Elsevier

Biochimie

Volume 94, Issue 3, March 2012, Pages 656-661
Biochimie

Research paper
Enzyme catalytic promiscuity: The papain-catalyzed Knoevenagel reaction

https://doi.org/10.1016/j.biochi.2011.09.018Get rights and content

Abstract

Papain as a sustainable and inexpensive biocatalyst was used for the first time to catalyze the Knoevenagel reactions in DMSO/water. A wide range of aromatic, hetero-aromatic and α,β-unsaturated aldehydes could react with less active methylene compounds acetylacetone and ethyl acetoacetate. The products were obtained in moderate to excellent yields with Z/E selectivities of up to 100:0. This case of biocatalytic promiscuity not only widens the application of papain to new chemical transformations, but also could be developed into a potentially valuable method for organic synthesis.

Graphical abstract

Papain was used for the first time to catalyze the Knoevenagel reaction of aromatic, hetero-aromatic and α,β-unsaturated aldehydes with less active methylene compounds acetylacetone and ethyl acetoacetate. The reaction gave moderate to good yields with Z/E selectivities of up to 100:0.

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Highlights

► Papain was used for the first time to catalyze Knoevenagel reaction in DMSO/water. ► The products were obtained in moderate to good yields. ► The effect of several kinds of reaction conditions was investigated.

Introduction

Knoevenagel reaction, as a facile and versatile method for the formation of carbon–carbon bond [1], has been commonly applied in the synthesis of chemicals and chemical intermediates such as carbocyclic as well as heterocyclic compounds of biological significance [2], coumarin derivatives, cosmetics, perfumes and pharmaceuticals [3]. Great efforts have been devoted to explore the effective catalysts, and elegant works have been described with high efficiency. Generally, these catalysts include bases [3], zeolites [4], ionic liquids [5], amino acids [6], [7] and some metal based Lewis acids [8], [9], [10]. Moreover, the Knoevenagel reactions involving 1,3-diketones have often been subjected to low yields or drastic reaction conditions, due to less activity of 1,3-diketones which tend to form stable six-membered cyclic enols [11], [12]. Therefore, the development of environmentally benign and cost-efficient catalysts for the Knoevenagel reactions of 1,3-diketones still maintains a significant challenge.

Over last three decades, enzymes as practical catalysts have been increasingly exploited for organic synthesis for their simple processing requirements, high selectivity and mild reaction conditions. Enzyme promiscuity means, in the broadest terms, one single active site of a given enzyme can catalyze different chemical transformation of natural or non-natural substrates [13], [14], [15], [16]. Although the promiscuous behaviors were originally thought to be rare events, a growing number of enzymes have been used to catalyze the formation of carbon–carbon and carbon–heteroatom bonds through some classic and widely used organic reactions [17], [18], [19]. Recently a lipase-catalyzed decarboxylative Knoevenagel reaction has been reported, which was the only example of enzymatic Knoevenagel reaction [20]. CAL-B (acrylic resin immobilized Candida antarctica lipase B) was used to catalyze decarboxylative Knoevenagel reaction of substituted aromatic aldehydes and β-ketoesters in CH3CN/H2O, and a primary amine was used as an additive to form a Schiff base in the course of the reaction. But very recently, the mechanism of lipase-catalyzed Knoevenagel reaction has been challenged [21]. Herein, we wish to report a novel discovery that papain could promote the direct Knoevenagel reaction without using additives, and aromatic, hetero-aromatic and α,β-unsaturated aldehydes could react with less active methylene compounds acetylacetone and ethyl acetoacetate resulting in moderate to good yields. Papain (EC 3.4.22.2) is a powerful proteolytic enzyme belonging to the cysteine protease family. Its enzymatic and physiological properties have been extensively studied. Papain is mainly produced from the extraction of Carica papaya latex. Nowadays, some protocols for the cloning and overexpression of papain using baculovirus/insect [22], yeast [23], [24] and bacteria [25], [26], [27] as expression host organisms have been reported. It is possible to obtain recombinant papain in substantial quantities for both basic research and industrial use. In this paper, papain was used for the first time to catalyze Knoevenagel reaction. It provides a novel case of enzyme catalytic promiscuity and might be a potential synthetic method for organic chemistry.

Section snippets

General information for the reagents

Papain (Sigma–Aldrich) from C. papaya (catalog number: 76220, ≥3 U/mg. 1 U corresponds to the amount of enzyme which hydrolyzes 1 μmol N-benzoyl-l-arginine ethyl ester (BAEE, Fluka No. 12880) per minute at pH 6.2 and 25 °C) was purchased from Sigma–Aldrich. Papain (Pangbo) from the latex of C. papaya (650 U/mg. One unit of activity was defined as the amount of the enzyme to produce TCA-soluble hydrolysis products from casein, which gives an absorbance value equivalent to 1 μg of tyrosine at

The catalytic activities of different proteinases in Knoevenagel reaction

Initial studies were undertaken using benzaldehyde and acetylacetone as a model reaction. We chose DMSO/water (Vwater/VDMSO = 0.15) as the medium, and the reaction was performed at 25 °C. The catalytic activities of 5 proteinases in Knoevenagel reaction were investigated using the model reaction (Table 1). The best result of 35% yield was achieved by using papain as the catalyst after 72 h (Table 1, entry 6). Other proteinases also exhibited varying degrees of catalytic activity in the

Conclusion

We describe here the first papain-catalyzed Knoevenagel reaction. The catalyst, with a safe, economical and environmentally benign quality, can catalyze the Knoevenagel reactions of a wide range of aromatic, hetero-aromatic and α,β-unsaturated aldehydes with less active methylene compounds acetylacetone and ethyl acetoacetate to give moderate to good yields. The influence of reaction conditions including solvents, water content, temperature and enzyme loading was investigated. The control

Acknowledgments

Financial support from the Natural Science Foundation Project of CQ CSTC (2009BA5051) is gratefully acknowledged.

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