U.S. patent application number 10/624640 was filed with the patent office on 2004-11-25 for synthesis and recovery of aspartame involving enzymatic deformylation step.
Invention is credited to Quaedflieg, Peter J.L.M., Sonke, Theodorus, Wagner, Adolf F.V..
Application Number | 20040234944 10/624640 |
Document ID | / |
Family ID | 56290158 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040234944 |
Kind Code |
A1 |
Quaedflieg, Peter J.L.M. ;
et al. |
November 25, 2004 |
Synthesis and recovery of aspartame involving enzymatic
deformylation step
Abstract
The invention relates to the synthesis of aspartame involving
enzymatic deformylation of an
N-formyl-.alpha.-L-aspartyl-L-phenylalanine compound by treatment
with an enzyme having formylmethionyl peptide deformylase activity
and having as a co-factor group 5 to 11 bivalent metal ions. The
invention also relates to selective preparation and recovery of
aspartame from a mixture of N-formyl-.alpha.- and
.beta.-L-aspartyl-L-phenylalanine compounds by treatment with such
enzyme. And finally, the invention relates to one-pot enzymatic
synthesis of aspartame from N-formyl-L-aspartic acid and L- or
D,L-phenylalanine methyl ester involving an enzymatic deformylation
reaction simultaneously with an enzymatic coupling reaction, as
well as to one-pot di- or oligopeptide synthesis by simultaneous
enzymatic coupling and deformylation reactions in general.
Inventors: |
Quaedflieg, Peter J.L.M.;
(Geleen, NL) ; Sonke, Theodorus; (Guttecoven,
NL) ; Wagner, Adolf F.V.; (Ludwigsburg, DE) |
Correspondence
Address: |
PILLSBURY WINTHROP, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Family ID: |
56290158 |
Appl. No.: |
10/624640 |
Filed: |
July 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10624640 |
Jul 23, 2003 |
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09886476 |
Jun 22, 2001 |
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6617127 |
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09886476 |
Jun 22, 2001 |
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PCT/NL99/00787 |
Dec 20, 1999 |
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60119077 |
Feb 8, 1999 |
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Current U.S.
Class: |
435/4 |
Current CPC
Class: |
C07K 5/0613 20130101;
C12P 41/007 20130101; C12N 9/80 20130101; C12P 21/02 20130101 |
Class at
Publication: |
435/004 |
International
Class: |
C12Q 001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 1998 |
EP |
98204373.9 |
Claims
1-29. (Canceled).
30. Method for the synthesis of di-or oligopeptides or derivatives
thereof from two starting materials, the first of which is an
N-formyl protected amino acid which is capable of undergoing an
enzymatic coupling reaction with a second amino acid or derivative
thereof, or with a di-or oligo-peptide or derivative thereof,
compound, wherein the N-formyl protecting group of the first
starting material is retained during the enzymatic coupling
reaction with the second starting material, whereby said protecting
group is cleaved off enzymatically, using an enzyme having
formylmethionyl peptide deformylase activity and having as a
co-factor bivalent metal ions chosen from the group of group 5 to
11 metals from the periodic system of elements, from the reaction
compound at a substantially higher, i. e. at least 10.times.
higher, rate than from the first starting material, and wherein two
enzymes are involved simultaneously for the enzymatic coupling
reaction between the starting materials and the enzymatic
deformylation of the reaction compound.
Description
[0001] The invention relates to a method for synthesis of
.alpha.-L-aspartyl-L-phenylalanine methyl ester (.alpha.-APM;
aspartame) involving enzymatic deformylation of an
N-formyl-.alpha.-L-aspartyl-L-phe- nylalanine compound. An
N-formyl-.alpha.-L-aspartyl-L-phenylalanine compound as meant
herein is understood to be either
N-formyl-.alpha.-L-aspartyl-L-phenylalanine or its methyl ester
(F-.alpha.-AP or F-.alpha.-APM). The invention also relates to a
method for preparation and recovery of .alpha.-APM from either (i)
a mixture of N-formyl-.alpha.- and
N-formyl-.beta.-L-aspartyl-L-phenylalanine or (ii) a mixture of
N-formyl-.alpha.- and N-formyl-.beta.-L-aspartyl-L-phenylala- nine
methyl ester (F-.alpha..beta.-AP or F-.alpha..beta.-APM) by
enzymatic deformylation. And finally, the invention relates to a
simple method for one-pot enzymatic synthesis of .alpha.-APM from
N-formyl-L-aspartic acid (F-Asp) and L- or D,L-phenylalanine methyl
ester (L- or D,L-PM) also involving an enzymatic deformylation
reaction. The latter combination of simultaneous enzymatic coupling
and deformylation reactions has wider and more general
applicability.
[0002] Aspartame (.alpha.-APM, L,L-form) is known to be a high
intensity artificial sweetener, having a sweetness which is about
200.times. as potent as the sweetness of sucrose, whereas its tast
properties are close to those of sucrose. The .beta.-form of APM
does not have sweet taste properties. .alpha.-APM is used for the
sweetening of various edible materials. Synthesis methods for
.alpha.-APM include chemical syntheses routes (e.g. by coupling of
the anhydride of F-Asp with L-Phe or L-PM) which are invariably
leading to mixtures of .alpha.- and .beta.-forms of F-APM in ratios
of about 70/30 to 80/20 wt./wt.). Apart from the required
deformylation, these chemical methods therefore also require
separation of the .alpha.-APM from .beta.-APM and large recycle
streams for recovery of .alpha.-APM in high purity and adequate
yield. The synthesis methods for .alpha.-APM also include enzymatic
coupling methods (e.g. by coupling of F-Asp or
N-benzyloxy-carbonyl-L-Asp, also known as Z-Asp, with D,L-PM or
L-PM). The enzymatic methods have the clear advantage that they
selectively yield the .alpha.-coupled L,L-product in protected
form. Although still needing a deprotection step, the enzymatic
coupling routes therefore do not require difficult separation of
.alpha.-APM from .beta.-APM nor large recycle streams for recovery
of .alpha.-APM in high purity and adequate yield. In the state of
the art processes for the synthesis of .alpha.-APM many of those
processes involve formyl-protection routes, and thus will need a
deformylation step in one of the final stages of the process.
Chemical deformylation, which is often performed in aqueous medium
containing methanol and a strong acid, has the disadvantage that
also demethylation of the phenylalanine methyl ester part of the
molecule will occur, and mixtures of many compounds will be
obtained in any subsequent methylation step. Enzymatic
deformylation is believed to take place under mild conditions, and
thus without simultaneous demethylation. However, so far no
suitable enzymatic deformylation process seems to be available.
[0003] Enzymatic deformylation of an
N-formyl-.alpha.-L-aspartyl-L-phenyla- lanine compound, namely of
F-.alpha.-APM, is disclosed in Example 11 of U.S. Pat. No.
4,668,625. This patent teaches that penicillin-acylases are not
suitable for removal of formyl-groups from oligopeptides. For
instance, in Example 11 the yield is described to be 20% after 36
hours of reaction at an extremely high concentration of active
enzyme as compared to the F-.alpha.-APM (namely 50 U of enzyme and
2 g of F-.alpha.-APM). However, even these results could not be
reproduced by the present applicants. This is, amongst other
things, because no clear disclosure is given of the Pseudomonas
strain used and of the method of isolating the enzyme having
penicillin-acylase activity therefrom.
[0004] Thus, there is need for an improved method of preparation
for synthesis of .alpha.-L-aspartyl-L-phenylalanine methyl ester
(.alpha.-APM; aspartame) involving enzymatic deformylation of an
N-formyl-.alpha.-L-aspartyl-L-phenylalanine compound, without the
disadvantages as mentioned above.
[0005] It now surprisingly has been found that enzymes having
formylmethionyl peptide deformylase activity (hereinafter for
convenience also represented by PDF or by PDF enzyme) can be used
in the synthesis of aspartame. The method for synthesis of
.alpha.-L-aspartyl-L-phenylalanine methyl ester by enzymatic
deformylation of an N-formyl-.alpha.-L-aspartyl- -L-phenylalanine
compound according to the present invention comprises treating
N-formyl-.alpha.-L-aspartyl-L-phenylalanine or its methyl ester
with an enzyme having formylmethionyl peptide deformylase activity
and having as a co-factor bivalent metal ions chosen from the group
of group 5 to 11 metals from the periodic system of elements.
[0006] The periodic system of elements (new IUPAC version) and the
group numbers as meant herein are presented in the Handbook of
Chemistry and Physics, 70th edition, CRC Press, 1989-1990, inner
page of cover. Bivalent metal ions from the group of group 5 to 11
metals are, for instance, V.sup.2+, Cr.sup.2+, Mn.sup.2+,
Fe.sup.2+, Co.sup.2+, Ni.sup.2+, Cu.sup.2+, Pd.sup.2+, and
Pt.sup.2+. Mn.sup.2+, Fe.sup.2+, Co.sup.2+ and Ni.sup.2+ are
preferred.
[0007] Preferably the amount of the bivalent metal ions should be
about equivalent to the number of moles of enzyme. Suitably the
molar ratio between these bivalent metal ions and the number of PDF
molecules is in the range of 0.6 to 1.4, preferably of 0.8 to 1.2,
and most preferred the amount of bivalent metal ions is equimolar
to the enzyme.
[0008] In case F-.alpha.-AP is used as a starting material, of
course, a final methylation step of the phenylalanine carboxylic
acid group needs to be carried out in order to obtain the desired
aspartame final product. Methods for such methylation are known to
the skilled man, e.g. from U.S. Pat. No. 4,946,988 and
EP-A-0468063.
[0009] Recovery of .alpha.-APM from the reaction mixture can be
done by any method known to the skilled man. Various methods of
crystallisation of aspartame have been described in the literature,
e.g. in EP-A-0091787, EP-A-0399605 and EP-A-0582351. Preferably the
recovery of .alpha.-APM is done at a pH near the iso-electric point
of .alpha.-APM, i.e. at a pH in the range of 3 to 7.
[0010] The present invention is in particular surprising as there
has not been any indication in the state of the art so far that PDF
enzymes are also suitable for deformylating terminal
N-formyl-L-aspartic acid residues in oligopeptides or
dipeptides.
[0011] Enzymes having formylmethionyl peptide deformylase activity
(PDF's) are widely available in nature. Mostly they are being
described as formylmethionine deformylases. It should be noticed,
however, that in the literature also other names are being used
instead of the name formylmethionine deformylase; in particular the
following names may be mentioned here: (poly)peptide deformylase,
N-formylmethionyl-aminoacyl-tR- NA deformylase,
N-formyl-L-methionine amidohydrolase,
N-formylmethionyl-aminoacyl-tRNA amidohydrolase.
[0012] The native PDF's in nature, e.g. in eubacteria, catalyse the
deformylation of the formyl group from the terminal N-methionine
residue in nascent polypeptides; more specifically, the PDF's
catalyse the hydrolysis of the N-formyl group from the N-terminal
L-methionine residue of nascent polypeptides synthesized by the
ribosomal protein synthesis machinery. However, no other practical
applications of these enzymes are known so far. On the contrary,
Rajagopalan et al. (Biochem. 36, 13910-8 (1997)) have established
that deformylases have strong sequence preference for methionine at
the N-terminus of peptide substrates and to a lesser extent for
norleucine at that site. It is thus surprising that these enzymes
can be used favourably in the synthesis of .alpha.-APM.
[0013] The PDF's are obtainable, for instance, from eubacteria, for
example Escherichia coli, Bacillus subtilis, Clostridium
beijerinckii, Clostridium acetobutylicum, Thermotoga maritima,
Thermus aguaticus, Thermus thermophilus, Calothrix PCC 7601,
Haemophilus influenzae, Bacillus stearothermophilus or Lactococcus
lactis. Preferably an enzyme from Escherichia coli is used.
[0014] The inventors have found that the PDF's in order to be able
to be used in the synthesis methods according to the present
invention require as a co-factor bivalent metal ions chosen from
the group of group 5 to 11 metals from the periodic system of
elements.
[0015] The reaction conditions for the enzymatic deformylation
according to the invention are not very critical. Any suitable
solvent system which is inert towards the PDF may be applied; such
solvents include aqueous systems (solutions or slurries) or aqueous
systems also containing a water-miscible organic solvent which is
inert under the reaction conditions. Aqueous systems, however, are
preferred. Also the concentration of the N-formyl compound is not
critical, and may be for instance in the range of about 10 to 1000
mM. It is not necessary that all of the N-formyl compound is
dissolved; part of it may be present as a slurry. The concentration
of the PDF likewise is not very critical, and usually will be at
0.001 to 100%, normally less than 30.0% by weight of the formyl
compound, e.g. at about 0.2 mM of PDF. The pH for the reaction
preferably is chosen in the range of 3.0 to 9.0, more preferably of
4.0 to 8.0. The temperature is not very critical, and suitably will
be in the range of to 50.degree. C., e.g. at about 37.degree. C.,
but for thermostable PDF enzymes higher temperatures may be
applied.
[0016] Until now a lot of work has been done with regard to the
isolation and identification of PDF's. The following paragraphs
give some more information on PDF's. Furtheron in this application,
it will be made clear how PDF's having bivalent metal ions as
required for the present invention can be obtained.
[0017] An example of a native PDF is the PDF-enzyme from E. coli.
This has been shown to be a monomeric enzyme having a length of 168
amino acids with a molecular mass of 19197 Dalton as calculated
from its amino acid sequence (minus the N-terminal methionine,
which is removed by a post-translational modification step). This
enzyme is made up of an active core domain composed of amino acids
1 to 147, and a C-terminal domain of amino acids 148-168; the
latter domain, however, is not essential for the deformylase
activity. The PDF coding gene from E. coli has been cloned (Mazel
et al., EMBO J., 13, 914-923, (1994)) and very successful
over-expression in E. coli of up to about 10.sup.2 to 10.sup.3
times of over-expression has been described using different
expression constructs (Groche et al., (Biochem. and Biophys. Res.
Comm., 246, 342-346 (1998)). Recently a comparison between the
three-dimensional structures of the Zn.sup.2+, Fe.sup.2+ and
Ni.sup.2+ containing E. coli PDF's has been published (Becker et
al., Nature Structural Biology, 5, 1998, 1053-1058).
[0018] Studies of PDF's, however, so far have been seriously
hampered for a very long period of time because of the extreme
lability of PDF's during purification, storage, and testing in
dilute form. Based on the results of the biochemical
characterisation studies with these extremely labile enzyme
preparations, it was assumed until recently (e.g. see Meinnel et
al., J. Mol. Biol., 262, 375-386, (1996)) that the native PDF
enzyme from E. coli contains one Zn.sup.2.sup.+ ion per enzyme
molecule. The PDF's still are being considered to be a distinct
family belonging to the zinc metalloprotease superfamily, but the
Zn.sup.2+ ions are not suitable as a co-factor in the method of the
present invention.
[0019] Recently it has been shown that PDF may be effectively
stabilized during purification, storage and testing for
formylmethionyl peptide deformylase activity (in dilute form of the
enzyme), by the addition of catalase. This and alternative
stabilizing measures led to the further insight (see Rajagopalan,
et al., J. Am. Chem. Soc., 119, 12418-12419 (1997), Becker et al.,
J. Biol. Chem., 273, 11413-6 (1998), Groche et al., (Biochem. and
Biophys. Res. Comm., 246, 342-346 (1998), and Ragusa et al., (J.
Mol. Biol., 280, 515-523 (1998)) that the presence of Zn.sup.2+ in
the earlier purified enzyme preparations from E. coli is mainly to
be attributed to the isolation methods used, and that the Zn.sup.2+
containing enzyme is (almost) completely catalytically inactive.
The addition of catalase, and/or alternative stabilising measures,
during the PDF purification procedure allows purification of the
native, catalytically fully active E. coli PDF, which could be
proven to contain one Fe.sup.2+ ion per enzyme molecule instead of
a Zn.sup.2+ ion. It has been found that taking such stabilizing
measures is also suitable during the methods for synthesis of
.alpha.-APM according to the present invention.
[0020] One of the measures which are suitable for stabilizing the
PDF is ensuring that the PDF's are being handled in an environment
having reduced O.sub.2 content, preferably under anaerobic
conditions. Under such conditions almost no reduction of molecular
oxygen to hydrogen peroxide takes place, with simultaneous
oxidation of the bivalent metal ion, such as Fe.sup.2+ Reduction of
the content of dissolved O.sub.2 can also be accomplished, by
enzymatic removal of thereof, for instance by using a glucose
oxidase/catalase system (Rajagopalan, P. T. R., and D. Pei, J.
Biol. Chem., 273, No. 35, 22305-22310, (1998)). The presence of
catalase prevents oxidation of the enzyme bound Fe.sup.2+ to
Fe.sup.3+, which renders the PDF catalytically inactive. Fe.sup.3+
is bound much more weakly than Fe.sup.2+, and is therefore readily
exchanged by Zn.sup.2+ for which the metal ion binding site of the
PDF has a much higher affinity. The net effect of such cascade of
events is inactivation of the PDF.
[0021] Stabilisation of PDF's also can be achieved by alternative
stabilising measures, namely by the addition/presence of other
stabilisation agents, for instance of trialkylphosphine compounds
or derivatives thereof; examples of such compouns are
triethylphosphine, tributylphosphine and TCEP
(tris-(2-carboxyethyl)-phosphine). Similarly as catalase, these
stabilisation agents easily react with H.sub.2O.sub.2 or other
peroxides. Further, inactivation of PDF's also can be prevented by
handling the PDF's at higher concentration, for instance at a PDF
concentration of at least 0.1 mg of PDF per ml, more preferably of
at least 1.0 mg/ml. The upper limit of the concentration of PDF is
not critical if practical concentrations are being used.
[0022] Exchange of the bivalent metal ions in the PDF's in order to
obtain PDF enzymes with a co-factor as necessary for the present
invention can be done by the various methods as described in Groche
et al., Biochem. Biophys. Res. Comm., 246, 342-346, (1998). These
methods include simple metal displacement by incubation of the
native enzyme in an excess of the desired bivalent metal ion, if
necessary preceeded by the preparation of the apoenzyme via
treatment of the native enzyme with a metal chelation compound.
Furthermore, the desired bivalent metal ion can already be
introduced in (at least part of the enzyme molecules) by using a
bacterial growth medium with an enhanced ratio of the desired
bivalent metal ion over Fe.sup.2+.
[0023] It is to be noticed that, instead of the enzymes used
according to the method of the invention, of course, also whole
cells, enzyme preparations, immobilised enzymes, etc. can be
applied which are having formylmethionyl peptide deformylase
activity. The terms enzyme, PDF, etc. as used herein, therefore
also include such other forms of active enzyme, including
genetically engineered mutants thereof, which for instance have
enhanced activity or selectivity in the deformylation reaction.
[0024] The enzymes to be used can be classified according to
standard classification schemes for enzyme activities. A very
important group of enzymes having formylmethionyl peptide
deformylase activity is classified as EC 3.5.1.27.
[0025] Preferably, the enzyme therefore is an enzyme having the
activity as described for EC 3.5.1.27 because excellent results are
being achieved in the deformylation with such enzymes. It should be
noticed, although until recently it was believed that the enzyme
coded as EC 3.5.1.31 is catalyzing a different reaction, but in the
meantime it has been shown that the enzymes known as EC 3.5.1.27
and EC 3.5.1.31 are coded for by exactly the same gene and have the
same activity. Therefore, as used herein, the term EC 3.5.1.27 is
encompassing not only EC 3.5.1.31, but likewise all other enzymes
having the same activity as described for EC 3.5.1.27.
[0026] Although the family of PDF's is composed of proteins with a
relatively low level of sequence identity, the 3D structures of the
members of this family appear closely related one to each other
with, in particular, the building of a common fold around the
bivalent metal ion and three signature sequences. As is described
(for PDF's indicated as PDF) by Wagner et al., J. Biol. Chem., 273,
11413-6 (1998), for many of these enzymes characteristically three
short amino acid stretches are present as strictly conserved
motifs, namely in that the enzymes contain the sequences (i) HEXXH,
(ii) EGCLS and (iii) GXGXAAXQ. In these sequences X represents any
natural amino acid, and standard one letter codes for amino acids
are used: A=alanine, C=cysteine, E=glutamic acid, G=glycine,
H=histidine, L=leucine, S=serine and Q=glutamine.
[0027] Preferably the method of the present invention is carried
out using a PDF which is obtainable from E. coli.
[0028] In particular the co-factor bivalent metal ions in the PDF
preferably are manganese, iron, cobalt and nickel ions. It has been
indicated hereinbefore how such exchange of co-factor bivalent
metal ions can be achieved.
[0029] Preferably in the method of the present invention PMdf's are
being used which contain Fe.sup.2+ and/or Ni.sup.2+ ions in the
active site because then highest activity and stability of the
enzymes is observed. Especially preferred are PDF's which contain
Ni.sup.2+ ions, because those enzymes are much more resistent
towards oxidation, and consequently more stable. As a consequence,
the taking of stabilising actions and/or addition/presence of a
stabilisation agent in such case is less important. Interestingly,
the exchange of Fe.sup.2+ for Ni.sup.2+ does not significantly
affect the enzyme's specific deformylating activity, which is at
the same high level as with the Fe.sup.2+ enzyme.
[0030] However, PDF's containing Fe.sup.2+ ions (about one molar
equivalent per molecule of enzyme) are most preferred, since they
do not require metal exchange as compared to their native
occurrence and because any remaining presence of the metal ions in
the final product to be obtained is not likely to cause any
problems, for instance toxicological problems. Of course, when
isolating the enzyme and when using it in the course of the present
invention, measures should be taken to avoid exchange of Fe.sup.2+
by Zn.sup.2+. Thus, preferably the bivalent metal ions are
Fe.sup.2+ ions and all treatments with the PDF enzyme are carried
out in the presence of a stabilisation agent.
[0031] The term "in the presence of a stabilisation agent" as used
here and hereinafter, is meant to include all measures shown above
for the stabilisation of the PDF's.
[0032] Preferably the stabilisation agent is catalase or a
trialkylphosphine compound or derivative, most preferably it is
catalase.
[0033] In the course of inventors' studies on the use of the PDF's
according to the invention in the synthesis of .alpha.-APM
surprisingly a further advantage of such use has been found. In
particular, it was observed that these enzymes are very
regioselective. In particular they have a very high activity for
deformylation of F-.alpha.-AP and F-.alpha.-APM, whereas they have
no or only low activity for deformylation of F-.beta.-AP or
F-.beta.-APM. Moreover, it has been found that presence of
F-.beta.-AP or F-.beta.-APM does not give any inhibition of the PDF
in the deformylation of F-.alpha.-AP and F-.alpha.-APM. These
respective activities towards the .alpha.- and .beta.-forms of F-AP
and of F-APM now have been found to differ by at least a magnitude
of 20.times. for F-AP, and by a magnitude of 200.times. or more for
F-APM. Thus, in a preferred embodiment of the present invention an
elegant and low-cost alternative is provided for the otherwise
difficult separation of the .alpha.- and .beta.-forms of AP and/or
APM in .alpha.-APM production routes using formyl protection.
[0034] In this preferred embodiment of the invention the
surprisingly found .alpha.-selectivity of the PDF enzymes according
to the invention is used in selectively recovering .alpha.-APM from
a mixture of F-.alpha.-APM and F-.beta.-APM, or in selectively
preparing .alpha.-AP from a mixture of F-.alpha.-AP and
F-.beta.-AP, followed by conversion of the .alpha.-AP into
.alpha.-APM, and recovering the .alpha.-APM.
[0035] In this embodiment of the invention either (i) a mixture of
N-formyl-.alpha.- and N-formyl-.beta.-L-aspartyl-L-phenylalanine
(F-.alpha..beta.-AP) or (ii) a mixture of N-formyl-.alpha.- and
N-formyl-.beta.-L-aspartyl-L-phenylalanine methyl ester
(F-.alpha..beta.-APM) is treated with an enzyme having
formylmethionyl peptide deformylase activity (PDF) and having as a
co-factor bivalent metal ions chosen from the group of group 5 to
11 metals from the periodic system of elements, with the formation
of .alpha.-L-aspartyl-L-phenylalanine or of its methyl ester,
respectively, whereby in case .alpha.-L-aspartyl-L-phenylalanine
(.alpha.-AP) is formed in the deformylation step a subsequent
methylation step of the phenylalanine carboxylic acid group is
carried out, and the .alpha.-L-aspartyl-L-phenylalanine methyl
ester (.alpha.-APM) is recovered.
[0036] The subsequent methylation step, if applicable, and the
recovery of .alpha.-APM are known to the skilled man, as has been
described hereinabove.
[0037] The selective preparation of .alpha.-AP(M) from a mixture of
F-.alpha.-AP(M) and F-.beta.-AP(M) in particular can suitably be
used in combination with chemical synthesis methods for AP(M) by
formyl-protection routes. Until the present invention the
formyl-deprotection step in such routes is troublesome and requires
long reaction times and, usually, a further esterification step.
Moreover, in such processes a mixture of at least eight compounds
(four .alpha.-compounds: .alpha.-APM, .alpha.-AP, .alpha.-AMP and
.alpha.-AMPM, and their corresponding .beta.-compounds; in AMP the
methyl ester group is present on the .beta.-carboxyl function of
the L-Asp moiety, and AMPM represents a dimethyl ester) is formed.
Recovery of .alpha.-APM in these processes needs to be done through
the intermediate selective precipitation and recovery of the
.alpha.-APM.HCl salt, whereas the other seven compounds
predominantly remain in solution and need to be recycled and
reconverted into their starting materials L-Asp and L-Phe (by
hydrolysis) and, after recovery thereof, again into .alpha.-APM.
This is disadvantageous because of the very long reaction times,
relatively low once-through yield of .alpha.-APM and low efficiency
on L-Asp and L-Phe due to losses in the recovery.
[0038] Preferably, in this second embodiment of the invention, the
PDF is an EC 3.5.1.27 enzyme because then excellent results are
being achieved. More preferably, the PDF contains the sequences (i)
HEXXH, (ii) EGCLS and (iii) GXGXAAXQ. In particular the PDF used is
obtainable from E. coli, and the bivalent metal ions are manganese,
iron, cobalt and nickel ions. It is even more preferred that the
bivalent metal ions are iron and/or nickel ions. Preferably the
bivalent metal ion is Fe.sup.2+ and all the treatments with the PDF
enzyme are carried out in the presence of a stabilisation agent. In
that case the stabilisation agent is preferably catalase or a
trialkylphosphine compound or derivative, and most advantageously
it is catalase.
[0039] For all further relevant remarks as to reaction conditions
etc. reference is made to the details given above in discussing the
first embodiment of the invention.
[0040] In another preferred embodiment of the invention the
enzymatic deformylation step according to the invention is
integrated into a novel one-pot enzymatic synthesis of .alpha.-APM.
In particular, in such embodiment N-formyl-L-aspartic acid (F-Asp)
is coupled enzymatically, using thermolysin as the coupling enzyme,
with L- or D,L-phenylalanine methyl ester (L- or D,L-PM), and
simultaneously, and in the same reaction vessel, the
N-formyl-.alpha.-L-aspartyl-L-phenylalanine methyl ester
(N-F-.alpha.-APM) formed by the coupling reaction is deformylated
by an enzyme having formylmethionyl peptide deformylase activity
(PDF) and having as a co-factor bivalent metal ions chosen from the
group of group 5 to 11 metals from the periodic system of elements
and being present in the reaction system for the enzymatic coupling
reaction.
[0041] It is noticed that the term "thermolysin" as used here and
hereinafter is intended to mean thermolysin, including any mutant
thereof, and any other enzyme which has suitable coupling activity
for said enzymatic coupling reaction.
[0042] Preferably, the .alpha.-APM so formed is recovered after the
reaction has proceeded till a conversion of more than 40%.
Conversion as meant here relates to the conversion of the L-PM
starting in the coupling step to the desired .alpha.-APM
endproduct. Without the simultaneous deformylating step the yield
of the enzymatic coupling reaction of F-Asp and L- or D,L-PM (which
is thermodynamically unfavourable due to the position of the
equilibrium of this reaction) is lying strongly on the side of the
substrates. Simultaneous presence of a PDF during the coupling
reaction results in a favourable shift to the right side thereof,
thereby directly leading to .alpha.-APM. In contrast, if no PDF is
used according to the present invention, troublesome other measures
need to be taken to shift said equilibrium and it is impossible to
synthesize the .alpha.-APM in a direct one-pot production method.
In the absence of the PDF direct yield of .alpha.-APM is zero.
Attempts to shift the equilibrium more favourably to the side of
synthesis (of F-.alpha.-APM, not of .alpha.-APM) already have been
made by creating reaction conditions where the F-.alpha.-APM formed
is precipitated in situ as an addition compound with L- or D-PM.
However, such formation of an addition compound requires relatively
high concentrations (and excess, 2 equivalents) of L-PM or D,L-PM.
In the latter case intermediate precipitation of a F-.alpha.-APM
adduct with L- and/or D-PM will occur (and in the former case of
the F-.alpha.-APM.L-PM addition compound). Therefore, surprisingly,
according to this third embodiment of the present invention
conversion with direct formation of .alpha.-APM has become
possible.
[0043] This advantageous third embodiment of the present invention
is only possible due to the surprising finding that the PDF's as
used herein do not have very significant deformylating activity
towards F-Asp as compared to their deformylating activity towards
the N-formyl-.alpha.-dipeptide derivatives or other (N-terminal)
N-formyl oligopeptides.
[0044] This is the more surprising as it is well known to the
skilled man that for most other deprotecting enzymes known in
peptide chemistry (e.g. PenG-acylases for the removal of
phenylacetic acid residues, or decarbamoylase enzymes for the
removal of carbamoyl groups) no such significant differences in
activity towards the protected monomeric and oligomeric compounds
are being found. On the contrary, the difference in activity
towards the protected monomeric and oligomeric compounds is either
almost absent (e.g. for PenG-acylases) or opposite, i.e. there is
much higher activity for the protected monomeric compound (e.g. for
decarbamoylases).
[0045] It is thus completely unexpected that the PDF's as used
according to the present invention would be suitable in the methods
of the present invention. The PDF's as used in the methods of the
present invention behave completely different from other
deacylating enzymes (or amidohydrolases).
[0046] This embodiment of the present invention is in particular
surprising as there has not been any indication in the state of the
art so far that PDF enzymes are suitable for deformylating terminal
N-formyl-L-aspartic acid residues in oligopeptides or dipeptides.
Moreover, the PDF's have significantly different deformylating
activities towards N-formyl amino acids and towards (N-terminal)
N-formyl oligopeptides. This enables a technically feasible process
with precipitation of .alpha.-APM without substantial formation of
by-products (e.g. of the diketopiperazine of .alpha.-APM), and
without need for large recycles, etc., in a one-pot single-step
process.
[0047] The reaction conditions for this third embodiment of the
present invention again are not very critical. As already mentioned
above for the first embodiment thereof, any suitable solvent system
which is inert towards the PDF may be applied; such solvents
include aqueous systems (solutions or slurries) or aqueous systems
also containing a water-miscible organic solvent which is inert
under the reaction conditions. Aqueous systems, however, are
preferred. Also the concentration of the N-formyl starting compound
(F-Asp) is not critical, and may be for instance in the range of
about 10 to 1000 mM. It is not necessary that all of the N-formyl
compound is dissolved; part of it may be present as a slurry. The
concentration of the PDF likewise is not very critical, and usually
will be at 0.001 to 100.0%, normally less than 30%, by weight of
the formyl compound, e.g. at about 0.2 mM of PDF. The pH for the
reaction preferably is chosen in the range of 3.0 to 9.0, more
preferably of 4.0 to 8.0 because then .alpha.-APM is formed without
any significant formation of by-products and the enzymes used are
being maintained at high stability and activity. If the .alpha.-APM
formed is precipitated there is even less risk for by-product
formation. The temperature is not very critical, and suitably will
be in the range of 10 to 50.degree. C., e.g. at about 37.degree.
C., but for thermostable PDF enzymes higher temperatures may be
applied.
[0048] In this third embodiment of the invention good results are
obtained if the PDF is an enzyme having the activity as described
for EC 3.5.1.27. Preferably, the PDF enzyme contains the sequences
of (i) HEXXH, (ii) EGCLS and (iii) GXGXAAXQ.
[0049] It is particularly advantageous in this third embodiment of
the invention, if the PDF enzyme has a deformylating activity
towards (oligo)peptides with N-formylmethionine at their
N-terminus, which is at least 10.times. higher, preferably at least
100.times. higher, and most preferred at least 200.times. higher
than its deformylating activity towards N-formyl methionine.
[0050] It is to be noticed that the deformylation activity of the
PDF's, in order to select most suitable PDF's, also may be
determined for (N-terminal) N-formyl compounds other than the
N-formylmethionine (oligo)peptides and their corresponding N-formyl
amino acids. The ratio between the deformylating activity values
obtained for such other N-formyl (oligo)peptides and amino acids
can be taken as an approximate measure for the suitability of the
specific PDF in this third embodiment.
[0051] This preferred embodiment of the invention may even have
more general applicability than for the synthesis of .alpha.-APM.
It is particularly advantageous when the enzymatic deformylation
reaction is simultaneously carried out with the enzymatic coupling
of an N-formyl amino acid (in the case of .alpha.-APM synthesis
this is F-Asp) and another amino acid or (oligo)peptide which is
unprotected at the terminal amino group (in the case of .alpha.-APM
synthesis this is L-PM).
[0052] Preferably, the method of the present invention is carried
out using a PDF which is obtainable from E. coli. In particular the
co-factor bivalent metal ions in the PDF preferably are Mn.sup.2+,
Fe.sup.2+ Co.sup.2+ and Ni.sup.2+ ions. It has been indicated
hereinbefore how such exchange of co-factor bivalent metal ions can
be achieved.
[0053] Preferably in this embodiment of the present invention PDF's
are being used which contain Fe.sup.2+ and/or Ni.sup.2+ ions in the
active site.
[0054] In particular, the bivalent metal ions are Fe.sup.2+ ions
and all treatments with the PDF enzyme are carried out in the
presence of a stabilisation agent. Preferably the stabilisation
agent is catalase or a trialkylphosphine compound or derivative,
most preferably it is catalase.
[0055] In addition to all abovementioned remarks as to reaction
conditions etc. it should be noticed that in this third embodiment
of the invention, in order to maintain the activity of both the
thermolysin coupling enzyme (which is an endoproteinase) and the
PDF at a sufficiently high level, taking one or more additional
measures may lead to better results. For instance, it may be
advisable to prevent any potential proteolytic degradation of the
PDF used by thermolysin. In particular, it can be advantageous to
use the thermolysin and/or the PDF in immobilized form.
Immobilization may be taken care of by any method available to the
skilled man. A suitable method is using the enzymes in the form of
so-called CLEC's ("cross-linked enzyme crystals"). Other methods
include use of so-called "crystalline enzyme" thermolysin and
PDF.
[0056] Alternatively, genetically engineered mutants of PDF's may
be used which have (a still acceptable, preferably unaltered or
even enhanced) activity towards the deformylation reaction but are
less prone to deactivation in the presence of thermolysin. These
mutants can be generated by a number of different approaches; for
instance, by site-directed mutagenesis, site-specific random
mutagenesis, regio-specific random mutagenesis, and completely
random mutagenesis; the latter form of mutagenesis is better known
as directed evolution. General applicable methods to perform these
different protein engineering approaches are well known to the
skilled man. If a random approach will be applied, the mutagenesis
cycle will need to be followed by selection of resistent and active
mutant(s), thereby leading to the identification of suitable
mutants that no longer contain the thermolysin accessible sites. To
obtain PDF mutants, which are completely resistent to thermolysin
degradation, a combination of different protein engineering
approaches and/or several rounds of random mutagenesis appears to
be the most effective procedure.
[0057] Vice versa mutants of thermolysin can be made having (a
still acceptable, but preferably unaltered or even enhanced)
coupling activity, but giving less inactivation of the PDF's.
[0058] Another suitable method for avoiding mutual deactivation of
the thermolysin and the PDF is the use of a physical barrier
between both enzymes, which prevents that both enzymes come into
direct contact while allowing almost unhindered transport of
reactants and products. An example of a suitable physical barrier
are dialysis membranes with a cut-off value of about 10 kDa, where
the thermolysin is present at one side of the membrane as well as
part of the substrates for the coupling reaction, and where the PDF
and part of the reaction products are present at the other side
thereof.
[0059] As a result of the above investigations regarding the
suitable one-pot enzymatic process for the synthesis of
.alpha.-APM, the inventors have found that the concept of such
one-pot process, i.e. a combination of an enzymatic coupling
reaction and simultaneous deformylation of the coupling product,
has wider and more general applicability than for the synthesis of
.alpha.-APM alone.
[0060] In its broadest scope the present invention therefore also
relates to novel one-pot syntheses of di- or oligopeptides or
derivatives thereof from two starting materials, the first of which
is an N-formyl protected amino acid which is capable of undergoing
an enzymatic coupling reaction with a second amino acid or
derivative thereof, or with a di- or oligo-peptide or derivative
thereof, thereby yielding an N-formyl protected reaction compound,
wherein the N-formyl protecting group of the first starting
material is retained during the enzymatic coupling reaction with
the second starting material, whereby said protecting group is
cleaved off enzymatically, using an enzyme having formylmethionyl
peptide deformylase activity and having as a co-factor bivalent
metal ions chosen from the group of group 5 to 11 metals from the
periodic system of elements, from the reaction compound at a
substantially higher, i.e. at least 10.times. higher, rate than
from the first starting material, and wherein two enzymes are
involved simultaneously for the enzymatic coupling reaction between
the starting materials and the enzymatic deformylation of the
reaction compound.
[0061] Examples of such combinations of enzymes, starting materials
(and protecting groups thereof), and final products are shown in
table 1 below:
1 TABLE 1 Starting materials Enzymes Final product A1.
N-Formyl-L-valine Aa. Thermolysin L-Valinyl-L- A2. L-Phenylalanine
Ab. PDF phenylalanine methyl ester methyl ester B1. N-Formyl-L- Ba.
Thermolysin L-Aspartyl-L- aspartic acid Bb. PDF phenylalanyl-L- B2.
L-Phenylalanyl-L- phenylalanine phenylalanine methyl methyl ester
ester C1. N-Formyl-L-aspar- Ca. Papain .alpha.-APM tic acid Cb. PDF
C2. L-Phenylalanine methyl ester D1. N-Formyl-L-aspar- Da.
Thermolysin L-Aspartyl-L- tic acid Db. PDF phenyl-alanyl D2.
L-Phenylalanine amide amide
[0062] The invention will now be illustrated by means of the
following Examples and Comparative Examples. However, the scope of
the present invention is by no means restricted by the Examples
shown.
[0063] Isolation of PDF(Fe.sup.2+)
[0064] For a detailed discussion of the methods used reference is
made to Groche et al., BBRC 246, 342-346 (1998). The following
paragraph gives a summary thereof; abbreviations are explained
below.
[0065] An EC 3.5.1.27 PDF(Fe.sup.2+) enzyme was isolated from
overproducing E. coli cells grown at 30.degree. C. in 1.61 TB
medium for 14-16 hours. About 13 g (wet weight) of cell paste were
suspended in 26 ml buffer (20 mM Hepes/KOH, 100 mM KF, pH 7.7
supplemented with 10 .mu.g/ml catalase from bovine liver
(Boehringer Mannheim) and 1 mM AEBSF, disintegrated by sonification
(Branson B12, 20 min) at 0.degree. C. and centrifuged at 200.000 g
for 1 hour. The clear supernatant (1.3 g of protein; according to
biurete reaction) was mixed with 1.3 ml 10%(w/v) Polymin G-35
(BASF) adjusted to pH 7.7 and centrifuged at 40.000 g for 10
minutes. The supernatant was applied to a 20 ml Met-Lys-Sepharose
column that had been equilibrated with 20 mM Hepes/KOH, 100 mM KF,
0.2 mM TCEP, pH 7.7. After washing with 120 ml of 20 mM Hepes/KOH,
100 mM KF, 0.2 mM TCEP, pH 7.7, the PDF(Fe.sup.2+) was eluted with
150 ml 20 mM Hepes/KOH, 100 mM KCl, 0.2 mM TCEP, pH 7.7. The
protein containing fractions were concentrated by ultrafiltration
using an Amicon PM10 membrane (yield: 140 mg protein, having an
activity of 800 IU/mg; assay conditions: 5 mM
formylmethionyl-alanine, 30.degree. C., pH=7.2). After adjustment
of the TCEP concentration to 1 mM and protein concentration to 40
mg/ml PDF(Fe.sup.2+) was stored frozen at -60.degree. C.
[0066] TB medium: 12 g/l of Bacto-Tryptone, Difco; 24 g/l of yeast
extract, Difco; 4 g/l of glycerole; 2.3 g/l of KH.sub.2PO.sub.4;
12.5 g/l of K.sub.2HPO.sub.4);
[0067] Hepes: N-2-hydroxyethylpiperazine-N'-2-ethane sulphuric
acid;
[0068] AEBSF: 2-aminoethyl-.beta.-benzene sulphonyl fluoride;
[0069] TCEP: tris-(2-carboxyethyl)-phosphine.
[0070] HPLC Analysis of the Starting Materials and Reaction
Products
[0071] Analysis of all compounds was carried out with a
reversed-phase HPLC column (Nucleosil 300-5 C.sub.18 4.6
mm.times.250 mm; Macherey-Nagel, Duren) using a linear gradient
(within 30 minutes) of 0 to 24% (vol/vol) acetonitrile in aqueous
0.1% (vol/vol) trifluoro-acetic acid at a flow rate of 1 ml/min and
at ambient temperature. All compounds were detected by
UV-spectrophotometry at a wavelength of 254 nm.
[0072] Deformylation Reactions
[0073] Deformylation reactions (Examples 1-3, and Comparative
Examples A-C) were performed as follows:
[0074] Compounds (100 mM) as indicated in Table 2 for each separate
Example were incubated in the presence of 100 mM aqueous
2-(N-morpholino)-ethanesulphonic acid (MES)/KOH buffer pH=6.2 and
250 mM KCl at 37.degree. C. The reactions of the Examples and
Comparative Examples A-C were started by the addition of
PDF(Fe.sup.2+), as prepared above, to a final concentration of 0.2
mM. At various times samples (5 .mu.l) were withdrawn and mixed
with 45 .mu.l of aqueous HClO.sub.4 (2% vol/vol final
concentration) to terminate the reaction in order to be able to
measure concentrations of deformylated compounds (containing a free
amino group). Following a brief centrifugation, the amount of
deformylated compounds in the supernatant was determined by
trinitrobenzene sulphonic acid (TNBS) according to the method of
Fields (Methods in Enzymology, 25, 464-468 (1972)), using
.epsilon..sub.420=21.2 mM.sup.-1cm.sup.-1. The amount of
(deformylated) compound each time was corrected for the intrinsic
amount of free amino compound which was detected without incubation
with the PDF. Identification of the reaction compounds was also
confirmed by HPLC analysis (as described above).
[0075] The results are summarized in table 2. The product yields
indicated are the yields after 10 hours of reaction time. It has
been shown in case of Examples 1 and 2 that the deformylation
reactions can be continued to a conversion of more than 90%.
[0076] In addition, the catalytic properties of the PDF in the
deformylation reactions of F-.alpha.-AP and F-.alpha.-APM (in
particular the initial reaction rates for deformylation in resp. 1
hour for F-.alpha.-APM and 3 hours for F-.alpha.-AP) were
determined by performing the reactions of Examples 1 and 2 at
various substrate concentrations from 2-140 mM. In particular, the
following catalytic properties of the enzyme were determined:
[0077] K.sub.M [mM]: Michaelis constant (this is the substrate
concentration at which the reaction rate is 50% of the maximum
reaction rate observed)
[0078] k.sub.cat [min.sup.-1]: turnover number
[0079] k.sub.cat/K.sub.M [M.sup.-1sec.sup.-1]: catalytic efficiency
(also called: specificity constant)
[0080] The values determined are also given in table 2.
[0081] The following Examples and Comparative Examples were
performed:
EXAMPLE 1
[0082] Use of PDF(Fe.sup.2+) for synthesis of .alpha.-APM from
F-.alpha.-APM
EXAMPLE 2
[0083] Use of PDF(Fe.sup.2+) for synthesis of .alpha.-AP from
F-.alpha.-AP
EXAMPLE 3
[0084] Use of PDF(Fe.sup.2+) for synthesis of .alpha.-APM from
F-.alpha..beta.-APM (Note: 100 mM F-.alpha.-APM, 25 mM
F-.beta.-APM)
COMPARATIVE EXAMPLE A: Use of PDF(Fe.sup.2+) for deformylation of
F-.beta.-APM
COMPARATIVE EXAMPLE B: Use of PDF(Fe.sup.2+) for deformylation of
F-.beta.-AP
COMPARATIVE EXAMPLE C: Use of PDF(Fe.sup.2+) for deformylation of
F-Asp
[0085]
2TABLE 2 Enzyme: PDF(Fe.sup.2+) Example or Comp. yield K.sub.M
k.sub.cat k.sub.cat/K.sub.M Example substrate product (%) [mM] :
[min.sup.-1] : [M.sup.-1sec.sup.-1] 1 F-.alpha.-APM .alpha.-APM
44.2 25.3 0.7 0.46 2 F-.alpha.-AP .alpha.-AP 21.6 12.3 0.2 0.27 3
*) F-.alpha..beta.-APM .alpha.-APM 44 0.46 .beta.-APM <0.1
<0.0001 A F-.beta.-APM .beta.-APM 0.2 <0.0001 B F-.beta.-AP
.beta.-AP 1.1 <0.0001 C F-Asp Asp 0.5 *) In this example no
inhibition by f-.beta.-APM of the enzyme activity towards
F-.alpha.-APM was observed.
COMPARATIVE EXAMPLE D:
[0086] use of penicillin-acylase for synthesis of .alpha.-APM from
F-.alpha.-APM.
[0087] This experiment was performed, slightly different from the
Examples described above, as follows:
[0088] 1 g of F-.alpha.-APM was dissolved in 25 ml of distilled
water and the pH was adjusted to 6,5 by the addition of 0.1 M
aqueous NaOH. Then, 0.2 g of an immobilized penicillin-acylase
preparation was added. The reaction mixture was allowed to stand
for 48 hours at 37.degree. C. At regular time intervals during the
reaction, samples were taken and analyzed by thin layer
chromatography using .alpha.-APM as a reference substance. In none
of the samples taken, formation of .alpha.-APM from F-.alpha.-APM
could be demonstrated.
EXAMPLE 4
[0089] One-pot enzymatic synthesis of .alpha.-APM from F-Asp and
L-PM.
[0090] 5 mg/ml of CLEC-thermolysin (PeptiCLEC-TR, from Altus
Biologics Inc., Cambridge, USA) was stirred at room temperature in
a buffer solution containing 100 mM MES/KOH (pH=6.2), 1.7 M NaCl,
10 mM CaCl.sub.2, 1 M of F-Asp and 100 mM of L-PM. The coupling
reaction started and continued for about 3-4 hours until a level of
the F-.alpha.-APM concentration was reached of about 11 mM. Then 8
mg/ml of PDF(Fe.sup.2+) as prepared above were added to the
reaction mixture and the reaction was continued for 20 more hours.
During said period the concentration of the F-.alpha.-APM decreased
to about 7 mM, whereas gradually formation of 19 mM of .alpha.-APM
was observed. At 24 hours the amount of L-PM was 55 mM and of L-Phe
formed 18 mM; at that time also 3 mM of the diketopiperazine of
.alpha.-APM was present. It is thus demonstrated that the
equilibrium of the coupling reaction is shifted to the right side
by the treatment with PDF(Fe.sup.2+) and that simultaneously
.alpha.-APM is formed in this one-pot process. It has been shown,
moreover, that the reaction is still proceeding after said 24
hours; thus, higher yields of .alpha.-APM can be achieved.
* * * * *