Communication pubs.acs.org/OPRD
Scaling-Up of “Smart Cosubstrate” 1,4-Butanediol Promoted Asymmetric Reduction of Ethyl-4,4,4-trifluoroacetoacetate in Organic Media Ralf Zuhse,† Christian Leggewie,‡ Frank Hollmann,§ and Selin Kara*,∥ †
CHIRACON GmbH, Biotechnologiepark, 14943 Luckenwalde, Germany Evocatal GmbH, Alfred-Nobel-Str. 10, 40789 Monheim am Rhein, Germany § Department of Biotechnology, Biocatalysis Group, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands ∥ Institute of Microbiology, Chair of Molecular Biotechnology, Technische Universität Dresden, 01062 Dresden, Germany ‡
S Supporting Information *
diol as a cosubstrate leads to a thermodynamically favorable lactone coproduct, which shifts the overall equilibrium towards more products. This approach offers a significant economic advantage by avoiding high surpluses of the cosubstrate and hence tedious product recovery from the excess amounts of cosubstrate/coproduct. This concept is also less harmful to the environment by 40-fold (based on 0.5 mol equiv of cosubstrate) reduction in the waste generated for >95% conversions.5 In addition to efficient cofactor regeneration, biocatalysis in nonaqueous media is another highlighted topic for enhancing productivities in enzymatic reactions. The use of nonaqueous media offers several advantages such as (i) high solubility of water-insoluble substrates, (ii) no stability issues for watersensitive compounds, (iii) no water-dependent side reactions, (iv) less wastewater generated, and (v) more straightforward downstream processing (DSP).6 Klibanov and co-workers6a,b,7 were the pioneers in the application of ADHs in predominantly organic media. Since then, the use of ADHs in organic media either by using whole-cells8 or isolated enzymes9 has gained increased interest in the research community. Hence, we became interested in the application of the “smart cosubstrate” approach for ADH-catalyzed bioreductions in predominantly organic media. Our experiments showed that only some amounts of water (2.5% v/v) in the form of buffer containing the cofactor were required for the biocatalyst’s activity. Regarding solvent hydrophobicity, log P value (logarithmic value of octanol−water partition coefficient) was reported as a significant parameter for optimizing biocatalytic reactions.10 However, it was also shown that log P value cannot be a direct criterion to choose an organic solvent, since solvent functionality11 also plays an important role. Hence various organic solvents with a broad log P ranging from 1.0 to 5.6 were screened, and among them, we chose methyl tert-butyl ether (MTBE) owing to its low boiling point and offering the highest enzyme activity. Overall, turnover numbers (TONs) of 64 000 for the enzyme and 960 for the NADH were achieved by using this approach.12
ABSTRACT: Biocatalytic asymmetric reduction of ethyl4,4,4-trifluoroacetoacetate under water-deficient reaction conditions using a “smart cosubstrate” 1,4-butanediol was demonstrated up to a 2 L scale. Substrate concentrations of 100 g/L were applied by using half-molar equivalent of 1,4-butanediol in methyl-tert-butylether (MTBE). Using this approach, full conversion of ethyl-4,4,4-trifluoroacetoacetate to the corresponding (S)-alcohol with an excellent enantiomeric excess (ee) of ≥99% was accomplished in 5 days. 150 g of isolated enantiopure product with high purity (94%) was obtained.
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INTRODUCTION The use of enzymes for the enantioselective organic synthesis has grown very fast during the past decades owing to the high selectivity of the enzymes.1 Especially alcohol dehydrogenases (ADHs) (also called ketoreductases or carbonyl reductases) have attracted attention because of their ability to catalyze highly selective reductions of carbonyl groups in, e.g., aldehydes, ketones, or keto esters.2 These synthetically useful reactions require hydride transfer from a reduced nicotinamide cofactor (NAD(P)H), a reversible reaction which makes an efficient cofactor regeneration highly desirable. First, cofactor regeneration is required for economic reasons as the stoichiometric use of NAD(P)H would be very costly. Second, an efficient cofactor regeneration system drives the thermodynamic equilibrium to the side of the product.3 For in situ regeneration of reduced nicotinamide cofactors in ADH-catalyzed reactions, the so-called “substrate-coupled” approach excels in simplicity, as only one biocatalyst is required for both production and regeneration reactions. However, in this approach, which represents a biocatalytic version of the well-known Meerwein−Ponndorf−Verley (MPV) reduction, reversibility, and the poor thermodynamic driving force of the reaction necessitate significant molar surplus (usually 10− 20 equiv) of the cosubstrate, e.g., ethanol and isopropanol.4 Recently, we have introduced a “smart cosubstrate” approach for NAD(P)H regeneration, whereby 1,4-butanediol (1,4-BD) was shown to be an efficient cosubstrate to promote NADHdependent biotransformations.5 The use of a lactone-forming © 2015 American Chemical Society
Received: November 28, 2014 Published: February 3, 2015 369
DOI: 10.1021/op500374x Org. Process Res. Dev. 2015, 19, 369−372
Communication
Organic Process Research & Development
The bioreduction of 1 coupled with 1,4-BD was performed in organic media at a 0.5 L scale (Table 1, entry 1). After 2.7 days,
Highly productive biotransformations originally developed at a milliliter-scale need to be further optimized during scaling-up processes to achieve large-scale production capacities. Considerations required for scaling-up of biocatalytic processes have been summarized in review articles.13 Large-scale application of ADHs either as whole-cells or as cell free preparations has been found in literature. For instance, Eli Lilly and Company established a multikilogram scale whole-cell based biocatalytic reduction of a prochiral ketone combined with a polymeric resin for in situ product removal. By this approach, product concentrations at 40 g/L could be achieved.14 Recently, Codexis, Inc. and Merck have demonstrated the application of ADHs for the enantioselective synthesis of alcohols up to the kilogram scale.15 Therein, the high stability of engineered ADHs against high amounts of organic solvents (∼70% v/v) allowed substrate concentrations of ∼100 g/L. In the present communication, we report scaling-up of ADHcatalyzed reductions from a few milliliter-scale to a liter-scale, whereby in situ cofactor regeneration was promoted by the “smart cosubstrate” 1,4-BD. The enzyme of choice was the ADH evo-1.1.200, commercial ADH preparation obtained from evocatal GmbH, as this enzyme excelled by its significant stability in organic media, which was demonstrated in our previous study.12
Table 1. Scaled-up reactions for the ADH-catalyzed reduction of 1 in organic mediaa composition (%) entry
V (L)
c(1) (g/L)
time (d)
1
S-(2)
1,4-BD
GBL
1
0.5
96
2
2
97
2.7 7 3 5
4 1.5 3 1
66 68 67 68
13 4 13 8
15 20 16 21
a
Reaction conditions: c(1) = 520/527 mM, c(1,4-BD) = 255/254 mM, c(NAD+) = 0.52 mM, c(evo 1.1.200) = 0.3 g/L, 2.5% (v/v) external water (200 mM Tris-HCl, pH 7.0), MTBE, 30 °C, and 1000 rpm.
96% conversion of 1 was achieved, giving the target alcohol product (S)-2 with an ee of ≥99%. The conversion reached 98% in 7 days. In both scales we observed that the increase in product formation (e.g., 66−68% and 67−68%) was lower than the increase in GBL (γ-butyrolactone) synthesis (e.g., 15−20% and 16−21%). This observation was attributed to the presence of an endogenous NADH oxidase,18 found in the crude enzyme preparation, which would promote oxidation of 1,4-BD to GBL without coupling it to reduction of the ketone. In addition it is worth mentioning that a decrease of about 5% of the total mass was detected over 7 days, which is most likely due to the evaporation of the reaction medium because of sampling during the several days. Hence, reduced reaction times would be highly desirable to reduce the number of samples. After the reaction workup, 87.5 g of yellowish oily product was collected, which was distillated for the purification of the alcohol product (Table 2).
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RESULTS AND DISCUSSION In our previous study, we had screened a broad range of substrates for ADH-catalyzed reductions coupled with 1,4-BD cosubstrate in organic media (milliliter-scale). The highest conversions were achieved when for the substrate cyclohexanone, 2-butanone, and ethyl-4,4,4-trifluoroacetoacetate were used, where in the latter the reduction product was detected with an excellent enantiomeric excess (ee) value of ≥99% (S).12 Encouraged by these results, we approached the bioreduction of ethyl-4,4,4-trifluoroacetoacetate promoted by the “smart cosubstrate” 1,4-BD (Scheme 1) for scaling-up. Based on the earlier work, MTBE was the solvent of choice for the experiments in this study.12
Table 2. Downstream processing for scaling-up the 0.5 L scale composition (%)
Scheme 1. ADH-catalyzed reduction of ethyl-4,4,4trifluoroacetoacetate (1) in nonaqueous media coupled with 1,4-BD as the cosubstrate a
0.5 L scale
1
S-(2)
1,4-BD
GBL
sub-fraction no. 1a sub-fraction no. 2b sub-fraction no. 3c distillation swamp
64 0 0 0
29 74 47 9
0 0 0 56
2 22 51 31
Distillation at 36−109 °C. bAt 109−114 °C. cAt 114 °C.
A clear separation of 1 and 1,4-BD was achieved, whereas the separation of the product 2 and GBL was not possible (22% of GBL found in the sub fraction no. 2, Table 2) under the distillation conditions applied. Therefore, in order to improve DSP, column head distillation was performed to purify the product of the following reaction. Next, the reaction was performed at 2 L scale (Table 1, entry 2). As a result, the bioreduction of 1 reached 96% conversion after 3 days, and full conversion was observed after 5 days. After the reaction was stopped, the organic medium was removed, and the resulting yellowish oily product (352 g) was further purified via distillation. Different from the previous reaction workup, the separation of 2 and GBL could be significantly improved (only 4% of GBL found in the fraction instead of 22% in sub fraction no. 2), which gave 150 g (about 70% yield from 1230 mmol of 1) of the isolated alcohol
As a rule of thumb, industrial biotransformations require substrate-to-enzyme ratios of >50 kg/kg for economic reasons.16 Different approaches are taken to fulfill this requirement such as immobilization of enzymes for reuse, protein engineering, and reaction engineering, etc.1e,17 In this study process optimization allows economic use of the target enzyme, i.e., the concentration of ∼0.3 g/L for a substrate concentration of 100 g/L leading to a substrate-to-enzyme ratio of >300. 370
DOI: 10.1021/op500374x Org. Process Res. Dev. 2015, 19, 369−372
Communication
Organic Process Research & Development
incubated enzyme, and the reaction mixture (577 mL) was kept at 30 °C and 1000 rpm using a magnetic stirrer. After 64 h, an aliquot (100 μL) was removed and mixed with MTBE (25 mL). After drying over anhydrous Na2SO4, the solvent was removed under reduced pressure (250 mbar, 40 °C) and analyzed by GC (8.5 mg sample dissolved in 1 mL dichloromethane). After 7 days the next sample was taken and prepared for analyzing by GC. Next, the organic phase was separated from the enzyme by simple decantation. The enzyme was rinsed once with MTBE (100 mL), the collected organic phase was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure (250 mbar, 40 °C) to give a yellowish oily compound (87.46 g). Then the compound was fractionally distillated under reduced pressure (70 mbar, 40 °C). Three fractions and a distillation swamp were obtained. Bioreduction, Scale-Up Step 2 (2 L). Portions of 16 g of the evo-1.1.200 enzyme preparation and 800 mg of NAD+ were incubated (at 25 °C and 300 rpm for 50 min) in 60 mL of TrisHCl buffer (200 mM, pH 7.0) in a sealed glass flask (4 L with three-neck). The substrate 1 (1230 mmol, 226.5 g) and the cosubstrate 1,4-BD (0.5 equiv., 613 mmol, 55.2 g) were prepared in MTBE (2100 mL). After 50 min, the substrates (from the stocks prepared in MTBE) were added to the incubated enzyme, and the reaction mixture (2334 mL) was kept at 30 °C and 1000 rpm using a magnetic stirrer. The first sample (100 μL) was taken after 72 h and mixed with MTBE (25 mL), followed by vigorously mixing and drying over anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure (250 mbar, 40 °C) and analyzed by GC (8.5 mg sample dissolved in 1 mL of dichloromethane). The next sample was taken after 5 days as similar given above and analyzed by GC. After 5 days, the solvent was separated from the enzyme by simple decantation. The enzyme was rinsed once with MTBE (100 mL), the collected organic phase was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure (250 mbar, 40 °C) to give a yellowish oily compound (352 g). Next, the compound was fractionally distillated under reduced pressure (70 mbar, 40 °C). Three fractions and a distillation swamp were obtained. The second fraction gave 150 g of yellowish oily compound containing 94% target product (S)-2 and 4% GBL.
Table 3. Downstream processing for scaling-up a 2 L scale composition (%) 2 L scale a
sub-fraction no. 1 sub-fraction no. 2b sub-fraction no. 3c distillation swamp a
1
S-(2)
1,4-BD
GBL
64 0 0 0
22 94 47 9
0 0 0 56
12 4 51 31
Distillation at 36−110 °C. bAt 111−112 °C. cAt 112−114 °C.
product (S)-2 (≥99% ee). Overall, we were pleased to report the productivity of the reaction as 250 kgproduct/kgfree enzyme, which lies in a range required for pharmaceuticals (100− 250 kgproduct/kgfree enzyme).19
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CONCLUSIONS “Smart cosubstrates” bear the potential of making biocatalytic reduction reactions more efficient and environmentally more benign by significantly reducing the cosubstrate loading. In this study we have demonstrated for the first time that the “smart cosubstrates” approach can be applied at preparative scale. A current limitation of the reaction system presented here lies in the need for destillative separation of the desired product and the GBL coproduct. This, however, may well be overcome by hydrolytic cleavage of the lactone coproduct yielding highly water-soluble γ-hydroxy acids, which can be separated from the product of interest via aqueous extraction. Further development of this procedure is currently ongoing in our laboratories. Today, examples on nonclassical (i.e., nonaqueous) reaction systems are largely limited to simple hydrolytic enzymes such as lipases while such systems for ADH-catalyzed reactions are comparably few. With the present study we have demonstrated that nonaqueous ADH-catalyzed reduction reactions are feasible on preparative scale making this catalyst type practical for organic synthesis.
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EXPERIMENTAL SECTION General Methods. All of the chemicals used for this study were of analytical-grade, available commercially, and used as received. The ADH evo-1.1.200, recombinantly expressed in Escherichia coli, was from evocatal GmbH (Monheim am Rhein, Germany). The reactions progress and chemical purity were evaluated by gas chromatography (GC) analyses using a Hydrodex β-6TBDM column (25 m × 0.25 mm). Temperature profile: rate [°C/min] = −, 25, 50; temperature [°C] = 60, 140, 225; hold [min] = 2, 10, 1. Equipment = Shimadzu GC-2010 Plus, pressure = 70.1 kPa; total flow = 55.1 mL/min; column flow = 1.01 mL/min; linear velocity = 27.8 cm/s; split = 50.7. Retention times = 1, 4.3 min; GBL, 7.5 min; (S)-2, 7.8 min; 1,4-BD, 10.7 min. Conversion values were determined based on the depletion of substrate concentrations, and the experiments were carried out once. Bioreduction, Scale-Up Step 1 (0.5 L). Samples of 4 g of the evo-1.1.200 enzyme preparation (lyophilized crude powder) and 202 mg of NAD+ were incubated in 15 mL of Tris-HCl buffer (50 mM, pH 7.0) for 50 min at 25 °C and 300 rpm in a sealed glass flask (1 L with three-neck). The substrate ethyl-4,4,4-trifluoroacetoacete 1 (300 mmol, 55.2 g) and the cosubstrate 1,4-BD (0.5 equiv., 152 mmol, 13.7 g) were prepared in MTBE (520 mL). After 50 min, the substrates (from the stocks prepared in MTBE) were added to the
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ASSOCIATED CONTENT
S Supporting Information *
Details on GC chromatogram and 1H NMR spectra. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: (+49) 0351-463-39517. Fax: (+49) 0351-463-39520. Email:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Deutsche Bundesstiftung Umwelt (DBU) for financial support of the project (AZ 13261). Dr. A. Bornadel is gratefully acknowledged for proofreading of the manuscript. 371
DOI: 10.1021/op500374x Org. Process Res. Dev. 2015, 19, 369−372
Communication
Organic Process Research & Development
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ABBREVIATIONS ADH, alcohol dehydrogenase; NADPH, nicotinamide adenine dinucleotide phosphate reduced form; NADH, nicotinamide adenine dinucleotide reduced form; MPV, Meerwein− Ponndorf−Verley; 1,4-BD, 1,4-butanediol; GBL, γ-butyrolactone; MTBE, methyl-tert-butylether; TON, turnover number; DSP, downstream processing
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REFERENCES
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DOI: 10.1021/op500374x Org. Process Res. Dev. 2015, 19, 369−372
