阅读说明：本技术 一种前列腺素中间体15α-羟基内酯的不对称还原合成方法 (Asymmetric reduction synthesis method of prostaglandin intermediate 15 alpha-hydroxy lactone ) 是由 陈芬儿 黄则度 竺科杰 孟歌 程荡 梁观峰 于 2021-01-27 设计创作，主要内容包括：本发明属于生物制药技术领域,具体为一种前列腺素中间体15α-羟基内酯的不对称还原合成方法。本发明包括：制备羰基还原酶的工程菌；制备工程菌的静息细胞悬浊液,并经过超声破碎或者加压破碎后得到含羰基还原酶的细胞上清液；经过蛋白纯化得到羰基还原酶的纯蛋白酶液；再与底物15-羰基前列腺素中间体(II)、葡萄糖脱氢酶、助溶剂、葡萄糖、辅因子混合,进行不对称羰基还原反应,制得15α-羟基前列腺素中间体(I)。所用羰基还原酶的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示。本发明可催化还原15-羰基前列腺素中间体(II)生成手性15α-羟基前列腺素中间体(I),所用生物催化剂方便易得,产物分离收率高、对映选择性优异。(The invention belongs to the technical field of biological pharmacy, and particularly relates to an asymmetric reduction synthesis method of a prostaglandin intermediate 15 alpha-hydroxy lactone. The invention comprises the following steps: preparing engineering bacteria of carbonyl reductase; preparing a resting cell suspension of the engineering bacteria, and obtaining cell supernatant containing carbonyl reductase after ultrasonic crushing or pressurized crushing; purifying the protein to obtain pure protease liquid of carbonyl reductase; then mixing with substrate 15-carbonyl prostaglandin intermediate (II), glucose dehydrogenase, cosolvent, glucose and cofactor to perform asymmetric carbonyl reduction reaction to prepare 15 alpha-hydroxy prostaglandin intermediate (I). The amino acid sequence of the carbonyl reductase is shown as SEQ ID NO.1 and SEQ ID NO. 2. The invention can catalyze and reduce the 15-carbonyl prostaglandin intermediate (II) to generate the chiral 15 alpha-hydroxy prostaglandin intermediate (I), the used biocatalyst is convenient and easy to obtain, the product separation yield is high, and the enantioselectivity is excellent.)

1. A prostaglandin intermediate 15 alpha-hydroxy lactone (I) asymmetric reduction synthesis method adopts carbonyl reductase as a catalyst, is used for asymmetric reduction of 15-carbonyl prostaglandin intermediate (II) to generate 15 alpha-hydroxy prostaglandin intermediate (I), and a reaction system comprises pure protease liquid of carbonyl reductase, substrate 15-carbonyl prostaglandin intermediate (II), glucose dehydrogenase, cosolvent, phosphate buffer, glucose and cofactor; the reaction formula of the asymmetric carbonyl reduction reaction is as follows:

in the formula, R¹Is hydrogen, C₁-C₁₀Alkyl or cycloalkyl, phenyl, benzyl, mono-or polysubstituted aryl or aralkyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, methyldiphenylsilyl, tetrahydropyranyl, formyl, C₁-C₁₀Alkyl acyl, benzoyl or biphenyl formyl, mono-or poly-substituted benzoyl or biphenyl formyl, R²Is C₁-C₁₀Alkyl or cycloalkyl, alkenyl, alkynyl, phenethyl or substituted phenethyl, phenoxymethylene or substituted phenoxymethylene;

the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO.1 or SEQ ID NO.2, or other carbonyl reductases which have an amino acid sequence with homology of more than 80% with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and keep catalytic activity.

2. The method according to claim 1, wherein the substrate concentration is 1.0 to 100g/L, the carbonyl reductase pure protein concentration is 0.2 g/L to 2g/L, the glucose dehydrogenase concentration is 0.1 g/L to 1 g/L, the glucose concentration is 5 to 750 g/L, and the cofactor concentration is 0 to 0.5mM in the asymmetric carbonyl reduction reaction.

3. The method according to claim 1, wherein the reaction temperature in the asymmetric carbonyl reduction reaction is 20 to 40%^oC, the pH of the reaction buffer is 5.0-8.0, and the buffer concentration is 100-250 mM.

4. The method according to claim 1, wherein in the asymmetric carbonyl reduction reaction, the cosolvent used is selected from dimethyl sulfoxide, N-dimethylformamide, and a solvent with high dielectric constant of N, N-dimethylacetamide; benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene aromatic solvents; n-hexane, cyclohexane nonpolar solvents; one or more of acetonitrile, ethyl acetate, dichloromethane, acetone, 1, 2-dichloroethane, methanol, ethanol, isopropanol polar solvent; the cosolvent accounts for 8-12% of the volume of the reaction system.

5. The method as claimed in claim 1, wherein in the reaction process, after the reaction is completed until the SFC detection substrate is completely exhausted, the target product is obtained by extracting 3-4 times by using equal volume of ethyl acetate, combining organic phases, washing 2 times by using saturated sodium chloride, drying by using anhydrous sodium sulfate and removing the organic solvent by reduced pressure distillation.

6. The method of claim 1, wherein the specific preparation process of the protease solution of carbonyl reductase is as follows:

(1) preparing engineering bacteria containing carbonyl reductase gene;

(2) preparing a resting cell suspension of the engineering bacteria;

(3) preparing a cell supernatant containing carbonyl reductase;

(4) preparing pure protease liquid of carbonyl reductase;

wherein the engineering bacteria contain plasmids pET28-KRED-WZL or pET28-SyADH, wherein the amino acid sequence of the carbonyl reductase KRED-WZL is shown as SEQ ID NO.1, the amino acid sequence of the carbonyl reductase SyADH is shown as SEQ ID NO.2, and the host cell is escherichia coli BL21(DE3).

7. The method of claim 6, wherein the preparation of the cell supernatant comprises: inoculating the engineering bacteria to a culture medium containing kanamycin, activating a shaking table, and expanding to OD₆₀₀When the value reaches 0.6-1.2, adding an inducer, continuing culturing, centrifugally collecting cells, resuspending by using buffer solution, crushing by ultrasound or pressurization, and centrifuging to obtain cell supernatant;

the inducer is IPTG, and the concentration of the inducer is 0.05mM-0.8 mM;

the culture conditions after adding the inducer are as follows: the culture temperature is 15-25 ℃, and the culture time is 8-24 h;

the buffer solution is potassium dihydrogen phosphate-dipotassium hydrogen phosphate; the buffer concentration is 30-300 mM.

8. The method of claim 7, wherein the method of preparing the pure protease solution comprises: mixing the cell supernatant with Ni resin, shaking for 20min in a shaking table, filtering to remove clear liquid, washing the resin with eluent containing low-concentration imidazole in 2-4 times of column volume, eluting pure protease with eluent containing high-concentration imidazole, collecting eluent with protein concentration greater than 3mg/mL, and mixing the eluates to obtain pure protease solution.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a synthesis method of a prostaglandin intermediate 15 alpha-hydroxy lactone.

Background

15 α -hydroxy lactones of formula (I) are known to be key intermediates in the synthesis of prostaglandins:

in the formula R¹Is hydrogen, C₁-C₁₀Alkyl or cycloalkyl, phenyl, benzyl, mono-or polysubstituted aryl or aralkyl, Trimethylsilyl (TMS), Triethylsilyl (TES), tert-butyldimethylsilyl (TBS), methyldiphenylsilyl (TBDPS), Tetrahydropyranyl (THP), formyl, C₁-C₁₀Alkyl acyl, benzoyl or biphenyl formyl, mono-substituted or poly-substituted benzoyl or biphenyl formyl and the like. R²Is C₁-C₁₀Alkyl or cycloalkyl, alkenyl, alkynyl, phenethyl or substituted phenethyl, phenoxymethylene or substituted phenoxymethylene.

The first reduction of the 15-carbonyl group of prostaglandins was by B.Samuelsson (R) ((R))Angew. Chem. Int. Ed. 1965, 4, 410) Report of sodium borohydride and sodium boron deuteride on PGE in deuteration experiments₁The metabolite of (b) undergoes carbonyl reduction of the lower side chain. E.j. Corey (J. Am. Chem. Soc., 1969, 91, 5675) On the basis of the original method, zinc borohydride is used for reducing the 15-site carbonyl group of the prostaglandin intermediate, a pair of epimers are obtained with high yield, and the ratio of the epimers to the prostaglandin intermediate is close to 1: 1. there is a significant waste of the other half isomer.

E. J. Corey(J. Am. Chem. Soc., 1971, 93, 1491;J. Am. Chem. Soc., 1972. 94, 8616) The trialkyl borane compound derived from (+) limonene and thiabendazole borane is adopted to carry out asymmetric reduction on the carbonyl group at the 15 th position of a prostaglandin intermediate, and the optimal result is 15 alpha: 15 beta = 92:8 by screening different substituents at the 11 th position.

E. J. Corey(J. Am. Chem. Soc., 1987, 109, 9725) A diastereoselectivity of 15 α: 15 β = 90:10 is obtained at room temperature by asymmetric reduction of the carbonyl group at position 15 of the prostaglandin intermediate with borane as a reducing agent using a catalytic amount of a Corey-Bakshi-Shibata (CBS) catalyst. Significant improvements over the previous stringent conditions have been achieved, but the results for diastereoselectivity remain unsatisfactory.

Chisa Ohta（J. Org. Chem. 2009, 74,8298) The subject group finds that (-) -diisopinocampheylchloroborane can well reduce the carbonyl group at the 15 th position of prostaglandin, the obtained diastereoselectivity result is superior to that of a CBS catalyst, and the diastereoselectivity result of 15 alpha: 15 beta = 95:5 can be obtained. However, this method also has the above-mentioned disadvantages: in order to obtain excellent diastereoselective results, the reaction needs to be carried out at low temperatures of-40 ℃.

There has also been some research into this reaction in the field of biocatalysis. K. Kieslich (US4247635) The Klebsiella pneumoniae ATCC 20110(Kloeckera jensenii ATCC 20110) has better carbonyl reduction selectivity on a 15-carbonyl prostaglandin intermediate with a lower side chain substituted by phenoxymethyl or substituted phenoxymethyl, and the isolation yield of the product reaches 60%. R, Diego (J. Mol. Cat. B: Enzym., 2015, 114, 7) Subject groupIt was found that a single microorganism, Pichia anomala, could simultaneously perform carbonyl reduction, double bond reduction and ester deprotection of the prostaglandin key intermediate, and finally yielded bimatoprost intermediate in 82% yield and 97% de and latanoprost intermediate in 62% yield and 97% de. However, since these yeast wild fungi contain a plurality of enzyme catalysts with different catalytic properties, such biotransformation processes involve a variety of different chemical reactions, and systematic and precise control of the transformation process is required to obtain a certain target product with high selectivity and high yield, but impurities of different degrees are inevitably generated.

Disclosure of Invention

The invention aims to overcome the defects of the existing chemical reduction technology and biological reduction technology and provide an enzymatic reduction synthesis method of prostaglandin intermediate 15 alpha-hydroxy lactone (I) with mild condition, high stereoselectivity and high yield.

The invention provides a reductive synthesis method of an enzyme-catalyzed prostaglandin intermediate 15 alpha-hydroxy lactone (I), which adopts carbonyl reductase (KRED) as a catalyst and is used for asymmetrically reducing the 15-carbonyl prostaglandin intermediate (II) to generate the 15 alpha-hydroxy prostaglandin intermediate (I), wherein a reaction system comprises pure protease liquid of the carbonyl reductase (KRED), a substrate 15-carbonyl prostaglandin intermediate (II), glucose dehydrogenase, a cosolvent, a phosphate buffer solution, glucose and a cofactor; the reaction formula of the asymmetric carbonyl reduction reaction is as follows:

in the formula, R¹Is hydrogen, C₁-C₁₀Alkyl or cycloalkyl, phenyl, benzyl, mono-or polysubstituted aryl or aralkyl, Trimethylsilyl (TMS), Triethylsilyl (TES), tert-butyldimethylsilyl (TBS), methyldiphenylsilyl (TBDPS), Tetrahydropyranyl (THP), formyl, C₁-C₁₀Alkyl acyl, benzoyl or biphenylyl, mono-or polysubstitutedBenzoyl or dibenzoyl and the like. R²Is C₁-C₁₀Alkyl or cycloalkyl, alkenyl, alkynyl, phenethyl or substituted phenethyl, phenoxymethylene or substituted phenoxymethylene.

Specifically, in the asymmetric carbonyl reduction reaction, the concentration of the substrate is 1.0-100g/L, the concentration of pure protein of carbonyl reductase (KRED) is 0.2-2 g/L, the concentration of glucose dehydrogenase is 0.1-1 g/L, the concentration of glucose is 5-750 g/L, and the concentration of cofactor is 0-0.5 mM.

Preferably, in the asymmetric carbonyl reduction reaction, the reaction temperature is 20-40 DEG^oAnd C, the pH value of the reaction buffer solution is 5.0-8.0. More preferably, the reaction temperature is 25-30 ℃, the pH of the reaction buffer is 6.5-7.5, and the buffer concentration is 100-250 mM.

In the asymmetric carbonyl reduction reaction, the cosolvent used is a solvent with high dielectric constant such as dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide and the like; aromatic solvents such as benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, and the like; nonpolar solvents such as n-hexane and cyclohexane; one or more polar solvents such as acetonitrile, ethyl acetate, dichloromethane, acetone, 1, 2-dichloroethane, methanol, ethanol, isopropanol, etc. The cosolvent accounts for 8-12% of the volume of the reaction system. The preferable solvent is dimethyl sulfoxide as a cosolvent, and the volume ratio of the dimethyl sulfoxide is 10%.

In the whole reaction process, after the reaction is carried out until the SFC detection substrate is completely exhausted, extracting for 3-4 times by using ethyl acetate with the same volume, combining organic phases, washing for 2 times by using saturated sodium chloride, drying by using anhydrous sodium sulfate, and removing the organic solvent by reduced pressure distillation to obtain the target product.

In the invention, the specific preparation process of the pure protease liquid of the carbonyl reductase (KRED) is as follows:

(1) preparing engineering bacteria containing carbonyl reductase (KRED) genes;

(2) preparing a resting cell suspension of the engineering bacteria;

(3) preparing a cell supernatant containing carbonyl reductase;

(4) preparing pure protease liquid of carbonyl reductase;

wherein, the amino acid sequence of the carbonyl reductase is shown as SEQ ID NO.1 or SEQ ID NO.2, or other carbonyl reductases which have an amino acid sequence with homology of more than 80% with the amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.2 and keep catalytic activity.

Wherein the engineering bacteria contain plasmid pET28-KRED-WZL or pET28-SyADH, wherein the amino acid sequence of the carbonyl reductase KRED-WZL is shown as SEQ ID NO.1, the amino acid sequence of the carbonyl reductase SyADH is shown as SEQ ID NO.2, and the host cell is escherichia coli BL21(DE3)

In the step (3), the preparation method of the cell supernatant comprises the following steps: inoculating the engineering bacteria to a culture medium containing kanamycin, activating a shaking table, and expanding to OD₆₀₀When the value reaches 0.6-1.2, adding an inducer, continuing culturing, centrifugally collecting cells, resuspending with a buffer solution, crushing by ultrasound or pressurization, and centrifuging to obtain the cell supernatant.

Preferably, the inducer is IPTG and the concentration of the inducer is 0.05mM-0.8 mM.

Preferably, the culture conditions after addition of the inducer are: the culture temperature is 15-25 ℃, and the culture time is 8-24 h.

Preferably, the buffer is potassium dihydrogen phosphate-dipotassium hydrogen phosphate; the buffer concentration is 30-300 mM.

In the step (4), the preparation method of the pure protease solution comprises the following steps: mixing the cell supernatant with Ni resin, shaking for 20min in a shaking table, filtering to remove clear liquid, washing the resin with eluent containing low-concentration imidazole in 2-4 times of column volume, eluting pure protease with eluent containing high-concentration imidazole, collecting eluent with protein concentration greater than 3mg/mL, and mixing the eluates to obtain pure protease solution.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention constructs engineering bacteria containing carbonyl reductase, applies the engineering bacteria to asymmetrically reduce 15-carbonyl prostaglandin intermediate (II) to generate 15 alpha-hydroxy prostaglandin intermediate (I), and provides a new biocatalysis manufacturing approach for preparing the important pharmaceutical intermediate;

(2) the invention adopts carbonyl reductase to carry out asymmetric carbonyl reduction reaction, the diastereoselectivity of the reaction is superior to that of chemical reduction, and the defect of low diastereoselectivity in the original chemical reduction way is overcome;

(3) the carbonyl reductase which is convenient and easy to obtain is used as a catalyst, and the whole reaction system is carried out under mild conditions, so that the low-temperature condition required in the chemical reduction process is avoided.

(4) Compared with the prior biotransformation technology, the invention adopts the simple and easily obtained escherichia coli engineering bacteria to realize the stereoselective reduction of the 15-carbonyl of the prostaglandin intermediate containing the side chains under different types, the reaction condition is simple, the complex regulation and control are not needed, the separation yield is excellent (80-97 percent), and the diastereoselectivity is excellent (94-98 percent de).

Drawings

FIG. 1 is SDS-PAGE electrophoresis chart of KRED-WZL protein pure enzyme and SyADH protein pure enzyme protein induced and expressed by genetically engineered bacteria. Wherein, M is nucleic acid Marker, 1: SyADH protein pure enzyme expressed by genetic engineering bacteria in an induction mode, 2: the KRED-WZL protein pure enzyme is induced and expressed by the genetic engineering bacteria.

FIG. 2-1 isRacemic-HPLC analysis of the hydroxyprostaglandin intermediate (Ia).

FIG. 2-2 is an HPLC analysis chart of dr value measured for 15. alpha. -hydroxyprostaglandin intermediate (Ia).

FIGS. 2-3 are 15 alpha-hydroxyprostaglandin intermediates (Ia)¹HNMR spectrogram.

FIGS. 2-4 are 15 alpha-hydroxyprostaglandin intermediates (Ia)¹³C NMR spectrum.

FIG. 3-1 isRacemic-HPLC analysis profile of hydroxyprostaglandin intermediate (Ib).

FIG. 3-2 is an HPLC analysis chart of dr value measured from 15. alpha. -hydroxyprostaglandin intermediate (Ib).

FIGS. 3-3 are 15 alpha-hydroxyprostaglandin intermediates (Ib)¹HNMR spectrogram.

FIGS. 3-4 are 15 alpha-hydroxyprostaglandin intermediates (Ib)¹³C NMR spectrum.

FIG. 4-1 isRacemic-hydroxy groupHPLC analysis of prostaglandin intermediate (Ic).

FIG. 4-2 is an HPLC analysis chart of dr value measured for 15 α -hydroxyprostaglandin intermediate (Ic).

FIGS. 4-3 are of 15 α -hydroxyprostaglandin intermediate (Ic)¹HNMR spectrogram.

FIGS. 4-4 are of 15 α -hydroxyprostaglandin intermediate (Ic)¹³C NMR spectrum.

FIG. 5-1 is a drawingRacemic-HPLC analysis of the hydroxyprostaglandin intermediate (Id).

FIG. 5-2 is a HPLC analysis chart of dr value measured for 15. alpha. -hydroxyprostaglandin intermediate (Id).

FIGS. 5-3 are 15 alpha-hydroxyprostaglandin intermediates (Id)¹HNMR spectrogram.

FIGS. 5-4 are of 15 alpha-hydroxyprostaglandin intermediates (Id)¹³C NMR spectrum.

Detailed Description

Example 1 construction, inducible expression, protein purification of carbonyl reductase Gene engineering bacteria

Transforming expression host BL21(DE3) with plasmid pET28-KRED-WZL or pET28-SyADH, inoculating BL21(DE3) -pET28-KRED-WZL or BL21(DE3) -pET28-SyADH into 5mL liquid LB test tube culture medium containing kanamycin resistance, activating the culture in a shaker at 37 ℃ for 8 hours, transferring the activated culture into liquid LB shake flask culture medium containing kanamycin resistance according to 1 percent of transfer amount, and performing constant temperature shaking culture in a fermentation culture medium for 3 hours at 37 ℃ and 200rpm until the thallus concentration reaches OD₆₀₀And when the concentration is 0.8, adding 0.1mM IPTG (final concentration), inducing at 18 ℃ for 18h, centrifuging at 8500rpm for 10min to collect cells, resuspending with 100mM potassium phosphate buffer solution with pH7.0, crushing for 15 min by 55% ultrasonic power, centrifuging to obtain cell supernatant containing KRED-WZL (or SyADH), loading with Ni resin, washing, eluting and collecting pure protease liquid containing KRED-WZL (or SyADH).

Example 2 preparation of 15 α -hydroxy prostaglandin intermediate (Ia)

To a 100 mL round bottom flask was added 103 mg (5 mM) of 15-oxo prostaglandin intermediate (IIa), 4mL (10% v/v) of DMSO (10 mM) as a final concentration of the co-substrate glucose, and NADP as a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 56mg/mL, reaction protein concentration of 1mg/mL) was taken, and then placed in a constant-temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product is analyzed by SFC, the dr value is 98:2, and column chromatography separation is carried out to obtain 93.3 mg of target product, the yield is as follows: 91 percent.¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 7.0 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), 7.20 – 7.08 (m, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.90 – 6.80 (m, 1H), 6.78 – 6.66 (m, 1H), 5.84 (dd, J = 15.6, 7.0 Hz, 1H), 5.74 (dd, J = 15.6, 5.0 Hz, 1H), 5.31 (q, J = 5.6 Hz, 1H), 5.15 – 4.94 (m, 1H), 4.53 (dq, J = 8.1, 4.2 Hz, 1H), 3.93 (dd, J = 9.4, 3.6 Hz, 1H), 3.88 – 3.76 (m, 1H), 2.95 – 2.75 (m, 3H), 2.68 – 2.58 (m, 1H), 2.54 (d, J = 16.0 Hz, 1H), 2.44 (d, J = 4.0 Hz, 1H), 2.28 (dd, J = 15.3, 4.1 Hz, 1H).¹³C NMR (101 MHz, CDCl3) δ 176.33, 165.93, 159.04, 146.11, 139.85, 134.97, 130.97, 130.85, 130.33, 130.19, 128.96, 128.25, 128.19, 127.30, 127.21, 121.56, 115.02, 113.08, 83.29, 78.98, 71.78, 70.06, 54.26, 42.71, 37.60, 34.97.

SFC analysis conditions: a chromatographic column: chiralpak IF-3 (4.6 mm. times.250 mm), mobile phase: CO 2₂MeOH = 75:25, flow rate: 3mL/min, sample size: 10 μ L, detection wavelength: 275nM, column temperature: 30 ℃, SFC pressure preparation: 140 bar, HPLC profile of the racemic product is shown in FIG. 2-1, and retention time of 15 α -hydroxyprostaglandin intermediate (Ia)18.618min, the retention time of the 15 β -hydroxyprostaglandin intermediate (Ia') was 21.765 min. The HPLC pattern of the carbonyl reduction product of the enzyme reaction is shown as 2-2, the main configuration is determined as 15 alpha-hydroxy prostaglandin intermediate (Ia), and the retention time is 18.834 min.

Example 3, preparation of 15 α -Hydroxyprostaglandin intermediate (Ib)

To a 50 mL round bottom flask was added 58 mg (10 mM) of 15-oxo prostaglandin intermediate (IIb), 1.2 mL (10% v/v) of DMSO (final concentration 20 mM) of glucose as a co-substrate, and final concentration 0.2mM of NADP⁺8.05 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 12mL, and 2.4mL (20% v/v) of the GDH cell supernatant, 300. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 20mg/mL, reaction protein concentration 0.5 mg/mL) was taken, and then placed in a constant temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product is analyzed by SFC, the dr value is 97:3, column chromatography separation is carried out to obtain 51.6 mg of target product, and the yield is as follows: 89 percent.¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 7.3 Hz, 2H), 7.52 – 7.43 (m, 2H), 7.43 – 7.35 (m, 1H), 7.28 – 7.23 (m, 2H), 7.02 – 6.92 (m, 1H), 6.86 (d, J = 8.2 Hz, 2H), 5.84 (dd, J = 15.7, 6.8 Hz, 1H), 5.75 (dd, J = 15.6, 5.0 Hz, 1H), 5.31 (q, J = 5.5 Hz, 1H), 5.17 – 4.97 (m, 1H), 4.61 – 4.46 (m, 1H), 3.96 (dd, J = 9.4, 3.6 Hz, 1H), 3.83 (dd, J = 9.4, 7.4 Hz, 1H), 2.96 – 2.74 (m, 3H), 2.71 – 2.44 (m, 3H), 2.28 (ddd, J = 15.5, 5.0, 1.9 Hz, 1H).¹³C NMR (101 MHz, CDCl₃) δ 176.43, 165.95, 158.30, 146.08, 139.87, 131.06, 130.74, 130.21, 129.57, 128.96, 128.24, 127.30, 127.20, 121.38, 114.60, 83.39, 79.12, 71.55, 70.22, 54.28, 42.70, 37.62, 35.03.

SFC analysis conditions: a chromatographic column: chiralpak AD-H (4.6 mm. times.250 mm), mobile phase: CO 2₂MeOH = 50:50, flow rate: 2 mL/min, sample size: 5 μ L, detection wavelength: 275nM, column temperature: 30 ℃, SFC pressure preparation: 130 bar, HPLC chromatogram of racemic product is shown in FIG. 3-1, retention time of 15 α -hydroxyprostaglandin intermediate (Ib) is 12.190 min, and retention time of 15 β -hydroxyprostaglandin intermediate (Ib') is 11.059 min. The HPLC pattern of the carbonyl reduction product of the enzyme reaction is shown as 3-2, the main configuration is determined as 15 alpha-hydroxy prostaglandin intermediate (Ib), and the retention time is 12.427 min.

EXAMPLE 4 preparation of 15 α -hydroxyprostaglandin intermediate (Ic)

To a 50 mL round bottom flask was added 116 mg (20 mM) of 15-oxo prostaglandin intermediate (IIc), 1.2 mL (10% v/v) of DMSO (40 mM) as a final concentration of the co-substrate glucose, and NADP as a final concentration of 0.2mM⁺8.05 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 12mL, and 2.4mL (20% v/v) of the GDH cell supernatant, 300. mu.L of KRED-WZL pure protease solution (protein concentration: 33 mg/mL, reaction protein concentration 0.8 mg/mL) in example 1 was taken, and then placed in a constant temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product is analyzed by SFC, the dr value is 99:1, and column chromatography separation is carried out to obtain 93.2 mg of target product, the yield is as follows: 80 percent.¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.51 – 7.43 (m, 2H), 7.43 – 7.36 (m, 1H), 7.28 – 7.23 (m, 2H), 7.22 – 7.09 (m, 3H), 5.70 (dd, J = 15.5, 5.6 Hz, 1H), 5.61 (dd, J = 15.5, 6.8 Hz, 1H), 5.27 (q, J= 5.7 Hz, 1H), 5.13 – 4.94 (m, 1H), 4.14 (q, J = 6.1 Hz, 1H), 2.90 – 2.73 (m, 3H), 2.73 – 2.56 (m, 3H), 2.51 (d, J = 17.5 Hz, 1H), 2.26 (ddd, J = 15.4, 5.3, 1.9 Hz, 1H), 1.87 – 1.79 (m, 2H).¹³C NMR (101 MHz, CDCl₃) δ 176.47, 165.99, 146.10, 141.59, 139.86, 136.07, 130.20, 128.96, 128.70, 128.45, 128.41, 128.25, 128.22, 127.30, 127.21, 125.96, 83.29, 79.06, 71.40, 54.06, 42.71, 38.68, 37.57, 34.92, 31.58.

SFC analysis conditions: a chromatographic column: chiralpak IF-3 (4.6 mm. times.250 mm), mobile phase: CO 2₂MeOH = 75:25, flow rate: 3mL/min, sample size: 5 μ L, detection wavelength: 275nM, column temperature: 30 ℃, SFC pressure preparation: 130 bar, HPLC chromatogram of racemic product is shown in FIG. 4-1, retention time of 15 α -hydroxyprostaglandin intermediate (Ic) is 15.418 min, and retention time of 15 β -hydroxyprostaglandin intermediate (Ic') is 12.547 min. The HPLC chromatogram of the carbonyl reduction product of the enzyme reaction is shown in 4-2, the main configuration is determined as 15 alpha-hydroxy prostaglandin intermediate (Ic), and the retention time is 14.928 min.

Example 5 preparation of 15 α -Hydroxyprostaglandin intermediate (Id)

To a 100 mL round bottom flask was added 178 mg (10 mM) of 15-oxo prostaglandin intermediate (IId), 4mL (10% v/v) of DMSO (30 mM) as a final concentration of the co-substrate glucose, and NADP as a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 16.8mg/mL, reaction protein concentration 0.3 mg/mL) was taken, and then placed in a constant temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product is analyzed by SFC, the dr value is 99:1, and the 173.2 mg target product is obtained by column chromatography separation, the yield is as follows: 97 percent.¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 7.1 Hz, 2H), 7.53 – 7.45 (m, 2H), 7.45 – 7.37 (m, 1H), 5.69 (dd, J = 15.5, 5.5 Hz, 1H), 5.62 (dd, J = 15.5, 6.8 Hz, 1H), 5.30 (q, J = 5.7 Hz, 1H), 5.18 – 5.00 (m, 1H), 4.12 (q, J = 6.1 Hz, 1H), 2.97 – 2.73 (m, 3H), 2.70 – 2.59 (m, 1H), 2.55 (d, J = 16.6 Hz, 1H), 2.28 (ddd, J = 15.4, 5.2, 1.9 Hz, 1H), 1.73 (d, J = 15.7 Hz, 2H), 1.59 – 1.41 (m, 2H), 1.38 – 1.26 (m, 5H), 0.92 – 0.83 (m, 3H).¹³C NMR (101 MHz, CDCl₃) δ 176.50, 165.97, 146.07, 139.88, 136.37, 130.19, 128.96, 128.36, 128.26, 128.23, 127.28, 127.19, 83.35, 79.13, 72.19, 54.05, 42.72, 37.58, 37.23, 34.94, 31.69, 24.98, 22.57, 14.02.

SFC analysis conditions: a chromatographic column: chiralpak IF-3 (4.6 mm. times.250 mm), mobile phase: CO 2₂MeOH = 75:25, flow rate: 3mL/min, sample size: 5 μ L, detection wavelength: 275nM, column temperature: 30 ℃, SFC pressure preparation: 130 bar, HPLC profile of the racemic product is shown in FIG. 5-1, retention time of 15 α -hydroxyprostaglandin intermediate (Id) is 9.106 min, and retention time of 15 β -hydroxyprostaglandin intermediate (Id') is 8.008 min. The HPLC pattern of the carbonyl reduction product of the enzyme reaction is shown as 5-2, the main configuration is determined as 15 alpha-hydroxy prostaglandin intermediate (Id), and the retention time is 9.336 min.

Example 6 preparation of 15 α -Hydroxyprostaglandin intermediate (Id)

To a 100 mL round bottom flask was added 89 mg (5 mM) of 15-oxo prostaglandin intermediate (IId), 4mL (10% v/v) of DMSO (10 mM) was added as a final concentration of the co-substrate glucose, and NADP was added to a final concentration of 0.2mM⁺Then, 19 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL and 8mL (20% v/v) of the GDH cell supernatant, and 8.52. mu.L of SyADH pure protease solution (protein concentration: 4.7mg/mL, reaction protein concentration: 1mg/mL) in example 1 was taken, and the resulting solution was stirred at 600 rpm in a thermostatic water bath at 30 ℃ for 33 hours. After the reaction is finished, extracting the mixture by using ethyl acetate3 times, the organic phases were combined, washed with saturated sodium chloride and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 76.6 mg of a target product, wherein the yield is as follows: 86 percent.¹H NMR，¹³C NMR and SFC analysis conditions were the same as in example 5.

EXAMPLE 7 preparation of 15 α -hydroxyprostaglandin intermediate (Ie)

To a 100 mL round bottom flask was added 750 mg (50 mM) of 15-oxo prostaglandin intermediate (IIe), 4mL (10% v/v) of DMSO (final concentration 150 mM) of glucose as a co-substrate, and a final concentration of 0.2mM NADP⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 84mg/mL, reaction protein concentration of 1.5 mg/mL) was taken, and then placed in a constant temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product is analyzed by SFC, the dr value is 99:1, column chromatography separation is carried out to obtain 705 mg of target product, and the yield is as follows: 94 percent.¹H NMR (400 MHz, CDCl₃) δ: 8.03-7.99 (m, 2H), 7.61-7.54 (m, 1H), 7.49-7.41 (m, 2H), 5.71-5.56 (m, 1H), 5.30-5.04 (m, 2H), 4.14-4.06 (m, 1H), 2.94-2.46 (m, 5H), 2.24 (ddd, 1H, J = 1.7, 5.2, 15.2 Hz), 1.70 (s, 1H), 1.56-1.42 (m, 2H), 1.36-1.17 (m,5H), 0.87 (m, J = 6.7 Hz, 3H).¹³C NMR (101 MHz, CDCl₃) δ: 176.30, 165.97, 136.31, 133.20, 129.50,129.38, 128.36, 128.23,83.24, 78.90, 72.13, 53.84, 42.63, 37.44, 37.13, 34.78, 31.64, 24.89, 22.44,13.94.

EXAMPLE 8 preparation of 15 α -hydroxyprostaglandin intermediate (If)

To a 100 mL round bottom flask was added 3.75 g (70 mM) of 15-oxo prostaglandin intermediate (IIf), 4mL (10% v/v) of DMSO was added as a 140 mM final concentration of the co-substrate glucose, and a final concentration of 0.5mM NADP⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 56mg/mL, reaction protein concentration of 1mg/mL) was taken, and then placed in a constant-temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. After concentration, the crude product is analyzed by SFC, the dr value is 99:1, column chromatography separation is carried out to obtain 3.5 g of target product, and the yield is as follows: 93 percent.¹H NMR (400 MHz, CDCl₃) δ 5.56 (dd, J = 15.3, 7.4 Hz, 1H), 5.41 (dd, J = 15.3, 8.5 Hz, 1H), 4.88 (td, J = 7.1, 3.2 Hz, 1H), 4.01 (q, J = 6.8 Hz, 1H), 3.90 (q, J = 7.8 Hz, 1H), 3.75 (s, 1H), 2.94 (s, 1H), 2.70 (dd, J = 18.1, 9.5 Hz, 1H), 2.62 – 2.46 (m, 2H), 2.39 (dd, J = 18.1, 1.7 Hz, 1H), 2.22 (q, J = 8.7 Hz, 2H), 1.90 (ddd, J = 14.7, 8.1, 3.3 Hz, 1H), 1.63 – 1.49 (m, 1H), 1.49 – 1.39 (m, 1H), 1.36 – 1.20 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 177.18, 137.01, 130.45, 82.55, 76.37, 73.01, 56.26, 42.48, 39.73, 37.18, 34.10, 31.76, 25.25, 22.71, 14.15.

Example 9 preparation of 15 α -hydroxy prostaglandin intermediate (Ig)

To a 100 mL round bottom flask was added 56mg (5 mM) of 15-oxo prostaglandin intermediate (IIg), 4mL (10% v/v) of DMSO (10 mM) as a final concentration of the co-substrate glucose, and NADP as a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant was collected715. mu.L of KRED-WZL purified protease solution (protein concentration: 28 mg/mL, reaction protein concentration: 0.5 mg/mL) in example 1 was stirred in a 30 ℃ thermostatic water bath at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 51 mg of a target product, wherein the yield is as follows: 90 percent.¹H NMR (400 MHz, CDCl₃) δ 5.56 (dd, J = 15.3, 7.4 Hz, 1H), 5.41 (dd, J = 15.3, 8.5 Hz, 1H), 4.88 (td, J = 7.1, 3.2 Hz, 1H),4.01 (q, J = 6.8 Hz, 1H), 3.90 (q, J = 7.8 Hz, 1H), 3.75 (s, 1H), 3.41 (s, 3H), 2.94 (s, 1H), 2.70 (dd, J = 18.1, 9.5 Hz, 1H), 2.62 – 2.46 (m, 2H), 2.39 (dd, J = 18.1, 1.7 Hz, 1H), 2.22 (q, J = 8.7 Hz, 2H), 1.90 (ddd, J = 14.7, 8.1, 3.3 Hz, 1H), 1.63 – 1.49 (m, 1H), 1.49 – 1.39 (m, 1H), 1.36 – 1.20 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H).

EXAMPLE 10 preparation of 15 α -hydroxyprostaglandin intermediate (Ih)

To a 100 mL round bottom flask was added 69 mg (5 mM) of 15-oxo prostaglandin intermediate (IIh), 4mL (10% v/v) of DMSO (10 mM) was added as a final concentration of the co-substrate glucose, and NADP was added to a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 67 mg/mL, reaction protein concentration of 1.2 mg/mL) was taken, and then placed in a constant temperature water bath at 40 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 61 mg of a target product, wherein the yield is as follows: 88 percent.¹H NMR (400 MHz, CDCl₃) δ 7.13-7.33 (m, 2H), 6.80 – 6.98 (m, 3H), 5.56 (dd, J = 15.3, 7.4 Hz, 1H), 5.41 (dd, J = 15.3, 8.5 Hz, 1H), 4.88 (td, J = 7.1, 3.2 Hz, 1H), 4.01 (q, J = 6.8 Hz, 1H), 3.90 (q, J = 7.8 Hz, 1H), 3.75 (s, 1H), 2.94 (s, 1H), 2.70 (dd, J = 18.1, 9.5 Hz, 1H), 2.62 – 2.46 (m, 2H), 2.39 (dd, J = 18.1, 1.7 Hz, 1H), 2.22 (q, J = 8.7 Hz, 2H), 1.90 (ddd, J = 14.7, 8.1, 3.3 Hz, 1H), 1.63 – 1.49 (m, 1H), 1.49 – 1.39 (m, 1H), 1.36 – 1.20 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H).

EXAMPLE 11 preparation of 15 α -Hydroxyprostaglandin intermediate (Ii)

To a 100 mL round bottom flask was added 216 mg (15 mM) of 15-oxo prostaglandin intermediate (IIi), 4mL (10% v/v) of DMSO (40 mM) as a final concentration of the co-substrate glucose, and NADP as a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 11.2 mg/mL, reaction protein concentration 0.2 mg/mL) was taken, and then placed in a constant temperature water bath at 40 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 186 mg of a target product, wherein the yield is as follows: 87 percent.¹H NMR (400 MHz, CDCl₃) δ 7.23-7.43 (m, 5H), 5.56 (dd, J = 15.3, 7.4 Hz, 1H), 5.41 (dd, J = 15.3, 8.5 Hz, 1H), 4.88 (td, J = 7.1, 3.2 Hz, 1H), 4.63 (s, 2H),4.01 (q, J = 6.8 Hz, 1H), 3.90 (q, J = 7.8 Hz, 1H), 3.75 (s, 1H), 2.94 (s, 1H), 2.70 (dd, J = 18.1, 9.5 Hz, 1H), 2.62 – 2.46 (m, 2H), 2.39 (dd, J = 18.1, 1.7 Hz, 1H), 2.22 (q, J = 8.7 Hz, 2H), 1.90 (ddd, J = 14.7, 8.1, 3.3 Hz, 1H), 1.63 – 1.49 (m, 1H), 1.49 – 1.39 (m, 1H), 1.36 – 1.20 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H).

EXAMPLE 12 preparation of 15 α -hydroxyprostaglandin intermediate (Ij)

To a 100 mL round bottom flask was added 71 mg (5 mM) of 15-oxo prostaglandin intermediate (IIj), 4mL (10% v/v) of DMSO (10 mM) was added as a final concentration of the co-substrate glucose, and NADP was added to a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 56mg/mL, reaction protein concentration of 1mg/mL) was taken, and then placed in a constant temperature water bath at 20 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 63 mg of a target product, wherein the yield is as follows: 90 percent.¹H NMR (400 MHz, CDCl₃) δ5.56 (dd, J= 15.3, 7.4 Hz, 1H), 5.41 (dd, J = 15.3, 8.5 Hz, 1H), 4.88 (td, J = 7.1, 3.2 Hz, 1H), 4.50-4.68 (m, 1H),4.01 (q, J = 6.8 Hz, 1H), 3.90 (q, J = 7.8 Hz, 1H), 3.75 (s, 1H), 3.64-3.47 (m, 2H), 2.94 (s, 1H), 2.70 (dd, J = 18.1, 9.5 Hz, 1H), 2.62 – 2.46 (m, 2H), 2.39 (dd, J = 18.1, 1.7 Hz, 1H), 2.22 (q, J = 8.7 Hz, 2H), 1.90 (ddd, J = 14.7, 8.1, 3.3 Hz, 1H), 1.74 – 1.49 (m, 7H), 1.49 – 1.39 (m, 1H), 1.36 – 1.20 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H).

EXAMPLE 13 preparation of 15 α -hydroxyprostaglandin intermediate (Ik)

To a 100 mL round bottom flask was added 76 mg (5 mM) of 15-oxo prostaglandin intermediate (IIk), 4mL (10% v/v) of DMSO, and a final concentration of 10mM adjuvantSubstrate glucose, final concentration 0.2mM NADP⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 7.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of SyADH pure protease solution (protein concentration: 56mg/mL, reaction protein concentration: 1mg/mL) in example 1 was taken, and then placed in a constant-temperature water bath at 25 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 65 mg of a target product, wherein the yield is as follows: 85 percent.¹H NMR (400 MHz, CDCl₃) δ 5.56 (dd, J = 15.5; 5.7 Hz, 1H), 5.45 (dd, J = 15.5; 7.5 Hz, 1H), 4.93 (td, J = 6.9; 2.3 Hz, 1H), 4.07 (q, J = 6.1 Hz, 1H), 3.97 (q, J = 5.8 Hz, 1H), 2.84-2.54 (m, 2H), 2.52-2.21 (m, 3H), 1.96 (ddd, J = 14.7; 5.4; 2.2 Hz, 1H), 1.67-1.20 (m, 8H), 0.88 (m, 12H), 0.04 (s, 6H).

EXAMPLE 14, 15 preparation of alpha-hydroxyprostaglandin intermediate (Il)

To a 100 mL round bottom flask was added 62 mg (5 mM) of 15-oxo prostaglandin intermediate (IIl), 4mL (10% v/v) of DMSO was added as a final concentration of 10mM of glucose as a co-substrate and 0.2mM of NADP as a final concentration⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 6.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 56mg/mL, reaction protein concentration of 1mg/mL) was taken, and then placed in a constant-temperature water bath at 25 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 53 mg of a target product, wherein the yield is as follows: 85 percent.¹H NMR (400 MHz, CDCl₃) δ 5.56 (dd, J = 15.3, 7.4 Hz, 1H), 5.41 (dd, J = 15.3, 8.5 Hz, 1H), 4.88 (td, J = 7.1, 3.2 Hz, 1H), 4.01 (q, J = 6.8 Hz, 1H), 3.90 (q, J = 7.8 Hz, 1H), 3.75 (s, 1H), 2.94 (s, 1H), 2.70 (dd, J = 18.1, 9.5 Hz, 1H), 2.62 – 2.46 (m, 2H), 2.39 (dd, J = 18.1, 1.7 Hz, 1H), 2.22 (q, J = 8.7 Hz, 2H), 2.02 (s, 3H), 1.90 (ddd, J = 14.7, 8.1, 3.3 Hz, 1H), 1.63 – 1.49 (m, 1H), 1.49 – 1.39 (m, 1H), 1.36 – 1.20 (m, 6H), 0.87 (t, J = 6.7 Hz, 3H).

Example 15 preparation of 15 α -hydroxy prostaglandin intermediate (Im)

To a 100 mL round bottom flask was added 75 mg (5 mM) of 15-oxo prostaglandin intermediate (IIm), 4mL (10% v/v) of DMSO (10 mM) as a final concentration of the co-substrate glucose, and NADP as a final concentration of 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 8.0) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 56mg/mL, reaction protein concentration of 1mg/mL) was taken, and then placed in a constant-temperature water bath at 35 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 61 mg of a target product, wherein the yield is as follows: 80 percent.¹H NMR (400 MHz, CDCl₃) δ: 8.03-7.99 (m, 2H), 7.61-7.54 (m, 1H), 7.49-7.41 (m, 2H), 5.71-5.56 (m, 1H), 5.30-5.04 (m, 2H), 4.14-4.06 (m, 2H), 2.94-2.46 (m, 4H), 1.80 (s, 3H), 1.56-1.42 (m, 2H), 1.36-1.17 (m,3H), 0.87 (d, J = 6.7 Hz, 3H).

EXAMPLE 16 preparation of 15 α -hydroxyprostaglandin intermediate (In)

Adding 15-carbonyl prostaglandin into 100 mL round bottom bottle296 mg (20 mM) of intermediate (IIn), 4mL (10% v/v) of DMSO (70 mM) as a final substrate, glucose as a co-substrate, and NADP as a final substrate, 0.2mM⁺26.8 mL of 100mM potassium phosphate buffer solution (pH = 6.5) was added to make the final reaction volume 40mL, and 8mL (20% v/v) of the GDH cell supernatant, 715. mu.L of the KRED-WZL pure protease solution of example 1 (protein concentration: 56mg/mL, reaction protein concentration of 1mg/mL) was taken, and then placed in a constant-temperature water bath at 30 ℃ and stirred at 600 rpm for reaction for 33 hours. After the reaction, the mixture was extracted with ethyl acetate 3 times, and the organic phases were combined, washed with saturated sodium chloride, and dried over anhydrous sodium sulfate. And (3) analyzing the concentrated crude product by using SFC, wherein the dr value is 99:1, and performing column chromatography separation to obtain 244 mg of a target product, wherein the yield is as follows: 80 percent.¹H NMR (400 MHz, CDCl₃) δ: 8.03-7.99 (m, 2H), 7.61-7.54 (m, 1H), 7.49-7.41 (m, 2H), 5.71-5.56 (m, 1H), 5.30-5.04 (m, 2H), 4.14-4.06 (m, 2H), 2.94-2.46 (m, 4H), 1.75-1.42 (m, 9H) 。

Sequence listing

<110> university of Compound Dan

<120> asymmetric reduction synthesis method of prostaglandin intermediate 15 alpha-hydroxy lactone

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 244

<212> PRT

<213> KRED-WZL

<400> 1

Met Asn Phe Thr Asp Lys Asn Val Ile Ile Thr Gly Gly Ser Ala Gly

1 5 10 15

Ile Gly Leu Ala Thr Ala Lys Lys Phe Ile Ala Lys Glu Ala Asn Val

20 25 30

Leu Val Thr Gly Arg Asn Thr Glu Ser Leu Asp Lys Ala Ser Val Thr

35 40 45

Ile Asn Ser Pro Lys Phe Lys Thr Leu Ala Ser Asp Ile Ser Lys Leu

50 55 60

Ala Asp Ile Ala Ala Leu Glu Lys Glu Val Ser Glu Ser Gly Lys Lys

65 70 75 80

Val Asp Val Leu Val Leu Asn Ala Gly Ile Ala Lys Gln Phe Ser Ile

85 90 95

Glu Glu Thr Thr Glu Glu Val Phe Asp Asp Leu Phe Asn Ile Asn Val

100 105 110

Lys Gly Leu Phe Phe Thr Leu Gln Lys Leu Ile Pro His Leu Ala Glu

115 120 125

Gly Ala Ser Ile Ile Leu Ile Ser Ser Gly Val Ser Val Ser Gly Tyr

130 135 140

Ala Gln Met Gly Ala Tyr Ala Ala Thr Lys Ser Ala Val Asp Ala Ile

145 150 155 160

Ala Arg Thr Ala Ala Ile Glu Leu Ala Asp Arg Lys Ile Arg Val Asn

165 170 175

Thr Val Ala Pro Gly Leu Thr Asp Thr Pro Met Asn Gln Gln Thr Pro

180 185 190

Glu Asp Ile Lys Asn Ala Ile Ala Ala Ala Val Pro Leu Lys Arg Ile

195 200 205

Gly Glu Ala Glu Glu Ile Ala Asn Ala Ile Val Phe Phe Ala Ser Ser

210 215 220

Glu Ala Ser Tyr Ile Ser Gly Ser Tyr Leu Ser Val Asp Gly Gly Val

225 230 235 240

Thr Ile Arg Arg

<210> 2

<211> 262

<212> PRT

<213> SyADH

<400> 2

Met Thr Thr Leu Pro Thr Val Leu Ile Thr Gly Ala Ser Ser Gly Ile

1 5 10 15

Gly Ala Thr Tyr Ala Glu Arg Phe Ala Arg Arg Gly His Asp Leu Val

20 25 30

Leu Val Ala Arg Asp Lys Val Arg Leu Asp Ala Leu Ala Ala Arg Leu

35 40 45

Arg Asp Glu Ser Gly Val Ala Val Glu Ala Leu Gln Ala Asp Leu Thr

50 55 60

Arg Pro Ala Asp Leu Ala Ala Val Glu Ile Arg Leu Arg Glu Asp Ala

65 70 75 80

Arg Ile Gly Ile Leu Ile Asn Asn Ala Gly Met Ala Gln Ser Gly Gly

85 90 95

Phe Val Gln Gln Thr Ala Glu Gly Ile Glu Arg Leu Ile Thr Leu Asn

100 105 110

Thr Thr Ala Leu Thr Arg Leu Ala Ala Ala Val Ala Pro Arg Phe Val

115 120 125

Gln Ser Gly Thr Gly Ala Ile Val Asn Ile Gly Ser Val Val Gly Phe

130 135 140

Ala Pro Glu Phe Gly Met Ser Ile Tyr Gly Ala Thr Lys Ala Phe Val

145 150 155 160

Leu Phe Leu Ser Gln Gly Leu Asn Leu Glu Leu Ser Pro Ser Gly Ile

165 170 175

Tyr Val Gln Ala Val Leu Pro Ala Ala Thr Arg Thr Glu Ile Trp Gly

180 185 190

Arg Ala Gly Ile Asp Val Asn Thr Leu Pro Glu Val Met Glu Val Asp

195 200 205

Glu Leu Val Asp Ala Ala Leu Val Gly Phe Asp Arg Arg Glu Leu Val

210 215 220

Thr Ile Pro Pro Leu His Val Ala Ala Arg Trp Asp Ala Leu Asp Gly

225 230 235 240

Ala Arg Gln Gly Leu Met Ser Asp Ile Arg Gln Ala Gln Ala Ala Asp

245 250 255

Arg Tyr Arg Pro Glu Ala

260