Betulinic acid amide was synthesized from the enzymatic reaction of betulinic acid and butylamine catalysed by Novozym 435. The effects of different reaction parameters, such as effect of reaction time, reaction temperature, amount of enzyme, and substrate molar ratio (betulinic acid : butylamine), were studied and conventionally optimised. Based on this study, the enzymatic synthesis of betulinic acid amide was found to be 64.6% at the optimum conditions of 24 h, 40°C, 100 mg enzyme, and 1 : 1 substrate molar ratio in 9 : 1 mixture of chloroform and hexane as solvent. The identification of final product was carried out using TLC, melting point, and FTIR and NMR showed the presence of betulinic acid amide.
Betulinic acid (BA), 3
Structure of betulinic acid.
Betulinic acid is a naturally occurring pentacyclic triterpenoid which has broad range of biological properties such as anticancer, anti-inflammatory, anti-HIV, antimicrobial, and antimalarial activities [
In the pharmaceutical industry, the medical uses of betulinic acid are strongly limited due to its poor solubility in water (0.02 mg/mL) which may contribute to the difficulty in absorption by the human body. It has been reported that the betulinic acid has three active sites, C-3 hydroxyl group, C-20 alkene, and C-28 carboxyl group which are suitable for derivatization. The modification or introduction of polar groups such as phthalates, amino acids, or sugar moieties at the C-3 and C-28 positions of betulinic acid in certain cases may increase its hydrosolubility and its anticancer activities [
The organic reaction method for the synthesis of 3-O-acyl betulinic acid ester has been described by Mukherjee et al. [
The aim of this study was to improve the percentage yield of enzymatic amidation reaction by optimising parameters such as reaction time, reaction temperature, enzyme amount, and substrate molar ratio. Figure
Enzymatic synthesis of betulinic acid amide (BAA).
Immobilised lipase (triacylglycerol hydrolase, EC 3.1.1.3; Novozym 435, 1000 PLU g-1) from
Betulinic acid was isolated from Malaysian
Betulinic acid (22.8 mg, 0.05 mmol) and butylamine (3.66 mg, 0.05 mmol) were placed in capped reaction vials. Chloroform and hexane (9 : 1) were added into the reaction mixture and stirred until all the reactants dissolved. Novozym 435 (100 mg) was then added into the reaction vials. Reaction mixtures were incubated at 40°C and 150 rpm for 24 h in a horizontal water bath shaker (Memmert WB14, Germany). Each reaction was repeated in triplicate and average values (mg) of products were reported. Control experiments were performed in the absence of enzyme.
Preliminary detection and identification of reaction products were carried out using Thin Layer Chromatography (TLC) with n-hexane/ethyl acetate (7 : 3, v/v) as the eluent. The plates were visualized under UV lamp and potassium permanganate (KMnO4) stain. The solvent was evaporated and chromatographed with gradient on silica gel 60 (n-hexane/ethyl acetate, 9 : 1–5 : 1 v/v). Then, the percentage of isolated yield was calculated.
IR spectrum was recorded using Perkin-Elmer FTIR model where samples were prepared by using KBR pellet. 1H spectra were recorded on BRUKER NMR spectrometer instrument operating at the frequency of 600 MHz. Samples weighed approximately 10–20 mg and were dissolved in deuterated solvent, CDCl3. The melting point of the reaction product was determined by using Electrothermal-MEL TEMP. For a pure compound, the difference between the temperature ranges was only about 1-2°C.
The reaction mixtures consist of betulinic acid (22.8 mg, 0.05 mmol), and butylamines (3.66 mg, 0.05 mmol) were placed in the capped reaction vials. Chloroform : hexane (10 mL) was added to the reaction mixture and stirred until all the reactants were dissolved. Novozym 435 (100 mg) was added and the incubation was carried out at 40°C and 150 rpm at different reaction times (4, 8, 12, 24, 48, and 72 h). The percentage yield (%) of betulinic acid amide (BAA) is defined as the number of moles of BAA produced over the initial number of moles of betulinic acid used.
The effect of different reaction temperatures (37, 40, 50, and 60°C) on BAA synthesis was studied.
The effect of different amounts of enzyme (50, 100, 150, 200, and 250 mg) on the BAA synthesis was investigated by varying the enzyme amount.
The reaction was repeated using betulinic acid with different molar ratios of butylamines (1 : 1, 1 : 2, 1 : 3, and 1 : 4) and placed in the capped reaction vials.
The BAA product obtained after purification appeared to be yellowish crystal with melting point in the range of 308–310°C. TLC analysis was carried out and the BAA product had
TLC spot.
1H proton NMR of BAA.
Figure
Effect of reaction time (reaction conditions: Novozym 435, 100 mg; temperature, 40°C; agitation speed, 150 rpm).
By increasing the reaction temperature, substrate solubility was found to be improving by reducing mass transfer limitations and making the substrate more available to the enzyme [
Effect of reaction temperature (reaction conditions: Novozym 435, 100 mg; time, 24 h; agitation speed, 150 rpm).
The effect of amount of enzyme was studied by varying the amount of enzyme added to the reaction mixture. Figure
Effect of amount of enzyme (reaction conditions: temperature, 40°C; time, 24 h; agitation speed, 150 rpm).
Figure
Effect of amount of substrate (reaction conditions: temperature, 40°C; time, 24 h; Novozym 435, 100 mg; agitation speed, 150 rpm).
The enzymatic synthesis of betulinic acid amide has been studied and focused on the effect of different operating variables (reaction time, reaction temperature, enzyme amount, and substrate molar ratio). It was shown that the catalytic activity increased by increasing the reaction time up to 24 h and also increasing the reaction temperature up to 40°C does not cause loss in the enzyme activity. The maximum percentage of betulinic acid amide produced was 64.6% with substrate molar ratio 1 : 1 and enzyme amount 100 mg for 24 h at 40°C. The identification structure of BAA was revealed by using TLC, MP, FTIR, and NMR spectroscopy.
It is an inhibitor that occupies the active site of an enzyme or the binding site of a receptor and prevents the normal substrate or ligand from binding.
It is the increase in the rate of a specified chemical reaction caused by an enzyme or other catalysts under specified assay conditions.
It is the enzyme changes in the structure of a neurotransmitter so that it is no longer recognized (inactive) by the receptor.
It is the point at which the concentrations of reactants and products do not change with time; forward and reverse reactions are equal so reactants and products are being created at the same rate.
It is the higher energy price (and slower reaction rate) due to the approach of larger atoms or groups in a chemical reaction, compared to a similar reaction involving smaller atoms or groups.
The authors declare that they have no competing interests.
The authors wish to thank Professor Dr. Faujan Hj Ahmad, all the staff in the Faculty of Applied Science of Universiti Teknologi MARA, and the staff in the Department of Chemistry of Universiti Putra Malaysia for the help in this study.