Biocatalytic racemisation of α-hydroxycarboxylic acids at physiological conditions

Article abstract of DOI:10.1039/b418786e

Biocatalytic racemisation of aliphatic, aryl-aliphatic and aromatic α-hydroxycarboxylic acids was accomplished using whole resting cells of Lactobacillus paracasei DSM 20207; the mild (physiological) reaction conditions ensured an essentially 'clean' isomerization in the absence of side reactions, such as elimination or decomposition. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b418786e

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Biocatalytic racemisation of a-hydroxycarboxylic acids at physiological
conditions{
Silvia M. Glueck, Barbara Larissegger-Schnell, Katrin Csar, Wolfgang Kroutil and Kurt Faber*
Received (in Cambridge, UK) 15th December 2004, Accepted 25th January 2005
First published as an Advance Article on the web 9th February 2005
DOI: 10.1039/b418786e
Prompted by reports on lactate racemase activity in anaerobic
rumen bacteria (Clostridium butylicum, Selenomonas ruminantium
and Megasphaera elsdenii10b), halophiles (Halobacterium and
Haloferax spp.11) and Lactobacillus spp.,12 we initiated a screening
for the respective enzymatic activity among both of the latter
genera for their ease of cultivation.{13 Whereas no activity could
be found among halophiles, several positive hits were identified
among Lactobacilli, the most active strain being Lactobacillus
paracasei DSM 20207.
Biocatalytic racemisation of aliphatic, aryl-aliphatic and aro-
matic a-hydroxycarboxylic acids was accomplished using whole
resting cells of Lactobacillus paracasei DSM 20207; the mild
(physiological) reaction conditions ensured an essentially ‘clean’
isomerization in the absence of side reactions, such as
elimination or decomposition.
Racemisation is an entropy-driven ‘downhill-reaction’ going in
hand with a loss of enantiomeric purity1 and is therefore generally
considered as an unwanted side-reaction rather than a synthetically
useful transformation. As a consequence, the controlled racemisa-
tion of organic compounds has been scarcely studied.2 It was only
recently that the need for ‘clean’ racemisation protocols had been
recognized, in particular due to the increasing demand for so-
called deracemisation processes, which allow the transformation of
a racemate into a single stereoisomeric product in 100% theoretical
yield.3,4 Detailed analysis of the (chemical) racemisation protocols
published so far2 reveal that typical reaction conditions employ
strongly acidic or basic conditions, which are incompatible with a
stereoselective (catalytic) in situ transformation and are thus of
limited use for dynamic kinetic resolutions. In order to circumvent
this limitation, enzymatic racemisation (taking place under
ambient reactions conditions) holds great potential.5
The racemisation of substrates 1–8 was tested using rehydrated
lyophilized (resting) cells of Lactobacillus paracasei DSM 20207
(Scheme 1, Table 1) in aqueous buffer at pH 6. Fast rates were
observed with the ‘natural’ substrate, lactate (S)-1. For reasons of
comparison, its relative rate was arbitrarily set as standard (100%).
Almost equally fast racemization of the straight-chain hexanoic
acid analogue (R)-2 (relative rate 80%) indicated a highly
desirable relaxed substrate tolerance of the system. However,
reduced rates were found with sterically demanding aliphatic
a-hydroxycarboxylic acids (S)-3 and (S)-414 bearing branched or
alicyclic side chains. On the other hand, good racemisation rates
were obtained with aryl-alkyl derivatives 5 and 6 (up to 59%
relative to lactate). 3-Phenyl-lactate (5) and aryl-substituted
derivatives thereof are frequently used chiral components of
pharmaceuticals, such as rhinovirus protease inhibitors15 and
During our studies directed towards the deracemisation of
a-hydroxycarboxylic acids by coupling lipase-catalyzed acyl-
transfer to the racemisation of the non-reacted substrate
enantiomer using mandelate racemase [EC 5.1.2.2]6 we encoun-
tered a stringent substrate limitation for the latter enzyme:
although mandelate racemase was very tolerant towards various
b,c-unsaturated a-hydroxycarboxylic acids,7 saturated (aliphatic)
substrate analogues were not accepted at all. The latter fact can be
explained by the lack of resonance stabilization of the correspond-
ing enolate intermediate within the active site of the enzyme.8
In order to extend the applicability of our deracemisation
protocol towards saturated a-hydroxycarboxylic acids, a matching
isomerase enzyme was required. Analysis of the biochemical
literature revealed the existence of a promising candidate: lactate
racemase [EC 5.1.2.1]. The respective enzymatic activity was
reported in various microbial strains;9 however, detailed knowl-
edge of the properties of this enzyme and its mode of action10 is
rather scarce. Furthermore, the biocatalytic racemisation of
saturated a-hydroxycarboxylic acids other than lactate has not
been studied to date.
Scheme 1 Biocatalytic racemisation of a-hydroxycarboxylic acids.
Table 1 Relative rates of racemisation of substrates 1–8
Substrate
R
Relative rate (%)a
(S)-1
(R)-2
(S)-3
(S)-4
(R)-5
(S)-5
(R)-6
(S)-6
(S)-7
(R)-8
a
-CH3
100
80
2
y1
59
21
47
35
25
24
-(CH2)3CH3
-CH(CH3)2
-CH2-cC6H11
-CH2Ph
-CH2Ph
-(CH2)2Ph
-(CH2)2Ph
-C6H5
-o-Cl-C6H4
{ Electronic supplementary information (ESI) available: experimental
section and chemical structures. See http://www.rsc.org/suppdata/cc/b4/
b418786e/
Relative rates were calculated from initial progress curves at
conversion of ¡5%; the racemisation rate of the ‘natural’ substrate
(S)-1 was arbitrarily set as standard (100%).
*Kurt.Faber@Uni-Graz.at
1904 | Chem. Commun., 2005, 1904–1905
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the Province of Styria and BASF AG is gratefully acknowledged.
R. Gaisberger (Graz) and Ciba Speciality Chemicals (Basel) are
thanked for the generous donation of chiral materials.
Silvia M. Glueck, Barbara Larissegger-Schnell, Katrin Csar,
Wolfgang Kroutil and Kurt Faber*
Department of Chemistry, Organic and Bioorganic Chemistry, Research
Centre Applied Biocatalysis, University of Graz, Heinrichstrasse 28,
A-8010 Graz, Austria. E-mail: Kurt.Faber@Uni-Graz.at;
Fax: +43-316-380-9840; Tel: +43-316-380-5332
Notes and references
1 E. L. Eliel, S. H. Wilen and M. P. Doyle, Basic Organic Stereochemistry,
Wiley, New York, 2001, pp. 277–278.
2 E. J. Ebbers, G. J. A. Ariaans, J. P. M. Houbiers, A. Bruggink and
B. Zwanenburg, Tetrahedron, 1997, 53, 9417.
3 K. Faber, Chem. Eur. J., 2001, 7, 5004.
Fig. 1 Time course of the biocatalytic racemisation of the enantiomers of
5 and 6.
natural antibiotic agents,16 and are particularly difficult to
racemise using conventional methods due to the ease of their
elimination, forming cinnamic acid. Compound (R)-6 is an
important building block for the synthesis of anti-hypertensive
agents and ACE-inhibitors.17 The remarkable flexibility of this
biocatalytic system was demonstrated by the fact that also
(S)-mandelate (7) was accepted at a fair rate (25%). Most
importantly, o-chloromandelate (8), which is a non-substrate for
mandelate racemase,7 was similarly well accepted (24%). The
(R)-enantiomer of the latter compound is a key intermediate for
the synthesis of anticoagulant agents used in cardiovascular
therapy (Clopidogrel/Plavix).18
4 O. Pamies and J.-E. Ba¨ckvall, Chem. Rev., 2003, 103, 3247.
5 B. Schnell, K. Faber and W. Kroutil, Adv. Synth. Catal., 2003, 345, 563.
6 U. T. Strauss and K. Faber, Tetrahedron: Asymmetry, 1999, 10, 4079.
7 U. Felfer, U. T. Strauss, W. Kroutil, W. M. F. Fabian and K. Faber,
J. Mol. Catal. B: Enzym., 2001, 15, 213.
8 V. M. Powers, C. W. Koo, G. L. Kenyon, J. A. Gerlt and
J. W. Kozarich, Biochemistry, 1991, 30, 9255; X. Prat-Resina,
M. Garcia-Viloca, A. Gonzalez-Lafont and J. M. Lluch,
ChemPhysChem, 2002, 4, 5365.
9 www.Brenda.Uni-Koeln.de.
10 (a) A. Cantwell and D. Dennis, Biochemistry, 1974, 13, 287; (b)
J. S. Pepple and D. Dennis, Biochim. Biophys. Acta, 1976, 429, 1036; (c)
T. Hino and S. Kuroda, Appl. Environ. Microbiol., 1993, 59, 255; (d)
S. B. Melville, T. A. Michel and J. M. Macy, FEMS Microbiol. Lett.,
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11 A. Oren, P. Gurevich and A. Silverman, Can. J. Microbiol., 1995, 41,
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12 C. Malleret, R. Lauret, S. D. Ehrlich, F. Morel-Deville and M. Zagorec,
Microbiology, 1998, 144, 3327; K. O. Stetter and O. Kandler, Arch.
Microbiol., 1973, 94, 221; T. Hiyama, S. Fukui and K. Kitahara,
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13 Detailed results on the screening and the full substrate spectrum will be
reported in a full paper.
14 (S)-3-Cyclohexyl-lactic acid (3) is a component of the E-selectin inhibitor
(S)-cHexLact-2-O-(3-Galb)(1A3)ddGlc(4A1)aFuc; see: T. Storz,
P. Dittmar, P. F. Fauquex, P. Marschal, W. U. Lottenbach and
H. Steiner, Org. Process Res. Dev., 2003, 7, 559.
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in Microbial Reagents in Organic Synthesis, ed. S. Servi, Kluwer,
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Close monitoring of the progress of racemisation over time for
both enantiomers of substrates 5 and 6 revealed that both of the
(R)-enantiomers were racemised more rapidly than the corres-
ponding (S)-counterparts (Fig. 1). Such ‘nonsymmetric’ kinetics is
not uncommon for enzyme-catalysed racemisation and is caused
by the difference in KM and kcat values of enantiomers.19 Overall,
the reactions proved to be essentially clean and less than y5% of
side products could be detected. It should be noted that attempts
to racemise 5 and 6 under conventional conditions (aq. pH 2–12,
100 uC, 48 h) were unsuccessful.
In summary, clean biocatalytic racemisation of structurally
diverse aliphatic, aryl-aliphatic and aromatic a-hydroxycarboxylic
acids was accomplished under mild conditions using resting cells of
Lactobacillus paracasei DSM 20207. The scope and limitations of
this novel biocatalytic activity, the nature of the actual enzyme(s)
involved and the mechanism of action responsible for this
racemisation is currently being studied in detail.
18 L. M. van Langen, F. van Rantwijk and R. A. Sheldon, Org. Process
Res. Dev., 2003, 7, 828.
19 M. A. Spies, J. J. Woodward, M. R. Watnik and M. D. Toney, J. Am.
Chem. Soc., 2004, 126, 7464; K. Shibata, K. Shirasuna, K. Motegi,
Y. Kera, H. Abe and R. Yamada, Comp. Biochem. Physiol., B:
Biochem. Mol. Biol., 2000, 126, 509.
This study was performed in cooperation with BASF AG
(Ludwigshafen) within the Competence Center Applied
Biocatalysis and financial support by the TIG, the City of Graz,
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 1904–1905 | 1905