Optimization of Lipase Catalyzed Synthesis of 3-O-benzoylbetulinic Acid
Betulinic acid derivatives were successfully synthesized from reactions of betulinic acid with benzoyl chloride and acetic anhydride using enzymes as biocatalyst in an organic solvent system. Preliminary detection of the reaction products were conducted using thin layer chromatography (TLC) and s...
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Format: | Thesis |
Language: | English English |
Published: |
2004
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Online Access: | http://psasir.upm.edu.my/id/eprint/301/1/549610_FS_2004_10.pdf http://psasir.upm.edu.my/id/eprint/301/ |
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Summary: | Betulinic acid derivatives were successfully synthesized from reactions of betulinic
acid with benzoyl chloride and acetic anhydride using enzymes as biocatalyst in an
organic solvent system. Preliminary detection of the reaction products were conducted
using thin layer chromatography (TLC) and subsequent quantitative studies were
conducted using gas chromatography. Characterizations of the products were further
conducted using (NMR), (MS) and (FTIR).
Initial screening of biological activities, revealed that the ester of the reaction of
betulinic acid with benzoyl chloride showed a good cytotoxicity activity while no
activity was detected for ester of reaction of betulinic acid and acetic anhydride. Thus,
the reaction of betulinic acid and benzoyl chloride was chosen for further studies.
Enzyme screening revealed that Novozyme was the most efficient biocatalyst for the
reactions which produced a good yield of 3-O-benzoylbetulinic acid at 45.3%. The
effects of various reaction parameters such as time of reaction, temperature, solvents used, amount of enzyme, mole ratio of substrates and initial water activity (aw) was
studied to determine optimal condition of 3-O-benzoylbetulinic acid synthesis. The
optimal conditions 3-O-benzoylbetulinic acid synthesis was obtained at incubation
time of 12 h; temperature 50°C; mole ratio of betulinic acid/benzoyl chloride, 4.0;
amount of lipase, 150 mg; organic solvent, chloroform and initial water activity (aw),
0.12.
Response surface methodology (RSM) was then used to study the interactive effects of
the reaction parameters. The best model for the reaction of 3-O-benzoylbetulinic acid
was quadratic model. Generally, increasing both reaction temperature and amount of
enzyme increased the percentage yields of products. However, a negative effect was
detected at higher temperature due to denaturation of enzymes. Increasing reaction
time and amount of enzyme improved the reactions, as did increasing reaction
temperature and amount of enzyme. High amount of enzyme showed a slightly
negative effect. In general, increasing the temperature and amount of enzyme resulted
in negative effects of the response. Optimal conditions derived from the RSM differed
slightly from those of the conventional “one-variable-at-a-time” approach due to the
latter’s inability to consider interactions among the parameters. The percentage yield
for the optimize reaction conditions derived from RSM was 48.5%.
Betulinic acid and its acylated product were subjected to cytotoxicity, anti-viral, antimicrobe
and anti-fungal assays in order to study the biological activities of this
compound. The cytotoxicity evaluation of these compounds against cancer cell
showed that 3-O-benzoylbetulinic acid from reaction of betulinic acid and benzoyl chloride (BCL) was the most active compound compared to betulinic acid (BA) and 3-
acetylbetulinic acid (BAA). BCL showed IC50 <5 @g/ml which indicated significant
cytotoxicity activity against all cancer cells. However, no activities were detected
against dengue viral and some microbes and fungi that were tested. |
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