Scale-up of a recombinant pig liver esterase-catalyzed desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate

Article abstract of DOI:10.1021/op500129e

A recombinant isoenzyme of pig liver esterase was used for the highly enantioselective desymmetrization of dimethyl cyclohex-4-ene-cis-1,2- dicarboxylate. The selected recombinant esterase showed a significant advantage in enantioselectivity over the comm

Full text of DOI:10.1021/op500129e

Article
pubs.acs.org/OPRD
Scale-Up of a Recombinant Pig Liver Esterase-Catalyzed
Desymmetrization of Dimethyl Cyclohex-4-ene-cis-1,2-dicarboxylate
Philipp Suss,†,§ Sabine Illner,‡ Jan von Langermann,‡ Sonja Borchert,† Uwe T. Bornscheuer,§
̈
Rainer Wardenga,*,† and Udo Kragl*,‡
†Enzymicals AG, Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany
‡Institute of Chemistry, University of Rostock, Albert-Einstein-Str. 3a, 18059 Rostock, Germany
§Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
S
* Supporting Information
ABSTRACT: A recombinant isoenzyme of pig liver esterase was used for the highly enantioselective desymmetrization of
dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate. The selected recombinant esterase showed a significant advantage in
enantioselectivity over the commonly used esterase from the mammalian source. The process was scaled up to yield 265 g of
product with a simplified pH control, and the target molecule was obtained with an enantiopurity of >99.5% ee.
Another major synthon is the target molecule (1S,2R)-1-
(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid 2a
((1S,2R)-monoester), which is a valuable intermediate for the
synthesis of many biologically active products, e.g., anticapsin,
(+)-aucantene, and cis-carbapenems (Scheme 1).15 Further
INTRODUCTION
■
The preparation of enantiopure building blocks gained
significant relevance in the industrial production of important
pharmaceuticals and agrochemicals during the past decades. For
example, about 39% of drugs sold worldwide in 2002 were
provided in an enantiomerically pure form.1 Originally the
separation of the enantiomers from the chemically synthesized
racemic mixture was preferred, e.g., by preparative chromatog-
raphy and (enantio)selective crystallization, which obviously
limits the maximum yield to 50% and results in low atom
efficiency and a significant waste problem. Asymmetric
synthesis processes in contrast allow a maximum theoretical
yield of 100%, and thus an impressive variety of chemical and
biocatalytical processes have been developed over the past
years.2 Biocatalysis has proven multiple times that higher
enantio- and regioselectivities are achieved at particularly mild
reaction conditions, facilitating a suppression of unwanted side
reactions, e.g., isomerization and racemization.3−6 For this
purpose the enzyme can be utilized in different forms: as
(partially) purified enzymes, free or in an immobilized enzyme
preparation, with cross-linking techniques, or even as whole
cells. The eventually applied enzyme form strongly depends on
the chosen specific process specifications.7 In addition, protein
stability, substrate specificity, and enantioselectivity of the
biocatalyst itself can be improved by molecular biology
techniques.8
Scheme 1. Target molecule (1S,2R)-monoester 2a is a very
useful intermediate for the production of various
pharmaceutically relevant products
potential applications are the creation of ligands for DC-SIGN
(useful in treatment of HIV infection), synthesis of β-
polypeptides against cytomegalovirus (Herpes), and the
synthesis of Tricyclic Core of Platencin (antibiotic to
MRSA).16−18
From the available pool of biocatalysts various enzymes have
repeatedly proven their value for the synthesis of chiral
synthons, especially at industrial scale.9−11 One major example
is the esterase from porcine liver, typically referred to as pig
liver esterase (PLE). PLE is a relatively low-cost enzyme that
shows a good stability and hydrolyzes a large range of
esters.12,13 Examples include the synthesis of precursors of
β3-receptor agonists, pyrethroids (from chrysanthemic acid
esters), the growth hormone secretagogue, and the anti-HIV-
drug (−)-FTC.7,12,14
Unfortunately, the classical preparation of pig liver esterase
includes an extraction from the animal tissue, which yields
varying mixtures of several isoenzymes.19,20 All of these
isoforms exhibit different enzyme characteristics, including
different stabilities and pH-dependencies and may also result in
opposite enantioselectivities, depending on the substrate.21−23
In addition, differences in the isoenzyme ratio may occur
Received: April 18, 2014
Published: June 23, 2014
© 2014 American Chemical Society
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between PLE production batches. Consequently the observed
enantiomeric excess of a reaction represents only a sum
parameter and is not always easily reproducible, which can
cause problems in large scale applications. The very unattractive
presence of several different PLE isoenzymes was ultimately
overcome by cloning and recombinant expression in safe non-
mammalian producer strains.24 Various authors reported the
expression in P. pastoris and in E. coli, which allows the use of
the full potential of selected isoenzymes, including high
enantioselectivity, but without side activities and regulatory
issues.25−28 In this contribution we report the successful
application of a recombinant pig liver esterase for the
desymmetrization reaction of dimethyl cyclohex-4-ene-cis-1,2-
dicarboxylate 1 (Scheme 2). From the available six recombinant
tion from diester 1. As expected, strong differences in activity
and selectivity were found between the isoenzymes (Figure 1).
Scheme 2. Comparison of differing enzyme preparations for
the desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-
dicarboxylate 1
Figure 1. Comparison of the specific activity towards dimethyl
cyclohex-4-ene-cis-1,2-dicarboxylate 1 (hydrolysis reaction). Condi-
tions: 25−50 mg PLE isoenzyme (lyophilizate), 0.2 mmol 1, 40 °C, 5
mL 5 mM phosphate buffer, pH 8, titration with 50 mM NaOH
solution.
ECS-PLE06 exhibits the highest enzymatic activity for the
desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarbox-
ylate, indicated as 100% relative activity. A reduced activity of
approximately 55% was found for ECS-PLE05, which is still
considerably high for the favored conversion of 1. The activities
of ECS-PLE03 and 04 are significantly lower, and they were
therefore excluded due to economic reasons. The remaining
isoenzymes ECS-PLE01 and 02 showed hardly any activity
towards 1 and were not favored as well. The enantioselectivity
of the available isoenzymes and thus the resulting synthetic
potential were additionally investigated with conversion
experiments on small scale (Figure 2).
pig liver esterases (ECS-PLE01−06) the most valuable
isoenzyme and the optimal reaction conditions were chosen.
The characteristic properties of the recombinant biocatalysts
were compared with their natural equivalent from the porcine
liver extract. Finally, the reaction was transformed into 250-g
scale, proving again the strong potential and large scale
applicability of recombinant pig liver esterases. Furthermore, a
simplified pH-control concept was used for this reaction, which
requires no additional equipment, e.g., titrator and pH
electrode. This self-regulating concept requires only standard
laboratory equipment, thus keeping overall costs low.
RESULTS AND DISCUSSION
■
Desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarbox-
ylate 1 allows the synthesis of two different enantiomers.
(1R,2S)-Monoester 2b can be achieved with the lipase B from
Candida antarctica as immobilized form Novozym 435 with
excellent enantioselectivity (>99.9%) and a yield of up to
99.8%.29 The other enantiomer, (1S,2R)-monoester 2a, was
repeatedly synthesized via a pig liver esterase-catalyzed reaction.
Unfortunately the reaction is not entirely enantioselective, and
a lower enantiomeric excess of 80−97% ee was obtained
(Scheme 2).12,30−36 The reduced enantioselectivity results from
the presence of several parallel hydrolytic reactions, each
independently catalyzed by different isoenzymes of pig liver
esterase. The products of highly enantioselective isoenzymes
are hereby mixed with products from less selective enzymes.
Thus, the use of a single, recombinantly expressed PLE
isoenzyme allows a highly enantioselective synthesis of 2a
(>99.5% ee).
Figure 2. Desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-
dicarboxylate 1 to (1S,2R)-monester 2a depending on the used pig
liver esterase isoenzyme. Conditions: 23 °C, 100 mM TRIS buffer pH
7.5, 10 mmol/L substrate; 4 U/mL recombinant PLE (according to
the standard activity assay).
In analogy to the results from the apparent reactivity the
ECS-PLE06 exhibited the highest reactivity in the conversion
experiments. After 120 min full conversion together with an
excellent enantiomeric excess of >99.5% was obtained. A
slightly lower rate of conversion was found for ECS-PLE05,
which is also in accordance with the previous results, Figure 1.
However, almost full conversion was also achieved after 120
Choice of Optimal PLE Isoenzyme. The available six PLE
isoenzymes (ECS-PLEs from Enzymicals AG) were screened
for the production of (1S,2R)-monoester 2a via desymmetriza-
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min, and an enantioselectivity of >99.5% was identified, which
is identical to that of ECS-PLE06. Interestingly, based on the
enzyme activity assay with p-nitrophenyl acetate, a similar
amount of the classical pig liver esterase (PLEClassical) yields a
significantly reduced reaction rate in comparison to that of
ECS-PLE05 and ECS-PLE06. After 60 min only 15%
conversion was possible, which was increased to only about
30% after 120 min. Most importantly, an enantiomeric excess of
nearly 96% ee was observed for the production of 2a in this
reaction, which is of course lower than the results obtained with
ECS-PLE05 and ECS-PLE06. This fact illustrates conclusively
the high synthetic advantage of recombinant PLEs. Further-
more, the other recombinant pig liver esterases showed no
synthetic value. The use of ECS-PLE03 yields only an almost
racemic product (3% ee) with 30% conversion after 120 min,
and the other recombinant PLEs showed no significant
conversion. Additional experiments with low amounts of
water-miscible solvents, which are known to alter the
characteristics of recombinant PLEs, were not successful for
the improvement of this catalytic reaction.37
increase to 25 °C partially improves the apparent enzyme
activity to a conversion of 47%. A further increase of the
reaction temperature to 40 °C yields full conversion after 30
min, which includes a fully enantioselective product formation.
Enzyme reactivity can be further improved with an increase of
reaction temperature to 60 °C. However, such high temper-
atures are not recommended for applications with higher
substrate concentrations and longer reaction times, because the
recombinant PLEs are less stable at >50 °C and above.25
Consequently, 40 °C was chosen as the optimal process
temperature. Noteworthy, excellent enantiomeric excesses
(>99.5% ee) were found within all investigated reactions,
which represent a robust selectivity of the enzyme.
Aside temperature, the pH of the reaction mixture is a
relevant parameter and therefore important in terms of
productivity and obtained enantiomeric excess. A selection of
representative experiments at different pH levels are shown in
Figure 4.
In conclusion, recombinant PLEs show impressive advan-
tages in comparison to the classical pig liver esterase
preparation. The highest reactivities and excellent enantiose-
lectivities were found for ECS-PLE06, which was consequently
chosen for the intended large scale-production of 2a. The lower
reactivity of the classical PLE is explainable by the presence of
different isoenzymes, as mentioned above. Here the recombi-
nant PLEs 1, 2, and 4 diminish the synthetic performance and
the presence of PLE 3, which yields only a racemic product,
causes a loss in enantioselectivity. Thus, only a lowered
reactivity and enantiomeric excess can be obtained with the
classical pig liver esterase.12
Choice of Reaction Conditions. The main enzyme
characteristics of recombinant PLEs were already reported
earlier in the literature.20,22,25 Unfortunately, specific process
data for the desymmetrization of 1 to 2a were not available
prior to this study. Thus, additional experiments were
conducted to define efficient process conditions for the ECS-
PLE06-catalyzed synthesis of 2a. In particular reaction
temperature and pH of the aqueous phase were of interest
for the attempted scale-up. The influence of reaction
temperature is exemplarily shown for ECS-PLE06 in Figure 3.
At 10 °C only an insufficient reactivity with an unsatisfactory
conversion of <10% after 30 min was observed. A temperature
Figure 4. Effect of pH on the ECS-PLE06-catalyzed conversion of
dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate. Conditions: 50 mM
phosphate buffer, 23 °C, 10 mM substrate; 4 U/mL enzyme
(according to standard activity assay).
It is clearly shown that the process is very robust with respect
to pH. No significant differences were found between results at
pH 7.5, 8.3, and 9.0. Full conversion with excellent
enantiomeric excess (>99.5% ee) were achieved at pH 8.3
and 9.0 after 60 min, and only a slightly longer reaction time
was required at pH 7.5. Similar results with a broad pH
optimum between 7.5 and 9.5 were reported by Lange et al.
with a recombinant pig liver esterase for the hydrolysis of
p-nitrophenyl acetate.25 Thus, a relatively stable pH control at
pH 8−9 will be sufficient for a large scale synthesis of 2a.
Scale-Up. Based on the initial experiments, the scale of the
reaction was gradually increased to facilitate the desymmetriza-
tion of 1 at higher substrate concentrations. Unfortunately,
such an increase yields the formation of a stoichiometric
amount of 2a, which is an acid and lowers the pH in the
aqueous solution. Thus, an efficient pH-adjustment technique is
essential to maintain optimal reaction conditions for the
biocatalyst throughout the reaction, typically by an external
addition of a base solution. Table 1, entries 1−4, shows
exemplarily the results via external pH controller (pH-stat
titration using NaOH solution). Reaction volumes from 20 to
630 mL with substrate concentration of 80−100 mM were
tested, and high conversions with excellent enantioselectivities
were obtained. Experiments at smaller scale (20−40 mL)
demonstrated clearly that the reaction allows full conversion
Figure 3. Effect of reaction temperature on the ECS-PLE06-catalyzed
conversion of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate. Condi-
tions: 50 mM phosphate buffer pH 7.5, 10 mM substrate; 4 U/mL
recombinant PLE (according to the standard activity assay).
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Table 1. Desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate at larger scale with pH-adjustment by an external
base solution or the acid scavenger NaHCO3
a
entry
T [°C]
V [L]
substrate [mM]
reaction time [h]
activity [U/mL]
conversion [%]
STY [g/(L·h)]
isolated yield [%]
pH-stat
1
2
3
4
25
25
40
40
0.02
0.02
0.04
0.63
100
100
80
10.0
3.9
3.8
5.7
3.0
4.8
>99
>99
>99
91
1.8
4.7
6.4
1.8
83
83
75
2.3
80
8.0
NaHCO3
5
6
7
8
25
40
40
40
0.04
0.04
0.04
8.80
80
80
6.5
1.2
1.2
4.0
3.0
6.1
>99
>99
>99
>99
2.3
12.3
30.7
9.2
91
74
82
200
200
15.2
5.5
a
Used volumetric enzyme activity; calculated from specific activity [U/mg] according to standard activity assay and enzyme amount [mg] per
volume [mL].
after 3.9 h at 25 °C, which can be further decreased to 2.3 h at
40 °C, representing a maximum space-time yield of 6.4 g/(L·h).
Unfortunately the application of larger volumes (0.63 L)
requires also larger amounts of NaOH for pH-adjustment,
which may result in a decrease of enzyme stability due to local
high-pH hot spots. For example only 91% conversion was
obtained at 630-mL scale, even with a prolonged reaction time
of 8 h. Apparently an insufficient mixing of the reaction
solution with the aqueous base for pH-adjustment at larger
scale causes strong fluctuation of pH and thus local enzyme
denaturation with a loss of enzyme activity. An adjustment of
the reactor geometry and base dosing may minimize this
problem but will require further investigations and still needs
higher and therefore uneconomic enzyme loadings to overcome
these limitations. The fluctuation of pH, especially at the early
stage of the reaction, is exemplarily shown in Figure 5.
maintains an optimal pH slightly above 8.38 Formed monoester
2a, a monocarboxylic acid, is rapidly neutralized by sodium
hydrogen carbonate. The formation of gaseous carbon dioxide
(CO2) can be visualized with a bubble counter and represents
also a rudimental reaction control. With this technique optimal
reaction conditions are easily maintained without any further
addition of secondary reactants (base solution). As shown in
Table 1, entries 5−8, higher substrate concentrations of up to
200 mM were successfully applied and allowed full conversion
at even shorter reactions times in comparison to the classical
technique. A maximum space-time yield of 30.7 g/(L·h) was
found, which is a 5.6-fold increase over the best example with a
classical pH-adjustment via addition of a base solution.
A preparative synthesis at larger scale was performed at 350 g
substrate scale (1.77 mol) with the prior defined optimal
reaction conditions, but with a slightly more economic enzyme
usage (entry 8). The required pH control was herein facilitated
with the presented simplified NaHCO3 protocol and allowed
full conversion of 1 to 2a after approximately 240 min (Figure
6).
Interestingly, initially produced 2a was obtained with lower
enantiomeric excess and slowly reached an excellent enantio-
meric excess of >99.5% ee when full conversion was achieved.
The initial presence of small amounts of racemic product 2,
from a nonenzymatic hydrolysis prior the enzymatic reaction
experiment, reduces the initial enantiomeric excess to ∼91%
(30 min). With the continuous progression of the fully
enantioselective biocatalytic reaction the enantiomeric excess
slowly increased to >99.5 ee.
The pH remained stable slightly above 8 throughout the
whole experiment. Formed CO2 from the acid-induced
decomposition of NaHCO3 was captured and visualized with
a gas bubble counter. After the full conversion was achieved, the
reaction was stopped with an excess of hydrochloric acid and
subjected to work-up. In total 265 g (82% yield) of enantiopure
(1S,2R)-monoester 2a was obtained, which corresponds to a
space time yield of 9.2 g/(L·h).
Figure 5. pH-dependent addition of a base solution causes fluctuations
of the pH, depending on the reactor geometry, especially during the
beginning of the reaction. Conditions: total volume 0.04 L, 80 mM
substrate, 40 °C, pH control with 0.5 M NaOH solution, pH
measurement near the buret; conversion was calculated from NaOH
consumption.
As an alternative other techniques for internal pH control
were evaluated, to overcome the limitations based on pH
fluctuation. High concentration buffers were excluded since
very high buffer concentrations would be required, which is
impractical due to costs and limited salt solubility. Fortunately,
weak bases such as CaCO3 or NaHCO3 were found to be
compatible with the enzymatic reaction. The highest potential
was demonstrated for sodium hydrogen carbonate (NaHCO3),
which exhibits a sufficient solubility in aqueous systems and
CONCLUSION
■
A novel and efficient process for the recombinant pig liver
esterase-catalyzed desymmetrization of 1 to (1S,2R)-1-
(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid 2a was
presented. For this purpose six commercially available
recombinant isoenzymes of pig liver esterase were investigated
within this study, and the most potent isoenzyme ECS-PLE06
was chosen. Optimal reaction conditions were determined and
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Figure 6. Scaled-up desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate. (Left) Temperature-controlled bioreactor with a total
■
□
●
capacity of 10 L. (Right) Time course of concentration of reagent 1 ( ), concentration of product 2 ( ), and ee ( ) as determined by GC analysis.
Conditions: 200 mM substrate, 5.5 U/mL ECS-PLE06, total volume 8.8 L, stirrer speed 300 rpm, 40 °C, pH control via acid scavenger NaHCO3.
eventually transferred for scale-up enabling very high space-
time yield. A simplified pH control, applying NaHCO3 as an
acid scavenger, was used, which stabilizes the pH at slightly
above 8 and requires no further equipment for dosing of a base.
A large scale synthesis yielded 265 g of enantiopure (1S,2R)-1-
(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid 2a. Further
optimization may include even higher substrate concentrations
or enhanced enzyme formulation. In addition, the presented
pH regulation concept with sodium hydrogen carbonate
NaHCO3 can be easily transferred to similar processes.
As shown in this study, recombinant pig liver isoenzymes can
be easily used at larger scales with high substrate concen-
trations. The enhanced enantiomeric excess of the target
substance 2a shows a significant advantage over the use of
classical preparations of pig liver esterases from mammalian
sources.
Standard Activity Assay. The activity of PLEs was
determined using p-nitrophenyl acetate (pNPA, 30 mM
dissolved in DMSO) with spectrophotometric quantification
of the p-nitrophenolate release over 100 s at 401 nm (ε30 °C
=
17 759 L/(mol·cm)) containing 850 μL of phosphate buffer
(pH 7.5, 50 mM), 50 μL of enzyme solution, and 100 μL of 30
mM p-nitrophenyl acetate in DMSO. One unit (U) of esterase
activity was defined as the amount of enzyme releasing 1 μmol
of p-nitrophenolate per minute under standard assay
conditions. In order to detect changes in absorbance we used
a UV−vis spectrometer (Specord 50, Analytic Jena AG, Jena
and Helios Alpha UV−vis spectrophotometer, Thermo
Scientific GmbH, Schwerte, Germany) equipped with a
reference cell including equal solutions without enzyme. Data
are reported as the mean
standard deviation of triplicate
measurements.
Isoenzyme Screening. Enzyme activity screening was
executed with a TitroLine 7000 from SI-analytics (Mainz,
Germany) in pH-Stat mode. A 100 mg portion of ECS-
PLE01−06 (lyophilized) was suspended in 15 mL of 5 mM
sodium phosphate buffer pH 8.0 at 40 °C. The reaction was
started by adding 45 mg (0.2 mmol) of diester 1. The activity of
the enzyme was calculated on the basis of the consumption of
50 mM sodium hydroxide solution. One unit (U) of enzyme
activity was defined as the amount of enzyme that hydrolyzes 1
μmol of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate 1 per
minute under the mentioned assay conditions.
Small scale desymmetrization experiments were carried out
in 5 mL glass vials with 10 mg of the respective isoenzyme in 4
mL of a buffered solution (0.1 M phosphate buffer, pH 7−8)
with 10 mM diester 1. The enzymatic hydrolysis was conducted
at 10, 23, and 40 °C. Samples (100 μL) were taken in intervals,
acidified with 5 μL of 6 M HCl (pH 2), and extracted with
EtOAc (200 μL). Conversion and enantiomeric excess of the
enzymatic reactions were obtained from analysis with gas
chromatography (see below).
Small-Scale Biocatalysis with ECS-PLE06 (pH-stat). A
100 mL round-bottom flask was charged with 40 mL of
deionized water, ECS-PLE06, and a magnetic stir bar. The
suspension was set to 40 °C and pretitrated to pH 8. The
reaction was started by adding 0.6 g (3.2 mmol) of 1 to the
reaction mixture containing 50 mM sodium phosphate buffer
pH 8.0. The solution was kept constant at pH 8 by addition of
0.5 M sodium hydroxide solution via an autoburet. After the
EXPERIMENTAL SECTION
■
General. All chemicals and solvents were purchased from
Alfa Aesar (Ward Hill, USA), Sigma-Aldrich (St. Louis, USA),
and Carl Roth GmbH & Co KG. (Karlsruhe, Germany) and
used as received. Deionized water was used throughout this
study.
Synthesis of Dimethyl Cyclohex-4-ene-cis-1,2-dicar-
boxylate 1. A 4 L round-bottom flask was charged with 500 g
(2.9 mol) of cis-cyclohex-4-ene-1,2-dicarboxylic acid, 2.4 L of
methanol, a magnetic stir bar, and a reflux condenser.
Subsequently 125 mL of concentrated sulfuric acid (98%)
was added dropwise to the stirred suspension. The reaction
mixture was heated to reflux and stirred for 48 h. The resulting
solution was partially evaporated under reduced pressure to
volume of 1 L. The concentrate was extracted three times with
1 L of MTBE. The combined organic layers were washed with
deionized water, neutralized with a concentrated aqueous
sodium hydrogen carbonate solution, and washed again with
deionized water. The ether was removed under reduced
pressure to give 495 g (yield 85%) of the clear oil 1. Details
are given in the Supporting Information.
Enzymes. Recombinant pig liver esterases ECS-PLE01 to
ECS-PLE06 are commercially available (as lyophilizate) from
Enzymicals AG (Greifswald, Germany), and the esterase from
porcine liver (mixture of isoenzymes as crude protein powder)
was purchased from Sigma-Aldrich (St. Louis, USA).
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addition of the required NaOH solution, the reaction was
stopped by addition of concentrated hydrochloric acid
(adjusted to pH 1−2). The precipitated protein was spun
down, and the aqueous layer extracted three times with 40 mL
of ethyl acetate. The combined organic layers were dried over
anhydrous MgSO4 and evaporated under reduced pressure to
give 0.56 g of 2 as yellowish oil (95% yield). After crystallization
the product was isolated as 0.49 g of white crystals with 83%
yield and excellent enantiomeric excess (>99.5% ee).
Small-Scale Biocatalysis with ECS-PLE06 (Sodium
Hydrogen Carbonate). A 100 mL round-bottom flask with
an integrated CO2-bubble counter was charged with 40 mL of
saturated sodium hydrogen carbonate solution, ECS-PLE06,
and a magnetic stirrer. The reaction was started by adding 0.6 g
(3.2 mmol) of 1 to the reaction mixture and stirring at 40 °C.
The process was monitored by TLC and monitored by a
bubble counter. After 70 min (TLC analysis indicated full
conversion) the reaction was stopped by adding concentrated
hydrochloric acid to pH 1−2, precipitated protein was removed
by centrifugation, and the resulting aqueous layer was extracted
three times with 40 mL of MTBE. The combined organic
phases were dried over anhydrous MgSO4 and evaporated
under reduced pressure to give 0.53 g of crystalline product
(91% yield, >99.5% ee).
NMR Analysis. 1H NMR and 13C NMR spectra were
recorded with a Bruker AVANCE (250 II) in CDCl3 or
DMSO-d6. Chemical shifts are reported in parts per million
relative to the solvent peak or TMS as an internal reference.
Splitting patterns are indicated as follows: s, singlet; d, doublet;
t, triplet; m, multiplet.
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: rainer.wardenga@enzymicals.com.
*E-mail: udo.kragl@uni-rostock.de.
Funding
The authors gratefully acknowledge financial support by the
Central Innovation Program SME (Zentrales Innovationsprog-
ramm Mittelstand, ZIM) of the Federal Ministry for Economic
Affairs and Energy (Bundesministerium fur Wirtschaft und
̈
Technologie, BMWi), grant number KF2622302AJ2.
Notes
Large-Scale Biocatalysis with ECS-PLE06 (Sodium
Hydrogen Carbonate). A stirred-tank reactor (STR) was
charged with 8.5 L of deionized water, 500 g of sodium
hydrogen carbonate, and ECS-PLE06. The suspension was
heated to 40 °C, and the reaction was started by adding 350 g
(1.77 mol) of 1 to the stirring solution. The pH was kept
constant at 8 during the whole process. After 4 h the reaction
mixture was heated to 95 °C to denature the protein.
Afterwards the suspension was acidified with concentrated
hydrochloric acid to pH 1. The precipitated protein was spun
down, and the aqueous layer was extracted three times with 8.8
L of MTBE (Cryofuge 8500i, Thermo Scientific, Schwerte,
Germany). The combined organic phases were dried over
MgSO4 and evaporated under reduced pressure to give 265 g
crystalline monoester 2 (82% yield, >99.5% ee), mp 65 °C;
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Ulf Menyes (Enzymicals AG) for useful
discussions and Sebastian Heim for his help in the experimental
work.
ABBREVIATIONS
■
TLC, thin layer chromatography; (−)-FTC, (−)-2′,3′-dideoxy-
5-fluoro-3′-thiacytidine; DC-SIGN, dendritic cell-specific inter-
cellular adhesion molecule-3-grabbing nonintegrin; MRSA,
methicillin-resistant Staphylococcus aureus
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■
22
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