Enantiomerically pure β-phenylalanine analogues from α-β-phenylalanine mixtures in a single reactive extraction step

Article abstract of DOI:10.1039/b921661h

An efficient and selective method for the extraction of α-amino acids in preference over their β-isomers using PdCl₂(PPh ₃)₂ was discovered, which enables the separation of product mixtures obtained in the enantioselective enzymatic formation of β-amino acids. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b921661h

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Enantiomerically pure b-phenylalanine analogues from a–b-phenylalanine
mixtures in a single reactive extraction stepw
a
ab
Bastiaan J. V. Verkuijl, Wiktor Szymanski, Bian Wu,b Adriaan J. Minnaard,*a
´
Dick B. Janssen,*b Johannes G. de Vries*ac and Ben L. Feringa*a
Received (in Cambridge, UK) 16th October 2009, Accepted 12th November 2009
First published as an Advance Article on the web 4th December 2009
DOI: 10.1039/b921661h
An efficient and selective method for the extraction of a-amino
acids in preference over their b-isomers using PdCl2(PPh3)2 was
discovered, which enables the separation of product mixtures
obtained in the enantioselective enzymatic formation of
b-amino acids.
resultant reaction mixture, however, consists of both a- and
b-amino acids. To the best of our knowledge, no convenient
system has been described so far for the separation of these
isomers. Although these enzymatic routes are particularly
attractive, the severe problems associated with isomer separation
presents a major limitation to the use of the PAM-catalyzed
synthetic methods.
The stereoselective synthesis of b-amino acids has attracted
increased attention in recent years as these compounds play a
key role in chemical biology, natural product synthesis and
drug delivery.1 Due to major advantages in the context of
sustainable and green chemistry, biocatalytic methods emerge
as valuable tools for the preparation of this class of
compounds.2 Most biocatalytic methods used for the preparation
of b-amino acids are based on the kinetic resolution of racemic
precursors, which severely limits their efficiency. Protocols
which are free of this limitation are scarce and rely on the use
of ammonia lyases,3 aminotransferases4 and aminomutases.5,6
Two variants of phenylalanine aminomutase (PAM)
catalysed conversions have been proposed for the synthesis
of aromatic b-amino acids (Scheme 1).
The separation of amino acid mixtures is mainly performed
by chromatographic methods or via crystallization.7 The use
of reactive liquid–liquid extraction8 could provide a facile
method for the purification of b-amino acids, which can also
be applied in a continuous fashion.9 Palladium(II) phosphine
complexes have proven their capability in complexation with
a-amino acids.10 Furthermore, we have recently shown that
chiral palladium phosphine complexes can separate the
enantiomers of racemic mixtures of a-amino acids using
enantioselective liquid–liquid extraction (ELLE). The
palladium complex can be fully recycled and re-employed in
a new extraction by treatment with aqueous HCl.11
During our studies on reactive extraction processes we
decided to investigate the possible use of the low-cost and
simple complex PdCl2(PPh3)2 as a host in the isomer selective
liquid–liquid extraction (ILLE) of mixtures of the a- and
b-phenylalanine isomers (Fig. 1). The initial results are
presented in Table 1.12 An excellent selectivity, with an aa–b
value of 47 is observed for the extraction of phenylalanine
regioisomers (entry 1). Phenylalanine analogues (entries 2–4)
with various substituents in the para position also show very
high selectivities with aa–b, ranging from 29 to 89.
Phenylalanine aminomutase can be used either for
isomerization of substituted a-phenylalanines to their
b-isomers (Scheme 1b)5 or to catalyse direct ammonia addition
to substituted cinnamic acids (Scheme 1a).6 In both cases
the products are obtained with excellent enantiopurity. The
One possible explanation for the difference in the distribution
+
is the higher pKa of the NH3 group of b-amino acids, when
compared to their a-counterparts. For example, pKa(b-Ala) =
10.24, as compared to pKa(a-Ala) = 9.69. The complexation
of the amino acid to the palladium host is supposed to involve
Scheme
1 PAM-catalyzed addition of ammonia to substituted
cinnamic acids (a) and isomerization of phenylalanine analogues (b).
a Organic and Molecular Inorganic Chemistry, University of
Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
E-mail: a.j.minnaard@rug.nl, b.l.feringa@rug.nl
b Department of Biochemistry, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, Nijenborgh 4,
9747 AG, Groningen, The Netherlands. E-mail: d.b.janssen@rug.nl
c DSM Pharmaceutical Products, Innovative Synthesis & Catalysis
P.O. Box 18, 6160, MD Geleen, The Netherlands.
E-mail: hans-jg.vries-de@dsm.com
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b921661h
Fig. 1 Schematic representation of ILLE. Symbols: : a-amino acid,
: b-amino acid, : host.
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 901–903 | 901

View Article Online
Table 1 ILLE of mixtures of a-Phe and b-Phe analogues
pH = 10.0 than at pH = 9.0. The aa–b is between 26 and 31 in
the range of pH 5.0–6.0. The optimum is reached at pH = 7.0
with aa–b = 4100. The increase in the distribution of b-Phe
results in the decrease in selectivity, and aa–b was found to be
4.5 at pH = 10.0.
D(org/aq)a
aa–b
b
Entry
1
Substrate
a-Phe
b-Phe
3.93
0.08
4.97
0.17
1.23
0.01
5.33
0.13
47
—
29
—
89
—
42
—
2
3
4
a-4Cl-Phe
b-4Cl-Ph
a-4F-Phe
b-4F-Phe
a-4MeO-Phe
b-4MeO-Phe
The distribution of both isomers might be controlled by
using additional equivalents of the host, while the operational
selectivity is kept constant at constant pH.16 The aa–b
remains 4100 when the host concentration is increased up
to 4 equiv. As a result, the b-Phe can be obtained in 95+ %
purity and 74% yield in a single extraction step.
Conditions: host = PdCl2(PPh3)2, [host] = 1.0 mM, [a-Phe] = [b-Phe] =
1.0 mM, organic phase = dcm, aqueous phase = doubly distilled
and [NaHCO3] = 2.0 mM, T = 6 1C.a The distribution D(org/aq) of
the substrate (AA) over the two phases is defined as the ratio of
Finally, we have investigated the use of an optimized
extraction system for the purification of b-Phe from a reaction
mixture obtained in
a PAM-catalyzed enantioselective
the concentrations of the substrate in the two phases (D = [AA]org
b
/
[AA]aq). The operational selectivity (aa–b) is defined as the ratio of
distribution of isomers (aa–b = Da-AA/Db-AA).
addition of ammonia to (E)-cinnamic acid (Scheme 1a).
Phenylalanine aminomutase (PAM) from Taxus chinensis
was obtained as recently described by Janssen et al.6 in high
purity.17 (E)-Cinnamic acid was used as a substrate in a
PAM-catalyzed amination reaction, as described before
(Scheme 1a).6 The ee values that were obtained for both
products were 499%. After initial workup,18 the concentrations
of a-Phe and b-Phe were found to be 0.27 mM and 0.20 mM,
respectively. The thus obtained mixture was subjected to an
ILLE process. Different concentrations of the host with
respect to [b-Phe]aq,0 were studied. The results are presented
in Table 2.
an anion exchange mechanism, where a chloride is exchanged by
a carboxylate function. In a buffered aqueous phase, the a-Phe
carboxylate will be predominant compared to the b-Phe
carboxylate,13 which will lead to a higher distribution of a-Phe.
Another cause for the difference in distribution between
a- and b-amino acids may arise from the different structures of
the formed complexes with the host. The supposed bidentate
interaction of the substrate with the palladium center10 would
result in a five membered ring for a-amino acids14 and a six
membered ring for b-amino acids,15 which could lead to a
difference in stability.
The distribution of a-Phe increases rapidly upon increase
of the host concentration. D(b-Phe) rises up to 0.40 at a
stoichiometry of 2.0. The aa–b is lower than in the case of
the preliminary studies (vide supra). This may be due to the
different ratio of a-Phe vs. b-Phe in the initial mixture.
Nevertheless, b-Phe can be obtained with 72% yield, 99% ee
and 96+ % purity with respect to a-Phe (entry 2). At higher
host stoichiometries, the selectivity decreases, as do the yield
and the purity of b-Phe.
To optimize the operational selectivity of the extraction of
a-Phe over b-Phe, the extraction of a-Phe from the aqueous
phases was performed at different pH values. The influence of
the pH of the aqueous phase on the distribution and the aa–b
of the ILLE of an equimolar mixture of a-Phe and b-Phe was
investigated over a pH range from 3.0 to 10.0. The results are
presented in Fig. 2.
In conclusion, the potential of palladium phosphine
complexes is demonstrated for the separation of a- and
b-phenylalanine analogues. By washing the aqueous solution
of the a- and b-amino acid with a dichloromethane solution of
the palladium phosphine complex, the a-phenylalanine is
extracted selectively from the aqueous phase, leaving the
b-isomer behind. In most cases, the distribution of the
a-phenylalanine analogues is several orders of magnitude
larger than that of the b-isomers. A possible explanation is
the difference in pKa of a- and b-Phe in combination with the
difference in stability of the formed complexes with the host.
In the pH range of 3.0–4.0 no significant distribution is
observed. At pH = 5.0, D(a-Phe) is 0.09 and increases to 3.17
at pH = 9.0. The distribution of b-Phe is not significant in the
range of pH 3.0–7.0 and increases from D(b-Phe) = 0.10 at
pH = 8.0 to D(b-Phe) = 0.61 at pH = 10.0. At pH = 10.0,
the b-Phe seems to strongly compete with a-Phe for complexation,
which causes the distribution of a-Phe to be slightly lower at
Table 2 Separation by ILLE of the a–b-isomer mixture of phenyl-
alanines synthesized using PAM
Yield (%)
(b-Phe)
Purity (%)
(b-Phe)
ee (%)
(b-Phe)
Entry
[host]/mM
aa–b
1
2
3
4
0.2
0.4
0.6
0.9
61.0
77.7
19.4
4.6
94
72
13
2
82
96
94
82
99+
99+
99+
99+
Fig. 2 The pH dependence of the ILLE of a-Phe and b-Phe. Grey
bars: D(a-Phe), white bars: D(b-Phe), ~: aa–b. Coordinates: D(org/aq)
(left) and aa–b (right). Conditions: host = PdCl2(tpp)2, [host] = 1.0 mM,
[a-Phe] = [b-Phe] = 1.0 mM, organic phase = dcm, aqueous phase =
100 mM phosphate buffer, T = 6 1C.
Host = PdCl2(tpp)2, [a-Phe]aq,0 = 0.27 mM, [b-Phe]aq,0 = 0.20 mM,
organic phase = dcm, aqueous phase = 90.0 mM phosphate buffer
(pH = 7.0), T = 6 1C. ee determined by chiral HPLC.
ꢀc
This journal is The Royal Society of Chemistry 2010
902 | Chem. Commun., 2010, 46, 901–903

View Article Online
In a single extraction step, it is possible to obtain the
b-phenylalanine with 495% purity, while other phenylalanine
analogues give similar results. The preparative example of
the synthesis of b-phenylalanine using the PAM-catalysed
amine addition to cinnamic acid yielded b-Phe in 72% yield,
96+ % purity and 99+ % ee.
4 J. Kim, D. Kyung, H. Yun, B. K. Cho, J.-H. Seo, M. Cha and
B. G. Kim, Appl. Environ. Microbiol., 2007, 73, 1772.
5 K. L. Klettke, S. Sanyal, W. Mutatu and K. D. Walker, J. Am.
Chem. Soc., 2007, 129, 6988.
6 (a) B. Wu, W. Szymanski, P. Wietzes, G. J. Poelarends,
B. L. Feringa and D. B. Janssen, ChemBioChem, 2009, 10, 338;
(b) W. Szymanski, B. Wu, B. Weiner, S. de Wildeman,
B. L. Feringa and D. B. Janssen, J. Org. Chem., 2009, DOI:
10.1021/jo901833y.
7 M. Breuer, K. Ditrich, T. Habicher, B. Hauer, M. Kesseler,
R. Sturmer and T. Zelinski, Angew. Chem., Int. Ed., 2004, 43, 788.
8 E. Eliel, S. Wilen and L. Mander, Miscellaneous Separation
Methods, in Stereochemistry of Organic Compounds, Wiley,
New York, NY, 1994, p. 416.
9 B. Schuur, A. J. Hallett, J. G. M. Winkelman, J. G. de Vries and
H. J. Heeres, Org. Process Res. Dev., 2009, 13, 911.
10 J. Bravo, C. Cativiela, J. E. Chaves, R. Navarro and
E. P. Urriolabeitia, Inorg. Chem., 2003, 42, 1006.
11 B. J. V. Verkuijl, A. J. Minnaard, J. G. de Vries and B. L. Feringa,
J. Org. Chem., 2009, 74, 6526.
12 For definitions, see caption of Table 1.
13 A difference in pKa of 0.55 will lead to a concentration difference
more than three times as high for the anions in a buffered solution.
14 D. Koch, W. Hoffmuller, K. Polborn and W. Beck, Z. Naturforsch.,
B: Chem. Sci., 2001, 56, 403.
The current system can be applied in large-scale processes
due to the possibility of Pd-complex recovery, which lowers
the operational costs and enables the continuous extraction of
unwanted isomers from reaction mixtures with a closed
circulation of the transition metal complex. Studies along
these lines are in progress.
This research was financially supported by the Netherlands
Organisation for Scientific Research (NWO) through the
Separation Technology program in cooperation with DSM
and Schering-Plough and by the B-Basic partner organizations
(www.b-basic.nl) through B-Basic,
a
public–private
NWO-ACTS programme (ACTS = Advanced Chemical
Technologies for Sustainability).
15 A. Bohm and D. Seebach, Helv. Chim. Acta, 2000, 83, 3262.
16 M. Steensma, N. J. M. Kuipers, A. B. De Haan and G. Kwant,
Chirality, 2006, 18, 314.
Notes and references
1 (a) E. Juaristi and V. Soloshonok, Enantioselective Synthesis of
b-Amino Acids, Wiley-Interscience, New York, 2nd edn, 2005;
(b) M. Liu and M. P. Sibi, Tetrahedron, 2002, 58, 7991;
(c) D. Seebach, A. K. Beck, S. Capone, G. Deniau, U. Groselj
and E. Zass, Synthesis, 2009, 1; (d) D. Seebach and J. Gardiner,
Acc. Chem. Res., 2008, 41, 1366; (e) R. P. Cheng, S. H. Gellman
and W. F. DeGrado, Chem. Rev., 2001, 101, 3219.
2 A. Liljeblad and L. T. Kanerva, Tetrahedron, 2006, 62, 5831.
3 B. Weiner, G. J. Poelarends, D. B. Janssen and B. L. Feringa,
Chem.–Eur. J., 2008, 14, 10094.
17 495% by SDS-PAGE with Coomassie staining.
18 As the conversion over 5 d was not complete, the mixture still
contained (E)-cinnamic acid. To avoid interference with the
isolation of b-Phe, (E)-cinnamic acid was removed by lowering
the pH to pH = 2.0 by addition of HCl (aq) and subsequent
extraction with dcm. The remaining a–b-Phe mixture was analyzed
by RP-HPLC and showed that (E)-cinnamic acid was removed
effectively. The a–b-Phe mixture was finally diluted 10 times with
100 mM aqueous phosphate buffer (pH = 7.0).
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 901–903 | 903