Biotechnological development of a practical synthesis of ethyl (S)-2-ethoxy-3-(p -methoxyphenyl)propanoate (EEHP): Over 100-fold productivity increase from yeast whole cells to recombinant isolated enzymes

Article abstract of DOI:10.1021/op200085k

The coupling of the enantioselective reduction catalyzed by Old Yellow Enzymes (OYEs), together with the in situ substrate feeding product removal (SFPR) concept, significantly improved the productivity of the g-scale preparation of ethyl (S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP), an important precursor of several PPAR-α/γ agonists, such as Tesaglitazar. The OYEs and the glucose dehydrogenase for cofactor regeneration were cloned, overexpressed in Escherichia coli, and purified. The synthetic sequence was completed by a NaClO₂ oxidation employing cheap and environmentally friendly conditions. The product was obtained in 94% yield and with an ee of 98% over the two steps.

Full text of DOI:10.1021/op200085k

ARTICLE
pubs.acs.org/OPRD
Biotechnological Development of a Practical Synthesis of Ethyl
(S)-2-Ethoxy-3-(p-methoxyphenyl)propanoate (EEHP): Over 100-Fold
Productivity Increase from Yeast Whole Cells to Recombinant
Isolated Enzymes
Matthias Bechtold,† Elisabetta Brenna,‡ Christian Femmer,† Francesco G. Gatti,*,‡ Sven Panke,†
Fabio Parmeggiani,*,‡ and Alessandro Sacchetti‡
†Department of Biosystems Science and Engineering (D-BSSE), ETH Z€urich, Mattenstrasse 26, CH-4058, Basel, Switzerland
‡Dipartimento di Chimica, Materiali e Ingegneria Chimica “G. Natta”, Politecnico di Milano, Via Mancinelli 7, I-20131, Milano, Italy
S Supporting Information
b
ABSTRACT: The coupling of the enantioselective reduction catalyzed by Old Yellow Enzymes (OYEs), together with the in situ
substrate feeding product removal (SFPR) concept, significantly improved the productivity of the g-scale preparation of ethyl (S)-2-
ethoxy-3-(p-methoxyphenyl)propanoate (EEHP), an important precursor of several PPAR-R/γ agonists, such as Tesaglitazar. The
OYEs and the glucose dehydrogenase for cofactor regeneration were cloned, overexpressed in Escherichia coli, and purified. The
synthetic sequence was completed by a NaClO2 oxidation employing cheap and environmentally friendly conditions. The product
was obtained in 94% yield and with an ee of 98% over the two steps.
’ INTRODUCTION
antidiabetic drugs of the PPAR-R/γ agonists family,5 active
against type 2 diabetes. The switch from a B.Y. whole cell
fermentative synthesis to a more industrially appealing recombi-
nant enzyme catalyzed process, combined with the in situ
substrate feeding product removal (SFPR) strategy,6 led to an
outstanding enhancement of productivity and addressed all the
above-mentioned issues.7
Nowadays, the chemical industry, besides the optimization
of the typical parameters characterizing a process, has to
satisfy the increasing societal requirement of more environ-
mentally compatible processes. Biocatalysis is doubtlessly the
most promising area in which to search for an answer to such a
challenging demand.
However, often the discovery of a valuable biotransformation
is quite far from a direct practical application. Many development
and optimization stages are in between, and the integration of
widely different competences (from the subject areas of chem-
istry, biotechnology, engineering, and so on) becomes manda-
tory. Among all bioconversions, the clearest example of such a
gap is provided by enantioselective baker’s yeast (B.Y.) mediated
reduction of prochiral activated CdC double bonds. Indeed,
even if thousands of reactions have been described so far,1 only a
few of them have been implemented at industrial scale.2
Despite the simplicity and cheapness of the setup, there are
several drawbacks from the downstream processing point of
view: (i) very low substrate concentrations tolerated by the
microorganism that lead to an intrinsically too low productivity;
(ii) difficult workup, due to the troublesome separation of
product from a huge amount of biomass; (iii) typically incom-
plete conversion and occurrence of side reactions, which imply
the use of industrially unappealing chromatographic steps; (iv)
the presence of enzymes with the same specific biocatalytic
activity which might have different enantioselectivity.
’ BAKER’S YEAST MEDIATED PROCESS
Recently, we reported a new synthesis of EEHP based on B.Y.
mediated reduction of aldehyde 2 to give the corresponding
saturated alcohol 3 in a good yield of 78% and an excellent ee of
99% (Scheme 1), using the in situ SFPR technique.4
The predominant bioconversions occurring within the resting
cell consist of the enantioselective anti addition of hydrogen to
the CdC double bond8 catalyzed by enoate reductase (ER)
enzymes and/or the reduction of the carbonyl group resulting
from one or more alcohol dehydrogenase (ADH) enzymes, as
illustrated in Scheme 2. Accordingly, the product distribution
typically observed at the end of the bioconversion (by 1H NMR)
is composed of a few percent of 2, over 80% of saturated alcohol
3, about 10% of allylic alcohol 4, and less than 10% of other side
products (mainly anisaldehyde coming from degradation of 2).
Even though this step compares well in terms of yield and
enantioselectivity with other reported metal-based reductions,3d,e
it suffers from (i) an extremely low productivity (0.39 g LÀ1 dÀ1),
(ii) a nonquantitative conversion, (iii) a complex purification
In this paper, we report the results of the development and
optimization of the bioconversion step that lies at the heart of a
practical process for the enantiospecific synthesis of ethyl (S)-2-
ethoxy-3-(p-methoxyphenyl)propanoate (EEHP),3,4 1, an
important pharmaceutical intermediate for the preparation of
Special Issue: INTENANT
Received: March 31, 2011
Published: August 01, 2011
r
2011 American Chemical Society
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|

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Scheme 1. Comparison between the Two Biocatalytic
Approaches to the Synthesis of EEHP
The ER activity test on the fractions collected according to the
chromatogram was not carried out spectrophotometrically on
typical model substrates like 2-cyclohexenone10 but directly in
biotransformation conditions with both NAD(P)H cofactors on
our substrate 2 to give 5, to be sure to find the specific activity.
Only the fractions corresponding to the first sharp peak of the
chromatogram showed an evident ER activity and a negligible
ADH activity. However, a still too large number of proteins were
present in the fraction (as proven by SDSÀPAGE analysis),
making the identification of the ER impossible at this stage.
Therefore, fractions containing the ER activity were pooled,
concentrated by centrifugal filtering units, and further separated
by means of a native PAGE, which was partially stained to reveal
the protein distribution and partially cut to enable (i) an activity
assay and (ii) MS/MS analysis of the present proteins (Figure 2).
The proteins in one slice of the gel which afforded 51%
conversion of 2, the highest value of all measured slices, were
digested and analyzed by MS/MS spectrometry. The bioinfor-
matics analysis (with ProteinLynx and Mascot search programs,
see Supporting Information) returned agreement with predicted
fragments from four proteins. Only one of them belongs to the
class of the oxidoreductases, the well-known Old Yellow Enzyme
2 (OYE2),11 widely studied for the bioreduction of activated
CdC double bonds.12 This result unambiguously rules out the
presence of novel ER activities. Moreover, it is known that
another homologue ER enzyme, named OYE3, is present in
B.Y.,10b but its expression level is so low13 that its activity could
not be detected in our enzyme identification procedure.
’ CLONING, OVEREXPRESSION, AND PURIFICATION
OF OYE2/OYE3
Although the IEX fraction already showed a remarkable
decrease in side activities, the isolation of the OYEs from yeast
cells is unconvenient because of their low expression levels.13
Therefore, the most practical approach consisted of cloning and
overexpressing each OYE, e.g., in the bacterial model host
Escherichia coli.
From the process development point of view, the direct use of
whole cells of E. coli overexpressing OYEs or its CFE as
biocatalysts without the need of any purification could be
convenient and desirable. Thus, we compared the activity of
the CFE obtained from a typical E. coli cloning strain (DH5R)
with that of yeast (Table 1). Unfortunately, very similar results
were obtained, indicating that also in E. coli interfering side
activities are present. In the attempt to find a host lacking
undesirableactivities towards our substrate, wetested the multiple-
deletion strain MDS41, which is deprived of 14% of its original
genome,14 but without success.
To ensure an easy and highly efficient purification, we selected
affinity purification via the well-established His-tag system. The
oye2 and oye3 genes were amplified by polymerase chain reaction
(PCR) from chromosomal DNA of BY4741; in the process the
stop codon was removed, and appropriate restriction sites were
introduced. Then each gene was inserted into the commercial
plasmid pET-30a bearing a C-terminal His-tag sequence. The
resulting plasmids were used to transform E. coli BL21 cells which
served as an expression host. Cultures of the two strains were
induced with IPTG, and the cells were harvested and disrupted
affording the corresponding CFEs, from which the recombinant
OYEs were isolated by immobilized metal affinity chromatogra-
phy (IMAC).
process based on the chemoselective oxidation of the undesired
allylic alcohol 4 to give the starting material (by MnO2), which
only then can be isolated from the product by chromatography,
and (iv) a useless and counterproductive reduction of the carbonyl
group (by ADH) since the final target is an ester, i.e., 1.
’ IDENTIFICATION OF ENZYMES WITH SUBSTRATE-
SPECIFIC ENOATE REDUCTASE (ER) ACTIVITY
Given that R-enolether aldehydes like 2 were reduced in
unusually high yields4 and are unconventional substrates, we
thought that it was worth investigating whether an unknown ER
activity could be identified.
For the enzyme identification procedure, instead of the
commercially available yeast strain that we employed for the
reduction, we chose a model S. cerevisiae strain (BY4741) since
its genome has been completely sequenced,9 facilitating later
assignment of genes to enzymes. Biotransformations of 2 run in
parallel with whole cells of each strain showed an identical
product distribution (by GC-MS analysis), validating the use of
the model yeast.
To identify the catalytic activities, the cell-free extract (CFE)
was prepared from a BY4741 culture and subjected to ion
exchange (IEX) chromatography on a quaternary ammonium
stationary phase for a preliminary separation of proteins based
on their different isoelectric points. A typical chromatogram is
shown in Figure 1.
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Scheme 2. Main Biocatalytic Reactions Occurring during the Biotransformation of 2 by (a) B.Y., (b) B.Y.-SFPR, (c) OYEs, and
(d) OYEs-SFPR
Figure 1. IEX chromatogram of yeast CFE. The peak which exhibited
ER activity is marked with an asterisk (*). Absorbance 280 nm (—),
Figure 2. Native PAGE separation of the active IEX fraction. Conver-
sions of 2 into 5 obtained from the activity tests are shown on the bands.
The slice portion submitted to the MS/MS analysis is highlighted.
M: protein marker lane. S: sample lane.
conductivity (À À), NaCl gradient 0À500 mM (À À).
3
’ COFACTOR REGENERATING SYSTEM
The progress curves registered for the conversion of 2 into 5
by OYEs in a batch reaction with overstoichiometric NAD(P)H
cofactors (Figure 3) show that OYE2 catalyzes the bioreduction
faster than OYE3 and that both enzymes exhibit a clear pre-
ference for NADPH, making the utilization of the latter more a
need than a choice, especially if a high productivity is sought.
To keep the incidence of cofactor costs very low, the setup of a
cofactor regeneration system is mandatory since it allows the use
of a catalytic amount of the latter, preferentially in the cheaper
oxidized form (NAD(P)+).
Among all types of NADPH regeneration systems that have
been reported so far, the most common and efficient is based
on the oxidation of glucose to give δ-gluconolactone, which in
turn hydrolyzes irreversibly to gluconic acid, driving the
reaction to completion.15 The oxidation of the sacrificial
substrate is catalyzed by a glucose dehydrogenase (GDH), as
illustrated in Scheme 3. The conversions of 2 at different
reaction times achieved using two efficient commercial GDHs
Table 1. Background Activity Tests on 2, Performed on CFEs
of Different Strains
product distribution (%)a
strain
2
3
4
5
subprod.
S. cerevisiae BY4741
E. coli DH5R
2
2
3
82
26
0
9
0
7
26
39
41
48
5
E. coli MDS41
11
a By GC-MS.
(from Pseudomonas sp. and from Thermoplasma acidophilum)
resulted satisfactory (Table 2). However, for reasons of
convenience and economy, we undertook the overexpression
and the purification of the regeneration enzyme as well.
Therefore, a gdh gene was amplified by PCR from the Bacillus
megaterium DSM509 strain, with primers designed on the
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Table 2. Comparison of the Performances of Three GDHs
with Respect to the Cofactors Employed and the Biotrans-
formation Timea
conversionc (%)
GDHb
PS
cofactor
NAD+
OYE
5 h
12 h
24 h
OYE2
OYE3
OYE2
OYE3
OYE2
OYE3
OYE2
OYE3
OYE2
OYE3
OYE2
OYE3
10.0
1.5
39.7
6.9
43.0
5.8
NADP+
NAD+
94.1
85.6
À
À
95.1
86.1
1.9
100
100
À
À
100
100
3.3
100
100
À
À
100
100
3.4
TH
BM
Figure 3. Progress curves for the conversion of 2 into 5 by OYEs with
overstoichiometric cofactors (OYE2/NADPH (b), OYE3/NADPH
(9), OYE2/NADH (O), OYE3/NADH (0)). Expt. cond.: 20 mM
HEPES buffer pH 7.0, 1 mM substrate, 1.1 mM NAD(P)H, 25 μg/mL of
OYE, 30 °C, 160 rpm. Conversions were determined by GC-MS.
NADP+
NAD+
0.6
1.6
1.8
NADP+
67.9
100
100
74.7
100
100
Scheme 3. Glucose/GDH Cofactor Regeneration System
a Expt. cond.: 50 mM phosphate buffer, pH 7.0, 5 mM substrate, 0.1 mM
NAD(P)+, 20 mM glucose, 4 U GDH (ref 17), 1% DMF, 30 °C, 160 rpm
(ref 18a and 19). b BM: recombinant from B. megaterium. PS: commer-
cial from Pseudomonas sp. TH: commercial from Thermoplasma acid-
ophilum. c By GC-MS.
even if it is known that some OYEs exhibit their maximum
activity around pH 8.0.18
In summary, it was decided to proceed with the optimiza-
tion of other parameters using OYE3, GDH-BM, NADP+, and
pH 7.0.
Anyway, the productivity (1.9 g LÀ1 À1, Table 3 entry 1),
d
obtained using such conditions with a typical substrate loading of
1.0 g LÀ1 19
remained unsatisfactory, although it improved
considerably with respect to that of yeast (0.39 g LÀ1 À1). In
,
known gdh sequence of the DSM319 strain, incorporating the
His-tag sequence in the forward primer (leading to an N-term-
inal His-tag). The purified PCR product was cloned into a
pKTS vector,16 and then the resulting pKTS-GDH plasmid
was introduced in E. coli BL21 (DE3) cells for overproduction.
A culture of the latter was induced with IPTG/anhydrotetra-
cycline and subjected to the same procedure described above
for the OYEs, yielding the purified recombinant GDH.17
In terms of performance, this new GDH from B. megaterium
(GDH-BM) compares well with its commercial counterparts
(Table 2), validating its use as a cofactor regenerating system,
with the additional advantage that it can be produced without
being dependent on a supplier.
d
the attempt to increase it further, we tested a higher substrate
loading (15.0 g LÀ1 vs 1.0 g LÀ1), but the conversions dropped
down, even employing higher cosolvent concentrations and/or
liquidÀliquid biphasic mixtures19d (entries 2À7).
The criterion adopted for our optimization process requires the
highest possible productivity with quantitative yields, to avoid any
purification procedure. Hence, although in the best case (entry 5)
we achieved a very high productivity (24.3 g LÀ1 À1), it is
d
impaired by a noncomplete conversion and therefore unaccepta-
ble. In contrast, by introducing the in situ SFPR strategy we
obtained rewarding results.7
The winning concept of SFPR developed by Vicenzi et al. at
Eli Lilly is based on maintaining both substrate and product
concentrations at a level not toxic for the microorganism and not
inhibiting the catalytic activity of enzyme deputed to the
biotransformation.6 To this purpose, the substrate, which is
typically lipophilic, is adsorbed on a hydrophobic resin, ensuring
its release into the aqueous medium at extremely low concentra-
tions; the same is true for the product, mostly adsorbed on the
resin as soon as it is formed. Such an absorption/desorption
equilibrium is almost instantaneous, and the concentration in the
aqueous phase can be easily adjusted by changing the ratio
between resin and substrate (Xr/s). Another interesting aspect
of this technique is that by lowering the concentrations using
higher Xr/s the enantioselectivity usually increases.20,4
’ OPTIMIZATION OF EXPERIMENTAL PARAMETERS
FOR THE BIOCONVERSION
Since quantitative conversions with both the OYEs after 12 h
were obtained, the necessity to achieve high conversions in the
shortest reaction time led us to set the latter to 12 h as a
reasonable trade-off. Although apparently at these conditions the
OYEs seem to give identical performances (Table 2), actually
OYE3 is slightly more stereoselective as proven by the optical
purity of 5 (85% ee with OYE2 vs 90% ee with OYE3, by chiral
GC). Disappointingly, both ee's results were lower than that
achieved with yeast whole cells (99% ee),4 most likely due to an
epimerization of 5 (Scheme 2). Consequently, we decided to
work at neutral pH to minimize such a potential racemization,
The results show clearly that by using a much lower Xr/s with
respect to those usually employed with the yeast (Xr/s 10À20)4,21
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Table 3. Optimization of the Substrate Loading, Xr/s, OYE3 Concentration, and Kind of Substrate for the Preparative Scale
Application
entry
substrate
loading (g LÀ1
)
cosolvent/biphasic system
Xr/s
OYE3 conc .(mg LÀ1
)
Conv.a (%)
eeb (%)
productivityc (g LÀ1 dÀ1
)
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
7
8
9
1
15
15
15
15
15
15
15
15
30
30
30
30
50
50
15
15
15
DMF 1%
DMF 1%
DMF 1%
DMF 5%
DMSO 5%
MTBE 20%
i-PrOAc 20%
resin
À
À
À
À
À
À
À
20
3
25
25
100
20
66
18
86
4
90
À
1.9
À
3
125
125
125
125
125
125
125
125
175
175
175
175
250
125
125
125
90
À
18.8
À
4
5
88
À
À
24.3
À
À
6
7
2
8
49
100
32
74
86
100
63
16
À
99
99
99
99
98
98
98
À
14.7
29.9
19.1
44.2
51.1
59.4
62.4
À
9
resin
10
11
12
13
14
15
16
17
18
resin
3
resin
3
resin
2
resin
1
resin
1
resin
1
resin
3
À
À
resin
3
À
À
À
À
resin
3
À
À
a By GC-MS. b By chiral GC. c Defined as the product of substrate loading, conversion, % eutomer (calculated from the ee) divided by the
biotransformation time.
5 days the alcohol 3 with a modest ee of 50%,7 indicating indeed
Chart 1. Non-Aldehyde-Based Substrates
that such a racemization occurs, and it is slightly faster than the
carbonyl reduction catalyzed by ADH. Moreover, a sample of
(S)-5, incubated at the same experimental conditions used in
the biotransformation, but without any enzyme, showed an
appreciable loss of optical purity, indicating that 5 epimerizes
spontaneously.
Another significant improvement of this process could have
resulted by cutting down the number of synthetic steps. Since in
the literature several examples in which OYEs catalyzed the
reduction of R,β-unsaturated methyl esters or acids have been
a quantitative conversion was achieved (entry 8 vs 9), thus
reported,19aÀc,22 we tested the acid 7 (precursor of 2, Scheme 1)
allowing us to further increase the loading from 15.0 to 30.0 g LÀ1
and its methyl ester 8 but without success (entries 16 and 17).
provided that OYE3 concentration is increased (entry 10 vs 11).
Even the activation of CdC double bonds provided by a stronger
The same trend was confirmed by the decrease of Xr/s from 3
electron-withdrawing group such as thiomethylester, i.e., 9,
down to 1 (entries 11À13), achieving in the best case a
resulted equally ineffective (entry 18).
productivity of 59.4 g LÀ1 dÀ1
.
However, such an approach cannot be reiterated once more at
a higher protein concentration (entry 14 vs 15) because too high
substrate loadings (e.g., 50.0 g LÀ1) require high amounts of
cosubstrate (glucose). In these conditions, significant protein
precipitation was observed, with a detrimental effect on the
conversion (entry 15).
Another beneficial effect that we recently observed in cou-
pling the SFPR methodology with isolated OYEs in reducing
R-substituted unsaturated aldehydes is that the ee improves with
respect to the corresponding homogeneous biotransformations.
Indeed, also in this case the ee increases from 90% to an excellent
99% (entry 1 vs 8) reaching the same value obtained with yeast.
Thus, we concluded that racemization of 2 occurs during the
biotransformation; however, in the presence of resin the product
is readily removed, and such a phenomenon is likely kinetically
suppressed.7
’ DEVELOPMENT OF THE OXIDATIVE STEP AND
G-SCALE IMPLEMENTATION OF THE TWO-STEP
PROCESS
The formation of aldehyde 5 instead of alcohol 3 is clearly
more profitable because it enables the application of a cheaper
and more environmentally compatible oxidative step with re-
spect to that used in the original synthesis (TEMPO/BAIB
system).4 Thus, the saturated aldehyde 5 was quantitatively
oxidized to the corresponding acid 6 by treatment with a solution
of NaClO2, phosphate buffer, and a chlorine scavenger (2-
methyl-2-butene) in t-BuOH/water (1:1), without any loss of
optical purity.23
Furthermore, we found it extremely convenient to oxidize
directly the aldehyde still adsorbed on the resin after previous
filtration from the enzyme solution and water washing of residual
cofactor and proteins. Indeed, after the usual acidÀbase workup
we isolated the acid in a 94% yield over the steps, with 98% ee.
In support of this hypothesis, the bioreduction of 2 with
fermenting B.Y. but without the SFPR methodology gave after
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Finally, we tested the bioconversion of 2 using the best setup
gas-cromatograph equipped with a 5973 mass detector and an
Agilent HP-5 (30 m  0.25 mm  0.25 μm) column. Method:
60 °C (1 min)/6 °C/min/150 °C (1 min)/12 °C/min/280 °C
(5 min). Chiral GC analyses were performed on a DANI HT
86.10 gas chromatograph equipped with a Varian Chirasil-Dex
CB (25 m  0.25 mm) column. Method: 75 °C (1 min)/3 °C/
min/119 °C (17 min)/30 °C/min/180 °C (5 min). Chiral
HPLC analyses were performed on a MerckÀHitachi L-4250
chromatograph equipped with a Chiralcel OD column and UV
detector (210 nm); mobile phase, n-hexane/i-PrOH 99:1; flow
rate, 0.6 mL/min. Optical rotations were determined on a Dr.
Kernchen Propol digital automatic polarimeter. TLC analyses
were performed on Merck Kieselgel 60 F254 plates.
Preliminary Whole Cell Experiments. Yeast cells (200 mg
CWW) were suspended in water (2.0 mL) containing glucose
(40 mg) and incubated at 30 °C with mechanical stirring. After
1 h the substrate solution (5.0 mL, 1.6 mM) was added, and
the reaction was monitored every few hours by withdrawal of a
500 μL sample, extraction with EtOAc (350 μL), and GC
analysis of the organic phase dried over Na2SO4.
(OYE3, GDH-BM, NADP+, Xr/s = 1, substrate loading 30 g LÀ1
,
pH 7.0, 30 °C) coupled with the oxidative step on a preparative
scale (1.0 g) affording 6 in almost quantitative yield (94%) and a
similar ee (98%) with an astonishing productivity of 55.6 g LÀ1 dÀ1
over the two steps.
’ CONCLUSIONS
To the knowledge of the authors, this is the first example of
OYE3 catalyzed bioreduction performed on g-scale and used for
the synthesis of APIs such as Tesaglitazar. The use of over-
expressed OYE3 combined with the in situ SFPR has allowed the
following improvements with respect to the original synthesis:
(i) the productivity increased 2 orders of magnitude from 0.39 to
59.4 g LÀ1 dÀ1; (ii) the conversion is quantitative, simplifying the
purification procedure; (iii) the ready recovery of products by
absorption on resin minimizes the epimerization process preser-
ving the high optical purity of 5; and (iv) the oxidative step is
carried out in cheaper and more environmentally compatible
conditions, making it much more feasible from the perspective of
an industrial scale-up.
Overexpression of the Enzymes in E. coli BL21 (DE3). A
5 mL culture in LB medium containing the appropriate antibiotic
(50 μg/mL of kanamycin for pET-30a, 100 μg/mL of ampicillin
for pKTS) was inoculated with a single colony from a fresh plate
and grown overnight at 37 °C and 220 rpm. This starter culture
was used to inoculate a 200 mL culture, which was in turn grown
overnight at the same conditions and used to inoculate a 1.5 L
culture. The latter was vigorously aerated at 37 °C and 220 rpm
until OD600 reached 0.4À0.5, and then enzyme expression was
induced by the addition of 0.1 mM IPTG (50 ng/mL of
anhydrotetracycline was also added in the case of the pKTS
plasmid). After 5À6 h the cells were harvested by centrifugation
(5000g, 20 min, 4 °C), resuspended in 50 mL of lysis buffer
(20 mM phosphate buffer pH 7.0, 300 mM NaCl, 10 mM
imidazole), and homogenized (Haskel high-pressure homo-
genizer). The CFE, after centrifugation (20 000g, 20 min,
4 °C), was chromatographed on an IMAC stationary phase
(Ni-Sepharose Fast Flow, GE Healthcare) with a mobile phase
composed of 20 mM phosphate buffer pH 7.0, 300 mM NaCl,
and a 10À300 mM imidazole gradient. Protein elution was
monitored at 280 nm, and the fractions were collected according
to the chromatogram and dialyzed twice against 1.0 L of 20 mM
phosphate buffer pH 7.0 (12 h each, 4 °C) to remove imidazole
and salts. Purified protein aliquots were stored frozen at À80 °C.
Screening-Scale Biotransformation of 2 into 5. The sub-
strate 2 (according to the desired substrate loading, see Table 3)
was dissolved in the required cosolvent or adsorbed on XAD
1180 resin (by adding the resin to a solution of the substrate in
Et2O and removing the solvent under reduced pressure). Either
dissolved or adsorbed, the substrate was then added to a solution
of glucose (4 equiv with respect to 2), NAD(P)+ (0.1 mM),
GDH (4 U), and OYE (25À250 μg/mL) in phosphate buffer
(1.0 mL, 50 mM, pH 7.0). The mixture was stirred for 12 h in an
orbital shaker (160 rpm, 30 °C). The resins were filtered on a
sieve, and both the resins and the aqueous phase were extracted
with EtOAc, centrifuging after every extraction (15 000g,
1.5 min). The combined organic solutions were dried on Na2SO4
and concentrated under reduced pressure, yielding 5 (or a
mixture of 5 and 2) as a yellowish oil.
Finally, someone might infer that even if the isolated enzymes
based process proved to be superior in terms of productivity and
efficiency with respect to its B.Y. mediated counterpart the latter
might be cheaper, because it does not require any effort for the
production of the noncommercially available enzymes. However,
the comparison between the quantities of biocatalyst (1:105,
enzymes vs B.Y.) and water (1:30) employed to process an equal
amount of substrate shows clearly that the whole cells based
route is highly unpractical on an industrial scale and therefore
economically unconvenient, even if B.Y. is extremely inexpen-
sive. Indeed, all the efforts (technological and economical)
required for the production of the isolated enzymes are largely
counterbalanced by the higher level of practicality and simplicity
introduced in the process.
’ EXPERIMENTAL SECTION
Materials and Strains. Chemicals, solvents, and commercial
enzymes were obtained from suppliers and used without further
purification. Compounds 2, 3, 4, 6, and 7 were prepared as
described in ref 4. A reference sample of racemic 5 was prepared
by DessÀMartin periodinane oxidation of 4.24 Strains were
obtained from the following sources: S. cerevisiae BY4741 from
EUROSCARF collection (Heidelberg, Germany), B. megaterium
DSM509 from DSMZ (German Collection of Microorganisms
and Cell Cultures), E. coli MDS42 from Scarab Genomics, and
E. coli BL21 (DE3) and E. coli DH5R from New England Biolabs.
General Methods. Yeast strains were grown in standard YPD
medium (10 g/L of yeast extract, 20 g/L of peptone, 20 g/L of
glucose) at 30 °C and E. coli strains in standard LB medium
(10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of NaCl) at
37 °C, with constant shaking at 220 rpm in both cases.
Analytical Methods. Protein concentration was determined
with the Bio-Rad Protein Assay reagent according to Bradford,25
using bovine serum albumine (BSA) as a standard. 1H and 13
C
NMR spectra were recorded on a Bruker ARX 400 spectrometer
(400 MHz 1H, 100.6 MHz 13C) in CDCl3 solution at rt, using
TMS as an internal standard for 1H and CDCl3 for 13C; chemical
shifts δ are expressed in ppm relative to TMS; J values are given
in Hz. GC-MS analyses were performed on an Agilent HP 6890
Data of (S)-2-Ethoxy-3-(4-methoxyphenyl)propanal
(S)-5. Prepared according to Table 3, entry 13. Colorless
oil: 99% purity by GC (tR 19.31 min); 98% ee by chiral GC
274
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Organic Process Research & Development
ARTICLE
20
(tR 30.6 min (S), 31.1 min (R)); [R]D = À68.7 (c 1.1,
’ ACKNOWLEDGMENT
1
CHCl3); H NMR (400 MHz, CDCl3) δ 9.68 (d, J = 2.3,
We thank Dr. Daniela Monti (ICRM-CNR, Milano) for her
collaboration and for providing samples of commercial GDHs.
We are grateful for the generous financial support from the E.U.
INTENANT project.
1H), 7.16 (d, J = 8.7, 2H), 6.84 (d, J = 8.7, 2H), 3.81À3.83 (m,
1H), 3.80 (s, 3H), 3.61 (mAB, 1H), 3.46 (mAB, 1H), 2.95 (mAB
,
1H), 2.86 (mAB, 1H), 1.20 (t, J = 7.0, 3H); 13C NMR (100.6
MHz, CDCl3) δ 203.5, 158.5, 130.4, 128.6, 113.9, 85.1, 66.5,
55.2, 35.8, 15.2. MS: m/z (%) 208 [M]+ (8), 179 (6), 151 (11),
121 (100), 91 (16).
’ REFERENCES
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0 °C in an ice bath under magnetic stirring. Then, a solution of
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’ ASSOCIATED CONTENT
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S
Supporting Information. Detailed enzyme identifica-
b
tion procedure, MS/MS analysis report, preparation of the
overexpressing strains, plasmid maps, amino acid sequence of
the GDH, SDSÀPAGE analysis of the purified proteins,
synthesis of substrates 8 and 9, and NMR spectra of 5. This
material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
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E.; Polizzi, K. M.; Bommarius, A. S. Adv. Synth. Catal. 2007, 349, 1521.
(b) Fryszkowska, A.; Toogood, H.; Sakuma, M.; Gardiner, J. M.;
Stephens, G. M.; Scrutton, N. S. Adv. Synth. Catal. 2009, 351, 2976.
Corresponding Author
*Tel.: +39 02 23993072. Fax: +39 02 23993080. E-mail:
francesco.gatti@polimi.it.
275
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