7064 J. Agric. Food Chem., Vol. 54, No. 19, 2006
Weitkamp et al.
to those described above for the transesterification of hydroxylated
cinnamic acid derivatives using Novozym 435 lipase preparation as
biocatalyst.
410 °C at 20 °C/min, and finally kept at 410 °C for 2 min. Injector
and detector temperatures were maintained at 380 °C.
Lipid extracts from transesterification reactions of methyl sinapate
with oleyl alcohol were separated on a 12 m × 0.22 mm i.d., 1 µm
HT5 AQ fused silica capillary column (SGE, Darmstadt, Germany)
with hydrogen as the carrier gas. The following temperature program
was used: initially at 100 °C (2 min), followed by linear programming
from 100 to 180 °C at 5 °C/min, then from 180 to 250 °C at 10 °C/
min, and finally from 250 to 410 °C at 20 °C/min (6 min).
Peaks in gas chromatograms were assigned by comparison of their
retention times with those of peaks from standards. Peak areas and
percentages were calculated using Hewlett-Packard GC ChemStation
software. For the determination of enzyme activities, small proportions
of methoxy compounds, which had been formed during carboxy-
methylation of hydroxycinnamic acids with diazomethane, were
calculated as the original hydroxy compounds.
In one set of experiments, transesterifications of methyl ferulate (62.4
mg, 0.3 mmol) were carried out with cis-9-octadecen-1-ol (80.4 mg,
0.3 mmol) catalyzed by Novozym 435 (100 mg) under partial vacuum
at 80 °C for 8 h. Thereafter, the biocatalyst was extracted twice with
diethyl ether, dried in vacuo (20 min, 40 °C), and used repeatedly for
nine consecutive reactions under identical conditions using fresh
substrate mixture each time. Loss of Novozym 435 enzyme preparation
was replaced by fresh catalyst.
Enzyme units were calculated from the initial rates (0.5, 1, or 4 h)
of transesterification of methyl or ethyl (hydroxy)cinnamates with fatty
alcohols. Similarly, enzyme units were determined for the transesteri-
fication of fatty acid methyl esters with 3-phenylpropanol analogues.
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 Aliquots were withdrawn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 compounds
were methylated (20 min, ∼5 °C) using a ∼0.2 M solution of
diazomethane in diethyl ether. The conversion by transesterification
was checked by TLC on 0.3 mm layers of silica gel H (VWR
International, Darmstadt, Germany), and spots were located by iodine
staining and, if required, by charring after spraying with 30% (v/v)
sulfuric acid followed by heating (200 °C). Isohexane-diethyl ether
(1:1) was used as solvent system as described previously (31). Similarly,
0.5 mm layers of silica gel H were used for the separation of reaction
products by preparative TLC. Reaction mixtures containing phenolic
hydroxy compounds were separated as described above. The various
fractions were scraped off the plates and extracted from silica gel using
water-saturated diethyl ether.
Purification. Alkyl 3-phenylpropanoates and 3-phenylpropyl al-
kanoates were extracted from the immobilized biocatalysts with diethyl
ether and purified by chromatography on a silica gel 60 (VWR
International) column (25 × 2 cm i.d.), using mixtures of isohexane-
diethyl ether as described previously (31). The purification of oleyl
ferulate is given as an example. Around 350 mg of the reaction mixture
dissolved in 1.5 mL of diethyl ether was applied to the column and
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 30 mL of isohexane-diethyl ether (7:3) yielded ∼30 mg of a
mixture of oleyl alcohol and oleyl ferulate, whereas 270 mg of oleyl
ferulate was obtained by elution with 30 mL of isohexane-diethyl ether
(1:1). In addition, the reaction products were purified by crystallization
from isohexane or mixtures of isohexane-diethyl ether (1:1).
Gas Chromatography (GC). Aliquots of esterification products
were removed from the reaction mixture, dissolved in diethyl ether,
and filtered through a 1.0 µm syringe filter to remove the lipase
catalysts. The filtrate was concentrated in a stream of nitrogen at 40-
50 °C, dissolved in diethyl ether, and treated with an ethereal solution
of diazomethane to convert small proportions of hydrolyzed carboxy
groups to the corresponding methyl esters. The resulting mixtures of
hydroxylated cinnamic acid methyl esters, unreacted alkanols, and
3-phenylpropanols as well as medium- and long-chain alkyl hydroxy-
cinnamates and 3-phenylpropyl alkanoates were 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 (33) to avoid coumarin formation during high-temperature GC.
Similarly, phenolic hydroxy groups of hydrocaffeic and caffeic acid
derivatives were (partially) methylated to improve FID response. A
Hewlett-Packard (Bo¨blingen, Germany) HP-5890 series II gas chro-
matograph equipped with a flame ionization detector was used.
Separations were carried out on a 15 m × 0.25 mm i.d., 0.1 µm Quadrex
400-1HT fused silica capillary column (Quadrex Corp., New Haven,
CT) using hydrogen as the carrier gas (column pressure ) 50 kPa).
The following temperature program was used to separate the various
compounds of reaction mixtures: initially at 120 °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
GC-MS Analyses. The fragmentation of the various alkyl 3-phenyl-
propanoates or 3-phenylpropyl alkanoates, formed by lipase-catalyzed
esterification reactions, was studied by GC-MS (EI mode) as described
previously (31).
RESULTS AND DISCUSSION
Phenolics, including hydroxylated cinnamic acids, gain
importance because of their antioxidant capacity and their
proposed beneficial effects on human health. Predominantly,
they appear as hydrophilic compounds in the hydrophilic phases
of foods. Lipophilization of hydroxylated cinnamic acids such
as p-coumaric and ferulic acids is, therefore, of great importance
to extend their field of applications to fatty food phases (14,
34). Lipase-catalyzed lipophilization of phenolic antioxidants,
such as hydroxylated cinnamic acids, by transesterification of
the corresponding methyl esters with fatty alcohols is of special
interest for their application as lipophilic antioxidants in oil-
based food. Similarly, compounds with inverse chemical
structure may be prepared from fatty acid methyl esters and
hydroxylated phenylpropan-1-ols. Recently, we have shown that
various medium- and long-chain hydroxycinnamic acid esters
can be efficiently prepared from hydroxycinnamic acid ana-
logues via lipase-catalyzed esterification with fatty alcohols. This
reaction is performed under environmentally friendly conditions,
particularly at moderate temperature and in the absence of
organic solvents and drying reagents such as molecular sieves
or sodium sulfate (31). In continuation of the above work we
have optimized this enzymatic method for the preparation of
lipophilic alkyl esters of hydroxylated cinnamic acids by using
transesterification of methyl or ethyl esters of hydroxylated
cinnamic acids with medium- or long-chain alcohols. Similarly,
compounds with inverse chemical structure such as 3-(4-
hydroxyphenyl)propyl alkanoates are prepared by lipase-
catalyzed transesterification of fatty acid methyl esters with 3-(4-
hydroxyphenyl)propan-1-ol or 3-(3,4-dimethoxyphenyl)propan-
1-ol.
The activity of three immobilized commercial lipases, that
is, Novozym 435, Lipozyme RM IM, and Lipozyme TL IM,
was checked for the transesterification of equimolar mixtures
of various short-chain alkyl (hydroxy)cinnamates and medium-
or long-chain alcohols. The conversions were performed for up
to 72 h at 80 °C under partial vacuum without solvent and drying
agent in direct contact with the reaction mixture using different
amounts of the respective enzyme. The time course of the
transesterification of methyl cinnamate with oleyl alcohol (cis-
9-octadecen-1-ol) catalyzed by the above lipases is shown in
Figure 3. The results of these experiments demonstrated that
immobilized lipase B from C. antarctica (Novozym 435) was
the biocatalyst with highest enzyme activity for the transesteri-
fication reactions. Lower transesterification activity was deter-