Asymmetric benzoin reaction catalyzed by benzoylformate decarboxylase

Article abstract of DOI:10.1016/S0957-4166(99)00516-9

Aromatic aldehydes are converted into benzoins by benzoylformate decarboxylase catalyzed C-C bond formation. The reaction affords (R)-benzoins with high enantiomeric excess and in good chemical yields. A broad range of aromatic aldehydes can be used as substrates in aqueous buffer or buffer/DMSO-solutions. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(99)00516-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 4769–4774
Asymmetric benzoin reaction catalyzed by benzoylformate
decarboxylase
Ayhan S. Demir,a,b Thomas Dünnwald,a Hans Iding,c Martina Pohl c and Michael Müller a,∗
aInstitut für Biotechnologie 2, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
bDepartment of Chemistry, Middle East Technical University, 06531 Ankara, Turkey
cInstitut für Enzymtechnologie der Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich, D-52426 Jülich,
Germany
Received 4 November 1999; accepted 15 November 1999
Abstract
Aromatic aldehydes are converted into benzoins by benzoylformate decarboxylase catalyzed C–C bond forma-
tion. The reaction affords (R)-benzoins with high enantiomeric excess and in good chemical yields. A broad range
of aromatic aldehydes can be used as substrates in aqueous buffer or buffer/DMSO-solutions. © 2000 Elsevier
Science Ltd. All rights reserved.
1. Introduction
Benzoin type 2-hydroxy ketones in general are important structural subunits in many biologically ac-
tive compounds and are also important synthons for stereoselective syntheses.1–5 The benzoin reaction,6
one of the oldest C–C bond forming reactions in organic chemistry, has been developed for classical
organic synthesis using non-chiral thiazolium7–9 and for stereoselective synthesis using chiral triazolium
salts10,11 as catalyst. Some other chemical methods for the enantioselective synthesis of 2-hydroxy
ketones, which are not based on C–C bond formation, have been evolved. Among them are the stereo-
selective oxidation of titanium enolates with dimethyldioxirane,12 of silyl enol ethers with chiral salen
complexes13 or fructose derived dioxirane14,15 and the stereoselective dihydroxylation of enolates with
N-sulfonyloxaziridines.16,17 As an alternative to chemical methods enantiomerically pure 2-hydroxy
ketones are prepared enzymatically by reduction of the corresponding α-diketone with baker’s yeast18,19
or an enzymatic kinetic resolution of the racemate of either 2-peroxo-,20 2-hydroxy-21 or 2-acetoxy
ketones.22,23
∗
Corresponding author. Tel: ++49-2461-613374; fax: ++49-2461-613870; e-mail: mi.mueller@fz-juelich.de
0957-4166/99/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00516-9
tetasy 3151

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The enzymatically catalyzed reaction should be a versatile stereoselective alternative to the classical
benzoin condensation.6 Benzoylformate decarboxylase (BFD, E.C. 4.1.1.7) has been found in bacteria
such as Pseudomonas putida, Acinetobacter calcoaceticus and Pseudomonas aeruginosa.24–26
The enzyme is involved in the degradation of aromatic compounds of the mandelate catabolism.
Only BFD from P. putida has been examined in detail so far. The coding gene was cloned27 and
the crystal structure published recently.28 The main enzymatic reaction catalyzed by BFD is the non-
oxidative decarboxylation of benzoylformate to benzaldehyde. BFD is a stable biocatalyst, which is
available in large-scale quantities by fermentation of either P. putida or from a recombinant E. coli-
strain carrying the BFD-gene.29 BFD is the most active thiamine diphosphate (ThDP)-dependent 2-
keto acid decarboxylase known so far which catalyzes both the decarboxylation (main reaction) and
the carboligation (side reaction) yielding either (R)-benzoin or (S)-2-hydroxy propanone derivatives with
high specific activities.29
The potential of BFD to catalyze C–C bond formation was first reported by Wilcocks and co-workers,30
who observed the formation of (S)-2-hydroxy-1-phenyl propanone ((S)-2-HPP) if benzoylformate was
decarboxylated in the presence of acetaldehyde using crude extracts of P. putida. Similar results were
obtained using benzaldehyde and acetaldehyde for carboligation.31 These results demonstrated that
BFD has the potential to synthesize chiral 2-hydroxy ketones using aldehydes without a previous
decarboxylation step. During this process (R)-benzoin is also obtained as a by-product in low yield.
We now present the first general synthesis of enantiomerically pure benzoin and substituted benzoin
derivatives from aromatic aldehydes via BFD catalyzed C–C bond formation.
2. Results and discussion
In our ongoing research on the BFD catalyzed formation of (S)-2-HPP we found that the benzalde-
hyde/acetaldehyde ratio is very important for the product distribution. Low concentration of benzalde-
hyde in the presence of excess acetaldehyde results in the formation of highly enantiomerically enriched
(S)-2-HPP.29 By either increasing the benzaldehyde concentration or decreasing the acetaldehyde con-
centration we observed the formation of (R)-benzoin. Performing the carboligation with benzaldehyde as
a sole substrate, (R)-benzoin is obtained in 20% yield and >99% ee. Optimization experiments (reaction
time, amount of enzyme, cofactor, and reaction medium) resulted in high yield of (R)-benzoin with >99%
ee using an aqueous buffer/DMSO-reaction system (Scheme 1).
Scheme 1.
Benzaldehyde was dissolved in a mixture of potassium phosphate buffer (50 mM, pH 7.0, containing
2.5 mM MgSO4 and 0.15 mM ThDP) and 30% DMSO. After addition of BFD the reaction was
accomplished for 48 h at room temperature and monitored by HPLC using a chiral stationary phase
column with authentic samples as references. After work up (R)-benzoin (ee >99%) was obtained in 70%
yield.
The reaction was carried out with different aromatic and heteroaromatic aldehydes and the correspon-
ding benzoin derivatives were obtained in almost enantiomerically pure form as summarized in Table 1.

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Table 1
Benzoins prepared by benzoylformate decarboxylase catalyzed enantioselective benzoin reaction

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Addition of DMSO increased the conversion rate, but had no effect on the enantiomeric excess of the
products. Analysis of 2-hydroxy ketones was performed by HPLC on a chiral stationary phase column
compared to racemic products, which were synthesized using classical chemical benzoin synthesis
methodology.32 The absolute configuration of the benzoin derivatives obtained was assigned to be (R)
according to correlation of the specific rotation of benzoin with data from the literature (Table 1) and to
HPLC data of commercially available enantiomers of benzoin.
As shown in Table 1, BFD accepts a broad range of different aromatic and heteroaromatic aldehydes,
which are bound to ThDP (Scheme 2) prior to ligation to a second aldehyde molecule. All (R)-benzoin
derivatives were obtained in high enantiomeric excess. The conversion rate was found to be a function of
the aldehyde structure, being maximal with benzaldehyde. Substituents with a +M-effect decreased the
conversion rate, whereas ortho-substituted benzaldehyde derivatives, except 2-fluorobenzaldehyde, were
only poorly accepted (Table 1).
Scheme 2.
As shown in Scheme 2, enzyme-bound ThDP catalyzes the formation of benzoins starting from
aldehydes. The mechanism was studied in detail with pyruvate decarboxylase, a ThDP-dependent
enzyme similar to BFD, and it may be assumed that BFD operates with a comparable mechanism.31
Catalysis involves initial deprotonation at C-2 to give the ylide 3, followed by nucleophilic addition to
the aldehyde (Scheme 2). The electron withdrawing thiazolium ring then allows deprotonation of C-2α of
the adduct 4 resulting in the enamine (‘active aldehyde’) 5 which attacks the second aldehyde selectively
from the less hindered side leading to an absolute configuration of (R) at the newly formed stereogenic
center. Release of benzoin 2 from the adduct 6 regenerates the ylide and completes the catalytic cycle.
The ongoing research on BFD-catalyzed enantioselective formation of 2-hydroxy ketones shows that
the binding of aromatic donor substrates to form the ‘active aldehyde’ 5 (Scheme 2) is preferred.
Analysis of the three-dimensional X-ray crystallographic structure of BFD28 suggests that this substrate’s
specificity may be due to stabilization by specific interactions in the active site of the enzyme.29 In
contrast to aliphatic acceptor aldehydes (like acetaldehyde) giving the corresponding (S)-2-HPP not
enantiomerically pure (90–92% ee) with benzaldehyde as a donor, aromatic acceptor aldehydes (like
benzaldehyde) should have only one possibility to fit into the cavity of the active site. Thus, the

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carboligation must be a consequence of a selective attack of the active aldehyde to benzaldehyde leading
to (R)-benzoins in enantiomerically pure form.
In summary, the method described here presents the first enzyme-catalyzed synthesis of enantiome-
rically pure (R)-benzoin derivatives in good yields. This work offers an attractive alternative synthesis of
enantiomerically pure (R)-benzoins compared to other published methods.7–17
3. Experimental
Pure BFD was obtained as described elsewhere.29 Preparative isolation of benzoins was carried out
by column chromatography on silica gel 60 (mesh size 40–63 µm). Analysis of benzoins was performed
using HPLC (HP series 1100, Hewlett Packard), fitted with a diode array detector, and equipped with a
chiral phase column (Chiralpack AD, Daicel Ltd). Optical rotations were measured on a polarimeter
241 (Perkin–Elmer). NMR spectra were recorded on an AMX 300 (Bruker Physik AG, Germany).
GC–MS spectra were determined on an HP 6890 series GC-system fitted with an HP 5973 mass
selective detector (Hewlett Packard; column HP-5MS, 30 m×250 µm; TGC (injector)=250°C, TMS (ion
source)=200°C, time programme (oven): T0 min=60°C, T3 min=60°C, T14 min=280°C (heating rate 20°C
min−1), T19 min=280°C).
3.1. Representative example for the synthesis of benzoin derivatives: (R)-benzoin 2a
Benzaldehyde (318 mg, 3 mmol) was dissolved in a mixture of DMSO (30 mL) and potassium
phosphate buffer (100 mL, 50 mM, pH 7.0, containing MgSO4 (2.5 mM) and ThDP (0.15 mM)). After
addition of BFD (450 U related to decarboxylation of benzoylformate) the reaction was allowed to stand
at room temperature for 48 h (the reaction was monitored by GC–MS and HPLC). The mixture was
extracted with dichloromethane (250 mL), the organic layer washed with water (25 mL) and brine (25
mL) and dried over Na2SO4. Evaporation of the solvent and purification of the crude product by column
chromatography (silica gel, dichloromethane) afforded 223 mg (70%) (R)-benzoin, ee >99%.
Acknowledgements
This work has been supported by the Deutsche Forschungsgemeinschaft in the scope of SFB 380. A.
S. D. expresses thanks for the Alexander von Humboldt Fellowship.
References
1. Coppola, G. M.; Schuster, H. F. α-Hydroxy Acids in Enantioselective Synthesis; VCH: Weinheim, 1997.
2. Gala, D.; DiBenedetto, D. J.; Clark, J. E.; Murphy, B. L.; Schumacher, D. P.; Steinman, M. Tetrahedron Lett. 1996, 37, 611.
3. Chaudhary, A. G.; Kingston, D. G. I. Tetrahedron Lett. 1993, 34, 4921.
4. Polonský, J. Forts. Chem. Org. Naturst. 1985, 47, 221.
5. Hanessian, S. Total Synthesis of Natural Products: The Chiron Approach; Pergamon Press: New York, 1983; Chapter 2.
6. Kuebrich, J. P.; Schowen, R. L.; Wang, M.; Lupes, M. E. J. Am. Chem. Soc. 1971, 93, 1214.
7. Stetter, H.; Rämsch, Y.; Kuhlmann, H. Synthesis 1976, 733.
8. Castells, J.; López-Calahorra, F.; Domingo, L. J. Org. Chem. 1988, 53, 4433.
9. Matsumoto, T.; Ohishi, M.; Inoue, S. J. Org. Chem. 1985, 50, 603.
10. Knight, R. L.; Leeper, F. J. J. Chem. Soc., Perkin Trans. 1 1998, 1891.
11. Enders, D.; Breuer, K. Helv. Chim. Acta 1996, 79, 1217.

4774
A. S. Demir et al. / Tetrahedron: Asymmetry 10 (1999) 4769–4774
12. Adam, W.; Müller, M.; Prechtl, F. J. Org. Chem. 1994, 59, 2358.
13. Adam, W.; Fell, R. T.; Stegmann, V. R.; Saha-Möller, C. R. J. Am. Chem. Soc. 1998, 120, 708.
14. Adam, W.; Saha-Möller, C. R.; Zhao, C.-G. Tetrahedron: Asymmetry 1998, 9, 4117.
15. Adam, W.; Fell, R. T.; Saha-Möller, C. R.; Zhao, C.-G. Tetrahedron: Asymmetry 1998, 9, 397.
16. Davies, F. A.; Chen, B.-C. Chem. Rev. 1992, 92, 919.
17. Hashiyama, T.; Morikawa, K.; Sharpless, K. B. J. Org. Chem. 1992, 57, 5067.
18. Nakamura, K.; Kondo, S.; Kawai, Y.; Hida, K.; Kitano, K.; Ohno, A. Tetrahedron: Asymmetry 1996, 7, 409.
19. Csuk, R.; Glänzer, B. I. Chem. Rev. 1991, 91, 49.
20. Adam, W.; Fell, R. T.; Hoch, U.; Saha-Möller, C. R.; Schreier, P. Tetrahedron: Asymmetry 1995, 6, 1047.
21. Adam, W.; Díaz, M. T.; Fell, R. T.; Saha-Möller, C. R. Tetrahedron: Asymmetry 1996, 7, 2207.
22. Demir, A. S.; Hamamci, H.; Tanyeli, C.; Akhmedov, I. M.; Doganel, F. Tetrahedron: Asymmetry 1998, 9, 1673.
23. Tanyeli, C.; Demir, A. S.; Dikici, E. Tetrahedron: Asymmetry 1996, 7, 2399.
24. Hegeman, G. D. Methods Enzymol. 1970, 17, 674.
25. Barrowman, M. M.; Harnett, W.; Scott, A. J.; Fewson, C. A.; Kusel, J. R. FEMS Microbiol. Lett. 1986, 34, 57.
26. Barrowman, M. M.; Fewson, C. A. Curr. Microbiol. 1985, 12, 235.
27. Tsou, A. Y.; Ransom, S. C.; Gerlt, J. A.; Buechter, D. D.; Babbitt, P. C.; Kenyon, G. L. Biochemistry 1990, 29, 9856.
28. Hasson, M. S.; Muscate, A.; McLeish, M. J.; Polovnikova, L. S.; Gerlt, J. A.; Kenyon, G. L.; Petsko, G. A.; Ringe, D.
Biochemistry 1998, 37, 9918.
29. Iding, H.; Dünnwald, T.; Greiner, L.; Liese, A.; Müller, M.; Siegert, P.; Grötzinger, J.; Demir, A. S.; Pohl, M. Chem. Eur. J.,
in press.
30. Wilcocks, R.; Ward, O. P.; Collins, S.; Dewdney, N. J.; Hong, Y.; Prosen, E. Appl. Environ. Microbiol. 1992, 58, 1699.
31. Iding, H.; Siegert, P.; Mesch, K.; Pohl, M. Biochim. Biophys. Acta 1998, 1385, 307.
32. Becker, H. G. O.; Berger, W.; Domschke, G.; Fanghänel, E.; Faust, J.; Fischer, M.; Gentz, F.; Gewald, K.; Gluch, R.; Mayer,
R.; Müller, K.; Pavel, D.; Schmidt, H.; Schollberg, K.; Schwetlick, K.; Seiler, E.; Zeppenfeld, G. Organikum; Barth Verlag:
Leipzig, Berlin, Heidelberg, 1993; p. 474, p. 547.