Chemoenzymatic one-pot synthesis of enantiopure L-arylalanines from arylaldehydes

Article abstract of DOI:10.1002/ejoc.200500902

Various enantiopure L-arylalanines can be prepared from the corresponding arylaldehydes in one-pot fashion by a combination of chemical and enzymatic reactions. The reagents used were (triphenyl-λ⁵- phosphanylidene)ethyl acetate (Wittig reactio

Full text of DOI:10.1002/ejoc.200500902

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200500902
Chemoenzymatic One-Pot Synthesis of Enantiopure
Arylaldehydes
L
-Arylalanines from
Csaba Paizs,[a,b] Adrian Katona, and János Rétey*
[a]
[a]
Keywords: Wittig reaction / Porcine liver esterase / Phenylalanine ammonia lyase / Sonification / Broad substrate specificity
Various enantiopure
L-arylalanines can be prepared from the
3-fluorophenyl and thiophen-2-yl. The pure L-enantiomers of
corresponding arylaldehydes in one-pot fashion by a combi- phenyl-, 4-chlorophenyl-, 3-fluorophenyl- and thiophen-2-yl-
nation of chemical and enzymatic reactions. The reagents
used were (triphenyl-λ -phosphanylidene)ethyl acetate (Wit-
tig reaction), porcine liver esterase (PLE), phenylalanine am-
monia lyase (PAL) from parsley and ammonia. As examples
the following aryl groups were used: phenyl, 4-chlorophenyl,
alanines were obtained after ion-exchange chromatography
in 88, 78, 72 and 91% yields, respectively, calculated from
the arylaldehydes.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
5
Introduction
Since most enzymes not only show strict reaction- and
enantioselectivity but are also specific to one or a few sub-
strates, their use in biocatalysis is rather limited. Enzymes
of broad substrate specificity, while maintaining other spe-
Both naturally occurring and unnatural -arylalanines
are frequent constituents of biologically and pharmaceuti-
cally important products such as protease inhibitors, an ex-
cificities, are therefore preferred as biocatalysts. Porcine
tremely important class of pharmaceuticals for treatment of
[4–7]
liver esterase (PLE)
(
and phenylalanine ammonia lyase
HIV, influenza or human cytomegalovirus.[
1–3]
Their enan-
[8]
PAL) fulfil these criteria.
tioselective synthesis by a simple method from commer-
cially available achiral and cheap starting materials is an
attractive goal, while the incorporation of unnatural aryla-
mino acids into pharmaceutically active compounds such
as antibiotics might circumvent resistance developed
against them in certain diseases.
Results and Discussion
Syntheses consisting of several steps usually require time-
consuming and expensive purification of the intermediates
Scheme 1. The one-pot synthesis of -arylalanines.
to remove side products or reagents that would impair the
next step. To avoid such problems, practically quantitative
conversions and compatible reagents and solvents are re-
quired, and in such a case a one-pot procedure should be
[
[
a] Institute of Organic Chemistry and Biochemistry, University of
Karlsruhe,
Richard-Willstätter-Allee, 76128 Karlsruhe, Germany
Fax: +49-721-6084823
E-mail: janos.retey@ioc.uka.de
b] Department of Biochemistry and Biochemical Engineering, achievable without purification of the intermediates. There
Babe s¸ -Bolyai University,
are several reports of chemoenzymatic one-pot syntheses
Arany János str. 11, 400028 Cluj-Napoca (Kolozsvár), Romania
for the preparation of enantiomerically pure compounds
Fax: +49-264-590818
[
9]
E-mail: paizs@chem.ubbcluj.ro
such as 4-amino-2-hydroxy acids or natural compounds
Eur. J. Org. Chem. 2006, 1113–1116
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1113

C. Paizs, A. Katona, J. Rétey
SHORT COMMUNICATION
Figure 1. (a) Elution diagram of the starting materials (blue trace) and the products of the Wittig (red trace) and PLE-mediated reactions
green trace). (b) Elution diagram of the products of the two enzymatic steps of the one-pot reaction: PLE-mediated (blue trace) and
(
PAL-catalysed (red trace). (c) Elution diagram of the isolated product of the one-pot reaction, -phenylalanine (blue trace) and (rac)-
phenylalanine as reference (red trace).
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Eur. J. Org. Chem. 2006, 1113–1116

Enantiopure -Arylalanines from Arylaldehydes
SHORT COMMUNICATION
such as (R)- and (S)-tembamide,[10] but no chemoenzymatic ture[16–19] on the substrate specificity of PAL we estimate
one-pot syntheses of amino acids have so far been reported. that at least 37 arylalanines can be prepared by the new
Since Wittig reactions between aryl aldehydes and (tri- method.
5
phenyl-λ -phosphanylidene)ethyl acetate commonly take
[
11]
place
in quantitative yields in aqueous solutions, subse-
quent conversions by PLE and PAL in the same pot might
be hoped to afford enantiopure -arylalanines in excellent Experimental Section
yields. Here we demonstrate with four examples that this is
indeed a powerful and quick procedure.
Materials and Methods: The NMR spectra were recorded on a
Bruker spectrometer operating at 400 MHz and 100 MHz at 25 °C.
EI-MS spectra were taken on a VG 7070E mass spectrometer op-
erating at 70 eV. HPLC analyses were conducted with a HP 1050
instrument and a Nucleodur 100–5 C8 ec column (0.46×25 cm),
the eluent was HCl (20 m) in water/acetonitrile (70:30, v/v) iso-
The reaction sequence and the aryl groups (a–d) chosen
are shown in Scheme 1. The progress of the reaction was
monitored after each step by HPLC to determine the best
conditions for quantitative conversions. When this had oc-
curred, the conditions (dilution, temperature, pH, ammonia cratic at a 1 mLmin flow rate. For each analysis 50 µL of the
concentration) were adjusted for the reagents catalysing the reaction mass suspension was diluted with 450 µL of a water/meth-
next step. For clarity the starting materials (blue trace), the anol mixture (50:50, v/v), dissolving the solid triphenylphosphane
Wittig products (red trace) and the products after hydrolysis
by PLE (green trace) are shown separately in Figure 1a. It
so happens that the Wittig reaction is not completely stere-
–
1
oxide. A Chirobiotic-T column (0.46×25 cm) was used for exami-
nation of the enantiomeric compositions of the isolated amino ac-
ids 4a–d with a methanol/water mixture (80:20, v/v) as eluent at
–
1
1
mLmin flow rate. Baseline enantiomeric separation of all race-
ospecific, the (E) and (Z) diastereomers being produced in
a ratio of 95:5. PLE is not specific for either one of these
diastereomers, as can be seen from the green trace, but (Z)-
cinnamic acid is inert to PAL. Figure 1b shows the HPLC
mic amino acids 4a–d was performed. Melting points were deter-
mined by the hot plate method and are uncorrected. Optical rota-
tions were determined on a Perkin–Elmer 201 polarimeter. All ana-
lytical and physical data for the isolated and purified -amino acids
analysis after PLE-mediated hydrolysis (blue trace) and af- are in good agreement with the literature data.[
14,20,21]
ter the PAL-catalysed ammonia addition (red trace). The
5
Aldehydes 1a–d, (triphenyl–λ –phosphanylidene)ethyl acetate, por-
elution diagrams are monitored at 205 nm; since the ab-
sorption of phenylalanine at this wavelength is much less
than those of (E)- and (Z)-cinnamic acids the areas under
cine liver esterase (PLE) and ion-exchange resin (Dowex 50X8)
were products of Aldrich, Fluka or Sigma.
the lines do not correspond to the real amounts of the prod- Recombinant PAL
ucts. The PAL reaction was stopped when the consumption
Wild-type phenylalanine ammonia lyase was overexpressed in
E. coli and purified as described, first according to Schuster and
of the (E)-cinnamate ceased. The final product, -phenylal-
anine was easily isolated on an ion-exchange column. The
procedure for the other arylalanines was similar and the
yields on the isolated arylalanines are shown in Table 1. Be-
side the yields, which varied between 72 and 91%, the abso-
lute configurations and the enantiomeric purities of the iso-
lated arylalanines were also determined. Figure 1c shows
the elution diagram from a chiral column of the produced
phenylalanine (blue trace) together with that of racemic
[22]
Rétey
Schulz.
and later with the improved method of Baedeker and
[23]
Synthesis of
L-Amino Acids from the Corresponding Aldehydes by a
One-Pot, Three-Step Reaction: A mixture of one of the aldehydes
5
1
a–d (5 mmol) and (triphenyl-λ -phosphanylidene)acetic acid ethyl
ester (1.91 g, 5.5 mmol) in water (15 mL) was vigorously stirred at
0 °C until the aldehyde had been completely transformed into the
cinnamate [mostly (E)-cinnamate (approx. 3 h, checked with
9
phenylalanine as a control (red trace). The expected  con- HPLC); in each case 3–5% (Z) isomer was also formed]. After the
figurations and the enantiopurities of all the arylalanines mixture had cooled to room temperature the pH of the solution
were confirmed by their optical rotations (data not shown), was adjusted to 8 with conc. ammonia solution and PLE (2 mg,
which were consistent with literature values.[
12–15]
2
3
8 iU) was added. The suspension was stirred and sonicated at 25–
0 °C while the pH of the solution was kept between 7 and 9 by
addition of conc. ammonia. After completion of the hydrolysis of
the cinnamic acid ester (approx. 6 h, checked with HPLC), water
and conc. ammonia were added to produce a 6  ammonia solu-
Table 1. Yields and reaction times for the one-pot synthesis of -
arylalanines.
Prod.[a]
Yield (%)[b]
2
tion and a 2 m cinnamate concentration. CO was bubbled
Subst.
Time (days)
through the system until the pH of the solution had been adjusted
to 10.2. After the addition of PAL (1 iU) the reaction mixture was
stirred under argon at 30 °C. The progress of the reaction was fol-
lowed by HPLC and when the formation of the -amino acid had
ceased the reaction mixture was degassed under reduced pressure,
the pH was adjusted to 1.5 with 5% HCl, and the solution was
filtered, heated to 90 °C for 10 min, cooled to room temperature,
filtered again and applied to a Dowex 50X8 cation-exchange resin
column.
1
1
1
1
a
b
c
-4a
-4b
-4c
-4d
88
78
72
91
2
2
3
4
d
[
a] ee Ͼ 98% for all the compounds. [b] Yields given are for the
isolated compounds.
In conclusion, we present a one-pot, high-yielding chem-
oenzymatic synthesis of enantiopure -arylalanines from Elution of the pure -enantiomers of the amino acids 4a–d was
the corresponding arylaldehydes. From earlier litera- achieved with 2  ammonia solution.
Eur. J. Org. Chem. 2006, 1113–1116
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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C. Paizs, A. Katona, J. Rétey
SHORT COMMUNICATION
[
[
[
10] A. Kamal, A. A. Shaik, M. Sandbhor, M. S. Malik, Tetrahe-
dron: Asymmetry 2004, 15, 3939–3944.
11] J. Dambacher, W. Zhao, A. El-Batta, R. Anness, C. Jiang, M.
Bergdahl, Tetrahedron Lett. 2005, 46, 4473–4477.
12] V. Viswanatha, V. J. Hruby, J. Org. Chem. 1980, 45, 2010–2012.
Acknowledgments
We thank Professor Georg E. Schulz for the plasmid of the modi-
fied PAL gene. Financial support from the European Commission,
the Deutsche Forschungsgemeinschaft and the Fonds der Chem-
ischen Industrie is gratefully acknowledged.
[13] E. L. Bennett, C. Niemann, J. Am. Chem. Soc. 1950, 72, 1806–
1807.
[
14] H. E. Smith, J. R. Neergaard, J. Am. Chem. Soc. 1997, 119,
116–124.
[
[
[
1] A. Dondoni, D. Perrone, M. T. Semola, J. Org. Chem. 1995,
0, 7927–7933.
2] J. Hertel, H. Struthers, C. B. Horj, P. W. Hruz, J. Biol. Chem.
004, 279, 55147–55152.
3] G. Gerona-Navarro, M. J. Pérez de Vega, M. T. García-Lopez,
G. Andrei, R. Snoeck, E. De Clercq, J. Balzarini, R. González-
Muñiz, J. Med. Chem. 2005, 48, 2612–2621.
4] H. J. Gais, G. Bülow, A. Zatorski, M. Jentsch, P. Maidonis, H.
Hemmerle, J. Org. Chem. 1989, 54, 5115–5122.
5] P. Arzel, V. Freida, P. Weber, A. Fadel, Tetrahedron: Asymmetry
[15] J. Meiwes, M. Schudok, G. Kretzschmar, Tetrahedron: Asym-
metry 1997, 8, 527–536.
[16] G. Renard, J. C. Guilleux, C. Bore, V. Malta-Valette, D. A. Ler-
ner, Biotechnol. Lett. 1992, 14, 673–678.
[17] A. Gloge, B. Langer, L. Poppe, J. Rétey, Arch. Biochem. Bio-
phys. 1998, 359, 1–7.
[18] W. Liu, (Great Lakes Chemical Co.) US P 5,981,239 (1999).
[19] A. Gloge, J. Zo n´ , Á. K o˝ vári, L. Poppe, J. Rétey, Chem. Eur. J.
2000, 6, 3386–3390.
[20] C. Xiong, W. Wang, C. Cai, V. J. Hruby, J. Org. Chem. 2002,
67, 1399–1402.
[21] J. Meiwes, M. Schudok, G. Kretzschmar, Tetrahedron: Asym-
metry 1997, 8, 527–536.
[22] B. Schuster, J. Rétey, FEBS Lett. 1994, 349, 252–254.
[23] M. Baedeker, G. E. Schulz, FEBS Lett. 1999, 457, 57–60.
Received: November 17, 2005
6
2
[
[
[
[
1999, 10, 3877–3881.
6] C. M. R. Ribiero, E. N. Passarto, E. C. S. Brenelli, Tetrahedron
Lett. 2001, 42, 6477–6479.
7] H. A. Sousa, C. A. M. Afonso, J. P. B. Mota, J. G. Crespo, Ind.
Eng. Chem. Res. 2003, 42, 5516–5525.
8] L. Poppe, J. Rétey, Curr. Org. Chem. 2003, 7, 1297–1315.
9] A. Sutherland, C. L. Willis, J. Org. Chem. 1998, 63, 7764–7769.
[
[
Published Online: January 19, 2006
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1113–1116