Note: Descriptions are shown in the official language in which they were submitted.
CA 02428163 2003-05-07
020261 AM / AL
1
Process for the enzymatic preparation of enantiomer-
enriched (3-amino acids
The present invention relates to a process for preparing
enantiomer-enriched 0-amino acids.
Optically active 13-aminocarboxylic acids occur in natural
substances such as alkaloids and antibiotics and their
isolation is increasingly acquiring interest, not least
because of their increasing importance as essential
intermediates in the preparation of pharmaceuticals (see,
inter alia: E. Juaristi, H. Lopez-Ruiz, Curr. Med. Chem.
1999, 6, 983-1004). Both the free form of optically active
(3-aminocarboxylic acids and their derivatives have
interesting pharmacological effects and.can also be used
in the synthesis of modified peptides.
The conventional racemate resolution by means of
diastereomeric salts (proposed route in: H. Boesch et al.,
Org. Proc. Res. Developm. 2001, 5, 23-27) and, in
particular, the diastereoselective addition of lithium
phenylethylamide (A. F. Abdel-Magid, J. H. Cohen, C. A.
Maryanoff, Curr. Med. Chem. 1999, 6, 955-970) have been
established as preparation methods for (3-aminocarboxylic
acids. The latter method is considered as intensively
researched and, despite numerous disadvantages occurring
in it, is preferably used. On the one hand, stoichiometric
amounts of a chiral reagent are needed, which is a big
disadvantage compared to catalytic asymmetrical methods.
In addition, expensive and, moreover, hazardous
auxiliaries, such as, for example, n-butyllithium are
needed to activate the stoichiometric reagent by
deprotonation. Moreover, the performance of the reaction
CA 02428163 2003-05-07
020261 AM / AL
2
at low temperatures of approximately -70 C is important
for a satisfactory stereoselectivity, which means a high
requirement relating to the reactor material, additional
costs and a high energy consumption.
Although the preparation of optically active 13-
aminocarboxylic acids biocatalytically plays only a
subordinate role at present, it is desirable, in
particular because of the economic and ecological
advantages of biocatalytic reactions. The use of
stoichiometric amounts of a chiral reagent is unnecessary
and, instead, small, catalytic amounts of enzymes are used
that are natural and environmentally-friendly catalysts.
Moreover, these biocatalysts, which are efficiently used
in aqueous medium, have, in addition to their catalytic
properties and their high efficiency the advantage, in
contrast to a multiplicity of synthetic metal-containing
catalysts, that it is not necessary to use metal-
containing, in particular heavy-metal-containing and
consequently toxic feedstocks.
In the prior art, for instance, the enantioselective N-
acylation of f-aminocarboxylic acids has already often
been reported.
Thus, L.T. Kanerva et al. describe in Tetrahedron:
Asymmetry, Vol. 7, No. 6, pages 1707-1716, 1996 the
enantioselective N-acylation of ethyl esters of various
cycloaliphatic P-aminocarboxylic acids with 2,2,2-
trifluoroethyl ester in organic solvents and lipase SP 526
from Candida antarctica or lipase PS from Pseudomonas
cepacia as biocatalyst.
CA 02428163 2003-05-07
020261 AID / AL
3
V.M. Sanchez et al. studied the biocatalytic racemate
resolution of ( )-ethyl 3-aminobutyrate (Tetrahedron:
Asymmetry, Vol. 8, No. 1, pages 37-40, 1997) with lipase
from Candida antarctica via the preparation of N-
acetylated P-aminocarboxylic esters.
EP-A-8 890 649 discloses a process for preparing optically
active amino acid esters from racemic amino acid esters by
enantioselective acylation with a carboxylic ester in the
presence of a hydrolase selected from the group comprising
amidase, protease, esterase and lipase, and subsequent
isolation of the unreacted enantiomers of the amino acid
esters.
WO-A-98/50575 relates to a process for obtaining a chiral
(3-aminocarboxylic acid or its corresponding ester by
bringing a racemic (3-aminocarboxylic acid, an acyl donor
and penicillin G acylase into contact under conditions for
stereoselectively acylating an enantomer of the racemic f3-
aminocarboxylic acid, in which the other enantiomer is
substantially not reacted and a chiral P-aminocarboxylic
acid is thus obtained. The reverse reaction sequence has
also been studied (V. A. Soloshonok, V. K. Svedas, V. P.
Kukhar, A. G. Kirilenko, A. V. Rybakova, V. A. Solodenko,
N. A. Fokina, 0. V. Kogut, I. Y. Galaev, E. V. Kozlova, I.
P. Shishkina, S. V. Galushko, Synlett 1993, 339-341; V.
Soloshonok, A. G. Kirilenko, N. A. Fokina, I. P.
Shishkina, S. V. Galushko, V. P. Kukhar, V. K. Svedas, E.
V. Kozlova, Tetrahedron: Asymmetry 1994, 5, 1119-1126; V.
Soloshonok, N. A. Fokina, A. V. Rybakova, I. P. Shishkina,
S. V. Galushko, A. E. Sochorinsky, V. P. Kukhar, M. V.
Savchenko, V. K. Svedas, Tetrahedron: Asymmetry 1995, 6,
1601-1610; G. Cardillo, A. Tolomelli, C. Tomasini, Eur. J.
CA 02428163 2003-05-07
020261 AM J AL
4
Org. Chem. 1999, 155-161). A disadvantage of this process
is the difficult working-up of the product mixture after
the enantioselective hydrolysis. After isolating the free
(3-aminocarboxylic acid, a mixture of phenylacetic acid and
N-phenylacetyl-p-aminocarboxylic acid is obtained that is
difficult to separate.
Their reaction with lipases has already been known for a
long time for obtaining enantiomer-enriched carboxylic
acids. In US5518903, this principle has been transferred
to N-protected R-amino acid esters, but with varying
success. Whereas only the corresponding benzyl ester of
racemic N-butoxycarbonyl-f-aminobutyric acid was resolved
highly enantioselectively by means of a lipase, the
remaining methyl esters or n-butyl esters used yielded
only ee values in the region of not more than 70% ee. In
this connection, it should be stated that, apparently,
going over from a corresponding methyl ester to an n-butyl
ester is accompanied by an impairment of the ee value of
the acid prepared. Thus, starting from the n-butyl ester
of N-Boc-p-aminobutyric acid, the ester hydrolysis with
the enzyme lipase from Asahi reduces after 8 days an ee
value of the corresponding acid of 45% ee in a yield of
37%. With the lipase PS supplied by Amano, a compound
enriched to 61% ee is obtained in the same reaction with a
yield of 41% at any rate within 7 days. In comparison
therewith, the corresponding methyl ester yields 70% ee.
From the results recently published by Faulconbridge et
al., it is to be inferred that the ester hydrolysis of
aromatic (3-amino acid ethyl esters at a pH of 8 with the
lipase PS supplied by Amano takes place with acceptable
yields and very good enantiomeric excesses (Tetrahedron
CA 02428163 2003-05-07
020261 AM / AL
Letters 2000, 41, 2679-81). The product is obtained with
an enantiomeric purity of up to 99% ee, but the synthesis,
which was performed exclusively in aqueous suspension, is
associated with some disadvantages. On the one hand, it
5 has been found that, although the crystallization is
selective under these conditions, the reaction per se
results, as documented in Comparison Example 2, in lower
ee values of 85.1% ee. All in all, this means, on the one
hand, a yield loss due to the formation of the undesirable
enantiomer and, on the other hand, it also entails the
problem that the ee value may easily also drop below 99%
ee or even below 98% ee as a function of slight process
fluctuations on a technical scale because of altered
crystallization conditions. As high an ee value as
possible of > 98% ee, in particular > 99% ee, is, however,
a requirement for pharmaceutical applications. In
addition, the performance in purely liquid medium would be
desirable as well as the performance in suspension in
order, for example, to be able to ensure a good enzyme
isolation by means of ultrafiltration. optimally, a high
ee value should likewise be produced in this step, which
cannot be achieved with the existing literature processes
(in this connection, see also Comparison Examples 1
and 2).
An enzymatic hydrolysis in the presence of single-phase
reaction media using organic solvents was reported by
Nagata et al. (S. Katayama, N. Ae, R. Nagata, Tetrahedron:
Asymmetry 1998, 9, 4295-4299). In that case, a cyclic (3-
amino acid ester was used. The best results with
enantioselectivities of 94% ee and conversions of 50% with
a reaction time of 20 h were achieved using a solvent
mixture composed of acetone (90%) and water (10%). Poorer
CA 02428163 2010-09-13
6
results were achieved with lower proportions of water. In
general, the use of readily water-soluble solvents has
proved superior compared with sparingly water-miscible
solvents. Thus, the use of diisopropyl ether as organic
medium, which was saturated with water and 20% acetone,
produces only an ee value of 58% ee. In contrast thereto,
a further readily water-soluble solvent proves suitable
(95% ee) in addition to acetone with THF, but in this case
long reaction times are needed (96 h) in order to achieve
a conversion that is to any extent complete.
Hitherto, this successful synthesis having the feature of
the presence of organic solvents has, however, remained
limited to the preparation of cyclic (3-amino acid esters.
Under the optimum conditions specified in the literature
for cyclic (3-amino acid esters (see above), drastically
lower yields and unacceptable, long reaction times are
obtained in the preparation of the desired target
compounds of the open-chain pendants (Comparison
Example 1).
The object of the present invention was therefore to
provide a further process for the enzymatic preparation of
(3-amino acids. In particular, the said process should
advantageously be usable on an industrial scale
economically as well as ecologically, i.e. be particularly
outstanding in regard to environmental compatibility,
industrial safety, ruggedness of the processing, the
space/time yield and selectivity.
These and further objects not mentioned in greater detail,
but obviously emerging from the prior art are achieved by
a process according to the present invention.
CA 02428163 2010-09-13
7
One embodiment of the present invention provides a process
for producing enantiomer-enriched N-unprotected, R-amino
acids by enzymatic hydrolysis of an enantiomeric mixture of
N-unprotected, R-amino acid esters with a hydrolase,
wherein the hydrolysis takes place in a two-phase system
composed of water and an organic solvent forming two phases
with water under a set of reaction conditions.
As a result of the fact that a process for preparing
enantiomer-enriched N-unprotected, in particular open-
chain R-amino acids is performed by enzymatic hydrolysis
of an enantiomeric mixture of N-unprotected, in particular
open-chain, R-amino acid esters with a hydrolase in a two-
phase system composed of water and an organic solvent
forming two phases under the given reaction conditions,
the object set is very surprisingly achieved, but, on the
other hand, in a no less advantageous way. In this
connection, not only are substantially higher reactivities
than in the hitherto known organic/aqueous systems are
surprisingly achieved, but also good enantioselectivities.
The reaction in the two-phase system can even be optimized
in such a way that the product is produced at >_99% ee
(Example 4). Moreover, it is interesting that the ee value
in accordance with Example 3 (89% ee) according to the
invention turns out to be markedly better compared with
the experiment in a purely aqueous system (Comparison
Example 2, 81.5% ee). In addition to the processing
advantages of organic solvents, this process consequently
has, moreover, the advantage of generating higher
enantioselectivities in the products compared with the
aqueous standard medium.
CA 02428163 2010-09-13
7a
Preferably, compounds of the general Formula (I)
NH2 O
R OR" (I)
R'
CA 02428163 2003-05-07
020261 AM / AL
8
where
R, R'' denote, independently of one another, (C1-C8) -alkyl,
(C2-C8) -alkenyl, (C2-C8) -alkynyl, (C3-C8) -cycloalkyl,
(C6-C18) -aryl, (C7-C19) -aralkyl, (C3-C18) -heteroaryl,
(C4-C19) -heteroaralkyl, ( (C1-C8) -alkyl) 1-3- (C3-C8) -cyc loalkyl ,
((C1-C8) -alkyl) 1-3- (C6-C18)-aryl, and ((C1-C8) -alkyl) 1-3 -
(C3-C18)-heteroaryl,
R' denotes H, (C1-C8) -alkyl, (C2-C8) -alkenyl, (C2-C8) -
alkynyl, (C3-C8) -cycloalkyl, (C6-C18) -aryl, (C7-C19) -aralkyl,
(C3-C18) -heteroaryl , ( C4-C19) -heteroaralkyl , ( (C1-C8) -
alkyl) 1-3- (C3-C8) -cycloalkyl, ((C1.-C8)-alkyl) 1-3- (C6-C18) -aryl,
and ((C1-C8) -alkyl) 1-3- (C3-C18) -heteroaryl,
in the process of the subject matter.
In principle, the person skilled in the art is free to
choose the appropriate ester group. He will base his
selection on economic and reaction-engineering aspects.
Favourable alcohols for forming the ester are, in
particular, those that can easily be removed from the
reaction mixture, optionally by distillation. Quite
particularly preferred in the method according to the
invention is the use of P-amino acid alkyl esters or ~i-
.amino acid aryl esters. Extremely preferred is the use of
appropriate n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl or tert-butyl esters.
The choice of the reaction parameters is likewise up to
the person skilled in the art. He will determine them
separately for the individual case on the basis of routine
experiments. At all. events, a pH value range of between 4
and 10, preferably between 6 and 9 and more preferably
between 7 and 8.5, is suitable for the enzymatic process
CA 02428163 2003-05-07
020261 AM / AL
9
of the subject matter. The lipase PS supplied by Amano has
proved particularly suitable around a pH of 8.
With regard to temperature, the same requirements exist in
principle as for the pH. Here, again, as optimum a
temperature as possible can be determined for the
individual case, depending on which enzyme functions most
optimally at which temperature. For enzymes from
thermophilic organisms, high temperatures of up to 100 C
are possible. Others, again, function optimally only at
<0 C to -15 C, possibly in an ice matrix. Preferably, the
temperature established during the reaction should be in
the range between 15 and 40 C and, more preferably, between
and 300C.
The choice of the enzyme to be used is the responsibility
15 of the person skilled in the art. Many suitable enzymes
can be selected from Enzyme Catalysis in Organic
Synthesis, Ed.: K. Drauz, H. Waldmann, VCH, 1995, page 165
and the literature cited therein. Preferably, a lipase is
taken for the ester hydrolysis, more preferably, the
20 lipase PS supplied by Amano from Pseudomonas cepacia is
used.
For the application, the polypeptide under consideration
can be used in free form as a homogeneously purified
compound or as an enzyme prepared as recombinant.
Furthermore, the polypeptide may also be used as a
constituent of an intact guest organism or in conjunction
with the digested cell material of the host organism
purified as much as desired.
The use of the enzymes in immobilized form is also
possible (Sharma B. P.; Bailey L. F. and Messing R. A.
CA 02428163 2003-05-07
020261 AM/ AL
(1982), Immobilisierte Biomaterialien - Techniken and
Anwendungen, Angew. Chem. 94, 836-852). Advantageously,
the immobilization takes place through lyophilization
(Paradkar, V. M.; Dordick, J. S. (1994), Aqueous-Like
5 Activity of a-Chymotrypsin Dissolved in Nearly Anhydrous
Organic Solvents, J. Am. Chem. Soc. 116, 5009-5010; Mori,
T.; Okahata, Y. (1997), A variety of lipi-coated glycoside
hydrolases as effective glycosyl transfer catalysts in
homogeneous organic solvents, Tetrahedron Lett. 38, 1971-
10 1974; Otamiri, M.; Adlercreutz, P.; Matthiasson, B.
(1992), Complex formation between chymotrypsin and ethyl
cellulose as a means to solubilize the enzyme in active
form in toluene, Biocatalysis 6, 291-305). Quite
particularly preferable is lyophilization in the presence
of surfactant substances, such as Aerosol OT or
polyvinylpyrrolidone or polyethylene glycol (PEG) or
Brij 52 (diethylene glycol monocetyl ether) (Kamiya, N.;
Okazaki, S.-Y.; Goto, M. (1997), Surfactant-horseradish
peroxidase complex catalytically active in anhydrous
benzene, Biotechnol. Tech. 11, 375-378).
Extremely preferred is immobilization on Eupergit , in
particular Eupergit C and Eupergit 250L (Rohm) (for a
summary see: E. Katchalski-Katzir, D. M. Kraemer, J. Mot.
Catal. B: Enzym. 2000, 10, 157). Equally preferred is the
immobilization on Ni-NTA in combination with the
polypeptide modified by attaching a His tag
(Hexahistidine) (Petty, K.J. (1996), Metal-chelate
affinity chromatography In: Ausubel, F.M. et al. eds.
Current Protocols in Molecular Biology, Vol. 2, New York:
John Wiley and Sons).
CA 02428163 2003-05-07
020261 AM / AL
11
Use as CLECs is likewise conceivable (St. Clair, N.; Wang,
Y.-F.; Margolin, A. L. (2000), Cofactor-bound cross-linked
enzyme crystals (CLEC) of alcohol dehydrogenase, Angew.
Chem. Int. Ed. 39, 380-383).
These measures can make it possible to generate, from
polypeptides that are unstable due to organic solvents,
those that can function in mixtures of aqueous and organic
solvents or entirely in organic medium.
The reaction of the subject matter can be performed in any
reaction vessel provided for the purpose. In detail, these
are normal batch reactors, loop reactors or an enzyme
membrane reactor (Bommarius, A. S.; Drauz, K.; Groeger,
U.; Wandrey, C.; Membrane Bioreactors for the Production
of Enantiomerically Pure a-Amino Acids, in: Chirality in
Industry (eds.: Collins, A. N.; Sheldrake, G. N.; Crosby,
J.) 1992, John Wiley & Sons, pages 371-397).
Suitable as organic phase that forms two phases with water
under the given reaction conditions, that is to say,
consequently, is insoluble or poorly water-soluble and can
be used in the method according to the invention, are all
the types of organic, insoluble or poorly water-soluble
solvents and also mixtures thereof. In particular, these
are ethers, ketones, esters, saturated or unsaturated,
linear or branched-chain hydrocarbons.
In this connection, methyl-tert-butyl ether (MTBE),
diisopropyl ether, ethyl acetate, hexane, heptane,
cyclohexane, methylcyclohexane and toluene, and also any
appropriate desirable mixtures thereof have proved
particularly suitable.
CA 02428163 2003-05-07
020261 AM / AL
12
If enzymes are used that exist in adsorbed form,
optionally on water-insoluble support materials and/or
minor constituents or stabilizers, it has proved
advantageous to isolate the insoluble support and/or minor
constituent or stabilizer prior to the use of the enzyme
in the reaction so that contamination of the product
produced with the insoluble support material of the enzyme
used does not occur, provided the separation of enzyme and
support is easily possible. For example, the lipase PS
supplied by Amano, which can advantageously be used, is
absorbed on silica supports. In this case, the aqueous
enzyme solution could therefore be filtered prior to
adding the reactants to the reaction medium in order to
remove the silicic acids from the reaction system. The
activities or processing stability of the enzyme do not,
as a rule, adversely affect this procedure.
Within the scope of the invention, "N-unprotected" is to
be understood as meaning that the (3-nitrogen atom of the
acid is not blocked by an N-protective group that is
stable under the reaction conditions. Regarded as such
are, in particular, the common protected groups, such as
Z, Boc, Fmoc, Eoc, Moc, acetyl, etc.
To be regarded as (C1-C8)-alkyl are methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, hexyl, heptyl or octyl together with
all the bonding isomers. These may be singly or multiply
substituted by (C1-C8)-alkoxy, (C1-Cg)-haloalkyl, OH,
halogen, NH2, NO2, SH or S- (C1-C8) -alkyl .
(C2-C8) -alkenyl is to be understood as meaning a (C1-C8)-
alkyl radical as depicted above, with the exception of
methyl, containing at least one double bond.
CA 02428163 2003-05-07
020261 AM / AL
13
(C2-C8) -aklynyl is to be understood as meaning a (C1-C8) -
alkyl radical as depicted above, with the exception of
methyl, containing at least one triple bond.
(C1-C8) -acyl is to be understood as meaning a (C1-C8) -alkyl
radical bonded to the molecule by means of a -C = O
function.
(C3-C8) -cycloalkyl is to be understood as meaning
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or
cycloheptyl radicals, etc. These may be substituted by one
or more halogens and/or N-, 0-, P-, S-atom-containing
radicals and/or may have N-, 0-, P-, S-atom-containing
radicals in the ring, such as, for example, 1-, 2-, 3-, 4-
piperidyl, 1-, 2-, 3-pyrrolidinyl, 2=-, 3-tetrahydrofuryl,
2-, 3-, 4-morpholinyl..These may be singly or multiply
substituted by (C1-C8)-alkoxy, (C1-C8)-haloalkyl, OH,
halogen, NH2, NO2, SH, S- (C1-C8) -alkyl, (C1-C8) -acyl,
(C1-C8) -alkyl.
A (C6-C18)-aryl radical is understood as meaning an
aromatic radical containing 6 to 18 carbon atoms. In
particular, these include compounds, such as phenyl,
naphthyl, anthryl, phenanthryl, biphenyl radicals. These
may be singly or multiply substituted by (C1-C8)-alkoxy,
(C1-C8) -haloalkyl, OH, halogen, NH2, NO2, SH, S- (C1-C8) -
alkyl, (C1-C8) -acyl, (C1-C8) -alkyl.
A (C7-C19) -aralkyl radical is a (C6-C18) -alkyl radical
bonded to the molecule by means of a (C1-C8)-alkyl radical.
(C1-C8)-alkoxy is a (C1-C8)-alkyl radical bonded to the
molecule under consideration by means of an oxygen atom.
CA 02428163 2003-05-07
020261 AM / AL
14
(C1-C8)-alkoxycarbonyl is a (C1-C8)-alkyl radical bonded to
the molecule under consideration by means of an -OC(O)
funktion. This is a synonym for the other oxycarbonyl
radicals.
(C1-C8)-haloalkyl is a (C1-C8)-alkyl radical substituted by
one or more halogen atoms.
A (C3-C18) -heteroaryl radical denotes, within the scope of
the invention, a five-, six- or seven-member aromatic ring
system containing 3 to 18 carbon atoms that contains
heteroatoms, such as, for example, nitrogen, oxygen or
sulphur in the ring. Regarded as such heteroaromatics are,
in particular, radicals, such as 1-, 2-, 3-furyl, such as
1-, 2-, 3-pyrrolyl, 1-,2-,3-thienyl, 2-, 3-, 4=pyridyl,
2-, 3-, 4-, 5-, 6-, 7-indolyl, 3-, 4-, 5-pyrazolyl, 2-,4-,
5-imidazolyl, acridinyl, quinolinyl, phenanthridinyl, 2-,
4-, 5-, 6-pyrimidinyl. These may be singly or multiply
substituted by (C1-C8)-alkoxy, (C1-C8)-haloalkyl, OH,
halogen, NH2, NO2, SH, S- (C1-C8) -alkyl, (C1-C8) -acyl,
(C1-C8) -alkyl.
(C4-C19) -heteroaralkyl is to be understood as meaning a
heteroaromatic system corresponding to the (C7-C19) -aralkyl
radical.
Suitable as halogens are fluorine, chlorine, bromine and
iodine.
The term enantiomer-enriched is to be understood as
meaning, within the scope of the invention, the proportion
of an enantiomer in the mixture with its optical antipodes
in a range of >50 % and <100 %.
CA 02428163 2010-09-13
The structures shown refer to all the possible
diastereomers and enantiomers and their mixtures that are
possible.
CA 02428163 2003-05-07
020261 AM / AL
16
Experimental Examples:
Comparison Example 1:
9.2 mmol of the racemic compound ethyl rac-3-amino-3-
phenylpropionate (1.79 g) are taken up in 50 ml of a
solvent mixture composed of 25 ml of water and 25 ml of
acetone as organic solvent component and the solution is
set to a pH of 8.2 by means of automatic pH adjustment
through adding 1 M sodium hydroxide solution (obtained
from Merck). On reaching a reaction temperature of 20 C,
200 mg of Amano lipase PS (Pseudomonas cepacia; obtained
through Amano Enzymes, Inc.) are added to start the
reaction. After a reaction time of 3, 5 and 24 hours, the
conversion rate formed of the (S)-3-amino-3-
phenylpropionic acid formed is determined. In this
process, a conversion of 1.8% after 3 hours, 2.0% after 5
hours and 5.5% after 24 hours is determined. The value of
the enantioselectivity was not determined because of the
unsatisfactory course of the reaction in view of the low
conversion. The conversion was determined by means of
HPLC.
Comparison Example 2:
9.2 mmol of the racemic compound ethyl rac-3-amino-3-
phenylpropionate (1.79 g) are taken up in 50 ml of water
and the solution is set to a pH of 8.2 by means of
automatic pH adjustment through adding 1 M sodium
hydroxide solution (obtained through Merck). In order to
dissolve the ester completely, 3 ml of acetone are also
added to the solution. On reaching a reaction temperature
of 20 C, 200 mg of Amano lipase PS (Pseudomonas cepacia;
obtained through Amano Enzymes, Inc.) are added to start
CA 02428163 2003-05-07
020261 AM / AL
17
the reaction. After a reaction time of 3 and 6 hours, the
conversion rate formed and also after 6 hours the
enantioselectivity of the (S) -3 -amino- 3 -phenylpropionic
acid formed are determined. In this process, a conversion
of 18.5% after 3 hours or 37.8% after 6 hours and an
enantioselectivity of 85.1% ee (after 6 hours) are
determined. The conversion and enantioselectivity were
determined by means of HPLC.
Example 3:
9.2 mmol of the racemic compound ethyl rac-3-amino-3-
phenylpropionate (1.79 g) are taken up in 50 ml of a two-
phase solvent mixture composed of 25 ml of water and 25 ml
of methyl tert-butyl ether (MTBE) as organic solvent
component and set to a pH of 8.2 by automatic pH
adjustment through adding 1 M sodium hydroxide solution
(obtained through Merck). on reaching a reaction
temperature of 20 C, 200 mg of Amano lipase PS (Pseudomonas
cepacia; obtained through Amano Enzymes, Inc.) are added
to start the reaction. After a reaction time of 3, 5 and
24 hours, the conversion rate formed of the (S)-3-amino-3-
phenylpropionic ac:Ld formed is determined. In this
process, a conversion of 23.5% after 3 hours or a
quantitative conversion of approximately :'_50% after 15
hours and an enantioselectivity of 89.0% ee (after 15
hours) are determined. The conversion. and
enantioselectivity were determined by means of HPLC.
Example 4:
81 ml of water are taken and 1.45 g of Amano lipase PS
(Pseudomonas cepacia; obtained through Amano Enzymes,
Inc.) are added thereto. The undissolved solid is then
CA 02428163 2003-05-07
020261 AM / AL
18
filtered off. 81 ml of methyl tert-butyl ether (MTBE) are
added as organic solvent component to the aqueous enzyme
solution resulting as filtrate. The two-phase system is
set to a pH of 8.2 by means of automatic pH adjustment
through adding 1 M sodium hydroxide solution (obtained
through Merck). On reaching a temperature of 20 C, 188.2
mmol of the racemic compound n-propyl rac-3-Amino-3-
phenylpropionate (39.0 g) are then added and the reaction
is started. The reaction time is 15 hours, a white
precipitate being produced that is composed of the desired
product (S)-3-amino-3-phenylpropionic acid. After a
reaction time of 15 hours, 160 ml of acetone are added to
complete the precipitation, stirring is continued for 45
minutes and the solid is filtered off. The solid is washed
several times with a little acetone and then dried in
vacuo. 12.91 g of the desired (S)-3-amino-3-
phenylpropionic acid are obtained, equivalent to a yield
of 41.6%. The enantioselectivity for the ;product is
99.6% ee. The enantioselectivity was determined by means
of HPLC. 98.8% was determined for the chemical purity
(determined by means of titration). The structure of the
product was additionally confirmed by means of NMR
spectroscopy.
