Tandem concurrent processes: One-pot single-catalyst biohydrogen transfer for the simultaneous preparation of enantiopure secondary alcohols

Full text of DOI:10.1021/jo802350f

SCHEME 1. Tandem ADH-Catalyzed Hydrogen Transfer
Concept
Tandem Concurrent Processes: One-Pot
Single-Catalyst Biohydrogen Transfer for the
Simultaneous Preparation of Enantiopure
Secondary Alcohols
Fabricio R. Bisogno,† Iva´n Lavandera,† Wolfgang Kroutil,‡
and Vicente Gotor*,†
Departamento de Qu´ımica Orga´nica e Inorga´nica, Instituto
UniVersitario de Biotecnolog´ıa de Asturias, UniVersity of
OViedo, 33006 OViedo, Spain, and Department of Chemistry,
Organic and Bioorganic Chemistry, UniVersity of Graz,
Heinrichstrasse 28, 8010 Graz, Austria
employing alcohol dehydrogenases (ADHs) has recently gained
increasing relevance.4 In these processes, a single enzyme
reduces/oxidizes the target substrate, sacrificing a small molecule
(cosubstrate) like 2-propanol/acetone as hydride donor/acceptor,
used in a huge molar excess (at least 10 equiv compared to 1
equiv substrate to afford conversions higher than 90%) due to
the reversible character of the reaction.
Vgs@fq.unioVi.es
ReceiVed October 27, 2008
Very recently, it has been described that small activated
ketones such as methyl acetoacetate5 or chloroacetone6 can be
employed as cosubstrates in ADH-catalyzed oxidation of
alcohols in near stoichiometric amount to achieve complete
conversion. Herein, we present a system in which the sacrificial
reaction has been turned into a highly valuable transformation,
resulting in a one-pot process combining activated ketones with
racemic sec-alcohols in order to concurrently obtain two
different optically enriched alcohols catalyzed by a single
enzyme, thus maximizing the atom efficiency enVironmental
factor E7 of the process, since no additional reagent is discarded.
Therefore, starting from a prochiral ketone and a racemic
alcohol, we can obtain two optically pure alcohols (Scheme 1).
Another advantage of this system is that the stereoselectivity
can be tuned by simply changing the biocatalyst employed.
In a first set of experiments, we studied the influence of the
ketone structure on some selected ADH-catalyzed reductions
A novel one-pot tandem biohydrogen transfer process to
concurrently obtain two enantiopure sec-alcohols is pre-
sented; thus, using a suitable single enzyme and a catalytic
amount of cofactor, several interesting building blocks could
be easily achieved in an enantiocomplementary fashion,
minimizing dramatically the quantity of reagents usually
employed in the “coupled-substrate” approach.
(3) Recent bibliography: (a) Modern Biooxidation. Enzymes, Reactions and
Applications; Schmid, R. D., Urlacher, V. B., Eds.; Wiley-VCH: Weinheim,
2007. (b) Arends, I. W. C. E. Catalytic Oxidations in Green Chemistry and
Catalysis; Sheldon, R. A., Arends, I. W. C. E., Hanefeld, U., Eds.; Wiley-VCH:
Weinheim, 2007. (c) Lenoir, D. Angew. Chem., Int. Ed. 2006, 45, 3206. (d)
Modern Oxidation Methods; Ba¨ckvall, J.-E., Ed.; Wiley-VCH: Weinheim, 2004.
(4) Recent bibliography: (a) Buchholz, S.; Gro¨ger, H. In Biocatalysis in the
Pharmaceutical and Biotechnology Industry; Patel, R. N., Ed.; CRC Press: Boca
Raton, 2007, p 757. (b) de Wildeman, S. M. A.; Sonke, T.; Schoemaker, H. E.;
May, O. Acc. Chem. Res. 2007, 40, 1260. (c) Moore, J. C.; Pollard, D. J.; Kosjek,
B.; Devine, P. N. Acc. Chem. Res. 2007, 40, 1412. (d) Goldberg, K.; Schroer,
K.; Lu¨tz, S.; Liese, A. Appl. Microbiol. Biotechnol. 2007, 76, 237. (e) Kroutil,
W.; Mang, H.; Edegger, K.; Faber, K. AdV. Synth. Catal. 2004, 346, 125. (f)
Kroutil, W.; Mang, H.; Edegger, K.; Faber, K. Curr. Opin. Chem. Biol. 2004, 8,
120. (g) Nakamura, K.; Matsuda, T. J. Org. Chem. 1998, 63, 8957.
(5) Peschko, C.; Stohrer, J. Enzyme catalyzed oxidation of secondary alcohols.
Wacker Chemie AG, Germany, DE 102006009743, A1 20070906; CAN 2007,
147, 321414.
Several (bio)catalytic methods to synthesize enantiopure
secondary alcohols have been developed in the past few years
to fulfill the increasing demand of this type of highly valuable
compounds.1 Among all methodologies described, stereoselec-
tive reduction of ketones2 and enantioselective oxidation of
racemic sec-alcohols3 using hydrogen transfer (HT) protocols
have extensively been studied because of the mild and simple
conditions employed in these transformations. In this context,
biocatalyzed HT (also called “coupled-substrate” approach)
† University of Oviedo.
‡ University of Graz.
(1) Breuer, M.; Ditrich, K.; Habicher, T.; Hauer, B.; Kesseler, M.; Stu¨rmer,
R.; Zelinski, T. Angew. Chem., Int. Ed. 2004, 43, 788.
(2) Recent reviews: (a) Wu, X.; Xiao, J. Chem. Commun. 2007, 2449. (b)
Ikariya, T.; Blacker, A. J. Acc. Chem. Res. 2007, 40, 1300. (c) Gladiali, S.;
Alberico, E. Chem. Soc. ReV. 2006, 35, 226. (d) Ikariya, T.; Murata, K.; Noyori,
R. Org. Biomol. Chem. 2006, 4, 393.
(6) Lavandera, I.; Kern, A.; Resch, V.; Ferreira-Silva, B.; Glieder, A.; Fabian,
W. M. F.; de Wildeman, S.; Kroutil, W. Org. Lett. 2008, 10, 2155.
(7) (a) Sheldon, R. A. Pure Appl. Chem. 2000, 72, 1233. (b) Trost, B. M.
Science 1991, 254, 1471.
1730 J. Org. Chem. 2009, 74, 1730–1732
10.1021/jo802350f CCC: $40.75 2009 American Chemical Society
Published on Web 01/12/2009

TABLE 1. Tandem Concurrent Biohydrogen Transfer Using Activated Ketones and 2-Octanola
Prelog ADH
anti-Prelog ADH
2a-k
3a
2a-k
3a
convb
ee (%)c
ee (%)c
enzyme
convb
ee (%)c d
ee (%)c
,
entry
ketone
enzyme
1
2
3
4
5
6
7
8
1a
1be
1c
ADH-A
ADH-A
ADH-A
ADH-A
ADH-Af
ADH-A
ADH-A
ADH-T
ADH-Te
ADH-Ae
ADH-Tf
81
57
97
85
93
89
91
73
78
>99 (R)d
>99 (R)d
>99 (R)d
>99 (R)d
99 (R)d
83 (R)
44 (R)
94 (R)
88 (R)
98 (R)
99 (R)
>99 (R)
70 (R)
72 (R)
94 (R)
>99 (R)
LB-ADH
LB-ADH
LB-ADH
LB-ADH
PR2
LB-ADH
LB-ADH
PR2
90
83
94
86
90
90
90
84
>99 (S)
>99 (S)
>99 (S)
>99 (S)
99 (S)
>99 (S)
>99 (S)
>99 (S)
98 (S)
79 (S)
92 (S)
88 (S)
>99 (S)
98 (S)
1d
1e
1fe
1g
1he
1i
>99 (R)d
>99 (R)d
>99 (R)d
>99 (S)
>99 (S)
90 (S)
9
10
11
g
1j
1k
85
>99
>99 (R)d
>99 (R)d
LB-ADH
87
>99
>99 (S)
>99 (S)
94 (S)
>99 (S)
LB-ADHh
a Enzyme (3-5 U); [1] 50 mM; [3a] 90-100 mM; [NAD(P)H] 1 mM. b Measured by achiral GC. c Measured by chiral GC or HPLC. d Switch in
Cahn-Ingold-Prelog priority (CIP). e [3a] 45 mM. f [3a] 180 mM. g Not appropriate ADH found. h [3a] 400 mM.
using a low excess of the cosubstrate (2 equiv of 2-propanol;
see Supporting Information). Thus, several ketones were reduced
using a Prelog (ADH-A from Rhodococcus ruber)8 or an anti-
Prelog [ADH from Lactobacillus breVis (LB-ADH)]9 enzyme.
It could be observed that non-activated ketones like acetophe-
none afforded 50% conversion. When para-substituted ac-
etophenones were reduced, electron-donating groups provided
low conversions (<30%), while electron-withdrawing substit-
uents afforded conversions of about 80%. Moreover, ketones
with an electron-withdrawing group at the R-position such as
R-chloroacetophenone furnished quantitative conversions. These
results can be explained by the different oxidation-reduction
potentials (∆E°) between the ketone/alcohol pair with regards
to the 2-propanol/acetone counterpart.10 It has been shown that
R-halohydrins are stabilized via intramolecular H-bonding
between the alcohol moiety and the halogen atom,11 therefore
preventing the ADH-catalyzed oxidation.6
Taking as an advantage the irreversibility of this HT, we
tested the concept in a one-pot tandem protocol to simulta-
neously obtain two enantiopure sec-alcohols (see scheme of
Table 1). In theory, an irreversible asymmetric reduction is
required to achieve a complete kinetic oxidative resolution, and
thus a molar amount of ketone to be reduced can be equal or
slightly higher than the molar amount of alcohol to be oxidized.
Thus, by mixing an activated ketone (1 equiv) with a racemic
alcohol (1.8-2 equiv), the selective reduction of the prochiral
compound plus the kinetic resolution of the racemate could be
achieved via HT by a single enzyme and a catalytic amount of
the pyrimidinic cofactor that is internally recycled. As a result
of the perfect selectivity shown by the biocatalysts utilized, the
hydride is abstracted from a single enantiomer of the racemic
alcohol and then exclusively transferred to one stereoface of
the prochiral ketone.
Therefore, several R-chloro, R-azido, and R-cyano ketones
(1a-k, Table 1) were purchased or synthesized and then
combined with racemic 2-octanol (3a). Except for the cyano
derivative 1i (entry 9), we were able to find a suitable Prelog
and anti-Prelog ADH to obtain enantioenriched or enantiopure
(R)- or (S)-3a using activated aliphatic and (hetero)aryl ketones,
which were reduced to the corresponding enantiopure alcohols
with very high yields. Compounds with electron-donating groups
in the phenyl ring (1b, entry 2) afforded lower conversions. By
simply changing the enzyme, enantiocomplementary products
could be achieved. Thus, ADH-A or LB-ADH were usually
employed, but in some cases Thermoanaerobacter sp. ADH
(ADH-T)12 or PR2 provided better results. As an example, we
scaled up the reaction of 2k with LB-ADH up to a substrate
concentration of 400 mM, showing the great robustness of the
system. The obtained R-activated alcohols are important precur-
sors of pharmaceutical compounds. For instance, (S)-2a is an
intermediate for the synthesis of fluoxetine, tomoxetine, and
nisoxetine,13 (R)-2b can be used as precursor of ꢀ-agonists like
octopamine or denopamine,14 and optically active 2k is a useful
chiral building block for the synthesis of different pharmaceu-
ticals.15
Several sec-alcohols were resolved using chloro ketone 1e
with ADH-A and 1k with LB-ADH (Table 2). Thus, aromatic
(3b-d), aliphatic (3e-f) such as sulcatol, or cycloalkyl (3g)
(8) This (S)-selective enzyme has a strong preference for NADH/NAD+. See:
(a) Edegger, K.; Gruber, C. C.; Poessl, T. M.; Wallner, S. R.; Lavandera, I.;
Faber, K.; Niehaus, F.; Eck, J.; Oehrlein, R.; Hafner, A.; Kroutil, W. Chem.
Commun. 2006, 2402. (b) Stampfer, W.; Kosjek, B.; Moitzi, C.; Kroutil, W.;
Faber, K. Angew. Chem., Int. Ed. 2002, 41, 1014.
(12) Findrik, Z.; Vasic´-Racki, D.; Lu¨tz, S.; Daussmann, T.; Wandrey, C.
Biotechnol. Lett. 2005, 27, 1087.
(13) Zhu, D.; Mukherjee, C.; Hua, L. Tetrahedron: Asymmetry 2005, 16,
3275.
(9) This (R)-selective enzyme shows a preference for NADPH/NADP+. See,
for instance: Wolberg, M.; Hummel, W.; Wandrey, C.; Mu¨ller, M. Angew. Chem.,
Int. Ed 2000, 39, 4306.
(10) Eckstein, M. F.; Peters, M.; Lembrecht, J.; Spiess, A. C.; Greiner, L.
AdV. Synth. Catal. 2006, 348, 1591.
(11) Goldstein, T.; Snow, M. S.; Howard, B. J. J. Mol. Spectrosc. 2006,
236, 1.
(14) Lee, D.-M.; Lee, J.-C.; Jeong, N.; Lee, K.-I. Tetrahedron: Asymmetry
2007, 18, 2662.
(15) Kizaki, N.; Yasohara, Y.; Hasegawa, J.; Wada, M.; Kataoka, M.;
Shimizu, S. Appl. Microbiol. Technol. 2001, 55, 590.
J. Org. Chem. Vol. 74, No. 4, 2009 1731

TABLE 2. Resolution of sec-Alcohols via ADH-Catalyzed Tandem
HT Mediated by r-Chloro Ketonesa
were synthesized by conventional reduction from the corresponding
ketones (NaBH4, MeOH, room temperature). All other reagents and
solvents were of the highest quality available. One unit (U) of ADH
reduces 1.0 µM of acetophenone to 1-phenylethanol per minute at
pH 7.5 and 30 °C in the presence of NAD(P)H. Flash chromatog-
raphy was performed using silica gel 60 (230-400 mesh).
General Procedure for the Tandem Concurrent Biohydro-
gen Transfer Using Activated Ketones and 2-Octanol. In a 1.5
mL Eppendorf vial, 3-5 U of commercially available ADH
(Lactobacillus breVis ADH, Rhodococcus ruber ADH-A, Ther-
moanaerobacter sp. ADH or PR2 ADH) in Tris-HCl buffer [50
mM, pH 7.5, 1 mM NAD(P)H, 1 mM MgCl2 for LB-ADH] were
mixed with both the racemic 2-octanol and the prochiral ketone
(1a-k) in a 1.8-2:1 molar ratio respectively (e.g., 90-100 mM
racemic 3a and 50 mM ketone) in a final volume of 0.6 mL. The
reaction was incubated at 30 °C with orbital rotation (150 rpm) for
24 h. Then, the reaction was stopped by extraction with ethyl acetate
(2 × 0.6 mL). The organic layer was separated by centrifugation
(2 min, 13,000 rpm) and dried (Na2SO4). Conversions and enan-
tiomeric excesses of the corresponding alcohols were determined
by GC or HPLC analysis using an achiral or chiral stationary phase,
respectively.
ADH-A
LB-ADH
1k
alcohol convb ee (%)c,d ee (%)c convb ee (%)c d ee (%)c
1e
3b-g
3b-g
,
3b
3c
3d
3e
3f
90
85
91
93
95
94
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
99 (R)
81 (R)
>99 (R)
95 (R)
>99 (R)
98 (R)
>99
94
99
97
>98
>98
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
87 (S)
>99 (S)
96 (S)
>99 (S)
>99 (S)
3g
General Procedure for the Resolution of sec-Alcohols via
ADH-Catalyzed Tandem HT Mediated by r-Chloro Ketones.
In a 1.5 mL Eppendorf vial, 3-5 U of commercially available ADH
(LB-ADH or ADH-A) in Tris-HCl buffer [50 mM, pH 7.5, 1 mM
NAD(P)H, 1 mM MgCl2 for LB-ADH] were mixed with both the
racemic alcohol (3b-g, 90-100 mM) and the prochiral ketone (1e
for ADH-A or 1k for LB-ADH, 50 mM) in a final volume of 0.6
mL. The reaction was incubated at 30 °C with orbital rotation (150
rpm) for 24 h. Then, the reaction was stopped by extraction with
ethyl acetate (2 × 0.6 mL). The organic layer was separated by
centrifugation (2 min, 13,000 rpm) and dried (Na2SO4). Conversions
and enantiomeric excesses of the corresponding alcohols were
determined by GC or HPLC analysis using an achiral or chiral
stationary phase, respectively.
a Enzyme (3-5 U); [1] 50 mM; [3a] 90-100 mM; [NAD(P)H] 1
mM. b Measured by achiral GC. c Measured by chiral GC. d Switch in
CIP.
derivatives could be successfully obtained in enantioenriched
form via tandem concurrent HT.
In summary, we have demonstrated a novel one-pot tandem
system to simultaneously obtain two enantiopure sec-alcohols
that possesses several advantages: conversion can be easily
controlled by the amount of racemic alcohol added, a single
biocatalyst and catalytic amount of cofactor are used, the
selectivity can be tuned by choosing the appropriate enzyme,
and the process can be scaled up.16 This is an elegant example
that shows how biocatalysis can be applied for the “clean”
synthesis of valuable enantiopure compounds maximizing the
atom efficiency.7
Acknowledgment. F.R.B. is supported by the Programme
Alꢀan, the European Union Program of High Level Scholarships
for Latin America (scholarship E07D402519AR). I.L. thanks
Principado de Asturias for personal funding (Clar´ın Program).
Financial support from the Spanish Ministerio de Ciencia e
Innovacio´n (MICINN, Project CTQ2007-61126/PPQ) is grate-
fully acknowledged.
Experimental Section
General. Alcohol dehydrogenases; ketones 1a, 1d, 1e, 1i, and
1k; racemic alcohols 3a, 3b, 3c, 3d, 3e, 3f, and 3g; and their
corresponding ketones were purchased from commercial sources.
R-Chloro ketones 1b, 1c, 1f, 1g, and 1j were synthesized following
modified protocols described in the literature.17 R-Azido ketone
1h was obtained as published before.8a Racemic alcohols 2a-k
Supporting Information Available: Experimental proce-
dures and analytics are detailed. This material is free of charge
via the Internet at http://pubs.acs.org.
JO802350F
(16) The selection of the substrates was based on their different physical
properties. Thus, ketones and aliphatic alcohols can be distilled, while the
employed aromatic alcohols can be separated using flash chromatography.
(17) (a) Mei, Y.; Bentley, P. A.; Du, J. Tetrahedron Lett. 2008, 49, 3802.
(b) Lee, J. C.; Bae, Y. H.; Chang, S. K. Bull. Korean Chem. Soc. 2003, 24, 407.
1732 J. Org. Chem. Vol. 74, No. 4, 2009