One-Pot Lipase-Catalyzed Aldol Reaction Combination of In Situ Formed Acetaldehyde

One-Pot Lipase-Catalyzed Aldol Reaction Combinationof In Situ Formed Acetaldehyde

Na Wang & Wei Zhang & Long-Hua Zhou &

Qing-Feng Deng & Zong-Bo Xie & Xiao-Qi Yu

Received: 7 May 2013 /Accepted: 6 August 2013 /Published online: 23 August 2013# Springer Science+Business Media New York 2013

Abstract A facile tandem route to α,β-unsaturated aldehydes was developed bycombining the two catalytic activities of the same enzyme in a one-pot strategy forthe aldol reaction and in situ generation of acetaldehyde. Lipase from Mucor mieheiwas found to have conventional and promiscuous catalytic activities for the hydrolysisof vinyl acetate and aldol condensation with in situ formed acetaldehyde. The firstreaction continuously provided material for the second reaction, which effectivelyreduced the volatilization loss, oxidation, and polymerization of acetaldehyde, as wellas avoided a negative effect on the enzyme of excessive amounts of acetaldehyde.After optimizing the process, several substrates participated in the reaction andprovided the target products in moderate to high yields using this single lipase-catalyzed one-pot biotransformation.

Keywords Lipase . Aldol condensation .One pot . Biocatalysis . Acetaldehyde . Promiscuity .

In situ

Introduction

Enzymes are widely used in modern organic synthesis because of their high activity,good selectivity, and mild reaction conditions [1–3]. In particular, biocatalytic promis-cuity, a new frontier that focuses on the ability of enzymes to catalyze alternativereactions differing from their natural reaction, has extended the application of enzymesin organic synthesis [4–6]. Various promising enzyme-catalyzed promiscuous reactionsinvolving transferase with lyase activity [7], hydrolase with lyase activity [8–12], andhydrolase with racemase activity [13] have been described in the past decade. Among

Appl Biochem Biotechnol (2013) 171:1559–1567DOI 10.1007/s12010-013-0435-4

Na Wang and Wei Zhang contributed equally to this work.

N. Wang (*) :W. Zhang : L.<H. Zhou : Q.<F. Deng : Z.<B. Xie :X.<Q. Yu (*)Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry,Sichuan University, Chengdu 610064, Chinae-mail: [email protected]: [email protected]

these enzymes, lipases are the most frequently used biocatalysts in organic synthesisbecause of their high stability, wide sources, and broad substrate specificity [14].Kapoor and Gupta [15] recently reviewed lipase promiscuity and its biochemicalapplications.

The aldol reaction is one of the most important carbon–carbon bond formationreactions in organic synthesis. Although numerous organocatalysts have been used inthis reaction [16–18], the application of enzymes performing this transformationoffers a green and convenient alternative to traditional chemical methods. Brannebyet al. [19] first reported that wild CAL-B (lipase from Candida antarctica) andSer105Ala mutant CAL-B have a catalytic activity for aldol reactions in 2003. Inour previous work, we have observed that several hydrolases display observableactivities for aldol addition [20–22] and reported the first lipase-catalyzed asymmet-ric aldol reaction between acetone and 4-nitrobenzaldehyde [23]. Guan et al. [24]also reported lipase-catalyzed aldol reaction of heterocyclic ketones with aldehydes,recently.

However, all these lipase-catalyzed aldol reactions are not involving acetaldehyde as asubstrate, which is a strong electrophile that can undergo a number of side reactions withother nucleophiles, such as self-polymerization and oxidation reaction. Moreover, acetal-dehyde is difficult to handle as a low-boiling-point liquid and may cause enzymedeactivation [25]. Therefore, in situ acetaldehyde generation by the corresponding sub-strates under mild conditions catalyzed by enzyme is an ideal method for further reaction.Majumder et al. [26] described the first aldol condensation reaction of in situ formedacetaldehyde with a tricyclic diketone catalyzed by CAL-B. María and de María [27]recently discussed the lipase-catalyzed in situ production of acetaldehyde followed byaldol condensation reaction catalyzed by benzaldehyde lyase. Similarly, Mifsud et al. [28]reported an oxidase-catalyzed in situ aldehyde generation for aldol addition reactionscatalyzed by D-fructose-6-phosphate aldolase. However, most of these cascade strategieshave been performed under the catalysis of two types of enzymes. Although multienzymecatalyzed transformations can be successfully accomplished, the incompatibility of dif-ferent enzymatic steps has become the biggest barrier to the development of multienzymeconversions [29]. Therefore, multi-step complicated processes can be better accomplishedwith single-enzyme catalysis in a one-pot strategy because this process avoids arduousseparation and purification of intermediates. However, studies on the combination ofconventional and promiscuous activities of a single enzyme in one-pot multi-step reac-tions are few. Wang et al. [30] reported protease-catalyzed Knoevenagel/intramoleculartransesterification reaction. Michael addition/acylation reaction can be performed in onepot and catalyzed by a single aminoacylase [29].

As a part of our ongoing interest in lipase-catalyzed promiscuous reactions forextending the application of lipase in organic synthesis, the combination of two catalyticactivities of one lipase in a one-pot strategy to prepare α,β-unsaturated aldehydes usingin situ prepared acetaldehyde (Scheme 1) was reported for the first time. The couplingof biocatalytic promiscuity and one-pot multi-step sequence based on a single enzymehas economic and environmental significance in organic synthesis. Lipase from Mucormiehei (MML) exhibited high activity for the aldol condensation reaction of aromaticaldehydes with acetaldehyde formed from the hydrolysis of vinyl acetate. The reactionwas optimized by investigating the influence of reaction conditions, including reactionmedia, temperature, enzyme concentration, and reaction time. This method was extendedto several aromatic aldehydes and the corresponding products, with moderate yieldsunder the catalysis of MML at 60 °C.

1560 Appl Biochem Biotechnol (2013) 171:1559–1567

Materials and Methods

Materials

Lipozyme® immobilized lipase from M. miehei (0.063 U/mg), Amano lipase from Mucorjavanicus (10 U/mg), lipase from porcine pancreas (6.8 U/mg), lipase from Candida rugosa(1,176 U/mg), lipase from Candida cylindracea (7.29 U/mg), and lipase from acrylic resinC. antarctica (≥10 U/mg) were purchased from Sigma-Aldrich. Pepsin, trypsin, and bovineserum albumin (BSA) were purchased from Aladdin Co., Ltd. (Shanghai, China). Unlessotherwise mentioned, all reagents were obtained from commercial suppliers and usedwithout further purification.

Analytical Methods

1H NMR and 13C NMR spectra were measured on the Bruker AM400 NMR Spectrometer(400 or 100 MHz, respectively) with CDCl3 as a solvent and were recorded in part permillion relative to tetramethylsilane. Thin-layer chromatography experiments wereperformed on glass-backed silica plates and visualized by UV detection. HPLC wasperformed with the Waters Associates equipment (Waters 2695 with 2998 Photodiode ArrayDetector) using a C18 column. Elution was performed with a mixture of methanol/water(55/45, v/v) at 0.8 ml/min.

General Procedure for Lipase-Catalyzed Aldol Reaction

A typical reaction mixture containing aromatic aldehydes (1.5 mmol), vinyl acetate (5 mL),H2O (5 mL), and Lipozyme® (400 mg) was incubated at 60 °C with 200 rpm. The reactionwas terminated by filtering off the enzyme after the indicated time. Silica gel was then addedto the reaction solution for evaporation. The obtained faint yellow powder was furtherpurified by flash column chromatography with ethyl acetate/petroleum ether (1:20 to 1:10)to obtain the product.

4-Nitrocinnamaldehyde

1H NMR (400 MHz, CDCl3): δ 9.78 (d, J=7.4 Hz, 1H), 8.30 (d, J=8.6 Hz, 2H), 7.74 (d,J=8.6 Hz, 2H), 7.54 (d, J=16.1 Hz, 1H), 6.73 (dd, J=16.1, 7.4 Hz, 1H); 13C NMR (100 MHz,CDCl3): δ 192.8, 148.8, 131.7, 129.1, 124.4.

Scheme 1 MML-catalyzed aldol condensation using an in situ generated acetaldehyde

Appl Biochem Biotechnol (2013) 171:1559–1567 1561

4-Cyanocinnamaldehyde

1H NMR (400 MHz, CDCl3): δ 9.77 (d, J=7.5 Hz, 1H), 7.74 (d, J=8.2 Hz, 2H), 7.67 (d,J=8.2 Hz, 2H), 7.48 (d, J=16.1 Hz, 1H), 6.78 (dd, J=16.1, 7.5 Hz, 1H); 13C NMR (100 MHz,CDCl3) δ 192.9, 149.5, 138.2, 132.8, 131.2, 128.7.

2-Nitrocinnamaldehyde

1H NMR (400 MHz, CDCl3): δ 9.79 (d, J=7.6 Hz, 1H), 8.12 (d, J=8.1 Hz, 1H), 8.05 (d,J=15.8 Hz, 1H), 7.72 (m, 2H), 7.62 (t, J=6.8 Hz, 1H), 6.64 (dd, J=15.8, 7.6 Hz, 1H); 13CNMR (100 MHz, CDCl3): δ 193.2, 147.4, 133.8, 132.7, 131.2, 129.1, 125.3.

4-Chlorocinnamaldehyde

1H NMR (400 MHz, CDCl3): δ 9.64 (d, J=7.6 Hz, 1H), 7.44 (d, J=8.3 Hz, 2H), 7.36 (t,J=10.5 Hz, 3H), 6.62 (dd, J=16.0, 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 193.3,151.0, 137.2, 132.5, 129.6, 129.4, 128.9.

4-Bromocinnamlaldehyde

1H NMR (400 MHz, CDCl3): δ 9.65 (d, J=7.6 Hz, 1H), 7.49 (m, 2H), 7.33–7.40 (m, 3H),6.64 (dd, J=7.6, 16.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 193.3, 151.0, 132.9, 132.3,129.8, 128.9, 125.6

6-(3-Oxoprop-1-en-1-yl)picolinaldehyde

1H NMR (400 MHz, CDCl3): δ 10.10 (s, 1H), 9.86 (d, J=7.7 Hz, 1H), 7.98 (d, J=5.9 Hz,2H), 7.75 (dd, J=6.1, 2.3 Hz, 1H), 7.60 (d, J=15.8 Hz, 1H), 7.22 (m, 1H); 13C NMR(100 MHz, CDCl3): δ 193.3, 193.1, 153.3, 153.2, 149.3, 138.1, 132.8, 127.7, 122.2.

Results and Discussion

The Catalytic Activities of Different Hydrolases in the One-Pot Aldol Reaction

Experiments were carried out to investigate the catalytic activity of various enzymes in two-step reactions, including conventional and promiscuous activities. The aldol reaction of 4-nitrobenzaldehyde with acetaldehyde generated from the transesterification of vinyl acetatewith i-PrOH in situ was examined. Several commercially available lipases displayed ob-servable activities for this reaction, and the highest yield of 35 % was achieved using MMLas a catalyst (Table 1, entry 3). Other hydrolases such as PPL and trypsin exhibited loweractivities. Some controlled experiments were conducted to demonstrate that the two catalyticactivities can be performed in a one-pot reaction system. Almost no product was detectedeven after 48 h when the reaction was performed without any biocatalyst (Table 1, entry 1).Denatured MML and protein-catalyzed experiments were also conducted to further elucidatethe promiscuous catalytic ability of certain hydrolases (Table 1, entries 9 and 10). Comparedwith their well-folded counterparts, denatured MML and BSA barely catalyzed the aldolreaction. These results indicated that the specific natural fold of lipase was responsible for itsability to catalyze the aldol reaction of 4-nitrobenzaldehyde with acetaldehyde.


To demonstrate each step of the multi-step reaction catalyzed by MML, the reactionwas divided into two steps to investigate the action of lipase. Firstly, incubating vinylacetate and i-PrOH with MML, acetaldehyde and isopropyl acetate were detected byGC/MS; however, neither was produced in the blank test. These results indicated thatthe generation of acetaldehyde in situ was an enzymatic reaction. After incubating themixture of vinyl acetate, i-PrOH, and MML for 24 h, MML was filtered, and 4-nitrobenzaldehyde was added to the reaction system for aldol condensation. However,no product was detected in the absence of enzyme for aldol reaction. These resultsshowed that the specific natural fold of lipase was responsible for its ability to catalyzethe aldol reaction of 4-nitrobenzaldehyde with acetaldehyde and that MML exerted acrucial catalytic effect on the aldol reaction.

Effects of Reaction Solvent

We then examined the influence of reaction media on the enzymatic tandem reaction. As shownin Table 2, the yields were markedly influenced by the reaction media. This tandem reactionwas promoted by water, and the highest yield was obtained as compared with others (Table 2,entry 7). Traditionally, acetaldehyde is known to deactivate lipases in the lipase-catalyzedtransesterification of vinyl esters and alcohols. However, various commercially availablemicrobial lipases showed different stabilities toward acetaldehyde. Recently, Franken et al.[25] described the mechanism of acetaldehyde-induced deactivation of lipases in detail. MMLwas more stable toward acetaldehyde formation, and lipase deactivation was not observed.

Generally, alcohols can provide better yields than other solvents owing to transesterificationwith vinyl acetate. In other words, vinyl acetate is the most commonly used acyl donor in thetransesterifications of alcohols because vinyl alcohol freed from the transesterification reactiontautomerizes to acetaldehyde. However, vinyl acetate was directly hydrolyzes acetaldehydecatalyzed by lipase rather than transesterification with alcohols in this one-pot reaction system.Chahinian et al. [31] investigated the kinetic behavior of lipase against the hydrolysis of vinylacetate and found that the lipases are highly active in the solution of vinyl acetate [31]. Thus,

Table 1 Catalytic activities of different enzymes

Entry Enzyme Yield (%)a

1 No enzyme 0

2 Lipase from acrylic resin Candida antarctica (CAL-B) 4

3 Lipase from Mucor miehei (MML) 35

4 Lipase from porcine pancreas (PPL) 13

5 Lipase from Candida rugosa (CRL) 0

6 Lipase from Mucor javanicus (MJL) 2

7 Pepsin 0

8 Trypsin 13

9 Bovine serum albumin 0

10 Denatured MMLb 0

For reaction conditions, the mixtures of 4-nitrobenzaldehyde (20 mg), enzyme (20 mg), vinyl acetate (1 mL),and i-PrOH (1 mL) were incubated at 50 °C with 200 rpm for 72 ha Yields were determined by HPLCb Pretreated with urea at 60 °C for 24 h


water was used as a solvent for further studies considering water as a kind of environment-friendly and “greener” solvent.

Effect of Enzyme Loading

The effect of enzyme loading on the reaction was investigated for the model reaction. As shownin Fig. 1, the yields were significantly influenced by different amounts of MML loading from 2to 80mg. However, once the amount ofMML loading exceeded 40mg, only a slight increase inyield was noted when more enzymes were loaded. Considering the cost of biocatalyst, 40 mg ofMML was chosen as the optimum quantity for the one-pot aldol reaction.

Effect of Temperature and Reaction Time

Reaction temperature also plays an important role in enzymatic reactions because of its effectson the catalytic properties and enzyme stability. The model reaction was then performed at three

Table 2 Effect of solvent on theMML-catalyzed tandem reaction

For reaction conditions, themixtures of 4-nitrobenzaldehyde(20 mg), MML (40 mg), vinylacetate (1 mL), and 1 mL or-ganic solvent were incubated at50 °C with 200 rpm for 72 haYields were determined byHPLC

Entry Solvent Yield (%)a

1 EtOH 33

2 n-PrOH 34

3 i-PrOH 39

4 n-BuOH 5

5 Phenylethanol 17

6 THF 22

7 H2O 53

8 Hexane 32

9 DMF 8

10 – 24

2 5 10 20 40 60 800

10

20

30

40

50

60

Yie

ld%

Enzyme loading/mg

Fig. 1 Effect of enzyme loading on theMML-catalyzed aldol reaction. For reaction conditions, the mixtures of 4-nitrobenzaldehyde (20 mg) and MML (2, 5, 10, 20, 40, 60, and 80 mg, respectively) in vinyl acetate (1 mL) anddeionized water (1 mL) were incubated at 50 °C with 200 rpm for 72 h. Yields were determined by HPLC


different temperatures (37 to 60 °C) and noted significant differences in yield among thedifferent temperatures. This reaction had strong temperature dependence, and better yield andreaction rates were achieved at 60 °C (Fig. 2). Higher temperatures were not screened,considering the high volatility of acetaldehyde. Consequently, 60 °C was chosen as theoptimum temperature in terms of yield.

0 20 40 60 80 100 120

-10

0

10

20

30

40

50

60

70

80

Yie

ld (

%)

Time (h)

37oC 50oC 60oC

Fig. 2 Time course of the MML-catalyzed aldol reaction in the different temperatures. For reaction condi-tions, the mixture of 4-nitrobenzaldehyde (20 mg), MML (40 mg), vinyl acetate (1 mL), and deionized water(1 mL) were incubated at different temperatures with 200 rpm for 72 h. Yields were determined by HPLC

Table 3 Effect of various substrates on MML-catalyzed tandem reaction

Entry Substrate Product Yield (%)a

1 78

2 38

3 48

4 45

5 66

6 45

Reaction conditions are as follows: aromatic aldehydes (1.5 mmol), vinyl acetate (5 mL), deionized water(5 mL), and MML (400 mg) at 60 °C for 5 daysa Yield of the isolated product after chromatography on silica gel


Scope of MML-Catalyzed One-Pot Tandem Reaction

The scope of the MML-catalyzed one-pot reaction was evaluated based on the previouslyoptimized reaction conditions. Various aromatic aldehydes as substrates reacted with the insitu produced acetaldehyde through an enzymatic tandem approach in moderate yields.Generally, benzaldehydes with electron-withdrawing substituents were better acceptorsand gave better yields. Heteroaromatic aldehydes also showed moderate yield (Table 3,entry 5). The expanding of more broad range of substrates is in progress.

Conclusion

A promiscuous enzymatic tandem reaction for the synthesis of α,β-unsaturated aldehydeswas developed. Lipase from M. miehei display displayed high activity for the aldol reactionof aromatic aldehydes to in situ produced acetaldehyde. This one-pot multi-step reactionconsisted of two relatively independent reactions and the combination of catalytic promis-cuity with its native activity of the hydrolysis of vinyl acetate. The first reaction continu-ously provided material for the second reaction, which effectively reduced volatilizationloss, oxidation, and polymerization of acetaldehyde, thereby avoiding the negative effect onthe enzyme of excessive amounts of acetaldehyde. After optimization, several substratesparticipated in the reaction and provided the target products in moderate to high yields usingthis single lipase-catalyzed one-pot biotransformation.

Acknowledgments We gratefully acknowledge the financial support of the National Program on Key BasicResearch Project of China (973 Program, 2013CB328900) and the National Natural Science Foundation ofChina (nos. 21001077 and 21021001). We also thank the Sichuan University Analytical and Testing Centerfor NMR analysis.

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One-Pot Lipase-Catalyzed Aldol Reaction Combination of In Situ Formed Acetaldehyde

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