Note: Descriptions are shown in the official language in which they were submitted.
CA 02459246 2004-03-02
1
ENZYMATIC PROCESS FOR THE ENANTIOMERIC RESOLUTION OF
AMINO ACIDS
The present invention relates to a novel
enzymatic process permitting the enantiomeric
resolution of amino acids in the form of the racemic
mixture.
Amino acids are often used in all kinds of
industries either, for example, as biologically active
compounds or as synthesis intermediates for the
preparation of compounds for pharmaceutical, chemical
or agricultural purposes in particular. Accordingly, it
became evident very quickly that it was often necessary
to be able to have available one or the other optically
active enantiomer of these amino acids. Numerous routes
for separating the enantiomers of pro-chiral amino
acids were therefore developed. In particular,
enzymatic processes permitting their enantiomeric
resolution were found to be an advantageous alternative
to the asymmetric synthesis approaches.
Thus it was that Soloshonok et al.
(Tetrahedron: Assymetry, Vol. 6(7), 1995, pp. 1601-
1610) employed an enzymatic process for enantiomeric
resolution of ~-amino acids in accordance with the
following reaction scheme:
CA 02459246 2004-03-02
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R'~'COOH
R waterlacetone (111 )
~COOH triethylamine
NHZ phenylacetyl chloride
-5°C; 2 hours O
Penicillin acylase
22-25°C, pH = 7.5
R
~COOH R
~COOH
HN
NH
2
O
Similarly, Topgi et al. (Bioorg. & Med. Chem.
1999, Vol. 7, pp. 2221-2229) elaborated a process for
resolving the (R) and (S) enantiomers of ethyl 3-amino-
4-pentynoate in enantiomerically pure form in
accordance with one of the following reaction schemes:
O
O O~'
H
R
R=TMS or H
Penicillin G amidohydrolase
phosphate buffer
O O
p O'~ ..
+ phenylacetic acid
i
H \ R HzN
R
(S) isomer (R) isomer
CA 02459246 2004-03-02
3
the initial phenylacetamide being obtained by acylating
the corresponding amine by reaction with phenylacetic
acid, or else:
U
O~
amine in racemic form
HzN
SiMej
Penicillin G amidohydrolase
phenylacetic acid
O O
O ~O"~ O~
H \ HzN
SiMe3 SiMe3
(R) isomer (S) isomer
The patent application WO 98/50575 describes
in more general terms a method of preparing a chiral
(3-amino acid, which comprises contacting a racemic
mixture of the said amino acid with an aryl donor and
the enzyme Penicillin G acylase (or amidohydrolase)
under conditions appropriate for stereoselectively
acylating one of the enantiomers of the racemic mixture
of the (3-amino acid to its corresponding N-acylated
derivative, the opposite enantiomer of the (3-amino acid
being obtained in an enantiomerically enriched form, in
accordance with the fol-lowing reaction scheme:
CA 02459246 2004-03-02
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O O
acyl donor
organic or aqueous solvents O
OR2 Rz
penicillin G acylase
HZN R~ H2N R~
O
Rz
O ~0
~N~R
H
the "acyl donor" in question being of general formula:
~3 _~4
in which R3 is selected from phenyl, phenoxy, amino,
various derivatives of phenyl, and pyridyl, and R4 is
selected from the substituents hydroxyl, alkoxy, alkyl,
alkenyl, alkynyl, haloalkyl, anyl, anylalkyl, from
sugars or steroids.
The patent application WO 98/50575 also
describes another alternative for preparing a chiral
(3-amino acid, which comprises contacting an amide in
racemic form with the enzyme Penicillin G acylase under
conditions appropriate for selectively deacylating one
of the enantiomers of the amide in racemic form to its
corresponding (3-amino acid, the opposite enantiomer of
the amide being obtained in an enantiomerically
CA 02459246 2004-03-02
enriched form, in accordance with the following
reaction scheme:
O
organic or aqueous solvents
R2
R penicillin G acylase
HzN~R~
O
O -0R2
-I-
R~N R
H
5
The processes discussed above, however,
exhibit the major drawback of proceeding, prior to or
simultaneously with the enzymatic step as such, via an
intermediate amide whose formula may be summarized as
follows:
OR'
aromatic nucleus,
preferably phenyl
Such an amide is insoluble in an aqueous
medium owing to the present of the aromatic nucleus,
which has drawbacks. For example, it is known that
certain enzymes are soluble in an aqueous medium and
CA 02459246 2004-03-02
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are often sensitive to the presence of organic
solvents. However, in order to be able to optimize the
yield of the enzymatic reaction, it is important to
have good solubility of the substrate relative to the
enzyme, in order that they may be in intimate contact.
It is primarily this drawback which the
present invention proposes to resolve. In effect, the
present invention relates, according to a first aspect,
to a novel process for separating enantiomers of an
amino acid, which consists in treating a racemic
mixture of the said amino acid with glutaric anhydride
and then with the enzyme glutaryl-7-ACA acylase so as
to recover one of the enantiomers of the said amino
acid, the other enantiomer remaining in the form of the
corresponding glutarylamide derivative.
This process is particularly advantageous
because the use of glutaric anhydride makes it possible
to proceed intermediately via a glutarylamide
derivative corresponding to the initial amino acid, the
glutaryl function conferring on the molecule its
solubility in an aqueous medium. Consequently, the
process of the present invention may be employed under
gentle reaction conditions and in an aqueous medium;
that is, in particular, without the use of an organic
cosolvent. -
The process of the present invention also
possesses the advantage of applying comprehensively to
all forms of amino acids (a, ~, y, etc). In the context
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of the present invention, the generic term "amino acid"
embraces not only the amino acids as such (that is, the
compounds having an amino function and an acid function
-COOH) but also the corresponding ester derivatives
(that is, the compounds for which the acid function is
replaced by an ester function COOR). Preferentially,
the process of the invention applies more specifically
to the amino acids of general formula (I):
NH~
COOR
R' (CH2)~
in which
- n is an integer selected from 0, 1, 2, 3, 4, 5 or 6,
- R represents a hydrogen atom or else an alkyl,
1'5 alkene, alkyne, cycloalkyl, aryl radical, a condensed
polycyclic hydrocarbon, or a heterocycle, all of these
radicals being optionally substituted,
- and R' represents an alkyl, alkene, alkyne,
cycloalkyl, aryl radical, a condensed polycyclic
hydrocarbon, a heterocycle, or else an oxy, thio,
sulphoxide or sulphonyl radical substituted by an
alkyl, aryl, cycloalkyl group or a heterocycle, all of
these radicals, moreover, being optionally substituted.
Thus in the ease where the amino acids are of
general formula (I), the process of the present
invention may be represented by the reaction scheme of
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Figure 1. This scheme represents a first step of
treatment with glutaric anhydride and a second step of
treatment with the enzyme glutaryl-7-ACA acylase. The
configuration of each of the products obtained, namely
the amino acid on the one hand and the glutarylamide
derivative on the other hand, depends on the nature of
the radical R'.
In the context of the present invention, the
alkyl, alkene and alkyne radicals contain generally
between 1 and 30 carbon atoms in a straight or branched
chain, without this being in any way limitative. This
also applies in the cases where these radicals are
substituents of other radicals. Preferably, these
radicals contain between 1 and 20 carbon atoms in a
straight or branched chain and, more preferably still,
between 1 and 10 carbon atoms in a straight or branched
chain. The alkyl radicals may be selected, for example,
from: methyl, ethyl, n-propyl, n-butyl, n-pentyl,
n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,
n-undecyl, n-dodecyl, n-tridecyl, n-pentadecyl,
n-hexadecyl, n-heptadecyl, n-octadecyl, isopropyl,
isobutyl, isopentyl, isohexyl, 3-methylpentyl,
neopentyl, neohexyl, 2,3,5-trimethylhexyl, sec-butyl,
tent-butyl, tert-pentyl. Preferred alkyl radicals are,
for example, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,
isopentyl, n-hexyl and isohexyl. The alkene radicals
may be selected, for example, from vinyl, 1-propenyl,
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allyl, butenyl, 2-methyl-1-propenyl, 2-methyl-
2-propenyl or 3-methyl-2-butenyl. The alkyne radicals
may be selected, for example, from ethynyl, 1-propynyl
or propargyl.
In the context of the present invention, the
cycloalkyl radicals generally contain between 3 and 12
carbon atoms. They are preferably selected from
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,
20 cycloundecyl and cyclododecyl. According to another
aspect of the present invention, the cycloalkyls may be
polycyclic. Preferably, these radicals are selected
from bicycloalkyls or tricycloalkyls.
According to the present invention, the term
"aryl" radical refers to monovalent aromatic
hydrocarbon radicals. Among the aryls, optionally
substituted phenyl is preferred.
In the context of the present invention, the
term condensed polycyclic hydrocarbon refers to
radicals selected preferably from pentalene, indene,
naphthalene, azulene, heptalene, biphenylene,
as-indacene, s-indacene, acenaphthylene, fluorene,
phenalene, phenanthrene, anthracene, fluoranthene,
acephenanthrylene, aceanthrylene, triphenylene, pyrene,
chrysene, naphthacene,..pleiadene, picene, perylene,
pentaphene, pentacene, tetraphenylene, hexaphene,
hexacene, rubicene, coronene, trinaphthylene,
heptaphene, heptacene, pyranthrene or ovalene.
CA 02459246 2004-03-02
In the context of the present invention, the
term "heterocycle" denotes monocyclic or fused
polycyclic compounds which contain one or more
heteroatoms, each ring being formed of 3 to 10 members.
5 The heterocycles according to the present invention
preferably contain between 1 and 3 heteroatoms selected
from oxygen, sulphur and nitrogen in a ring formed of
3 to 10 members. The heterocycles of the present
invention are selected preferably from thiophene,
10 benzo[b]thiophene, naphtho[2,3-b]thiophene,
thianthrene, furan, 2 H-pyran, isobenzofuran,
2 H-chromene, xanthene, phenoxathiine, 2 H-pyrrole,
pyrrole, imidazole, pyrazole, pyridine, pyrazine,
pyrimidine, pyridazine, indolizine, isoindole,
3 H-indole, indole, 1 H-indazole, purine,
w 4 H-quinolizine, isoquinoline, quinoline, phthalazine,
1,8-naphthyridine, quinoxaline, quinazoline, cinnoline,
pteridine, 4a H-carbazole, carbazole, ~-carboline,
phenanthridine, acridine, perimidine,
1,7-phenanthroline, phenazine, phenarsazine,
isothiazole, phenothiazine, isoxazole, furazan,
phenoxazine, isochromane, pyrrolidine, 02-pyrroline,
imidazolidine, ~2-imidazoline, pyrazolidine,
O3-pyrazoline, piperidine, piperazine, indoline,
isoindoline, quinuclidine and morpholine.
In the context of the present invention, when
the various radicals as defined above are substituted,
the said substituent or substituents are selected in
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general from halogen atoms, aryl, heterocycle,
hydroxyl, alkoxy, aryloxy, thiol, alkylthio, arylthio,
alkyl sulphoxide, aryl sulphoxide, alkylsulphonyl,
arylsulphonyl, cyano, vitro, sulphonamide,
alkylsulphonamide and arylsulphonamide groups,
depending on the nature of the radical. Preferably, the
various radicals as defined above are substituted 1, 2
or 3 times. According to a preferred aspect, the
halogen atoms are selected from chlorine, fluorine,
bromine and iodine. Where the alkyl radicals are
substituted by halogen atoms, the atoms in question are
preferably fluorine atoms. The number of fluorine
substituents is preferably 1, 2, 3, 4, 5, 6 or 7. For
example, the group in question may be a trifluoromethyl
group.
The amino acids of the present invention are
preferably selected from the compounds of general
formula (I) in which n is an integer selected from 0,
1, 2 or 3, R represents a hydrogen atom or else an
alkyl or aryl radical and R' is as defined above.
More preferably still, the amino acids of the
present invention are selected from the compounds of
general formula (I) in which n is an integer equal to
0, 1 or 2, R represents a hydrogen atom or else an
alkyl function and R' i.s selected from optionally
substituted heterocycles or aryls. In the latter case,
the aryl and/or heterocycle radicals are preferably
substituted 1, 2 or 3 times.
CA 02459246 2004-03-02
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The enzyme glutaryl-7-ACA acylase has already
been used as a catalyst in numerous industrial
processes, particularly for the hydrolysis of ~-lactams
such as N-glutaryl-7-aminoacetoxycephalosporinic acid.
It may be derived from numerous microorganisms, for
example of the genus Acinetobacter, Arthrobacter,
Bacillus, Pseudomonas, Stenotrophomonas or else
Xanthomonas, in accordance with the techniques which
are well known to the person skilled in the art, an
enzyme specialist. The enzyme glutaryl-7-ACA acylase
may also be obtained commercially, for example, from
Roche Diagnostic GmbH (Roche Molecular Biochemicals,
Standhofer Strasse 116, D-68305 Mannheim) or else from
Recordati S.p.A. (Stabilimento di opera, Via Lambro 38,
25 I-20090 Opera (MI)).
The enzyme glutaryl-7-ACA acylase may be used
in various forms, without this modifying its
stereospecificity and its stereoselectivity. For
example, glutaryl-7-ACA acylase may be used in soluble
or else immobilized form. In this second case, the
enzyme is generally immobilized in accordance with
techniques which are well known to the person skilled
in the art. For example, the enzyme may be contained in
polymeric gels or else attached to solid supports by
covalent bonding, cross-linking, adsorption or
encapsulation. Appropriate supports which are commonly
used are, for example, porous glass, porous ceramics,
synthetic polymers (for example polystyrene, polyvinyl
CA 02459246 2004-03-02
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alcohols, polyethylene, polyamides or polyacrylamides),
or polymers of natural origin (for example cellulose).
Through the use of the enzyme glutaryl-7-ACA
acylase it is possible to obtain for each enantiomer a
high enantiomeric excess ("ee"), in particular greater
than or equal to 90~, or even greater than or equal to
95~, and preferably greater than or equal to 99~. In
the context of the present invention, the "enantiomeric
excess" refers to the ~ excess of one of the
enantiomers relative to the racemic mixture. More
specifically, the enantiomeric excess is calculated as
follows:
f R~ - [s]
ee (%) - * l 00 = % (R) - % tS)
LR~ -~- IS]
1;5
where [R] and [S] represent the concentration of (R)
and (S) enantiomer respectively.
Generally, the amount of enzymes employed
relative to the total amount of initial amino acid
(substrate) is between 1 and 100 units (U) per mmole of
substrate, and preferably between 10 and 40 units per
mmole of substrate. 2 unit of enzyme corresponds to the
amount of enzyme required to hydrolyse 1 ~,mol of
N-glutaryl-7-aminoacetoxycephalosporinic acid per
minute under standard pH and temperature conditions
which are known to the person skilled in the art.
CA 02459246 2004-03-02
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According to the invention, the reaction is
carried out in an optionally buffered aqueous medium.
In this case, the aqueous buffer, with a concentration
of between 10 mM and 200 mM, may be selected from
acetate buffers which can be used at a pH of between 5
and 6.5 or phosphate buffers which can be used at a pH
of between 6.5 and 8, or else pyrophosphate buffers
which can be used at a pH of between 8 and 9.
The process of the invention is therefore
implemented in a medium in which the pH is monitored
and adjusted to between 6 and 9. Preferably, the pH of
the reaction medium is monitored and adjusted speci-
fically to between 7.5 and 8.5, and more preferably
still between 8 and 8.5. The pH may be monitored with
the aid of a pH-scat by the addition of an acid such
as, for example, hydrochloric acid, sulphuric acid or
phosphoric acid and a base such as, for example, sodium
hydroxide, potassium hydroxide or aqueous ammonia.
The treatment of the amino acids in
accordance with the invention with glutaric anhydride
is implemented at a temperature of between 20°C and
40°C, and preferably between 25°C and 35°C. Moreover,
the second step, employing the enzyme glutaryl-7-ACA
acylase, is carried out at a temperature of between
10°C and 50°C, and preferably 25°C and 35°C.
Lastly, the reaction time varies
comprehensively between 1 hour and 100 hours and
depends in particular on the amino acid concerned and
CA 02459246 2004-03-02
on the en2yme concentration. Generally, the reaction is
left to proceed for as long as is needed to obtain the
desired enantiomer with a satisfactory enantiomeric
excess. The amount of chiral amino acid obtained and
5 the value of the enantiomeric excess are monitored
using the conventional techniques known to the person
skilled in the art. Advantageously, monitoring is
carried out by means of HPLC (High Performance Liquid
Chromatography).
10 According to the present invention, the (R)
and (S) enantiomers obtained by the process described
above may be separated easily, owing to the fact that
one is present in the form of an amine which is soluble
in an aqueous medium and the other is present in the
15 form of a solid amide. Consequently, a further subject
of the present invention relates to the process as set
out above which comprises as a subsequent step the
separation of the (R) and (S) enantiomers.
The separation of the said (R) and (S)
enantiomers may be carried out easily by virtue of
conventional techniques which are known to the person
skilled in the art. This is done, for example, by
filtration, extraction, chromatography or
crystallization.
Where the des-ire is to isolate the other
enantiomer in the amino acid form and not in the
glutarylamide derivative form, the said enantiomer of
the glutarylamide derivative which has been isolated in
CA 02459246 2004-03-02
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accordance with the process described above is then
hydrolysed so as to recover the corresponding amino
acid in the enantiomeric form. It should be noted that
this process is advantageous since the hydrolysis
allows the stereochemistry of the compound employed to
be retained and does not lead to racemization of the
chiral glutarylamide derivative. Where the amino acids
are of general formula (I), this additional step may be
represented by the reaction scheme according to
Figure 2.
The hydrolysis is carried out in accordance
with conventional techniques which are known to the
person skilled in the art. The hydrolysis in question
may in particular be an acidic or a basic hydrolysis.
In the latter case, it is carried out, for example, in
w the presence of a base such as sodium hydroxide, at a
temperature between 50°C and 90°C and under atmospheric
pressure. However, any other operating conditions which
are also suitable and known to the person skilled in
the art may be used.
The present invention is useful in the sense
that it allows amino acids in racemic form to be
resolved, thereby making it possible to have available
one or the other enantiomer of the said amino acid, the
said enantiomers constituting, in particular, synthesis
intermediates.
By way of illustration, the present invention
makes it possible, for example, to have available (S)-
CA 02459246 2004-03-02
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3-amino-3-phenylpropanoic acid, which constitutes an
intermediate in the synthesis of the compound of
general formula:
H H
N\ /N
/ O I / N N H
N
O O
which is a VLA4 receptor antagonist involved in
particular in asthmatic disorders.
As well as the preceding provisions, the
present invention also embraces features and advantages
which will emerge from the examples which follow, and
which should be considered as illustrative of the
invention without limiting its scope.
FTGURES
Figure 1: Schematic representation of the 15t step of
treatment of an amino acid of general formula (I)
according to the present invention with glutaric
anhydride and of the 2nd step of treatment with the
enzyme glutaryl-7-ACA acylase. The configuration of
each of the products obtained, namely the amino acid on
CA 02459246 2004-03-02
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the one hand and the glutarylamide derivative on the
other hand, depends on the nature of the radical R'.
Figure 2: Schematic representation of the step of
transforming the chiral glutarylamide derivative into
its corresponding chiral amino acid by hydrolysis.
s~~r amror _~ a
Example 1; Resolution of 3-amino-3-(4'-nitrophenyl)-
propanoic acid in the form of the racemic mixture
a) Acylation of racemic 3-amino-3-(4'-nitrophenyl)-
propanoic acid
40.6 g of racemic 3-amino-3-(4'-nitrophenyl)-
propanoic acid are dissolved in 200 ml of distilled
water and 55 ml of triethylamine. 29.4 g of glutaric
anhydride are added in small portions and the reaction
mixture is stirred for 1 hour. The reaction mixture is
then acidified with 8.2 ml of 950 (weight/volume)
sulphuric acid. The precipitate thus obtained is
filtered off, washed 3 times with 15 ml of distilled
water and dried to constant weight under vacuum at
55°C. This gives 52.84 g of racemic 3-(glutarylamide)-
3-(4'-nitrophenyl)propanoic acid.
The nature of the product obtained was determined by
HPLC (High Performance Liquid Chromatography).
CA 02459246 2004-03-02
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b) Enzymatic deacylation using crystalline glutaryl-7-
ACA acylase in suspension (100 ml reactor)
20 g of the acid obtained in the preceding
step are dissolved in 75 ml of distilled water. The pH
of the suspension is adjusted to 8.2 by adding 12.2 ml
of 30~ (weight/volume) sodium hydroxide. 826 mg
(626 units) of a suspension of crystalline glutaryl-7-
ACA acylase are added to this solution. The reaction
mixture is stirred at 35°C for 51 hours and at a pH
which is monitored and adjusted to between 7.9 and 8.1.
At the end of the reaction, the reaction mixture is
cooled to 20°C and the pH is adjusted to 7.0 by adding
5N hydrochloric acid. 20 ml of ethanol are then added.
The resulting precipitate of (R)-3-amino-3-(4'-nitro-
phenyl)propanoic acid is filtered off, washed 3 times
with 30 ml of ethanol and dried to constant weight
under vacuum at 45°C. This gives 5.65 g of (R)-3-amino-
3-(4'-nitrophenyl)propanoic acid with an enantiomeric
excess of greater than 99~.
The mother liquor is acidified with 17.5 ml
of 5N hydrochloric acid. The precipitate which forms is
filtered off and washed twice with 10 ml of distilled
water. The filter cake obtained is dried to constant
weight under vacuum at 45°C. This gives 8.25 g of (S)-
3-(glutarylamide)-3-(4'~-nitrophenyl)propanoic acid and
(R)-3-(glutarylamide)-3-(4'-nitrophenyl)propanoic acid
in a 91:9 ratio.
CA 02459246 2004-03-02
The nature of the products obtained was determined by
HPLC.
c) Enzymatic deacylation using immobilized glutaryl-7-
5 ACA acylase obtained from Roche Diagnostic GmbH
(100 ml reactor)
20.5 g of the acid obtained in step a) are
dissolved in 75 ml of distilled water. The pH of the
suspension is adjusted to 8.2 by adding 11.9 ml of 30~
10 (weight/volume) sodium hydroxide. 6.2 g (632.4 units)
of moist immobilized glutaryl-7-ACA acylase (Roche
Diagnostic GmbH) are added to this solution. The
reaction mixture is stirred at 35°C for 18 hours and at
a pH which is monitored and adjusted to between 7.9 and
I5 8.1. At the end of the reaction, the pH of the reaction
mixture is adjusted to 9.5 by addition of 30~
(weight/volume) sodium hydroxide and the volume of the
reaction mixture is adjusted to 200 ml so as to
dissolve the (R)-3-amino-3-(4'-nitrophenyl)propanoic
20 acid produced. The immobilized enzyme is removed by
filtration and the pH of the mother liquor is adjusted
to 7.0 by addition of 5N hydrochloric acid. Then 50 ml
of ethanol are added to the reaction mixture. The
precipitated (R)-3-amino-3-(4'-nitrophenyl)propanoic
acid is filtered off, v~ashed with 10 ml of ethanol and
dried to constant weight under vacuum at 45°C. This
gives 5.375 g of (R)-3-amino-3-(4'-nitrophenyl)-
t
CA 02459246 2004-03-02
21
propanoic acid with an enantiomeric excess equal to
98 0 .
The mother liquor is acidified with 20 ml of
5N hydrochloric acid. The precipitate which forms is
filtered off and washed twice with 15 ml of distilled
water. The filter cake is dried to constant weight
under vacuum at 45°C. This gives 6 g of (S)-3-
(glutarylamide)-3-(4'-nitrophenyl)propanoic acid in a
94:6 ratio.
The nature of the products obtained was
determined by HPLC.
d) Enzymatic deacylation using crystalline glutaryl-7-
ACA acylase in suspension (500 ml reactor)
100 g of racemic 3-(glutarylamide)-3-(4'-
nitrophenyl)propanoic acid obtained as in step a) are
dissolved in 400 ml of distilled water. The pH of the
suspension is adjusted to 8.2 by adding 60 ml of 30~
(weight/volume) sodium hydroxide. 6.4 mg (4653 units)
of a suspension of crystalline glutaryl-7-ACA acylase
are added to this solution. The reaction mixture is
stirred at 35°C for 30 hours and at a pH which is
monitored and adjusted to between 7.9 and 8.1. At the
end of the reaction, the reaction mixture is cooled to
20°C and the pH is adjusted to 13 by addition of 25 ml
of 300 (weight/volume) sodium hydroxide so as to
dissolve the (R)-3-amino-3-(4'-nitrophenyl)propanoic
acid formed. The insoluble particles are filtered and
CA 02459246 2004-03-02
22
the pH of the mother liquor is adjusted to 7.0 by
addition of 27 ml of 36~ hydrochloric acid. Then 100 ml
of ethanol are added to the reaction mixture. The
precipitate of (R)-3-amino-3-(4'-nitrophenyl)propanoic
acid obtained is filtered off, washed twice with 100 ml
of ethanol and dried to constant weight under vacuum at
45°C. This gives 28.87 g of (R)-3-amino-3-(4'-
nitrophenyl)propanoic acid with an enantiomeric excess
equal to 97~.
The nature of the product obtained was
determined by HPLC.
Example 2: Resolution of 3-amino-3-phenylpropanoic acid
in the form of the racemic mixture
a) Acylation of racemic 3-amino-3-phenylpropanoic acid
488 g of racemic 3-amino-3-phenylpropanoic
acid are dissolved in 2 litres of distilled water
containing 236 g of sodium hydroxide in the form of
pellets. 437.5 g of glutaric anhydride are added in
small portions to the reaction mixture over 1 hour,
with stirring and at 20°C. After 1 hour, the reaction
mixture is acidified with 236 ml of 950 (weight/volume)
sulphuric acid and cooled to 10°C. The precipitate thus
formed is filtered and..-then washed 3 times with 600 ml
of distilled water. The moist filter cake thus
obtained, weighing 1610 g, contains 602 g of racemic
CA 02459246 2004-03-02
23
3-glutarylamide-3-phenylpropanoic acid. The nature of
the products obtained was determined by HPLC.
b) Enzymatic deacylation using crystalline glutaryl-7-
ACA acylase in suspension (5 litre reactor)
1575 g of the moist filter cake obtained
before, containing 589 g of racemic 3-glutarylamide-3-
phenylpropanoic acid, are dissolved in 1610 ml of
distilled water. The pH of the suspension is adjusted
to 8.0 by adding 440 ml of 30~ (weight/volume) sodium
hydroxide. 52.6 g (32,000 units) of a suspension of
crystalline glutaryl-7-ACA acylase are added to this
solution. The reaction mixture is stirred at 35°C for
41 hours and at a pH which is monitored and adjusted to
between 7.9 and 8.1. At the end of the reaction, the
reaction mixture is cooled to 20°C and the pH is
adjusted to Z3 by addition of 25 ml of 30~
(weight/volume) sodium hydroxide so as to dissolve the
(R)-3-amino-3-(4'-nitrophenyl)propanoic acid produced.
The insoluble particles are filtered off and the pH of
the mother liquor is adjusted to 7.0 by addition of
27 ml of 36~ hydrochloric acid.
The resulting precipitate of (R)-3-amino-3-
phenylpropanoic acid is filtered off, washed with
150 ml of distilled water and dried to constant weight
under vacuum at 45°C. This gives 96.9 g of (R)-3-amino-
3-phenylpropanoic acid with an enantiomeric excess
equal to 98~.
CA 02459246 2004-03-02
24
The mother liquor is acidified with 180 ml of
95~ (weight/volume) sulphuric acid. The precipitate
thus formed is filtered off and washed twice with
400 ml of cooled distilled water. The filter cake is
dried to constant weight under vacuum at 45°C. This
gives 314.6 g of (S)-3-(glutarylamide)-3-phenylpro-
panoic acid and (R)-3-(glutarylamide)-3-phenylpropanoic
acid in a 90:10 ratio.
The nature of the products obtained was
determined by HPLC.
c) Deacylation of (S)-3-glutarylamide-3-phenylpropanoic
acid by basic hydrolysis
557.2 g of a mixture of (S)-3-glutarylamide-
3-phenylpropanoic acid and (R)-3-glutarylamide-3-
phenylpropanoic acid in a 90:10 ratio, obtained in
preceding step b), are distilled in 3.91 litres of
distilled water and 1.65 litres of 300 (weight/volume)
sodium hydroxide. The reaction mixture is stirred at
70°C for 4 days.
The reaction mixture is then cooled to 15°C.
The product obtained is precipitated with adjustment of
the pH to 6.9 by addition of 3,21 litres of 370
(weight/volume) hydrochloric acid, filtered off and
dried to constant weight under vacuum at 45°C. This
gives 202.4 g of (S)-3-amino-3-phenylpropanoic acid and
(R)-3-amino-3-phenylpropanoic acid with an enantiomeric
excess of greater than 98~.
CA 02459246 2004-03-02
25
The nature of the products obtained was
determined by HPLC.
