Indian Journal of Biochemistry & Biophysics Vol. 39, August 2002, pp. 259-263

Pseudomonas c.epacia lipase - mediated transesterification reactions of h ydrocinnamates

K Priya, T Venugopal and Anju Chadha*

Department of Chemistry, Indian Institute of Technology - Madras, Chennai 600 036, India

Received 10 May 2002; revised and accepted 11 June 2002

Use of lipase from Pseudomonas cepacia in transesterifcation reactions of ethyl hydrocinnamate with different alcohols has been examined. Among the alcohols tested, viz., n-butanol, iso-amyl alcohol, benzyl alcohol, n-octanol and 1-phenylethanol , only n-butanol yielded the transesterified product. Among the solvents tested, viz., n-heptane, n-hexane,

. cyc\ohexane, toluene, diisopropylether and n-butanol , the initial rate of transesterification proceeded in the order cyC\ohexane > n-heptane > n-hexane > diisopropylether > n-butanol > toluene. Using hexane as the solvent and a substrate to enzyme ratio of I :5, the substrate to alcohol ratio was varied to maximize the yield. n-Butyl hydrocinnamate was obtained in 92% yield in 48 hr by employing a I: I (wtlwt) ratio of ethyl hydrocinnamate to lipase and a 1:5 (vol/vol) ratio of ethyl hydrocinnamate to n-butanol in hexane.

Introduction Lipases are enzymes extensively used in organic synthesis, particularly as chiral catalysts l

. The natural substrates of these enzymes are triglycerides, i.e. esters of glycerol and fatty acids. These enzymes, which belong to the class of hydrolases are also known to accept a wide range of unnatural substrates e.g. esters of carboxylic acids and are also used for the synthesis of esters. Our work on lipases originated with lipase-mediated resolutions of compounds containing secondary alcohol groups - both by hydrolysis and by transesterification2

, . Further on, in the process of making esters of cinnamic and hydrocinnamic acids intermediates in other synthetic schemes in our laboratory, we explored the possibility of esterification by a biocatalytic method. Hydrocinnamates have also been used for . the protection of chymotrypsin from denaturation by ethanol and urea4

. Methyl hydrocinnamate is a precursor for the synthesis of 1,3,4,9-tetrahydropyrano [3,4-b] indole-I-acetic acid, which finds use as an analgesic, inflammation inhibitor and antipyretic5

. Ethyl hydrocinnamate has been used for the synthesis of HIV -1 protease inhibitors6

.

Hydrocinnamic acid esters also find application in the flavor and fragrance industr/.

A survey of the literature revealed that even though there are numerous examples of the use of lipases in

*Corresponding author. Telephone: 257 8258; Fax: 2578241 ; E-mail : anjuc@iitm.ac.in

resolution of racemic esters, alcohols and acids, they have not been used as extensively in forming simple (achiral) esters except using their natural substrates viz., fatty acids8

• There are not many reported procedures of the use of lipases for esterification of small organic acids9

.1O

• Lipozyme, a fungal lipase from Mucor meihei, is a commercially available immobilized enzyme, which is known to catalyze ester synthesis with a variety of carboxylic acids ll

.

Esterifications of carboxylic acids are equilibrium reactions generally performed at high temperatures in the presence of an acid or base as catalyst using a large excess of either of the reactants i.e. acid or alcohol. However, the yields are often poor and necessitate the removal of water formed during the course of the reaction which is generally accomplished by doing the reaction at high temperature and using a Dean-Stark apparatus. Though several new chemical catalysts have been employed to improve the yield of esterification reactions, they share the common disadvantage of high temperature requirements and th use of inorganic compounds as catalysts pose elaborate waste management problems I2

•13

. Unlike in esterification reactions, water is not formed in transesterification reactions. Chemical transesterifications are also equilibrium reactions done in the presence of a large excess of ester or an alcohol and a catalyst, which is an acid, base or inorganic compound 14. Ester to ester transformation is of particular importance in reactions where the carboxylic acids are labile, moistare

260 INDIAN J. BIOCHEM. BIOPHYS ., VOL. 39, AUGUST 2002

senSItive, difficult to isolate and pose problems of solubility. The read ily available methyl and ethyl es ters serve as convenient starting materials for the preparation of higher esters.

Biocatalyst-mediated transesterification methods are also known but are used most often for resolution of racemic acids and alcohols l

,2.3,15. In this study, we describe the transesterification of ethyl hydrocinnamate with various alcohols by Pseudolllonas cepacia lipase and optimization of the reaction parameters. Interestingly, ethyl c innamate was not a good substrate for this enzyme.

Materials and Methods Solvents and alcohols were glass distilled prior to

use and were not dried further. Pseudomonas cepacia lipase (lipase PS) provided by Amano Pharmaceutical Co., Nagoya, Japan was used as such without any purifica tion . IH-NMR spectra were recorded on a lEOL GSX-400 (400MHz) spectrometer in CDCI3

wi th tetramethylsilane as the internal standard. Cinnamic acid , recrystallized from ethanol was reduced using hydrogen and Pd/C (S %). Standard esters viz., ethyl and n-butyl hydrocinnamate, were prepared by the esterification of hydrocinnamic acid with ethanolln-butanoi in sod ium-dried toluene using p -toluene sulphonic acid as the catalyst and a Dean­Stark apparatus to remove the water formed in the reaction. The products, thus obtained were characterized by IH-NMR and compared with the I iterature data 16.

Typical Procedure for Transesterification of Ethyl hydrocinnamate

Ethyl hydrocinnamate (l00 mg, 0.S6 mmol), alcohol (2.8 mmol) and lipase PS (l00 mg) in 10 mJ

c: o

100

80

'f! 60 ~ c: 8 40 ~

20

20 40 60 80 . 100 120

Time (hours)

Fig. I-Effect of substrate to enzyme ratio on the transesteriticalion of ethylhydrocinnamate using n-butanol

of hexane were incubated at 30°C or 60°C in an orbital shaker set at 2S0 rpm. Aliquots were drawn at regular intervals of time and analyzed on a Nucon Gas Chromatograph (S700 series) using FID (flame ionization detector). A SE-30 column operating isothermally at 200°C was used to separate and identify the n-butyl hydrocinnamate ester formed enzymatically. The injector and detector were maintained at 21S°C. After completion of the reaction , the lipase was filtered off. The filtrate was concentrated and purified by column chromatography. The product obtained was characterized by comparing the IH-NMR spectrum obtained with that of the standard 16.

Results and Discussion To begin with, SOO mg (2.8 mmole) of ethyl

hydrocinnamate (1), referred to as the substrate, ]1-

butanol (1.3 ml , 14 mmole) and lipase from Pseudomonas cepacia (l00 mg) were taken in hexane (20 ml) and stirred using a magnet ic stirrer. The reaction was monitored every 10-12 hr. Formation of the transesterified product (2) was observed after four days. The reaction did not go to completion even after 10 days. The product (obtained in 3S% yield) was characterized by NMR. Instead of treating this as an inefficient reaction and discarding it, we experimented with the reaction parameters.

Effect of substrate to enzyme ratio The effect of substrate (ethyl hydrocinnamate) to

enzyme ratio (wtlwt) on the transesterification rate is shown in Fig. 1 while the initial rate obtained is listed

in Table 1. The initial rate of 1.1xlO'i Ilmol/hr. mg of enzyme for a substrate:enzyme ratio of I :0 .6, doubled to 2.3xlO,lllmol/hr.mg of enzyme when the amount of enzyme was increased to get a substrate: enzyme ratio

Table I-Effect of substrate to enzyme ratio on the initial rate of transesteritication of ethyl hydrocinnarnate with n-butanol

Substrate: Enzyme ratio

1:0.1 1:0 .2 1:0 .3 1:0,4 1:0.6 1:1 1:1.5 1:2

Initial rate* (x 1O' IJ,.lmol/hr.mg of enzyme)

0.14 0.49 0.6 0.7 1.1 2.3 2.6 2.8

*Initial rate is defined as formation of n-butyl hydrocinnamate in J,.lmollhr.mg of enzyme

PRIY A et al .: TRANSESTERIFICATION REACTIONS OF HYDROCINNAMATES 261

of 1: 1. Only a marginal improvement in initial rate (2.3 to 2.8xlO- 1 Ilmollhr.mg of enzyme) was observed, when the amount of enzyme was further doubled to get a ratio 1 :2. A steady increase in the transesterification rate is observed on increasing the amount of enzyme, as more of it is now available for catalysis. This is true until the ratio of 1: 1. However, further increase in the concentration of the enzyme does not elicit any improvement in the rate, as the concentration of the substrate is limiting. A ratio of 1: 1 (substrate to enzyme, wt/wt) was chosen for further studies as a 92% conversion was obtained in 48 hr. In all these experiments, the solvent used was hexane and the ratio of substrate to alcohol was mai ntained as 1:5 (mole/mole). An important feature in all these experiments is that no apparent decrease in amount of n-butyl hydrocinnamate was observed even after 5 days. It can be seen from various literature reports that the choice of the substrate to enzyme ratio depends on the system and varies from 1 :0.5 to 1 :i7

•

The relative purity of the lipase preparation used also decides the amount used for the reaction.

Effect of solvent Fig. 2 shows the effect of different solvents on the

transesterification reaction, while the initial rate observed for the various solvents is listed in Table 2. The choice of the organic solvent is an important criterion in lipase catalyzed transesterifications. It has been reported that lipase catalyzed reactions are generally favored in solvents with increasing hydrophobicit/ 8

•19

• Log P, defined as the logarithm of the partition coefficient of the given compound between octanol and water has been proposed as a quantitative measure of solvent polarity. Generally solvents with log P ~ 4 are hydrophobic and show

t: o

100

80

.~ 60

~ t: o U 40 ';f!.

~:::0.~ r,...-.Q=ij g

~ - a_a

. 0-----

//-;;=:~::;~pYlether -_heptane - "'- cyclohexane -o-toluene

o -0- n-buUnol

20 40 60 80 100 120

Time (hours)

Fig. 2-Effect of solvent on the transesterification of ethyl hydrocinnamate using n- butanol

maximum catalytic activity, while solvents having log P ::; 2 are hydrophilic and hence not expected to be very suitable for biocatalysis and solvents with log P values between 2 and 4 are much less hydrophilic but not predictable about their suitability for biocatalysis2o. At higher solvent log P values there is no direct correlation with increasing enzyme activity. Best yields have been reported for an esterification reaction run in n-hexane (log P=3.5) as compared to those run in n-heptane (log P=4.0) and isooctane (log P=4.5)21 .

In the case of transesterification of ethyl hydrocinnamate, a similar trend was seen. The initial rate for the transesterification of ethyl hydrocinnamate was 3, 2.6 and 2.3 x 10-1 Ilmole / hr.mg of enzyme for cyclohexane (log P=3.44), heptane (log P = 4.5) and hexane (log P = 4) respectively. In all these cases a conversion of >90% (mole to mole) was observed in 48 hr. Even though the initial rate of transesterification in diisopropyl ether (log P=1.57) was less than that observed in hexane, heptane and cyc\ohexane, a maximum of 92% conversion (mole to mole) was observed in 120 hr. In the case of aromatic and chlorinated solvents, biocompatibility with the enzyme is important and is system specific, hence their suitability in biocatalysis is also unpredictable. The transesterification reaction of ethyl hydrocinnamate with n-butanol was slow in toluene with an initial rate of 0.57 x 10-1 Ilmoie / hr.mg of enzyme. In solvents like acetone, acetonitrile, and THF erratic results were obtained. When n-butanol (log P=0.32) was used as both a solvent and nuc\eophile, the initial rate was 1.2 x 10-1

Ilmole/hr.mg of enzyme and a maximum· mole conversion of 95% was observed in 120 hr. In all these experiments, the ratio of substrate to alcohol was maintained as 1:5 (mole/mole) and the ratio of substrate to enzyme was 1: 1 (wt/wt). Modeling .

Table 2-Effect of reaction medium on the initial rate of transesterification of ethyl hydrocinnarnate with n-butanol

Solvent Log P

Heptane 4.5 Hexane 4.0 Cyclohexane 3.44 Toluene 2.73 Diisopropylether 1.52 n-Butanol 0.84

Initial rate* (x I O-Illmollhr. mg of enzyme)

2.6 2.3 3

0.57 2.1 1.2

*Initial rate is defined as formation of n-butyl hydrocinnamate in flmollhr.mg of enzyme

262 INDIAN J. BIOCHEM . BIOPHYS., VOL. 39, AUGUST 2002

Pseudomonas cepacia Lipase .. 1 2

(Yield: 92 %)

Scheme 1-Transesterification reaction between ethyl hydrocinnamate and II-butanol catalyzed by Pseudomonas cepacia lipase

studies have been done to offer a rationale for the solvent effects and to predict the equilibrium conversions22

. The various parameters that govern the enzyme activity in different solvents are not completely known. Hexane was chosen as the solvent for further studies due to its ready availability and ease of removal.

Effect of temperature A marked increase in the initial rate of

transesterification was observed from 2.3 to 3.5 x1O-1

)lmole/hr.mg of enzyme, when the temperature was increased from 33 to 60°C. However, after 48 hr irrespective of the temperature employed a maximum mole conversion of 97-98% was achieved for all the reactions. In all these experiments, the solvent used was hexane, the ratio of substrate to alcohol was 1:5 (mole/mole) and that of substrate to enzyme was 1: 1 (wUwt).

Other parameters Sonication of the enzyme suspension has been

shown to enhance reaction rates in some cases23.

Sonication of the reaction mixture for 6 hr (60 watts, Branson 121 OR-MTH) prior to addition of the substrate did not indicate any appreciable change in percent conversIOn of the product formed. Transesterification reaction performed using recovered lipase PS showed a conversion of only 50% after 48 hr as against 90% for the fresh sample of enzyme. However, when lipase PS, immobilized on celite (prepared by a known procedure23

) was used, a conversion of 94% was achieved in 96 hr and this immobilized enzyme could be reused thrice with only a modicum loss « 5%) of enzyme activity .

Effect of other alcohols The other alcohols used for the transesterification

of ethyl hydrocinnamate were iso-amyl alcohol , benzyl alcohol and n-octanol. 1-Phenylethanol was

also tried with a view to resolve the racemic alcohol. None of these reactions yielded any product even after 10 days (Scheme 1).

Conclusion This study has shown the optimization of important

parameters viz., enzyme to substrate ratio, choice of solvent and temperature, which are vital for effecting biocatalytic reactions in good yield. Ethyl hydrocinnamate (1) can be converted to n-butyl hydrocinnamate (2) In a lipase PS mediated transesterification reaction with n-butanol in 92% isolated yield at 60°C in 48 hr using hexane as solvent.

Acknowledgement Financial support from the Department of Science

and Technology, India (SP/Sl/G-06/99) is gratefully acknowledged.

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PRIY A et al.: TRANSESTERIFICA TlON REACTIONS OF HYDROCINNAMATES 263

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