Overcoming thermodynamic and kinetic limitations of aldolase-catalyzed reactions by applying multienzymatic dynamic kinetic asymmetric transformations

Article abstract of DOI:10.1002/anie.200604142

(Figure Presented) Dynamic and successful: The asymmetric synthesis of 2-amino-1-phenylethanol was achieved by aminomethylation of benzaldehyde in the presence of the two enzymes L-threonine aldolase and L-tyrosine decarboxylase in a novel one-pot, two-enzyme process (see scheme). A modified method with three enzymes led to the enantioenriched amino alcohol in very high yield.

Full text of DOI:10.1002/anie.200604142

Communications
DOI: 10.1002/anie.200604142
Biocatalysis
Overcoming Thermodynamic and Kinetic Limitations of Aldolase-
Catalyzed Reactions by Applying Multienzymatic Dynamic Kinetic
Asymmetric Transformations**
Johannes Steinreiber, Martin Schürmann, Michael Wolberg, Friso van Assema,
Christoph Reisinger, Kateryna Fesko, Daniel Mink, and Herfried Griengl*
Nature uses enzymes for both the formation and cleavage of
C C bonds within aldols.[1] More than 40 aldolases have been
À
characterized to date, and their application offers biocatalytic
access to a-hydroxycarbonyl compounds.[2] However, the
usefulness of this transformation for synthetic purposes is
often limited by the position of the equilibrium (thermody-
namic limitation).[3] Furthermore, kinetic limitations also
exist, which means that the reaction rates for the formation
of the stereoisomers are sometimes not sufficiently different
to ensure high stereoselectivities.
Herein we show how these limitations can be overcome
for the studied transformation—the synthesis of b-hydroxy-a-
amino acids—by the application of threonine aldolases (TAs)
to catalyze the reaction of glycine with appropriate aldehydes.
All glycine-dependent aldolases require pyridoxal-5’-phos-
phate (PLP) to catalyze the reversible aldol reaction of
glycine and the acceptor aldehyde. We describe herein the
transformation of benzaldehyde to phenylserine (1,
Scheme 1) as a model system. Several natural l- and d-
Scheme 1. Synthesis of l-phenylserine (1) using l-threonine aldolase.
threonine aldolases (lTA/dTA, EC 4.1.2.5)[4] and a recently
engineered dTA (starting from alanine racemase)[5] have
been discovered so far, and it has been shown that all TAs can
transform a variety of aromatic and aliphatic aldehydes.[6] If a
tenfold excess of glycine over benzaldehyde is used (lTA,
optimized conditions) an 85% yield is achieved, since the
equilibrium is shifted to the side of the aldol.[7] The process
can be optimized by continual removal of the product to
ensure high yields.[8]
A still better approach to overcome the thermodynamic
limitations is to pull the transformation to the aldol side of the
equilibrium by an irreversible reaction of the initially formed
b-hydroxyamino acid 1. lTA, dTA, and most enzymes
involved in amino acid metabolism/catabolism are highly
Ca selective (l/d), while the Cb selectivity is disappointingly
low for both lTA and dTA (kinetic limitation, syn/anti).[6] In
recent studies we showed that the kinetic limitation was
overcome by using dTA from Alcaligenes xylosoxidans,[9]
which yielded diastereomerically pure d-syn-1.[7] For lTA,
the enantioselectivity at the carbon atom bearing the amino
groupis high ( ee > 99%), whereas the selectivity for the
transformation of the aldehyde to a hydroxy groupis low; this
[*] Dr. J. Steinreiber, Dr. C. Reisinger, Prof. H. Griengl
Research Centre Applied Biocatalysis
Petersgasse 14, 8010 Graz (Austria)
Fax: (+43)316-873-8740
E-mail: herfried.griengl@a-b.at
Homepage: http://www.a-b.at
Dr. M. Schürmann, F. van Assema, Dr. D. Mink
DSM Pharmaceutical Products
Advanced Synthesis, Catalysis & Development
P.O. Box 18, 6160 MD Geleen (The Netherlands)
Dr. M. Wolberg
DSM Pharma Chemicals, ResCom
Donaustaufer Strasse 378, 93055 Regensburg (Germany)
leads to a diastereomeric ratio l-syn-1/l-anti-1 of 60:40.[7]
consecutive and Cb-selective stepwould also helpto over-
come this limitation.
A
K. Fesko
Institute of Organic Chemistry
Graz University of Technology
Stremayrgasse 16, 8010 Graz (Austria)
l-Amino acid decarboxylases are known to be highly
substrate specific. Only l-tyrosine decarboxylase (l-TyrDC,
PLP-dependent) was shown to tolerate an additional hydroxy
groupin the b position and to be tolerant towards aromatic
substituents.[10] l-TyrDC is highly selective with respect to the
NH2-bound carbon atom, and only the l enantiomer is
transformed. Thus, l-TyrDC can be utilized to resolve
chemically synthesized dl-syn-1 into 2-amino-1-phenyletha-
nol ((R)-2) in a maximum yield of 50%, with d-syn-1 left
behind.[10] The addition of dTA enabled the untransformed
diastereomer (d-syn-1) to be recycled by degrading it to
benzaldehyde and glycine in a so-called parallel kinetic
resolution process.[11,12]
[**] The Österreichische Forschungsförderungsgesellschaft (FFG), the
Province of Styria, the Styrian Business Promotion Agency (SFG),
the City of Graz, the Fonds zur Förderung der wissenschaftlichen
Forschung (FWF, project W901-B05 DK Molecular Enzymology),
and the European Commission (within the framework of a Marie-
Curie Industrial Host Fellowship to M.S. and M.W.) are acknowl-
edged for financial support. We would like to thank Marcel
Wubbolts, Theo Sonke, and Kurt Faber for stimulating discussions,
as well as to Karl Gruber and Michael Uhl for the model of
l-threonine aldolase in the Table of Contents graphic.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Chemie
However, besides this simple resolution of dl-syn-1, the
thermodynamic limitations can be overcome by a combina-
tion of an lTA-catalyzed aldol reaction with an in situ
decarboxylation using l-TyrDC. As a consequence, the
aldol equilibrium is shifted to the l-aldol products, thus
resulting in high conversions. l-TyrDC (from Enterococcus
faecalis V583)[13] and lTA (from Pseudomonas putida)[14]
were cloned and overexpressed in Escherichia coli. In our
model reaction we investigated the unprecedented bienzy-
matic synthesis of 2 from benzaldehyde and glycine by
utilizing a reversible lTA-catalyzed aldol reaction (forming
the intermediate product l-syn-1/l-anti-1) with subsequent
irreversible decarboxylation (Scheme 2). The product of this
amino alcohol (R)-2 (91%) was complete after 58 h.
(Scheme 3). Hence, the combination of lTA and l-TyrDC
promises to be suitable for the quantitative conversion of
aldehydes into 1,2-amino alcohols. Interestingly, the moder-
Scheme 3. Conversion of benzaldehyde into the intermediates l-syn-1/
l-anti-1 and the final product (R)-2. Reaction conditions: benzaldehyde
100 mm, glycine 1m, lTA 38 U, l-TyrDC 0.4 U, PLP 50 mm, pH 5.5,
258C; yield, diastereomeric ratio (d.r.), and enantiomeric ratio (e.r.)
were determined by HPLC.
ate Cb selectivity of lTA (R/S 60:40) in the aldol reaction was
slightly altered (R/S 43:57) in the bienzymatic process. This
finding can be explained by the fact that l-syn-1 (2S,3R) is
converted into (R)-2 at a faster rate than is l-anti-1 (2S,3S) to
(S)-2, thus shifting the syn/anti ratio of the intermediate
products l-syn-1/l-anti-1.
This approach resulted in the low selectivity at the
b position of 2 being improved to 89:11 (R/S) by stereoselec-
tive decarboxylation. The stereomeric ratios of l-1 (d.r.) and
2 (e.r.) were constant throughout the reaction, which implies
that the dynamic process is efficient.
The resolution of dl-syn-1 by using the described one-pot,
two-enzyme DYKAT is not possible because of the strict
l selectivity of lTA and l-TyrDC. Only l-syn-1 is trans-
formed in a so called kinetic asymmetric transformation
(KAT), which leaves the d enantiomer behind. High ee values
(> 99%) of the amino alcohol (R)-2 can consequently be
obtained, but only in a maximum yield of 50%. A novel one-
pot, three-enzyme protocol (Scheme 4) was investigated to
overcome this limitation, but with the high selectivity
retained. The initial KAT (l-TyrDC) of l-syn-1 yielded
Scheme 2. Synthesis of 2-amino-1-phenylethanol (2) using l-threonine
aldolase and l-tyrosine decarboxylase.
formal one-pot aminomethylation of benzaldehyde retains
the hydroxy-substituted stereogenic center, while “losing” the
second center—as the amino function is transformed into a
terminal group. By this means, the moderate Cb selectivity of
the aldolase is drastically improved because of the high syn/
anti selectivity of l-TyrDC (10:1, see the Supporting Infor-
mation). The reaction sequence described is to the best of our
knowledge the first example of a bienzymatic
resolution of diastereomers. A similar process
involving an organocatalytic protocol was referred
to as a dynamic kinetic asymmetric transformation
(DYKAT).[15,16] For dynamic processes it is of
utmost importance that the equilibration (ka1 and
ka2) is much faster than the irreversible step( kb1 and
kb2) so as to obtain yield high selectivities and to
reach the theoretical yield of 100%.[17] Equilibra-
tion studies using lTA showed the syn–anti inter-
conversion of 1 to be fast (see the Supporting
Information).
In a first approach to overcome the thermody-
namic limitation by this means we tested the
compatibility of lTA and l-TyrDC. Indeed, the
equilibrium reaction of benzaldehyde and glycine to
form l-syn-1/l-anti-1 was successfully shifted
towards the product side by the irreversible decar-
Scheme 4. Theoretical outcome for the synthesis of (R)-2 by using a KAT/DYKAT
protocol (88% ee: 94% (R)-2, 6% (S)-2). [a] Estimated Cb selectivity of the coupled
boxylation. The conversion of benzaldehyde into
the intermediate l-syn-1/l-anti-1 (8%) and the final lTA/l-TyrDC reaction: 89:11 (R/S; see Scheme 3).
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Communications
enantiopure (R)-2 (50%). Addition of the aldolases lTA and
DYKAT protocol starting from dl-syn-1 gave enantiopure
amino alcohol (R)-2 in good yield. Currently, we are testing
other acceptor compounds in this novel one-pot amino-
methylation to obtain valuable aromatic 1,2-amino alcohols.
dTA[9] after 24 h resulted in a three-enzyme DYKAT (l-
TyrDC, lTA/dTA) which converted the remaining d-syn-1
(50%) into benzaldehyde and glycine, and thereafter into l-
syn-1/l-anti-1 and finally (R)-2/(S)-2. Theoretically, the quan-
titative resolution of dl-syn-1 by this protocol with delayed
addition of lTA and dTA should give an ee value of 88% for
amino alcohol 2 (R, Scheme 3), when the results of the one-
pot, two-enzyme approach are considered (R/S 89:11,
Scheme 3). The following application of this protocol
showed that enantioenriched 2 (82% ee R) was obtained in
only 44% yield at pH 5.5 if all three enzymes were added at
the beginning (Table 1, entry 1). The low yield might be
Received: October 9, 2006
Published online: January 24, 2007
Keywords: aldol reaction · amino alcohols · biocatalysis · lyases ·
.
threonine aldolase
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Table 1: Synthesis of (R)-2 by using a KAT/DYKAT protocol.[a]
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Entry
pH
Yield 2 [%]
(5 h)
ee (R) [ %]
(5 h)
Yield 2 [%]
(80 h)
ee (R) [ %]
(80 h)
1[b]
2[c]
5.5
5.5
6.0
6.0
6.5
6.5
7.0
7.0
7.5
7.5
5.5
23
46
15
39
13
35
3
25
<1
10
80
>99
72
>99
80
>99
>99
>99
>99
>99
n.d.[f]
44
67
47
57
28
58
11
50
4
82
>99
82
>99
80
>99
82
>99
>99
>99
>99
3[b]
4[c]
5[b]
6[c]
7[b]
8[c]
9[b]
10[c]
11[c,d]
24
n.d.[f]
58[e]
[8] M. Bechthold, S. Makart, M. Heinemann, S. Panke, J. Biotech-
nol. 2006, 124, 146 – 162.
[9] J. Q. Liu, M. Odani, T. Yasuoka, T. Dairi, N. Itoh, M. Kataoka, S.
Shimizu, H. Yamada, Appl. Microbiol. Biotechnol. 2000, 54, 44 –
51.
[a] Conditions: 1-mL reaction volume, dl-syn-1 100 mm, glycine 900 mm,
PLP 50 mm, MnCl2 50 mm, 258C, lTA 19 U, dTA 6 U, l-TyrDC 0.4 U; yield
and ee values were determined by HPLC. [b] Addition of lTA/dTA at t=
0 h. [c] Addition of lTA/dTA after t=24 h. [d] 5-mL reaction scale.
[e] Yield of isolated product after t=100 h. [f] n.d.=not determined.
[10] T. Nikaido, N. Kawada, T. Hamatani, Y. Ueda (Daicel Chemical
Industries), WO 9523869, 1995; T. Nikaido, N. Kawada, T.
Hamatani, Y. Ueda (Daicel Chemical Industries), US 5731175,
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T. Hamatani, Y. Ueda (Daicel Chemical Industries),
US 5874613, 1999.
[11] S. Ito, T. Nikaido, A. Matsuyama (Daicel Chemical Industries),
JP 46076, 2001.
[12] For reviews about parallel kinetic resolution, see a) J. R. Dehli,
V. Gotor, Chem. Soc. Rev. 2002, 31, 365 – 370; b) E. Vedejs, M.
Jure, Angew. Chem. 2005, 117, 4040 – 4069; Angew. Chem. Int.
Ed. 2005, 44, 3974 – 4001.
[13] a) T. Boerresen, N. K. Klausen, L. M. Larsen, H. Soerensen,
Biochim. Biophys. Acta 1989, 993, 108 – 115; b) N. Connil, Y.
Le Breton, X. Dousset, Y. Auffray, A. Rince, H. Prevost, Appl.
Environ. Microbiol. 2002, 68, 3537 – 3544.
[14] J. Q. Liu, S. Ito, T. Dairi, N. Itoh, S. Shimizu, H. Yamada, Appl.
Microbiol. Biotechnol. 1998, 49, 702 – 708.
[15] DYKAT: a) asymmetric allyllic alkylation: B. M. Trost, Chem.
Pharm. Bull. 2002, 50, 1 – 14, and references therein; b) chemo-
enzymatic desymmetrization of 1,3-diols: M. Edin, J. Steinreiber,
J.-E. Bꢀckvall, Proc. Natl. Acad. Sci. USA 2004, 101, 5761 – 5766.
[16] Organocatalytic DYKAT: E. Reyes, A. Cordova, Tetrahedron
Lett. 2005, 46, 6605 – 6609.
explained by inhibitory effects of MnCl2 or dTA on l-TyrDC;
these effects will be investigated in more detail in the near
future. However, delayed addition of lTA/dTA (after 24 h)
yielded enantiopure 2 (Table 1, entries 2 and 11, ee > 99% R)
as well as improved conversion (HPLC 67%, isolated product
58%). Similar results were obtained at pH 6.0 and 6.5
(Table 1, entries 3–6). Higher pH values (7.0 and 7.5,
entries 7–10) resulted in a decreased yield because of the
low activity of l-TyrDC at pH > 6.5 (optimum pH: 5.5). The
excellent ee values of 2 together with the good yields (pH 5.5–
6.5) show that all three enzymes collaborate well. Further-
more, the kinetic limitation—low Cb selectivity in the lTA-
catalyzed reaction—is overcome and enantiopure (R)-2 is
obtained in yields of over 50%.
In summary, the protocols described herein overcome the
thermodynamic (DYKAT) and the kinetic (KAT/DYKAT)
limitations of reactions catalyzed by l-threonine aldolase.
The first bienzymatic DYKAT involves an aldolase with low
selective and a decarboxylase with high diastereoselectivity.
By applying this approach we were able to shift the aldol
reaction of glycine and benzaldehyde to quantitative con-
version and to improve the low Cb selectivity. A KAT/
[17] a) O. Pamies, J.-E. Baeckvall, Chem. Rev. 2003, 103, 3247 – 3261;
b) H. Pellissier, Tetrahedron 2003, 59, 8291 – 8327.
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