Enzyme catalytic promiscuity: lipase catalyzed synthesis of substituted 2H-chromenes by a three-component reaction

Abstract

A new method for the synthesis of substituted 2H-chromenes catalyzed by lipase was reported in the first time. Besides providing a simple and efficient method for the synthesis of 2H-chromenes, this study extends the applicability of lipase in organic synthesis.

---

Enzyme catalytic promiscuity, the ability of an enzyme's active site to catalyze more than one type of chemical transformation,¹ can widen the applications of the existing catalysts and provide novel synthesis pathways. Hydrolases, especially lipases, play a key role in this area due to their broad specificity and excellent stability in organic solvents.² It's well known that carbon–carbon bond-forming reactions are fundamental in organic synthesis. In recent years, many reports indicate that lipase is a useful and efficient catalyst to create carbon–carbon bonds in organic synthesis.^3,4 Some elegant works of lipase catalytic promiscuity in the carbon–carbon bond-forming reactions (aldol condensation, Morita–Baylis–Hillman reaction, Michael addition, Markovnikov addition, and Knoevenagel reaction, et al.) have been reported in the last decades.^5–8

2H-Chromenes are ubiquitous structural motifs in a wide range of biologically active compounds which display various pharmacological activities, such as anti-HIV, antidiabetic, anticancer, antioxidative and antiviral agents.^9–13 Many methods have been reported for the synthesis of 2H-chromene core structure, such as catalytic Petasis reaction of salicylaldehydes, Pd-catalyzed ring closure of 2-isoprenyl phenols, intramolecular cyclization of Wittig intermediates, Baylis–Hillman reaction of 2-hydroxy benzaldehydes with methyl vinyl ketones, electrocyclic ring closure of vinylquinone derivatives, proline-catalyzed multicomponent reaction and phototransformations of several multichromophoric substrates containing 1,2-dibenzoylalkene moieties.^14–20 However, most of these methods have encountered some drawbacks like inconvenient substrate, low yield of product, long reaction time and complicated reaction process. To the best of our knowledge, the enzymatic synthesis of substituted 2H-chromenes has not been reported yet. Herein we reported a simple method for the synthesis of substituted 2H-chromenes catalyzed by lipase (Scheme 1).

Scheme 1 Lipase catalyzed synthesis of substituted 2H-chromenes.

Initially, four lipases were applied to catalyze the synthesis of tertiary alkoxy chromene (4a) and the results are summarized in Table 1. It can be found that all the selected lipases can catalyze the three-component reaction and porcine pancreas lipase (PPL) produced the highest yield (89%). Neither the denatured enzyme nor bovine serum albumin (BSA) could catalyze the reaction, which indicated that the reaction should be catalyzed by the active lipase. Obviously PPL showed higher catalytic activity in the selected reaction condition. Therefore, we chose PPL as the catalyst for further study.§

Table 1 Effect of enzyme sources on the synthesis of substituted 2H-chromenes^a

Entry	Enzyme	Yield^b (%)
a Reaction condition: salicylaldehyde (1 mmol), acetoacetone (1 mmol), methanol (10 mmol), N,N-dimethylformamide (5 mL), enzyme (20 mg), water content (30% v/v), 55 °C, 48 h.b Yields are given for isolated products after column chromatography.c ND: not detected.d PPL was denatured by heating it to 100 °C for 1 h in N,N-dimethylformamide.
1	Porcine pancreas lipase (PPL)	89
2	Candida antarctica lipase B (CALB)	56
3	Pseudomonas sp. lipase (PSL)	63
4	C. rugosa lipase (CRL)	43
5	Bovine serum albumin (BSA)	ND^c
6	Porcine pancreas lipase (denatured)^d	ND^c
7	No enzyme	ND^c

---

It is widely accepted that the active site of lipase, so-called catalytic triad Asp-His-Ser, plays a key role in the promiscuous catalysis.²¹ Based on our observations, we hypothesized a possible mechanistic pathway of the lipase-catalyzed synthesis of 2H-chromene skeleton (Scheme 2). Firstly, the substrate acetoacetone was deprotonated by the catalytic triad of lipase forming an enolate ion (a). Secondly, another substrate salicylaldehyde accepted the proton from the catalytic triad and simultaneously connected the enolate ion (a) to obtain an intermediate (b) with the forming a carbon–carbon bond. Then, the key compound (c) of Knoevenagel condensation could be formed by dehydration. Finally, in the active center of lipase, a rearrangement of the key compound (c) was made to form a more stable benzopyrylium cation (d), which reacts with methanol to form the methoxy product (4). It is important to point out that the substituted 2H-chromene (4) obtained in this study was racemic, which suggested that lipase didn't take part in the final step of this three-component reaction. These results might support our hypothesized mechanism to some extent. Further study to verify this hypothesis is underway and will be published in the future.

Scheme 2 Proposed mechanism of the lipase-catalyzed synthesis of substituted 2H-chromenes.

Organic solvent could influence the enzyme catalytic performance.²² In order to improve the catalytic behavior of PPL, six organic solvents were screened for the three-component reaction and the results are listed in Table 2. The results clearly indicated that the catalytic activity of PPL was tremendously 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.²³ According to the results, the highest yield was obtained while DMF (N,N-dimethylformamide) was selected as the reaction media.

Table 2 Effect of solvent on the synthesis of substituted 2H-chromenes^a

Entry	Solvent	log P^b	Yield^c (%)
a Reaction condition: salicylaldehyde (1 mmol), acetoacetone (1 mmol), methanol (10 mmol), solvent (5 mL), PPL (20 mg), water content (30% v/v), 55 °C, 48 h.b log P is defined as the partition coefficient of a given compound in a two-phase system of water and octanol.c Yields are given for isolated products after column chromatography.
1	N,N-Dimethylformamide	−1.04	89
2	Dioxane	−0.27	72
3	Dimethyl sulfoxide	−1.35	68
4	Tetrahydrofuran	0.46	53
5	Acetonitrile	−0.33	28
6	Ethyl acetate	0.73	19

---

Generally, a certain amount of water is needed to maintain the proper conformation of enzyme.²⁴ As shown in Fig. 1, the relationship between the yields of tertiary alkoxy chromene and the water content exhibited a hill-shaped curve and the highest yield (89%) was obtained when the water content was 30% v/v. Further increase or decrease of the water content would result in a decrease of yield which indicated that the active conformation of PPL can be regulated by water obviously.

Fig. 1 Effect of water content on the synthesis of substituted 2H-chromenes. Reaction condition: salicylaldehyde (1 mmol), acetoacetone (1 mmol), methanol (10 mmol), DMF (5 mL), PPL (20 mg), water content (10–60% v/v), 55 °C, 48 h. Yields are given for isolated products after column chromatography.

We investigated the effect of temperature on the catalytic performance of PPL for this reaction. The results shown in Fig. 2 indicated that the yield was greatly improved by raising the temperature, and reached the best yield of 89% at 55 °C after 48 h. However, the yield would decrease by further increasing the temperature. When the reaction temperature elevated, the collision chance between enzyme and substrate molecules increased, which might help to form enzyme–substrate complexes and then improve the reaction rate.²⁵ But the high temperature (above 55 °C) might lead to the denaturation of enzyme and thus slow the reaction rate. So, 55 °C was chosen as the optimum temperature in this study.

Fig. 2 Effect of temperature on the synthesis of substituted 2H-chromenes. Reaction condition: salicylaldehyde (1 mmol), acetoacetone (1 mmol), methanol (10 mmol), DMF (5 mL), PPL (20 mg), water content (30% v/v), temperature (15–65 °C), 48 h. Yields are given for isolated products after column chromatography.

To explore the scope and feasibility of this three-component reaction, a series of alcohols were examined under the optimal conditions.

As shown in Table 3, the reaction rate was the fastest when methanol was used as the substrate and it became slower when the alcohol with longer carbon chain was used as substrate. When the length of carbon chain was longer than pentanol, the product of 2H-chromene could not be detected. A longer alcohol may be more difficult to enter the active site of the enzyme due to its steric strain.²⁶ Furthermore, we investigated the reaction using the aromatic alcohol and branch alcohol (phenylmethanol, 2-phenylethanol and isopropanol) as substrate and found the reactions could also proceed efficiently to obtain the corresponding products (4e, 4f, 4g) in good yields (58–79%).

Table 3 Lipase catalyzed synthesis of substituted 2H-chromenes with different alcohols^a

Entry	Alcohol	Product	Yield^b (%)
a Reaction condition: salicylaldehyde (1 mmol), acetoacetone (1 mmol), alcohol (10 mmol), DMF (5 mL), PPL (20 mg), water content (30% v/v), 55 °C, 48 h. Yields are given for isolated products after column chromatography.b Yields are given for isolated products after column chromatography.c ND: not detected.
1	Methanol	4a	89
2	Ethanol	4b	82
3	Butanol	4c	66
4	Pentanol	4d	45
5	Hexanol	—	ND^c
6	Isopropanol	4e	71
7	tert-Butanol	—	ND^c
8	Phenylmethanol	4f	58
9	2-Phenylethanol	4g	79
10	Phenol	—	ND^c

---

In conclusion, we have demonstrated for the first time that lipase can catalyze the synthesis of substituted 2H-chromenes with good yields (45–89%). It provides a new case of lipase catalytic promiscuity and extends the utility of lipase in organic synthesis. Furthermore, this study supplied a simple and one pot protocol for the synthesis of alkoxy chromene in synthetic organic chemistry. However, the yields were not very satisfied and the reusability of catalyst should be considered in this method. It's well known that immobilization is a useful tool to improve enzyme features (activity, specificity, reusability, stability, etc.) in modern biotechnology.^27–31 In order to improve the performance of lipase, a study adopting the technique of immobilization is underway and will be reported in due course.

Acknowledgements

We gratefully acknowledge the National Natural Science Foundation of China (no. 21172093, 31070708, 21072075) and the Natural Science Foundation of Jilin Province of China (no. 201115039, 20140101141JC) for the financial support.

Notes and references

1. I. Nobeli, A. D. Favia and J. M. Thornton, Nat. Biotechnol., 2009, 27, 157.
2. E. Busto, V. Gotor-Fernández and V. Gotor, Chem. Soc. Rev., 2010, 39, 4504.
3. C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck and P. Berglund, J. Am. Chem. Soc., 2003, 125, 874.
4. M. Svedendahl, K. Hult and P. Berglund, J. Am. Chem. Soc., 2005, 127, 17988.
5. M. T. Reetz, R. Mondiere and J. D. Carballeira, Tetrahedron Lett., 2007, 48, 1679.
6. M. Svedendahl, K. Hult and P. Berglund, J. Am. Chem. Soc., 2005, 127, 17988.
7. W. B. Wu, N. Wang, J. M. Xu, Q. Wu and X. F. Lin, Chem. Commun., 2005, 2348.
8. Y. F. Lai, H. Zheng, S. J. Chai, P. F. Zhang and X. Z. Chen, Green Chem., 2010, 12, 1917.
9. N. Iwata, N. Wang, X. Yao and S. Kitanaka, J. Nat. Prod., 2004, 67, 1106.
10. R. Korec, K. H. Sensch and T. Zoukas, Arzneim. Forsch., 2000, 50, 122.
11. F. Cassidy, M. J. Evan, M. S. Hadel, A. H. Halodij, P. E. Leach and G. Sternp, J. Med. Chem., 1992, 35, 1623.
12. J. H. Kwak, H. E. Kang, J. K. Jung, H. Kim, J. Cho and H. Lee, Arch. Pharmacal Res., 2006, 29, 728.
13. K. C. Nicolaou, J. A. Pfefferkorn and G.-Q. Cao, Angew. Chem., Int. Ed., 2000, 39, 734.
14. Q. Wang and M. G. Finn, Org. Lett., 2000, 2, 4063.
15. M. Iyer and G. R. Trivedi, Synth. Commun., 1990, 20, 1347.
16. T. Hanamoto, K. Shindo, M. Matsuoka, Y. Kiguchi and M. Kondo, J. Chem. Soc., 2000, 1, 3082.
17. P. T. Kaye and X. W. Nocanda, J. Chem. Soc., 2000, 1, 1331.
18. K. A. Parker and T. L. Mindt, Org. Lett., 2001, 3, 3875.
19. D. Ramaiah, P. M. Scaria, D. R. Cyr, P. K. Das and M. V. George, J. Org. Chem., 1988, 53, 2016.
20. L. C. Rao, H. M. Meshram, N. S. Kumar, N. N. Rao and N. J. Babu, Tetrahedron Lett., 2014, 55, 1127.
21. I. Lavandera, S. Fernández, J. Magdalena, M. Ferrero, R. J. Kazlauskas and V. Gotor, ChemBioChem, 2005, 6, 1381.
22. E. N. Xun, X. L. Lv, W. Kang, J. X. Wang, H. Zhang, L. Wang and Z. Wang, Appl. Biochem. Biotechnol., 2012, 168, 697.
23. Z. Wang, R. Wang, J. Tian, B. Zhao, X. F. Wei, Y. L. Su, C. Y. Li, S. G. Cao, T. F. Ji and L. Wang, J. Asian Nat. Prod. Res., 2010, 12, 56–63.
24. Y. S. Lin, P. Y. Wang, A. C. Wu and S. W. Tsai, J. Mol. Catal. B: Enzym., 2011, 68, 245.
25. R. Tian, C. H. Yang, X. F. Wei, E. N. Xun, R. Wang, S. G. Cao, Z. Wang and L. Wang, Biotechnol. Bioprocess Eng., 2011, 16, 337.
26. E. Brenna, F. G. Gatti, A. Manfredi, D. Monti and F. Parmeggiani, Adv. Synth. Catal., 2012, 354, 2859.
27. M. Fernández-Fernández, M. Á. Sanromán and D. Moldes, Biotechnol. Adv., 2013, 31, 1808.
28. O. Barbosa, R. Torres, C. Ortiz, A. Berenguer-Murcia, R. C. Rodrigues and R. Fernandez-Lafuente, Biomacromolecules, 2013, 14, 2433.
29. R. C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres and R. Fernández-Lafuente, Chem. Soc. Rev., 2013, 42, 6290.
30. K. Hernandez and R. Fernandez-Lafuente, Enzyme Microb. Technol., 2011, 48, 107.
31. C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente and R. C. Rodrigues, Adv. Synth. Catal., 2011, 353, 2885.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra03367a

‡ These authors contributed equally to this work.

§ A typical enzymatic procedure of the reaction: PPL was added to a 50 mL round-bottom flask containing salicylaldehyde (1 mmol), acetoacetone (1 mmol), alcohol (10 mmol) and DMF (5 mL). The suspension was maintained at 55 °C and shaken at 200 rpm for 48 h (the reaction was monitored by TLC). The reaction was stopped by extracting the reaction system with ethyl acetate (3 × 15 mL) and washed with brine solution. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product thus was purified by column chromatography on silica gel using ethyl acetate–petroleum ether. All the isolated products were well characterized by their ¹H-NMR spectral analysis.

---