Efficient tandem biocatalytic process for the kinetic resolution of aromatic β-amino acids

Article abstract of DOI:10.1002/adsc.201000035

We describe a simple and efficient enzymatic tandem reaction for the preparation of enantiomerically pure β-phenylalanine and its analogues from the corresponding racemates. In this process, phenylalanine aminomutase (PAM) catalyzes the stereoselective isomerization of (R)-β-phenylalanines to (S)-a-phenylalanines, which are in situ transformed to cinnamic acids by phenylalanine ammonia lyase (PAL). Preparative scale conversions are done with a mutated PAM with enhanced catalytic activity.

Full text of DOI:10.1002/adsc.201000035

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DOI: 10.1002/adsc.201000035
Efficient Tandem Biocatalytic Process for the Kinetic Resolution
of Aromatic b-Amino Acids
a
a,b
c
d
´
Bian Wu, Wiktor Szymanski, Stefaan de Wildeman, Gerrit J. Poelarends,
Ben L. Feringa,b and Dick B. Janssena,*
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
a
Fax : (+31)-50-363-4165; phone: (+31)-50-363-4008; e-mail: D.B.Janssen@rug.nl
Department of Organic and Molecular Inorganic Chemistry, Stratingh Institute for Chemistry, University of Groningen,
Nijenborgh 4, 9747 AG, Groningen, The Netherlands
DSM Pharmaceutical Products, P.O. Box 18, 6160 MD, Geleen, The Netherlands
Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius
Deusinglaan 1, 9713 AV, Groningen, The Netherlands
b
c
d
Received: January 15, 2010; Published online: May 28, 2010
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201000035.
Abstract: We describe a simple and efficient enzy-
matic tandem reaction for the preparation of enan-
tiomerically pure b-phenylalanine and its analogues
from the corresponding racemates. In this process,
phenylalanine aminomutase (PAM) catalyzes the
stereoselective isomerization of (R)-b-phenylala-
nines to (S)-a-phenylalanines, which are in situ
transformed to cinnamic acids by phenylalanine am-
monia lyase (PAL). Preparative scale conversions
are done with a mutated PAM with enhanced cata-
lytic activity.
lipases.[3] For example, Fꢀlçp et al. reported the use of
Candida antarctica lipase A for the kinetic resolution
of aromatic aliphatic/aromatic b-amino esters,[4]
heteroACHTUNGTRNEUNG
aromatic b-amino esters[5] and ethyl 3-amino-3-
(4-cyanophenyl)propanoate.[6] Although these meth-
ods are generally of practical value, lipases with
higher catalytic activity, enantioselectivity and chemo-
selectivity, as well as better compatibility in organic
solvent, are necessary. Recently, the application of
phenylalanine aminomutase (PAM) for the enzymatic
synthesis of (R)-b-arylalanines has been reported and
two catalytic routes have been developed. Walker and
co-workers showed that b-phenylalanines can be ob-
tained by PAM-catalyzed isomerization of the corre-
sponding a-isomers[7] and our group has demonstrated
that PAM can catalyze the amination of (E)-cinnamic
acids to produce aromatic b-amino acids.[8]
Keywords: b-amino acids; biotransformations;
enantioselectivity; phenylalanine aminomutase;
phenylalanine ammonia lyase; tandem reactions
PAM exhibits extremely high enantioselectivity to-
wards (R)-b-amino acids. Therefore we decided to ex-
Enantiomerically pure b-amino acids are present in a plore the use of this enzyme as a potential catalyst for
number of natural products that possess a wide range the kinetic resolution of rac-b-phenylalanines. In this
of biological activities.[1] The synthesis of b-amino process, the (R)-b-phenylalanines would be converted
acids is a challenging target for organic chemists. into enantiopure (S)-a-phenylalanines, leaving behind
Thus far, a number of chemical methods have been enantiopure (S)-b-phenylalanines (Figure 1 A). The
developed for the synthesis of b-amino acids based on main advantage of such a procedure would be its sim-
classical resolution, stoichiometric use of chiral auxil- plicity, mild reaction conditions and cofactor inde-
iaries and homologation.[1] A considerable number of pendence. However, the bottleneck of this strategy is
catalytic asymmetric methods have also been pro- the equilibrium between the a- and b-phenylalanine
posed.[2] Biocatalytic routes, which would not require (roughly 1:1) which is reached in the PAM-catalyzed
high catalyst loadings, could fulfill the increasing isomerization reaction,[9] causing incomplete conver-
demand for environmentally friendly synthetic meth- sion of the enantiomer preferred by PAM. To shift
ods. Until recently, most of the biocatalytic ap- this equilibrium, a second enzyme is required, that
proaches to prepare enantiopure b-amino acids relied can effectively convert (S)-a-phenylalanine, the prod-
on kinetic resolution with hydrolytic enzymes, usually uct of the isomerization reaction. Such an enzymatic
Adv. Synth. Catal. 2010, 352, 1409 – 1412
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Bian Wu et al.
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Figure 1. A) Resolution of racemic b-phenylalanines using PAM and PAL. B) Proposed mechanism for MIO-based enzymes.
The cinnamate product will be released in a lyase-catalyzed reaction, while a mutase will catalyze the ammonia readdition
as shown.
tandem reaction based on shifting an equilibrium has
also been explored for preparation of enantiopure ep- the kinetic resolution of (R,S)-b-phenylalanine at
oxide from halo ketones.[10]
2 mM concentration was carried out using wild-type
To establish a proof of principle for this approach,
Phenylalanine ammonia lyase (PAL) catalyzes the PAM in the absence of PAL. When approximately
conversion of various (S)-a-phenylalanines to cinnam- 27% of b-phenylalanine was converted, (R)-b-phenyl-
ic acids.[11] Both PAL and PAM rely on a protein-de- alanine and (S)-a-phenylalanine reached an equilibri-
rived cofactor, 4-methylideneimidazol-5-one (MIO), um and the conversion of (R)-b-phenylalanine
which is generated autocatalytically from three active stopped (Figure 2, upper panel). The ee of (S)-b-phe-
site residues, Ala-Ser-Gly, forming an MIO signature nylalanine reached a value of only ~40%. In a paral-
motif.[12] The catalytic role of the MIO cofactor has lel reaction, PAL was added and the reaction pro-
been disputed, but most evidence suggests that it ceeded to ~55% substrate conversion after 48 h yield-
makes a covalent adduct with the ammonia and the ing enantiopure (S)-b-phenylalanine (99% ee)
amine substrate (Figure 1 B).[12e] The similarity in re- (Figure 2, lower panel). This result corresponds well
action conditions and complementarity in substrate to the expected conversion pattern and proves that
specificity between PAM and PAL make the latter an PAL is needed for the complete conversion of (R)-b-
ideal candidate for driving the PAM-catalyzed kinetic phenylalanine.
resolution to completion.
To investigate the scope of the catalytic system, a
In this communication, we report a new approach variety of compounds were tested. In order to acceler-
to prepare a series of (S)-b-phenylalanines in which ate the reaction, a PAM mutant Q319M, which pos-
PAM is used in combination with PAL from Rhodo- sesses higher activity towards b-amino acids and was
sporidium toruloides. In the Scheme shown in prepared on the basis of the similarity of PAM to
Figure 1 A, PAM catalyzes the conversion of (R)-b- PAL[13] and tyrosine aminomutase,[14,15] was used in-
phenylalanines [(R)-1] to their corresponding a-iso- stead of wild-type PAM. We found that several b-phe-
mers [(S)-2], which undergo PAL-catalyzed elimina- nylalanines can be converted by PAM Q319M with a
tion of ammonia to form cinnamic acids 3. The intro- reasonable rate and their corresponding (S)-a-isomers
duction of the latter enzyme should overcome the un- are good substrates of PAL. Hence, these compounds
favourable equilibrium, since the second reaction is could be processed by the tandem reaction described
quasi-irreversible and the reverse reaction requires a here (Figure 3).
very high concentration of ammonia (>1M). In addi-
The kinetic resolution of compounds 1 was per-
tion, the cinnamic acid products can be easily separat- formed on an analytical scale. For compound 1a, less
ed from the remaining (S)-b-phenylalanines.
PAL was added since it is the natural substrate. The
conversion and ee were determined by chiral HPLC
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Adv. Synth. Catal. 2010, 352, 1409 – 1412

Efficient Tandem Biocatalytic Process for the Kinetic Resolution of Aromatic b-Amino Acids
with 24 h time intervals. All five compounds were suc-
cessfully resolved, giving the corresponding enantio-
pure (S)-b-phenylalanines at approximately 50% con-
version (Table 1). The E-values that were observed
with all the remaining enantiomers are sufficiently
high to be practically useful.
Having found excellent enantioselectivity and good
conversion, we set up a preparative experiment to ki-
netically resolve racemic b-phenylalanine 1a. The
preparative scale reaction was performed by incubat-
ing 50 mg of racemic b-phenylalanine with 7 mg of
PAM Q319M (0.03 mol%) and 0.5 mg of PAL
(0.002 mol%) in 30 mL buffer. To simplify product
isolation, phosphate buffer was used, instead of
Tris·HCl buffer. After 72 h, the (R)-b-phenylalanine
was completely transformed. The product was puri-
fied on a Dowex 50W X8 cation exchange resin
column, to give 48% isolated yield of pure (S)-b-phe-
nylalanine [(S)-1a] with an excellent ee of>99%.
In summary, a novel system was developed for the
kinetic resolution of racemic b-amino acids using an
aminomutase (PAM) coupled with an ammonia lyase
(PAL). Various aromatic b-amino acids were resolved
in high yields and with excellent enantioselectivities.
The synthetic applications of PAM to obtain enantio-
pure (R)-b-phenylalanines have been explored previ-
ously. By introducing PAL into the reaction system,
PAM can also be used for the preparation of (S)-b-
amino acids. Since different PALs possess varying
substrate spectra,[17] the substrate range of this
tandem process can be broadened by using other
PALs (e.g., PAL from Petroselinum crispum).[13] Con-
sidering the mild reaction conditions, the fact that no
expensive cofactors are required and the simple
downstream-processing, this newly introduced shift-
moving enzymatic system provides an attractive
method for the preparation of useful chiral building
blocks.
Figure 2. PAM-catalyzed resolution of rac-b-phenylalanine
(2 mM), without (upper panel) and with (lower panel) PAL.
&
^
*
, (R)-a-phenylalanine; , (S)-a-phenylalanine; , (R)-b-
~
phenylalanine; , (S)-b-phenylalanine.
Experimental Section
Expression and Purification of PAM and PAL
The genes coding for PAM (T. chinensis) and PAL (R. toru-
loides) with optimized codon usage for E. coli were synthe-
Figure 3. Substrates tested for the PAM/PAL system.
Table 1. PAM Q319M/PAL-catalyzed kinetic resolution of substrates 1.[a]
Substrate
Time
PAM Q319M (mol%)
PAL (mol%)
Configuration of 1
Conversion
ee of (S)-1
E
1a
1b
1c
1d
1e
72 h
72 h
48 h
48 h
96 h
0.03
0.03
0.03
0.06
0.09
0.002
0.02
0.02
0.02
0.02
S
S
S
S
S
52%
51%
51%
52%
50%
99%
99%
99%
98%
97%
116
>200
>200
91
>200
[a]
30 mmol of racemic b-phenylalanines were incubated with a suitable amount of purified PAM Q319M and PAL[16] in 3 mL
Tris·Cl buffer (20 mM, pH 8.8, with 3% glycerol) at 378C.
Adv. Synth. Catal. 2010, 352, 1409 – 1412
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Bian Wu et al.
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sized by DNA 2.0. The construction of the PAM expression
vector was described before.[8a] Similarly, the PAL gene was
cloned into the pBAD-His vector (Invitrogen) at the same
cloning sites (NdeI/HindIII) as PAM. The wild-type PAM,
the Q319M mutant and PAL were expressed in E. coli
TOP10 as N-terminal hexahistidine fused proteins. The pu-
rification of the protein was achieved by one step Ni-based
metal affinity chromatography. After purification, the
enzyme was desalted into a storage buffer (20 mM Tris·HCl,
1 mM DTT, 25% glycerol, pH 8.8) and stored at À808C.
The purity of the protein was analyzed by SDS-PAGE and
the protein concentration was determined by the Bradford
assay.
7991–8035; c) D. Seebach, A. K. Beck, S. Capone, G.
Deniau, U. Groselj, E. Zass, Synthesis 2009, 1–32.
´
[2] B. Weiner, W. Szymanski, D. B. Janssen, A. J. Min-
naard, B. L. Feringa, Chem. Soc. Rev. 2010, 39, 1656–
1691.
[3] a) A. Liljeblad, L. T. Kanerva, Tetrahedron 2006, 62,
5831–5854; b) V. Gotor-Fernꢂndez , V. Gotor, Curr.
Opin. Drug. Discov. Devel. 2009, 12, 784–797.
[4] S. Gedey, A. Liljeblad, F. Fꢀlçp, L. T. Kanerva, Tetrahe-
dron: Asymmetry, 2001, 12, 105–110.
[5] M. Solymꢂr, F. Fꢀlçp, L. T. Kanerva, Tetrahedron:
Asymmetry 2002, 13, 2383–2388.
[6] M. Solymꢂr, L. T. Kanerva, F. Fꢀlçp, Tetrahedron:
Asymmetry 2004, 15, 1893–1897.
Preparative Scale Kinetic Resolution of Racemic
[7] K. L. Klettke, S. Sanyal, W. Mutatu, K. D. Walker, J.
b-Phenylalanine
Am. Chem. Soc. 2007, 129, 6988–6989.
´
[8] a) B. Wu, W. Szymanski, P. Wietzes, S. de Wildeman,
The preparative scale reaction was performed by incubating
50 mg of racemic b-phenylalanine with 7 mg of PAM
Q319M (0.03 mol%) and 0.5 mg of PAL (0.002 mol%) in
30 mL phosphate buffer (20 mM, pH 8.8, with 3% glycerol).
The reaction mixture was filter-sterilized after the addition
of the enzyme, and kept at 378C. Every 24 h, samples were
taken from the reaction mixture to examine the conversion
of the (R)-b-phenylalanine. After 72 h, the (R)-b-phenylala-
nine was completely transformed and the reaction was
stopped by adjusting the pH to 1.5. The reaction mixture
was then applied to a Dowex 50W X8 cation exchange resin
column, pre-conditioned by washing with 2M aqueous am-
monia, 1M aqueous HCl and water. The column was
washed with H2O to remove the undesired compounds, such
as cinnamic acid, buffer components and glycerol. (S)-b-
Phenylalanine was eluted with 2M ammonia solution. After
lyophilization, (S)-b-phenylalanine was obtained as white
solid; yield: 24 mg.
G. J. Poelarends, B. L. Feringa, D. B. Janssen, ChemBio-
Chem 2009, 10, 338–344; b) W. Szymanski, B. Wu, B.
Weiner, S. de Wildeman, B. L. Feringa, D. B. Janssen, J.
Org. Chem. 2009, 74, 9152–9157.
[9] W. Mutatu, K. L. Klettke, C. Foster, K. D. Walker, Bio-
chemistry 2007, 46, 9785–9794.
[10] a) T. M. Poessl, B. Kosjek, U. Ellmer, C. C. Gruber, K.
Edegger, K. Faber, P. Hildebrandt, U. T. Bornscheuer,
W. Kroutil, Adv. Synth. Catal. 2005, 347, 1827–1834;
b) B. Seisser, I. Lavandera, K. Faber, J. H. Lutje Spel-
berg, W. Kroutil, Adv. Synth. Catal. 2007, 349, 1399–
1404; c) J. H. Schrittwieser, I. Lavandera, B. Seisser, B.
Mautner, W. Kroutil, Eur. J. Org. Chem. 2009, 14,
2293–2298.
[11] K. R. Hanson, E. A. Havir, Arch. Biochem. Biophys.
1970, 141, 1–17.
[12] a) D. Rçther, D. Merkel, J. Rꢃtey, Angew. Chem. 2000,
112, 2592–2594; Angew. Chem. Int. Ed. 2000, 39, 2462–
2464; b) C. L. Steele, C. L. Chen, C. L. Dougherty,
C. L. Li, S. Hofstead, K. S. Lam, Z. H. Xing, S. J.
Chiang, Arch. Biochem. Biophys. 2005, 438, 1–10;
c) K. D. Walker, K. Klettke, T. Akiyama, R. Croteau, J.
Biol. Chem. 2004, 279, 53947–53954; d) S. Bartsch,
U. T. Bornscheuer, Angew. Chem. 2009, 121, 3412–
3415; Angew. Chem. Int. Ed. 2009, 48, 3362–3365;
e) H. A. Cooke, C. V. Christianson, S. D. Bruner, Curr.
Opin. Chem. Biol. 2009, 13, 453–461.
Supporting Information
A list of used compounds and stereochemical analysis of the
products are presented in the Supporting Information.
Acknowledgements
The project is financially supported by the Netherlands Min-
istry of Economic Affairs and the B-Basic partner organiza-
tions (www.b-basic.nl) through B-Basic, a public-private
NWO-ACTS program (ACTS=Advanced Chemical Technol-
ogies for Sustainability). The authors thank Dr. O. May and
Dr. B. Kaptein from DSM for helpful discussions.
[13] H. Ritter, G. E. Schulz, Plant Cell, 2004, 16, 3426–
3436.
[14] a) C. V. Christianson, T. J. Montavon, G. M. Festin,
H. A. Cooke, B. Shen, S. D. Bruner, J. Am. Chem. Soc.
2007, 129, 15744–15745; b) C. V. Christianson, T. J.
Montavon, S. G. Van Lanen, B. Shen, S. D. Bruner, Bio-
chemistry 2007, 46, 7205–7214.
[15] B. Wu and D. B. Janssen, unpublished data.
[16] Purity>95%, determined by SDS-polyacrylamide gel
electrophoresis with Coomassie staining.
[17] L. Poppe, J. Rꢃtey, Angew. Chem. 2005, 117, 3734–
3754; Angew. Chem. Int. Ed. 2005, 44, 3668–3688.
References
[1] a) E. Juaristi, V. Soloshonok, Enantioselective Synthesis
of b-Amino Acids, Wiley-Interscience, New York, 2nd
edn., 2005; b) M. Liu, P. Sibi, Tetrahedron 2002, 58,
1412
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Adv. Synth. Catal. 2010, 352, 1409 – 1412