Recombinant Pig Liver Esterase-Catalyzed Synthesis of (1S,4R)-4-Hydroxy-2-cyclopentenyl Acetate Combined with Subsequent Enantioselective Crystallization

Article abstract of DOI:10.1021/acs.oprd.6b00093

The recombinant pig liver esterase catalyzed hydrolysis of cis-1,4-diacetoxy-2-cyclopentene forming (1S,4R)-4-hydroxy-2-cyclopentenyl acetate was investigated and realized at preparative scale. Relevant reaction conditions were examined and optimized to achieve full conversion with an enantiomeric excess of about 86% ee. Enantiopure product was then obtained after enantioselective crystallization, which required further studies of the solid phase behavior, including its binary melting point phase diagram.

Full text of DOI:10.1021/acs.oprd.6b00093

Article
pubs.acs.org/OPRD
Recombinant Pig Liver Esterase-Catalyzed Synthesis of (1S,4R)‑4-
Hydroxy-2-cyclopentenyl Acetate Combined with Subsequent
Enantioselective Crystallization
Janine Hinze,† Philipp Suss,‡,§ Silja Strohmaier,†,∥ Uwe T. Bornscheuer,§ Rainer Wardenga,*,‡
̈
and Jan von Langermann*,†
†University of Rostock, Institute of Chemistry, Albert-Einstein-Str. 3a, 18059 Rostock, Germany
‡Enzymicals AG, Walther-Rathenau-Str. 49a, 17489 Greifswald, Germany
§University of Greifswald, Institute of Biochemistry, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
∥School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia
S
* Supporting Information
ABSTRACT: The recombinant pig liver esterase catalyzed hydrolysis of cis-1,4-diacetoxy-2-cyclopentene forming (1S,4R)-4-
hydroxy-2-cyclopentenyl acetate was investigated and realized at preparative scale. Relevant reaction conditions were examined
and optimized to achieve full conversion with an enantiomeric excess of about 86% ee. Enantiopure product was then obtained
after enantioselective crystallization, which required further studies of the solid phase behavior, including its binary melting point
phase diagram.
specific, reproducible characteristics.13−15 A recent example
highlights the desymmetrization of dimethyl cyclohex-4-ene-cis-
1,2-dicarboxylate yielding (1S,2R)-1-(methoxycarbonyl)-
1. INTRODUCTION
In the past decades biotransformations became a highly
valuable technique for the production of important chemicals
cyclohex-4-ene-2-carboxylic acid. Herein classical PLE from
on an industrial scale. Major examples include the synthesis of
(bio)ethanol,1 high fructose corn syrup (HFCS),2 acrylamide,3
its natural sources achieves only an enantiomeric excess
between 80 and 97% ee, due to contrary enantioselectivities
different classes of antibiotics, and numerous other fine
chemicals.4 Within these systems the use of biocatalysts offers
of the respective isoenzymes, as mentioned above. In contrast,
the use of a recombinant isoenzyme preparations facilitates the
several significant advantages in comparison to classical
chemical processes, e.g., milder reaction conditions, less toxic
direct synthesis of the enantiopure product (>99.5% ee).16,17
In this contribution we present the successful combination of
reagents, and the use of water as solvent. Typically moderate to
a partially enantioselective rPLE-catalyzed hydrolysis of cis-1,4-
high regio- and enantioselectivities are achieved with such
diacetoxy-2-cyclopentene 1 and a subsequent enantioselective
biocatalysts including high atom efficiencies and simplified
crystallization at preparative scale. The resulting product
downstream processing. The biocatalysts itself can also be
(1S,4R)-4-hydroxy-2-cyclopentenyl acetate 2a is an important
chiral building block for the preparation of prostaglandins and
further improved by modern molecular biology techniques to
optimize the entire production process.5 In comparison,
other active natural products, such as cyclopentanoids (Figure
classical resolutions of racemic mixtures, e.g., by crystallization
1). Reaction temperature, pH, and cosolvent influence were
or chromatographic methods facilitates only a maximum yield
investigated and optimized to facilitate full conversion of the
enzymatic reaction with the highest achievable product
enantiomeric excess of 86% ee.
In addition, the solid phase behavior of the target substrate
2a was determined with respect to its binary melting point
phase diagram (BPD) and X-ray powder diffraction (XRPD) to
of ≤50% including even lower atom efficiencies and high waste
streams.2,6−8
Within the group of industrially relevant enzymes pig liver
esterase (PLE, esterase from porcine liver) is one of the most
frequently used esterases in synthetic chemistry. The broad
substrate range of PLE allows diverse applications of regio- and
ensure optimal enantiomeric purity after enantioselective
stereoselective hydrolytic reactions in asymmetric synthesis and
kinetic resolution processes.9−12 Unfortunately the natural
crystallization. Herein enantiomeric substances exhibit in
general three basic types of phase behavior: racemic compound
source of this enzyme yields a preparation-dependent variable
mixture of various PLE isoenzymes with significantly different
(true racemate), conglomerate (simple eutectic), and pseudo-
racemates (solid solutions). Target compound 2a belongs to
substrate specificities, which consequently result in a mixture of
the group of racemic compounds and thus exhibits a crystalline
enzymatic activities and thus limits the usability of PLE for
racemate with both enantiomers in equal and ordered
production purposes. Moreover, regulatory issues may prevent
the use of such a PLE-preparation for product synthesis.
Fortunately, the use of recombinant PLEs (rPLEs) overcomes
such problems and allows the utilization in synthesis with
Received: March 21, 2016
© XXXX American Chemical Society
A
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Figure 1. Schematic representation of the binary (A) and ternary (B) phase diagram of a racemic compound behavior; * − eutectic(s), RS − solid
(R)-enantiomer, SS − solid (S)-enantiomer, RSS − solid racemic mixture, liq − liquid.
Scheme 1. rPLE-Catalyzed Synthesis of Enantiopure (1S,4R)-4-Hydroxy-2-cyclopentenyl Acetate and Potential Products
(Spinosyn Analogue,18 Corey Lactone,19 and ent-15-epi-F2t-Isoprostane20)
quantities in its crystal structure. Within the binary melting
point diagram of such systems two symmetrical eutectics are
found, as shown in Figure 1A. Enantioselective crystallization is
herein thermodynamically only feasible from a supersaturated
enantioenriched solution within the two-phase-region at the
respective side of the enantiomer, highlighted red and blue in
Figure 1B. Consequently the enantiomeric excess of the
original enzyme-derived solution needs to be higher than the
respective eutectic of 2a to facilitate the formation of pure solid
enantiomer (Figure 1).21,22
2. RESULTS AND DISCUSSION
From cis-1,4-diacetoxy-2-cyclopentene 1 the undesired counter-
enantiomer (1R,4S)-4-hydroxy-2-cyclopentenyl acetate can be
Figure 2. Comparison of specific activities (black) and the obtained
enantiomeric excesses (gray) of the six ECS-PLE isoenzyme
preparations. Reaction conditions: 0.25−15.5 mg/mL PLE isoenzyme
(lyophilizate), 2.5 mg (13.5 μmol) 1, 1.5 mL 100 mM phosphate
obtained with high enantiomeric excess by hydrolysis with
immobilized Candida antarctica lipase B (CalB).18 In contrast,
pig liver esterase converts 1 to the desired enantiomer (1S,4R)-
4-hydroxy-2-cyclopentenyl acetate 2a with an enantiomeric
excess of 86% ee.19,23 For full enantiopurity a subsequent
enantioselective crystallization step is required (Scheme 1).
2.1. Choice of Optimal PLE Isoenzyme. In this study six
commercially available isoenzymes (ECS-PLE01-06 from
Enzymicals AG) were investigated toward their specific activity
and enantioselectivity for the hydrolysis of cis-1,4-diacetoxy-2-
cyclopentene 1 (Figure 2).
Significant differences between the individual isoenzymes
were found for the selective hydrolysis of cis-1,4-diacetoxy-2-
cyclopentene 1. This behavior is based on different sequences
and the respective structures of the proteins, which were
already reported in detail.24,25 ECS-PLE01, 02, 04, and 05
buffer pH 7.5, 40 °C, 120 min, Negative enantiomeric excesses
represents the enantioselectivity toward the other enantiomer 2b.
exhibit very poor specific activities of 0.8, 2.0, 3.7, and 10.0 U/g,
respectively. For all these enzymes only moderate enantiose-
lectivities were found, including a reverse enantioselectivity
using ECS-PLE04 and 05 toward (1R,4S)-4-hydroxy-2-cyclo-
pentenyl acetate. Thus, these four isoenzymes with their
insufficient substrate specificity and selectivity were excluded
from further experiments. Noteworthy, excellent enantioselec-
tivities were observed for some of these isoenzymes for the
conversion of other substrates. For example, ECS-PLE05
demonstrates satisfying enzymatic activity for the conversion
B
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of cyclohex-4-ene-cis-1,2-dicarboxylate.16 In this presented case
ECS-PLE03 (89 U/g) and ECS-PLE06 (153 U/g) exhibited
the highest specific activity for the intended hydrolysis of 1 with
similarly high enantiomeric excesses of about 81% ee and 83%
ee. Aside specific activity and enantioselectivity, the stability of
the chosen isoenzymes is very important for future application.
Consequently, enzyme activity was investigated over time to
ensure sufficient catalyst stability within a preparative process
(Figure 3).
Figure 4. Effect of pH (A) and reaction temperature (B) on
conversion (graph) and enantiomeric excess (squares) for the ECS-
PLE06-catalyzed hydrolysis of cis-1,4-diacetoxy-2-cyclopentene 1.
Reaction conditions: (A) 2 U/mL ECS-PLE06 (as lyophilizate,
according to the standard activity assay), 9.2 mg (50 μmol) 1, 5 mL of
100 mM phosphate buffer, 25 °C, 120 min. (B) 0.25 U/mL ECS-
PLE06 (as lyophilizate, according to the standard activity assay), 2.5
mg (13.5 μmol) of 1, 1.5 mL of 100 mM phosphate buffer pH 7.5, 120
min.
Figure 3. Comparison of the remaining relative enzyme activity of
ECS-PLE03 (squares) and ECS-PLE06 (triangles) at 50 °C.
Experimental conditions: ECS-PLE03 and ECS-PLE06 were stored
in 100 mM phosphate buffer pH 7.5 at 50 °C; activity was determined
via p-NPA standard assay; activity at 0 h was set to 100%.
As shown herein, ECS-PLE06 possesses at 50 °C a consistent
stable enzyme activity over 23 h and a remaining activity of 24%
after 168 h, which translates to a half-life stability of 91 h. In
contrast, ECS-PLE03 lost activity significantly faster with a
remaining activity of 19% after only 4 h, which converts to a
very low half-life stability of only 1.7 h. This reason for the
different stabilities is currently unknown. Consequently,
isoenzyme ECS-PLE06 was preferred in this study for the
hydrolysis of 1. Nevertheless, ECS-PLE03 with its high activity
and selectivity could be favored for other substrates.
2.2. Reaction Conditions. Several changes of enantio-
selectivity and reaction velocity by modification of the reaction
media were reported for pig liver esterase. In order to obtain
optimal parameter for the enzymatic hydrolysis of 1 the
influence of pH, reaction temperature, and cosolvents on the
performance of the investigated rPLEs were examined. The
results are summarized in Figures 4 and 5.
Figure 5. Effect of cosolvents on the ECS-PLE06-catalyzed on
conversion (graph) and enantiomeric excess (squares) for the ECS-
PLE06-catalyzed hydrolysis of cis-1,4-diacetoxy-2-cyclopentene 1.
Reaction conditions: 0.25 U/mL ECS-PLE06 (lyophilizate, according
to the standard activity assay), 2.5 mg (13.5 μmol) of 1, 200 mM
cosolvent, 1.5 mL of 100 mM phosphate buffer pH 7.5, 50 °C, 120
min.
substrate was hydrolyzed after 2 h, while >80% conversion was
achieved at 50 °C. Most importantly, a relative high
enantiomeric excess of 83% remains nearly constant at all
reaction temperatures. Furthermore, ECS-PLE06 shows a
constant relative enzyme activity over 24 h at this temperature
(Figure 3). Consequently, a reaction temperature of 50 °C was
chosen for all following experiments within this study.
Moreover, the effects of cosolvents were also considered in
this study since positive effects of certain solvents were recently
reported, e.g., Smith et al. found an improved enantioselectivity
for PLE isoenzymes by applying a selection of cosolvents.26
However, in the present study only negative effects were found
for the hydrolysis of 1. Drastic losses of enantioselectivity and
enzyme activity of ECS-PLE06 were detected using THF, iso-
propanol, and tert-butanol as cosolvents with very low
conversions of 30% and below. Methanol, ethanol, DMSO,
First, the pH-dependence (Figure 4) was investigated at
different pH levels. At pH 7, pH 7.5, and pH 8, similar
conversions and enantiomeric excesses were found. In all cases
(1S,4R)-4-hydroxy-2-cyclopentenyl acetate 2a was formed with
87% ee, except for pH 9 with a much lower ee of 68%. This
effect is caused by the autohydrolysis of the substrate at
elevated pH conditions. To avoid the unintended auto-
hydrolysis the pH level was set to pH 7.5 for all further
experiments, which is relatively similar to an early report of
Brusehaber et al.23 In addition, the effect of reaction
̈
temperature (Figure 4B) was examined at lower enzyme
concentration, since the influence of higher temperatures on
ECS-PLE06 has already been reported.16 As shown in Figure
4B, an increase of reaction temperature improves as expected
the conversion of 1 to 2a. At 30 °C only one-third of the
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Table 1. rPLE-Catalyzed Hydrolysis of cis-1,4-Diacetoxy-2-cyclopentene at Larger Scale with pH-Adjustment by an External
Base Solution
a
b
entry
V (L)
substrate (mM)
activity (U/mL)
reaction time (min)
ee (%)
conversion (%)
STY [g/(L·h)]
1
2
3
4
5
6
7
8
0.02
0.02
0.1
100
100
100
100
100
200
500
230
20.0
10.0
10.0
5.0
11.0
36.7
88
89
87
85
83
84
84
91
98
>99
97
95
90
91
76.0
23.0
22.1
22.1
10.2
14.1
11.2
7.7
37.5
0.02
0.02
0.02
0.02
2.4
36.7
2.5
75.0
5.0
110.0
240.0
240.0
c
10.0
5.0
63
94
a
b
The substrate was not soluble at higher concentrations, and thus an emulsion was formed. Used volumetric enzyme activity; calculated from
c
specific activity [U/mg] according to standard activity assay and enzyme amount [mg] per volume [mL]. Due to an extremely slowing down of the
reaction velocity, the reaction was interrupted after 240 min.
and 1,4-dioxane gave only moderate conversions of up to 65%
(DMSO) with a maximum enantiomeric excess of 81%
(methanol). In conclusion, a usage of organic cosolvents was
not applicable for the scale-up of the investigated ECS-PLE06
catalyzed process.
2.3. Scale-up. After the selection of the catalyst isoenzyme
ECS-PLE06 and the optimal process conditions of pure
phosphate buffer pH 7.5 and 50 °C, the hydrolysis of 1 was
scaled-up. Due to the higher substrate amount and therefore
higher acid formation, an efficient pH-adjustment by an
external base solution was required.
To optimize the process regarding substrate concentration
and enzyme activity, several parameters were investigated, and
the conducted reactions at larger scale are summarized in Table
Figure 6. Binary phase diagram (BPD) of 4-hydroxy-2-cyclopentenyl
1. In all cases enantiomeric excesses up to 91% ee were
achieved with excellent space-time-yields. Substrate concen-
trations between 100−200 mM were successfully converted
with high conversion (entries 1−6), while even higher
concentrations of 500 mM significantly inhibited ECS-PLE06
(entry 7). A multigram-scale was eventually performed at 230
mM to ensure optimal efficiency of the biocatalyst (entry 8, see
also Experimental Section for the detailed preparation) with an
enantiomeric excess of 91% ee and a space-time-yield of 7.7 g/
(L·h).
acetate 2.
2.4. Enantioselective Crystallization. Solid Phase
Behavior. Since only an unsatisfactory enantiomeric excess
was obtained from the enzyme-catalyzed preparation of 2a, a
further enantioselective crystallization step was necessary for
the preparation of the enantiopure compound. For this purpose
more details about the solid phase behavior of the chiral target
substance were required to understand and optimize the
subsequent crystallization process. Especially the binary phase
diagram of 4-hydroxy-2-cyclopentenyl acetate 2 is herein
important since it highlights the eutectics of the investigated
chiral system. Consequently solid mixtures of different mole
fractions of 2 between the racemic mixture and the pure
enantiomer were investigated by differential scanning calorim-
etry (DSC), and the results were used to construct the binary
phase diagram (Figure 6). A racemic compound behavior was
found (further details about this solid phase behavior are
mentioned in the Introduction) with a eutectic at a mole
fraction of 0.725 (45% ee). Since such chiral systems are
symmetrically with respect to the racemic mixture, the other
eutectic is positioned at 0.275 toward the other undesired
enantiomer (not shown in Figure 7). Moreover, the theoretical
calculation of the liquidus curve applying the simplified
Figure 7. XRPD-results of enantiopure (1S,4R)-4-hydroxy-2-cyclo-
pentenyl acetate 2a, racemic 4-hydroxy-2-cyclopentenyl acetate, and
their mixtures close to the eutectic of 0.725.
1 and 2 in the Experimental Section) gave a relatively good
agreement with the experimental values.
The position of the eutectic is relevant for the intended
crystallization, because it represents the lower limit of the final
crystallization process. Enantioselective crystallization is only
feasible until the eutectic composition is reached in the
depleted mother liquor. Below this point the racemic mixture
crystallizes, which obviously reduces the enantiopurity of the
desired solid product (see also Figure 1).
In addition, the racemic compound behavior was also
confirmed by X-ray powder diffraction (XRPD) (Figure 7).
Characteristic diffraction angles of the pure enantiomer are ca.
15, 18, 23, and 24° and for the racemate at about 22, 25, 27,
and 28°. In the measured diffractograms, no additional signals
equations of Schroder-van-Laar and Prigogine-Defay (see eqs
̈
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were found within all investigations of the pure materials and
mixtures under any circumstances; thus, significant poly-
morphism of 2 is fortunately not present. Furthermore, no
solvate behavior of any kind was found in this study. Additional
XRPD results are presented in the Supporting Information file
(Figure SI-4).
Crystallization of Enantiopure (1S,4R)-4-Hydroxy-2-cyclo-
pentenyl Acetate. In total, three enantioselective crystalliza-
tions were performed to improve the enantiomeric excess of 2.
For the first crystallization entry 5 and 6 of the scale-up
reactions were used. Scale-up 1 to 4 and 7 were merged and
crystallized in a second experiment. The crude product (Table
1 Entry 8) of the multigram scale was crystallized in a third
experiment. Data and results of the three crystallizations are
shown in Table 2.
cyclopentenyl acetate 2a and all applied solvents were
purchased from Sigma-Aldrich (Munich, Germany). All
chemicals were used as received. Deionized water was used
throughout this study.
Enzymes. The recombinant pig liver esterases, ECS-PLE01
to ECS-PLE06, are commercially available (as lyophilizate)
from Enzymicals AG (Greifswald, Germany). Classical pig liver
esterase (mixture of isoenzymes from the natural source, crude
protein powder) was purchased from Sigma-Aldrich (St. Louis,
USA).
Standard Activity Assay. The activity of recombinant and
classical PLEs was determined from the hydrolysis of p-
nitrophenyl acetate (pNPA, 30 mM dissolved in DMSO) with
spectrophotometric quantification of p-nitrophenolate release
over 120 s at 30 °C and 400 nm (ε25°C = 13190 L/(mol·cm)).
The assay solution contained 850 μL of 50 mM phosphate
buffer pH 7.5, 50 μL of enzyme solution and 100 μL 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 the UV/vis-spectrometer
Specord 50 (Analytic Jena AG, Jena, Germany) and Helios
Alpha UV/vis-spectro-photometer (Thermo Scientific GmbH,
Schwerte, Germany) were used. All measurements were
executed with a reference cell including an equal solution
Table 2. Enantioselective Crystallization of (1S,4R)-4-
Hydroxy-2-cyclopentenyl Acetate 2a
initial ee final ee
remaining
ee (%)
theoretical max.
yield (%)
practical
yield (%)
entry
(%)
(%)
1
2
3
83
86
91
≥99.5
98
71.0
54.0
71.0
69
75
84
33
65
44
99
With an initial enantiomeric excess of 83%, the first
crystallization facilitated pure (1S,4R)-4-hydroxy-2-cyclopen-
tenyl acetate 2a (ee ≥ 99.5%) with a moderate yield of 33%
(entry 1). Since an enantiomeric excess of 71% ee remained in
the mother liquor, this process was terminated too soon, and
the theoretical maximum yield of 66% was not achieved. In
comparison, the second crystallization started with an
enantiomeric excess of 86% ee, yielding 2 with an enantiomeric
excess of 98% ee at an adequate yield of 63% (theoretical
maximum of 75%). The crystallization of crude product 2 (42
g, see also Experimental Section for details in implementation)
required an additional recrystallization step to remove
unreacted substrate 1 and a significant amount of protein
impurities. Consequently only 44%, based on the mass of the
crude product, were obtained from the enantioselective
crystallization. Nevertheless, the subsequent enantioselective
crystallization was successful, and an enantiomeric excess of
99% ee was achieved (entry 3). Further studies will target an
improved purification strategy from the reaction solution.
without enzyme. Data are reported as mean
deviation of triplicate measurements.
standard
Isoenzyme Screening. 0.25−15.5 mg of ECS-PLE01-06
(lyophilized) or commercial available PLE were suspended in
1.5 mL of 100 mM phosphate buffer pH 7.5 at 40 °C. The
reaction was started by adding 2.5 mg (13.5 μmol) of 1. After 2
h the resulting reaction mixture was extracted with ethyl acetate
and conversion and enantiomeric excess of the enzymatic
reactions obtained from analysis with gas chromatography (see
also below). Small scale desymmetrization experiments were
carried out with the respective isoenzyme (0.25 U/mL) in 1.5
mL of a buffered solution (100 mM phosphate buffer) with 2.5
mg (13.5 μmol) of 1. The enzymatic hydrolysis was conducted
at pH 7, pH 7.5, pH 8, and pH 9 at 25 °C. pH measurements
were examined with a pH 3210 pH-meter from WTW
(Weilheim, Germany), using a pH glass electrode model
IJ44C and calibrated with technical buffer from WTW. The
reaction was examined at 30, 40, and 50 °C at pH 7.5.
Cosolvents (methanol, ethanol, iso-propanol, tert-butylalcohol,
1,4-dioxane, DMSO, and THF) were determined at pH 7.5 and
50 °C. After 2 h the reaction was stopped by extracting the
reactants with ethyl acetate. Conversion and enantiomeric
excess of the enzymatic reactions were obtained from analysis
with gas chromatography (see below).
Small-Scale Biocatalysis with ECS-PLE06. A 100 mL
round-bottom flask was charged with 20 mL of 100 mM
phosphate buffer pH7.5, ECS-PLE06, and a magnetic stir bar.
The suspension was set to 50 °C. The reaction was started by
adding 368 mg (2 mmol) of 1 to the reaction mixture. The
solution was kept constant at pH 7.5 by addition of 0.5 M
sodium hydroxide solution via an autoburet. After the addition
of the required NaOH solution, the reaction was stopped by
extraction with twice volume of ethyl acetate. The precipitated
protein was removed by filtration of both phases and the
aqueous layer extracted three to five times with 20 mL ethyl
acetate. The combined organic layers were evaporated under
reduced pressure to give (1S,4R)-4-hydroxy-2-cyclopentenyl
acetate 2a (ee = 86%).
3. CONCLUSION
In this contribution the successful desymmetrization of cis-1,4-
diacetoxy-2-cyclopentene by recombinant pig liver esterase
isoenzyme was investigated at larger scale. 50 °C and pH 7.5
were found to be the optimal reaction conditions and a
desymmetrization reaction at preparative scale was performed.
Herein recombinant pig liver esterase isoenzymes provide a
powerful and more reproducible alternative to classical pig liver
esterase preparations.
For the subsequent enantioselective crystallization, the solid
phase behavior of 4-hydroxy-2-cyclopentenyl acetate was
determined including its binary phase diagram. The subsequent
enantioselective crystallizations yielded several gram of
enantiopure (1S,4R)-4-hydroxy-2-cyclopentenyl acetate 2a.
4. EXPERIMENTAL SECTION
General. cis-1,4-Diacetoxy-2-cyclopentene 1 was purchased
from Chiracon (Luckenwalde, Germany), (1S,4R)-4-hydroxy-2-
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Crystallization. Crude product 2a was dissolved in n-
heptane, conditioned to 50 °C, and continuously cooled to 35
°C with 10 K/min. Seed crystals were added until crystal
growth occurred. Afterward the solution temperature was
lowered to 10 °C with 20 K/min. The obtained product was
filtered off and dried, yielding (1S,4R)-4-hydroxy-2-cyclo-
pentenyl acetate with excellent enantiomeric excess (>99% ee).
Multigram Scale. For the preparation of enantiopure
(1S,4R)-4-hydroxy-2-cyclopentenyl acetate 2a at multigram
scale, 78 g (0.4 mol) of 1 were converted with 5 U/mL ECS-
PLE06 in 2.4 L of 100 mM phosphate buffer pH 7.5 at 50 °C.
The solution was kept constant at pH 7.5 by addition of 0.5 M
potassium hydroxide solution via an autoburet. The reaction
was followed via TLC (cyclohexane−2-propanol 90:10 v/v
stained with KMnO4). The reaction was stopped by adding 5 L
of ethyl acetate and to precipitate the protein 1.5 L of an
acetone:2-propanol mixture (2:1 v/v) was added. The whole
mixture was stirred for 15 min afterward treated with Celite545
to become a well filterable solid. The solid precipitate was
filtered off, and remaining aqueous layer was extracted twice
with 2.5 L of ethyl acetate. The organic layers were combined
and dried over MgSO4. The solvent was removed under
reduced pressure to give 42 g of a brown crude product. The
crude product was recrystallized in 400 mL of n-heptane:ethyl
acetate (80:20 v/v) to give 20 g of 2a. For the enantioselective
crystallization crude product 2a was dissolved in 400 mL of n-
heptane−ethyl acetate (90:10 v/v) and conditioned to 50 °C.
Subsequent the solution was continuously cooled from 50 to 10
°C with 10 K/min. Seed crystals were added until crystal
growth occurred. The obtained product was filtered off and
dried, yielding 16.7 g of (1S,4R)-4-hydroxy-2-cyclopentenyl
acetate with an enantiomeric excess of 99%.
Measurements covered a 2Θ range from 5° to 40° with a step
size of 0.02°.
Binary Phase Diagram. The shown liquidus curves of the
enantiomer and the racemate were calculated according to the
simplified equations of Schroder-van-Laar and Prigogine-Defay
̈
(eq 1 and eq 2) with x = mole fraction, R = gas constant, Tf,E,
Tf,R = melting temperature of enantiomer and racemic
compound, and ΔHf,E, ΔHf,R = enthalpy of fusion of
enantiomer and racemic compound (see also Supporting
Information). The theoretical eutectic was calculated from
the intersection of the liquidus lines. Polymorphism of the
target compound was not found within this study.
⎛
⎝
⎞
⎠
ΔHf,E
R
1
1
⎜
⎜
⎟
⎟
ln x =
−
T
T
f,E
f
(1)
(2)
⎛
⎝
⎞
⎠
2ΔHf,R
1
1
⎜
⎜
⎟
⎟
ln 4x(1 − x) =
−
R
T
T
f,R
f
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.oprd.6b00093.
■
S
Summary of HPLC chromatograms, further XRPD
results of the investigated target compound, and NMR
data (DOCX)
AUTHOR INFORMATION
Corresponding Authors
*E-mail (R.W.): rainer.wardenga@enzymicals.com.
*E-mail (J.v.L.): jan.langermann@uni-rostock.de.
■
Analytical Methods. Conversion and the corresponding
enantiomeric excess of 2a were quantified by GC-FID using a
Thermo Scientific Trace 1310 gas chromatograph fitted with a
Chrompack Chiralsil-Dex-CB column (25 m × 0.25 mm × 0.25
μm). Injector temperature: 250 °C, detector temperature: 250
°C, temperature gradient: start at 67 °C, rise with 10 °C/min to
150 °C and hold for 2 min, rise with 15 °C/min to 200 °C, and
hold for 3 min. Retention times: 8.4 min (1S,4R)-4-hydroxy-2-
cyclopentenyl acetate 2a, 8.6 min (1R,4S)-4-hydroxy-2-cyclo-
pentenyl acetate, and 8.8 min cis-1,4-diacetoxy-2-cyclopentene
1 (see also Supporting Information).
Funding
Financial support is gratefully acknowledged by the Central
Innovation Program SME (Zentrales Innovationsprogramm
MittelstandZIM) by the Federal Ministry for Economic
Affairs and Energy (Bundesministerium fur Wirtschaft and
̈
TechnologieBMWi), grant number: KF2622302AJ2.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
NMR Analysis. 1H NMR and 13C NMR-spectra were
recorded with a Bruker AVANCE (300 MHz) in CDCl3.
Chemical shifts (δ) are reported in parts per million relative to
the solvent peak. Splitting patterns are indicated as follows: s,
singlet; d, doublet; t, triplet; m, multiplet; quint, quintet; dt,
double triplet; dd, double doublet.
Differential Scanning Calorimetry (DSC) Measure-
ments. DSC experiments were performed on a Mettler
Toledo DSC 823e. The start temperature was 25 °C, the final
temperature was 130 °C, and a heating rate of 2 K/min was
applied. In order to produce controlled mixtures for phase
diagram determination, weighed amounts of enantiomer were
mixed and dissolved in acetone, and after evaporation of the
solvent, the solid was grounded to a fine powder. 10−15
milligrams of the respective mixtures were used for DSC
experiments.
The authors thank Prof. Dr. Udo Kragl (University of Rostock)
and Dr. Ulf Menyes (Enzymicals AG) for useful discussions as
well as Philipp Thiele (University of Rostock) for the
implementation of XRPD measurements.
ABBREVIATIONS
■
BPD, binary phase diagram; DSC, differential scanning
calorimetry; ECS-PLE, recombinant pig liver esterase produced
by Enzymicals AG (Greifswald, Germany); XRPD, X-ray
powder diffraction
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