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Plant-mediated stereoselective biotransformations in natural deep eutectic solvents
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Plant-mediated stereoselective biotransformations in natural deep eutectic solvents ⁎
Manuela Panića, Maja Majerić Elenkovb, Marin Rojeb, Marina Cvjetko Bubaloa, , Ivana Radojčić Redovnikovića a b
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia
A R T I C L E I N F O
A B S T R A C T
Keywords: Asymmetric reduction Carrot root Enantioselective hydrolysis Natural deep eutectic solvents
The challenge of chiral chemical production lies in the preparation of chiral building blocks in accordance to green chemistry principles. Thus, the aim of this study was to associate the application of whole-cell biocatalysis (plant cells) and the use of environmentally friendly solvents (NADES) for enantioselective preparation of chiral molecules. For that purpose, reduction of 1-(3,4-dimethylphenyl)ethanone and hydrolysis of ( ± )-1-phenylethyl acetate by carrot root in cholinium-based eutectic mixtures as solvents were successfully conducted. The type of hydrogen bond donor and the amount of water present in NADESs influenced the conversion and/or enantiomeric excess in both enantioselective reactions. In conclusion, inversion of the enantioselectivity exhibited by the biocatalyst towards 1-(3,4-dimethylphenyl)ethanone was noticed by the addition of different amounts of water to NADES. Overall, the results suggest that NADES possess good biocompatibility with plant cells and can be applied as green reaction medium for plant-mediated bioreduction.
1. Introduction Introduction of green chemistry principles into preparation of various commercial products, such as chiral building blocks for drug preparation, is becoming an imperative in scientific community. The basics of green chemistry are summarized in twelve principles [1] and suggesting that chemical products and processes should be designed in such way to reduce or completely remove the application and creation of harmful and dangerous reagents, catalyst, and products, with the unique goal to protect the environment not by cleaning up, but by inventing new chemical processes that do not pollute. There are numerous tools of green chemistry, such as use of alternative solvents and environmentally benign raw (or renewable) materials, use of alternative energy sources (e.g. microwaves, ultrasound), performing reactions by (bio)catalysis, that are nowadays intensively studied [2]. Biotechnology methods fit logically to the goals of green chemistry meaning that the use of the biological methods and materials for industrial manufacturing is an excellent foundation to create an environmentally friendly process. In the same way, biocatalysis – the use of whole cells, cell extracts, or purified enzymes in organic synthesis – fits perfectly into green chemistry because this the “bio” part of biocatalysis makes it environmentally friendly (reactions are performed with non-toxic and biodegradable catalysts under mild operational and
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environment-friendly conditions), while the “catalysis” part enables conversion of many substrates to products by eliminating the need for stoichiometric reagents. Biocatalytic approach also ensures catalyzing otherwise difficult transformations in high regio-, chemo- and enantioselective manner and eliminates multiple steps involved in complex chemical syntheses (reduced waste and hazards, improved yields, and cut costs) [3]. Due to properties such as non-volatility, non-flammability, low toxicity and biodegradability, natural deep eutectic solvents (NADES) in recent years are considered as new green solvents. Besides green character, one of the distinctive feature of these solvents is the possibility to modify their structure in order to adjust their physicochemical properties for certain application. Since the number of possible chemical structures of these solvents is vast, the possibility of their design for specific applications makes them very interesting for use in synthetic chemistry, the field of organometallics [4–6], solar technology [7], photosynthesis [8], electrochemistry, making nanomaterials, biocatalysis [9] and separation and analysis of various compounds [10,11]. During last 10 years NADES have started to be assessed as tools for biocatalysis, either as solvents or as separative agents, to overcome challenging workup procedures. A number of enzymes like epoxide hydrolase, protease and lipase, peroxidase in many cases, have shown enhanced activity and stability in the presence of cholinium- or
Corresponding author. E-mail address: [email protected] (M.C. Bubalo).
https://doi.org/10.1016/j.procbio.2017.12.010 Received 31 October 2017; Received in revised form 18 December 2017; Accepted 21 December 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Panić, M., Process Biochemistry (2017), https://doi.org/10.1016/j.procbio.2017.12.010
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ethylammonium-based NADES paired with alcohols, acids or amides as hydrogen bond donors [12,13], while whole-cell biocatalysis has been scarcely studied [14–17]. Based on aforementioned, the aim of this study is to associate the application of whole-cell biocatalysis (plant cells) and the use of environmentally friendly solvents (NADES) as two important green approaches for the preparation of interesting chiral intermediates. Furthermore, used NADES greatly differ in their physical properties such as viscosity, density and polarity which could significantly influence the biocatalysis efficiency [18]. For that purpose two enantioselective reactions catalyzed by carrot root in cholinium-based eutectic mixtures as solvent were conducted: reduction of 1-(3,4-dimethylphenyl)ethanone and hydrolysis of ( ± )-1-phenylethyl acetate.
Table 1 List of NADES used in this study with corresponding price ($ kg−1) and pH values for all tested water solutions of NADES.
2. Experimental 2.1. General 1-(3,4-Dimethylphenyl)ethanone, ( ± )-1-phenylethyl acetate, (S (−))-1-phenylethanol, (S)-1- (3,4-dimethylphenyl)ethanol and all chemicals for NADES synthesis (choline chloride, glucose, xylose, xylitol, glycerol and ethylene glycol) were purchased from Sigma–Aldrich (purity of ≥99%) and used without further purification. Organic solvents used were of analytical grade and supplied from Merck, Germany. Carrot (Daucus carota L.) was obtained from a local market. Carrot root was peeled with a knife to remove all traces of dirt and skin from the cortex. Vegetable meat was then washed with pure water and maintained in a 5% sodium hypochlorite aqueous solution for 20 min there after they were washed with ethanol and cut into small thin slices (approx. 5 mm × 5 mm × 2 mm) under a sterile laminar flow cabinet. Absolute configurations were determined by chiral GCMS analysis using reference compounds. Chiral gas chromatography analyses were performed on Shimadzu QP2010PLUS instrument equipped with Beta DEX 225 capillary chiral column (30 m × 0.25 mm × 0.25 μm) and MS detector.
NADES
Abbreviation
Molar ratio
Price of solventa($ kg−1)
Water content (%, w/w)
pH
Choline chloride: glucose
ChGlc
1:1
54.72
30 50 80
5.2 4.0 4.1
Choline chloride: xylose
ChXyl
2:1
63.99
30 50 80
3.8 4.1 4.3
Choline chloride: xylitol
ChXylol
5:2
90.95
30 50 80
6.6 6.2 5.2
Choline chloride: glycerol
ChGly
1:2
48.28
30 50 80
3.3 3.5 3.9
Choline chloride: ethylene glycol
ChEG
1:2
43.72
30 50 80
7.1 6.0 5.2
a The price of solvents was estimated according to the website of Fisher scientific (United States).
conversioning crude products. Crude products were resuspended with 100 μL n-heptane and analysed by a gas chromatograph with following temperature programs: for reduction – injector (220 °C), detector (200 °C), column temperature: 105 °C, 1 min, 105 °C–155 °C (2 °C min−1); for hydrolysis injector (220 °C), detector (200 °C), column temperature: 80 °C (2 min), 80 °C–140 °C (5 °C min−1). Helium was used as a carrier gas at a flow rate of 96.9 mL min−1. Quantification of data was done by the calibration with standard samples. For reactions performed in n-heptane, aliquots of 50 μL were directly analysed by a gas chromatograph. The hydrolysis reaction performed in ChGlc containing 80% of water and reduction reaction performed in ChEG containing 30% of water in which conversion and enantiomeric excess were the highest, were monitored at specific time intervals. Conversion and enantiomeric excess (ee) of 1-(3,4-dimethylphenyl)ethanone or ( ± )-1-phenylethyl acetate were calculated as described in Vandenberge et al. [19]. All experiments were repeated at least three times.
2.2. Preparation of NADES Choline chloride (ChCl) was dried in the vacuum concentrator (Savant SPD131DDA SpeedVac Concentrator, Thermo scientific, USA) at 60 °C for 24 h before use. NADES were synthesized at a certain ratio of ChCl to hydrogen bond donor (HBD) (ethylene glycol, glucose, glycerol, xylitol and xylose) to obtain liquids at room temperature (Table 1). The mixture of ChCl and HBD was stirred in a flask at 50 °C for 2 h. Additionally, different NADES solutions in water were prepared by dilution of a certain volume of NADES in deionized water (water solutions of NADES containing 30%, 50% and 80% of water (w/w) were prepared). The pH values for each NADES were determined by a 405-DPAS pH-electrode (Mettler Toledo, Zagreb). The measuring range of pH was 0–12 in temperature range 0–100 °C.
3. Results and discussion The purpose of conducted experiments was to comprise two green chemistry tools into the preparation of chiral intermediates: (i) enantioselective whole cell (carrot root) biocatalysis and (ii) application of green solvents (NADES). Carrot root was chosen as biocatalyst since it is cheap, widespread and easily available, and represents a valuable model in plant-mediated biocatalytic transformations of organic compounds [19–21]. Blank experiments without the biocatalysts were also performed in all tested NADES, and no conversion was observed within reaction times used in this study. The reduction of simple prochiral aromatic ketone 1-(3,4-dimethylphenyl)ethanone to corresponding chiral alcohols (1S)-(3,4-dimethylphenyl)ethanol and (1R)-(3,4-dimethylphenyl)ethanol (Fig. 1S), and enantioselective hydrolysis of ( ± )-1-phenylethyl acetate to (S)-1-phenylethanol and (R)-1-phenylethanol (Fig. 2S) can either serve as simple model reactions for enantioselective hydrolysis/reduction, or as interesting reactions from commercial point of view since the optically active 1-phenylethanol (especially (R)-1-phenylethanol) and 1-(3,4-dimethylphenyl)ethanol are used as chiral building block and synthetic intermediate in fine chemical, pharmaceutical and agrochemical industries [20]. In addition, there are no published data on plant-mediated biocatalytic transformations within NADES solvents and we were curious to
2.3. Reduction and hydrolysis by carrot root All experiments were carried out in properly sealed test tubes at 25 °C placed horizontally on a vortex mixer at 85 rpm. To 3.5 g (reduction) or 5 g (hydrolysis) of carrot root cut into pieces 15 mL of appropriate solvent (pure water or NADES) and finally 1-(3,4-dimethylphenyl)ethanone (0.008 mol L−1) or ( ± )-1-phenylethyl acetate (0.004 mol L−1) was added. Preliminary experiments indicated that 72 h (reduction) and 48 h (hydrolysis) period is sufficient for achievement of the chemical equilibrium in all solvents tested. For reactions performed in NADES, substrates and products were extracted with nheptane. The biphasic mixture containing 500 μL of reaction aliquots and 4500 μL of n-heptane was strongly shaken for 5 min and analysed by a gas chromatograph as indicated below. The organic phase was collected and n-heptane was evaporated under reduced pressure, 2
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Fig. 1. Conversion (a) and enantiomeric excess (b) of asymmetric reduction of 1-(3,4)-dimethylphenyl)ethanone to (S)-1-(3,4-dimethylphenyl)ethanol and (R)-1-(3,4-dimethylphenyl) ethanol by carrot root in 0.015 L NADES containing 30–80% of water (w/w), and pure water. Reaction conditions: 0.008 M 1-(3,4)-dimethylphenyl)ethanone; 3.5 g carrot root; 72 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD.
3.1. Asymmetric reduction of acetophenone by carrot root in NADES
investigate the behavior of plant enzymes responsible for hydrolysis and reduction of organic compounds in those kind of media. Five different choline-based NADES with sugar (glucose and xylose), sugar alcohols (xylitol), or polyalcohols (glycerol and ethylene glycol) as hydrogen bond donor (HBD) were chosen as solvents. NADES selected were weakly acidic to neutral (pH 3.3–7.1) since it is well known that plant enzymes are able to perform reactions under mild conditions (pH and temperature), with remarkable chemo-, regio-, and stereoselectivity [20] (Table 1). Accordingly, widespread NADES with organic acid as HBD (pH < 1) were not taken into consideration [15,18]. In accordance to green synthesis approach, selected NADES are cheap in comparison to organic solvents commonly used in biocatalysis (e.g. used NADES are more than 1.5 times cheaper from n-heptane) and exhibit low cytotoxicity with EC50 > 2000 mg L−1 [22–24]. In this work NADES were used as aqueous solutions with 30–80% of water (w/w) since enzymes need a certain amount of water for their activity, whereby addition of water to NADES also allows coping with the noticeable mass transfer problem caused by the relatively high viscosity of NADES [15,18]. Carrot root-catalyzed reduction and hydrolysis with a lower percentage of water in ChXyl (10%) were also conducted, however, conversions were < 10% due to the high viscosity of NADES, as mentioned before. Enantiomeric excess for (1S)-(3,4-dimethylphenyl)ethanol was 75.27% and for (R)-1-phenylethanol was 33.72% which is not significantly different (p < 0.05) in comparison to bioreductions performed in NADES containing 30% of water. Reactions were also conducted in pure water as a referent solvent.
Bioreductions mediated by functionally intact cells obtained directly from cut portions of plants are gaining attention as plant cell enzymes are able to catalyze reduction of prochiral ketones in high regio- and stereospecific manner, without the need for coenzyme regeneration [21]. Herein, the experimental setup for asymmetric reduction of 1-(3,4-dimethylphenyl)ethanone was very simple: substrate 1-(3,4-dimethylphenyl)ethanone to a suspension of carrot root cut into pieces in NADES or pure water was added and shaken at room temperature for 3 days and then analysed by GC–MS. As it can be observed, the presence of NADES in water, regardless of the percentage of NADES in aqueous solutions, led to much lower conversions than those observed in pure water (91.3%) (Fig. 1a). Conversions obtained have not exceeded 50% for the reactions performed in all tested NADES (10.8–49.9%). Also, it was observed that the type of HBD present in NADES greatly influenced the conversion. Regarding the type of HBD, the best conversion was obtained with glucose followed by xylose > glycerol > xylitol > ethylene glycol (Fig. 1a). Higher bioreduction conversions obtained in medium containing sugar as HBD is consistent with current literature suggesting that the presence of auxiliary substrates (e.g. sugar) for cofactor-recycling system could reduce the problem of co-factor consumption and, therefore, greatly enhance the bioconversion [15,25]. Furthermore, increasing water content in NADES positively influenced the conversions and the highest conversions were observed in 3
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Fig. 2. Time courses of carrot root-catalyzed asymmetric reduction of 1-(3,4)-dimethylphenyl)ethanone to (S)-1-(3,4-dimethylphenyl)ethanol and (R)-1-(3,4-dimethylphenyl)ethanol in 0.015 L ChGlc containing 80% of water. Reaction conditions: 0.008 M 1-(3,4)-dimethylphenyl)ethanone; 3.5 g carrot root; 72 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD.
Only bioreaction in ChGlc containing 30% of water (w/w) with the pH 5.2 was not subject to this rule, indicating that in this case, some other issues such as the type of HBD and water content could be crucial for determining enantiomeric excess rather than pH value of NADES.
NADES containing 80% of water. Previous publications also indicated that reduction conversions decline when high concentrations of NADES in water was used. As mentioned, high content of NADES may obstruct the mass transfer and could also cause entrapment of the substrate within the NADES through H-bonding (minimized availability of the substrate) [14,15,18]. The pH value of reaction solution also plays a crucial role in biotransformation processes by altering the ionic state of the substrate and charging enzymes thus leading to changes in enzyme’s activity and enantioselectivities [26]. This is especially interesting when the substrate could be transformed by several isoenzymes with different enantioselectivity at different pH [21]. The lower conversions detected running the reduction in different NADES mixtures may also be due to a lower enzyme affinity towards the substrate [13,16]. For reactions performed in water high enantioselectivity (95.6% ee) giving the (S) alcohol was observed, indicating that the reaction follows Prelog’s rule [27]. Reactions in moderate to highly diluted NADES (≥50% of water) followed the same rule, while in concentrated NADES (30% of water) (R) alcohol was being dominant (ee from 32.66% to 75.27%). For example, by changing of the water content in ChXyl solution from 80% to 30% it is possible to obtain S-alcohol or R-alcohol with a very high ee, 80.28% and 75.27%, respectively (Fig. 1b). Cvjetko Bubalo et al. and Maugeri and Domínguez De María [14,15], who also noticed similar effect during performing baker yeast-mediated reduction of β-ketoester, and Vitale et al. during performing baker yeastmediated reduction of ketones [16], noted that by changing the proportion of the NADES added, a complete inversion of enantioselectivity could be observed, indicating that some S-enantioselective enzymes could be inhibited by NADES. Additionally, a time course of the reduction in ChGlc containing 80% of water, in which enantiomeric excess was the highest, was monitored (Fig. 2). Moreover, it was noticed that the inversion of the configuration may depend on the pH value of tested NADES. When the pH of solvent was in the range 3.9–6.0 (R)-alcohol configuration was dominant while in outer values of this range S-alcohol was mostly produced. Similar behavior has been observed in other studies with several possible explanations: solubility of the compounds in media, rapid interconversion of enantiomers through a pH-dependent enolization process and/or the presence of more than one acetophenone-reducing protein [28–30].
3.2. Enantioselective hydrolysis of ( ± )-1-phenylethyl acetate by carrot root in NADES Potential of plant cells to catalyze hydrolysis of various ester is less known and understood than the reduction of prochiral compounds, however, several reports on this topic motivated us to study the potential of NADES-assisted enantioselective hydrolysis of chiral aromatic ester ( ± )-1-phenylethyl acetate [19,31,32]. Briefly, the substrate ( ± )-1-phenylethyl acetate to a suspension of carrot root cut into pieces in NADES water was added, shaken at room temperature for 2 days and analysed by GC–MS. For all tested NADES aqueous solutions obtained conversions were relatively high (> 79.97%), with the highest hydrolysis conversions obtained in NADES with 80% of water (89.96%–98.95%), similar to those observed in water (99.49%) (Fig. 3a). Though it seems that there is no obvious connection between NADES structure and hydrolysis conversion, the enantiomeric excess was found to be correlated with water content in NADES solution, being in the range from 3.47–39.04% (with (R)-alcohol configuration being dominant) (Fig. 3b). In all tested NADES decrease in water content led to higher enantioselectivity of reaction, while carrot root in water as a media did not have a preference for either the (R)- or (S)-enantiomer, resulting in the formation of a racemic mixture. It is largely unknown which types of hydrolytic enzymes could be involved in this reaction, although some authors imply that lipases (enzymes that produce free fatty acids in plant cell which are incorporated in the mixed micelles) could be responsible for enantioselective hydrolysis [19]. Vandenberghe et al. [19], who also studied hydrolysis of ( ± )-1-phenylethyl acetate by different plants suggested that the enzyme responsible for the reaction was not stereoselective and transformed both (R) and (S) substrate enantiomers into the corresponding secondary alcohols. However, since there are differences in ee values regarding HBD in NADES used, observed changes in the enantioselectivity could also be 4
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Fig. 3. Conversion (a) and enantiomeric excess (b) of enantioselective hydrolysis of ( ± )-1-phenylethyl acetate to (S)-1-phenylethanol and (R)-1-phenylethanol by carrot root in 0.015 L NADES containing 30–80% of water (w/w) and pure water. Reaction conditions: 0.004 M ( ± )-1-phenylethyl acetate; 5 g carrot root; 48 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD. *50/50 (R/S).
two or more lipases with different stereospecificity of which lipases with the preference for (R) enantiomer converts the substrate with a higher rate. Irrespective of the explanation for this phenomenon, the fact that it is possible to obtain high concentration of (R)-1-phenyl ethanol by stopping the reaction after 2 h makes this plant-mediated approach for stereoselective hydrolysis attractive.
explained by the presence of several lipases with different enantioselectivity that transform either (R) or (S) ester, that are inhibited differently by NADES used. Similar conclusion was reported by Maugeri and Domínguez De María [14] who studied baker’s yeast mediated reduction of ethyl acetoacetate: authors implied that changes in the enantioselectivity of baker’s yeast in NADES with different water content were created as a result of the inhibition or even complete knock out of several oxidoreductases, whereas others remained active. The reaction in ChEG containing 30% of water, in which conversion and enantiomeric excess were the highest, was monitored at specific time intervals (Fig. 4). In the first 2 h (R)-1-phenyl ethanol with 73.00% ee and 67.63% conversion was produced. After that, enantiomeric excesses gradually decreased in next 46 h (no change in ee was observed afterwards). If our thesis about several enzymes able to convert the chiral acetate is correct, observed phenomenon could be explained by
4. Conclusion In summary, we successfully conducted two enantioselective reactions: reduction of 1-(3,4-dimethylphenyl)ethanone and hydrolysis of ( ± )-1-phenylethyl acetate by carrot root in cholinium-based eutectic mixtures as solvent. To our knowledge this is first report that NADES could also serve as solvent for plant cells biocatalyst process opening its new possible application. However, impact of NADES on plant cell 5
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Fig. 4. Time courses of carrot root-catalyzed enantioselective hydrolysis of ( ± )-1-phenylethyl acetate to (S)-1-phenylethanol and (R)-1-phenylethanol in 0.015 L ChEG containing 30% of water. Reaction conditions: 0.004 M ( ± )-1-phenylethyl acetate; 5 g carrot root; 48 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD.
biocatalysis cannot be simply explained by a single interpretation involving a complicated interaction between them. Therefore, is worth to further investigate the mechanisms behind it to gain sufficient knowledge about the influence of NADES on plant cell that we could use this approach in bigger scale.
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Short communication
Plant-mediated stereoselective biotransformations in natural deep eutectic solvents ⁎
Manuela Panića, Maja Majerić Elenkovb, Marin Rojeb, Marina Cvjetko Bubaloa, , Ivana Radojčić Redovnikovića a b
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia Ruđer Bošković Institute, Bijenička cesta 54, HR-10000 Zagreb, Croatia
A R T I C L E I N F O
A B S T R A C T
Keywords: Asymmetric reduction Carrot root Enantioselective hydrolysis Natural deep eutectic solvents
The challenge of chiral chemical production lies in the preparation of chiral building blocks in accordance to green chemistry principles. Thus, the aim of this study was to associate the application of whole-cell biocatalysis (plant cells) and the use of environmentally friendly solvents (NADES) for enantioselective preparation of chiral molecules. For that purpose, reduction of 1-(3,4-dimethylphenyl)ethanone and hydrolysis of ( ± )-1-phenylethyl acetate by carrot root in cholinium-based eutectic mixtures as solvents were successfully conducted. The type of hydrogen bond donor and the amount of water present in NADESs influenced the conversion and/or enantiomeric excess in both enantioselective reactions. In conclusion, inversion of the enantioselectivity exhibited by the biocatalyst towards 1-(3,4-dimethylphenyl)ethanone was noticed by the addition of different amounts of water to NADES. Overall, the results suggest that NADES possess good biocompatibility with plant cells and can be applied as green reaction medium for plant-mediated bioreduction.
1. Introduction Introduction of green chemistry principles into preparation of various commercial products, such as chiral building blocks for drug preparation, is becoming an imperative in scientific community. The basics of green chemistry are summarized in twelve principles [1] and suggesting that chemical products and processes should be designed in such way to reduce or completely remove the application and creation of harmful and dangerous reagents, catalyst, and products, with the unique goal to protect the environment not by cleaning up, but by inventing new chemical processes that do not pollute. There are numerous tools of green chemistry, such as use of alternative solvents and environmentally benign raw (or renewable) materials, use of alternative energy sources (e.g. microwaves, ultrasound), performing reactions by (bio)catalysis, that are nowadays intensively studied [2]. Biotechnology methods fit logically to the goals of green chemistry meaning that the use of the biological methods and materials for industrial manufacturing is an excellent foundation to create an environmentally friendly process. In the same way, biocatalysis – the use of whole cells, cell extracts, or purified enzymes in organic synthesis – fits perfectly into green chemistry because this the “bio” part of biocatalysis makes it environmentally friendly (reactions are performed with non-toxic and biodegradable catalysts under mild operational and
⁎
environment-friendly conditions), while the “catalysis” part enables conversion of many substrates to products by eliminating the need for stoichiometric reagents. Biocatalytic approach also ensures catalyzing otherwise difficult transformations in high regio-, chemo- and enantioselective manner and eliminates multiple steps involved in complex chemical syntheses (reduced waste and hazards, improved yields, and cut costs) [3]. Due to properties such as non-volatility, non-flammability, low toxicity and biodegradability, natural deep eutectic solvents (NADES) in recent years are considered as new green solvents. Besides green character, one of the distinctive feature of these solvents is the possibility to modify their structure in order to adjust their physicochemical properties for certain application. Since the number of possible chemical structures of these solvents is vast, the possibility of their design for specific applications makes them very interesting for use in synthetic chemistry, the field of organometallics [4–6], solar technology [7], photosynthesis [8], electrochemistry, making nanomaterials, biocatalysis [9] and separation and analysis of various compounds [10,11]. During last 10 years NADES have started to be assessed as tools for biocatalysis, either as solvents or as separative agents, to overcome challenging workup procedures. A number of enzymes like epoxide hydrolase, protease and lipase, peroxidase in many cases, have shown enhanced activity and stability in the presence of cholinium- or
Corresponding author. E-mail address: [email protected] (M.C. Bubalo).
https://doi.org/10.1016/j.procbio.2017.12.010 Received 31 October 2017; Received in revised form 18 December 2017; Accepted 21 December 2017 1359-5113/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Panić, M., Process Biochemistry (2017), https://doi.org/10.1016/j.procbio.2017.12.010
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ethylammonium-based NADES paired with alcohols, acids or amides as hydrogen bond donors [12,13], while whole-cell biocatalysis has been scarcely studied [14–17]. Based on aforementioned, the aim of this study is to associate the application of whole-cell biocatalysis (plant cells) and the use of environmentally friendly solvents (NADES) as two important green approaches for the preparation of interesting chiral intermediates. Furthermore, used NADES greatly differ in their physical properties such as viscosity, density and polarity which could significantly influence the biocatalysis efficiency [18]. For that purpose two enantioselective reactions catalyzed by carrot root in cholinium-based eutectic mixtures as solvent were conducted: reduction of 1-(3,4-dimethylphenyl)ethanone and hydrolysis of ( ± )-1-phenylethyl acetate.
Table 1 List of NADES used in this study with corresponding price ($ kg−1) and pH values for all tested water solutions of NADES.
2. Experimental 2.1. General 1-(3,4-Dimethylphenyl)ethanone, ( ± )-1-phenylethyl acetate, (S (−))-1-phenylethanol, (S)-1- (3,4-dimethylphenyl)ethanol and all chemicals for NADES synthesis (choline chloride, glucose, xylose, xylitol, glycerol and ethylene glycol) were purchased from Sigma–Aldrich (purity of ≥99%) and used without further purification. Organic solvents used were of analytical grade and supplied from Merck, Germany. Carrot (Daucus carota L.) was obtained from a local market. Carrot root was peeled with a knife to remove all traces of dirt and skin from the cortex. Vegetable meat was then washed with pure water and maintained in a 5% sodium hypochlorite aqueous solution for 20 min there after they were washed with ethanol and cut into small thin slices (approx. 5 mm × 5 mm × 2 mm) under a sterile laminar flow cabinet. Absolute configurations were determined by chiral GCMS analysis using reference compounds. Chiral gas chromatography analyses were performed on Shimadzu QP2010PLUS instrument equipped with Beta DEX 225 capillary chiral column (30 m × 0.25 mm × 0.25 μm) and MS detector.
NADES
Abbreviation
Molar ratio
Price of solventa($ kg−1)
Water content (%, w/w)
pH
Choline chloride: glucose
ChGlc
1:1
54.72
30 50 80
5.2 4.0 4.1
Choline chloride: xylose
ChXyl
2:1
63.99
30 50 80
3.8 4.1 4.3
Choline chloride: xylitol
ChXylol
5:2
90.95
30 50 80
6.6 6.2 5.2
Choline chloride: glycerol
ChGly
1:2
48.28
30 50 80
3.3 3.5 3.9
Choline chloride: ethylene glycol
ChEG
1:2
43.72
30 50 80
7.1 6.0 5.2
a The price of solvents was estimated according to the website of Fisher scientific (United States).
conversioning crude products. Crude products were resuspended with 100 μL n-heptane and analysed by a gas chromatograph with following temperature programs: for reduction – injector (220 °C), detector (200 °C), column temperature: 105 °C, 1 min, 105 °C–155 °C (2 °C min−1); for hydrolysis injector (220 °C), detector (200 °C), column temperature: 80 °C (2 min), 80 °C–140 °C (5 °C min−1). Helium was used as a carrier gas at a flow rate of 96.9 mL min−1. Quantification of data was done by the calibration with standard samples. For reactions performed in n-heptane, aliquots of 50 μL were directly analysed by a gas chromatograph. The hydrolysis reaction performed in ChGlc containing 80% of water and reduction reaction performed in ChEG containing 30% of water in which conversion and enantiomeric excess were the highest, were monitored at specific time intervals. Conversion and enantiomeric excess (ee) of 1-(3,4-dimethylphenyl)ethanone or ( ± )-1-phenylethyl acetate were calculated as described in Vandenberge et al. [19]. All experiments were repeated at least three times.
2.2. Preparation of NADES Choline chloride (ChCl) was dried in the vacuum concentrator (Savant SPD131DDA SpeedVac Concentrator, Thermo scientific, USA) at 60 °C for 24 h before use. NADES were synthesized at a certain ratio of ChCl to hydrogen bond donor (HBD) (ethylene glycol, glucose, glycerol, xylitol and xylose) to obtain liquids at room temperature (Table 1). The mixture of ChCl and HBD was stirred in a flask at 50 °C for 2 h. Additionally, different NADES solutions in water were prepared by dilution of a certain volume of NADES in deionized water (water solutions of NADES containing 30%, 50% and 80% of water (w/w) were prepared). The pH values for each NADES were determined by a 405-DPAS pH-electrode (Mettler Toledo, Zagreb). The measuring range of pH was 0–12 in temperature range 0–100 °C.
3. Results and discussion The purpose of conducted experiments was to comprise two green chemistry tools into the preparation of chiral intermediates: (i) enantioselective whole cell (carrot root) biocatalysis and (ii) application of green solvents (NADES). Carrot root was chosen as biocatalyst since it is cheap, widespread and easily available, and represents a valuable model in plant-mediated biocatalytic transformations of organic compounds [19–21]. Blank experiments without the biocatalysts were also performed in all tested NADES, and no conversion was observed within reaction times used in this study. The reduction of simple prochiral aromatic ketone 1-(3,4-dimethylphenyl)ethanone to corresponding chiral alcohols (1S)-(3,4-dimethylphenyl)ethanol and (1R)-(3,4-dimethylphenyl)ethanol (Fig. 1S), and enantioselective hydrolysis of ( ± )-1-phenylethyl acetate to (S)-1-phenylethanol and (R)-1-phenylethanol (Fig. 2S) can either serve as simple model reactions for enantioselective hydrolysis/reduction, or as interesting reactions from commercial point of view since the optically active 1-phenylethanol (especially (R)-1-phenylethanol) and 1-(3,4-dimethylphenyl)ethanol are used as chiral building block and synthetic intermediate in fine chemical, pharmaceutical and agrochemical industries [20]. In addition, there are no published data on plant-mediated biocatalytic transformations within NADES solvents and we were curious to
2.3. Reduction and hydrolysis by carrot root All experiments were carried out in properly sealed test tubes at 25 °C placed horizontally on a vortex mixer at 85 rpm. To 3.5 g (reduction) or 5 g (hydrolysis) of carrot root cut into pieces 15 mL of appropriate solvent (pure water or NADES) and finally 1-(3,4-dimethylphenyl)ethanone (0.008 mol L−1) or ( ± )-1-phenylethyl acetate (0.004 mol L−1) was added. Preliminary experiments indicated that 72 h (reduction) and 48 h (hydrolysis) period is sufficient for achievement of the chemical equilibrium in all solvents tested. For reactions performed in NADES, substrates and products were extracted with nheptane. The biphasic mixture containing 500 μL of reaction aliquots and 4500 μL of n-heptane was strongly shaken for 5 min and analysed by a gas chromatograph as indicated below. The organic phase was collected and n-heptane was evaporated under reduced pressure, 2
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Fig. 1. Conversion (a) and enantiomeric excess (b) of asymmetric reduction of 1-(3,4)-dimethylphenyl)ethanone to (S)-1-(3,4-dimethylphenyl)ethanol and (R)-1-(3,4-dimethylphenyl) ethanol by carrot root in 0.015 L NADES containing 30–80% of water (w/w), and pure water. Reaction conditions: 0.008 M 1-(3,4)-dimethylphenyl)ethanone; 3.5 g carrot root; 72 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD.
3.1. Asymmetric reduction of acetophenone by carrot root in NADES
investigate the behavior of plant enzymes responsible for hydrolysis and reduction of organic compounds in those kind of media. Five different choline-based NADES with sugar (glucose and xylose), sugar alcohols (xylitol), or polyalcohols (glycerol and ethylene glycol) as hydrogen bond donor (HBD) were chosen as solvents. NADES selected were weakly acidic to neutral (pH 3.3–7.1) since it is well known that plant enzymes are able to perform reactions under mild conditions (pH and temperature), with remarkable chemo-, regio-, and stereoselectivity [20] (Table 1). Accordingly, widespread NADES with organic acid as HBD (pH < 1) were not taken into consideration [15,18]. In accordance to green synthesis approach, selected NADES are cheap in comparison to organic solvents commonly used in biocatalysis (e.g. used NADES are more than 1.5 times cheaper from n-heptane) and exhibit low cytotoxicity with EC50 > 2000 mg L−1 [22–24]. In this work NADES were used as aqueous solutions with 30–80% of water (w/w) since enzymes need a certain amount of water for their activity, whereby addition of water to NADES also allows coping with the noticeable mass transfer problem caused by the relatively high viscosity of NADES [15,18]. Carrot root-catalyzed reduction and hydrolysis with a lower percentage of water in ChXyl (10%) were also conducted, however, conversions were < 10% due to the high viscosity of NADES, as mentioned before. Enantiomeric excess for (1S)-(3,4-dimethylphenyl)ethanol was 75.27% and for (R)-1-phenylethanol was 33.72% which is not significantly different (p < 0.05) in comparison to bioreductions performed in NADES containing 30% of water. Reactions were also conducted in pure water as a referent solvent.
Bioreductions mediated by functionally intact cells obtained directly from cut portions of plants are gaining attention as plant cell enzymes are able to catalyze reduction of prochiral ketones in high regio- and stereospecific manner, without the need for coenzyme regeneration [21]. Herein, the experimental setup for asymmetric reduction of 1-(3,4-dimethylphenyl)ethanone was very simple: substrate 1-(3,4-dimethylphenyl)ethanone to a suspension of carrot root cut into pieces in NADES or pure water was added and shaken at room temperature for 3 days and then analysed by GC–MS. As it can be observed, the presence of NADES in water, regardless of the percentage of NADES in aqueous solutions, led to much lower conversions than those observed in pure water (91.3%) (Fig. 1a). Conversions obtained have not exceeded 50% for the reactions performed in all tested NADES (10.8–49.9%). Also, it was observed that the type of HBD present in NADES greatly influenced the conversion. Regarding the type of HBD, the best conversion was obtained with glucose followed by xylose > glycerol > xylitol > ethylene glycol (Fig. 1a). Higher bioreduction conversions obtained in medium containing sugar as HBD is consistent with current literature suggesting that the presence of auxiliary substrates (e.g. sugar) for cofactor-recycling system could reduce the problem of co-factor consumption and, therefore, greatly enhance the bioconversion [15,25]. Furthermore, increasing water content in NADES positively influenced the conversions and the highest conversions were observed in 3
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Fig. 2. Time courses of carrot root-catalyzed asymmetric reduction of 1-(3,4)-dimethylphenyl)ethanone to (S)-1-(3,4-dimethylphenyl)ethanol and (R)-1-(3,4-dimethylphenyl)ethanol in 0.015 L ChGlc containing 80% of water. Reaction conditions: 0.008 M 1-(3,4)-dimethylphenyl)ethanone; 3.5 g carrot root; 72 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD.
Only bioreaction in ChGlc containing 30% of water (w/w) with the pH 5.2 was not subject to this rule, indicating that in this case, some other issues such as the type of HBD and water content could be crucial for determining enantiomeric excess rather than pH value of NADES.
NADES containing 80% of water. Previous publications also indicated that reduction conversions decline when high concentrations of NADES in water was used. As mentioned, high content of NADES may obstruct the mass transfer and could also cause entrapment of the substrate within the NADES through H-bonding (minimized availability of the substrate) [14,15,18]. The pH value of reaction solution also plays a crucial role in biotransformation processes by altering the ionic state of the substrate and charging enzymes thus leading to changes in enzyme’s activity and enantioselectivities [26]. This is especially interesting when the substrate could be transformed by several isoenzymes with different enantioselectivity at different pH [21]. The lower conversions detected running the reduction in different NADES mixtures may also be due to a lower enzyme affinity towards the substrate [13,16]. For reactions performed in water high enantioselectivity (95.6% ee) giving the (S) alcohol was observed, indicating that the reaction follows Prelog’s rule [27]. Reactions in moderate to highly diluted NADES (≥50% of water) followed the same rule, while in concentrated NADES (30% of water) (R) alcohol was being dominant (ee from 32.66% to 75.27%). For example, by changing of the water content in ChXyl solution from 80% to 30% it is possible to obtain S-alcohol or R-alcohol with a very high ee, 80.28% and 75.27%, respectively (Fig. 1b). Cvjetko Bubalo et al. and Maugeri and Domínguez De María [14,15], who also noticed similar effect during performing baker yeast-mediated reduction of β-ketoester, and Vitale et al. during performing baker yeastmediated reduction of ketones [16], noted that by changing the proportion of the NADES added, a complete inversion of enantioselectivity could be observed, indicating that some S-enantioselective enzymes could be inhibited by NADES. Additionally, a time course of the reduction in ChGlc containing 80% of water, in which enantiomeric excess was the highest, was monitored (Fig. 2). Moreover, it was noticed that the inversion of the configuration may depend on the pH value of tested NADES. When the pH of solvent was in the range 3.9–6.0 (R)-alcohol configuration was dominant while in outer values of this range S-alcohol was mostly produced. Similar behavior has been observed in other studies with several possible explanations: solubility of the compounds in media, rapid interconversion of enantiomers through a pH-dependent enolization process and/or the presence of more than one acetophenone-reducing protein [28–30].
3.2. Enantioselective hydrolysis of ( ± )-1-phenylethyl acetate by carrot root in NADES Potential of plant cells to catalyze hydrolysis of various ester is less known and understood than the reduction of prochiral compounds, however, several reports on this topic motivated us to study the potential of NADES-assisted enantioselective hydrolysis of chiral aromatic ester ( ± )-1-phenylethyl acetate [19,31,32]. Briefly, the substrate ( ± )-1-phenylethyl acetate to a suspension of carrot root cut into pieces in NADES water was added, shaken at room temperature for 2 days and analysed by GC–MS. For all tested NADES aqueous solutions obtained conversions were relatively high (> 79.97%), with the highest hydrolysis conversions obtained in NADES with 80% of water (89.96%–98.95%), similar to those observed in water (99.49%) (Fig. 3a). Though it seems that there is no obvious connection between NADES structure and hydrolysis conversion, the enantiomeric excess was found to be correlated with water content in NADES solution, being in the range from 3.47–39.04% (with (R)-alcohol configuration being dominant) (Fig. 3b). In all tested NADES decrease in water content led to higher enantioselectivity of reaction, while carrot root in water as a media did not have a preference for either the (R)- or (S)-enantiomer, resulting in the formation of a racemic mixture. It is largely unknown which types of hydrolytic enzymes could be involved in this reaction, although some authors imply that lipases (enzymes that produce free fatty acids in plant cell which are incorporated in the mixed micelles) could be responsible for enantioselective hydrolysis [19]. Vandenberghe et al. [19], who also studied hydrolysis of ( ± )-1-phenylethyl acetate by different plants suggested that the enzyme responsible for the reaction was not stereoselective and transformed both (R) and (S) substrate enantiomers into the corresponding secondary alcohols. However, since there are differences in ee values regarding HBD in NADES used, observed changes in the enantioselectivity could also be 4
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Fig. 3. Conversion (a) and enantiomeric excess (b) of enantioselective hydrolysis of ( ± )-1-phenylethyl acetate to (S)-1-phenylethanol and (R)-1-phenylethanol by carrot root in 0.015 L NADES containing 30–80% of water (w/w) and pure water. Reaction conditions: 0.004 M ( ± )-1-phenylethyl acetate; 5 g carrot root; 48 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD. *50/50 (R/S).
two or more lipases with different stereospecificity of which lipases with the preference for (R) enantiomer converts the substrate with a higher rate. Irrespective of the explanation for this phenomenon, the fact that it is possible to obtain high concentration of (R)-1-phenyl ethanol by stopping the reaction after 2 h makes this plant-mediated approach for stereoselective hydrolysis attractive.
explained by the presence of several lipases with different enantioselectivity that transform either (R) or (S) ester, that are inhibited differently by NADES used. Similar conclusion was reported by Maugeri and Domínguez De María [14] who studied baker’s yeast mediated reduction of ethyl acetoacetate: authors implied that changes in the enantioselectivity of baker’s yeast in NADES with different water content were created as a result of the inhibition or even complete knock out of several oxidoreductases, whereas others remained active. The reaction in ChEG containing 30% of water, in which conversion and enantiomeric excess were the highest, was monitored at specific time intervals (Fig. 4). In the first 2 h (R)-1-phenyl ethanol with 73.00% ee and 67.63% conversion was produced. After that, enantiomeric excesses gradually decreased in next 46 h (no change in ee was observed afterwards). If our thesis about several enzymes able to convert the chiral acetate is correct, observed phenomenon could be explained by
4. Conclusion In summary, we successfully conducted two enantioselective reactions: reduction of 1-(3,4-dimethylphenyl)ethanone and hydrolysis of ( ± )-1-phenylethyl acetate by carrot root in cholinium-based eutectic mixtures as solvent. To our knowledge this is first report that NADES could also serve as solvent for plant cells biocatalyst process opening its new possible application. However, impact of NADES on plant cell 5
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Fig. 4. Time courses of carrot root-catalyzed enantioselective hydrolysis of ( ± )-1-phenylethyl acetate to (S)-1-phenylethanol and (R)-1-phenylethanol in 0.015 L ChEG containing 30% of water. Reaction conditions: 0.004 M ( ± )-1-phenylethyl acetate; 5 g carrot root; 48 h; 25 °C. Each data point represents the mean of triplicate experiments; error bars represent the SD.
biocatalysis cannot be simply explained by a single interpretation involving a complicated interaction between them. Therefore, is worth to further investigate the mechanisms behind it to gain sufficient knowledge about the influence of NADES on plant cell that we could use this approach in bigger scale.
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