Accepted Manuscript

Title: Lipase-catalyzed Knoevenagel condensation betweenα,�-unsaturated aldehydes and active methylene compounds

Author: Zhi Wang Chun-Yu Wang Hao-Ran Wang HongZhang Ya-Lun Su Teng-Fei Ji Lei Wang

PII: S1001-8417(14)00138-7DOI: http://dx.doi.org/doi:10.1016/j.cclet.2014.03.036Reference: CCLET 2922

To appear in: Chinese Chemical Letters

Received date: 15-1-2014Revised date: 7-3-2014Accepted date: 11-3-2014

Please cite this article as: Z. Wang, C.-Y. Wang, H.-R. Wang, H. Zhang, Y.-L. Su, T.-F.Ji, L. Wang, Lipase-catalyzed Knoevenagel condensation between rmalpha,rmbeta-unsaturated aldehydes and active methylene compounds, Chinese Chemical Letters(2014), http://dx.doi.org/10.1016/j.cclet.2014.03.036

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Graphical Abstract Lipase-catalyzed Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene compounds

Zhi Wang a，#, Chun-Yu Wang c，#, Hao-Ran Wang a, Hong Zhang a, Ya-Lun Su b, Teng-Fei Ji b,*, Lei Wang a,* a Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, College of Life

Science, Jilin University, Changchun 130023, China b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia

Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

c State key Laborarory of Supramolecular Structure and Materials, Jilin University, Changchun 130023, China

CHO

O O+

LipaseO

O

A simple and efficient Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene compounds is reported. This reaction can be catalyzed by PPL (lipase from porcine pancreas) with satisfied yields (49%-92%) and moderate Z/E selectivity. Original article

Lipase-catalyzed Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene compounds

Zhi Wang a，#, Chun-Yu Wang c，#, Hao-Ran Wang a, Hong Zhang a, Ya-Lun Su b,

Teng-Fei Ji b,*, Lei Wang a, ∗ a Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, College of life science, Jilin University, Changchun 130023, China; b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; c State key laborarory of supramolecular structure and materials, Jilin University, Changchun 130023, China;

———

∗ Corresponding authors. E-mail addresses: jitf@imm.ac.cn; w_lei@jlu.edu.cn

# These authors contributed equally to this work.

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1. Introduction Enzyme catalytic promiscuity is a property shown by many enzymes [1]. It refers to the ability of an

enzyme of catalyzing more than one type of chemical transformation and has only recently been exploited for synthetic applications [2-5]. As a part of our research to explore the applications in this new area, we are focusing on the Knoevenagel condensation, which is a useful and widely employed method of producing α,β-enones/enoesters in organic synthesis. The Knoevenagel condensation, discovered by E. Knoevenagel in 1896, is widely used both in the academic environment and in the chemical pharmaceutical and perfume industries [6-9]. Generally, these condensations can be carried out in the presence of bases or Lewis acids [10-12]. In addition, some methods like electrochemistry, microwave and ultrasound irradiation have also been reported for the Knoevenagel condensation [13-15]. However, these methods suffer from numerous limitations such as hazardous solvents, high reaction temperatures or harsh reaction conditions.

Recently, it has been found that hydrolases can catalyze the Knoevenagel condensation. For example, lipase from porcine pancreas (PPL) was also used to catalyze the Knoevenagel condensation between substituted benzaldehydes and methyl cyanoacetate in moderate yields (30%-85%), and PPL tolerated a variety of functional groups on the aromatic ring [16]. However, much still remains to be done on the applications of hydrolase-catalyzed Knoevenagel condensation. It is well known that the Knoevenagel condensations are generally carried out with aromatic aldehydes as substrates. Only a limited number of examples of α,β-unsaturated aldehydes as starting materials have been reported. Hu et al. reported that papain could catalyze the Knoevenagel reactions in DMSO/water between the substituted cinnamaldehydes and active methylene compounds (acetylacetone and ethyl acetoacetate). The products were obtained in moderate yield (42%-86%) and low Z/E selectivity [17]. The application of lipase in this type of Knoevenagel condensation has not been reported so far. This prompted us to further study the Knoevenagel condensations catalyzed by lipase between α,β-unsaturated aldehydes and active methylene compounds. In the current study, we investigated the lipase-catalyzed Knoevenagel condensation with crotonaldehyde (1a) and acetoacetone (2a) as the substrates (Scheme 1).

2. Experimental 2.1 Materials Porcine pancreas lipase (lipase type II) was purchased from Sigma (Beijing, China). C. rugosa lipase

(CRL) was purchased from Sigma (St Louis, MO). Pseudomonas sp. lipase (PSL) was purchased from Amano Pharmaceutical Co. Ltd. (Japan). Candida antarctica lipase B (CAL-B) was kindly donated by

ARTICLE INFO ABSTRACT

Article history: Received 15 January 2014 Received in revised form 7 March 2014 Accepted 11 March 2014 Available online Keywords: Lipase Knoevenagel condensation α,β-Unsaturated aldehyde Active methylene compound

A simple and efficient Knoevenagel condensation between α,β-unsaturated aldehydes and active methylene compounds is reported. Notably, this condensation can be catalyzed by PPL (lipase from porcine pancreas) with satisfied yields (49%-92%). Moreover, PPL induces moderate Z/E selectivity in the Knoevenagel condensation.

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Novo Nordisk Industries (China). Bacillus subtilis lipase (BSL2) was over expressed from Bacillus subtilis strain IFFI10210 in our laboratory according to the method we have reported [18]. Aeropyrum pernix esterase (APE1547) was produced from a hyperthermophilic archaeon strain, then purified and lyophilized [19]. These enzymes were used after lyophilization for enzymatic reaction without further purification. 4-Crotonaldehyde was purchased from Sigma. Other reagents were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Commercially available reagents and solvents were used without further purification. NMR spectra were recorded on an Inova 500 (1H, 500 MHz) spectrometer. ESI-MS was performed on an Agilent 1100 LC/MSD.

2.2 Lipase-catalyzed Knoevenagel condensation

The reaction was performed at 40 oC for 120 h in a round bottom flask contained α,β-unsaturated

aldehyde (1 mmol), active methylene compound (1 mmol), tert-butyl alcohol (5 mL), deionized water (20% v/v) and PPL powder (20 mg). After completion, the reaction mixture was evaporated and obtained the crude product of condensation, then purified by silica gel chromatography, the data of 1H NMR and ESI-MS of all products are given in Supporting information. All experiments were repeated three times and the final results were based on the average value.

3. Results and discussion

It’s well known that the catalytic activity of a lipase depends mainly on the type and origin of the lipase [20]. In this study, five kinds of lipases were screened to catalyze this Knoevenagel condensation. It could be found (Table 1) that all the selected lipases can catalyze the condensation. Among the tested lipases, PPL produced the highest yield (48%).

Many reports have shown that organic media is one of the most important factors influencing the enzyme catalytic performance [21]. In this study, the effects of reaction solvents in the Knoevenagel condensation between crotonaldehyde (1a) and acetoacetone (2a) were investigated in Table 2. The results clearly indicated that the catalytic activity of PPL was significantly influenced by the solvent. It’s believed that the solvent can lead to a conformational change of the enzyme and then affect the enzyme activity. However, the relationship between the value of log P and enzyme activity is not clear so far and need to be further investigated. According to the results, the highest yield was obtained while tert-Butyl alcohol was selected as the reaction media.

It is known that the enzyme needs a certain amount of water to maintain its proper conformation [22]. Therefore, Water content strongly affects the catalytic behavior of an enzyme in non-aqueous media. As shown in Fig. 1, the relationship between the yields of the condensation products and the water content exhibited a bell-shaped curve and the highest yield (83%) was obtained when the water content was 20% (v/v). Further increase or decrease of water content resulted in a decline of enzyme activity. When the water content was low, the conformation of PPL was excessively rigid, which made the “induced-fit” process of PPL difficult and led to the low enzyme activity. However, the enzyme conformation in organic media with high water content may be too flexible, which may also decrease the activity of the enzyme [23].

A set of reactions were carried out with a variety of active methylene compounds under the optimal conditions. The two α,β-unsaturated aldehydes chosen were crotonaldehyde (1a) and cinnamaldehyde (1b), respectively. As shown in Table 3, the yields of targeted products were lower when 1b was selected as the substrate. It might be because cinnamaldehyde (1b) is more difficult to enter the active site of the enzyme due to the steric hindrance. As for the active methylene compounds, methyl cyanoacetate exhibited the highest activity in the condensation. It might be attributed to the specific substrate selectivity of enzyme based on the structure geometry and the reactivity of substrate. Additionally, satisfactory Z/E selectivity was obtained for the products from active methylene compounds (2b-2d). It is worth pointing out that PPL exhibits a higher Z/E selectivity than previously reported papain in the Knoevenagel condensations [17].

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4. Conclusion In conclusion, we have investigated the Knoevenagel condensation between α,β-unsaturated aldehydes

and active methylene compounds. Notably, this simple, efficient, and environmentally-benign method is very clean and can give satisfied yields (49-92%). Moreover, PPL can produce moderate Z/E selectivity of the products. With these significant advantages, this method has great potential for future applications. This method represents a contribution to the realm of synthetic organic chemistry and might extend the utility of enzyme promiscuity.

Acknowledgments

The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 21172093, 31070708, 21072075), the Natural Science Foundation of Jilin Province of China (No. 201115039, 20140101141JC) and the Scientific Research Fund of Jilin University (No. 450060326007).

References

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Fig. 1 Effect of water content on the Knoevenagel reaction between crotonaldehyde and acetoacetone. Reaction conditions: 1a (1mmol), 2a (1mmol), tert-butyl alcohol (5 mL), water content (0-30% v/v) and PPL (20 mg) at 40 oC for 120 h. Scheme 1 Knoevenagel condensation between crotonaldehyde (1a) and acetoacetone (2a) Table 1 Effect of enzyme sources on the Knoevenagel reaction between crotonaldehyde (1a) and acetoacetone (2a)a.

Entry Lipase Yield (%) 1 PPL 45 2 CaLB 32 3 BSL 31 4 PSL 15 5 CRL 22 6 APE1547 28 7 No

enzyme trace

aReaction conditions: 1a (1 mmol), 2a (1 mmol), i-propanol (5 mL) and enzyme (20 mg) at 40 oC for 120 h. Table 2 Effect of solvents on the Knoevenagel reaction between crotonaldehyde (1a) and acetoacetone (2a) Entry Solvent Log P Yield (%)

1 i-Propanol -0.07 45 2 tert-Butyl alcohol 0.58 59 N,N-

Dimethylformamide -1.0 36

3 Dimethyl sulfoxide -1.4 51 4 Acetonitrile -0.33 24

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5 Chloroform 1.61 17 aReaction conditions: 1a (1 mmol), 2a (1 mmol), organic solvent (5 mL) and enzyme (20 mg) at 40 oC for 120 h. Table 3 The PPL catalyzed Knoevenagel reactions between α,β-unsaturated aldehydes and active methylene compounds. a Entry

Aldehyde active methylene compound

Yield (E/Z)

1 1a 3aa 83% 2 1b

OO

2a 3ab 78%

3 1a 3ba 72% (93:7)

4 1b

O

OO

2b

3bb 61% (66:34)

5 1a 3ca 51% (91:9)

6 1b

O

OO

2c

3cb 49% (76:24)

7 1a 3da 92% (100:0)

8 1b

NCO

O

2d 3db 89% (100:0)

9 1a 3ea 54% 10 1b O O

OO

2e 3eb 49% aReaction conditions: α,β-unsaturated aldehyde (1 mmol), active methylene compound (1 mmol), tert-butyl alcohol (5 mL), water content (20% v/v) and PPL (20 mg), at 40 oC for 120 h.