WO2002079485A1 - Process for producing (r)-2-hydroxy-1-phenoxypropane derivative while preventing the formation of transfer by- product - Google Patents
Process for producing (r)-2-hydroxy-1-phenoxypropane derivative while preventing the formation of transfer by- product Download PDFInfo
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- WO2002079485A1 WO2002079485A1 PCT/JP2002/003138 JP0203138W WO02079485A1 WO 2002079485 A1 WO2002079485 A1 WO 2002079485A1 JP 0203138 W JP0203138 W JP 0203138W WO 02079485 A1 WO02079485 A1 WO 02079485A1
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- An object of the present invention is to provide a (R) -2-hydroxy-1-phenoxypropane derivative useful as an intermediate of a drug, and in particular, a 2,3-difluoro-6-nitro-1-nitro [useful as an intermediate of a synthetic antibacterial agent.
- (R)-(2-Hydroxypropyl)] oxy] Benzene can be obtained by using an enzyme derived from a microorganism, a culture of a microorganism capable of producing the enzyme, or a processed product thereof, and using the resulting phenoxyacetone as an inexpensive raw material.
- An object of the present invention is to provide a method for efficiently producing a hydroxy-11-hydroxypropane derivative.
- the present inventors have conducted intensive studies and, as a result, have found that the transfer reaction can be suppressed by controlling the pH, temperature conditions, and the like during the reduction reaction, and have completed the present invention.
- R represents a phenyl group which may have a substituent
- the R-selective reduction of the phenoxyacetone derivative represented by the above formula (1) using a culture of a microorganism capable of producing an enzyme or a treated product thereof yields the general formula (2);
- R represents the same group as described above
- R represents the same group as described above
- the by-product amount of the ⁇ -1-hydroxy-12-phenoxypropane derivative is 2% or less as a ratio in the product.
- R 2-Hydroxy-1-phenyl; related to the production of nonoxypropane derivatives.
- the present invention provides the above-mentioned production method, wherein the by-product amount of the (R) -1-hydroxy-2-phenoxypropane derivative is 1% or less in the product; (R) -1-hydroxy-2- The above-mentioned production method, wherein the by-produced amount of the phenoxypropane derivative is 0.5% or less in the product; the above-mentioned production method, wherein R is a 2,3-difluoro-6-nitrophenyl group.
- the present invention provides the above-mentioned production method wherein the reduction is carried out under the condition of ⁇ 2 to 7; the production method wherein the reduction is carried out under the condition of ⁇ 2 to 6.5; and adjusting the ⁇ by using a weak base.
- the above method wherein the weak base is ammonia, carbonate or phosphate;
- the above-mentioned production method which is ammonium carbonate, sodium carbonate, calcium carbonate, disodium hydrogen phosphate or dihydrogen hydrogen phosphate; the above-mentioned production method in which the reduction is carried out at 10 to 36 ° C; the substrate concentration is 5% ( w / v) The above-mentioned production method.
- the present invention provides the above-mentioned production method, wherein the enzyme is derived from Candida maris (C andida maris); the above-mentioned production method, wherein the enzyme is derived from Candida maris (C andida maris I FO 10003);
- the present invention relates to the above-mentioned production method, wherein the microorganism capable of producing the enzyme is Escherichiacoli HB101 (NT FPG) accession number FERM BP-71117.
- R represents a phenyl group which may have a substituent.
- substituents include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, a nitro group, a nitroso group, a cyano group, an amino group, a hydroxyamino group, an alkylamino group having 1 to 10 carbon atoms, and a carbon number of 2 ⁇ 10 dialkylamino groups, N-protected amino groups, azide groups, trifluoromethyl groups, carboxynore groups, honoleminolele groups, acetinole groups, benzoyl groups, hydroxynole groups, carbon number :! An alkyloxy group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, an alkylthio group having 1 to 10 carbon atoms, and the like.
- alkylamino group having 1 to 10 carbon atoms as the substituent examples include, for example, methyl and ethyl. , N-propyl, isopropyl, n-butyl, isobutynole, sec-butyl, tert-butylinole, pentyl, n-hexyl, heptyl, octyl, noel, decyl and the like.
- the alkyl group may be selected so that the total carbon number of the two alkyl groups is 2 to 10.
- examples of the C 1-10 acryloxy group include a formyloxy group, an acetyloxy group, a propionyloxy group, a ptyryloxy group, a valeryloxy group, a bivaloyloxy group and a hexanoloxy group.
- protecting groups for N-protected amino groups include, for example, Protective 'Groups'in' Organic Synthesis, 2nd edition (Protective Group Organic Synthesis, 2nd Ed.) And Ao dora double. Protective groups described in Theodora W.
- aralkyl-type protecting groups such as a benzyl group, a phenethyl group, and a triphenylmethyl group; a methanol phenol group, a trifluoromethyl phenol group, a benzene phenol group, a benzene phenol group, and a phenol group.
- o-Nitrobenzenesnolephoninole group m-2-nitrobenzens / lefonyl group, s-lefonyl-type protecting group such as benzenesulfo- / le-group; methoxycarbonyl group, ethoxycarbol Protective groups such as phthaloyl, acetyl, chloroacetyl, trifluoroacetyl, bivaloyl, benzoyl and the like; and the like.
- a protecting group such as a methoxycaponyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, and more preferably a benzyloxycarbonyl group.
- the substituent is preferably a halogen such as a fluorine atom, a nitro group, a cyano group, or the like, and more preferably a fluorine atom, a nitro group, or the like.
- the number of the substituents is 0 to 3, preferably 3.
- the phenoxyacetone derivative represented by the general formula (1) which is a raw material of the present invention, is, for example, 2-acetoeroxy-1,3,4-difluorobenzene, and 2,3-difluoro-6-nitro. It is known that phenol can be easily synthesized by reacting with chloroacetone in the presence of a strong base (JP-A-61-246151).
- the (R) -1-hydroxy-2-phenoxypropane derivative represented by the general formula (3) by-produced by the rearrangement reaction is, for example, 2,3-difluoro-6-nitro [[(R ) — (1—Hydroxyisopropyl)] benzene]
- 2,3,4-trifluoroetrobenzene is reacted under basic conditions with (R) — 1,2 monopropanediol It is known that synthesis is possible by such a method (Japanese Patent Application Laid-Open No. 2-178287).
- Examples of the enzyme derived from a microorganism having the ability to R-selectively reduce the hepatic group used in the present invention include an enzyme derived from a microorganism belonging to the genus Candida, preferably Candida maris. (C andida maris), and more preferably an enzyme derived from Candida's Maris (C andida maris I FO 1 0003), in view of the strength of its reducing ability to the phenoxyacetone derivative (1). It is.
- the microorganism capable of producing the enzyme may be any of a wild strain or a mutant strain.
- a microorganism induced by a genetic technique such as cell fusion or genetic manipulation can also be used.
- a microorganism capable of producing the above-mentioned enzyme, which has been genetically engineered, may be, for example, a step of isolating and / or purifying the enzyme and determining a part or all of the amino acid sequence of the enzyme, based on the amino acid sequence.
- the above-mentioned transformed cells the above-mentioned transformed cells wherein the plasmid is pNTFPG, the above-mentioned transformed cells wherein the transformed cells are Escherichia coli, and the like. More preferably, Escherichiaco 1 i HB101 (pNTFPG) accession number FERM BP-71 17 is mentioned. This is located at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Tsukuba-Higashi, Ibaraki, Japan, on April 11, 2012. Deposited internationally under a treaty.
- AIST National Institute of Advanced Industrial Science and Technology
- the culture medium for the microorganism capable of producing the enzyme is not particularly limited as long as the microorganism can grow.
- carbon sources include sugars such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids and esters thereof such as oleic acid and stearic acid, and oils such as rapeseed oil and soybean oil.
- the cultivation is performed aerobically, and can usually be performed at a culturing time of about 5 to 120 hours, a pH of the medium of 3 to 9, and a culturing temperature of 10 to 50 ° C.
- the immobilization can be performed by a method well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, an entrapment method, etc.).
- suitable solvent include water or a mixed solvent of water and an organic solvent such as toluene, ethyl acetate, and hexane, and preferably water.
- the acceptable amount of (R) -1-hydroxy-2-phenoxypropane derivative as a by-product is the ratio in the product, that is, the amount of transfer product Z (the amount of target product + the amount of transfer product) (Amount) X 100% is 2% or less, preferably 1% or less, more preferably 0.5% or less (wherein, the transition form is (R) -1-hydroxy-2-phenoxypropane). Represents a derivative, and the intended substance is (R) -2-hydroxy-11-phenoxypropane derivative).
- the reaction conditions for suppressing the above by-products are as follows: to avoid contact with the base as much as possible, adjust the pH from acidic to near neutral using a weak base, and react at a relatively low temperature. Is desirable. In addition, it is desirable to shorten the reaction time as much as possible, for example, by using a substrate concentration as high as possible or using an enzyme having a strong reducing ability. Further, it is desirable to gradually add a base (to avoid local strong alkaline conditions). These reaction conditions may be used alone as long as the by-product amount of the (R) -1-hydroxy-2-phenoxypropane derivative is 2% or less in the product. , Or two or more of them may be used in combination.
- the pH in the above reduction reaction is usually 2 to 8, preferably 2 to 7, more preferably 2 to 6.5, and still more preferably 5.0 to 6.5.
- a strong base such as sodium hydroxide can be used if other conditions (temperature, substrate concentration, enzyme used, etc.) are sufficiently appropriate.
- ammonia carbonic acid
- a weak base such as a carbonate such as ammonium, sodium carbonate and calcium carbonate
- a weak base such as a phosphate such as dihydrogen phosphate and sodium phosphate sodium is preferably used, and more preferably ammonium carbonate and the like are used.
- the reaction temperature in the above reduction reaction is usually 10 to 36 ° C, preferably 15 to 30 ° C, more preferably 15 to 27 ° C.
- the substrate concentration in the above reduction reaction is usually 1% (w / v) as a percentage of the weight (g) of the phenoxyacetone derivative (1) as the substrate with respect to the solvent volume (ml) used at the start of the reaction. ), Preferably at least 5% (w / v), more preferably at least 10% (w / v). Also, the base is adjusted to satisfy the substrate concentration. The quality can be added together or continuously. Examples of the solvent used at the start of the reaction include water, a mixed solvent of water and an organic solvent, and the like. The volume of the culture solution, buffer, etc. shall be included in the solvent volume.
- the reaction time in the above-mentioned reduction reaction varies depending on the type and concentration of the enzyme, microorganism or its processed substance, and the substrate used, but it is usually 1 to 96 hours, preferably 1 to 96 hours.
- the above-described reduction reaction can greatly reduce the amount of expensive coenzyme used by using a commonly used NADH regeneration system in combination.
- a typical NADH regeneration system for example, a method using gnorecose dehydrogenase and glucose can be mentioned.
- a culture of a transformed microorganism into which a gene for a carbohydrate reductase gene and a gene for an enzyme (eg, glucose dehydrogenase) having the ability to regenerate a coenzyme dependent on the enzyme are introduced into the same host microorganism; If the same reaction as above is carried out using a product or the like, it is not necessary to separately prepare an enzyme source required for coenzyme regeneration, so that (R) — 2-hydroxyl-one-fuoxy Propane derivatives can be produced.
- an enzyme eg, glucose dehydrogenase
- the (R) -2-hydroxy-11-phenoxypropane derivative generated by the above reduction reaction can be purified by a conventional method.
- the (R) -2-hydroxy-1-phenoxypropane derivative may be subjected to treatment such as centrifugation or filtration to remove suspended cells such as bacterial cells, if necessary. Then, after extraction with an organic solvent such as ethyl acetate or toluene, the organic solvent is removed under reduced pressure, and further purified by distillation under reduced pressure or a process such as mouth chromatography.
- ERM BP—7117 was sterilized in a 500 ml volume flask with 100 ml medium (tryptone 16 g, yeast extract 10 g, sodium chloride 5 g, water 1 Littoner was inoculated at pH 7.0 before sterilization, and cultured with shaking at 37 ° C for 13 hours. After centrifuging 100 ml of the obtained culture solution, the cells without the supernatant were suspended in 5 ml of 50 mM phosphate buffer (pH 6.5), and then disrupted by sonication. Thus, a cell-free extract was prepared.
- 100 ml medium tryptone 16 g, yeast extract 10 g, sodium chloride 5 g, water 1 Littoner was inoculated at pH 7.0 before sterilization, and cultured with shaking at 37 ° C for 13 hours. After centrifuging 100 ml of the obtained culture solution, the cells without the supernatant were suspended in 5 ml of 50 mM phosphate buffer (pH 6.5), and then
- Metabolite by-product (%) Ab p / (Ap + Ab p) X I 00
- Peak area of 2-acetonyloxy 3,4 difluoro mouth-trobenzene Peak area of 2,3-difluoro-6-tro [[(R)-(2-hydroxypropynole)] oxy] benzene
- Example 1 0.1 ml of the cell-free extract used in Example 1 was added to 1.9 ml of 0.5 M phosphate buffer (pH 6.5), 23 mg of gnorecose, 0.1 mg of NAD and 0.1 mg of NAD. Ninoreokishi 3, was added 4-difluoromethyl O b nitrobenzene 2 Omg (substrate concentration 1% (w / v)) , 1M 1 ⁇ 2 ⁇ 1 0 4 1 with water) ⁇ : 6. adjusted to 5 Meanwhile, the reaction was carried out at 27, 30, 33, 36 or 40 ° C. with stirring for 67 hours.
- Example 2 0.5 ml of the cell-free extract used in Example 1, 49.5 ml of deionized water, 5.9 g of dalcose, 5.6 mg of NAD, 2-acetonyloxy 3,4-difluoro Add 5 g of nitrobenzene (substrate concentration 10% (w / v)) and 20% (w / v) in aqueous ammonium carbonate solution; adjust the H to 6.5, and react for 32 hours with stirring at 30 ° C Went. At 6 hours and 20 hours after the start of the reaction, 5.6 mg of NAD was added, respectively.
- Escherichiacoli HB101 accession number F ERM BP—5835 (producing reductase from Candida magnoliae) was sterilized in a 500-ml flask with a 100-ml medium (tryptone 16). g, 10 g of yeast extract, 5 g of Shiridani sodium, 1 liter of water, pH 7.0 before sterilization), and cultured with shaking at 37 ° C for 20 hours.
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Abstract
It is intended to provide a process for efficiently producing an (R)-2-hydroxy-1-phenoxypropane derivative with high qualities, which is highly useful as a drug intermediate, from an inexpensive material. A (R)-2-hydroxy-1-phenoxypropane derivative with high qualities is produced by a reaction of R-selectively reducing a phenoxyacetone derivative, which is an inexpensive material, with the use of a microorganism-origin enzyme or an optionally processed culture of a microorganism capable of producing this enzyme while controlling the reaction pH value, temperature conditions, etc. so as to prevent the formation of an (R)-1-hydroxy-2-phenoxypropane derivative as a by-product.
Description
明細書 Specification
転移体の副生を抑制した (R) _2—ヒドロキシー 1—フエノキシプロパン誘導 体の製造法 技術分野 Method for producing (R) _2-hydroxy-1-phenoxypropane derivative with suppressed by-product formation
本発明は、 医薬品中間体として有用な (R) — 2—ヒドロキシー 1一フエノキ シプロパン誘導体、 とりわけ、 合成抗菌剤の中間体として有用な 2, 3ージフル オロー 6—-トロ一 [ [ (R) — (2—ヒ ドロキシプロピル) ] ォキシ] ベンゼ ンの製造法に関する。 背景技術 INDUSTRIAL APPLICABILITY The present invention relates to (R) —2-hydroxy-11-phenoxypropane derivative, which is useful as a pharmaceutical intermediate, and in particular, 2,3-difluoro-6-toro-1 [-(R) —, which is useful as an intermediate for a synthetic antibacterial agent. (2-hydroxypropyl)] oxy] benzene. Background art
従来、 微生物等を用いて、 フエノキシアセトン誘導体を (R) 一 2—ヒドロキ シー 1一フエノキシプロパン誘導体に還元する方法としては、 以下の方法等が知 られている。 Conventionally, the following methods are known as a method for reducing a phenoxyacetone derivative to (R) 12-hydroxy-11-phenoxypropane derivative using a microorganism or the like.
1) 糸状菌類を用いて、 2, 3—ジフルオロー 6—二トロー [ [ (R) — (2 ーヒドロキシ^ °口ピル) ] ォキシ] ベンゼンを製造する方法 (特開平 3— 183 489号公報) 。 1) A method of producing 2,3-difluoro-6-nitro [[(R)-(2-hydroxy ^ ° pyr)] oxy] benzene using a filamentous fungus (Japanese Patent Application Laid-Open No. 3-183489).
2) コリネバタテリゥム (Co r y n e b a c t e r i um) 属に属する微生 物を用いて、 2, 3—ジフルオロー 6—ニトロ一 [ [ (R) — (2—ヒドロキシ プロピ ] ォキシ] ベンゼンを製造する方法 (特開平 5— 68577号公報) 。 2) A method for producing 2,3-difluoro-6-nitro-[[(R)-(2-hydroxypropy] oxy] benzene using a microorganism belonging to the genus Corynebacterium ( JP-A-5-68577).
3) キャンディダ (C a n d i d a) 属由来のカルボ-ル還元酵素を用いて、 (R) — 2—ヒドロキシー 1一フエノキシプロパン誘導体を製造する方法 (WO 3) A method for producing (R) —2-hydroxy-11-phenoxypropane derivative using a carbon reductase derived from the genus Candida (Candida) (WO
00/37666) 。 00/37666).
しかしながら、 上記 1) 及び 2) の方法では、 得られる (R) — 2—ヒ ドロ キシー 1一フエノキシプロパン誘導体の光学純度が低い、 または反応の際の基質 濃度が極めて低い等、 実用上問題があった。 一方、 我々は、 上記 3) に示した方 法により、 10%以上の基質濃度で反応ができ、 目的物を光学純度 99% e. e. 以上で得ることができる効率的な製造法を既に報告している。 一般に、 1, 2— ジオール類の 1位ァシル基が塩基性条件にて 2位に転移しゃすいことは良く知ら
れているが (Au s t. J. Ch em. (1998) , 5 1 (6) , 455) 、 置換基を有してもよいフエニル基が 1位から 2位に転移することは知られていな かった。 However, in the methods 1) and 2), the obtained (R) -2-hydroxy-1-phenoxypropane derivative has a low optical purity or a very low substrate concentration during the reaction. There was a problem. On the other hand, we have already reported an efficient production method that can react at a substrate concentration of 10% or more by the method shown in 3) above and can obtain the target product with an optical purity of 99% ee or more. I have. In general, it is well known that the 1-position acyl group of 1,2-diols transfers to the 2-position under basic conditions. (Aust. J. Chem. (1998), 51 (6), 455), but it is known that an optionally substituted phenyl group transfers from the 1-position to the 2-position. I didn't.
ところが、 (R) — 2—ヒ ドロキシ一 1一フエノキシプロパン誘導体と、 転 移体である (R) — 1ーヒドロキシー 2—フエノキシプロパン誘導体を分離でき る精密な分析方法を確立し、 上記還元反応を詳細に検討した結果、 還元反応条件 下にて、 置換基を有してもよいフエニル基の転移反応が進行し、 (R) — 1ーヒ ドロキシー 2—フエノキシプロパン誘導体が副生していることが確認された。 ( R) — 1ーヒドロキシー 2—フエノキシプロパン誘導体は、 その構造が (R) — 2—ヒ ドロキシー 1—フエノキシプロパン誘導体と類似しているため、 物理的な 手法による除去精製は非常に困難であり、 医薬品中間体として望まれる高品質の (R) —2—ヒドロキシー 1一フエノキシプロパン誘導体を製造する上での新た な課題となっていた。 発明の要約 However, we established a precise analytical method that can separate (R) -2-hydroxy-11-phenoxypropane derivative and the transfer product (R) -1-hydroxy-2-phenoxypropane derivative. As a result of a detailed study of the above reduction reaction, the transfer reaction of a phenyl group which may have a substituent proceeds under the reduction reaction conditions, and the (R) -1-hydroxy-2-phenoxypropane derivative is obtained. It was confirmed that by-products were produced. (R) — 1-Hydroxy-2-phenoxypropane derivative has a structure similar to that of (R) — 2-hydroxy 1-phenoxypropane derivative, and is very difficult to remove and purify by physical means. It was difficult and had a new problem in producing high quality (R) -2-hydroxy-11-phenoxypropane derivatives desired as pharmaceutical intermediates. Summary of the Invention
本発明の目的は、 医薬品の中間体として有用な (R) —2—ヒドロキシ一 1— フエノキシプロパン誘導体、 とりわけ、 合成抗菌剤の中間体として有用な 2, 3 ージフルオロー 6—ニトロ一 [ [ (R) - (2—ヒ ドロキシプロピル) ] ォキシ ] ベンゼンを、 微生物由来の酵素あるいは該酵素の産生能を有する微生物の培養 物あるいは該処理物を用いて、 安価原料であるフエノキシアセトン誘導体を R選 択的に還元することにより製造する方法において、 フエエル基の転移による (R ) —1ーヒドロキシー 2—フエノキシプロパン誘導体の副生を抑制し、 高品質の (R) 一 2—ヒドロキシー 1一フ ノキシプロパン誘導体を効率的に製造する方 法を提供することにある。 An object of the present invention is to provide a (R) -2-hydroxy-1-phenoxypropane derivative useful as an intermediate of a drug, and in particular, a 2,3-difluoro-6-nitro-1-nitro [useful as an intermediate of a synthetic antibacterial agent. (R)-(2-Hydroxypropyl)] oxy] Benzene can be obtained by using an enzyme derived from a microorganism, a culture of a microorganism capable of producing the enzyme, or a processed product thereof, and using the resulting phenoxyacetone as an inexpensive raw material. A method for producing a derivative by R-selective reduction, in which by-products of (R) -1-hydroxy-2-phenoxypropane derivative due to transfer of a fuel group are suppressed, and high-quality (R) 12- An object of the present invention is to provide a method for efficiently producing a hydroxy-11-hydroxypropane derivative.
本発明者らは上記に鑑み、 鋭意検討した結果、 還元反応時の p H及び温度条件 等を制御することにより、 転移反応を抑制できることを見いだし、 本発明を完成 するに至った。 In view of the above, the present inventors have conducted intensive studies and, as a result, have found that the transfer reaction can be suppressed by controlling the pH, temperature conditions, and the like during the reduction reaction, and have completed the present invention.
即ち、 本発明は、 一般式 (1) ;
That is, the present invention provides a compound represented by the general formula (1):
(式中、 Rは置換基を有してもよいフエ二ル基を表す) で表されるフエノキシァ セトン誘導体のカルボ二ル基を R選択的に還元する能力を有する微生物由来の酵 素あるいは該酵素の産生能を有する微生物の培養物あるいは該処理物を用いて、 前記式 (1 ) で表されるフエノキシアセトン誘導体を R選択的に還元することに より、 一般式 (2 ) ; (Wherein, R represents a phenyl group which may have a substituent) or an enzyme derived from a microorganism having the ability to R-selectively reduce the carboxy group of a phenoxyaceton derivative represented by the formula: The R-selective reduction of the phenoxyacetone derivative represented by the above formula (1) using a culture of a microorganism capable of producing an enzyme or a treated product thereof yields the general formula (2);
(式中、 Rは前記と同じ基を表す) で表される (R) — 2—ヒドロキシ一 1ーフ エノキシプロパン誘導体を製造する方法において、 一般式 (3 ) ; (Wherein R represents the same group as described above). A method for producing a (R) -2-hydroxy-1-phenoxypropane derivative represented by the following general formula (3):
Ηα 人 (3) Ηα people (3)
(式中、 Rは前記と同じ基を表す) で表される (ί — 1ーヒドロキシ一 2—フ ェノキシプロパン誘導体の副生量が生成物中の比率として 2 %以下であることを 特徴とする (R) — 2—ヒドロキシ一 1—フ; ノキシプロパン誘導体の製造法に 関する。 (Wherein, R represents the same group as described above) (wherein the by-product amount of the ί-1-hydroxy-12-phenoxypropane derivative is 2% or less as a ratio in the product. R) — 2-Hydroxy-1-phenyl; related to the production of nonoxypropane derivatives.
また、 本発明は、 (R) — 1—ヒドロキシー 2—フエノキシプロパン誘導体の 副生量が生成物中の比率として 1 %以下である上記製造法; (R) — 1ーヒドロ キシ— 2—フエノキシプロパン誘導体の副生量が生成物中の比率として 0 . 5 % 以下である上記製造法;前記 Rが 2 , 3—ジフルオロー 6—ニトロフエニル基で ある上記製造法に関する。 Further, the present invention provides the above-mentioned production method, wherein the by-product amount of the (R) -1-hydroxy-2-phenoxypropane derivative is 1% or less in the product; (R) -1-hydroxy-2- The above-mentioned production method, wherein the by-produced amount of the phenoxypropane derivative is 0.5% or less in the product; the above-mentioned production method, wherein R is a 2,3-difluoro-6-nitrophenyl group.
さらに、 本発明は、 還元を ρ Η 2〜 7の条件下で行う上記製造法;還元を ρ Η 2〜6 . 5の条件下で行う上記製造法;弱塩基を用いて ρ Ηを調整する上記製造 法;弱塩基が、 アンモニア、 炭酸塩または燐酸塩である上記製造法;弱塩基が、
炭酸アンモニゥム、 炭酸ナトリウム、 炭酸カルシウム、 燐酸水素ニナトリウム、 または、 憐酸水素二力リゥムである上記製造法;還元を 10〜36 °Cの条件下で 行う上記製造法;基質濃度が 5% (w/v) 以上である上記製造法に関する。 また、 本発明は、 前記酵素が、 キヤンディダ 'マリス (C a n d i d a ma r i s) 由来である上記製造法;前記酵素が、 キャンディダ 'マリス (C a n d i d a ma r i s I FO 10003) 由来である上記製造法;前記酵素の産 生能を有する微生物が E s c h e r i c h i a c o l i HB 101 ( NT FPG) 受託番号 FERM B P— 71 1 7である上記製造法に関する。 発明の詳細な開示 Further, the present invention provides the above-mentioned production method wherein the reduction is carried out under the condition of ρΗ2 to 7; the production method wherein the reduction is carried out under the condition of ρΗ2 to 6.5; and adjusting the ρΗ by using a weak base. The above method, wherein the weak base is ammonia, carbonate or phosphate; The above-mentioned production method, which is ammonium carbonate, sodium carbonate, calcium carbonate, disodium hydrogen phosphate or dihydrogen hydrogen phosphate; the above-mentioned production method in which the reduction is carried out at 10 to 36 ° C; the substrate concentration is 5% ( w / v) The above-mentioned production method. In addition, the present invention provides the above-mentioned production method, wherein the enzyme is derived from Candida maris (C andida maris); the above-mentioned production method, wherein the enzyme is derived from Candida maris (C andida maris I FO 10003); The present invention relates to the above-mentioned production method, wherein the microorganism capable of producing the enzyme is Escherichiacoli HB101 (NT FPG) accession number FERM BP-71117. Detailed Disclosure of the Invention
以下に本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
前記式 (1) 、 (2) および (3) において、 Rは置換基を有してもよいフ ュニル基を表す。 置換基としては、 例えば、 フッ素原子、 塩素原子、 臭素原子、 ヨウ素原子等のハロゲン、 ニトロ基、 ニトロソ基、 シァノ基、 アミノ基、 ヒドロ キシァミノ基、 炭素数 1〜 10のアルキルァミノ基、 炭素数 2〜 10のジアルキ ルァミノ基、 N—保護ァミノ基、 アジド基、 トリフルォロメチル基、 カルボキシ ノレ基、 ホノレミノレ基、 ァセチノレ基、 ベンゾィル基、 ヒ ドロキシノレ基、 炭素数:!〜 1 0のアルキルォキシ基、 炭素数 1〜 10のァシルォキシ基、 炭素数 1〜 10のァ ルキルチオ基等が挙げられる。 In the above formulas (1), (2) and (3), R represents a phenyl group which may have a substituent. Examples of the substituent include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, a nitro group, a nitroso group, a cyano group, an amino group, a hydroxyamino group, an alkylamino group having 1 to 10 carbon atoms, and a carbon number of 2 ~ 10 dialkylamino groups, N-protected amino groups, azide groups, trifluoromethyl groups, carboxynore groups, honoleminolele groups, acetinole groups, benzoyl groups, hydroxynole groups, carbon number :! An alkyloxy group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, an alkylthio group having 1 to 10 carbon atoms, and the like.
上記置換基としての炭素数 1〜10のアルキルアミノ基、 炭素数 1~ 10のァ ルキルォキシ基、 炭素数 1~10のアルキルチオ基における炭素数 1〜10のァ ルキル部分としては、 例えばメチル、 ェチル、 n—プロピル、 イソプロピル、 n —プチル、 イソブチノレ、 s e c—プチル、 t e r t—プチノレ、 ペンチル、 n—へ キシル、 ヘプチル、 ォクチル、 ノエル、 デシル等が挙げられる。 また、 炭素数 2 〜10のジアルキルアミノ基においては、 2つのアルキル基の合計炭素数が 2〜 10となるようにアルキル基を選択すればよい。 さらに、 炭素数 1〜 10のァシ ルォキシ基としては、 ホルミルォキシ基、 ァセチルォキシ基、 プロピオ二ルォキ シ基、 プチリルォキシ基、 バレリルォキシ基、 ビバロイルォキシ基、 へキサノィ ルォキシ基等が挙げられる。
また、 N—保護ァミノ基の保護基としては、 例えば、 プロテクティブ'グルー プス 'イン 'オーガニック ·シンセシス第 2版 (P r o t e c t i v e Gr o u p s i n O r g a n i c S y n t h e s i s, 2 n d E d. ) 、 ァォ ドラ ダブリュ. グリーン (Th e o d o r a W. G r e e n) 著、 ジョン ' ウィリー■アンド 'サンズ (J OHN WI LEY & SONS) 出版、 19 90年の 309頁〜 384頁に記載された保護基が挙げられる。 具体的には、 ベ ンジル基、 フエネチル基、 トリフエニルメチル基等のァラルキル型保護基;メタ ンスノレホニノレ基、 トリフノレオロメタンスノレホニノレ基、 ベンゼンスノレホニノレ基、 ー トノレエンスノレホェノレ基、 o—ニ トロベンゼンスノレホニノレ基、 m—二 トロべンゼ ンス /レホニル基、 ; —-ト口ベンゼンスルホ -/レ基等のス /レホニル型保護基;メ トキシカルボニル基、 エトキシカルボ-ル基、 t e r t—プトキシカルボニル基、 ベンジロキシカルボニル基等の力ルバメート型保護基; フタロイル基、 ァセチル 基、 クロロアセチル基、 トリフルォロアセチル基、 ビバロイル基、 ベンゾィル基 等のァセチル型保護基が挙げられる。 好ましくは、 メ トキシカ ポニル基、 エト キシカルボニル基、 t e r t一ブトキシカルポニル基、 ベンジロキシカルポ-ル 基等の力ルバメート型保護基であり、 より好ましくはべンジロキシカルボニル基 である。 Examples of the alkylamino group having 1 to 10 carbon atoms as the substituent, an alkyloxy group having 1 to 10 carbon atoms, and an alkyl moiety having 1 to 10 carbon atoms in the alkylthio group having 1 to 10 carbon atoms include, for example, methyl and ethyl. , N-propyl, isopropyl, n-butyl, isobutynole, sec-butyl, tert-butylinole, pentyl, n-hexyl, heptyl, octyl, noel, decyl and the like. In the dialkylamino group having 2 to 10 carbon atoms, the alkyl group may be selected so that the total carbon number of the two alkyl groups is 2 to 10. Further, examples of the C 1-10 acryloxy group include a formyloxy group, an acetyloxy group, a propionyloxy group, a ptyryloxy group, a valeryloxy group, a bivaloyloxy group and a hexanoloxy group. Examples of protecting groups for N-protected amino groups include, for example, Protective 'Groups'in' Organic Synthesis, 2nd edition (Protective Group Organic Synthesis, 2nd Ed.) And Ao dora double. Protective groups described in Theodora W. Green, published by John Wiley & Sons, 1990, pp. 309-384, 1990. Specifically, aralkyl-type protecting groups such as a benzyl group, a phenethyl group, and a triphenylmethyl group; a methanol phenol group, a trifluoromethyl phenol group, a benzene phenol group, a benzene phenol group, and a phenol group. o-Nitrobenzenesnolephoninole group, m-2-nitrobenzens / lefonyl group, s-lefonyl-type protecting group such as benzenesulfo- / le-group; methoxycarbonyl group, ethoxycarbol Protective groups such as phthaloyl, acetyl, chloroacetyl, trifluoroacetyl, bivaloyl, benzoyl and the like; and the like. Can be Preferably, it is a protecting group such as a methoxycaponyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, and more preferably a benzyloxycarbonyl group.
上記の置換基としては、 好ましくはフッ素原子等のハロゲン、 ニトロ基、 シ ァノ基等であり、 より好ましくはフッ素原子、 ニトロ基等である。 また、 置換基 の数としては、 0〜3個が挙げられ、 好ましくは 3個である。 The substituent is preferably a halogen such as a fluorine atom, a nitro group, a cyano group, or the like, and more preferably a fluorine atom, a nitro group, or the like. In addition, the number of the substituents is 0 to 3, preferably 3.
従って、 置換基を有してもよいフエニル基としては、 好ましくは、 例えばフエ 二ノレ基、 2—シァノフエ-ノレ基、 2—エトロフエ-/レ基、 4一-トロフエ二ノレ基、 2, 3—ジフルオロー 6—ニトロフエニル基等が挙げられ、 特に好ましくは 2, 3ージフルオロー 6—二トロフエニル基等が挙げられる。 Accordingly, the phenyl group which may have a substituent is preferably, for example, a phenyl group, a 2-cyanophen group, a 2-ethrophen // group, a 4-trofenynole group, a 2, 3 —Difluoro-6-nitrophenyl group and the like, and particularly preferably 2,3 difluoro-6-nitrophenyl group and the like.
本発明の原料である一般式 (1) で表されるフエノキシアセトン誘導体は、 例 えば、 2—ァセトエルォキシ一 3, 4—ジフルォロェトロベンゼンの場合、 2, 3—ジフルオロー 6—二トロフエノールを、 強塩基存在下、 クロ口アセトンと反 応させることにより、 容易に合成できる事が知られている (特開昭 6 1— 246 1 5 1号公報) 。
また、 転移反応により副生する一般式 (3) で表される (R) — 1—ヒドロキ シー 2—フエノキシプロパン誘導体は、 例えば、 2, 3—ジフルオロー 6—二ト ロー [ [ (R) — (1—ヒ ドロキシイソプロピル) ] 才キシ] ベンゼンの場合、 2, 3, 4—トリフルォロエトロベンゼンを、 塩基性条件下で、 (R) — 1, 2 一プロパンジオールと反応させることにより合成が可能である事が知られている (特開平 2— 1 78287号公報) 。 The phenoxyacetone derivative represented by the general formula (1), which is a raw material of the present invention, is, for example, 2-acetoeroxy-1,3,4-difluorobenzene, and 2,3-difluoro-6-nitro. It is known that phenol can be easily synthesized by reacting with chloroacetone in the presence of a strong base (JP-A-61-246151). The (R) -1-hydroxy-2-phenoxypropane derivative represented by the general formula (3) by-produced by the rearrangement reaction is, for example, 2,3-difluoro-6-nitro [[(R ) — (1—Hydroxyisopropyl)] benzene] In the case of benzene, 2,3,4-trifluoroetrobenzene is reacted under basic conditions with (R) — 1,2 monopropanediol It is known that synthesis is possible by such a method (Japanese Patent Application Laid-Open No. 2-178287).
本発明に用いる上記力ルポ二ル基を R選択的に還元する能力を有する微生物 由来の酵素としては、 キャンディダ属 (C a n d i d a) に属する微生物由来の 酵素が挙げられ、 好ましくはキャンディダ ·マリス (C a n d i d a ma r i s ) 由来の酵素であり、 フエノキシアセトン誘導体 (1) に対する還元能力の強 さの点から、 より好ましくはキャンディダ 'マリス (C a n d i d a ma r i s I FO 1 0003) 由来の酵素である。 Examples of the enzyme derived from a microorganism having the ability to R-selectively reduce the hepatic group used in the present invention include an enzyme derived from a microorganism belonging to the genus Candida, preferably Candida maris. (C andida maris), and more preferably an enzyme derived from Candida's Maris (C andida maris I FO 1 0003), in view of the strength of its reducing ability to the phenoxyacetone derivative (1). It is.
また、 上記酵素の産生能を有する微生物としては、 野生株または変異株のい ずれでもあり得る。 あるいは細胞融合または遺伝子操作等の遺伝学的手法により 誘導される微生物も用いられ得る。 遺伝子操作された上記酵素の産生能を有する 微生物は、 例えば、 これらの酵素を単離及び zまたは精製して酵素のアミノ酸配 列の一部または全部を決定する工程、 このァミノ酸配列に基づいて酵素をコード する D N A配列を得る工程、 この D N Aを他の微生物に導入して組み換え微生物 を得る工程、 及び、 この組み換え微生物を培養して本酵素を得る工程を含有する 方法により得ることができる (WOO 1/05 996) 。 In addition, the microorganism capable of producing the enzyme may be any of a wild strain or a mutant strain. Alternatively, a microorganism induced by a genetic technique such as cell fusion or genetic manipulation can also be used. A microorganism capable of producing the above-mentioned enzyme, which has been genetically engineered, may be, for example, a step of isolating and / or purifying the enzyme and determining a part or all of the amino acid sequence of the enzyme, based on the amino acid sequence. It can be obtained by a method comprising a step of obtaining a DNA sequence encoding an enzyme, a step of introducing this DNA into another microorganism to obtain a recombinant microorganism, and a step of culturing the recombinant microorganism to obtain the present enzyme ( WOO 1/05 996).
上記酵素の産生能を有する微生物としては、 例えば、 上記酵素をコードする DN A及び該酵素が依存する補酵素を再生する能力を有する酵素をコードする D NAを有するプラスミドで形質転換された形質転換細胞等が挙げられる。 好まし くは、 前記補酵素を再生する能力を有する酵素がグルコース脱水素酵素である上 記形質転換細胞、 前記グルコース脱水素酵素がバシラス 'メガテリゥム (B a c i 1 1 s me g a t e r i um) 由来である上記形質転換細胞、 前記プラス ミドが pNTFPGである上記形質転換細胞、 形質転換細胞が大腸菌である上記 形質転換細胞等が挙げられる。 より好ましくは、 E s c h e r i c h i a c o 1 i HB 101 (pNTFPG) 受託番号 FERM BP—71 1 7が挙げら
れ、 これは日本国茨城県つくば巿東 1丁目 1番地 1中央第 6にある独立行政法人 産業技術総合研究所特許生物寄託センターに、 平成 1 2年 4月 1 1日付で、 プダ ぺスト条約に基づいて国際寄託されている。 Examples of the microorganism capable of producing the enzyme include, for example, a transformant transformed with a plasmid having DNA encoding the enzyme and a DNA encoding an enzyme capable of regenerating a coenzyme on which the enzyme depends. Cells and the like. Preferably, the above-mentioned transformed cell, wherein the enzyme having the ability to regenerate the coenzyme is glucose dehydrogenase, wherein the glucose dehydrogenase is derived from Bacillus megaterium (Bacillus sme gaterium). The above-mentioned transformed cells, the above-mentioned transformed cells wherein the plasmid is pNTFPG, the above-mentioned transformed cells wherein the transformed cells are Escherichia coli, and the like. More preferably, Escherichiaco 1 i HB101 (pNTFPG) accession number FERM BP-71 17 is mentioned. This is located at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Tsukuba-Higashi, Ibaraki, Japan, on April 11, 2012. Deposited internationally under a treaty.
本発明において、 上記酵素の産生能を有する微生物の為の培養培地は、 その微 生物が増殖し得るものである限り特に限定されない。 例えば、 炭素源として、 グ ルコース、 シユークロース等の糖質、 エタノール、 グリセロール等のアルコール 類、 ォレイン酸、 ステアリン酸等の脂肪酸及びそのエステル類、 菜種油、 大豆油 等の油類等;窒素源として、 硫酸アンモ-ゥム、 硝酸ナトリウム、 ペプトン、 力 ザミノ酸、 コーンスティープリカ一、 ふすま、 酵母エキス等;無機塩類として、 硫酸マグネシウム、 塩化ナトリゥム、 炭酸カルシウム、 燐酸水素二力リゥム、 燐 酸二水素カリウム等;他の栄養源として、 麦芽エキス、 肉エキス等を含有する通 常の液体培地が使用できる。 培養は好気的に行い、 通常、 培養時間 5 ~ 1 2 0時 間程度、 培地の p H 3 ~ 9、 培養温度 1 0〜 5 0 °Cで行い得る。 In the present invention, the culture medium for the microorganism capable of producing the enzyme is not particularly limited as long as the microorganism can grow. Examples of carbon sources include sugars such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids and esters thereof such as oleic acid and stearic acid, and oils such as rapeseed oil and soybean oil. Ammonium sulfate, sodium nitrate, peptone, strength zamino acid, corn steep liquor, bran, yeast extract, etc .; inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, sodium hydrogen phosphate, potassium dihydrogen phosphate And the like; a normal liquid medium containing malt extract, meat extract and the like as other nutrient sources can be used. The cultivation is performed aerobically, and can usually be performed at a culturing time of about 5 to 120 hours, a pH of the medium of 3 to 9, and a culturing temperature of 10 to 50 ° C.
本発明の還元反応は、 適当な溶媒中に、 基質であるフエノキシアセトン誘導体、 補酵素 NAD及び上記微生物の培養物またはその処理物等を添加し、 p H調整下 で攪拌することにより行い得る。 ここで、 「微生物の培養物」 とは、 菌体を含む 培養液あるいは培養菌体を意味し、 「その処理物」 とは、 例えば、 粗抽出液、 凍 結乾燥微生物体、 アセトン乾燥微生物体、 またはそれら菌体の破砕物等を意味す る。 さらに、 当該微生物の培養物及びその処理物は、 酵素自体あるいは菌体のま ま公知の手段で固定化されて用いられ得る。 固定化は、 当業者に周知の方法 (例 えば架橋法、 物理的吸着法、 包括法等) で行い得る。 また、 上記適当な溶媒とし ては、 例えば水、 または、 水と、 トルエン、 酢酸ェチル、 へキサン等の有機溶剤 との混合溶媒等が挙げられ、 好ましくは水である。 The reduction reaction of the present invention is carried out by adding a substrate, a phenoxyacetone derivative, a coenzyme NAD, a culture of the above microorganism or a processed product thereof, to an appropriate solvent, and stirring the mixture under pH adjustment. obtain. Here, the term “culture of microorganisms” means a culture solution or cultured cells containing cells, and the “treated product” includes, for example, crude extract, freeze-dried microorganisms, and acetone-dried microorganisms. Or a crushed product of the cells. Furthermore, the culture of the microorganism and the processed product thereof can be used by immobilizing the enzyme itself or the bacterial cells by known means. The immobilization can be performed by a method well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, an entrapment method, etc.). Examples of the suitable solvent include water or a mixed solvent of water and an organic solvent such as toluene, ethyl acetate, and hexane, and preferably water.
上記還元反応においては、 置換基を有してもよいフエニル基の転移反応が非 常に起こり易い為、 (R ) — 1ーヒドロキシー 2—フエノキシプロパン誘導体が 副生する傾向にある。 この (R) — 1—ヒドロキシ一 2—フエノキシプロパン誘 導体は、 目的物である (R) — 2—ヒドロキシー 1一フエノキシプロパン誘導体 と構造が非常に類似している為、 除去精製が困難な化合物である。 よって、 医薬 品中間体として望まれる高品質の (R) — 2—ヒドロキシー 1一フエノキシプロ
パン誘導体を取得する為には、 転移体である (R) — 1—ヒドロキシー 2—フエ ノキシプロパン誘導体の副生を最小限に抑制する必要がある。 In the above reduction reaction, since a transfer reaction of a phenyl group which may have a substituent is very likely to occur, a (R) -1-hydroxy-2-phenoxypropane derivative tends to be by-produced. This (R) -1-hydroxy-1-phenoxypropane derivative has a very similar structure to the target product, (R) -2-hydroxy-11-phenoxypropane derivative. Is a difficult compound. Therefore, the high-quality (R) -2-hydroxy-11-phenoxypro desired as a pharmaceutical intermediate In order to obtain a pan derivative, it is necessary to minimize the by-product of the trans (R) -1-hydroxy-2-phenoxypropane derivative.
ここで、 (R) — 1—ヒドロキシー 2—フエノキシプロパン誘導体の副生量 として許容できる量としては、 生成物中の比率、 即ち、 転移体生成量 Z (目的物 生成量 +転移体生成量) X 100として、 2%以下であり、 好ましくは 1%以下、 より好ましくは 0. 5%以下である (ここで、 転移体とは (R) — 1—ヒドロキ シー 2—フエノキシプロパン誘導体を表し、 目的物とは (R) — 2—ヒドロキシ 一 1一フエノキシプロパン誘導体を表す) 。 Here, the acceptable amount of (R) -1-hydroxy-2-phenoxypropane derivative as a by-product is the ratio in the product, that is, the amount of transfer product Z (the amount of target product + the amount of transfer product) (Amount) X 100% is 2% or less, preferably 1% or less, more preferably 0.5% or less (wherein, the transition form is (R) -1-hydroxy-2-phenoxypropane). Represents a derivative, and the intended substance is (R) -2-hydroxy-11-phenoxypropane derivative).
上記副生物を抑制する為の反応条件としては、 できるだけ塩基との接触を避 ける為、 弱塩基を用いて pHを酸性から中性付近に調整しながら、 比較的低い温 度にて反応させることが望ましい。 また、 基質濃度をできるだけ高くする、 還元 能力の強い酵素を用いる等、 反応時間をできるだけ短くして行うことが望ましい。 さらに、 塩基を徐々に添加する (局所的に強アルカリ条件とならないようにする ) ことが望ましい。 なお、 (R) — 1—ヒドロキシ _ 2—フエノキシプロパン誘 導体の副生量を、 生成物中の比率として 2%以下にする限りは、 これら反応条件 は、 単独で用いてもよいし、 2種以上を組み合わせて用いてもよい。 The reaction conditions for suppressing the above by-products are as follows: to avoid contact with the base as much as possible, adjust the pH from acidic to near neutral using a weak base, and react at a relatively low temperature. Is desirable. In addition, it is desirable to shorten the reaction time as much as possible, for example, by using a substrate concentration as high as possible or using an enzyme having a strong reducing ability. Further, it is desirable to gradually add a base (to avoid local strong alkaline conditions). These reaction conditions may be used alone as long as the by-product amount of the (R) -1-hydroxy-2-phenoxypropane derivative is 2% or less in the product. , Or two or more of them may be used in combination.
上記還元反応における p Hとしては、 通常 2〜8、 好ましくは 2〜 7、 より 好ましくは 2〜6. 5、 さらに好ましくは 5. 0〜6. 5である。 The pH in the above reduction reaction is usually 2 to 8, preferably 2 to 7, more preferably 2 to 6.5, and still more preferably 5.0 to 6.5.
pH調整を行うために添加する塩基としては、 他の条件 (温度、 基質濃度、 使用酵素等) が十分に適切であれば、 水酸化ナトリゥム等の強塩基も使用しうる 力 例えば、 アンモニア ;炭酸アンモニゥム、 炭酸ナトリウム、 炭酸カルシウム 等の炭酸塩;燐酸水素二力リゥム、 燐酸水素ニナトリゥム等の燐酸塩等の弱塩基 が好ましく用いられ、 より好ましくは炭酸アンモユウム等が用いられる。 As a base to be added for pH adjustment, a strong base such as sodium hydroxide can be used if other conditions (temperature, substrate concentration, enzyme used, etc.) are sufficiently appropriate. For example, ammonia; carbonic acid A weak base such as a carbonate such as ammonium, sodium carbonate and calcium carbonate; a weak base such as a phosphate such as dihydrogen phosphate and sodium phosphate sodium is preferably used, and more preferably ammonium carbonate and the like are used.
上記還元反応における反応温度としては、 通常10~36°〇、 好ましくは 1 5〜30°C、 より好ましくは 1 5〜27°Cである。 The reaction temperature in the above reduction reaction is usually 10 to 36 ° C, preferably 15 to 30 ° C, more preferably 15 to 27 ° C.
上記還元反応における基質濃度としては、 反応開始時に使用する溶媒容量 ( m l) に対する、 基質であるフエノキシアセトン誘導体 (1) の重量 (g) の百 分率として、 通常 1% (w/v) 以上、 好ましくは 5% (w/v) 以上、 より好 ましくは 10% (w/v) 以上である。 また、 当該基質濃度を満たすように、 基
質を一括または連続的に添カ卩し得る。 なお、 上記反応開始時に使用する溶媒とし ては、 水、 または、 水と有機溶剤との混合溶媒等が挙げられるが、 上記基質濃度 を算出する場合には、 上記溶媒以外に、 用いた抽出液、 培養液、 緩衝液等の容量 も溶媒容量に含めるものとする。 The substrate concentration in the above reduction reaction is usually 1% (w / v) as a percentage of the weight (g) of the phenoxyacetone derivative (1) as the substrate with respect to the solvent volume (ml) used at the start of the reaction. ), Preferably at least 5% (w / v), more preferably at least 10% (w / v). Also, the base is adjusted to satisfy the substrate concentration. The quality can be added together or continuously. Examples of the solvent used at the start of the reaction include water, a mixed solvent of water and an organic solvent, and the like. The volume of the culture solution, buffer, etc. shall be included in the solvent volume.
上記還元反応における反応時間としては、 用いる酵素、 微生物またはその処 理物、 基質の種類や濃度によって異なるが、 通常 1〜 9 6時間、 好ましくは 1 ~ The reaction time in the above-mentioned reduction reaction varies depending on the type and concentration of the enzyme, microorganism or its processed substance, and the substrate used, but it is usually 1 to 96 hours, preferably 1 to 96 hours.
4 8時間、 より好ましくは 1〜3 2時間、 さらに好ましくは 1〜2 4時間である。 また、 上記還元反応は、 一般に用いられる NAD H再生系を組み合わせて用い る事により、 高価な補酵素の使用量を大幅に減少させ得る。 代表的な N AD H再 生系としては、 例えば、 グノレコース脱水素酵素及びグルコースを用いる方法が挙 げられる。 48 hours, more preferably 1-32 hours, and even more preferably 1-24 hours. In addition, the above-described reduction reaction can greatly reduce the amount of expensive coenzyme used by using a commonly used NADH regeneration system in combination. As a typical NADH regeneration system, for example, a method using gnorecose dehydrogenase and glucose can be mentioned.
さらに、 カルボ-ル還元酵素遺伝子及びこの酵素が依存する補酵素を再生す る能力を有する酵素 (例えばグルコース脱水素酵素) の遺伝子を同一宿主微生物 内に導入した形質転換微生物の培養物またはその処理物等を用いて、 上記同様の 反応を行えば、 別途に補酵素の再生に必要な酵素源を調製する必要がないため、 より低コストで (R) — 2—ヒ ドロキシー 1一フユノキシプロパン誘導体が製造 され得る。 Further, a culture of a transformed microorganism into which a gene for a carbohydrate reductase gene and a gene for an enzyme (eg, glucose dehydrogenase) having the ability to regenerate a coenzyme dependent on the enzyme are introduced into the same host microorganism; If the same reaction as above is carried out using a product or the like, it is not necessary to separately prepare an enzyme source required for coenzyme regeneration, so that (R) — 2-hydroxyl-one-fuoxy Propane derivatives can be produced.
上記還元反応により生じた (R) — 2—ヒドロキシー 1一フエノキシプロパン 誘導体は、 常法により精製され得る。 例えば、 (R) — 2—ヒドロキシー 1ーフ エノキシプロパン誘導体は、 微生物等を用いた場合には、 必要に応じて遠心分離、 濾過等の処理を施して菌体等の懸濁物を除去し、 次いで酢酸ェチル、 トルエン等 の有機溶媒で抽出した後、 有機溶媒を減圧下で除去し、 さらに減圧蒸留またはク 口マトグラフィ一等の処理を行うことにより、 精製され得る。 発明を実施するための最良の形態 The (R) -2-hydroxy-11-phenoxypropane derivative generated by the above reduction reaction can be purified by a conventional method. For example, if microorganisms are used, the (R) -2-hydroxy-1-phenoxypropane derivative may be subjected to treatment such as centrifugation or filtration to remove suspended cells such as bacterial cells, if necessary. Then, after extraction with an organic solvent such as ethyl acetate or toluene, the organic solvent is removed under reduced pressure, and further purified by distillation under reduced pressure or a process such as mouth chromatography. BEST MODE FOR CARRYING OUT THE INVENTION
以下に実施例を挙げ、 本発明をさらに具体的に説明するが、 本発明はこれら実 施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
(実施例 1 )
E s c h e r i c h i a c o 1 i HB 101 ( p N T F P G) 受託番号 F ERM BP— 7117を、 500 m 1容坂ロフラスコ中で滅菌した 100 m 1 の培地 (トリプトン 16 g、 ィーストエキス 10 g、 塩化ナトリウム 5 g、 水 1 リットノレ、 滅菌前 pH7. 0) に接種し、 37°Cで 13時間振とう培養した。 得 られた培養液 100m lを遠心分離操作にかけ、 上清を除いた菌体を 50 mM燐 酸力リゥム緩衝液 ( p H 6. 5) 5m lに懸濁した後、 超音波破砕することによ り無細胞抽出液を調製した。 (Example 1) Escherichiaco 1 i HB101 (p NTFPG) Accession No. F ERM BP—7117 was sterilized in a 500 ml volume flask with 100 ml medium (tryptone 16 g, yeast extract 10 g, sodium chloride 5 g, water 1 Littoner was inoculated at pH 7.0 before sterilization, and cultured with shaking at 37 ° C for 13 hours. After centrifuging 100 ml of the obtained culture solution, the cells without the supernatant were suspended in 5 ml of 50 mM phosphate buffer (pH 6.5), and then disrupted by sonication. Thus, a cell-free extract was prepared.
この無細胞抽出液 0. lm 1に、 各種 pHに調整した 0. 5M燐酸カリウム緩 衝液 (pH5. 5、 6. 0、 6. 5、 7. 0、 7. 5または 8. 0) 1. 9 m 1、 グルコース 23mg、 NAD 0. 11 m g及び 2—ァセトニルォキシー 3 , 4 - ジフルォロニトロベンゼン 2 Omgを添加し (基質濃度 1% (w/v) ) 、 1M K2HPO 4水溶液で p Hを調整しつつ、 30 °Cで攪拌下、 67時間反応を行 つた。 反応開始より 5時間目、 30時間目及び反応終了時に反応液の一部を分取 し、 酢酸ェチルで抽出、 減圧濃縮した。 得られた濃縮物を HP LCにより分析し (カラム: CO SMO S I L 5 C 8 -MS (ナカライテスク社製、 0. 46 X 25 cm) 2本連結、 力ラム温度: 40 °C、 溶離液:ァセトニトリル , 0. 1 % 燐酸水溶液 = 25/75、 流速: 1m l /分、 検出波長: 210 nm、 溶出時間 : 2—ァセトニルォキシ一 3, 4ージフルォロニト口ベンゼン 64分、 2, 3 —ジフノレオロー 6—二トロー [ [ (R) - (2—ヒドロキシプロピル) ] ォキシ ] ベンゼン 52分、 2, 3—ジフルオロー 6—エトロ一 [ [ (R) 一 (1ーヒ ドロキシイソプロピル) ] 才キシ] ベンゼン 56分) 、 変換率及び転移体 2 , 3—ジフルオロー 6—-トロー [ [ (R) — (1—ヒドロキシイソプロピル) ] ォキシ] ベンゼンの副生量を、 下記式に従い算出した。 結果を表 1に示す。 変換率 (%) = (Ap+Ab p) / (A s +Ap+Ab p) X 100 0.5 M of this cell-free extract was added to 0.5 M potassium phosphate buffer adjusted to various pH (pH 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0) 1. 9 ml, glucose 23 mg, NAD 0.11 mg and 2-acetonyloxy 3,4-difluoronitrobenzene 2 Omg (substrate concentration 1% (w / v)), 1M K 2 HPO 4 aqueous solution The reaction was carried out for 67 hours with stirring at 30 ° C. while adjusting the pH of the solution. At 5 hours, 30 hours and at the end of the reaction, a part of the reaction solution was separated, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained concentrate was analyzed by HP LC (column: CO SMO SIL 5 C8-MS (manufactured by Nacalai Tesque, 0.46 x 25 cm), connected in series, column temperature: 40 ° C, eluent: Acetonitrile, 0.1% aqueous solution of phosphoric acid = 25/75, Flow rate: 1 ml / min, Detection wavelength: 210 nm, Elution time: 2-acetonyloxy-1,3,4-difluoronitrile benzene 64 minutes, 2,3-diphnoleol 6-2 Trow [[(R)-(2-hydroxypropyl)] oxy] benzene 52 minutes, 2,3-difluoro-6-etro-1 [[(R) -1 (1-hydroxypropyl)]] benzene 56 minutes ), Conversion rate, and by-product amount of 2,3-difluoro-6-trow [[(R)-(1-hydroxyisopropyl)] oxy] benzene were calculated according to the following formula. Table 1 shows the results. Conversion rate (%) = (Ap + Ab p) / (A s + Ap + Ab p) X 100
転移体副生量 (%) =Ab p/ (Ap +Ab p) X I 00 Metabolite by-product (%) = Ab p / (Ap + Ab p) X I 00
As : 2—ァセトニルォキシー 3, 4ージフルォ口-トロベンゼンのピーク面積 Ap : 2, 3—ジフルオロー 6—-トロー [ [ (R) — (2—ヒドロキシプロピ ノレ) ] ォキシ] ベンゼンのピーク面積 As: Peak area of 2-acetonyloxy 3,4 difluoro mouth-trobenzene Ap: Peak area of 2,3-difluoro-6-tro [[(R)-(2-hydroxypropynole)] oxy] benzene
Ab p : 2, 3—ジフルオロー 6—二トロ _ [ [ (R) — (1ーヒドロキシイソ
プロピル) ] ォキシ] ベンゼンのピーク面積 Ab p: 2,3-Difluoro-6-nitro _ [[(R) — (1-Hydroxyiso Propyl)] Doxy] Benzene peak area
反 J心 pH 変換率 (%) 転移体副生量 (%) Anti-J heart pH conversion (%) Metabolite by-product (%)
5hrs 30hrs 67hrs 5hrs 30hrs 67hrs 5hrs 30hrs 67hrs 5hrs 30hrs 67hrs
5.5 83.7 83.7 84.1 0.09 0.25 0.37 5.5 83.7 83.7 84.1 0.09 0.25 0.37
6.0 99.6 99.7 99.8 0.14 0.38 0.85 6.0 99.6 99.7 99.8 0.14 0.38 0.85
6.5 99.9 >99.9 >99.9 0.30 1.03 2.18 6.5 99.9> 99.9> 99.9 0.30 1.03 2.18
7.0 99.7 99.6 99.8 0.70 1.98 4.86 7.0 99.7 99.6 99.8 0.70 1.98 4.86
7.5 99.6 99.9 99.8 1.67 5.42 12.15 7.5 99.6 99.9 99.8 1.67 5.42 12.15
8.0 99.8 99.9 99.9 4.20 29.29 34.54 8.0 99.8 99.9 99.9 4.20 29.29 34.54
(実施例 2) (Example 2)
実施例 1で用いた無細胞抽出液 0. 1 m 1に、 0. 5 M燐酸力リゥム緩衝液 ( pH6. 5) 1. 9m 1、 グノレコース 23mg、 NAD 0. l lmg及ぴ 2—ァ セトニノレオキシー 3, 4—ジフルォロニトロベンゼン 2 Omgを添加し (基質濃 度 1% (w/v) ) 、 1M 1^2^1?04水溶液で1)^:を6. 5に調整しつつ、 27、 30、 33、 36または 40°Cにて攪拌下、 67時間反応を行った。 反応 開始より 5時間目、 30時間目及び反応終了時に反応液の一部を分取し、 酢酸ェ チルで抽出、 減圧濃縮した。 得られた濃縮物を、 実施例 1と同様の方法で分析し、 変換率及び転移体 2, 3—ジフルオロー 6—-トロー [ [ (R) — (1ーヒドロ キシイソプロピル) ] ォキシ] ベンゼンの副生量を算出した。 結果を表 2に示す。 0.1 ml of the cell-free extract used in Example 1 was added to 1.9 ml of 0.5 M phosphate buffer (pH 6.5), 23 mg of gnorecose, 0.1 mg of NAD and 0.1 mg of NAD. Ninoreokishi 3, was added 4-difluoromethyl O b nitrobenzene 2 Omg (substrate concentration 1% (w / v)) , 1M 1 ^ 2 ^ 1 0 4 1 with water) ^: 6. adjusted to 5 Meanwhile, the reaction was carried out at 27, 30, 33, 36 or 40 ° C. with stirring for 67 hours. At 5 hours, 30 hours, and at the end of the reaction, a part of the reaction solution was collected, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained concentrate was analyzed in the same manner as in Example 1 to determine the conversion and the conversion of the 2,3-difluoro-6-tro [[(R) — (1-hydroxyisopropyl)] oxy] benzene The biomass was calculated. Table 2 shows the results.
表 2 Table 2
JR. '心 m W 拇率 (%) 転移体副生量! ¾ JR. 'Heart m W Thumb rate (%) Metabolite by-product amount!
(°C) 5hrs 30hrs 67hrs 5hrs 30hrs 67hrs (° C) 5hrs 30hrs 67hrs 5hrs 30hrs 67hrs
27 95.6 96.3 98.7 0.26 0.65 1.43 27 95.6 96.3 98.7 0.26 0.65 1.43
30 99.9 >99.9 >99.9 0.30 1.03 2.18 30 99.9> 99.9> 99.9 0.30 1.03 2.18
33 99.8 99.8 99.7 0.71 1.32 3.12 33 99.8 99.8 99.7 0.71 1.32 3.12
36 99.5 99.6 99.7 0.87 1.96 4.80 36 99.5 99.6 99.7 0.87 1.96 4.80
40 99.1 99.6 99.8 0.90 3.31 8.09 40 99.1 99.6 99.8 0.90 3.31 8.09
(実施例 3 ) (Example 3)
実施例 1で用いた無細胞抽出液 0. 5 m 1に、 脱ィオン水 49. 5 m 1、 ダル コース 5. 9 g、 NAD 5. 6mg、 2—ァセトニルォキシー 3, 4—ジフノレオ 口-トロベンゼン 5 gを添加し (基質濃度 10% (w/v) ) 、 30% (w/v
) 水酸化ナトリウム水溶液で p Hを 6. 5に調整しつつ 30 °Cで攪拌下、 32時 間反応を行った。 反応開始から 6時間目及ぴ 20時間目にそれぞれ NAD 5. 6 mgを添カ卩した。 反応終了後、 反応液をトルエンで抽出、 減圧濃縮し、 黄色油状 の 2, 3—ジフルオロー 6 トロー [ [ (R) _ (2—ヒドロキシプロピル) ] ォキシ] ベンゼン濃縮物を得た。 この濃縮物を、 実施例 1と同様の方法により 分析したところ、 変換率は 99. 9 %以上、 転移体 2, 3—ジフルォロ一 6—二 トロー [ [ (R) — (1ーヒドロキシイソプロピル) ] ォキシ] ベンゼンの副生 量は 1. 29%であった。 また、 上記濃縮物の一部をカラムクロマトグラフィー により精製し、 ジニト口ベンゾィル化した後、 HP LC分析 (カラム: C h i r a 1 c e 1 OD— H (ダイセル化学社製、 0. 46 X 25 c m) 、 溶離液:へ キサン Zィソプロパノール = 50/50、 流速: lm 1Z分、 温度: 40°C、 検 出波長: 254 nm、 溶出時間: R体ェナンチォマー 34分、 S体ェナンチ ォマー 27分) を行ったところ、 光学純度は 99. 9%e.e.以上であった。 (実施例 4) 0.5 ml of the cell-free extract used in Example 1, 49.5 ml of deionized water, 5.9 g of dalcose, 5.6 mg of NAD, 2-acetonyloxy 3,4-diphnoleole -Add 5 g of trobenzene (substrate concentration 10% (w / v)), 30% (w / v ) The reaction was carried out for 32 hours with stirring at 30 ° C while adjusting the pH to 6.5 with an aqueous sodium hydroxide solution. At 6 hours and 20 hours after the start of the reaction, 5.6 mg of NAD was added to the mixture. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily 2,3-difluoro-6 tro [[(R) _ (2-hydroxypropyl)] oxy] benzene concentrate. When this concentrate was analyzed by the same method as in Example 1, the conversion was 99.9% or more, and the transition product 2,3-difluoro-1-6-2 tro [[(R) — (1-hydroxyisopropyl) [Oxy] Benzene by-product amount was 1.29%. In addition, a part of the above concentrate was purified by column chromatography and dinitrated benzoylated, and then subjected to HP LC analysis (column: Chira 1 ce 1 OD-H (0.46 x 25 cm, manufactured by Daicel Chemical). , Eluent: Hexane Z-isopropanol = 50/50, Flow rate: lm 1Z min, Temperature: 40 ° C, Detection wavelength: 254 nm, Elution time: R-enantiomer 34 min, S-enantiomer 27 min) As a result, the optical purity was 99.9% ee or more. (Example 4)
実施例 1で用いた無細胞抽出液 0. 5 m 1に、 脱イオン水 49. 5 m 1、 ダル コース 5. 9 g、 NAD 5. 6mg、 2—ァセトニルォキシー 3 , 4—ジフルォ ロニトロベンゼン 5 gを添加し (基質濃度 10% (w/v) ) 、 20% (w/v ) 炭酸アンモユウム水溶液で; Hを 6. 5に調整しつつ、 30°Cで攪拌下、 32 時間反応を行つた。 反応開始から 6時間目及び 20時間目にそれぞれ NAD 5. 6mgを添加した。 反応終了後、 反応液をトルエンで抽出、 減圧濃縮し、 黄色油 状の 2, 3ージフルオロー 6一二トロー [ [ (R) — (2—ヒ ドロキシプロピル ) ] ォキシ] ベンゼン濃縮物を得た。 この濃縮物を、 実施例 1と同様の方法によ り分析したところ、 変換率は 99. 9 %以上、 転移体 2, 3—ジフルォロ一 6— ニトロ一 [ [ (R) — (1—ヒ ドロキシイソプロピル) ] ォキシ] ベンゼンの副 生量は 0. 38%であった。 また、 上記濃縮物の一部をカラムクロマトグラフィ 一により精製し、 ジニトロベンゾィル化した後、 HPLC分析 (実施例 3と同様 ) を行ったところ、 光学純度は 99. 9%e.e.以上であった。
(実施例 5 ) 0.5 ml of the cell-free extract used in Example 1, 49.5 ml of deionized water, 5.9 g of dalcose, 5.6 mg of NAD, 2-acetonyloxy 3,4-difluoro Add 5 g of nitrobenzene (substrate concentration 10% (w / v)) and 20% (w / v) in aqueous ammonium carbonate solution; adjust the H to 6.5, and react for 32 hours with stirring at 30 ° C Went. At 6 hours and 20 hours after the start of the reaction, 5.6 mg of NAD was added, respectively. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily 2,3-difluoro-612-tallow [[(R)-(2-hydroxypropyl)] oxy] benzene concentrate. . When this concentrate was analyzed by the same method as in Example 1, the conversion was 99.9% or more, and the transition product 2,3-difluoro-1-6-nitro [[(R) — (1-H The amount of by-products of droxyisopropyl)] oxy] benzene was 0.38%. Further, part of the concentrate was purified by column chromatography one after dinitrobenzoyl I le of was subjected to HPLC analysis (as in Example 3), optical purity 99. 9% e. E. More Met. (Example 5)
実施例 1で用いた無細胞抽出液 1. 25 m 1に、 脱イオン水 23. 75 m 1 s グルコース 14. 6 NAD 2. 8 mgを添カ卩したものに対し、 2—ァセトニ ルォキシ一 3, 4ージフルォロニトロベンゼン 0. 25 g (基質濃度 1% (w/ V) ) 、 1. 25 g (基質濃度 5% (w/v) ) 、 2. 5 g (基質濃度 10 % ( w/v) ) 、 5. O g (基質濃度 20% (w/v) ) または 12. 5 g (基質濃 度 50% (w/v) ) を添加し、 20% (w/v) 炭酸アンモニゥム水溶液で p Hを 6. 5に調整しつつ、 30°Cにて攪拌下、 30時間反応を行った。 反応開始 から 6時間目及び 20時間目にそれぞれ NAD 2. 8mgを添加した。 反応終了 後、 反応液をトルエンで抽出、 減圧濃縮し、 黄色油状の 2, 3—ジフルオロー 6 一-トロー [ [ (R) _ (2—ヒドロキシプロピル) ] ォキシ] ベンゼン濃縮物 を得た。 この濃縮物を、 実施例 1と同様の方法により分析し、 変換率及ぴ転移体 2, 3—ジフルオロー 6—二トロー [ [ (R) — (1—ヒドロキシイソプロピル ) ] ォキシ] ベンゼンの副生量を算出した。 また、 上記濃縮物の一部をカラム力 ラムクロマトグラフィーにより精製し、 ジニトロベンゾィル化した後、 HPLC 分析 (実施例 3と同様) を行ったところ、 光学純度はいずれも 99. 9 %e.e.K 上であった。 結果を表 3に示す。 The cell-free extract used in Example 1 and 1.25 mg of deionized water 23.75 m 1 s glucose 14.6 NAD 2.8 mg were added to 25 ml of 2-acetonyloxyl 3 , 4 difluoronitrobenzene 0.25 g (substrate concentration 1% (w / v)), 1.25 g (substrate concentration 5% (w / v)), 2.5 g (substrate concentration 10% (w / v)) / v)), 5.Og (substrate concentration 20% (w / v)) or 12.5 g (substrate concentration 50% (w / v)) and add 20% (w / v) ammonium carbonate The reaction was carried out for 30 hours with stirring at 30 ° C. while adjusting the pH to 6.5 with an aqueous solution. At 6 hours and 20 hours after the start of the reaction, 2.8 mg of NAD was added, respectively. After the completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily concentrate of 2,3-difluoro-6-1-tro [[(R) _ (2-hydroxypropyl)] oxy] benzene. This concentrate was analyzed in the same manner as in Example 1 to obtain the conversion and the by-product of the transition product 2,3-difluoro-6-twotro [[(R) — (1-hydroxyisopropyl)] oxy] benzene. The amount was calculated. A part of the above concentrate was purified by column chromatography, subjected to dinitrobenzoylation, and analyzed by HPLC (same as in Example 3). The optical purity was 99.9% eeK in all cases. Was on. Table 3 shows the results.
表 3 基質濾度 変換率 転移体副生量 Table 3 Substrate filtration conversion rate Transfer by-product amount
(%(w/v)) (%) (%) (% (w / v)) (%) (%)
1 >99.9 1.27 1> 99.9 1.27
5 >99.9 0.42 5> 99.9 0.42
10 >99.9 0.36 10> 99.9 0.36
20 >99.9 0.28 20> 99.9 0.28
50 99.9 0.20 50 99.9 0.20
(実施例 6) (Example 6)
実施例 1で用いた無細胞抽出液 2. 5 m 1に、 脱ィオン水 20. 2 m 1、 ダル コース 25. 3 gs NAD 7. 3 m g及び 2—ァセトニルォキシー 3 , 4—ジフ ルォ口-トロベンゼン 25 gを添加し (基質濃度 110% (w/v) ) 、 20% (wZv) 炭酸アンモユウム水溶液で pHを 6. 5に調整しつつ、 27°Cで攪拌
下、 21時間反応を行った。 反応開始から 6時間目及び 19時間目にそれぞれ N AD 7. 2mgを添加した。 反応終了後、 反応液をトルエンで抽出、 減圧濃縮し、 黄色油状の 2, 3—ジフルオロー 6—二トロー [ [ (R) — (2—ヒドロキシプ 口ピル) ] ォキシ] ベンゼン濃縮物を得た。 この濃縮物を実施例 1と同様の方法 により分析したところ、 変換率は 99. 9%以上、 転移体 2, 3—ジフルオロー 6—二トロー [ [ (R) — ( 1ーヒドロキシイソプロピル) ] ォキシ] ベンゼン の副生量は 0. 04%であった。 また、 上記濃縮物の一部をカラムカラムクロマ トグラフィ一により精製し、 ジニトロベンゾィル化した後、 HP LC分析 (実施 例 3と同様) を行ったところ、 光学純度は 9 9. 9%e.e.以上であった。 EXAMPLE cell-free extract 2. 5 m 1 used in 1, de Ion water 20. 2 m 1, Dar course 25. 3 g s NAD 7. 3 mg and 2 § Seto sulfonyl O Kishi 3, 4-diphenyl Add 25 g of roto-torobenzene (substrate concentration 110% (w / v)) and stir at 27 ° C while adjusting the pH to 6.5 with 20% (wZv) aqueous ammonium carbonate solution The reaction was carried out for 21 hours. At 6 hours and 19 hours after the start of the reaction, 7.2 mg of NAD was added, respectively. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily concentrate of 2,3-difluoro-6-nitro [[(R)-(2-hydroxypropyl)] oxy] benzene. . When this concentrate was analyzed by the same method as in Example 1, the conversion was 99.9% or more, and the transition product 2,3-difluoro-6-twotro [[(R)-(1-hydroxyisopropyl)] oxy ] The amount of benzene by-product was 0.04%. A part of the above concentrate was purified by column chromatography, subjected to dinitrobenzoylation, and subjected to HP LC analysis (same as in Example 3). The optical purity was 99.9% ee. That was all.
(比較例 1 ) (Comparative Example 1)
E s c h e r i c h i a c o l i HB 101 (p NT S 1 G) 受託番号 F ERM BP— 5835 (キャンディダ ·マグノリエ由来の還元酵素を生産する ) を、 500m l容坂ロフラスコ中で滅菌した 100m lの培地 (トリプトン 1 6 g、 ィーストエキス 10 g、 塩ィ匕ナトリウム 5 g、 水 1リツトル、 滅菌前 pH 7. 0) に接種し、 37°Cで 20時間振とう培養した。 得られた培養液 50ml に、 2ーァセトニノレォキシ一 3 , 4—ジフルォロニ ト ロベンゼン 2. 5 g、 グノレ コース 2. 97 g、 NADP 2. 9mgを添加し (基質濃度 5 % (w/v) ) 、 5 N水酸化ナトリゥム水溶液で p Hを 6. 5に調整しつつ、 30°Cで攪拌下、 2 3時間反応を行った。 反応終了後、 反応液をトルエンで抽出、 減圧濃縮し、 黄色 油状の 2, 3—ジフルオロー 6—エトロ一 [ [ (R) — (2—ヒドロキシプロピ ル) ] ォキシ] ベンゼン濃縮物を得た。 この濃縮物を実施例 1と同様の方法によ り分析したところ、 変換率は 80%、 転移体 2, 3—ジフルオロー 6—二トロー [ [ (R) — (1—ヒドロキシイソプロピ /レ) ] ォキシ] ベンゼンの副生量は 2. 28%であった。 また、 上記濃縮物の一部をカラムカラムクロマトグラフィーに より精製し、 ジュトロベンゾィル化した後、 HPLC分析 (実施例 3と同様) を 行ったところ、 光学純度は 99. 0%e.e.であった。 産業上の利用可能性
本発明の方法により、 医薬品中間体として極めて有用な、 高品質の (R) - 2 ーヒドロキシ一 1一フエノキシプロパン誘導体を、 安価な原料から効率よく製造 する事ができる。
Escherichiacoli HB101 (pNTS1G) accession number F ERM BP—5835 (producing reductase from Candida magnoliae) was sterilized in a 500-ml flask with a 100-ml medium (tryptone 16). g, 10 g of yeast extract, 5 g of Shiridani sodium, 1 liter of water, pH 7.0 before sterilization), and cultured with shaking at 37 ° C for 20 hours. To 50 ml of the obtained culture medium, 2.5 g of 2-acetoninoleoxy-1,3,4-difluoronitrobenzene, 2.97 g of gnorecose, and 2.9 mg of NADP were added (substrate concentration 5% (w / v)), while adjusting the pH to 6.5 with a 5 N aqueous sodium hydroxide solution, the reaction was carried out at 30 ° C for 23 hours with stirring. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily concentrate of 2,3-difluoro-6-etro [[(R)-(2-hydroxypropyl)] oxy] benzene. When this concentrate was analyzed by the same method as in Example 1, the conversion was 80%, and the transition product 2,3-difluoro-6-two-trough [[(R)-(1-hydroxyisopropyl) /]] The amount of benzene by-product was 2.28%. A part of the above concentrate was purified by column chromatography, converted into dutrobenzoyl, and subjected to HPLC analysis (same as in Example 3). The optical purity was 99.0% ee. there were. Industrial applicability By the method of the present invention, a high-quality (R) -2-hydroxy-11-phenoxypropane derivative extremely useful as a pharmaceutical intermediate can be efficiently produced from inexpensive raw materials.
Claims
(式中、 Rは置換基を有してもよいフエエル基を表す) で表されるフエノキシァ セトン誘導体のカルボエル基を R選択的に還元する能力を有する微生物由来の酵 素あるいは該酵素の産生能を有する微生物の培養物あるいは該処理物を用いて、 前記式 (1 ) で表されるフエノキシアセトン誘導体を R選択的に還元することに より、 一般式 (2 ) ; (Wherein, R represents a fuel group which may have a substituent) An enzyme derived from a microorganism having the ability to R-selectively reduce the carboxy group of the phenoxyacetone derivative represented by R-selective reduction of the phenoxyacetone derivative represented by the above formula (1) using a culture of the microorganism having the formula (1) or a treated product thereof, whereby the general formula (2);
(式中、 Rは前記と同じ基を表す) で表される (R ) — 2—ヒドロキシ _ 1ーフ (Wherein R represents the same group as described above).
'口パン誘導体を製造する方法において、 一般式 (3 ) ; 'In the method for producing mouth bread derivatives, the general formula (3):
R R
(式中、 Rは前記と同じ基を表す) で表される (R) — 1ーヒドロキシ一 2—フ ェ ンプロパン誘導体の副生量が生成物中の比率として 2 %以下であることを 特徴とする (R) — 2—ヒドロキシー 1—フエノキシプロパン誘導体の製造法。 (Wherein, R represents the same group as described above), wherein the amount of by-product of the (R) -1-hydroxy-12-phenpropane derivative is 2% or less as a ratio in the product. (R) — A method for producing 2-hydroxy-1-phenoxypropane derivatives.
2 . ( R) 一 1—ヒドロキシ一 2—フエノキシプロパン誘導体の副生量が生成 物中の比率として i %以下である請求の範囲 1記載の製造法。 2. The process according to claim 1, wherein the by-product amount of the (R) -11-hydroxy-12-phenoxypropane derivative is i% or less as a ratio in the product.
3 . (R) 一 1—ヒドロキシ一 2—フエノキシプロパン誘導体の副生量が生成 物中の比率として 0 . 5 %以下である請求の範囲 2記載の製造法。
3. The production method according to claim 2, wherein the by-product amount of the (R) -11-hydroxy-12-phenoxypropane derivative is 0.5% or less as a ratio in the product.
4. 前記 Rが 2, 3—ジフルオロー 6 _ニトロフエニル基であることを特徴と する請求の範囲 1〜 3のいずれかに記載の製造法。 4. The method according to any one of claims 1 to 3, wherein R is a 2,3-difluoro-6-nitrophenyl group.
5. 還元を pH2〜 7の条件下で行う請求の範囲 1〜4のいずれかに記載の製 造法。 5. The production method according to any one of claims 1 to 4, wherein the reduction is performed under conditions of pH 2 to 7.
6. 還元を pH2〜6. 5の条件下で行う請求の範囲 5記載の製造法。 6. The production method according to claim 5, wherein the reduction is carried out under a condition of pH 2 to 6.5.
7. 弱塩基を用いて p Hを調整する請求の範囲 1 ~ 6のいずれかに記載の製造 法。 7. The production method according to any one of claims 1 to 6, wherein pH is adjusted using a weak base.
8. 弱塩基が、 アンモエア、 炭酸塩または燐酸塩であることを特徴とする請求 の範囲 7記載の製造法。 8. The method according to claim 7, wherein the weak base is ammoair, carbonate or phosphate.
9. 弱塩基が、 炭酸アンモニゥム、 炭酸ナトリウム、 炭酸カルシウム、 燐酸水 素ニナトリウム、 または、 燐酸水素二力リゥムである請求の範囲 8記載の製造法。 9. The production method according to claim 8, wherein the weak base is ammonium carbonate, sodium carbonate, calcium carbonate, disodium hydrogenphosphate, or dihydrogen phosphate.
10. 還元を 10〜36 °Cの条件下で行う請求の範囲 1〜9のいずれかに記載 の製造法。 10. The production method according to any one of claims 1 to 9, wherein the reduction is performed under a condition of 10 to 36 ° C.
11. 基質濃度が 5% (w/v) 以上であることを特徴とする請求の範囲 1〜 10のいずれかに記載の製造法。 11. The method according to any one of claims 1 to 10, wherein the substrate concentration is 5% (w / v) or more.
12. 前記酵素が、 キャンディダ'マリス (C a n d i d a ma r i s) 由 来である請求の範囲 1〜 1 1のいずれかに記載の製造法。 12. The production method according to any one of claims 1 to 11, wherein the enzyme is derived from Candida's Maris (Candi damaris).
1 3. 前記酵素が、 キャンディダ .マリス (C a n d i d a ma r i s I FO 1 0003) 由来である請求の範囲 1 2記載の製造法。
13. The method according to claim 12, wherein the enzyme is derived from Candida maris IFO 10003.
14. 前記酵素の産生能を有する微生物が E s c h e r i c h i a c o l i HB 101 (pNTF PG) 受託番号 F ERM B P— 71 17である請求の 範囲 1〜 1 3のいずれかに記載の製造法。
14. The method according to any one of claims 1 to 13, wherein the microorganism capable of producing the enzyme is EscherichiaciacoliHB101 (pNTF PG) accession number F ERMBP-7117.
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03183489A (en) * | 1989-12-13 | 1991-08-09 | Dai Ichi Seiyaku Co Ltd | Production of optically active propoxybenzene derivative |
JPH04267890A (en) * | 1991-02-20 | 1992-09-24 | Dai Ichi Seiyaku Co Ltd | Production of (r)-2-propoxybenzene derivative |
JPH0568577A (en) * | 1990-12-11 | 1993-03-23 | Mercian Corp | Production of propoxybenzen derivative |
WO2000037666A1 (en) * | 1998-12-18 | 2000-06-29 | Kaneka Corporation | Process for producing (r)-2-hydroxy-1-phenoxypropane derivative |
JP2002085085A (en) * | 2000-09-11 | 2002-03-26 | Daicel Chem Ind Ltd | Method for producing (r)-propoxybenzene derivative |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03183489A (en) * | 1989-12-13 | 1991-08-09 | Dai Ichi Seiyaku Co Ltd | Production of optically active propoxybenzene derivative |
JPH0568577A (en) * | 1990-12-11 | 1993-03-23 | Mercian Corp | Production of propoxybenzen derivative |
JPH04267890A (en) * | 1991-02-20 | 1992-09-24 | Dai Ichi Seiyaku Co Ltd | Production of (r)-2-propoxybenzene derivative |
WO2000037666A1 (en) * | 1998-12-18 | 2000-06-29 | Kaneka Corporation | Process for producing (r)-2-hydroxy-1-phenoxypropane derivative |
JP2002085085A (en) * | 2000-09-11 | 2002-03-26 | Daicel Chem Ind Ltd | Method for producing (r)-propoxybenzene derivative |
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