 4, and mix at -80°C to -70°C for 70 min to 80 min; S42. Triethylamine was added, and the mixture was reacted at 20℃~30℃ for 2h~3h. The reaction was quenched with water, extracted with dichloromethane, washed successively with hydrochloric acid, water, sodium bicarbonate solution and water, dried, filtered and concentrated, and purified to obtain compound 5. The molar ratio of compound 4, oxalyl chloride and triethylamine is 1:(1~2):(4~5).

[0010] Furthermore, step S50 includes: S51. Mix (R)-2-acetoxy-1,1,2-triphenylethanol with tetrahydrofuran, cool to below -78°C in a nitrogen atmosphere, add diisopropylaminolithium, and mix at 0°C for 1 h to obtain a lithium salt solution. S52. Mix magnesium bromide with tetrahydrofuran, cool to below -78°C in a nitrogen atmosphere, add lithium salt solution, mix at -78°C for 1 hour, and then cool to -115°C to obtain a premixed solution; S53. Compound 5 was mixed with tetrahydrofuran and added dropwise to the premixed solution at -115℃. The reaction was allowed to proceed for 2-3 hours. The reaction was quenched with acetic acid and saturated sodium bicarbonate solution. The mixture was then extracted, washed, dried, filtered, concentrated, and purified to obtain compound 6. The molar ratio of compound 5, (R)-2-acetoxy-1,1,2-triphenylethanol, diisopropylaminolithium and magnesium bromide is 1:(3~5):(7~9):(7~9).

[0011] Furthermore, step S60 includes: S61. Mix compound 6 with methanol, add sodium methoxide and react for 7-9 hours; S62. Add solid ammonium chloride, concentrate under reduced pressure to remove methanol, add saturated ammonium chloride solution, extract, dry, filter, concentrate, and purify to obtain compound 7.

[0012] Furthermore, step S70 includes: Compound 7, imidazole and N,N-dimethylformamide were mixed, tert-butyldimethylchlorosilane was added, and the mixture was stirred at 20℃~30℃ for 8h~12h. Sodium bicarbonate aqueous solution was added, and the mixture was extracted, dried, filtered, concentrated and purified to obtain compound 8. Step S80 includes: Compound 8 was mixed with methanol, and lithium hydroxide aqueous solution was added dropwise. The mixture was stirred at 20℃~30℃ for 1h~3h, purified, and dried to obtain compound 9. Step S90 includes: Coenzyme A was dissolved in a 1:1 volume ratio of tetrahydrofuran / water solution, compound 9 and BOP were added, and the mixture was stirred at 20℃~30℃ for 1 min~5 min. Potassium carbonate was added, and the mixture was stirred at 20℃~30℃ for 8 h~12 h. The mixture was purified and dried to obtain compound 10. Step S100 includes: Compound 10 was dissolved in a 1:1 formic acid / water solution and mixed at 20°C to 30°C for 6 to 10 hours. After purification and drying, compound 11 was obtained. Step S110 includes: Compound 11 was dissolved in a 1:1 volume ratio of tetrahydrofuran / water solution, and palladium on carbon catalyst was added. The mixture was then placed in a hydrogen atmosphere and reacted at 20°C to 30°C for 8 to 12 hours. The mixture was then filtered, concentrated, and dried to obtain 6-methyl-3,5-dioxooctanoyl-CoA.

[0013] The present invention also proposes a 6-methyl-3,5-dioxooctyl coenzyme, which is prepared according to the aforementioned method for preparing 6-methyl-3,5-dioxooctyl coenzyme A.

[0014] This invention also proposes the application of 6-methyl-3,5-dioxooctyl-CoA in the preparation of QS-21, wherein the preparation method of QS-21 includes: QA-TriR-FRXX, 6-methyl-3,5-dioxooctyl-CoA, ACT2 enzyme and buffer were mixed and reacted at 28℃~32℃ for 4 h~6 h, and then purified to obtain QS-21. The 6-methyl-3,5-dioxooctanoyl-CoA includes the 6-methyl-3,5-dioxooctanoyl-CoA as described above, or is prepared according to the preparation method of 6-methyl-3,5-dioxooctanoyl-CoA as described above.

[0015] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: 1. Breakthrough in intermediate preparation bottleneck: For the first time, nine-carbon coenzyme A was successfully prepared and identified through chemical synthesis, solving the core problem that the intermediate is unstable and cannot be obtained in vitro, and providing a stable substrate for in vitro acyl transfer; 2. C9 activity verification: The purified ACT2 expressed in the mammalian cell system can achieve accurate acyl transfer, thus proving the correctness of the intermediate; 3. Strong reaction controllability: Chemically synthesized nine-carbon coenzyme A is used directly in vitro as a donor, without relying on the host's endogenous metabolic system. The reaction conditions are mild and controllable, and the product yield and purity are stable. 4. Outstanding technological innovation: The in vitro acyl transfer reaction of QS-21 was achieved for the first time, and an in vitro catalytic system of "chemical synthesis intermediate + mammalian cell expression enzyme" was established, laying the foundation for the development of the total synthesis process of QS-21; 5. Great potential for large-scale production: Chemically synthesized nine-carbon coenzyme A can be prepared in batches, the mammalian cell expression system is mature and easy to scale up, the enzyme can be used immediately after purification, the operation is simple and the cost is controllable, making it suitable for industrial applications. Attached Figure Description

[0016] Figure 1 The chemical equation diagram is shown for the preparation method of 6-methyl-3,5-dioxooctanoyl coenzyme A provided in Example 1 of this invention. Figure 2 The chemical equation diagram for the preparation method of QS-21 provided in Example 2 of this invention; Figure 3 This is a GS-MS result diagram of the QS-21 precursor intermediate provided in Example 2 of this invention; Figure 4 This is a GS-MS result diagram of the QS-21 precursor intermediate of repeat group 1 provided in Example 2 of the present invention; Figure 5This is a GS-MS result diagram of the QS-21 precursor intermediate of repeat group 2 provided in Example 2 of the present invention; Figure 6 This is the HPLC result chromatogram of experimental group 1 provided in Example 2 of this invention; Figure 7 This is another HPLC result chromatogram of experimental group 1 provided in Example 2 of the present invention; Figure 8 This is the HPLC result chromatogram of replicate group 1 provided in Example 2 of the present invention; Figure 9 This is an HPLC result chromatogram of replicate group 2 provided in Example 2 of the present invention. Detailed Implementation

[0017] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings, which illustrate embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be thorough and complete.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0019] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0020] This invention prepares and identifies an unstable nine-carbon coenzyme A intermediate (6-methyl-3,5-dioxooctanoyl coenzyme A) through chemical synthesis. Functional expression of the BAHD family acyltransferase ACT2 is achieved using an insect cell sf9 expression system. After ultrasonic lysis, the protein ACT2 is purified by nickel column chromatography. Using the chemically synthesized nine-carbon coenzyme A as the acyl donor and the QS-21 precursor intermediate (QA-TriR-FRXX) as the acyl acceptor, the first step of the acyl transfer reaction is completed in vitro to generate the QS-21 intermediate (QA-TriR-FRXX-C9) with a nine-carbon acyl group linked at C-28.

[0021] I. Core Components and Fabrication: 1. Chemical synthesis of nine-carbon coenzyme A: Starting from 2-methylbutyric acid, 6-methyl-3,5-dioxooctanoyl coenzyme A was chemically synthesized through activation, condensation, and coenzyme A coupling steps. The results were analyzed by LC-MS / MS. 1 Its structure and purity (≥95%) were determined by techniques such as HNMR. 2. Catalytic enzyme and expression system: Acyltransferase ACT2 (derived from Quillajasaponaria, gene accession number OQ241420) contains a functional domain responsible for recognizing the nine-carbon coenzyme A donor and the QS-21 precursor receptor; the specific source of ACT2 is not limited. In some embodiments of the present invention, insect cells sf9 are selected as the expression host to ensure the correct folding and activity maintenance of ACT2. 3. Reaction buffer system: 50 mM PB (pH 7.5), 10 mM MgCl2, 5 mM DTT, 10% glycerol.

[0022] Example 1 This embodiment provides a nine-carbon coenzyme A, see reference. Figure 1 Its preparation method includes the following steps: first step: (3S,4S)-6-[(tert-butyldimethyl)siloxy]-4-hydroxy-3-methylhex-1-ene (compound 2): Weigh out potassium tert-butoxide (2.48 g, 24.0 mmol, 1.2 eq), dissolve it in THF (40 mL), and neutralize. Cool the solution to -78°C. Add cis-2-butene solution (15 mL, 167 mmol, 8.3 eq), followed by n-butyllithium (1.6 M / L, 15.5 mL, 24.8 mmol, 1.24 eq). After the addition is complete, stir at -78°C for 5 minutes, then raise the temperature to -45°C and stir for 20 minutes. Cool the orange solution to -78°C and add (+)-B-diisomenthyl methoxyborane (9.05 g, 28.6 mmol, 1.43 eq) dissolved in 50 mL of tetrahydrofuran. Add the solution dropwise over approximately 20 minutes. A white solution is obtained. Stir at -78°C for 45 minutes. A boron trifluoride diethyl ether compound (4.4 mL, 34.2 mmol, 1.71 eq) was added dropwise to the reaction solution. After 5 minutes, a solution of 3-(tert-butyldimethylsiloxy) propionaldehyde 1 (3.76 g, 20.0 mmol, 1.0 eq) in THF (12 mL) was added. The mixture was stirred at -78°C for 8 hours. Then, an aqueous solution of NaOH (30 mL, 3N) was added and the mixture was stirred at room temperature for 15 hours. The reaction mixture was neutralized with HCl in a dilute solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried (sodium sulfate), filtered, and the solvent was removed under reduced pressure. Rapid silica gel chromatography (hexane / ethyl acetate, 9:1) yielded compound 2 (4.34 g) as a colorless oil.

[0023] LCMS(ESI)m / z:CalcdforC 13 H 28 O2Si(M+H)245.2, found245.2. Step Two: (3S,4S)-4-(benzyl)oxy-6-[(tert-butyldimethyl)silyl]oxy-3-methylhex-1-ene (compound 3): A solution of compound 2 (2.6 g, 12 mmol, 1.0 eq) and benzyl bromide (10.0 mL, 81 mmol, 6.7 eq) in THF / DMF (2:1, 30 mL) was cooled to 0 °C. NaHMDS (1.0 M, 18 mL, 18 mmol, 1.5 eq) was added dropwise to the reaction mixture. The reaction mixture was heated to room temperature and stirred at this temperature for 2.5 h. The reaction was quenched with saturated ammonium chloride solution (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed, dried over brine (30 mL) (Na₂SO₄), filtered, and concentrated. Rapid column chromatography on silica gel (hexane / dichloromethane 1:1) gave compound 3 (0.33 g, 91% yield) as a colorless oil.

[0024] Step 3: (3S,4S)-4-benzyloxy-6-hydroxy-3-methylhex-1-ene (compound 4): Compound 3 (0.63 g, 1.886 mmol, 1.0 equivalence) was dissolved in THF (5.5 mL) solution and stirred at room temperature for 5 min. Tetrabutylammonium fluoride (1.0 M THF solution, 2.3 mL, 2.3 mmol, 1.2 eq) was added. The reaction was allowed to proceed at room temperature for 40 min, and then the tetrahydrofuran was removed by concentration. The residue was dissolved in dichloromethane (10 mL), washed with ammonium chloride aqueous solution (10 mL × 2) and water (10 mL), dried (Na₂SO₄), filtered, and concentrated. Silica gel chromatography yielded compound 4 as a colorless oil (0.39 g, 94% yield). 1 HNMR (600MHz, Chloroform- d )δ7.38–7.27(m,5H),5.92–5.85(m,1H),5.13–5.04(m,2H),4.65(d, J =11.3Hz, 1H), 4.52(d, J =11.3Hz,1H),3.79–3.67(m,2H),3.54(ddd, J =8.9,5.5,3.6Hz,1H),2.68–2.58(m,1H),1.80–1.67(m,2H),1.06(d, J =6.9Hz, 3H). Step 4: (3S,4S)-3-benzyloxy-4-methylhex-5-enal (compound 5): Oxaloyl chloride (1.35 mL, 11.94 mmol, 1.4 eq) was dissolved in DCM (30 mL), cooled to -78 °C, and DMSO (1.70 mL, 23.88 mmol, 2.8 eq) was added. The mixture was stirred for 15 minutes. Substance 4 (1.87 g, 8.53 mmol) was dissolved in dichloromethane (30 mL), and the mixture was stirred at -78 °C for 75 minutes. Triethylamine (4.8 mL, 34.12 mmol, 4.0 eq) was added. The mixture was then heated to room temperature and reacted for 2.5 hours. The reaction was quenched with water (30 mL) and extracted with dichloromethane (3 × 30 mL). The combined organic layers were washed successively with 50 mL of 1% HCl, 50 mL of 5% NaHCO3 solution, and 100 mL of water, dried (Na2SO4), filtered, concentrated, and purified by silica gel column chromatography to give a colorless oil 5 (1.77 g, 95% yield).

[0025] 1 HNMR(600MHz, CDCl3)δ9.79(dd,1H,J=2.0,1.5Hz),7.35-7.25(m,5H),5.83(m,1H),5.12(m,1H),5.09(m,1H),4.61(A B,1H,J=11.6Hz),4.56(AB,1H,J=11.6Hz),3.88(ddd,1H,J=7.8,5.9,4.0Hz),2.7-2.51(m,3H),1.08(d,3H,J=6.8Hz). Step 5: Compound 6: (R)-2-acetoxy-1,1,2-triphenylethanol (2.46 g, 7.15 mmol, 1.2 eq) was added to a reaction flask, followed by the addition of THF (20 mL). The mixture was evacuated and purged with nitrogen three times. The reaction mixture was cooled to -78 °C, and LDA (14.3 mmol, 2.4 eq) was added, but the mixture did not dissolve completely. The reaction mixture was heated to 0 °C, and the mixture was stirred at this temperature for one hour. MgBr2 (14.3 mmol, 2.4 eq) was added to THF (80 mL), and the mixture was evacuated and purged with nitrogen three times. The reaction mixture was cooled to -78 °C to obtain a lithium salt solution. The freshly prepared lithium salt solution was added to the suspension, and the mixture was stirred at -78 °C for one hour, then cooled to -115 °C. A THF solution of compound 5 (5 mL) was added dropwise at -115 °C, and the reaction was allowed to proceed for 2 hours. The reaction was then quenched with AcOH (1.5 mL) and saturated NaHCO3 solution (20 mL). The two separate aqueous layers were extracted with dichloromethane. The organic layer was dried (Na2SO4), filtered and concentrated, and purified by silica gel column chromatography to obtain intermediate 6 (dr 4:1) of the corresponding β-hydroxy ester.

[0026] LCMS(ESI)m / z:CalcdforC 36 H 38 O5(M-18+H)533.2,found533.2. Step 6: Compound 7: Intermediate 6 (3.3 g, 6.0 mmol) was dissolved in methanol (100 mL), and NaOMe (1 M, 6.0 mL, 6.0 mmol, 1.0 eq) was added at room temperature and reacted for 8 hours. 10.0 g of solid NH4Cl was added to the reaction mixture, and the methanol was removed by concentration under reduced pressure. A saturated NH4Cl solution was added, and the mixture was extracted with dichloromethane (100 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by silica gel column chromatography yielded a colorless liquid intermediate 7 (1.54 g, 89% yield).

[0027] 1 HNMR(600MHz,cdcl3)δ7.35-7.28(5H,m),5.95,-5.89(1H,m),5.11-5.07(3H,m),4.70-4.69(2H,d),4.48,-4.46(2H,d),4.22,-4 .21(1H,m),4.20-4.19(1H,m),2.69-2.68(2H,m),2.49-2.39(4H,m),1.69-1.63(4H,m),1.28-1.26(1h,m),1.24,-1.03(6H,dt). LCMS(ESI)m / z:CalcdforC 17 H 24 O4(M+H)293.2,found293.2. Step 7: Compound 8: Intermediate 7 (1.5 g, 5.14 mmol) and imidazole (0.56 g, 8.224 mmol) were added to DMF (20 mL) at room temperature. Then, TBSCl (1.82 g, 11.822 mmol) was added, and the mixture was stirred at room temperature for 10 hours. A solution of NaHCO3 (0.3 g) dissolved in H2O (80 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (4 × 80 mL). The organic phases were combined, dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography to obtain intermediate 8 (1.7 g, 82% yield), a colorless oily intermediate.

[0028] 1HNMR (600MHz, CDCl3) δ7.38-7.24(m,5H),5.89(m,1H),5.07(d,1H,J=1.3Hz),5.04(dt,1H,J=5.8,1.5Hz),4.65(AB,1H,J=11.4Hz),4.45(AB,1H,J=11. 4Hz),4.3(m,1H),3.65(s,3H),3.51(m,1H),2.64(m,1H),2.49(m,2H),1.74 -1.57(m,2H),1.04(d,3H,J=6.7Hz),0.87(s,9H),0.05(s,3H),0.03(s,3H). LCMS(ESI)m / z:CalcdforC 23 H 38 O4Si (M+H)407.2, found407.2. Step 8: Compound 9: Intermediate 8 (0.8 g, 1.97 mmol, 1.0 eq) was dissolved in methanol (4 mL), the reaction was cooled to 0 °C, and LiOH was added dropwise. . H2O (165 mg, 3.94 mmol, 2 eq) was dissolved in a solution of H2O (4 mL), and the mixture was heated to room temperature and reacted for 2 hours. The reaction mixture was purified, lyophilized, and yielded product 9 (500 mg, 64.7% yield).

[0029] 1 HNMR(600MHz, CDCl3)δ7.34-7.26(5H),5.91-5.85(1H,m),5.09(1H,m),5.07,(1H,m), 4.63(1H,s),4.61(1H,s),4.47(1H,s),4.45(1H,s),4.29-4.26(1H,m),3.44,-3.41(1 H,m),2.61-2.60(1H,m),2.52,-2.48(1H,m),2.42-2.39(1H,m),1.74,-1.72(1H,m),1 .70-1.67(1H,m),1.03(1H,dd),1.02(1H,dd),0.88(9H,s),0.07(3H,s),0.06(3H,s). LCMS(ESI)m / z:CalcdforC 22 H 36 O4Si(M+H)393.2, found393.2. Step 9: Compound 10: Coenzyme A (975 mg, 1.25 mmol, 1.0 eq) was weighed and dissolved in a THF / H₂O (5 mL, 1:1) mixture. Intermediate 9 (500 mg, 1.25 mmol, 1.0 eq) and BOP (1.100 g, 2.1 mmol, 1.6 eq) were added. The mixture was stirred at room temperature for 2 minutes, and K₂CO₃ (350 mg, 2.5 mmol, 2.0 eq) was added. The mixture was stirred overnight at room temperature. After purification, the solution was lyophilized to give intermediate 10 (500 mg, ~35% yield) as a white solid. 1 HNMR(600MHz,D2O)δ8.35(1H,S),8.30(8H,S),8.00(1H,S),7.11(4H,S),5.94(1H,S),5.62,(1H,S),4.73(2H,S)4.43(1H,S),4.13(1H, S),3.91-3.74(4H,m),3.4-3.14(8H,m),2.82,-2.74(2H,d),2.43-2.28(5H,d),1.44(2H,S),0.76(5H,S),0.61(12H,S),-0.21(6H,S). LCMS(ESI)m / z:CalcdforC 43 H 66 N7O 19 P3SSi(M+H)1138.3, found1138.3. Step 10 Compound 11: Compound 10 (500 mg, 1.25 mmol, 1.0 eq) was weighed and dissolved in a mixed solvent of HCOOH / H2O (5 mL, 1:1), and stirred at room temperature for 8 hours. The reaction was purified and lyophilized to give a white solid intermediate 11 (300 mg, ~67% yield).

[0030] LCMS(ESI)m / z:CalcdforC 37 H 52 N7O 19 P3S(M+H)1028.2,found1028.2. Step 11: Compound 12: Intermediate 11 (200 mg) was weighed and dissolved in a THF / H₂O (2.0 mL, 1:1) mixture. Pd / C (20 mg) was added to the reaction mixture, and the mixture was evacuated three times by nitrogen and three times by hydrogen. The reaction was carried out overnight at room temperature and 15 psi. The mixture was filtered, concentrated, reversed-phase prepared, and lyophilized to give a white solid product, COA-C9 (120 mg, ~72.7% yield).

[0031] 1 HNMR(400MHz,D2O)δ8.51(1H,S),8.31(2H,S),8.24(1H,S),6.11-6.10,(1H,d),4.77-4.76(2H,m),4. 51(1H,S),4.17-4.10(1H,m),3.94(1H,s),.3.78-3.74(2H,dd),3.63,-3.59(2H,dd),3.50-3.47(2H,d d),3.38,-3.35(2H,t),3.27-3.24(2H,t),3.03,-2.91(3H,td),2.80,-2.75(1H,dd),2.71,-2.66(1H, dd),2.36-2.33(2H,t),,1.60-1.562H,t),1.33-1.25(2H,m),1.10-1.05(1H,m),0.85,-0.69(12H,m). LCMS(ESI)m / z:CalcdforC 30 H 52 N7O 19 P3S(M+H), found939.23. Example 2 This embodiment proposes a method for preparing QS-21, see reference. Figure 2 This includes the following steps: Reaction system preparation: Add 2 μL of QS-21 precursor intermediate (molecular weight: 1513.59) substrate (final concentration 0.2 mM), ACT2 protein (final concentration 0.5 mg / mL), 1 μL of 1M MgCl2, and 50 μL of CoA-C9 donor (molecular weight 939) provided in Example 1 (final concentration 0.4 mM) to the centrifuge tube in sequence, and add PB buffer to a total volume of 200 μL and mix well.

[0032] Isothermal reaction: The above reaction system was incubated in a shaking incubator at 30℃ for 16 hours. After the reaction was completed, 400 μL of methanol was added to the reaction system and mixed thoroughly to terminate the reaction. (Molecular weight of the reaction product: 1685.81) Product testing 100 μL of the sample after the reaction was terminated in Example 2 was filtered through a 0.22 μm filter membrane, and the molecular weight of the product was verified by GS-MS (column: C18, size 4.6 mm × 250 mm). At the same time, the product content was detected by HPLC.

[0033] Experimental group 1: GS-MS results are as follows Figure 3 As shown, the precursor intermediate of QS-21 has a molecular weight of 1513.59. The precursor intermediate reacts with the donor to generate a product with a molecular weight of 1685.81. HPLC data is as follows... Figure 6 The peak elution time of the precursor intermediate of QS-21 in group CK is shown to be 12.4. Figure 7 The substrate and donor react to form a new peak, with the product peak appearing at 14.95.

[0034] Repeat group 1: GS-MS results are as follows Figure 4 As shown, the precursor intermediate reacts with the donor to generate a product with a molecular weight of 1685.81. HPLC results are as follows. Figure 8 The substrate and donor react to form a new peak, with the product peak appearing at 14.95.

[0035] Repeat group 2: GS-MS results are as follows Figure 5 As shown, the precursor intermediate reacts with the donor to generate a product with a molecular weight of 1685.81. HPLC results are as follows. Figure 9 The substrate and donor react to form a new peak, with the product peak appearing at 14.95.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0037] In summary, the technical solution of this application has the following beneficial technical effects: 1. Breakthrough in intermediate preparation bottleneck: For the first time, nine-carbon coenzyme A was successfully prepared and identified through chemical synthesis, solving the core problem that the intermediate is unstable and cannot be obtained in vitro, and providing a stable substrate for in vitro acyl transfer; 2. C9 activity verification: The purified ACT2 expressed in the mammalian cell system can achieve accurate acyl transfer, thus proving the correctness of the intermediate; 3. Strong reaction controllability: Chemically synthesized nine-carbon coenzyme A is used directly in vitro as a donor, without relying on the host's endogenous metabolic system. The reaction conditions are mild and controllable, and the product yield and purity are stable. 4. Outstanding technological innovation: The in vitro acyl transfer reaction of QS-21 was achieved for the first time, and an in vitro catalytic system of "chemical synthesis intermediate + mammalian cell expression enzyme" was established, laying the foundation for the development of the total synthesis process of QS-21; 5. Great potential for large-scale production: Chemically synthesized nine-carbon coenzyme A can be prepared in batches, the mammalian cell expression system is mature and easy to scale up, the enzyme can be used immediately after purification, the operation is simple and the cost is controllable, making it suitable for industrial applications.

Claims

1. A method for preparing 6-methyl-3,5-dioxooctanoyl-CoA, characterized in that, include: S10. (+)-B-diisomentheptyl methoxyborane, Z-crotonyllithium, 3-(tert-butyldimethylsiloxy)propionaldehyde and solvent were mixed and reacted under alkaline conditions. After neutralization with acidic solution, the mixture was extracted, washed, dried, filtered, and purified to obtain compound 2. S20. Compound 2, benzyl bromide, sodium di(trimethylsilyl)aminomethylene and solvent were mixed and reacted. The reaction was quenched by adding saturated ammonium chloride solution. The mixture was extracted, washed, dried, filtered and concentrated, and purified to obtain compound 3. S30. Compound 3, tetrabutylammonium fluoride and solvent are mixed and reacted, the solvent is concentrated and removed, the mixture is redissolved in dichloromethane, washed, dried, filtered and concentrated, and purified to obtain compound 4. S40. Compound 4, oxalyl chloride, triethylamine and solvent were mixed and reacted. The reaction was quenched with water, extracted, washed, dried, filtered and concentrated, and purified to obtain compound 5. S50. Compound 5, (R)-2-acetoxy-1,1,2-triphenylethanol, diisopropylaminolithium, magnesium bromide and solvent were heated and mixed under an inert atmosphere, and then cooled and mixed. The reaction was quenched by adding acetic acid and saturated sodium bicarbonate solution. The mixture was extracted, washed, dried, filtered and concentrated, and purified to obtain compound 6. S60. Compound 6, methanol, sodium methoxide and solvent are mixed and reacted. Ammonium chloride is added to quench the reaction. The mixture is then extracted, washed, dried, filtered, concentrated and purified to obtain compound 7. S70. Compound 7, imidazole, tert-butyldimethylchlorosilane and solvent were mixed and reacted, and sodium bicarbonate solution was added. The mixture was then extracted, dried, filtered, concentrated and purified to obtain compound 8. S80. Compound 8, methanol, and lithium hydroxide solution were mixed and reacted, purified, and dried to obtain compound 9; S90. Compound 9, coenzyme A, BOP, potassium carbonate and solvent were mixed and reacted, purified and dried to obtain compound 10; S100. Compound 10 is mixed with an aqueous solution of formic acid, reacted, purified, and dried to obtain compound 11; S110. Compound 11, palladium on carbon catalyst and solvent were mixed and reacted in a hydrogen atmosphere. The mixture was then filtered, concentrated and dried to obtain 6-methyl-3,5-dioxooctanoyl-CoA. The reaction flow for the preparation of 6-methyl-3,5-dioxooctanoyl-CoA is shown below: 。 2. The method for preparing 6-methyl-3,5-dioxooctanoyl-CoA according to claim 1, characterized in that, Step S10 includes: S11. Mix potassium tert-butoxide with tetrahydrofuran, cool to below -78°C, add cis-2-butene and 1.6 M / L n-butyllithium, mix, and then mix at -50°C to -40°C for 20 min to 30 min. S12. Add (+)-B-diisomenthyl methoxyborane dropwise to the solution after the mixed reaction within 20 min, mix at -80℃ to -70℃ for 40 min to 50 min, add 20 mmol of tetrahydrofuran solution of 3-(tert-butyldimethylsiloxy)propionaldehyde, and mix at -80℃ to -70℃ for 6 h to 10 h. S13. Add 3M NaOH aqueous solution, mix at 20℃~30℃ for 12h~16h, add hydrochloric acid and mix, extract, wash, dry, filter, purify, and obtain compound 2; The molar ratio of potassium tert-butoxide, cis-2-butene, and n-butyllithium is 1:(6~8):(1~2). The molar ratio of potassium tert-butoxide, (+)-B-diisomenthyl methoxyborane and 3-(tert-butyldimethylsilyloxy)propionaldehyde is 1:(1~1.5):(1~2).

3. The method for preparing 6-methyl-3,5-dioxooctanoyl-CoA according to claim 1, characterized in that, Step S20 includes: S21. Mix compound 2, benzyl bromide, and tetrahydrofuran, cool to below 0°C, add sodium di(trimethylsilyl)aminodiaminodimethyl ... S22. Extract, wash, dry, filter, and purify to obtain compound 3; The molar ratio of compound 2, benzyl bromide and sodium di(trimethylsilyl)amino is 1:(6~7):(1~2).

4. The method for preparing 6-methyl-3,5-dioxooctanoyl-CoA according to claim 1, characterized in that, Step S30 includes: S31. Mix compound 3 with tetrahydrofuran, add tetrabutylammonium fluoride, mix at 20℃~30℃ for 30min~60min, concentrate to remove tetrahydrofuran, and obtain concentrated residue; S32. Dissolve the concentrated residue in dichloromethane, wash with ammonium chloride aqueous solution and water respectively, dry, filter, concentrate and purify to obtain compound 4; The molar ratio of compound 3 to tetrabutylammonium fluoride is 1:(1~2).

5. The method for preparing 6-methyl-3,5-dioxooctanoyl-CoA according to claim 1, characterized in that, Step S40 includes: S41. After mixing oxalyl chloride and dichloromethane, cool to below -78°C, add dimethyl sulfoxide and mix, add compound 4, and mix at -80°C to -70°C for 70 min to 80 min; S42. Triethylamine was added, and the mixture was reacted at 20℃~30℃ for 2h~3h. The reaction was quenched with water, extracted with dichloromethane, washed successively with hydrochloric acid, water, sodium bicarbonate solution and water, dried, filtered and concentrated, and purified to obtain compound 5. The molar ratio of compound 4, oxalyl chloride and triethylamine is 1:(1~2):(4~5).

6. The method for preparing 6-methyl-3,5-dioxooctanoyl-CoA according to claim 1, characterized in that, Step S50 includes: S51. Mix (R)-2-acetoxy-1,1,2-triphenylethanol with tetrahydrofuran, cool to below -78°C in a nitrogen atmosphere, add diisopropylaminolithium, and mix at 0°C for 1 h to obtain a lithium salt solution. S52. Mix magnesium bromide with tetrahydrofuran, cool to below -78°C in a nitrogen atmosphere, add lithium salt solution, mix at -78°C for 1 hour, and then cool to -115°C to obtain a premixed solution; S53. Compound 5 was mixed with tetrahydrofuran and added dropwise to the premixed solution at -115℃. The reaction was allowed to proceed for 2-3 hours. The reaction was quenched with acetic acid and saturated sodium bicarbonate solution. The mixture was then extracted, washed, dried, filtered, concentrated, and purified to obtain compound 6. The molar ratio of compound 5, (R)-2-acetoxy-1,1,2-triphenylethanol, diisopropylaminolithium and magnesium bromide is 1:(3~5):(7~9):(7~9).

7. The method for preparing 6-methyl-3,5-dioxooctyl-CoA according to claim 1, characterized in that, Step S60 includes: S61. Mix compound 6 with methanol, add sodium methoxide and react for 7-9 hours; S62. Add solid ammonium chloride, concentrate under reduced pressure to remove methanol, add saturated ammonium chloride solution, extract, dry, filter, concentrate, and purify to obtain compound 7.

8. The method for preparing 6-methyl-3,5-dioxooctyl-CoA according to claim 1, characterized in that, Step S70 includes: Compound 7, imidazole and N,N-dimethylformamide were mixed, tert-butyldimethylchlorosilane was added, and the mixture was stirred at 20℃~30℃ for 8h~12h. Sodium bicarbonate aqueous solution was added, and the mixture was extracted, dried, filtered, concentrated and purified to obtain compound 8. Step S80 includes: Compound 8 was mixed with methanol, and lithium hydroxide aqueous solution was added dropwise. The mixture was stirred at 20℃~30℃ for 1h~3h, purified, and dried to obtain compound 9. Step S90 includes: Coenzyme A was dissolved in a 1:1 volume ratio of tetrahydrofuran / water solution, compound 9 and BOP were added, and the mixture was stirred at 20℃~30℃ for 1 min~5 min. Potassium carbonate was added, and the mixture was stirred at 20℃~30℃ for 8 h~12 h. The mixture was purified and dried to obtain compound 10. Step S100 includes: Compound 10 was dissolved in a 1:1 formic acid / water solution and mixed at 20°C to 30°C for 6 to 10 hours. After purification and drying, compound 11 was obtained. Step S110 includes: Compound 11 was dissolved in a 1:1 volume ratio of tetrahydrofuran / water solution, and palladium on carbon catalyst was added. The mixture was then placed in a hydrogen atmosphere and reacted at 20°C to 30°C for 8 to 12 hours. The mixture was then filtered, concentrated, and dried to obtain 6-methyl-3,5-dioxooctanoyl-CoA.

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