Quantitative transformation of racemic 2-hydroxy acids into (R)-2-hydroxy acids by enantioselective oxidation with glycolate oxidase and subsequent reduction of 2-keto acids with D-lactate dehydrogenase

Article abstract of DOI:10.1016/S0957-4166(97)00637-X

The enzymatic resolution of chiral 2-hydroxy acids 1 by enantioselective oxidation with molecular oxygen in the presence of glycolate oxidase from spinach (Spinacia oleracea) and subsequent asymmetric reduction of 2-oxo acids 2 with D-lactate dehydrogenase from Lactobacillus leichmannii leads to enantiomerically pure (R)-2-hydroxy acids in up to 89% yield based on the racemate.

Full text of DOI:10.1016/S0957-4166(97)00637-X

Pergamon
Tetrahedron: Asymmetry 9 (1998) 351–355
Quantitative transformation of racemic 2-hydroxy acids into
(
R)-2-hydroxy acids by enantioselective oxidation with glycolate
oxidase and subsequent reduction of 2-keto acids with D-lactate
dehydrogenase
a
b,∗
Chantu R. Saha-Möller and Peter Schreier b
a
Waldemar Adam, Michael Lazarus,
a
Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
b
Received 24 November 1997; accepted 16 December 1997
Abstract
The enzymatic resolution of chiral 2-hydroxy acids 1 by enantioselective oxidation with molecular oxygen in
the presence of glycolate oxidase from spinach (Spinacia oleracea) and subsequent asymmetric reduction of 2-oxo
acids 2 with D-lactate dehydrogenase from Lactobacillus leichmannii leads to enantiomerically pure (R)-2-hydroxy
acids in up to 89% yield based on the racemate. © 1998 Elsevier Science Ltd. All rights reserved.
Optically active 2-hydroxy acids are important building blocks for the asymmetric synthesis of
1
a
1b
1c
2
3
glycols, halo esters and epoxides. Several chemical and enzymatic methods have been described
4
previously on the synthesis of optically active α-hydroxy acids. Recently, we have reported the
enzymatic resolution of chiral 2-hydroxy carboxylic acids by enantioselective oxidation with molecular
oxygen, catalyzed by the glycolate oxidase (GOX) from spinach (Spinacia oleracea). This novel
enzymatic transformation affords optically active 2-hydroxy acids in excellent ee values. Nevertheless,
the enantioselective oxidation of the (S)-2-hydroxy acid to corresponding 2-oxo acid 2 (Scheme 1,
Method A) provides only a maximum yield of 50% for the (R)-2-hydroxy acid, based on the racemate.
For synthetic applications and economic reasons it would be desirable to prepare the (R)-2-hydroxy acids
quantitatively from their racemic precursors by a biocatalytic process.
The microbial D-lactate dehydrogenase (D-LDH, E.C. 1.1.1.28), which catalyzes in vivo the NADH-
5
dependent interconversion of pyruvate and D-lactate in anabolic and catabolic pathways, is a useful
enzyme in organic synthesis; it reduces a range of 2-oxo acids to the corresponding (R)-2-hydroxy acids.6
Therefore, we have coupled the glycolate–oxidase-catalyzed oxidation of racemic 2-hydroxy acids with
the asymmetric reduction of 2-oxo acids by D-lactate dehydrogenase from Lactobacillus leichmannii
∗
Corresponding author. E-mail: Lazarus@pzlc.uni-wuerzburg.de
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(97)00637-X

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W. Adam et al. / Tetrahedron: Asymmetry 9 (1998) 351–355
Scheme 1. Oxidation of 2-hydroxy acids with molecular oxygen catalyzed by the glycolate oxidase from spinach (Spinacia
oleracea) (Method A) and reduction of 2-oxo acids by the D-lactate dehydrogenase (Methods B and C)
for the efficient oxidoreductative transformation to (R)-2-hydroxy acids (Scheme 1, Method B and C).
Herein, we report for the first time such a tandem biocatalytic transformation of racemic 2-hydroxy acids.
The results of the biocatalytic transformation of the 2-hydroxy acids 1a–e are given in Table 1, wherein
conversion and enantiomeric excess of the known6 2-hydroxy carboxylic acids 1 were assessed by gas
chromatography. The enzymatic oxidation of the racemic 2-hydroxy acids 1 with glycolate oxidase by
–9
4
molecular oxygen were carried out as reported earlier. Subsequently, the D-lactate dehydrogenase was
added together with catalytic amounts of NADH for enantioselective reduction of the 2-oxo acid 2 to
corresponding (R)-2-hydroxy acid 1. D-Lactate dehydrogenase contains an air-sensitive thiol group, but
it is stable when used in an inert atmosphere in the presence of reducing agents such as dithiothreitol to
prevent autooxidation. The cosubstrate NADH is recycled by the system formate/formate dehydrogenase,
so that the product, CO , of the regeneration system does not complicate the workup.
2
The results in Table 1 show that for the one-pot reaction (Method B) of the 2-hydroxy acids 1a–c
with the glycolate oxidase and the D-lactate dehydrogenase the (R)-2-hydroxy acids 1a–c are obtained in
moderate to high enantiomeric excesses (ee values 67–91%) and 84–93% yields, based on the racemate
(
Table 1, entries 1–4, 8, 9, 12, 13). However, the (R)-2-hydroxy acids 1a–c could not be obtained
enantiomerically pure by this method; presumably the gylcolate oxidase catalyzes the reduction of the
-oxo acids 2a–c to the (S)-2-hydroxy acids 1a–c under these anaerobic conditions. Thus, we have carried
2
out the reaction of 2a according to Method B, but without the addition of D-LDH (Table 1, entry 5).
Indeed, the GOX-catalyzed reduction of the 2-oxo acid 2a to the enantiomerically pure (S)-2-hydroxy
acid 1a at 100% conversion reveals that the glycolate oxidase and the D-lactate dehydrogenase compete
under anaerobic conditions for the 2-oxo acid 2 as substrate. To circumvent this loss in enantioselectivity,
we have modified our biocatalytic transformation of the racemic 2-hydroxy acids 1. After the enzymatic
resolution of the 2-hydroxy acids 1a,b with glycolate oxidase, the acids 1a,b and 2a,b were separated
from the aqueous enzyme solution and added to the D-lactate dehydrogenase medium (Method C). In this
way, the (R)-2-hydroxy acids 1a,b may be obtained essentially enantiomerically pure at 85–89% yield,
based on the racemate (Table 1, entries 6, 7, 10, 11).
Furthermore, we have investigated the enzymatic resolution of the mandelic acid 1d and the phenyl-
lactic acid (1e) with glycolate oxidase. While the mandelic acid 1d was not accepted by the glycolate
oxidase (Table 1, entry 14), the phenyllactic acid 1e was oxidized by molecular oxygen in the presence
of glycolate oxidase (Table 1, entries 15, 16). Nonetheless, the enzyme activity towards this substrate is
remarkably diminished. Thus, the enzymatic resolution of the phenyllactic acid 1e may not be achieved
with affordable amounts of the enzyme and within a reasonable reaction time (Table 1, entry 16).
In summary, we have shown that the tandem enzymatic transformation of racemic 2-hydroxy acids
1
with glycolate oxidase and D-lactate dehydrogenase from Lactobacillus leichmannii leads to enan-
tiomerically pure (R)-2-hydroxy acids 1 in up to 89% yield based on the racemate. Thus, this novel

W. Adam et al. / Tetrahedron: Asymmetry 9 (1998) 351–355
353
Table 1
Conversion of racemic 2-hydroxy acids into (R)-2-hydroxy acids catalyzed by the glycolate oxidase
(
Method A) and the D-lactate dehydrogenase (Methods B and C)
biocatalytic method enables the clean preparation of enantiomerically pure (R)-2-hydroxy acids 1 almost
quantitatively from the racemic precursors.
1. Experimental
1.1. Materials
The glycolate oxidase (GOX) and the catalase were purchased from Sigma. D-Lactate dehydrogenase
(
D-LDH) and formate dehydrogenase (FDH) were obtained as gift samples from Boehringer Mannheim
and used without further purification. The racemic 2-hydroxy acids 1a–e were purchased from Fluka and
Sigma.

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W. Adam et al. / Tetrahedron: Asymmetry 9 (1998) 351–355
1.2. General procedure for the enzymatic resolution of the 2-hydroxy acids 1d–e with glycolate oxidase
(Method A)
These reactions were performed in an Amicon Ultrafiltration Cell (Model 8050), in which the filtration
membrane was substituted by a Teflon sheet. The glycolate oxidase was added to 50 mL of a solution
pH 7.8) of the 2-hydroxy acid 1 (cf Table 1), Tris(hydroxymethyl)-aminoethane (tris-HCl, 0.1 M),
(
flavin mononucleotide (FMN, 1 mM) and catalase (1600 IU). The resulting mixture was stirred at 15°C
under O (0.4 MPa). Aliquots (1 mL) were removed and worked up to monitor the progress of substrate
2
conversion. For this purpose, the solution was acidified with 6 N hydrochloric acid (pH 3) and extracted
with ethyl ether (3×5 mL). The combined organic phases were dried over Na SO and the solvent was
2
4
evaporated under reduced pressure (20°C, 17 Torr). The acids 1 and 2 were converted to their methyl
esters by treatment with diazomethane and submitted to GC analysis.
1.3. General procedure for the quantitative transformation of the racemic 2-hydroxy acids 1 with
glycolate oxidase and D-lactate dehydrogenase
Method B: The enzymatic resolution of the racemic 2-hydroxy acids 1a–c was performed as described
above. At 50% conversion, the buffer was adjusted to pH 7.5. After bubbling nitrogen gas through the
solution for 15 min, the D-lactate dehydrogenase (cf Table 1) was added, together with ammonium
formate (1.5 equiv. based on the 2-oxo acid 2), tris–HCl (0.1 M), dithiothreitol (1 mM), FDH (13–25
IU) and catalytic amounts of NAD (0.002 mmol). The resulting mixture was stirred at room temperature
under N (0.4 MPa). Aliquots (1 mL) were removed and worked up as described above to monitor the
2
progress of substrate conversion. After the complete conversion of the 2-oxo acid 2, the mixture was
extracted with ethyl ether (3×25 mL) and the combined organic layers were dried over Na SO . The
2
4
solvent was removed (20°C, 17 Torr) and the 2-hydroxy acid 1a–c was obtained in 84–93% yields.
Method C: The enzymatic resolution of the racemic 2-hydroxy acids 1a and 1b was performed as
described under Method A. At 50% conversion, the solution was acidified with 6 N hydrochloric acid
(
pH 3) and extracted with ethyl ether (3×25 mL). The combined organic phases were dried over Na SO
2
4
and the solvent was evaporated under reduced pressure (20°C, 17 Torr) to yield the 2-hydroxy acids 1a,b
and the 2-oxo acids 2a,b as a crude mixture in 92–96% yield. The acids were added to a solution (pH 7.5)
of ammonium formate (1.5 equiv. based on the 2-oxo acid 2), tris–HCl (0.1 M), dithiothreitol (1 mM), D-
LDH (cf Table 1), FDH (13 U) and a catalytic amount of NAD (0.002 mmol). The resulting mixture was
stirred at room temperature (ca. 20°C) under N (0.4 MPa). Aliquots (1 mL) were removed and worked
2
up as described above to monitor the progress of substrate conversion. After complete conversion of the
2
-oxo acid 2, the mixture was extracted with ethyl ether (3×25 mL) and the combined organic layers
were dried over Na SO . The solvent was removed (20°C, 17 Torr) and the 2-hydroxy acid 1a,b was
2
4
obtained in 85–89% yields.
Acknowledgements
This work was supported by the Bayerische Forschungsstiftung (Bayerischer Forschungsverbund
Katalyse-FORKAT), the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 347 ‘Selektive
Reaktionen metallaktivierter Moleküle’) and the Fonds der Chemischen Industrie. We thank Boehringer
Mannheim for the generous gift of enzymes.

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