Synthesis of enantiopure chloroalcohols by enzymatic kinetic resolution

Article abstract of DOI:10.1039/b613937j

3-Alkenyl and heteroaryl chloroalcohols have been obtained in excellent enantiomeric excess (>99%) by enzymatic kinetic resolution using the haloalcohol dehalogenase HheC. Yields were close to the theoretical maximum for all substrates employed. Furthermo

Full text of DOI:10.1039/b613937j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of enantiopure chloroalcohols by enzymatic kinetic resolution†
Robert M. Haak,a Chiara Tarabiono,b Dick B. Janssen,b Adriaan J. Minnaard,*a Johannes G. de Vries*a,c and
Ben L. Feringa*a
Received 26th September 2006, Accepted 2nd November 2006
First published as an Advance Article on the web 11th December 2006
DOI: 10.1039/b613937j
3-Alkenyl and heteroaryl chloroalcohols have been obtained in excellent enantiomeric excess (>99%)
by enzymatic kinetic resolution using the haloalcohol dehalogenase HheC. Yields were close to the
theoretical maximum for all substrates employed. Furthermore, the applicability of this methodology
on multigram scale has been established.
Introduction
Results and discussion
Haloalcohol dehalogenases are enzymes that catalyze the inter-
conversion of haloalcohols and epoxides.1 HheC is a haloalcohol
dehalogenase produced by Agrobacterium radiobacter AD1, a soil-
dwelling bacterium that is able to use halogen-containing organic
compounds as its sole carbon source. The enzyme was discovered
some years ago.2 Recently, its structure, kinetics and catalytic
mechanism have been elucidated.3,4 The substrate binds near
a catalytic triad (Ser132-Tyr145-Arg149), of which the tyrosine
residue activates the hydroxy group of the haloalcohol. Concurrent
SN2-type attack on the vicinal carbon atom by the hydroxy group
leads to ring closure and expulsion of the halide anion.3,4
Notably, the biocatalytic potential of HheC has been the
subject of investigation5 and its substrate scope was found to
be remarkable.6 HheC catalyzes the reversible ring-closure of
haloalcohols to form epoxides, as well as the irreversible ring-
opening of epoxides with a number of non-halide nucleophiles,
such as cyanide, nitrite, and azide.
We chose to concentrate on chloroalcohols functionalized with
unsaturated and heteraromatic moieties, since these are especially
versatile synthetic scaffolds. The compounds prepared10 and
studied are summarized in Scheme 1.
Here, we report the preparation in enantiomerically pure form
of functionalized vicinal chloroalcohols. These are highly valuable
and frequently used building blocks in synthesis.
Scheme 1 Synthesis of functionalized chloroalcohols.10
A number of strategies have been used to prepare these
compounds in enantiomerically pure form. Chloroalcohols 5–
7 have been prepared by asymmetric transfer hydrogenation of
the corresponding chloroketones.7 Furthermore, enantiomerically
pure 6 has been obtained by lipase-catalyzed kinetic resolution
of the racemic chloroalcohol,8 and a route to enantiomerically
pure 5 by Red-Al reduction of the enantiomerically pure alkyne is
known.9 Enantiomerically pure 1–4 have not been reported before,
and a general, convenient, and highly enantioselective method for
preparing enantiopure 1–7 is lacking.
Kinetic resolution on analytical scale
We studied the synthesized chloroalcohols as substrates for
HheC, initially on analytical scale. The results of this screening
are summarized in Table 1. Enzymatic activity towards each
of the substrates is expressed both as initial enzyme activity
(lmol·min−1·mg−1 of enzyme) and as turnover frequency (s−1).11
In the last column the selectivity factor E is given for each of the
substrates.
aDepartment of Organic and Molecular Inorganic Chemistry, Stratingh
Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The
Netherlands. E-mail: B.L.Feringa@rug.nl; Fax: +31 50 363 4296; Tel: +31
50 363 4235
For the linear substrates, a clear trend in reactivity can be
observed: the shorter the chain, the faster the enzymatic conver-
sion. This is expected on the basis of previous observations.6 We
attribute the observed reactivity pattern to increasing difficulty
of the substrate to fit in the active site of the enzyme in a reactive
conformation as it gets bulkier. Based on such steric arguments, the
reactivity pattern of substrates 1–5 can be rationalized. Differences
in substrate binding, influenced by steric factors, may also partly
explain the marked difference in reactivity between the otherwise
comparable substrates 6 and 7.
bDepartment of Biochemistry, Groningen Biomolecular Sciences and Biotech-
nology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Gronin-
gen, The Netherlands
cDSM Research, LS-ACS & D, P.O. Box 18, 6160, MD, Geleen, The
Netherlands
† Electronic supplementary information (ESI) available: Experimental
procedures and full spectroscopic data for compounds 1–7. See DOI:
10.1039/b613937j
318 | Org. Biomol. Chem., 2007, 5, 318–323
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Table 1 HheC-catalyzed kinetic resolutions of substrates 1–7 on analyt-
ical scalea
Init. enz. activ.b/
Entry R =
lmol·min−1·mg−1 TOF/s−1c Ed
1
2
48
29
22.4
13.5
>200
177
3
4
5
8
12
10
3.7
5.6
4.7
>200
102
>200
6
7
47
11
21.9
5.1
>200
65
a General conditions: 0.2 mmol scale, 10 mM in Tris-sulfate pH 8.1. b Initial
enzyme activity (lmol of product per min per mg of HheC). c Per enzyme
subunit. d Obtained by fitting measured data points (concentration vs.
time) against the mathematical curves for competitive Michaelis–Menten
kinetics using MicroMathꢀR ScientistꢀR .
Fig. 1 Formulae for competitive Michaelis–Menten kinetics displayed by
HheC (1a and b), for the enzymatic selectivity E (2), and an example of a
typical progress curve for enzymatic conversion of substrate 1.
The enantioselectivity of these transformations is high in all
cases, and even the lowest E observed (65, Table 1, entry 7) is
excellent for a kinetic resolution.
All of the HheC-catalyzed ring closure reactions described here,
show competitive Michaelis–Menten kinetics.12,13 Initially, epoxide
formation of the slower reacting enantiomer is inhibited by the
faster reacting enantiomer. When all fast-reacting enantiomer has
been consumed, ring closure of the other enantiomer starts to
take place, sometimes at an appreciable rate. This behaviour is
illustrated in Fig. 1.14
Preparative scale reactions were performed on 2.0 mmol scale at
low concentration (10 mM) in Tris-sulfate buffer, i.e., analogous
to the analytical reactions. In this manner it proved possible to
isolate enantiomerically pure chloroalcohols (ee > 99%) in fair
to high yields (Table 2, entries 1–4). Our attempts to isolate also
the produced epoxides failed, since it turned out that they rapidly
hydrolyze in situ to form the corresponding diols.
It was attempted to suppress this hydrolysis by performing the
reaction in a two-phase system (Table 2, entries 5 and 6), but
even in those cases hydrolysis proved inevitable. The role of this
spontaneous hydrolysis will be examined in more detail further
on.
In formulae 1a and 1b, depicted in Fig. 1, R and S represent
R
the concentrations of both enantiomers, VmaxR, VmaxS, Km and
S
Km are the relevant Michaelis–Menten parameters, and kc is the
first-order rate constant of chemical hydrolysis. After fitting these
equations by numerical integration to the obtained data points,15
the E-value was calculated from formula 2 (Fig. 1).16
As is shown in Fig. 1, the reaction rate of the slow-reacting (S)-
enantiomer can be appreciable. This is the case especially for the
linear substrates 1–4. Naturally, this behavior should be taken into
account when these reactions are to be performed on preparative
scale (vide infra).
Accordingly, our efforts focused on obtaining the enantiomer-
ically pure halohydrins, which were isolated in good yields and
with excellent enantioselectivities (Table 2).
The absolute configuration of the remaining chloroalcohols was
S in all cases, in agreement with previous studies which showed that
HheC is R-selective for most substrates.5,6 A variety of methods
was used to elucidate the absolute configuration of the slow-
reacting enantiomer of the chloroalcohols. For (S)-(E)-1-chloro-
4-phenyl-but-3-en-2-ol ((S)-5) the absolute configuration could be
deduced from a crystal structure (CCDC 605888).17 (S)-6 and (S)-
7 could be correlated to known compounds by the sign of their
optical rotation.7,8 Finally, (S)-1, (S)-3, and (S)-4 were converted to
their saturated analogues, for which specific rotations are known.18
Kinetic resolution on preparative scale
Having established that this enzymatic kinetic resolution is highly
efficient, we set out to transform substrates 1 and 3–7 on a
preparative scale. In view of the excellent selectivities, our initial
aim was to isolate both the enantiomerically pure remaining
chloroalcohol and the produced epoxide.
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Table 2 Enzymatic kinetic resolutions of unsaturated and heteroaromatic vicinal chloroalcohols on preparative scale
Entry
Substrate
R =
Chloroalcohol (yielda, ee, conf.)
40%, >99%, S
Diol (yielda)
N.i.c
1
2
1b
3d
31%, >99%, S
N.i.c
3
4
4e
5d
29%, >99%, S
47%, >99%, S
24%f
19%f
5
6
6g
7h
42%, 98.5%, S
47%i, >99%, S
N.i.c
49%i
a Isolated yield (based on 50% maximum). b 1.0 mmol scale, 10 mM in Tris-sulfate buffer pH 8.1. c Not isolated. d 2.0 mmol scale, 10 mM in Tris-sulfate
buffer pH 8.1. e 1.5 mmol scale, 10 mM in Tris-sulfate buffer pH 8.1. f Mixture of diols. g 16 mmol scale, 1 : 1 toluene–Tris-sulfate pH 8.1. h 120 mmol
scale, 1 : 10 toluene–Tris-sulfate pH 8.1. i Crude yield.
We were especially interested in the possibilities of performing
this resolution as a preparative procedure. Interestingly, it was
possible to perform this kinetic resolution on multigram scale
(Table 2, entries 5 and 6). For instance, starting from 20.9 g of
racemic 2-chloro-1-thiophen-2-yl-ethanol (7), there was obtained
9.8 g (94% of the theoretical yield) of (S)-7 with an excellent ee of
>99%. No modifications of the enzyme were needed to achieve
these results. To avoid the use of excessive amounts of buffer
solution, the latter two reactions were performed in a two-phase
system of toluene and Tris-buffer.
Scheme
ethanol.
2
Incomplete conversion of (R)-2-chloro-1-(4-nitrophenyl)-
Conclusions
Role of spontaneous hydrolysis
The spontaneous hydrolysis of the epoxide products mentioned
earlier was initially perceived as a drawback of the system.
However, comparison of our results with those previously reported
for the related compound 2-chloro-1-phenyl-ethanol, made us
reconsider.
As illustrated in Scheme 2, when a kinetic resolution is
performed on 2-chloro-1-(4-nitrophenyl)-ethanol, the affinity of
HheC is larger for the (R)- than for the (S)-enantiomer. Hence,
the (R)-enantiomer is converted first. However, the Keq of the
equilibrium between (R)-2-chloro-1-(4-nitrophenyl)-ethanol and
(R)-2-(4-nitrophenyl)-oxirane is 40 mM,6 which indicates that a
few percent of the (R)-enantiomer will always remain present.
Therefore the maximum ee of the remaining chloroalcohol will be
about 95%.5,6
Presumably, in our system the spontaneous hydrolysis of the
formed epoxide pulls the equilibrium to the right side, thus
ensuring excellent ee’s of the remaining chloroalcohols.
Interestingly, in the case of substrate 7 (Table 2, entry 6)
hydrolysis proceeds with almost complete racemization, resulting
from equal rates of hydrolysis on the terminal and internal carbon
atom of the (enantiomerically pure) epoxide.19
In conclusion, a highly efficient kinetic resolution protocol was
developed for functionalized vicinal chloroalcohols. The majority
of these compounds had not been reported before in their enan-
tiomerically pure form. Various unsaturated and heteroaromatic
chlorohydrins were resolved in high yields and with excellent enan-
tioselectivities. This resolution was shown to be effective on multi-
gram scale, rendering it highly practical as a preparative method.
Experimental
General
Starting materials were purchased from Aldrich or Acros and used
as received unless stated otherwise. All solvents were reagent grade
and dried and distilled prior to use. Demineralized water was used
in the preparation of all aqueous solutions.
Column chromatography was performed on silica gel (Aldrich
60, 230–400 mesh). TLC was performed on silica gel 60/Kiesel-
guhr F254 or neutral aluminium oxide 60 F254 where indicated.
1H and 13C NMR spectra were recorded on a Varian VXR300
1
(299.97 MHz for H, 75.48 MHz for 13C) or a Varian AMX400
320 | Org. Biomol. Chem., 2007, 5, 318–323
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(399.93 MHz for 1H, 100.59 MHz for 13C) spectrometer in
CDCl3 unless stated otherwise. Chemical shifts are reported in
d values (ppm) relative to the residual solvent peak (CHCl3, 1H =
7.24, 13C = 77.0). Carbon assignments are based on APT 13C
experiments. Splitting patterns are indicated as follows: s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).
Mass spectra (HRMS) were performed on an Jeol JMS-
600H. GCMS spectra were recorded on a Hewlett Packard
HP6890 equipped with a HP1 column and an HP 5973 Mass
Selective Detector.
GC analysis was performed on a Shimadzu GC-17A or a
Hewlett Packard HP6890 spectrometer equipped with the columns
indicated for each compound separately.
HPLC analysis was performed on a Shimadzu HPLC system
equipped with two LC-10AD vp solvent delivery systems, a DGU-
14A degasser, a SIL-10AD vp auto injector, an SPD-M10A vp
diode array detector, a CTO-10A vp column oven, and an SCL-
10A vp system controller using the columns indicated for each
compound separately.
57; chiral GC: CP Chiralsil Dex CB, 25 m × 0.25 mm × 0.25 lm,
He-flow: 1.0 mL min−1, 120 ◦C isothermic, Tr = 12.6 min (S), Tr =
13.0 min (R).
1-Chloro-octa-3,5-dien-2-ol (4). Obtained as a colorless oil
(1.29 g; 8.0 mmol; 79%) after flash chromatography (pentane–
1
Et2O 7 : 1, Rf = 0.26); H NMR (CDCl3) d 6.30 (dd, J = 15.0,
10.3 Hz, 1H), 6.02 (dd, J = 15.0, 10.3 Hz, 1H), 5.79 (dt, J = 15.0,
6.6 Hz, 1H), 5.54 (dd, J = 15.4, 5.9 Hz, 1H), 4.35 (br, 1H), 3.60
(ddABX, J = 11.0, 3.7 Hz, DmAB = 47.5 Hz, 1H), 3.48 (ddABX, J =
11.0, 7.3 Hz, DmAB = 47.5 Hz, 1H), 2.23 (d, J = 3.7 Hz, 1H), 2.09
(dt, J = 13.9, 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13C NMR
(CDCl3) d 138.4 (d), 133.4 (d), 128.1 (d), 128.0 (d), 72.1 (d), 49.6
(t), 25.6 (t), 13.2 (q); MS (EI+) m/z = 162 (M+), 160 (M+), 111,
93, 55; HRMS (EI+) calculated: 160.06549, measured: 160.06621;
chiral GC: CP Chiralsil Dex CB, 25 m × 0.25 mm × 0.25 lm,
He-flow: 1.0 mL min−1, 125 ◦C isothermic, Tr = 14.8 min (S),
Tr = 15.6 min (R).
(E)-1-Chloro-4-phenyl-but-3-en-2-ol (5)7,9,10,21,22
.
Obtained af-
Optical rotations were measured on a Schmidt + Haensch
Polartronic MH8 using a 10 cm cell.
ter flash chromatography (pentane–Et2O 5 : 1, Rf = 0.24) as a
colorless oil (2.34 g; 12.8 mmol; 64%), which crystallized upon
1
standing; H NMR (CDCl3) d 7.20–7.40 (m, 5H), 6.71 (dd, J =
Synthesis of substrates 1–7
16.1, 1.1 Hz, 1H), 6.19 (dd, J = 16.1, 6.0 Hz, 1H), 4.52 (br m, 1H),
3.71 (ddABX, J = 11.0, 3.7 Hz, DmAB = 48.9 Hz, 1H), 3.58 (ddABX
,
Substrates 1–7 were synthesized according to a literature
procedure.10 Spectroscopic data and chromatographic separation
conditions will now follow for each of the substrates.
J = 11.0, 7.3 Hz, DmAB = 48.9 Hz, 1H), 2.38 (d, J = 4.4 Hz, 1H);
13C NMR (CDCl3) d 136.0 (s), 132.7 (d), 128.6 (d), 128.1 (d), 127.2
(d), 126.6 (d), 72.3 (d), 49.6 (t); MS (EI+) m/z = 184 (M+), 182
(M+), 133, 115, 105; HRMS (EI+) calculated: 182.0498, measured:
182.0507; chiral HPLC: Chiralcel OD, 40 ◦C, heptane–IPA 92 : 8,
1.0 mL min−1, Tr = 11.5 min (S), Tr = 16.0 min (R).
1-Chloro-pent-3-en-2-ol (1)20. Obtained as a colorless oil
(2.21 g; 18.3 mmol; 73%) after flash chromatography (pentane–
1
Et2O 4 : 1); H NMR (CDCl3) d 5.76–5.80 (m, 1H), 5.48 (dd,
J = 15.4, 6.6 Hz, 1H), 4.26 (m, 1H), 3.58 (ddABX, J = 11.0, 3.7 Hz,
DmAB = 45.0 Hz, 1H), 3.47 (ddABX, J = 11.0, 7.3 Hz, DmAB = 45.0 Hz,
1H), 2.17 (d, J = 4.0 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H); 13C NMR
(CDCl3) d 129.7 (d), 129.2 (d), 72.3 (d), 49.6 (t), 17.7 (q); MS (EI+)
m/z = 122 (M+), 120 (M+), 107, 105, 71, 53, 41; chiral GC: Chi-
raldex B-PM, 30 m × 0.25 mm × 0.25 lm, He-flow: 1.1 mL min−1,
80 ◦C isothermic, Tr = 10.2 min (S), Tr = 10.9 min (R).
2-Chloro-1-fur-2-yl-ethanol (6)7,8
.
Obtained as a light yellow
oil (1.87 g; 12.7 mmol; 64%) after flash chromatography (pentane–
Et2O 4 : 1, gradient to 3 : 1, Rf, 3 : 1 = 0.40); for the resolution on 2.3 g
scale, different preparations were combined; 1H NMR (CDCl3) d
7.39 (s, 1H), 6.36 (s, 2H), 4.93 (m, 1H), 3.83 (m, 2H), 2.53 (d, J =
5.5 Hz, 1H); 13C NMR (CDCl3) d 152.6 (s), 142.6 (d), 110.4 (d),
107.6 (d), 68.0 (d), 47.7 (t); MS (EI+) m/z = 148 (M+), 146 (M+),
97; chiral GC: Chiraldex G-TA, 30 m × 0.25 mm × 0.25 lm, He-
1-Chloro-hex-3-en-2-ol (2). Obtained as a colorless oil (1.77 g;
◦
flow: 0.5 mL min−1, 120 C isothermic, Tr = 5.1 min (R), Tr =
13.2 mmol; 66%) after flash chromatography (pentane–Et2O 6 : 1,
1
gradient to 4 : 1); H NMR (CDCl3) d 5.84 (dtd, J = 15.8, 6.2,
5.4 min (S).
1.1 Hz, 1H), 5.44 (ddt, J = 15.4, 6.6, 1.5 Hz, 1H), 4.28 (br, 1H),
2-Chloro-1-thiophen-2-yl-ethanol (7)7,23
.
Obtained as a color-
3.59 (ddABX, J = 11.0, 3.7 Hz, DmAB = 46.1 Hz, 1H), 3.47 (ddABX
,
less oil (2.86 g; 17.6 mmol; 88%) after flash chromatography
(pentane–Et2O 4 : 1, Rf = 0.33); for the 20 g scale resolution, this
compound was prepared analogously (64%); 1H NMR (CDCl3) d
7.29 (dd, J = 5.1, 1.1 Hz, 1H), 7.03 (ddd, J = 3.7, 1.1, 0.7 Hz, 1H),
6.99 (dd, J = 5.1, 3.7 Hz, 1H), 5.15 (ddd, J = 8.1, 4.0, 0.7 Hz, 1H),
3.80 (ddABX, J = 11.4, 4.0 Hz, DmAB = 27.4 Hz, 1H), 3.72 (ddABX, J =
11.4, 8.1, DmAB = 27.4 Hz, 1H), 2.81 (br, 1H); 13C NMR (CDCl3) d
143.2 (s), 126.9 (d), 125.4 (d), 124.7 (d), 70.2 (d), 50.4 (t); MS (EI+)
m/z = 164 (M+), 162 (M+), 113; HRMS (EI+, for C6H737ClOS)
calculated: 163.9877, measured: 163.9881; chiral GC: Chiraldex◦B-
PM, 30 m × 0.25 mm × 0.25 lm, He-flow: 1.1 mL min−1, 135 C
isothermic, Tr = 14.2 min (S), Tr = 14.8 min (R).
J = 11.0, 7.3 Hz, DmAB = 46.1 Hz, 1H), 2.22 (d, J = 3.7 Hz, 1H),
2.06 (qdd, J = 7.3, 6.6, 1.5 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H); 13
C
NMR (CDCl3) d 136.4 (d), 127.0 (d), 72.3 (d), 49.7 (t), 25.2 (t),
13.1 (q); MS (EI+) m/z = 134 (M+), 105, 85, 67, 55; HRMS (EI+)
calculated: 134.0498, measured: 134.0491; chiral GC: Chiraldex
B-TA, 30 m × 0.25 mm × 0.25 lm, He-flow: 1.0 mL min−1, 85 ◦C
isothermic, Tr = 16.7 min (S), Tr = 18.0 min (R).
1-Chloro-oct-3-en-2-ol (3)10,21
. Obtained as a colorless oil
(903 mg; 5.55 mmol; 56%) after flash chromatography (pentane–
1
Et2O 6 : 1, Rf = 0.32); H NMR (CDCl3) d 5.79 (dtd, J = 15.4,
7.0, 1.1 Hz, 1H), 5.44 (ddt, J = 15.4, 6.6, 1.5 Hz, 1H), 4.2–4.35
(m, 1H), 3.58 (ddABX, J = 11.0, 3.7 Hz, DmAB = 45.0 Hz, 1H), 3.47
(dd, J = 11.0, 7.7 Hz, DmAB = 45.0 Hz, 1H), 2.19 (d, J = 4.2 Hz,
1H), 1.99–2.09 (br, 2H), 1.2–1.4 (br, 4H), 0.87 (t, J = 7.3 Hz, 3H);
13C NMR (CDCl3) d 135.2 (d), 127.9 (d), 72.4 (d), 49.9 (t), 31.9
(t), 31.0 (t), 22.1 (t), 13.9 (q); MS (EI+) m/z = 162 (M+), 113, 95,
Production and purification of the enzyme
Halohydrin dehalogenase was expressed in E. coli MC1061. The
hheC gene was amplified by PCR from pGEFHheC and cloned
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into pBAD/Myc-HisA between NcoI and PstI sites. Plasmid DNA
was transformed by electroporation to E. coli cells, which were
then plated out on LB plates containing ampicillin and incubated
overnight at 30 ◦C. A preculture was started by inoculating 100 mL
of TB containing 50 lg mL−1 ampicillin with the transformants
from a plate to a starting OD600 of 0.1. After overnight incubation
at 30 ◦C, the preculture was diluted in 1 L of TB, containing
50 lg mL−1 ampicillin, 2.5 mM betaine, 0.5 M sorbitol and
0.02% arabinose, and the culture was incubated for two days
at 37 ◦C. The cells were centrifuged, washed, and resuspended
in 50 mL of TEMG buffer (10 mM Tris-SO4, 1 mM EDTA,
1 mM b-mercaptoethanol, and 10% glycerol, pH 7.5) containing a
protease inhibitor cocktail (Complete Protease Inhibitor Cocktail
Tablets, Roche). Cells were broken by sonication and the extract
was centrifuged (50 000 rpm, 45 min, 4 ◦C). The supernatant was
applied on a 50 mL Q-Sepharose anion exchange column and
elution was carried out with a gradient of 0 to 0.45 M ammonium
sulfate in TEMG. The collected fractions that displayed enzymatic
activity were pooled and concentrated. The enzyme was stored at
4 ◦C or −20 ◦C.
product(s) obtained were purified by column chromatography,
using the conditions described for the racemic substrates.
Isolated yields and ee’s
Specific conditions for the resolutions of substrates 1 and 3–5
are outlined in Table 3. Substrates 6 and 7 were resolved on a
larger scale, in a two-phase system consisting of toluene in addition
to Tris-sulfate buffer. Although some enzyme deactivation was
observed under these conditions, it remained possible to perform
these transformations using very low catalyst loadings: 1.5·10−4
mol% and 3.0·10−4 mol% for 6 and 7, respectively.
Resolution of substrate 6. To a mixture of 50 mL Tris-sulfate
(2M, pH 8.1) and 50 mL toluene was added a 1 : 1 w/w solution
of racemic 6 (2.32 g, 16.1 mmol) in DMF. Then, 225 lg HheC
were added. Since after 8 h the conversion turned out to proceed
slower than expected, another 255 lg HheC were added, followed
by another 176 lg after 32 h (total amount of enzyme: 656
lg). The reaction was stopped after 48 h. Flash chromatography
(SiO2, eluent pentane–Et2O 4 : 1, Rf(chloroalc) = 0.33) yielded 989 mg
(6.75 mmol, 42%) of (S)-5 with an ee of 98.5%.
Resolution of substrate 7. A 1 : 1 v/v solution of racemic 7
(20.9 g; 129 mmol) in DMF was added to a mixture of 1 L
Tris-sulfate (1M, pH 8.1) and 100 mL toluene. Subsequently, a
solution containing 2.68 mg of active HheC was added, followed
by another 1.99 mg after 7 h, 0.88 mg after 24.5 h, 1.395 mg after
33.5 h, 1.395 mg after 54 h, 1.53 mg after 76 h, and 0.396 mg
after 79.5 h (total amount of enzyme: 10.266 mg). The reaction
was worked up after 4 d by extraction with toluene, yielding 9.8 g
(60.3 mmol, 47%) of crude (S)-7 with an ee of >99%. To extract
the formed diol from the reaction mixture, the residual aqueous
layer was evaporated, and the resulting salt slurry extracted with
dimethoxypropane and ethyl acetate, yielding 9.1 g (63.1 mmol,
49%) of almost racemic 1-thiophen-2-yl-ethane-1,2-diol.
26
General procedure for enzymatic kinetic resolution on
analytical scale
To 20 mL of Tris-SO4 buffer (100 mM, pH 8.1) at room
temperature, 200 lL of a 1 M stock solution of substrate in DMF
were added. Then, 20 lL of a solution of HheC in TEMG24 were
added.25 Periodically, 1.0 mL aliquots were taken from the reaction
mixture, which were extracted with 1.0 mL of toluene containing
5.0 mM of dodecane as an internal standard. The resulting organic
solutions were then analyzed by chiral GC.
In the case of substrate 5, another internal standard was used
(cinnamyl alcohol, present in the reaction mixture instead of the
extraction solvent) as well as another extraction solvent, heptane.
Reactions with this substrate were analyzed by chiral HPLC.
1-Thiophen-2-yl-ethane-1,2-diol.
1H NMR (CDCl3) d 7.26–
7.24 (m, 1H), 7.00–6.96 (m, 2H), 5.03 (dd, J = 7.3, 3.7 Hz, 1H),
3.83–3.72 (m, 2H), 3.05 (br, 1H), 2.50 (br, 1H).
General procedure for enzymatic kinetic resolution on
preparative scale
Determination of absolute configuration
Typically, reactions were performed analogous to the procedure
described for the kinetic resolutions on analytical scale, but on a
scale of 1.0–2.0 mmol. This general procedure is for a reaction
on 2.0 mmol scale. To 200 mL of Tris-sulfate buffer (100 mM,
pH 8.1) at room temperature, 2.0 mL of a 1 M stock solution
of substrate in DMF were added. Then, 50 lL of a solution of
HheC in TEMG were added.25 When the reaction had finished, the
mixture was extracted with diethyl ether (or ethyl acetate if the aim
was to isolate the formed diol as well), the combined organic layers
dried on Na2SO4, filtered, and the solvents evaporated. The crude
The absolute configurations of the remaining enantiomers of the
chloroalcohols were determined using several methods.
For (S)-(E)-1-chloro-4-phenyl-but-3-en-2-ol (5), a crystal struc-
ture (Cl used as heavy atom) could be obtained17 (CCDC 605888,
CIF available as supplementary information).
Suitable crystals were obtained by slow diffusion of pentane
into a concentrated solution of (S)-5 in diethyl ether.
(S)-6 and (S)-7 could be correlated to known compounds by the
sign of their optical rotation.7,8
Table 3
Substrate
Scale/mmol
Enzyme/lg
Reaction time/h
Isolated yielda (mg, mmol, %)
(S)-1
(S)-3
(S)-4
(S)-5
1.0
2.0
1.5
2.0
200
250
300
740b
5
16
66
3
47, 0.40, 40
100, 0.62, 31
70, 0.44, 29
173, 0.95, 47
a Ee’s were >99% in each case. b A large amount of enzyme was used to shorten the reaction time.
322 | Org. Biomol. Chem., 2007, 5, 318–323
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6: [a]2D0 = +29.2 (c 0.452, CHCl3) → S (lit. (S)-6 [a]2D0 = +23.0 (c
0.52, CHCl3)).7
Spelberg, L. Tang, R. M. Kellogg and D. B. Janssen, Tetrahedron:
Asymmetry, 2004, 15, 1095; J. H. Lutje Spelberg, L. Tang, M. Van
Gelder, R. M. Kellogg and D. B. Janssen, Tetrahedron: Asymmetry,
2002, 13, 1083; L. Tang, J. E. T. Van Hylckama Vlieg, J. H. Lutje
Spelberg, M. W. Fraaije and D. B. Janssen, Enzyme Microb. Technol.,
2002, 30, 251; J. H. Lutje Spelberg, J. E. T. Van Hylckama Vlieg, T.
Bosma, R. M. Kellogg and D. B. Janssen, Tetrahedron: Asymmetry,
1999, 10, 2863.
6 M. Majeric´ Elenkov, B. Hauer and D. B. Janssen, Adv. Synth. Catal.,
2006, 348, 579; G. Hasnaoui, J. H. Lutje Spelberg, E. De Vries, L.
Tang, B. Hauer and D. B. Janssen, Tetrahedron: Asymmetry, 2005, 16,
1685; J. H. Lutje Spelberg, J. E. T. Van Hylckama Vlieg, L. Tang, D. B.
Janssen and R. M. Kellogg, Org. Lett., 2001, 3, 41.
7: Identified as S-7 using the sign of rotation (+). (Lit. (S)-7
[a]2D0 = +28.5 (c 0.53, CHCl3)).7
Compounds 1, 3, or 4 had not been described before in their
enantiomerically pure form. Therefore, they were converted by
hydrogenation to their saturated analogues, for which optical
rotations are known.18 Hydrogenation using Pd/C as a catalyst
proved unsatisfactory, therefore Wilkinson’s catalyst was em-
ployed (Reaction 1).
7 T. Hamada, T. Torii, K. Izawa and T. Ikariya, Tetrahedron, 2004, 60,
7411.
8 Z. Gercek, D. Karakaya and A. S. Demir, Tetrahedron: Asymmetry,
2005, 16, 1743.
9 T. Schubert, W. Hummel and M. Mueller, Angew. Chem., Int. Ed., 2002,
41, 634.
10 M. Lautens, M. L. Maddess, E. L. O. Sauer and S. G. Ouellet, Org.
Lett., 2002, 4, 83.
11 Per enzyme subunit, i.e. per active site. NB The active form of HheC is
a tetramer consisting of four identical subunits (28 kD per monomer);
each of the four monomers is catalytically active.
(1)
12 A. Fersht, Enzyme Structure and Mechanism, W. H. Freeman and
The unsaturated chloroalcohol (1 mmol) was dissolved in
4 mL methanol together with 5 mol% Wilkinson’s catalyst, and
this mixture was stirred until the catalyst had dissolved. After
various vacuum–N2 cycles, the reaction mixture was put under
an atmosphere of H2 (25 bar) and was allowed to react overnight.
Then it was filtered over a plug of silica, the solvent evaporated and
the residue analyzed. When the product was positively identified
as the saturated chloroalcohol, the crude material was purified by
flash chromatography, and the optical rotation measured.
company, New York, 2nd edn, 1985.
13 J. L. L. Rakels, B. Romein, A. J. J. Straathof and J. J. Heijnen, Biotechnol.
Bioeng., 1994, 43, 411.
14 In this case, the fast-reacting enantiomer reacts first. For an example
of the opposite behaviour, see: J. H. Lutje Spelberg, R. Rink, R. M.
Kellogg and D. B. Janssen, Tetrahedron: Asymmetry, 1998, 9, 459.
15 The computer program MicroMathꢀR ScientistꢀR was used.
16 C. S. Chen, Y. Fujimoto, G. Girdaukas and C. Sih, J. Am. Chem. Soc.,
1982, 104, 7294.
17 C10H11ClO, Mr = 182.65, orthorhombic, P212121, a = 4.9735(7), b =
3
−3
˚
˚
10.531(2), c = 17.521(3) A, V = 917.7(3) A , Z = 4, Dx = 1.322 g cm
,
F(000) = 384, l = 3.63 cm−1, k(MoKa) = 0.71073 A, T = 100(1) K, 7911
reflections measured, GooF = 1.078, wR(F2) = 0.0698 for 2109 unique
reflections and 153 parameters and R(F) = 0.0311 for 1994 reflections
obeying Fo 4.0 r(Fo) criterion of observability. The asymmetric unit
consists of one molecule of the title compound; the molecules are linked
into an infinite one-dimensional chain by O–H · · · O intermolecular
bonds. The chiral center of C9 showed the S-configuration‡.
18 T. Sakai, K. Wada, T. Murakami, K. Kohra, N. Imajo, Y. Ooga, S.
Tsuboi, A. Takeda and M. Utaka, Bull. Chem. Soc. Jpn., 1992, 65, 631.
19 For background literature on this phenomenon, see for instance: N. W.
Boaz, Tetrahedron: Asymmetry, 1995, 6, 15; H. E. Audier, J. F. Dupin
and J. Jullien, Bull. Soc. Chim. Fr., 1968, 3844.
˚
8 (from 1): [a]2D0 = +1.3 (c 4.6, CHCl3) → S (lit. (S)-8 [a]D20
=
+1.1 (c 2.9, CHCl3)).18
9 (from 3): [a]2D0 = +1.1 (c 6.5, CHCl3) → S (lit. (S)-9 [a]D20
+1.4 (c 3.1, CHCl3)).18
=
9 (from 4): Identified as (S)-9 using the sign of rotation (+).18
Acknowledgements
A. Meetsma is gratefully acknowledged for elucidating the crystal
structure of (S)-5. We thank A. J. A. Gayet, H. J. Heeres,
G. N. Kraai, C. Kronenburg, M. Schreuder Goedheijt, and
T. Sonke for valuable discussions, and T. D. Tiemersma-Wegman
for technical support. Financial support from NWO, the Ministry
of Economic Affairs, DSM, and N. V. Organon, administered
through the IBOS program, is gratefully acknowledged.
20 This compound has been reported in: A. N. Pudovik and B. E.
Ivanov, Zh. Obshch. Khim., 1956, 26, 1910, (Engl. Ed., 1956, 26, 2129);
Ponomarew et al., Zh. Obshch. Khim., 1957, 27, 1226, (Engl. Ed., 1957,
27, 1309).
21 M. Lautens and M. L. Maddess, Org. Lett., 2004, 6, 1883.
22 O. Grummitt and R. M. Vance, J. Am. Chem. Soc., 1950, 72, 2669; I. E.
Muskat and L. B. Grimsley, J. Am. Chem. Soc., 1930, 52, 1574.
23 A. Kamal, G. B. R. Khanna, R. Ramu and T. Krishnaji, Tetrahedron
Lett., 2003, 44, 4783; C. Corral, V. Darias, M. P. Ferna´ndez-Tome´, R.
Madron˜ero and J. Del R´ıo, J. Med. Chem., 1973, 16, 882; H. Hopff and
R. Wandeler, Helv. Chim. Acta, 1962, 45, 982.
References
1 N. Kasai, T. Suzuki and Y. Furukawa, J. Mol. Catal. B: Enzym., 1998,
4, 237.
2 A. J. Van den Wijngaard, D. B. Janssen and B. Witholt, J. Gen.
Microbiol., 1989, 135, 2199.
24 The buffer solution consisted of 0.15 M (NH4)2SO4 in TEMG (10 mM
Tris-sulfate, pH 7.5, 3 mM EDTA, 0.1% 2-mercaptoethanol, 10%
glycerol).
25 The solution contained 3.0–6.0 mg ml−1 of active enzyme. Thus, the
amount of HheC used was 60–120 mg for analytical scale resolutions
and 150–300 mg for preparative scale resolutions. The actual amount
of active HheC in each individual resolution was checked prior to
the reaction by a spectrophotometric assay, according to a method
described in: J. H. Lutje Spelberg, L. Tang, M. Van Gelder, R. M.
Kellogg and D. B. Janssen, Tetrahedron: Asymmetry, 2002, 13, 1083.
26 M. P. Sibi and R. Sharma, Synlett, 1992, 497.
3 R. M. De Jong, J. J. W. Tiesinga, A. Villa, L. Tang, D. B. Janssen
and B. W. Dijkstra, J. Am. Chem. Soc., 2005, 127, 13338; R. M. De
Jong, H. J. Rozeboom, K. H. Kalk, L. Tang, D. B. Janssen and B. W.
Dijkstra, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2002, 58, 176;
J. E. T. Van Hylckama Vlieg, L. Tang, J. H. Lutje Spelberg, T. Smilda,
G. J. Poelarends, T. Bosma, A. E. J. Van Merode, M. W. Fraaije and
D. B. Janssen, J. Bacteriol., 2001, 183, 5058.
4 L. Tang, A. E. J. Van Merode, J. H. Lutje Spelberg, M. W. Fraaije
and D. B. Janssen, Biochemistry, 2003, 42, 14057; L. Tang, J. H. Lutje
Spelberg, M. W. Fraaije and D. B. Janssen, Biochemistry, 2003, 42,
5378.
5 L. Tang, D. E. Torres Pazmin˜o, M. W. Fraaije, R. M. De Jong, B. W.
Dijkstra and D. B. Janssen, Biochemistry, 2005, 44, 6609; J. H. Lutje
‡ CCDC reference number 605888. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b613937j
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The Royal Society of Chemistry 2007
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