Organic Process Research & Development
Article
suspension was stirred until a homogeneous solution was
achieved (appr. after 12 h). After that, the reaction mixture was
cooled to 0 °C with an ice-bath and subsequently 1.2 mL of
thionyl chloride was added over 15 min. The reaction was
allowed to warm to room temperature and stirred overnight at
room temperature. The resulting solution was partially
evaporated to approximately 3 mL and extracted 3 times with
crystalline (1S,2R)-1-(methoxycarbonyl)cyclohex-4-ene-1-car-
boxylic acid.
Enzymes. Recombinant pig liver esterase ECS-PLE06 is
commercially available as lyophilized powder from Enzymicals
AG (Greifswald, Germany).
Standard Activity Assay. The activity of PLE was
determined using p-nitrophenyl acetate (pNPA, 30 mM
dissolved in DMSO) with spectrophotometric quantification
of the p-nitrophenolate release over 60 s at 401 nm (ε30 °C =
3
mL 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 organic phase was dried over anhydrous
magnesium sulfate and evaporated under reduced pressure to
give 2.49 g (13.0 mmol; 77%) of the dimethyl cyclohex-4-ene-
cis-1,2-dicarboxylate 2 as colorless oil.
1
7 759 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
p-nitrophenolate per minute under standard assay conditions.
In order to detect changes in absorbance, a UV−vis
spectrometer (Helios Alpha UV−vis spectrophotometer,
Thermo Scientific GmbH, Schwerte, Germany) was used.
Analytical Methods. Gas Chromatography. Conversion
and the corresponding enantiomeric excess of 3 were quantified
by GC-FID using a CP3800 equipped with an autosampler
CP8400 (all formerly Varian, now Agilent, Santa Barbara,
U.S.A.) and a Chrompack ChiralsilDex-CB column (25 m ×
Sequential Multistep One-Pot Synthesis of (1S,2R)-1-
Methoxycarbonyl)cyclohex-4-ene-1-carboxylic Acid
Small-Scale). A 50 mL round-bottom flask was charged
(
(
with 0.49 g (3.2 mmol) of cis-cyclohex-4-ene-1,2-dicarboxylic
anhydride, 0.63 mL (15.6 mmol) of methanol, and a magnetic
stirrer. The suspension was stirred until a homogeneous
solution was achieved (appr. after 12 h). After that, the reaction
mixture was cooled to 0 °C with an ice-bath, and subsequently,
0.234 mL (3.23 mmol) of thionyl chloride was added over 15
0
.25 mm × 0.25 μm). The injector temperature was set to 220
min. The reaction was allowed to warm up to RT and stirred
until full conversion to the diester was observed (TLC). To this
solution, 39 mL of deionized water and sodium hydrogen
carbonate was added, until the mixture reached a pH of 8.3.
Then, 220 U (5.5 U/mL) of ECS-PLE06 was added to the
stirring solution, and the temperature was maintained at 40 °C.
After all diester was hydrolyzed by ECS-PLE06 (controlled by
TLC), the reaction was heated to 95 °C for 10 min and
acidified with hydrochloric acid to pH 1. The reaction mixture
was centrifuged at 6000 g for 10 min to precipitate the
denaturated protein. The remaining aqueous layer was
extracted with three portions of 40 mL of MTBE. The
combined organic layers were dried over anhydrous magnesium
sulfate and evaporated under reduced pressure to give 0.49 g
°
1
2
C, the detector temperature to 250 °C. Temperature gradient:
30 °C for 12 min, rise with 10 °C/min to 170 °C, and hold for
0 min. Retention times: 23.9 min (1R,2S)-1-(methoxycarbon-
yl)-cyclohex-4-ene-2-carboxylic acid 3 and 24.8 min (1S,2R)-2-
(
methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid.
Thin-Layer Chromatography. The reactions were moni-
tored by TLC, if required (cyclohexane:2-propanol, 95:5) RF:
diester 2, 0.6; monoester 3, 0.3; diacid 1a or anhydride 1b, 0.2.
Plates were stained with KMnO solution (3 g of KMnO , 20 g
4
4
of K CO , 5 mL of 5% NaOH, 300 mL of H O).
2
3
2
AUTHOR INFORMATION
■
(
(
2.7 mmol, 84%) of enantiopure crystalline (1S,2R)-1-
methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid.
*
*
Sequential Multistep One-Pot Synthesis of (1S,2R)-1-
Methoxycarbonyl)cyclohex-4-ene-1-carboxylic Acid
Scale-up). A 1 L jacked glass reactor with overhead stirrer
Present Address
(
(
#
Institute for Biomedical Engineering, Rostock University
Medical Center, Friedrich-Barnewitz Str. 4, 18119 Rostock,
Germany
was charged with 19.8 g (0.13 mol) of cis-cyclohex-4-ene-1,2-
dicarboxylic anhydride and 25.8 mL (0.64 mol) of methanol.
The suspension was stirred at RT until a homogeneous solution
resulted (appr. after 12 h). After that, the reaction mixture was
cooled to 0 °C, and subsequently, 9.5 mL (0.131 mmol) of
thionyl chloride was added over 120 min. The reaction was
allowed to warm up to RT and stirred until full conversion to
the diester was observed (TLC). To this solution, 595 mL of
deionized water and sodium hydrogen carbonate was added,
until the mixture reached a pH of 8.3. Then 3.5 kU (5.5 U/mL)
of ECS-PLE06 was added to the stirring solution, and the
temperature was maintained at 40 °C. After all diester was
hydrolyzed by PLE06 (controlled by TLC), the reaction was
heated to 95 °C for 10 min and acidified with hydrochloric acid
to pH 1. The reaction mixture was centrifuged at 6000 g for 10
min to precipitate the denaturated protein. The remaining
aqueous layer was extracted with three portions of 650 mL of
MTBE. The combined organic layers were dried over
anhydrous magnesium sulfate and evaporated under reduced
pressure to give 18.9 g (0.103 mol, 79%) of enantiopure
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
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 and
̈
Technologie, BMWi), grant no. KF2622302AJ2.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ECS-PLE, recombinant pig liver esterase; TLC, thin layer
chromatography
D
Org. Process Res. Dev. XXXX, XXX, XXX−XXX