Biocatalytic Racemization of R-Hydroxycarboxylic Acids
encountered a stringent substrate limitation for the latter
enzyme: Although mandelate racemase was very toler-
ant toward various â,γ-unsaturated R-hydroxycarboxylic
Less is known about the enzyme properties and its mode
of action: Although lactate racemase from Clostridium
butylicum,25 Lactobacillus sake, and L. curvatus was
purified and biochemically characterized to a certain
extent, detailed proof for the assumption on its mecha-
nism of action through an internal hydride shift involving
an R-carbonyl enzyme-bound intermediate is still miss-
ing. Most important, the biocatalytic racemization of
saturated R-hydroxycarboxylic acids other than lactate
has not been studied to date.28
26
27
1
1
acids, saturated (aliphatic) substrate analogues were
not accepted at all. The latter fact can be explained by
the lack of resonance stabilization of the corresponding
enolate intermediate within the active site of the en-
1
2,13
zyme.
Furthermore, severe steric hindrance was
11
observed for ortho-substituted mandelate derivatives.
To extend the applicability of our deracemization protocol
toward saturated aliphatic and arylaliphatic R-hydroxy-
carboxylic acids, which are lacking the minimum struc-
tural requirements of mandelate racemase, i.e., at least
one CdC bond in the â,γ-position, a matching isomerase/
racemase enzyme was required.
Results and Discussion
In search of a suitable racemase activity applicable to
a broad spectrum of substrates encompassing aliphatic
and arylaliphatic R-hydroxycarboxylic acids, which could
not be isomerized with use of mandelate racemase, a
screening was initiated based on the data for lactate
racemase discussed above. Thus, rehydrated (resting)
cells of a representative set of Lactobacilli (20 strains),
Lactococci (9 strains), and halophilic organisms, such as
Halococcus (7 strains) and halobacteria (3 strains, Ha-
lobacterium, Haloferax, and Haloarcula), were screened
for their ability to racemize R-hydroxycarboxylic acids in
aqueous buffer at pH 6. To cover a reasonably wide
substrate spectrum, straight-chain, branched, and cyclic
aliphatic R-hydroxycarboxylic acids [(S)-1,2, (R)-3, and
(S)-4-6] were chosen. Special emphasis was put on aryl-
alkyl derivatives, such as phenyl-lactates and 4-phenyl-
Due to the fact that the vast majority of biochemical
processes are stereospecific, Nature has faced little need
for racemization and, as a consequence, “racemases” are
a small group of enzymes, which have been biochemically
classified as subgroup [EC 5.1.X.X] among the diverse
14-16
and heterogeneous group of isomerases.
Despite their
rare occurrence in Nature, their importance in synthetic
organic chemistry lies in the fact that they often can
catalyze “chemically impossible” isomerization reactions.
Careful analysis of the biochemical literature on race-
mases acting on R-hydroxycarboxylic acids revealed the
existence of a promising candidate: Lactate racemase
1
7
[EC 5.1.2.1].
The biochemical data on lactate racemase available to
2
-hydroxybutanoates [(S)-7-12 and (R)-7,8], since com-
date are somewhat scattered and divers. The respective
enzymatic activity was reported (or assumed) in various
microbial strains in context with their ability to produce
pounds of this type are frequently used as chiral building
blocks for the synthesis of pharmacologically active target
molecules. For instance, (S)-3-cyclohexyl lactate (6) was
(or degrade) D-lactate through an L-specific lactate path-
X
found to be an essential component of sialyl Lewis -
way. The biodegradation/formation of D-lactate from the
L-isomer via lactate racemase was identified in particular
among (anaerobic) rumen bacteria, such as Megasphaera
analogues, which are currently tested as inhibitors of
29
E-selectin for the treatment of inflammatory disorders.
(R)-4-Phenyl-2-hydroxybutanoate (8), which has been
1
8,19
20
elsdenii
and Selenomonas ruminantium. Similar
30
prepared by kinetic resolution, asymmetric bioreduc-
degradation pathways were found in Staphylococcus
tion,3
1,32
microbial stereoinversion, and asymmetric
33
2
1
22,23
aureus, Lactobacillus sakei,
and halophilic Archaea,
24
synthesis34 is an important intermediate for the synthesis
of a wide range of ACE inhibitors.
such as Haloferax volcanii and various Haloarcula spp.
3
-Phenyl lactate (7) and derivatives thereof are fre-
(
11) Felfer, U.; Strauss, U. T.; Kroutil, W.; Fabian, W. M. F.; Faber,
quently used components of pharmaceuticals and natural
K. J. Mol. Catal. B: Enzym. 2001, 15, 213.
antibiotic agents.3
5,36
Among them, the p-hydroxy ana-
(
12) Powers, V. M.; Koo, C. W.; Kenyon, G. L.; Gerlt, J. A.; Kozarich,
J. W. Biochemistry 1991, 30, 9255. Prat-Resina, X.; Garcia-Viloca, M.;
Gonzalez-Lafont, A.; Lluch, J. M. ChemPhysChem 2002, 4, 5365.
(13) For a review on the substrate tolerance of mandelate racemase
(24) Oren, A.; Gurevich, P.; Silverman, A. Can. J. Microbiol. 1995,
41, 302.
and a general substrate model see: Felfer, U.; Goriup, M.; Koegl, M.;
Wagner, U.; Faber, K.; Kroutil, W. Adv. Synth. Catal. Submitted for
publication.
(25) Cantwell, A.; Dennis, D. Biochemistry 1974, 13, 287. Pepple,
J. S.; Dennis, D. Biochim. Biophys. Acta 1976, 429, 1036.
(26) Hiyama, T.; Fukui, S.; Kitahara, K. J. Biochem. 1968, 64, 99.
(27) Stetter, K. O.; Kandler, O. Arch. Microbiol. 1973, 94, 221.
(28) For a preliminary communication see: Glueck, S. M.; Laris-
segger-Schnell, B.; Csar, K.; Kroutil, W.; Faber, K. Chem. Commun.
2005, 1904.
(
(
(
(
14) Adams, E. Adv. Enzymol. Relat. Areas Mol. Biol. 1976, 44, 69.
15) Glaser, L. The Enzymes 1972, 6, 355.
16) Tanner, M. E. Acc. Chem. Res. 2002, 35, 237.
17) Synonyms for lactate racemase are lacticoracemase, hydroxy
acid racemase, and lactic acid racemase. No sequence or X-ray
structure for lactate racemase or hydroxy acid racemase is available
to date. www.Brenda.Uni-Koeln.de.
(29) Storz, T.; Dittmar, P.; Fauquez, P. F.; Marschal, P.; Lottenbach,
W. U.; Steiner, H. Org. Proc. Res. Dev. 2003, 7, 559.
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Technol. 2002, 30, 673.
(31) Schmidt, E.; Blaser, H.-U.; Fauquex, P. F.; Sedelmeier, G.;
Spindler, F. In Microbial Reagents in Organic Synthesis; Servi, S., Ed.;
Kluwer: Dordrecht, The Netherlands 1992; pp 377-388.
(32) Oda, S.; Inada, Y.; Kobayashi, A.; Ohta, H. Biosci. Biotechnol.
Biochem. 1998, 62, 1762.
(33) Chadha, A.; Baskar, B. Tetrahedron: Asymmetry 2002, 13, 1461.
(34) Blaser, H.-U.; Burkhardt, S.; Kirner, H. J.; M o¨ ssner, T.; Studer,
M. Synthesis 2003, 1679.
(
18) Hino, T.; Kuroda, S. Appl. Environ. Microbiol. 1993, 59, 255.
19) Hino, T.; Shimada, K.; Maruyama, T. Appl. Environ. Microbiol.
(
1
1
3
994, 60, 1827.
(
20) Melville, S. B.; Michel, T. A.; Macy, J. M. FEMS Microbiol. Lett.
987, 40, 289.
21) Stockland, A. E.; San Clemente, C. L. J. Bacteriol. 1969, 100,
47.
22) Malleret, C.; Lauret, R.; Ehrlich, S. D.; Morel-Deville, F.;
Zagorec, M. Microbiology 1998, 144, 3327.
(
(
(
23) No lactate racemase activity was found in Lactobacillus casei
NRRL-B445 and L. delbrueckii ATCC 11842, see: Hj o¨ rleifsdottir, S.;
Seevaratnam, S.; Holst, O.; Mattiasson, B. Curr. Microbiol. 1990, 20,
(35) For antifungal activity see: Str o¨ m, K.; Sj o¨ gren, J.; Broberg, A.;
Schn u¨ rer, J. Appl. Environ. Microbiol. 2002, 68, 4322.
(36) For anti-Listeria activity see: Dieuleveux, V.; van der Pyl, D.;
Chataud, J.; Gueguen, M. Appl. Environ. Microbiol. 1998, 64, 800.
2
87. Ragout, A.; Psece de Riuz Holgado, A.; Oliver, G.; Sineriz, F.
Biochimie 1989, 71, 639.
J. Org. Chem, Vol. 70, No. 10, 2005 4029