Efficient lipase-catalyzed synthesis of new lipid antioxidants based on a catechol structure

Article abstract of DOI:10.1016/j.tet.2005.05.100

Lipid antioxidants phenolic saturated fatty acid esters were synthesized in high yields and short reaction times using the corresponding ethyl fatty acid esters, lipase from Candida Antarctica, vacuum and no solvent. Phenolic esters with mono- and polyunsaturated fatty acids (EPA and DHA) were also prepared.

Full text of DOI:10.1016/j.tet.2005.05.100

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Tetrahedron 61 (2005) 7654–7660
Efficient lipase-catalyzed synthesis of new lipid antioxidants based
on a catechol structure
*
´
˜
A. Torres de Pinedo, P. Penalver, D. Rondon and J. C. Morales
Department of Chemistry, Puleva Biotech, Camino de Purchil, 66, 18004 Granada, Spain
Received 10 March 2005; revised 30 May 2005; accepted 31 May 2005
Available online 17 June 2005
Abstract—Lipid antioxidants phenolic saturated fatty acid esters were synthesized in high yields and short reaction times using the
corresponding ethyl fatty acid esters, lipase from Candida Antarctica, vacuum and no solvent. Phenolic esters with mono- and
polyunsaturated fatty acids (EPA and DHA) were also prepared.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
most of the phenolic derivatives posses high solubility in
aqueous solutions and it is very desirable for the food
industry to be able to make them soluble in fats and oils.14
Oxygen is necessary for life of most biological systems.
Paradoxically, the exposition to oxygen leads to oxidative
stress by the formation of free radicals that react easily with
other molecules in the cell, and may alter cellular
mechanisms, being a possible cause of some inflammatory
or cardiovascular diseases or even cancer and aging.1–3
Similarly, oxidation affects deterioration of food, specially
items with a high lipid fraction in their composition.
Antioxidants are used by nature to counteract the effect of
oxidation,4 and new synthetic antioxidants are also been
developed against the effects of oxidative stress.
The use of lipases in non-aqueous solvents has been
previously described for the preparation of phenolic acid
esters. Guyot et al.15 and Stamatis et al.16 reported
enzymatic esterification of phenolic acids and fatty alcohols
with lipase CAL-B from Candida Antarctica, but in both
cases very long reaction times were needed to obtain
reasonable yields. Twu et al.17 partially solved this problem
for the esterification of hydroxyphenylpropionic acid and
octanol, where high yields were obtained after 58 h.
Buisman et al.18 first investigated the esterification of
phenols with carboxylic fatty acids and reported the
synthesis of hydroxytyrosol with octanoic acid in hexane.
They obtained acylation at the primary hydroxyl group of
Phenols are a large family of natural compounds that are
able to donate the hydrogen atom of the phenolic OH to the
free radicals, thus stopping the propagation chain during the
oxidation process. Phenolic antioxidants have been studied
due to their biological relevance. Hydroxytyrosol, a
component of olive oil phenols, has been shown to inhibit
human low-density lipoprotein (LDL) oxidation (a critical
step in atherosclerosis),5,6 to inhibit platelet aggregation7
and to possess anti-inflammatory,8 anticancer9 and cell
protection properties.10 Lipid antioxidants have been
prepared from natural antioxidants, since they would be
able to prevent lipid peroxidation of cell membranes11,12
and may also prevent oxidation of biologically important
polyunsaturated fatty acids. Eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA) were covalently attached
to ascorbic acid to protect them from oxidation.13 Moreover,
hydroxytyrosol with moderate yield after long reaction time.
´
19
Gonzalez-Alcudia et al. reported the synthesis of
hydroxytyrosol acetate in presence of pancreatic lipase in
ethyl acetate after 48 h of reaction with 86% yield.
We present the enzymatic esterification of a series of di-
ortho-phenolic compounds, including hydroxytyrosol, with
several fatty acids using Novozym 435w (Fig. 1). High
yields were obtained in short reaction times for all the
phenolic esters prepared with saturated fatty acids by using
no solvent and applying vacuum during the reaction (Fig. 2).
Moderate to good yields were obtained for esters containing
Keywords: Lipid antioxidants; Phenols; Fatty acid ester; Hydroxytyrosol.
*
Corresponding author. Tel.: C34 958 240320; fax: C34 958 240160;
e-mail: jcmorales@pulevabiotech.es
Figure 1. Structures of phenol antioxidants under study.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.05.100

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Figure 2. Structures of phenolic saturated fatty acid esters synthesized.
monounsaturated and polyunsaturated fatty acids under the
same reaction conditions (Fig. 3).
of the reaction was quite simple. The reaction mixture was
dissolved in acetonitrile, the lipase filtered, and the
acetonitrile phase washed with hexane to eliminate excess
of ethyl palmitate. Finally, it was concentrated and purified
on a short silica column. When these new reaction
conditions were extended to the other phenols in the series,
high yields were obtained except for compound 8, similarly
to what happened under the previous reaction conditions
(see Table 1).
2. Results and discussion
We prepared the phenolic alcohols (1–4) by reduction from
their corresponding carboxylic acids as reported by Capasso
et al.20 Acylation of these phenols was carried out with
palmitic acid and lipase Novozym 435w following
conditions reported by Buisman et al.18 except that
acetonitrile was used as solvent instead of hexane, due to
solubility problems. We obtained the corresponding esters
(5–8) in moderate yields (Table 1).
Next, we extended the study to other saturated fatty acids to
probe the influence of the length of the fatty acid in the
esterification reaction using the conditions with ethyl fatty
ester, no solvent and vacuum. We observed lower yields of
ester formation for a short chain fatty acid, such as butyrate,
than for palmitate, except for phenols 3 and 4, where similar
or better yields were obtained. High yields were obtained
when ethyl stearate was used as the acylating agent in the
reaction, similar to those for ethyl palmitate, except for
compound 1, possibly due to its low solubility in the
reaction media. The effect of the length of the saturated
carboxylic acid is not very clear. Actually, contradictory
In order to improve the yields of the acylations obtained in
acetonitrile, we tried ethyl esters as the acylating agents and
also as the solvent. When 3,4-dihydroxybenzylic alcohol 1
was reacted with ethyl palmitate as the solvent using
Novozym 435w at 37 8C, the reaction went almost to
completion after 14 h and the corresponding ester was
obtained with 98% yield after column purification. Work-up
Figure 3. Structures of phenolic unsaturated and polyunsaturated fatty acid esters synthesized.

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7656
A. Torres de Pinedo et al. / Tetrahedron 61 (2005) 7654–7660
Table 1. Reaction yields for phenolic saturated fatty acid esters
Phenol
Acylating agent
Product
Yield (%)
3,4-Dihydrobenzylic alcohol 1
Palmitic acida
Palmitic acida
Palmitic acida
Palmitic acida
Ethyl palmitateb
Ethyl palmitateb
Ethyl palmitateb
Ethyl palmitateb
Ethyl butyrateb
Ethyl butyrateb
Ethyl butyrateb
Ethyl butyrateb
Ethyl stearateb
Ethyl stearateb
Ethyl stearateb
Ethyl stearateb
5
6
7
8
5
6
7
8
9
10
11
12
13
14
15
16
75
81
85
69
98
98
97
71
75
59
97
96
74
94
97
72
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
3,4-Dihydrobenzylic alcohol 1
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
3,4-Dihydrobenzylic alcohol 1
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
3,4-Dihydrobenzylic alcohol 1
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
a Reaction conditions: phenol-fatty acid ratio, 1:2, Novozym 435w, acetonitrile, 37 8C, 16 h.
b Reaction conditions: phenol/ethyl fatty acid ester ratio, 1:30, Novozym 435w, no solvent, 37 8C, 6–16 h.
results are also found in the literature when Candida
Antarctica lipase is used to esterify ascorbic acid with
3. Experimental
saturated fatty acids. Stamatis et al.16 found a decrease in
yields for ester formation with increasing chain length,
whereas Yan et al.21 found just the opposite effect. It is
important to note that, in both cases, acetone or tert-butanol
were used as the solvent and that long reaction times were
needed to obtain reasonable yields.
Chemicals were purchased from Sigma-Aldrich and used
without further purification. The immobilized lipase from
Candida Antarctica (Novozym 435w) was a gift from
Novozymes A/S Spain. Thin-layer chromatography (TLC)
was performed on precoated silica gel 60 Alugram SIL/
UV254 from Macherey Nagel. FAB Mass spectra were
collected on a Hewlett-Packard 5988 spectrometer, using a
Fisons VG platform or a Fisons VG Autospec-Q. NMR
experiments were performed on a Bruker AM-300 and a
Bruker AMX-300, operating at 300 MHz for 1H and
75 MHz for 13C, and on a Bruker AMX-400, operating at
400 MHz for 1H and 100 MHz for 13C. CDCl3 was used as
solvent. Chemical shifts are expressed in d (parts per
million) using the solvent as internal reference.
Finally, we studied the phenolic ester formation with
the ethyl esters of the biologically relevant oleic,
eicosapentaenoic (EPA), and docosahexaenoic (DHA)
acids. Lower yields were obtained for oleic than for stearic
ethyl ester for all four phenolic compounds in the series,
except for compound 4 that maintained the same yields for
both (see Table 2). A more dramatic decrease in yields
(29–51%) was obtained for EPA and DHA ethyl esters when
reacted with phenols 1, 2, and 4. Actually, the side
reaction of hydrolysis of the corresponding fatty acid
ethyl esters to the carboxylic acid was quite fast in these
cases as observed by TLC. Surprinsingly, reaction with
compound 3 resulted in 67 and 97% of the ester formation
with DHA and EPA, respectively. Probably, the fact that the
primary alcohol is at three-carbon distance from the
phenolic structure in compound 3 helps the ester reaction
to compete with the hydrolysis reaction of the polyunsatu-
rated ethyl esters.
3.1. Synthetic procedure
A suspension of the alcohol (0.4 mmol), Novozym 435w
(40 mg) in ethyl fatty acid ester (12 mmol) was stirred
vigorously under vacuum (5–10 mmHg) at 37 8C for
4–16 h. Then, acetonitrile (75 mL) was added, lipase
filtered, and the crude washed with hexane (3!25 mL).
The acetonitrile phase was concentrated to dryness under
reduced pressure and finally purified in a short silica column
using flash chromatography (hexane/diethyl ether 10:1, 2:1
Table 2. Reaction yields for phenolic mono- and polyunsaturated fatty acid esters
Phenol
Acylating agent
Product
Yield (%)
3,4-Dihydrobenzylic alcohol 1
Ethyl oleate
Ethyl oleate
Ethyl oleate
Ethyl oleate
Ethyl eicosapentaenoate
Ethyl eicosapentaenoate
Ethyl eicosapentaenoate
Ethyl eicosapentaenoate
Ethyl docosahexaenoate
Ethyl docosahexaenoate
Ethyl docosahexaenoate
Ethyl docosahexaenoate
17
18
19
20
21
22
23
24
25
26
27
28
64
81
87
72
33
51
97
32
32
43
67
29
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
3,4-Dihydrobenzylic alcohol 1
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
3,4-Dihydrobenzylic alcohol 1
2-(3,4-Dihydroxyphenyl)-ethanol 2
3-(3,4-Dihydroxyphenyl)-propanol 3
3,4-Dihydroxycinnamylic alcohol 4
Reaction conditions: phenol-carboxylic ethyl ester ratio, 1:30, Novozym 435w, no solvent, 37 8C, 4–16 h.

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A. Torres de Pinedo et al. / Tetrahedron 61 (2005) 7654–7660
7657
or 1:1 depending on the polarity of final compound) to yield
the phenolic fatty acid ester. In the case of the reactions with
butyrate ethyl ester, no final chromatography was needed.
All compounds were O95% pure as observed by H NMR
spectroscopy.
1.25 (m, 24H, –CH2–), 0.87 (t, JZ6.3 Hz, 3H, –CH3); 13C
NMR (75 MHz, CDCl3): d 174.2 (–COO–), 134.0 (Ph-
CH]CH–), 121.3 (Ph-CH]CH–), 143.9, 143.8, 129.9,
120.2, 115.5, 113.3 (Ar), 65.2 (–CH2OCO–), 34.4, 32.0,
30.4, 29.8, 29.7, 29.5, 29.4, 29.3, 29.2, 25.1, 22.8 (–CH2–),
14.2 (–CH3); HRMS FABC calcd 427.2824 for
C25H40O4Na [MCNa]C, found 427.2819.
1
3.2. HPLC procedure
Monitoring by HPLC was carried out using 2695 Alliance
Waters separation system and a 2996 photodiode array
detector. The column was a Symmetry C18, 250!4.0 mm,
5 mm particle size, protected with a C18 precolumn. The
mobile phase A was acetonitrile and B was 1% acetic acid in
water. Gradient started on 92% A for 4 min, then linear
ramp to 100% A in 6 more min, maintaining 100% A for
8 min, and returning to starting conditions in 2 min. Flow
rate was 1 mL/min and detection was achieved at 280 nM.
3.2.5. 3,4-Dihydroxybenzylic butyrate 9. 75%. Trans-
parent syrup; 1H NMR (300 MHz, CDCl3): d 6.88 (d,
JZ1.8 Hz, 1H, ar), 6.83 (d, JZ8.0 Hz, 1H, ar), 6.79 (dd,
JZ8.0, 1.7 Hz, 1H, aro), 4.99 (s, 2H, PhCH2OOC–), 2.31 (t,
JZ7.4 Hz, 2H, –OOC–CH2–), 1.65 (h, JZ7.4 Hz, 2H,
OOC–CH2–CH2), 0.92 (t, JZ7.4 Hz, 3H, –CH3); 13C NMR
(75 MHz, CDCl3): d 174.5 (–COO–), 144.0, 143.7, 128.9,
121.5, 115.9, 115.4 (Ar), 66.3 (PhCH2OCO–), 36.4 (–OOC–
CH2–), 18.5 (–OOC–CH2–CH2), 13.7 (–CH3); HRMS
FABC calcd 222.0892 for C12H14O4Na [MCNa]C,
found 222.0892.
3.2.1. 3,4-Dihydroxybenzylic palmitate 5. 98%. White
1
solid; H NMR (300 MHz, CDCl3): d 6.89 (d, JZ1.5 Hz,
1H, ar), 6.84 (d, JZ8.1 Hz, 1H, ar), 6.79 (dd, JZ8.0,
1.7 Hz, 1H, ar), 4.99 (s, 2H, PhCH2OOC–), 2.32 (t,
JZ7.4 Hz, 2H, –OOC–CH2–), 1.61 (q, JZ6.0 Hz, 2H,
OOC–CH2–CH2), 1.25 (m, 24H, –CH2–), 0.87 (t, JZ
6.4 Hz, 3H, –CH3); 13C NMR (75 MHz, CDCl3): d 175.7
(–COO–), 145.2, 144.9, 130.3, 122.9, 117.2, 116.7 (Ar),
67.5 (PhCH2OCO–), 35.9 (–OOC–CH2–), 33.3, 31.1, 31.0,
30.8, 30.7, 30.6, 30.5, 26.3, 24.1 (–CH2–), 15.5 (–CH3);
HRMS FABC calcd 401.2668 for C23H38O4Na [MCNa]C,
found 401.2672.
3.2.6. 2-(3,4-Dihydroxyphenyl) ethyl butyrate 10. 59%.
Transparent syrup; 1H NMR (300 MHz, CDCl3): d 6.78 (d,
JZ8.1 Hz, 1H, ar), 6.73 (d, JZ1.5 Hz, 1H, ar), 6.63 (dd,
JZ8.0, 1.5 Hz, 1H, ar), 6.19 (1s (w), 1H, Ph-OH), 6.0 (1s
(w), 1H, Ph-OH), 4.23 (t, JZ7.1 Hz, 2H, –CH2OOC–), 2.79
(t, JZ7.1 Hz, 2H, Ph-CH2–), 2.27 (t, JZ7.5 Hz, 2H,
–OOC–CH2–), 1.65 (h, JZ7.4 Hz, 2H, –OOC–CH2–CH2),
0.92 (t, JZ7.4 Hz, 3H, –CH3); 13C NMR (75 MHz, CDCl3):
d 174.9 (–COO–), 143.9, 142.6, 130.5, 121.3, 115.9, 115.5
(Ar), 65.3 (–CH2OCO–), 36.4 (–OOC–CH2–), 34.5 (Ph-
CH2–), 18.5 (–OOC–CH2–CH2), 13.7 (–CH3); HRMS
FABC calcd 247.0946 for C12H16O4Na [MCNa]C,
found 247.0947.
3.2.2. 2-(3,4-Dihydroxyphenyl) ethyl palmitate 6. 98%.
White solid; 1H NMR (300 MHz, CDCl3): d 6.78 (d,
JZ8.1 Hz, 1H, ar), 6.73 (d, JZ1.5 Hz, 1H, ar), 6.63 (dd,
JZ8.0, 1.5 Hz, 1H, ar), 4.23 (t, JZ7.1 Hz, 2H, –CH2OOC–),
2.80 (t, JZ7.1 Hz, 2H, ar-CH2–), 2.28 (t, JZ7.5 Hz, 2H,
–OOC–CH2–), 1.58 (q, JZ6.1 Hz, 2H, –OOC–CH2–CH2),
1.25 (m, 24H, –CH2–), 0.87 (t, JZ6.4 Hz, 3H, –CH3); 13C
NMR (75 MHz, CDCl3): d 174.5 (–COO–), 143.7, 142.4,
130.7, 121.4, 116.0, 115.5 (Ar), 65.1 (–CH2OCO–), 34.6,
34.5, 32.0, 29.8, 29.7, 29.7, 29.5, 29.4, 29.3, 29.2, 25.0,
24.8, 22.8 (–CH2–), 14.2 (–CH3); HRMS FABC calcd
415.2824 for C24H40O4Na [MCNa]C, found 415.2819.
3.2.7. 3-(3,4-Dihydroxyphenyl) propyl butyrate 11. 97%.
Transparent syrup; 1H NMR (300 MHz, CDCl3): d 6.77 (d,
JZ8.0 Hz, 1H, ar), 6.68 (d, JZ1.5 Hz, 1H, ar), 6.58 (dd,
JZ8.0, 1.6 Hz, 1H, ar), 4.07 (t, JZ6.6 Hz, 2H, –CH2OOC–),
2.55 (t, JZ7.4 Hz, 2H, ar-CH2–), 2.30 (t, JZ7.5 Hz, 2H,
–OOC–CH2–), 1.89 (q, JZ6.7 Hz, 2H, –CH2–), 1.65 (h, JZ
7.5 Hz, 2H, –OOC–CH2–CH2), 0.95 (t, JZ7.4 Hz, 3H,
–CH3); 13C NMR (75 MHz, CDCl3): d 174.6 (–COO–),
143.8, 141.9, 134.2, 120.8, 115.6, 115.4 (Ar), 63.9
(–CH2OCO–), 36.4 (–OOC–CH2–), 31.5, 30.3, 18.6
(–CH2–), 13.7 (–CH3); HRMS FABC calcd 261.1103 for
C13H18O4Na [MCNa]C, found 261.1107.
3.2.3. 3-(3,4-Dihydroxyphenyl) propyl palmitate 7. 97%.
White solid; 1H NMR (300 MHz, CDCl3): d 6.76 (d,
JZ8.0 Hz, 1H, ar), 6.68 (d, JZ1.8 Hz, 1H, ar), 6.58 (dd,
JZ8.0, 1.8 Hz, 1H, ar), 4.06 (t, JZ6.6 Hz, 2H, –CH2OOC–),
2.55 (t, JZ7.4 Hz, 2H, ar-CH2–), 2.30 (t, JZ7.5 Hz, 2H,
–OOC–CH2–), 1.88 (q, JZ6.7 Hz, 2H, –CH2–), 1.65 (q, JZ
7.2 Hz, 2H, –OOC–CH2–CH2), 1.24; 13C NMR (75 MHz,
CDCl3): d 174.5 (–COO–), 143.7, 141.8, 134.3, 120.8,
115.6, 115.5 (Ar), 63.8 (–CH2OCO–), 34.5, 32.0, 31.5, 30.4,
29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 25.1, 22.8 (–CH2–),
14.2 (–CH3); HRMS FABC calcd 429.2981 for
C25H42O4Na [MCNa]C, found 429.2977.
3.2.8. 3,4-Dihydroxycinnamyl butyrate 12. 96%. Trans-
parent syrup; H NMR (300 MHz, CDCl3): d 6.92 (s, 1H,
1
ar), 6.80 (s, 2H, ar), 6.51 (d, JZ15.8 Hz, 1H, Ph-CH]CH–
CH2–), 6.09 (dt, JZ15.8, 6.6 Hz, 1H, Ph-CH]CH–CH2–),
4.69 (d, JZ6.5 Hz, Ph-CH]CH–CH2–), 2.32 (t, JZ7.4 Hz,
2H, –OOC–CH2–), 1.66 (h, JZ7.4 Hz, 2H, –OOC–CH2–
CH2), 0.95 (t, JZ7.4 Hz, 3H, –CH3); 13C NMR (75 MHz,
CDCl3): d 174.1 (–COO–), 134.1 (Ph-CH]CH–), 121.4
(Ph-CH]CH–), 144.0, 143.8, 129.8, 120.2, 115.5, 113.3
(Ar), 65.3 (–CH2OCO–), 36.4 (–OOC–CH2–), 18.6 (–CH2–),
13.7 (–CH3); HRMS FABC calcd 259.1105 for
C13H16O4Na [MCNa]C, found 259.1109.
3.2.4. 3,4-Dihydroxycinnamyl palmitate 8. 71%. White
solid; 1H NMR (300 MHz, CDCl3): d 6.92 (s, 1H, ar), 6.81
(s, 2H, ar), 6.51 (d, JZ15.8 Hz, 1H, Ph-CH]CH–CH2–),
6.09 (dt, JZ15.8, 6.6 Hz, 1H, Ph-CH]CH–CH2–), 4.69 (d,
JZ6.5 Hz, Ph-CH]CH–CH2–), 2.33 (t, JZ7.4 Hz, 2H,
–OOC–CH2–), 1.63 (q, JZ7.0 Hz, 2H, –OOC–CH2–CH2),
3.2.9. 3,4-Dihydroxybenzylic stearate 13. 74%. White
1
solid; H NMR (300 MHz, CDCl3): d 6.88 (d, JZ1.8 Hz,
1H, ar), 6.83 (d, JZ8.1 Hz, 1H, ar), 6.78 (dd, JZ8.0,