Haloalkane dehalogenase catalysed desymmetrisation and tandem kinetic resolution for the preparation of chiral haloalcohols

Article abstract of DOI:10.1016/j.tet.2012.06.059

Six different bacterial haloalkane dehalogenases were recombinantly produced in Escherichia coli, purified, and used to catalyse the conversion of prochiral short-chain dihaloalkanes and a meso dihaloalkane, yielding enantioenriched haloalcohols. A two-reaction one-enzyme process was established in which the desymmetrisation of a dihaloalkane is followed by kinetic resolution of the chiral haloalcohol that is produced in the first step. In case of 1,3-dibromo-2-methylpropane and 1,3-dibromo-2-phenylpropane, an increase of the enantiomeric excess of the respective haloalcohol was observed in time, leading to ee values of >97%, with analytical yields of 24 and 52%, respectively. The results show that haloalkane dehalogenases can be used for the production of highly enantioenriched haloalcohols and that in some cases product enantiopurity can be improved by allowing a two-step one-enzyme tandem reaction.

Full text of DOI:10.1016/j.tet.2012.06.059

Tetrahedron 68 (2012) 7645e7650
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Haloalkane dehalogenase catalysed desymmetrisation and tandem kinetic
resolution for the preparation of chiral haloalcohols
a
a
a
,
,
*
Alja Westerbeek , Jan G.E. van Leeuwen , Wiktor Szymanski b, Ben L. Feringa b, Dick B. Janssen a
ꢀ
a Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
b Center for Systems Chemistry, Stratingh Institute for Chemistry and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG,
Groningen, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Six different bacterial haloalkane dehalogenases were recombinantly produced in Escherichia coli, pu-
rified, and used to catalyse the conversion of prochiral short-chain dihaloalkanes and a meso diha-
loalkane, yielding enantioenriched haloalcohols. A two-reaction one-enzyme process was established in
which the desymmetrisation of a dihaloalkane is followed by kinetic resolution of the chiral haloalcohol
that is produced in the first step. In case of 1,3-dibromo-2-methylpropane and 1,3-dibromo-2-
phenylpropane, an increase of the enantiomeric excess of the respective haloalcohol was observed in
time, leading to ee values of >97%, with analytical yields of 24 and 52%, respectively. The results show
that haloalkane dehalogenases can be used for the production of highly enantioenriched haloalcohols
and that in some cases product enantiopurity can be improved by allowing a two-step one-enzyme
tandem reaction.
Received 1 March 2012
Received in revised form 6 June 2012
Accepted 14 June 2012
Available online 23 June 2012
Keywords:
Biocatalysis
Kinetic resolution
Desymmetrisation
Enantioselective
Haloalkane dehalogenase
Haloalcohols
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
process (Scheme 1). In such a system, the change of the enantio-
meric excess of intermediate 2 over time depends on the ratios of
Enantiopure 1,2-haloalcohols (halohydrins) and 1,3-haloalcohols
are chiral intermediates for the preparation of several important
classes of compounds. Halohydrins can be converted into lactones1
and epoxides,2,3 which are versatile building blocks in organic syn-
thesis.4e7 For example, indene bromohydrin is a precursor for the
anti-HIV protease inhibitor Indinavir,4 and 1,3-haloalcohols are pre-
the kinetic constants.16 If one enantiomer of the intermediate is
formed faster in the first step (k1>k2), and consumed slower in the
second reaction (k4>k3), an increase of enantiomeric excess over
time is expected,17 unlike in a normal kinetic resolution, where
product ee decreases over time, especially when the E-value is not
very high. In the opposite case (k3>k4), the enantiomeric excess of
the intermediate will decrease, and it is possible that towards the
end of the conversion the enantiomer of 2 that is not the preferred
product in the first step predominates.18
cursors of b
-amino acids8 and 1,3-aminoalcohols,9 which are used in
the synthesis of bioactive compounds and as chiral ligands,10 re-
spectively. Current biocatalytic routes towards enantiopure 1,2- and
1,3-haloalcohols include haloperoxidase-mediated conversion of
unsaturated compounds to vicinal halohydrins,11 kinetic resolution
of halohydrins using a Pseudomonas strain,12 and lipase-catalysed
resolution of racemic alcohols and esters.13e15 We are interested in
exploring the possibility of employing isolated enzymes for the
preparation of these compounds, using enantioenrichment methods
that donot solely relyonkinetic resolution, e.g., by takingprochiralor
meso compounds as starting materials.
Scheme 1. Reaction scheme for the tandem conversion of a prochiral or meso diha-
loalkane 1 to chiral haloalcohol 2 and prochiral or meso diol 3.
Since haloalcohols can in principle be produced by enzymatic
hydrolysis of dihaloalkanes, their preparation could be facilitated
by the use of a tandem desymmetrisation/kinetic resolution
Applications of such single-enzyme tandem reactions were
predominantly investigated using hydrolases.16e18 Wang and co-
workers16 have shown that both porcine pancreatic lipase (PPL)
and pig liver esterase (PLE) can perform a conversion of prochiral
diesters with subsequent enhancement of the enantiomeric excess
* Corresponding author. Nijenborgh 4, 9747 AG, Groningen, The Netherlands. Fax:
þ31 50 363 4165; e-mail address: d.b.janssen@rug.nl (D.B. Janssen).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.06.059

7646
A. Westerbeek et al. / Tetrahedron 68 (2012) 7645e7650
Table 1
of the monoester product by conversion of one enantiomer to the
diol. In another study, lipase from Pseudomonas cepacia was used
for the preparation of a cyclic monoester in a tandem reaction from
the diester.17 Other tandem desymmetrisation reactions19 were
based on the use of two dehydrogenases or two transaminases with
complementary enantioselectivities. By enzymatically converting
one enantiomer of an alcohol (or amine) to a prochiral ketone,
followed by back-conversion of this achiral intermediate to the
alcohol (or amine) using an enzyme with opposite product enan-
tioselectivity, full conversion to obtain enantiopure alcohol20,21 or
amine22 could be achieved.
The potential of haloalkane dehalogenases in stereoselective
biocatalytic reactions was recently recognised and was employed in
the preparation of chiral esters,23 bromoalkanes24 and hal-
oamides25,26 via kinetic resolution. In the current study, we ex-
plored the possibility of using haloalkane dehalogenases in
a tandem reaction as described in Scheme 1. We tested the activity
of haloalkane dehalogenases from five different sources for the
activity and stereoselectivity with selected prochiral and meso
compounds 1ae1e (Fig. 1).
Kinetic parameters for conversion of prochiral/meso compounds with purified de-
halogenases, measured in 50 mM TriseSO4, pH 8.2, at room temperature
Enzyme Kinetic parameters 1a
1b
1c
0.34
1.6
4.7
1d
1e
DhaA
DbjA
LinB
KM
kcat
kcat/KM
>2.0
>0.07
0.031
4.8
0.047
0.01
<0.15
0.36
>2.3
N.D.
<0.015
N.D.
KM
kcat
kcat/KM
>2.2
>0.05
0.025
N.D.
>1.0
0.17
0.76
4.5
N.D.
N.D.
<0.002
kobs¼0.005 >0.9
N.D.
0.87
KM
kcat
kcat/KM
0.41
0.97
2.4
N.D.
N.D.
<0.002
0.19
1.4
7.3
0.17
1.4
8.4
N.D.
ꢁ 0.8
N.D.
DhaA31 KM
>1.1
>0.19
0.14
1.7
0.47
0.28
0.021 <0.15
1.6 0.22
77 >1.5
N.D.
ꢁ 0.4
N.D.
kcat
kcat/KM
DhlA
KM
kcat
kcat/KM
N.D.
N.D.
N.D.
N.D.
<0.002 <0.002
>1.1
>0.017
0.015
>1.3
0.06
0.050 <0.002
N.D.
N.D.
DmlA
KM
kcat
N.D.
N.D.
N.D.
N.D.
>1.0
>0.015 >0.4
>1.3
N.D.
N.D.
kcat/KM
<0.002 <0.002
0.015
0.34
<0.002
KM in mM, kcat and kobs in sꢀ1 and kcat/KM¼mMꢀ1 sꢀ1. KM and kcat values had an RSD
of ꢂ20%. For compound 1e, reactions were performed in 20% DMSO at maximum
solubility of 0.5 mM. N.D.: not determined because saturation was not obtained due
to low substrate solubility.
1,2,3-Trichloropropane (TCP, 1b) is a toxic xenobiotic compound
that is used in chemical synthesis and occurs as a side-product in
the synthesis of epichlorohydrin and 1,3-dichloropropene. Im-
proper disposal of TCP led to several cases of spreading of this
pollutant into water.32 TCP occurs mainly as a waste compound, but
its enantioselective conversion would yield a valuable chiral
building block.33 Hydrolysis of 1b using DhaA was improved using
directed eviolution,34 which led to a DhaA variant with six muta-
tions (DhaA31).31 This variant possesses a 28-fold higher catalytic
efficiency towards compound 1b than wild-type DhaA (Table 1), in
agreement with data reported by Damborsky and co-workers.31
The activity of the other enzymes with 1b was very low or un-
detectable (Table 1), although low activity of LinB for compound 1b
was reported earlier under different conditions.35
The DhaA31 mutant also has by far the highest catalytic effi-
ciency and affinity for tribromopropane 1c. The catalytic rates of
DhaA and LinB are in the same range, whereas the other enzymes
are much less active with 1c. Tribromopropane 1c is a better sub-
strate than trichloropropane 1b for all the haloalkane dehaloge-
nases, in agreement with the lower stability of the carbon-bromine
bond.
With 1,3-dibromo-2-methylpropane (1d), high affinities and
catalytic efficiencies were found for DhaA, DbjA, LinB and DhaA31.
Again both DmlA and DhlA possessed much lower activity. To in-
crease the solubility (0.5 mM) of 1,3-dibromo-2-phenylpropane
(1e) in the reaction medium DMSO (20% v/v) was added. Com-
pound 1e was only converted by LinB and DhaA31, in agreement
with the observation that LinB reacts best with bulkier compounds,
like its original substrate 1,3,4,6-tetrachloro-1,4-cyclohexadiene.29
In summary, LinB and DhaA31 are the best enzymes for con-
version of compounds 1ae1e. Under the experimental conditions,
DhaA31 converts all of the tested substrates. LinB shows measur-
able conversion of all compounds with rates of about 1 sꢀ1, except
for 1,2,3-trichloropropane (1b), for which the enzyme has ex-
tremely low activity. DhaA and DbjA also convert 3e4 substrates,
but DmlA and DhlA both have poor activity with all tested
compounds.
Fig. 1. Prochiral and meso substrates (1ae1e) together with the products of their single
and double dehalogenation (2 and 3, respectively).
2. Results and discussion
2.1. Enzyme properties
Haloalkane dehalogenases used in this study were from Xan-
thobacter autotrophicus GJ10 (DhlA),27 Rhodococcus rhodochrous
NCIMB13064 (DhaA),28 Sphingomonas paucimobilis UT26 (LinB),29
Bradyrhizobium japonicum USDA110 (DbjA)30 and Mesorhizobium
loti MAFF303099 (DmlA).30 DhaA31 is a mutant of DhaA with en-
hanced catalytic activity for 1,2,3-trichloropropane.31 Haloalkane
dehalogenase genes were expressed with an N-terminal His-tag
under control of an L-arabinose inducible promoter in E. coli
TOP10. Typically, around 50 mg of enzyme was obtained from a 1 L
of overnight induced culture after purification by FPLC using Ni-
NTA columns (GE Healthcare). The purity of the isolated enzymes
was >95% as judged by SDS-polyacrylamide gel electrophoresis.
The kinetic parameters kcat and KM and the catalytic efficiency
(kcat/KM) for the haloalkane dehalogenase catalysed conversions of
compounds 1ae1e were determined (Table 1). Due to the low
substrate solubility, measurement of accurate values of KM and kcat
values was not possible for all of the substrates, since saturation of
the reaction rate could not be obtained with some compounds. In
such cases, only the catalytic efficiency is reported. For all sub-
strates, activity with at least one of the tested enzymes was found.
meso-2,3-Dibromobutane (1a) was converted by four of the six
tested enzymes. Of these, LinB possessed the highest catalytic ef-
ficiency. The catalytic efficiency found with DhaA31 is 5-fold higher
than with DbjA and DhaA, yet still an order of magnitude lower
than with LinB.

A. Westerbeek et al. / Tetrahedron 68 (2012) 7645e7650
7647
2.2. Enantioselectivity
the second reaction. This makes this process unattractive for
a tandem desymmetrisation and kinetic resolution of 2a.
The graph in Fig. 2B shows the conversion of 1b by LinB, which
was chosen because it provided the product 2b with the highest
enantiomeric excess (Table 2). The conversion of 1b to 2b is rela-
tively slow (Table 1). The enantiomeric excess of 2b is 58% at the
beginning, and slightly decreases to 50% during the course of the
reactions. Enantiopure dichloropanol 2b is not obtained, which
renders LinB unsuitable for its production. Next, the conversion of
1b by DbjA (Fig. 2C) and DhlA (Fig. 2D) was attempted. When DbjA
was used, the enantiomeric excess of 2b stayed the same over the
whole reaction time, while the concentration of 2b was decreasing.
For DhlA, a small kinetic resolution effect was observed, as the e.e.
of 2b slightly raised from 50 to 58%, but the enantioselectivity was
too low and the conversion proceeded very slowly.
During the conversion of 1c to 3c by DhaA, the first reaction was
very fast (Fig. 2E). (R)-2c is the preferred product of the desym-
metisation step, but is also the preferred enantiomer for the second
reaction leading to the diol 3c. This second reaction even inverts the
enantiomeric excess as conversion proceeds, since (S)-2c domi-
nated at the end of the reaction. The unfavourable ratio of the ki-
netic constants (Scheme 1) makes this reaction unsuitable for
production of enantiopure 2c.
Fig. 2F shows the changes of the concentrations of 1d and 2d
during the course of conversion by DbjA. The desymmetrisation of
tribromopropane 1d was fast, but gave an enantiomeric excess of
only 28% for (S)-2d. During the course of the second reaction, this
value went up slowly to 97% after 300 min, as the non-preferred
enantiomer (R)-2d is the preferred substrate for the reaction to
diol 3d by DbjA. This confirms the beneficial effect of the enzyme-
catalysed kinetic resolution on the e.e. of 2d, even though the an-
alytical yield at the e.e. of 97% is low, only 24%.
For 3-bromo-2-methylpropan-1-ol (2d), the commercially
available enantiomers were used to measure the catalytic effi-
ciencies. The determined E-value with DbjA is only 6 (Table 3),
which is in agreement with the time-course experiment (Fig. 2D)
where (R)-2d is the preferred substrate for the formation of the diol
3d.
Fig. 3 shows the changes in enantiomeric excess of 2e and
concentration of 1e and 2e during the course of the reaction cata-
lysed by LinB. The first reaction of 1e to 2e is very fast and produced
(S)-2e with an e.e. of 64%. In the course of the much slower second
conversion of 2e to the diol 3e the e.e. of (S)-2e increased to 98%
after 20 h, as (R)-2e is the preferred enantiomer for the second
reaction, resulting in kinetic resolution of 2e. The final analytical
yield of the reaction was 52%.
In the literature, various yields and ees were described for
tandem desymmetrisation/kinetic resolution processes, ranging
from 15% (95% ee)16 to 51% (95% ee)17 or 55% (99% ee),37 i.e., in the
same range as the values we found here. Further improvement
would require enzymes with higher enantioselectivity in the first
step or better complementarity in the tandem conversion. The
engineering of enzyme variants with enhanced enantioselectivity
has been intensively explored.38,39 Haloalkane dehalogenases be-
The enantioselectivity of the conversion of haloalkanes 1 to
haloalcohols 2 was determined by measuring the enantiomeric
excess of 2 after a certain reaction time (Table 2 and Table 4 in
Experimental section).
Table 2
Enantiomeric excess of products 2 obtained in haloalkane dehalogenase catalysed
conversion of substrates 1
Enzyme/product
2aa
2b
2c
2d
2e
DhaA
DbjA
LinB
DhaA31
DmlA
DhlA
88%
85%
4.4%
64%
N.D.
N.D.
18% (R)
39% (R)
52% (R)
17% (R)
6% (S)
42% (R)
15% (R)
24% (R)
6% (S)
18% (R)
3% (R)
41% (R)
70% (S)
30% (R)
19% (R)
39% (R)
49% (R)
N.D.
N.D.
79% (S)
57% (S)
N.D.
49% (R)
N.D.
N.D.: not determined, because of low activity.
a
For compound 2a the absolute configuration was not determined.
Table 3
Kinetic parameters of conversion of 3-bromo-2-methylpropan-1-ol (2d) with DbjA
kcat (sꢀ1
)
Km (mM)
kcat/KM (mMꢀ1 sꢀ1
)
E-value
(R)-Enantiomer
(S)-Enantiomer
>0.7
>0.11
>2.5
>2.3
0.30
0.047
6
Table 4
Incubation times, amount of enzyme and substrate concentrations used in
incubations
Enzyme (amount) 1a (1 mM) 1b (2 mM) 1c (1 mM) 1d (1 mM) 1e (0.5 mM)a
DhaA (0.64 mg)
DbjA (0.56 mg)
LinB (0.48 mg)
30 min
5 min
30 min
90 min
90 min
90 min
20 min
90 min
90 min
20 min
20 min
20 min
3 min
60 min
60 min
60 min
60 min
60 min
60 min
90 min
90 min
d
d
60 minb
60 minc
d
DhaA31 (0.64 mg) 30 min
DmlA (1.3 mg)
DhlA (0.62 mg)
d
d
d
a
DMSO (20% v/v) was added to enhance the solubility.
Divergent amount of LinB: 0.24 mg.
Divergent amount of DhaA31: 0.32 mg.
b
c
The enantiomeric excess of 2a was high in the reactions cata-
lysed by DbjA and DhaA. The ee value measured for LinB product
was very low. For the dichloropropanol 2b, moderate ee values
were found; here the highest value was obtained with LinB. The ee
values for 2c were low to moderate, with the highest value of 42%
found with DbjA. This enzyme provided also the best result for 2d.
The enantiomeric excess of 2e was only determined for LinB and
DhaA31, which both gave values above 50%.
2.3. Tandem conversions for kinetic resolution of
haloalcohols
We proceeded to study the possibility of using haloalkane
dehalogenases in a tandem desymmetrisation/kinetic resolution
process (Scheme 1), taking advantage of the fact that haloalkane
dehalogenases have been described to convert haloalcohols to di-
ols.36 Based on the results presented in Table 2, we chose the most
promising combinations of enzymes and substrates to perform
reactions in which the concentrations of 1 and 2 and the enantio-
meric excess of 2 were monitored in time.
Fig. 2A shows the DbjA-catalysed conversion of 1a to 2a, which
was converted further to the diol 3a. The enantiomeric excess of 2a
started at 85%, but this value decreased as the predominantly
formed enantiomer of product 2a is also the preferred substrate for
long to the a/b hydrolase fold family of proteins, and several
members of this class of enzymes were subjected to engineering
studies40 aimed at enhancing enzyme enantioselectivity, including
lipases,41e44 esterases,38 and epoxide hydrolases.45,46 For hal-
oalkane dehalogenases, it has been shown that the enantiose-
lectivity can also be enhanced by structure-based protein
engineering.24 Furthermore, directed evolution has yielded com-
plementary mutants with enhanced selectivity in asymmetric
conversion of trichloropropane 1b to the enantioenriched 2,3-
dichloropropanols.33 In addition to protein engineering, expres-
sion of putative dehalogenases identified by genome sequence
analysis is providing dehalogenase variants with new

7648
A. Westerbeek et al. / Tetrahedron 68 (2012) 7645e7650
A
B
1.0
0.8
0.6
0.4
0.2
0.0
100
80
60
40
20
0
2.0
1.5
1.0
0.5
0.0
100
80
60
40
20
0
0
50
100
150
200
0
200 400 600 800 1000
Time (min)
Time (min)
C
D
100
80
60
40
20
0
100
80
60
40
20
0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0
50 100 150 200 250 300
0
100
200
300
Time (h)
Time (h)
E
F
1.0
0.8
0.6
0.4
0.2
0.0
100
50
0
1.0
0.8
0.6
0.4
0.2
0.0
100
80
60
40
20
0
-50
-100
0
200
400
600
0
50 100 150 200 250 300
Time (min)
Time (min)
Fig. 2. Tandem conversion of 1ae1d followed in time. Concentrations of substrates 1 (6) and haloalcohols 2 (C) are depicted, together with the enantiomeric excess of hal-
oalcohols 2 (-, broken line). (A) Substrate 1a, conversion with DbjA, (B) substrate 1b, conversion with LinB, (C) substrate 1b, conversion with DbjA, (D) substrate 1b, conversion
with DhlA, (E) substrate 1c, conversion with DhaA, (F) substrate 1d, conversion with DbjA.
0.5
0.4
0.3
0.2
0.1
0.0
100
80
60
40
20
0
conversions and tandem reactions of meso and prochiral com-
pounds explored here.
3. Conclusions
0.5
0.4
0.3
0.2
0.1
0.0
100
80
60
40
20
0
In this paper we report the activity of haloalkane dehalogenases
towards prochiral and meso dihaloalkanes 1ae1e. For four com-
pounds, ee values from 52 to 88% were obtained in asymmetric
conversions. The possibility of a sequential desymmetrisation and
kinetic resolution, catalysed by the same enzyme, was demon-
strated for products 2ae2e. The activity of haloalkane dehaloge-
nases in conversions of substrates 1 allowed the use of 6e23% (w/
w) of the catalyst. Although the reported yields were modest, the
results comprise the first use of haloalkane dehalogenases for such
a tandem process.
0
10
20
t (min)
30
40
50
0
200
400
600
800
1000
1200
Time (min)
Fig. 3. Tandem conversion of rac-1,3,-dibromo-2-phenylpropane (1e) by LinB followed
over time. The concentration of 1e (6) and 2e (C) are depicted, together with the
enantiomeric excess of 2e (-, dotted line). The inset shows the first 50 min of the
conversion.
4. Experimental section
4.1. General
selectivities.47e49 The combined use of genomics-based enzyme
discovery and structure-based engineering would thus allow the
development of a toolbox of dehalogenase variants tailored for
specific biocatalytic processes including the type of asymmetric
1H NMR spectra were recorded at 300 or 400 MHz with CDCl3 as
solvent. 13C NMR spectra were obtained at 75.4 or 100.59 MHz in
CDCl3. Chemical shifts were determined relative to the residual

A. Westerbeek et al. / Tetrahedron 68 (2012) 7645e7650
7649
solvent peaks (CHCl3,
d
¼7.26 ppm for 1H,
d
¼77.0 for 13C). The fol-
4.4. Determination of absolute configuration
lowing abbreviations are used to indicate signal multiplicity: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Enantiomeric excess determinations were performed by capillary
GC analysis or HPLC analysis using a flame ionisation detector
or UV-detection (all in comparison with racemic products, column
and conditions further specified in relevant experiment
descriptions). Optical rotations were measured in CHCl3 on
a polarimeter with a 10 cm cell (c given in g/100 mL). Flash
chromatography was performed on silica gel. Solutions were
dried with MgSO4 or Na2SO4 and concentration steps were
conducted with a rotary evaporator.
For compounds 2b and 2c, the absolute configuration was de-
termined by transformation into the epihalohydrin18 and compar-
ison with a commercial sample. For compound 2d, this was done by
comparison with a commercial sample. The absolute configuration
of compound 2e was determined by isolation of the enzymatic
reaction product and comparison of optical rotation with a litera-
ture value ([
a
]
D þ12.8 (c 0.67, CHCl3) for 68% ee), lit.52 ([
D ꢀ21.7 (c
a
]
1.8, CHCl3) for the (R)-enantiomer).
4.5. Enzyme production
Compounds1a,1c, 3d and 3ewere purchased from SigmaeAldrich,
compounds 1b and 2b were purchased at TCI Europe, and compound
2c was purchased at Acros Organics. Racemic compound 2a was
obtained according to a literature procedure.50
For construction of the haloalkane dehalogenase gene expres-
sion plasmid, the genes were cloned into a pBAD vector, with ad-
dition of a DNA segment encoding an N-terminal hexahistidine tag.
Precultures were started by inoculating 5 mL of LB medium with E.
coli TOP10 transformants from glycerol stocks and incubated
overnight at 37 ꢃC, while shaking. The next day, 2 L of LBamp me-
dium was inoculated with 1% of an overnight culture and incubated
at 37 ꢃC with shaking. Enzyme expression was induced by adding
4.2. General procedure 1: preparation of 1,3-dibromides from
1,3-diols
N-Bromosuccinimide (2.2 mmol) was added at 0 ꢃC to a mixture
of diol (1.0 mmol) and triphenylphosphine (2.2 mmol) in dichloro-
methane (10.0 mL). The cooling bath was removed and the reaction
mixture was stirred at room temperature for 30 min. The solvent
was evaporated and the product was purified by column chroma-
0.002% (w/v) L-arabinose at when the OD600 reached 0.6. Cultures
were incubated overnight with shaking at 30 ꢃC, except for the
culture aimed at expression of LinB, which was incubated at 17 ꢃC.
After growth, the cells were harvested, broken by sonication,
centrifuged and the supernatant with the haloalkane dehaloge-
nases was used for purification using three in line His-Trap Ni-NTA
columns from GE Healthcare attached to an AKTA purifier system.
The purified enzymes were concentrated using an Amicon ultra-
tography (Silicagel, 40e63 mm, pentane).
Compound1d: 1,3-dibromo-2-methylpropane. Yield: 71%. Col-
ourless oil. 1H NMR (300 MHz, CDCl3):
d
1.15 (d, 3H, 3J¼7.2 Hz, CH3),
2.22e2.44 (m, 1H, CH), 3.45 (dd, 2J¼10.2 Hz, 3J¼6.0 Hz, 2H,
2ꢄCH2Br), 3.53 (dd, 2J¼10.2 Hz, 3J¼5.1 Hz, 2H, 2ꢄCH2Br); 13C NMR
filtration cell and stored in TMG buffer (10 mM TriseSO4, 1 mM b-
(50 MHz, CDCl3):
(70), 55 (100).
d
18.1,37.2, 37.9; MS (EIþ) m/z 216 (Mþ, 15), 135
mercaptoethanol and 10% (v/v) glycerol, pH 7.5). Protein concen-
trations were determined with Bradford reagent (Biorad) using
bovine serum albumin as the standard. The purified enzyme was
analysed using SDS-polyacrylamide gel electrophoresis.
Compound1e: 1,3-dibromo-2-phenylpropane. Yield: 66%. Col-
ourless oil. 1H NMR (400 MHz, CDCl3):
d
3.38 (quintet, 3J¼6.6 Hz,
1H, CH), 3.73 (dd, 2J¼10.4 Hz, 3J¼6.4 Hz, 2H, 2ꢄCH2Br), 3.80 (dd,
2J¼10.4 Hz, 3J¼6.8 Hz, 2H, 2ꢄCH2Br), 7.22e7.38 (m, 5H, ArH); 13C
4.6. Enzymatic reactions
NMR (50 MHz, CDCl3):
d 35.8, 49.2, 127.8, 128.2, 129.0, 139.8;
MS (EIþ) m/z 278 (Mþ, 10), 197 (30), 183 (50), 117 (40), 104 (100),
91 (55).
Kinetic parameters were determined using a halide release as-
say. Assays were done at room temperature in 200 mL 50 mM
Reference racemic compound rac-2e was obtained by per-
forming general procedure 1 with 1 equiv of both N-bromosucci-
nimide and triphenylphosphine. The compound was obtained as
TriseSO4, pH 8.2. For compound 1e, 20% of DMSO was added to
enhance the solubility to 0.5 mM. Samples were taken at different
times and after adding mercuric thiocyanate and ferric ammonium
sulfate the absorption was measured at 460 nm.27,53 The initial
rates of halide release at different substrate concentrations were
plotted in a MichaeliseMenten graph in order to determine the
kinetic parameters.
Time-course experiments were done as follows. To 10 mL of
a solution of compound 1 in 50 mM TriseSO4, pH 8.2, enzyme
was added and the mixture was incubated at 30 ꢃC (for details, see
Table 5). At various times, 1 mL samples were taken and extracted
with a solvent containing dodecane as internal standard. The sam-
ples were dried with MgSO4. The enantiomeric excess values for
haloalcohols 2ae2c were determined by Chiral GC and for 2d and 2c
by chiral HPLC, as described above. For the reactions with
a colourless oil. 1H NMR (400 MHz, CDCl3):
d 3.18e3.25 (m, 1H, CH),
3.66 (dd, 2J¼10.0 Hz, 3J¼6.4 Hz, 2H, CH2Br), 3.95 (m, 2H, CH2OH),
7.22e7.38 (m, 5H, ArH), consistent with the literature.51
4.3. Determination of enantiomeric excess with single time
point measurements
The enantiomeric excess values described in Table 1 were de-
termined by adding enzyme (0.24e1.3 mg) to 10 mL of a solution of
substrate (0.5e2 mM) in 50 mM Tris, pH 8.2. The mixture was in-
cubated at 30 ꢃC, for 3e90 min, depending on enzyme activity.
Ee values of the haloalcohols 2 were determined by chiral
chromatography analysis. For compound 2a: chiral GC, Chiraldex G-
TA (1 mL/min, isothermal, 42 ꢃC), retention times (min) 36.9 and
Table 5
38.3. For compound 2b: chiral GC, Hydrodex-
isothermal, 140 ꢃC), retention times (min) 8.2 (S) and 8.6 (R). For
compound 2c: chiral GC, Hydrodex- -TBDAc (1 mL/min, iso-
b-TBDAc (1 mL/min,
Details of the tandem conversions of compounds 1
Compound
1a
1b
1b
1b
1c
1d
1e
b
thermal, 140 ꢃC), retention times (min) 9.6 (S) and 9.9 (R). For
compound 2d: chiral HPLC, after transformation into p-nitro-
benzoyl ester following literature procedure,52 Chiralcel OJ-H
(heptane/i-PrOH 97:3, v/v, flow 0.5 mL/min), retention times
(min) 36.3 (R) and 39.0 (S). For compound 2e: chiral HPLC, Chiralcel
OD-H (heptane/i-PrOH, 93:7, v/v, flow 0.5 mL/min), retention times
(min) 19.4 (R) and 20.2 (S).
Enzyme (mg)
DbjA LinB
DbjA DhlA DhaA DbjA
LinB
(0.56) (0.48) (5.0) (5.0) (0.64) (0.50) (0.24)
Solution (mL)
Substrate (mM)
Substrate (mg)
10
1
2.2
10
2
2.9
20
1
2.9
20
1
2.9
10
1
2.8
20
1
4.3
30
0.5
4.2
Extraction solvent (mL) CHCl3 CHCl3 CHCl3 CHCl3 CHCl3 CH2Cl2 Et2O
(0.5) (0.5) (0.5) (0.5) (0.5) (1.5)
(2.0)
2
Sample vol. (mL)
1
1
1
1
1
2

7650
A. Westerbeek et al. / Tetrahedron 68 (2012) 7645e7650
ꢀ
25. Westerbeek, A.; Szymanski, W.; Wijma, H. J.; Marrink, S. J.; Feringa, B. L.;
compounds 1a, 1b and 1c, the conversion was determined with the
same GC program, whereas for 1d and 1e a different GC programwas
used. For 1d: AT5 GC column (80 ꢃC/130 ꢃC (10 ꢃC/min), 130 ꢃC for
1 min, 130 ꢃC/80 ꢃC (20 ꢃC/min), retention times 3.8 min (2d) and
4.4 min (1d)). For 1e: AT5 GC column (80 ꢃC/180 ꢃC (10 ꢃC/min),
180 ꢃC for 1 min, 180 ꢃC/80 ꢃC (20 ꢃC/min), retention times:
10.9 min (2e); 11.9 min (1e)).
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