Stereoselective prostereogenic 3-oxo ester reduction mediated by a novel yeast alcohol dehydrogenase derived from Kluyveromyces marxianus CBS 6556

Article abstract of DOI:10.1002/adsc.200600379

A yeast alcohol dehydrogenase (ADH) has been purified up to a purification factor value of 21,731-fold from Kluyveromyces marxianus CBS 6556. The purification procedure consisted of two chromatographic steps (DEAE-anion exchange and affinity chromatography). The optimal pH was 7, its optimal temperature was 40°C and its co-factor was NADPH. This novel ADH efficiently mediated the reduction of 3-oxo esters with a high degree of stereoselectivity, providing chiral alcohols having the (S) absolute configuration at the newly formed stereogenic centre by delivering the hydride from the re-face of the prochiral carbonyl compounds.

Full text of DOI:10.1002/adsc.200600379

FULL PAPERS
DOI: 10.1002/adsc.200600379
Stereoselective Prostereogenic 3-Oxo Ester Reduction Mediated
by a Novel Yeast Alcohol Dehydrogenase Derived from
Kluyveromyces marxianus CBS 6556
a
a
a,
Maria Grazia Perrone, Ernesto Santandrea, Antonio Scilimati, *
and Christoph Syldatk
a
b
Dipartimento Farmaco-Chimico, Università di Bari, Via E.Orabona 4, 70125 Bari, Italy
Fax : (+39)-080-544-2231; phone: (+39)-080-544-2762; e-mail: ascilimati@farmchim.uniba.it
Universität Karlsruhe (TH), Engler-Bunte-Institut, Lehrstuhl für Technische Biologie, Engler-Bunte-Ring 1,
b
76131 Karlsruhe, Germany
Received: July 26, 2006
Abstract: A yeast alcohol dehydrogenase (ADH) has reoselectivity, providing chiral alcohols having the
been purified up to a purification factor value of (S) absolute configuration at the newly formed ste-
2
6
1,731-fold from Kluyveromyces marxianus CBS reogenic centre by delivering the hydride from the
556. The purification procedure consisted of two re-face of the prochiral carbonyl compounds.
chromatographic steps (DEAE-anion exchange and
affinity chromatography). The optimal pH was 7, its
optimal temperature was 408C and its co-factor was Keywords: alcohol dehydrogenase; asymmetric bio-
NADPH. This novel ADH efficiently mediated the reduction; fermentation; Kluyveromyces marxianus;
reduction of 3-oxo esters with a high degree of ste- 3-oxo esters
Introduction
cheese whey as a cheap source of nutrients for its cul-
[
11]
tivation. It has been investigated for the prepara-
Nowadays the use of biological catalysts in the syn- tion of useful intermediates in the synthesis of phar-
[
12]
thesis of pharmaceuticals, fine chemicals and chiral in- maceuticals
but very few works have been pub-
lished describing its usefulness in the asymmetric re-
Most of the work, published in the last thirty years in duction of prostereogenic ketones and kinetic resolu-
[
1]
termediates for drug development is very common.
[
10,13–21]
this field, has been focused on finding biocatalytic sys- tion.
tems, microorganisms or isolated enzymes, with the
Herein, we report the partial purification of an al-
ability of promoting the formation of new stereogenic cohol dehydrogenase (ADH) from Kluyveromyces
centres with high stereoselectivity in achiral mole- marxianus CBS 6556, responsible for such a highly
cules.
One of the most investigated processes is the asym- comparison with an ADH enzyme isolated from a dif-
enantioselective reduction of 1 (Scheme 1) and its
[
2–8]
metric bioreduction of prostereogenic ketones.
ferent strain of the same yeast, showing a reverse ste-
Many research groups have focused their attention on reochemical preference in the bioreduction of ethyl
[
14]
searching for microorganisms able to accomplish such acetoacetate and ethyl 2-(phthalimido)methyl-3-ox-
[
15–16]
a biotransformation, which may represent a valuable obutanoate (3, Scheme 2).
alternative to the deeply investigated Saccharomyces
[9]
cerevisiae.
Recently, we reported that the yeast Kluyveromyces Results and Discussion
marxianus CBS 6556 reduced ethyl 2-(4-chlorophen-
oxy)-3-oxobutanoate (1) affording nearly enantiomer- The reduction of ethyl 2-(4-chlorophenoxy)-3-oxobu-
ically pure ethyl (2R,3S)-2-(4-chlorophenoxy)-3-hy- tanoate (1) was performed using growing and resting
[
10]
droxybutanoate (2, Scheme 1).
marxianus is not a widely used biocatalyst in organic (Scheme 1). The results are summarised in Table 1.
synthesis as yet. This yeast is known for its capacity of Such results prompted us to develop a method for
Kluyveromyces cells of Kluyveromyces marxianus CBS 6556
[10]
growing on lactose as a sole carbon and energy the isolation and purification of the alcohol dehydro-
source, which presents the possibility of utilizing genase (ADH) involved in the above-mentioned bio-
Adv. Synth. Catal. 2007, 349, 1111 – 1118
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Maria Grazia Perrone et al.
FULL PAPERS
Scheme 1. Bioreduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate (1).
Scheme 2. Bioreduction of ethyl 2-(phthalimido)methyl-3-
oxobutanoate (3) by Kluyveromyces marxianus KCTC 7155.
Table 1. Results of growing and resting cells of Kluyveromy-
ces marxianus CBS 6556-mediated reduction of ethyl 2-(4-
chlorophenoxy)-3-oxobutanoate (1).
Figure 1. pH optimization: crude extract (500 mL), 0.1M
KH PO (at the defined pH value) (500 mL), 3 mM NADPH
[10]
2
4
[a]
[b]
[c]
(34.2 mL) and 1 (11 mL) in absolute ethanol (c=0.1 mM)
were incubated at 308C. Reaction progress was monitored
by GC analysis.
Reaction con- Reaction
Yield
[%]
de
ee(2R,3S)
[%]
ditions
time [h]
[%]
Growing cells
Resting cells
8
1.5
73
84
>99
>99
86
97
[
[
a]
b]
Yield refers to the product isolated by chromatography.
Diastereoisomeric excesses (syn>anti pair) determined
1
by H NMR.
[
c]
Enantiomeric excesses were determined by HPLC.
reduction. Before starting the purification procedure,
attempts were performed on the crude extract, ob-
tained by mechanical disruption of Kluyveromyces
marxianus CBS 6556 cells, to assess the best pH, tem-
perature and conditions for the bioreduction of ethyl
2
-(4-chlorophenoxy)-3-oxobutanoate (1). As shown in
Figure 1, 2 was quantitatively obtained when 0.1M
KH PO buffer solution (pH 7.0) was employed. In
Figure 2. Temperature optimization: crude extract (500 mL),
0.1M KH PO (500 mL) (pH 7.0), 3 mM NADPH (34.2 mL)
2
4
2
4
this case, the reaction was complete within 45 min. and 1 (11 mL) in absolute ethanol (c=0.1 mM) were incu-
Reaction times exceeded this time at all other pH bated at the defined temperature. Reaction progress was
values.
Once the best pH value had been found, the biore-
duction was performed at different temperatures
Figure 2). As it can be seen, at 408C the reaction was tion factor value of 362. The specific activity was mea-
complete within 30 min. At the temperatures above sured by monitoring the reduction of 2-(4-chlorophe-
08C the reaction rate was lower. noxy)-3-oxobutanoate (1) with GC (Scheme 1). The
The isolation/purification procedure consisted of active fractions were then further purified by an affin-
monitored by GC analysis.
(
4
two steps, performed by chromatography using first ity chromatography, using a 2’5’-ADP Sepharose 4B
an anion exchange and then an affinity resin affinity resin, which shows high affinity towards
(
Table 2).
NADPH-dependent enzymes. Specific activity in-
In the first step, the crude extract was partially pu- creased to 4,781 U/mg, leading to a 21,731-fold total
rified over a DEAE-Sepharose anion exchange resin. purification. However, in spite of the very high purifi-
The specific activity increased from 0.22 U/mg (in the cation factor value, the desired enzyme was found to
crude extract) to 79.6 U/mg protein, with a purifica- be only partially purified, since three spots were pres-
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Adv. Synth. Catal. 2007, 349, 1111 – 1118

Stereoselective Prostereogenic 3-Oxo Ester Reduction
FULL PAPERS
Table 2. Purification steps of the novel ADH from Kluyver-
omyces marxianus CBS 6556, detected in fermentative re-
duction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate (1).
The absolute configuration of 2 formed in the bio-
reduction performed in the presence of K. marxianus
was established to be (2R,3S) by single crystal X-ray
analysis of the corresponding acid (quantitatively ob-
[a]
[d]
Step
Protein
mg]
Total[
Specific ac- Purification
[c]
[
10]
b]
tained by treating 2 with KOH) (Figure 4). All the
other absolute configurations were assessed by HPLC
and single crystal X-ray analysis of authentic samples
[
units
tivity [U/
mg]
A
T
E
N
(fold)
[
Unit]
Crude ex- 42
tract
9.24
0.22
79.6
–
[22]
prepared from threonines as previously described.
The observed stereoselectivity is different from the
findings of Koh, who patented a novel ADH isolated
from Kluyveromyces marxianus KCTC 7155, which
produced a mixture of ethyl (2R,3R)- and (2S,3R)-2-
DEAE Se-
pharose
5
398
76.5
362
2
’5’-ADP
0.016
4,781
21,731
Sepharose
(
phthalimido)methyl-3-hydroxybutanoate (4) when
4B
catalysing the reduction of its precursor ethyl 2-
[
[
[
[
a]
Protein content was determined using the Bradford
method (see Experimental Section).
Total units were determined by multiplying specific activ-
ity by protein content.
Specific activity at each step was determined by a spec-
trophotometric assay.
(
(
phthalimido)methyl-3-oxobutanoate
Scheme 2).
(3)
[15–16]
b]
We then investigated the stereochemical behaviour
of the ADH isolated, by using our purification proto-
col from Kluyveromyces marxianus CBS 6556, in the
c]
d]
Purification factor value is the ratio between specific ac- bioreduction of 3. This reaction was performed under
tivity at a given step and specific activity of the crude ex- different conditions. The substrate 3 was first incubat-
tract.
ed in the presence of growing cells of Kluyveromyces
marxianus CBS 6556 which gave 4 with 20% diaste-
reomeric excess (de) and enantiomeric excess
ent in the active fraction, as evidenced by SDS-PAGE ee(2R,3S)/(2S,3R) =22% and ee(2R,3R)/(2S,3S) =82% (Fig-
analysis. ure 5B).
The obtained enzyme solution was used to perform Absolute configurations were assessed by compari-
the bioreduction of 1. As expected, only (2R,3S)-2 son with HPLC data reported by Koh and co-work-
[
3]
[15]
formed (Figure 3). These results suggest that the ers. As reported in the mentioned work the main
identified enzyme is accountable for the highly stereo- formed stereoisomers had absolute configurations
selective reduction of 1 observed in the presence of (2R,3R) and (2R,3S), even though a different strain of
[
10]
Kluyveromyces marxianus CBS 6556 whole cells.
the microorganism was used.
Figure 3. (A) HPLC chromatogram of diastereoisomeric mixture of ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate (2) ob-
tained by NaBH reduction of ethyl 2-(4-chlorophenoxy)-3-oxobutanoate (1), and (B) HPLC chromatogram of ethyl (2R,3S)
4
2-(4-chlorophenoxy)-3-hydroxybutanoate (2) from reduction of racemic ethyl 2-(4-chlorophenoxy)-3-oxobutanoate (1) car-
ried out in the presence of the enzyme sample obtained after two step anion-exchange/affinity chromatography within 1
hour (Table 2).
Adv. Synth. Catal. 2007, 349, 1111 – 1118
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Ethyl 2-(phthalimido)methyl-3-oxobutanoate (3),
was also reduced in the presence of bakerꢁs yeast,
which is known to generally produce (S)-3-hydroxy
[
3,23]
compounds by reducing 3-oxoesters.
these conditions the expected products were indeed
formed [de=29%, ee(2R,3S)/(2S,3R) >99% and
ee(2R,3R)/(2S,3S) >99%] (Figure 6).
In fact, under
Interestingly, the reduction of 3 carried out with the
enzyme isolated from Kluyveromyces marxianus CBS
6556 under the above described conditions yielded
the product with opposite stereochemistry, (2R,3S)
and (2S,3S) being the main stereoisomers formed
(
9
de=35%, ee(2R,3S)/(2S,3R) =88% and ee(2R,3R)/(2S,3S)
9%) (Figure 7).
In this study, Kluyveromyces marxianus CBS 6556
>
Figure 4. ORTEP view of the asymmetric unit with the
atomic numbering scheme of (2R,3S)-2-(4-chlorophenoxy)-
was selected for its outstanding reducing properties
towards keto esters of our interest when compared
3-hydroxybutanoic acid. Thermal ellipsoids probability level
at 30%.
Figure 5. (A) HPLC chromatogram of diastereoisomeric mixture of ethyl 2-(phthalimido)methyl-3-hydroxybutanoate (4) ob-
tained by NaBH reduction of ethyl 2-(phthalimido)methyl-3-oxobutanoate (3), and (B) HPLC chromatogram of ethyl 2-
4
(
phthalimido)methyl-3-hydroxybutanoate (4) obtained carrying out the bioreduction of 3 in the presence of growing Kluy-
veromyces marxianus CBS 6556 cells.
Figure 6. HPLC chromatogram of ethyl 2-(phthalimido)methyl-3-hydroxybutanoate (4) obtained from bioreduction of 3 in
the presence of bakerꢁs yeast.
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Stereoselective Prostereogenic 3-Oxo Ester Reduction
FULL PAPERS
Figure 7. HPLC chromatogram of ethyl 2-(phthalimido)methyl-3-hydroxybutanoate (4) obtained from bioreduction of 3 per-
formed in the presence of the ADH isolated from Kluyveromyces marxianus CBS 6556 following the procedure summarized
in Table 2, and detailed in the Experimental Section.
[
16]
with other yeast strains, including bakerꢁs yeast. The (de=60%).
These data led us to the conclusion
isolated enzyme shows an improvement in the rate of that the enzyme we have identified and partially puri-
the reduction of 1 and allows the quantitative conver- fied from Kluyveromyces marxianus CBS 6556 is a
sion of the keto ester avoiding the formation of by- different ADH endowed with opposite stereochemical
products as observed when performing the reaction preference with respect to the one already reported,
with different yeast strains: the use of whole cell cata- and that the yeast Kluyveromyces marxianus CBS
lyst could have the disadvantage of the presence of an 6556 constitutes a useful tool for the preparation of
enzymatic pool competing with the dehydrogenase af- enantiomerically pure ethyl (2R,3S)-2-(4-chlorophen-
[
23,24]
fording undesidered products.
Obtaining enantiopure compounds is a challenging
oxy)-3-hydroxybutanoate (2).
Wider investigations are currently in progress
task in organic chemistry, due to elevated cost of the aimed at exploiting the substrate specificity and syn-
reactant and/or starting materials. On the other hand, thetic applications of Kluyveromyces marxianus CBS
the preparation of optically active compounds is 6556 and the novel ADH, also compared to other
highly important in various synthetic preparative or- well-known biocatalysts able to mediate the bioreduc-
ganic chemistry areas. In this sense, the possibility of tion reaction. Further work is also ongoing to defini-
a readily available reducing agent capable of reducing tively purify the novel identified ADH from Kluyver-
substrates like 1 in quantitative yield, high enantio- omyces marxianus CBS 6556.
meric excess and in short times proves essential.
Experimental Section
Conclusions
A Labfors Fermenter, equipped with all features necessary
for a long-term sterile operations, with 3.6-L total volume
and 2.5-L working volume was used. All the major variables
related with the fermentation processes were monitored and
An NADPH-dependent alcohol dehydrogenase
(
ADH) was partially purified from Kluyveromyces
marxianus CBS 6556.
It is able to perform the bioreduction of 1 yielding
the nearly enantiopure 3-hydroxy ester 2 [de>99%,
ee(2R,3S) =97%]. The best identified reaction condi-
stored by computer.
[25]
Ethyl 2-(4-chlorophenoxy)-3-oxobutanoate (1)
ethyl 2-(phthalimido)methyl-3-oxobutanoate (3) were pre-
and
[15]
tions were 408C in 0.1M KH PO buffer solution
2
4
pared as previously reported. Spectroscopic and analytical
(
pH 7.0). Under these conditions, the reaction was data of ethyl 2-(4-chlorophenoxy)-3-hydroxybutanoate (2)[25]
complete within 30 min.
and ethyl 2-(phthalimido)methyl-3-hydroxybutanoate (4)
On the other hand, the reduction of 3-oxo ester 3 are consistent with those already reported. Reaction prog-
[15]
ress was monitored by TLC and GC analysis. Thin-layer
performed by using our ADH (Table 2 and Figure 1),
produced ethyl (2R,3S)- and (2S,3S)-2-(phthalimido)-
methyl-3-hydroxybutanoate (4) as the main stereo-
chromatography (TLC) was performed on silica gel sheets
with a fluorescent indicator (Stratochrom SIF, 60 F254
Merck); TLC spots were observed under ultraviolet light or
isomers
[de=35%,
ee(2R,3S)/(2S,3R) =88%
and
visualised with I vapour. GC analyses were performed by
2
ee(2R,3R)/(2S,3S) >99%]. These results are different from
those obtained by Koh and co-workers, who used
an ADH from Kluyveromyces marxianus KCTC 7155
which produces a mixture of (2R,3R)-4 and (2S,3R)-4 Hewlett Packard 6890–5793 MSD. Column chromatography
using an HP5 column (methyl silicone gum; 30 m”
[
15]
0
.25 mm”250 mm film thickness) on a Hewlett Packard 5890
model, Series II. GC-MS analyses were performed on a
with a diastereoisomeric ratio (2R,3R)/(2S,3R)=80:20 was performed using silica gel Merck 60 (0.063–0.200 mm).
AHCTREUNG
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Maria Grazia Perrone et al.
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1
H NMR spectra were recorded in CDCl on a Varian Mer-
were extracted by adding ethyl acetate (1 mL) and then cen-
3
cury 300 MHz spectrometer.
trifuged at 10,000 g for 5 min. The supernatant (organic
phase) was separated, dried over anhydrous Na SO4 and
The ees and absolute configurations of the products were
determined by HPLC analysis performed on a Perkin–
Elmer 200 series with a UV/Vis detector 785 A and a com-
mercially available Chiralcel OD (Daicel) column under iso-
cratic conditions employing n-hexane/2-propanol=98:2 as
mobile phase, flow rate 1 mL/min, l=230 nm for 2 and n-
hexane/2-propanol=95:5, flow rate 1 mL/min, l=230 nm
2
then analysed by GC and TLC. The reaction was stopped
after 24 h. The content of the flask was centrifuged and the
supernatant extracted three times with ethyl acetate.
Purified ADH-mediated reduction: NADPH (6 mg,
0
.007 mmol) in 10 mM phosphate buffer pH 6.8 (25 mL) and
3
(0.2 mmol) were added to the enzyme solution obtained
by affinity chromatography (5 mL, Table 2). The suspension
was shaken at 308C for 11 days, monitoring the progress of
the reaction by GC and TLC. The crude product was ex-
tracted with ethyl acetate after saturation of the suspension
with NaCl. The organic phase was dried over anhydrous
Na SO and the solvent evaporated under reduced pressure.
for 4. The retention time (t ) of each stereoisomer was de-
R
termined by using as t 8 the retention time of the mobile
R
phase prepared replacing i-PrOH with i-PrOH-d revealed
8
by the refractive index detector.
Optical density was measured at 620 nm by a Perkin–
Elmer UV/Vis Lambda Bio 20 spectrophotometer.
2
4
DEAE Sepharose and 2’5’-ADP Sepharose 4B were pur-
chased from Amersham Biosciences.
Determination of Protein Content
The protein content was determined by using the method of
Bradford, calibrated by bovine serum albumin (BSA) as a
standard (Bio-Rad Protein Assay kit).
[26]
Microorganism Sources
Bakerꢁs yeast used to reduce the 3-oxoesters 1 and 3 was
supplied by Lievitalia. Kluyveromyces marxianus CBS 6556
was obtained from a public type culture collection (CBS).
Evaluation of Enzyme Activity
The enzyme activity was evaluated using both spectrophoto-
metric (method A) and GC (method B) method.
Culture Medium
Method A: spectrophotometric assay: The enzyme activi-
ty was determined by monitoring the decrease of absorb-
ance at 340 nm during the reduction of ethyl 2-(4-chlorophe-
noxy)-3-oxobutanoate (1). The standard assay mixture con-
tained, in a total volume of 1.0 mL, the enzyme solution
The microorganisms were cultivated under aerobic condi-
tions in a medium containing 0.3% yeast extract, 0.3% malt
extract, 0.5% peptone, 1% glucose. 2% Agar-Agar was
added to the same medium for cells preservation on agar
slants.
(
5
0.89 mL), 1 (0.01 mL of 100 mM ethanol solution of 1:
0 mg 1 in 2 mL EtOH) and 1 mM NADPH (0.1 mL, solu-
tion prepared in KH PO buffer at pH 6.5).
Procedures for the Bioreduction of Ethyl 2-
2
4
(
Phthalimido)methyl-3-oxobutanoate (3)
Method B: GC assay: The enzyme activity was deter-
mined by measuring the percentage of product 2 formation
after 1 h shaking suspension at 408C. The standard assay
mixture contained the enzyme solution (1 mL), 1 (11 mL,
Bakerꢀs yeast-mediated reduction: Bakerꢁs yeast (17 g) was
dispersed to give a smooth paste in tap water (32 mL). Ethyl
2
-(phthalimido)methyl-3-oxobutanoate (3) (200 mg) was
then added to the suspension which was then stirred at
50 rpm and kept at 378C. The reaction progress was moni-
1
00 mM ethanol solution of 1: 50 mg of 1 in 2 mL EtOH)
and NADPH (35 mL) equimolar with 1.
2
tored by GC analysis: 0.5 mL samples were extracted by
adding ethyl acetate (1 mL) and then centrifuged at 10,000 g
for 5 min. The supernatant (organic phase) was separated,
dried over anhydrous Na SO and then analysed by GC and
SDS-PAGE Analysis
Purification steps were analysed using SDS-PAGE on 10%
polyacrylamide gels, and the protein bands were stained
2
4
TLC. The reaction was stopped after 24 h. The reaction mix-
ture was extracted several times with EtOAc. The extracts
were combined and dried over anhydrous Na SO . A yellow
with either Coomassie Brilliant Blue R-250 or silver stain
ꢂ
(
Sigma ProteoSilver Silver Stain Kit).
2
4
oil was obtained after removal of the solvent under reduced
pressure.
Kluyveromyces marxianus CBS 6556 whole growing
cells-mediated reduction: Cells preserved on agar slants at
Purification
Purifications were performed at 48C.
4
8C were used to inoculate 250-mL flasks containing the
Buffers: Buffer A. 0.01M KH PO (pH 6.5). Buffer B.
2
4
cultivation medium (100 mL). The flasks were incubated
0.01M KH PO (pH 6.5), 0.4M NaCl. Buffer C. 0.01M
2 4
aerobically at 308C on an orbital shaker and stirred at
KH PO (pH 6.5), 2M NaCl.
2 4
2
(
50 rpm. 250-mL flasks containing the cultivation medium
100 mL) were then inoculated with 5 mL of the 24-h-old
suspension and incubated in the same conditions for 24 h. A
-L flask containing the cultivation medium (400 mL) was
Step 1. Extraction: Kluyveromyces marxianus (24.5 g, wet
mass weight) obtained from fermentation was washed twice
with Buffer A (60 mL) and then centrifuged at 7,500 g for
5 min. The wet mass was suspended in Buffer A to a final
concentration of 60% w/vand the cells were disrupted at
08C with 0.8 mm glass beads for 10 min. Glass beads were
removed and the suspension was centrifuged at 12,500 g for
20 min at 08C.
1
then inoculated with 5 mL of the latter suspension and incu-
bated for 24 h and 3 (200 mg) dissolved in ethanol (1 mL)
was added when the culture had the optical density of 7.2.
The reaction was monitored by GC analysis: 0.5 mL samples
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Adv. Synth. Catal. 2007, 349, 1111 – 1118

Stereoselective Prostereogenic 3-Oxo Ester Reduction
FULL PAPERS
Step 2. DEAE-Sepharose chromatography: The crude ex-
tract (42.5 mL) was passed through a DEAE-Sepharose
column, equilibrated with Buffer A. The adsorbed enzyme
was eluted for the first 60 min only with Buffer A and then
with a mixture of Buffer A and Buffer B (0–33% Buffer B
in 30 min; 33% Buffer B for 20 min; 33–100% Buffer B in
[2] C. J. Sih, C. S. Chen, Angew. Chem. Int. Ed. Engl. 1984,
23, 570–578.
[3] S. Servi, Synthesis 1990, 1–25.
[4] R. Csuk, B. I. Glanzer, Chem. Rev. 1991, 91, 49–97.
[5] O. P. Ward, C. S. Young, Enzyme Microb. Technol.
1990, 12, 482–493.
30 min). The active fractions were collected and diluted with
[6] T. Ema, H. Yagasaki, N. Okita, M. Takeda, T. Sakai,
Buffer A to 50% v/v (final volume 100 mL).
Step 3. 2’5’-ADP Sepharose 4B: The enzyme solution
Tetrahedron 2006, 62, 6143–6149.
[7] K. Nakamura, Y. Kawai, T. Miyai, S. Honda, N. Nakaji-
ma, A. Ohno, Bull. Chem. Soc. Jpn. 1991, 64, 1467–
1470.
[8] K. Nakamura, R. Yamanaka, T. Matsuda, T. Harada,
Tetrahedron: Asymmetry 2003, 14, 2659–2681.
(
100 mL) was passed through a 2’5’-ADP Sepharose 4B
column and equilibrated with Buffer A. The adsorbed
enzyme was eluted for the first 60 min with Buffer A and
then with a mixture of Buffer A and Buffer C (0–33%
Buffer C in 20 min; 33% Buffer C for 30 min; 33–100%
Buffer C in 30 min). The active fractions were collected.
[
9] I. Kaluzna, T. Matsuda, A. K. Sewell, J. D. Stewart, J.
Am. Chem. Soc. 2004, 126, 12827–12832.
[
10] M. G. Perrone, E. Santandrea, A. Scilimati, C. Syldatk,
V. Tortorella, F. Capitelli, V. Bertolasi, Tetrahedron:
Asymmetry 2004, 15, 3511–3517.
X-Ray Analysis
To establish the absolute configuration at C-7 and C-9 in an
unambiguous manner, suitable crystals were grown and sub-
jected to single crystal X-ray analysis, using a Nonius Kappa
CCD area detector diffractometer equipped with a fine
[
[
[
11] S. Grba, V. Stehlik-Tomas, D. Stanzer, N. Vah cˇ i c´ , A.
Skrlin, Chem. Biochem. Eng. Q. 2002, 16, 13–16.
12] B. Rosche, M. Breuer, B. Hauer, P. L. Rogers, Biotech-
nology Lett. 2003, 25, 841–845.
13] M. G. Perrone, E. Santandrea, A. Scilimati, C. Syldatk,
V. Tortorella, Tetrahedron: Asymmetry 2004, 15, 3511–
ˇ
focus sealed graphite-monochromated Mo-K radiation (l=
a
0.71073 Š). Data for (2R,3S)-2-(4-chlorophenoxy)-3-hydrox-
ybutanoic acid were collected at 293(2) K. Data reduction
and cell refinement were carried out with the programs
3
517.
[
27]
[28]
[14] J. B. Ribeiro, J.Mol. Cat. B: Enzymatic 2003, 24–
25,121–124.
15] H. Y. Koh, Y.-S. Han, J.-H. Cha, W. H. Kim, J.-S. Lee,
DENZO and COLLECT. The structure was solved by
[29]
the direct methods procedure of SIR97, while the refine-
ment processes were carried on full matrix least squares
technique using SHELXL-97.
[
[
30]
H.-C. Yun, US Patent 170,844, 2003.
Detailed crystal data and
[31]
[16] J. H. Cha, A. N. Pae, K. I. Choi, Y. S. Cho, W. H. Kim,
Y. S. Han, H.-C. Yun, J. Lee, H. Y. Koh, E. Lee, Bio-
technology Lett. 2002, 24, 1695–1698.
geometrical parameters have been deposited (cif file).
The asymmetric unit of (2R,3S)-2-(4-chlorophenoxy)-3-hy-
droxybutanoic acid with the atomic numbering scheme is
depicted in Figure 4.
[
17] F. Molinari, E. G. Occhiato, F. Aragozzoni, A. Guarna,
Tetrahedron: Asymmetry 1998, 9, 1389–1394.
18] F. Molinari, C. Bertolini, F. Aragozzoni, Annali di Mi-
crobiologia ed Enzimologia 1997, 47, 131–137.
Pertinent crystallographic data for (2R,3S)-2-(4-chloro-
phenoxy)-3-hydroxybutanoic acid: C H ClO , M =230.64
[
1
0
11
4
r
ꢀ
3
g·cm , orthorhombic, space group: P2 2 2, a=13.2109(4),
1
1
3
[19] F. Molinari, K. Solange Cavenago, A. Romano, D.
b=24.8989(9), c=6.8380(2) Š, cell volume=2244.75(12) Š ,
ꢀ
3
ꢀ1
Romano, R. Gandolfi, Tetrahedron: Asymmetry 2004,
Z=8, T=293(2) K, 1 =1.365 g·cm , m=0.331 mm , q
c
1
5, 1945–1947.
20] F. Molinari, R. Gandolfi, European Patent EP 1413627,
004.
range=2.258-27.528, hkl indices ꢀ17ꢁhꢁ17,ꢀ31ꢁkꢁ32,
[
[
ꢀ
8ꢁlꢁ8, reflections (measured)=12,009, reflections
2
(
unique)=5031, reflections (unique [F >2s
ACHTREUNG
{jF j}]): 2563,
o
o
21] J. B. Ribeiro, L. M. Andrade de Sousa, M. da Volta
Soares, M. da Conceiżo, Klaus V. Ramos, F. Radler
de Aquino Neto, C. A. Mansour Fraga, S. G. Ferreira
Leite, Y. Cordeirod, O. A. C. Antunes, Tetrahedron:
Asymmetry 2006, 17, 984–988.
Rint =0.052, 344 parameters, R /wR (all data): 0.1433/0.1059,
1
2
R /wR2 (I>2s(I)): 0.0529/0.0824, Flack parameter=
1
3
ꢀ
0.02(8), largest diff. peak/hole: 0.162/ꢀ0.245 e·Š .
[
[
[
[
22] M. G. Perrone, E. Santandrea, L. Di Nunno, A. Scili-
mati, V. Tortorella, F. Capitelli, V. Bertolasi, Tetrahe-
dron: Asymmetry 2005, 16, 783–792.
Acknowledgements
23] M. G. Perrone, E. Santandrea, A. Scilimati, V. Tortorel-
Work carried out under the framework of the National Proj-
ect “Progettazione, Sintesi e Valutazione Biologica di Nuovi
Farmaci Cardiovascolari” supported by the Ministero dell’U-
niversità e della Ricerca (MUR, Rome). Thanks are also due
to the University of Bari and CNR-Istituto di Chimica dei
Composti OrganoMetallici-ICCOM/Sezione di Bari (Italy).
la, F. Capitelli, V. Bertolasi, Tetrahedron: Asymmetry
2
004, 15, 3501–3510.
24] M. G. Perrone, E. Santandrea, A. Scilimati, C. Syldatk,
V. Tortorella, Tetrahedron: Asymmetry 2005, 16, 1473–
1
477.
25] M. G. Perrone, E. Santandrea, N. Dell’Uomo, F. Gian-
nessi, F. M. Milazzo, A. F. Sciarroni, A. Scilimati, V.
Tortorella, Eur. J. Med. Chem. 2005, 40, 143–154.
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