Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media

Article abstract of DOI:10.1021/ol0272139

(Matrix presented) An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has been developed. In this biphasic reaction media, which is advantageous for reactions at higher subs

Full text of DOI:10.1021/ol0272139

ORGANIC
LETTERS
2
003
Vol. 5, No. 2
73-176
Practical Asymmetric Enzymatic
Reduction through Discovery of a
Dehydrogenase-Compatible Biphasic
Reaction Media†
1
,
‡
,§
‡
|
Harald Gr o1 ger,* Werner Hummel,* Stefan Buchholz, Karlheinz Drauz,
§
‡
‡
§
Tien Van Nguyen, Claudia Rollmann, Hendrik H u1 sken, and Kofi Abokitse
Degussa AG, Project House Biotechnology, P.O. Box 1345, 63403 Hanau, Germany,
Institut f u¨ r Enzymtechnologie der Heinrich-Heine-UniVersit a¨ t, Forschungszentrum
J u¨ lich, Stetternicher Forst, 52426 J u¨ lich, Germany, and Degussa AG,
BU Fine Chemicals, FC-TRM, P.O. Box 1345, 63403 Hanau, Germany
harald.groeger@degussa.com
Received October 31, 2002
ABSTRACT
An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has
been developed. In this biphasic reaction media, which is advantageous for reactions at higher substrate concentrations, both enzymes
(
alcohol dehydrogenase and FDH from Candida boidinii) remain stable. The reductions with poorly water-soluble ketones were carried out at
substrate concentrations of 10−200 mM, and the optically active (S)-alcohols were formed with moderate to good conversions and with up to
99% ee.
>
The enzymatic reductive amination by means of an enzyme-
coupled in situ cofactor regeneration according to Scheme
feature of this process is the use of the formate dehydroge-
nase (FDH) from Candida boidinii for the in situ regeneration
of the cofactor NADH. The extension of this attractive
1
is a powerful tool for the preparation of enantiomerically
1
pure R-amino acids. This has been demonstrated by Bom-
marius et al. in the industrial synthesis of L-tert-leucine and
2
Scheme 1. Production of L-tert-Leucine via Reductive
related R-amino acids, which runs on a ton scale. A key
Amination
*
Corresponding authors. E-mail address for W.H.: w.hummel@fz-
juelich.de.
†
Dedicated to Professor Dr. Heribert Offermanns on the occasion of
his 65th birthday.
‡
Degussa AG, Project House Biotechnology.
Heinrich-Heine-Universit a¨ t.
Degussa AG, BU Fine Chemicals.
§
|
(1) (a) Hummel, W.; Sch u¨ tte, H.; Schmidt, E.; Wandrey, C.; Kula, M.-
R. Appl. Microbiol. Biotechnol. 1987, 26, 409-416. (b) Krix, G.; Bom-
marius, A. S.; Drauz, K.; Kottenhahn, M.; Schwarm, M.; Kula, M.-R. J.
Biotechnol. 1997, 53, 29-39.
(2) (a) Bommarius, A. S.; Drauz, K.; Hummel, W.; Kula, M.-R.;
Wandrey, C. Biocatalysis 1994, 10, 37-47. (b) Schultz, C.; Gr o¨ ger, H.;
Dinkel, C.; Drauz, K.; Waldmann, H. In Applied Homogeneous Catalysis
with Organometallic Compounds, 2nd ed.; Cornils, B., Herrmann, W. A.,
Eds.; VCH-Wiley: Weinheim, Germany, 2002; Vol. 2, Chapter 3.2.1.
1
0.1021/ol0272139 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/01/2003

3
,4
enzymatic concept toward the synthesis of optically active
aqueous phase with an organic solvent. Organic and aqueous
phases are separated by a hydrophobic membrane. However,
although good space-time yields in the range of 60-104 g
5-7
alcohols is highly desirable, but still some drawbacks exist
that limit practicability and large-scale applicability. This
method falls short of others although numerous efficient
alcohol dehydrogenases with excellent (R)- and (S)-enantio-
specificities have been developed3 and are, at least in part,
already commercially available.
A main limitation is the low solubility of hydrophobic
ketone substrates in aqueous media. Therefore, reactions are
usually carried out at a very low substrate concentration of
e5-10 mM, which leads to nonsatisfactory volumetric
productivities. Consequently, even on a lab scale, the
practicability of the reduction of hydrophobic ketones via
enzymatic in situ cofactor regeneration is obviously limited
despite its high potential. The presence of an organic solvent
could improve the solubility of poorly water-soluble ketones
but generally causes severe enzyme deactivation. In particu-
lar, this is known for the formate dehydrogenase from C.
-
1
-1
10a,b
L
day are obtained,
the reaction is limited by the
solubility of the ketone in water, which is often below 5-10
mM. Thus, the development of alternative reaction concepts
for the asymmetric reduction with isolated enzymes still
represents a challenge.
In this communication, we report the first example of an
asymmetric enzymatic reduction of poorly water-soluble
ketones, including an in situ recycling of the cofactor NADH
with FDH, which runs in the “direct” presence of an organic
solvent at higher substrate concentrations. Such suitable
biphasic reaction media allowing reductions at higher
substrate concentrations represent a significant process
improvement. The principles of this process are summarized
in Scheme 2. This novel type of a biocatalytic synthesis of
,4
8
9
boidinii which is sensitive to organic solvents. Several kinds
of alcohol dehydrogenases were found to be unstable in the
presence of organic solvents, too. The best solution so far is
represented by a continuous process with an enzyme-
membrane reactor.10 This efficient “three-loop” concept is
based on an enzymatic reaction in pure aqueous media, a
separation of the aqueous phase from the enzyme via
ultrafiltration, and subsequent continuous extraction of the
Scheme 2. Concept of Enzymatic Reduction in Biphasic Media
(
3) It should be mentioned at this stage that numerous efficient (S)- and
(
R)-specific alcohol dehydrogenases have already been found. For selected
contributions, see: (a) Bradshaw, C. W.; Hummel, W.; Wong, C.-H. J.
Org. Chem. 1992, 57, 1532. (b) Bogin, O.; Peretz, M.; Burstein, Y. Protein
Sci. 1997, 6, 450-458. (c) Korkhin, Y.; Kalb, A. J.; Peretz, M.; Bogin, O.;
Burstein, Y.; Frolow, F. J. Mol. Biol. 1998, 278, 967-981. (d) Holt, P. J.;
Williams, R. E.; Jordan, K. N.; Lowe, C. R.; Bruce, N. C. FEMS Microbiol.
Lett. 2000, 190, 57-62. (e) Matsuda, T.; Harada, T.; Nakamura, K. Chem.
Commun. 2000, 1367-1368. (f) Riebel, B.; Hummel, W. Biotechnol. Lett.
2
001, 23, 231-234 (g) Schubert, T.; Hummel, W.; Kula, M.-R.; M u¨ ller,
M.; Eur. J. Org. Chem. 2001, 4181-4187. (h) Stampfer, W.; Kosjek, B.;
Moitzi, C.; Kroutil, W.; Faber, K. Angew. Chem. 2002, 114, 1056-1059.
(
i) Hummel, W.; Abokitse, K.; Drauz, K.; Rollmann, C.; Gr o¨ ger, H. AdV.
Synth. Catal. 2003, in press.
4) For reviews about biocatalytic reduction in general, see: (a) Hummel,
(
W. AdV. Biochem. Eng./Biotechnol. 1997, 58, 146-184. (b) Faber, K.
optically active (S)-alcohols possesses the following crite-
ria: (i) an aqueous-organic solvent system in which both
enzymes (carbonyl reductase and FDH from C. boidinii for
the cofactor regeneration) remain stable; (ii) good solubility
of poorly water-soluble ketones, which led to high substrate
concentrations of up to 200 mM; (iii) a simple reaction
protocol for lab-scale applications, comprising an economi-
cally attractive processing procedure, which allows a simple,
flexible, and fast preparation of optically active alcohols; and
Biotransformations in Organic Chemistry, 4th ed.; Springer-Verlag: Berlin,
2
000; Chapter 2.2.3, pp 192-194.
5) For selected contributions of an alternative approach via asymmetric
(
metal-catalyzed hydrogenation of ketones, see: (a) Burk, M. J.; Hems, W.;
Herzberg, D.; Malan, C.; Zanotti-Gerosa, A. Org. Lett. 2000, 2, 4173-
4
176. (b) Ohkuma, T.; Koizumi, M.; Yoshida, M.; Noyori, R. Org. Lett.
2
000, 2, 1749-1751. (c) Ohkuma, T.; Takeno, H.; Honda, Y.; Noyori, R.
AdV. Synth. Catal. 2001, 343, 369-375. (d) Review: Noyori, R.; Okhuma,
T. Angew. Chem., Int. Ed. 2001, 40, 40-73.
(6) For selected contributions of an alternative approach via asymmetric
whole-cell biocatalytic reduction of ketones, see: (a) Yasohara, Y.; Kizaki,
N.; Hasegawa, J.; Wada, M.; Kataoka, M.; Shimizu, S. Tetrahedron:
Asymmetry 2001, 12, 1713-1718. (b) See ref 3e. (c) See ref 3h.
(iv) a robust process with potential for technical-scale
(7) For selected contributions of an alternative approach via asymmetric
applications in the future.
reduction of ketones based on substrate-coupled cofactor regeneration,
see: (a) Wolberg, M.; Ji, A. G.; Hummel, W.; M u¨ ller, M. Synthesis 2001,
The first key step was the development of a suitable
reaction medium that guarantees a high solubility of ketones,
as well as a high stability of the enzymes. As mentioned
above, the stability of the FDH from C. boidinii as the most
sensitive enzyme component turned out to be a critical issue.
9
1
37-942. (b) Schubert, T.; Hummel, W.; M u¨ ller, M. Angew. Chem. 2002,
14, 656-659.
(8) Properties of the cofactor-regenerating enzyme FDH from C. boidinii
have been improved remarkably by the Kula group using protein engineer-
ing. Thus, a stable and efficient formate dehydrogenase is now available
on a large scale for NADH regeneration; see: Slusarczyk, H.; Felber, S.;
Kula, M.-R.; Pohl, M. Eur. J. Biochem. 2000, 267, 1280-1289.
(9) Despite its great technical potential and applicability, the FDH from
(10) (a) Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransforma-
tions; Wiley-VCH: Weinheim, Germany, 2000; pp 103-106. (b) Kruse,
W.; Hummel, W.; Kragl, U. Recl. TraV. Chim. Pays-Bas 1996, 115, 239-
243. (c) For a review of this method and other large-scale applica-
tions of oxidoreductases, see: Hummel, W. TIBTECH 1999, 17, 487-
492.
C. boidinii was found to be sensitive to the presence of organic solvents.
The general problem of rapid deactivation of enzymes other than hydrolases
in the presence of organic solvents has been previously described in several
contributions, e.g.: In Kruse, W.; Kragl, U.; Wandrey, C. DE 4436149,
1
996.
174
Org. Lett., Vol. 5, No. 2, 2003

We focused on this specific enzyme as the preferred FDH
for cofactor regeneration due to its low price, large-scale
availability, and proven technical applicability. A screening
Table 1. Reduction in Biphasic Media with Different
Substrates
8
of polar solvents showed a strong deactivation of the FDH
8
(
(
mutant C23S, C262A) even in the presence of only 10%
v/v) of the organic solvent component (Figure 1). In
a
General procedure: at a reaction temperature of 30 °C, 10 U of (S)-
alcohol dehydrogenase from R. erythropolis (expression in E. coli; see also
ref 3i) and 10 U of formate dehydrogenase from C. biodinii (mutant: C23S,
C262A; expression in E. coli; see also ref 8) were added to a solution of
Figure 1. Stability of the FDH in aqueous-organic media
activities were measured after ca. 3 days for n-hexane, 2 days for
n-heptane, and 2 days or less for the other solvents; for details, see
(
0
.5 mmol of the ketone component, 2.5 mmol of sodium formate (171.6
mg) and 0.1 mmol of NADH (70.2 mg) in a solvent mixture, consisting of
0 mL of n-heptane and 40 mL of a phosphate buffer (50 mM; pH 7.0).
Supporting Information).
1
After the reaction mixture was stirred for 21 h, the organic phase was
separated and the aqueous phase was extracted with 3 × 5 mL of methyl
tert-butyl ether. The collected organic phases are dried over magnesium
sulfate, and after filtration and evaporation of the volatile components in
addition, typical solvents for a biphasic media failed, e.g,
MTBE, which led to a remarkable decrease of the activity
after 2 days. A very rapid loss of activity was found when
ethyl acetate was used. In this case, a (nearly) complete
deactivation was observed within only 6 h. Nonpolar
aromatic hydrocarbons, e.g., toluene, deactivate FDH re-
markably, too.
vacuo, the resulting oily crude product was analyzed with respect to the
conversion (via NMR and HPLC). b Enantiomeric excess (ee) was deter-
mined by chiral GC chromatography.
p-chloroacetophenone was converted into the optically active
(
S)-enantiomer, (S)-4a, with >99% ee (69% conversion;
entry 1).
A conversion of 65% and a slightly lower enantioselec-
However, we were pleased to find that in the presence of
10% (v/v) n-hexane, a long-term nondestabilization was
achieved (Figure 1). Even after 66-69 h, activities of 92
and 90% remained for aqueous solutions containing 10 and
tivity of 97% ee were observed when using p-bromoaceto-
phenone as a starting material (entry 2). The asymmetric
reduction of phenoxyacetone proceeded quantitatively under
formation of (S)-4c with an enantioselectivity of >99% ee
(entry 3). Compounds of type 4c or derivatives thereof are
known to be biologically active, thus representing pharma-
ceutically interesting molecules.
20% (v/v) n-hexane, respectively. Notably, other aliphatic
hydrocarbons, e.g., n-heptane, also were found to be suitable
organic solvents, and the stability was maintained at an
increased amount of the organic solvent, e.g., n-heptane, of
up to 60% (v/v).
The high degree of stability independent of the amount
of organic solvent guarantees sufficient flexibility with
respect to the fine-tuning of the reaction later in the process
development step. In addition, the large-scale available (S)-
selective alcohol dehydrogenase (ADH) from Rhodococcus
These studies, which proved the applicability of the new
reaction system for preparative conversions, were followed
by further investigation of the new reaction media at higher
substrate concentrations in order to reach high volumetric
productivities. As a model reaction, the reduction of p-
chloroacetophenone in the presence of the (S)-ADH from
3i
erythropolis, which we have developed very recently, turned
out to be stable for long periods under the same conditions.
Thus, a suitable solvent system was found in which both
enzymes remain stable.
3
i
R. erythropolis was investigated. Increased substrate con-
centrations from 10 mM (69% conversion) up to 100 mM
led to comparable conversions (Figure 2). At 20 and 40 mM
concentrations, slightly improved conversions of 77 and 75%,
respectively, were obtained. Even at a 100 mM concentration,
a good conversion of 74% was still achieved. An analogous
reaction with subsequent chromatographic purification of the
crude product gave the desired product (S)-4a with a yield
With this enzyme-compatible reaction media in hand,
preparative conversions were carried out as a next step. We
were pleased to find that good conversions accompanied by
excellent enantioselectivities were obtained with a variety
of aromatic ketone substrates. Examples are shown in Table
1. In the presence of an (S)-ADH from R. erythropolis,
Org. Lett., Vol. 5, No. 2, 2003
175

higher volumetric productivities can be obtained with this
new reaction medium.
In conclusion, a practical and efficient asymmetric reduc-
tion of ketones via enzymatic in situ cofactor regeneration
was found that runs at high substrate concentrations in the
direct presence of an organic solvent. The general applicabil-
ity of this reaction system as well as the simple access to
optically active alcohols on a lab scale (and possibly on a
technical scale in the future) are further advantages. Cur-
rently, several downstream process improvements comprising
recycling issues are under investigation.
Acknowledgment. This work was supported by the
Bundesministerium f u¨ r Bildung und Forschung (Biotech-
nologie 2000-Nachhaltige BioProduktion; Project “Entwick-
lung eines biokatalytischen und nachhaltigen Verfahrens zur
industriellen Herstellung enantiomerenreiner Amine und
Alkohole unter besonderer Ber u¨ cksichtigung der Atom o¨ kon-
omie”).
Supporting Information Available: Protocol for the
investigation of the enzymatic stabilities in aqueous-organic
solutions (according to Figure 1) as well as experimental
protocols and data referring to Table 1 and Figure 2,
respectively. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 2. Conversions at increased substrate concentrations.
of 67% (for experimental details, see Supporting Informa-
tion). A further increase of the substrate concentration up to
200 mM gave a still satisfactory conversion of 63%. Thus,
OL0272139
176
Org. Lett., Vol. 5, No. 2, 2003