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Enzyme-Catalyzed Henry Reaction in Choline Chloride-Based Deep Eutectic Solvents
Enzyme-Catalyzed Henry Reaction in Choline Chloride-Based Deep Eutectic Solvents
Journal of Microbiology and Biotechnology. 2016. Jan, 26(1): 80-88
Copyright © 2016, The Korean Society For Microbiology And Biotechnology
  • Received : July 01, 2015
  • Accepted : October 05, 2015
  • Published : January 28, 2016
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About the Authors
Xuemei Tian
College of Chemistry, Jilin University, Changchun 130012, P.R. China
Suoqin Zhang
College of Chemistry, Jilin University, Changchun 130012, P.R. China
Liangyu Zheng
College of Life Sciences, Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, P.R. China
lyzheng@jlu.edu.cn

Abstract
The enzyme-catalyzed Henry reaction was realized using deep eutectic solvents (DESs) as a reaction medium. The lipase from Aspergillus niger (lipase AS) showed excellent catalytic activity toward the substrates aromatic aldehydes and nitromethane in choline chloride:glycerol at a molar ratio of 1:2. Addition of 30 vol% water to DES further improved the lipase activity and inhibited DES-catalyzed transformation. A final yield of 92.2% for the lipase AS-catalyzed Henry reaction was achieved under optimized reaction conditions in only 4 h. In addition, the lipase AS activity was improved by approximately 3-fold in a DES–water mixture compared with that in pure water, which produced a final yield of only 33.4%. Structural studies with fluorescence spectroscopy showed that the established strong hydrogen bonds between DES and water may be the main driving force that affects the spatial conformation of the enzyme, leading to a change in lipase activity. The methodology was also extended to the aza-Henry reaction, which easily occurred in contrast to that in pure water. The enantioselectivity of both Henry and aza-Henry reactions was not found. However, the results are still remarkable, as we report the first use of DES as a reaction medium in a lipase-catalyzed Henry reaction.
Keywords
Introduction
The Henry reaction is an important and classical C–C bond-formation reaction, which is an essential element of synthetic organic chemistry [7 , 16 , 18 , 19] . The reaction is the coupling of a nucleophilic nitroalkane with an electrophilic aldehyde or ketone to produce a synthetically useful β-nitro alcohol [18] . The resulting β-nitro alcohols have been employed in the synthesis of many key intermediates of biologically active compounds, such as natural products, insecticides, fungicides, and antibiotics [2 , 3 , 6 , 16] . The Henry reaction can occur using most popular organic bases, including carbonates, bicarbonates, alkali metal hydroxides, alkoxides, and organic nitrogen bases [18] . Apart from bases, some other catalysts include the rare earth metal alkoxides, rare earth hexamethyldisilazides, and binaphthol-rare earth metal complexes [18] . The main drawbacks of the synthetic reactions are the formation of unwanted byproducts that pollute the environment [4] . Therefore, the determination of mild reaction conditions that prevent the formation of unwanted byproducts is necessary.
Biocatalysts show immense advantages, including mild reaction conditions, simple separation, good selectivity, and high yields, as efficient and environmentally friendly biotransformation tools in organic synthesis [27] . A growing number of biocatalysts have been found to be capable of catalyzing synthetic reactions, deviating from their natural functions. Exploiting biocatalyst promiscuity is believed to have great potential in expanding synthetic methodologies [13] . Some biocatalysts show good catalytic activity for the Henry reaction. Although some enzymes, such as hydroxynitrile lyase, are able to induce some degree of enantioselectivity [14 , 17] , the use of aminoacylase, transglutaminase or lipase generally leads to poor enantiocontrol [5 , 24 - 26] . In this case, enantioselectivity is only obtained by coupling the biocatalytic Henry reaction to the enzymatic kinetic resolution of the racemic mixture [18] . However, even when low enantioselectivity may be achieved, biocatalytic processes present clear advantages from an environmental point of view. Some biocatalysts have been described to promote the Henry reaction in organic solvents, biphasic systems composed of water and organic solvents, or in aqueous medium [25 , 26] . However, the environmental harm from organic solvents is unavoidable. In addition, the long reaction time, high reaction temperature, and low enzymatic activity are unfavorable. Therefore, developing new enzymatic methods for the Henry reaction in eco-friendly and effective reaction media is of great importance.
An environmentally friendly, enzyme-promoted procedure for the Henry reaction was first studied using water-in-[Bmim][PF 6 ] microemulsions as the reaction medium. The Amano acylase from Aspergillus oryzae showed good catalytic activity for the addition reactions of nitromethane with a series of aromatic aldehydes; however, reaching the ideal reaction yield was still time-consuming (48 h) [29] . Although ionic liquids are regarded as potential environmentally friendly alternatives to conventional organic solvents owing to their unique properties, such as excellent solubility as well as thermal and chemical stabilities, their high price and operational difficulties limit their usage [20 , 22] .
In recent years, new types of solvents, namely, deep eutectic solvents (DESs), have been used as reaction media [1 , 23] . DESs, similar to ionic liquids, have a low melting point, low volatility, and high thermal stability. In addition, DESs are biodegradable, non-toxic, inexpensive, and simple to prepare. DESs consist of an ammonium or phosphonium salt, such as choline chloride, and a hydrogen-bond donor (HBD), such as urea or glycerol. The properties of DESs can be easily changed by adjusting the type and molar ratio of the salt and HBD [10] . Although the strong hydrogen bonding involved in this kind of solvent might be extremely denaturing for enzymes, some studies have demonstrated that some hydrolases can maintain their high activities in DESs, and the DESs can even improve the catalytic activity of the enzymes [8 , 10 , 12] . However, only a very few of reaction types such as esterification or transesterification dealing with their use in DESs have been reported so far [10 , 12] . Thus, the applications of DESs in other enzyme-catalyzed reactions have attracted increasing research attention.
In this study, we introduced DESs in an enzyme-catalyzed Henry reaction to promote the reaction. We report the first examples of biocatalytic Henry reactions in DES-based medium and provide a rapid, eco-friendly synthesis strategy for nitro alcohol compounds.
Materials and Methods
- Materials
Lipase from Aspergillus niger (Lipase AS “Amano”) was donated by Amano Enzyme Ltd., Japan. Choline chloride (ChCl) was bought from J & K Scientific Ltd., China. Benzaldehyde, 2-nitrobenzaldehyde, 3-nitrobenzaldehyde, and 4-nitrobenzaldehyde were purchased from Shanghai Darui Fine Chemical Ltd., China. 2,4-Dinitrobenzaldehyde and ethylene glycol (EG) were purchased from Energy Chemical, China. 4-Bromobenzaldehyde and 4-methylbenzaldehyde were bought from Aladdin Industrial Co., China. Nitromethane was purchased from Sinopharm Chemical Reagent Ltd., China. Glycerol (Gly) and Urea were purchased from Changchun Baijin Biotechnology Ltd, China. All organic solvents were reagent grade and employed without further purification.
- Preparation of Deep Eutectic Solvents
In this study, DESs were prepared according to the procedures reported in the literature [1 , 10] . The preparation involved reaction of choline chloride with urea, glycerol, or ethylene glycol at 80℃ until a c lear solution was obtained. The p repared DESs were cooled and used for the enzyme-catalyzed Henry reaction without any purification.
- General Procedure for Lipase AS-Catalyzed Henry Reaction in DES
4-Nitrobenzaldehyde (0.15 mmol) and nitromethane (1.5 mmol) were added to DES (0.7 ml) in a sealed flask. The lipase AS (10 mg) was then added to the reaction mixture. The resulting mixture was shaken at 25℃ under nitrogen protection. When the aldehyde substrate was entirely converted, water (0.7 ml) was added into the reaction mixture to reduce the viscosity of the reaction system, and dichloromethane was used for the extraction. The resulting extracts were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was dissolved in minimal ethyl acetate and loaded onto a silica gel column. The products were monitored on silica gel plates (Qingdao Haiyang Chemical Co., Ltd., China) using UV light for spot detection. All the purified products were eluted with ethyl acetate:petroleumether (1:6 (v/v)). The samples were collected, concentrated, and weighed to calculate the reaction yield. The authenticity of the organic compounds prepared during the study was confirmed by 300 MHz nuclear magnetic resonance (Mercury-300B; Varian, USA). The enantiomeric excesses of the Henry products were analyzed by high-performance liquid chromatography (HPLC, Acme 9000; Young Lin Instrument Co., Ltd.) using a Daicel Chiralpack AD-H or Daicel Chiralpack OD-H column. The elution solvent was a mixture of n -hexane and isopropanol. The flow rate was 1.0 ml/min, and the detection was achieved under UV light at 210 nm. The retention times for all the products were confirmed based on their standard (purity >99.5%). Control reactions without enzyme were carried out under the same conditions. The NMR and HPLC spectra for all the products are shown in the Supplementary material.
- General Procedure for Lipase AS-Catalyzed Aza-Henry Reaction in DES with Water
The aza-Henry reaction of N -Ts (Ts = p -tosyl) imines (0.15 mmol) coupling with nitromethane (2.25 mmol) was catalyzed by lipase AS (10 mg) in 0.7 ml of ChCl:Gly (1:2) with different vol% water. The resulting mixture was shaken at 25℃ under nitrogen protection. When the N -Ts imines substrate was entirely converted, water (0.7 ml) was added into the reaction mixture to reduce the viscosity of the reaction system, and ethyl acetate was used to extract the products. The resulting extracts were dried over anhydrous Na 2 SO 4 a nd c oncentrated u nder r educ ed p ressure. After the samples were dissolved in 2.0 ml of acetonitrile, a 20 μl aliquot of the sample was obtained and analyzed by HPLC (Acme 9000, Young Lin Instrument Co., Ltd.) using an Eclipse XDB-C18 column. The elution solvent was a 50:50 mixture of acetonitrile and water. The flow rate was 1.0 ml/min, and the detection was achieved under UV light at 254 nm. The retention time of the corresponding nitro amine product was 7.7 min. The yields of nitro amine products were confirmed by external standard method based on their standard (purity >99.5%). Control reactions without enzyme were carried out under the same conditions. The NMR and HPLC spectra for the product are shown in the Supplementary material.
- Fluorescence Spectroscopy
Fluorescence measurements were carried out using a RF-5301 PC spectrofluorophotometer. Enzyme solution was prepared by dissolving lipase AS (20 mg) in deionized water (1.0 ml), and then a 100 μl enzyme solution was mixed with DES (ChCl: Gly 1:2) and water to reach a final water content that ranged from 20 to 100 vol%. The enzyme samples prepared were detected by their fluorescence spectrum. The fluorescence was recorded at an excitation wavelength of 280 nm, and the detection wavelength ranged from 300 to 540 nm. A blank medium without enzyme was subtracted to discount the influence of the DES and water on the enzyme fluorescence spectrum.
Results and Discussion
- Effects of Various DESs on Lipase AS-Catalyzed Henry Reaction
A model reaction of 4-nitrobenzaldehyde with nitromethane catalyzed by lipase AS was selected as a standard ( Fig. 1 ) to determine whether DESs could be a suitable medium for enzyme-catalyzed Henry reactions. We selected three DESs, including ChCl:Gly, ChCl: EG, and ChCl:Urea, to observe their effects on a lipase AS-catalyzed Henry reaction. The molar ratio of ChCl and HBD was set at 1:2 to ensure that the formed DES was liquid at our reaction temperature (25°C). The possible catalytic effect of DES medium was studied as control experiments to ensure the accuracy of the experimental results. The results show that the Henry reaction could proceed in DES without any catalyst. Thus, DES might play an important role in the Henry reaction through hydrogen bonding interactions with nitromethane and aldehyde group. The final yields of enzyme-catalyzed Henry reactions were calculated based on the control experiments. As shown in Table 1 , the lipase AS-catalyzed Henry reaction occurred in all of the selected DESs to different extents depending on the nature and molar ratio of DES components ( i.e ., ChCl and HBD). This finding was attributed to possible variations in the network formed by hydrogen bonds, leading to different behaviors of their constituents. When ChCl:Gly was used as the reaction medium, the total reaction yields of both the lipase AS-catalyzed reaction and the control experiment decreased with the increase in glycerol content; this finding might be attributed to the resulting high viscosity of DES, thereby hindering the substrate from accessing the enzyme active site and decreasing the reaction rate [25] . The best final reaction yield of 32.3% was obtained with ChCl:Gly (1:2) as the reaction medium. Similar results were also observed when ChCl:EG was employed as the reaction medium, but the DES-catalyzed transformation was much stronger than that in ChCl:Gly, leading to a low final reaction yield of lipase AS-catalyzed Henry reaction (13.7% to 15.5%). For ChCl:Urea, we found that the yield of lipase AS-catalyzed Henry reaction was similar to that of DES-catalyzed transformation. This phenomenon could be explained by a previous research of Singh et al. [23] , in which the ChCl:Urea itself was found to be a highly effective catalyst of the Henry reaction, restricting lipase activity. From the above results, we concluded that the lipase AS-catalyzed Henry reaction can be achieved in DES, but DES-catalyzed transformation was the major obstacle for its application. Thus, other strategies are needed to control the DES-catalyzed transformation.
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Lipase AS-catalyzed Henry reaction of 4-nitrobenzaldehyde and nitromethane.
Effects of various DESs on lipase AS-catalyzed Henry reactiona.
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aReaction conditions: Lipase AS (10 mg), 4-nitrobenzaldehyde (0.15 mmol), nitromethane (1.5 mmol), and DES (0.7 ml) were stirred at 25℃ bTotal yields of Henry reaction without enzyme. cTotal yields of Henry reaction with enzyme. dFinal yields of enzyme-catalyzed Henry reaction.
- Effects of Water Content on Lipase AS-Catalyzed Henry Reaction in DES
Enzymes require a specific amount of water to maintain their activities. In this experiment, water was introduced in DES to determine its effect on the lipase AS-catalyzed Henry reaction. From Fig. 2 , DES-catalyzed transformation was controlled effectively, and the rates of lipase AS-catalyzed reactions were drastically accelerated with the increase in added water. The maximum final reaction yields of 90.3% and 91.5% were achieved when 30 and 40 vol% water, respectively, were added to ChCl:Gly (1:2), which only needed 4 h to reach such high reaction yields. However, adding more than 40 vol% water decreased the lipase AS-catalyzed conversions, although the DES-catalyzed transformation was weak. When pure water was selected as the reaction medium for the lipase AS-catalyzed Henry reaction, the final reaction yield was only 33.4% in the same reaction conditions as that in the DES–water mixture. Similar results were observed when ChCl:EG was used as the reaction medium, and a high final yield of 82% was reached with 50 vol% water in ChCl:EG (1:2).
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Effects of water content on lipase AS-catalyzed Henry reactions of 4-nitrobenzaldehyde and nitromethane in (A) ChCl:Gly (1:2) and (B) ChCl:EG (1:2).

Total yields of Henry reaction without enzyme. Total yields of Henry reaction with enzyme. Final yields of enzyme-catalyzed Henry reaction.

The above results indicate that ChCl-based DES with water can be an effective reaction medium to enhance lipase AS-catalyzed Henry reaction. This phenomenon might be explained by the formation of strong interactions (hydrogen bonds) between water and DES, thus making DES a suitable solvent rather than a catalyst. Consequently, the hydrogen bonds formed in the DES–water mixtures constitute the main driving force affecting the spatial conformation of the enzyme, further leading to a change in lipase activity [9 , 28] . Fluorescence spectroscopy results of lipase AS in ChCl:Gly (1:2) with water are shown in Fig. 3 . The decrease of water content in DES can trigger a λmax redshift; a significant λmax redshift from 334 nm to 339nm was observed when 30 vol% or 40 vol% water was added to DES, which agreed well with the experimental data. This result suggested that an appropriate amount of water in DES can trigger a subtle change in the microenvironment of the amino acid residues in lipase AS and is capable of providing the enzyme with a slightly more relaxed tertiary structure, possibly facilitating the lipase-catalyzed Henry reaction. Excessive water (>40 vol%) might constitute a diffusion barrier for the substrates, thus decreasing both lipase AS- and DES-catalyzed reaction yields. Controlling the water content for lipase-catalyzed Henry reactions in a DES system is therefore important.
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Fluorescence spectroscopy of lipase AS in ChCl:Gly (1:2) with water.
- Optimization of the Reaction Parameters for Lipase AS-Catalyzed Henry Reaction in DES–Water Mixture
The lipase AS catalytic system in the DES–water mixture gave the best results. Hence, the reaction was examined further to determine the optimum reaction conditions.
First, lipase AS-catalyzed Henry reactions between 4-nitrobenzaldehyde and nitromethane in ChCl:Gly (1:2) with 30 vol% water were investigated at temperatures ranging from 20℃ to 30℃. Fig. 4 illustrates the effects of temperature on the time-course yields. The time-course experiments showed that the total reaction yields of both DES- and lipase-catalyzed reactions increased with the increase in temperature and reaction time. The reaction rate was faster when the reaction temperature was set at 30℃ than that at 25℃ and 20℃, and a high lipase AS-catalyzed total yield of 95% was achieved in 3 h. However, the DES-catalyzed reaction total yield also reached 20.2%, which was greater than that at 20℃ and 25℃, leading to a lipase AS-catalyzed final yield of only 74.8%. This phenomenon could be explained by the lower viscosity of DES at high temperatures, thus minimizing mass transfer issues. When the reaction was carried out at 20℃, a low level of DES-catalyzed reaction was observed; however, the reaction needed at least 10 h to reach a final yield of 83.2%. Thus, 25℃ was selected as the optimum reaction temperature, in which a high final yield of 90% was achieved in only 4 h.
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Effects of temperature on the time-course yields of lipase AS-catalyzed Henry reactions in ChCl:Gly (1:2) with 30 vol% water.

Total yields of Henry reactions without enzyme at 20℃ (), 25℃ (), and 30℃ (). Total yields of Henry reactions with enzyme at 20℃ (), 25℃ (), and 30℃ (). Final yields of enzyme-catalyzed Henry reactions at 20℃ (▲), 25℃ (●), and 30℃ (■).

Substrate molar ratios of 4-nitrobenzaldehyde and nitromethane were then optimized, and the results are shown in Fig. 5 A. The total yields of DES-catalyzed reactions decreased and the total yields of lipase AS-catalyzed reactions improved greatly with the increasing of the substrate molar ratio from 1:2 to 1:15. Further increase in the substrate molar ratio to 1:20 decreased the total reaction yield of the lipase-catalyzed reaction. A maximum final reaction yield of 92.2% was achieved at a substrate molar ratio of 1:15. The additive amount of lipase AS is an important parameter that needs to be optimized as well. As shown in Fig. 5 B, the final reaction yields increased with the increase in the additive amount of lipase AS. A high reaction yield of 91.2% was obtained when 10 mg of the enzyme was added. Further increase in the amount of enzyme to 15 mg did not improve the reaction yield. This result might be caused by the aggregation of the enzyme at high concentrations, reducing the accessibility of enzyme particles to the substrates. The aggregation of the enzyme might be attributed to the high viscosity of DES solvent, preventing the enzyme from dispersing efficiently in the reaction system. Similar results have been observed by other groups [30 , 31] .
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Optimization of the substrate molar ratio and lipase AS amount.

() Effects of substrate molar ratios of 4-nitrobenzaldehyde and nitromethane on the lipase AS-catalyzed Henry reaction in ChCl:Gly (1:2) with 30 vol% water. Total yields of Henry reaction without enzyme; ◆ Total yields of Henry reaction with enzyme; Final yields of enzyme-catalyzed Henry reaction. () Effects of enzyme additive amount on the lipase-catalyzed Henry reaction in ChCl:Gly (1:2) with 30 vol% water.

In summary, the lipase AS-catalyzed Henry reaction achieved a 92.2% final reaction yield under the following optimized reaction conditions: reaction temperature of 25℃, substrate molar ratio of 1:15, enzyme amount of 10 mg, and reaction time of 4 h.
- Lipase AS-Catalyzed Henry Reaction of Nitromethane with Other Aromatic Aldehydes
Under the optimized conditions, other aromatic aldehydes were used in the lipase AS-catalyzed Henry reaction to show the generality and expand the scope of this methodology. The results in Table 2 indicate that the reaction system was sensitive to electronic effects, and the benzaldehydes with electron-withdrawing substituents can effectively participate in the reaction. In general, nitro-substituted benzaldehydes furnished nitro alcohol with final reaction yields well above 87.7%. Notably, 4-nitrobenzaldehyde and 2, 4-dinitrobenzaldehyde produced 92.2% and 91.7% final yields, respectively. This result may be because electron-withdrawing groups can enhance the electrophilicity of the carbonyl carbon in the aldehydes, facilitating the reaction. The reaction yields were lower with halo-substituted benzaldehydes than those with nitro-substituted benzaldehydes, which further confirmed that the strong electron-withdrawing groups on the benzene ring had beneficial effects on the Henry reaction. Lipase AS-catalyzed Henry reactions using benzaldehydes with electron-withdrawing group as substrate in the presence of ChCl:Gly (1:2) containing 30 vol% water were superior to that in pure water. In addition, the reaction rates could be greatly improved to provide the corresponding products with satisfactory yields in a short reaction time (4 h). By contrast, aromatic aldehydes containing an electron-donating group gave relatively low yields. When para -methoxy-substituted benzaldehyde was used as the substrate, the final reaction yield was only 9.6%. Moreover, no product was created when para -methyl-substituted benzaldehyde was used as the substrate, because electron-donating groups render carbonyl carbons in aldehydes less electrophilic. Similar to the benzaldehydes bearing an electron-donating group, no product was found when benzaldehyde with no substituent was used as the reaction substrate when either DES–water or pure water was used as the reaction medium, although it contains relatively small steric hindrance of alkyls.
Lipase AS-catalyzed Henry reaction of nitromethane with other aromatic aldehydesa.
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aReaction conditions: lipase AS (10 mg), aldehydes (0.15 mmol), nitromethane (2.25 mmol), and solvents (0.7 ml) were stirred at 25℃. N.R. means no reaction. bFinal yields of enzyme-catalyzed Henry reaction. cThe reaction was performed at 37℃
- Extension of Methodology to Lipase AS-Catalyzed Aza-Henry Reaction
Given the development of the Henry reactions catalyzed by lipase in DES–water, we further extended the methodology to the aza-Henry reaction to provide a powerful and an efficient method for the synthesis of β-nitro amines, which can be readily converted into 1,2-diamines and α-amino acids. Such molecules are important building blocks in most natural product syntheses [15] . Various catalysts, including metal complexes and organocatalysts, have been developed for the aza-Henry reaction [11 , 21 , 32] . However, no reports have focused on enzyme-catalyzed aza-Henry reactions, especially when N -sulfonyl-protected imines were used as the substrates.
In this study, the aza-Henry reaction of N -Ts (Ts = p -tosyl) imines coupling with nitromethane to produce the corresponding nitro amines was selected as the model reaction. The results of lipase AS-catalyzed aza-Henry reactions are summarized in Table 3 . The lipase-catalyzed aza-Henry reaction was allowed to proceed first in pure water, but the reaction did not initiate at all. The reaction rapidly progressed when DES was added, indicating that DES is indeed important for the reaction to progress, even though the reaction rate was slow. Similar to the lipase AS-catalyzed Henry reaction in DES, the aza-Henry reaction occurred in ChCl:Gly (1:2) without any catalyst, and a reaction yield of 17.1% was reached. Based on the control experiments, the final reaction yield of 15.8% for the lipase-catalyzed aza-Henry reaction was obtained. Addition of water in ChCl:Gly (1:2) affected the reaction in the same way, evidently decreasing the DES-catalyzed transformation and accelerating the lipase AS-catalyzed reaction. A high final reaction yield of 38.7% was obtained at 30 vol% water content in ChCl:Gly (1:2). Although a longer reaction time (40 h) was required and lower yields were obtained compared with that of the lipase AS-catalyzed Henry reaction, the results are still remarkable. This study is the first to investigate the lipase-catalyzed aza-Henry reaction in DES. Further optimization work is in progress.
Lipase AS-catalyzed aza-Henry reaction ofN-Ts imines coupling with nitromethane in ChCl:Gly (1:2) with different water content.a
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aReaction conditions: Lipase AS (10 mg), N-Ts imines (0.15 mmol), nitromethane (2.3 mmol), and DES solvent (0.7 ml) were stirred at 25℃. N.R. means no reaction. bTotal yields of aza-Henry reaction without enzyme. cTotal yields of aza-Henry reaction with enzyme. dFinal yields of enzyme-catalyzed aza-Henry reaction.
In summary, we have developed a simple, environmentally friendly, and efficient catalytic system by using lipase AS in DES–water mixtures for the synthesis of nitro alcohol compounds. In this study, DES was first introduced in the lipase-catalyzed Henry reaction. Addition of a certain amount of water to DES remarkably improved the enzyme activity, and the reaction time was greatly shortened. Fluorescence analysis suggested that the improvement of the reaction rate of the lipase AS-catalyzed Henry reaction was mainly caused by the synergistic action of DES and water due to the extensive hydrogen bond network throughout the solvent. By optimizing the reaction parameters, an excellent yield of 92.2% was achieved in a very short reaction time of 4 h. The methodology was also extended to the aza-Henry reaction. Although enantioselectivity for Henry and aza-Henry reactions was not found, we deemed that enantioselectivity can be improved by site-directed mutagenesis or variation of the reaction media. To the best of our knowledge, this study is the first to report on a biocatalyst that can effectively catalyze Henry and aza-Henry reactions in DES.
Acknowledgements
The authors are grateful for the financial support provided by the National Natural Science Foundation of China (Nos. 21372098 and 20802025), Jilin Provincial Science and Technology Sustentation Program (Nos. 201215033 and 20110436), Basic Operating Expenses, and 985 Project at Jilin University.
References
Abbott AP , Boothby D , Capper G , Davies DL , Rasheed RK 2004 Deep eutectic solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic liquids. J. Am. Chem. Soc. 126 9142 - 9147    DOI : 10.1021/ja048266j
Borah JC , Gogoi S , Boruwa J , Kalita B , Barua NC 2004 A highly efficient synthesis of the C-13 side-chain of taxol using Shibasaki's asymmetric Henry reaction. Tetrahedron Lett. 45 3689 - 3691    DOI : 10.1016/j.tetlet.2004.02.150
Bunnage ME , Davies SG , Goodwin CJ , Ichihara O 1994 An expeditious asymmetric-synthesis of allophenylnorstatine. Tetrahedron 50 3975 - 3986    DOI : 10.1016/S0040-4020(01)89672-1
Busto E , Fernandez VG , Gotor V 2011 Protein-mediated nitroaldol addition in aqueous media Catalytic promiscuity or unspecific catalysis? Org. Process Res. Dev. 15 236 - 240    DOI : 10.1021/op100130c
Busto E , Gotor-Fernandez V , Gotor V 2010 Hydrolases: catalytically promiscuous enzymes for non-conventional reactions in organic synthesis. Chem. Soc. Rev. 39 4504 - 4523    DOI : 10.1039/c003811c
Colak M , Aral T , Hosgoren H , Demirel N 2007 Synthesis of novel c hiral Schiff-base l igands a nd t heir a pplic ation in asymmetric nitro aldol (Henry) reaction. Tetrahedron Asymmetry 18 1129 - 1133    DOI : 10.1016/j.tetasy.2007.04.025
Davis AV , Driffield M , Smith DK 2001 A dendritic active site: catalysis of the Henry reaction. Org. Lett. 3 3075 - 3078    DOI : 10.1021/ol016197x
Duranda E , Lecomtea J , Baréaa B , Piomboa G , Dubreucqb E , Villeneuvea P 2012 Evaluation of deep eutectic solvents as new media forCandida antarcticaB lipase catalyzed reactions. Process Biochem. 47 2081 - 2089    DOI : 10.1016/j.procbio.2012.07.027
Durand E , Lecomte J , Baréa B , Villeneuve P 2014 Towards a better understanding of how to improve lipase-catalyzed reactions using deep eutectic solvents based on choline chloride. Eur. J. Lipid Sci. Technol. 116 16 - 23    DOI : 10.1002/ejlt.201300246
Durand E , Lecomte J , Villeneuve P 2013 Deep eutectic solvents: synthesis, application, and focus on lipase-catalyzed reactions. Eur. J. Lipid Sci. Technol. 115 379 - 385    DOI : 10.1002/ejlt.201200416
Gao FF , Zhu J , Tang Y , Deng MZ , Qian CT 2006 Asymmetric aza-Henry reaction of N-tosyl imine. Chirality 18 741 - 745    DOI : 10.1002/chir.20315
Gorke JT , Srienc F , Kazlauskas RJ 2008 Hydrolase-catalyzed biotransformations in deep eutectic solvents. Chem. Commun. 10 1235 - 1237    DOI : 10.1039/b716317g
Hult K , Berglund P 2007 Enzyme promiscuity: mechanism and applications. Trends Biotechnol. 25 231 - 238    DOI : 10.1016/j.tibtech.2007.03.002
Khadjawi MG , Purkarthofer T , Skranc W , Griengl H 2007 Hydroxynitrile lyase-catalyzed enzymatic nitroaldol (Henry) reaction. Adv. Synth. Catal. 349 1445 - 1450    DOI : 10.1002/adsc.200700064
Knudsen KR , Risgaard T , Nishiwaki N , Gothelf KV , Jørgensen KA 2001 The first catalytic asymmetric aza-Henry reaction of nitronates with imines: a novel approach to optically active β-nitro-α-amino acid and alpha, betadiamino acid derivatives. J. Am. Chem. Soc. 123 5843 - 5844    DOI : 10.1021/ja010588p
Le ZG , Gou LT , Jiang GF , Yang XB , Liu HQ 2013 Henry reaction catalyzed by lipase A fromAspergillus niger. Green Chem. Lett. Rev. 6 277 - 281    DOI : 10.1080/17518253.2013.818721
Matsumoto K , Asakura S 2014 Albumin-mediated asymmetric nitroaldol reaction of aromatic aldehydes with nitromethane in water. Tetrahedron Lett. 55 6919 - 6921    DOI : 10.1016/j.tetlet.2014.10.109
Milner SE , Moody TS , Maguire AR 2012 Biocatalytic approaches to the Henry (nitroaldol) reaction. Eur. J. Org. Chem. 16 3059 - 3067    DOI : 10.1002/ejoc.201101840
Mridula P , Jyoti B K , R uli B 2009 H enry r eac tion in environmentally benign methods using imidazole as catalyst. Green Chem. Lett. Rev. 2 249 - 253    DOI : 10.1080/17518250903410074
Naushad M , Alothman ZA , Khan AB , Ali M 2012 Effect of ionic liquid on activity, stability, and structure of enzymes: a review. IntJ. Biol. Macromol. 51 555 - 560    DOI : 10.1016/j.ijbiomac.2012.06.020
Okino T , Nakamura S , Furukawa T , Takemoto Y 2004 Enantioselective aza-Henry reaction catalyzed by a bifunctional organocatalyst. Org. Lett. 6 625 - 627    DOI : 10.1021/ol0364531
Sheldon R 2001 Catalytic reactions in ionic liquids. Chem. Commun. 23 2399 - 2407    DOI : 10.1039/b107270f
Singh BS , Lobo HR , Shankarling GS 2012 Choline chloride based eutectic solvent: magical catalytic system for carbon–carbon bond formation in the rapid synthesis of β-hydroxy functionalized derivatives. Catal. Commun. 24 70 - 74    DOI : 10.1016/j.catcom.2012.03.021
Svedendahl M , Hult K , Berglund P 2005 Fast carboncarbon bond formation by a promiscuous lipase. J. Am. Chem. Soc. 127 17988 - 17989    DOI : 10.1021/ja056660r
Tang RC , Guan Z , He YH , Zhu W 2010 Enzyme-catalyzed Henry (nitroaldol) reaction. J. Mol. Catal. B Enzym. 63 62 - 67    DOI : 10.1016/j.molcatb.2009.12.005
Wang JL , Li X , Xie HY , Liu BK , Lin XF 2010 Hydrolase-catalyzed fast Henry reaction of nitroalkanes and aldehydes in organic media. J. Biotechnol. 145 240 - 243    DOI : 10.1016/j.jbiotec.2009.11.022
Wenda S , Illner S , Mell A , Kragl U 2011 Industrial biotechnology - the future of green chemistry? Green Chem 13 3007 - 3047    DOI : 10.1039/c1gc15579b
Wu BP , Wen Q , Xu H , Yang Z 2014 Insights into the impact of deep eutectic solvent on horseradishperoxidase: activity, stability and structure. J. Mol. Catal. B Enzym. 101 101 - 107    DOI : 10.1016/j.molcatb.2014.01.001
Xia WJ , Xie ZB , Jiang GF , Le ZG 2013 The Henry reaction in [Bmim][PF6]-based microemulsions promoted by acylase. Molecules 18 13910 - 13919    DOI : 10.3390/molecules181113910
Zhang Y , Wang N , Xie ZB , Zhou LH , Yu XQ 2014 Ionic liquid as a recyclable and efficient medium for lipase-catalyzed asymmetric cross aldol reaction. J. Mol. Catal. B Enzym. 110 100 - 110    DOI : 10.1016/j.molcatb.2014.10.008
Zhao H , Zhang C , Crittle TD 2013 Choline-based deep eutectic solvents for enzymatic preparation of biodiesel from soybean oil. J. Mol. Catal. B Enzym. 85-86 243 - 247    DOI : 10.1016/j.molcatb.2012.09.003
Zhou H , Peng D , Qin B , Hou ZR , Liu XH , Feng XM 2007 Highly enantioselective aza-Henry reaction ofN-tosyl imines catalyzed byN,N-dioxide-Cu(I) complexes. J. Org. Chem. 72 10302 - 10304    DOI : 10.1021/jo702200u