JPH01141600A - Production of optically active 3-hydroxypyrrolidine derivative - Google Patents

Abstract

PURPOSE:To selectively produce an optically active 3-hydroxypyrrolidine derivative by stereoselectively hydrolyzing a mixture of (R)- and (S)-N-benzyl-3- acyloxypyrrolidines with a microorganism, etc. CONSTITUTION:A mixture of (R)- and (S)-N-benzyl-3-acyloxypyrrolidines expressed by formula I (X represents H or 1-17C alkyl or alkenyl group) is prepared. A microorganism of the genus Pseudomonas, Mucor, etc., or an enzyme derived from the above-mentioned microorganism is reacted with the mixture to optically resolve the mixture into an optically active N-benzyl-3- hydroxypyrrolidine expressed by formula II and an optically active N-benzyl-3- acyloxypyrrolidine expressed by formula III (X is same as formula I) which is sterically antipodal thereto. The respective optically active compounds are then separated and collected.

Description

Detailed Description of the Invention (Industrial Application Field) (In the formula, X represents hydrogen or a substituted or unsubstituted alkyl group or alkenyl group having 1 to 17 carbon atoms.)
R) and (8) -N-benzyl-3-acyloxypyrrolidine mixture is stereoselectively hydrolyzed to form the general formula [
1 [] A microorganism or enzyme having stereoselective esterase activity that produces optically active N-benzyl-3-hydroxypyrrolidine represented by the general formula [ID (i
t) and (S) -N-benzyl-3-acyloxypyrrolidine mixture to form optically active N-benzyl-
3-Hydroxypyrrolidine [I[] and optically active N-benzyl-3-acyloxypyrrolidine represented by the general formula [white (in the formula, X is the same as above)] which is sterically opposite to this, Optically active N-benzyl-3-hydroxypyrrolidine and its enantiomer optically active N-benzyl-3-hydroxypyrrolidine, which is characterized by dividing and separating and collecting each optically active compound.
The present invention relates to a method for producing 3-acyloxypyrrolidine.

Optically active N-benzyl-3 produced by the present invention
-Hydroxypyrrolidine and N-benzyl-3-acyloxypyrrolidine are useful synthetic intermediates for pharmaceuticals, agricultural chemicals, and the like.

(Prior art and problems) Regarding the production method of optically active (S) -N-benzyl-3-hydroxypyrrolidine, a method of synthesizing L-malic acid as a raw material (Japanese Patent Application Laid-Open No. 61-63652) is known. However, since it is partially racemized, it is necessary to further perform optical resolution with D-mandelic acid to improve optical purity. In addition, a method of reducing N-benzyl-8-pyrroline using an asymmetric reducing agent (→-diisopiinocampheylborane) is known [Journal of American Chemical Society (J, Am , Oh
em, Soc, ), ios, 2049.1986].

In addition, for (S)-3-hydroxypyrrolidine that does not have a benzyl group at the N-position, a method of deriving it from L-glutamic acid (Synthetake Communication, 16.1815
．． 1986), and the reverse (R) form is obtained by decarboxylating trans-4-hydroxy-L-proline (Japanese Patent Application Laid-Open No. 6O-23328), which is synthesized by benzylation. It is also possible. However, these methods are difficult to consider as economical industrial production methods because they require long steps and special reagents.

The present invention provides optically active N-benzyl-3-hydroxypyrrolidine represented by the formula [ID] and optically active N-benzyl-3-acyloxypyrrolidine represented by the general formula [1'], which are useful as synthetic raw materials for pharmaceuticals, etc. It is economically produced by optical resolution by stereoselectively hydrolyzing a mixture of (R) and (S) -N-benzyl-3-acyloxypyrrolidine, which can be easily chemically synthesized, using microorganisms or enzymes. It is intended for this purpose.

(Means and effects for solving the problem) First, the inventor,
reported that racemic (R8)-N-benzyl-3-acyloxypyrrolidine [Il], which is a mixture of equal amounts of the (R) and (8) isomers, is a novel compound that has never been reported before. However, we discovered that this product is inexpensive and can be easily synthesized by reacting (R5)-N-benzyl-3-hydroxypyrrolidine, which can be easily synthesized from L-malic acid, with a fatty acid halide, etc. A patent application was filed. Next, the present inventors discovered that this new compound (R) and (S)
-N-benzyl-3-acyloxypyrrolidine mixture [
1] was considered and investigated by asymmetric hydrolysis using microorganisms or enzymes for optical resolution.

As a result, we discovered the existence of microorganisms and enzymes that can stereoselectively hydrolyze the compound [ID] to produce an optically active compound [■]. 3-Hydroxypyrrolidine [■
] and optically active N-benzyl-3-acyloxypyrrolidine [I'], which is sterically antipodal. In addition, optically active N-benzyl-3-acyloxypyrrolidine [1']fi can be further hydrolyzed using alkaline hydrolysis, microorganisms or enzymes discovered in the present invention, or reverse without stereoselectivity to achieve stereoconfiguration. It is possible to obtain optically active N-benzyl-3-hydroxypyrrolidine retaining the (R) and (8) N-benzyl-3-hydroxy pyrrolidines using microorganisms or enzymes with any stereospecificity. Both pyrrolidine enantiomers can be obtained simultaneously.

The invention of this production method enables the production of both enantiomers of optically active N-benzyl-3-hydroxypyrrolidine and optically active N-benzyl-3-acyloxypyrrolidine more economically than conventional methods. became.

01) and (S)-N-benzyl-3-acyloxypyrrolidine represented by the general formula [1] used as the substrate of the present invention are racemic bodies that are a mixture of equal amounts of the (R) and (8) bodies. However, a mixture containing a larger amount of either the (R) isomer or the (8) isomer may also be used. Further, as the substituent X, hydrogen, a linear or branched alkyl group having 1 to 17 carbon atoms, an alkenyl group, unsubstituted, or substituted with halogen or the like can be used. For example, methyl group, ethyl group, ethylene group, n-propyl group, isopropyl group, 1-propylene group, n-butyl group, isobutyl group, 1-butylonone group, n-pentyl group,
n-hexyl group, n-heptyl group, n-octyl group, n
-nonacyl group, n-decyl group.

n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecyl group.

Straight chain or branched unsubstituted alkyl groups such as n-octadecyl group, alkenyl groups, etc., such as chloromethyl group, dichloromethyl group, trifluoromethyl group, 2-chloroethyl group, 3-chloropropyl group.

Examples include halogen-substituted alkyl groups such as 4-chlorobutyl group.

Compound El) was stereoselectively hydrolyzed to obtain optically active [I
Microorganisms or enzymes that have stereoselective esterase activity that produce [I] can be easily found by conducting the following screening.

For example, as a specific example, 10 mJ of a nutrient medium in which microorganisms can grow is placed in a large test tube, seedlings of the test bacteria are planted, and then cultured with shaking at 25 to 35°C for 1 to 3 days. Next, 2π (W/V) of (R8)-N-benzyl-3-butyryloxypyrrolidine was added to this culture solution, and
The reaction is carried out at 0 to 35°C for 1 to 3 days while adjusting the pH to 5 to 7 with NaOH. To progress the hydrolysis, the reaction solution was adjusted to pf-L2.0 with sulfuric acid, the acetic acid produced was extracted with twice the amount of ethyl acetate, and this was analyzed by gas chromatography (filling material, Shimadzu FAL-M 6%/Simalite, φ0. 3×1
00 cm column, temperature 140°C). The time-dependent changes in each reaction solution are observed, and a strain that exhibits an extremely slow reaction is selected for the first time when about half the amount of acetic acid produced relative to the added substrate is produced. Next, change the reaction scale to 500 mJ, perform the same cultivation and hydrolysis as above, and after completion, use 500 ml of dichloromethane! Extract 4 times and remove solvent under reduced pressure. This was loaded onto a silica gel column (Wakogel C-200, 250, F),
Unreacted N-benzyl-3-butyryloxypyrrolidine was eluted with hegisan/ethyl acetate (10/2). Furthermore, N-benzyl-3-hydroxypyrrolidine produced is eluted with ethyl acetate. By concentrating both components, oily optically active N-benzyl-3-butyryloxypyrrolidine and optically active N-benzyl-3-hydroxypyrrolidine are obtained.

When optically active N-benzyl-3-butyryloxypyrrolidine is hydrolyzed with NaOH, it becomes optically active N-benzyl-3-hydroxypyrrolidine without loss of optical activity, which is purified by distillation and compared to By measuring the optical rotation, it can be easily determined whether the microorganism has stereoselective esterase activity or not. Screening for enzymes originating from each dish can be carried out in the same manner by dissolving 0.5I of the enzyme in 10 ml of 0.1 M phosphate buffer instead of the microbial culture solution. In this way, microorganisms or enzymes that can stereoselectively hydrolyze N-benzyl-3-butyryloxypyrrolidine can also stereoselectively hydrolyze each of the 14 N-benzyl-3-acyloxypyrrolidines mentioned above. Shows hydrolytic activity.

(B, ) and (8) -N-benzyl-3-acyloxypyrrolidine mixtures were stereoselectively hydrolyzed and (R)
-CIO and (s)-CI'] Examples of microorganisms that have stereoselective esterase activity include microorganisms belonging to the genus Pseudomonas, Mucor, and Alcaligenes.
s) 1FO3081, Pseudomonas-7: Pseudomonas aerugi-
nosa) I F 0 3080, Mucor javanicus I
FO4569, Alcaligenes eutrophus (Ale
al igenseutrophus) ATCC1
7697 etc. can be used.

The culture solution itself can be used for these microorganisms, but enzymes can also be extracted and purified from the culture solution or bacterial cells. Also, commercially available enzymes can be used directly.

For example, lipoprotein lipase (LPL Amano 3, derived from Pseudomonas genus, manufactured by Ohno Pharmaceutical), lipase M-AP
10 (derived from the genus Mucor, manufactured by Ohno Seiyaku Aji), Lipase PL (derived from the genus Alcaligenes, manufactured by Meito Sangyo ■), and the like are suitable for this purpose.

On the other hand, a mixture of (R) and (8) -N-benzyl-3-acyloxypyrrolidine was stereoselectively hydrolyzed, and (
8) -Cl] and (R) -C as a microorganism having stereoselective esterase activity that gives white
There are microorganisms belonging to the genus Phycomyces, etc., and more specifically, Rhizopus jabonicus (Rhizopus j.
aponius) I F O4758, Rhizopus delemer (Rhizopus delemer) I
FO4697, Phycomyces nitens (Phyco
myees n1tens) I F 0 5694,
etc. are available.

The culture solution itself can be used for these microorganisms, but enzymes can also be extracted and purified from the culture solution or bacterial cells. Also, commercially available enzymes can be used directly.

For example, Lipase I'N (derived from Phycomyces sp., manufactured by Wako Pure Chemical Industries, Ltd.), Lipase (derived from Rhizops sp., manufactured by Seikagaku Kogyo ■), Lipase "Saiken J100 (derived from Rhizopus sp., manufactured by Osaka Bacteria Research Institute ■), Neurase" (derived from the genus Rhizopus, manufactured by Tenfu Pharmaceutical) are suitable for this purpose.

In the hydrolysis reaction, substrate [I] is added to a microbial culture solution, enzyme extract, or enzyme aqueous solution at a rate of 0.5 to 75π (W/V).
Addition is carried out at a concentration range of 10°C to 55°C with stirring. The amount of enzyme depends on the enzyme activity, but usually [
I] in a range from equivalent to 1/1000th.

The PLL of the reaction is carried out in the range of 5.0 to 8.5, but if the pH changes during the hydrolysis reaction, it is desirable to maintain the pH at the optimum level with an appropriate acid or alkaline aqueous solution.

In the reaction, microorganisms or enzymes can be immobilized on a water-insoluble carrier and used repeatedly.

For immobilization of microorganisms, for example, acrylamide polymer, urethane polymer, ethylene glycol derivative polymer,
Examples include methods using carrageenan, calcium alginate, and the like. In addition, Amberlite XAD is used to immobilize enzymes.
-7, Amberlight XAD-2, Diaion HP2
Examples include methods using synthetic adsorbents such as 0, Diaion HP2MG, and Octyl Sepharose 0L-4B.

Substrate [I] generally has a low solubility in water, but this does not interfere with the reaction as long as it is sufficiently stirred.

However, in order to speed up the reaction, acetone,
Hydrophilic solvents such as ethanol, surfactants, etc. may be added to the extent that they do not interfere with the reaction.

The reaction was monitored by analyzing the organic acid produced by hydrolysis using gas chromatography, and using 0.5 of the [N] added.
The reaction may be terminated when an equivalent amount of organic acid is produced.

As a method for separating the hydrolyzate [1] and the remaining [I'], a hydrophobic organic solvent such as hexane is used.

Cyclohexane, petroleum ether, methylene chloride.

Using chloroform, carbon tetrachloride, etc., pll4.0-8
．． From the reaction solution adjusted to the range of 5, [1'] containing only white or some [11] is extracted and separated, and if necessary, [1'] and [1'] are separated by silica gel chromatography (separated with hexane-acetone solvent). 11 can be separated to obtain pure products. Furthermore, the residue remaining in the reaction solution can be completely recovered by adjusting the pH of the reaction solution to 9-14 mW with NaOH or the like, and then extracting with the above-mentioned solvent. What should be noted here is that [white] undergoes hydrolysis under alkaline conditions, so it is necessary to start with alkaline conditions (13 and [1').
] is not good because it causes a decrease in the optical purity of [13].

[Separation of white and [Seal] can be carried out using the above-mentioned silica gel chromatography, other inorganic adsorbents such as Florisil, alumina, zeolite, etc., or synthetic adsorbents such as Amberlite XAD-7, Amberlite XAD-2,
Diamond Ion I-[P 20, Diamond Ion FIP2M
G, minutes using Octyl-Sepharose 0L-4B, etc.!
You can also do J.

If it has a long carbon chain (white), it can be separated by distillation.

Since it has no optical activity, it can be used as a raw material for synthesis, but NaOH, KOH, Ca (OH)
It is also possible to easily lead to [II] while retaining the optical activity by chemically hydrolyzing it with an alkali such as No. 2, or using an esterase or microorganism with different stereoselectivity.

The optical purity of [11 and [white] can be measured by the following method. [The white color was hydrolyzed by the above method and converted to [I]. [II] was reacted with an equimolar amount of p-toluenesulfonyl chloride in methylene chloride to tosylate, and then subjected to high performance liquid chromatography (f( I'T, C) (Column: Chiral Cell OB (manufactured by JASCO Corporation) φ0.46
25CrIt1 eluent Hexane:isopropyl/-
20: 1), flow rate 1.5 ml/min, detection 221 nrn, retention time (R) body: 36.7 min, (
S) body 51.3 minutes).

(Examples) Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited only to these Examples.

Example 1 100 mJ of 0.1M phosphate buffer (pH 7,'0)
To the solution, 0.1y of riboprotein lipase (LPL Amano 3, derived from Pseudomonas sp., manufactured by Ohno Pharmaceutical Co., Ltd.) and 10.9% of racemic (R8)-N-benzyl-3-butyryloxypyrrolidine were added, and the pH was adjusted to 7 with hydrochloric acid. After adjusting the temperature to 0, the reaction was carried out at 40°C for 28 hours with stirring. The reaction solution is 1 00
m! ! After extracting with hexane three times and removing the solvent under reduced pressure,
Oily (8)-N-benzyl-3-butyryloxypyrrolidine 4.95.9 was obtained. Next, the extraction residue was Na
Adjust the pH to 13 with OH solution, 100ml each! After extracting three times with hexane and removing the solvent under reduced pressure, the oily (R)-N-
Benzyl-8-hydroxypyrrolidine 8.42Ii was obtained. (8) Distillation of -N-benzyl-3-butyryloxypyrrolidine under reduced pressure (182-187°C 7amHy)
A colorless and transparent purified product of I, 4.5y was obtained. The specific optical rotation of this product is C (2) 719.2° (C; 5, MeOH)
The NMR, IR, and spectra were consistent with the standard, and the elemental analysis values were consistent with the calculated values.

Next, add 50ml of IN-Na0TI to the crystal! was added and hydrolyzed under heating to produce 50ml of methylene chloride!
Extracted three times. The solvent was removed under reduced pressure, and 84
1 g of (8)-N-benzyl-3-hydroxypyrrolidine was obtained. This was distilled under reduced pressure at 111℃/2'
mmH5t), 3.00.9% of a colorless oil was obtained. The NMR and 1-liter spectrum of this product match the standard product,
The elemental analysis values were in good agreement with the calculated values. Specific rotation is [α]
−8.79° (C=5, MeOH). Optical purity measured by high performance liquid chromatography is 1
It was 00% e, e. On the other hand, the specific optical rotation of (R)-N-benzyl-3-hydroxypyrrolidine after distillation is (a
] +8.73° (C=5, MeOH), optical purity is 99
%e, e.

Examples 2-14 100 mJ of 0.1 M phosphate buffer (p I-I
7.0) with racemic (IL8)-N-benzyl-3-
Add 45.5 E mol each of acyloxypyrrolidine, and further add 0 ml of lipoprotein lipase (LPL Amano 3).
．． 1-1. After adding Oy and adjusting p■ to 7.0 with hydrochloric acid,
The reaction was carried out at 40° C. for 24 to 72 hours with stirring. The reaction was stopped when 50π of the added substrate was hydrolyzed, and the unreacted (8) was extracted 5 times with 100rn/methylene chloride.
A mixture of -N-benzyl-3-acyloxypyrrolidine and hydrolyzed (R)-N-benzyl-3-hydroxypyrrolidine was obtained.

After removing the solvent under reduced pressure, it was loaded onto a silica gel column (Wako C200, 2ooy) and eluted with hexane-ethyl acetate (10:2 to 5:10).
-N-benzyl-3-acyloxypyrrolidine and (R)
-N-benzyl-3-hydroxypyrrolidine was obtained. (S)-N-benzyl-3-acyloxypyrrolidine was hydrolyzed in the same manner as in Example 1, and then its optical purity was measured. These results are shown in Table 1.

Example 15 Lipoprotein lipase (T, PT, Amano 3) 2
y in 0.05M phosphate buffer (pH 7,0) 40me
Amberlite XAD-7 was suspended at 40.9
The enzyme was adsorbed onto the resin by stirring overnight. Next, the resin was filtered and washed with about 500 m/m of 0.05M phosphate buffer (pH 7.0) to remove unadsorbed enzyme. This enzyme adsorption resin was packed in a column (φ2, 2 x 15 cm), and while keeping it at 33℃, (RS')-N-benzyl-3
- Loaded with 5 g of hexanoyloxypyrrolidine. Next, add 0.05M phosphate buffer (p)I 7.0) to
oOm and ff were run to elute (R)-N-benzyl-3-hydroxypyrrolidine. Then add 500 m of hexane
unreacted (8)-N-benzyl-3-hexanoyloxypyrrolidine was eluted.

The solution containing (R)-N-benzyl-3-hydroxypyrrolidine was concentrated under reduced pressure to 50 mJ, and NaOH was added to prt.
After ts, 100m! ! of methylene chloride three times. After removing the solvent under reduced pressure, it was distilled under reduced pressure to obtain (R) as a colorless oil.
-N-benzyl-3-hydroxypyrrolidine 1.32.
I got an F. The specific optical rotation of this object is [α] + 3.75° (
C=5, MeOH), and the optical purity by HPLC analysis was 99πe, e.

On the other hand, (8')-N-benzyl-3-hexanoyloxypyrrolidine was purified by distillation under reduced pressure (BL) at 160°C/8 mmHji) after removing the solvent from hexane under reduced pressure. 21. I got V. The specific optical rotation of this material is Cαl −15,8° (C=5, MeO[()
Show this. Further hydrolysis under alkaline conditions (8)
-N--benzyl-3-hydroxypyrrolidine, which was tosylated) [The optical purity was measured by PLC and found to be 100πe, e.

Examples 16-22 Glucose 2π, yeast extract 0.5π, meat extract 0
, 3%, peptone 0.3%, olive oil 1% (pH 7,
0) Nutrient liquid 1500rr +/- consisting of the composition of 8
Place in a 1 volume mini jar, sterilize at 120°C for 20 minutes, inoculate with the microorganisms shown in Table 2, and incubate at 30°C for 40 hours with 1vv aeration.
m, stirring at 500 rpm. Then p■ [7
．． 0, (R8)-N-benzyl-3-hexanoyloxypyrrolidine 5y was added, and the mixture was reacted at 30°C for 48 hours. After the reaction, the bacterial cells were removed by centrifugation, and the supernatant was extracted three times with equal amounts of hexane to recover unreacted N-benzyl-3-hexanoyloxypyrrolidine.

Next, the extraction residue was adjusted to pI (18) with NaOH solution and extracted three times with an equal amount of methylene chloride to obtain hydrolyzed N-benzyl-3-hydroxypyrrolidine.

After removing the solvent under reduced pressure, clear colorless N-benzyl-3-hydroxypyrrolidine was obtained by distillation, and after tosylation,
The optical purity was measured by HPLO and the results were as shown in Table 2.

Table 2 Examples 23 to 28 10.9 of (R8)-N-benzyl-3-hexanoyloxypyrrolidine was added to 100 rR of 0.1 M phosphate buffer (pH 7,0), and the commercially available compounds shown in Table 3 were added. The enzyme was added and the reaction was carried out at 40° C. for 22 to 120 hours with stirring.

The reaction solution was extracted three times with equal amounts of hexane to recover unreacted N-benzyl-3-hexanoyloxypyrrolidine. The pH was then adjusted to 13 with Na0FI solution, extracted three times with an equal volume of methylene chloride, and the hydrolyzed N-benzyl-
3-Hydroxypyrrolidine was extracted and separated. The solvent was removed under reduced pressure and then purified by distillation to obtain N-benzyl-3-hydroxypyrrolidine as a colorless transparent oil. After tosylating this, the optical purity was measured by HPLC and the results are shown in Table 3.

Table 3

Claims (9)

[Claims] (1) General formula [I] ▲ Numerical formulas, chemical formulas, tables, etc. are available▼ [I] [In the formula, X represents hydrogen, a substituted or unsubstituted alkyl group having 1 to 17 carbon atoms, or an alkenyl group. ] Indicated by (
R) and (S)-N-benzyl-3-acyloxypyrrolidine mixture was stereoselectively hydrolyzed to form the general formula [I
I]▲There are mathematical formulas, chemical formulas, tables, etc.▼The microorganism or enzyme having stereoselective esterase activity that produces optically active N-benzyl-3-hydroxypyrrolidine shown in [II] is shown by the general formula [I]. (R) and (S)-N-benzyl-3-acyloxypyrrolidine mixture to form optically active N-benzyl-3-hydroxypyrrolidine [II] and the sterically opposite general formula [II]. I'] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I'] (In the formula, X is the same as above) A method for producing optically active N-benzyl-3-hydroxypyrrolidine and/or its enantiomer optically active N-benzyl-3-acyloxypyrrolidine, which comprises separating and collecting an optically active compound. (2) Compound [II] has the optical activity (R)-N-benzyl-3-hydroxy represented by the formula (R)-[II] ▲There are mathematical formulas, chemical formulas, tables, etc.▼(R)-[II] pyrrolidine, and the compound of general formula [I'] is (
S)-[I'] ▲There are mathematical formulas, chemical formulas, tables, etc.▼(S)-[I'] (X is the same as above) Optically active (S)-N-benzyl-3-acyloxypyrrolidine The manufacturing method according to claim 1. (3) The production method according to claim 1 or 2, wherein the microorganism or enzyme having stereoselective esterase activity is a microorganism belonging to the genus Pseudomonas, Mucor, or Alcaligenes, or an enzyme derived from the microorganism. . (4) Compound [II] is an optically active (S)-N-benzyl-3-hydroxypyrrolidine represented by the formula (S)-[II] ▲There are mathematical formulas, chemical formulas, tables, etc.▼(S)-[II] The compound with the general formula [I ′] has the optical activity represented by (R)-[I ′] ▲There are mathematical formulas, chemical formulas, tables, etc.▼(R)-[I ′] (X is the same as above) The method for producing (R)-N-benzyl-3-acyloxypyrrolidine according to claim 1. (5) The production method according to claim 1 or 4, wherein the microorganism having stereoselective esterase activity or the enzyme is a microorganism belonging to the genus Rhizopus or the genus Fucomyces, or an enzyme derived from the microorganism. (6) The production method according to any one of claims 1 to 5, wherein the enzyme is lipase. (7) The production method according to any one of claims 1 to 6, in which a microorganism or enzyme having stereoselective esterase activity is immobilized on a water-insoluble carrier. (8) (R) and (S)- represented by the general formula [I]
Optically active N-benzyl-3-hydroxypyrrolidine [II] obtained by stereoselectively hydrolyzing an N-benzyl-3-acyloxypyrrolidine mixture with a microorganism or enzyme and optically active N-benzyl-3- When separating and collecting [II] and [I'] from a mixture with acyloxypyrrolidine [I'], separate them by column chromatography using an inorganic or synthetic adsorbent as a carrier, and collect each. A manufacturing method according to any one of claims 1 to 7, characterized in that: (9) (R) and (S)- represented by the general formula [I]
Optically active N-benzyl-3-hydroxypyrrolidine [II] obtained by stereoselectively hydrolyzing an N-benzyl-3-acyloxypyrrolidine mixture with a microorganism or enzyme and optically active N-benzyl-3- When [II] and [I ′] are separated and collected from a mixture with acyloxypyrrolidine [I ′], [I ′] is extracted using a hydrophobic solvent and separated from [II], and each is separated from [II]. 8. A manufacturing method according to any one of claims 1 to 7, characterized in that the product is collected.