Lipophilic (hydroxy)phenylacetates by solvent-free lipase-catalyzed esterification and transesterification in vacuo

Article abstract of DOI:10.1021/jf8002224

Various long-chain alkyl (hydroxy)phenylacetates were prepared in high yield by lipase-catalyzed transesterification of the corresponding short-chain alkyl hydroxyphenylacetates and fatty alcohols in equimolar ratios. The reactions were performed in vacuo at moderate temperatures in the absence of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from Candida antarctica (Novozym 435) was the most effective biocatalyst for the various transesterification reactions. Generally, Novozym 435-catalyzed transesterifications of short-chain alkyl (hydroxy)phenylacetates with long-chain alcohols led to higher conversions and enzyme activities than the corresponding esterifications. For example, the transesterification activity was up to 4-fold higher than the esterification activity for the formation of oleyl 4-hydroxy-3-methoxyphenylacetate using Novozym 435 as a biocatalyst. The relative transesterification activities were as follows: phenylacetate > 3-methoxyphenylacetate ≈ 4- methoxyphenylacetate > 4-hydroxy-3-methoxyphenylacetate > 3-hydroxyphenylacetate ≈ 4-hydroxyphenylacetate ? 2-methoxyphenylacetate ? 3,4-dihydroxyphenylacetate. With respect to the position of methoxy and hydroxy substituents, the transesterification activity of Novozym 435 decreased in the order meta ≈ para ? ortho. Compounds with inverse chemical structures, for example, tyrosyl oleate, were obtained by Novozym 435-catalyzed esterification and transesterification of fatty acids and their methyl esters, respectively, with 2-phenylethan-1-ols. In contrast to the transesterifications of short-chain alkyl (hydroxy)phenylacetates with fatty alcohols, higher conversions and enzyme activities were observed for the Novozym 435-catalyzed esterifications of (hydroxy)phenylethanols with long-chain fatty acids than the corresponding transesterifications with fatty acid methyl esters.

Full text of DOI:10.1021/jf8002224

J. Agric. Food Chem. 2008, 56, 5083–5090 5083
Lipophilic (Hydroxy)phenylacetates by Solvent-Free
Lipase-Catalyzed Esterification and
Transesterification in Vacuo
PETRA WEITKAMP, NIKOLAUS WEBER, AND KLAUS VOSMANN*
Max-Rubner-Institut, Bundesforschungsinstitut fu¨r Erna¨hrung und Lebensmittel, Piusallee 68-76,
D-48147 Mu¨nster, Germany
Various long-chain alkyl (hydroxy)phenylacetates were prepared in high yield by lipase-catalyzed
transesterification of the corresponding short-chain alkyl hydroxyphenylacetates and fatty alcohols
in equimolar ratios. The reactions were performed in vacuo at moderate temperatures in the absence
of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from
Candida antarctica (Novozym 435) was the most effective biocatalyst for the various transesterification
reactions. Generally, Novozym 435-catalyzed transesterifications of short-chain alkyl (hydroxy)phe-
nylacetates with long-chain alcohols led to higher conversions and enzyme activities than the
corresponding esterifications. For example, the transesterification activity was up to 4-fold higher
than the esterification activity for the formation of oleyl 4-hydroxy-3-methoxyphenylacetate using
Novozym 435 as a biocatalyst. The relative transesterification activities were as follows: phenylacetate
> 3-methoxyphenylacetate ≈ 4-methoxyphenylacetate > 4-hydroxy-3-methoxyphenylacetate >
3-hydroxyphenylacetate ≈ 4-hydroxyphenylacetate . 2-methoxyphenylacetate . 3,4-dihydroxyphe-
nylacetate. With respect to the position of methoxy and hydroxy substituents, the transesterification
activity of Novozym 435 decreased in the order meta ≈ para . ortho. Compounds with inverse
chemical structures, for example, tyrosyl oleate, were obtained by Novozym 435-catalyzed esterifi-
cation and transesterification of fatty acids and their methyl esters, respectively, with 2-phenylethan-
1-ols. In contrast to the transesterifications of short-chain alkyl (hydroxy)phenylacetates with fatty
alcohols, higher conversions and enzyme activities were observed for the Novozym 435-catalyzed
esterifications of (hydroxy)phenylethanols with long-chain fatty acids than the corresponding
transesterifications with fatty acid methyl esters.
KEYWORDS: 2-(4-Hydroxyphenyl)ethyl oleate (tyrosyl oleate); palmityl 4-hydroxyphenylacetate; oleyl
4-methoxyphenylacetate; immobilized lipase B; Candida antarctica; Novozym 435; solvent-free; esterifi-
cation; transesterification
INTRODUCTION
further improved by esterification with fatty alcohols leading
to an increase of lipophilicity (13–17). The range of applications
of such lipophilic antioxidants may be extended by their possible
use as additives for food and technical applications (14, 18, 19).
Similarly, compounds with inverse chemical structures such as
fatty acid esters of hydroxyphenylethanols, for example, tyrosyl
alkanoates, also may have antioxidant and biological activities
as known for alkyl hydroxyphenylacetates (13, 20).
Common plant-derived foods only contain low proportions
of lipophilic antioxidants. Generally, the ratio of lipophilic to
hydrophilic antioxidant capacity is <1:10 in fruits and vegetables
(1). Plant antioxidants include ω-(hydroxyphenyl)alkanoic acids
such as hydroxybenzoic, hydroxycinnamic, and hydroxyphe-
nylacetic acids (2–7). The analogous ω-(hydroxyphenyl)al-
kanols, for example, 4-hydroxyphenylethanol (tyrosol) and
3-hydroxytyrosol, have also been detected in fruits and veg-
etables, particularly olives (5, 8–10). Beneficial effects on health
have been attributed to the antioxidant capacity of such plant
phenolic acids, particularly against oxidative attacks by radical-
scavenging activity (9, 11, 12). Both the antioxidant capacity
and the bioavailability of several of these compounds may be
Enzymatic esterification of hydroxyphenylalkanoic acids may
be of advantage over chemical esterification for the preparation
of lipophilic alkyl hydroxyphenylalkanoates, particularly for
food use, particularly because no hazardous chemicals are used
for the preparation. Until now, enzymatic esterification and
transesterification procedures requiring organic solvents or using
the alcohol component as the solvent have been reported for
the preparation of various alkyl esters of hydroxyphenylalkanoic
acids including alkyl cinnamates, phenylacetates, and benzo-
* To whom correspondence should be addressed. Tel: +49-251-
48167-0. Fax: +49-251-48167-60. E-mail: klaus.vosmann@mri.bund.de.
10.1021/jf8002224 CCC: $40.75  2008 American Chemical Society
Published on Web 06/10/2008

5084 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Weitkamp et al.
concentrated to dryness, dissolved in 2 mL of dichloromethane, and
filtered through a 0.45 µm PTFE syringe filter to separate the biocatalyst.
An aliquot of the filtrate was analyzed as given below. Similar reaction
conditions were used for the preparation of phenylethyl alkanoates with
inverse chemical structures. As a typical example, oleic acid (84.6 mg,
0.3 mmol) was esterified with 2-(4-hydroxyphenyl)ethanol (41.4 mg,
0.3 mmol) under identical conditions as described above for the
transesterification of hydroxylated phenylacetic acid derivatives using
Novozym 435 lipase preparation as a biocatalyst.
Similarly, the conversions of analogous compounds were studied
under competitive conditions using equimolar mixtures of, for example,
methyl 4-hydroxyphenylacetate + methyl 4-hydroxyhydrocinnamate
or methyl 4-hydroxybenzoate + methyl 4-hydroxyhydrocinnamate.
Blanks were performed under standard assay conditions without lipase.
Enzyme units were calculated from the initial rates (usually 0.5 h)
of esterification or transesterification reactions. One unit of enzyme
activity was defined as the amount of enzyme (g) that produced 1 µmol/
min of the respective alkyl (hydroxy)phenylacetate or fatty acid
(hydroxy)phenylethyl ester.
Thin-Layer Chromatography (TLC). Aliquots were withdrawn
from the reaction mixtures, and free carboxy groups of compounds
were methylated (∼15 min, 20 °C) using a ∼0.2 M solution of
diazomethane in diethyl ether (34). The conversion by esterification or
transesterification was checked by TLC on 0.3 mm layers of silica gel,
and spots were located by iodine staining and, if required, by charring
after spraying with 30% (v/v) sulfuric acid solution followed by heating
(200 °C). A mixture of iso-hexane-diethyl ether (1:1) was used as a
solvent system as described previously (29, 30).
Purification. Alkyl 2-phenylacetates and 2-phenylethyl alkanoates
were extracted from the immobilized biocatalysts with diethyl ether
and purified by chromatography on a silica gel 60 (VWR International)
column (25 cm × 2 cm i.d.) using mixtures of i-hexane-diethyl ether
as eluents as described previously (29, 30). The purification of oleyl
4-hydroxyphenylacetate is given as an example. The reaction mixture
(187 mg; conversion 95% as determined by GC) dissolved in 1.5 mL
of diethyl ether was applied to the column and eluted first with 20 mL
of iso-hexane and then subsequently with 20 mL portions of various
iso-hexane-diethyl ether mixtures (97:3, 95:5, 9:1, 8:2, and 7:3). Elution
with 20 mL of iso-hexane-diethyl ether (7:3) yielded oleyl 4-hydrox-
yphenylacetate (isolated yield, 148 mg and 79.1%; purity, 99%). After
column chromatography, various alkyl 2-phenylacetates and 2-phenyl-
ethyl alkanoates containing saturated alkyl or acyl moieties, respectively,
were crystallized from iso-hexane or a mixture of iso-hexane-diethyl
ether (1:1).
GC. Aliquots of esterification and transesterification products were
removed from the reaction mixture, extracted, and filtered as described
above. The filtrate was concentrated in a stream of nitrogen at 40-50
°C, dissolved in diethyl ether/methanol (10:1, v/v), and treated with
an ethereal solution of diazomethane to convert carboxylic acids to
the corresponding methyl esters. The resulting mixture of methyl
phenylacetates, unreacted 1-alkanols, 2-phenylethanols, and fatty acid
methyl esters as well as medium- and long-chain alkyl phenylacetates
or 2-phenylethyl alkanoates were analyzed by GC. The hydroxy groups
of methyl 2-hydroxyphenylacetate and methyl 3,4-dihydroxyphenylac-
etate were partly methylated to the methoxy groups by treatment with
an ethereal solution of diazomethane in the presence of catalytic
amounts of silica gel (35) in order to improve FID response. The
following relative retention times (RRt) were found using oleyl alcohol
and methyl oleate, respectively, as reference compounds: oleyl phe-
nylacetate, 1.81; oleyl 2-, 3-, and 4-methoxy phenylacetates, 2.00, 2.04,
and 2.08, respectively; oleyl 3-hydroxy- and 4-hydroxyphenylacetates,
2.10 and 2.19, respectively; oleyl 4-hydroxy-3-methoxyphenylacetate,
2.26; oleyl 3,4-dihydroxyphenylacetate, 2.35; oleyl 4-hydroxyphenoxy-
acetate, 2.31; oleyl 2-(1-naphthyl)acetate, 2.38; oleyl 2-(2-naphthy-
l)acetate, 2.45; 2-phenylethyl oleate, 1.78; 2-(1-naphthyl)ethyl oleate,
2.33; 2-(2-naphthyl)ethyl oleate, 2.34; 2-(2-hydroxyphenyl)ethyl oleate,
2.01; 2-(3-hydroxyphenyl)ethyl oleate, 2.05; 2-(4-hydroxyphenyl)ethyl
oleate, 2.06; and 2-(4-hydroxy-3-methoxyphenyl)ethyl oleate, 2.14.
Derivatization of Reaction Products by Base-Catalyzed Trans-
methylation. Under standard conditions, 2 mL of 0.5 M methanolic
sodium methylate solution was added to 5-10 mg of the long-chain
ates (21–29). Recently, we have described procedures for the
lipase-catalyzed esterification and transesterification of (hy-
droxy)cinnamic acids and methyl (hydroxy)cinnamates, respec-
tively, with medium- or long-chain alcohols, which was
performed with equimolar amounts of reactants at moderate
temperatures. Neither hazardous chemicals nor solvents nor
drying agents were used in the reaction mixtures, and water or
methanol was removed under reduced pressure (29, 30). This
method was much more efficient than other enzymatic esteri-
fication and transesterificatioin procedures described earlier (14).
The aim of the present work was to extend this simple and
environmentally friendly lipase-catalyzed preparation of lipo-
philic medium- and long-chain alkyl (hydroxy)cinnamates to
the preparation of the analogous (hydroxy)phenylacetates by
using esterification and transesterification reactions. Addition-
ally, compounds with inverse chemical structures such as
(hydroxy)phenylethyl alkanoates were prepared by esterification
and transesterification of fatty acids and their methyl esters,
respectively, with (hydroxy)phenylethanols (29–33).
MATERIALS AND METHODS
General Section. Methyl 2-methoxyphenylacetate, methyl 3,4-
dihydroxyphenylacetate, and methyl 2-(1-naphthyl)acetate were pre-
pared from the corresponding carboxylic acids by the reaction with
diazomethane (34). All other chemicals and substrates were com-
mercially available. Immobilized lipase preparations from Candida
antarctica (lipase B and Novozym 435), Rhizomucor miehei (Lipozyme
RM IM), and Thermomyces lanuginosus (Lipozyme TL IM) were kindly
provided by Novozymes (Bagsvaerd, Denmark).
Gas chromatography (GC) was carried out on a 15 m × 0.25 mm
i.d., 0.1 µm J&W DB-5HT fused silica capillary column (Agilent
Technologies, Waldbronn, Germany) using hydrogen as the carrier gas
(column pressure 80 kPa). The following temperature program was
used to separate the various compounds of reaction mixtures: initially
at 100 °C for 1 min, followed by linear programming from 100 to 280
°C at 10 °C/min, and finally kept at 280 °C for 1 min. Injector and
flame ionization detector (FID) temperatures were maintained at 380
°C. Peaks in gas chromatograms were assigned by comparison of their
retention times with those of peaks from standard preparations, which
were identified by GC-MS (see below). Peak areas and percentages
were calculated using Hewlett-Packard GC ChemStation software. For
the determination of enzyme activities, small proportions of methoxy
compounds that had been formed during methoxylation of, for example,
hydroxyphenylacetic acids with diazomethane were calculated as the
original hydroxy compounds.
An Agilent Technologies (Bo¨blingen, Germany) HP-6890 N gas
chromatograph equipped with an autosampler and FID was used for
gas chromatographic analyses. GC-MS analyses were carried out on a
Hewlett-Packard model 5890 series II/5989A apparatus equipped with
a 0.1 µm DB-1HT fused silica capillary column (J&W Scientific), 15 m
× 0.25 mm i.d., using the electron ionization (EI or CI mode, 70 eV)
mode. The carrier gas was He at a flow rate of 1.0 mL × min-1. The
column temperature was initially kept at 100 °C for 2 min and then
programmed from 100 to 320 at 10 °C × min-1, and the final
temperature was held for 8 min. Other operating conditions were split/
splitless injector in split mode (split 1:10; temperature, 320 °C), interface
temperature (320 °C), and ion source temperature (250 °C).
Lipase-Catalyzed Reactions. As a typical example, methyl 4-hy-
droxyphenylacetate (49.8 mg, 0.3 mmol) was transesterified with cis-
9-octadecen-1-ol (80.4 mg, 0.3 mmol) in the presence of 12.5-50 mg
of immobilized Novozym 435 lipase by magnetic stirring in a screw-
capped reaction tube. The tube was placed in a 100 mL Schlenk reaction
vessel under partial vacuum (∼80 kPa) at 80 °C in the dark for periods
up to 72 h with water trapping in the gas phase using potassium
hydroxide pellets. This moderate vacuum (∼80 kPa) was used to prevent
substantial loss of substrates. The experiments were performed in
duplicate (n ) 2). Samples of the reaction products were withdrawn at
various intervals and extracted with diethyl ether. The extracts were

Lipophilic (Hydroxy)phenylacetates
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5085
phenylacetic acid with oleyl alcohol is far higher than that of
Lipozyme RM IM or Lipozyme TL IM. Similar results were
previously obtained for the esterification and transesterification
of cinnamic acid derivatives (29, 30). Novozym 435 was used,
therefore, as a biocatalyst for all subsequent esterification and
transesterification reactions.
Effects of Enzyme and Temperature. The time course of
the lipase-catalyzed esterification of 4-hydroxyphenylacetic acid
with oleyl alcohol (cis-9-octadecen-1-ol) at 80 °C using different
amounts (6.3, 12.5, and 25 mg) of Novozym 435 as a biocatalyst
is shown in Figure 2. It is obvious from these results that the
conversion rate clearly increases with increasing amounts of
the immobilized lipase added to the reaction mixture.
Figure 3 shows the lipase-catalyzed transesterification of
methyl 3-methoxyphenylacetate with oleyl alcohol at different
temperatures (20, 45, and 80 °C) using Novozym 435 as a
biocatalyst. These results demonstrate that increasing the
temperature from 20 to 80 °C leads to an around 6-fold increase
of the conversion rate as observed at 0.5 h (Table 1).
Figure 1. Chemical structures of alkyl phenylacetates (1-8), alkyl
phenoxyacetates (9), and alkyl naphthylacetates (15 and 16) as well as
of structurally similar inverse phenylethyl alkanoates (10-14) and
naphthylethyl alkanoates (17 and 18) prepared by lipase-catalyzed
esterification and transesterification.
Effects of Substituents. Figure 4 shows the time course of
the Novozym 435-catalyzed esterification of 3-hydroxy- and
4-hydroxyphenylacetic acids as well as the Novozym 435-
catalyzed transesterification of methyl 3-hydroxy- and 4-hy-
droxyphenylacetates both with oleyl alcohol. It is evident from
these studies and further results shown in Table 1 that the
enzyme activity of the biocatalyst is far higher for the transes-
terification as compared to the esterification of, for example,
methyl 4-hydroxyphenylacetate and 4-hydroxyphenylacetic acid
(323 vs 165 units). Moreover, it is obvious from Figure 4A
that the esterification rate of 4-hydroxyphenylacetic acid is
higher than that of 3-hydroxyphenylacetic acid, whereas the
transesterification rates of the methyl esters of both hydrox-
yphenylacetic acids are quite similar (Figure 4B). It is worth
noting that the analogous 2-hydroxyphenylacetic acid and its
methyl ester are not esterified and transesterified at all, whereas
a phenoxy group as present in 4-hydroxyphenoxyacetic acid
does not change the esterification activity of Novozym 435 as
compared to 4-hydroxyphenylacetic acid (Table 1).
Figure 5 and Table 1 demonstrate that the enzyme activities
of Novozym 435 for the transesterification of the various methyl
methoxyphenylacetates with oleyl alcohol depend on the posi-
tion of the 2-, 3-, or 4-methoxy substituent. Under the conditions
described, transesterification activity is quite similar for methyl
phenylacetates with methoxy substituents at the 3- and 4-posi-
tions, whereas sterical hindrance by the bulky 2-methoxy group
decreases the transesterification activity of Novozym 435 to a
large extent.
alkyl (hydroxy)phenylacetates or (hydroxy)phenylethyl alkanoates and
transmethylated (34). Derivatization mixtures of phenolic compounds
were acidified before extracting. The extracts (1-2 µL) were directly
injected into the gas chromatograph. GC separation of the derivatization
products consisting of methyl (hydroxy)phenylacetates and long-chain
alcohols or fatty acid methyl esters and (hydroxy)phenylethanols was
carried out as described above. Base-catalyzed transmethylation was
used to analyze the chemical structure of the lipophilic phenolic reaction
products.
GC-MS Analyses. The fragmentation of the various alkyl 2-phe-
nylacetates or 2-phenylethyl alkanoates formed by lipase-catalyzed
esterification reactions was studied by GC-MS. Compounds containing
carboxy groups were analyzed after derivatization to the corresponding
methyl esters using an etherial solution of diazomethane and purification
by preparative TLC or column chromatography. The molecular ions
(EI mode) were as follows: oleyl phenylacetate, 386 [M]+; oleyl 2-,
3-, and 4-methoxyphenylacetate, 416 [M]+; oleyl 3-hydroxy- and
4-hydroxyphenylacetate, 402 [M]+; lauryl 4-hydroxyphenylacetate, 320
[M]+; palmityl 4-hydroxyphenylacetate, 376 [M]+; oleyl 4-hydrox-
yphenylacetate, 402 [M]+; oleyl 3,4-dihydroxyphenylacetate, 418 [M]+;
oleyl 4-hydroxyphenoxyacetate, 418 [M]+; oleyl 2-(1-naphthyl)acetate
and oleyl 2-(2-naphthyl)acetate, 436 [M]+; 2-phenylethyl oleate, 386
[M]+; and 2-(4-hydroxy-3-methoxyphenyl)ethyl oleate, 432 [M]+. Mole
peaks of the following compounds were determined in CI mode with
methane as the reagent gas (other GC-MS conditions were identical):
2-(2-hydroxyphenyl)ethyl oleate, 402 [M]+; 2-(3-hydroxyphenyl)ethyl
oleate, 402 [M]+; 2-(4-hydroxyphenyl)ethyl oleate (tyrosyl oleate), 402
[M]+; and 2-(1-naphthyl)ethyl oleate and 2-(2-naphthyl)ethyl oleate,
436 [M]+.
The influence of the position of bulky substituents was also
studied by esterification and transesterification of naphthylacetic
acids and methyl naphthylacetates. Figure 6 shows the effect
of the position of the carboxymethylene group of 1-naphthyl-
and 2-naphthylacetic acid as well as methyl 1-naphthyl- and
methyl 2-naphthylacetate on the lipase-catalyzed esterification
and transesterification with oleyl alcohol using Novozym 435
as a biocatalyst. The esterification activity of Novozym 435 is
higher for 2-naphthylacetic acid than for 1-naphthylacetic acid,
and similarly, the transesterification activity is higher for methyl
2-naphthylacetate than for methyl 1-naphthylacetate. It is
obvious from these results that the sterical hindrance in position
1 of the naphthalene backbone decreases esterification and
transesterification rates. Moreover, the data given in Table 1
again indicate that the transesterification activity of Novozym
435 is higher for both methyl 1-naphthyl- and methyl 2-naph-
RESULTS
The enzyme activities of three immobilized commercial
lipases, that is, Novozym 435, Lipozyme RM IM, and Lipozyme
TL IM, were studied for the esterification of various phenylacetic
acids as well as the transesterification of their methyl or ethyl
esters with medium- and long-chain alcohols (Figure 1 and
Table 1). In addition, enzyme activities were determined for
the esterification and transesterification of 2-(1-naphthyl)acetic
and 2-(2-naphthyl)acetic acids as well as their methyl esters,
respectively, with long-chain alcohols. The conversions were
performed with equimolar mixtures of substrates for up to 72 h
at 20-80 °C under partial vacuum (∼80 kPa) without solvent
and drying agent in the reaction mixture using different amounts
of the respective lipase. The results given in Table 1 show that
the enzyme activity of Novozym 435 for the esterification of

5086 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Weitkamp et al.
Table 1. Enzyme Activities of Immobilized Candida antarctica Lipase B (Novozym 435) for the Esterification and Transesterification of Various 2-Phenylacetic
Acids and Their Methyl Esters, Respectively, with Medium- or Long-Chain Alcohols as Well as Fatty Acids and Their Methyl Esters with Various
2-Phenylethanols
phenylacetic acids and methyl
medium- and long-chain reaction Novozym 435 temperature maximum conversion enzyme activities
(°C)
alcohols or 2-phenylethanols productsa (mg/assay) (mol%) after (h) (units/g) after (h)b
(SEM
(n ) x)
phenylacetates or fatty acids and
fatty acid methyl esters
esterification of 2-phenylacetic acid derivatives
phenylacetic acidc
cis-9-octadecen-1-ol
1
12.5
40
20
80
80
45
80
80
80
80
80
80
80
95 (1), 96 (4)
95 (8)
77 (48)
92 (8), 97 (24)
94 (1)
ND
721
439
21
207
624
(31.0 (n ) 2)
(22.3 (n ) 2)
(0.8 (n ) 2)
(15.4 (n ) 2)
(12.9 (n ) 2)
(n ) 2)
(4.9 (n ) 2)
(1.4 (n ) 2)
(32 (n ) 4)
(5.8 (n ) 2)
(0.9 (n ) 2)
(12.0 (n ) 2)
2-methoxyphenylacetic acid
3-methoxyphenylacetic acid
4-methoxyphenylacetic acid
2-hydroxyphenylacetic acid
3-hydroxyphenylacetic acid
4-hydroxyphenylacetic acidd
4-hydroxyphenoxyacetic 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
2
3
4
12.5
12.5
12.5
25
5
6c
9
25
25
25
73 (24)
95 (48)b, 94 (4)e
175
165
151
145
16
91 (8)f
4-hydroxy-3-methoxyphenylacetic acid cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
7
15
16
12.5
50
50
88 (24), 94 (48)
96 (48)
94 (2), 98 (72)
1-naphthylacetic acid
2-naphthylacetic acid
155
transesterification of methyl or ethyl 2-phenylacetate derivatives
methyl phenylacetate
cis-9-octadecen-1-ol
1
12.5
80
20
80
80
45
80
80
45
20
45
80
80
80
80
80
80
80
80
94 (4)
91(8)
96 (4)
97 (2)
35 (48)
79 (48)
94 (4)
94 (8)
80 (8)
89 (8)
ND
87 (4), 89 (24)
94 (4), 96 (8)
93 (8), 95 (24)
93 (8), 95 (48)
92 (4), 94 (24)
94 (4)
653
315
39
(22.2 (n ) 2)
(8.9 (n ) 2)
(7.2 (n ) 2)
(14.4 (n ) 2)
(0.17 (n ) 2)
(1.2 (n ) 2)
(36.8 (n ) 2)
(15.1 (n ) 2)
(14.6 (n ) 2)
(0.2 (n ) 2)
(n ) 2)
(3.4 (n ) 2)
(3.9 (n ) 2)
(1.8 (n ) 2)
(0.8 (n ) 2)
(22.5 (n ) 2)
(28.7 (n ) 4)
(0.1 (n ) 2)
methyl 1-naphthylacetate
methyl 2-naphthylacetate
methyl 2-methoxyphenylacetate
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
15
16
2
50
50
12.5
164
2.6
19g
670
430
149
418
methyl 3-methoxyphenylacetate
cis-9-octadecen-1-ol
3
4
12.5
methyl 4-methoxyphenylacetate
methyl 2-hydroxyphenylacetate
methyl 2-(3-hydroxyphenyl)acetate
methyl 4-hydroxyphenylacetate
methyl 4-hydroxyphenylacetate
methyl 4-hydroxyphenylacetateh
ethyl 4-hydroxyphenylacetate
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
dodecan-1-ol
hexadecan-1-ol
cis-9-octadecen-1-ol
cis-9-octadecen-1-ol
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
5
6a
6b
6c
6c
7
313
439
297
323
485
572
7
ethyl 4-hydroxy-3-methoxyphenylacetate cis-9-octadecen-1-ol
methyl 3,4-dihydroxyphenylacetate
cis-9-octadecen-1-ol
8
100
30 (48)
transesterifications under competitive conditions
methyl 4-hydroxyphenylacetate +
methyl 4-hydroxyhydrocinnamate
methyl 4-hydroxybenzoate +
methyl 4-hydroxyhydrocinnamate
methyl 4-hydroxyphenylacetate
cis-9-octadecen-1-ol
12.5
12.5
12.5
80
80
80
343
116
1.9
214
445
313
256
(14.0 (n ) 2)
(9.1 (n ) 2)
(0.17 (n ) 2)
(40.6 (n ) 2)
(21.8 (n ) 2)
(47 0.5 (n ) 2)
(51.1 (n ) 2)
cis-9-octadecen-1-ol
dodecan-1-ol
+ hexadecan-1-ol
+ cis-9-octadecen-1-ol
esterification of 2-phenylethanol derivatives
oleic acid
oleic acid
oleic acid
oleic acid
2-phenylethanol
10
17
18
11
12.5
80
20
80
94 (0.5)
89 (8)
94 (1)
96 (0.5)
97 (8)
96 (1)
82 (4)
81 (8)
93 (4)
95 (4)
87 (24)
99 (8)
753
462
587
193
221
169
194
237
551
697
92
(5.8 (n ) 2)
(10.5 (n ) 2)
(30.4 (n ) 4)
(0.1 (n ) 2)
(15.7 (n ) 2)
(18.4 (n ) 2)
(10.5 (n ) 2)
(9.6 (n ) 3)
(1.6 (n ) 2)
(4.3 (n ) 2)
(9.9 (n ) 6)
(17.2 (n ) 2)
2-(1-naphthyl)ethanol
2-(2-naphthyl)ethanol
2-(2-hydroxyphenyl)ethanol
12.5
50
12.5
50
80
25
80
45
45
80
45
80
12.5
12.5
12.5
oleic acid
oleic acid
2-(3-hydroxyphenyl)ethanol
2-(4-hydroxyphenyl)ethanol
12
13b
oleic acid
2-(4-hydroxy-3-methoxyphenyl)
ethanol
14
12.5
286
transesterification of 2-phenylethanol derivatives
methyl oleate
2-phenylethanol
10
12.
45
20
80
80
80
80
80
80
80
89 (4)
85 (8)
87 (2)
72 (2), 80 (48)
89 (24)
91 (8)
90 (24)
58 (8), 63 (24)
68 (8), 69 (24)
595
263
630i
189
160
225
146
184i
180i
(5.0 (n ) 2)
(14.7 (n ) 2)
(11.7 (n ) 2)
(10.6 (n ) 2)
(14.3 (n ) 4)
(3.8 (n ) 2)
(3.7 (n ) 2)
(5.3 (n ) 4)
(20.8 (n ) 2)
triolein
2-phenylethanol
10
11
12
13a
13b
13b
12.5
25
25
25
25
25
25
methyl oleate
methyl oleate
methyl palmitate
methyl oleate
triolein
2-(2-hydroxyphenyl)ethanol
2-(3-hydroxyphenyl)ethanol
2-(4-hydroxyphenyl)ethanol
2-(4-hydroxyphenyl)ethanol
2-(4-hydroxyphenyl)ethanol
2-(4-hydroxyphenyl)ethanol
sunflower oil
a Numbering is according to the chemical structures given in Figure 1. b Standard assay conditions, if not otherwise indicated are as follows: 0.3 mmol of carboxylic
acid or methyl ester + 0.3 mmol of alcohol; immobilized lipase/assay, 12.5 mg; 80 °C; 80 kPa; and 0.5 h. c After subtraction of blank ()2.3 mol% oleyl 2-phenylacetate
within 0.5 h at 80 °C), no conversion was detected using Lipozyme RM IM and Lipozyme TL IM, respectively. d After subtraction of blank ()5 mol% oleyl 4-hydroxyphenylacetate
within 8 h at 80 °C or 12 mol% oleyl 4-hydroxyphenylacetate within 24 h at 80 °C). e Fifty milligrams of Novozym 435. f n ) 2. g After 8 h. h Blank ) 0.4 mol% oleyl
i
4-hydroxyphenylacetate within 48 h at 80 °C. Molar ratio triglyceride:(hydroxy)phenylethanols (1:3). SEM, standard error of mean; ND, not detected.

Lipophilic (Hydroxy)phenylacetates
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5087
Figure 5. Effect of the position of 2-methoxy- (0), 3-methoxy- (4), and
4-methoxy-substituents (O) of methyl methoxyphenylacetates (0.3 mmol
each) on the lipase-catalyzed transesterification with oleyl alcohol (0.3
mmol) at 45 °C and 80 kPa using Novozym 435 (12.5 mg) as a biocatalyst;
n ) 2 for each.
Figure 2. Lipase-catalyzed esterification of 0.3 mmol of 4-hydroxyphe-
nylacetic acid with 0.3 mmol of oleyl alcohol at 80 °C and 80 kPa using
different amounts (4, 6.3 mg; 0, 12,5 mg; and ], 25 mg) of Novozym
435 as a biocatalyst; n ) 2 for each.
Figure 6. Effect of the position of the carboxymethylene group of
1-naphthyl- ([) and 2-naphthylacetic acid (]) (0.3 mmol each) as well
as methyl 1-naphthyl- (9) and 2-naphthylacetate (0) (0.3 mmol each) on
the lipase-catalyzed esterification and transesterification, respectively, with
0.3 mmol of oleyl alcohol at 80 °C and 80 kPa using Novozym 435 (50
mg/assay) as a biocatalyst; n ) 2 for each.
Figure 3. Lipase-catalyzed transmethylation of 0.3 mmol of methyl
3-methoxyphenylacetate with 0.3 mmol of oleyl alcohol at different
temperatures (4, 20 °C; 0, 45 °C; and ], 80 °C) and 80 kPa using
Novozym 435 (12.5 mg) as a biocatalyst; n ) 2 for each.
Figure 4. Time course of lipase-catalyzed (A) esterification of 3-hydroxy-
(9) and 4-hydroxyphenylacetic acid (2) (0.3 mmol each; 25 mg Novozym
435/assay) as well as (B) transesterification of methyl 3-hydoxy- (0) and
4-hydroxyphenylacetate (4) (0.3 mmol/each; 12.5 mg Novozym 435/assay)
with oleyl alcohol at 80 °C and 80 kPa; n ) 2 for each.
Figure 7. Effects of methoxy- and hydroxy-substituents of phenylacetates
on the Novozym 435-catalyzed transesterification of 0.3 mmol of ethyl
4-hydroxyphenylacetate (]) (12.5 mg of Novozym 435), 0.3 mmol of ethyl
4-hydroxy-3-methoxyphenylacetate (0) (12.5 mg of Novozym 435), and
0.3 mmol of methyl 3,4-dihydroxyphenylacetate (4) (100 mg of Novozym
435) with 0.3 mmol of oleyl alcohol, each at 80 °C and 80 kPa; n ) 2
for each.
thylacetate as compared to the esterification activity for 2-naph-
thylacetic acid and, particularly, 1-naphthylacetic acid.
Very different enzyme activities were found for the Novozym
435-catalyzed transesterification of various methyl or ethyl
phenylacetates with oleyl alcohol. For example, Table 1 shows
that a 4-hydroxy substituent as compared to a 4-methoxy group
decreased the enzyme activity as is shown for the transesteri-
fication of methyl 4-hydroxyphenylacetate and methyl 4-meth-
oxyphenylacetate, respectively, with oleyl alcohol. Under similar
reaction conditions, introduction of a further hydoxy group into
methyl 4-hydroxyphenylacetate as in methyl 3,4-dihydroxyphe-
nylacetate again led to both substantially decreased transesteri-
fication activity (323 vs 7 units/g) and maximum conversion
(94 vs 30 mol%; Table 1 and Figure 7). Moreover, the time
course of the Novozym 435-catalyzed transesterification of ethyl
4-hydroxyphenylacetate and ethyl 4-hydroxy-3-methoxypheny-
lacetate both with oleyl alcohol is shown in Figure 7. It is
obvious from these results and the data shown in Table 1
thatsin contrast to an additional 3-hydroxy group as shown
abovesthe presence of an additional 3-methoxy group as in
4-hydroxy-3-methoxyphenylacetate did not decrease maximum
conversion of the substrate and rather increased transesterifi-
cation activity of Novozym 435.
Effect of Chain-Length of the Phenylalkanoyl Moiety.
Figure 8 and Table 1 show the enzyme activities of Novozym
435 lipase under competitive conditions for the transesterifica-
tion of oleyl alcohol with methyl 4-hydroxyphenylacetate and
methyl 4-hydroxyphenylpropionate (Figure 8A) as well as with

5088 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Weitkamp et al.
Figure 10. Effect of the position of hydroxy substituents of 2-phenyle-
thanols on the lipase-catalyzed (A) esterification (12.5 mg of Novozym
435; 45 °C) of 2-hydroxy- ([), 3-hydroxy- (9), or 4-hydroxy-phenylethanol
(2) (0.3 mmol each) with 0.3 mmol of oleic acid as well as (B)
transesterification (25 mg of Novozym 435; 80 °C) of 2-hydroxy- (]),
3-hydroxy- (0), or 4-hydroxy-phenylethanol (4) (0.3 mmol each) with 0.3
mmol of methyl oleate, respectively; n ) 2 for each.
Figure 8. Transesterification activities of immobilized Novozym 435 lipase
(12.5 mg) under competitive conditions for the conversions of (A) methyl
4-hydroxyphenylpropionate (4-hydroxyhydrocinnamate) (1) and methyl
4-hydroxyphenylacetate (2) as well as (B) methyl 4-hydroxyphenylpropi-
onate (4-hydroxyhydrocinnamate) (1) and methyl 4-hydroxybenzoate (3),
0.15 mmol, each with 0.3 mmol of oleyl alcohol at 0.5 h, 80 °C, and 80
kPa; n ) 2 for each.
acid using Novozym 435 as a biocatalyst to study the influence
of the position of bulky substituents on the conversion to the
corresponding oleic acid esters. As expected, the results of these
experiments show that the esterification rate was far higher for
the conversion of 2-(1-naphthyl)ethanol as compared to 2-(2-
naphthyl)ethanol (Table 1).
Figure 10 shows the effect of the position of hydroxy
substituents at the phenyl moiety on the Novozym 435-catalyzed
esterification of 2-hydroxy-, 3-hydroxy-, or 4-hydroxyphenyle-
thanol with oleic acid (Figure 10A) as well as the corresponding
transesterification with methyl oleate (Figure 10B). Again,
esterification is preferred over transesterification, and the
esterification activity of Novozym 435 increases in the following
order for these three phenylethyl alcohols: 3-hydroxy- >
2-hydroxy- > 4-hydroxyphenylethanol. It is worth noting that
2-hydroxyphenylethanol was esterified and transesterified with
oleic acid and methyl oleate, respectively, whereas no conver-
sion was found for the analogous reactions of 2-hydroxyphe-
nylacetic acid and the corresponding methyl ester with oleyl
alcohol as described above. 2-(4-Hydroxy-3-methoxyphenyl)e-
thanol was also esterified with oleic acid in high yield using
Novozym 435 as a biocatalyst (Table 1). The enzyme activity
of Novozym 435 is distinctly lower for the transesterification
of 2-hydroxy-, 3-hydroxy-, or 4-hydroxyphenylethanols with
methyl oleate as compared to the esterification with oleic acid.
Under similar conditions, transesterification of 4-hydroxyphe-
nylethanol with triolein or sunflower oil led to moderate
conversions only (Table 1), most probably because of side
reactions such as transesterification/re-esterification, as was
obvious from substantial concentrations of mono- and diglyc-
erides in the reaction mixture.
Figure 9. Esterification and transesterification of 0.3 mmol of 2-phenyle-
thanol with 0.3 mmol of oleic acid (]) and 0.3 mmol of methyl oleate
(0), respectively, at 20 °C and 80 kPa using Novozym 435 (12.5 mg/
assay) as a biocatalyst; n ) 2 for each.
methyl 4-hydroxyphenylpropionate and methyl 4-hydroxyben-
zoate (Figure 8B). Obviously, the transesterification activity
of Novozym 435 for these three compounds increases in the
following order: methyl 4-hydroxyphenylacetate > methyl
4-hydroxyphenylpropionate (4-hydroxyhydrocinnamate) . meth-
yl 4-hydroxybenzoate. It is also evident from Table 1 that lauryl
alcohol (1-dodecanol) is the preferred substrate when an
equimolar mixture of medium- and long-chain alcohols is
transesterified with 4-hydroxyphenylacetate under competitive
conditions as compared to palmityl alcohol (1-hexadecanol) and
oleyl alcohol.
Phenylethyl Alkanoates with Inverse Structure. In addition
to the preparation of various alkyl phenylacetates by esterifi-
cation and transesterification of phenylacetic acid derivatives
with long-chain alcohols, the corresponding 2-phenylethan-1-
ol derivatives were reacted with fatty acids or fatty acid methyl
esters to form the corresponding lipophilic fatty acid esters of,
for example, 4-hydroxyphenylethanol (tyrosol) having inverse
chemical structure as compared to the alkyl phenylacetates
described above. Figure 9 and Table 1 show the esterification
and transesterification of 2-phenylethanol with oleic acid and
methyl oleate, respectively, at 20 °C using Novozym 435 as a
biocatalyst. In contrast to our data on the esterification and
transesterification of phenylacetic acid and methyl phenylacetate,
respectively, these results demonstrate that esterification of
2-phenylethanol with oleic acid is preferred over transesterifi-
cation of 2-phenylethanol with methyl oleate for the formation
of 2-phenylethyl oleates. Similarly, the analogous 2-(1-naph-
thyl)ethanol or 2-(2-naphthyl)ethanol was esterified with oleic
DISCUSSION
Recently, we have shown that various medium- and long-
chain alkyl (hydroxy)cinnamates can be efficiently prepared
from hydroxycinnamic acids and their analogues via lipase-
catalyzed esterification or transesterification with fatty alcohols
under environmentally friendly conditions. This implies the use
of nonactivated reactants in an equimolar ratio as starting
materials, reduced pressure for the removal of reaction water
or methanol, moderate temperatures, and avoiding organic
solvents and drying reagents (29, 30). In continuation of the
above studies, we have applied these enzymatic esterification
and transesterification reactions to the preparation of lipophilic

Lipophilic (Hydroxy)phenylacetates
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5089
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alkyl esters of (hydroxy)phenylacetic acids such as oleyl
4-hydroxyphenylacetate. In a similar manner, the inverse fatty
acid esters of (hydroxy)phenylethanols were prepared, for
example, 2-(4-hydroxyphenyl)ethyl oleate (tyrosol oleate). High
conversions (mostly g90%) were obtained within relatively
short reaction periods using equimolar mixtures of acids or
methyl esters and alcohols, which reduces costs and simplifies
purification. In many cases, this method is superior to various
other enzymatic preparations, which utilize high excess of
substrates, long reaction times, drying reagents, and, in part,
toxic solvents (for a review see ref 14).
In addition, the following recommendations can be given for
the preparation of alkyl phenylacetates and phenylethyl al-
kanoates: (i) Esterification and transesterification activities of
Novozym 435 exceed by far those of Lipozyme RM IM and
Lipozyme TL IM. (ii) The transesterification activity of No-
vozym 435 is generally higher for the preparation of alkyl
phenylacetates, whereas the esterification activity is higher for
the preparation of phenylethyl alkanoates. (iii) An increase of
temperature and amount of Novozym 435 raise the esterification
and transesterification rates. (iv) Introduction of a methoxy and,
particularly, a hydroxy substituent at the phenyl moiety de-
creases esterification and transesterification rates. Methoxy and,
particularly, hydroxy substituents in the 2-position of the phenyl
moiety reduce esterification and transesterification rates far
higher than in the 3- or 4-position, which is probably caused
by sterical hindrance and/or hydrogen bonds (36). (v) In contrast
to many chemical esterification procedures, the primary aliphatic
hydroxy group of (hydroxy)phenylethan-1-ols is regioselectively
esterified using Novozym 435 as a biocatalyst. The two specific
binding sites of C. antarctica lipase B for alkyl and acyl moieties
may explain the very different esterification and transesterifi-
cation activities observed for the various analogous phenylac-
etate and phenylethanol substrates. These results are consistent
with earlier findings on the esterification and transesterification
of benzoic, phenylacetic, and, particularly, cinnamic acid
derivatives (14, 28–30, 36).
Phenolics predominantly appear as polar antioxidants in the
hydrophilic phases of foods. Lipophilization of phenolics is,
therefore, of great importance to extend their field of applications
to fatty phases in oil-based foods as well as multiphase food
systems containing both lipophilic and hydrophilic phases (14, 28).
Lipase-catalyzed lipophilization of antioxidative phenylalkanoic
acids such as hydroxylated benzoic, phenylacetic, and cinnamic
acids and their short-chain alkyl esters by esterification and
transesterification, respectively, with fatty alcohols may be of
special interest for food and nonfood applications. (Hydrox-
y)phenylalkyl fatty acid esters with inverse chemical structures
obtained by lipase-catalyzed esterification and transesterification
of fatty acids and fatty acid methyl esters, respectively, with
(hydroxy)phenylethanols also have lipophilic properties and may
be used for similar applications.
(19) Adams, T. B.; Cohen, S. M.; Doull, J.; Feron, V. J.; Goodman,
J. I.; Marnett, L. J.; Munro, I. C.; Portoghese, P. S.; Smith, R. L.;
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C.; Pliakis, E.; Samiotki, M.; Panayotou, G.; Antonopoulou, S.
Biological activity of acetylated phenolic compounds. J. Agric.
Food Chem. 2007, 55, 80–89.
Supporting Information Available: Gas chromatograms of
reaction mixtures of the transesterification of methyl 4-hydrox-
yphenylacetate with oleyl alcohol leading to oleyl 4-hydrox-
yphenylacetate and of the esterification of 2-(4-hydroxyphe-
nyl)ethanol (tyrosol) with oleic acid leading to 2-(4-hydroxy-
phenyl)ethyl oleate (tyrosyl oleate). This material is available
free of charge via the Internet at http://pubs.acs.org.
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Received for review January 22, 2008. Revised manuscript received
April 3, 2008. Accepted April 12, 2008.
JF8002224