METHOD FOR PRODUCING cis-5-HYDROXY-2-PIPERIDINECARBOXYLIC
ACID DERIVATIVE, AND METHOD FOR PURIFYING cis-5-HYDROXY-2- PIPERIDINECARBOXYLIC ACID
TECHNICAL FIELD
[0001]
The present invention relates to an industrial method for producing a
10 derivative of cis-5-hydroxy-2-piperidinecarboxylic acid (which is also called cis-5-
hydroxypipecolic acid). The present invention also relates to a method for purifying
cis-5-hydroxy-2-piperidinecarboxylic acid.
BACKGROUND ART
15 [0002]
cis-5-Hydroxy-2-piperidinecarboxylic acid is a useful intermediate for
pharmaceuticals. However, since this compound has 2 asymmetric carbons, 4 kinds
of isomers are present, and selective synthesis of a single kind of isomer, or single
kind of diastereomer, is very difficult. Thus, improvement of the purity by
20 separation of unnecessary isomers, or purification, by chemical conversion is
necessary.
[0003]
For example, methods for obtaining cis-5-hydroxy-2-piperidinecarboxylic
acid by introduction of a hydroxyl group to 2-piperidinecarboxylic acid by
25 hydroxylation reaction using a microorganism or enzyme have been reported.
However, it has been reported that a compound having a substituted hydroxyl group
at another position, such as a 3-position-hydroxylated compound, may be produced
2
as a by-product in addition to the compound of interest, cis-5-hydroxy-2-
piperidinecarboxylic acid (Non-patent Document 1). Separation of the by-product
is not described in the document. A method in which 5-hydroxy-2-
piperidinecarboxylic acid is synthesized from 5-hydroxylysine by enzymatic reaction
has also been reported (Patent Document 1), but the document does not descri5 be
separation of the generated cis/trans isomers. In a report describing synthesis of 5-
hydroxy-2-piperidinecarboxylic acid from 5-hydroxylysine by a method similar to the
method of Patent Document 1 (Patent Document 2), isomers are separated using an
ion-exchange column. Since this method requires use of excessive amounts of a
10 filler and eluent with respect to the substrate, the method is not realistic from the
viewpoint of industrial production.
Thus, there is no known method by which a necessary stereoisomer can be
selectively obtained with high purity from cis-5-hydroxy-2-piperidinecarboxylic acid
synthesized using a microorganism or enzyme.
15 [0004]
There are known chemical synthesis methods using, as a material, L-amino
acid, in which the stereochemistry of one of the two asymmetric carbons, the 2-
position carbon, can be fixed. For example, a method using L-pyroglutamic acid as
a material (Patent Document 3) has been reported. This method requires use of an
20 iridium catalyst, which is expensive, for formation of a piperidine ring, which is
problematic. Methods using L-glutamic acid as a material (Non-patent Documents
2 and 3) and a method using a proline derivative as a material (Non-patent Document
4) have also been reported, but all of these methods require use of a diazo compound,
which is highly risky, and also require a multistep complex process. Moreover, for
25 separation of (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid, the reaction is carried
out via 5-oxo-2-piperidinecarboxylic acid to preferentially obtain the (2S,5S)
compound by its reduction (Non-patent Document 4). This requires separation
3
using a silica gel column for removal of impurities such as isomers. Because of the
load of this process, this method is not industrially satisfactory.
[0005]
It is also known that (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid and its esters are lactonized to give benzyl (1S,4S)-5-aza-5 2-
oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate (Non-patent Documents 4 and 5).
These documents on lactonization do not mention about impurities such as isomers,
and the effects of purification by these methods have been unclear. Non-patent
Document 4 describes that benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
10 carboxylate was obtained as an oily substance after purification with a silica gel
column. Non-patent Document 5 describes that crystallization of benzyl (1S,4S)-5-
aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate occurs, but, since the
lactonization is carried out using cis-5-hydroxy-2-piperidinecarboxylic acid as a
material, behavior of impurities such as isomers and the effect of purification are not
15 clear. There is a report describing separation of a cis/trans mixture of methyl Nbenzyloxycarbonyl-
5-hydroxy-2-piperidinecarboxylate by acid treatment (Non-patent
Document 3), but, since the compound was a carboxylic acid ester, it was impossible
to separate lactone obtained from the cis isomer from the ester of the trans isomer by
a simple method such as solvent extraction. Moreover, Non-patent Document 3
20 does not describe the yield in this process. When the present inventors studied this
method, the yield of the lactone was low because of occurrence of a remarkable side
reaction between lactone obtained from the cis isomer (benzyl (1S,4S)-5-aza-2-oxa-3-
oxobicyclo[2.2.2]octane-5-carboxylate)) and the residual trans isomer ((2S,5R)-Nbenzyloxycarbonyl-
5-hydroxy-2-piperidinecarboxylic acid ester).
25 As described above, there are problems to be solved for industrial production
of highly pure cis-5-hydroxy-2-piperidinecarboxylic acid by an inexpensive method.
4
PRIOR ART DOCUMENTS
[Patent Documents]
[0006]
Patent Document 1: JP 4590981 B
Patent Document 2: JP 2010-5 88395 A
Patent Document 3: WO 2010/126820
[Non-patent Documents]
[0007]
Non-patent Document 1: Adv. Synth. Catal., 2011, 353, 1375.
10 Non-patent Document 2: Chem. Commun., 1996, 349.
Non-patent Document 3: Tetrahedron Lett., 1988, 29, 2231.
Non-patent Document 4: Tetrahedron: Asym., 2006, 17, 2479.
Non-patent Document 5: Rec. Trav. Chim. Pays-Bas, 1959, 78, 648.
15 SUMMARY OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0008]
The present invention aims to provide a method for purifying cis-5-hydroxy-
2-piperidinecarboxylic acid, which is a useful intermediate for pharmaceuticals, and a
20 method for producing its derivative.
MEANS FOR SOLVING THE PROBLEMS
[0009]
The present inventors intensively studied to solve the problems described
25 above. As a result, the present inventors solved the problems by a process of
reacting low-purity cis-5-hydroxy-2-piperidinecarboxylic acid containing impurities
with an acid halide and/or acid anhydride to induce a cis-5-hydroxy-2-
5
piperidinecarboxylic acid derivative, thereby solving the problems.
In a more specific mode, the present inventors discovered that impurities such
as isomers can be separated by reacting cis-5-hydroxy-2-piperidinecarboxylic acid
with an acid halide and/or acid anhydride to convert the cis-5-hydroxy-2-
piperidinecarboxylic acid into a lactone whose amino group is protected, and the5 n
performing crystallization and/or solvent extraction. That is, studies by the present
inventors revealed that the cis-5-hydroxy-2-piperidinecarboxylic acid derivative in
the present invention can be selectively converted to lactone by reaction with an acid
halide and/or acid anhydride, and that this lactone can be crystallized under specific
10 conditions. The present inventors then developed a method in which the lactone is
crystallized to achieve removal of impurities such as isomers having low
crystallizability.
In another specific mode, the present inventors discovered that impurities
such as isomers having a carboxyl group can be removed by solvent extraction after
15 reacting cis-5-hydroxy-2-piperidinecarboxylic acid with an acid halide and/or acid
anhydride, and then with an alcohol in the presence of an acid catalyst to convert the
cis-5-hydroxy-2-piperidinecarboxylic acid into an ester whose amino group is
protected. That is, the present inventors studied to develop a method in which, in
the presence of an acid catalyst, a cis-5-hydroxy-2-piperidinecarboxylic acid
20 derivative is selectively converted into lactone, and the lactone is reacted with an
alcohol present in the reaction system to efficiently and selectively convert the cis
isomer into an ester while suppressing a side reaction, followed by removing residual
impurities such as isomers having a carboxyl group by solvent extraction.
The present inventors then discovered that highly pure cis-5-hydroxy-2-
25 piperidinecarboxylic acid can be obtained by hydrolyzing the resulting lactone and/or
ester to give cis-N-protected-5-hydroxy-2-piperidinecarboxylic acid, and then
removing the protective group for the amino group, thereby completing the present
6
invention.
[0010]
That is, the present invention can be summarized as follows.
[1] A method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative, the method comprising a step of converting cis-5-hydroxy-5 2-
piperidinecarboxylic acid into a compound(s) of Formula (1) and/or Formula (2)
(wherein R1 represents a protective group for an amino group, and R2 represents a
C1-C6 alkyl group).
[0011]
10
[0012]
[2] The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative according to [1], comprising a step of reacting cis-5-hydroxy-2-
piperidinecarboxylic acid with an acid halide and/or acid anhydride to convert the
15 cis-5-hydroxy-2-piperidinecarboxylic acid into a compound of Formula (1).
[3] The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative according to [1], comprising a step of reacting cis-5-hydroxy-2-
piperidinecarboxylic acid with an acid halide and/or acid anhydride, and then with an
alcohol in the presence of an acid catalyst, to convert the cis-5-hydroxy-2-
20 piperidinecarboxylic acid into a compound of Formula (2).
[4] A method for regenerating cis-5-hydroxy-2-piperidinecarboxylic acid,
the method comprising the steps of:
converting cis-5-hydroxy-2-piperidinecarboxylic acid into a compound(s) of
Formula (1) and/or Formula (2) (wherein R1 represents a protective group for an
7
amino group, and R2 represents a C1-C6 alkyl group); and
converting the compound(s) of Formula (1) and/or Formula (2) into cis-5-
hydroxy-2-piperidinecarboxylic acid.
[0013]
5
[0014]
[5] The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative according to any one of [1] to [3], wherein the cis-5-hydroxy-2-
piperidinecarboxylic acid is cis-5-hydroxy-2-piperidinecarboxylic acid synthesized
10 by bacterial reaction and/or enzymatic reaction.
[6] The method for regenerating cis-5-hydroxy-2-piperidinecarboxylic acid
according to [4], wherein the cis-5-hydroxy-2-piperidinecarboxylic acid is cis-5-
hydroxy-2-piperidinecarboxylic acid synthesized by bacterial reaction and/or
enzymatic reaction.
15 [7] A method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid, the
method comprising a step of reacting a mixture containing cis-5-hydroxy-2-
piperidinecarboxylic acid and an impurity with an acid halide and/or acid anhydride,
or with an acid halide and/or acid anhydride and an alcohol, to convert the cis-5-
hydroxy-2-piperidinecarboxylic acid into a compound(s) of Formula (1) and/or
20 Formula (2), separating the compound(s) and then converting the separated
compound(s) into cis-5-hydroxy-2-piperidinecarboxylic acid.
[0015]
8
[0016]
[8] The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to [7], wherein the impurity is 2-piperidinecarboxylic acid or an analogue
thereof5 .
[9] The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to [8], wherein the 2-piperidinecarboxylic acid or an analogue thereof is
trans-5-hydroxy-2-piperidinecarboxylic acid.
[10] The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
10 according to any one of [7] to [9], wherein the step of separating the compound(s) of
Formula (1) and/or Formula (2) is carried out by crystallization or solvent extraction.
[11] The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to any one of [7] to [10], wherein the mixture containing cis-5-hydroxy-2-
piperidinecarboxylic acid and an impurity is a mixture synthesized by bacterial
15 reaction and/or enzymatic reaction.
EFFECT OF THE INVENTION
[0017]
By the method of the present invention, highly pure cis-5-hydroxy-2-
20 piperidinecarboxylic acid and its derivatives can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018]
Fig. 1 is a schematic diagram illustrating routes for purifying cis-5-hydroxy-2-
9
piperidinecarboxylic acid from cis-5-hydroxy-2-piperidinecarboxylic acid that may
contain an impurity(s). The route 1 is a route in which cis-5-hydroxy-2-
piperidinecarboxylic acid is reacted with an acid halide and/or acid anhydride to give
a compound of Formula (1) directly or via cis-N-protected-5-hydroxy-2-
piperidinecarboxylic acid, and the compound is then converted into cis-5-hydroxy-5 2-
piperidinecarboxylic acid. The route 2 is a route in which cis-N-protected-5-
hydroxy-2-piperidinecarboxylic acid is reacted with an acid catalyst to give a
compound of Formula (1), and the compound is then converted into cis-5-hydroxy-2-
piperidinecarboxylic acid by hydrolysis. The route 3 is a route in which cis-N10
protected-5-hydroxy-2-piperidinecarboxylic acid is reacted with an alcohol in the
presence of an acid catalyst to give a compound of Formula (2), and the compound is
then converted to cis-5-hydroxy-2-piperidinecarboxylic acid by hydrolysis.
Fig. 2. is a diagram showing the result of HPLC analysis of hydroxylysines
obtained with recombinant lysine hydroxylases.
15 Fig. 3 is a diagram showing the result of HPLC analysis of hydroxylysines
obtained with recombinant lysine hydroxylases.
DESCRIPTION OF THE EMBODIMENTS
[0019]
20 The present invention is described below in detail.
In the present invention, examples of the cis-5-hydroxy-2-
piperidinecarboxylic acid include (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid,
(2R,5R)-5-hydroxy-2-piperidinecarboxylic acid, and mixtures thereof. The cis-5-
hydroxy-2-piperidinecarboxylic acid may be a racemic compound. The cis-5-
25 hydroxy-2-piperidinecarboxylic acid may be forming a salt with an acid or base.
[0020]
The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
10
derivative of the present invention comprises a step of converting cis-5-hydroxy-2-
piperidinecarboxylic acid into a compound(s) of Formula (1) and/or Formula (2).
[0021]
In Formula (1) and Formula (2), R1 represents a protective group for an ami5 no
group, and specific examples of the protective group include the following groups.
However, the protective group is not limited to these examples.
Examples of the protective group for the amino group include acyl groups
such as formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl,
10 propionyl, benzoyl, and 4-chlorobenzoyl; alkoxycarbonyl groups such as
methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, and
allyloxycarbonyl; arylalkyl groups such as benzyl, 4-methoxybenzyl, 4-bromobenzyl,
and 1-phenethyl; and sulfonyl groups such as methanesulfonyl, p-toluenesulfonyl,
and 2-nitrobenzenesulfonyl.
15 [0022]
Among these, acyl groups and alkoxycarbonyl groups are preferred since
these groups can be easily removed; acetyl, chloroacetyl, trifluoroacetyl, benzoyl,
tert-butoxycarbonyl, and benzyloxycarbonyl are more preferred; and acetyl, tertbutoxycarbonyl,
and benzyloxycarbonyl are still more preferred since these are
20 industrially inexpensive. The protective group is especially preferably
benzyloxycarbonyl since benzyloxycarbonyl can be removed by hydrogenation
without leaving a non-volatile component, and the load of purification of cis-5-
hydroxy-2-piperidinecarboxylic acid can therefore be reduced.
[0023]
11
In Formula (2), R2 represents a C1-C6 alkyl, and specific examples of R2
include primary alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, and hexyl;
secondary alkyl groups such as isopropyl, isobutyl, isopentyl, cyclopentyl, and
cyclohexyl; and tertiary alkyl groups such as tert-butyl. Among these, methyl, ethyl,
isopropyl, butyl, and tert-butyl are preferred, and ethyl and isopropyl are mor5 e
preferred.
[0024]
In the present invention, the cis-5-hydroxy-2-piperidinecarboxylic acid
derivative may be the compound(s) of Formula (1) and/or Formula (2)
10 itself/themselves.
Specific examples of the compound of Formula (1) include benzyl 5-aza-2-
oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate, tert-butyl 5-aza-2-oxa-3-
oxobicyclo[2.2.2]octane-5-carboxylate, and 5-acetyl-5-aza-2-oxa-3-
oxobicyclo[2.2.2]octane.
15 Specific examples of the compound of Formula (2) include methyl cis-N-
(benzyloxycarbonyl)-5-hydroxy-2-piperidinecarboxylate, ethyl cis-N-
(benzyloxycarbonyl)-5-hydroxy-2-piperidinecarboxylate, isopropyl cis-N-
(benzyloxycarbonyl)-5-hydroxy-2-piperidinecarboxylate, methyl cis-N-(tertbutyloxycarbonyl)-
5-hydroxy-2-piperidinecarboxylate, ethyl cis-N-(tert20
butyloxycarbonyl)-5-hydroxy-2-piperidinecarboxylate, and isopropyl cis-N-(tertbutyloxycarbonyl)-
5-hydroxy-2-piperidinecarboxylate.
[0025]
The cis-5-hydroxy-2-piperidinecarboxylic acid derivative may be a compound
obtained by chemical conversion of a compound(s) of Formula (1) and/or Formula
25 (2).
Specific examples of such a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative include carboxylic acids such as cis-N-(benzyloxycarbonyl)-5-hydroxy-2-
12
piperidinecarboxylic acid and cis-N-(tert-butyloxycarbonyl)-5-hydroxy-2-
piperidinecarboxylic acid; esters such as benzyl cis-N-(benzyloxycarbonyl)-5-
hydroxy-2-piperidinecarboxylate and benzyl cis-N-(tert-butyloxycarbonyl)-5-
hydroxy-2-piperidinecarboxylate; carboxylic acid amides such as cis-N-
(benzyloxycarbonyl)-5-hydroxy-2-piperidinecarboxylic acid amide, cis-N-(tert5 -
butyloxycarbonyl)-5-hydroxy-2-piperidinecarboxylic acid amide, benzyl cis-5-
hydroxy-2-(methylcarbamoyl)piperidine-1-carboxylate, tert-butyl cis-5-hydroxy-2-
(methylcarbamoyl)piperidine-1-carboxylate, benzyl cis-2-(1-tertbutyloxycarbamoylpyrrolidine-
3-ylcarbamoyl)-5-hydroxypiperidine-1-carboxylate,
10 tert-butyl cis-2-(1-benzyloxycarbamoylpyrrolidine-3-ylcarbamoyl)-5-
hydroxypiperidine-1-carboxylate, benzyl cis-2-(1-tert-butyloxycarbamoylpiperidine-
4-ylcarbamoyl)-5-hydroxypiperidine-1-carboxylate, and tert-butyl cis-2-(1-
benzyloxycarbamoylpiperidine-4-ylcarbamoyl)-5-hydroxypiperidine-1-carboxylate.
[0026]
15 In one mode of the method of the present invention, cis-5-hydroxy-2-
piperidinecarboxylic acid is reacted with an acid halide and/or acid anhydride to
protect the amino group of the cis-5-hydroxy-2-piperidinecarboxylic acid, and
subsequent lactonization of the resultant causes its conversion into a compound of
Formula (1).
20 [0027]
The acid halide and/or acid anhydride is/are not limited as long as the acid
halide and/or acid anhydride can protect the amino group and allow(s) the
lactonization. Examples of the acid halide include acid chlorides and acid bromides.
In view of ease of handling, acid chlorides are preferred. Examples of the acid
25 anhydride include carboxylic acid anhydrides, and sulfonic acid anhydrides. In
view of ease of handling and the cost, carboxylic acid anhydrides are preferred.
Specific examples of the acid halide and/or acid anhydride include acylating
13
agents such as formic acid-acetic anhydride, acetic anhydride, acetyl chloride,
chloroacetyl chloride, dichloroacetyl chloride, trichloroacetyl chloride, trifluoroacetic
anhydride, propionyl chloride, benzoyl chloride, 4-chlorobenzoyl chloride, acetyl
bromide, propionyl bromide, and benzoyl bromide; alkoxycarbonylating agents such
as di-tert-butyldicarbonate, benzyloxycarbonyl chloride, allyloxycarbonyl chloride5 ,
benzyloxycarbonyl bromide, and allyloxycarbonyl bromide; and sulfonylating agents
such as methanesulfonyl chloride, p-toluenesulfonyl chloride, 2-nitrobenzenesulfonyl
chloride, methanesulfonyl bromide, p-toluenesulfonyl bromide, and 2-
nitrobenzenesulfonyl bromide.
10 Among these, acylating agents and alkoxycarbonylating agents are preferred.
Acetic anhydride, chloroacetyl chloride, trichloroacetyl chloride, trifluoroacetic
anhydride, di-tert-butyldicarbonate, benzyloxycarbonyl chloride, and
allyloxycarbonyl chloride are more preferred since these can be easily removed after
the protection of the amino group. Acetic anhydride, di-tert-butyldicarbonate, and
15 benzyloxycarbonyl chloride are still more preferred since these are industrially
inexpensive. Benzyloxycarbonyl chloride is especially preferred since it can be
removed by hydrogenation without leaving a non-volatile component, and the load of
purification of cis-5-hydroxy-2-piperidinecarboxylic acid can therefore be reduced.
[0028]
20 In the present invention, two or more kinds of acid halide and/or acid
anhydride may be used. In cases where two or more kinds of acid halide and/or acid
anhydride are used, these may be added to the reaction system at once, but it is
preferred to use different kinds of acid halide and/or acid anhydride for in each the
process of amino group protection and the process of lactonization. For example, it
25 is preferred to carry out a method in which benzyloxycarbonyl chloride is used for
the protection of the amino group, and acetic anhydride is used for the lactonization.
In cases where the protection of the amino group and the lactonization are carried out
14
in a single reactor, a single kind of acid halide and/or acid anhydride is preferably
used for suppression of by-products having different protective groups. In cases
where the protection of the amino group and the lactonization are carried out in
separate reactors, two or more kinds of acid halide and/or acid anhydride are
preferably used in view of optimization of the reactions and cost reduction. I5 n
particular, the protection of the amino group and the lactonization are preferably
carried out in a single reactor since the number of reactors required for the production
can be decreased, and the cost can therefore be reduced.
[0029]
10 The amount of the acid halide and/or acid anhydride to be used is usually 1 to
10 molar equivalents, preferably 1.2 to 5 molar equivalents, more preferably 1.5 to 3
molar equivalents relative to the total amount of the cis-5-hydroxy-2-
piperidinecarboxylic acid and the amine compound to be protected. The acid halide
and/or acid anhydride may be added dividedly in a plurality of times.
15 [0030]
In the reaction with the acid halide and/or acid anhydride, a base may be used,
if necessary. Specific examples of the base include, but are not limited to, tertiary
amines such as triethylamine, diisopropylethylamine, N-methylmorpholine,
quinuclidine, and 1,4-diazabicyclo[2.2.2]octane; pyridines such as pyridine, 4-
20 dimethylaminopyridine, and 2,6-lutidine; organic strong bases such as 1,8-
diazabicyclo[5.4.0]undeca-7-ene and tetramethylguanidine; metal amides such as
lithium diisopropylamide and sodium hexamethyldisilazide; alkyl metals such as nbutyllithium,
sec-butyllithium, tert-butyllithium, and isopropylmagnesium bromide;
metal hydrides such as sodium hydride and calcium hydride; metal alkoxides such as
25 sodium methoxide, sodium ethoxide, sodium tert-butoxide, and potassium tertbutoxide;
carbonates such as sodium hydrogen carbonate and potassium carbonate;
phosphates such as potassium phosphate and sodium hydrogen phosphate;
15
hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, and
calcium hydroxide; and cyanides such as sodium cyanide and potassium cyanide.
Preferred bases vary depending on the protective reagent to be used. In
cases where a preferred protective reagent, that is, acetic anhydride, di-tertbutyldicarbonate,
or benzyloxycarbonyl chloride, is used, the base is preferabl5 y
tertiary amines, pyridines, carbonates, or hydroxides. In cases where the especially
preferred protective reagent, that is, benzyloxycarbonyl chloride, is used, the base is
preferably a hydroxide, which is inexpensive.
In cases where a base is used, the amount of base is preferably added such
10 that the pH of the reaction mixture becomes 7 to 12. In cases where water is used as
the solvent, the pH of the reaction mixture herein means the pH of the layer
containing water. In cases where a solvent other than water is used, the pH of the
reaction mixture herein means the pH of the layer which contains water when an
equal volume of water is added to the reaction mixture. In cases where water is
15 used as the solvent, hydrolysis of the compound of Formula (1) may occur when the
basicity of the reaction mixture is too strong. The pH is therefore more preferably 7
to 12, especially preferably 7 to 11.
[0031]
Examples of the reaction solvent include water; esters such as ethyl acetate
20 and butyl acetate; alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-
butanol, 2-ethyl-1-hexanol, and 2-butanol; ethers such as diethyl ether, di-n-butyl
ether, diisopropyl ether, di-n-butyl ether, tert-butyl methyl ether, tetrahydrofuran, and
dioxane; ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone;
nitriles such as acetonitrile; amides such as dimethylformamide, dimethylacetamide,
25 and N-methylpyrrolidinone; sulfones such as dimethylsulfoxide and sulfolane;
hydrocarbons such as hexane, heptane, and toluene; and mixed solvents of two or
more of these. Among these, water, and mixed solvents containing water and one
16
or more of the above solvents, are preferred. In particular, in cases where the cis-5-
hydroxy-2-piperidinecarboxylic acid is synthesized by bacterial reaction and/or
enzymatic reaction, the cis-5-hydroxy-2-piperidinecarboxylic acid is obtained as a
solution whose solvent is mainly water. Therefore, in view of simplicity of the
production process, it is preferred to use the solution as it is, or to concentrate part 5 of
the solution, for carrying out the reaction using water as a main solvent.
[0032]
The reaction temperature is usually -20°C to 100°C, preferably -10°C to 50°C.
Since reaction at high temperature may cause decomposition of reagents and products,
10 the reaction temperature is more preferably 0°C to 30°C.
[0033]
The protection of the amino group and the lactonization are preferably carried
out by adding an acid halide and/or acid anhydride (the route 1 in Fig. 1). It is
especially preferred to carry out the protection of the amino group and the
15 lactonization using a single acid halide and/or acid anhydride in a single reactor in
view of simplicity of the operation (the upper formula in the route 1 in Fig. 1).
[0034]
The protection of the amino group and the lactonization may also be carried
out under different conditions (the route 2 in Fig. 1). That is, the amino group may
20 be protected by addition of the acid halide and/or acid anhydride, and then an acid
catalyst may be used to perform the lactonization. In cases where the protection of
the amino group and the lactonization are carried out under different reaction
conditions, it is preferred to suppress production of the compound of Formula (1) at
the stage of protection of the amino group. The protection of the amino group can
25 be selectively and effectively carried out by using the acid halide and/or acid
anhydride in the same molar amount as that of the amino group, or by adding the acid
halide and/or acid anhydride dividedly such that excessive presence of the acid halide
17
and/or acid anhydride relative to the amino group is avoided, and then stopping the
reaction when the material cis-5-hydroxy-2-piperidinecarboxylic acid has almost
disappeared. In cases where the protection of the amino group is carried out in a
solvent containing water, the compound of Formula (1) generated by lactonization, if
any, can be hydrolyzed by a method such as making the pH of the reaction mixtur5 e
strongly basic, increasing the temperature of the reaction mixture to a temperature of
not less than room temperature, performing the reaction for long time, or a
combination of one or more of these methods, and, as a result, the amino-protected
compound can be obtained. By subsequently reacting the amino-protected
10 compound with an acid catalyst, the compound of Formula (1) can be obtained.
[0035]
The compound of Formula (1) produced can be isolated by a method such as
the crystallization described below.
[0036]
15 In another mode of the method of the present invention, cis-5-hydroxy-2-
piperidinecarboxylic acid is reacted with an acid halide and/or acid anhydride, and
then with an alcohol in the presence of an acid catalyst, to convert the cis-5-hydroxy-
2-piperidinecarboxylic acid into a compound of Formula (2) (the route 3 in Fig. 1).
By the reaction with an alcohol in the presence of an acid catalyst, the compound of
20 Formula (1) produced by the reaction with an acid halide and/or acid anhydride is
converted to the compound of Formula (2). In this process, in cases where
compounds having a carboxyl group other than the compound of Formula (1),
especially the 2-piperidinecarboxylic acid or an analogue thereof described later, are
contained as impurities, these compounds are not converted to lactone like the
25 compound of Formula (1). Therefore, the compound derived from cis-5-hydroxy-2-
piperidinecarboxylic acid can be selectively converted to the compound of Formula
(2), which is an ester, while the other compounds are left in the reaction system as
18
carboxylic acids. Thereafter, by extraction with a basic aqueous solution, the
compound of Formula (2) can be separated into the organic layer, while the
impurities other than the compound of Formula (2) can be separated into the aqueous
layer.
[5 0037]
The alcohol used herein is not limited as long as it is a C1-C6 alcohol.
Examples of the alcohol include primary alcohols such as methanol, ethanol, 1-
propanol, 1-butanol, 1-pentanol, and 1-hexanol; secondary alcohols such as 2-
propanol, 2-butanol, cyclopentanol, and cyclohexanol; and tertiary alcohols such as
10 tert-butanol. Among these, secondary alcohols are preferred, and 2-propanol is
more preferred since it is inexpensive, has a low boiling point, and can be easily
removed. In cases where a primary alcohol is used, a side reaction may occur to
cause direct conversion of a carboxylic acid into its ester in the presence of an acid
catalyst, resulting in low effect of removal of impurities. In cases where a tertiary
15 alcohol is used, reactivity with the compound of Formula (1) is low. Therefore, the
compound of Formula (2), which is a secondary alcohol, may react with the
compound of Formula (1) to partially form a dimer, resulting in low yield of the
compound of Formula (2). Thus, in the present invention, a secondary alcohol is
preferably used.
20 An ester of the above-described alcohol may be added in the presence of an
acid catalyst and water so that hydrolysis reaction of the ester allows the alcohol to be
present in the reaction system, and the alcohol may be reacted with the cis-Nprotected-
5-hydroxy-2-piperidinecarboxylic acid in the presence of an acid catalyst.
The alcohol, or the ester which generates the alcohol, may be used in an
25 excess amount, and the molar amount of the alcohol or ester to be used is usually 1 to
500 times the molar amount of the cis-5-hydroxy-2-piperidinecarboxylic acid. In
cases where the amount of the alcohol or ester is too small, a side reaction in which
19
the compound of Formula (2) reacts with the compound of Formula (1) may occur to
form a dimer, while in cases where the amount of the alcohol or ester is too large, the
effect of the acid catalyst decreases. Thus, the molar amount of the alcohol or ester
is preferably 2 to 100 times, more preferably 3 to 50 times, especially preferably 5 to
20 times the molar amount of the cis-5-hydroxy-2-piperidinecarboxylic aci5 d
[0038]
Examples of the acid catalyst include inorganic acids such as hydrochloric
acid, sulfuric acid, nitric acid, phosphoric acid, and polyphosphoric acid; sulfonic
acids such as p-toluenesulfonic acid, methanesulfonic acid, and
10 trifluoromethanesulfonic acid; and carboxylic acids such as acetic acid, formic acid,
trifluoroacetic acid, trichloroacetic acid, benzoic acid, and oxalic acid. Among
these, sulfonic acids are preferred since they have high solubility in organic solvents
and sufficiently strong acidity for allowing the reaction to proceed. p-
Toluenesulfonic acid and methanesulfonic acid are more preferred since they are
15 industrially inexpensive.
The molar amount of the acid catalyst to be added is usually 0.001 to 10 times
the molar amount of the cis-5-hydroxy-2-piperidinecarboxylic acid. In cases where
the molar amount of the acid catalyst is too small, the reaction proceeds slowly, while
the molar amount of the acid catalyst is too large, the load of the post treatment is
20 large. The molar amount of the acid catalyst is therefore preferably 0.01 to 5 times,
more preferably 0.02 to 1 times, especially preferably 0.05 to 0.5 times the molar
amount of the cis-5-hydroxy-2-piperidinecarboxylic acid.
[0039]
Examples of the reaction solvent include organic acids such as formic acid
25 and acetic acid; esters such as ethyl acetate and butyl acetate; alcohols such as
methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-ethyl-1-hexanol, and 2-
butanol; ethers such as diethyl ether, di-n-butyl ether, diisopropyl ether, di-n-butyl
20
ether, tert-butyl methyl ether, tetrahydrofuran, and dioxane; ketones such as acetone,
methyl ethyl ketone, and methyl isobutyl ketone; nitriles such as acetonitrile; amides
such as dimethylformamide, dimethylacetamide, and N-methylpyrrolidinone;
sulfones such as dimethylsulfoxide and sulfolane; hydrocarbons such as hexane,
heptane, and toluene; and mixed solvents of two or more of these. Among these5 ,
solvents which do not show coordination of a proton are preferred since they do not
weaken the activity of the acid catalyst. More specifically, hydrocarbons are
preferred. In addition, organic acids, which themselves are acids, and esters, which
produce acids by hydrolysis, are preferred. Toluene is more preferred because of its
10 low cost, high reaction rate, and high solubility of acid catalysts therein.
[0040]
The reaction temperature is usually 0°C to 150°C, preferably 20°C to 120°C.
Reaction at higher temperature may cause a side reaction, but reaction at lower
temperature is slow and takes a long time. The reaction temperature is therefore
15 more preferably 40°C to 80°C.
[0041]
The compound of Formula (2) generated can be isolated by a method such as
the extraction method using an organic solvent described later.
[0042]
20 The compounds of Formulae (1) and (2) can be converted to cis-N-protected-
5-hydroxy-2-piperidinecarboxylic acid by treatment with a base.
[0043]
Examples of the base to be used herein include hydroxides such as sodium
hydroxide, potassium hydroxide, lithium hydroxide, and calcium hydroxide;
25 phosphates such as sodium phosphate, potassium phosphate, and calcium phosphate;
carbonates such as lithium carbonate, sodium carbonate, potassium carbonate,
magnesium carbonate, and calcium carbonate; and hydrogen carbonates such as
21
sodium hydrogen carbonate and potassium hydrogen carbonate. The base is
preferably a hydroxide.
The molar amount of the base to be used is usually 0.1 to 10 times, preferably
0.5 to 5 times, more preferably 0.9 to 3 times the total molar amount of the
compounds of Formulae (1) and (5 2).
[0044]
By carrying out deprotection of the amino group of the cis-N-protected-5-
hydroxy-2-piperidinecarboxylic acid, cis-5-hydroxy-2-piperidinecarboxylic acid (cis-
5-hydroxypipecolic acid) can be produced. In particular, in cases where R1 in the
10 Formulae (1) and (2) is a benzyloxycarbonyl group, deprotection of the amino group
can be carried out by hydrogenation reaction. The hydrogenation reaction can be
carried out by, for example, using a palladium carbon catalyst. By such a reaction,
the cis-5-hydroxy-2-piperidinecarboxylic acid can be regenerated.
[0045]
15 The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid of the
present invention is a method in which highly pure cis-5-hydroxy-2-
piperidinecarboxylic acid is obtained by purification from cis-5-hydroxy-2-
piperidinecarboxylic acid containing an impurity(s).
[0046]
20 Examples of the impurity include 2-piperidinecarboxylic acid or an analogue
thereof, amino acids, peptides, sugars, and fatty acids. Since, among these, 2-
piperidinecarboxylic acid or an analogue thereof, amino acids, and peptides have an
amino group(s) and carboxyl group(s), these can be hardly separated from the desired
cis-5-hydroxy-2-piperidinecarboxylic acid of interest by an ordinary purification
25 method such as ion-exchange resin purification. In particular, since 2-
piperidinecarboxylic acid or an analogue thereof has the same skeleton as cis-5-
hydroxy-2-piperidinecarboxylic acid, its separation is very difficult, and
22
establishment of removal process is very important.
Examples of the 2-piperidinecarboxylic acid or an analogue thereof include
isomers of cis-5-hydroxy-2-piperidinecarboxylic acid; and 2-piperidinecarboxylic
acid and analogues thereof. Examples of the isomers of cis-5-hydroxy-2-
piperidinecarboxylic acid include stereoisomers such as trans isomers, and structura5 l
isomers having hydroxyl groups at different positions. It should be noted that
optical isomers of cis-5-hydroxy-2-piperidinecarboxylic acid are not included in the
impurity of the present invention.
Examples of the trans isomers include trans-5-hydroxy-2-
10 piperidinecarboxylic acid, that is, (2S,5R)-5-hydroxy-2-piperidinecarboxylic acid,
(2R,5S)-5-hydroxy-2-piperidinecarboxylic acid, and mixtures of these. Examples of
the structural isomers include cis-2-hydroxy-2-piperidinecarboxylic acid, trans-2-
hydroxy-2-piperidinecarboxylic acid, cis-3-hydroxy-2-piperidinecarboxylic acid,
trans-3-hydroxy-2-piperidinecarboxylic acid, and trans-4-hydroxy-2-
15 piperidinecarboxylic acid. It should be noted that cis-4-hydroxy-2-
piperidinecarboxylic acid is not included in the impurity of the present invention.
Examples of the 2-piperidinecarboxylic acid and analogues thereof include 2-
piperidinecarboxylic acid and 1,2,3,4-tetrahydro-2-pyridinecarboxylic acid.
Examples of the amino acids include essential amino acids such as proline,
20 lysine, and isoleucine, and unnatural amino acids such as 3-hydroxyproline, 4-
hydroxyproline, and 5-hydroxylysine.
Examples of the peptides include dipeptides, oligopeptides, and proteins.
Examples of the sugars include glucose, gluconic acid, sodium gluconate,
potassium gluconate, and calcium gluconate.
25 Examples of the fatty acids include C2-C4 short-chain fatty acids, C5-C12
medium-chain fatty acids, long-chain fatty acids having not less than 13 carbon
atoms, and glycerol esters.
23
[0047]
A mixture containing cis-5-hydroxy-2-piperidinecarboxylic acid and an
impurity(s) is reacted with an acid halide and/or acid anhydride, or with an acid
halide and/or acid anhydride and an alcohol, to convert the cis-5-hydroxy-2-
piperidinecarboxylic acid into a compound(s) of Formula (1) and/or Formula (2)5 .
Subsequently, the compound(s) is/are separated by crystallization, solvent extraction,
and/or the like and then treated with a base to convert the compound(s) into cis-Nprotected-
5-hydroxy-2-piperidinecarboxylic acid. By subsequently carrying out
deprotection of the amino group, cis-5-hydroxy-2-piperidinecarboxylic acid which
10 the impurity has been removed can be obtained.
That is, cis-5-hydroxy-2-piperidinecarboxylic acid can be separated from the
impurity utilizing specific lactonization of the cis-5-hydroxy-2-piperidinecarboxylic
acid. Even in cases where the impurity other than the above-described impurities is
contained, the cis-5-hydroxy-2-piperidinecarboxylic acid can be separated also from
15 the impurity.
In the present description, the crystallization includes not only ordinary
crystallization by adding a poor solvent, acid, base, or the like to a solution, or
azeotropically removing a good solvent such as water, to decrease solubility of the
desired product and recover the product as crystals; but also recrystallization in
20 which crude crystals that have once been obtained are dissolved in an appropriate
solvent and then recrystallized. The crystallization may be promoted by addition of
seed crystals to the solution.
In the crystallization of the compound of Formula (1), crystallization is
preferably carried out for a crude product of the compound of Formula (1) obtained
25 by the reaction with the acid halide and/or acid anhydride. This is because a small
amount of acidic component(s) generated from the acid halide and/or acid anhydride,
and/or other components derived from reagents, may remain to induce crystallization.
24
[0048]
Examples of the crystallization solvent for the crystallization treatment
include water; organic acids such as acetic acid and propionic acid; esters such as
ethyl acetate, isopropyl acetate, and butyl acetate; alcohols such as methanol, ethanol,
1-propanol, 2-propanol, 1-butanol, 2-ethyl-1-hexanol, and 2-butanol; ethers such a5 s
diethyl ether, di-n-butyl ether, diisopropyl ether, methyl-tert-butyl ether, di-n-butyl
ether, tetrahydrofuran, and dioxane; ketones such as acetone, methyl ethyl ketone,
and methyl isobutyl ketone; nitriles such as acetonitrile; aliphatic hydrocarbons such
as hexane and heptane; aromatic hydrocarbons such as toluene and xylene; and
10 mixed solvents of two or more of these. Among these, alcohols, aliphatic
hydrocarbons, aromatic hydrocarbons, and mixed solvents of two or more of these
are preferred since solubility of the compound(s) of Formula (1) and/or Formula (2)
in these solvents is sufficiently low. Aliphatic hydrocarbons, aromatic
hydrocarbons, and mixed solvents of two or more of these are more preferred, and
15 hexane, heptane, toluene, and mixed solvents of two or more of these are especially
preferred.
[0049]
Examples of the organic solvent to be used for the extraction include waterinsoluble
solvents, for example, esters such as ethyl acetate, isopropyl acetate, and
20 butyl acetate; ethers such as diethyl ether, methyl-tert-butyl ether, di-n-butyl ether,
diisopropyl ether, di-n-butyl ether, tert-butyl methyl ether, and tetrahydrofuran;
aliphatic hydrocarbons such as hexane and heptane; and aromatic hydrocarbons such
as toluene and xylene.
[0050]
25 The above-described method is especially useful in cases where cis-5-
hydroxy-2-piperidinecarboxylic acid is to be purified from a mixture of cis-5-
hydroxy-2-piperidinecarboxylic acid and trans-5-hydroxy-2-piperidinecarboxylic
25
acid.
[0051]
That is, in a mode of the purification method of the present invention (the
route 1 or route 2 in Fig. 1), a mixture of cis-5-hydroxy-2-piperidinecarboxylic acid
and trans-5-hydroxy-2-piperidinecarboxylic acid is reacted with an acid halide and/5 or
acid anhydride to convert the cis-5-hydroxy-2-piperidinecarboxylic acid into the
compound of Formula (1). The resulting compound is then separated by
crystallization and/or extraction with an organic solvent, and then treated with a base
to convert the compound into cis-N-protected-5-hydroxy-2-piperidinecarboxylic acid.
10 By subsequently carrying out deprotection of the amino group, cis-5-hydroxy-2-
piperidinecarboxylic acid separated from the trans isomer can be obtained.
In such cases, the acid halide and/or acid anhydride is/are preferably used in
an excessive amount over 1 molar equivalent relative to the total amount of the cis-5-
hydroxy-2-piperidinecarboxylic acid and the trans-5-hydroxy-2-piperidinecarboxylic
15 acid. The amount of the acid halide and/or acid anhydride is usually 1 to 10 molar
equivalents, preferably 1.2 to 5 molar equivalents, more preferably 1.5 to 3 molar
equivalents. In cases where both an acid halide and an acid anhydride are used,
these are added such that their total amount exceeds 1 molar equivalent relative to the
total amount of the cis-5-hydroxy-2-piperidinecarboxylic acid and the trans-5-
20 hydroxy-2-piperidinecarboxylic acid.
By the reaction with the acid halide and/or acid anhydride, the cis-5-hydroxy-
2-piperidinecarboxylic acid is converted to the compound of Formula (1). On the
other hand, the trans-5-hydroxy-2-piperidinecarboxylic acid remains in the reaction
system without being converted, or is converted to the mixed acid anhydride of trans-
25 5-hydroxy-2-piperidinecarboxylic acid described below. This mixed acid anhydride
can be converted into the trans-5-hydroxy-2-piperidinecarboxylic acid again by post
treatment. Thus, its separation by crystallization reaction is possible.
26
[0052]
In another mode of the purification method of the present invention (the route
3 in Fig. 1), a mixture of cis-5-hydroxy-2-piperidinecarboxylic acid and trans-5-
hydroxy-2-piperidinecarboxylic acid is reacted with an acid halide and/or aci5 d
anhydride to protect the amino group. Subsequently, the cis-N-protected-5-
hydroxy-2-piperidinecarboxylic acid generated is reacted with an alcohol (or an ester
that generates an alcohol) in the presence of an acid catalyst to allow conversion into
the compound of Formula (2) via the compound of Formula (1). The resulting
10 compound is separated by extraction with an organic solvent, and then treated with a
base to allow conversion into cis-N-protected-5-hydroxy-2-piperidinecarboxylic acid.
By subsequently carrying out deprotection of the amino group, cis-5-hydroxy-2-
piperidinecarboxylic acid separated from the trans isomer can be obtained.
In such cases, the acid halide and/or acid anhydride is/are preferably used in
15 an amount of about 1 molar equivalent relative to the total amount of the cis-5-
hydroxy-2-piperidinecarboxylic acid and the trans-5-hydroxy-2-piperidinecarboxylic
acid.
By performing the reaction with the acid halide and/or acid anhydride to
protect the amino group, and then with the alcohol (or ester that generates an alcohol)
20 in the presence of the acid catalyst, the cis-5-hydroxy-2-piperidinecarboxylic acid is
converted into the compound of Formula (2), while the trans-5-hydroxy-2-
piperidinecarboxylic acid is converted to the trans-N-protected-5-hydroxy-2-
piperidinecarboxylic acid described below. Thus, their separation is possible by
extraction with an organic solvent.
27
[0053]
cis-5-hydroxy-2-piperidinecarboxylic acid containing an impurity(s)
cis-5-hydroxy-2-piperidinecarboxylic acid and trans-5-hydroxy-2-
may be those synthesized by bacterial reaction and/5 or
enzymatic reaction. For example, by the method described in Example 29 of JP
mixture of cis-5-hydroxy-2-piperidinecarboxylic acid and trans-5-
hydroxy-2-piperidinecarboxylic acid cis-5-hydroxy-2-
piperidinecarboxylic acid containing an impurity(s) cis-5-hydroxy-
10 2-piperidinecarboxylic acid and trans-5-hydroxy-2-
also be products recovered after separation of a cis-5-hydroxy-2-piperidinecarboxylic
acid derivative by the method of the present invention.
[0054]
In cases where cis-5-hydroxy-2-piperidinecarboxylic acid is purified by the
15 cis-5-hydroxy-2-piperidinecarboxylic acid
containing an impurity(s) cis-5-hydroxy-2-piperidinecarboxylic acid
and trans-5-hydroxy-2- synthesized by bacterial reaction
and/or enzymatic reaction, the obtained cis-5-hydroxy-2-piperidinecarboxylic acid is
preferably further subjected to adsorption purification using activated carbon and/or
20 crystallization using a solvent containing water to remove impurities such as a
colored substance, to further increase purity of the cis-5-hydroxy-2-
piperidinecarboxylic acid.
[0055]
As the activated carbon, an arbitrary known activated carbon may be used,
25 and examples of the known activated carbon include coal-based, wood-based,
28
coconut shell-based, and resin-based activated carbons. The activated carbon may
also be prepared by activating a material activated carbon such as a coal-based,
wood-based, coconut shell-based, or resin-based material activated carbon by a
method such as gas activation, steam activation, or chemical activation using zinc
chloride, phosphoric acid, or the like5 .
Specific examples of the activated carbon include Calgon CPG, Calgon CAL,
Calgon SGL, Diasorb W, Diahope MS10, Diahope M010, Diahope MS16, Diahope
6MD, Diahope 6MW, Diahope 8ED, Diahope ZGN4, and CENTUR, manufactured
by Calgon Mitsubishi Chemical Corporation; GAC, GAC PLUS, GCN PLUS, C
10 GRAN, RO, ROX, DARCO, CN, SX, SX PLUS, SA, SX, PK, and W, manufactured
by Norit Japan Co., Ltd.; GW, GWH, GLC, 4GC, KW, PW, and PK, manufactured
by Kuraray Chemical Co., Ltd.; HC-30S, GL-30S, 4G-3S, PS, and PC, manufactured
by Tsurumicoal Co., Ltd.; P, W, CW, SG, SGP, S, GB, CA, and K, manufactured by
Futamura Chemical Co., Ltd.; Shirasagi KL, Shirasagi W2C, Shirasagi WH2C,
15 Shirasagi W5C, Shirasagi WH5C, Shirasagi WH5X, Shirasagi XS7100H-3,
Carboraffin, Shirasagi A, Shirasagi C, and Shirasagi M, manufactured by Japan
EnviroChemicals Ltd.; and Hokuetsu CL-K, Hokuetsu HS, and Hokuetsu KS,
manufactured by Ajinomoto Fine-Techno Co., Inc.
By this operation, impurities showing absorption within the wavelength range
20 of 400 nm to 800 nm can be removed.
[0056]
As the solvent containing water to be used for the crystallization, a watersoluble
organic solvent can be used. Specific examples of the solvent include
organic acids such as acetic acid and propionic acid; esters such as ethyl acetate;
25 alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol; ethers
such as tetrahydrofuran and dioxane; ketones such as acetone, methyl ethyl ketone,
and diethyl ketone; nitriles such as acetonitrile; amides such as dimethylformamide,
29
dimethylacetamide, and N-methylpyrrolidinone; sulfones such as dimethylsulfoxide
and sulfolane; and mixed solvents of two or more of these. It is also preferred to
further increase purity of the cis-5-hydroxy-2-piperidinecarboxylic acid by carrying
out crystallization of the cis-5-hydroxy-2-piperidinecarboxylic acid using the abovedescribed
solvent. Among these, organic acids, alcohols, and ketones, whos5 e
solubility in water is higher, are preferred. Alcohols and ketones are more preferred,
and ethanol and acetone are especially preferred.
EXAMPLES
10 [0057]
The present invention is described below in more detail by way of Examples.
However, the present invention is not limited by these Examples.
[0058]
The quantitative analyses in the Examples were carried out by HPLC (High
15 Performans Liquid Chromatography) under the following conditions.

Column: Astec CLC- , manufactured by
SUPELCO
Mobile phase: 2 mmol/L aqueous copper sulfate solution
20 Flow rate: 1.0 mL/minute
Column temperature: 45°C
Detection wavelength: UV 254 nm

Column: L-column (4.6 mm × 250 mm, 5
25 Evaluation and Research Institute, Japan
Mobile phase:
A: 0.1 wt% aqueous trifluoroacetic acid solution
30
B: methanol
gradient (concentration of B): 0 minute, 20% 2 minute, 20% 10
minute, 80% 20 minute, 80%
Flow rate: 1.0 mL/minute
Column temperature: 5 40°C
Detection wavelength: UV 200 nm

Column: ZORB
manufactured by Agilent Technologies
10 Mobile phase:
A: 0.1 wt% aqueous phosphoric acid solution
B: acetonitrile
gradient (concentration of B): 0 minute minute, 5
minute 12 minute, 80%
15 Flow rate: 1.0 mL/minute
Column temperature: 40°C
Detection wavelength: UV 210 nm

Column: COSMOSIL 5C18-AR-II (4.6 mm × 150 mm), manufactured by
20 Nacalai Tesque, Inc.
Mobile phase: 50 mmol/L phosphate buffer (pH 2.7)
Flow rate: 1.0 mL/minute
Column temperature: 40°C
Detection wavelength: UV 340 nm
25
Column: SUMICHIRAL OA-6100 (4.6 mm × 250 mm), manufactured by
Sumika Chemical Analysis Service, Ltd.
31
Mobile phase: 1 mmol/L copper sulfate
Flow rate: 1.0 mL/minute
Column temperature: 30°C
Detection wavelength: UV 254 nm

Column: CLC-D (4.6 mm × 150 mm), manufactured by SUPELCO
Mobile phase: 2 mmol/L copper sulfate
Flow rate: 1.0 mL/minute
Column temperature: 30°C
10 Detection wavelength: UV 254 nm
[0059]
[Reference Example 1]
[Preparation Example of Recombinant Escherichia coli (E. coli) JM109/pKW32
15 (dpkA, aip, gdh, kr), in Which N-Methyl-L-amino Acid Dehydrogenase (Hereinafter
Referred to as DpkA), L-Amino Acid Oxidase (Hereinafter Referred to as AIP),
Glucose-1-dehydrogenase (Hereinafter Referred to as GDH), and Amino Acid
Racemase (hereinafter referred to as KR) Are Co-expressed]
(1) Cloning of Genes
20 Based on a gene sequence of dpkA (SEQ ID NO:1) encoding DpkA
(GenBank Accession No. BAD89743, SEQ ID NO:2) derived from Pseudomonas
putida(P. putida), primers for amplifying the full-length sequence of the dpkA gene,
dpkA_F (SEQ ID NO:9) and dpkA_R (SEQ ID NO:10), were designed and
synthesized. PCR was carried out using chromosomal DNA of P. putida as a
25 template according to a conventional method, to obtain a DNA fragment of about 1.0
kbp.
A gene sequence of aip (SEQ ID NO:3) encoding a protein AIP (SEQ ID
32
NO:4), which has the amino acid sequence of L-amino acid oxidase derived from
Scomber japonicus (GenBank Accession No.CAC00499) with the exception that the
signal peptide is removed and methionine is added, was designed and artificially
synthesized. Primers for amplifying the full-length sequence of the aip gene, aip_F
(SEQ ID NO:11) and aip_R (SEQ ID NO:12), were designed and synthesized. PC5 R
was carried out according to a conventional method, to obtain a DNA fragment of
about 1.5 kbp.
Based on a gene sequence of gdh (SEQ ID NO:5) encoding a protein (SEQ ID
NO:6) having the amino acid sequence of GDH derived from Bacillus subtilis
10 (GenBank Accession No.NP_388275) with the exception that the 96th amino acid
residue, glutamic acid, is substituted with alanine, primers for amplifying the fulllength
sequence of the gdh gene, gdh_F (SEQ ID NO:13) and gdh_R(SEQ ID
NO:14), were designed and synthesized. PCR was carried out according to a
conventional method, to obtain a DNA fragment of about 0.8 kbp.
15 Based on a gene sequence of kr (SEQ ID NO:7) encoding KR derived from P.
putida (GenBank Accession No. NP_745855, SEQ ID NO:8), primers for amplifying
the full-length sequence of the rk gene, kr_F (SEQ ID NO:15) and kr_R (SEQ ID
NO:16), were designed and synthesized. PCR was carried out using chromosomal
DNA of P. putida as a template according to a conventional method, to obtain a
20 DNA fragment of about 1.2 kbp.
[0060]
(2) Preparation of Expression Plasmid
Each of the DNA fragments obtained in (1) was digested with restriction
enzymes EcoRI and XbaI, and introduced downstream of the trc promoter in a
25 MunI/XbaI digest of a plasmid pKW32, which is described in WO 2012/029819,
using a Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.), to
obtain pKW32dpkA, pKW32aip, pKW32gdh, and pKW32kr, respectively.
33
Subsequently, pKW32aip was digested with SpeI and NdeI to obtain a DNA
fragment of about 2.4 kbp containing aip, and the resulting DNA fragment was
introduced downstream of dpkA in the ring-opened (linear) plasmid of about 4.2 kbp
obtained by digesting pKW32dpkA with XbaI and NdeI, to obtain pKW32 (dpkA,
aip)5 .
pKW32gdh was digested with SpeI and NdeI to obtain a DNA fragment of
about 1.7 kbp containing gdh, and the resulting DNA fragment was introduced
downstream of aip in the ring-opened (linear) plasmid of about 5.7 kbp obtained by
digesting pKW32 (dpkA, aip) with XbaI and NdeI, to obtain pKW32 (dpkA, aip,
10 gdh).
Finally, pKW32kr was digested with SpeI and NdeI to obtain a DNA fragment
of about 2.1 kbp containing kr, and the resulting DNA fragment was introduced
downstream of gdh in the ring-opened (linear) plasmid of about 6.5 kbp obtained by
digesting pKW32 (dpkA, aip, gdh) with XbaI and NdeI, to obtain pKW32 (dpkA, aip,
15 gdh, kr).
[0061]
(3) Preparation of Expressing Strain
Using the plasmid pKW32 (dpkA, aip, gdh, kr) obtained in (2), E. coli JM109
(manufactured by Takara Bio Inc.) was transformed according to a conventional
20 method, to obtain recombinant E. coli JM109/pKW32 (dpkA, aip, gdh, kr).
[0062]
[Reference Example 2]
[Preparation Example of 5-Hydroxy-2-piperidinecarboxylic Acid Reaction Mixture]
25 To a 1-L jar fermenter (manufactured by ABLE Corporation, type BMJ), 45 g
of 5-hydroxylysine hydrochloride (230 mmol, prepared according to a method
described in Bull. Chem. Soc. Jpn., 1962, 35, 2006), 2.27 mL of Adekanol LG-109,
34
and 67.34 g (374 mmol) of glucose were added, and dissolved in water. Thereafter,
20 wt% aqueous sodium hydroxide solution was added dropwise thereto until the pH
became 8. To the resulting liquid, bovine-liver-derived catalase (manufactured by
Wako Pure Chemical Industries, Ltd.) was added at 2000 U/L; NADP+
(manufactured by Oriental Yeast Co., Ltd.) was added at 0.2 mmol/L; and wet cell5 s
of the recombinant E. coli JM109/pKW32 (dpkA, aip, gdh, kr) prepared in Reference
Example 1 were added at 25 g/L; followed by adding water to the resulting mixture
to a liquid volume of 566 mL. The reaction was allowed to proceed at 30°C, a
stirring rate of 500 rpm, and an aeration rate of about 1 L/min for 43 hours. During
10 the reaction, the pH was kept at 8 by adding 20 wt% aqueous sodium hydroxide
solution dropwise. Exactly the same operations were carried out twice thereafter,
and the reaction mixtures obtained by the 3 rounds of operations (using 135 g of 5-
hydroxylysine hydrochloride, 690 mmol) were combined. The pH of the reaction
mixture was adjusted to 2.5 using 6 mol/L sulfuric acid. Thereafter, 20 wt%
15 aqueous sodium hydroxide solution was added dropwise to adjust the pH to 4, and
the resulting liquid was centrifuged at 10,000 rpm for 20 minutes to remove insoluble
impurities. The obtained supernatant was passed through a microfiltration
membrane (manufactured by Asahi Kasei Corporation), and then through an
ultrafiltration membrane (manufactured by Asahi Kasei Corporation), to remove
20 impurities. Analysis of 5-hydroxy-2-piperidinecarboxylic acid contained in the
resulting solution was carried out by HPLC under the conditions of . As
a result, the amount of (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid produced was
43.9g (302 mmol; yield, 44%), and the amount of (2S,5R)-5-hydroxy-2-
piperidinecarboxylic acid produced was 29.7g (205 mmol; yield, 30%).
25
Example 1
[0063]
35
<1-1> Production of Benzyl (1S,4S)-5-Aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate
To1153 g of a reaction mixture containing 20.7 g (143 mmol) of (2S,5S)-5-
hydroxy-2-piperidinecarboxylic acid and 18.0 g (124 mmol) of (2S,5R)-5-hydroxy-2-
piperidinecarboxylic acid [(2S,5S):(2S,5R) = 53.6:46.4 (molar ratio)] obtaine5 d
according to the method of Reference Example 2, 10 mol/L aqueous sodium
hydroxide solution (60 mL) was added to adjust the pH from 3.65 to 10.81. The
resulting liquid was concentrated under reduced pressure, to obtain 422.4 g of a
slurry solution. In each of 2 flasks, 211.2 g of the obtained slurry was placed, and
10 the internal temperature was decreased to 5°C to 7°C, followed by carrying out the
following operations for each flask.
[The following is description of the operations for one of the flasks. Each flask
contained 10.4 g (72 mmol) of (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid and
9.0 g (62 mmol) of (2S,5R)-5-hydroxy-2-piperidinecarboxylic acid.]
15 [0064]
To the flask, 18.8 mL (133 mmol) of benzyloxycarbonyl chloride was added
dropwise, and 13.3 mL (133 mmol) of 10 mol/L aqueous sodium hydroxide solution
was then added to adjust the pH from about 9 to 10, followed by allowing the
reaction to proceed at a temperature within the range of 10°C to 20°C for 10 minutes.
20 To the resulting liquid, 18.8 mL (133 mmol) of benzyloxycarbonyl chloride was
added dropwise, and 13.5 mL (135 mmol) of 10 mol/L aqueous sodium hydroxide
solution was then added to adjust the pH from 9 to 11, followed by allowing the
reaction to proceed at a temperature within the range of 10°C to 20°C for 3 hours.
To the resulting liquid, 19 mL of water was added, and 6.2 mL (44 mmol) of
25 benzyloxycarbonyl chloride was then added dropwise, followed by allowing the
liquid to gradually warm to room temperature. To the resulting liquid, 200 mL of
toluene was added, and 4 mL of 10 mol/L aqueous sodium hydroxide solution (40
36
mmol) was then added to adjust the pH to 11, followed by separating the organic
layer. To the aqueous layer, 100 mL of toluene was added to perform re-extraction,
and the aqueous layer was separated to obtain the organic layer. This organic layer
was combined with the previously obtained organic layer. The resulting mixture
was washed with 10 mL of water, followed by separating the resulting organic layer5 .
From the organic layer obtained from each of the two flasks, the solvent was
removed by distillation under reduced pressure at 35°C, to obtain 64.9 g of a yellow
oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
10 containing 45 wt% benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate (111 mmol; yield, 78% with respect to the (2S,5S)-5-hydroxy-2-
piperidinecarboxylic acid), 15 wt% (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid=benzyloxyformic acid=anhydride (23 mmol; yield, 19%
with respect to the (2S,5R)-5-hydroxy-2-piperidinecarboxylic acid), 36 wt% benzyl
15 alcohol, and 5 wt% toluene. Thus, the conversion of the material 5-hydroxy-2-
piperidinecarboxylic acid, which contained (2S,5S) and (2S:5R) at a ratio of
(2S,5S):(2S:5R) = 53.6:46.4 (molar ratio), to benzyl (1S,4S)-5-aza-2-oxa-3-
oxobicyclo[2.2.2]octane-5-carboxylate allowed effective removal of compounds
having the stereochemistry of (2S,5R), and, by this, the purity of compounds having
20 the stereochemistry of (2S,5S) could be increased to (2S,5S):(2S:5R) = 82.8:17.2
(molar ratio).
[0065]
The resulting crude product was cooled to 10°C to 15°C, and 36 mL of
toluene and 36 mL of hexane were added thereto. As a result, precipitation of white
25 solids occurred. The precipitated white solids were collected by filtration, and
washed by sprinkling 100 mL of hexane thereon. The resulting white solids were
dried under reduced pressure at room temperature, to obtain 20.7 g of white solids.
37
As a result of 1H-NMR analysis, the white solids were found to be a mixture
containing 92.5 wt% benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate (yield, 51% with respect to the (2S,5S)-5-hydroxy-2-piperidinecarboxylic
acid) and 7.5 wt% (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
acid=benzyloxyformic acid=anhydride. By the crystallization, the purity 5 of
compounds having the stereochemistry of (2S,5S) could be increased to
(2S,5S):(2S:5R) = 92.5:7.5 (molar ratio).
1H-NMR (400 MHz, CDCl3)
-1.85 (1H, m), 2.02-2.25 (3H, m), 3.52 (1H, d, J=11.9 Hz), 3.70 (1H, dt,
10 J=12.2, 3.3Hz), 4.70-4.86 (2H, m), 5.12-5.21 (2H, m), 7.32-7.40 (5H, m).
[0066]
<1- Production of Benzyl (1S,4S)-5-Aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate
To 80 g of a reaction mixture containing 1.74 g (12.0 mmol) of (2S,5S)-5-
15 hydroxy-2-piperidinecarboxylic acid and 1.01 g (7.0 mmol) of (2S,5R)-5-hydroxy-2-
piperidinecarboxylic acid [(2S,5S):(2S,5R)=63.2:36.8 (molar ratio)] obtained
according to the method of Reference Example 2, 10 mol/L aqueous sodium
hydroxide solution was added to adjust the pH from 3.65 to 9.8. The resulting
liquid was concentrated under reduced pressure, and 38 g of a solution was obtained.
20 To the solution, 3.1 mL of 2-propanol was added. The internal temperature of the
obtained solution was set to about 25°C. To the solution, 3.2 g (18.8 mmol) of
benzyloxycarbonyl chloride was added dropwise, and 2.3 g (22.6 mmol) of 40 wt%
aqueous sodium hydroxide solution was further added, followed by allowing the
reaction to proceed at a temperature within the range of 20°C to 25°C for 30 minutes.
25 To the resulting liquid, 3.2 g (18.8 mmol) of benzyloxycarbonyl chloride was added
dropwise, and 2.3 g (22.6 mmol) of 40 wt% aqueous sodium hydroxide solution was
further added, followed by allowing the reaction to proceed at a temperature within
38
the range of 20°C to 25°C for 30 minutes. Thereafter, the operation of adding 1.6 g
(9.4 mmol) of benzyloxycarbonyl chloride dropwise to the resulting liquid, adding
1.1 g (11.3 mmol) of 40 wt% aqueous sodium hydroxide solution thereto, and then
allowing the reaction to proceed at a temperature within the range of 20°C to 25°C
for 30 minutes was repeated 3 times. Subsequently, the reaction mixture wa5 s
cooled to 0°C to 5°C, and crystals precipitated in the reaction system were collected
by filtration. The obtained crystals were dried under reduced pressure at 40°C to
45°C to obtain 2 g of white crystals.
As a result of 1H-NMR analysis, the obtained crystals were found to be benzyl
10 (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate (80 wt%; yield, 56%
with respect to the (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid).
[0067]
<1- Production of Benzyl (1S,4S)-5-Aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate
15 To 160 g of a reaction mixture containing 3.26 g (22.5 mmol) of (2S,5S)-5-
hydroxy-2-piperidinecarboxylic acid and 2.24 g (15.4 mmol) of (2S,5R)-5-hydroxy-2-
piperidinecarboxylic acid [(2S,5S):(2S,5R)=59.4:40.6 (molar ratio)] obtained
according to the method of Reference Example 2, 10 mol/L aqueous sodium
hydroxide solution was added to adjust the pH from 3.65 to 9.5. The resulting
20 liquid was concentrated under reduced pressure, and 74 g of a solution was obtained.
To the solution, 6.0 mL of 2-propanol was added. The internal temperature of the
obtained solution was set to about 25°C. To the solution, 6.4 g (37.6 mmol) of
benzyloxycarbonyl chloride was added dropwise, and 4.5 g (45.2 mmol) of 40 wt%
aqueous sodium hydroxide solution was further added, followed by allowing the
25 reaction to proceed at a temperature within the range of 20°C to 25°C for 30 minutes.
To the resulting liquid, 6.4 g (37.6 mmol) of benzyloxycarbonyl chloride was added
dropwise, and 4.5 g (45.2 mmol) of 40 wt% aqueous sodium hydroxide solution was
39
further added, followed by allowing the reaction to proceed at a temperature within
the range of 20°C to 25°C for additional 30 minutes. Thereafter, 3.2 g (18.8 mmol)
of benzyloxycarbonyl chloride was added dropwise to the reaction mixture, and 2.3 g
(22.6 mmol) of 40 wt% aqueous sodium hydroxide solution was further added
dropwise thereto, followed by allowing the reaction to proceed at a temperatur5 e
within the range of 20°C to 25°C for 30 minutes. Thereafter, 1.9 g (11.1 mmol) of
benzyloxycarbonyl chloride was added dropwise to the reaction mixture, and 1.4 g
(14.0 mmol) of 40 wt% aqueous sodium hydroxide solution was added dropwise
thereto, followed by allowing the reaction to proceed at a temperature within the
10 range of 20°C to 25°C for 30 minutes. To the resulting reaction mixture, 71 mL of
toluene was added, and liquid separation was carried out. To the extracted organic
layer, 13 mL of water and 6.2 g of 40 wt% aqueous sodium hydroxide solution were
added, and liquid separation was carried out again. The obtained aqueous layer, in
an amount of 32 g, was divided into two aliquots, and subjected to the purification
15 methods A and B.
[0068]
Purification Method A: Addition of Acetic Anhydride
To 16 g of the aqueous layer obtained in <1-
anhydride was added. The obtained crystals were collected by filtration, and dried
20 under reduced pressure at 40°C to 45°C. The dried crystals appeared white, and
their weight was 1.5 g.
The obtained crystals were analyzed by HPLC under the conditions of
. As a result, the obtained crystals were found to be benzyl (1S,4S)-5-
aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate (100 wt%; yield, 50% with
25 respect to the (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid).
[0069]
Purification Method B: Extraction under Acidic Conditions
40
To 16 g of the aqueous layer obtained in <1- , 30 mL of water was added,
and 5 mol/L hydrochloric acid was added thereto to adjust the pH in the system to
1.07. Thereafter, 55 mL of ethyl acetate was added to the resulting mixture, and
liquid separation was carried out. Subsequently, 55 mL of ethyl acetate was added
to the obtained aqueous layer, and liquid separation was carried out again. T5 he
obtained organic layers were combined, and concentrated under reduced pressure
until the weight decreased to half of the original weight. To the obtained organic
layer, 2.9 g (28.1 mmol) of triethylamine and 2.9 g (28.1 mmmol) of acetic anhydride
were added at 20°C to 25°C. After stirring the resulting mixture for 30 minutes,
10 0.57 g (5.6 mmol) of triethylamine and 0.57 g (5.6 mmmol) of acetic anhydride were
added thereto at 20°C to 25°C. After stirring the resulting reaction mixture for 30
minutes, 20 g of saturated aqueous sodium hydrogen carbonate solution was added
thereto. After liquid separation, 71 mL of toluene was added to the obtained
aqueous layer, and the operation of extraction was carried out again.
15 The obtained organic layers were combined, and concentrated under reduced
pressure. Cooling of the obtained concentrate to 0°C to 5°C caused precipitation of
crystals. The obtained crystals were collected by filtration, and then dried under
reduced pressure at 40°C to 45°C. The dried crystals were white, and their weight
was 2.4 g. The obtained crystals were analyzed by HPLC under the conditions of
20 . As a result, the obtained crystals were found to be benzyl (1S,4S)-5-
aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate (98 wt%; yield, 70% with respect
to the (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid).
[0070]
<1-2> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
25 Acid
To a flask, 20.0 g (92.5 wt%, 71 mmol) of the benzyl (1S,4S)-5-aza-2-oxa-3-
oxobicyclo[2.2.2]octane-5-carboxylate obtained in <1-1> and 60 mL of ethanol were
41
added, and 76.5 mL of 1 mol/L aqueous sodium hydroxide solution was added
thereto at room temperature, followed by stirring the mixture for 1 hour. The
solvent was removed under reduced pressure at 35°C to 40°C, to obtain 61.2 g of a
yellow oily substance. To the resulting substance, 100 mL of ethyl acetate was
added, and the organic layer was separated. To the obtained aqueous layer, 14 m5 L
of 5 mol/L hydrochloric acid was added to adjust the pH to 1.2. The aqueous layer
was subjected to extraction using 200 mL of ethyl acetate twice, and the obtained
organic layer was washed with 2 mL of water. After separation of the organic layer,
the solvent was removed under reduced pressure at 35°C to 40°C, to obtain 24.0 g of
10 a pale yellow oily substance.
As a result of 1H-NMR analysis, this pale yellow oily substance was found to
be (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid (purity, 76
wt%; 65 mmol; yield, 92%). The peak for (2S,5R)-N-benzyloxycarbonyl-5-
hydroxy-2-piperidinecarboxylic acid was not found at all by 1H-NMR.
15 1H-NMR (400 MHz, CDCl3, rotational isomer mixture)
-1.39 (1H, m), 1.70-1.85 (1H, m), 1.98-2.06 (1H,m), 2.27-2.39 (1H, m), 2.80
(0.4H, t, J=11.8 Hz), 2.87 (0.6H, t, J=11.6 Hz), 3.61-3.72 (1H, m) 4.18-4.33 (1H, m),
4.81-4.88 (0.4H, m), 4.93-4.98 (0.6H, m), 5.12-5.21 (2H, m), 7.28-7.40 (5H, m).
[0071]
20 <1-3> Production of (2S,5S)-5-Hydroxy-2-piperidinecarboxylic Acid
To a flask, 18.0 g (64.5 mmol) of the (2S,5S)-N-benzyloxycarbonyl-5-
hydroxy-2-piperidinecarboxylic acid obtained in <1-2>, 90 mL of ethanol, and 2.5 g
of 10% palladium carbon (manufactured by N. E. Chemcat Corporation, PE-type,
55.3%, containing water) were added, and hydrogenation was carried out under
25 normal temperature and pressure. Three hours later, 22 mL of water was added
thereto, and hydrogenation was carried out under normal temperature and pressure.
Disappearance of the starting material could be confirmed 8.5 hours later. The
42
palladium carbon was removed by filtration, and 100 mL of water was sprinkled
thereon for washing. From the obtained filtrate, the solvent was removed under
reduced pressure at 45°C to 55°C, to obtain 11.6 g of a white slurry.
The obtained slurry was analyzed by HPLC under the conditions of . As a result, the slurry was found to contain 5-hydroxy-2-piperidinecarboxyli5 c
acid with a purity of 65 wt% in an amount of 64.5 mmol and yield of 81%. The
isomer ratio was as follows: (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid:(2S,5R)-
5-hydroxy-2-piperidinecarboxylic acid=98.8:1.2. The material used in <1-1> had a
purity of as low as (2S,5S):(2S,5R)=53.7:46.3 (molar ratio), and the purity was
10 increased to (2S,5S):(2S,5R)=98.8:1.2 (molar ratio) by the method of the present
invention.
[0072]
<1-4> Activated Carbon Treatment, and Crystallization
To a flask, 0.38 g of the slurry obtained in <1-3> (purity, 65 wt%; 1.7 mmol),
15 2.5 mL of water, 0.025 g of an activated carbon manufactured by Calgon Mitsubishi
Chemical Corporation, CAL, and 0.025 g of an activated carbon manufactured by
Calgon Mitsubishi Chemical Corporation, 6ED, were added. The resulting mixture
was stirred at room temperature for 1 hour, and the active carbons were then removed
by filtration, followed by concentrating the aqueous solution to 0.5 mL. While the
20 concentrate was stirred, 5.0 mL of ethanol was added dropwise thereto, and the
resulting mixture was cooled to 5°C. As a result, crystals gradually precipitated.
While the temperature was kept at 5°C, the stirring was continued for 1 hour, and 2.0
mL of acetone was added dropwise thereto at the constant temperature of 5°C,
followed by stirring the resulting mixture for additional 0.5 hour. Thereafter, the
25 precipitated crystals were collected by filtration, and 1.0 mL of an ice-cooled
ethanol/acetone solution (=5/2 volume ratio) was sprinkled thereon for washing.
The crystals were then dried under reduced pressure at 60°C, to obtain 0.25 g (purity,
43
98 wt%; yield, 99.2%) of crystals.
The obtained crystals were analyzed by HPLC under the conditions of
. Based on the result, the isomer ratio of 5-hydroxy-2-
piperidinecarboxylic acid contained in the obtained crystals was as follows: (2S,5S)-
5-hydroxy-2-piperidinecarboxylic acid:(2S,5R)-5-hydroxy-2-piperidinecarboxyli5 c
acid=99.5:0.5.
Example 2
[0073]
10 <2-1> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
Acid and (2S,5R)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic Acid
To 124 mL of a reaction mixture containing 0.723 g (4.98 mmol) of (2S,5S)-
5-hydroxy-2-piperidinecarboxylic acid and 0.830 g (5.72 mmol) of (2S,5R)-5-
hydroxy-2-piperidinecarboxylic acid obtained according to the method of Reference
15 Example 2 [(2S,5S):(2S,5R)=46.5:53.5 (molar ratio)], 2.88 mL (36 mmol) of 50 wt%
aqueous sodium hydroxide solution was added, and the resulting mixture was
concentrated at a bath temperature of 40°C under a reduced pressure of 50 hPa, to
obtain 132 g of a solution. In a water bath, 3.1 mL (22 mmol) of benzyloxycarbonyl
chloride and 0.44 mL (5.5 mmol) of 50 wt% aqueous sodium hydroxide solution
20 were dividedly added to the obtained solution while the pH was adjusted to about 9.
After leaving the resulting solution to stand overnight, 60 mL of ethyl acetate and
0.88 mL (11 mmol) of 50 wt% aqueous sodium hydroxide solution were added
thereto, followed by carrying out filtration through Celite. The organic layer was
separated, and the aqueous layer was washed with ethyl acetate. To the aqueous
25 layer, 2.9 mL of concentrated hydrochloric acid (33 mmol) was added to adjust the
pH to 3. Thereafter, extraction with ethyl acetate was carried out 3 times. The
solvent was removed from the resulting organic layer under reduced pressure at 35°C,
44
to obtain 2.44 g of a yellow oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 80 wt% (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid (6.91
mmol; yield, 65%; (2S,5S):(2S,5R)=4:6 (molar ratio)), 12 wt% ethyl acetate, and 5 nd 8
wt% acetic acid.
[0074]
<2-2> Production of Benzyl (2S,5S)-3-Oxo-2-oxa-5-azabicyclo[2.2.2]octane-5-
carboxylate
10 To a flask, 2.35 g of the (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained in <2-1> [6.72 mmol, (2S,5S):(2S,5R)=4:6 (molar
ratio)], 10 mL of toluene, and 116 mg (0.67 mmol) of p-toluenesulfonic acid
monohydrate were added, and the reaction was allowed to proceed at 60°C for 2
15 hours. After cooling the resulting reaction product to room temperature, 30 mL of
ethyl acetate, 5 mL of water, and 7 mL of 1 mol/L sodium hydroxide solution were
added thereto. As a result, the pH of the aqueous layer became 9. After separating
the aqueous layer, re-extraction was carried out with ethyl acetate. The obtained
organic layer was washed with saturated aqueous sodium bicarbonate solution and
20 saturated brine. The resulting organic layer was concentrated to obtain 0.44 g of a
brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 75 wt% benzyl (2S,5S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate (1.27 mmol; yield, 19%), 17 wt% ethyl acetate, and 9 wt% toluene.
25 In the material N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid,
(2S,5S) and (2S,5R) were contained at (2S,5S):(2S,5R)=4:6 (molar ratio). By its
conversion to benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate,
45
compounds having the stereochemistry of (2S,5R) could be effectively removed.
However, since the yield of benzyl (1S,4S)-5-aza-2-oxa-3-
oxobicyclo[2.2.2]octane-5-carboxylate was 19%, the (2S,5S) isomer was obtained in
a moderate yield of about 50%. It has been observed that a side reaction occurred
between benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylat5 e
generated by the reaction and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid left unreacted in the system, to cause esterification. This
is assumed to be the cause of the moderate yield.
[0075]
10 <2-3> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
Acid
To a flask, 0.44 g (purity, 75 wt%; 1.27 mmol) of the benzyl (1S,4S)-5-aza-2-
oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate obtained in <2-2>, 2.6 mL of methanol,
and 2.6 mL of 1 mol/L aqueous sodium hydroxide solution were added, and left to
15 stand overnight. To the resulting reaction mixture, 1.3 mL of 2 mol/L hydrochloric
acid was added to adjust the pH to 4, and extraction with ethyl acetate was then
carried out. The resulting organic layer was concentrated to obtain 0.33 g of a
brown oily substance. The substance was then purified by silica gel
chromatography to obtain 0.25 g of (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
20 piperidinecarboxylic acid (0.88 mmol; yield, 69%).
Example 3
[0076]
<3-1> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
25 Acid and (2S,5R)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic Acid
To a flask, 0.76 g of (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid and
(2S,5R)-5-hydroxy-2-piperidinecarboxylic acid [5.24 mmol, (2S,5S):(2S,5R)=7:3
46
(molar ratio)], and 6.3 mL of 1 mol/L aqueous sodium hydroxide solution were
added, and 0.93 mL (5.3 mmol) of benzyloxycarbonyl chloride was added to the
resulting mixture under ice-cooling. The temperature of the mixture was then
slowly increased to room temperature, and 2 mL of tetrahydrofuran and 5.2 mL of 1
mol/L aqueous sodium hydroxide solution were dividedly added thereto while the 5 pH
was adjusted to about 9. After leaving the resulting mixture to stand overnight, the
organic layer was separated, and the aqueous layer was washed with ethyl acetate.
To the aqueous layer, 3.2 mL of 2 mol/L hydrochloric acid was added to adjust the
pH to 3, and the resulting mixture was extracted 3 times with ethyl acetate. The
10 resulting organic layer was concentrated to obtain 0.79 g of a yellow oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 76 wt% (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid (2.15
mmol; yield, 41%; (2S,5S):(2S,5R)=7:3 (molar ratio)), and 24 wt% ethyl acetate.
15 [0077]
<3-2> Production of Benzyl (2S,5S)-5-Aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate
To a flask, 0.79 g of the (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
20 piperidinecarboxylic acid obtained in <3-1> (purity, 76 wt%; 2.15 mmol;
(2S,5S):(2S,5R)=7:3 (molar ratio)), 5 mL of toluene, and 49 mg (0.28 mmol) of ptoluenesulfonic
acid monohydrate were added, and the reaction was allowed to
proceed at 60°C for 2 hours. After cooling the resulting reaction product to room
temperature, 10 mL of ethyl acetate, 3 mL of water, and 1.2 mL of 1 mol/L sodium
25 hydroxide solution were added thereto. As a result, the pH of the aqueous layer
became 7. After separating the aqueous layer, re-extraction was carried out with
ethyl acetate. The obtained organic layer was washed with saturated aqueous
47
sodium bicarbonate solution and saturated brine. The resulting organic layer was
concentrated to obtain 0.34 g of a brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 62 wt% benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate (0.80 mmol; yield, 37%), 21 wt% (2S,5S)-N-benzyloxycarbonyl-5 5-
hydroxy-2-piperidinecarboxylic acid (0.25 mmol; yield, 12%), and 17 wt% toluene.
In the material N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid,
(2S,5S) and (2S,5R) were contained at (2S,5S):(2S,5R)=7:3 (molar ratio). By its
conversion to benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate,
10 compounds having the stereochemistry of (2S,5R) could be effectively removed.
The total yield of benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate and (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid
was 49%, and the (2S,5S) isomer was obtained in a moderate yield of about 70%.
Similarly to <2-1>, a side reaction occurred between benzyl (1S,4S)-5-aza-2-
15 oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate generated by the reaction and (2S,5R)-
N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid left unreacted in the
system, to cause esterification. However, since the amount of (2S,5R)-Nbenzyloxycarbonyl-
5-hydroxy-2-piperidinecarboxylic acid in the starting material
was small, the (2S,5S) isomer could be selectively, and relatively efficiently
20 recovered.
[0078]
<3-3> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
Acid
To a flask, 0.34 g of the oily substance obtained in <3-2> [containing 0.80
25 mmol benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate and 0.25
mmol (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid], 2 mL of
methanol, and 1.9 mL of 1 mol/L aqueous sodium hydroxide solution were added,
48
and left to stand overnight. To the resulting reaction mixture, 2 mL of 2 mol/L
hydrochloric acid was added to adjust the pH to 4, and extraction with ethyl acetate
was then carried out. The resulting organic layer was concentrated to obtain 0.39 g
of a brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixtur5 e
containing 76 wt% (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
acid (1.05 mmol; yield, 100%; (2S,5S):(2S,5R)=87:13 (molar ratio)), 23 wt% ethyl
acetate, and 1 wt% acetic acid.
[0079]
10 <3-4> Production of (2S,5S)-5-Hydroxy-2-piperidinecarboxylic acid
To a flask, 0.39g of the (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained in <3-3> (purity, 77 wt%; 1.06 mmol), 2 mL of
methanol, and 61 mg of 10% palladium carbon (manufactured by N. E. Chemcat
Corporation, PE-type, 55.3%, containing water) were added, and hydrogenation was
15 carried out under normal temperature and pressure for 3 hours. The palladium
carbon was removed by filtration through Celite, and washing with methanol-water
was carried. The obtained filtrate was concentrated to obtain 0.21 g of (2S,5S)-5-
hydroxy-2-piperidinecarboxylic acid as a pale yellow oily substance.
Example 4
20 [0080]
<4-1> Production of Benzyl (1S,4S)-5-Aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
carboxylate and Ethyl (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylate
To a flask, 0.26 g of (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
25 piperidinecarboxylic acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained according to the method of <2-1> (purity, 82
wt%; 0.76 mmol; (2S,5S):(2S,5R)=5:5 (molar ratio)), 1 mL of ethyl acetate, and 5 L
49
(0.08 mmol) of methanesulfonic acid were added, and the reaction was allowed to
proceed at 60°C for 3 hours. After cooling the resulting reaction product to room
temperature, 10 mL of ethyl acetate and 1 mL of 1 mol/L sodium hydroxide solution
were added thereto. As a result, the pH of the aqueous layer became about 10.
After separating the aqueous layer, re-extraction was carried out with ethyl acetate5 .
The obtained organic layer was dried over magnesium sulfate. The resulting
organic layer was concentrated to obtain 0.12 g of a pale brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 26 wt% benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-
10 carboxylate (0.11 mmol; yield, 15%), 52 wt% ethyl (2S,5S)-N-benzyloxycarbonyl-5-
hydroxy-2-piperidinecarboxylic acid (0.20 mmol; yield, 26%), 13 wt% ethyl acetate,
and 8 wt% toluene.
It is thought that, in the present Example, a small amount of ethanol generated
from ethyl acetate reacted with benzyl (1S,4S)-5-aza-2-oxa-3-
15 oxobicyclo[2.2.2]octane-5-carboxylate, to generate ethyl (2S,5S)-Nbenzyloxycarbonyl-
5-hydroxy-2-piperidinecarboxylate. In the material Nbenzyloxycarbonyl-
5-hydroxy-2-piperidinecarboxylic acid, (2S,5S) and (2S,5R) were
contained at (2S,5S):(2S,5R)=5:5 (molar ratio). As a result of 1H-NMR analysis and
HPLC analysis (under the conditions of ), it could be confirmed that the
20 isomer ratio of the ethyl N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylate
was as follows: (2S,5S):(2S,5R)>10:1 (molar ratio). Thus, compounds having the
stereochemistry of (2S,5R) could be effectively removed. Since the total yield of
benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate and ethyl
(2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylate was 41%, the yield
25 of the (2S,5S) isomer was about 80%. Thus, the (2S,5S) isomer could be selectively
and efficiently recovered.
[0081]
50
<4-2> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
Acid
To a flask, 0.12 g of the oily substance obtained in <4-1> (containing 0.11
mmol of benzyl (1S,4S)-5-aza-2-oxa-3-oxobicyclo[2.2.2]octane-5-carboxylate and
0.20 mmol of ethyl (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-5 2-
piperidinecarboxylate), 1 mL of ethanol, and 0.66 mL of 1 mol/L aqueous sodium
hydroxide solution were added, and left to stand overnight. The reaction mixture
was then concentrated, and 0.7 mL of 1 mol/L hydrochloric acid was added thereto to
adjust the pH to 3, followed by carrying out extraction with ethyl acetate. The
10 resulting organic layer was concentrated under reduced pressure to obtain 0.11 g of a
brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 84 wt% (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
acid (0.33 mmol, quantitative, (2S,5S):(2S,5R)=10:1 (molar ratio)), 11 wt% ethyl
15 acetate, and 5 wt% toluene.
[0082]
<4-3> Production of (2S,5S)-5-Hydroxy-2-piperidinecarboxylic Acid
To a flask, 0.11 g of the (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained in <4-2> (purity, 82 wt%, 0.33 mmol), 1 mL of
20 methanol, and 18 mg of 10% palladium carbon (manufactured by N. E. Chemcat
Corporation, PE-type, 55.3%, containing water) were added, and hydrogenation was
carried out under normal temperature and pressure for 9 hours. The palladium
carbon was removed by filtration through Celite, and washing with methanol-water
was carried. The obtained filtrate was concentrated to obtain 58 mg of (2S,5S)-5-
25 hydroxy-2-piperidinecarboxylic acid as a pale yellow oily substance
((2S,5S):(2S,5R)=10:1 (molar ratio), 1H-NMR analysis).
51
Example 5
[0083]
<5-1> Production of Ethyl (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylate
To a flask, 0.55 g of (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-5 2-
piperidinecarboxylic acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained according to the method of <2-1> (purity, 78
wt%; 1.52 mmol; (2S,5S):(2S,5R)=3:7 (molar ratio)), 2 mL of toluene, 0.5 mL of
L (0.15 mmol) of methanesulfonic acid were added, and the
10 reaction was allowed to proceed at 40°C for 5 hours.
The reaction mixture was analyzed by HPLC under the conditions of , and the following result was obtained: ethyl (2S,5S)-N-benzyloxycarbonyl-5-
hydroxy-2-piperidinecarboxylate:ethyl (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylate:(2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
15 piperidinecarboxylic acid:(2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid=24:8:7:57.
Under the assumption that the material and products have the same absorption
coefficient, the degree of conversion of the (2S,5S) isomers was about 77%, and the
degree of conversion of the (2S,5R) isomers was about 12%. Thus, the (2S,5S)
20 isomers showed a higher degree of esterification.
[0084]
<5- S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylate
To a flask, 0.62 g of (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
25 piperidinecarboxylic acid and (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained according to the method of <2-1> (purity, 78
wt%; 1.72 mmol; (2S,5S):(2S,5R)=3:7 (molar ratio)), 2 mL of toluene, 0.5 mL of 2-
52
propanol, and 11 added, and the
reaction was allowed to proceed at 50°C for 5 hours. After cooling the resulting
reaction product to room temperature, 3 mL of toluene and 1.2 mL of 1 mol/L
sodium hydroxide solution were added thereto. As a result, the pH of the aqueous
layer became about 9. After separating the aqueous layer, re-extraction was carrie5 d
out with toluene. The resulting organic layer was concentrated to obtain 0.17 g of a
pale brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 91 wt% isopropyl N-benzyloxycarbonyl-5-hydroxy-2-
10 piperidinecarboxylate (0.47 mmol; yield, 27%; (2S,5S):(2S,5R)=10:1 (molar ratio)),
and 9 wt% toluene.
In the present Example, the material N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid contained (2S,5S) and (2S,5R) at (2S,5S):(2S,5R)=3:7
(molar ratio). Based on the result of 1H-NMR analysis, the isomer ratio of the
15 isopropyl N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylate was follows:
(2S,5S):(2S,5R)=10:1 (molar ratio). Thus, compounds having the stereochemistry
of (2S,5R) could be effectively removed. Since the yield of the isopropyl (2S,5S)-Nbenzyloxycarbonyl-
5-hydroxy-2-piperidinecarboxylate was 27%, the yield of the
(2S,5S) isomer was about 80%. Thus, the (2S,5S) isomer could be selectively and
20 efficiently recovered.
[0085]
<5-2> Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
Acid
To a flask, 0.17 g of the isopropyl (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
25 piperidinecarboxylate obtained in <5-
methanol, and 0.71 mL of 1 mol/L aqueous sodium hydroxide solution were added,
and the reaction was allowed to proceed at 60°C for 4 hours. After washing the
53
reaction mixture with toluene, 0.45 mL of 2 mol/L hydrochloric acid was added
thereto to adjust the pH to 3, and extraction with ethyl acetate was carried out. The
resulting organic layer was concentrated to obtain 0.17 g of a brown oily substance.
As a result of 1H-NMR analysis, this oily substance was found to be a mixture
containing 70 wt% N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic aci5 d
(0.41 mmol; yield, 88%; (2S,5S):(2S,5R)=7:1 (molar ratio)), 26 wt% ethyl acetate,
and 4 wt% toluene.
[0086]
<5-3> Production of (2S,5S)-5-Hydroxy-2-piperidinecarboxylic Acid
10 To a flask, 0.17 g of the (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-
piperidinecarboxylic acid obtained in <5-2> (purity, 70 wt%; 0.41 mmol;
(2S,5S):(2S,5R)=7:1 (molar ratio)), 1 mL of methanol, and 22 mg of 10% palladium
carbon (manufactured by N. E. Chemcat Corporation, PE-type, 55.3%, containing
water) were added, and hydrogenation was carried out under normal temperature and
15 pressure for 3 hours. The palladium carbon was removed by filtration through
Celite, and washing with methanol-water was carried. The obtained filtrate was
concentrated to obtain 89 mg of (2S,5S)-5-hydroxy-2-piperidinecarboxylic acid as a
pale yellow oily substance.
20 [0087]
[Reference Example 3]
<3-1> Cloning of Lysine Hydroxylase Gene
Based on a gene sequence (hyl-1, SEQ ID NO:17) encoding an L-argininehydroxylase
VioC homologue Hyl-1 derived from the Flavobacterium johnsoniae
25 NBRC14942 strain (GenBank Accession No. ABQ06186, SEQ ID NO:18), primers
for amplifying the full-length sequence of the hyl-1 gene, hyl1_F (SEQ ID NO:29)
and hyl1_R (SEQ ID NO:30), were designed and synthesized. Using chromosomal
54
DNA of Flavobacterium johnsoniae as a template, PCR was carried out according to
a conventional method, to obtain a DNA fragment of about 1.0 kbp.
In addition, VioC homologues derived from the Kineococcus radiotolerans
NBRC101839 strain, Chitinophaga pinensis NBRC15968 strain, Chryseobacterium
gleum NBRC15054 strain, and Niastella koreensis NBRC106392 strain wer5 e
designated Hyl-2 (GenBank Accession No.ABS05421, SEQ ID NO:20), Hyl-3
(GenBank Accession No.ACU60313, SEQ ID NO:22), Hyl-4 (GenBank Accession
No.EFK34737, SEQ ID NO:24), and Hyl-5 (GenBank Accession No.AEV99100,
SEQ ID NO:26), respectively. Based on gene sequences encoding the enzymes,
10 (hyl-2 (SEQ ID NO:19), hyl-3 (SEQ ID NO:21), hyl-4 (SEQ ID NO:23), and hyl-5
(SEQ ID NO:25)), primers for amplifying the full-length sequence of each gene were
designed and synthesized. Primers hyl2_f (SEQ ID NO:31) and hyl2_r (SEQ ID
NO:32) for hyl-2, primers hyl3_f (SEQ ID NO:33) and hyl3_r (SEQ ID NO:34) for
hyl-3, primers hyl4_f (SEQ ID NO:35) and hyl4_r (SEQ ID NO:36) for hyl-4, and
15 primers hyl5_f (SEQ ID NO:37) and hyl5_r (SEQ ID NO:38) for hyl-5 were
synthesized, and PCR was carried out using chromosomal DNA of each strain
according to a conventional method. Each reaction produced a DNA fragment of
about 1.0 kbp.
Each of the 5 kinds of DNA fragments obtained was digested with restriction
20 enzymes NdeI and XhoI, and ligated into NdeI/XhoI-digested pET21a (Novagen)
according to a conventional method, to obtain pEHYL1, pEHYL2, pEHYL3,
pEHYL4, and pEHYL5, respectively.
A gene sequence (hyl-6, SEQ ID NO:27) encoding Hyl-6 (GenBank
Accession No. ABS05421, SEQ ID NO:28), which was derived from a marine
25 actinobacterium PHSC20C1, was artificially synthesized, and inserted into
pJExpress401 (DNA2.0) to prepare a plasmid pJHYL6.
Subsequently, E. coli (Eschelichia coli) BL21(DE3) (manufactured by
55
Invitrogen) was transformed with each of the resulting plasmids according to a
conventional method, to obtain recombinant E. coli BL21(DE3)/pEHYL1,
BL21(DE3)/pEHYL2, BL21(DE3)/pEHYL3, BL21(DE3)/pEHYL4,
BL21(DE3)/pEHYL5, and BL21(DE3)/pJHYL6. In order to obtain bacterial cells
expressing the genes, each type of recombinant E. coli was cultured at 30°C usi5 ng
liquid LB medium supplemented with ampicillin and a lac promoter inducer, and
collected after 20 hours of the culture.
[0088]
<3-2> Confirmation of Lysine Hydroxylase Activity by Resting-cell Reaction
10 In a plastic tube, 5 mmol/L L-lysine, 10 mmol/L 2-oxoglutaric acid, 1 mmol/L
L-ascorbic acid, 0.1 mmol/L iron sulfate, and recombinant E. coli obtained by the
method according to Reference Example <3-1> were mixed to provide a reaction
mixture such that the turbidity OD600 was 10. The reaction was allowed to proceed
in 0.5 mL of the prepared mixture at 30°C at pH 7.0 for 3 hours. The reaction
15 product was derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA),
and hydroxylysine was analyzed by HPLC under the conditions of . As a
result, as shown in Fig. 2 and Fig. 3, it could be confirmed that BL21(DE3)/pEHYL2
and BL21(DE3)/pJHYL6 produced a compound corresponding to the retention time
of a standard product of 3-hydroxylysine, 8.04 minutes. It could also be confirmed
20 that BL21(DE3)/pEHYL1, BL21(DE3)/pEHYL3, BL21(DE3)/pEHYL4, and
BL21(DE3)/pEHYL5 produced a compound corresponding to the retention time of a
standard product of 4-hydroxylysine, 8.16 minutes.
[0089]
<3-3> Synthesis of (2S,3S)-3-Hydroxylysine
25 To a 1-L jar fermenter, 35 mL of 1 mol/L potassium phosphate buffer (pH
7.0), 304 mL of desalted water, 1.28 g of L-lysine hydrochloride, 2.05 g of 2-
oxoglutaric acid, 0.14 g of sodium L-ascorbate, 0.02 g of iron sulfate, 0.35 g of
56
Adekanol LG109, and 8 g of wet cells of recombinant E. coli BL21(DE3)/pEHYL2
obtained by the method according to Reference Example <3-1> were mixed together,
and the reaction was allowed to proceed at 30°C, pH 7.0, a stirring rate of 500 rpm,
and an aeration rate of 2.0 vvm for 17 hours. Completion of the reaction was
judged by confirming disappearance of the peak for L-lysine by carrying out HPL5 C
analysis under the conditions of . From the liquid after the completion
of the reaction, bacterial cells and bacterial debris were removed by centrifugation
and microfiltration, to obtain 390 g of a filtrate.
After passing 390 g of the filtrate through an ion-exchange resin column
10 (DIAION (registered trademark) SK-1B (Type H), 60.0 g), washing was carried out
with water, followed by elution with an aqueous solution containing 150 mmol of
ammonia. The ammonia eluate was concentrated to obtain 1.0 g of (2S,3S)-3-
hydroxylysine (6.17 mmol; yield, 88%).
1H-NMR (400MHz, D2 -1.58 (2H, m), 1.63-1.73 (1H, m), 1.74-1.88 (1H,
15 m), 2.93-3.04 (2H, m), 3.47 (1H, d, J=4.3 Hz), 3.89 (1H, dt, J=8.4, 4.5 Hz)
[0090]
<3-4> Determination of Stereochemistry of (2S,3S)-3-Hydroxylysine
To a flask, 8.3 mg (0.051 mmol) of (2S,3S)-3-hydroxylysine obtained by the
method according to Reference Example <3-3>, 0.26 ml of 1 mol/L aqueous sodium
20 hydroxide solu L (0.13 mmol) of benzyloxycarbonyl chloride were
added, and the resulting mixture was stirred at room temperature for 1 hour. To the
mixture, 0.26 mL L (0.13
mmol) of benzyloxycarbonyl chloride were further added, and the reaction was
allowed to proceed overnight at room temperature. Subsequently, 0.5 mL of
25 tetrahydrofuran was added to the reaction mixture, and the reaction was allowed to
proceed at 60°C for additional 2 hours. After cooling the reaction mixture to room
temperature, 95 mg of sodium hydroxide was added thereto, and the reaction was
57
allowed to proceed overnight at room temperature. The reaction mixture was
washed twice with toluene- L of concentrated
hydrochloric acid was added to the reaction mixture to make the reaction mixture
strongly acidic. After 3 times of washing with ethyl acetate, the aqueous layer was
extracted 4 times with 1-butanol. The 1-butanol layer was dried over anhydr5 ous
magnesium sulfate, and then concentrated, to obtain 14.6 mg of (4S,5S)-5-(3-
benzyloxycarbonylaminopropyl)-2-oxo-4-oxazolidinecarboxylic acid (0.045 mmol;
yield, 89%).
The stereochemistry of the obtained (4S,5S)-5-(3-
10 benzyloxycarbonylaminopropyl)-2-oxo-4-oxazolidinecarboxylic acid was determined
by NOESY measurement. The NOESY measurement was carried out using an
AVANCE DRX500 spectrometer manufactured by Bruker (equipped with a
CryoProbe) at 25°C with a mixing time of 0.8 msec. As a chemical shift reference,
3.31 ppm for methanol was used. The result of NOESY measurement was as
15 follows.
[0091]
[0092]
Since a cross peak was found between the 3-position hydrogen atom (H4) and
20 4-position hydrogen atom (H5), but was not found between the 4- -
position, the substituents at the 4-position and the 5-position could be confirmed to
have the cis configuration. Since the absolute configuration of the lysine used in the
enzymatic reaction was S, it could be confirmed that the 5-(3-
benzyloxycarbonylaminopropyl)-2-oxo-4-oxazolidinecarboxylic acid obtained in the
58
present Reference Example has the stereochemistry of (4S,5S), and that 3-
hydroxylysine as its material has the stereochemistry of (2S,3S).
1H-NMR (400 MHz, MeOH-d4 -1.53 (3H, m, H ,H x2), 1.59-1.68 (1H, m,
H ), 3.02-3.08 (2H, m, H ), 3.60-3.64 (1H, m, H4), 3.93-4.00 (1H, m, H5), 4.90-5.02
(2H, m, Bn), 7.18-7.28 (5H, m, Bn)5 .
[0093]
[Reference Example 4]
Production of (2S,3S)-3-Hydroxypipecolic Acid [(2S,3S)-3-Hydroxy-2-
10 piperidinecarboxylic Acid]
In a plastic tube, 0.75 mL of 1 mol/L tris hydroxymethyl aminomethane
buffer (pH 8.0), 9.21 mL of desalted water, 86 mg of the (2S,3S)-3-hydroxylysine
obtained in Reference Example <3-3>, 0.083 mL of 50 mmol/L NADPH, 0.7 mL of
1.0 mol/L glucose, and 1.25 mL of a 100-g/L suspension of the recombinant E. coli
15 JM109/pKW32 (dpkA, aip, gdh, kr) obtained in Reference Example 1 were mixed
together, and the reaction was allowed to proceed at 30°C at pH 8.0 at a stirring rate
of 1000 rpm for 20 hours. Completion of the reaction was judged by confirming
disappearance of the peak for (2S,3S)-3-hydroxylysine by HPLC analysis under the
conditions of . From the liquid after the completion of the reaction,
20 bacterial cells and bacterial debris were removed by centrifugation, to obtain 10.5 g
of a supernatant.
After passing 10.5 g of the supernatant through an ion-exchange resin column
(DIAION (registered trademark) SK-1B (Type H), 4.0g), washing was carried out
with water, followed by elution with an aqueous solution containing 16.4 mmol of
25 ammonia. The ammonia eluate was concentrated to obtain 255 mg of a solid brown
substance. As a result of NMR analysis, this solid substance was found to be a
mixture containing 20 wt% (2S,3R)-3-hydroxypipecolic acid (0.35 mmol; yield,
59
66.3%) and 80 wt% tris hydroxymethyl aminomethane.
1H-NMR (400 MHz, D2 -1.56 (2H, m), 1.73-1.85 (2H, m), 2.71-2.79 (1H,
m), 3.04-3.11 (1H, m), 3.23 (1H, d, J=7.6Hz), 3.79-3.86 (1H, m).
Example 5 6
[0094]
Production of (2S,5S)-N-Benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic Acid
To a flask, 78.7 mg (0.542 mmol) of (2S,5S)-5-hydroxy-2-
piperidinecarboxylic acid and 2.8 mg (0.019 mmol) of (2S,5R)-5-hydroxy-2-
10 piperidinecarboxylic acid obtained according to the method of Example 1, and 98.5
mg of (2S,3S)-3-hydroxy-2-piperidinecarboxylic acid obtained according to the
method of Reference Example 4 (purity, 20 wt%, 0.136 mmol; containing 78.8 mg of
tris hydroxymethyl aminomethane) were added, and 0.200 mL of water and 0.674 mL
of 2 mol/L aqueous sodium hydroxide solution were added thereto at room
15 temperature. After adding 0.190 mL (1.35 mmol) of benzyloxycarbonyl chloride
dropwise to the resulting mixture, 0.500 mL of 2 mol/L aqueous sodium hydroxide
solution was added thereto to adjust the pH from 9 to 10. The reaction was allowed
to proceed at room temperature for 25 minutes, and 0.098 mL (0.697 mmol) of
benzyloxycarbonyl chloride was then added dropwise to the resulting reaction
20 mixture, followed by adding 0.400 mL of 2 mol/L aqueous sodium hydroxide
solution thereto to adjust the pH from 10 to 11. The reaction was further allowed to
proceed at room temperature for 40 minutes, and 0.098 mL (0.697 mmol) of
benzyloxycarbonyl chloride was added dropwise thereto, followed by allowing the
reaction to proceed at room temperature for 2 hours. To the resulting reaction
25 mixture, 3 mL of toluene was added, and liquid separation was then carried out. To
the extracted organic layer, 0.500 mL of water was added, and liquid separation was
carried out again. The resulting organic layer was concentrated to obtain 330 mg of
60
a pale yellow oily substance. To this oily substance, 2 mL of ethanol, 1.1 mL of 1
mol/L aqueous sodium hydroxide solution, and 0.3 mL of water were added, and the
reaction was allowed to proceed at room temperature for about 1 hour. The
resulting reaction mixture was concentrated, and ethanol was evaporated.
Thereafter, 1 mL of ethanol, 0.3 mL of water, 0.1 mL of 1 mol/L aqueous sodi5 um
hydroxide solution, and 0.1 mL of 2 mol/L aqueous sodium hydroxide solution were
added thereto, and the reaction was allowed to proceed at room temperature for 2
hours. The reaction mixture was concentrated, and ethanol was evaporated,
followed by carrying out liquid separation using 3 mL of toluene. To the obtained
10 aqueous layer, 1 mL of water and 0.75 mL of 1 mol/L hydrochloric acid were added
to adjust the pH of the aqueous solution to 1. The aqueous layer was subjected
twice to extraction with 4 mL of ethyl acetate, and the resulting organic layer was
concentrated to obtain 92.0 mg of a pale yellow oily substance.
As a result of 1H-NMR analysis, this pale yellow oily substance was found to
15 contain 80.5 wt% (2S,5S)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic
acid (0.265 mmol; yield, 49%) and 19.5 wt% ethyl acetate. No peak was found at
all for either (2S,5R)-N-benzyloxycarbonyl-5-hydroxy-2-piperidinecarboxylic acid or
(2S,3S)-N-benzyloxycarbonyl-3-hydroxy-2-piperidinecarboxylic acid by 1H-NMR.
20 INDUSTRIAL APPLICABILITY
[0095]
The present invention can be used as a method for purifying cis-5-hydroxy-2-
piperidinecarboxylic acid, which is useful as an intermediate for pharmaceuticals,
and can also be used as a method for producing its derivatives.

CLAIMS
1. A method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative, said method comprising a step of converting cis-5-hydroxy-2-
piperidinecarboxylic acid into a compound(s) of Formula (1) and/or Formula (5 2)
(wherein R1 represents a protective group for an amino group, and R2 represents a
C1-C6 alkyl group).
2. The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
10 derivative according to claim 1, comprising a step of reacting cis-5-hydroxy-2-
piperidinecarboxylic acid with an acid halide and/or acid anhydride to convert said
cis-5-hydroxy-2-piperidinecarboxylic acid into a compound of Formula (1).
3. The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative according to claim 1, comprising a step of reacting cis-5-hydroxy-2-
15 piperidinecarboxylic acid with an acid halide and/or acid anhydride, and then with an
alcohol in the presence of an acid catalyst, to convert said cis-5-hydroxy-2-
piperidinecarboxylic acid into a compound of Formula (2).
4. A method for regenerating cis-5-hydroxy-2-piperidinecarboxylic acid, said
method comprising the steps of:
20 converting cis-5-hydroxy-2-piperidinecarboxylic acid into a compound(s) of
Formula (1) and/or Formula (2) (wherein R1 represents a protective group for an
amino group, and R2 represents a C1-C6 alkyl group); and
converting the compound(s) of Formula (1) and/or Formula (2) into cis-5-
hydroxy-2-piperidinecarboxylic acid.
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5. The method for producing a cis-5-hydroxy-2-piperidinecarboxylic acid
derivative according to any one of claims 1 to 3, wherein said cis-5-hydroxy-2-
piperidinecarboxylic acid is cis-5-hydroxy-2-piperidinecarboxylic acid synthesized
by bacterial reaction and/or enzymatic reacti5 on.
6. The method for regenerating cis-5-hydroxy-2-piperidinecarboxylic acid
according to claim 4, wherein said cis-5-hydroxy-2-piperidinecarboxylic acid is cis-
5-hydroxy-2-piperidinecarboxylic acid synthesized by bacterial reaction and/or
enzymatic reaction.
10 7. A method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid, said
method comprising a step of reacting a mixture containing cis-5-hydroxy-2-
piperidinecarboxylic acid and an impurity with an acid halide and/or acid anhydride,
or with an acid halide and/or acid anhydride and an alcohol, to convert the cis-5-
hydroxy-2-piperidinecarboxylic acid into a compound(s) of Formula (1) and/or
15 Formula (2), separating said compound(s) and then converting the separated
compound(s) into cis-5-hydroxy-2-piperidinecarboxylic acid.
8. The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to claim 7, wherein said impurity is 2-piperidinecarboxylic acid or an
20 analogue thereof.
9. The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to claim 8, wherein said 2-piperidinecarboxylic acid or an analogue thereof
is trans-5-hydroxy-2-piperidinecarboxylic acid.
10. The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to any one of claims 7 to 9, wherein said step of separating the
5 compound(s) of Formula (I) and/or Formula (2) is carried out by crystallization or
solvent extraction.
1 1. The method for purifying cis-5-hydroxy-2-piperidinecarboxylic acid
according to any one of claims 7 to 10, wherein said mixture containing cis-5-
hydroxy-2-piperidinecarboxylic acid and an impurity is a mixture synthesized by
10 bacterial reaction and/or enzymatic reaction.