Here, we focused on a simple enzymatic epoxidation of alkenes using lipase and phenylacetic acid. The immobilised
Epoxides are an important class of compounds in many industrial processes and often made by the epoxidation of alkenes [
Many of these epoxides are synthesised using epoxidising agents such as metal catalysts or strong mineral acids; however, the yields of epoxide tend to be low and accompanied by side product formation as well as corrosion problems [
Prilezhaev epoxidation of alkenes with a peroxy acid is the most common method used in research laboratories and industries nowadays [
Lately, we reported some of our findings on epoxidation of alkene using phenylacetic acid [
1-Nonene (98%), 1-heptene (97%), styrene (99%), cyclohexene (99%) and 1-methylcyclohexene (97%), cyclohexene oxide (standard), and chloroform-
In a typical experiment, epoxidation was carried out in a 50 mL capacity round bottom flask. Initially, alkene (1-nonene) (0.6 mmol) was evenly mixed in chloroform (10 mL) to form a constant solution. Neatly, phenylacetic acid (8.8 mmol) and Novozym 435 (1.7% wt/wt, 19.9 mg) were added to the mixture. The reaction was initiated by adding H2O2 (30% wt/wt, 4.4 mmol) in one step using an autotitrator (Metrohm, New Zealand). Ultimately, the reaction was carried out in a water bath shaker (Hotech Instrument, Taiwan) at temperature (35°C), time (12 h), and speed (250 rpm). For all experiments, the epoxide was synthesised at least in duplicate.
Samples were withdrawn at appropriate time intervals for a quantitative analysis. A 0.1 mL sample was diluted 100 times by mixing 9.9 mL of ethyl acetate (HPLC grade) and filtered using 0.45
Analysis of epoxide yield was performed by using GC (Agilent Technology model 7890, GC system) coupled with a mass spectrometer, model 5975 C inert-MSD, with triple axis detector operated in the electron emission (EI) mode. The compound was separated on a (30.0 m × 0.25 mm) HP-5 ms column coated with 0.25
The isolation of phenylacetic acid was performed using a 125 mL separating funnel. Initially, the reaction mixture was washed with distilled water to remove the remaining H2O2. After the phase separation, the organic layer was extracted out and dried over 5% (w/w) of Na2SO3 and Na2SO4, respectively. The crude mixture was purified by silica gel column chromatography with a mobile phase containing hexane and ethyl acetate (3 : 2). The purified combined fractions were evaporated under vacuum by rotary evaporator (Buchi, Switzerland). The phenylacetic acid was then characterised with GC-MS, 1H, and 13C NMR and compared with the reported data.
Identification of final epoxide product was performed using spectroscopic analysis and matched with the reported data. The epoxide was periodically characterised by FT-IR (PerkinElmer-model 1650, USA) and GC-MS (Agilent Technology, USA) (see Supplementary Material available online at
In the enzyme-catalysed epoxidation reaction, H2O2 acts as oxidising agent and converts the carboxylic acid into peroxy acid. In this study, the reaction was initiated using lipase Novozym 435 to catalyse the production of phenylacetic peroxy acid (the oxygen carrier) via the perhydrolysis of phenylacetic acid. Scheme
Mechanism of lipase-catalysed perhydrolysis reaction [
The lipase catalytic site contains a catalytic triad composed of a histidine (His), serine (Ser), and an aspartate (Asp)/glutamic acid residue [
In the initial step, the lone pair electrons on the N atom of the histidine have the potential to accept the hydroxyl proton from the serine. This is due to a carboxyl residue of the aspartate/glutamic acid that forms a hydrogen bond with the imidazole ring of the histidine, making the N atom mentioned above be very electronegative. The serine residue makes a nucleophilic attack on a carbonyl carbon of the acyl group that belongs to the substrate (R1COOH) to form an intermediate known as tetrahedral intermediate
In the second step, the bond connecting carbon and oxygen atoms in the carboxylic acid is broken. The covalent electron on the oxygen thus moves to attack the hydrogen of the histidine and breaks the N–H linkage. The histidine group enhances the proton transfer from the hydroxyl group resulting in the release of a water molecule (HOH). When a H2O2 molecule comes, it attacks the carbonyl carbon of the serine complex resulting in the movement of the
The
Mechanism of Prilezhaev epoxidation of alkene.
It is interesting to underline that all chemo enzymatic epoxidation reactions performed in the presence of phenylacetic acid gave good results, probably due to lipase stabilisation in mild phenylacetic acid. The summary of the epoxidation of different alkenes with Novozym 435, H2O2, and phenylacetic acid is shown in Table
Novozym 435-mediated epoxidation of alkenes.
Entry | Alkene | Epoxide | Yield/%a |
---|---|---|---|
1 |
|
|
97 |
2 |
|
|
99 |
3 |
|
|
75 |
4 |
|
|
85, 90b |
5 |
|
|
95 |
The yield of styrene oxide is lower than aliphatic epoxides, which was probably due to acid-catalysed hydrolysis and isomerisation of styrene oxide to diol and benzaldehyde, respectively. This phenomenon is similar to other lipase-catalysed epoxidation systems [
It was worth mentioning that some authors formerly have reported that the lipase is not able at all to perform epoxidation reaction in one single addition of H2O2 [
Subsequent to epoxidation of various alkenes, recyclability of phenylacetic acid was studied in order to reduce the acid waste produced during the epoxidation reaction. In this study, phenylacetic acid was successfully isolated and purified from the reaction mixture and subjected to characterization with GC-MS and NMR. The GC chromatogram of epoxidation of 1-nonene using recycled phenylacetic acid is shown in Figure
The GC chromatogram of epoxidation of 1-nonene using recycled phenylacetic acid.
Only a small amount of recycled phenylacetic acid (0.3 g) was obtained after a series of isolation and purification steps. The result demonstrated that the highest yield of 90% 1-nonene oxide was achieved when phenylacetic acid was reused for the epoxidation reaction. This proved that phenylacetic acid is a very efficient perhydrolysis substrate as it can be reused for several epoxidation reactions.
In brief, the epoxidation reaction by lipase-mediated perhydrolysis of phenylacetic acid is a practical method, especially suitable for acid sensitive alkenes. The recyclability result exemplifies the importance of perhydrolysis substrate selection in chemo-enzymatic epoxidation of alkenes as this would control the cost of production and also promote the eco-friendly chemo-enzymatic epoxidation needs on both laboratory and industrial scales.
Remarkably, the new developed system was efficient and convenient, since the epoxidation of the tested alkenes was rapid in producing the respective epoxides with yields above 90%. This implied the promise of practical application of this methodology. In particular, the epoxidation of styrene was found to be over 90% in 12 h as compared to other systems using dimethyl carbonate and ethyl acetate, which required at least 6 h dosing of H2O2 and additional 16 h of stirring time [
In addition, our method required 50% less H2O2 concentration and 20 h less time but produced almost 7% higher epoxide in comparison with the epoxidation of styrene in lactone [
1-Nonene oxide: the analytical procedure was performed as reported by Abdulmalek et al. [
Overall, the use of phenylacetic acid gave epoxide ranging from reasonable to good yields when compared to those reported when using strong carboxylic acids such as acetic acid and formic acid. Moreover, the recyclability result is very overwhelming, rendering this epoxidation method one of the best methods ever described. The method also revealed that only a mild operating temperature (35°C) and a milligram-scale of Novozym 435 (19.9 mg) were required to give higher catalytic activity towards the acid peroxidation process and limit the enzyme deactivation by H2O2. In this way, we found that the scope of the reaction can be extended particularly for industrial application.
The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.
The authors would like to thank the financial support from the Ministry of Higher Education (MOHE) through FRGS and a Graduate Research Fellowship (GRF) from the Universiti Putra Malaysia (UPM).