Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of acetophenone derivatives: Effect of charge density

Article abstract of DOI:10.1039/c2ob06870b

In an effort to study the effect of substituent groups of the substrate on the alcohol dehydrogenase (ADH) reductions of aryl-alkyl ketones, several derivatives of acetophenone have been evaluated against ADHs from Lactobacillus brevis (LB) and Thermoanaerobacter sp. (T). Interestingly, ketones with non-demanding (neutral) para-substituents were reduced to secondary alcohols by these enzymes in enantiomerically pure form whereas those with demanding (ionizable) substituents could not be reduced. The effect of substrate size, their solubility in the reaction medium, electron donating and withdrawing properties of the ligand and also the electronic charge density distribution on the substrate molecules have been studied and discussed in detail. From the results, it is observed that the electronic charge distribution in the substrate molecules is influencing the orientation of the substrate in the active site of the enzyme and hence the ability to reduce the substrate.

Full text of DOI:10.1039/c2ob06870b

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PAPER
Investigation of asymmetric alcohol dehydrogenase (ADH) reduction of
acetophenone derivatives: effect of charge density†
Hemantkumar G. Naik,*a Bahar Yeniad,a Cor E. Koninga and Andreas Heisea,b
Received 7th November 2011, Accepted 30th April 2012
DOI: 10.1039/c2ob06870b
In an effort to study the effect of substituent groups of the substrate on the alcohol dehydrogenase (ADH)
reductions of aryl–alkyl ketones, several derivatives of acetophenone have been evaluated against ADHs
from Lactobacillus brevis (LB) and Thermoanaerobacter sp. (T). Interestingly, ketones with non-
demanding (neutral) para-substituents were reduced to secondary alcohols by these enzymes in
enantiomerically pure form whereas those with demanding (ionizable) substituents could not be reduced.
The effect of substrate size, their solubility in the reaction medium, electron donating and withdrawing
properties of the ligand and also the electronic charge density distribution on the substrate molecules have
been studied and discussed in detail. From the results, it is observed that the electronic charge distribution
in the substrate molecules is influencing the orientation of the substrate in the active site of the enzyme
and hence the ability to reduce the substrate.
reductions for the synthesis of chiral monomers in order to
Introduction
access polymers with defined chiral compositions.25–27 Unfortu-
Enantiopure alcohols are valuable intermediates and have found
important applications in the synthesis of pharmaceuticals and
other fine chemicals.1–10 The asymmetric reduction of prochiral
ketones is one of the straightforward approaches used to access
enantiomerically pure alcohols. In this regard, a variety of chiral
metal complexes have been developed as catalysts for asym-
metric ketone reductions.11–16 These methods predominantly use
toxic metals and expensive metal hydrides, which require special
reaction conditions. In the search for alternatives, several biocata-
lytic methods for stereoselective reduction of ketones have been
developed,17–19 which hold great potential with respect to
environmental compatibility and catalytic efficiency.
Dehydrogenases are non-heme redox enzymes which catalyze
hydrogen-transfer reactions in the presence of a coenzyme as
hydrogen donor or acceptor. A number of different dehydrogen-
ases (isolated from different sources) have been utilized for the
asymmetric reduction of carbonyl functionalities. Stereoselective
reduction of ketones using alcohol dehydrogenases (ADH) has
become an important method for industrial preparation of opti-
cally pure alcohols.20–24 We have started to investigate ADH
nately, most of the biocatalytically applicable ADHs show a
rather narrow substrate pattern: preferentially ketones which bear
small, non-ionizable, non polar, non-demanding substituent
functional groups have been reduced successfully.28 The
influence of different functional substituent groups on the enzy-
matic reduction of ketones has not yet been studied systemati-
cally. There is thus an increasing interest in understanding the
effect of substituent groups on the biocatalytic reduction of
ketones, which will eventually help in understanding the
reduction mechanism and the development of suitable enzymatic
reduction strategies for substituted ketones.
We therefore synthesized a series of acetophenone derivatives
with different demanding substituent functional groups. These
ketones were reduced by ADH enzymes from Lactobacillus
brevis (R selective) and Thermoanaerobacter sp. (S selective) and
the effect of different parameters on the reduction investigated in
detail. To the best of our knowledge, the available literature on
the ADH reductions does not satisfactorily disclose the effect of
different functional groups on the biocatalytic reduction. More-
over, not much has been explored on the substrate specificity of
ADHs from Lactobacillus brevis (ADH-LB) and Thermoanaero-
bacter sp (ADH-T). Correlating substrate profiles of the ADHs
with their sequential and structural information would provide
valuable insight into the understanding of how these enzymes
control activity and enantioselectivity. With this perspective in
mind, we have studied substrate specificity and enantioselectivity
for the carbonyl reductases from Lactobacillus brevis and
Thermoanaerobacter sp with various ketones of diverse structures.
aTechnische Universiteit Eindhoven, Laboratory of Polymer Chemistry,
Den Dolech 2, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
E-mail: h.g.naik@tue.nl, hemantnaik@rediffmail.com;
Fax: +31 40 246 3966; Tel: +31 40 247 4942
bDublin City University, School of Chemical Sciences, Glasnevin,
Dublin 9, Ireland
†Electronic supplementary information (ESI) available: Spectral data of
reaction products. See DOI: 10.1039/c2ob06870b
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us to study the effect of different parameters on the enzymatic
reduction of acetophenone derivatives in detail. The major
factors influencing the reduction according to the literature37 are
pH of the medium, temperature of the reaction, the size of the
substrate and their solubility in the reaction medium. The effect
of pH and reaction temperature on the enzymatic reduction of
ADH-LB and ADH-T has already been studied extensively.38,39
In the literature it is reported that although the activity of these
enzymes increases with increase in temperature the half-life of
the enzyme decreases steadily. Hence, an optimum temperature
of 37 °C was chosen for the reaction. An increase in reaction
temperature to 42 °C for ketones IV to X did not improve the
reaction (results not shown). All reactions were carried out at pH
7.0 due to the stability and reactivity of the ketones and enzymes
at lower and higher pH (note: ADH-LB has two activity maxima
at pH 5.5 and 9.0). The other obvious parameters, which
influence the reaction, are the size of the substrate and their solu-
bility in the reaction medium. Complete insolubility of the sub-
strate in the reaction medium or substrates bigger than the cavity
of enzyme will prevent reduction. Whether these parameters can
explain the results obtained for the investigated substituted
ketones will be discussed in the following.
Results and discussion
The R-alcohol dehydrogenase (RADH) from Lactobacillus
brevis (ADH-LB) is a NADP-dependent member of the extended
superfamily of short-chain dehydrogenases/reductases (SDRs)29–31
(enzyme class EC 1.1.1.2, CAS 9031-72-532). It is a homotetra-
meric enzyme32,33 with 251 amino acid residues and a molecular
mass of 26 627 Da33,34 or 22.5 kDa per subunit depending on
the source. ADH-LB was discovered during a search for bio-
technologically interesting alcohol dehydrogenases.35 The active
site of the enzyme contains a four member catalytic tetrad
(Asn113, Ser142, Tyr155 and Lys159).36 The complimentary
S-alcohol dehydrogenase from Thermoanaerobacter sp.
(ADH-T) is a NADP dependent alcohol dehydrogenase from the
family of thermophillic enzymes with 352 amino acid series.
The binding site for the substrate for ADH-LB is reported
to be formed by the nicotinamide moiety of NAD(P) and a
hydrophobic patch on the enzyme surface, more precisely at
the interface of helix αG and the loop between strand βE and
helix αF.34
Scheme 1 shows the library of the functional ketones
employed in the systematic study of ADH reductions. Initially
the ketones were all reduced in a PBS buffer (pH 7.4)/IPA sol-
ution using ADH-LB and ADH-T in presence of NADPH as
cofactor at 37 °C.25,27 The progress of the reaction was moni-
Effect substrate size
1
tored by H NMR spectroscopy and gas chromatography (GC).
The stereoselectivity of the enzymes was further analyzed by
chiral GC yielding the enantiomeric excess.
It is well known that all enzymatic reactions are performed
through interaction of the active site of the enzyme with the
ligands or substrate molecules. These active sites are normally
buried inside the enzyme and are only accessible through a small
channel called cavity. The size and shape of these cavities play a
crucial role in the binding specificity.40 For the ketone to be
reduced it must first reach the active site through the cavity and
then the carbonyl group of the ketone should bind to the active
site of the ADH through a ternary-complex with the co-substrate
NADPH. Hence, the size of the ketone is a very important factor
in the enzymatic reduction.41 However, proteins are not rigid but
are in dynamic flexible state and ligand binding causes variation
in protein binding cavity volume (PCV).42 Binding of the ligand
to a protein may cause conformational changes that align the
residues involved in binding in their correct orientation and
hence changes in cavity volume.43–45 The size of the substrate
does influence the binding specificity and hence the enantios-
electivity or stereospecificity46 but does not reject the substrate
completely from binding to the active site. From the results of
reduction of ketones of different sizes against ADH-LB and
ADH-T presented in Table 1, it seems that though we cannot
completely rule out the effect of size, the enzymatic reduction
did not show any dependence on size of the ketone substrate at
least in this study.
In the first attempt to reduce ketones from I to IX, only
ketones I, II and III could be reduced successfully by ADH-LB
and ADH-T whereas ketones from IV to X could not be reduced
at all. Changes in temperature, pH and ionic strength of the reac-
tion medium did not improve the results. Although all substrates
are from the same family of acetophenone derivatives with only
difference in p-substituent functional group, the results on enzy-
matic reductions were completely contradictory. This prompted
The ADH-LB has a large hydrophobic cavity, next to the nico-
tinamide ring of the co-factor, leading to the active site. The
volume of the cavity has been determined to be ∼310 Å3 using
Q-SiteFinder47 and pocket-finder.48 The phenyl ring of substrate
interacts with hydrophobic side-chains (Ala93, Leu152, Val195,
Leu198 and Met205) and with the aromatic ring of Tyr189,
while its carbonyl group forms a hydrogen bond (distance 3.3 Å)
with the terminal hydroxyl group of Tyr155, which is the most
conserved residue of the whole SDR superfamily and the
Scheme 1 Various
employed in the reduction by ADH-LB and ADH-T.
para-substituted
acetophenone
derivatives
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Table 1 Enzymatic reduction of acetophenone derivatives of different
molecular size by ADT-LB and ADH-T enzymes
Table 2 Solubility of different ketones in the buffer medium and
different methods used for the ADH-LB and ADH-T reductions
Ref. no.
Ketone
Molecular sizea (Å3)
Conversion (%) by NMR
Solubility in
Method
Solvent used in
Method Ba
Conversion
(%) by NMR
Ketone buffer medium useda
1
2
3
4
5
6
7
8
I
138.14
144.07
147.07
168.15
165.84
187.49
210.23
219.59
219.78
234.78
306.78
309.64
99
0
0
97
99
0
26
0
VIII
X
II
V
I
Soluble
Sparingly
soluble
Sparingly
soluble
Sparingly
soluble
Sparingly
soluble
Sparingly
soluble
A & B
A & B
Ethylacetate
Ethylacetate
0
99b
III
IX
XII
IV
XIII
VI
VII
V
II
A & B
A & B
A & B
A & B
B
Ethylacetate
Ethylacetate
97b
99b
0
III
VI
9
15
0
0
Ethylacetate,
diisopropylether
Ethylacetate
10
11
12
XII
IV
26c
0
0
Insoluble
Ethylacetate,
diisopropylether
Ethylacetate
a The sizes of the ketones were calculated by SPARTAN program after
minimization of energy of the structure.
XIII
Insoluble
B
15c
a Method A: Consists of 20% solution of 2-propanol in 50 mM PBS
buffer (pH 7.0); Method B: Consists of 50/50 mixture of an organic
solvent (ethylacetate or dichloromethane or diisopropylether) and 20%
solution of 2-propanol in 50 mM PBS buffer (pH 7.0). b The conversion
data obtained from method A. c The product was not isolated (see Fig. 4
and 5 in ESI†).
established catalytic base of the oxidative reaction direction.36
The molecular size of all the ketones, in the present study, is less
than ∼310.0 Å3 as calculated by SPARTAN program49 after
applying Hartee–Fock model of density functional theory and
minimization of energy. The active cavity therefore has sufficient
space to accommodate all ketone substrates investigated in this
study. However, ketones I to III with molecular size ranging
between ∼140.0 Å3–170.0 Å3 were successfully reduced by
ADH-LB as well as by ADH-T, whereas ketones VIII and X
although smaller than 150.0 Å3 molecular sizes were not
reduced. Moreover, ketone XII and XIII with molecular sizes
higher than 200 Å3 were reduced to some extent. This shows
that not only the size determines the reactivity but that there
might be an influence of the nature of the substrate on the
reaction.
did not observe any change in results by changing the reaction
condition to a two-phase system with an organic solvent.
This indicates that solubility is also not the criterion which is
influencing the reaction as the use of a suitable two-phase
solvent system will aid the reaction in case the substrate is inso-
luble in the reaction medium. For XII and XIII the reaction
stopped at conversions of 26 and 15%, respectively and further
addition of NADPH or enzyme did not increase conversion.
We speculate that due to the reversibility of the reaction an
equilibrium position under these conditions has been reached.
Effect substrate solubility
Effect of electron withdrawing or electron donating property of
substituent
The other important criterion that has significant influence on
the reaction is the solubility of the substrate ketones. Practically
the enzymatic reactions are generally carried out in a buffer sol-
ution to maintain the optimal conditions considering enzyme
stability. However, the majority of ketones of interest are highly
hydrophobic, and thus possesses low solubility in aqueous
media leading to low substrate concentrations ranging from <5 to
10 mM. One of the traditional methods used to overcome this
drawback is the usage of two phase system in which a co-solvent
is be added to raise the substrate solubility in the reaction
mixture.50–56 This co-solvent can either be water-miscible57–60
resulting in a one-phase system or, it can be water-immisci-
ble50,51,61 leading to an aqueous–organic two-phase system.
Table 2 summarizes the effect of solubility of different ketones
used in the present study on the enzymatic reduction. From the
results it is observed that there is no specific trend on the
reduction with solubility. Ketones I, II and III, though sparingly
soluble in the buffer medium, are quantitatively reduced whereas
V though soluble (and VI sparingly soluble) in the medium
remain unreduced. However, XIII, which is a solid and is insolu-
ble in the medium is reduced to an appreciable amount using the
two-phase system with ethylacetate. In case of other ketones, we
In a recent study on effects of para-substituted acetophenones on
the catalytic activity of 3α-hydroxysteroid dehydrogenase
(3α-HSD) from rat liver (Rattus norvegicus) Uwai et al. showed
that the electron donating/withdrawing property of para-substitu-
ent group influence the rate of reduction.62 The introduction of
an electron withdrawing group increases the rate of reduction
whereas an electron donating reduces. This is further supported
by a study on the reduction of substituted acetophenones against
a carbonyl reductase from Candida magnolia by Zhu et al.63,64
This is because the electron donating substituent group at
the para-position suppresses the delocalization of the partially
polarized aryl-conjugated carbonyl (CvO) > C–O bond either
by resonance or by induction whereas an electron withdrawing
substituent promotes the delocalization and hence the reduction.
However, in the present study we did not observe any relation
between the property of the p-substituent and the reaction. Of the
ketones II, III, IV, VII, IX and XIII with electron withdrawing
functional groups at the para-position to the carbonyl group in
conjugation with aromatic ring only ketones II, III, XIII were
successfully reduced. Again ketones XI and XII although
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Table 3 Enzymatic reduction of acetophenone derivatives of different
p-substituted functional group by ADT-LB and ADH-T enzymes
Ketone
Property of p-substituent
Conversion (%) by NMR
I
II
None
99
97
99
0
Electron withdrawing
Electron withdrawing
Electron withdrawing
Electron withdrawing
Electron withdrawing
Electron withdrawing
Mildly electron donating
Mildly electron donating
Mildly electron donating
Strongly electron donating
Strongly electron donating
Strongly electron donating
III
IV
VII
IX
XIII
V
VI
XII
VIII
X
0
0
15a
0
0
26a
0
0
89
XI
a The product is not isolated (see Fig. 4 and 5 in ESI†).
Fig. 1 Calculated electrostatic density potential (a) (in kJ mol−1 at the
default isovalue 0.002 electron per Bohr3) and local ionization density
(b) (in kJ mol−1 at the default isovalue 20.0 electron per Bohr3) surfaces
showing the electron charge distribution on the molecule for ketone
III (A), ketone XI (B).
contain strongly electron donating functional groups were suc-
cessfully reduced whereas reduction of ketones V, VI, VIII and
X, also containing electron donating groups, were unsuccessful
(Table 3). These observations are also supported by earlier
literature.65–67
Electronic charge distribution
If we carefully analyze the results and compare them with the
nature of the p-substituent groups of the substrate ketones, the
ketones with a neutral functional group at the para-position were
reduced successfully while reduction of ketones with ionically
demanding functional groups were unsuccessful. These observa-
tions suggest that there is indeed a strong electronic effect of the
nature of the para-substituent on the catalytic activity of alcohol
dehydrogenases from Lactobacillus brevis and Thermoanaero-
bacter sp but not through the electron withdrawing or donating
properties.
Fig. 2 Calculated electrostatic density potential (a) (in kJ mol−1 at the
default isovalue 0.002 electron per Bohr3) and local ionization density
(b) (in kJ mol−1 at the default isovalue 20.0 electron per Bohr3) surfaces
showing the electron charge distribution on the molecule for ketone
IV (A), ketone VI (B).
Tidor et al.68,69 observed that the electrostatic interactions
between ligand and biomolecules play an important role in deter-
mining binding affinities and specificities. The electrostatic inter-
action between the receptor protein and a ligand was described
as a screened Coulombic interaction between two charges sepa-
rated by an effective distance.70 Therefore, the electrostatic inter-
action between the ligand and protein hugely depends on the
charge density which is simply electron charge distribution on
the ligand. Careful observation of the structure of the aceto-
phenone derivatives (I to XIII) and their results suggests that a
higher electronic charge density far away from the carbonyl
group of acetophenone may be playing a critical role in the
reduction of the substrates. Therefore the direct information
about the electronic charge distribution of the ligand molecule
should provide complete insight into its interaction with the
enzyme. The electrostatic potential map, an overlaying of a
quantity called the electrostatic potential (energy of interaction
of a point positive charge with the nuclei and electrons of a
molecule, depends on the location of the point positive charge)
onto the electron density, is valuable for describing overall mole-
cular charge distribution.49 Another important parameter
which provides valuable information on the molecular charge
distribution is the local ionization potential map which is an
overlay of the energy of electron removal (“ionization”) onto the
electron density.
The electrostatic potential and local ionization potential sur-
faces for the acetophenone derivatives in the present study have
been determined using SPARTAN program49 and are presented
in Fig. 1 and 2. The figures show the electrostatic density poten-
tial map which is a direct representation of distribution of elec-
tron charge density throughout the molecule and local ionization
density surface which is again a measure of charge distribution
at any location around the molecule. The color red depicts
regions of most negative electrostatic potential, while the color
blue depicts the regions of most positive electrostatic potential.
Intermediate colors represent intermediate values: red < orange <
yellow < green < blue.
4964 | Org. Biomol. Chem., 2012, 10, 4961–4967
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The Fig. 1A and 1B show the electrostatic density potential
and local ionization density maps for ketones III and XI which
were reduced easily by the ADH enzymes under study. From the
figures it is observed that there is a high electron density (low
ionization potential energy = red color) around the carbonyl
group of the ketones. In contrast, Fig. 2A and 2B show the elec-
trostatic density potential and local ionization density maps of
ketones IV and VI, which remain unreduced by the enzymes in
this study. In this case it is observed that the electron density is
situated at the para-position, far away from the carbonyl group
of the ketone. Similar observations were noticed for rest of the
ketones (see Fig. 1–3 in ESI†).
In case of ketone IV the local ionization density map (Fig. 2A
(b)) gives valuable information on the electron charge distri-
bution though the density potential map (Fig. 2A(a)) does not
show a clear information. The same is true for ketone XI where
the density potential map (Fig. 1B(a)) provides complete infor-
mation about the charge distribution over the molecule though
the local ionization density map (Fig. 1B(b)) does not show a
clear difference. Therefore electrostatic density potential and
local ionization density maps together provide the information
on the electronic charge distribution on a molecule.
These observations strongly suggest that the electron charge
density might be playing an important role on the enzymatic
reduction of ketones. With this information we hypothesize that
the molecule may be entering the cavity of the enzyme with the
part of the molecule having higher electron charge density facing
the active site of the enzyme. Therefore in case of ketones I, II,
III and XI, it is the carbonyl group with higher electron density
which is facing the active site while the molecule is entering the
cavity. This allows the carbonyl group of ketone to bind to the
active site and get reduced. However, in case of ketones IV, V
and VI it is the para-substituent group with higher electron
density which is facing the active site while the molecule is
entering the cavity of the enzyme. This leaves the carbonyl
group of these ketones far away from binding to the active site
and hence it cannot be reduced.
influences the binding. In the present study, we observed that
there is a charge complementarity difference between enzyme
and reactive group of some of the substrates induced by their
electronic charge distribution. Thus, the electronic charge distri-
bution in the molecules might be influencing the orientation of
the substrate in the active site of the enzyme and hence the
ability to reduce the substrate. Further investigations on
influence of electron charge density and also the actual mecha-
nism by which the nature of the substituent influences the
enzyme activity are being studied and will be reported in our
upcoming manuscript. These studies will help in the develop-
ment of a suitable enzymatic reduction strategy for substituted
acetophenones.
Experimental
Materials
Commercial reagents were used as received. Solvents for reac-
tions (distilled THF, Et2O; commercial benzene, toluene and
CH2Cl2) were filtered over columns of dried alumina under a
positive pressure of argon. Solvents for extractions and flash
column chromatography were of technical grade and were dis-
tilled prior to use. The 4-vinylacetophenone (II) was synthesized
as described previously.25 Acetophenone (I), 4-ethynylaceto-
phenone (III), N-(4-acetylphenyl)acetamide (IX) and 4-hydroxy
acetophenone (VIII) were purchased from Acros. 4-Aminoaceto-
phenone (X), 1-(4-methoxyphenyl)ethanone (XI), 1-(4-tert-
butylphenyl)ethanone (XII), 1-(biphenyl-4-yl)ethanone (XIII),
2-chloroethyl benzene, sodium azide (NaN3), glycidylmethacry-
late (GMA), pMDETA, aluminum oxide (Al2O3), copper(I)
bromide (Cu(I)Br), cesium carbonate (CsCO3), triethylamine
(TEA), hydrochloric acid (HCl) and NaHCO3 were purchased
from Aldrich whilst ammonium chloride (NH4Cl), methacryloyl
chloride and monochlorodiethyleneglycol were obtained from
Fluka and all used as received. N-(4-Acetylphenyl)methacryl-
amide (IV), 1-(4-(2-(2-hydroethoxy)ethoxy)phenyl)ethanone (V),
2-(2-(4-acetylphnoxy)ethoxy)ethyl methacrylate (VI) and 5-(4-
(4-acetylphenyl)-1H-1,2,3-triazol-1-yl)-4-hydroxy-2-methylpent-
1-en-3-one (VII), were synthesized in our lab. NADPH and
Conclusions
alcohol dehydrogenase from Lactobacillus brevis (4100 U mL−1
(ADH-LB) and Thermoanaerobacter sp. (331
mL−1
)
)
U
From the analysis of the various factors like solubility, electronic
effects and molecular size that could explain substrate accep-
tance by the alcohol dehydrogenase enzymes from Lactobacillus
brevis and Thermoanaerobacter sp. it is more evident that non-
polar para substituents are favoured over polar or ionisable para
substituents. With these results we conclude that there is an
effect of the nature of the substituent group on the reduction of
aromatic ketones using alcohol dehydrogenases from Lacto-
bacillus brevis and Thermoanaerobacter sp, which were enantio-
complimentary ketoreductases giving (R-) and (S-) enantiomers
of the same ketone, making them useful in the synthesis of
enantiopure alcohol intermediates of pharmaceutical and agri-
cultural interest. The binding of substrates to the specificity
pocket of an enzyme involves a combination of chemical forces
including hydrogen bonds and electrostatic, hydrophobic, and
steric interactions. Shape and charge complementarity between
enzyme and substrate have been proposed as keys to enzyme
function. The difference in any of these complementarities
(ADH-T) were purchased from Julich Chiral Solutions GmbH,
a Codexis company, Germany. All the solvents are obtained
from Biosolve.
Computational details
The SPARTAN 06 program49 running on Workstation Intel®
Core™2 Quad CPU Q9550 2.66 GHz processor with 4GB RAM
under Windows XP operating system was used to carry out HF
and DFT-B3LYP calculations. The geometries were fully opti-
mized at the DFT B3LYP level of theory with a 6-311G*
basis set and using an ab-initio HF method with 6-311G* basis
set. Electrostatic density potential and local ionization density
surfaces were calculated at both HF/6-31G* and DFT/B3LYP/
6-31G* levels. The electrostatic density potential surfaces are
represented in kJ mol−1 at default isovalue 0.002 electron per
Bohr.
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K. Ramig, S. Swaminathan, V. W. Rosso, S. K. Pack, B. T. Lotz,
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Enantioselective reductions of ketones using alcohol
dehydrogenase
General procedure A: (in a homogeneous medium)
Using alcohol dehydrogenase from LB (ADH-LB). 1 g of the
substrate (ketone) was dissolved/suspended in a reaction mixture
of 2-propanol (40 mL) and PBS buffer solution (pH 7.4,
160 mL) containing 20 mM NADPH and 0.5 mM MgCl2 and
maintained at 37 °C with uniform mixing. The enzyme ADH-02
(50 μL, 4100 U mL−1) was then added to the reaction mixture
and the mixture was allowed to stir overnight. The progress
of the reaction was monitored by TLC and Chiral GC (Varian
430-GC) measurements and the mixture was treated with excess
of water and extracted in methyl t-butyl ether. The organic layer
was washed with brine, dried over magnesium sulfate and con-
centrated under reduced pressure.
Using alcohol dehydrogenase from T (ADH-T). Substrates were
reduced by ADH-05 using the procedure similar to the one used
for ADH-02 enzyme but without MgCl2. In brief, 1 g of the sub-
strate (ketone) was dissolved/suspended in a reaction mixture of
2-propanol (40 mL) and PBS buffer solution (pH 7.4, 160 mL)
containing 20 mM NADPH and maintained at 37 °C with
uniform mixing, followed by the addition of ADH-05 enzyme
(285 μL, 331 U mL−1). The reaction mixture was stirred over-
night and the progress of the reaction was monitored by TLC
and Chiral GC (Varian 430-GC) measurements. The mixture was
treated with excess water and extracted in methyl t-butyl ether.
The organic layer was washed with brine, dried over magnesium
sulfate and concentrated under reduced pressure.
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General procedure B: (in a heterogeneous medium). In this
method, the substrates were reduced by the respective enzymes
in a heterogeneous organic–aqueous biphasic medium to
improve the solubility of the poorly water-soluble or water inso-
luble ketones. The reaction conditions were similar to those in
procedure A for both the enzymes, except that the medium con-
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organic solvent (dichloromethane, diisopropylether, ethylacetate
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Acknowledgements
The authors thank The Netherlands Organization for Scientific
Research (NWO-Echo, Project Number: 10009001) for the
financial support of this research. This work forms part of the
Research Program of the Dutch Polymer Institute (Project
number 684). AH is a SFI Stokes Senior Lecturer (07/SK/
B1241). The authors also thank A.R.A. Palmans of Macro-
Organic Chemistry, Eindhoven University of Technology for her
kind technical support in this work.
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