JPH03191797A - Enzymatic optical resolution of d,l-amines - Google Patents

Abstract

PURPOSE:To carry out the subject enzymatic optical resolution of D,L-amines with high selectivity by reacting one optically active isomer alone of the D,L- amines with a substrate ester in a solvent mainly composed of organic solvents using an immobilized enzyme. CONSTITUTION:An amine represented by formula I is made to a reaction with a substrate ester expressed by formula II in the presence of an immobilized enzyme of protease, lipase, etc., in a solvent mainly composed of organic solvents. The above-mentioned reaction is carried out ordinarily at 4-80 deg.C, preferably at 20-50 deg.C. Immediately after the above-mentioned reaction, the solvent is distilled off, thus obtaining an optically active amide with high selectivity. In the above-mentioned enzymatic optical resolution method of the D,L-amines using the enzyme-fixed carrier, the extraction, dialysis, etc., for removal of the enzyme are not required at all after the reaction. In addition, the above- mentioned reaction is free from the reverse reaction or the side reactions as the solvent composition such as water content can be kept optimal. A high yield and a high optical yield can be realized therefor.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention uses an immobilized enzyme in a solvent mainly consisting of an organic solvent to highly selectively target only one optically active form of L-amines. The present invention relates to a method for enzymatic optical resolution of D,L-amines by reacting with a substrate ester to obtain an optically active amide and the other optically active amine.

(Prior art) Synthesis of optically active organic compounds is very important in the development of physiologically active compounds.
ry March), Advanced Organi 7ri
Chemistry (^advanced OrganicC
(McGraw-Hill; 1977) 2nd edition, p. 106.
++netric 5 synthesis) and same as above 10
The optical resolution method described on page 8 is known.

However, conventional methods have the disadvantages that the same mole of optically active substrate is required to separate racemates or to produce one optically active form, and the optical yield of the optically active form is very low. are doing. Moreover, although many examples of application to carbonyl compounds, carboxylic acids, and olefins are known, at present, not much is known about the synthesis of optically active amines or the separation of optically active amines.

On the other hand, in addition to the conventional synthesis of optically active substances using chemical methods, the synthesis of optically active substances using enzymatic reactions has become active in recent years. Its advantages over chemical methods include high selectivity, i.e., high optical yield, mild reaction conditions, little side reactions, and only the presence of a catalytic amount of enzyme. Since this method can only be applied to hydrolysis reactions, redox reactions, etc., it has been difficult in terms of optical synthesis of amines. However, as a new method to overcome the limitations of such enzymatic methods, enzymatic reactions in organic solvents have been investigated, and peptide synthesis has become possible.
et al. added protease or lipase powder to an organic solvent and succeeded in obtaining ε-peptide, which was not produced at all in an aqueous enzymatic reaction, by the reaction between an L-lysine ester and a phenylalanine ester. * (T
etrahedron Letters, 1988.5
487).

Furthermore, by developing these findings, we succeeded in obtaining optically active amides by adding subtilisin or lipase powder to an organic solvent and reacting D and L amines with substrate esters (Journal of
American Chemical 5ociet
y.

1989.111.3094).

However, with such powder methods, it is difficult to isolate the target product after the reaction is completed, the enzyme as a catalyst is not effectively used because the enzyme is not dissolved, and alcohols produced during the reaction are difficult to isolate. is difficult to remove,
There are many problems, such as the fact that the amount of water present in the system is not uniform, so that reverse reactions occur simultaneously, reducing the yield of the target product.

Particularly in enzymatic reactions in organic solvents, it is important to control the amount of water in the reaction system. Too much moisture may promote reverse reactions and rearrangement reactions. On the other hand, in order to maintain enzyme activity, it is thought that an amount of water called so-called esotential water is absolutely necessary. In other words, in the active state, a certain amount of water exists in the enzyme and its vicinity, and the question is how to efficiently supply substrate molecules that are difficult to dissolve in water to the enzyme in such a hydrophilic environment. This is an important issue.

(Problem to be solved by the invention) By enzymatic method using enzyme powder in organic solvent.

The optical resolution method for L-amines is superior to conventional chemical methods, but as mentioned above, it is difficult to isolate the product, the efficiency of enzyme utilization is low, and the reverse reaction is difficult. The purpose of the present invention is to The aim is to provide new means to solve such problems.

(Means for Solving the Problems) These objects of the present invention were achieved by reacting D,L-amines and substrate esters in the presence of an immobilized enzyme.

The D,L-amines and substrate esters used in the present invention are represented by the following general formulas (I) and 0, respectively.

Ht N CHR” R” COR
'1 (1) (II) R1 of the amines represented by the general formula (I) is preferably a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, and a substituted or unsubstituted alkyl group that may form a ring. The group is preferably an alkyl group, a phenyl group, a halogen atom or an alkoxy group. Particularly preferable R1 has 1 to 1 carbon atoms.
4 straight chain or branched alkyl group. Rt of the amines represented by the general formula (1) is preferably an alkyl group having 1 to 15 carbon atoms, an aryl group, or a carbamoyl group, and the substituent thereof is an alkyl group, an aryl group,
A halogen atom or an alkoxy group is preferred. Particularly preferred as R2 are linear or branched alkyl groups having 2 to 8 carbon atoms, aryl groups having 6 to 10 carbon atoms, aryl groups having 8 to 12 carbon atoms, or heterocyclic alkyl groups or carbon atoms. It represents a carbamoyl group of numbers 1 to 5.

R3 of the substrate ester represented by the general formula CI+) is preferably an alkyl group or an aryl group having 1 to 10 carbon atoms, and the substituent is an alkyl group, a halogen atom,
Aryl groups are preferred. Particularly preferred R3 is an alkyl group having 1 to 5 carbon atoms or an aryl group having 6 to 10 carbon atoms. R4 is preferably a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, or a substituted or unsubstituted alkyl group having 6 to 16 carbon atoms, particularly preferably an unsubstituted or unsubstituted alkyl group having 1 to 5 carbon atoms. Represents an alkyl group substituted with a chloro atom or a fluoro atom.

Many of the D,L-amines represented by the general formula (1) are commercially available and can be easily obtained, but the imine form is preferable to the ketone form shown by the following general formula (II), which is the precursor thereof. After obtaining (IV), catalytic reduction or L i A
Easily synthesized by IHa reduction.

CIII), (IV)
(1) Alternatively, the substituted product (V
After obtaining l), it can be easily synthesized by reducing it with hydrazine (this reaction is famous as Gabriel 5 synthesis).

The ester represented by the general formula (II) is commercially available and can be easily obtained, but it can also be easily synthesized by a condensation reaction of carboxylic acids and alcohols.

Specific examples of compounds represented by general formula (I) and general formula (I[) are shown below, but the invention is not limited thereto.

(1) (1) HzN　COCJs CH3 C.I.

CI)-(3) HJ　CHHCHz)4cHs CHl c, n.

Hs (1)-(5) HJ　CHC+oHt+ Hi CH3 H3 CH2 O F3 H1F (II) (4) C4H9C0CzHnC1 1 2 HI Hs (II) (1) CH2-C-OCRs 1 (II) (2) CsHsCOCH3 II (II) (3) C1HsC-OCHxCFs 1 (II) (13) C3HtC-OCHICH mushroom CF2 1 (n) (14) CsH++C-0CtHaCj! 1 [11) (15) CsHtC-OCHtC11tOH 1 (If) (16) CHsCOCJ* 1 C.I.

The enzyme used in the immobilized enzyme used in the present invention is protease or lipase. An example of protease is α
- Chymotrypsin, subtilisin, papain, promelain, prolysin, thermolysin, etc.
In particular, subtilisin is most preferred. As an example of lipase, lipoprotein lipase is most preferred.

In the method of the present invention, immobilized versions of these enzymes are used. The powder or molded particles for immobilization are not particularly limited as long as they are resistant to the organic solvent used (
, natural or synthetic polymers and inorganic materials can be used. Examples of these are shown below. Natural materials include powders or molded particles of cellulose (for example, trade name Cellulofine) and its derivatives such as carboxymethyl cellulose, sulfopropyl cellulose, sulfated cellulose, diethylaminoethyl cellulose, and iminodiacetic acid derivatized cellulose. Synthetic polymers include polyvinyl alcohol, Examples include poly (2-hydroxyethyl) methacrylate, polystyrene, polymethyl methacrylate, polyacrylamide, other vinyl polymers, and copolymers thereof [commercial products include, for example, Amberlite, % Amberlyst (Am
berlyst), Dowex, etc. Sephacryl (5ephacryl), Copal (Toyopearl), Sphelon (Sph)
Examples include carriers for liquid chromatography such as Eron).

Examples of inorganic materials include glass, porous ceramics, and metal oxides.

Enzymes can be immobilized on these carriers by conventional methods such as physical adsorption, inclusion, crosslinking, and covalent bonding. For these methods, for example, Me
thods in Enzymology vol.

34", "same, vol, 44", "same, vol,
135” (more than ^cades+ic Press),
”Iss+obilized Enzymesand
Ce1ls” (Adaa+ Hilger, 1
987) and “immobilized biocatalyst” (partly edited by Chibata)
Kodansha, 1986). However, the immobilization method is not limited to these (for example, it is also possible to use a method in which the enzyme is simply held on a porous carrier (in addition, in the following, the enzyme is , immobilized on particles such as molded particles are called enzyme-immobilized carriers).

In the method of the present invention, the enzyme can be used after being immobilized and dried by freeze-drying, or after being gradually replaced with the organic solvent used.

The immobilized enzyme used in the present invention also includes enzymes immobilized on membranes. The material of the enzyme-immobilized membrane is not particularly limited as long as it is insoluble in the organic solvent system used, but examples include cellulose, chitosan, glucomannan, polyvinyl alcohol, and ethylene-vinyl alcohol. Polymer, polyethylene, polypropylene,
Polytetrafluoroethylene, polyimide, ceramic, and the like are preferably resistant to a wide range of organic solvent systems, and among these, membranes containing cellulose as a main component are particularly preferred because of their low nonspecific adsorption of proteins.

The shape of the membrane may be flat, hollow, or tubular, and the membrane thickness is preferably 1011 m or more from the viewpoint of membrane strength.A wide range of pore sizes can be used, but microscopic pores are particularly preferred from the viewpoint of enzyme utilization efficiency. A membrane with pores is preferred, with a diameter of 0.1μ to 20μ
Particularly preferred are filtration membranes (microfilters, filter papers, etc.) with an average pore diameter of (for example, filter paper), but so-called asymmetric membranes with varying pore sizes are particularly preferred.

A flow method at normal pressure may be used to bring the enzyme-immobilized membrane into contact with the organic solvent, but in order to increase the reaction efficiency of the enzyme by speeding up the movement of the substrate solution, pressure is applied to bring the enzyme-immobilized membrane into contact with the organic solvent. A method of passing an organic solvent is more preferred.

Methods for immobilizing the enzymes mentioned above on membranes include cross-linking between enzymes using a multifunctional reagent, and immobilizing enzymes on membranes using methods such as covalent bonding, electrostatic interaction, and physical adsorption. It can be fixed by almost the same operation as when fixing to a carrier.

The organic solvent that can be used in the present invention is not particularly limited, but examples include the following. Solvents that mix with water include methanol, ethanol, ethylene glycol,
Glycerol, acetone, dioxane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, etc. Also, solvents that are difficult to mix with water include n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, 5ec.
-butyl alcohol, t-butyl alcohol, 2-methyl-2-butanol, n-amyl alcohol, 1so-
Amyl alcohol, t-amyl alcohol, n-hexanol, 3-methyl 3-pentanol, n-heptatool, n-octatool, diethyl ether, diisopropyl ether, ethyl acetate, n-butyl acetate, methylene chloride, chloroform, tetra Chloride-* element, n-hexane,
Examples include n-hebutane, n-octane, isooctane, cyclohexane, benzene, toluene, and xylene. These organic solvents are selected depending on the combination with the enzyme used, but N-, N-
Dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, solvents that are difficult to mix with water include t-butyl alcohol, 2-methyl-2-butanol, 1so-amyl alcohol, t-amyl alcohol, 3-methyl-3- Pentanol, methylene chloride and toluene are used. Particularly preferred among these is N,N-dimethylformamide.

-Amyl alcohol, t-amyl alcohol, 3-methyl-3-pentanol, toluene are used.

These solvents can be used in combination, and can also contain water at a concentration of 10% or less and less than the saturation solubility.

It is preferable that the concentration of the starting materials used in the method of the present invention is as high as possible because of the rapid reaction rate; these are usually used at concentrations within the solubility in their respective solvents - for boat fishing, from 1mM to 2M, usually from 1QmM to It is preferable to use about 0.5M.

The amount of immobilized enzyme used in the present invention is not particularly limited; the higher the concentration used, the shorter the reaction time will be.
This also depends on the specific activity of the immobilized enzyme. In general, on a dry weight basis, the weight of enzyme per milli-o1 of restarting material is 1 to 10 g, preferably about 10 to 5 g. The molar ratio of the compounds represented by ) is 10:1 to 1:10, preferably 3:1 to 1:3.

The method using the enzyme-immobilized carrier of the present invention can be carried out, for example, by suspending a dried immobilized enzyme in a solvent containing both raw materials represented by general formulas (1) and (II) and stirring the suspension. After the completion of the reaction, the product solution can be easily taken out by operations such as filtration or decantation. Alternatively, the reaction can be carried out by filling a column with an immobilized enzyme suspended in an organic solvent used for the reaction, and flowing the organic solvent containing the starting material into the packed bed. The use of such a method is industrially advantageous in that the reaction can be carried out continuously.

The reaction temperature is usually 4°C to 80°C, preferably 20°C to 5°C.
It is 0°C.

The contact time (reaction time) between the substrate solution and the immobilized enzyme is usually about 5 minutes to about 100 hours, but this greatly depends on the type and concentration of the substrate, the amount and activity of the immobilized enzyme, temperature, etc. It is.

When using the enzyme-immobilized membrane of the present invention, the flow rate at which the substrate solution permeates through the enzyme-immobilized membrane is approximately 0.03~
Approximately 2 m/min・d is preferable, and the reaction temperature is 10 to 90°C.
is preferred.

Biotechnology is a suitable device for use.
andBioengineering vol.X
I, p 366 (1969), S, 5ouri
Written by rajan rReverse 0slIlosisJ
, LogosPress Lim1ted 5cie
ntific Publication (1970)
Examples include devices described in pI) 25-27 and Japanese Patent Publication No. 62-53151.

(Effect of the invention) In the enzymatic optical resolution method of D,L-amines using the enzyme-immobilized carrier of the present invention, there is no need for extraction operations or dialysis to remove the enzyme afterwards, and the solvent is removed immediately after the reaction. The product is obtained by distillation. Furthermore, since the solvent composition such as water content can be maintained at optimum conditions, reverse reactions and side reactions can be suppressed, and high reaction yields and optical yields can be achieved.

The enzymatic optical resolution method of D,L-amines using the enzyme-immobilized membrane of the present invention also eliminates the need for an operation to remove the enzyme. Furthermore, since the enzyme is uniformly immobilized on the inner surface of the micropores, the utilization efficiency of the enzyme is very high and efficient. The amount of water that affects the reverse reaction and the alcohols produced by the reaction are uniformly distributed throughout the membrane, so the reverse reaction is effectively prevented.

Furthermore, by forcibly feeding the substrate into the micropores under a certain pressure, it is possible to supply the substrate without causing diffusion, preventing a decrease in reaction yield due to the progress of the reverse reaction, and producing products with poor solubility. Effects such as prevention of enzyme reaction inhibition caused by substances and by-products and an increase in production rate due to the effect of promoting access of substrates with low water solubility to the vicinity of the enzyme can be obtained.

(Example) Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 0.025M HE made by water-cooling Nezumi Awamon Sada ■Taiki Mei Ai Subtilisin 100■
PES buffer 3+d (pH 7, 5, 5M NaB
0.5 g of dried Amberlite XAD-7 resin (porous polymer beads from Rohm and Haas) was added. After shaking slowly for 12 hours at 4°C, add 50% glutaraldehyde solution to 0.
．． 2- was added and shaken at 4°C for 3 hours to immobilize the enzyme. Then 0.1M HEPES buffer pi ('7.
5) to remove unfixed subtilisin.

Furthermore, thermolysin and riboprotein lipase were immobilized by the same operation.

Example 2 Isolation and purification [α]. were measured and compared with literature value data and shown in Table 1.

Comparative Example 1 100μ of enzyme powder was added to the same substrate solution (20-) as described in Example 2, and the mixture was heated in a flask at 25°C to 45°C for 244°C.
I spent a lot of time. After the reaction solution was filtered, the solvent was distilled off under reduced pressure and the amide compound was separated by silica gel chromatography.

The obtained data are shown in Table 1 as a comparative example.

The enzyme-immobilized carrier was packed into a glass column with an inner diameter of 1c11,
D,L-amines (general formula CI
]) 4mM and substrate ester (general formula C1C11)
) The solvent solution (20d) of 4 was permeated and circulated for 24 hours at a flow rate of about 1-7 minutes at 25°C to 45°C. After distilling off the solvent of the reaction solution under reduced pressure, the amide compound was separated by silica gel chromatography. Table 1 shows that the method of permeating and circulating the reaction solution using the immobilized carrier of the present invention has a higher yield and lower yield with the same enzyme and reaction substrate than the known method (comparative example) in which powdered enzyme is added to the solvent. It can be seen that the optical yield is improved.

In addition, in the experimental examples of the present invention (-1 to Arashi 5), since there is no enzyme in the reaction solution, there is no need to remove the enzyme in the post-treatment step, and the progress of the reverse reaction in this step No reduction in reaction yield occurred.

Example 3 (1) Preparation of a fixed armpit of an imperial plate (34 g of p-nitrophenoxychloroformate and 100 g of acetonitrile were added to a 1200 d cellulose microfilter FR40 (manufactured by Fuji Photo Film).

Pyridine 30- is added dropwise while stirring under water cooling, followed by shaking at 40°C for 30 minutes. Thereafter, the membrane was thoroughly washed with methanol and acetone to obtain an activated cellulose membrane.

Subsequently, subtilisin 100w was added to 16mM CaCjg.
0.05M borate buffer pH 8.0 containing 50-
dissolve in The above solution was passed through an activated cellulose membrane set in a 47-φ filter holder suitable for furnaces at a rate of about 3 to 7 minutes at 4°C using a peristaltic pump, and the subtilisin was immobilized by circulation for 5 hours. In order to remove remaining active groups, a 0.1 M ethanolamine aqueous solution was circulated at 4° C. for 2 hours, and finally, the membrane was sufficiently washed by permeating the buffer solution to obtain a subtilisin-immobilized cellulose microfilter.

A cellulose microfilter on which thermolysin (100 .mu.m) and lipoprotein lipase (100 .mu.m.) were immobilized was prepared in exactly the same manner.

Example 4 The thermolysin fixed membrane prepared in Example 3 was set in a filter hole cedar (47 m-φ), and an organic solvent solution of D, L-amines [134 mM and substrate ester (II) 4 mM] was added using a helister pump. (20sd) was permeated and circulated at 25°C to 45°C, and the amide compound produced after 24 hours was isolated and quantified. The flow rate was approximately 3-7 minutes. The experimental results are shown in Table 2.

Comparative Example 2 An amide compound was produced and separated by the same method as described in Comparative Example 1, and the obtained data are shown in Table 2 as a comparative example.

From Table 2, the enzymatic optical resolution method of L-amines using the enzyme-immobilized membrane of the present invention has a higher yield of amidation than the known method (comparative example) in which powdered enzyme is added to an organic solvent. It can be seen that the yield and optical yield are improved. In addition, it became unnecessary to remove the enzyme after the reaction, and it was possible to prevent a reverse reaction due to contamination of the enzyme during the reaction process.

Claims (3)

[Claims] (1) D, characterized in that an optically active amide form is obtained with high selectivity by reacting D,L-amines with a substrate ester form in a solvent mainly consisting of an organic solvent in the presence of an immobilized enzyme. , a method for enzymatic optical resolution of L-amines. (2) The method for enzymatic optical resolution of D,L-amines according to claim 1, wherein the immobilized enzyme is an enzyme immobilized on powder or molded particles. (3) The method for enzymatic optical resolution of D,L-amines according to claim 1, wherein the immobilized enzyme is an enzyme immobilized on a membrane.