A Study of Green Enzyme Catalysed Sonochemical Oxidation-Reduction of Ketone/s Using UV-Vis Spectrophotometry
Shrikant R. Kulkarni
Vishwakarma Institute of Technology, Bibwewadi, Pune
*Corresponding Author E-mail: shrikant.kulkarni@vit.edu
ABSTRACT:
The oxidation-reduction of various ketones to alcohols and vice versa has so far been brought about by different chemical reagents (catalysts) which many a times specially synthesized for accomplishing selectivity in the reaction as well as to enhance the rate of reaction and further to bring about substantial improvement in the yield. However, there are limitations to these kind of initiatives e.g. reduction of Acetophenone in presence of Sodium Borohydride yields hardly about 47.3% of (S)-1-phenylethanol. However, these days efforts are underway to evolve at reagents which are more and more eco-friendly or green, and use of for instance enzymes derived from plants is gaining momentum and has a comparative advantage too over earlier initiatives which make use of catalysts which are not eco-friendly. Use of enzyme like D. Carota extracted from Carrot in this case to bring about oxidation-reduction of ketone/s is instrumental in enhancing the rate of reaction under the influence of sonic waves as well as hike in yield of reaction against conventional methods. The initiative in this case is a green one. Apart from Ketones, oxidation-reduction of β-ketoesters, cyclic ketones, azido ketones, open chain ketones like 2-butanone, 2-pentanone, etc. can also be brought about on similar lines with similar kind of effects using D. Carota enzyme.
The oxidation-reduction is brought about by sonication and further kinetics of the reaction is constantly monitored using UV-Vis spectrophotometry at 200, 270 nm in particular variation in absorbance with depletion in concentration of Ketone/s, attributed to π → π* transition due to –C=O group and Benzene band due to presence of Aromatic ring resply.
KEYWORDS: Oxidation-reduction, Enzyme, Sonication, Ketones, Spectrophotometry
In the search for practical ways, we turned our attention to environmentally friendly biocatalytic methods.1 These methods usually make use of isolated enzymes or microorganisms to prepare optically active compounds. In recent years plant cell cultures and whole plant cells have also been used for this purpose for performing synthetic transformations with high enantioselectivity.2 Recently some results on the reduction of substituted acetophenones,3 including seleno acetophenones,[3c] using whole fungal cells have been obtained. In this communication, we focused initially our attention on the potential of Daucus carota root (carrot) as biocatalyst.4a 6. As far as we know this is the first attempt to perform a biotransformation using plant cells. In addition, in order to explore the potential of D. carota root we decided to evaluate also the bioreduction of acetophenone to afford the corresponding chiral alcohols.
The reaction is based on the utilization of carrot root as enzymatic source (alcohol dehydrogenase and their cofactors) to reduce the pro chiral organochalcogeno acetophenones3 to the chiral organochalcogeno-a-methylbenzyl alcohol4. Compound4 would present several potential uses.
Carrot cells accumulate 6-methoxymellein as the phytoalexin upon the invasion of pathogenic microorganisms7, and bio- synthesis of this compound is catalyzed by two inducible en-zymes, 6-hydroxymellein synthase 8,9 and 6-hydroxymellein- O-methyltransferase10.
2. MATERIALS AND METHODS:
Instrumentation:
A Shimadzu 1650 UV (Shimadzu, Japan) spectrophotometer with 1 cm matched quartz cells was used for the estimation. A Sonicator UP200S (Hielsche, Germany) was used for the sonication, homogenization and furthering the redox reaction of Acetophenone.
Chemicals and reagents:
All the reagents were of analytical grade. Millipore water was used throughout the experiment.
Standard Preparation:
Accurately weighed quantity (5 mg ) of Acetophenone was dissolved separately in 10 ml of water in a 50 ml volumetric flask. The solution was shaken vigorously and the volume was adjusted up to the mark with Millipore water to obtain a stock solution on sonication for 5 minutes;. The solution was then filtered using vacuum filtration. For the selection of analytical wavelength, the stock solutions of Acetophenone was separately diluted in Millipore water, to get concentrations of 20 μg/ml each, and scanned in the wavelength range of 200-400 nm. From the spectrum of standard solution of Acetophenone, wavelengths 200 nm and 270 nm as two characteristic wavelengths were selected. For calibration curves, stock Solution of Acetophenone was appropriately diluted to obtain concentration range of 1-10 μg/ml and 5- 40 μg/ml respectively. The absorbance was measured at these two wavelengths and calibration curve was plotted.
Sample preparation:
For the estimation of acetophenone from the extract, known quantity of carrot was cut and finely divided, weighed. Quantity equivalent to 5 mg of the same was transferred to 50 ml volumetric flask. The solution was shaken vigorously and the volume was adjusted up to the mark with Millipore water. The solution was then filtered using vacuum filtration with 0.2µ filter paper. Absorbance of this solution was measured at appropriate wavelengths.
d) Precision and accuracy:
All measurements were made very meticulously at all stages right. The precision and accuracy studies were performed on replicate assays on the same day (within-run-precision) over three consecutive days (between-run-precision) at different level of concentrations of Acetophenone. The precision was calculated using relative standard deviation (RSD).
Spectrophotometer Sonicator conditions:
conditions:
System : UV – 1650PC System: Ultrasonic
(Shimadzu Make) Processor (UP200S)
Software : UV Probe Frequency : 0.5 cycles
Source : 50 W Deuterium Lamp Amplitude : 60%
Wavelength Range:190 – 1100 nm
Detector : Silicon Photodiode
3. RESULTS AND DISCUSSION:
To begin with standard solution of Acetophenone was subjected to spectroscopic analysis and a UV spectrum was obtained as shown in Fig.1 on calibrating the instrument using standard procedure.
Fig.1 UV spectrum of Acetophenone
The reduction of Acetophenone was brought about by sonication and progress of the reaction was followed by measuring the absorbance of the reaction mixture during sonication by immersing sonotrode in to the reaction mixture and putting it into operation , taking out the requisite amount of reaction mixture at regular time interval to see how far depletion in concentration of Acetophenone has taken place as the absorbance value was found to decline with time and thereby justifying the Beer’s law which states that absorbance is directly proportional to concentration. It shows the reduction of Acetophenone has progessed over the time. Further, the downward trend in absorbance and consequently concentration was witnessed at two different wavelengths which are characteristic ones for Acetophenone. The corresponding plots plotted in Fig. 2 and 3 reflects on the same.
The progress of the redox reaction took place in a smooth manner and the reduction took place substantially and the yield of the alcohol on reduction of ketone was of the order of 95% which was reflected from the Concentration v/s Time plot in Fig.2 and 3.
Fig.2 Depletion in concentration of Acetophenone with time at 200nm
Fig.3 Depletion in concentration of Acetophenone with time at 270nm
4. CONCLUSIONS:
The reduction of ketone namely, acetophenone has been brought about successfully using an enzyme, D.Carota obtained from Carrot under the influence of Sonication. The reaction in true sense is green or eco-friendly one. The percentage yield of alcohol obtained is of high order which is in vicinity of 95%. The UV-Visible Spectrophotometry can be successfully employed for following the progress of such reactions. By employing similar technique β-ketoesters, cyclic ketones, azido ketones, open chain ketones like 2-butanone, 2-pentanone, etc. can also be transformed into corresponding alcohols. Further work holds a lot much of promise.
5. ACKNOWLEDGEMENT:
Author would like to express his sincere thanks and gratitude to Vishwakarma Institute of Technology, Pune for its kind support and encouragement in pursuing the research initiative.
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Received on 15.10.2010 Modified on 29.10.2010
Accepted on 04.11.2010 © AJRC All right reserved
Asian J. Research Chem. 4(2): February 2011; Page 293-295