The interaction of heteroaryl-acrylates and alanines with phenylalanine ammonia-lyase from parsley

Article abstract of DOI:10.1002/chem.200501034

Acrylic acids and alanines substituted with heteroaryl groups at the β-position were synthesized and spectroscopically characterized (UV, HRMS, ¹H NMR, and ¹³C NMR spectroscopy). The heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl and contained the alanyl side chains either at the 2- or 3-positions. While the former are good substrates for phenylalanine ammonia lyase (PAL), the latter compounds are inhibitors. Exceptions are thiophen-3-yl-alanine, a moderate substrate and furan-3-yl-alanine, which is inert. Possible reasons for these exceptions are discussed. Starting from racemic het eroaryl-2-alanines their D-enantiomers were prepared by using a stereodestructive procedure. From the heteroaryl-2- acrylates, the L-enantiomers of the heteroaryl-2-alanines were prepared at high ammonia concentration. These results can be best explained by a Friedel - Crafts-type electrophilic attack at the aromatic part of the substrates as the initial step of the PAL reaction.

Full text of DOI:10.1002/chem.200501034

FULL PAPER
DOI: 10.1002/chem.200501034
The Interaction of Heteroaryl-Acrylates and Alanines with Phenylalanine
Ammonia-Lyase from Parsley
[
a, b]
[a]
[a]
Csaba Paizs,
Adrian Katona, and Jµnos RØtey*
Abstract: Acrylic acids and alanines
substituted with heteroaryl groups at
the b-position were synthesized and
spectroscopically characterized (UV,
HRMS, H NMR, and C NMR spec-
troscopy). The heteroaryl groups were
furanyl, thiophenyl, benzofuranyl, and
benzothiophenyl and contained the
alanyl side chains either at the 2- or 3-
positions. While the former are good
substrates for phenylalanine ammonia-
lyase (PAL), the latter compounds are
eroaryl-2-alanines their d-enantiomers
were prepared by using a stereode-
structive procedure. From the heteroar-
yl-2-acrylates, the l-enantiomers of the
heteroaryl-2-alanines were prepared at
high ammonia concentration. These re-
sults can be best explained by a Frie-
del–Crafts-type electrophilic attack at
the aromatic part of the substrates as
the initial step of the PAL reaction.
inhibitors. Exceptions are thiophen-3-
yl-alanine, a moderate substrate and
furan-3-yl-alanine, which is inert. Possi-
ble reasons for these exceptions are
discussed. Starting from racemic het-
1
13
Keywords: biosynthesis · enantiose-
lectivity
·
enzyme catalysis
·
Friedel–Crafts
Introduction
more highly resolved structure of PAL obtained from pars-
ley (Petroselinum crispum) is consistent with the Friedel–
[4]
Phenylalanine ammonia-lyase (PAL) is an important plant
enzyme, as its product, (E)-cinnamic acid is the precursor of
lignines, coumarins, and flavonoids. Its mechanism of action
has been the subject of great interest. . PAL and HAL (his-
tidine ammonia-lyase) contain the unique superelectrophilic
prosthetic group 5-methylene-3,5-dihydroimidazol-4-one
Crafts like attack of MIO at the phenyl group of phenylala-
nine.
[5]
Another interesting property of PAL is its ability to react
with a large number of arylalanines, which makes it a useful
[1]
[6]
enantiospecific biocatalyst. Originally it was believed that
the ammonia eliminations both by PAL and HAL were irre-
versible. In this case, enantioselectivity for the l-amino acids
would allow preparation of the d-amino acids by a stereo-
destructive procedure. However, at a surprisingly high am-
monia concentration (~6m) both the PAL and HAL reac-
tions can be run in the reverse direction, that is, the enantio-
selective addition of ammonia can be achieved on a prepa-
rative scale. Thus the pure l-enantiomers of various halo-
gen-substituted phenylalanines (Cl, F), pyridyl and
pyrimidyl alanines etc. have been prepared by the reverse
(
MIO) which is able to attack the phenyl or imidazolyl
group of their substrates. Thereby, the b-protons of the
amino acids are activated allowing their abstraction by an
enzymatic base, a step, which is followed by elimination of
ammonia. This mechanism is initiated by a Friedel–Crafts-
type attack (Scheme 1).
[2]
The X-ray structure of HAL was solved in 1999; howev-
[3]
er, the structure of PAL has only recently published. The
[7]
PAL reaction. Here we describe the synthesis of various
acrylic acids, substituted with heterocyclic aromatic groups,
and the corresponding amino acids both in the racemic and
enantiomerically pure form. They are either substrates or in-
hibitors of PAL.
[
a] Dr. C. Paizs, A. Katona, Prof. Dr. J. RØtey
Institute of Organic Chemistry and Biochemistry
University of Karlsruhe, Richard-Willstätter-Allee
7
6128 Karlsruhe (Germany)
Fax : (+49)721-608-4823
E-mail: janos.retey@ioc.uka.de
[
b] Dr. C. Paizs
Department of Biochemistry and Biochemical Engineering
Babes-Bolyai University, Arany Jµnos str. 11
Results
4
00028 Cluj-Napoca (Kolozsvµr) (Romania)
Synthesis of the heteroaryl- acrylic acids and racemic amino
acids: The starting materials for the synthesis of the acrylic
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2006, 12, 2739 – 2744
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2739

a final step the protecting group was removed and the phe-
nylalanine analogues were isolated by precipitation at their
isoelectric point (Scheme 2).
Determination of the kinetic constants for the heteroaryl
alanines: The kinetic measurements are based on the UV
spectrophotometrical determination of the concentration of
the acrylates formed. First, their extinction coefficients (e)
were determined in Tris-buffer (0.1m, pH 8.8) at wave-
lengths (l) for which the corresponding amino acids do not
show absorption. The enzymatic assays were carried out in
1
mL cuvettes in the above mentioned buffer at 308C in
presence of 25 mg of PAL with the substrate concentration
varying between 0.5–2.5 mm. In Table 1 the maximal velocity
(
Vmax) values relative to that of phenylalanine and the
values of the Michaelis constant (K ) are listed. For amino
m
acids in which the alanine group is attached to the hetero-
aromatic ring at the 2-position, PAL could catalyze their
transformation into acrylates; however, the reaction rates
generally were lower than for the parent compound, only
rac-thiophen-2-yl alanine reacts approximately at the same
rate as phenylalanine.
The behavior of the amino acids in which the alanyl por-
tion is attached to the heteroaromatic ring at position 3 was
different. Only rac-thiophen-3-yl-alanine (7d) was a poor
substrate for PAL. In addition, we investigated the interac-
tion of the nonsubstrate amino acids with PAL, by kinetic
measurements on the parent reaction, monitoring the forma-
tion of cinnamate at 290 nm. While rac-furan-3-yl-alanine
(
(
7c) appeared not to interact with PAL, rac-benzofuran-
7g) and rac-benzothiophen-3-yl alanines (7h) proved to be
strong competitive inhibitors. Moreover, l-7g and l-7h were
Scheme 1. Mechanism of the PAL-
mediated reaction involving a Friedel–
Crafts-type attack of the MIO group
at the phenyl moiety of phenylalanine.
and amino acids were the corre-
sponding heteroaryl aldehydes.
These compounds were trans-
5
formed with triphenyl-l -phos-
phanilidene ethyl acetate into
the corresponding acrylic acid
esters, which were subsequently
hydrolyzed in presence of KOH
to yield the desired cinnamates.
Alternatively, the aldehydes
were transformed into chloro-
methylene derivatives; this was
followed by their coupling with
N-protected diethyl amino mal-
onate. Mild basic hydrolysis of
the diethyl esters formed pro-
duced N-protected amino ma-
lonic acids, which were decar-
Scheme 2. Synthesis of the racemic arylamino acids and acrylic acids. i) NaBH
azole/CH Cl ; iii) NaH, XCONH(CO Et) /DMF, 608C; iv) 10% KOH, 20 h; v) toluene/xylene, reflux, 2 h;
vi) 10% LiOH, 608C, 2.5 h; vii) 18% HCl, reflux, 4 h; viii) Ph
4 3 2
/CH OH; ii) SOCl , benzotri-
2
2
2
2
3
P=CHCO
2
Et/toluene, reflux, 2 h; ix) 10% KOH,
boxylated in boiling toluene. In reflux.
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Heteroaryl-Acrylates and Alanines
FULL PAPER
Table 1. Kinetic constants for the PAL-mediated reactions.
Substrate
K
m
[mm]
K
i
[mm]
V
max/Vmax Phe
l-Phe
rac-7a
rac-7b
rac-7d
rac-7e
rac-7 f
rac-7g
l-7g
32.01Æ2
–
–
–
–
–
–
1
76.39Æ3
0.34
1.01
0.16
0.49
0.14
1
1
1
1
134.96Æ3
18.24Æ1
48.38Æ2
116.63Æ4
–
–
–
–
25Æ3
13Æ2
16Æ3
7Æ2
rac-7h
l-7h
approximately doubly as strong inhibitors as the racemates,
indicating that the d-amino acids do not interact with the
enzyme.
Difference UV spectroscopy: The difference UV spectra of
wild-type- and the MIO-less mutant Ser202Ala-PAL were
recorded in the absence and in the presence of the competi-
tive inhibitor rac-7g. Earlier, it was shown that the differ-
ence UV spectra of the above mentioned protein pair exhib-
its a discrete maximum between 305 and 310 nm, which was
assigned to the cross-conjugate double bond system of
Figure 1. The UV difference spectra show no interaction between inhibi-
tor rac-7g and MIO. a) Blue trace: wt-PAL in Tris-HCl buffer/S202A-
PAL in Tris-HCl buffer as the blank; red trace: rac-7g and wt-PAL in
Tris-HCl buffer/rac-7g and S202A-PAL in Tris-HCl buffer as the blank.
b) Blue trace: rac-7g and wt-PAL in Tris-HCl buffer/wt-PAL in Tris-HCl
buffer as a blank; red trace: rac-7g in Tris-HCl buffer/Tris-HCl buffer as
a blank.
[8]
MIO. This maximum also appeared unaltered when the
difference spectra were recorded in the presence of rac-7g
(
Figure 1a). In addition, the dif-
Table 2. Preparative enantioselective synthesis of heteroaryl amino acids catalyzed by PAL.
ference spectra of wild-type
PAL in presence of rac-7g, with
wild-type PAL as blank, were
identical to the UV spectra of
rac-7g (Figure 1b).
[
a]
[b]
D
Substrate Product
Yield [%]
PAL [iU] Time [days] [a]
[
[
11]
12]
(
(
(
E)-9a
E)-9b
E)-9e
l-7a
l-7b
l-7e
l-7 f
d-7a
d-7b
d-7d
d-7e
d-7 f
66
89
54
49
44
45
45
43
44
6
3
2
4
4
4
2
7
3
3
À41.88 in H
À30.38 in H
2
2
O, 25 8C (À41.88 in H
O, 20 8C (À30.38 in H
2
2
O, 25 8C)
O, 20 8C)
6
6
6
À14.58 in CH
3 2
CO H, 208C
(E)-9 f
rac-7a
rac-7b
rac-7d
rac-7e
rac-7 f
À23.88 in CH CO H, 208C
3
2
[
[
[
11]
2.5
2
+41.78 in H
+31.88 in H
+42.68 in H
2
2
2
O, 25 8C (+41.78 in H
O, 25 8C (+31.88 in H
O, 25 8C (+42.68 in H
2
2
2
O, 25 8C)
O, 25 8C)
O, 25 8C)
13]
12]
Biocatalysis: There are some
examples for using PAL or
Rhodotorula graminis and Rho-
dotorula glutinis cells in the
preparative scale production of
l-amino acids starting from the
corresponding acrylates. As far
7.5
6
+14.48 in CH
+23.88 in CH
3
CO
CO
2
H, 208C
H, 208C
6
3
2
[
a] >98% ee for all the compounds (ee=enantiomeric excess). [b] Yields are given for the isolated com-
pounds.
as we know PAL was not used for the preparation of d-
amino acids by enantioselective kinetic resolution of the
racemates. Exploiting the results obtained by the kinetic
measurements, we performed the synthesis of d-7a,b,d–f by
incubating the corresponding racemates with different
amounts of PAL at 308C (Table 2, Scheme 3) in Tris-buffer
(
0.1m, pH 8.8). The progress of the reaction was monitored
Scheme 3. PAL-mediated reactions for the preparation of enantiopure d-
and l-heteroaryl alanines.
by HPLC with a Chirobiotic-T column (Figure 2). When the
l-amino acids were completely transformed into acrylates
the reaction was stopped by adjusting the pH to 1.5 and the
acrylates were filtered off. The enzyme was precipitated by
heating the mixture to 908C, followed by filtration of the
turbid solution. The d-amino acids were isolated and puri-
fied from the filtrate by ion exchange chromatography.
For the production of the l-amino acids, acrylic acids
action and the workup procedure were conducted as de-
scribed above (Figure 3).
Discussion
9
a,b,e,f were incubated with PAL (Table 2, Scheme 3) in
The synthesis of the acrylic acids substituted with heteroaryl
groups and the corresponding racemic amino acids was
ammonia (6m) buffered at pH 10.2 by bubbling CO ; the re-
2
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www.chemeurj.org
2741

J. RØtey et al.
reaction with Rhodotorula gra-
minis cells resuspended in a
buffered 9m ammonia solution,
it was found that while furan-3-
yl-acrylate is inert, thiophen-2-
yl-acrylate is a good substrate
for the preparation of the cor-
[9]
responding l-amino acid.
The binding pocket of PAL is
more selective for the benzofur-
an- and benzothiophenyl ala-
nines. The 2-yl isomers (7e and
7
f) are good substrates, while
the 3-yl isomers (7g and 7h)
are strong competitive inhibi-
tors. For the 2-yl isomers, MIO
can be attached to C-3 in the
aromatic ring (Figure 4c) and
ammonia elimination follows.
In contrast, both ortho positions
with respect to the alanyl group
of the 3-yl isomers were not ac-
cessible by MIO. The quaterna-
ry C-4 is sterically hindered to
Figure 2. The elution diagram for PAL-catalyzed enantiodestructive synthesis of d-thiophen-2-yl-alanine. PAL
and rac-amino acid, Tris-HCl buffer (pH 8.8): blue trace: after 30 min; red trace: after 2 days.
a
Friedel–Crafts-type attack,
while C-2 is too far from the
prosthetic group (Figure 4d).
Difference UV spectroscopy
also indicated that the latter
isomers can bind strongly, but
only in the conformation that
cannot be attacked by MIO.
The stereodestructive prepa-
ration of enantiopure d-aryl-
amino acids by PAL is possible
because the reaction is only re-
versible at extremely high am-
Figure 3. Elution diagram for the synthesis of l-thiophen-2-yl-alanine.
rather conventional. Some of them had been described in
the early literature (see references in the Supporting infor-
mation), but their characterization by modern spectroscopic
methods (UV, high resolution H- and C-NMR spectrosco-
py, and MS) was not available. This is now provided in the
Supporting Information.
monia concentrations (>4m). The Km for ammonia for the
reverse reaction has been determined to be 4.4m at pH 8.8
[10]
and 2.6m at pH 10.
Thus, about 90% of the possible
1
13
yields of the d-amino acids were achieved (Table 2). This is
the first time that these d-amino acids were prepared by the
PAL reaction.
To test whether the electrophilic attack of MIO at the
aromatic ring is a prerequisite for the acceptance by PAL,
the alanyl side-chain was attached at the 2- or 3-position of
the heteroaryl group. Most of the latter isomers were inhibi-
tors rather than substrates of PAL. One exception is thio-
phen-3-yl-alanine (7d) which is, in contrast to furan-3-yl ala-
nine (7c), a reluctant substrate of PAL. This may be due to
the decreased electronegativity of sulphur compared with
oxygen. In other words, sulphur is similar to carbon and 7d
can be bound by the active site in a conformation in which
the attack by MIO is possible (Figure 4a and b). By contrast,
the furanyl group of 7c seems to be too polar to bind in the
alternative conformation. Therefore 7c is neither a substrate
nor an inhibitor. Also in a whole cell mediated biocatalytic
Four l-aryl amino acids could be prepared by conducting
the PAL reaction in the reverse direction with a 6m concen-
tration of ammonia. As shown in Figure 3 and Table 2, the
yields were good to moderate and the l-amino acids were
enantiomerically pure. The latter could be determined by
D
using a chiral column and/or by a comparison of their [a]
[11–13]
values with those in the literature
(Figures 2 and 3, and
Table 2). In contrast to the whole cell procedure using Rho-
[14]
dotorula glutinis cells for PAL from parsley, l-7d was a
poor substrate. However, it could not be prepared by the re-
verse PAL reaction, but rac-7d was used to obtain the
d-enantiomer by the stereodestructive forward reaction.
In conclusion, furanyl, thiophenyl, benzofuranyl, and ben-
zothiophenyl groups attached to acrylyl and alanyl side
2742
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Heteroaryl-Acrylates and Alanines
FULL PAPER
Figure 4. Model of the active site of PAL with various arylalanines arranged in accordance with the Friedel–Crafts-like mechanism.
(
1d),
benzofuran,
benzothiophene,
ortho-hydroxyacetophenone,
chains were examined as substrates or inhibitors of PAL.
The results support the mechanism involving a Friedel–
Crafts attack as the first step. In addition the usefulness of
PAL as biocatalyst for the preparation of enantiopure het-
erocyclic arylamino acids is shown.
5
ethylchloroacetate, triphenyl-l -phosphanilidene ethyl acetate, all inor-
ganic reagents, and solvents were products of Aldrich or Fluka. All sol-
vents were purified and dried by standard methods as required.
Recombinant PAL: The wild-type- and mutant Ser202Ala phenylalanine
ammonia-lyases were overexpressed in E. coli and purified, first accord-
ing to the method described by Schuster and RØtey and later by using
[
15]
[
16]
the improved method described by Baedeker and Schulz.
Kinetic
measurements and enzymatic reactions were performed at 308C.
Experimental Section
Kinetic measurements
m
Determination of Vmax and K values for substrate amino acids: The ki-
1
13
Analytical methods: The H- and C NMR spectra were recorded on a
netic constants were determined by using a 1 mL cuvette and were re-
corded at intervals of 1 min over 5 min. The UV absorptions of the acryl-
ates produced were recorded at wavelengths for which the corresponding
amino acids show only low absorption. After incubation of the enzyme
Bruker spectrometer operating at 400 and 100 MHz, respectively, at
58C. EI-MS were taken on a VG 7070E mass spectrometer operating at
0 eV. HPLC analyses were conducted with a HP 1050 instrument by
using a Chirobiotic-T column (0.46”25 cm) and a mixture of methanol
2
7
(
25 mg) in 0.1m Tris-HCl buffer at pH 8.8 and 308C for 5 min, various
À1
and water as the eluent at 1 mLmin flow rate. Retention times of the
amounts of amino acids were added with the substrate concentration of
the enzymatic assays varied between 05–2.5 mm.
enantiomers and the used eluents are presented in the Supporting Infor-
mation. For kinetic measurements, a Varian Cary 3E UV-VIS spectro-
photometer was used. TLC was carried out by using Merck Kieselgel
i
Determination of K for inhibitor amino acids: Following the procedure
described above, the PAL reaction was monitored by measuring the ab-
sorption of the produced cinnamate at intervals of 1 min at 290 nm. The
phenylalanine concentration was varied with three different concentra-
tions of the inhibitors (0.1, 0.2, and 0.5 mm). The kinetic parameters are
shown in Table 1.
6
0F254 sheets. Spots were visualized by treatment with 5% ethanolic
phosphomolybdic acid solution and heating. Preparative chromatographic
separations were performed by using column chromatography on Merck
Kieselgel 60 (0.063–0.200 mm). Melting points were determined by a hot
plate method and are uncorrected. Optical rotations were determined on
a Perkin–Elmer 201 polarimeter and [a]
D
values are given in units of
Difference UV spectroscopic measurements: UV difference spectra were
measured at an enzyme concentration of 0.5 mg”mL and inhibitor con-
À1
2
À1
1
0
degcm g
.
À1
Reagents and solvents: Furan-2-carbaldehyde (1a), thiophene-2-carb-
aldehyde (1b), furan-3-carbaldehyde (1c), thiophene-3-carbaldehyde
centration of 0.5 mm in 0.1m Tris-HCl buffer at pH 8.8 and 308C from 260
to 350 nm with 1 mL quartz cuvettes.
Chem. Eur. J. 2006, 12, 2739 – 2744
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2743

J. RØtey et al.
Enzymatic synthesis of enantiopure amino acids
[
[
[
1] For a recent review see: L. Poppe, J. RØtey, Angew. Chem. 2005,
Kinetic resolution of racemic amino acids catalyzed by PAL: PAL was
added to a solution of one of the amino acids rac-7a,b,d–f (0.5 mmol) in
Tris-HCl buffer (0.1m, pH 8.8, 40 mL), and the reaction mixture was stir-
red under argon at 308C. The progress of the reaction was followed by
HPLC. When the l-enantiomer was completely consumed, the pH of the
solution was adjusted to 1.5 with 5% HCl (filtered in case of 7e and f),
and then the solution was heated to 908C for 10 min, cooled to room
temperature, filtered, and applied to a Dowex 50X8 cation exchange
resin column. The elution of the pure d-enantiomer of the amino acids
was achieved by using 2m ammonia solution. Reaction times, quantity of
the enzyme in each case, and the yields of the products are presented in
Table 2.
1
17, 3734–3754; Angew. Chem. Int. Ed. 2005, 44, 3668–3688.
2] T. F. Schwede, M. Bädeker, M. Langer, J. RØtey, G. E. Schulz, Pro-
tein Eng. 1999, 12, 151–153.
3] J. C. Calabrese, D. B. Jordan, A. Boodhoo, S. Sariaslani, T. Vannelli,
Biochemistry 2004, 43, 11403–11416.
[
[
4] H. Ritter, G. E. Schulz, Plant Cell 2004, 16, 3426–3436.
5] B. Schuster, J. RØtey, Proc. Natl. Acad. Sci. USA 1995, 92, 8433–
8
437.
6] A. Gloge, B. Langer, L. Poppe, J. RØtey, Arch. Biochem. Biophys.
998, 359, 1–7.
7] A. Gloge, J. ZoÇ, . Kðvµri, L. Poppe, J. RØtey, Chem. Eur. J. 2000,
, 3386–3390.
8] D. Rçther, D. Merkel, J. RØtey, Angew. Chem. 2000, 112, 2592–
594; Angew. Chem. Int. Ed. 2000, 39, 2462–2464.
9] W. Liu, (Great Lakes Chemical Co.) US P-5981239, 1999.
[
[
[
[
1
6
Synthesis of l-amino acids from cinnamates by PAL-mediated enantiose-
lective ammonia addition: CO
cinnamates 9a,b,d,e (0.675 mmol) in half-concentrated ammonia solution
20 mL) until the pH of the solution was adjusted to 10.2. After the addi-
2
was bubbled into a solution of one of the
2
(
[
[
[
[
[
10] S. Viergutz, L. Poppe, A. Tomin, J. RØtey, Helv. Chim. Acta 2003,
6, 3601–3612.
11] G. Renard, J. C. Guilleux, C. Bore, V. Malta-Valette, D. A. Lerner,
Biotechnol. Lett. 1992, 14, 673–678.
12] G. Kretzschmar, H. U. Hoppe, H. Wicke, M. Schlingmann, R.
Keller, B. Sachse, (Hoechst AG, Germany), DE-3820302A1, 1989.
13] J. Meiwes, M. Schudok, G. Kretzschmar, Tetrahedron: Asymmetry
tion of PAL, the reaction mixture was stirred under argon at 308C. The
progress of the reaction was followed by HPLC and when the formation
of the l-amino acids had stopped, the reaction mixture was degassed
under reduced pressure. The pH was adjusted to 1.5 with 5% HCl, fil-
tered, and the solution was then heated to 908C for 10 min, cooled to
room temperature, filtered again, and applied to a Dowex 50X8 cation
exchange resin column. Elution of the pure l-enantiomer of the amino
acids was achieved by using 2m ammonia solution. Reaction times, quan-
tity of the enzyme in each case, and the yields of the products are pre-
sented in Table 2.
8
1
997, 8, 527–536.
14] C. Doebler, H. J. Kreuzfeld, H. W. Krause, M. Michalik, Tetrahe-
dron: Asymmetry 1993, 4, 1833–1842.
15] B. Schuster, J. RØtey, FEBS Lett. 1994, 349, 252–254.
16] M. Baedeker, G. E. Schulz, FEBS Lett. 1999, 457, 57–60.
[
[
Acknowledgements
We thank Professor Georg E. Schulz and Dr. Mathias Baedeker for a
plasmid of the modified PAL gene. Financial support by the Deutsche
Forschungsgemeinschaft, the European Commission, and the Fonds der
Chemischen Industrie is also gratefully acknowledged.
Received: August 23, 2005
Published online: January 18, 2006
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Chem. Eur. J. 2006, 12, 2739 – 2744