Synthesis of monoterpene esters by alcoholysis reaction with Mucor miehei lipase in a solvent-free system

Article abstract of DOI:10.1007/s11746-998-0079-7

The syntheses of geranyl acetate and citronellyl acetate by alcoholysis reaction catalyzed by immobilized lipase from Mucor miehei was studied for the first time in a solvent-free system. Reactions were carried out at a terpene alcohol/acyl donor molar ratio of 1:5 with Lipozyme at 10% of the total weight of the reactants in a solvent-free system. Incubations were carried out at 55 to 60 °C for ethyl and butyl acetates as acyl donors, whereas for methyl acetate the incubation temperature was 40 to 45 °C. Excess concentration of acyl donor increases the percentage of geranyl acetate and citronellyl acetate, while excess of terpene alcohol concentration decreases the same. Yields from 75 to 77% molar conversion (90 to 98% conversion, w/w) were obtained after 8 to 28 h of reaction time.

Full text of DOI:10.1007/s11746-998-0079-7

J8258
Synthesis of Monoterpene Esters by Alcoholysis Reaction
with Mucor miehei Lipase in a Solvent-Free System
T. Chatterjee and D.K. Bhattacharyya*
Department of Chemical Technology, University Colleges of Science and Technology,
University of Calcutta, Calcutta 700009, India
ABSTRACT: The syntheses of geranyl acetate and citronellyl geranyl esters by direct esterification with an immobilized li-
acetate by alcoholysis reaction catalyzed by immobilized lipase
from Mucor miehei was studied for the first time in a solvent-
free system. Reactions were carried out at a terpene alcohol/
acyl donor molar ratio of 1:5 with Lipozyme at 10% of the total
weight of the reactants in a solvent-free system. Incubations
were carried out at 55 to 60°C for ethyl and butyl acetates as
acyl donors, whereas for methyl acetate the incubation temper-
ature was 40 to 45°C. Excess concentration of acyl donor in-
creases the percentage of geranyl acetate and citronellyl ac-
etate, while excess of terpene alcohol concentration decreases
the same. Yields from 75 to 77% molar conversion (90 to 98%
pase was also low (20). These problems have been overcome
by lipase-catalyzed transesterification reactions with other
acyl donors, such as acetic anhydride (21), isopropenyl ac-
etate (8), and triacetin (8,12). Chulalaksananukul et al. (11)
studied the lipase-catalyzed transesterification of geraniol with
C1, C2, C3, C4, and higher acetates as acyl donors. The reac-
tions were carried out in the presence of organic solvents be-
cause the solvent hydrophobicity influenced enzymatic activ-
ity (22). Yee and Akoh (21) reported quite high yields for the
Pseudomonas sp. lipase-catalyzed transesterification of ger-
conversion, w/w) were obtained after 8 to 28 h of reaction time. anyl acetate in the presence of organic solvents (petroleum
JAOCS 75, 651–655 (1998).
ether, hexane, and pentane) of log P values ≥3.0 (log P is the
partition coefficient between water and octanol).
KEY WORDS: Citronellol, citronellyl acetate, geraniol,
geranyl acetate, immobilized lipase, Mucor miehei, transesteri-
fication.
The alcoholysis reaction of terpene alcohols and esters of
short-chain acids and alcohols to make terpene alcohol esters
has not been studied adequately. Two important points need
to be considered in the synthesis of terpene alcohol esters by
alcoholysis reactions. Because the acetates act as solvents, it
Short-chain monocarboxylic acid esters of terpene alcohols becomes imperative to examine whether the alcoholysis reac-
are important fragrance compounds, generally synthesized by tion can be conducted without using any hydrocarbon sol-
the direct addition of a lower organic acid to terpenic olefins vents. Oguntimein et al. (23) synthesized geraniol esters by
under nonaqueous conditions in the presence of an acid cata- direct esterification in a solvent-free system with Candida
lyst or a strong cation exchange resin (1). The esters of ter- antarctica lipase. This paper presents alcoholysis reactions
pene alcohols are also synthesized by direct esterification of with Mucor miehei lipase in the absence of any organic sol-
terpene alcohols with short-chain acids by chemical methods. vent, which, to the best knowledge of the authors, have not
In recent years, enzymatic syntheses with lipases (triacylglyc- been reported before. We also present here an isolation
erol hydrolases E.C. 3.1.1.3) as catalysts have been used to process for the recovery of the terpene alcohol esters in pure
produce a number of commercially important flavor esters in form. These two aspects are important for commercial syn-
anhydrous organic solvents (2–6), both by transesterification thesis of terpene alcohol esters.
(7–13) and by direct esterification (3–5,14,15).
The present study deals with the synthesis of acetate esters
The nature of the acyl donor used plays an important role of geraniol and citronellol by lipase-catalyzed alcoholysis in
in the transesterification or esterification methodologies in the a solvent-free system and investigates the effects of substrate
presence of lipase as catalyst. Thus, yields of geranyl acetate concentration (terpene alcohols/acyl donor molar ratio), reac-
and citronellyl acetate were low when acetic acid was used as tion temperature, and reaction period.
an acyl donor in both transesterification and esterification re-
actions (14,16,17). A distinct reduction in the pH of the mi-
croenvironment of the lipases by acetic acid actually interfered
MATERIALS AND METHODS
with their aqueous layer (18,19). Moreover, the yield of some Materials. All alcohols, esters, and solvents were commer-
cially available and were used without further purification.
*To whom correspondence should be addressed at Department of Chemical
The immobilized lipase from M. miehei (Lipozyme IM) was
Technology, University Colleges of Science and Technology, University of
obtained from Novo-Nordisk A/S (Bagsvaerd, Denmark).
Calcutta, 92, Acharya Prafulla Chandra Road, Calcutta 700009, India.
Silicic acid (60 to 120 mesh) for column chromatography was
E-mail: dkb@cucc.ernet.in.
Copyright © 1998 by AOCS Press
651
JAOCS, Vol. 75, no. 5 (1998)

652
T. CHATTERJEE AND D.K. BHATTACHARYYA
purchased from Loba Chemicals (Calcutta, India). Silica gel
G for thin-layer chromatography (TLC) was purchased from
Tara Chemicals (Calcutta, India). Deuterated chloroform
1
(CDCl3) for H NMR spectrometry was purchased from
Aldrich Chemicals Co. Ltd. (Gillingham, United Kingdom).
Alcoholysis method. Ester synthesis was carried out by
magnetically stirring the reactants in a 50-mL standard-joint
conical flask, fitted with a condenser and a CaCl2 guard tube.
With methyl acetate, the reaction was carried out at varying
alcohol/ester molar ratios (1:1, 1:3, and 1:5) with immobi-
lized M. miehei lipase (Lipozyme) at 10% of the total weight
of the reactants. The weight of terpene alcohols was always
kept constant, and only the weight of acyl donors was varied.
Incubations were carried out at 55 to 60°C when ethyl acetate
and butyl acetate were used as acyl donors. The incubation
temperature was kept between 40 and 45°C with methyl ac-
etate. The optimal alcohol/ester molar ratio for use with
methyl acetate was used in the reaction involving ethyl and
butyl acetates as acyl donors.
R
R
SCHEME 1
Extraction and analysis. At the end of the incubation pe-
riod, each reaction mass was filtered under vacuum to remove
the Lipozyme. The enzyme-free material from the methyl ac-
etate reaction product was extracted with distilled water (3 ×
15 mL) to remove unreacted methyl acetate and methanol.
The water-washed organic phase, which contained the terpene
ester, was next dried over anhydrous sodium sulfate. The
dried material was then subjected to column chromatography
on silicic acid (60 to 120 mesh) for quantitative isolation of
the esters of terpene alcohols.
After the reactions with ethyl acetate and butyl acetate, the
Lipozyme was separated by vacuum filtration. The reaction
products were next subjected to vacuum distillation to remove
the unreacted acyl donors and the liberated alcohols, viz.,
ethanol and butanol. The completeness of the removal of acyl
donors was tested by gas–liquid chromatography (HP 5890A;
Hewlett-Packard, Avondale, PA), equipped with a flame-ion-
ization detector. A 10% DEGS (Hewlett-Packard) column
was used. Oven, injector, and detector temperatures were
140°C (isothermal), 200°C, and 210°C, respectively. The car-
rier gas was nitrogen (flow rate 30 mL/min).
The crude products, free from acyl donors, were then sub-
jected to column chromatography on silicic acid (60 to 120
mesh) with 100 mL n-hexane/diethyl ether (99:1, vol/vol).
The solvents were then evaporated on a water bath, followed
by applying vacuum at ambient temperature (30°C) to give
pure geranyl and citronellyl acetates as colorless oils
(Scheme 1). The purity of the products was confirmed by
TLC and by using spectral methods, viz., Fourier transform
infrared (FTIR) spectroscopy and proton nuclear magnetic
resonance (1H NMR) spectrometry.
Qualitative analyses were carried out by TLC. Samples
were diluted 1:5 (vol/vol) with chloroform, and the diluted
samples were used for TLC analysis on glass plates (20 × 20
cm) with a 0.2-mm layer of silica gel G. The plates were de-
veloped with n-hexane/diethyl ether (70:30, vol/vol). All
spots were identified by strong iodine absorption. The Rf val-
ues of the esters were 0.92 for geranyl acetate and 0.90 for
citronellyl acetate, respectively.
The infrared (FTIR) spectra were recorded on a Perkin-
Elmer 1600 Fourier transform spectrometer (Norwalk, CT)
with the samples spread between NaCl plates. The 1H NMR
spectra were determined in deuterated chloroform with a
Bruker AM-300L spectrometer (Fallenden, Switzerland) that
was operated at a frequency of 300 MHz with tetramethyl
silane (TMS) as internal standard.
Quantitation. Geranyl acetate and citronellyl acetate
were quantitated by standard column chromatographic
techniques on silicic acid (60 to 120 mesh). The percentage
yields of esters (molar yield percentage) with respect to alco-
hols for different incubation times were calculated from the
weight of esters produced and the weight of alcohols used in
the reactions, as well as from their respective molecular
weights.
RESULTS AND DISCUSSION
Identification of products by spectral method. The spectral
data of the starting alcohols and the product esters are shown
in Table 1. The FTIR spectra of geraniol and citronellol
showed absorption of hydroxyl groups at 3334 and 3333
cm−1, respectively. The FTIR of the acetates of geraniol and
citronellol lacked absorption for the hydroxyl group at 3333
to 3334 cm−1 but showed absorption at 1741 cm−1, as ex-
pected for the ester carbonyl group. Similarily, from the 1H
NMR data, the resonance signal at δ 3.73 ppm for geraniol
and δ 3.68 ppm for citronellol, which are due to methylene
protons (–CH2) of the –CH2OH end group, disappeared in the
acetates and were replaced by δ 4.57 ppm for geranyl acetate
and δ 4.55 ppm for citronellyl acetate. The increase in δ val-
ues in the products, compared to the starting alcohols, is due
to formation of the acetate (–OCOCH3) group, which shifts
the methylene protons to higher frequency (24).
The FTIR and 1H NMR data were compared with the stan-
JAOCS, Vol. 75, no. 5 (1998)

LIPASE-CATALYZED SYNTHESIS OF MONOTERPENE ESTERS BY ALCOHOLYSIS
653
TABLE 1
Spectral Data of Geraniol, Geranyl Acetate, Citronellol , and Citronellyl Acetatea
FTIR νmax
(cm −1 a
1H NMR
Mol. wt.
(M+)
Name of compound
Geraniol
)
[δ (ppm); J (Hz)]
3334, 2958, 2924,
1458, 1373, 1057
5.47 (t, 1H, J = 6)
5.15 (t, 1H, J = 6)
4.2 (d, 1H), 3.73
(q, 2H), 2.08 (m,
4H), 1.77 (s, 6H),
1.64 (s, 3H)
154
Geranyl acetate
2924, 2365, 1741,
1447, 1374, 1235,
1024
5.39 (t, 1H, J = 6)
5.06 (t, 1H, J = 3)
4.57 (d, 2H, J = 6),
2.1 (s, 3H), 2.03
(m, 4H), 1.69 (s,
6H), 1.62 (s, 3H)
196
(C:H:O
73.31:
10.02:
16.52)
Citronellol
3333, 2958, 2924,
1456, 1377, 1057,
1008
5.13 (t, 1H, J = 6)
4.13 (q, 1H), 3.68
(m, 2H), 2.61(m,
2H), 2.02 (m, 2H),
1.71 (s, 6H), 1.60
(m, 1H), 1.40 (m,
2H), 0.91 (m, 3H)
156
Citronellyl acetate
2957, 2361, 1741,
1452, 1373, 1238,
1035, 829
5.06 (t, 1H, J = 6)
4.55 (t, 2H, J = 8)
2.02 (m, 2H), 2.00
(s, 3H), 1.65 (s,
6H), 1.55 (br s,
1H), 1.30 (m, 4H),
0.89 (m, 3H)
198
(C:H:O
70.60:
10.44:
18.64)
aFTIR, Fourier transform infrared; NMR, nuclear magnetic resonance.
dard samples, which conclusively indicate the formation of ratio of 1:5 for terpene alcohol/acyl donor (methyl acetate).
geranyl and citronellyl acetates by alcoholysis. However, in the present study, substantial conversion was
Substrate molar ratio. The effect of substrate concentra- achieved in 8 h for both geranyl acetate and citronellyl acetate
tion on the synthesis of flavor esters with M. miehei has been by immobilized lipase from M. miehei. The lipase-catalyzed
reported (25). Inhibitory effect of geraniol on M. miehei li- alcoholysis of geraniol with propyl acetate as acyl donor,
pase (IM 20) activity was observed by Chulalaksananukul et which is regarded as the best acyl donor for geranyl acetate
al. (11) and by Claon and Akoh (3), although the degree of synthesis, has resulted in 85% yield after 3 d of incubation
inhibition depends mainly on the type of enzyme used (26).
(11). Langrand et al. (17) reported the yield for the same reac-
Initially, we studied the alcoholysis reaction at 1:1 and 1:3 tion, with isoamyl acetate as the substrate, to be 24% after 24
terpene alcohol/ester (methyl acetate) molar ratios, and ger- h of incubation. The alcoholysis reactions, when conducted
anyl acetate and citronellyl acetate were obtained in poor for longer periods, viz., 18 and 28 h, did not increase the yield
yields. The molar percentage conversions of geraniol acetate of the products, but showed a slight decline. We concluded
were 16.54 and 19.25% for geraniol/methyl acetate molar ra- that the highest yields of geranyl acetate and citronellyl ac-
tios of 1:1 and 1:3, respectively, whereas for citronellyl ac- etate could be achieved in 8 h, which would make the process
etate, the molar conversions were 23.85 and 25.25%, even attractive and hence commercially exploitable.
after 8 h of incubation. By increasing the terpene alcohol/ester
molar ratio to 1:5, the yield became as high as 77.92 mol%
after 8 h of incubation (Table 2). Hence, for both monoter-
pene esters yields improved as the concentration of acyl
ACKNOWLEDGMENTS
The financial support of the University Grant Commission (West
Bengal, India) during the study is gratefully acknowledged. We are
donors was increased and that of terpene alcohol was de- also grateful to Ananya Dan of the Department of Polymer Science
and Technology for performing the FTIR spectra of the compounds.
creased.
Time course of reaction. The time course of lipase-cat-
REFERENCES
alyzed synthesis of geranyl acetate and citronellyl acetate is
shown in Table 2. About 42% geraniol and 44% citronellol
conversions to acetates were attained in 4 h by using a molar
1. Nigam, S.C., S.N. Bannore, H.N. Subbarao, and Sukh Dev, A
Novel Process for Conversion of Terpenic Olefins to the Corre-
JAOCS, Vol. 75, no. 5 (1998)

654
T. CHATTERJEE AND D.K. BHATTACHARYYA
TABLE 2
Yield in Molar Percentage of Esters by Lipase-Catalyzed Alcoholysis Reactions of Geraniol
and Citronellol with Different Acyl Donors
Alcohol
(x)
Acyl donor
(y)
Molar ratio
(x:y)
Reaction
temperature (°C)
Reaction
time (h)
Conversiona
(mol%)
Geraniol
Methyl acetate
1:1
1:3
1:5
1:5
1:5
40 to 45
8
8
4
8
18
16.54
19.25
42.44
76.43
76.07
Geraniol
Geraniol
Ethyl acetate
Butyl acetate
1:5
55 to 60
55 to 60
40 to 45
8
18
28
76.62
75.97
75.32
1:5
8
18
28
77.14
76.22
71.07
Citronellol Methyl acetate
1:1
1:3
1:5
1:5
1:5
8
8
4
8
18
23.85
25.25
44.25
77.92
76.51
Citronellol Ethyl acetate
Citronellol Butyl acetate
1:5
55 to 60
55 to 60
8
18
28
77.48
76.11
75.68
1:5
8
18
28
76.52
77.11
76.84
aConversion = (wt. of terpene ester obtained × M.W. of terpene alcohol) (initial wt. of terpene alco-
/
hol × M.W. of terpene ester) × 100.
sponding Alcohols, Ethers or Esters of Perfumery Value, in 11th
International Congress of Essential Oils, Fragrance and
sis by Lipase-Catalyzed Transesterification, Ibid. 17:67–70
(1995).
Flavours, Vol. 5, 1989, pp. 113–118.
2. de Castro, H.F., W.A. Anderson, M. Moo-Young, and R.L.
Legge, Partitioning of Water During the Production of Terpene
10. Yee, L.N., C.C. Akoh, and R.S. Phillips, Pseudomonas sp. Cat-
alyzed Synthesis of Geranyl Esters by Transesterification, J. Am.
Oil Chem. Soc. 72:1407–1408 (1995).
Esters Using Immobilized Lipase, in Biocatalysis in Non-Con- 11. Chulalaksananukul, W., J.S. Condoret, and D. Combes, Geranyl
ventional Media, edited by J. Tramper, M.H. Vermule, H.H.
Beefstink, and U. von Stockar, Elsevier Science Publishers
B.V., New York, 1992, pp. 475–482.
Acetate Synthesis by Lipase-Catalyzed Transesterification in
n-Hexane, Enzyme Microb. Technol. 14:293–298 (1992).
12. Claon, P.A., and C.C. Akoh, Lipase-Catalyzed Synthesis of Pri-
mary Terpenyl Acetates by Transesterification: Study of Reac-
tion Parameters, J. Agric. Food Chem. 42:2349–2352 (1994).
13. Rizzi, M., P. Stylos, and M. Reuss, A Kinetic Study of Immobi-
lized Lipase Catalyzing the Synthesis of Isoamyl Acetate by
Transesterification in n-Hexane, Enzyme Microb. Technol. 14:
709–714 (1992).
3. Claon, P.A., and C.C. Akoh, Enzymatic Synthesis of Geraniol
and Citronellol Esters by Direct Esterification in n-Hexane,
Biotechnol. Lett. 15:1211–1216 (1993).
4. Manjon, A., J.L. Iborra, and A. Arocas, Short-Chain Flavor
Ester Synthesis by Immobilized Lipase in Organic Media, Ibid.
13:339–344 (1991).
5. Marlot, C., G. Langrand, C. Triantaphylides, and J. Baratti, 14. Okumura, S., M. Iwai, and Y. Tsujisaka, Synthesis of Terpene
Ester Synthesis in Organic Solvent Catalyzed by Lipases Immo-
bilized on Hydrophilic Supports, Ibid. 7:647–650 (1985).
Alcohol Esters by a Lipase, Biochim. Biophys. Acta 575:
156–165 (1979).
6. Nishio, T., and M. Kamimura, Ester Synthesis in Various Or- 15. Gillies, B., H. Yamazaki, and D.W. Armstrong, Production of
ganic Solvents by Three Kinds of Lipase Preparations Derived
from Pseudomonas fragi, Agric. Biol. Chem. 52:2631–2632
(1988).
Flavour Esters by Immobilized Lipase, Biotechnol. Lett. 9:
709–714 (1987).
16. Iwai, M., S. Okumura, and Y. Tsujisaka, Synthesis of Terpene
Alcohol Esters by Lipase, Agric. Biol. Chem. 33:2731–2732
(1980).
7. Gray, C.J., J.S. Narang, and S.A. Barker, Immobilization of Li-
pase from Candida cylindracea and Its Use in the Synthesis of
Menthol Esters by Transesterification, Enzyme Microb. Tech- 17. Langrand, G., N. Rondot, C. Triantaphylides, and J. Baratti,
nol. 12:800–807 (1990).
8. Akoh, C.C., and P.A. Claon, Lipase-Catalyzed Synthesis of Ter-
Short Chain Ester Synthesis by Microbial Lipases, Biotechnol.
Lett. 12:581–586 (1990).
pene Esters by Transesterification in n-Hexane, Biotechnol. Lett. 18. Iwai, M., S. Okumura, and Y. Tsujisaka, Application of Micro-
16:235–240 (1994).
9. Yee, L.N., C.C. Akoh, and R.S. Phillips, Terpene Ester Synthe-
bial Lipase to the Determination of the Fatty Acid Distribution
in Natural Fats, Agric. Biol. Chem. 44:1203–1208 (1988).
JAOCS, Vol. 75, no. 5 (1998)

LIPASE-CATALYZED SYNTHESIS OF MONOTERPENE ESTERS BY ALCOHOLYSIS
655
19. Welsh, F., and R.E. Williams, Lipase-Mediated Production of 24. Kemp, W., Organic Spectroscopy, 3rd edn., Macmillan Press,
Flavour and Fragrance Esters from Fusel Oil, J. Food Sci. 54:
1565–1568 (1989).
20. Chulalaksananukul, W., J.S. Condoret, O. Delorme, and R.M.
Willemot, Kinetic Study of Esterification by Immobilized Li-
pase in n-Hexane, FEBS Lett. 276:181–184 (1990).
Hampshire, 1994, p. 172.
25. Chulalaksananukul, W., J.S. Condoret, and D. Combes, Geranyl
Acetate Synthesis by Lipase-Catalyzed Transesterification in
Supercritical Carbon Dioxide, Enzyme Microb. Technol. 15:
691–698 (1993).
21. Yee, L.N., and C.C. Akoh, Enzymatic Synthesis of Geranyl Ac- 26. Welsh, F.W., and R.E. Williams, Lipase-Mediated Production
etate by Transesterification with Acetic Anhydride as Acyl
Donor, J. Am. Oil Chem. Soc.73:1379–1384 (1996).
of Ethyl Butyrate in Nonaqueous Systems, Ibid. 12:743–748
(1990).
22. Katchalski-Katzir, E., Medium and Biocatalytic Engineering, in
Biocatalysis in Non-Conventional Media, edited by J. Tramper,
M.H. Vermule, H.H. Beefstink, and U. von Stockar, Elsevier
Science Publishers B.V., New York, Vol. 8, 1992, pp. 3–9.
23. Oguntimein, G.B., W.A. Anderson, and M. Moo-Young, Syn-
thesis of Geraniol Esters in a Solvent-Free System Catalysed by
Candida antarctica Lipase, Biotechnol. Lett. 17:77–82 (1995).
[Received October 1, 1996; accepted December 8, 1997]
JAOCS, Vol. 75, no. 5 (1998)