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Abstract

A crude extract from earthworms was used as an environmentally friendly and easily accessible biocatalyst for asymmetric direct aldol reactions. The β-hydroxy carbonyl compounds could be prepared in yields of up to 94%, with ee values of up to 98% and dr of up to >99:1. A wide range of substrates could participate in the reaction. This earthworm catalyst was also efficient in the large-scale reaction, providing product in excellent yield and good selectivity. The protocol as an example gives inspiration for the development of sustainable catalysts from nature, which also provides a potential possibility for application of earthworm catalyst in industrial production.

Keywords

earthworm extract asymmetric aldol reaction biocatalysis large-scale synthesis

Introduction

In view of the importance of safety, health, and environmental issues, the use of nonpolluting, environmentally friendly, and easy-to-handle catalysts for reactions has long been a pursuit of chemists.¹ Nature is an important source of catalysts, but the use of natural catalysts in organic synthesis is very limited. Therefore, to find easy methods for use of environmentally benign natural catalysts is highly desirable. Some crude extracts of organisms have been employed as biocatalysts because of their simple and convenient preparation, environmental friendliness, and cost-effectiveness.^2–5 Earthworms, just like humans, rely on bacteria in their intestines to oxidize and convert plants and minerals into nutrients.⁶ Among the enzymes that are secreted by the alimentary tract of earthworms, a large number of serine proteases are most studied.⁷ These enzymes are stable and strongly resistant to organic solvents and detergents, and they retain full activity for years at room temperature.⁸ To use earthworm enzymes expediently, we developed a practical way to get crude extract of earthworms as an all-natural catalyst that avoids expensive and time-consuming enzyme purification. Our group recently reported that crude extract of earthworms, as a versatile, environmentally friendly, and sustainable biocatalyst, could catalyze various organic reactions including Mannich, aldol, Biginelli, Henry, aza-Diels-Alder reactions, and domino reactions for the synthesis of coumarin derivatives.^9,10

The asymmetric direct aldol reaction as one of the most powerful carbon-carbon bond formation methods has become one of the central study issues in organic synthesis.¹¹ In recent years, a lot of successful metal catalysts and organocatalysts for direct asymmetric aldol reactions have been reported with high efficiency and enantioselectivity,¹² including some methods under solvent-free mechanochemical conditions,^13–15 yet the development of eco-friendly and sustainable catalysts for the asymmetric aldol reaction is still a significant challenge. There are some reports about enzyme-catalyzed direct asymmetric aldol reactions using aldolases or some promiscuous enzymes.^16–21 However, it is still difficult to find a cost-efficient and simple procedure for biocatalytic direct asymmetric aldol reaction for scale-up.²² Thus, we think it is still necessary to further explore the application of earthworm extract for the asymmetric direct aldol reaction and especially for the large-scale reaction, although we have concisely reported the use of earthworm extract in asymmetric direct aldol reaction in which eight examples were investigated.⁹ Herein, we report detailed studies of earthworm extract-catalyzed asymmetric direct aldol reaction with largely expanded substrates, in which 18 new examples were included and a scale-up reaction was tested. This study further demonstrates the excellent utility of earthworm extract for asymmetric direct aldol reaction.

Materials and Methods

Materials

Live earthworms (Eisenia foetida) were purchased from Tianjin Cheng Gong earthworm culture farm (Tianjin, China). The crude earthworm extract was prepared according to our previously reported method.⁹ All reagents were purchased from commercial suppliers and used without further purification.

General Procedure for the Synthesis of Compounds 3

A round-bottom ﬂask was charged with the crude earthworm extract (75 mg), aldehyde 1 (0.50 mmol), ketone 2 (7.50 mmol), and MeCN (0.85 mL), to which deionized water (0.15 mL) was introduced. The resultant mixture was stirred at 30 °C and monitored by thin-layer chromatography. Upon completion of the reaction, the earthworm extract was filtrated off, and ethyl acetate (20 mL) was employed to wash the filter cake. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (petroleum ether/ethyl acetate = 5/1~3/1) to give the product.

Results and Discussion

First, a cheap and readily obtainable crude earthworm extract was prepared from E. foetida according to our previously reported method.⁹ The aldol reaction of 4-cyanobenzaldehyde (1a) and cyclohexanone (2a) was selected as a model reaction. The crude earthworm extract was used as a biocatalyst to catalyze the model aldol reaction in MeCN, and it was found that this extract displayed a spectacular catalytic activity toward the model aldol reaction, giving the product in a good yield of 78% with a good enantioselectivity of 82% ee for the anti-isomer (anti:syn = 78:22; Table 1 , entry 1). As a comparison, a commercially available lumbrokinase was used as a catalyst for the model aldol reaction, which gave the product in a moderate yield of 56% with 73% ee for the anti-isomer (anti:syn = 73:27; Table 1 , entry 2). Next, some control experiments were conducted to confirm the catalytic effect of the earthworm extract on the model aldol reaction ( Table 1 , entries 3–7). A blank experiment in the absence of catalyst gave the product in only a trace amount ( Table 1 , entry 3), demonstrating that the earthworm extract indeed catalyzed the model aldol reaction in an asymmetric manner. As a contrast, the reactions catalyzed by the nonenzyme proteins bovine serum albumin and egg white albumin were also conducted, which gave the product in yields of 35% and 60%, respectively; however, nearly no enantioselectivity was observed in these two cases ( Table 1 , entries 4 and 5). The results indicated that although nonenzyme proteins had the ability to catalyze the model aldol reaction, they did not have enantioselectivity. We then conducted another control experiment in which the earthworm extract was pretreated with urea (a common protein denaturing agent) at 100 °C for 24 h, and this urea-pretreated earthworm extract was used to catalyze the model reaction. It gave the product in a low yield of 37% with a low selectivity of 51:49 dr and 24% ee ( Table 1 , entry 6), demonstrating that the urea-pretreated earthworm extract lost most activity and selectivity on the model reaction. To exclude the effect of urea on the reaction, urea was used in the model reaction, and only a trace amount of product was observed ( Table 1 , entry 7). The above control experiments confirmed that the model aldol reaction was indeed catalyzed by the earthworm extract, and the enzyme(s) in the extract may be responsible for this catalytic activity.

Table 1.

Control Experiments and Solvent Screening.^a

Entry	Catalyst	Solvent	Time (h)	Yield (%)^b	dr (anti:syn)^c	ee (%; anti)^c

1	Earthworm extract	MeCN	120	78	78:22	82
2	Lumbrokinase	MeCN	120	56	73:27	73
3	None	MeCN	120	Trace	—	—
4	Bovine serum albumin	MeCN	120	35	35:65	3
5	Egg white albumin	MeCN	120	60	37:63	3
6	Earthworm extract pretreated with urea at 100 °C^d	MeCN	120	37	51:49	24
7	Urea	MeCN	120	Trace	—	—
8	Earthworm extract	CHCl₃	136	72	71:29	80
9	Earthworm extract	Methyl phenyl ether	136	17	91:9	80
10	Earthworm extract	Toluene	136	28	72:28	78
11	Earthworm extract	CH₂Cl₂	136	54	69:31	77
12	Earthworm extract	n-Butyl acetate	120	38	62:38	77
13	Earthworm extract	1,4-Dioxane	120	75	70:30	76
14	Earthworm extract	Solvent-free	120	74	57:43	75
15	Earthworm extract	Methyl tert-butyl ether	120	54	65:35	75
16	Earthworm extract	THF	120	72	59:41	73
17	Earthworm extract	Cyclohexane	136	51	60:40	73
18	Earthworm extract	EtOAc	136	62	58:42	69
19	Earthworm extract	i-PrOH	136	72	67:33	64
20	Earthworm extract	DMSO	96	77	62:38	58
21	Earthworm extract	EtOH	120	55	60:40	50
22	Earthworm extract	H₂O	120	26	62:38	48
23	Earthworm extract	MeOH	136	50	57:43	44
24	Earthworm extract	Phosphate buffer (pH 7)	136	73	52:48	26

Unless otherwise noted, all reactions were performed with 1a (0.5 mmol), 2a (5 equiv), deionized water (0.05 mL), solvent (0.95 mL), and catalyst (50 mg) at 30 °C.

Isolated yield.

Determined by chiral high-performance liquid chromatography analysis.

The earthworm extract (50 mg) in urea solution (0.28 M; urea [50 mg] in deionized water [3 mL]) was stirred at 100 °C for 24 h, and then water was removed under reduced pressure.

In addition, we explored effect of different solvents on the earthworm extract catalyzed model aldol reaction ( Table 1 , entries 1 and 8–24). The reaction medium had an obvious influence on this reaction. It seems likely that the reaction in low- to moderate-polarity solvents displayed better stereoselectivity than in high-polarity solvents. For instance, the reaction in MeCN, CHCl₃, methyl phenyl ether, toluene, CH₂Cl₂, n-tutyl acetate, 1,4-dioxane, methyl tert-butyl ether, THF, cyclohexane, or EtOAc ( Table 1 , entries 1, 8–13, and 15–18) gave product with 69% to 82% ee, while the reaction in i-PrOH, DMSO, EtOH, H₂O, or MeOH ( Table 1 , entries 19–23) gave product with 44% to 64% ee. Moreover, the reaction under solvent-free conditions gave product in a good yield of 74% with 75% ee but with a low dr (57:43 anti:syn; Table 1 , entry 14). The reaction in water gave product in a low yield of 26% with a low selectivity (48% ee, 62:38 dr; Table 1 , entry 22). When phosphate buffer (pH 7, 0.2 M) was used as a solvent, the reaction provided product in a good yield of 73% but with the lowest enantioselectivity of 26% ee and the lowest diastereoselectivity (52:48 dr, anti:syn; Table 1 , entry 24). Among the tested solvents, the best yield of 78% and the highest enantioselectivity of 82% ee for anti isomer (78:22 dr, anti:syn) were obtained in MeCN ( Table 1 , entry 1). Thus, we selected MeCN as the solvent for the aldol reaction catalyzed by the earthworm extract.

As water contents are essential for the stereoselectivity and activity of enzymes in organic solvents,^23,24 the influence of water contents on the earthworm extract catalyzed model aldol reaction was investigated ( Table 2 ). The model reaction in MeCN (analytical purity) without addition of water gave the product in a yield of 73% with a low ee of 64% ( Table 2 , entry 1). The best yield of 85% was obtained with 74% ee when the water content was 20% ( Table 2 , entry 5). When the water content exceeded 25%, the yield and ee declined sharply ( Table 2 , entries 7 and 8). Taking into consideration both yield and ee, we chose 15% as the optimum water content, which gave a good yield of 83% with 78% ee and 76:24 (anti:syn) dr value ( Table 2 , entry 4).

Table 2.

Screening of Water Content.^a

Entry	H₂O (%)	Yield (%)^b	dr (anti:syn)^c	ee (%; anti)^c

1	0	73	67:33	64
2	5	78	77:23	77
3	10	77	76:24	79
4	15	83	76:24	78
5	20	85	77:23	74
6	25	83	71:29	59
7	30	59	71:29	63
8	40	45	68:32	59

Reaction conditions: 1a (0.5 mmol), 2a (5 equiv), deionized water (0–0.40 mL), and earthworm extract (50 mg) in MeCN (1.00–0.60 mL) at 30 °C for 120 h.

Isolated yield.

Determined by chiral high-performance liquid chromatography analysis.

To further improve the stereoselectivity and yield of the model reaction, the molar ratio of the substrates was screened ( Table 3 , entries 1–6). When the molar ratio of 4-cyanobenzaldehyde (1a) to cyclohexanone (2a) was 1:1, only 48% yield with 77% ee was obtained ( Table 3 , entry 1). An excellent yield of 96% with a good ee of 86% was achieved when the molar ratio increased to 1:15 (1a:2a; Table 3 , entry 5). Further increasing the molar ratio to 1:20 (1a:2a) led to a decrease in both the yield and ee ( Table 3 , entry 6). Thus, 1:15 (1a:2a) was chosen as the optimum molar ratio for the reaction. Next, the influence of the earthworm extract loading on the model reaction was investigated ( Table 3 , entries 5, 7, and 8). The results showed that the yield of the reaction was decreased from 96% to 65% as the amount of earthworm extract decreased from 50 mg to 25 mg ( Table 3 , entries 5 and 7). Further increasing the amount of earthworm extract to 75 mg resulted in a better diastereoselectivity ( Table 3 , entry 8). Therefore, 75 mg of earthworm extract was selected as an optimal catalyst loading for the reaction (0.5 mmol scale in 1.0 mL MeCN/water).

Table 3.

Screening of Substrate Molar Ratio and Catalyst Loading.^a

Entry	Earthworm Extract (mg)	1a:2a	Yield (%)^b	dr (anti:syn)^c	ee (%; anti)^c

1	50	1:1	48	72:28	77
2	50	1:3	71	78:22	82
3	50	1:5	79	81:19	85
4	50	1:10	95	78:22	82
5	50	1:15	96	78:22	86
6	50	1:20	71	82:18	85
7	25	1:15	65	84:16	86
8	75	1:15	96	83:17	86

Reaction conditions: 1a (0.5 mmol), 2a (1–20 equiv), earthworm extract (25–75 mg), deionized water (0.15 mL), and MeCN (0.85 mL) at 30 °C for 120 h.

Isolated yield.

Determined by chiral high-performance liquid chromatography analysis.

Subsequently, the influence of temperature on the earthworm extract catalyzed model aldol reaction was investigated ( Table 4 , entries 1–7). It was shown that the yield increased from 56% to 96% as the temperature rose from 15 °C to 30 °C ( Table 4 , entries 1–4). Meanwhile, increasing the temperature from 15 °C to 45 °C led to a decrease in both the diastereoselectivity (from 89:11 to 60:40 [anti:syn]) and enantioselectivity (from 91% ee to 63% ee; Table 4 , entries 1–7). As a compromise between yield and stereoselectivity, 30 °C was chosen as an optimum temperature for the reaction.

Table 4.

Influence of Temperature on the Model Aldol Reaction.^a

Entry	Temperature (°C)	Yield (%)^b	dr (anti:syn)^c	ee (%; anti)^c

1	15	56	89:11	91
2	20	81	88:12	89
3	25	92	87:13	88
4	30	96	80:20	86
5	35	95	76:24	82
6	40	95	69:31	74
7	45	93	60:40	63

Reaction conditions: 1a (0.5 mmol), 2a (15 equiv), earthworm extract (75 mg), deionized water (0.15 mL), and MeCN (0.85 mL) for 120 h.

Isolated yield.

Determined by chiral high-performance liquid chromatography analysis.

To investigate the generality and scope of this earthworm extract catalyzed direct asymmetric aldol reaction, several other substrates were tested ( Table 5 ). The earthworm extract exhibited excellent catalytic activity and stereoselectivity toward aldol reaction, and satisfactory results were obtained with yields of up to 94%, dr of up to >99:1 (anti:syn), and ee of up to 98% ( Table 5 , entries 7, 17, 5, and 9). Various substituted aromatic and heteroaromatic aldehydes could be used as aldol acceptors, and cyclic, heterocyclic, and acyclic ketones could all be used as aldol donors. Generally, the aromatic aldehydes containing 1 or 2 electron-withdrawing group(s) exhibited higher reactivity than those containing an electron-donating group ( Table 5 , entries 1–10). Also, substituent position on benzaldehydes had some impact on the diastereoselectivity of the reaction. For instance, when reacting with cyclohexanone or tetrahydro-4H-thiopyran-4-one, the benzaldehydes with a substituent on the o-position gave higher dr values than those with a substituent on the m- or p-position ( Table 5 , entries 1–3, 4, 5, 9, 10, 15, and 16), probably because of the steric hindrance of substituents. Furthermore, the most sterically hindered substrates 2, 6-dichlorobenzaldehyde gave the excellent diastereoselectivity of 98:2 ( Table 5 , entry 7). The reaction of heteroaromatic aldehyde 2-thienylaldehyde with cyclohexanone afforded the desired product in an acceptable yield with moderate selectivity ( Table 5 , entry 11). When cycloheptanone was employed to react with 4-cyanobenzaldehyde, the product was obtained in a low yield of 24% with a low enantioselectivity of 22% ee but with a high diastereoselectivity of 98:2 dr (anti:syn; Table 5 , entry 13). Linear chain ketone acetone gave a low yield of 14% with 62% ee when reacting with 4-cyanobenzaldehyde ( Table 5 , entry 14). In addition, it was promising that the heterocyclic ketones containing sulfur or nitrogen were also available as substrates in this biocatalytic process giving corresponding products in satisfactory yields with moderate to good selectivities ( Table 5 , entries 15–18). It seems that the sulfur-containing heterocyclic ketones gave better selectivity than nitrogen-containing ketones. For all the tested substrates, the anti-isomers were obtained as the major products with a different degree of enantioselectivity but only low or no enantioselectivity for syn-isomers was observed.

Table 5.

Substrate Scope and Large-Scale Reaction.^a

Entry	R¹ R²	R³	Product	Time (h)	Yield (%)^b	dr (anti:syn)^c	ee (%; anti)^c

1	-(CH₂)₄-	3-NO₂C₆H₄	3a	92	91	92:8	89
2	-(CH₂)₄-	2-NO₂C₆H₄	3b	115	65	95:5	89
3	-(CH₂)₄-	4-NO₂C₆H₄	3c	92	90	89:11	87
4	-(CH₂)₄-	3-ClC₆H₄	3d	170	61	85:15	91
5	-(CH₂)₄-	2-ClC₆H₄	3e	188	59	>99:1	89
6	-(CH₂)₄-	4-BrC₆H₄	3f	120	30	78:22	92
7	-(CH₂)₄-	2,6-Cl₂C₆H₃	3g	187	94	98:2	71
8	-(CH₂)₄-	4-MeC₆H₄	3h	239	38	88:12	92
9	-(CH₂)₄-	4-MeOC₆H₄	3i	220	17	85:15	98
10	-(CH₂)₄-	2-MeOC₆H₄	3j	192	44	99:1	76
11	-(CH₂)₄-	2-thienyl	3k	216	41	71:29	77
12	-(CH₂)₄-	C₆H₅	3l	142	45	68:32	79
13	-(CH₂)₅-	4-CNC₆H₄	3m	219	24	98:2	22
14	Me, H	4-CNC₆H₄	3n	144	14	—	62
15	-(CH₂)₂SCH₂-	3-NO₂C₆H₄	3o	187	69	84:15	86
16	-(CH₂)₂SCH₂-	2-NO₂C₆H₄	3p	137	31	96:4	90
17	-(CH₂)₂NBocCH₂-	4-NO₂C₆H₄	3q	130	94	68:32	71
18	-(CH₂)₂NBocCH₂-	3-NO₂C₆H₄	3r	164	68	69:31	68
19 (scale-up)^d	-(CH₂)₄-	3-NO₂C₆H₄	3a	92	90	92:8	92

Reaction conditions: aldehyde 1 (0.5 mmol), ketone 2 (15 equiv), earthworm extract (75 mg), deionized water (0.15 mL), and MeCN (0.85 mL) at 30 °C.

Isolated yield.

Determined by chiral high-performance liquid chromatography analysis.

Reaction conditions: 3-nitrobenzaldehyde (20 mmol), cyclohexanone (15 equiv), earthworm extract (3.0 g), deionized water (2.0 mL), and MeCN (38.0 mL) at 25 °C.

Finally, it is notable that this earthworm catalyst is also efficient in large-scale aldol reaction. For example, the reaction between 3-nitrobenzaldehyde and cyclohexanone could be easily performed on a large scale of 20 mmol, which gave an excellent yield of 90% with 92% ee and 92:8 dr (anti:syn; Table 5 , entry 19).

In summary, a crude extract from the earthworm species E. foetida as an eco-friendly and sustainable biocatalyst for asymmetric direct aldol reaction was described in detail. The protocol has a wide range of substrates with cyclic, heterocyclic, and acyclic ketones as aldol donors and various substituted aromatic and heteroaromatic aldehydes as aldol acceptors. The corresponding product β-hydroxy carbonyl compounds could be prepared in yields of up to 94% with ee values of up to 98% and dr of up to >99:1 (anti:syn). This procedure is also large-scaleable. Especially, the main advantages of this biocatalyst are its simple preparation, easy availability, and cheapness. The procedure does not require any additional cofactors or special equipment. Using the vast potential of abundant and sustainable natural sources as catalysts may become a significant alternative approach for large-scale synthetic chemical transformations.

Footnotes

Acknowledgements

We acknowledge members of the Guan lab for their contribution to the extraction of earthworm enzymes.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was financially supported by the National Natural Science Foundation of China (No. 21472152 and No. 21672174) and the Basic and Frontier Research Project of Chongqing (cstc2015jcyjBX0106).

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Earthworms as a Biocatalyst: In Asymmetric Aldol Reactions

Abstract

Keywords

Introduction

Materials and Methods

Materials

General Procedure for the Synthesis of Compounds 3

Results and Discussion

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Supplementary Material