Solvent-free lipase-catalyzed preparation of long-chain alkyl phenylpropanoates and phenylpropyl alkanoates

Article abstract of DOI:10.1021/jf060052t

An enzymatic method was developed for the preparation of medium- or long-chain alkyl 3-phenylpropenoates (alkyl cinnamates), particularly alkyl hydroxy- and methoxy-substituted cinnamates such as oleyl p-coumarate and oleyl ferulate. The various alkyl cin

Full text of DOI:10.1021/jf060052t

J. Agric. Food Chem. 2006, 54, 2969−2976 2969
Solvent-free Lipase-Catalyzed Preparation of Long-Chain Alkyl
Phenylpropanoates and Phenylpropyl Alkanoates
KLAUS VOSMANN, PETRA WEITKAMP, AND NIKOLAUS WEBER*
Institut fu¨r Lipidforschung, Bundesforschungsanstalt fu¨r Erna¨hrung und Lebensmittel, Piusallee 68-76,
D-48147 Mu¨nster, Germany
An enzymatic method was developed for the preparation of medium- or long-chain alkyl 3-phenyl-
propenoates (alkyl cinnamates), particularly alkyl hydroxy- and methoxy-substituted cinnamates such
as oleyl p-coumarate and oleyl ferulate. The various alkyl cinnamates were formed in high to moderate
yield by lipase-catalyzed esterification of cinnamic acid and its analogues with fatty alcohols in vacuo
at moderate temperatures in the absence of drying agents and solvents. Immobilized Candida
antarctica lipase B was the most effective biocatalyst for the various esterification reactions. The
relative esterification activities were of the following order: dihydrocinnamic > cinnamic > 3-meth-
oxycinnamic > dihydrocaffeic ≈ 3-hydroxycinnamic > 4-methoxycinnamic > 2-methoxycinnamic >
4-hydroxycinnamic > ferulic ≈ 3,4-dimethoxycinnamic > 2-hydroxycinnamic acid. With respect to
the position of the substituents at the phenyl moiety, the esterification activity increased in the order
meta > para > ortho. Rhizomucor miehei lipase demonstrated moderate esterification activity.
Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-
hydroxyphenyl)propyl oleate, were also obtained in high yield by esterification of fatty acids with the
corresponding 3-phenylpropan-1-ols.
KEYWORDS: Alkyl cinnamates; alkyl 3-(hydroxyphenyl)propenoates; oleyl coumarates; alkyl 3-(meth-
oxyphenyl)propenoates; oleyl ferulate; palmityl ferulate; 3-phenylpropyl alkanoates; 3-(4-hydroxyphenyl)
oleate; immobilized Candida antarctica lipase B; lipase-catalyzed esterification
INTRODUCTION
Mendez (25) demonstrated the occurrence of dihydrocinnamic
acid derivatives, for example, dihydro-p-coumaric and dihy-
droferulic acids, in bracken fern (Pteridium aquilinum). Al-
though hydroxy cinnamic acids esters of polysaccharides occur
most commonly in plants, various other esters of cinnamic acid
and its analogues including medium- or long-chain alkyl esters
have also been detected, for example, alkyl ferulates (Figure
1), which are predominantly found in the cork layers of several
plant species (26-32). In addition, similar compounds with
inverse chemical structure, that is, 3-(4-hydroxyphenyl)propenyl
(p-coumaryl) alkanoates, were isolated from apple (Malus
domestica) fruits (33, 34).
Minor constituents of plants are gaining importance due to
their proposed health benefits (1, 2). For example, epidemiologi-
cal and nutritional studies suggest that the consumption of foods
containing high proportions of phenolic antioxidants may
prevent chronic diseases such as coronary heart disease and
colorectal cancer (3-5). The beneficial effects of phenolic
antioxidants, such as 3-(hydroxyphenyl) propenoates (hydroxy
cinnamic acids), on health have been attributed to their
antioxidant capacity, particularly their ability to protect low-
density lipoproteins from oxidative attack (6). The antioxidant
capacity and biological availability of several of these com-
pounds may be further improved by increasing their lipophilicity
(7-12), and the range of applications of such lipophilic
antioxidants may be extended by their possible use as additives
for food and nutraceutical (12, 13) as well as cosmetic and
technical applications (14-18).
Various methods are known for the chemical esterification
of cinnamic acid and its analogues, for example, esterification
of hydroxy cinnamic acids or their tetrahydropyranyl derivatives
with various alcohols in the presence of N,N-dicyclohexylcar-
bodiimide in tetrahydrofuran or pyridine (9, 35). Moreover,
esterification of hydroxy cinnamic acids including ferulic acid
with alkanols in the presence of acid catalysts such as sulfuric
acid or p-toluenesulfonic acid has been described using benzene,
toluene, or chlorobenzene as the solvents (18). Sitostanyl ferulate
was prepared by esterification of acetylated ferulic acid with
sitostanol in the presence of N,N-dicyclohexylcarbodiimide and
4-(dimethylamino)pyridine (36). Enzymatic esterification is
preferred, however, over chemical esterification for the prepara-
Hydroxy cinnamic acids and their methoxy derivatives such
as 4-hydroxycinnamic (p-coumaric), ferulic, caffeic and sinapic
acids are common constituents of plants, particularly of cereals
such as rice, rye, and wheat, in which they are predominantly
esterified to cell wall polysaccharides (19-24). Recently,
* Corresponding author. Tel.: +49-251-48167-0. Fax: +49-251-48167-
60. E-mail: niweber@uni-muenster.de.
10.1021/jf060052t CCC: $33.50
© 2006 American Chemical Society
Published on Web 03/28/2006

2970 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Vosmann et al.
(Biocatalysts, Pontypridd, Mid Glamorgan, U.K.), Rhizopus arrhizus,
Rhizopus niVeus, Rhizopus jaVanicus (all from Fluka), and Fusarium
oxysporum (Lipopan F BG, Novozymes), and pectinase from Aspergil-
lus niger (Serva, Heidelberg, Germany) as well as plant proteases/
esterases including bromelain from Ananassa comosus, papain from
Carica papaya, and ficin from Ficus carica (all from Sigma).
Lipase-Catalyzed Reactions. As a typical example, 4-methoxycin-
namic acid (53.4 mg, 0.3 mmol) was esterified with cis-9-octadecen-
1-ol (80.4 mg, 0.3 mmol) in the presence of 50 mg of the various lipase
preparations by magnetic stirring in a screw-capped tube in vacuo at
80 °C for periods of up to 144 h with water-trapping in the gas phase
using potassium hydroxide pellets. A moderate vacuum (80 kPa) was
used to prevent substantial loss of substrates. Samples of the reaction
products were withdrawn at various intervals, extracted with diethyl
ether, and filtered through a 0.45 µm syringe filter to remove the
biocatalyst. An aliquot of the filtrate was analyzed as given below.
Similar reaction conditions were used for the preparation of
phenylpropyl alkanoates with inverse chemical structure. As a typical
example, oleic acid (84.8 mg, 0.3 mmol) was esterified with cinnamyl
alcohol (40.2 mg, 0.3 mmol) under identical conditions as described
above for the esterification of cinnamic acid derivatives using Novozym
435 lipase preparation as biocatalyst.
Enzyme units were calculated from the initial rates (4 or 24 h) of
esterification of the various 3-phenylpropanoic and 3-phenylpropenoic
acids with alkanols as well as the corresponding esterification of fatty
acids with various 3-phenylpropanol and 3-phenylpropenol derivatives.
One unit of enzyme activity was defined as the amount of enzyme
(grams) that produced 1 µmol of the respective alkyl or phenylpropyl
ester per minute.
Thin-Layer Chromatography (TLC). Aliquots were withdrawn
from the reaction mixtures, and free carboxy groups of the various
compounds of the reaction mixture were methylated using a solution
of diazomethane in diethyl ether. The conversion was checked by TLC
on 0.3 mm layers of silica gel H (E. Merck, Darmstadt, Germany),
and spots were located by iodine staining and charring by spraying
with 30% sulfuric acid followed by heating (200 °C).
The different methods of development (A and B) used led to the
following Rf values of the various compounds: (A) isohexane-diethyl
ether (1:1), one run to the top, long-chain alkyl cinnamates and
dihydrocinnamates, 0.55-0.6; long-chain methoxy-substituted 3-phe-
nylpropyl alkanoates, 0.5-0.55; long-chain 3-(3,4-dimethoxy-phenyl)-
propyl alkanoates, 0.4; fatty acid methyl esters, 0.75; saturated and
unsaturated long-chain fatty alcohols, 0.3; unesterified fatty acids and
3-phenylpropanoic acids, 0.4-0.45; (B) isohexane-diethyl ether (1:
1), developed up to half of the plate, followed by the same solvent
system to the top, long-chain alkyl 3-(hydroxyphenyl)propenoates, 0.6-
0.65; medium- and long-chain alkyl ferulates, 0.5; long-chain 3-(4-
hydroxyphenyl)propyl oleate, 0.6; 3-phenylpropan-1-ol (dihydrocin-
namyl alcohol) and 3-phenylpropen-1-ol (cinnamyl alcohol) 0.3; 3-(4-
hydroxyphenyl)propan-1-ol and 3-(3,4-dimethoxyphenyl)propan-1-ol,
0.1-0.15; unesterified fatty acids and 3-phenylpropanoic acids, 0.45-
0.55.
Similarly, 0.5 mm layers of silica gel H were used for the separation
of reaction products by preparative TLC. The various fractions were
scraped off the plates and extracted from silica gel using water-saturated
diethyl ether. Reaction mixtures containing phenolic hydroxy com-
pounds were separated under similar conditions as described above
using method B.
Purification by Crystallization. Alkyl 3-phenylpropanoates and
3-phenylpropyl alkanoates were extracted from the immobilized bio-
catalysts with diethyl ether and purified by column chromatography
and/or crystallization from isohexane or mixtures of isohexane-diethyl
ether (1:1). The following melting points were determined using a
Kofler heating block: oleyl 2-hydroxycinnamate, 37-39 °C; palmityl
2-hydroxycinnamate, 84-85 °C; palmityl 3-hydroxycinnamate, 78-
79 °C; oleyl 4-hydroxycinnamate, 38-39 °C; palmityl 4-hydroxycin-
namate, 89-90 °C; palmityl 2-methoxycinnamate, 46-47 °C; palmityl
3-methoxycinnamate, 47-48 °C; palmityl 4-methoxycinnamate, 59-
60 °C; lauryl ferulate, 45-48 °C; palmityl ferulate, 61-63 °C; oleyl
ferulate, 30-33 °C; oleyl dihydrocaffeate, 21-23 °C; oleyl caffeate,
78-79 °C; cinnamyl palmitate (3-phenylprop-2-en-1-yl palmitate), 45-
Figure 1. Chemical structures of the alkyl cinnamate (alkyl 3-phenylpro-
penoate) derivative oleyl ferulate and the structurally similar inverse
dihydrocinnamyl (3-phenylpropyl) alkanoate derivative 3-(4-hydroxyphenyl)-
propyl oleate.
tion of hydroxy cinnamic acids esters that may be used in foods.
Little is known about lipase-catalyzed esterification of cinnamic
acid derivatives, particularly hydroxy cinnamic acids (12).
Enzymatic esterification procedures requiring organic solvents
or using the alcohol component as the solvent have been
reported for the preparation of short- and medium-chain alkyl
esters of hydroxy cinnamic acids including alkyl ferulates (37-
43). Similarly, the corresponding compounds with inverse
chemical structure, that is, 3-phenylpropyl and 3-phenylpropenyl
alkanoates, may be formed by esterification of fatty acids with
various dihydrocinnamyl and cinnamyl alcohol derivatives (3-
phenylpropan-1-ols and 3-phenylpropen-1-ols). Such “retro”
compounds may also be of interest because they are expected
to have antioxidant properties equivalent to those shown for
alkyl hydroxycinnamates (33, 34).
The aim of the present work was to develop a simple and
environmentally friendly method for the preparation of various
lipophilic medium- and long-chain alkyl 3-phenylpropanoates,
particularly hydroxy- and methoxycinnamates, by esterification
of 3-phenylpropanoic and 3-phenylpropenoic acids and their
analogues with medium- or long-chain fatty alcohols as well
as of the structurally inverse 3-phenylpropyl alkanoates by
esterification of medium- or long-chain fatty acids with 3-phe-
nylpropan-1-ols using lipases as biocatalysts. The esterification
reactions were performed at moderate temperatures without
solvents or drying agents using reduced pressure to remove
reaction water.
MATERIALS AND METHODS
Materials. 1-Dodecanol, 1-hexadecanol, and cis-9-octadecen-1-ol
(oleyl alcohol) as well as trans-cinnamic acid [(E)-3-phenylpropenoic
acid], dihydrocinnamic acid (3-phenylpropanoic acid), 2- and 3-hy-
droxy- as well as 2-, 3-, and 4-methoxycinnamic acids, 4-hydroxy-3-
methoxycinnamic acid (ferulic acid), 3,4-dihydroxycinnamic acid
(caffeic acid), 3,4-dihydroxydihydrocinnamic acid (dihydrocaffeic acid),
4-hydroxy-3,5-dimethoxycinnamic acid (sinapic acid), methyl trans-
cinnamate, cinnamyl alcohol (3-phenylpropen-1-ol), dihydrocinnamyl
alcohol (3-phenylpropan-1-ol), 3-(4-hydroxyphenyl)propan-1-ol, and
3-(3,4-dimethoxy)propan-1-ol were obtained from Sigma-Aldrich-Fluka
(Deisenhofen, Germany). 4-Hydroxycinnamic acid (p-coumaric acid)
was a product of Alfa Aesar (Karlsruhe, Germany). For comparison,
trans-ferulic acid was partially isomerized to cis-ferulic acid by UV
irradiation in methanolic solution (44).
Immobilized lipase preparations from Candida antarctica (lipase B,
Novozym 435), Rhizomucor miehei (Lipozyme RM IM), and Thermo-
myces lanuginosus (Lipozyme TL IM) were kindly provided by
Novozymes, Bagsvaerd, Denmark. Various other enzymes were also
checked for esterification activity using cinnamic acid derivatives and
cis-9-octadecen-1-ol as the substrates, including lipases from Candida
rugosa (Sigma), Candida lipolytica (Fluka), Chromobacterium Viscosum

Preparation of Alkyl Phenylpropanoates
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2971
46 °C; 3-(4-hydroxyphenyl)propyl palmitate, 60-61 °C; 3-(3,4-
dimethoxyphenyl)propyl palmitate, 35-36 °C. Other long-chain alkyl
cinnamates or cinnamyl alkanoates such as oleyl cinnamate, oleyl
dihydrocinnamate, oleyl 3-hydroxycinnamate, oleyl 2-methoxycin-
namate, oleyl 3-methoxycinnamate, oleyl 4-methoxycinnamate, oleyl
3,4-dimethoxycinnamate, 2-ethylhexyl ferulate, oleyl sinapate, cinnamyl
oleate, 3-phenylpropyl palmitate, 3-(4-hydroxyphenyl)propyl oleate are
liquids at room temperature.
Column Chromatography. As a typical example, the reaction
mixture resulting from the esterification of ferulic acid with oleyl
alcohol (around 400 mg) was dissolved in 2 mL of diethyl ether and
applied to a 25 × 2 cm i.d. silica gel 60 column (Merck). The column
was eluted first with 30 mL of isohexane and then with 30 mL portions
of various isohexane-diethyl ether mixtures (95:5, 9:1, 8:2, 7:3).
Elution with isohexane-diethyl ether (1:1) yielded around 300 mg of
oleyl ferulate, which was crystallized from isohexane-diethyl ether
(1:1).
Gas Chromatography (GC). Aliquots of esterification products
were removed from the reaction mixture, dissolved in dichloromethane,
and filtered through a 0.45 µm PTFE syringe filter to remove the lipase
catalysts. The filtrate was concentrated, dissolved in diethyl ether, and
treated with an ethereal solution of diazomethane to convert the
unreacted carboxy groups to the corresponding methyl esters. The
resulting mixture of methyl esters, unreacted alkanols or 3-phenylpro-
panols, and alkyl cinnamates or 3-phenylpropyl alkanoates was analyzed
by GC. The phenolic hydroxy group of 2-hydroxycinnamic acid
derivatives was methylated to the 2-methoxy compounds by treatment
with an ethereal solution of diazomethane in the presence of catalytic
amounts of silica gel (45) to avoid coumarin formation during high-
temperature GC. A Hewlett-Packard (Bo¨blingen, Germany) HP-5890
series II gas chromatograph equipped with a flame ionization detector
was used. Separations were carried out on a 15 m × 0.25 mm i.d., 0.1
µm 400-1HT fused silica capillary column (Quadrex Corp., New Haven,
CT) using hydrogen as the carrier gas (column pressure of 50 kPa).
Three different temperature programs were used to separate the
various compounds of reaction mixtures: (method A) initially at 150
°C for 2 min, followed by linear programming from 150 to 180 °C at
2 °C/min, then from 180 to 360 °C at 20 °C/min, and finally kept at
360 °C for 2 min; (method B) initially at 140 °C for 2 min, followed
by linear programming from 140 to 180 °C at 5 °C/min, then from
180 to 250 °C at 10 °C/min (5 min isothermally), then from 250 to
410 °C at 20 °C/min, and finally kept at 410 °C for 2 min; and (method
C) initially at 80 °C for 2 min, followed by linear programming from
80 to 280 °C at 10 °C/min, and finally kept at 280 °C for 5 min. Injector
and detector temperatures were maintained at 380 °C. Retention times
of the various reaction products and starting materials are given in Table
1.
Peaks in gas chromatograms were assigned by comparison of their
retention times with those of peaks from TLC fractions that had been
identified by GC-MS. Response factors of FID were determined using
purified compounds, and percentages of peak areas were calculated
using a Hewlett-Packard GC ChemStation software. For the determi-
nation of enzyme activities, small proportions of methoxy compounds
that had been formed during carboxymethylation of hydroxycinnamic
acids with diazomethane were calculated as the original hydroxy
compounds.
Reversed Phase High-Performance Liquid Chromatography
(RP-HPLC). Aliquots of purified reaction products were dissolved in
diethyl ether, filtered as described for GC, concentrated, and dissolved
in acetonitrile. Samples were analyzed for their composition by RP-
HPLC as follows: the HPLC system consisted of a Merck-Hitachi pump
L-6200 (E. Merck) equipped with a column oven (VDS Optilab, Berlin,
Germany) set at 30 °C, a Knauer K 2501 UV-vis detector (Knauer,
Berlin, Germany) set to 220 nm, and a PL-ELS 2100 (Polymer
Laboratories, Darmstadt, Germany) ELSD detector (thermostated to
30 °C for acetonitrile), which were used in series. UV and mass traces
were monitored and evaluated in a Knauer Eurochrom for Windows,
Preparative Version, data acquisition unit (Knauer).
Table 1. GC Retention Times of Various Medium- and Long-Chain
Alkyl 3-Phenylpropanoates, Alkyl 3-Phenylpropenoates, and
3-Phenylpropyl Alkanoates and Starting Materials
alkyl 3-phenylpropanoates, alkyl 3-phenylpropenoates,
and 3-phenylpropyl alkanoates as well as
starting materialsa
retention
time
(min)
Method Ab
methyl dihydrocinnamate
methyl cinnamate
1.0
1.2
methyl 2-methoxycinnamate
methyl 3-methoxycinnamate
methyl 4-methoxycinnamate
methyl 3-hydroxycinnamate
methyl 4-hydroxycinnamate
oleyl alcohol
1.6
1.6
1.8
1.8
1.9
6.6
oleyl dihydrocinnamate
oleyl cinnamate
12.5
13.8
13.6
13.7
13.8
13.3
13.4
oleyl 2-methoxycinnamate
oleyl 3-methoxycinnamate
oleyl 4-methoxycinnamate
oleyl 3-hydroxycinnamate
oleyl 4-hydroxycinnamate
Method Bb
methyl ferulate
lauryl alcohol
0.9
1.0
palmityl alcohol
3.6
oleyl alcohol
5.5
lauryl ferulate
palmityl ferulate
oleyl cis-ferulate
oleyl trans-ferulate
oleyl sinapate
15.3
18.6
19.7
20.3
19.0
Method Cb
3-phenylpropan-1-ol
cinnamyl alcohol
1.6
2.1
3-(4-hydroxyphenyl)propan-1-ol
3-(3,4-dimethoxyphenyl)propan-1-ol
methyl oleate
6.2
6.8
10.7
17.4
17.7
19.2
19.9
3-phenylpropyl oleate
3-phenylpropenyl oleate (cinnamyl oleate)
3-(4-hydroxyphenyl)propyl oleate
3-(3,4-dimethoxyphenyl)propyl oleate
a Carboxylic acids were converted to the methyl esters by reaction with
diazomethane. b For details see Materials and Methods.
Germany) and precolumn using (method A) acetonitrile isocratically
at a flow rate of 1.0 mL/min, (method B) acetonitrile isocratically at a
flow rate of 0.6 mL/min, and (method C) methanol-water-acetic acid
(50:50:0.5) isocratically at a flow rate of 1 mL/min and a UV detector
set at 320 nm. Injections (around 20-60 µg) of the reaction products
were carried out with a Rheodyne 7161 sample injector (Cotati, CA)
equipped with a 20-µL sample loop. Peak areas and percentages were
calculated using the Eurochrom software. Retention times of the various
reaction products and starting materials are given in Table 2.
GC-MS Analyses. The fragmentation of the various alkyl 3-phen-
ylpropanoates or 3-phenylpropyl alkanoates formed by lipase-catalyzed
esterification reactions was studied by GC-MS (EI mode). Compounds
containing carboxy groups were analyzed after derivatization to the
corresponding methyl esters using an ethereal solution of diazomethane
and purification by preparative TLC or column chromatography. GC-
MS analyses were carried out on a Hewlett-Packard model 5890 series
II/5989A apparatus equipped with a 15 m × 0.25 mm i.d., 0.25 µm
Rtx-5MS fused silica capillary column (Restek Germany, Bad Hom-
burg, Germany), using the electron impact ionization (EI, 70 eV) mode.
Alternatively, separations were carried out on a 15 m × 0.25 mm i.d.,
0.1 µm 400-1HT (Quadrex Corp.) fused silica capillary column. The
separation conditions were identical for both columns. The carrier gas
was He at a flow rate of 1.0 mL/min. The column temperature was
initially kept at 140 °C for 2 min and then programmed from 140 to
180 °C at 5 °C/min, held there for 5 min, and then programmed from
180 to 360 °C at 20 °C/min; the final temperature was held for 9 min.
Other operating conditions were split/splitless injector in split mode
Long-chain alkyl 3-phenylpropanoates, alkyl 3-phenylpropenoates,
and 3-phenylpropyl alkanoates were separated on a 250 mm × 4 mm
i.d., 5 µm LiChrospher RP-18e column (Phenomenex, Aschaffenburg,

2972 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Vosmann et al.
natural phenolic acids occurring ubiquitously in all parts of
plants. These hydroxylated cinnamic acids gain importance
because of their antioxidant capacity and their beneficial effects
on human health. Phenolics including hydroxylated cinnamic
acids predominantly occur as hydrophilic compounds. Their use
as food additives is limited, therefore, to hydrophilic phases in
foods. Lipophilization of hydroxy cinnamic acids derivatives
is of great importance in extending their field of applications
to fatty food phases (12).
Table 2. RP-HPLC Retention Times of Various Medium- and
Long-Chain Alkyl 3-Phenylpropanoates, Alkyl 3-Phenylpropenoates,
and 3-Phenylpropyl Alkanoates and Starting Materials
alkyl 3-phenylpropanoates, alkyl 3-phenylpropenoates,
3-phenylpropyl alkanoates, and starting materialsa
retention
time (min)
Method Ab
oleyl dihydrocinnamate
oleyl cinnamate
oleyl 2-methoxycinnamate
oleyl 3-methoxycinnamate
oleyl 4-methoxycinnamate
oleyl 3,4-dimethoxycinnamate
oleyl ferulate
14.9
16.6
15.7
15.0
14.2
11.7
4.7
Lipase-catalyzed lipophilization of phenolic antioxidants,
particularly hydroxycinnamic acids and similar compounds with
analogous or inverse chemical structure (Figure 1), by esteri-
fication of various 3-phenylpropanoic acids with long-chain fatty
alcohols or of fatty acids with various 3-phenylpropanols is of
special interest for their application as lipophilic antioxidants
in oil-based food and feed as well as in cosmetic and technical
products. Recently, we have shown that plant steryl and stanyl
esters can be efficiently prepared from sterols and stanols via
lipase-catalyzed esterification with fatty acids under environ-
mentally friendly conditions, particularly in the absence of
organic solvents and drying reagents such as molecular sieves
or sodium sulfate (46, 47). In continuation of the above work,
we have developed an enzymatic method for the preparation of
lipophilic alkyl esters of 3-phenylpropanoic (dihydrocinnamic)
and 3-phenylpropenoic (cinnamic) acid derivatives by esterifi-
cation with medium- or long-chain fatty alcohols. Similarly,
compounds with inverse chemical structure, that is, 3-phenyl-
propyl alkanoates, are prepared by lipase-catalyzed esterification
of long-chain fatty acids with various 3-phenylpropan-1-ols. The
reactions proceed in vacuo at moderate temperature in the
absence of organic solvents and drying reagents using im-
mobilized lipases as biocatalysts.
methyl ferulate
2.7
oleyl alcohol
5.9
3-(4-hydroxyphenyl)propyl oleate
3-(4-hydroxyphenyl)propyl palmitate
3-phenylpropyl (dihydrocinnamyl) palmitate
3-phenylpropenyl (cinnamyl) palmitate
3-(3,4-dimethoxyphenyl)propyl palmitate
8.7
9.0
15.0
13.2
10.7
Method Bb
oleyl 2-hydroxycinnamate
oleyl 3-hydroxycinnamate
oleyl 4-hydroxycinnamate
2-ethylhexyl ferulate
dodecyl ferulate
18.6
17.5
17.4
5.4
9.3
palmityl ferulate
oleyl ferulate
oleyl dihydrocaffeate
oleyl caffeate
oleyl sinapate
18.3
17.3
12.5
14.2
15.4
Method Cb
cis-ferulic acidc
6.0
5.6
trans-ferulic acidc
a Carboxylic acids were converted to the methyl esters by reaction with
diazomethane. b For details see Materials and Methods. c Separated as free
carboxylic acids.
Enzyme Screening. Various esterases, particularly lipases,
were checked for the esterification reaction of 4-methoxycin-
namic acid with oleyl alcohol (cis-9-octadecen-1-ol) as the
screening system. The conversions were performed for 24 h at
50 and/or 80 °C in vacuo (80 kPa) without solvent using 50
mg of the respective enzyme preparation. The results of these
experiments demonstrated that immobilized lipase B from
Candida antarctica (Novozym 435) was the biocatalyst with
highest enzyme activity for the above esterification reaction (data
not shown). A similar esterification activity was determined for
immobilized lipase from Rhizomucor miehei (Lipozyme RM
IM), whereas all other enzymes of the screening, including
immobilized lipase from Thermomyces lanuginosus (Lipozyme
TL IM), lipases from Candida rugosa, Candida lipolytica,
Chromobacterium Viscosum, Rh. arrhizus, Rh. niVeus, Rh.
jaVanicus, F. oxysporum, and pectinase from Aspergillus niger
as well as plant esterases/proteases including papaya (Carica
papaya) latex, bromelain from Ananassa comosus, and ficin
from F. carica, demonstrated very low esterification activity,
if any, under the screening conditions described above (data
not shown).
(split 1:10, temperature 360 °C), interface temperature of 360 °C, and
ion source temperature of 200 °C.
For example, the following molecular ions and important mass
fragments were observed: m/z oleyl cinnamate 398 [M]+, 149 [M -
C18H33O]+, 131 [M - C18H35O2]+; oleyl dihydrocinnamate 400 [M]+,
149 [M - C18H35]+, 133 [M - C18H35O]+; oleyl 2-hydroxycinnamate,
414 [M]+, 165 [M - C18H33]+, 146 [M - C18H36O]+; 3-hydroxy- and
4-hydroxycinnamates 414 [M]+, 164 [M - C18H34]+, 147 [M -
C18H35O]+; palmityl 2-hydroxy-, 3-hydroxy-, and 4-hydroxycinnamates
388 [M]+, 164 [M - C16H32]+, 147 [M - C16H33O]+; palmityl
2-methoxy-, 3-methoxy-, and 4-methoxycinnamates 402 [M]+, 178 [M
- C16H32]+, 161 [M - C16H33O]+; oleyl 2-methoxy-, 3-methoxy-, and
4-methoxycinnamates 428 [M]+, 178 [M - C18H34]+, 161 [M -
C18H35O]+; oleyl 3,4-dimethoxycinnamate 458 [M]+, 208 [M -
C18H34]+, 191 [M - C18H35O]+; oleyl sinapate 474 [M]+, 224 [M -
C18H34]+, 207 [M - C18H35O]+, 167 [M - C20H35O2]+; 2-ethylhexyl
ferulate 306 [M]+, 194 [M - C8H16]+, 177 [M - C8H16O]+; dodecyl
ferulate 362 [M]+, 194 [M - C12H24]+, 177 [M - C12H25O]+; palmityl
ferulate 418 [M]+, 194 [M - C16H32]+, 177 [M - C16H33O]+; oleyl
ferulate 444 [M]+, 194 [M - C18H34]+, 177 [M - C18H35O]+; oleyl
dihydrocaffeate 432 [M]+, 182 [M - C18H34]+, 165 [M - C18H35O]+;
oleyl caffeate 430 [M]+, 180 [M - C18H34]+, 163 [M - C18H35O]+;
3-phenylpropyl oleate 400 [M]+, 118 [M - C18H34O2]+; 3-phenylpropyl
palmitate 374 [M]+, 118 [M - C16H32O2]+; 3-phenylprop-2-enyl
palmitate (cinnamyl palmitate) 372 [M]+, 239 [M - C9H9O]+, 133 [M
- C16H31O]+, 117 [M - C16H31O2]+; 3-(3,4-dimethoxyphenyl)propyl
palmitate 434 [M]+, 196 [M - C16H30O]+, 178 [M - C16H31O2]+; 3-(4-
hydroxyphenyl)propyl oleate 416 [M]+, 134 [M - C18H34O2]+; 3-(4-
hydroxyphenyl)propyl palmitate 390 [M]+, 134 [M - C16H32O2]+.
Lipase Specificities. The time course of the esterification of
cinnamic acid with oleyl alcohol at 80 °C using three com-
mercial immobilized lipases, Novozym 435, Lipozyme RM IM,
and Lipozyme TL IM, is shown in Figure 2. It is obvious from
these results that Novozym 435 shows the highest conversion
rates, whereas moderate and very low esterification rates were
obtained for Lipozyme RM IM and Lipozyme TL IM, respec-
tively, as biocatalysts.
RESULTS AND DISCUSSION
Figure 3 shows the time course of the esterification of various
dihydrocinnamic (3-phenylpropanoic) and cinnamic (3-phenyl-
propenoic) acids with oleyl alcohol at 80 °C in vacuo using
3-Phenylpropenoic (cinnamic) acid analogues such as 4-hy-
droxycinnamic (p-coumaric), ferulic, and sinapic acids are

Preparation of Alkyl Phenylpropanoates
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2973
(3-phenylprop-2-enoic acid) leads to a large decrease of lipase
activity (Figure 3A); (c) introduction of a methoxy group
(Figure 3B) or a hydroxy group (Figure 3C) on the phenyl
ring leads to further loss of lipase activity; and (d) the presence
of more than one methoxy and/or hydroxy group in the substrate
molecule (e.g., ferulic, caffeic, and sinapic acids) results in an
additional reduction of enzyme activity as demonstrated in
Figure 3D and Table 3.
Table 3 shows the enzyme activities of three commercial
immobilized lipases and maximum conversions for the esteri-
fication of various dihydrocinnamic and cinnamic acids with
medium- or long-chain fatty alcohols and of fatty acids with
various 3-phenylpropanols and 3-phenylpropenols under dif-
ferent reaction conditions. These data demonstrate that im-
mobilized lipase B from C. antarctica (Novozym 435) is
generally superior to immobilized lipase preparations from Rh.
miehei (Lipozym RM IM) and, particularly, Th. lanuginosus
(Lipozyme TL IM) in the production of alkyl dihydrocinnamates
and cinnamates. Together with the results shown in Figure 3
the following relative esterification activities are found for
Novozym 435: dihydrocinnamic > cinnamic > 3-methoxycin-
namic > dihydrocaffeic ≈ 3-hydroxycinnamic > 4-methoxy-
cinnamic > 2-methoxycinnamic > 4-hydroxycinnamic > ferulic
≈ 3,4-dimethoxycinnamic > 2-hydroxycinnamic acid. With
respect to the position of substituents on the phenyl moiety,
the esterification activity of Novozym 435 increases in the order
3- > 4- > 2-position (meta > para > ortho) (Figure 3B,C). It
is worth noting that the enzyme activity of Lipozyme RM IM
is similar to or even higher than that of Novozym 435 for the
esterification of meta-substituted cinnamic acids including
3-hydroxy-, 3-methoxy-, and 3,4-dimethoxycinnamic acids with
oleyl alcohol. However, conversions found for Novozym 435-
catalyzed esterifications of cinnamic acid analogues are clearly
higher than those observed for Lipozyme RM IM-catalyzed ones
(Table 3). The Novozym 435-catalyzed esterification of long-
chain fatty acids with various 3-phenylpropanols generally
produces the corresponding 3-phenylpropyl alkanoates in high
yield (Table 3).
Figure 2. Time course of the formation of oleyl cinnamate by esterification
of cinnamic acid with oleyl alcohol catalyzed by various immobilized
microbial lipases (
TL IM). Reaction conditions: molar ratio of cinnamic acid to oleyl alcohol,
1:1; 50 mg of immobilized lipase; 80 C. Values are the mean of two
], Novozym 435; 0, Lipozyme RM IM; 4, Lipozyme
°
determinations. For details see Materials and Methods.
Reaction Conditions. Generally, enzyme activity for the
esterification of cinnamic acid and its analogues with oleyl
alcohol was highest using immobilized lipase B from C.
antarctica (Novozym 435) at 80 °C in vacuo. Moderate
esterification activity is observed for immobilized lipase from
Rh. miehei (Lipozyme RM IM) and rather low activity for
immobilized lipase from Th. lanuginosus (Lipozyme TL IM)
(Table 3 and Figure 2). All other lipases and esterases screened
as described above show far lower esterification activities, if
any (data not shown).
Figure 3. Time course of the formation of oleyl esters of cinnamic acid
and its analogues including
cinnamate (A); , 2-methoxy-,
namate (B); and , 2-hydroxy-,
]
, oleyl dihydrocinnamate, and
, 3-methoxy-, and , 4-methoxycin-
, 3-hydroxy-, and, , 4-hydroxycin-
0, oleyl
]
]
0
0
4
4
namate (C), by Novozym 435-catalyzed esterification of dihydrocinnamic,
cinnamic, methoxycinnamic, and hydroxycinnamic acids with oleyl alcohol.
Time course of the formation of oleyl ferulate (D) by esterification of ferulic
acid with oleyl alcohol using different amounts of Novozym 435 lipase
(], 50 mg; 0, 100 mg; 4, 250 mg). Values are the mean of two
determinations. For details see Materials and Methods.
Depending on the reaction conditions, conversions to alkyl
dihydrocinnamates and cinnamates as well as 3-phenylpropyl
alkanoates of up to 98 mol % were obtained by esterification
of various dihydrocinnamic and cinnamic acids with medium-
and long-chain fatty alcohols such as lauryl, palmityl, and oleyl
alcohol or of 3-phenylpropanols with long-chain fatty acids such
as palmitic and oleic acids catalyzed by Novozym 435 lipase
in vacuo. Increasing the reaction temperature from 60 to 80 °C
increased the enzyme activity for the formation of various oleyl
3-phenylpropanoates by 2-6-fold as shown for the esterification
of cinnamic and dihydro-, methoxy-, and hydroxycinnamic acids
with oleyl alcohol (Table 3).
Novozym 435 as biocatalyst. In particular, the formation of oleyl
dihydrocinnamate and oleyl cinnamate (Figure 3A), various
oleyl methoxy- and hydroxycinnamates, for example, 2-meth-
oxy-, 3-methoxy-, and 4-methoxycinnamates (Figure 3B),
2-hydroxy-, 3-hydroxy-, and 4-hydroxycinnamates (Figure 3C),
and oleyl ferulate (4-hydroxy-3-methoxycinnamate) (Figure
3D), by Novozym 435-catalyzed esterification of the corre-
sponding cinnamic acids with oleyl alcohol is demonstrated over
a period of 72 or 144 h. It is obvious from these results that (a)
highest esterification activity is observed with dihydrocinnamic
acid (3-phenylpropanoic acid) as the substrate (Figure 3A); (b)
introduction of a >CdC< double bond to form cinnamic acid
Under the reaction conditions described, equimolar mixtures
of substrates or relatively low excess of one of the substrates is
necessary to obtain acceptable yields of methoxy- and/or
hydroxy-substituted alkyl cinnamates, for example, oleyl 4-hy-

2974 J. Agric. Food Chem., Vol. 54, No. 8, 2006
Vosmann et al.
Table 3. Enzyme Activities of Various Lipases for the Esterification of 3-Phenylpropenoic (Cinnamic) Acid and Its Analogues with Medium- or
Long-Chain Saturated and Unsaturated Fatty Alcohols and for the Esterification of Alkanoic Acids with 3-Phenylpropanol Analogues
3-phenylpropenoic (cinnamic) acid
and its analogues and fatty acids
medium- and long-chain fatty
alcohols and 3-phenylpropanols
max conversiona
(mol %) after [h]
enzyme activitya
(units/g)
±
(n
SEM
lipase
)
x)
dihydrocinnamic acid
dihydrocinnamic acid
dihydrocinnamic acid
dihydrocinnamic acid
cinnamic acid
cinnamic acid
cinnamic acid
cinnamic acid
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
2-ethylhexan-1-ol
Novozym 435
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Novozym 435
Lipozyme RM IM
Lipozyme TL IM
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Lipozyme RM IM
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
Novozym 435
98 [4]
98b
22.9c
22.8d
2.8
±
±
0.28 (n
1.6 (n
0.03 (n
0.05 (n
0.48 (n
0.79 (n
0.64 (n
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
2)
2)
2)
2)
3)
2)
2)
2)
3)
2)
2)
2)
4)
2)
3)
2)
3)
2)
2)
2)
2)
2)
2)
2)
4)
2)
2)
2)
2)
4)
2)
3)
2)
3)
5)
3)
2)
4)
2)
2)
2)
2)
2)
2)
2)
2)
2)
2)
2)
2)
2)
96 [72]c
91 [4]
±
±
±
±
±
57 [72]
94 [24]
96 [72]c
26 [72]
5 [72]
51 [72]
12 [72]c
7 [72]
<1 [72]
96 [48]
95 [72]c
58 [72]
4 [72]
94 [72]
22 [72]c
35 [72]
2 [72]
8 [144]
<1 [144]
<1 [144]
94 [144]
28 [144]
<1 [144]
65 [144]
10 [144]
<1 [144]
25 [72]
4 [72]c
6.0
2.7c
3.1
ndi
−
(n
2-methoxycinnamic acid
2-methoxycinnamic acid
2-methoxycinnamic acid
2-methoxycinnamic acid
3-methoxycinnamic acid
3-methoxycinnamic acid
3-methoxycinnamic acid
3-methoxycinnamic acid
4-methoxycinnamic acid
4-methoxycinnamic acid
4-methoxycinnamic acid
4-methoxycinnamic acid
2-hydroxycinnamic acid
2-hydroxycinnamic acid
2-hydroxycinnamic acid
3-hydroxycinnamic acid
3-hydroxycinnamic acid
3-hydroxycinnamic acid
4-hydroxycinnamic acid
4-hydroxycinnamic acid
4-hydroxycinnamic acid
3,4-dimethoxycinnamic acid
3,4-dimethoxycinnamic acid
3,4-dimethoxycinnamic acid
3,4-dimethoxycinnamic acid
ferulic acid
ferulic acid
ferulic acid
ferulic acid
ferulic acid
ferulic acid
ferulic acid
dihydrocaffeic acid
caffeic acid
sinapic acid
oleic acid
palmitic acid
oleic acid
palmitic acid
oleic acid
palmitic acid
oleic acid
palmitic acid
0.9
±
0.15 (n
ndc
nd
−
−
−
(n
(n
(n
nd
4.9
±
±
±
0.56 (n
0.15 (n
0.39 (n
1.8c
5.6
nd
− (n
1.8
±
±
±
0.25 (n
0.01 (n
0.14 (n
0.3c
0.9
nd
nd
nd
nd
3.3
1.9
nd
0.6
−
−
−
−
(n
(n
(n
(n
±
±
0.40 (n
0.26 (n
− (n
0.01 (n
0.03 (n
±
±
0.6
nd
−
(n
0.06 (n
(n
0.26 (n
(n
0.3
±
±
ndc
1.3
−
27 [72]
2 [72]
nd
2.0
−
73 [144]
27 [72]
15 [72]
25 [72]
82 [144]f
94 [144];g 98 [48]h
27 [72]f
30 [72]
33 [144]
23 [144]g
79 [4]
85 [24]
93 [4]
93 [4]
90 [4]
96 [8]
±
±
±
±
±
±
±
±
0.17 (n
0.03 (n
0.04 (n
0.03 (n
0.03 (n
0.02 (n
0.01 (n
0.15 (n
0.01 (n
dodecan-1-ol
hexadecan-1-ol
0.3e
0.3e
0.4e
0.5e,f
0.4e,g
0.3e,f
3.4
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cinnamic alcohol
0.1g
nd
−
(n
0.21 (n
2.3 (n
0.03 (n
19.7
11.5
23.3
23.3
22.4
22.0
21.8
19.4
±
±
±
±
±
±
cinnamic alcohol
3-phenylpropan-1-ol
3-phenylpropan-1-ol
3-(4-hydroxyphenyl)propan-1-ol
3-(4-hydroxyphenyl)propan-1-ol
3-(3,4-dimethoxyphenyl)propan-1-ol
3-(3,4-dimethoxyphenyl)propan-1-ol
0.19 (n
0.61 (n
0.25 (n
2.6 (n
3.2 (n
91 [8]
85 [8]
±
±
a Standard assay conditions, if not otherwise indicated: 0.3 mmol of carboxylic acid
b Twelve and a half milligrams of Novozym 435. c Maximum conversion and enzyme activity at 60
+
0.3 mmol of alcohol; immobilized lipase/assay, 50 mg; 80
°C; 80 kPa; 4 h.
°
C. d Enzyme activity after 2 h: 45 units/g. e Enzyme activity after 24
h. f One hundred milligrams of immobilized lipase preparation. g Two hundred and fifty milligrams of Novozym 435. h Molar ratio ferulic acid to oleyl alcohol, 1:2 or 2:1; 250
mg of Novozym 435. i nd, not determined.
droxycinnamate and oleyl ferulate (Table 3), which differs from
the enzyme-catalyzed esterification methods developed by others
(12, 37-43). Moreover, the procedure described above for the
Novozym 435-catalyzed esterification of various substituted
cinnamates including hydroxycinnamates requires much less
time as compared to those reported by others (37, 39-42). Large
amounts of immobilized lipase B from C. antarctica (Novozym
435) are necessary, however, to obtain satisfactory yields,
particularly of alkyl cinnamates bearing more than one methoxy
or hydroxy group (Table 3 and Figure 3). This was found in
previous studies as well (12, 37, 39, 40). Further research will
deal with experiments reducing the enzyme concentration for
the preparation of methoxy- and hydroxy-substituted alkyl
dihydrocinnamates and cinnamates as well as 3-phenylalkyl
alkanoates.
The molecular ions and typical mass fragments of the
esterification products, that is, alkyl cinnamates and cinnamyl
alkanoates, obtained by GC-MS confirmed the chemical struc-
tures of these compounds as given above. These data agree well
with those given in the literature (32).
In conclusion, we have developed a lipase-catalyzed esteri-
fication procedure for the preparation of lipophilic alkyl
cinnamates and similar compounds with inverse chemical
structure, particularly hydroxy- and/or methoxy-substituted alkyl

Preparation of Alkyl Phenylpropanoates
J. Agric. Food Chem., Vol. 54, No. 8, 2006 2975
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Received for review January 6, 2006. Revised manuscript received
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