Biocatalytic asymmetric hydrogen transfer.

Full text of DOI:10.1002/1521-3773(20020315)41:6<1014::AID-ANIE1014>3.0.CO;2-6

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These results demonstrate that both aldol enantiomers could
be accessed through aldol or retro-aldol reactions using the
same antibody 84G3. In order to assign the absolute config-
urations, the enantiopure products were synthesized by
independent chemical asymmetric synthesis for compounds
9, 11, and 13.[9] The absolute configuration of compound 7 was
assigned by analogy with compound 9.
In conclusion, we described here the first aldolase antibody,
ab84G3, capable of rerouting the regioselectivity of a series of
cross aldol reactions which led to the formation of the
otherwise disfavored products. This new reactivity highlights
the scope of the reactive immunization strategy developed by
the groups of Lerner and Barbas for catalyst design. This work
further increases the repertoire and efficiency of antibody-
catalysed aldol reactions. Further studies on the reactivity of
ab84G3 are currently in progress.
Biocatalytic Asymmetric Hydrogen Transfer**
Wolfgang Stampfer, Birgit Kosjek, Christian Moitzi,
Wolfgang Kroutil,* and Kurt Faber
Driven by the increased public awareness on hazards
derived from chemical production, environmentally benign
oxidation methods have gained increasing importance.[1] In
this context, the reduction of ketones at the expense of a
sacrificial secondary alcohol and the corresponding reverse
reaction (known since the 1920s as the Meerwein Ponn-
dorf Verley reduction (MPVRed) and Oppenauer-oxidation
(OOx), respectively, constitute typical ™green∫ redox reac-
tions.[2] As a consequence, they have been re-investigated
recently[3] to replace the previously employed metal alkoxides
with catalysts showing improved efficiency,[3c] better recover-
ability,[3g] avoiding aldol condensation as side reaction,[3f] and
water-soluble analogues.[3b] Asymmetric variants have been
pursued by using enantioselective hydride-transfer methods
based on chiral transition metal complexes[3e] or chiral hydride
sources.[3a]
All biocatalytic methods for the asymmetric hydrogen
transfer are based on alcohol dehydrogenases requiring
nicotinamide cofactors. They have several advantages over
the chemical methods, such as 1) their intrinsic asymmetry,
2) absence of side reactions, such as aldol condensation, and
3) they operate under essentially mild reaction conditions.
However, their large-scale application has been impeded by
the requirement for cofactor-recycling.[4] Since the sacrificial
secondary alcohol used as cosubstrate (for MPVRed) or the
carbonyl compound (for OOx) has to be employed in excess
to drive the reaction from equilibrium towards completion,
cosubstrate inhibition is common in such a ™coupled-sub-
strate∫ approach based on the use of a single enzyme.[5]
Although this drawback has been surmounted to some extent
by using a second dehydrogenase, which is highly specific for
the sacrificial cosubstrate,[6] these so-called ™coupled-en-
zyme∫ methods are rather complex and require the handling
of isolated enzymes and cofactor(s). As a consequence,
biochemical MPVReds and OOxs on a large scale are limited
by the use of fermenting cells[7] and/or low (co)substrate
concentration(s).[8]
Received: October 24, 2001 [Z18117]
[1] General reviews on the catalytic enantioselective aldol reaction:
a) E. M. Carreira in Comprehensive Asymmetric Catalysis, Vol. 3
(Eds.: E. N. Jacobsen, A. Pfaltz, H. Yamamoto), Springer, Heidelberg,
1999, chap. 29-1; b) S. G. Nelson, Tetrahedron:Asymmetry 1998, 9, 357;
H. Grˆger, E. M. Vogl, M. Shibasaki, Chem. Eur. J. 1998, 4, 1137; T. D.
Machajewski, C.-H. Wong, Angew. Chem. 2000, 112, 1406; Angew.
Chem. Int. Ed. 2000, 39, 1352.
[2] a) H. J. M. Gijsen, L. Qiao, W. Fitz, C.-H. Wong, Chem. Rev. 1996, 96,
443; b) B. List, R. A. Lerner, C. F. Barbas III, J. Am. Chem. Soc. 2000,
122, 2395; H. Grˆger, J. Wilken, Angew. Chem. 2001, 113, 545; Angew.
Chem. Int. Ed. 2001, 40, 529; c) K. Sakthivel, W. Notz, T. Bui, C. F.
Barbas III, J. Am. Chem. Soc. 2001, 123, 5260.
[3] a) J. Wagner, R. A. Lerner, C. F. Barbas III, Science 1995, 270, 1797;
b) R. Bjˆrnestedt, G. Zhong, R. A. Lerner, C. F. Barbas III, J. Am.
Chem. Soc. 1996, 118, 11720; c) G. Zhong, T. Hoffmann, R. A. Lerner,
S. Danishefsky, C. F. Barbas III, J. Am. Chem. Soc. 1997, 119, 8131;
d) C. F. Barbas III, A. Heine, G. Zhong, T. Hoffmann, S. Gramatikova,
R. Bjˆrnestedt, B. List, J. Anderson, E. A. Stura, I. A. Wilson, R. A.
Lerner, Science 1997, 278, 2085; e) T. Hoffmann, G. Zhong, B. List, D.
Shabat, J. Anderson, S. Gramatikova, R. A. Lerner, C. F. Barbas III, J.
Am. Chem. Soc. 1998, 120, 2768; f) G. Zhong, D. Shabat, B. List, J.
Anderson, S. C. Sinha, R. A. Lerner, C. F. Barbas III, Angew. Chem.
1998, 110, 2609; Angew. Chem. Int. Ed. 1998, 37, 2481; g) B. List, D.
Shabat, G. Zhong, J. M. Turner, A. Li, T. Bui, J. Anderson, R. A.
Lerner, C. F. Barbas III, J. Am. Chem. Soc. 1999, 121, 7283.
[4] a) G. Zhong, R. A. Lerner, C. F. Barbas III, Angew. Chem. 1999, 111,
3957; Angew. Chem. Int. Ed. 1999, 38, 3738; for synthetic applications
using ab84G3, see: b) S. C. Sinha, J. Sun, G. P. Miller, M. Wartmann,
R. A. Lerner, Chem. Eur. J. 2001, 7, 1691; c) S. C. Sinha, J. Sun, G. P.
Miller, C. F. Barbas III, R. A. Lerner, Org. Lett. 1999, 10, 1623.
[5] For the regioselectivity of the aldol condensations of 2-butanone and
2-pentanone in the presence of ab38C2, see ref. [3a].
We have recently isolated a highly enantioselective secon-
dary-alcohol dehydrogenase[9] from Rhodococcus ruber
DSM 44541, which is exceptionally stable towards organic
solvents. The activity of the enzyme remains high at concen-
trations of up to 20% (v/v) acetone and 50% (v/v) 2-propanol.
This activity enables the use of the enzyme for MVRed and
OOx in the ™coupled-substrate∫ approach. For preparative-
[6] For aldol condensations with a-hydroxyacetone in the presence of
ab38C2, see for example ref. [3e] and: B. List, D. Shabat, C. F.
Barbas III, R. A. Lerner, Chem. Eur. J. 1998, 4, 881; D. Shabat, B.
List, R. A. Lerner, C. F. Barbas III, Tetrahedron Lett. 1999, 40, 1437.
[7] Tetrahydrothiophen-3-one has been reported to be a donor substrate
for ab38C2 (see ref. [3e]) but there is no discussion regarding
regioselectivity.
[8] a) J. Uenishi, H. Tomozane, M. Yamato, Tetrahedron Lett. 1985, 29,
3467; b) J. Uenishi, H. Tomozane, M. Yamato, J. Chem. Soc. Chem.
Commun. 1985, 717.
[*] Dipl.-Ing. Dr. W. Kroutil, Dipl.-Ing. W. Stampfer, Mag. B. Kosjek,
C. Moitzi, Prof. Dr. K. Faber
Department of Chemistry, Organic and Bioorganic Chemistry
University of Graz
Heinrichstrasse 28, 8010 Graz (Austria)
Fax : (‡43)316-380-9840
[9] V. Maggiotti, J. B. Wong, R. Razet, A. R. Cowley, V. Gouverneur,
unpublished results.
E-mail: Wolfgang.Kroutil@uni-graz.at
[**] This study was performed within the Spezialforschungsbereich ™Bio-
katalyse∫, and financial support by the Fonds zur Fˆrderung der
wissenschaftlichen Forschung (Vienna, project No. F115) and the
Federal Ministry of Science (Vienna) is gratefully acknowledged.
1014
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Table 1. Results of the biocatalytic reduction of various ketones employing
lyophilized whole cells of Rhodococcus ruber DSM 44541 with 50% (v/v)
2-propanol in buffer.
scale applications, whole lyophilized or resting cells can be
used as a readily available alternative.
Reduction mode: Whole lyophilized cells of Rhodococcus
ruber DSM 44541[10, 11] grown on a standard medium (glucose,
peptone, yeast extract) without enzyme induction catalyzed
the reduction of 1a in aqueous buffer at pH 7.5 with excellent
enantioselectivity to furnish (S)-sulcatol (2a; Scheme 1) with
Substrate
t [h]
ee (Product) [%]
Conversion [%]
1a
1b
1c
22
22
22
22
22
22
22
96
> 99 (S)
> 99 (S)
> 99 (S)
97( S)
> 99 (S)
> 99 (S)
> 99 (S)
> 99 (S)
7
0
91
81
7
65
92
82
67
3-octanone
2-decanone
1-cyclohexyl ethanone
1-(naphth-2-yl) ethanone
oct-3-en-2-one
9
1a (1.0 g, 125 gLÀ1, 0.99 molLÀ1) was reduced at 248C within
24 h employing lyophilized whole cells of Rhodococcus ruber
DSM 44541 (0.3 g) in phosphate buffer (pH 7.5, 50 mm)
containing 50% 2-propanol to give (S)-sulcatol 2a in 67%
yield and >99% ee.[16, 17]
Scheme 1. Biocatalytic oxidation/reduction of secondary alcohols/ketones
employing lyophilized cells in buffer and cosubstrate [20% (v/v) acetone/
50% (v/v) 2-propanol].
Oxidation mode: Encouraged by these results, we exam-
ined the applicability of the system for the reverse reaction,
that is, the oxidation of rac secondary alcohols at the expense
of acetone as sacrificial cosubstrate.[18] Indeed, whole cells of
Rhodococcus ruber DSM 44541 exclusively oxidized the
S enantiomer from racemic substrates rac-2a, rac-2b, and
rac-2c (Table 2). The relative rate of oxidation for (S)-2b was
about 55 times higher than that of (S)-2c. Optimization of the
system showed that the oxidation rate for rac-2c measured at
different acetone concentrations reached a maximum at 5%
(v/v) of acetone (Figure 2). However, the reaction ceased
before the maximum obtainable conversion of 50% (for a
kinetic resolution) was reached. Complete reaction (con-
version ˆ 50%) occurred when the acetone concentration was
increased to 20% (v/v), however, a reduction of reaction rate
was observed, and preliminary results indicated that enzyme
inhibition occurred. A compromise between a maximum
reaction rate and minimum enzyme inhibition was found, the
best results were obtained with an acetone concentration of
5% (v/v) at start followed by continuous addition throughout
the reaction reaching a final acetone concentration of 20%
(v/v).[19]
an ee value of >99%, while the reaction was driven towards
completion by addition of 2-propanol. When the cosubstrate
concentration was gradually increased,[12] the reaction rate
reached a maximum at 50% (v/v) of 2-propanol (Figure 1).[13]
To explore the limits of the productivity of the system, the
substrate concentration was varied by keeping the concen-
tration of 2-propanol at a constant level of 50% (v/v). A broad
optimum for the substrate concentration was found within a
range of 0.5 to 1.0 molLÀ1, which corresponds to 76 gLÀ1 to
126 gLÀ1 for this substrate (Figure 1).[14, 15]
The flexibility of this system was demonstrated by the
reduction of substrates containing an aromatic moiety or a,b-
unsaturated ketones (Table 1) to furnish the corresponding
alcohol with excellent ee values.
A preparative-scale reaction was repeated at high substrate
and cosubstrate concentration. Without further optimization,
Table 2. Results of the biocatalytic kinetic resolution by oxidation of
secondary alcohols employing lyophilized whole cells of Rhodococcus
ruber DSM 44541.
Substrate
Relative Enantio-
Conversion ee [%]
activity[a] selectivity E[b] [%]
rac-2a
rac-2b
1000
813
374
338
178
62
15
3
2.6
> 100
> 100
> 100
> 100
49.0
49.4
46.798.5(
(49.795.7
98.2
49.3
44.4
33.0(
36.9
97.2(R)
97.8(R)
R)
rac-(E)-3-octen-2-ol
rac-4-phenyl-2-butanol
cyclopentanol
rac-1-(naphth-2-yl)ethanol
rac-2c
R)
> 100
> 100
2.749.5
ꢀ 1
98.2(R)
77.8(R)
R)
rac-3-octanol
rac-4-octanol
0
[a] The relative activity for 1a was arbitrarily set to 1000, which
corresponds to 1.55 mmol transformed substrate hÀ1 gÀ1 lyophilized cells.
[b] Enantioselectivity was calculated from ee values and conversion: E ˆ
{ln[(1 À c)(1 À ee)]}/ln[(1 À c)(1 ‡ ee)]. All experiments were performed
employing Rhodococcus ruber DSM 44541 with 20% (v/v) acetone in
phosphate buffer at 248C and at pH 8.0.
Figure 1. Activity a of whole cells of Rhodococcus ruber DSM 44541 for
the reduction of 1a in phosphate buffer (pH 7.5) at different concentrations
!
*
of 2-propanol ( ) and at different substrate concentration 1a [ , 50% (v/v)
2-propanol].
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1015

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for reduction; pH 8.0 for oxidation) for 30 min at
308C. 2-Propanol or acetone, respectively, was added
at a certain concentration. The substrate concentra-
tion was constant for all experiments (1a, 126 gLÀ1
;
rac- 2c, 18 gLÀ1). Samples were shaken in Eppendorf
vials at 130 rpm and 248C on a rotary shaker for 2.3
(1a) or 4 h (rac-2c), respectively. The reactions were
quenched by addition of ethyl acetate (0.6 mL)
followed by centrifugation.
The optimum for the substrate concentration for the
reduction [oxidation] was determined by rehydrating
whole lyophilized cells of Rhodococcus ruber
DSM 44541 (20 mg) [30 mg] in phosphate buffer
(650 mL, 50 mm, pH 7.5/reduction, pH 8.0/oxidation)
for 30 min at 308C. 2-Propanol [acetone] was added at
a concentration of 50% (v/v) [20% (v/v)]. Substrate
1a [rac-2b] was added and the reaction shaken at 248C
at 130 rpm for 3 h.
Figure 2. Activity a of whole cells of Rhodococcus ruber DSM 44541 for the oxidation of rac-2c
The enantiomeric excess was determined on a Chrom-
pack Chirasil Dex column (25 m  0.32 mm  0.25 mm,
H2): 2a: 808C isotherm, 3.4 min (S), 3.7min ( R); 2c:
1008C/5 min 128C/min 1608C/0 min, 6.4 min (R),
!
in phosphate buffer (pH 8.0) at different concentrations of acetone ( ) and for the oxidation of
*
rac-2b at different substrate concentration ( ) at 20% (v/v) acetone.
7.6 min (S);
a
G-PN column (30 m  0.32 mm,
Surprisingly, no maximum was reached when we tried to
optimize the substrate concentration for rac-2b at a fixed
acetone concentration of 20% (v/v) (Figure 2); the reaction
rate increased continuously with increasing substrate concen-
tration of up to 1.8 molLÀ1, which corresponds to 237gL À1 for
rac-2-octanol 2b. Beyond this value, no reliable data could be
obtained as the mixture turned very viscous.
These results were confirmed on a preparative scale by
kinetic resolution of rac-2-octanol (1.0 g, concentration
111 gLÀ1, 0.84 molLÀ1) by oxidation using 0.6 g of lyophilized
Rhodococcus cells and a final acetone concentration of 20%
(v/v) in buffer at 248C. Within 24 h, a gas chromatography (GC)
conversion of 48.8% was reached and unreacted (R)-2-octanol
was isolated in 48% yield and 96% ee together with 2-octa-
none (37% yield).[20] The enantioselectivity of the reaction
was calculated as E > 100 (for a definition of E see Table 2).
On the one hand, w-phenylalkan-2-ols and allylic alcohols,
as well as cyclopentanol are very good substrates (Table 2),
while secondary alkanols having a more remote hydroxy
moiety in the (w-2) or (w-3) position are oxidized with lower
activity.
NG 9908-08, H2) was used for the determination of 2b as its acetate,
9.8 min (S), 10.2 min (R). The absolute configuration of (S)-1a and (S)-(E)-
3-octen-2-ol was proven by comparison of the optical rotation with the
literature,[23] and that of (R)-2b and (R)-2c by co-injection with commer-
cially available nonracemic samples on chiral GC.
Received: October 1, 2001 [Z17999]
[1] a) R. A. Sheldon, Pure Appl. Chem. 2000, 72, 1233 1246; b) R. A.
Sheldon, CHEMTECH 1994, 38 47; c) K. Sato, M. Aoki, J. Takagi, K.
Zimmermann, R. Noyori, Bull. Chem. Soc. Jpn. 1999, 72, 2287 2306;
d) K. Sato, M. Hyodo, M. Aoki, X.-Q. Zheng, R. Noyori, Tetrahedron
2001, 57, 2469 2476.
[2] C. F. de Graauw, J. A. Peters, H. van Bekkum, J. Huskens, Synthesis
1994, 1007 1017.
[3] a) E. J. Campbell, H. Zhou, S. T. Nguyen, Org. Lett. 2001, 3, 2391
2393; b) A. N. Ajjou, Tetrahedron Lett. 2001, 13 15; c) T. Ooi, Y.
Itagaki, T. Miura, K. Maruoka, Tetrahedron Lett. 1999, 40, 2137 2138;
d) J. C. van der Waal, P. J. Kunkeler, K. Tan, H. van Bekkum, J. Catal.
1998, 173, 74 83; e) F. Touchard, M. Bernard, F. Fache, F. Delbecq, V.
Guiral, P. Sautet, M. Lemaire, J. Organomet. Chem. 1998, 567, 133
136; f) K. G. Akamanchi, B. A. Chaudhari, Tetrahedron Lett. 1997, 38,
6925 6928; g) P. Leyrit, C. McGill, F. Quignard, A. Choplin, J. Mol.
Catal. A 1996, 112, 395 400.
[4] a) R. Devaux-Basseguy, A. Bergel, M. Comtat, Enzyme Microb.
Technol. 1997, 20, 248 258; b) W. Hummel, Adv. Biochem. Eng. 1997,
58, 145 184.
[5] K. Faber, Biotransformations in Organic Chemistry, 4th ed., Springer,
Heidelberg, 2000, pp. 178 183.
To our knowledge, this is the first report of a preparative-
scale biocatalytic oxidation of secondary alcohols using
acetone as a cosubstrate, which is presumably because the
oxidation of alcohols is thermodynamically unfavored and
cosubstrate[21] or product[22] inhibition are common phenom-
ena for this type of reaction. In addition, acetone is known to
deactivate enzymes.
[6] D. Vasic-Racki, M. Jonas, C. Wandrey, W. Hummel, M.-R. Kula, Appl.
Microbiol. Biotechnol. 1989, 31, 215 222.
[7] a) H. I. Perez, H. Luna, N. Manjarrez, A. SolÌs, Tetrahedron:
¬
Asymmetry 2001, 12, 1709 1712; b) G. Fantin, M. Fogagnolo, A.
Medici, P. Pedrini, S. Fontana, Tetrahedron:Asymmetry 2000, 11,
The biocatalytic redox-system described here is a simple
and flexible method, at high substrate concentration, for the
Meerwein Ponndorf Verley reduction of ketones and its
reverse counterpart, the Oppenauer oxidation of secondary
alcohols by a simple switch of cosubstrate. The method is
highly regio- and enantioselective, and it represents essen-
tially ™clean∫ chemistry.
¬
2367 2373; c) H. I. Pe rez, H. Luna, N. Manjarrez, A. SolÌs, M. A.
Nunƒez, Biotechnol. Lett. 1999, 21, 855 858; d) M. Fogagnolo, P. P.
Giovannini, A. Guerrini, A. Medici, P. Pedrini, N. Colombi, Tetrahe-
dron:Asymmetry 1998, 9, 2317 2327; e) H. Ohta, H. Fujiwara, G.
Tsuchihashi, Agric. Biol. Chem. 1984, 48, 317 322.
[8] In general, the substrate concentration was below 0.15 molLÀ1 and
cosubstrate concentration was below 3% (v/v); see: a) K. Nakamura,
Y. Inoue, T. Matsuda, I. Misawa, J. Chem. Soc. Perkin Trans. 1 1999,
2397 2402; b) M. Wolberg, W. Hummel, C. Wandrey, M. M¸ller,
Angew. Chem. 2000, 112, 4476 4478; Angew. Chem. Int. Ed. 2000, 39,
4306 4308; c) A. Goswami, R. L. Bezbaruah, J. Goswami, N.
Borthakur, D. Dey, A. K. Hazarika, Tetrahedron:Asymmetry 2000,
11, 3701 3709; d) D. R. Griffin, F. Yang, G. Carta, J. L. Gainer,
Biotechnol. Prog. 1998, 14, 588 593; for higher (co)substrate
Experimental Section
Optimum of cosubstrate concentration: Cells of Rhodococcus ruber DSM
44541 (40 mg) were rehydrated in phosphate buffer (400 mL, 50 mm, pH 7 .5
1016
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concentrations see: e) C. Heiss, R. S. Phillips, J. Chem. Soc. Perkin
Trans. 2000, 2821 2825 [15% (v/v) cosubstrate 2-propanol,
Compartmentalization of a Gadolinium
1
Complex in the Apoferritin Cavity: A Route To
Obtain High Relaxivity Contrast Agents for
Magnetic Resonance Imaging**
0.12 molLÀ1 substrate]; f) A. Matsuyama, H. Yamamoto, N. Kawada,
Y. Kobayashi, J. Mol. Catal.
B
2001, 11, 513 521 (0.3 molLÀ1
‡
substrate, no details on the NAD /NADH recycling system);
g) H. D. Simpson, D. A. Cowan, Protein Pept. Lett. 1997, 4, 25 31
[11% (v/v) cosubstrate 2-propanol, 0.44 molLÀ1 substrate].
Silvio Aime,* Luca Frullano, and
Simonetta Geninatti Crich
‡
[9] Enzyme purification showed that at least four NAD /NADH-depend-
ent active fractions are present in the cells, of which only one had a
stability towards acetone and 2-propanol comparable to that of the
The search for high relaxivities continues to be a central
item in the development of paramagnetic contrast agents
(CA) for magnetic resonance imaging (MRI).[1 3] This class of
diagnostic agents is mainly represented by highly stable
GdIII chelates since the GdIII ion, with its seven unpaired
electrons, provides both a very high magnetic moment and a
long electronic relaxation time. The observed relaxivity r1 for
the free GdIII chelates may be considered as the sum of three
contributions arising from the water molecule(s) directly
coordinated to the paramagnetic ion (inner-sphere term),[1, 4]
from water molecules hydrogen bonded at the surface of the
complex (second coordination sphere term),[5] and from water
molecules diffusing in the proximity of the complex (outer-
sphere term).[1, 4] By using the theory of paramagnetic
relaxation, the design of improved systems has been pursued
through the optimization of the determinants of each
contribution to the overall relaxivity. However, it was found
early on that the observed relaxivity could only be explained if
further contributions are operative when interaction with the
surface of the protein occurs. For example, the binding of
GdDOTP (H8DOTP ˆ 1,4,7,10-tetrakis(methylenephosphon-
ic acid)-1,4,7,10-tetraazacyclododecane), which does not con-
tain any inner-sphere water molecules, to human serum
albumin (HSA) causes a relaxation enhancement of approx-
imately five times.[1, 6] It was straightforward to assign such
additional contributions to water molecules and exchangeable
protons on the surface of the protein in the proximity of the
binding site(s) of the paramagnetic complex. Clearly the
microenvironment of the paramagnetic chelate is highly
relevant to the determination of the relaxation-enhancing
capability of a given GdIII chelate. We deemed it of interest to
explore new routes for the attainment of high relaxivities by
exploiting such ™protein surface∫ effects. The aim was to
design systems containing a large proteic surface for inter-
action with the paramagnetic complex that would affect a
large number of hydration water molecules and mobile
protons, which, in turn, would exchange with the bulk solvent
and act as amplifiers of the presence of the CA. One way to
deal with a large surface is to design a spherical compartment
in which the CA is trapped while the water molecules are free
‡
whole cells. The other NAD /NADH depending dehydrogenases
were either unstable or could not use 2-propanol/acetone as cosub-
strate. However, all dehydrogenases displayed the same stereoprefer-
ence.
[10] Lyophilized cells can be stored at ‡48C for several months without
noticeable loss of activity.
[11] For growth conditions see: W. Kroutil, M. Mischitz, K. Faber, J. Chem.
Soc. Perkin Trans. 1 1997, 3629 3636.
[12] All optimization experiments were performed at 308C.
[13] Whole lyophilized cells lost 50% of their activity in 30% 2-propanol
after 35 h, in 50% 2-propanol already after 3 h.
[14] Since the solubility of most substrates in aqueous buffer is limited,
apparent substrate concentrations are used throughout this study
which correspond to molsubstrate/total volume of the system.
[15] The reason for the decrease of activity at high substrate concentration
may be inhibition, deactivation, or limitations of the substrate
transport (diffusion, transport into the cell). However, the reason
was not clarified for whole cells because of the complexity of the
system, but will be elucidated for the isolated enzyme.
[16] This corresponds to
a
productivity of 26 mmol product hÀ1 LÀ1
employing 0.3 g whole lyophilized cells, or 2.3 g product gÀ1 cells.
Literature values for g product gÀ1 whole cells for reductions are at
0.006 ggÀ1[7d] or 0.02 ggÀ1 [8c]
.
[17] The moderate isolated yield results from the limited half-life of whole
cells at 50% (v/v) 2-propanol and may be increased by either using
more cells or by starting at a lower concentration of 2-propanol [e.g.
30% (v/v)] and by continuously increasing it.
[18] The use of different aldehydes/ketones such as, octanal, propanal,
isobutyraldehyde, cyclohexanone, cyclobutanone, cyclopentanone,
octan-2-one, chloroacetone, and others for cofactor recycling has
been reported, although no detailed data are available: see ref. [8a]; J.
Grunwald, B. Wirz, M. P. Scollar, A. M. Klibanov, J. Am. Chem. Soc.
1986, 108, 6732 6734; K. Nakamura, Y. Inoue, A. Ohno, Tetrahedron
Lett. 1994, 35, 4375 4376; A. M. Snijder-Lambers, E. N. Vulfson, H. J.
Doddema, Recl. Trav. Chim. Pays-Bas 1991, 100, 226 230; C.
¡
Gorrebeeck, M. Spanoghe, D. Lanens, G. L. Lemiere, R. A. Dom-
misse, J. A. Lepoivre, F. C. Alderweireldt, Recl. Trav. Chim. Pays-Bas
1991, 110, 231 235; H. Weenen, R. L. G. M. Boog, W. Apeldoorn,
Proceedings of the international conference of Bioflavour 95 (Dijon,
France), 1995, pp. 375 380.
[19] Whole lyophilized cells lost 50% of their activity in 20% acetone after
40 h.
[20] This corresponds to
a
productivity of 18 mmol product hÀ1 LÀ1
employing 0.6 g whole lyophilized cells or 0.8 g product gÀ1 cells. A
literature value for g product gÀ1 cells of a comparable reaction is
0.01 g product gÀ1 cells.[8a]
[21] J.-O. Winberg, J. S. McKinley-McKee, Biochem. J. 1988, 251, 223 227.
[22] W. Hummel, Trends Biotechnol. 1999, 17, 487 492, and references
cited therein; ™Dehydrogenases in the synthesis of chiral compounds∫:
M.-R. Kula, U. Kragl in Stereoselective Biocatalysis (Ed.: R. N. Patel),
Marcel Dekker, New York, 2000, pp. 839 866.
[23] K. Nakamura, T. Matsuda, J. Org. Chem. 1998, 63, 8957 8964; T.
Fujisawa, H. Kohama, K. Tajima, T. Sato, Tetrahedron Lett. 1984, 25,
5155 5156.
[*] Prof. S. Aime, Dr. L. Frullano, Dr. S. Geninatti Crich
Dipartimento di Chimica I.F.M.
¡
Universita di Torino
via P. Giuria 7, 10125 Torino (Italy)
Fax : (‡39)11-670-7855
E-mail: aime@ch.unito.it
[**] Financial support from Bracco Imaging SpA, MUIR (PRIN), and
CNR (Target Project on Biotechnology and L95/95) is gratefully
acknowledged. This work has been carried out under the scheme of
the EU-COST D18 framework.
Angew. Chem. Int. Ed. 2002, 41, No. 6
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
1433-7851/02/4106-1017 $ 17.50+.50/0
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