JP2003002872A - Method for preparation of lysine derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for industrial preparation of an optically active lysine derivative which is useful as an intermediate of medicines. SOLUTION: After protecting an amino group or the amino group together with a carboxyl group of optically active 2-amino-6-methyl-6-nitroheptanoic acid with a protecting group, the nitro group is reduced to synthesize a 6, 6- dimethyl-lysine derivative. Then, the 6, 6-dimethyl-lysine derivative is reacted with an acetic acid derivative.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a specific optically active lysine derivative useful as a pharmaceutical intermediate.

[0002]

2. Description of the Related Art General formula (3)

[0003]

[Chemical formula 26]

[* Represents an asymmetric carbon, and P ₁ and P ₂ each independently represent a protecting group for an amino group or a hydrogen atom (except when P ₁ and P ₂ are simultaneously a hydrogen atom). ), Or P ₁ and P ₂ together represent an amino-group protecting group, and P ₄ represents a hydrogen atom or a carboxyl-group protecting group. ] The optically active 6,6-dimethyl lysine derivative represented by or general formula (5)

[0005]

[Chemical 27]

[*, P ₁ and P ₂ have the same meanings as described above, R ₁ represents an alkyl group having 1 to 6 carbon atoms or an aralkyl group having 7 to 12 carbon atoms, and P ₅ is a hydrogen atom or A protective group for a carboxyl group is shown. ] The optically active lysine derivative represented by the above is a compound useful as a pharmaceutical intermediate.

For example, a compound having an asymmetric carbon configuration of S-configuration is represented by the following formula (25) which is useful as an antihypertensive agent having inhibitory activity against angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP). To be an important intermediate for pharmaceutical compounds (Journal of Medicinal Chemistry, Vol. 42, 305-311)
Page, 1999 [Journal of Medicinal Chemistry, 19
99, 42, 305-311]).

[0008]

[Chemical 28]

As a method for producing the pharmaceutical compound represented by the general formula (25), Journal of Medicinal Chemistry, Vol. 42, pp. 305-311, 1999 [Journal of Medicinal Chemistry, 1999, 42, 305- 31
1] discloses a production method represented by the following scheme using (S) -2-phthalimido-6-hydroxyhexanoic acid as a starting material.

[0010]

[Chemical 29]

However, this method has many steps,
A simpler production method is required for industrial production.

By the way, it is known that a 5-substituted hydantoin can be used as a starting material and directly acted on a microbial enzyme system to produce an optically active amino acid. When a microbial enzyme system is used, an enzyme (hydantoinase) that hydrolyzes a 5-substituted hydantoin compound to produce an N-carbamyl amino acid, and the produced N-carbamyl amino acid is optically selectively degraded to obtain an optical activity. An enzyme that produces amino acids (N-carbamyl amino acid hydrolase) is required.

Conventionally, as a method for producing a D-amino acid using a microorganism originally containing these two kinds of enzymes, or a substance containing these enzymes, Pseudomonas (Pseu) is used.
domonas) method using bacteria (Japanese Patent Publication No. 56-0030)
34), a method using a bacterium of the genus Agrobacterium (Japanese Patent Laid-Open No. 03-019696) and the like are known.

As a method for producing the L-amino acid, a method using a bacterium of the genus Flavobacterium (Japanese Patent Publication No. 56-008749) and a method using a bacterium of the genus Bacillus (Japanese Patent Laid-Open No. 63-1987) 248
95), a method using a Pseudomonas genus bacterium (JP-A-01-071476), and a method using an Arthrobacter genus bacterium (J. Biotechno).
l. 46, p. 63, 1996) etc. are known.

Furthermore, hydantoinase and N of various bacteria
It has been reported that an optically active amino acid can be produced by isolating carbamyl amino acid hydrolase gene DNA and expressing it in E. coli (Biotechnol. Prog. 16: 564, 2000). Year, or Patent No. 2902112).

The substrate specificity of these hydantoinase or N-carbamyl amino acid hydrolase is broad to a certain extent, and by combining these two enzymes within the range of substrate specificity, a natural or non-natural optically active substance having optical activity can be obtained. It has been detailed that various amino acids can be produced from 5-substituted hydantoin compounds (Enzyme Catalysisin
Organic Synthesis, K. Drauz et al., Volume 1, Chapter B2.4, 40
9-431 pages, VCH Publishing, 1995). However, since the optically active amino acids that can be produced are limited in their substrate specificity, it is not possible to produce the corresponding optically active amino acids from all 5-substituted hydantoin compounds. For example, Microbacterium liquefaciens AJ394 capable of producing L-tryptophan in high yield from racemic 5-indolylmethylhydantoin.
0 strain (formerly Flavobacterium sp.
acterium sp.). ) Acts well on a 5-substituted hydantoin having an aromatic ring and can produce many kinds of L-form aromatic amino acids such as L-phenylalanine and L-tyrosine, but 5-methylhydantoin, 5-sec-butylhydantoin, 5-
It is known to be aromatic-specific because it does not act on carboxymethylhydantoin and 5-carboxyethylhydantoin at all and does not produce the corresponding L-alanine, L-isoleucine, L-aspartic acid and L-glutamic acid. (Agric. Biol. Chem. 51, 729, 1987).

[0017]

The object of the present invention is to provide a method for industrially producing an optically active lysine derivative represented by the above general formulas (3) and (5), which is useful as a pharmaceutical intermediate. It is in.

[0018]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, have been industrially excellent as a novel method for producing the compounds represented by the general formulas (3) and (5). I found a simple reaction sequence. That is, after protecting the amino group of optically active 2-amino-6-methyl-6-nitroheptanoic acid and optionally a carboxyl group with a protecting group, the nitro group is reduced to give 6,6 represented by the general formula (3). -A process for synthesizing a dimethyl lysine derivative and further reacting the 6,6-dimethyl lysine derivative with an acetic acid derivative to synthesize an optically active lysine derivative represented by the general formula (5) is achieved. Found and completed the present invention.

More specifically, the present invention includes the following contents.

[1] Expression (1)

[0021]

[Chemical 30]

[* Indicates an asymmetric carbon. ] The amino group or the amino group and carboxyl group of the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the general formula (2)

[0023]

[Chemical 31]

[* Represents an asymmetric carbon, and P ₁ and P ₂ each independently represent an amino-protecting group or a hydrogen atom (except when P ₁ and P ₂ simultaneously become a hydrogen atom). ), Or P ₁ and P ₂ together represent an amino-group protecting group, and P ₃ represents a hydrogen atom or a carboxyl-group protecting group. ] The optically active amino acid derivative represented by

[0025]

[Chemical 32]

[*, P ₁ and P ₂ have the same meaning as described above, and P ₄ represents a hydrogen atom or a protecting group for a carboxyl group. ] The optically active 6,6-dimethyl lysine derivative or its salt represented by these are obtained, These are represented by general formula (4)

[0027]

[Chemical 33]

[Y ₁ represents a leaving group, R ₁ represents ₁ carbon atom
Is an alkyl group having 6 to 6 or an aralkyl group having 7 to 12 carbon atoms. ] The general formula (5) characterized by reacting with an acetic acid derivative represented by

[0029]

[Chemical 34]

[*, P ₁ , P ₂ and R ₁ have the same meanings as described above, and P ₅ represents a hydrogen atom or a protecting group for a carboxyl group. ] The manufacturing method of the optically active lysine derivative represented by these, or its salt. [2] The production method according to [1], wherein the reduction is catalytic reduction using a transition metal catalyst and a metal sulfate. [3] The production method according to [1], wherein the reduction is catalytic reduction using a palladium catalyst and ferrous sulfate. [4] Either P ₁ or P ₂ is a tert-butoxycarbonyl group and the other is a hydrogen atom, and P ₃ , P ₄ and P
The production method according to [1], wherein ₅ is a methyl group and R ₁ is a tert-butyl group. [5] Optically active 2-amino-6 represented by the above formula (1)
-Protecting the amino group or amino group and carboxyl group of methyl-6-nitroheptanoic acid or a salt thereof with a protecting group to give an optically active amino acid derivative represented by the above general formula (2), and reducing the nitro group. A method for producing the optically active 6,6-dimethyllysine derivative represented by the above general formula (3) or a salt thereof, comprising: [6] The production method according to [5], wherein the reduction is catalytic reduction using a transition metal catalyst and a metal sulfate. [7] The production method according to [5], wherein the reduction is catalytic reduction using a palladium catalyst and ferrous sulfate. [8] The production method according to [5], wherein _{one of} P _{1 and} P ₂ is a tert-butoxycarbonyl group, the other is a hydrogen atom, and P ₃ and P ₄ are methyl groups. [9] 6,6 represented by the general formula (3) characterized by reducing the amino acid derivative represented by the general formula (2)
-A method for producing a dimethyllysine derivative or a salt thereof. [10] The production method according to [9], wherein the reduction is catalytic reduction using a transition metal catalyst and a metal sulfate. [11] The production method according to [9], wherein the reduction is catalytic reduction using a palladium catalyst and ferrous sulfate. [12] The production method according to [9], wherein _{one of} P _{1 and} P ₂ is a tert-butoxycarbonyl group, the other is a hydrogen atom, and P ₃ and P ₄ are methyl groups. [13] Optical activity represented by the general formula (3) 6,6-
The dimethyl lysine derivative or a salt thereof is converted to the above general formula (4)
A method for producing an optically active lysine derivative represented by the above general formula (5) or a salt thereof, which comprises reacting with an acetic acid ester derivative represented by: [14] Either P ₁ or P ₂ is a tert-butoxycarbonyl group, the other is a hydrogen atom, P ₄ and P ₅ are methyl groups, and R ₁ is a tert-butyl group [1
3] The manufacturing method as described above. [15] Optically active 2-amino-represented by the above formula (1)
6-methyl-6-nitroheptanoic acid has the following (a) to
The manufacturing method according to [1], which is manufactured through the step (c): (a) General formula (6)

[0031]

[Chemical 35]

[Y ₂ represents a leaving group. ] 4-represented by
Methyl-4-nitropentane derivative and general formula (7)

[0033]

[Chemical 36]

[R ₂ and R ₃ each independently have 1 to 6 carbon atoms.
Is an alkyl group or an aralkyl group having 7 to 12 carbon atoms. ] By reacting with 2-acylaminomalonic acid diester represented by the general formula (8)

[0035]

[Chemical 37]

[R ₂ and R ₃ have the same meanings as described above. ] Obtaining 2-acylamino-2- (4-methyl-4-nitropentyl) malonic acid diester represented by the formula: (b) 2-acylamino-2- represented by the general formula (8).
The (4-methyl-4-nitropentyl) malonic acid diester is hydrolyzed and decarboxylated to give the compound of formula (9)

[0037]

[Chemical 38]

A step of obtaining 2-acetylamino-6-methyl-6-nitroheptanoic acid (racemic form) represented by: (c) 2-acetylamino-6-methyl-6 represented by formula (9) -A step of reacting nitroheptanoic acid (racemic form) with an acylase to obtain the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1) or a salt thereof. [16] Optically active 2-amino-6- represented by formula (1)
Methyl-6-nitroheptanoic acid has the following steps (d)-
The manufacturing method according to [1], which is manufactured through (g): (d) Formula (10)

[0039]

[Chemical Formula 39]

5- (4-methyl-4-nitropentylidene) hydantoin represented by the formula

[0041]

[Chemical 40]

Step of obtaining 5- (4-methyl-4-nitropentyl) hydantoin represented by: (e) Hydrolyzing 5- (4-methyl-4-nitropentyl) hydantoin represented by formula (11) Decompose and formula (12)

[0043]

[Chemical 41]

2-amino-6-methyl-6- represented by
Step of obtaining nitroheptanoic acid (racemic form); (f) 2-amino-6-methyl-represented by formula (12)
Amino group of 6-nitroheptanoic acid is acylated to obtain a compound represented by the general formula (13):

[0045]

[Chemical 42]

[R ₄ represents a methyl group or a phenyl group. ] 2-Acylamino-6-methyl-6- represented by
Step of obtaining nitroheptanoic acid (racemic form); (g) 2-acylamino-6 represented by general formula (13).
-A step of reacting methyl-6-nitroheptanoic acid (racemic form) with an acylase to obtain the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by formula (1) or a salt thereof. [17] Any of the compounds represented by the formula (14) and the general formulas (15) to (18) (including an optically active substance and a racemate) or a salt thereof:

[0047]

[Chemical 43]

[P ₁ and P ₂ each independently represent a protecting group for an amino group or a hydrogen atom (provided that P ₁ and P ₂ are simultaneously hydrogen atoms), or P ₁ and P ₂ are integral with each other. Represents an amino-protecting group (provided that P ₁ and P ₂
Except for the case where the groups together represent a phthaloyl group), P _3a
Represents a protecting group for a carboxyl group, and P ₄ represents a hydrogen atom or a protecting group for a carboxyl group. ]. [18] Any compound represented by formulas (19) to (23) or a salt thereof:

[0049]

[Chemical 44]

.. [19] Any of the compounds represented by the formulas (10) and (11) and the general formulas (6), (8) and (24) (the compounds represented by the formulas (8) and (24) are optical). (Including active form and racemate) or salts thereof:

[0051]

[Chemical formula 45]

[Y ₂ represents a leaving group (excluding chlorine atom), R ₄ represents a methyl group or a phenyl group, and R ₂ and R
₃ is each independently an alkyl group having 1 to 6 carbon atoms or 7
~ 12 aralkyl groups are shown. ]. [20] Optically active 2-amino-represented by the above formula (1)
6-Methyl-6-nitroheptanoic acid is produced through the following step (h): [1]: (h) General formula (11)

[0053]

[Chemical formula 46]

5- (4-methyl-4-nitropentyl) hydantoin represented by
An optically active compound represented by the formula (1) after being optically and selectively hydrolyzed with at least one selected from the group consisting of hydantoinase and N-carbamyl amino acid hydrolase.
A step of obtaining amino-6-methyl-6-nitroheptanoic acid or a salt thereof. [21] Microorganisms, processed products of microorganisms, sources of hydantoinase and N-carbamyl amino acid hydrolase are genus Agrobacterium, genus Bacillus or microbacterium (Microba).
cterium) bacterium, [20]. [22] The origin of the microorganisms, processed products of the microorganisms, hydantoinase and N-carbamyl amino acid hydrolase is a bacterium of the genus Agrobacterium, and optically active 2-amino-6-methyl-6-nitroheptanoic acid. Alternatively, the salt thereof is D-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof, [20]. [23] The origin of the microorganism, the processed product of the microorganism, the hydantoinase and the N-carbamyl amino acid hydrolase is a bacterium of the genus Microbacterium or Bacillus, and is optically active 2-amino-6-methyl. -6-Nitroheptanoic acid or a salt thereof is L-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof, [20].

The present invention will be described in detail below.

In the formula in the present invention, P ₁ and P ₂ each independently represent a protecting group for an amino group or a hydrogen atom. However, P
_{The case where 1} and P ₂ simultaneously become a hydrogen atom (that is, when the amino group has no protecting group) is excluded. Or P ₁
And P ₂ together represent an amino-protecting group. The protecting group for the amino group is not particularly limited. For example, Protecting Groups in Organic Chemistry, 2nd Edition, John Willy & Sons, 1991 [Protecting Groups in Organic Chemis
try 2nd edition (John Wiley & Sons, Inc. 1991))
The protecting groups and the like described in 1 can be used. A typical protecting group is a benzyloxycarbonyl group (Z
Group), 9-fluorenylmethyloxycarbonyl group (F
moc group), tert-butoxycarbonyl group (Boc
Group), a methoxycarbonyl group (Moc group), and the like. When P ₁ and P ₂ together represent an amino-group protecting group, examples of the protecting group include a phthaloyl group. As P ₁ and P ₂ in the present invention, it is particularly preferable that either one is a tert-butoxycarbonyl group and the other is a hydrogen atom.

In the formula in the present invention, P ₃ , P ₄ and P ₅
Represents a hydrogen atom or a protective group for a carboxyl group. The protective group for the carboxyl group is not particularly limited, and for example,
Examples thereof include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Specifically, examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, and a phenyl group. In the present invention, a methyl group is particularly preferable as the protective group for the carboxyl group represented by P ₃ , P ₄ and P ₅ .

In the formula in the present invention, P _3a represents a carboxyl group-protecting group, which has the same _{meaning as} the carboxyl group-protecting group represented by P ₃ .

In the formula in the present invention, R ₁ has ₁ carbon atom
Is an alkyl group having 6 to 6 or an aralkyl group having 7 to 12 carbon atoms. As the alkyl group having 1 to 6 carbon atoms,
Examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group and a t-butyl group. Examples of the aralkyl group having 7 to 12 carbon atoms include a benzyl group. R ₁ is especially tert
-Butyl group is preferred.

In the formula in the present invention, R ₂ and R ₃ each independently represent an alkyl group having 1 to 6 carbon atoms or an aralkyl group having 7 to 12 carbon atoms. 1 to 1 carbon atoms
Examples of the alkyl group of 6 include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group,
Examples thereof include an isobutyl group, a sec-butyl group, a t-butyl group and the like. Examples of the aralkyl group having 7 to 12 carbon atoms include a benzyl group. As R ₂ and R ₃ , an alkyl group having 1 to 6 carbon atoms is preferable, an alkyl group having 1 to 4 carbon atoms is more preferable, and an ethyl group is particularly preferable. Further, it is preferable that R ₂ and R ₃ are the same.

In the present invention, Y ₁ in formula (4) and Y ₂ in formula (6) each represent a leaving group. The leaving group refers to an atom or an atomic group that is released from the reaction substrate in a substitution reaction, an elimination reaction, etc., for example, chlorine, bromine, iodine, paratoluenesulfonyloxy group, mesyloxy group, trifluoromethanesulfonyloxy group, Alkyl or phenyl carbonate group or 1 carbon atom
Examples thereof include saturated or unsaturated acyloxy groups and the like. As a preferable leaving group, a bromine atom or an iodine atom can be mentioned.

A typical compound preferably produced by the production method of the present invention is a compound represented by the general formula (3), Nα-t-butoxycarbonyl-6, represented by the following formula (23). 6-dimethyl-L-lysine methyl ester can be mentioned, and as the compound represented by the general formula (5), Nα-t-butoxycarbonyl-Nε-t-butoxycarbonylmethyl-6 represented by the following formula (26). , 6
-Dimethyl-L-lysine methyl ester can be mentioned.

[0063]

[Chemical 47]

The method for producing the amino acid derivative represented by the general formula (1), which is the starting material in the present invention, will be described.

The amino acid derivative represented by the general formula (1) can be synthesized, for example, from the three routes (I), (II) and (III) shown in the following reaction scheme.

[0066]

[Chemical 48]

[*, R ₂ , R ₃ , R ₄ and Y ₂ have the same meanings as described above. ]

The case of route (I) will be described.

(I) 2-Nitropropane and methyl acrylate are heated to reflux by adding a catalytic amount of potassium fluoride in a suitable solvent such as ethanol. Methyl 4-methyl-4-nitropentanoate can be obtained by concentrating (or distilling) the reaction solution and extracting it (Bretin of the Chemical Society of Japan, 1
966, 39, No. 11, 2549-2551: Bu
lletin of the Chemical Society of Japan 1966, 39
(11), 2549-2551). The obtained methyl 4-methyl-4-nitropentanoate can be isolated / purified by a conventional method such as distillation or chromatography, but it can usually be used in the next step without isolation / purification. .

(Ii) Methyl 4-methyl-4-nitropentanoate obtained as described above is added with 2 equivalents or more of a reducing agent such as sodium borohydride in a suitable solvent such as ethanol, Room temperature to reflux temperature, preferably 40
Stir at 1 to 24 hours, preferably 2 to 5 hours at ℃ to 70 ℃. Concentrate (or evaporate) the reaction mixture
Then, 4-methyl-4-nitropentanol can be obtained by the extraction operation. The obtained 4-methyl-4-nitropentanol can be isolated and purified by a conventional method such as distillation or chromatography, but usually it can be used in the next reaction without isolation and purification.

(Iii) Substituting the leaving group for the hydroxyl group of 4-methyl-4-nitropentanol to obtain the 4-methyl-4-nitropentane derivative represented by the general formula (6).
This can be done by methods known to those skilled in the art. For example, when substituting a halogen atom such as a bromine atom or an iodine atom, which is a preferable leaving group, the following method can be used.

4-methyl-4-nitropentanol,
For example, in a suitable solvent such as methylene chloride, a tertiary amine such as triethylamine is added in an amount of about 1 to 3 equivalents, preferably 1.
About 2 to 2 equivalents, and a sulfonyl halide represented by the general formula (27) (preferably methanesulfonyl chloride) is added at about 1 to 3 equivalents, preferably about 1.1 to 2 equivalents, and at room temperature to about 0 ° C. Stir for about 30 minutes to 2 hours. Then, 4-methyl-4-nitropentanolsulfonic acid ester represented by the general formula (28) can be obtained by an extraction operation. The obtained 4-methyl-4-nitropentanol sulfonic acid ester is usually used after being isolated and purified by a conventional method such as crystallization and chromatography.

Next, 4-methyl-represented by the formula (28)
The 4-nitropentanol sulfonic acid ester is reacted with an alkali metal halide to obtain 4-methyl-4-nitropentyl halide represented by the formula (29). The alkali metal halide is preferably sodium iodide or sodium bromide. For example, 4-methyl-4-nitropentanol sulfonic acid ester represented by the formula (28) is added in an appropriate solvent such as acetone at about 0 ° C to 30 ° C,
It is preferably about 15 ° C to 25 ° C and about 1 to 10 equivalents, preferably about 4 to 5 equivalents of an alkali metal halide is added to the mixture for about 6 to 24 hours, preferably about 10 hours.
Stir for about 12 hours. The reaction solution is concentrated (or evaporated) and extracted to obtain 4-methyl-4-nitropentyl halide represented by the general formula (29). Obtained 4
-Methyl-4-nitropentyl halide can be isolated and purified by a conventional method such as chromatography, but usually it can be used for the next reaction without isolation and purification.

[0074]

[Chemical 49]

[Wherein R ₅ represents an alkyl group having 1 to 3 carbon atoms or a phenyl group which may have a substituent (preferably an alkyl group having 1 to 3 carbon atoms), and X ₁ and X ₂ represents a halogen atom. ]

(Iv) 4-methyl-4 represented by the formula (6)
-A nitropentane derivative (preferably 4-methyl-4-nitropentyl halide represented by the general formula (29)) and a 2-acetylaminomalonic acid diester represented by the general formula (7) are reacted to produce the above-mentioned general compound. 2 represented by equation (8)
-Acetylamino-2- (4-methyl-4-nitropentyl) malonic acid diester (racemic) is obtained.

As the 2-acetylaminomalonic acid diester represented by the general formula (7), 2-acetamidomalonic acid diethyl ester in which R ₂ and R ₃ are ethyl groups is particularly preferably used. For example, sodium ethoxide and 2-acetamidomalonic acid diester are dissolved in a suitable solvent such as ethanol, 4-methyl-4-nitropentyl derivative is added, and the mixture is heated at 50 ° C to about 3 to 2 at reflux temperature.
React for about 4 hours. The reaction liquid is represented by the general formula (8) by concentrating (or distilling) the reaction liquid and performing an extraction operation
Acetylamino-2- (4-methyl-4-nitropentyl) malonic acid diester (racemic form) can be obtained (Tetrahedron, 1985, Vol. 41, No. 22, 5).
307-5331: Tetrahedron 1985, 41 (22), 530.
See 7-5311). The obtained 2-acetylamino-2- (4
-Methyl-4-nitropentyl) malonic acid diester (racemic form) can be isolated and purified by a conventional method such as chromatography, but it is usually used for the next reaction without isolation and purification. it can.

(V) The 2-acetylamino-2- (4-methyl-4-nitropentyl) malonic acid diester represented by the formula (8) is, for example, a mixed solvent of water and a water-soluble solvent such as ethanol. In it, alkali such as potassium hydroxide is added and heated to reflux to give 2-acetylamino-2- (4-methyl-4-nitropentyl) malonic acid, and then acid such as hydrochloric acid or sulfuric acid is added to decarboxylate . 2-Acetylamino-6-methyl-6-nitroheptanoic acid (racemate) represented by the formula (9) can be obtained by concentrating and crystallization of the reaction solution (Journal of Medicinal.
Chemistry, Volume 42, pp. 305-311, 1999.
Year: Journal of Medicinal Chemistry, 1999, 42, 305-
See 311). The obtained 2-acetylamino-6-methyl-6-nitroheptanoic acid (racemic form) is usually used after being isolated and purified by a conventional method such as crystallization and chromatography.

(Vi) 2-Acetylamino-6-methyl-6-nitroheptanoic acid (racemic form) represented by the formula (9)
Then, an acylase is allowed to act to obtain the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1). The acylase is not particularly limited, and commercially available acylases such as those manufactured by Amano Co. can be used. For example, a pH of 5-10, preferably 6
2-acetylamino-6-methyl-6- prepared in
L in a water solvent in which nitroheptanoic acid (racemic form) is dissolved
-Add acylase and stir at preferably 30 to 40 ° C for about 3 hours to 2 days. The pH is adjusted to about 1 to 3 to precipitate the unreacted D-form of the formula (9), which is separated by filtration,
The mother liquor was concentrated to dryness and the like to give 2-amino-6-methyl-6-
An L-form of nitroheptanoic acid is obtained. When D-acylase is used, D-amino 2-amino-6-methyl-6-nitroheptanoic acid can be obtained (Journal of.
Medicinal Chemistry, Volume 42, 305-31
Page 1, 1999: Journal of Medicinal Chemistry, 1
See 999, 42, 305-311).

On the other hand, as another method, 2-acetylamino-6-methyl-6-nitroheptanoic acid (racemic form) is deacetylated with D-acylase to deacetylate the D-form, and the unreacted L-form under acidic conditions. It is also possible to obtain the L-form of 2-amino-6-methyl-6-nitroheptanoic acid by crystallizing, separating the crystals, heating under reflux in an acidic aqueous solution, deacetylating and concentrating to dryness.

The obtained optically active 2-amino-6-methyl-6-nitroheptanoic acid can be isolated and purified by a conventional method such as crystallization and chromatography, but it is usually isolated and purified. It can be used in the next reaction without any treatment.

The case of route (II) will be described.

(Vii) Examples of the method for producing 4-methyl-4-nitropentanal include the following two production methods.

(Vii-1) Acrylonitrile and 2-nitropropane are reacted in the presence of hexadecyltrimethylammonium chloride in an alkaline aqueous solvent such as an aqueous sodium hydroxide solution. Then, 4-methyl-
Obtain 4-nitrovaleronitrile (European Journal of Organic Chemistry (199
8), Volume 2, pages 355-357: European Journal o.
f Organic Chemistry (1998), (2), 355-357).
The obtained 4-methyl-4-nitrovaleronitrile can be isolated and purified by a conventional method such as crystallization and chromatography, but usually it can be used for the next reaction without isolation and purification. .

4-methyl-4-nitrovaleronitrile is heated at about 0 ° C to -78 ° C, preferably at about -40 ° C to -60 ° C in a suitable solvent such as methylene chloride with a reducing agent such as diisobutylaluminum hydride. After reduction at temperature, it is treated with an acid such as hydrochloric acid or sulfuric acid. Then, 4-methyl-4-nitropentanal is obtained by extraction.
The obtained 4-methyl-4-nitropentanal was distilled,
Although it can be isolated and purified by a conventional method such as chromatography, it can usually be used in the next reaction without isolation and purification.

(Vii-2) 2-Methyl-4-nitropentanal can also be obtained by adding 2-nitropropane and acrolein to a solution in which methanol and sodium metal are dissolved (JP-A 01-305056). See the bulletin). The obtained 4-methyl-4-nitropentanal is usually used after being isolated and purified by a conventional method such as distillation or chromatography.

(Viii) Next, 4-methyl-4-nitropentanal and hydantoin are reacted to obtain 5- (4-methyl-4-nitropentylidene) hydantoin (see JP-A-11-140076).

For example, 4-methyl-4-nitropentanal and hydantoin are heated and refluxed in a mixed solvent of water and a water-soluble solvent such as acetonitrile or isopropyl alcohol in the presence of a base such as sodium carbonate or potassium carbonate.
The reaction is preferably carried out for about 3 to 5 days. Then, by extraction operation, 5- (4-methyl-4-nitropentylidene)
You can get Hydantoin.

In this reaction, firstly 5- (1-hydroxy-
4-Methyl-4-nitropentyl) hydantoin is produced as an intermediate product, and finally the desired 5- (4-methyl-4-nitropentylidene) hydantoin is obtained.

Further, 5- (1-hydroxy-4-methyl-4-nitropentyl) hydantoin was isolated, the hydroxyl group was protected with methanesulfonyl chloride or the like, and 5- (1-
Obtained as methanesulfonyloxy-4-methyl-4-nitropentyl) hydantoin, etc., which was then obtained from 1,8-diazabicyclo [5.4.0] undec-7-ene (DB
U) and the like to give the desired 5-
It is also possible to obtain (4-methyl-4-nitropentylidene) hydantoin.

The obtained 5- (4-methyl-4-nitropentylidene) hydantoin can be isolated and purified by a conventional method such as crystallization and chromatography.
It can be used in the next reaction without purification.

(Viv) Next, 5- (4-methyl-4-nitropentylidene) hydantoin is reduced using a transition metal catalyst such as palladium to give 5- (4-methyl-4-nitropentyl) hydantoin. obtain. For example, 5- (4-
Methyl-4-nitropentylidene) hydantoin is added to a mixed solvent of water and a water-soluble solvent such as methanol or ethanol, and usually palladium carbon is added in an amount of 0.1 to 5 mol%, preferably 0.5 to 2 mol%, and hydrogen gas is added. And react for 1 to 12 hours, preferably 2 to 5 hours. The reaction solvent can be concentrated to dryness to give 5- (4-methyl-4-nitropentyl) hydantoin. The obtained 5- (4-methyl-4-nitropentyl) hydantoin can be isolated and purified by a conventional method such as chromatography, but it can also be used in the next reaction without isolation and purification.

(X) Next, 5- (4-methyl-4-nitropentyl) hydantoin was hydrolyzed with alkali to give 2-
Amino-6-methyl-6-nitroheptanoic acid is obtained (see JP-A-11-140076). For example, 5-
In an aqueous solution in the presence of an alkali such as 1 to 3 equivalents of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, calcium hydroxide or barium hydroxide, preferably with respect to (4-methyl-4-nitropentyl) hydantoin. At 20 to 200 ° C., preferably 100 to 150 ° C.
5- (4-methyl-4-nitropentyl) hydantoin is hydrolyzed. Then, 2-amino-6-methyl-6-nitroheptanoic acid can be obtained by concentrating the reaction solution to dryness. The obtained 2-amino-6-methyl-6-nitroheptanoic acid can be isolated / purified by a conventional method such as crystallization or chromatography, but usually it is used in the next reaction without isolation / purification. Can be used.

(Xi) Next, 2-amino-6-methyl-6-
Amino group of nitroheptanoic acid is acylated to obtain 2-acylamino-6-methyl-6-nitroheptanoic acid (racemic compound) represented by the formula (13) (JP-A-11-14007).
No. 6 publication). For example, 2-amino-6-methyl-6
-Nitroheptanoic acid in an aqueous solution, acetyl chloride,
An acyl halide such as benzoyl chloride or an acid anhydride such as acetic anhydride or benzoic anhydride is preferably used in an amount of 1 to 2 equivalents, and the pH is higher than pH 7, preferably pH 8 to 1
1, and the acylation is usually performed at 0 to 80 ° C., preferably 0 to 30 ° C. to give 2-acylamino-6-methyl-6.
-Nitroheptanoic acid (racemic form) can be obtained.
The obtained 2-acylamino-6-methyl-6-nitroheptanoic acid is usually used after being isolated and purified by a conventional method such as crystallization and chromatography.

(Xii) By reacting 2-acylamino-6-methyl-6-nitroheptanoic acid obtained as described above with an acylase in the same manner as described above, a compound represented by the formula (1) is obtained. The optically active 2-amino-6-methyl-6-nitroheptanoic acid can be obtained.

Optically Active 2-Amino-Represented by Formula (1)
6-methyl-6-nitroheptanoic acid can be prepared by the following route (II
It can also be manufactured by I). That is, the general formula (11)
The 5- (4-methyl-4-nitropentyl) hydantoin represented by the formula (1) is optically selective using one or more selected from the group consisting of microorganisms, processed products of microorganisms, hydantoinase, and N-carbamyl amino acid hydrolase. Hydrolyzed to give optically active 2-amino-6- of formula (1)
This is a route for obtaining methyl-6-nitroheptanoic acid or a salt thereof.

The case of this route (III) will be described.

Optically active 2-amino-represented by the formula (1)
6-methyl-6-nitroheptanoic acid has the general formula (11):
The 5- (4-methyl-4-nitropentyl) hydantoin represented by the formula (1) is optically selective using one or more selected from the group consisting of microorganisms, processed products of microorganisms, hydantoinase, and N-carbamyl amino acid hydrolase. Can be obtained by hydrolysis.

The origin of the microorganism, the processed product of the microorganism, the hydantoinase and the N-carbamyl amino acid hydrolase used at this time is Agrobacterium (Ag.
It is preferably a bacterium of the genus robacterium, genus Bacillus or bacterium of the genus Microbacterium.

Furthermore, microorganisms, and processed products of microorganisms,
The origins of hydantoinase and N-carbamyl amino acid hydrolase are derived from Agrobacterium.
m) is a genus bacterium and is optically active 2-amino-6-methyl-
6-nitroheptanoic acid or a salt thereof is preferably D-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof.

Furthermore, microorganisms, and processed products of microorganisms,
The origins of hydantoinase and N-carbamyl amino acid hydrolase are derived from Microbacteriu
m) or a bacterium of the genus Bacillus, wherein optically active 2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof is L-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof. It is preferably a salt.

5- (4-methyl-) represented by the formula (11)
4-Nitropentyl) hydantoin can be prepared by the method of route (II). 5-prepared
(4-Methyl-4-nitropentyl) hydantoin is
It may be purified and isolated by a conventional method, or may be in the form as it exists in the reaction solution. 5-
(4-Methyl-4-nitropentyl) hydantoin is
It may be a mixture of D-form and L-form, or may contain either one. In the case of a mixture of D-form and L-form, the mixing ratio is arbitrary.

The microorganism, the treated product of the microorganism, the hydantoinase, or the N-carbamyl amino acid hydrolase used may be one which can optically and selectively hydrolyze 5- (4-methyl-4-nitropentyl) hydantoin. Good.

Optically selective hydrolysis means that the 5- (4-methyl-4-nitropentyl) hydantoin represented by the formula (11) is hydrolyzed to obtain an optically active compound represented by the formula (1).
-Amino-6-methyl-6-nitroheptanoic acid or a salt thereof, which is a reaction capable of obtaining D-form or L-form
It is a reaction in which one of the two bodies, 2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof, is produced.

Microorganisms, processed products of microorganisms, and hydantoinase act on 5- (4-methyl-4-nitropentyl) hydantoin, which optically hydrolyzes it to give optically active N-carbamyl-2-amino-. 6-Methyl-6-nitroheptanoic acid, i.e. N-carbamyl-L-2-
Amino-6-methyl-6-nitroheptanoic acid, or N
-Including carbamyl-D-2-amino-6-methyl-6-nitroheptanoic acid. N-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid is optically and selectively used as 2-amino-6 for microorganisms and treated products of microorganisms.
Even when there is no activity of converting to -methyl-6-nitroheptanoic acid, or when only hydantoinase catalyzes the optically selective reaction, the enzymatic reaction with N-carbamyl amino acid hydrolase or the enzyme-containing product is continued. By performing various hydrolysis treatments or chemical hydrolysis treatments with nitrous acid, optically active 2-amino-6-methyl-6-nitroheptanoic acid can be produced in high yield while maintaining the optical activity. Is.

The microorganism, the treated product of the microorganism, and N-carbamylamino acid hydrolase act on N-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, and hydrolyze this optically and selectively. Optically active 2-amino-6-
Includes those capable of producing methyl-6-nitroheptanoic acid. 5- (4-Methyl-4)
-Nitropentyl) hydantoin has no activity for optically selective conversion into N-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, or only N-carbamyl amino acid hydrolase is an optically selective reaction. Even in the case of catalyzing the above, by performing enzymatic hydrolysis treatment or chemical hydrolysis treatment with hydantoinase or a substance containing the enzyme in advance, optically active 2-amino-6-methyl- This is because 6-nitroheptanoic acid can be produced.

Therefore, the microorganisms and the treated products of the microorganisms used in the present invention act on 5- (4-methyl-4-nitropentyl) hydantoin, which optically hydrolyzes it to give optically active N- Any substance may be used as long as it has a hydantoinase capable of producing carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, and although the hydantoinase has no optical selectivity, it produces N-carbamyl-2- Having an optically selective N-carbamyl amino acid hydrolase capable of optically hydrolyzing amino-6-methyl-6-nitroheptanoic acid to produce optically active 2-amino-6-methyl-6-nitroheptanoic acid Anything may be used as long as it is one.

The microorganism referred to in the present invention may be cultivated by any culturing method such as liquid culturing and solid culturing as long as it has a necessary ability, and the culturing liquid itself or live bacteria collected from the culturing liquid. Any body may be used. The microorganism of the present invention may be a strain newly isolated from the natural world such as soil or plant, or may be a strain artificially bred by treatment with a mutagenic agent or recombinant DNA technology. .

The method for culturing the microorganism in the present invention can be carried out using a medium usually used in this field, that is, a medium containing a carbon source, a nitrogen source, inorganic salts, trace metal salts, vitamins and the like. In addition, depending on the type of microorganism or the culture conditions, 0.05 to
Optical activity by adding a 5-substituted hydantoin compound such as 5-indolylmethylhydantoin or 5-isopropylhydantoin of about 1.0 g / dl.
It is also possible to promote the production activity of -amino-6-methyl-6-nitroheptanoic acid. Substrate into the cells 5-
In order to enhance the permeability of (4-methyl-4-nitropentyl) hydantoin, a surfactant such as Triton X or Tween or an organic solvent such as toluene or xylene can be used. As a specific substance used as the medium component, for example, the carbon source is not limited as long as the microorganism to be used is available, for example, glucose, sucrose, fructose, glycerol, maltose, acetic acid, etc., or a mixture thereof. Can be used. As a nitrogen source,
Ammonium sulfate, ammonium chloride, urea, yeast extract, meat extract, corn steep liquor, casein hydrolyzate, etc., or a mixture thereof can be used. As a specific medium composition, for example, glucose 0.5%, ammonium sulfate 0.5%, powdered yeast extract 1.0%, peptone 1.0%, KH ₂ PO ₄ 0.1%, K ₂ HPO _{4
0.3%, MgSO 4 · 7H 2} O 0.05%, FeSO
_{4 · 7H 2 O0.001%, MnSO} 4 · 5H 2 O 0.0
A medium (pH 7.0) containing 01% and the like can be mentioned.

The culture temperature is usually within the range in which the microorganism to be used grows, that is, 20 to 45 ° C., but preferably 25 to 37 ° C. The pH of the medium is adjusted within the range of 3 to 11, preferably 4 to 8. The aeration conditions are set aerobically or anaerobically to conditions suitable for the growth of the microorganisms to be used, but aerobic conditions are usually preferred. The culturing time was optically active 2-amino-6-methyl-6-
The time is not particularly limited as long as it is a time for which nitroheptanoic acid is efficiently produced, but it is usually 12 to 144 hours, preferably 24 to 96 hours.

A preferable example of the microorganism of the present invention is Agrobacterium (Agrobacterium) for producing (R) -2-amino-6-methyl-6-nitroheptanoic acid.
In order to produce (S) -2-amino-6-methyl-6-nitroheptanoic acid by a microorganism belonging to the genus Bacteria, microorganisms belonging to the genus Microbacterium and the genus Bacillus are listed. To be

As these microorganisms, specifically, for example, to produce (R) -2-amino-6-methyl-6-nitroheptanoic acid, Agrobacterium sp. (Agrobacterium sp.) AJ 11220 strain (formerly Pseudomonas hydantoinophilum (Pseudomona
s hydantoinophilum). ) Is mentioned. Agrobacterium sp. (Agrobacterium
sp.) AJ 11220 strain was established on December 20, 1977 by the former Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology (currently,
A microorganism that has been deposited at the Patent Biological Depository Center of the National Institute of Advanced Industrial Science and Technology (AIST) and has been given the deposit number FERM-P4347. Then, it was transferred to an international deposit under the Budapest Treaty on June 27, 2001, and deposited. It is a microorganism assigned with the number FERMBP-7645. To produce (S) -2-amino-6-methyl-6-nitroheptanoic acid, Microbacterium liquefaciens AJ39 is used.
40 [previously classified as Aureobacterium liquefaciens. ], Bacillus SP. (Bacillus sp.) AJ
12299 is mentioned. Microbacterium liquefaciens AJ39
40 shares on June 27, 1975, again Bacillus SP. (Bacillus sp.) AJ12299 strain is 198
On July 5, 2006, it was deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry (currently, Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology), with deposit number FER.
A microorganism to which M-P3135 and FERM-P8837 have been assigned, which was then transferred to an international deposit under the Budapest Treaty on June 27, 2001, and the deposit number is FE.
It is a microorganism provided with RM BP-7644 and FERM BP-7646.

The treated product of the microorganism of the present invention is obtained by subjecting the microorganism of the present invention to physical treatment such as ultrasonic wave, glass beads, French press, freeze-drying, enzymatic treatment with lytic enzyme,
It means that it has been treated by a chemical treatment with an organic solvent or a surfactant. Even a product obtained by subjecting the culture solution of the microorganism of the present invention or living cells to the treatment described above is a treated product of the microorganism of the present invention as long as it has a necessary ability. Furthermore, if the treated product obtained by these treatments is a crude fractionated enzyme or a purified enzyme prepared by a conventional method (liquid chromatography, ammonium sulfate fractionation, etc.) and has the required ability, this is also the It is a treated product of the microorganism of the invention.

A preferable source of the processed product of the microorganism is, for example, to produce (R) -2-amino-6-methyl-6-nitroheptanoic acid, a microorganism belonging to the genus Agrobacterium is used. , (S)-
To produce 2-amino-6-methyl-6-nitroheptanoic acid, Microbacterium
Examples include microorganisms belonging to the genus Bacillus.

The origin of the treated product of the microorganism means the microorganism before the treatment. However, a trait transformed with the gene by isolating a hydantoinase gene from a microorganism having a hydantoinase activity or an N-carbamylamino acid hydrolase gene from a microorganism having an N-carbamylamino acid hydrolase activity The origin of the treated product of the microorganism obtained by treating the transformant is the microorganism from which the gene was isolated.

A hydantoinase which optically hydrolyzes a hydantoin compound can be obtained as follows.
For example, D- which produces an N-carbamyl-D-amino acid
As a bacterium having a hydantoinase, a bacterium belonging to the genus Bacillus having a thermostable enzyme is known, and as an example, Bacillus stearothermophilus (Bacillus st.
A hydantoinase or a hydantoinase-containing fraction may be prepared from Earothermophilus) ATCC31195 etc. (Appl. Microbiol. Biotechnol. 43, 270, 1995). The ATCC 31195 strain is an American Type Culture Collection.
Culture Collection, Address 12301 Parklawn Drive, Rock
ville, Maryland 20852, United States of America)
Can be obtained from. In addition, L-hydantoinase which acts specifically on the L-form hydantoin compound is, for example, Bacillus sp. (Bacillus sp.) AJ12
Its existence is known to the 299 strain (Japanese Patent Laid-Open No. 63-24 / 1988).
894).

Hydantoinase, which has no optical selectivity, is a microbacteriophage required by Microbacteri.
um liquefaciens) AJ3912 [formerly Aureobacterium liqi
uefaciens). Before that, Flavobacterium sp. (Flavobacterium sp.). ] And the above Microbacterium liquefaciens AJ3
In addition to 940 (Japanese Patent Publication No. 56-008749), for example, Arthrobacter auresense
aurescens) is known to exist (J. Biotechno
Volume 61, page 1, 1998). 197 for AJ3912 strain
Deposited on June 27, 1993 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (currently, Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology) with deposit number FERM-
A microorganism to which P3133 has been added, and then 200
It is a microorganism that was transferred to international deposit under the Budapest Treaty on June 27, 1 and was given the deposit number FERMBP-7643.

N-carbamyl amino acid hydrolase which selectively hydrolyzes N-carbamyl amino acid in D-form is
For example, the above Agrobacterium sp. (Agroba
cterium sp.) AJ 11220 strain is known to exist (Japanese Patent Publication No. 56-003034). Also N
-N- which selectively hydrolyzes carbamyl amino acid in L-form
Carbamyl amino acid hydrolase can be obtained, for example, from the above Microbacterium liquefaciens.
m liquefaciens) AJ3912 (Japanese Patent Publication No. 56-008)
749), Bacillus S. (Bacillus s)
p.) Its existence is known to AJ12299.

Preferred sources of hydantoinase and N-carbamyl amino acid hydrolase are, for example, Agrobacterium for producing (R) -2-amino-6-methyl-6-nitroheptanoic acid (D-form). It is a microorganism belonging to the genus Agrobacterium. on the other hand,
To generate (S) -2-amino-6-methyl-6-nitroheptanoic acid (L-form), Microbacterium (Mic
It is a microorganism belonging to the genus robacterium and the genus Bacillus.

The source of hydantoinase means the source of acquisition of hydantoinase. The origin of the hydantoinase recovered by crushing the cells of a microorganism having hydantoinase activity is the microorganism. However, the origin of the hydantoinase recovered from the transformant transformed with the gene by isolating the hydantoinase gene from the microorganism having the hydantoinase activity is the microorganism from which the gene was isolated.

The source of N-carbamyl amino acid hydrolase means the source of obtaining N-carbamyl amino acid hydrolase. N- recovered by crushing cells of a microorganism having N-carbamyl amino acid hydrolase activity
The origin of the carbamyl amino acid hydrolase is the microorganism. However, the N-carbamyl amino acid hydrolase gene was isolated from a microorganism having N-carbamyl amino acid hydrolase activity, and the origin of the N-carbamyl amino acid hydrolase recovered from the transformant transformed with the gene. Is the microorganism from which the gene was isolated.

The substrate, 5- (4-methyl-4-nitropentyl) hydantoin, can be used in a reaction system containing cells isolated after culturing a microorganism, a treated product thereof, hydantoinase or N-carbamyl amino acid hydrolase. , Is added collectively, intermittently or continuously within a concentration range where the production of optically active 2-amino-6-methyl-6-nitroheptanoic acid is not suppressed. For addition method,
It can be added directly during the culture of the microorganism. At the time of addition, it may be mixed with an organic solvent or a surfactant for the purpose of increasing solubility or promoting dispersion. Further, for the purpose of continuing or accelerating the metabolism of microorganisms, it may be added by mixing with a medium component such as a carbon source or a nitrogen source.

When a microorganism, a treated product of the microorganism, a hydantoinase, or an N-carbamyl amino acid hydrolase is subjected to the reaction, these are entrapped in carrageenan gel or polyacrylamide, or immobilized on a membrane such as polyethersulfone or regenerated cellulose. It is also possible to use.

The reaction system of the present invention comprises a hydantoin compound and a microorganism, a treated product of the microorganism, hydantoinase, or
The reaction liquid containing N-carbamyl amino acid hydrolase may be allowed to stand or stir for 8 hours to 5 days while adjusting the temperature to an appropriate temperature of 25 to 40 ° C. and keeping the pH at 5 to 9.

The amount of (R)-or (S) -2-amino-6-methyl-6-nitroheptanoic acid in the culture solution or reaction solution can be rapidly measured by a well-known method. . For example, “CROWN” manufactured by Daicel Chemical Industries
High performance liquid chromatography using an optical resolution column such as PAK CR (+) ”may be used. Thus, the (R)-or (S) -2-amino-6-methyl-6-nitroheptanoic acid accumulated in the culture solution or the reaction solution is used by collecting it from the culture solution or the reaction solution by a conventional method. be able to. Collection from the culture solution or the reaction solution requires well-known means usually used in this field in such a case, for example, operations such as filtration, centrifugation, vacuum concentration, ion exchange or adsorption chromatography, and crystallization. It is used in combination as appropriate.

Next, the production method of the present invention shown in the following scheme will be described.

[0127]

[Chemical 50]

[*, P ₁ , P ₂ , P ₃ , P _3a , P ₄ , P ₅ ,
Y ₁ and R ₁ have the same meanings as described above. ]

First, the amino group of the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1) or the amino group and the carboxyl group are protected by a protecting group and represented by the general formula (2). The method for producing the optically active amino acid derivative will be described.

The protecting group may be protected by first protecting the amino group or the carboxyl group. The carboxyl group does not necessarily have to be protected in the reduction reaction, and the compound represented by the general formula (5) may not be protected depending on the purpose. When it is protected, it is preferable to protect it before the reduction reaction, but it can be protected in any subsequent process. Note that the claims of the present application include aspects of such a process.

To protect the amino group, an amino group protecting reagent such as an alkoxycarbonylating reagent, an acylating reagent or a sulfonylating reagent may be allowed to act in the presence of a base, if necessary.

For example, the compound represented by the formula (1) or (30) is dissolved in advance in a suitable solvent by adding a suitable base if necessary, and then an alkoxycarbonylating reagent, an acylating reagent or a sulfonylation reagent. An amino group protecting reagent such as a reagent is added.

The reagent for protecting the amino group is not particularly limited, and not only reagents commonly used in peptide synthesis but also functional groups such as an alkoxycarbonyl group, an acyl group and a sulfonyl group for introducing arbitrary substituents. Any compound having a group can be used.

Examples of such an amino group protecting reagent include methoxycarbonyl chloride, ethoxycarbonyl chloride, t-butoxycarbonyl chloride, benzyloxycarbonyl chloride, di-t-butyl dicarbonate, and tetrahydrofuran-3-yl chloride. Alkoxycarbonylating reagents such as oxycarbonyl, acylating reagents such as acetic anhydride, acetyl chloride, benzoyl chloride and trifluoroacetic anhydride, sulfonyls such as methanesulfonyl chloride, trifluoromethanesulfonyl chloride, benzenesulfonyl chloride and p-toluenesulfonyl chloride. A chemical reagent and the like can be mentioned. In particular, it is preferable to protect with a t-butoxycarbonyl group, and in this case, the protecting reagent is preferably di-
t-Butyl dicarbonate can be used.

The amino group-protecting reagent can be used in an amount of usually 1 to 1.5 equivalents, preferably 1.1 to 1.3 equivalents, relative to the compound to be protected. Similarly, the base can be used usually in 1 to 3 equivalents, preferably 1.5 to 2 equivalents.

As the solvent used for protecting the amino group, water, methanol, ethanol, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, toluene, or the like, or a mixed solvent thereof, or the like, can be used a suitable solvent depending on the reagent. .

Examples of the base include pyridine, triethylamine, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, etc., disodium monohydrogen phosphate, dipotassium monohydrogen phosphate and the like. In the case of the compound represented by the general formula (1), sodium hydroxide and potassium hydroxide are particularly preferable, and in the case of the compound represented by the general formula (30), sodium hydrogen carbonate and potassium hydrogen carbonate are preferable. .

The reaction time varies depending on the reagent used and the reaction temperature, but as an example, when t-butoxycarbonylation is carried out using di-t-butyl dicarbonate, 4
The reaction is completed in a few minutes to 2 hours at 0 ° C., and in a few minutes to 10 hours when the reaction is performed at room temperature.

When protecting the carboxyl group, it can be usually carried out by esterification. For example, in the case of the compound represented by the general formula (1), for example, alcohol and thionyl chloride may be mixed in advance, and then the compound represented by the general formula (1) may be added and reacted. Further, for example, in the case of the compound represented by the general formula (31), for example, an esterifying agent such as dimethylformamide dimethylacetal,
Alternatively, the carboxyl group can be esterified using a base and an alkylating agent.

As the solvent used for esterification of the carboxyl group, methanol, ethanol, t-butanol, benzyl alcohol, toluene, tetrahydrofuran, dichloromethane or the like or a mixed solvent thereof or the like is used. A suitable solvent can be used depending on the ester group to be introduced.

When esterifying the carboxyl group of the compound represented by the general formula (1), for example, in a corresponding alcohol (for example, in the case of forming methyl ester, in methanol), usually 2 to 3 equivalents of hydrochloric acid gas are used. , 1 to 2 equivalents of acetyl chloride, or 1 to 2 equivalents of p-toluenesulfonic acid can be added to introduce the ester group.

It is also possible to react the alcohol with about 1 to 1.2 equivalents of thionyl chloride and then add the compound represented by the general formula (1). The reaction time varies depending on the reagents used and the reaction temperature, but as an example, methanol-
When methyl esterification is performed using thionyl chloride,
The reaction is usually completed at 60 ° C. in about 3 hours to 5 hours.

In the case of the compound represented by the general formula (31),
For example, it can be performed by a method of esterification using an esterifying agent such as dimethylformamide dimethylacetal in an amount of about 1.5 to 2.5 equivalents. Further, in the presence of about 1 to 1.5 equivalents of a base (for example, an amine such as cyclohexylamine or an alkali metal salt such as cesium carbonate), about 1 to 1.5 equivalents of an alkyl halide (for example,
It is also possible to use a method in which methyl iodide or benzyl bromide) is used for protection. The reaction time varies depending on the reagent used and the reaction temperature, but as an example, when methyl esterification is carried out using dimethylformamide dimethylacetal in dichloromethane, it is usually 20 at room temperature.
The reaction is completed in about 24 hours.

Next, a step of reducing the nitro group of the optically active amino acid derivative represented by the general formula (2) to obtain the optically active 6,6-dimethyllysine derivative represented by the general formula (3) or a salt thereof. Will be described.

The reduction of the nitro group is carried out in a suitable alcohol solvent such as methanol, ethanol or isopropyl alcohol and a mixed solvent of these alcohols and water, preferably by the combination of (i) a metal and a metal sulfate or a metal chloride. It can be carried out by reduction, (ii) combination of a transition metal catalyst with a metal sulfate or metal chloride, or (iii) catalytic reduction with a transition metal catalyst.

(I) Reduction by combination of metal and metal sulfate or metal chloride will be described.

Examples of preferable metals include iron and zinc. Preferred metal sulfates include ferrous sulfate, copper sulfate, sodium hydrogen sulfate and the like. Examples of preferable metal chlorides include zinc chloride and cobalt chloride.
The metal sulfate and the metal chloride may be used as a mixture.

As a particularly preferred combination, a combination of iron with ferrous sulfate and / or sodium hydrogensulfate can be mentioned.

For example, 1 to 40 iron is added to the optically active amino acid derivative represented by the general formula (2) in the above solvent.
1 to 20 equivalents of ferrous sulfate (and / or sodium hydrogensulfate), preferably 20 to 30 equivalents of iron,
1 part ferrous sulfate (and / or sodium hydrogen sulfate)
The reduction reaction can be carried out by stirring with 0 to 15 equivalents, preferably in the temperature range of room temperature to 50 ° C. The pressure may be normal pressure. The time required for reduction varies depending on the added metal, metal sulfate, etc., but is usually 6 to
The range is 72 hours.

(Ii) The catalytic reduction using a combination of a transition metal catalyst and a metal sulfate or metal chloride will be described.

Preferable transition metal catalysts include palladium catalysts, platinum catalysts and the like. These transition metal catalysts can be used in the form of palladium carbon, palladium hydroxide, platinum dioxide, platinum carbon and the like.

Examples of preferable metal sulfates include ferrous sulfate and copper sulfate. Examples of preferable metal chlorides include zinc chloride and cobalt chloride. The metal sulfate and the metal chloride may be used as a mixture.

As a particularly preferable combination, a combination of a palladium catalyst and ferrous sulfate can be mentioned.

For example, with respect to the optically active amino acid derivative represented by the general formula (2), the palladium catalyst in the above solvent is usually 0.5 to 10 mol%, preferably 1 to 3 mo.
1%, usually 1 to 10 equivalents of ferrous sulfate, preferably 2 to 5 equivalents of iron, hydrogen pressure is usually 1 to 20 atm, preferably 1 to 5 atm, reaction temperature is usually room temperature to 100 ℃, The reduction reaction can be carried out preferably by stirring in the temperature range of room temperature to 40 ° C. The time required for reduction depends on the catalyst to be added, temperature, pressure, etc.,
It is usually in the range of 3 to 48 hours.

(Iii) The catalytic reduction using a transition metal catalyst will be described.

Preferred transition metal catalysts include palladium catalysts, platinum catalysts and the like. These transition metal catalysts can be used in the form of palladium carbon, palladium hydroxide, platinum dioxide, platinum carbon and the like.

For example, with respect to the optically active amino acid derivative represented by the general formula (2), the palladium catalyst in the above solvent is usually 0.5 to 10 mol%, preferably 1 to 3 mo.
1%, the hydrogen pressure is usually 10 to 30 atm, preferably 15 to 20 atm, the reaction temperature is usually 50 to 100 ° C,
The reduction reaction can be carried out preferably by stirring in the temperature range of 70 ° C to 90 ° C. The time required for the reduction varies depending on the catalyst to be added, etc., but is usually 3 to 48.
It is a range of time.

The reducing method (i) can be carried out at a relatively low temperature and low pressure (for example, normal temperature and normal pressure).
Requires a relatively large amount of metal such as iron. A relatively small amount of transition metal may be used in the reduction method (iii), but it requires a reaction at a relatively high temperature and a high pressure. Since the reduction method of (ii) requires a relatively small amount of transition metal or the like and can be carried out at a relatively low temperature and a low pressure (for example, normal temperature and a normal pressure), the reduction method of (i) and (iii) above can be used. It is an industrially superior manufacturing method than the method.

The reaction solvent containing the optically active 6,6-dimethyllysine derivative represented by the general formula (3) thus obtained is concentrated (or evaporated) by concentration under reduced pressure or the like.
Then, using an organic solvent such as ethyl acetate, toluene and dichloromethane or a mixed solvent such as toluene-isopropyl alcohol, water and a base such as sodium carbonate and sodium hydrogen carbonate are further added to adjust the pH to about 8 to 9 for extraction. To do. The optically active 6,6-dimethyllysine derivative can be obtained by distilling off the organic solvent layer by concentration under reduced pressure or the like. Further, it can be further purified by a conventional method such as chromatography.

Next, the optically active 6,6-dimethyllysine derivative represented by the general formula (3) or a salt thereof is reacted with the acetic acid ester derivative represented by the general formula (4). The step of obtaining the optically active lysine derivative represented by (5) or a salt thereof will be described.

The acetic acid ester derivative represented by the general formula (4) can be easily synthesized from the α-hydroxyacetic acid ester derivative. For example, trifluoromethanesulfonyloxyacetic acid benzyl ester can be obtained by adding α-hydroxyacetic acid benzyl ester in dichloromethane in the presence of pyridine to give trifluoromethanesulfonyloxyacetic acid benzyl ester (Angevante Chemie, 1986, 98).
Vol., 264: Angewandte Chemie 1986, 98, 264). Note that, for example, the α-halogenoacetic acid ester derivative is commercially available from Sigma-Aldrich Japan KK or the like and can be easily obtained.

The 6,6-dimethyllysine derivative represented by the general formula (3) is dissolved in a suitable solvent, and the acetic ester derivative is added in the presence of a base to react. Examples of the solvent include acetonitrile, methanol, ethanol, isopropanol, ethyl acetate, methyl t-butyl ether, toluene and the like. As the base, a tertiary amine such as triethylamine, diethylisopropylamine or diisopropylethylamine is used, and is usually used in an amount of 2 to 10 equivalents, preferably 3 to 4 equivalents based on the 6,6-dimethyllysine derivative. The acetic acid ester derivative is usually used at 1 to 2 equivalents, preferably around 1.2 equivalents, usually 0 ° C to 40 ° C, preferably room temperature, usually 12 to 48 hours, preferably 18 to 24 hours.
Stir for about an hour. The reaction solution is concentrated (or evaporated) by concentration under reduced pressure or the like, and extraction is performed using an organic solvent such as ethyl acetate, toluene, dichloromethane or a mixed solvent such as toluene-isopropyl alcohol. Then, the organic solvent layer is distilled off under reduced pressure to obtain the optically active lysine derivative represented by the general formula (5).

Further, the optically active lysine derivative represented by the general formula (5) is converted into tetrahydrofuran, dichloromethane,
In a state of being dissolved in a solvent such as chloroform, acetone, acetonitrile or the like, an acid such as methanesulfonic acid or paratoluenesulfonic acid is usually added in an amount of 0.8 to 1.5 equivalents, preferably 1.0 to 1.1 equivalents. After addition, ethyl acetate, diethyl ether, petroleum ether, methyl tert-butyl ether, tert-butyl acetate, isopropyl acetate,
By adding a poor solvent such as hexane, cyclohexane, methylcyclohexane, heptane, toluene, xylene, and methylisobutylketone, a salt is precipitated (crystallized), separated and dried to obtain a compound represented by the general formula (5). Crystals of an optically active lysine derivative salt can be obtained.

Further, the optically active lysine derivative represented by the general formula (5) was converted into ethyl acetate, diethyl ether, petroleum ether, methyl tert-butyl ether, acetic acid ter.
In a state of being dissolved in a solvent such as t-butyl, isopropyl acetate, hexane, cyclohexane, methylcyclohexane, heptane, toluene, xylene, methyl isobutyl ketone, an acid such as methanesulfonic acid or paratoluenesulfonic acid is usually added to .8 to 1.5 equivalents, preferably 1.0 to 1.1 equivalents to precipitate salts (crystallization)
It is also possible to obtain crystals of the optically active lysine derivative salt represented by the general formula (5) by separating and drying.

[0165]

The present invention will be described in detail below with reference to examples, but these examples do not limit the present invention.

<Reference Example 1> Methyl 4-methyl-4-nitropentanoate 25 g (0.28 mol) of 2-nitropropane was dissolved in 140 ml of ethanol to give 25.3 m of methyl acrylate.
1 (0.28 mol), potassium fluoride 1.63 g
(0.028 mol) was added and the mixture was heated under reflux for 4 hours. After cooling, ethanol was distilled off by concentration under reduced pressure, and 100 ml of ethyl acetate and 50 ml of water were added for extraction. The ethyl acetate was distilled off under reduced pressure to obtain 40.6 g of the desired compound. (Yield 8
2.5%) ¹ H-NMR (CDCl ₃ ) δppm: 1.60 (s, 6H), 2.22-2.38 (m, 4H),
3.70 (s, 3H)

Reference Example 2 Methyl 4-methyl-4-nitropentanol 4-methyl-4-nitropentanoate 17.5 g
After dissolving (0.1 mol) in 200 ml of ethanol, 5
After cooling to ℃, 7.55 g of sodium borohydride (0.2
mol) was added. The mixture was stirred at room temperature for 1 hour and at 50 ° C. for 3 hours. After adjusting the pH to 3 by adding 100 ml of water and hydrochloric acid, ethanol was distilled off by concentration under reduced pressure. Ethyl acetate 250
ml and 100 ml of water were added for extraction, and the extract was washed with saturated saline. Ethyl acetate was distilled off by concentration under reduced pressure to obtain 14.7 g of the target compound. (Yield 91.2%) ¹ H-NMR (CDCl ₃ ) δppm: 1.5 (m, 2H), 1.61 (s, 6H), 1.95-2.
05 (m, 2H), 3.65 (t, 2H)

Reference Example 3 4-methyl-4-nitropentanol methanesulfonic acid ester 4-methyl-4-nitropentanol 4.3 g (29.
2 mmol) was dissolved in 100 ml of methylene chloride, 6.1 ml (43.8 mmol) of triethylamine was added, and the mixture was ice-cooled. Methanesulfonyl chloride 2.94 ml
(38.0 mmol) was added, and the mixture was stirred under ice cooling for 1 hour.
It was washed with 50 ml of water, 50 ml of 0.5 mol / L hydrochloric acid, 50 ml of 5% aqueous sodium hydrogen carbonate solution and 50 ml of saturated saline. Methylene chloride was distilled off by concentration under reduced pressure, and the residue was crystallized with 4 ml of ethyl acetate and 20 ml of n-hexane. After filtration, it was dried under reduced pressure at 40 ° C. to obtain 6.17 g of the desired compound. (Yield 93.8%) ¹ H-NMR (CDCl ₃ ) δppm: 1.62 (s, 6H), 1.70-1.80 (m, 2H),
2.05 (dd, 2H), 3.06 (s, 3H), 4.23 (t, 2H) mass spectrum m / e: 243 (M + NH ₄ ⁺ )

<Example 1> -Iodo-4-methyl-4-nitropentyl 4-methyl-4-nitropentanol methanesulfonic acid ester 3.38 g (15 mmol) was added to acetone 50.
8.7 g of sodium iodide (58 mmo)
1) was added and the mixture was stirred overnight. 20 ml of water was added, acetone was distilled off by concentration under reduced pressure, and 50 ml of ethyl acetate was added for extraction. 5% sodium thiosulfate aqueous solution 20m
1, washed with 20 ml of a saturated saline solution, and concentrated under reduced pressure to remove ethyl acetate to obtain 3.84 g of the desired compound. (Yield 99.6%) ¹ H-NMR (CDCl ₃ ) δppm: 1.60 (s, 6H), 1.74-1.86 (m, 2H),
2.02 (dd, 2H), 3.18 (t, 2H)

<Example 2> 2-Acetylamino-2- (4-methyl-4-nitropentyl) malonic acid diethyl ester 20 ml of 20% sodium ethoxide in 10 ml of ethanol.
74 g (11 mmol) and 2-acetamidomalonic acid diethyl ester 2.39 g (11 mmol) were added and dissolved, and 4-methyl-4-nitropentyl iodide 2.57 was added.
5 g of ethanol (8 ml) of g (10 mmol) was added.
Heated to reflux for hours. Ethanol was distilled off by concentration under reduced pressure, 50 ml of ethyl acetate was added, and the mixture was washed with 20 ml of water and 20 ml of saturated saline. Ethyl acetate was distilled off by concentration under reduced pressure, and the residue was purified by silica gel column chromatography to obtain 2.89 g of the desired compound. (Yield 83.
4%) ¹ H-NMR (CDCl ₃ ) δppm: 1.00-1.14 (m, 2H), 1.25 (t, 6H),
1.54 (s, 6H), 1.89 (dd, 2H), 2.05 (s, 3H), 2.33 (dd, 2H),
1.44 (q, 4H) mass spectrum m / e: 347 (MH ⁺ )

<Example 3> 2-Acetylamino-6-methyl-6-nitroheptanoic acid 2-acetylamino-2- (4-methyl-4-nitropentyl) malonic acid diethyl ester 193.5 g (55)
8.5 mmol) was dissolved in 270 ml of ethanol, and 51 g (781.9 mmol) of potassium hydroxide was added to 250 parts of water.
It was dissolved in ml and added. After stirring at 90 ° C. for 1 hour, 51 g (781.9 mmol) of potassium hydroxide dissolved in 250 ml of water was added, and the mixture was stirred at the same temperature for 2 hours.
Cool to 40 ° C., add concentrated hydrochloric acid to adjust pH to 1.5,
Stir overnight at 80 ° C. Ethanol was distilled off by concentration under reduced pressure and ice-cooled. The precipitated crystals were separated and washed with ethyl acetate 20
The slurry was washed with 0 ml and dried under reduced pressure. 99.6 g of the target compound was obtained. (Yield 72%) ¹ H-NMR (CDCl ₃ ) δppm: 1.23-1.44 (m, 2H), 1.57 (s, 6H),
1.66-1.78 (m, 2H), 1.91 (t, 2H), 2.04 (s, 3H), 4.53 (dd, 1
H) mass spectrum ^{m / e: 245 (MH -} )

Example 4 (S) -2-Amino-6-methyl-6-nitroheptanoic acid 2-acetylamino-6-methyl-6-nitroheptanoic acid 49.5 g (200 mmol) was added with 246 ml of water. In addition, 30% sodium hydroxide was added to adjust pH to 8.87 and dissolved. L-acylase (4.95 g) and anhydrous cobalt chloride (0.27 g) were added to adjust the pH to 9, and the mixture was stirred overnight at room temperature. The pH was adjusted to 1.5 by adding concentrated hydrochloric acid, and the mixture was ice-cooled. The precipitated crystals were separated by filtration, the filtrate was washed twice with 250 ml of a mixed solution of toluene-isopropyl alcohol 1: 1, and the aqueous layer was concentrated under reduced pressure. 100 ml of methanol was added for concentration, and 100 ml of methanol was added for concentration. As a result, 20.5 g of the desired product was obtained. ¹ H-NMR (DMSO-d ₆ ) δppm: 1.19-1.42 (m, 2H), 1.54 (s, 6H),
1.68-1.76 (m, 2H), 1.86 (t, 2H), 3.53 (t, 1H)

Example 5 (S) -2-Amino-6-methyl-6-nitroheptanoic acid methyl ester hydrochloride (S) -2-Amino-6-methyl-6-nitroheptanoic acid 20.5 g Was dissolved in 177 ml of methanol, and 7.34 ml of thionyl chloride was slowly added dropwise under ice cooling. The mixture was heated under reflux for 2.5 hours, and methanol was concentrated under reduced pressure to obtain 21.9 g of the desired product. ¹ H-NMR (CDCl ₃ ) δppm: 1.35-1.50 (m, 2H), 1.60 (s, 6H),
1.92-2.08 (m, 4H), 3.82 (s, 3H), 4.18 (m, 1H)

Example 6 (S) -2-t-Butoxycarbonylamino-6-methyl-6-nitroheptanoic acid methyl ester (S) -2-Amino-6-methyl-6-nitroheptanoic acid methyl ester 21.9 g of ester hydrochloride was added to 88.
It was dissolved in 5 ml and 40 ml of water, and the pH was adjusted to 7.5 with a saturated aqueous sodium hydrogen carbonate solution. 21.9 g (100.4 mmol) of di-t-butyl dicarbonate was dissolved in 44 ml of methanol and added, and the mixture was added at room temperature for 1 hour and at 40 ° C. for 2 hours.
Stir for hours. After distilling off the methanol by concentration under reduced pressure, 400 ml of ethyl acetate was added for extraction, and 0.5mo
The mixture was washed with 100 ml of 1 / l hydrochloric acid, 100 ml of saturated aqueous sodium hydrogen carbonate solution and 100 ml of saturated saline solution, and dried over anhydrous magnesium sulfate. Anhydrous magnesium sulfate was removed by filtration and concentrated under reduced pressure to obtain 25.7 g of the desired product.
(Yield 80.5%) ¹ H-NMR (CDCl ₃ ) δppm: 1.23-1.37 (m, 2H), 1.43 (s, 9H),
1.58-1.68 (s + m, 8H), 1.92 (t, 2H), 3.75 (s, 3H), 4.30 (b
r.1H), 5.02 (br, 1H) mass spectrum m / e: 319 (MH ⁺ )

Example 7 (S) -2-t-Butoxycarbonylamino-6-methyl-6-nitroheptanoic acid (S) -2-Amino-6-methyl-6-nitroheptanoic acid 10.25 g (50.2 mmol) in methanol 40
ml, dissolved in 25 ml of water, and 10.92 g (50.2 mmol) of di-t-butyl dicarbonate was added to methanol 2
It was dissolved in 0 ml and added, and the mixture was stirred at 40 ° C. for 1 hour. Furthermore, 5.46 g (25.1 g) of di-t-butyl dicarbonate
mmol) in 10 ml of methanol and add 4
The mixture was stirred at 0 ° C for 2.5 hours. Methanol was distilled off by concentration under reduced pressure, 200 ml of ethyl acetate and 6N hydrochloric acid were added, and p
It was adjusted to H2.0 and extracted. The extract was washed with 100 ml of water and 100 ml of saturated saline and dried over anhydrous magnesium sulfate. The solvent was distilled off by concentration under reduced pressure to obtain 7.66 g of the desired product. ¹ H-NMR (CDCl ₃ ) δppm: 1.30-1.42 (m, 2H), 1.47 (s, 6H),
1.55 (s, 9H), 1.63-1.78 (m, 2H), 1.88-1.98 (m, 2H), 4.33
(Br, 1H), 5.00 (br, 1H)

Example 8 (S) -2-t-Butoxycarbonylamino-6-methyl-6-nitroheptanoic acid methyl ester (S) -2-t-Butoxycarbonylamino-6-methyl-6- 3.90 g of nitroheptanoic acid (12.81 mm
ol) was dissolved in 20 ml of dichloromethane and 2.6 ml of dimethylformamide dimethyl acetal (19.2
2 mmol) was added and the mixture was stirred at room temperature for 20 hours. Furthermore, dimethylformamide dimethyl acetal 1.74 m
1 (12.8 mmol) was added and stirred for 4 hours. 0.
5N hydrochloric acid 30 ml, saturated sodium hydrogen carbonate aqueous solution 30
It was washed with 30 ml of saturated saline. The solvent was distilled off by concentration under reduced pressure to obtain 2.53 g of the desired product. (Yield 62%)

Example 9 Nα-t-butoxycarbonyl-6,6-dimethyl-
L-lysine methyl ester (S) -2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoic acid methyl ester 10.05 g
(31.6 mmol) was dissolved in 200 ml of methanol, 8.81 g (157.8 mmol) of iron powder and 40.5 g (145.6 mmol) of ferrous sulfate heptahydrate were added, and the mixture was stirred at 40 ° C. for 17 hours. . Because the raw material remained,
After removing insoluble matter by filtration, 8.81 g of iron powder
(157.8 mmol), ferrous sulfate heptahydrate 40.
5 g (145.6 mmol) was added and the mixture was stirred at 40 ° C. for 7 hours. After removing insolubles by filtration, methanol was removed by concentration under reduced pressure, and then 300 ml of ethyl acetate and 10 ml of water were added.
The pH was adjusted to 9 by adding 0 ml of a 10% sodium carbonate aqueous solution, and extraction was performed. The ethyl acetate was distilled off by concentration under reduced pressure to obtain 5.01 g of the desired product. (Yield 55.0%) ¹ H-NMR (CDCl ₃ ) δppm: 1.10 (s, 6H), 1.46 (s, 9H), 1.56-
1.68 (m, 2H), 1.72 (m, 2H), 3.75 (s, 3H), 4.30 (br, 1H), 5.
05 (br, 1H) ¹³ C-NMR (CDCl ₃ ) δppm: 19.30, 27.31, 28.74, 32.29, 4
2.82, 48.82, 51.24, 52.29, 78.89, 154.45, 172.44 Mass spectrum m / e: 289 (MH ⁺ )

Example 10 Nα-t-butoxycarbonyl-6,6-dimethyl-
L-lysine methyl ester (S) -2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoic acid methyl ester 500 mg
(1.57 mmol) methanol 8 ml, water 0.5 m
Dissolved in 1, 50% wet 5% palladium carbon 219m
g and ferrous sulfate heptahydrate 1.32 g (4.75 m)
mol) was added and the mixture was stirred at room temperature for 3 hours under a hydrogen pressure of 3 atm.
After removing palladium on carbon by filtration, methanol was distilled off by concentration under reduced pressure, and ethyl acetate was 50 ml and water was 50 m.
pH was adjusted to 10 by the addition of 1 and sodium carbonate and extracted. After the layer separation, the aqueous layer was separated into toluene-isopropanol (1:
1) Re-extracted with 100 ml. The organic layers were combined and the solvent was distilled off by concentration under reduced pressure to obtain 350 mg of the desired product. (Yield 77.3%)

<Example 11> Nα-t-butoxycarbonyl-6,6-dimethyl-
L-lysine methyl ester (S) -2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoic acid methyl ester 300 mg
(0.94 mmol) is dissolved in 10 ml of methanol,
Add 0.15 g of 50% wet 5% palladium on carbon,
The mixture was stirred at hydrogen pressure of 20 atm and 80 ° C. for 24 hours. Palladium carbon was removed by filtration, and methanol was removed by concentration under reduced pressure to obtain 219 mg of the desired product. (Yield 80.8
%)

<Example 12> Nα-t-butoxycarbonyl-Nε-t-butoxycarbonylmethyl-6,6-dimethyl-L-lysine methyl ester methanesulfonate Nα-t-butoxycarbonyl-6,6- Dimethyl-L
-Lysine methyl ester 1.0 g (3.46 mmol)
Was dissolved in 30 ml of acetonitrile, 1.60 ml (9.19 mmol) of diisopropylethylamine, and 0.812 g (4.15 m) of bromoacetyl t-butyl ester.
mol) was added and the mixture was stirred at room temperature for about 20 hours. Acetonitrile was distilled off by concentration under reduced pressure, 30 ml of ethyl acetate was added to remove insoluble matter by filtration, and 15 ml of water was added twice to 1 times.
It was washed with 15 ml of 0% sodium carbonate and dried over anhydrous sodium sulfate. After removing sodium sulfate by filtration, 0.2 ml of methanesulfonic acid was added and stirred at room temperature. The precipitated crystals are separated and dried under reduced pressure to give the target product as 1.1.
8 g was obtained. (Yield 68.5%) ¹ H-NMR (DMSO-d ₆ ) δppm: 1.22 (s, 6H), 1.25-1.43 (m + s, 11
H), 1.49-1.68 (s + m, 13H), 2.30 (s, 3H), 3.63 (s, 3H), 3.
87 (m, 2H), 3.98 (m, 1H) ¹³ C-NMR (DMSO-d ₆ ) δppm: 19.63, 22.37, 27.63, 28.14,
30.80, 36.60, 41.90, 51.70, 53.16, 59.12, 78.23, 8
3.09, 155.58, 166.41, 173.06 Mass spectrum m / e: 403 (MH ⁺ )

Reference Example 4 4-Methyl-4-nitrovaleronitrile Acrylonitrile 6.0 g (113 mmol), 2-nitropropane 12.0 g (135 mmol), and hexadecyltrimethylammonium chloride 2.0 g were added to give 0.2%.
The mixture was stirred overnight in 200 ml of 1N aqueous sodium hydroxide solution at room temperature. After that, the layers were separated, and 50 ml of methylene chloride was added to the aqueous layer for extraction twice. The combined organic layers were washed with 30 ml of saturated brine, the solvent was distilled off, and the oil was dried to give 4-methyl-4-oil-4-methyl-4-. Nitrovaleronitrile 10.4 g (7
3 mmol) was obtained. (Yield 64.6%) ¹ H-NMR (CDCl ₃ ) δppm: 1.65 (s, 6H), 2.28-4.46 (m, 4H)

Reference Example 5 4-Methyl-4-nitropentanal 4-methyl-4-nitrovaleronitrile 10.4 g (1
3 mmol) in 100 ml of methylene chloride,
100 ml of a 1 M diisobutylaluminum hydride-hexane solution was added dropwise at 50 ° C., and after stirring for 0.5 hours, 8 ml of water was gradually added to obtain 5.0 mg of magnesium sulfate.
g and filtered. Then, after adding 50 ml of 1N hydrochloric acid and stirring for 0.5 hours, the layers were separated, the organic layer was washed with saturated saline, and then the solvent was distilled off and dried to give oily 4-methyl-4.
6.9 g (48 mmol) of -nitropentanal was obtained. ¹ H-NMR (CDCl ₃ ) δppm: 1.60 (s, 6H), 2.25 (t, 2H), 2.52
(t, 2H), 9.79 (s, 1H)

Example 13 5- (4-methyl-4-nitropentylidene) hydantoin oil 4-methyl-4-nitropentanal 4.0 g (2
8 mmol), hydantoin 4.0 g (40 mmo
l) and 50 g of 2.8 g (20 mmol) of sodium carbonate
% Acetonitrile water 200 ml, refluxed and stirred for 3 days. The reaction solution was concentrated, ethyl acetate was added, and the extraction operation was performed 3 times. The organic layer was concentrated to dryness to obtain 5.15 g (13.9 mmol) of 5- (4-methyl-4-nitropentylidene) hydantoin. (Yield 50.0%) ¹ H-NMR (DMSO-d ₆ ) δppm: 1.56 (s, 6H), 2.00 (m, 2H), 2.13
(m, 2H), 5.42 ( t, 1H) Mass spectrum ^{m / e: 226 (MH -} )

<Example 14> 5- (4-methyl-4-nitropentylidene) hydantoin 20.8 g (210 mmol) of hydantoin and 7.2 g (70 mmol) of sodium carbonate were added with 70 ml of water and heated to 85 ° C. Dissolved. 20.0 g (140 mmol) of 4-methyl-4-nitropentanal was dissolved in 130 ml of isopropyl alcohol and added. After heating under reflux for 2 hours, isopropyl alcohol was distilled off under reduced pressure, and 20 ml of water and concentrated hydrochloric acid were added to adjust the pH to 8. The precipitated crystals were separated, dried and 5- (1-hydroxy-4-methyl-4-nitropentylidene) hydantoin 27.3.
g was obtained. 19.4 g (78.9 mmol) of this compound
Was dissolved in 60 ml of pyridine, 7.9 ml (102.6 mmol) of methanesulfonyl chloride was added under ice cooling, and the mixture was stirred at room temperature for 3 hours. 50 ml of water was added and the mixture was extracted 3 times with 50 ml of ethyl acetate. The extracts were combined and washed 4 times with 50 ml of 1 mol hydrochloric acid. After the layers were separated, the organic layer was concentrated to dryness to give 5- (1-methanesulfonyloxy-4-methyl-4).
20.5 g of (nitropentylidene) hydantoin were obtained.
This compound (5.74 g, 17.8 mmol) was added to tetrahydrofuran (80 ml) and 1,8-diazabicyclo [5.
4.0] Undeca-7-ene 2.7 ml (17.8 mm
ol) was added and the mixture was stirred at room temperature for 4 hours. Tetrahydrofuran was concentrated under reduced pressure, and 50 ml of ethyl acetate and 80 ml of 0.5M hydrochloric acid were added for extraction. After separating the layers, ethyl acetate was concentrated to dryness to obtain 4.0 g (17.6 mmol) of 5- (4-methyl-4-nitropentylidene) hydantoin (yield 61.6%).

Example 15 5- (4-Methyl-4-nitropentyl) hydantoin 5- (4-methyl-4-nitropentylidene) hydantoin 3.15 g (13.9 mmol) of methanol and 60% of methanol
ml and 10 ml of water were added, and 2.2 g of palladium carbon and hydrogen were added for reduction. After the reaction, palladium carbon was removed by filtration, concentrated to dryness, and decanted with water and dichloromethane to obtain 1.1 g of 5- (4-methyl-4-nitropentyl) hydantoin. ¹ H-NMR (DMSO-d ₆ ) δppm: 1.18-1.30 (m, 2H), 1.52 (s, 6H),
2.55-2.70 (m, 2H), 2.83-2.90 (m, 2H), 3.96-4.01 (m, 1H) Mass spectrum ^{m / e: 228 (MH -} )

Example 16 5- (4-methyl-4-nitropentyl) hydantoin 5- (4-methyl-4-nitropentylidene) hydantoin 25.85 g (114 mmol) in methanol 20
Add 0 ml, add 27% aqueous sodium hydroxide and add p.
It was adjusted to H10 and dissolved. 1.1 g of palladium on carbon,
Hydrogen was added for reduction. After the reaction, palladium carbon was filtered off, and methanol was distilled off by concentration under reduced pressure to obtain 50 ml of water.
Was added and the pH was adjusted to 2 with concentrated hydrochloric acid. The precipitated crystals were separated and dried to give 5- (4-methyl-4-nitropentyl) hydantoin 19.02 g (83 mmol) (yield 72.9%).

In the examples below, 2-amino-6-
Quantification of methyl-6-nitroheptanoic acid was carried out by the column "Inertosyl ODS-2" (φ4.6 x GL Science).
High performance liquid chromatography (HPLC) using 250 mm). The analysis conditions are as shown below. Mobile phase: 30 mM phosphoric acid aqueous solution / methanol = 8/2
(V / V) Flow rate: 1.0 ml / min. Column temperature: 50 ° C Detection: UV210nm

The optical purity of 2-amino-6-methyl-6-nitroheptanoic acid was measured by the optical resolution column "CROWNPAK CR (+)" (φ4.6 ×, manufactured by Daicel Chemical Industries, Ltd.).
250 mm) was used for high performance liquid chromatography. The analysis conditions are as shown below. Mobile phase: Perchloric acid aqueous solution (pH 1.8) / acetonitrile = 8/2 (V / V) Flow rate: 1.0 ml / min. Column temperature: 50 ° C. Detection: UV210 nm Under these conditions, (R) -2-amino-6-methyl-6
-Nitroheptanoic acid can be fractionally quantified at a retention time of 4.4 minutes, and (S) -2-amino-6-methyl-6-nitroheptanoic acid at a retention time of 6.1 minutes.

Example 17 Glucose 0.5%, ammonium sulfate 0.5%, powdered yeast extract 1.0%, peptone 1.0
%, KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.3%, M
_{gSO 4 · 7H 2 O 0.05%} , FeSO 4 · 7H 2 O
_{0.001%, MnSO 4 · 5H 2} O 0.001%, D
L-5-indolylmethylhydantoin 0.25% (p
The medium containing H7.0) was dispensed into a 500 ml Sakaguchi flask by 50 ml, and sterilized at 120 ° C. for 10 minutes. After cooling, it was cultured at 30 ° C. for 24 hours on an agar plate medium containing 0.5% glucose, 0.5% ammonium sulfate, 1.0% powdered yeast extract and 1.0% peptone. liquefaciens) AJ
One platinum loop of 3940 was inoculated and shaken aerobically at 30 ° C. for 20 hours. After that, centrifuge operation (12,000
The cells were collected for 10 minutes), suspended in 40 ml of 100 mM Tris-HCl buffer (pH 8.0), and then centrifuged again to obtain washed cells. An equal weight of the same Tris-HCl buffer was added to the washed cells to prepare a cell suspension. Next, 100 mg of DL-5- (4-methyl-4-nitropentyl) hydantoin was weighed for 16 m.
1 of 100 mM Tris-HCl buffer (pH 8.0) was suspended and used as a substrate solution. To the substrate solution, 4 ml of the cell suspension was added and incubated at 37 ° C. 2-amino-6-methyl-6-nitroheptanoic acid produced in the reaction solution after 6, 20, 30, 44, 68 and 96 hours of the reaction is shown in Table 1.

[0190]

[Table 1]

Example 18 70 ml of the reaction solution was prepared in the same manner as in Example 17, and the reaction was carried out for 120 hours. After the reaction, the reaction solution (containing 2-amino-6-methyl-6-nitroheptanoic acid 207 mg) was diluted to 200 ml with water,
The cells were removed by centrifugation (12,000 g, 10 minutes). Then, the supernatant was added to a cation exchange resin column (Amberlite IR-120B, total column 2.6 cm).
× (20 cm in length), flow (flow rate 3 ml / min), and
Amino-6-methyl-6-nitroheptanoic acid was adsorbed. After washing the column with 500 ml of water, 300 ml of 2% ammonia water was passed through the column (flow rate 3 ml / min),
2-Amino-6-methyl-6-nitroheptanoic acid was eluted. The eluent is a rotary evaporator at 40 ° C.
Was concentrated under reduced pressure to 3 ml, and acetone was added dropwise thereto to give 2-amino-6-methyl-6-nitroheptanoic acid as an ammonium salt (dry matter weight: 130 mg). Obtained 2-amino-6-methyl-6
-Nitroheptanoic acid is an optical resolution column CROWN
When subjected to HPLC analysis using PAK CR (+), it was found to be L-form (S-form), and its optical purity was 99% e. e. That was all.

<Example 19> Glucose 0.5%, ammonium sulfate 0.5%, powdered yeast extract 1.0%, peptone 1.0
%, KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.3%, M
_{gSO 4 · 7H 2 O 0.05%} , FeSO 4 · 7H 2 O
_{0.001%, MnSO 4 · 5H 2} O 0.001%, D
L-5-indolylmethylhydantoin 0.25% (p
H7.0) to agar plates containing 0.5% glucose
%, Ammonium sulfate 0.5%, powdered yeast extract 1.0%, peptone 1.0%, and Agrobacterium sp. Cultured at 30 ° C. for 24 hours. (Agrobacterium
sp.) AJ 11220 strain is applied to the entire surface of the plate medium,
It was cultivated aerobically at 30 ° C. for 24 hours. After that, the bacterial cells were scraped off, and 100 mM Tris-HCl buffer solution (p
H8.0) to obtain a bacterial cell suspension. Next, DL-5
-(4-Methyl-4-nitropentyl) hydantoin 1
Weigh out 00 mg and 50 mg of sodium sulfite, and measure 8 m.
1 of 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) was added, and this was used as a substrate solution. To the substrate solution, 2 ml of the cell suspension was added and incubated at 30 ° C. After reaction for 1.5, 3, 6 and 20 hours, 2- produced in the reaction solution
Amino-6-methyl-6-nitroheptanoic acid is shown in Table 2. After the reaction for 20 hours, the reaction solution is diluted with water, and the sterilized body fluid is used as an optical resolution column CROWNPAK CR.
When subjected to HPLC analysis using (+), it was found to be D-form (R-form), and its optical purity was 94%.
e. e. Met.

[0193]

[Table 2]

Example 20 Maltose 2%, ammonium sulfate 0.5%, powdered yeast extract 1.0%, peptone 1.0
%, KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.3%, M
_{gSO 4 · 7H 2 O 0.05%} , FeSO 4 · 7H 2 O
_{0.001%, MnSO 4 · 5H 2} O 0.001%, D
L-5-isopropylhydantoin 0.2% (pH 7.
50 ml of the medium containing 0) in a 500 ml Sakaguchi flask.
It was dispensed in increments of 1 and sterilized at 120 ° C for 10 minutes. After cooling
30 on agar plate medium containing 0.5% glucose, 0.5% ammonium sulfate, 1.0% powdered yeast extract, 1.0% peptone
Bacillus sp. Cultured at 24 ° C for 24 hours. (Bacill
us sp.) One platinum loop of AJ12299 was inoculated and aerobically shake-cultured at 30 ° C. for 20 hours. Thereafter, the cells were collected by centrifugation (12,000 g, 10 minutes), and the cells were collected in 100 mM potassium phosphate buffer (pH 7.0) 4
After suspending in 0 ml, the same centrifugation operation was performed again to obtain washed bacterial cells. An equal weight of the same potassium phosphate buffer was added to the washed cells to prepare a cell suspension. Next, DL-5
(4-Methyl-4-nitropentyl) hydantoin 1.
25 g / dl, glucose 1.25 g / dl, MgSO
The ₄ · 7H ₂ O 0.05g / dl was added to 100mM potassium phosphate buffer (pH7.0) to prepare a substrate solution. To 4 ml of this substrate solution, 1 ml of the bacterial cell suspension was added, and the mixture was incubated aerobically at 30 ° C. in a test tube for 48 hours while shaking. After the reaction, reaction liquid 2.0m
0.4 ml of concentrated hydrochloric acid under ice cooling, and 300 mM N
0.2 ml of aNO ₂ was added, and the mixture was allowed to stand at 4 ° C. for 24 hours, and N-carbamyl-produced by the reaction with bacterial cells.
2-Amino-6-methyl-6-nitroheptanoic acid was decarbamylated. When the concentration of 2-amino-6-methyl-6-nitroheptanoic acid in the reaction solution after decarbamyl was measured, it was 0.51 g / dl. The optical activity was measured to find that it was L-form (S-form), and the optical purity was 99.
% E. e. That was all.

Reference Example 6 Flavobacterium sp. (Flavobacterium sp.) AJ3912 strain and AJ3
As a result of re-identification, strain 940 was identified as Microbacterium liquefaciens (formerly Aureobacte).
rium liquefaciens)]. In other words, the Burgers Manual, which is a taxonomy of bacteria
The following physiological properties were shown as a result of conducting a physiological property test in the light of Obterdeminative Bacteology Vol. 1 (9th edition 1994, published by William & Wilkins).

[0196]

Reference Example 7 Pseudomonas hydantoinophilum AJ 1
As a result of re-identification, the 1220 strain was identified as Agrobacterium sp. (Agrobacterium sp.).
That is, the taxonomy of bacteria, the Burgers Manual of Determinative Bacteology No. 1.
As a result of conducting a physiological property test against Volume 1 (9th edition, 1994, published by William & Wilkins), the following physiological properties were shown.

[0198] test Cell morphology 0.8 × 1.5-2.0 μm Gram stain − Spore − Motility +: periodontal hair Catalase + Oxidase + O / F test − Nitrate reduction + Indole production − Glucose acidification − Arginine dihydrolase- Urease + Esculin hydrolysis + Gelatin hydrolysis − β-galactosidase + Substrate utilization     Glucose +     L-arabinose +     D-mannose +     D-mannitol +     N-acetyl-D-glucosamine +     Maltose +     Potassium gluconate −     n-capric acid-     Adipic acid −     dl-malic acid +     Sodium citrate −     Phenyl acetate-

[0199]

INDUSTRIAL APPLICABILITY According to the present invention, the optically active lysine derivative represented by the above general formulas (3) and (5), which is useful as a pharmaceutical intermediate, can be produced by an industrial method.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07C 271/22 C07C 271/22 C07D 233/76 C07D 233/76 C12P 13/04 C12P 13/04 41/00 41/00 H // C07B 53/00 C07B 53/00 F 61/00 300 61/00 300 (C12P 41/00 C12R 1:07 C12R 1:07) 1:01 (C12P 41/00 C07M 7:00 C12R 1:01) C07M 7:00 (72) Inventor Ikumasa Onishi 1-1, Suzuki-cho, Kawasaki-ku, Kanagawa Prefecture Ajinomoto Co., Inc. Amino Science Research Institute (72) Inventor Masaki Naito Suzuki, Kawasaki-ku, Kawasaki-shi, Kanagawa 1-1 Ajinomoto Co., Inc. Kawasaki Plant (72) Inventor Kunisuke Izawa 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Amino Science Research Institute (72) Person Kenzo Yokozeki 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa F-Term (reference) inside Amino Science Research Center, Ajinomoto Co., Inc. 4B064 AE03 CB01 CB24 CD27 DA01 4H006 AA01 AA02 AC48 AC52 BA19 BA25 BA36 BA55 BA61 BE20 BS10 BU22 BV22 BV72 RA06 RB34 4H039 CA71 CB40

Claims (23)

[Claims] 1. Formula (1): [* Indicates an asymmetric carbon. ] The amino group of the optically active 2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof represented by general formula (2) is prepared by protecting the amino group or the amino group and the carboxyl group with a protecting group. [* Indicates an asymmetric carbon, and P ₁ and P ₂ each independently represent an amino-protecting group or a hydrogen atom (provided that P ₁ and P ₂ are
₁ and P ₂ are simultaneously hydrogen atoms), or P ₁ and P ₂ together represent an amino-group protecting group, and P ₃ represents a hydrogen atom or a carboxyl-group protecting group. ] The optically active amino acid derivative represented by the formula [3] is prepared by reducing the nitro group. [*, P ₁ and P ₂ have the same meanings as described above, and P ₄ represents a hydrogen atom or a protecting group for a carboxyl group. ] The optically active 6,6-dimethyllysine derivative represented by the formula or a salt thereof is obtained, which is represented by the general formula (4): [Y ₁ represents a leaving group, R ₁ represents an alkyl group having 1 to 6 carbon atoms or an aralkyl group having 7 to 12 carbon atoms. ] A general formula (5) characterized by reacting with an acetic acid ester derivative [*, P ₁ , P ₂ and R ₁ have the same meanings as described above, and P ₅
Represents a hydrogen atom or a protective group for a carboxyl group. ] The manufacturing method of the optically active lysine derivative represented by these, or its salt. 2. The production method according to claim 1, wherein the reduction is catalytic reduction using a transition metal catalyst and a metal sulfate. 3. The production method according to claim 1, wherein the reduction is catalytic reduction using a palladium catalyst and ferrous sulfate. 4. One of P _{1 and} P ₂ is a tert-butoxycarbonyl group and the other is a hydrogen atom, and P ₃ and P ₄
And P ₅ is a methyl group, and R ₁ is a tert-butyl group. 5. The formula (1): [* Indicates an asymmetric carbon. ] The amino group of the optically active 2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof represented by the general formula (2) is protected with a protecting group to give a compound of the general formula (2): [* Indicates an asymmetric carbon, and P ₁ and P ₂ each independently represent an amino-protecting group or a hydrogen atom (provided that P ₁ and P ₂ are
₁ and P ₂ are simultaneously hydrogen atoms), or P ₁ and P ₂ together represent an amino-group protecting group, and P ₃ represents a hydrogen atom or a carboxyl-group protecting group. ] The optically active amino acid derivative represented by the formula [3], wherein the nitro group is reduced. [*, P ₁ and P ₂ have the same meanings as described above, and P ₄ represents a hydrogen atom or a protecting group for a carboxyl group. ] The manufacturing method of the optically active 6,6-dimethyl lysine derivative or its salt represented by these. 6. The production method according to claim 5, wherein the reduction is catalytic reduction using a transition metal catalyst and a metal sulfate. 7. The production method according to claim 5, wherein the reduction is catalytic reduction using a palladium catalyst and ferrous sulfate. 8. The production method according to claim 5, wherein _{one of} P _{1 and} P ₂ is a tert-butoxycarbonyl group, the other is a hydrogen atom, and P ₃ and P ₄ are methyl groups. 9. A compound represented by the general formula (2): [* Indicates an asymmetric carbon, and P ₁ and P ₂ each independently represent an amino-protecting group or a hydrogen atom (provided that P ₁ and P ₂ are
₁ and P ₂ are simultaneously hydrogen atoms), or P ₁ and P ₂ together represent an amino-group protecting group, and P ₃ represents a hydrogen atom or a carboxyl-group protecting group. ] The general formula (3) characterized by reducing the amino acid derivative represented by: [*, P ₁ and P ₂ have the same meanings as described above, and P ₄ represents a hydrogen atom or a protecting group for a carboxyl group. ] The manufacturing method of the 6,6- dimethyl lysine derivative or its salt represented by these. 10. The production method according to claim 9, wherein the reduction is catalytic reduction using a transition metal catalyst and a metal sulfate. 11. The production method according to claim 9, wherein the reduction is catalytic reduction using a palladium catalyst and ferrous sulfate. 12. _{One of} P _{1 and} P ₂ is tert-
The production method according to claim 9, wherein the butoxycarbonyl group, the other is a hydrogen atom, and P ₃ and P ₄ are methyl groups. 13. A compound represented by the general formula (3): [* Indicates an asymmetric carbon, and P ₁ and P ₂ each independently represent an amino-protecting group or a hydrogen atom (provided that P ₁ and P ₂ are
₁ and P ₂ are simultaneously hydrogen atoms), or P ₁ and P ₂ together represent an amino-group protecting group, and P ₄ represents a hydrogen atom or a carboxyl-group protecting group. ] The optically active 6,6-dimethyllysine derivative represented by the formula or a salt thereof is represented by the general formula (4): [Y ₁ represents a leaving group, R ₁ represents an alkyl group having 1 to 6 carbon atoms or an aralkyl group having 7 to 12 carbon atoms. ] The compound represented by the general formula (5): [*, P ₁ , P ₂ and R ₁ have the same meanings as described above, and P ₅
Represents a hydrogen atom or a protective group for a carboxyl group. ] The manufacturing method of the optically active lysine derivative represented by these, or its salt. 14. Either P ₁ or P ₂ is tert-
The production method according to claim 13, wherein the butoxycarbonyl group, the other is a hydrogen atom, P ₄ and P ₅ are methyl groups, and R ₁ is a tert-butyl group. 15. The optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1) is produced through the following steps (a) to (c). Production method according to 1: (a) General formula (6) [Y ₂ represents a leaving group. ] 4-methyl-4- represented by
Nitropentane derivatives and general formula (7): [R ₂ and R ₃ each independently represent an alkyl group having 1 to 6 carbon atoms or an aralkyl group having 7 to 12 carbon atoms. ] By reacting with a 2-acylaminomalonic acid diester represented by the general formula (8) [R ₂ and R ₃ have the same meanings as described above. ] 2-
Step of obtaining acylamino-2- (4-methyl-4-nitropentyl) malonic acid diester; (b) 2-acylamino-2-represented by the general formula (8).
The (4-methyl-4-nitropentyl) malonic acid diester is hydrolyzed and decarboxylated to give the compound of formula (9): A step of obtaining 2-acetylamino-6-methyl-6-nitroheptanoic acid (racemic body) represented by: (c) 2-acetylamino-6-methyl-6-nitroheptane represented by formula (9) A step of reacting an acid (racemic form) with an acylase to obtain an optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1) or a salt thereof. 16. The optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1) is produced through the following steps (d) to (g). Production method described in 1: (d) Formula (10) The 5- (4-methyl-4-nitropentylidene) hydantoin represented by the formula is reduced to give the compound of the formula (11): A step of obtaining 5- (4-methyl-4-nitropentyl) hydantoin represented by: (e) hydrolyzing 5- (4-methyl-4-nitropentyl) hydantoin represented by formula (11) Formula (12) A step of obtaining 2-amino-6-methyl-6-nitroheptanoic acid (racemate) represented by: (f) 2-amino-6-methyl-represented by formula (12)
The amino group of 6-nitroheptanoic acid is acylated to give a compound of the general formula (13): [R ₄ represents a methyl group or a phenyl group. ] The step of obtaining 2-acylamino-6-methyl-6-nitroheptanoic acid (racemate) represented by: (g) 2-acylamino-6 represented by general formula (13)
-A step of reacting methyl-6-nitroheptanoic acid (racemic form) with an acylase to obtain the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by formula (1) or a salt thereof. 17. The formula (14) and the general formulas (15) to
Any of the compounds represented by (18) (including optically active compounds and racemates) or a salt thereof: [P ₁ and P ₂ each independently represent a protecting group for an amino group or a hydrogen atom (except when P ₁ and P ₂ simultaneously become a hydrogen atom), or P ₁ and P ₂ together form an amino group. Represents a protecting group for a group (except when P ₁ and P ₂ together represent a phthaloyl group), P _3a represents a protecting group for a carboxyl group, and P ₄ represents a protecting group for a hydrogen atom or a carboxyl group. Show. ]. 18. A compound represented by any of formulas (19) to (23) or a salt thereof: . 19. A compound represented by any of formulas (10) and (11) and general formulas (6), (8) and (24) (compounds represented by formula (8) and formula (24)) Include optically active forms and racemates) or salts thereof: [Y ₂ represents a leaving group (excluding chlorine atom), R ₄ represents a methyl group or a phenyl group, and R ₂ and R ₃ each independently represent an alkyl group having 1 to 6 carbon atoms or 7 to 12 carbon atoms. The aralkyl group of is shown. ]. 20. The production according to claim 1, wherein the optically active 2-amino-6-methyl-6-nitroheptanoic acid represented by the formula (1) is produced through the following step (h). Method: (h) General formula (11) The 5- (4-methyl-4-nitropentyl) hydantoin represented by the formula (1) is optically selective using one or more selected from the group consisting of microorganisms, processed products of microorganisms, hydantoinase, and N-carbamyl amino acid hydrolase. Hydrolyzed to give optically active 2-amino-6- of formula (1)
A step of obtaining methyl-6-nitroheptanoic acid or a salt thereof. 21. The origin of a microorganism, a processed product of the microorganism, a hydantoinase and an N-carbamyl amino acid hydrolase is Agrobacterium.
Genus, genus Bacillus or microbacterium
21. The method according to claim 20, which is a bacterium of the genus (Microbacterium). 22. The origin of the microorganism, the processed product of the microorganism, the hydantoinase and the N-carbamyl amino acid hydrolase is Agrobacterium.
Optically active 2-amino-6-methyl-6-
Nitroheptanoic acid or its salt is D-2-amino-6
21. The method according to claim 20, which is -methyl-6-nitroheptanoic acid or a salt thereof. 23. The origin of the microorganism, the processed product of the microorganism, the hydantoinase and the N-carbamyl amino acid hydrolase is Microbacterium.
A genus or bacterium of the genus Bacillus, wherein optically active 2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof is L-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof. Claim 2 characterized by the above.
The method described in 0.