JP2002000295A - Method for producing optically active glycerol α-monocarboxylic acid ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing an optically active glycerol α-monocarboxylate which is a useful production intermediate for medicines, etc. SOLUTION: This method for producing the optically active glycerol α- monocarboxylate is characterized by esterifying glycerol with an acyl donor in the presence of an enzyme having an activity in synthesizing the optically active glycerol α-monocarboxylate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an optically active glycerol α-monocarboxylate useful as a raw material or an intermediate of a drug or the like.

[0002]

2. Description of the Related Art As a chemical synthesis method of an optically active glycerol derivative, a method of obtaining glycerol acetonide in the S-form from D-mannitol (J. Org. Chem., 43, 4876,
(1978)), a method for obtaining L-ascorbic acid (J. Am. S
oc. Chem., 102, 6304, (1980)). However, these methods are not practical because the number of steps is large and the operation is complicated. A method of optically resolving racemic glycerol acetonide, glycerol cyclic carbonate, and the like using a resolving agent containing a polysaccharide derivative as an active ingredient is also known (JP-A-61-122283). In this case, a large amount of the solvent is consumed, so that it is difficult to become an economical method.

As a biological synthesis method, a method of stereoselectively hydrolyzing a carboxylic acid ester of racemic glycerol ketal by utilizing an enzyme such as lipase (EP55)
6909, US Pat. No. 4,931,339, etc.) or a method for stereoselectively esterifying (JP-A-2-27097, JP-A-5-192186)
And the like, but only half of the racemic substrate is used, which is not economical.

On the other hand, a method of converting a prochiral 2-O-substituted glycerol into an optically active monocarboxylic acid ester by utilizing a transesterification reaction of lipase (Tetrahedron. Let
t., 29, 5173 (1988)), but the process of converting glycerol to a 2-O-substituted product is complicated and has an economical problem.

As a method for obtaining optically active glycerol acetonide, a method is known in which only one optical isomer of racemic glycerol acetonide is oxidized by a microorganism (EP244912, USP4956285, EP412585, etc.). , And regeneration of coenzymes and the like, and it is hard to be an industrial production method.

There are many reports on the synthesis of monoacylglycerol from glycerol and an acyl donor in the presence of lipase, but the yield and optical purity are low, making it difficult to produce an industrial process (Enzyme. Microb. Tech
nol., 17, 578 (1995), JP-A-11-113590).

[0007]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an efficient method for producing optically active glycerol α-monocarboxylic acid ester.

[0008]

Means for Solving the Problems In order to solve the above problems,
The present inventors have conducted intensive studies on a method for producing an optically active glycerol α-monocarboxylic acid ester.As a result, in the presence of a specific enzyme, when glycerol is reacted with an acid anhydride as an acyl donor, an efficient optical reaction is achieved. It has been found that active glycerol α-monocarboxylic acid ester is formed, and the present invention has been completed.

That is, the present invention relates to an optically active glycerol α-monocarboxylic acid ester obtained by esterifying glycerol with an acyl donor in the presence of an enzyme capable of synthesizing optically active glycerol α-monocarboxylic acid ester. The method for producing an optically active glycerol α-monocarboxylate, wherein the compound represented by the general formula (II) is used as an acyl donor.

Embedded image (In the formula, R is a substituted or unsubstituted aliphatic hydrocarbon group or aromatic hydrocarbon group.)

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The acyl donor used in the present invention is preferably an active acyl donor, and examples thereof include an acid anhydride of an aliphatic or aromatic carboxylic acid represented by the general formula (III).

Embedded image (In the formula, R is a substituted or unsubstituted aliphatic hydrocarbon group or aromatic hydrocarbon group.)

In the general formula (III), the aliphatic hydrocarbon group represented by R may be either saturated or unsaturated, and preferably has 1 to 17 carbon atoms. Examples of the aromatic hydrocarbon group represented by R include an aryl group such as phenyl and t-butylphenyl, an arylalkyl group, and an arylalkenyl group such as phenylallyl. In the aliphatic hydrocarbon group and the aromatic hydrocarbon group represented by R, part or all of the hydrogen atoms bonded to the carbon atoms may be substituted with a suitable substituent. Examples of the substituent of the aliphatic hydrocarbon group include halogen such as chlorine. Examples of the substituent of the aromatic hydrocarbon group include halogen such as alkyl and chlorine, and nitro. Specifically, acetic anhydride, propionic anhydride, n-anhydride
Butyric acid, iso-butyric anhydride, n-valeric anhydride, iso-valeric anhydride,
Examples thereof include acid anhydrides such as pivalic anhydride, maleic anhydride, benzoic anhydride, and phthalic anhydride.

The enzyme used in the present invention is not particularly limited as long as it has an activity of synthesizing optically active glycerol α-monocarboxylate. For example, the genus Aspergillus, Rhizopus (Rhi
genus, genus Penicillium, genus Geotrichum, genus Mucor, genus Rhizomucor, genus Humicola, genus Pseudomonas, Burkhodelia (Bu)
rkholderia), Candida, Brevibacterium, Corynebacterium, Nocardia,
Genus Cedecea, Bacillus, Proteus, Methylobacterium
An enzyme produced by a microorganism belonging to the genus bacterium or the like and having an ability to produce optically active glycerol α-monocarboxylic acid ester can be used.

[0013] Lipase AK [Pseudomonas fluorescens (Pseud
omonas fluorescens)), lipase D (Rhizopus
Delema (from Rhizopus delemar)), Lipase PS (from Pseudomonas cepacia),
Lipase F-AP (from the genus Rhizopus sp.), Lipase G (Penicillium
camembertii)), Lipase GC-20 (Geotricam
Lipase PZ-6 (from the genus Aspergillus sp.), Lipase M-AP (from the genus Mucor (Mucor sp.), Amano Pharmaceutical Co., Ltd.), Novozyme 435 (Candida・ Antactica (from Candida antarctica), manufactured by Novo), Kirazyme cf, Kirazyme C2, Kirazyme L-1 (from Burkholderia (Burkholderia) genus), Kirazyme L-2 (from Candida (Candida) genus), Kirazyme L -6 (derived from the genus Pseudomonas sp.), Kirazyme L-8
(Derived from the genus Humicola, manufactured by Roche Diagnostics), lipozyme (Rhizomucor
miehei), manufactured by Fulka].

In the present invention, it is also possible to use, as it is, a culture, a cell or a treated product of the above-mentioned microorganism having the above-mentioned optically active glycerol α-monocarboxylic acid ester-producing ability. Examples of such microorganisms include Cedecea davisae JCM1685, Brevibacterium protophomiae IFO12, which can be obtained as a strain to be distributed.
128, Corynebacterium aquaticum
terium aquaticum) IFO12154, Nocardia corallina IFO3338, Pseudomonas
Maltophila (Pseudomonas maltophila) IFO12690, Pseudomonas chlororafis
phis) IFO3523, Bacillus stearothermophilus (B
acillus stearothermophillus IAM1035, Bacillus subtilis JCM1465T, Methylobacterium radiotolerance
lerans) IAM12099, Proteus vulgaris (Proteus
vulgaris) IAM12003 etc. can be used.

When a commercially available enzyme is used as the enzyme, the enzyme can be used as it is or immobilized on a suitable carrier by a conventional method. When a microorganism is used, a culture obtained by culturing the microorganism in a medium can be used as it is, or a cell obtained by collecting cells from the culture by centrifugation or the like or a processed product thereof can be used. Examples of the treated cells include freeze-dried cells, crushed cells, cell-free extracts, crude enzymes or purified enzymes obtained from the cell-free extracts by separation operations such as gel filtration and ion exchange chromatography. . Moreover, it is also possible to immobilize and use the cells or the treated cells by a conventional method. When the enzyme has a property of being out of the cells, it can be recovered from the culture supernatant by a conventional method such as lyophilization or a method using an organic solvent.

In the present invention, a solvent is not particularly required for the reaction, but the reaction system can be made uniform by adding an organic solvent. The organic solvent used is not particularly limited, but specifically, for example, tetrahydrofuran, acetonitrile, 1,4-dioxane, acetone, triethylamine and the like can be used alone or in combination.

The amount of the acyl donor used in the present invention is not particularly limited as long as an optically active glycerol α-monocarboxylic acid ester can be synthesized.
0.1 to 5 mol is preferable for 1 mol of glycerol. The reaction temperature varies depending on the stability of the enzyme used and the like, but is usually 0 to 80 ° C.

The resulting optically active glycerol α-monocarboxylic acid ester can be isolated from the reaction solution by a conventional method, for example, by a known method such as extraction, distillation and column chromatography.

When the optical purity of the glycerol α-monocarboxylate produced by the enzymatic reaction is not sufficient,
The optical purity can be increased by recrystallization.

[0020]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Immobilized Kirazyme L-2, cf, C
2 (manufactured by Roche Diagnostics) and 10 mg of anhydrous sodium sulfate were added to 2 ml of a 1,4-dioxane solution containing 50 mM glycerol and benzoic anhydride, and the mixture was added at 30 ° C.
And reacted for 48 hours. The optical purity and quantification of the produced glycerol α-monobenzoate were performed by high performance liquid chromatography using a Chiralpak AD-RH column (manufactured by Daicel Chemical Industries, Ltd.). A mixture of methanol and a 5 mM phosphoric acid aqueous solution (60:40) was used as a carrier, the flow rate was 1 ml / min, and the detection was performed at UV 254 nm. As a result, glycerol α-
The production of monobenzoate was 35.9 mM and the optical purity was 47.6.
% ee (R-form).

Example 2 100 mM glycerol and 20
To 2 ml of a 1,4-dioxane solution containing 0 mM benzoic anhydride, 10 mg of immobilized chirazymes L-2, cf, and C2 and 0.2 g of anhydrous sodium sulfate were added as in Example 1, and the mixture was shaken at 30 ° C. for 30 hours. The reaction was performed. The production of glycerol α-monobenzoate was 53.5 mM, and the optical purity was 75.4% ee (R-form).

Example 3 10 mmol in a 500 ml Erlenmeyer flask
Of glycerol and 10 mmol of the acid anhydride shown in Table 1 were dissolved in 100 ml of 1,4-dioxane. To this solution, add 500 mg of immobilized Kirazyme L-2, cf, C2, and add
The reaction was performed by shaking for a time. 10 mmol of (+)-in the reaction solution
Camphorsulfonic acid and 20 mmol of acetone dimethyl acetal were added and reacted at room temperature for 16 hours to protect the diol moiety with acetonide. After the reaction solution was concentrated under reduced pressure, the residue was dissolved in 50 ml of ethyl acetate, and the ethyl acetate solution was washed successively with saturated aqueous sodium bicarbonate (50 ml × 3) and saturated saline (50 ml × 1). It was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The target compound was further isolated and purified from the residue by silica gel chromatography, and the structure was determined by ¹ H-NMR and ¹³ C-NMR.
Table 1 shows the isolation yield of each α-monoacylglycerol acetonide.

[0024]

[Table 1]

The results of NMR analysis are as follows. <Acetylsolketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm 4.36-4.30 (m, 1H), 4.15 (dd, 1H, J = 4.5, 11.6
Hz), 4.09 (dd, 1H, J = 4.5, 10.6 Hz), 4.07 (dd, 1H,
J = 6.1, 12.1 Hz), 3.74 (dd, 1H J = 6.1,6.1 Hz), 2.
10 (s, 3H), 1.44 (s, 3H), 1.38 (s, 3H). 13 C-NMR (CDCl 3, 90 MHz) δppm 171.0, 109.9, 73.6, 66.3, 64.8, 26.6, 25.3,
20.8.

<Propionyl solketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm = 4.36-4.29 (m, 1H), 4.18 (dd, 1H, J = 4.5, 1)
5.5Hz), 4.12-4.07 (m2H), 3.75 (dd, 1H J = 6.2,8.4),
2.38 (q, 2H, J = 7.6 Hz), 1.44 (s, 3H), 1.37 (s, 3
H), 1.15 (t, 3H, J = 7.6 Hz) ¹³ C-NMR (CDCl ₃ , 90 MHz) δppm 174.2, 109.8, 73.6, 66.2, 64.6, 27.3, 26.6,
25.3, 9.0

<N-butyroyl solketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm 4.35-4.29 (m, 1H), 4.20-4.06 (m, 2H), 3.74
(dd, 1H, J = 6.1,8.5 Hz), 2.35-2.29 (m, 2H), 1.69-
1.62 (m, 2H), 1.44 (s, 3H), 1.37 (s, 3H), 0.95 (t,
3H, J = 7.5 Hz). 13 C-NMR (CDCl 3, 90 MHz) δppm 173.4, 109.8, 73.6, 66.2, 64.4, 35.9, 26.6,
25.3, 18.3, 13.5.

<Iso-butyroyl solketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm 4.32 (5th, 1H, 6.9 Hz), 4.18-4.05 (m, 2H),
3.76 (dd, 1H, 6.3,8.4 Hz), 2.60 (7th, 1H, J = 7.0
Hz), 1.44 (s, 3H), 1.37 (s, 3H), 1.18 (d, 6H, J = 9.
^{2 Hz). 13 C-NMR} (CDCl 3, 90 MHz) δppm 176.9, 109.7, 73.6, 66.3, 64.3, 33.9, 26.6,
25.3, 18.9.

<N-Pentanoyl solketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm 4.37-4.28 (m, 1H), 4.16 (dd, 1H, J = 4.8, 11.
6), 4.11-4.06 (m, 2H), 3.74 (dd, 1H, J = 6.2, 8.4
H), 2.35 (t, 2H, J = 7.3 Hz), 1.67-1.58 (m, 2H), 1.4
4 (s, 3H), 1.37 (s, 3H), 0.92 (t, 3H, J = 4.3Hz). 13 C-NMR (CDCl 3, 90 MHz) δppm 173.6, 10.8, 73.6, 66.3, 64.5, 33.8, 26.9, 2
6.7, 254, 22.2, 13.7.

<Iso-pentanoyl solketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm 4.32 (5th, 1H, J = 5.0 Hz), 4.16 (dd, 1H, J =
4.6, 11.5 Hz), 4.12-4.03 (m, 2H), 3.75 (dd, 1H, J =
6.2, 8.4 Hz), 2.23 (d, 2H, J = 6.8 Hz), 2.11 (7th, 1
H, J = 6.8 Hz), 1.44 (s, 3H), 1.37 (s, 3H), 0.91 (d,
6 H, J = 6.6Hz). 13 C-NMR (CDCl 3, 90 MHz) δppm 172.9, 10.8, 73.6, 66.3, 64.4, 43.1, 26.7, 2
5.6, 25.4, 22.3.

<Pivaloyl solketal> ¹ H-NMR (CDCl ₃ , 400 MHz) δppm 4.30 (5th, 1H, J = 5.7 Hz), 4.13 (dd, 1H, J =
5.0, 29.5 Hz), 4.11 (dd, J = 5.0, 6.5 Hz), 4.07
(dd, 1H, J = 6.4, 8.3 Hz), 3.73 (dd, 1H, J = 6.1, 8.3
Hz), 1.44 (s, 3H ), 1.36 (s, 3H), 1.22 (s, 9H). 13 C-NMR (CDCl 3, 90 MHz) δppm 178.2, 109.6, 73.6, 66.3, 64.1, 38.8, 27.1,
26.6, 25.4.

[0032]

According to the present invention, an optically active glycerol α-monocarboxylate useful as a raw material or an intermediate of a drug or the like can be economically provided.

Claims (4)

[Claims] 1. A method for producing an optically active glycerol α-monocarboxylic acid ester by esterifying glycerol with an acyl donor in the presence of an enzyme capable of synthesizing optically active glycerol α-monocarboxylic acid ester. A process for producing an optically active glycerol α-monocarboxylic acid ester, comprising using a compound represented by the general formula (I) as an acyl donor. Embedded image (In the formula, R is a substituted or unsubstituted aliphatic hydrocarbon group or aromatic hydrocarbon group.) 2. An acyl donor comprising acetic anhydride, n-propionic anhydride, n-butyric anhydride, iso-butyric anhydride, n-valeric anhydride,
The method for producing an optically active glycerol α-monocarboxylate according to claim 1, wherein the ester is iso-valeric anhydride, pivalic anhydride or benzoic anhydride. 3. The optically active glycerol α according to claim 1, wherein the enzyme having the activity of synthesizing the optically active glycerol α-monocarboxylate is derived from a microorganism.
-A method for producing a monocarboxylic acid ester. 4. The method according to claim 1, wherein the microorganism is Aspergillus.
Genus, Rhizopus, Penicillium
genus, Geotrichum genus, Mucor genus, Rhizomucor genus, Humicola genus, Pseudomonas
Genus, genus Burkholderia, genus Candida, Brevibacteriu
m), Corynebacterium, Nocardia, Cedecea,
The method for producing an optically active glycerol α-monocarboxylate according to claim 3, which is at least one selected from the group consisting of Bacillus, Proteus, and Methylobacterium.