U.S. patent application number 13/212826 was filed with the patent office on 2011-12-08 for production of monatin enantiomers.
This patent application is currently assigned to CARGILL, INCORPORATED. Invention is credited to Dean Brady, Subash Buddoo, Amanda Louise Rousseau, Lucia H. Steenkamp, Paul A. Steenkamp.
Application Number | 20110300282 13/212826 |
Document ID | / |
Family ID | 40508799 |
Filed Date | 2011-12-08 |
United States Patent
Application |
20110300282 |
Kind Code |
A1 |
Brady; Dean ; et
al. |
December 8, 2011 |
PRODUCTION OF MONATIN ENANTIOMERS
Abstract
Methods for preferentially hydrolyzing one stereoisomer of an
isoxazoline diester over another, as well as an enzyme for
facilitating the preferential hydrolysis are provided. Also
provided are methods for providing mixtures of (RR) and (RS)
monatin as well as (SS) and (SR) monatin, which methods can include
the step of stereoselectively hydrolyzing an isoxazoline
diester.
Inventors: |
Brady; Dean; (Midrand,
ZA) ; Steenkamp; Lucia H.; (Boksburg, ZA) ;
Rousseau; Amanda Louise; (Bedford Gardens, ZA) ;
Buddoo; Subash; (Randburg, ZA) ; Steenkamp; Paul
A.; (Boksburg, ZA) |
Assignee: |
CARGILL, INCORPORATED
Wayzata
MN
|
Family ID: |
40508799 |
Appl. No.: |
13/212826 |
Filed: |
August 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11865403 |
Oct 1, 2007 |
8003361 |
|
|
13212826 |
|
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Current U.S.
Class: |
426/548 ;
435/118; 435/121; 435/19 |
Current CPC
Class: |
C07D 413/06 20130101;
C07D 209/12 20130101 |
Class at
Publication: |
426/548 ;
435/118; 435/121; 435/19 |
International
Class: |
C12P 17/16 20060101
C12P017/16; A23L 1/236 20060101 A23L001/236; C12Q 1/44 20060101
C12Q001/44 |
Claims
1. A process comprising: stereoselectively hydrolyzing a compound
of Formula II using Carboxyestrase NP enzyme: ##STR00016## wherein:
R.sup.1 and R.sup.2 are independently C.sub.1-10 alkyl; to form at
least one of a compound of Formula IIIa: ##STR00017## or a compound
of Formula IIIa' ##STR00018## and a compound of Formula IIb:
##STR00019##
2. The process of claim 1, wherein R.sup.1 and R.sup.2 are both
ethyl.
3. The process of claim 1, further comprising converting the
compound of Formula IIIa or IIIa' or both into a compound of
Formula Ia: ##STR00020##
4. The process of claim 3, wherein said converting comprises: (a)
hydrolyzing the compound of Formula IIIa or IIIa' to form a
compound of Formula IVa: ##STR00021## and (b) and hydrogenating the
compound of Formula IVa.
5. The process of claim 4, wherein said hydrogenating forms a
mixture of a compound of Formula Ia and a compound of Formula Ic:
##STR00022## and the process, further comprises separating the
compound of Formula Ia from the compound of Formula Ic.
6. The process of claim 1, further comprising converting the
compound of Formula IIb into a compound of Formula Ib:
##STR00023##
7. The process of claim 6, wherein said converting comprises: (a)
hydrolyzing the compound of Formula IIb to form a compound of
Formula IVb: ##STR00024## (b) and hydrogenating the compound of
Formula IVb.
8. The process of claim 7, wherein said hydrogenating forms a
mixture of a compound of Formula Ib and a compound of Formula Id:
##STR00025## and the process further comprises separating the
compound of Formula Ib from the compound of Formula Id.
9. The process of claim 5, further comprising: (a) incorporating
the compound of Formula Ia into a food composition.
10. A process comprising: hydrolyzing an isoxazoline diester
composition comprising a mixture of seteroisomers using
carboxylesterase NP enzyme.
11. A method for identifying enzymes useful in the production of
steroisomerically-pure or stereisomerically-enriched monatin
compositions, comprising: screening hydrolytic enzymes for
steroselective hydrolytic activity on a isoxazoline diester,
wherein the screening comprises selecting a hydrolytic enzyme,
forming a reaction mixture comprising the selected enzyme and an
isoxazoline diester, providing conditions under which a hydrolysis
reaction of the isoxazoline diester is expected to proceed, and
analyzing the reaction mixture for presence of
stereoisomerically-pure or stereoisomerically-enriched monatin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/865,403, filed Oct. 1, 2007, which application is herein
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention is in the field of organic and
biocatalytic synthesis.
BACKGROUND
[0003] Monatin is a high-intensity sweetener having the chemical
formula:
##STR00001##
[0004] Because of various naming conventions, monatin is also known
by a number of alternative chemical names, including:
2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid;
4-amino-2-hydroxy-2-(1H-indol-3-ylmethyl)-pentanedioic acid;
4-hydroxy-4-(3-indolylmethyl)glutamic acid; and,
3-(1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl)indole.
[0005] Monatin contains two chiral centers leading to four
potential stereoisomeric configurations. The R,R configuration (the
"R,R stereoisomer" or "(R,R)-monatin"); the S,S configuration (the
"S,S stereoisomer" or "(S,S)-monatin"); the R,S configuration (the
"R,S stereoisomer" or "(R,S)-monatin"); and the S,R configuration
(the "S,R stereoisomer" or "(S,R)-monatin"). The different
stereoisomers of monatin have different sweetening characteristics.
For example, (S,S)-monatin is approximately 50-200 times sweeter
than sucrose by weight, while (R,R)-monatin is approximately
2000-2400 times sweeter than sucrose by weight.
[0006] Certain isomeric forms of monatin can be found in the bark
of roots of the Schlerochiton ilicifolius plant located
predominantly in the Limpopo region, but also in Mpumalanga and the
North West Province of South Africa. However, the concentration of
the monatin present in the dried bark, expressed as the indole in
its acid form, has been found to be about 0.007% by mass. See U.S.
Pat. No. 4,975,298. The exact method by which monatin is produced
in the plant is presently unknown.
[0007] At least in part because of its sweetening characteristic
and utility in food applications (including beverages), it is
desirable to have an economic source of monatin. Furthermore,
because of the different sweetening characteristics of the
different stereoisomers, it is desirable to have an economic source
of a single stereoisomer of monatin, such as the R,R stereoisomer.
Thus, there is a continuing need to develop methods for the
production of monatin in stereoisomerically-pure or
stereoisomerically-enriched form.
SUMMARY
[0008] The invention provides methods for the production of the
high intensity sweetener monatin from an enantiomerically-pure or
enantiomerically-enriched isoxazoline ester.
[0009] In some embodiments of the invention, an isoxazoline diester
undergoes enantioselective hydrolysis to produce
enantiomerically-pure or enantiomerically-enriched isoxazoline
monoester and enantiomerically-pure or enantiomerically-enriched
isoxazoline diester. In some embodiments, the enantioselective
hydrolysis preferentially acts on the isoxazoline diester
"R"-isomer over the isoxazoline diester "S"-isomer resulting in
enantiomerically-pure or enantiomerically-enriched R-isoxazoline
monoester and enantiomerically-pure or enantiomerically-enriched
S-isoxazoline diester. The monoester and diester can then be
separated, and once separated each can then be converted to its
corresponding monatin isomers. Thus, for example, where the enzyme
is stereospecifically active for R-isoxazoline diester, a mixture
of the R,R and R,S forms of monatin can be produced from the
R-isoxazoline monester and a mixture of the S,S and S,R forms of
monatin can be produced from the S-isoxazoline diester. In some
embodiments, the resultant R,R and R,S forms of monatin are
separated from one another, for example by physical means known in
the art. In some embodiments the resultant S,S and S,R forms of
monatin are separated from one another, for example by physical
means known in the art.
[0010] In some embodiments of the invention, methods of identifying
enzymes useful for producing stereoisomerically-pure or
stereoisomerically-enriched monatin compositions is provided. In
some embodiments, the method comprises screening hydrolytic enzymes
for stereoselective hydrolytic activity on an isoxazoline diester.
The screening comprises selecting a hydrolytic enzyme, such as
hydrolases, including microbial lipases and esterases, forming a
reaction mixture comprising the selected enzyme and an isoxazoline
diester, providing conditions under which a hydrolysis reaction of
the isoxazoline diester would be expected to occur, and analyzing
the reaction mixture for the presence of isoxazoline monoester in
enantiomerically-pure or enantiomerically-enriched form and/or
isoxazoline diester in enantiomerically-pure or
enantiomerically-enriched form.
[0011] The specification including the figures, describe certain
embodiments of the invention. A person of ordinary skill should
realize, however, from the description therein that the invention
is capable of modifications in various aspects, all without
departing from the spirit and scope of the invention. Accordingly,
the specification and figures are to be regarded as illustrative in
nature and not restrictive.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 is an LC-chromatogram of the reaction of isoxazoline
diester (Formula II, R.sup.1 and R.sup.2 are ethyl) with
carboxyesterase NP.
[0013] FIG. 2 is a chiral HPLC chromatogram of the carboxy esterase
NP-catalyzed reaction of isoxazoline diester (Formula II, R.sup.1
and R.sup.2 are ethyl) giving (R,R)- and (R,S)-monatin after
hydrogenation.
DESCRIPTION
[0014] This disclosure provides methods for stereoselectively
hydrolyzing a stereoisomeric mixture of isoxazoline diester. This
disclosure also provides enzymes capable of preferentially
hydrolyzing one isoxazline diester isomer over another isoxazoline
diester. This disclosure also provides methods of identifying
enzymes capable of stereoselectively hydrolyzing isomeric mixtures
of isoxazoline diesters.
[0015] This disclosure provides methods for producing mixtures of
(R,R) and (R,S) monatin as well as mixtures of (S,S) and (S,R)
monatin. The disclosure also provides methods for producing single
isomers of monatin. In some embodiments, the method comprises using
an enzyme to preferentially hydrolyze one isoxazoline diester
isomer over another into a monoester, separating the monoester from
the diester, converting the isoxazoline monoester into a mixture of
monatin diastereomers and resolving those diastereomers using
physical means known in the art, and converting the isoxazoline
diester into a mixture of monatin diastereomers and resolving those
diastereomers using physical means known in the art.
[0016] Each reference herein to a molecule containing chiral
centers, refers to all stereoisomeric forms of the molecule, unless
otherwise specified. Although each stereoisomer is a distinct
compound, in practice a mixture of stereoisomers is often referred
to as "a compound", such as e.g., "the compound (.+-.)isoxazoline
diethylester" or "isoxazoline diethylester". Similarly, each
structural depiction of a molecule herein containing chiral centers
represents all stereoisomeric forms of the molecule, unless
otherwise specified, for example through use of wedge diagrams to
show three-dimensional conformation.
[0017] Also, unless otherwise specified or unless otherwise clear
from the context, references to "R,R monatin" or "S,S monatin" mean
the single stereoisomer of monatin or a mixture enriched in the
specified stereoisomer. "Enriched" means that the mixture includes
a higher ratio of designated stereoisomer to non-designated
stereoisomer as compared to the original mixture from which it was
obtained.
[0018] A single stereoisomer can be differentiated from a
stereoisomerically-enriched mixture of stereoisomers by referring
to the former as a "single stereoisomer" or "single isomer" or
"single enantiomer," as appropriate. Thus, for example, unless
otherwise specified or unless otherwise clear from the context,
"(S,S) monatin" indicates the single stereoisomer of monatin with S
configuration at each stereogenic center, or a mixture enriched in
which (S,S) monatin.
[0019] Additionally, each compound formula designated with a Roman
numeral will follow a convention herein of meaning a single
stereoisomer or a mixture enriched in that stereoisomer when
followed by a lower case letter. For example, reference to "a
compound" of Formula II:
##STR00002##
refers to an isomeric mixture of the compound shown. (The use of
"(.+-.)" is included for clarity to designate that the indicated
formula includes a mixture of isomeric forms.) Similarly, reference
to "a compound" of Formula IIa:
##STR00003##
refers to both the single enantiomer shown and to a mixture
enriched in that enantiomer, unless otherwise specified or unless
otherwise clear from the context. (The use of "(R)" is included for
to designate that the indicated formula represents the R isomer or
a mixture enriched in the R isomer.) Typically, for the compounds
described herein, the letter "a" will designate compounds having an
"R" configuration, or where the compound includes two chiral
centers "a" designates the "R,R" or "R,S" configurations; and, "b"
will designate compounds having an "S" configuration or where two
chiral centers are present, the "S,S" or "S,R" configurations.
[0020] Unless otherwise specified, the terms "include," "includes,"
"including" and the like are intended to be open-ended. Thus, for
example, "include" means "include but are not limited to."
[0021] Except as otherwise noted, the articles "a," "an," and "the"
mean "one or more."
[0022] As used herein, the term "about" encompasses the range of
experimental error that occurs in any measurement. Unless otherwise
stated, all measurements are presumed to have the word "about" in
front of them even if the word "about" is not expressly used.
[0023] The term "alkyl" as employed herein by itself or as part of
another group refers to both straight and branched chain saturated
radicals of up to 10 carbons, unless the chain length is otherwise
limited, such as methyl, ethyl, propyl, isopropyl, butyl, s-butyl,
t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,
4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl and
the like.
[0024] An overall process according to an embodiment of the
invention is illustrated in Schemes 1a and 1b, below.
##STR00004##
##STR00005##
[0025] As Scheme 1a illustrates, the selected enzyme
stereoselectively hydrolyzes one isomer over another isomer, and in
the embodiment illustrated the R-isomer is preferentially
hydrolyzed over the S-isomer. Although Scheme 1a indicates that the
R-isomer may undergo hydrolysis at both esters, it is believed that
the selected enzyme will largely cleave only one of the esters. For
example, it is believed that the carboxylesterase NP (DSM) largely
cleaves the --CO.sub.2R.sup.2 ester. In particular, an experiment
carried out on a mixed methyl/ester diester showed that while the
carboxylesterase enzyme was able to cleave both esters, greater
than about 75% of the product was the compound of Formula IIIa'.
There was approximately 10% of the other regioisomer present in the
reaction.
[0026] In some embodiments, the present invention is directed to a
process comprising: hydrolyzing a compound of Formula II:
##STR00006##
wherein:
[0027] R.sup.1 and R.sup.2 are independently C.sub.1-10 alkyl;
to form a compound of Formula IIIa and/or IIIa':
##STR00007##
and a compound of Formula IIb:
##STR00008##
The compounds of Formula IIIa/IIIa' and IIb are useful in the
production of (R,R)-inonatin and (S,S)-monatin, respectively.
[0028] Useful values of R.sup.1 and R.sup.2 include alkyl,
particularly C.sub.1-6 alkyl, more particularly C.sub.1-4 alkyl.
Examples of useful values of R.sup.1 and R.sup.2 include methyl,
ethyl, propyl, isopropyl and butyl, especially methyl and ethyl. In
some embodiments, R.sup.1 and R.sup.2 are both ethyl.
[0029] The compound of Formula II can be synthesized by methods
known in the art. For example, synthesis of the compound of Formula
II in which R.sup.1 is methyl and R.sup.2 is ethyl is described in
C. W. Holzapfel, Synth. Comm. 24:3197-3211 (1994). See also U.S.
Pat. No. 5,128,482.
[0030] It will be apparent to one of ordinary skill in the art that
the compound of Formula IIb is "formed" from the compound of
Formula II by depletion of the other enantiomer. That is, if the
compound of Formula II is recognized as the mixture of the compound
of Formula IIa and the compound of Formula IIb (for example, a
racemic mixture of the compound of Formula IIa and the compound of
Formula IIb), then the preferential conversion of IIa to IIIa
results in enrichment of the starting racemate in the unhydrolyzed
enantiomer. Thus, the starting racemate II becomes a "compound of
Formula IIb" using the previously described nomenclature.
[0031] One of ordinary skill in the art will also appreciate that
few, if any, stereospecific reactions perfectly discriminate
between stereoisomers. Thus, hydrolysis of a compound of Formula II
may result in the formation of a compound of Formula Mb:
##STR00009##
via undesired hydrolysis of IIb. Such a result is contemplated by
the present invention, provided that the amount of the single
enantiomer of Formula IIIb formed is less than the amount of the
target single enantiomer formed (i.e., Formula IIIa and/or
IIIa').
[0032] Hydrolysis of the compound of Formula II may be accomplished
enzymatically or non-enzymatically. In an embodiment, enzymatic
hydrolysis may be accomplished using, the carboxy esterase,
Carboxyesterase NP. The enzyme activity Carboxylesterase NP was
first discovered in Bacillus thai 1-8, which was deposited under
the accession number CBS 679.85 (Yuki, S., 1967, Jpn. J. Genet. 42
p 251). The gene for Carboxylesterase NP was subsequently cloned
into Bacillus subtilis 1-85/pNAPT-7, which was deposited under the
accession number CBS 673.86 (W J Quax and C. P. Broekhuizen 1994.
Development of a new Bacillus carboxyl esterase for use in the
resolution of chiral drugs. Journal Applied Microbiology and
Biotechnology 41: 425-431). This was the organism used in the
present studies. The gene was also cloned and deposited as Bacillus
subtilis 1A-40/pNAPT-8 (CBS 674.86), and Bacillus subtilis
1A-40/pNAPT-7 (CBS 675.86). The gene sequence for Carboxylesterase
NP has been published (Melloney J. Droge, Rein Bos and Wim J. Quax
(2001). Paralogous gene analysis reveals a highly enantioselective
1,2-O-isopropylideneglycerol caprylate esterase of Bacillus
subtilis. Eur. J. Biochem. 268: 3332-3338).
[0033] The products of the enzymatic reaction, for example,
compounds of Formula IIIa and Formula IIb, can be separated using
chromatographic techniques. One embodiment of the method of the
invention is exemplified in FIG. 1. As shown on FIG. 1, isoxazoline
diester (Formula II, R.sup.1 and R.sup.2 are ethyl) was
enzymatically reacted with carboxyesterase NP and the products were
separated and analyzed by LC-MS (liquid chromatography-mass
spectrometry) chromatography (here, exemplified using a Waters
Platform LC-MS system).
[0034] Further or alternatively, taking advantage of the different
reactivities of carboxylic acids and esters (see March and Larock,
supra) one of the compounds of Formula IIIa or Formula IIb may be
transformed into a different compound prior to separation from the
other. For example, borane reduces carboxylic acids much more
readily than it does esters (see, e.g., March at pages 1544-46).
Thus, the mixture can be subjected to reducing conditions using
borane that will selectively reduce the compound of Formula IIIa to
its corresponding alcohol. This alcohol can then be separated from
the compound of Formula IIb by standard techniques, such as
chromatography. The present invention contemplates any such
separation of the compound of Formula IIIa, or a compound derived
from it, from the compound of Formula IIb, or a compound derived
from it, following the selective hydrolysis described above, as
"separating the compound of Formula IIIa from the compound of
Formula IIb" as a means to obtaining enantiomerically pure or
enantiomerically enriched (R,R)- or (S,S)-monatin.
[0035] In some embodiments, the present invention further comprises
converting the compound of Formula IIIa into a compound of Formula
Ia:
##STR00010##
i.e., into (R,R)-monatin.
[0036] In some embodiments, conversion of the compound of Formula
IIIa into a compound of Formula Ia may be accomplished by
[0037] (a) hydrolyzing the compound of Formula IIIa to form a
compound of Formula IVa:
##STR00011##
and
[0038] (b) hydrogenating the compound of Formula IVa.
[0039] Useful reagents for hydrolysis are well known to those of
ordinal skill in the art and are described in, e.g., M. B. Smith
and J. March, March's Advanced Organic Chemistry, 5th ed., New
York: John Wiley & Sons, Inc., 2001 ("March"), particularly at
pages 469-74; and R. C. Larock, Comprehensive Organic
Transformations, 2nd ed., New York: John Wiley & Sons, Inc.,
1999 ("Larock"), particularly at pages 1959-68. Examples of useful
reagents for hydrolysis include Group IA and IIA alkoxides such as
LiOH, NaOH, KOH and Ba(OH).sub.2. Other useful reagents include
Sm/I.sub.2/MeOH and MgI.sub.2. Methyl esters may also be cleaved
with, e.g., (Na.sub.2CO.sub.3 or K.sub.2CO.sub.3)/MeOH/H.sub.2O,
NaO.sub.2/DMSO, KSCN/DMF, EtSH/(AlCl.sub.3 or AlBr.sub.3),
Me.sub.2SI(AlCl.sub.3 or AlBr.sub.3), (Li or Na)SeCH.sub.3/DMF,
NaCN/HMPA, (LiI or LiBr)/DMF, LiI/(NaOAc or NaCN)/DMF, BCl.sub.3,
AlI.sub.3 or MeSiCl.sub.3. In some embodiments, hydrolysis is
carried out using KOH in an alcoholic solvent such as MeOH or
EtOH.
[0040] In addition, hydrolysis of the compound of Formula IIIa into
a compound of Formula IVa can be accomplished using a hydrolase,
and especially a carboxyesterase, that is capable of accepting a
compound of the Formula IIIa as a product and of producing a
compound of Formula IVa.
[0041] Useful conditions for hydrogenation are well known to those
of ordinary skill in the art and are described in, e.g., March and
Larock, supra. In some embodiments, hydrogenation is carried out
using H.sub.2 and a sponge nickel catalyst.
[0042] If the hydrogenation of the compound of Formula IVa is not
done stereoselectively, it will form a mixture of a compound of
Formula Ia and a compound of Formula Ic:
##STR00012##
See, for example, FIG. 2, showing a chiral HPLC chromatogram of the
carboxyesterase NP-catalyzed reaction of isoxazoline diester
(Formula II, R.sup.1 and R.sup.2 are ethyl) giving (R,R)- and
(R,S)-monatin after hydrogenation.
[0043] The compounds of Formula Ia and Formula Ic can then
separated from each other, e.g., by selective crystallization.
These compounds are related as diastereomers, and the separation of
diastereomers is well-known and considered routine in the art. See,
e.g., D. Kozma (ed.), CRC Handbook of Optical Resolutions via
Diastereomeric Salt Formation, CRC Press: Boca Raton (2002).
[0044] In some embodiments, the present invention further comprises
converting the compound of Formula IIb into a compound of Formula
Ib:
##STR00013##
[0045] In some embodiments, conversion of the compound of Formula
IIb into a compound of Formula Ib may be accomplished by (a)
hydrolyzing the compound of Formula IIb to form a compound of
Formula IVb:
##STR00014##
and
[0046] (b) hydrogenating the compound of Formula IVb.
[0047] If the hydrogenation of the compound of Formula IVb is not
done stereoselectively, it will form a mixture of a compound of
Formula Ib and a compound of Formula Id:
##STR00015##
The compounds of Formula Ib and Formula Id can then be separated
from each other. These compounds are related as diastereomers, and,
as discussed above, the separation of diastereomers is well-known
and considered routine in the art.
[0048] Certain processes of the invention are illustrated in the
following examples. While multiple embodiments are disclosed
herein, still other embodiments of the present invention may become
apparent to those skilled in the art from review of the entirety of
this specification. As should be realized from the description
herein, the invention is capable of modifications in various
aspects, all without departing from the spirit and scope of the
present invention. Accordingly, the drawing and entirety of the
description are to be regarded as illustrative in nature and not in
a limiting sense.
EXAMPLES
Example 1
Screening of Hydrolytic Enzymes for Isoxazoline Diester
[0049] The screening is clone in 0.1 M sodium phosphate buffer pH
7.1% Triton X-100 is added to the buffer and then isoxazoline
diester is added slowly at 37.degree. C. until saturation is
reached.
[0050] Aliquots of 1.5 ml of the above solution are placed in
Eppendorff tubes and different hydrolytic enzymes are added to the
tubes. A microspatula tip of the powdered enzymes is used or 100
.mu.l of the liquid enzymes. The reactions are incubated for 20
hours on a vibrating shaker at an appropriate temperature,
generally, room temperature.
[0051] Following the incubations, the reaction mixtures are
centrifuged at 10,000 rpm for 2 minutes to remove any insoluble
material. The samples are then analysed on HPLC. A Phenomenex Luna
C18 column is used as a linear gradient consisting of eluent A:
0.1% H.sub.3PO.sub.4 in TEA pH 3.5 and eluent B: 100%
acetonitrile.
Example 2
Evaluation of Carboxylesterase NP (DSM) Activity for Isoxazoline
Diester
[0052] Only Carboxylesterase NP, was tested for activity according
to the protocol in Example 1. The HPLC yielded a single product
peak, and corresponding to enantiopure R-monatin according to
chiral-HPLC and LC-MS following hydrogenation and workup.
Example 3
Synthesis of Monatin
[0053] Carboxylesterase NP was re-tested on 10 ml scale in order to
do further analytical work.
[0054] 10 ml of a 0.1 M sodium phosphate buffer containing 1%
Triton X-100 was placed in a test tube. 50 mg of substrate was
added and either 50 mg of the dry enzyme or 1 ml of the liquid
enzyme. The tube was sealed and the reaction shaken at 180 rpm for
20 hours at 40.degree. C. (to prevent microbial growth). The
unreacted substrate was removed by centrifugation at 3000 rpm for 3
minutes and any excess substrate removed by extraction with an
equal volume of ethyl acetate. The aqueous phase was the hydrolysed
with KOH and then hydrogenated to yield monatin.
[0055] The reaction resulted in R'R' monatin as well as R'S'
monatin in approximately equal amounts as determined by achiral and
chiral analysis as well as LS-MS. FIG. 2 is a chiral HPLC
chromatogram of the carboxy esterase NP-catalyzed reaction of
isoxazoline diester (Formula II, R.sup.1 and R.sup.2 are ethyl)
giving (R,R)- and (R,S)-monatin after hydrogenation.
Example 4
Enzymatic Hydrolysis of Isoxazoline Diester with Carboxylesterase
NP
[0056] The reaction was done in 0.1 M phosphate buffer containing
1% Triton X-100 at 37.degree. C. and pH 7. The substrate
isoxazoline diester was added to 200 mL of the buffer, and the
mixture was stirred until a saturated solution was reached. 200
.mu.L of the liquid enzymes Carboxylesterase NP (DSM) was added to
2 mL of reaction mixture. The reaction was agitated at 40.degree.
C. for 20 hours at 230 rpm. At the end of the reaction, the
reaction mixture was transferred to an Eppendorf tube and
centrifuged to remove any undissolved material. The supernatant was
analyzed on a Waters Platform LC-MS system. The reaction mixture
was extracted with ethyl acetate to remove unreacted substrate,
leaving the hydrolyzed product in the aqueous reaction mixture. The
chromatographic separation, the output is shown in FIG. 1, was on a
Waters Alliance 2695 HPLC system and a Phenomenex Gemini C18 column
(250 min.times.2.1 mm (5 .mu.m)). The starting eluent was 95% water
containing 10 mM ammonium acetate (pH 4.5 with acetic acid) and 5%
acetonitrile. These conditions were maintained for 5 minutes
followed by a linear gradient to 100% acetonitrile at 15 minutes,
and then maintained at 100% acetonitrile for 2 minutes. The column
was then returned to initial conditions and allowed to stabilize
for 7 minutes. The total run time was 30 minutes. The flow rate was
0.2 mL/min and the column temperature was kept stable at 40.degree.
C. The mass spectrometer was operated in electrospray mode,
utilising +/- voltage switching. The analysis conditions can be
summarised as follows:
[0057] Capillary voltage 2.5 kV; Cone voltage 25 V; Extractor lens
voltage 1 V; RF lens voltage 0.5 V; Source block temperature
120.degree. C.; desolvation temperature 450.degree. C.; Mass range
scanned 100-400 Daltons (0.5 seconds cycle time). Nitrogen was used
as nebulization and desolvation gas at a flow rate of 75 and 575
l/hr respectively. A Waters 2996 Photo Diode Array (PDA) detector
was used to optimize the chromatographic separation and was used in
scan mode covering the 200-600 nm wavelength range.
Example 5
Chemical Hydrolysis and Hydrogenation
[0058] The reaction was carried out in a 20 mL pressure reactor.
The reaction mixture as produced in Example 2 (10 mL volume
containing 25 mg of monoester) was treated with ethanol (1.5 mL)
and the pH was adjusted to 12 with potassium hydroxide (200 mg).
The resulting mixture was stirred at room temperature for 30
minutes, after which analysis by HPLC showed complete hydrolysis to
the diacid. To this mixture was added a sponge nickel catalyst
(A-7063, 50 mg) as a wet paste containing about 50% water. The
reactor was sealed, evacuated and purged with hydrogen gas three
times, after finally pressurizing the vessel to 5 bar with
hydrogen. The reaction was continued for 60 minutes under these
conditions with stirring, and monitored by HPLC.
[0059] Having now fully described this invention, it will be
understood by those of ordinary skill in the art that the same can
be performed within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any embodiment thereof. All patents and
publications cited herein are fully incorporated by reference
herein in their entireties.
* * * * *