JPH0694441B2 - DL-threo-2- (3,4-di-lower alkyloxy-α-hydroxybenzyl) alkyl isovalerate and process for producing the same - Google Patents

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to D-useful as a synthetic intermediate in the production of DL-threo-3-isopropylmalic acid.
The present invention relates to an alkyl L-threo-2- (3,4-di-lower alkyloxy-α-hydroxybenzyl) isovalerate and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION 3-Isopropylmalate dehydrogenase, an enzyme that catalyzes the third step of the leucine biosynthesis system, is widely distributed in the microbial world, and D-threo-3-isopropylmalic acid is a substrate for this enzyme. . 3-Isopropylmalic acid has two asymmetric carbon atoms in the molecule and has four stereoisomers, namely D-threo, L-threo, D-erythro and L-erythro. The substrate is a D-threo body, and several methods for obtaining it have been attempted. The biosynthesis method is shown, for example, in Methods in Enzymology 17A Vol. 791-793 (Academic Press, 1970). For example, a 20 l culture tank is used to obtain a crude product. Is separated and purified using a column to obtain 15 to 30 g of unnecessary isomers and 3 to 6 g of the desired product, and the yield is not sufficient. The target product is a mixture of D-erythro form and D-threo form. Synthetic chemistry methods are described in, for example, the journal Biochemistry, Vol. 1, No. 6, 1157-11.
As shown on page 61 (1962), for example, starting from ethyl 2-bromoisovalerate, it seems that the target product is obtained with a yield of about 5% through several steps using unfavorable reagents. It has become. However, it is stated that it is difficult to separate it from the by-produced 2-isopropyl substitution product,
And what was obtained as a target product was a mixture of the above-mentioned four stereoisomers.

[0003]

As described above, the conventional production method is inefficient, and the intended product obtained does not contain necessary D-threo in abundance. Or a mixture containing the other three stereoisomers and only 1/4 of which was D-threo. Needless to say, the production efficiency should be good and the method should be industrially practical, and even if the other stereoisomer which is not required is simultaneously produced, the required D-threo isomer, that is, It has been extremely demanded to obtain a product containing more DL-threo-isopropylmalic acid.

As a result of various studies in response to this demand, the present inventor has invented and completed a novel method for stepping a useful synthetic route. Recently, in the field of genetic engineering, gene cloning technology has advanced and has become an extremely popular method. There are three enzymes involved in the bacterial leucine biosynthesis system, and their respective genes are leuA, leuC, and leuB in the order of their involvement. Of these, 3-isopropylmalate dehydrogenase (enzyme number 1.1.1.
The gene that makes 85) is Roy B. Roy B and genes homologous thereto are widely present in microorganisms such as bacteria and fungi and plants, and are genes that are easy to handle and can be easily cloned. In Escherichia coli used as a host for cloning, defective strains such as leucine and tryptophan are often used, and among them, defective strains of Leu B are stable and easy to handle, and many types are widely used. Therefore, Roy B is a particularly essential material for training when attempting cloning in genetic engineering. Also, when cloning a gene of a special bacterium such as a highly thermostable bacterium, Loy B is often the first gene to be cloned.

D-threo-3-isopropylmalic acid, which is a substrate of 3-isopropylmalate dehydrogenase, which is an enzyme that catalyzes the third step of the leucine biosynthesis system, is
Although it is indispensable for elucidating and identifying the species difference of gene LeuB of this enzyme, there is no preferable production method as described above, and therefore no commercial product is available, which is an obstacle to smoother development of genetic engineering. Is. The development of genetic engineering in this field is to improve what was previously done by a fermentation method with poor productivity by utilizing gene recombination technology, and to carry out a large-scale biosynthesis of the essential amino acid leucine by a bioreactor. Is expected.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides DL-threo-
It is an invention of alkyl 2- (3,4-di-lower-alkyloxy-α-hydroxybenzyl) isovalerate. Further, the present invention is to react an alkyl isovalerate with 3,4-di-lower alkyloxybenzaldehyde in the presence of lithium diisopropylamide to coexist with hexamethylphosphorus triamide in aldol condensation. Characterized by DL-threo-2- (3,4-di-lower alkyloxy-
It is an invention of a method for producing an α-hydroxybenzyl) alkyl isovalerate. That is, the present inventor is earnestly searching for a method for producing DL-threo-3-isopropylmalic acid which is synthetically efficient and industrially easy to carry out, and the novel compound of the present invention and the production thereof are being studied. The present invention has been completed by finding a method and obtaining a novel method for producing DL-threo-3-isopropylmalic acid via the method.

The final target compound is D-threo-3-isopropylmalic acid, but since it is not produced in vivo, its L
The body coexists. The D-form and the L-form can be separated from each other in research, but industrially, the coexistence of the L-form does not hinder practical use, so this book should not bear the financial burden. The invention is directed to a process for making DL-threo-3-isopropylmalic acid.

Since the compounds involved in the present invention have two or more chiral moieties and their isomers are called diastereomers, they cannot be perfectly represented on the paper, but according to the customary practice. The formula of the method for producing DL-threo-3-isopropylmalic acid using the compound and the production method of the present invention is as follows.

[0009]

[Formula 1]

[0010]

[Formula 2]

[0011]

[Formula 3]

[0012]

[Formula 4] (Here, R ¹ , R ² , and R ³ are the same or different lower alkyl groups, and R ⁴ is an alkyl group or an aryl group.) In the above formula, compound [1]

[0013]

[Chemical 1]

Is an alkyl isovalerate, compound [A]

[0015]

[Chemical 2]

Is a 3,4-di-lower alkyloxybenzaldehyde, compound [2]

[0017]

[Chemical 3]

DL-threo-2- (3,4-di-lower alkyloxy-α-hydroxybenzyl) alkyl isovalerate, compound [3]

[0019]

[Chemical 4]

Is an O-acylated DL-threo-2- (3,4-di-lower alkyloxy-α-hydroxybenzyl) alkyl isovalerate, compound [4]

[0021]

[Chemical 5]

Is an O-acylated DL-threo-3-isopropylmalate monoalkyl, compound [5]

[Chemical 6] Is DL-threo-3-isopropylmalic acid.

Here, the compounds [2], [3], and [4]
Is a novel compound that has not been published in the literature. The method of the present invention is
Compound [1] was converted into lithium diisopropylamide (L
Compound), which is characterized by allowing hexamethylphosphorus triamide (referred to as HMPA) to coexist in the case of reacting with 3,4-di-lower-alkyloxybenzaldehyde in the presence of DA) to effect aldol condensation. 2] is a manufacturing method. In order to obtain DL-threo-3-isopropylmalic acid, the hydroxy group of the compound [2] is protected with an acyl group to give a compound [3], and then ruthenium trichloride or ruthenium dioxide is used as a catalyst. By reacting with an oxidizing agent capable of oxidizing, for example, alkali periodate,
Compound [4] is obtained. During this period, it is usual to separate and remove a small amount of by-product erythro-form, to hydrolyze the ester by reacting compound [4] with alkali hydroxide, or to purify each compound. is there. Then, if the compound [4] is hydrolyzed, the final target compound D
L-threo-3-isopropylmalic acid [5] is obtained.

Further, the manufacturing method of the present invention will be described in detail. LDA is prepared by a conventional method. After adding a commercially available hexane solution of butyllithium to diisopropylamine in an ether type solvent such as tetrahydrofuran (referred to as THF) in an atmosphere of an inert gas such as argon, the reaction was carried out at the time of cooling, and then HMP was added thereto.
Add A. Then, an ether type solvent solution of the compound [1] is slowly added dropwise while maintaining the temperature at about -50 ° C. After the stirring reaction, a solution of 3,4-di-lower alkyloxybenzaldehyde in an ether type solvent is added. After completion of the reaction as monitored by thin layer chromatography, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with a solvent such as n-hexane or ether, the extract was washed with water and dried, and if necessary, HMPA was removed through a short column of silica gel and then the solvent was removed. Is distilled off to obtain a mixed product of threo body and erythro body. The yield is over 80%. In this mixed product, the ratio of threo body to erythro body was 4: 1 and the threo body was overwhelmingly large, which is the effect of the method of the present invention and HMPA coexisting. HMP
In the absence of coexistence of A, the ratio is usually conversely 1: 5 to 7. Then, the mixture is passed through a silica gel column using a mixed solvent such as ether and hexane or ethyl acetate and hexane to separate the threo body and the erythro body. The properties of both isomers will be described in more detail in Examples, but the threo form, that is, the compound [2], is an oil, and the erythro form is a crystalline form. The compound [2] is dissolved in pyridine, acetic anhydride is added, and the mixture is reacted at room temperature or under cooling for several hours to one day and then the reaction solution is poured into ice water and extracted with an appropriate solvent such as ether or hexane to wash the extract solution. Then, the compound [3] is obtained by drying. This is dissolved in a solvent such as ether or carbon tetrachloride, and this solution is further added to an appropriate aqueous mixed solvent, and then an appropriate oxidizing agent such as alkali periodate is added to the catalyst, ruthenium trichloride or Giving ruthenium dioxide or the like, the reaction is continued until the spot of the raw material (ie, compound [3]) on the thin layer chromatogram disappears. The compound [4] obtained by removing the solvent is reacted with an aqueous alkali hydroxide solution at room temperature to hydrolyze the ester. After the reaction, a solvent (ether, ethyl acetate, etc.) is extracted with hydrochloric acid, washed with water and dried, and then the solvent is removed to obtain a crystal of compound [5].

[0025]

In the present invention, the mechanism by which coexistence of HMPA gives the overwhelming amount of the desired isomer should be understood in a purely academic way and should not be confirmed at this stage. . However, according to the aldol condensation of the present invention,
Working in coexistence is an essential requirement. Examples of the present invention will be shown below, but the present invention is not limited thereto.

[0026]

【Example】

Example 1. Synthesis of DL-threo-2- (3,4-dimethoxy-α-hydroxybenzyl) isovalerate ethyl In a 1-liter four-necked flask, add 39.4 g of diisopropylamine and 100 ml of THF under an argon stream and stir at 0-5 ° C. did. In this state, 142.2 g of a 15% hexane solution of butyllithium was dropped, and after the dropping, stirring was continued for 30 minutes, and 133.5 g of HMPA was further added. Next, set the internal temperature to about -50 ° C.
To this, a solution prepared by dissolving 42.2 g of ethyl isovalerate in 100 ml of THF was dropped, and stirring was continued for 30 minutes after the dropping, and further, in this state, 59.2 g of 3,4-dimethoxybenzaldehyde was dissolved in 200 ml of THF. After dropping, the mixture was stirred at 0 ° C. for 30 minutes. After the reaction was completed, the temperature of the reaction solution was returned to room temperature, and 500 ml of hexane was added to the extraction, and the hexane layer was extracted twice with 300 ml of saturated NH ₄ Cl aqueous solution once.
It was washed twice with 300 ml of N HCl and twice with 300 ml of distilled water in this order. After washing, the hexane layer was dried over Na ₂ SO ₄ and the solvent was distilled off under reduced pressure to give a yellow oil (87.4 g, yield 84%).
Got "Mixture of threo body and _{erythro-form" NMR (CCl 4): δ} 0.85~1.25 (m, 9H, (C H 3) 2 CH -, - CH
₂ C H ₃ ), 1.45 to 2.25 (m, 1H, (CH ₃ ) ₂ C H- ), 2.30 to 2.60
(T, 1H, iPr-CH-), 2.95 to 3.50 (broad, 1H, -OH), 3.70
~ 3.75 (s, 6H, -C ₆ H ₃ (OC H ₃ ) ₂ ), 3.85 ~ 4.20 (q, 2H, -C H _{2
CH 3), 4.55~4.85 (d,} 1H, HO-C H -), 6.65~6.85ppm
(M, 3H, benzene ring proton). Next, column chromatography (silica gel [Wakogel C-300, Wako Pure Chemical Industries, Ltd. product name]), developing solvent
The isomers were separated with hexane: ethyl acetate = 2: 1) to obtain 68.2 g of the target threo compound as a pale yellow oily substance (yield of isomer separation step: 78%). NMR (CDCl ₃ ): δ 0.75 to 1.30 (m, 9H, (C H ₃ ) ₂ CH-, -C
H ₂ C H ₃ ), 1.50 to 2.05 (m, 1H, (CH ₃ ) ₂ C H- ), 2.35 to 2.65
(T, 1H, iPr-CH-), 3.00 to 3.60 (broad, 1H, -OH), 3.75
~ 3.85 (s, 6H, -C ₆ H ₃ (OC H ₃ ) ₂ ), 3.85 ~ 4.25 (q, 2H, -C H _{2
CH 3), 4.75~4.95 (d,} 1H, HO-C H -), 6.70~7.00ppm
(M, 3H, benzene ring proton)

Reference Example 1. (a) O-acetyl-DL-threo-2- (3,4-dimethoxy-α-
Synthesis of ethyl hydroxybenzyl) isovalerate In a 500 ml four-necked flask, 27 g of ethyl DL-threo-2- (3,4-dimethoxy-α-hydroxybenzyl) isovalerate obtained in Example 1 and 200 ml of pyridine were added. In addition, it stirred at 0-5 degreeC. In this state, 46.4 g of acetic anhydride was added and stirred for 1 hour, the temperature was further raised to 25 to 30 ° C., and the reaction was carried out for 7 hours with stirring. After completion of the reaction, the reaction solution was poured into 200 ml of ice water, and 300 ml of a hexane-ethyl acetate solution (hexane: ethyl acetate = 2: 1) was added thereto for extraction. After the extraction operation was performed 3 times, the organic layers were combined and washed with 500 ml of 1N HCl twice, 200 ml of 5% NaHCO ₃ twice, and 200 ml of saturated saline once in this order. After washing, the organic layer is dried over Na ₂ SO ₄ , and the solvent is distilled off under reduced pressure,
27 g (yield 87.5%) of a pale yellow oily substance was obtained. NMR (CCl ₄ ): δ 0.75 to 1.00 (d, 6H, (C H ₃ ) ₂ CH-),
1.05 to 1.45 (t, 3H, -CH ₂ C H ₃ ), 1.45 to 2.00 (m, 1H, (CH ₃ )
₂ C H- ), 1.85 ~ 2.00 (s, 3H, CH ₃ COO-), 2.60 ~ 2.95 (d
d, 1H, iPr-CH - ), 3.75~3.85 (m, 6H, -C 6 H 3 (OC H 3) 2),
3.90 to 4.30 (q, 2H, -C H ₂ CH ₃ ), 5.75 to 5.95 (d, 1H, CH ₃ CO
OC H- ), 6.65 ~ 6.85 ppm (m, 3H, benzene ring protons).

(B) Synthesis of O-acetyl-DL-threo-3-isopropylmalate monoethyl O-acetyl-DL-threo-2- (3) obtained in the previous step (a) was placed in a 1-liter four-necked flask. , 4-dimethoxy-α-hydroxybenzyl) ethyl isovalerate 27 g, CCl ₄ 200 ml, CH ₃ CN 200 ml,
300 ml of distilled water was added, and the mixture was stirred and dissolved at 5-8 ° C.
In this state, 0.4 g of RuCl ₃ and 56.8 g of NaIO ₄ were added to this, and after stirring and reacting for 10 minutes, 100 g of NaIO ₄ was added and the reaction was stirred for 10 minutes. Add 100 g of NaIO ₄ at 5-8 ° C and add 30
After the reaction with stirring for 1 minute, the reaction temperature was raised to 25 to 30 ° C. and the reaction was carried out with stirring for 7 hours. After completion of the reaction, isopropyl alcohol 10
The oxidation reaction was stopped by adding 0 ml, and NaIO _{3 was} filtered through Celite.
And RuCl ₃ were removed. A hexane-ethyl acetate solution (hexane: ethyl acetate = 2: 1) was added to the filtrate for extraction. After performing the extraction operation three times, the organic layers were combined and washed with 500 ml of distilled water. The organic layer was dried over Na2SO4, and the solvent was evaporated under reduced pressure to give a brown oil (16.7 g, yield 8
5%). NMR (CDCl ₃ ): δ 0.80 to 1.10 (d, 6H, (C H ₃ ) ₂ CH-),
1.15 ~ 1.50 (t, 3 H, -CH ₂ C H ₃ ), 1.70 ~ 2.50 (m, 1H, (C
_{H 3) 2 C H -)} , 2.05~2.20 (s, 3H, CH 3 COO -), 2.65~2.90
(M, 1H, iPr-CH-), 3.95 to 4.45 (q, 2H, -C H ₂ CH ₃ ), 5.15
~ 5.40 (d, 1H, CH ₃ COO-C H- ), 8.30 ~ 8.65ppm (s, 1H, -CO
OH).

(C) Synthesis of DL-threo-3-isopropylmalic acid In a 500 ml four-necked flask, KOH 22.4 g and distilled water 20
0 ml was added, and the mixture was stirred and dissolved at 25 to 30 ° C., and 16.7 g of O-acetyl-DL-threo-3-isopropylmalate monoethyl obtained in the previous step (b) was added thereto, and the mixture was stirred and reacted for 1 hour. After the reaction was completed, 250 ml of 2N HCl was added to the reaction solution to adjust the pH to 2 or less. 300 ml of ethyl acetate was added to this and extraction was performed. After performing the extraction operation three times, the organic layers were combined, dried over Na ₂ SO ₄ , and the solvent was distilled off under reduced pressure to obtain 11 g of brown crystals (yield 92%). The crude crystals (5 g) were dissolved in water (100 ml), washed with hexane (100 ml) five times, and the aqueous layer was concentrated to dryness to give yellow crystals (4.4 g). 4.4 g of the yellow crystals was added to 200 ml of 1,2-dichloroethane, heated under reflux for 10 minutes, cooled to 10 ° C., and the crystals were collected by filtration (yield 3.9
g). The crystals (3.9 g) were further added to 1,2-dichloroethane (500 ml), heated under reflux for 10 minutes, allowed to cool to 25 ° C., and the crystals were collected by filtration to give white crystals (3.2 g, yield 64%). NMR (acetone-d ₆ ): δ 0.90 to 1.45 (d, 6H, (C H ₃ ) _2-
CH-), 1.80 to 2.55 (m, 1H, (CH ₃ ) ₂ -C H- ), 2.55 to 2.90
(Dd, 1H, iPr-CH-), 4.35 ~ 4.65 (d, 1H, HO-C H- ), 6.50
~ 9.00ppm (broad, 3H, -COOH × 2, -OH).

[0030]

INDUSTRIAL APPLICABILITY The production of DL-threo-3-isopropylmalic acid was far too powerless to obtain the target isomer in both the conventional biosynthesis and chemical synthesis. The efficiency was low and it could not be industrialized at all. The present invention is expected to provide an epoch-making intermediate and a production method for obtaining this object, show relatively mild conditions and excellent efficiency, and facilitate industrial production. is there.

Claims (2)

[Claims] 1. An alkyl DL-threo-2- (3,4-dilower alkyloxy-α-hydroxybenzyl) isovalerate. 2. A reaction of alkyl isovalerate with 3,4-di-lower alkyloxybenzaldehyde in the presence of lithium diisopropylamide to allow aldol condensation to cause coexistence of hexamethylphosphorus triamide. A method for producing an alkyl DL-threo-2- (3,4-di-lower alkyloxy-α-hydroxybenzyl) isovalerate, which is characterized. [0001]