Organic Process Research & Development
Article
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
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S
* Supporting Information
This material is available free of charge via the Internet at
AUTHOR INFORMATION
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Corresponding Authors
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
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We thank Dr. Ulf Menyes (Enzymicals AG) for useful
discussions and Sebastian Heim for his help in the experimental
work.
ABBREVIATIONS
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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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dx.doi.org/10.1021/op500129e | Org. Process Res. Dev. 2014, 18, 897−903