Biocatalytic enantioselective synthesis of N-substituted aspartic acids by aspartate ammonia lyase

Article abstract of DOI:10.1002/chem.200801407

The gene encoding aspartate ammonia lyase (asp B) from Bacillus sp. YM55-1 has been cloned and over-expressed, and the recombinant enzyme containing a C-terminal HiS₆ tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the HiS₆ tag does not affect AspB activity. The enzyme processes L-aspartic acid, but not D-aspartic acid, with a k_m of ≈15 mM and a K_cat, of ≈ 40s^-1. By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The k_cat values are comparable to those observed for the AspB-catalyzed addition of ammonia to fumarate (≈90 s^-1), whereas the Km values are only slightly higher. The products of the enzyme-catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97% ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.

Full text of DOI:10.1002/chem.200801407

DOI: 10.1002/chem.200801407
Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by
Aspartate Ammonia Lyase
[
a]
[b]
[c]
[a]
Barbara Weiner, Gerrit J. Poelarends,* Dick B. Janssen,* and Ben L. Feringa*
Abstract: The gene encoding aspartate
ammonia lyase (aspB) from Bacillus
sp. YM55-1 has been cloned and over-
expressed, and the recombinant
enzyme containing a C-terminal His6
tag has been purified to homogeneity
and subjected to kinetic characteriza-
tion. Kinetic studies have shown that
the Michael addition of hydroxylamine,
hydrazine, methoxylamine, and methyl-
amine to fumarate. Both hydroxyla-
mine and hydrazine were found to be
excellent substrates for AspB. The kcat
values are comparable to those ob-
served for the AspB-catalyzed addition
catalyzed addition of hydrazine, me-
thoxylamine, and methylamine to fu-
marate were isolated and characterized
by NMR spectroscopy and HPLC anal-
ysis, which revealed that AspB catalyz-
es all the additions with excellent enan-
tioselectivity (>97% ee). Its broad nu-
cleophile specificity and high catalytic
activity make AspB an attractive
enzyme for the enantioselective synthe-
sis of N-substituted aspartic acids,
which are interesting building blocks
for peptide and pharmaceutical synthe-
sis as well as for peptidomimetics.
À1
of ammonia to fumarate (ꢀ90 s ),
the His tag does not affect AspB activ-
whereas the K values are only slightly
6
m
ity. The enzyme processes l-aspartic
higher. The products of the enzyme-
acid, but not d-aspartic acid, with a K
m
À1
of ꢀ15 mm and a k of ꢀ40 s . By
cat
Keywords: amino acids · biotrans-
using this recombinant enzyme in the
reverse reaction, a set of four N-substi-
tuted aspartic acids were prepared by
formations
· enzyme catalysis ·
kinetics · lyases
Introduction
Aspartate ammonia lyases (also referred to as aspartases;
EC 4.3.1.1) are microbial enzymes that play a key role in ni-
trogen metabolism by catalyzing the reversible elimination
of ammonia from l-aspartate to yield fumarate (Scheme 1).
Scheme 1. General reaction of aspartase.
Several related aspartases have been purified and character-
ized from Gram-positive and -negative bacteria, including
E. coli, Hafnia alvei, Pseudomonas fluorescens, Bacillus
[
a] B. Weiner, Prof. Dr. B. L. Feringa
Department of Organic and Molecular Inorganic Chemistry
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
Fax : (+31)50-363-4296
[1]
[2]
[3]
[
4]
[5]
subtilis, and Bacillus sp. YM55-1. The aspartase from E.
coli (AspA) has been studied most extensively, and its crys-
[6]
E-mail: b.l.feringa@rug.nl
tal structure has been elucidated. AspA functions as a ho-
motetramer with each monomer consisting of 478 amino
acid residues, and the enzyme is allosterically activated by
its substrate (l-aspartate) and Mg ions, which are required
for activity at alkaline pH. It is proposed that AspA carries
out an anti elimination reaction in which an active-site base
abstracts a proton from C-3 of l-aspartate to form an
[
b] Dr. G. J. Poelarends
Department of Pharmaceutical Biology
Groningen Research Institute of Pharmacy, University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
Fax : (+31)50-363-3000
2
+
E-mail: g.j.poelarends@rug.nl
[
c] Prof. Dr. D. B. Janssen
Department of Biochemistry, University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
Fax : (+31)50-363-4165
[7]
enzyme-stabilized enolate intermediate. This forms upon
elimination of ammonia the product, fumarate. The rate-de-
termining step is the cleavage of the carbonÀnitrogen bond,
E-mail: d.b.janssen@rug.nl
which may be facilitated by a general acid that protonates
the leaving ammonia group. Although several active-site res-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801407.
10094
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 10094 – 10100

FULL PAPER
idues have been investigated by site-directed mutagenesis,
kinetic analysis, and chemical modification, the essential cat-
alytic base and presumed catalytic acid have not yet been
region of the expression vector pBAD/Myc-His A, which re-
sulted in the construct pBAD(AspB-His). Sequencing of the
A
T
E
N
cloned gene verified that no mutations had been introduced
during the amplification and cloning procedures. The aspB
[6–8]
identified.
[
9]
AspA is one of the most specific enzymes known. Ex-
tensive studies over the last 80 years have shown that no
other substrates can replace l-aspartic acid in the deamina-
tion reaction.
semialdehyde is deaminated by AspA, but the enzyme is at
the same time irreversibly deactivated. In contrast, many
competitive inhibitors have been reported and these all re-
quire a carboxylic acid or similarly charged functional group
at each end of the carbon chain. Interestingly, Emery re-
ported in 1963 that hydroxylamine is an alternative nucleo-
phile for aspartase, but the N-hydroxyaspartic acid produced
gene in pBAD(AspB-His) is under transcriptional control of
A
H
E
N
the araBAD promoter and recombinant aspartase containing
the C-terminal fusion peptide was produced upon induction
with arabinose in soluble and active form in E. coli TOP10.
Expression of the aspB gene was most efficient when cells
were cultivated at 378C and when 0.04% (w/v) arabinose
was used. The recombinant enzyme was purified by a one-
[10]
Only the suicide substrate l-aspartate-b-
[11]
step protocol (the C-terminal His tag forms a metal binding
6
[
7]
site for affinity purification on metal-chelating resin), which
typically provided 20–30 mg of homogeneous enzyme per
liter of culture. The His -tagged AspB was found to migrate
6
[
12]
was too unstable for isolation.
The high selectivity of
during non-denaturing polyacrylamide gel electrophoresis
with a similar mobility to that of native AspB carrying no
fusion tag (G. J. Poelarends (2008), unpublished results),
which suggests that gross conformational changes do not
occur and that the homotetrameric quaternary structure of
AspA for its natural substrates limits the practical applica-
tion of this enzyme. The reverse reaction catalyzed by
AspA, that is, the amination of fumarate, is used commer-
cially in the industrial production of the artificial sweetener
aspartame
ester).
(N-l-a-aspartyl-l-phenylalanine
An excess of ammonia is used in this process to
1-methyl
native AspB is maintained in the His -tagged enzyme.
6
[13,14]
A mixture containing His -tagged AspB and l-aspartic
6
1
drive the equilibrium from fumarate towards l-aspartic acid.
The yields are usually quantitative and l-aspartic acid is ob-
tained with an enantiomeric excess of >99%.
acid was monitored by H NMR spectroscopy to verify that
the product of the reaction is fumarate, as previously report-
ed for the aspartase enzyme from E. coli. The enzymatic
conversion of l-aspartic acid yields fumarate, as indicated
by a singlet at d=6.39 ppm. This spectrum is identical to an
In our studies we have focussed on aspartase (AspB)
[5]
from the thermophilic bacterium Bacillus sp. YM55-1. This
aspartase is an interesting enzyme for industrial application
because of its high activity, relative thermostability, and lack
of allosteric regulation by substrate or metal ions. The crys-
[16]
independent sample, which confirms that fumarate is the
product of the AspB-catalyzed conversion of l-aspartic acid.
The rate of deamination of l-aspartate by His -tagged AspB
6
[15]
tal structure of AspB was elucidated in 2003 by Fujii et al.
was monitored spectrophotometrically by following the for-
The overall topology and active-site structure of AspB are
similar to those observed in AspA and fumarase C (FumC)
from E. coli, which confirms its membership in the aspar-
tase/fumarase superfamily of enzymes. Like AspA, AspB
functions as a homotetramer in which each subunit is com-
posed of 468 amino acid residues. To date, there is no crystal
structure available of AspB (or any other aspartase) com-
plexed to a substrate or product, and details of the catalytic
mechanism have not yet been elucidated.
mation of fumarate at 240 nm in 50 mm NaH PO buffer
2
4
À1
(pH 8.5) at 258C. A kcat value of (40Æ7) s and a Km of
(15Æ2) mm were found, which results in a k /K of ꢀ2.7”
cat
m
3
À1 À1
10 m s . Similar kinetic parameters have previously been
[5]
found for purified AspB in its native form.
Because the C-terminal fusion peptide does not interfere
with the structural and enzymatic properties of AspB, and
because purification of His -tagged AspB is very efficient,
6
this AspB variant has been used for all of the experiments
described below.
Herein, we report the cloning and overexpression of the
aspB gene in E. coli and the efficient one-step affinity pu-
rification of the recombinant His -tagged enzyme. By using
H NMR spectroscopy, the purified enzyme was extensively
Screening for alternative substrates: Amino acids: Several
amino acids were tested as potential substrates of AspB.
The deamination reactions were monitored by using a col-
orimetric assay that follows ammonia production upon incu-
6
1
screened for its ability to process alternative substrates. We
have identified four amines that can replace ammonia as the
substrate in the AspB-catalyzed Michael addition to fuma-
rate. The resulting N-substituted aspartic acids are interest-
ing building blocks for peptide synthesis and peptidomimet-
ics.
bation with 25 mm substrate in 100 mm Na HPO4 buffer
2
(pH 9.0) at 378C. The compounds d-aspartic acid, l-cysteine,
l-histidine, l-phenylalanine, l-glutamine, l-tyrosine, l-
serine, l-alanine, l-valine, l-leucine, l-threonine, l-lysine, a-
methyl-dl-aspartic acid, b-methyl-dl-aspartic acid, l-gluta-
A
H
E
N
Results and Discussion
mate, b-alanine, b-dl-aminobutyric acid, b-asparagine, b-
phenylalanine, and b-leucine were not processed by AspB.
This screening thus failed to identify any alternative amino
acid substrate that can replace l-aspartic acid. The specifici-
ty for l-aspartic acid has also been clearly demonstrated for
Expression, purification, and characterization of AspB: The
aspB gene was amplified from plasmid pUCBA and cloned
in-frame with both the initiation ATG start codon and the
sequences that code for the myc epitope and polyhistidine
[7]
the corresponding aspartase (AspA) from E. coli.
Chem. Eur. J. 2008, 14, 10094 – 10100
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10095

B. Feringa, D. B. Janssen, G. J. Poelarends et al.
Nucleophiles: It has previously been determined that E. coli
aspartase can catalyze the reverse reaction, the trans addi-
tion of ammonia to fumarate, with high stereoselectivity, ex-
clusively yielding l-aspartic acid. This observation prompt-
ed us to examine whether AspB catalyzes the stereoselec-
tive amination of fumarate and whether different nucleo-
philes can be used in this enzyme-catalyzed Michael addi-
tion reaction (Scheme 2). The addition reactions were
later stage were rather complex, which we assume results
from chemical decomposition of the N-hydroxyaspartic acid.
The instability of this class of compounds has previously
[7]
[12]
been reported by Emery
and Ottenheijm and Her-
[17]
A
H
R
U
G
spectrum recorded 1 day after the addition of enzyme
showed hardly any conversion of the starting material, but
after 2 weeks of incubation at pH 7.0, the spectrum showed
complete loss of the signals corresponding to fumarate
(
Table 1, entry 2) and the formation of new signals corre-
sponding to the expected N-methoxyaspartic acid (d=2.39
dd, 1H), 2.58 (d, 1H), 3.59 (s, 3H), 3.85 ppm (dd, 1H); see
(
Scheme 2. AspB-catalyzed addition of various nucleophiles to fumarate.
the Supporting Information). We repeated the screening at
pH 8.0, and it was established that after 1 week the reaction
was complete (Table 1, entry 3). When hydrazine was used
as the nucleophile, complete disappearance of the starting
material and the formation of the expected 2-hydrazinosuc-
cinic acid (d=2.31 (dd, 1H), 2.49 (dd, 1H), 3.49 ppm (dd,
1H); see the Supporting Information) was observed after in-
cubation for 1 day (Table 1, entry 4). By using methylamine
as the nucleophile, the reaction was complete after 1 week
of incubation at pH 8.0 and the expected N-methylaspartic
acid (d=2.62 (s, 3H), 2.88 (d, 2H), 3.75 ppm (dd, 1H); see
the Supporting Information) was formed (Table 1, entry 5).
The data clearly show that AspB efficiently processes
small substituted amines such as hydroxylamine and hydra-
zine, but displays very low (methoxylamine and methyl-
1
monitored by H NMR spectroscopy under a variety of con-
ditions, including different enzyme concentrations and dif-
ferent pH values (7.0, 8.0, and 9.5). Representative conver-
sions for each reaction are summarized in Table 1. Interest-
ingly, we observed a robust activity of AspB with hydroxyl-
A
C
H
T
R
E
U
N
G
amine and hydrazine, and a small but significant activity
with methoxylamine and methylamine. In the absence of
AspB, these amines do not react with fumarate.
Table 1. Addition of various nucleophiles to fumarate catalyzed by AspB
[
a]
in phosphate buffer in an NMR tube with a volume of 0.6 mL.
Entry
Nucleophile
pH
Time
Conversion [%]
A
H
R
U
G
[
b]
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
H
2
NOH
7.0
7.0
8.0
7.0
8.0
8.0
9.5
8.0
7.0
7.0
8.0
7.0
8.0
8.0
9.5
8.0
8.0
9.5
8.0
20 min
12 days
6 days
1 days
7 days
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
0
0
0
[
[
c]
c]
with larger amine nucleophiles. Small charged nucleophiles
(azide, cyanide, and cyanate) are also not processed. Taken
together, these observations suggest that the nucleophile
binding pocket of AspB is designed to bind small amine
compounds and excludes any charged or large nucleophiles.
In the following sections we describe the results of more
detailed studies of the AspB-catalyzed addition reactions of
four alternative nucleophiles, including the steady-state ki-
netic parameters and characterization of the generated N-
substituted aspartic acid products.
MeONH
MeONH
2
2
[
c]
H
MeNH
EtNH
EtNH
EtNH
NaN
NaCN
NaCN
2
NNH
2
d]
[
2
[
d]
2
2
2
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
14 days
[
[
d]
e]
[
c]
3
[
[
c]
1
1
1
1
1
1
1
1
1
1
c]
[
c]
c]
NaOCN
NaOCN
[
[
[
[
d]
d]
e]
Gly
Gly
Gly
Kinetic measurements: The rate of amination of fumarate
catalyzed by AspB was measured by following the decrease
in absorbance at 270 nm in phosphate buffer (pH 8.0) at
228C. Both hydroxylamine and hydrazine are good sub-
strates for the enzyme and the kinetic parameters are sum-
marized in Tables 2 and 3. The data clearly show that AspB
processes both amines with similar catalytic efficiency.
Whereas the kcat values are comparable to those observed
for the AspB-catalyzed addition of ammonia to fumarate,
[
[
[
d]
d]
e]
formamide
formamide
formamide
0
[
a] Unless indicated otherwise, 250 mmol nucleophiles, 25.0 mmol fuma-
rate, and 50 mm phosphate buffer in D O were used. [b] 1.12 mmol AspB,
00 mmol nucleophile, 20.0 mmol fumarate. [c] 9.34 mmol AspB.
d] 8.95 mmol AspB. [e] 17.9 mmol AspB.
2
2
[
the K values are slightly higher (1.8-fold for hydroxylamine
m
Hydroxylamine appears to be the best alternative sub-
and 3.6-fold for hydrazine, Table 2). As a result, the k /Km
values are 1.6- and 3.4-fold lower for hydroxylamine and hy-
drazine compared with ammonia, respectively.
The kinetic parameters also show that similar Km values
for fumarate are found for the three AspB-catalyzed amina-
tion reactions (Table 3). The kcat values obtained with a
fixed concentration of amine nucleophile (Table 3) are
slightly lower than those obtained with a fixed and saturat-
cat
1
strate for AspB (Table 1, entry 1). The H NMR spectrum
recorded 20 min after the addition of the enzyme showed
the complete disappearance of the signals corresponding to
fumarate (d=6.41 ppm) and the formation of new signals
corresponding to the expected N-hydroxyaspartic acid (d=
3
.65 (dd, 1H), 2.50 (dd, 1H), 2.29 ppm (dd, 1H); see the
Supporting Information). The NMR spectra recorded at a
10096
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Chem. Eur. J. 2008, 14, 10094 – 10100

Enantioselective Synthesis of Aspartic Acids
FULL PAPER
Table 2. Kinetic parameters for the AspB-catalyzed addition of different
amine nucleophiles to fumarate.
A
T
E
N
xyaspartic acid showed the same unstable behavior. After
week of incubation, the signals in the H NMR spectrum
[
a]
1
1
Entry
Nucleophile
K
m
k
cat
À1
kcat/K
m
[Lmol s ]
corresponding to N-hydroxyaspartic acid were no longer
present. Ottenheijm and Herscheid previously described N-
hydroxy acids as highly unstable. The N-hydroxy group is
readily converted into the corresponding nitroso compound
either by air oxidation or disproportionation, which subse-
quently undergoes rapid decarboxylation to the aldoxime.
However, the presumed decomposition products could nei-
ther be isolated nor identified.
À1 À1
[mm]
A
H
R
U
G
]
[
b]
[17]
1
2
3
NH
3
85Æ40
89Æ12
99Æ5.5
94Æ7.6
1040
654.3
304.1
[
b]
H
H
2
NOH
NNH
151Æ22
[
c]
2
2
308Æ49
[
a] Steady-state kinetic parameters were measured using a fixed concen-
tration of 20 mm fumarate in 50 mm phosphate buffer (pH 8.0) at 228C.
Errors are standard deviations. [b] [AspB]=0.041 mm. [c] [AspB]=
0
.082 mm.
As the decomposition of N-hydroxyaspartic acid is initiat-
ed by the oxidation of its hydroxy functionality, protection
of the hydroxy group should give a stable compound. Sever-
al attempts to protect the hydroxy group through benzyla-
tion, silylation, and acylation failed and could not overcome
the rapid decomposition. In an alternative approach, the car-
Table 3. Kinetic parameters for the AspB-catalyzed amination of fuma-
rate using different nucleophiles at a fixed concentration.
[
a]
Entry
Nucleophile
K
m
k
cat
À1
kcat/K
m
[Lmol s ]
À1 À1
[mm]
A
H
R
U
G
]
[
b]
1
2
3
NH
3
1.61Æ0.76
2.78Æ0.56
1.54Æ0.24
59Æ17
92Æ11
31Æ6.8
36414
33166
20193
[
b]
boxylic acid groups were esterified with concentrated H SO4
H
H
2
NOH
NNH
2
[
c]
2
2
in MeOH overnight, which yielded only 4% of the product
[
a] Steady-state kinetic parameters were measured at fixed nucleophile
concentrations ([NH ]=200 mm, [H NOH]=400 mm, and [H NNH ]=
50 mm) in 50 mm phosphate buffer (pH 8.0) at 228C. Errors are standard
deviations. [b] [AspB]=0.0179 mm. [c] [AspB]=0.0358 mm.
with 40% ee. The harsh conditions probably lead to racemi-
zation at the stereogenic center. Mild esterification with tri-
methylsilyldiazomethane also failed.
3
2
2
2
7
To establish the enantiomeric excess of the enzymatically
produced N-hydroxyaspartic acid, hydrogenolysis of the NÀ
O bond of N-hydroxyaspartic acid with a catalytic amount
[18]
ing concentration of fumarate (Table 2).
Nevertheless,
of PtO and a few drops of acetic acid was performed. This
2
these observations suggest that the three different amine nu-
cleophiles are optimally positioned in the active site of
AspB to undergo an amination reaction, and that the bind-
ing and positioning of fumarate is not significantly affected
by the binding of the different amine nucleophiles.
yielded (S)-aspartic acid, which was isolated in 80% yield
[19]
and 97% ee (Scheme 3). To summarize, we could identify
optically active N-hydroxyaspartic acid as the product of the
AspB-catalyzed addition of hydroxylamine to fumarate, but
failed to stabilize and isolate this interesting product.
Under the conditions used, the observed initial rates for
the AspB-catalyzed addition of methoxylamine or methyla-
mine to fumarate were too low to measure accurate kinetic
parameters. Although a structural basis for this observation
is not yet known, the lower rates observed with methoxyl-
A
C
H
T
R
E
U
N
G
amine and methylamine suggest that these slightly larger
amines are not optimally bound in the active site of AspB
to undergo an addition reaction.
Isolation and characterization of the products of the AspB-
catalyzed amination reactions: The AspB-catalyzed amine
additions to fumarate were performed routinely in 5 mm
phosphate buffer at pH 8.0 and 378C. The enzyme concen-
tration was varied depending on the rate of the reaction. To
isolate and characterize N-hydroxyaspartic acid, the addition
of hydroxylamine to fumarate was scaled up to 1.0 mmol of
substrate. By using 9.34 mmol (0.9 mol%) of enzyme the re-
action was complete within 15 min, as assessed both by UV
Scheme 3. Enantioselective hydrazine and hydroxylamine addition to fu-
marate and hydrogenolysis to l-Asp.
The enzymatic addition of hydrazine to fumarate was
scaled up to 5.0 mmol. The stable product 2-hydrazinosuc-
cinic acid was successfully purified in quantitative yield by
ion-exchange chromatography on cationic Dowex 50 resin.
To determine the enantiomeric excess, the crude 2-hydrazi-
nosuccinic acid was reduced with a catalytic amount of PtO2
to give (S)-aspartic acid, which was purified by ion-exchange
chromatography on cationic Dowex 50 resin. Subsequent
HPLC analysis established the product to have 99% ee
(Scheme 3).
1
analysis and H NMR spectroscopy. Several attempts to
purify the enzymatically formed N-hydroxyaspartic acid by
ion-exchange column chromatography on cationic Dowex 50
resin, SPE-SCX cation exchange, and basic Amberlite IRA
140 all failed. Identification of N-hydroxyaspartic acid as the
product of the AspB-catalyzed addition of hydroxylamine to
1
fumarate was established by H NMR spectroscopy by com-
parison with chemically synthesized N-hydroxyaspartic acid.
Both enzymatically and chemically synthesized N-hydro-
Chem. Eur. J. 2008, 14, 10094 – 10100
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10097

B. Feringa, D. B. Janssen, G. J. Poelarends et al.
As the enzymatic addition of methoxylamine to fumarate
occurs slowly, we used a high concentration of biocatalyst
nogenic amino acids are interesting building blocks for pep-
tidomimetics, synthetic enzymes, and pharmaceuticals. For
example, 2-hydrazinosuccinic acid is an important structural
(
1
1.8 mol% of AspB). The reaction was scaled up to
.0 mmol and was allowed to proceed for 1 week until fuma-
[23]
unit because it represents a turn mimic in peptides, and
several peptides containing this structural motif show antibi-
rate was completely converted. The resulting N-methoxyas-
partic acid could not be purified by chromatography on a
cationic Dowex 50 resin. Therefore the crude methoxyas-
partic acid was esterified using TMSCl in MeOH, which
yielded N-methoxyaspartic dimethyl ester in 11% yield over
two steps with an enantiomeric excess >99% (Scheme 4).
[24]
otic activity. When included in peptides, N-methylaspartic
acid leads to enhanced proteolytic stability and an increase
[25]
in lipophilicity.
Although this investigation has set the stage for the devel-
opment of a biocatalytic process for the stereoselective syn-
thesis of N-substituted aspartic acid derivatives, the biocata-
[26]
lytic scope of AspB is rather limited. Based on the struc-
tural characterization of the enzyme, we have started pro-
tein engineering experiments aimed at evolving AspB activi-
ty towards a broader range of amine compounds.
Experimental Section
Scheme 4. Enantioselective addition of methoxylamine to fumarate and
esterification.
Construction of the expression vector for the production of AspB: The
aspB gene was amplified by PCR by using two synthetic primers, the
[
5]
coding sequence for the aspartase in plasmid pUCBA as the template,
and PCR reagents supplied in the Expand High Fidelity PCR system fol-
lowing the protocol supplied with the system (F. Hoffman-La Roche,
Ltd.). The forward primer (5’-ATACCATGGATACCGATGTTCG-3’)
contains a NcoI restriction site (in bold) followed by 13 bases that corre-
spond to the coding sequence of the aspB gene. The reverse primer (5’-
CATAAGCTTTTTTCTTCCAGCAATTCC-3’) contains a HindIII re-
striction site (in bold) followed by 18 bases that correspond to the com-
plementary sequence of the aspB gene. The resulting PCR product and
the pBAD/Myc-His A vector (Invitrogen) were digested with NcoI and
HindIII restriction enzymes, purified, and ligated using T4 DNA ligase.
Aliquots of the ligation mixture were transformed into competent E. coli
TOP10 cells. Transformants were selected at 378C on LB/ampicillin
plates. Plasmid DNA was isolated from several colonies and analyzed by
restriction analysis for the presence of the insert. The cloned aspB gene
was sequenced to verify that no mutations had been introduced during
the amplification of the gene. The newly constructed expression vector
The enzymatic addition of methylamine gave full conver-
sion of fumarate after 6 days of incubation by using
1.4 mol% of AspB. The N-methylaspartic acid was purified
by column chromatography on silica gel to give a yield of
[
20]
9
5% and the pure acid had >99% ee (Scheme 5). Deter-
2
0
mination of the optical rotation gave a value of [a] =
D
+
24.5 (c=0.26 in 1n HCl), which corresponds to an S con-
[21]
figuration according to literature.
was named pBAD
Expression and purification of AspB-His
duced in E. coli TOP10 using the pBAD expression system. Fresh TOP10
cells containing pBAD(AspB-His) were collected from a LB/ampicillin
A
H
R
U
G
6
: The AspB enzyme was pro-
Scheme 5. Enantioselective AspB-catalyzed addition of methylamine to
fumarate.
plate using a sterile loop and used to inoculate 1 L of a LB/ampicillin
medium that contained 0.04% (w/v) arabinose. After overnight growth
at 378C and 200 rpm, the cells were harvested by centrifugation (10 min
at 6000 rpm at 48C in a JA-10 rotor) and stored at À208C until further
use.
The biocatalytic production of these four N-substituted
aspartic acids has previously been demonstrated for 3-meth-
[22]
ylaspartate ammonia lyase (methylaspartase). In contrast
to that study, we have presented a detailed analysis of these
reactions, including an HPLC analysis of the products and
the determination of kinetic parameters.
In a typical purification experiment, cells of three 1 L cultures were
thawed, combined, and suspended in lysis buffer (15 mL, 50 mm
NaH PO , 300 mm NaCl, 10 mm imidazole, pH 8.0). Cells were disrupted
2 4
by sonication for 10”1 min (with 3–5 min rest in between each cycle) at
a 60 W output after which unbroken cells and debris were removed by
centrifugation (30 min at 15000 rpm at 48C in a JA17 rotor). The super-
natant was filtered through a 0.45 mm pore diameter filter and incubated
with nickel nitriloacetic acid (4”1 mL slurry in small columns at 48C for
Conclusion
2
nights), which had previously been equilibrated with lysis buffer. The
nonbound proteins were eluted from the column by gravity flow. The col-
umns were first washed with lysis buffer (10 mL per column) and then
We have exploited the nucleophile promiscuity of aspartase
(
AspB) from Bacillus sp. YM55-1 for catalyzing stereoselec-
with buffer A (50 mm NaH
0 mL per column). Retained proteins were eluted with buffer B (50 mm
NaH PO , 300 mm NaCl, 250 mm imidazole, pH 8.0; 3.0 mL per column).
2 4
PO , 300 mm NaCl, 20 mm imidazole, pH 8.0;
1
tive amination reactions to produce the N-substituted as-
partic acids (S)-N-hydroxyaspartic acid, (S)-2-hydrazinosuc-
cinic acid, (S)-N-methylaspartic acid, and N-methoxyaspart-
ic acid with excellent enantioselectivities. These non-protei-
2
4
Fractions (ꢀ0.5 mL) were analyzed by SDS-PAGE on gels containing
1
2% acrylamide, and those that contained purified aspartase were
À1
pooled and concentrated to a protein concentration of about 9.4 mgmL
10098
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 10094 – 10100

Enantioselective Synthesis of Aspartic Acids
FULL PAPER
2
3
in 50 mm NaH
2
PO
4
(pH 8.0) using an Amicon-stirred cell equipped with
(500 MHz, D
CH ), 2.49 (dd,
(dd,
O): d=36.9 (CH
ESI): m/z: 149.1 [M+1] , 171.1 [M+Na] , 147.1 [MÀ1] .
2
O): d=2.31 (dd,
J
A
T
E
G
J
ACHTREUNG
2
3
ACHTREUNG
a YM30 (30000 MW cut-off) ultrafiltration membrane. The purified
enzyme was stored at À808C until further use.
Colorimetric assay for ammonia detection: The deamination of potential
amino acid substrates was monitored by following ammonia production
upon incubation with the different compounds. Accordingly, an appropri-
ate amount of enzyme was incubated in a microtitre plate with an amino
2
J(2,2)=16.1 Hz, J(2,1)=4.5 Hz, 1H; CH
2
A
H
R
U
G
3
3
ACHTREUNG
13
J
A
H
R
N
(1,2)=8.5 Hz,
J
D
(
2
2
2
+
+
N-Methylaspartic acid: A solution of 6n HCl was added to a solution of
methylamine (40% in water, 4.20 mL, 50.0 mmol) in 5 mm phosphate
buffer in a Greiner tube until pH 8.0 was reached. A 500 mm stock solu-
tion of fumarate (10 mL, 5.00 mmol) at pH 8.0 was added, and the Grein-
er tube was filled up to 50 mL with 5 mm phosphate buffer (pH 8.0).
After addition of AspB (400 mL, 0.072 mmol) from a frozen stock, the
tube was shaken at 100 rpm at 378C for 7 days. The reaction was fol-
lowed to completion, as confirmed spectrophotometrically until no ab-
sorption at 240–270 nm was observed. The solvent was evaporated and
the white residue purified by flash chromatography on silica gel (HOAc/
2 4
acid (150 mL of a 25 mm solution) in 100 mm Na HPO buffer (pH 9.0).
After incubation of the plate at 378C for 18 h, a drop of 1.5m trichloro-
acetic acid followed by 100 mL of Nesslerꢁs reagent were added. A red-
brown color indicated the presence of AspB activity.
Kinetic studies: To determine the kinetic parameters for the AspB-cata-
lyzed deamination of l-aspartate, the kinetic assays were performed at
2
58C by following the increase in absorbance at 240 nm, which corre-
À1
À1
sponds to the formation of fumarate (e=2530m cm ). An aliquot of
AspB was diluted in 50 mm NaH PO buffer (pH 8.5) to yield a final
enzyme concentration of 0.08 mm and incubated for 60 min at 258C. Sub-
sequently, a 1 mL portion was transferred to a 10 mm quartz cuvette and
the enzyme activity was assayed by the addition of a small quantity (1–
EtOAc/MeOH/H
philization, the product (95%)
2
O=3:3:3:2). After evaporation of the solvent and lyo-
2
4
[
29]
20
was obtained as white gum. [a]D
=
1
+
2
24.6 (c=0.256 in 1n HCl); H NMR (300 MHz, D O): d=2.62 (s, 3H;
3
3
ACHTREUNG
CH
3
), 2.88 (d,
(1,2)=8.8 Hz, 1H; CH); C NMR (125 MHz, D
4.7 (CH ), 60.1 (CH), 172.9 (CO H), 176.8 ppm (CO
2
J
A
H
R
U
G
), 3.75 ppm (dd,
O): d=31.2 (CH
H); MS (ESI):
in H O/MeOH
J
3
13
J
A
H
R
U
G
2
2
1
0 mL) of sodium l-aspartate from a stock solution. The stock solution
3
3
was made up in 50 mm NaH PO buffer (pH 8.5). The concentrations of
l-aspartate used in the assay ranged from 5–100 mm.
2
4
+
m/z: 146.2 [MÀ1] ; HPLC (Astec CLC-L, 2 mm CuSO
4
2
À1
9
0:10, flow 1.0 mLmin
,
408C): 9.2 (l-N-MeAsp), 11.4 min (d-N-
To determine the kinetic parameters for the AspB-catalyzed addition of
amines to fumarate, the kinetic assays were performed at 228C and the
decrease in absorbance was followed at 270 nm, which corresponds to the
MeAsp); 99.5% ee.
N-Methoxyaspartic acid: Methoxylamine hydrochloride (4.18 g,
50.0 mmol) was dissolved in 5 mm phosphate buffer (10 mL) and 5n
NaOH was added until tube pH 8.0 was reached. A 500 mm stock solu-
tion of fumarate (10 mL, 5.00 mmol) at pH 8.0 was added and the Grein-
er tube was filled up to 50 mL with 5 mm phosphate buffer (pH 8.0).
After addition of AspB (500 mL, 0.090 mmol) from a frozen stock, the
tube was shaken at 100 rpm at 378C. The progress of the reaction was
monitored spectrophotometrically. After 9 days, no absorption was ob-
served at 240–270 nm, which indicated that the reaction had been com-
À1
À1 [27]
depletion of fumarate (e=555m cm ).
for fumarate, an aliquot of AspB was diluted in 50 mm NaH
buffer (pH 8.0) containing a fixed concentration of amine (ammonia
00 mm, hydroxylamine 400 mm, hydrazine 750 mm, all titrated to pH 8.0)
To determine the values of
K
m
2
PO
4
2
to yield a final enzyme concentration of 0.0179 mm (0.0358 mm when using
hydrazine). Subsequently, a 1 mL portion was transferred to a 10 mm
quartz cuvette and the enzyme activity was assayed by the addition of a
small quantity of fumarate from a stock solution made up in 50 mm
NaH
2
PO
4
buffer (pH 8.0). The concentrations of fumarate used in the
for the
pleted. The solvent was evaporated and the white residue lyophilized and
1
assay ranged from 0.10–3.75 mm. To determine the values of K
amine nucleophiles, the incubation mixtures contained various concentra-
tions of nucleophiles ranging from 20–571 mm, 0.041 mm of AspB
m
used without further purification. H NMR (400 MHz, D
2
O): d=2.39
2
3
2
ACHTREUNG
(dd,
15.2 Hz, J
(1,2)=7.6 Hz, J
34.5 (CH ), 61.5, 63.0, 179.9 (CO
J
A
H
R
U
G
J
A
H
R
U
2
), 2.58 (d,
), 3.85 ppm (dd, J-
(1,2)=5.2 Hz, 1H; CH); C NMR (75.4 MHz, D O): d=
H), 180.2 ppm (CO H).
J(2,2)=
3
3
2
3
13
(
0.081 mm when using hydrazine), and 50 mm phosphate buffer (pH 8.0).
A
H
R
N
2
A 350 mL portion of this solution was transferred to a 1 mm quartz cuv-
ette, and reactions were started by adding 20 mm of fumarate. The kinetic
data were fitted by nonlinear regression data analysis using the Grafit
2
2
N-Methoxyaspartic acid dimethyl ester: A suspension of the crude
lyophilized N-methoxyaspartic acid (5.00 mmol) containing buffer salts in
MeOH (18 mL) was cooled with ice and TMSCl (4.00 mL, 31.5 mmol,
6.3 equiv) was added dropwise. A precipitate formed and the solution
was stirred at room temperature overnight. After removal of the solvent,
the product was purified by flash chromatography on silica gel (pentane/
[
28]
program.
General procedure for the nucleophile screening: The nucleophile
screening was carried out on a Varian Inova 500 NMR spectrometer
using a pulse sequence for selective presaturation of the water signal.
The reactions were performed in a NMR tube with a volume of 550 mL
at room temperature, and H NMR spectra were recorded after 1 day,
and 1, 2, and 3 weeks. A sample initially contained 9.34 or 8.95 mmol of
2
0
EtOAc=2:1) to yield a colorless oil (0.107 g, 0.560 mmol, 11%). [a] =
D
1
1
2
À3.6 (c=1.4 in CHCl
3 3
); H NMR (300 MHz, CDCl ): d=2.71 (dd, J-
3
2
ACHTREUNG
3
A
H
R
U
(2,2)=16.3 Hz, J
A
H
R
U
G
2
), 2.83 (dd, J
AspB, 0.25 mmol of fumarate, and 10 equivof nucleophile. The nucleo-
A
H
R
U
G
2
), 3.51 (s, 3H; OCH ), 3.71 (s, 3H; CO
3
2
3
3
3
ACHTREUNG
phile was dissolved in 50 mm phosphate buffer containing D
to reduce the water signal in the spectrum. The total volume was adjust-
ed to 550 mL by using buffer in D O. At the same time a blank sample
2
O in order
2
3
J
A
H
R
U
G
J
1
3
2
2
2
3
3
3
2
+
+
without AspB was monitored. The reactions were monitored at different
pH values (pH 7.0, 8.0, and 9.5).
2
Me); MS (EI): m/z: 191 [M] , 160 [MÀOMe] , 132
+
[MÀCO
2
Me] ; HRMS: calcd. for C
6
H
10NO
4
: 160.0610; found: 160.0612;
À1
HPLC (Chiralpak OD-H, heptane/iPrOH 95:5, flow 0.5 mLmin ):
2
-Hydrazinosuccinic acid: A solution of 6n HCl was added to a solution
2
1.9 min; 99% ee.
of hydrazine (35% in water, 5.20 mL, 50.0 mmol) in 5 mm phosphate
buffer in a Greiner tube until pH 8.0 was reached. A 500 mm stock solu-
tion of fumarate (10 mL, 5.00 mmol) at pH 8.0 was added and the Grein-
er tube was filled up to 25 mL with 5 mm phosphate buffer (pH 8.0).
After addition of AspB (80.0 mL, 0.014 mmol) from a frozen stock, the
tube was shaken at 95 rpm at 378C. The reaction was allowed to continue
to completion, as confirmed spectrophotometrically by the disappearance
of the absorption at 240–270 nm. The solvent was evaporated and the
oily residue was purified by ion-exchange column chromatography on a
N-Hydroxyaspartic acid: A solution of 5n NaOH was added to a solution
of hydroxylamine hydrochloride (0.69 g, 10.0 mmol) in 5 mm phosphate
buffer in a Greiner tube until pH 8.0 was reached. A 1m stock solution of
fumarate (1 mL, 1.00 mmol) at pH 8.0 was added and the Greiner tube
was filled up to 15 mL with 5 mm phosphate buffer at pH 8.0. After the
addition of AspB (50 mL, 0.009 mmol) from a frozen stock, the tube was
shaken at 100 rpm at 378C for 30 min. The solvent was evaporated and a
white gum obtained. H NMR (400 MHz, D
15.8 Hz,
4.2 Hz, 1H; CH
1
2
2
O): d=2.29 (dd,
J
J
A
C
H
T
R
E
U
N
G
(2,2)=
+
3
2
ACHTREUNG
3
cationic Dowex 50 resin (H , 20–50 mesh, washed with water) by elution
J
A
H
R
N
(2,1)=9.0 Hz, 1H; CH
2
), 2.50 (dd,
J
(2,2)=15.8 Hz,
ACHTREUNG
(2,1)=
3
3
ACHTREUNG
with 2.5% NH
tion, the product (0.799 g, 5.39 mmol, 108%)
tively as a colorless gum. [a] =À16.1 (c=0.557 in 1N HCl); H NMR
3
solution. After evaporation of the solvent and lyophiliza-
2
) 3.65 ppm (dd,
J(1,2)=8.6 Hz, J(1,2)=4.6 Hz, 1H;
A
H
R
U
G
[
29]
was obtained quantita-
CH). Attempts at purification by ion-exchange chromatography led to
decomposition of the material.
2
0
1
D
Chem. Eur. J. 2008, 14, 10094 – 10100
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10099

B. Feringa, D. B. Janssen, G. J. Poelarends et al.
Acknowledgements
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[
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van Assema and M. Boesten (DSM, Geleen) for technical support. We
thank Dr. Y. Kawata, Tottori University, Japan, for the kind gift of plas-
mid pUCBA. This research was supported by the Bio-Based Sustainable
Industrial Chemistry (B-BASIC) programme of NWO-ACTS in The
Netherlands. G.J.P. was supported by a VENI grant from the Division of
Chemical Sciences of the Netherlands Organization of Scientific Re-
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29] The yield was determined by H NMR spectroscopy because residu-
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Received: July 11, 2008
Published online: October 8, 2008
3
10100
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 10094 – 10100