Synthesis of Lipophilic Antioxidants by a Lipase-B-Catalyzed Addition of Peracids to the Double Bond of 4-Vinyl-2-methoxyphenol

Article abstract of DOI:10.1021/acs.jafc.5b03551

4-Vinyl guaiacol (2) was lipophilized through the electrophilic addition of peracids to its vinylic double bond. Those peracids were formed in situ, by the Candida antarctica lipase-B-assisted perhydrolysis of carboxylic acids ranging from C2 to C18, in hydrogen peroxide solution. The addition of peracids with 4-8 carbons in their alkyl chains led to the formation of two regioisomers, with the prevalence of hydroxyesters bearing a primary free hydroxyl (4c-4e). This prevalence became more pronounced when peracids with longer alkyl chains (C10-C18) were used. In this case, only isomers 4f-4h were formed. The antioxidant activity of the resulting hydroxyesters was assessed by means of the conjugated autoxidizable triene (CAT) assay, and it was found out that the 4-vinyl guaiacol antioxidant activity was significantly increased by grafting alkyl chains with 2-8 carbons.

Full text of DOI:10.1021/acs.jafc.5b03551

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Article
Synthesis of Lipophilic Antioxidants by a Lipase B Catalyzed
Addition of Peracids to the Double Bond of 4-Vinyl-2-Methoxyphenol
Erika Zago, Erwann Durand, Nathalie Barouh, Jérôme Lecomte, Pierre Villeneuve, and Chahinez AOUF
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03551 • Publication Date (Web): 04 Oct 2015
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Journal of Agricultural and Food Chemistry
Synthesis of Lipophilic Antioxidants by a Lipase B Catalyzed Addition of
Peracids to the Double Bond of 4-Vinyl-2-Methoxyphenol
Erika Zago,a Erwann Durand,a Nathalie Barouh,a Jérôme Lecomte,a Pierre villeneuve a and
Chahinez Aouf b*
a: CIRAD, UMR IATE, Fꢀ34398 Montpellier, France.
b: INRA, UMR 1083 SPO, Fꢀ34060 Montpellier, France.
*Corresponding author: Chahinez Aouf
2 place Pierre viala, 34060 Montpellier, France
+(33) 4 99 61 24 54
aouf@supagro.inra.fr
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Abstract
4ꢀvinyl guaiacol 2 was lipophilized through the electrophilic addition of peracids to its vinylic
double bond. Those peracids were formed in situ; by the Candida antarctica lipase B assisted
perhydrolysis of carboxylic acids ranging from C2 to C18, in hydrogen peroxide solution. The
addition of peracids with 4 to 8 carbons in their alkyl chains led to the formation of two
regioisomers with the prevalence of hydroxyesters bearing a primary free hydroxyl (4c to 4e).
This prevalence became more pronounced when peracids with longer alkyl chains (C10 to
C18) were used. In this case, only isomers 4f to 4h were formed. The antioxidant activity of
the resulting hydroxyesters was assessed by means of CAT assay and it was found out that the
4ꢀvinyl guaiacol antioxidant activity was significantly increased by grafting alkyl chains with
2 to 8 carbons.
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Keywords: lipophilization, vinylphenols, electrophilic addition, antioxidant activity.
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Introduction
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Ubiquitous in plant kingdom and widely distributed in our diet, phenolics are the most
abundant secondary metabolites of plants, with more than 8000 phenolic structures currently
known. Ranging from simple molecules such as phenolic acids to highly polymerized
substances such as tannins, they have been considered as excellent natural antioxidants.1ꢀ3
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Antioxidants are increasingly of interest in food, cosmetic and pharmaceutical industries. For
instance, in food applications, they retard the oxidative degradation of unsaturated fats and
oils, preserving the food quality and improving its nutritional value.4, 5 However, the major
drawback of the use of phenolic antioxidants in oil based food processing, is their poor
lipophilic character. Hence, grafting a lipophilic moiety to these hydrophilic compounds could
be a good alternative to enhance their solubility and antioxidant properties in lipidic media.
This transformation commonly known as lipophilization is a widely recognized method for
expending the applications of phenolic derivatives to fats/oil and lipidꢀcontaining foods and
formulations.
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Within the phenolic compounds family, hydroxycinnamic acids (phenolic acids) and their
derivatives which occur abundantly in fruit, vegetables, cereals and spices, possess interesting
biological properties6 and antioxidant potency.7, 8 Several studies, dealing with the enzymatic
lipophilization of these phenolics by esterification and transesterification reactions, have been
reported. These studies mainly focused on the catalytic role of lipase B from Candida
antarctica in the esterification of phenolic acids with aliphatic alcohols in organic solvents 9-11
12-14
or solvent free media.
However, many of these lipase catalysed esterifications exhibited
low conversion yields and/or reaction rates (except with cinnamic acid), or led to high yield
only when ethanol and activated vinyl hydroxycinnamates were used as substrates.
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Beside esterification and transesterification, lipase B from Candida antartica (CALꢀB) is an
excellent catalyst for a wide range of chemical reactions15-17 including, among others, olefins
epoxidation. Indeed, in the presence of carboxylic acids, it acts as a perhydrolase which
catalyzes the reversible formation of peracids in aqueous hydrogen peroxide solutions.18 To
achieve the epoxidation reaction, the electrophilic peracids, produced in situ, have to carry the
oxygen to the nucleophilic partner, the alkenes. However, in the presence of strongly
nucleophilic alkenes, peracids are able to modify their usual function as oxygen carrier.
Indeed, it was reported by Chen et al. 19 that the reaction of Oꢀbenzyl ferulic acid with metaꢀ
chloroperbenzoic acid (mCPBA) in dichloromethane, resulted in the peracid 1,2ꢀaddition to
the phenolic compound double bond. This electrophilic addition was attributed to the presence
of the Oꢀbenzyl and Oꢀmethyl substituents on the aromatic ring, which increase the electronic
density and thus the nucleophilic property of the double bond.
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Within the hydroxycinnamic acids derivatives, vinylphenols are perfectly tailored to undergo
such reactions. Indeed, the vinyl side chain conjugated to the phenolic ring (which mostly
bears electronꢀreleasing groups) enhances the vinyl double bond nucleophilicity and peracids
addition might be envisioned. 4ꢀvinylphenol derivatives such as 4ꢀvinylcatechol, 4ꢀvinyl
guaiacol and canolol (4ꢀvinyl syringol) are natural compounds widespread in plant kingdom20,
21. In industry, these substances are treated as food additives and are approved as flavouring
agents by regulatory agencies.22 They are also involved in resins, elastomers and adhesives
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production.
However, no study has examined the possibility to produce lipophilic
antioxydants from vinylphenols.
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In the present work, the strong nucleophilic character of vinylphenols has been exploited to
propose an original way for lipophilization by means of peracids. As a vinylphenols
representative, 4ꢀvinyl guaiacol 2 was selected to undergo this transformation.
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Thus, The perhydrolysis activity of lipase B from Candida antarctica
contributed to
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produce a set of peracids, with different alkyl chain lengths, which were grafted by
electrophilic addition to the 4ꢀvinyl guaiacol double bond. Subsequently, the antioxidant
activity of 4ꢀvinyl guaiacol and its resulting hydroxyesters was evaluated using the conjugated
autoxidizable triene (CAT) assay. The synthesized hydroxyesters can be used as effective
antioxidants in multiphase food products and pharmaceuticals.
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Material and methods
Chemicals
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Vanillin (99%), malonic acid (99%), piperidine (≥99.5%), butanoic acid (≥99%), hexanoic
acid (≥98%), octanoic acid(≥98%), decanoic acid (≥98%), dodecanoic acid (≥98%),
octadecanoic acid (≥95%), tertꢀbutyldimethysilyl chloride (97%), imidazole (≥99%),
immobilised lipase B from Candida antarctica (novozym 435), hydrogen peroxide (30 wt.%
in H2O), Tung oil from Aleurites fordii seeds (average MW = 872 g.molꢀ1), Phosphate buffer
solution pH 7.2 (PBS), polyoxyethylene(23)lauryl ether (Brij 35, estimated MM = 1198
g.molꢀ1), (±)ꢀ6ꢀHydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ2ꢀcarboxylic acid (Trolox) were
purchased
from
SigmaꢀAldrich
France.
2,2′ꢀAzobis(2ꢀmethylpropionamidine)
dihydrochloride (AAPH) was obtained from Wako Chemical, (Neuss, Germany), Glacial
acetic acid was purchased from Merck France and hexadecanoic acid was supplied by
SEPPIC France. All solvents were analytical or HPLC grade (SigmaꢀAldrich France) and
were used as received.
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All the reactions were monitored by TLC performed on silica gel 60 F254 (provided by
Merck). Excepted for 2, all compounds were dissolved in CDCl3 and analysed using a Brucker
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400 MHz spectrometer, operating at 400 MHz for H and 100 MHz for C. Chemical shifts
1
were referenced using residual internal CH3Cl signals at 7.26 ppm and 77.0 ppm for H and
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13C respectively. Compound 2 solution in DMSOꢀd6 was analysed on Agilent VNRMS DD2
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500MHz spectrometer, operating at 500,05 MHz for H and 125.75 MHz for 13C, using a
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5mm indirect detection Zꢀgradient probe. The chemical shifts were reported to that of internal
DMSO at 2.5 ppm and 39.5 ppm for 1H and 13C respectively. Assignments of both proton and
carbon resonances, identification and structure characterization of products were performed
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using both 1D and 2D NMR spectrum analysis, homonucluear H and heteronuclear H/13C
experiments. Elemental analyses were performed by combustion on Flash EA 1112
(thermoFinnigan 2003). HRMS analyses were performed by a XevoꢀG2ꢀXS QꢀTof (from
Waters). Statistical analysis (Oneꢀway ANOVA) of CAT values was done using JMP
software v.12. (SAS Institute, USA). The means were then compared using Student’s tꢀtest.
The significance level α was set to 0.05.
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Protection of the phenolic hydroxyl of 4-vunyl guaiacol 2
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Under argon, a solution of tertbuthyldimethylsilyl chloride (1.2 mmol, 0.18 g) in DMF (2 mL)
was added dropwise to a stirred solution of 4ꢀvinyl guaiacol 2 (1 mmol, 0.15 g) and imidazole
(2.5 mmol, 0.17 g) in DMF (3 mL). Stirring was maintained 26 h at room temperature and
then water (25 mL) and ethyl acetate (25 mL) were added. The aqueous layer was separated
and extracted with ethyl acetate (2x25 mL). The combined organic layers were washed with
brine (30 mL), dried over anhydrous MgSO4 and concentrated under reduced pressure. Flash
chromatography purification (petroleum ether/ethyl acetate 90:10) afforded:
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Tert-butyl(2-methoxy-4-vinylphenoxy)dimethylsilane 3: colourless oil, 85% yield (0.83
1
mmol). H NMR (400 MHz, CDCl3) δ = 0.18 (s, 6H, H12), 1.02 (s, 9H, H10), 3.84 (s, 3H,
H9), 5.14 (dd, J= 10.8, 0.91 Hz, 1H, H8), 5.61 (dd, J= 17.6, 0.91 Hz, 1H, H8), 6.65 (dd, J=
17.6, 10.9 Hz, 1H, H7), 6.81 (d, J= 8.1 Hz, 1H, H6), 6.88 (dd, J= 8.2, 1.9 Hz, 1H, H5), 6.94
13
(s, 1H, H2) ppm. C NMR (100 MHz, CDCl3) δ = 4.6 (C12), 18.4 (C11), 25.7 (C10), 55.4
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(C9), 109.6 (C2), 111.7 (C8), 119.4 (C6), 120.4 (C5), 131.7 (C1), 136.7 (C7), 145.1 (C4),
151.0 (C3) ppm. [M+ H+] calcl. 265.1504, found. 265.1510. C15H24O2Si calcd. C 68.13, H
9.15, Si 10.62; found. C 70.02, H 8.89, Si 11.31.
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Lipophilization of the silylated vinyl phenol 3
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In 50 mL round bottom flask equipped with magnetic stirrer, 2 mmol of carboxylic acid, 3
mmol of hydrogen peroxide 30% (0.34 mL) and Novozym 435 (10 wt% relative to the weight
of the silylated vinyl phenol, 0.026 g) were mixed in 2 mL of toluene. After 30 min at 40°C, a
solution of compound 3 (1 mmol, 0.26 g) in toluene (1 mL) was added to the reaction medium
over 2 h. The reaction was monitored by TLC. After the complete consumption of the peracid,
catalyst beads were removed from the reaction mixture by filtration, 30 mL of water and 30
mL of ethyl acetate were added to the filtered reaction mixture. The aqueous layer was
separated and extracted with ethyl acetate (2x30 mL). The combined organic layers were
washed with brine (40 mL), dried over anhydrous MgSO4 and concentrated under reduced
pressure. The phenolic group deprotection was performed on the crude products.
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Deprotection of the phenolic hydroxyl
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The crude product formed from the reaction of compound 3 with peracids (1 mmol) was
diluted in 2 mL of dry THF and the solution was cooled down to 0°C. Under argon, a
solution of TBAF (1.2 mmol) in 2 mL of dry THF was added dropwise to the previous
solution. After 3h at 0°C, water (25 mL) and ethyl acetate (25 mL) were added. The aqueous
layer was separated and extracted with ethyl acetate (2x25 mL). The combined organic layers
were washed with brine (30 mL), dried over anhydrous MgSO4 and concentrated under
reduced pressure. Flash chromatography purification (petroleum ether/ethyl acetate 90:10 to
60:40) afforded βꢀhydroxyesters 4a to 4h and αꢀhydroxyesters 5b to 5e. For example, the
electrophilic addition of octanoic peracid to compound 3, gave rise:
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2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)ethyl octanoate 4d. Yellow oil, 46% yield (0.45
mmol).1H NMR (400 MHz, CDCl3) δ = 0.88 (t, J= 6.9 Hz, 3H, H17), 1.26ꢀ1.1.29 (m, 8H,
H16ꢀH13), 1.62ꢀ1.66 (m, 2H, H12), 2.38 (t, J= 7.40 Hz, 2H, H11), 3.78 (dd, J= 11.90, 4.30
Hz, 1H, H9α), 3.86 (m, 1H, H9β), 3.90 (s, 3H, H7), 5.65 (s, 1H, OH aliphatic), 5.79 (dd, J=
7.60, 4.30 Hz, 1H, H8), 6.86 (d, J= 8.46 Hz, 1H, H6), 6.89 (d, J= 7.80 Hz, 1H, H5), 6.91 (s,
13
1H, H2) ppm. C NMR (100 MHz, CDCl3) δ = 14.0 (C17), 22.5 (C16), 25.0 (C12), 28.8
(C15), 29.0 (C14), 31.6 (C13), 34.5 (C11), 55.9 (C7), 66.0 (C9), 76.5 (C8), 109.5 (C2), 114.5
(C5), 119.8 (C6), 129.0 (C1), 145.8 (C4), 146.5 (C3), 173.5 (C10) ppm. [M+ H+] calcl.
311.1821, found. 311.1835. C17H26O5 calcd. C 65.78, H 8.44; found. C 65.67, H 8.51.
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2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)ethyl octanoate 5d. Yellow oil, 30% yield (0.29
mmol).1H NMR (400 MHz, CDCl3) δ = 0.89 (t, J= 6.9 Hz, 3H, H17), 1.28ꢀ1.33 (m, 8H, H16ꢀ
H13), 1.64 (m, 2H, H12), 2.36 (t, J= 7.50 Hz, 2H, H11), 3.90 (s, 3H, H7), 4.16 (dd, J= 11.50,
3.58 Hz, 1H, H9α), 4.25 (dd, J= 11.50, 3.50 Hz, 1H, H9β), 4.89 (dd, J= 8.40, 3.40 Hz, 1H,
H8), 5.66 (s, 1H, OH aliphatic), 6.86 (dd, J= 8.50, 1.80 Hz, 1H, H6), 6.89 (d, J= 8.10 Hz, 1H,
13
H5), 6.94 (s, 1H, H2) ppm. C NMR (100 MHz, CDCl3) δ = 14.0 (C17), 22.35 (C16), 24.9
(C12), 28.8 (C15), 29.0 (C14), 31.6 (C13), 34.2 (C11), 55.9 (C7), 69.2 (C9), 73.3 (C8), 108.5
(C2), 114.3 (C5), 119.2 (C6), 131.8 (C1), 145.6 (C4), 146.7 (C3), 174.0 (C10) ppm. [M+ H+]
calcl. 311.1821, found. 311.1817. C17H26O5 calcd. C 65.78, H 8.44; found. C 65.91, H 8.36.
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The other NMR data are displayed in the supplementary information.
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Determination of the antioxidant capacity using the Conjugated Autoxidizable Triene
(CAT) assay.
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Prior to use, commercial tung oil was stripped (removing of polar compounds including
tocopherols) as follows. In a glass column packed with 25 g of alumina, 25 ml of a tung oil
solution in nꢀhexane (200 mg.mLꢀ1) were eluted followed by 25 mL of hexane. Then, hexane
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was evaporated under vacuum and the stripped tung oil was aliquoted into brown glass tubes,
inerted under nitrogen stream, and stored at −18 °C until use. All experiments were performed
under shelter from light, as much as possible.
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Fresh solutions of Trolox and phenolic at concentrations ranging from 0.4 to 4.0 mmol.Lꢀ1
were prepared in chloroform/methanol (2:1), then diluted in methanol in order to obtain a
concentration range of 0.1 to 1.0 µmol.Lꢀ1, and stored in amber vials. 50 µL of these solutions
were transferred to a 96ꢀwell microplate (Greiner, Frickenhausen, Germany) which was then
preheated to 37 °C and shook in a thermostated shaker (PHMT Grant Instruments Ltd.,
Shepreth, England) for 5 min at 1200 rpm.
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Twentyꢀfive milliliters of PBS containing Brij 35 (34 µmol.Lꢀ1) were added to 5 mg of
stripped tung oil into an amber glass vial. Thereafter, this preꢀmixture was vortexed for 10
seconds prior to homogenization in an Ultra Turrax homogenizer (Janke & Kunkel, Staufen,
Germany) at 2400 rpm for 90 seconds. Each well of the microplate was supplemented with
100 µL of this emulsion. To enhance repeatability, the microplate was immediately preꢀheated
and maintained under stirring in a thermostated shaker (PHMT Grant Instruments Ltd.) at 37
°C for 1 min at 1200 rpm.
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Fifty microliters of a solution of AAPH in PBS (4 mmol.Lꢀ1), prepared immediately before
reading, were added into wells. Finally, each well contained a final volume of 200 µL, with
115 µmol.Lꢀ1 tung oil, 17 µmol.Lꢀ1 Brij 35, 1 mmol.Lꢀ1 of AAPH and antioxidants at various
concentrations (0.25 to 1.0 µmol.Lꢀ1). The kinetics of the reaction were immediately assessed
by monitoring absorbance decay at 273 nm. Measurements were taken every minute within 6
hours at 37 ± 0.5 °C with shaking the plate 5 seconds before each reading, using a microplate
reader Model M1000 (Tecan, Groedig, Austria) equipped with software Magellan.
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All measurements were performed in triplicate and reported as average ± SD. To normalize
data, the raw absorbance values were expressed in relative absorbance according to equation
1:
ꢀꢁ
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Relative absorbance = ꢀꢂ
Eq.1
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Where At and A0 are the absorbances read at time t (min) and t0 (min), respectively. The area
under the curve (AUC) corresponding to the decay of relative absorbance was calculated
thanks to equation 2:
ꢀꢆ
ꢀꢂ
ꢀꢇ
ꢀꢂ
ꢀꢈꢋꢂ
ꢀꢂ
196
ꢃꢄꢅ = 1 +
+
+ ⋯ + ꢀꢈꢉꢊ
+
Eq.2
ꢀꢂ
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The chart area provided by an antioxidant sample was then calculated using the difference
between AUC in the presence of a sample of antioxidant (AUCsample) and the blank (AUCblank
)
corresponding to a solution without antioxidant. Trolox was used as internal calibration
standard. Thus, the antioxidant capacity (CAT value) of a sample was given in Trolox
equivalents and calculated by equation 3:
(ꢑꢛꢓꢔꢏ ꢛꢝ ꢙꢚꢛꢓꢛꢜ)
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CAT value = (ꢌꢍꢎ ꢏꢐꢑꢒꢓꢔꢕꢌꢍꢎ ꢖꢓꢐꢗꢘ)
×
Eq.3
(ꢌꢍꢎ ꢙꢚꢛꢓꢛꢜꢕꢌꢍꢎ ꢖꢓꢐꢗꢘ)
(ꢑꢛꢓꢔꢏ ꢛꢝ ꢏꢐꢑꢒꢓꢔ)
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Where AUCsample, AUCblank and AUCTrolox are the areas in the graph where the protection of
tung oil occurs in the presence of a sample, the antioxidantꢀfree solution and the solution
containing the Trolox, respectively. The CAT value is expressed in moles of Trolox per moles
of the tested compound, i.e. in Trolox equivalents.
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Results and discussion
In order to produce antioxidant 4ꢀvinylꢀguaiacol derivatives with increased lipophilicity, the
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following fourꢀstep synthesis route was adopted: i) 4ꢀvinylꢀguaiacol 2 production from the
reaction of vanillin ii) The protection of the compound 2 phenolic hydroxyl by silylꢀ
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etherification (to avoid the undesirable oxidation or polymerisation sideꢀreactions). iii) The
electrophilic addition of the in situ generated peracids (with different chain length) to the
protected 4ꢀvinylꢀguaiacol 3. iv) The phenolic hydroxyls deprotection.
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Synthesis of 4-vinyl-guaiacol 2 and protection of its hydroxyl group
Generally, the availability of vinylphenols in natural sources does not meet the industrial
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requirements. Hence, several chemical syntheses of these phenolic compounds, including
catalytic dehydrogenation of ethylphenols, Wittig reaction or the hydroxycinnamic acids
decarboxylation, have been described25. However, a majority of these procedures required
expensive reagents and harsh conditions to eventually give rise to the products in low yields.
Recently, an efficient synthesis of these compounds by KnoevenagelꢀDoebner25 reaction in
mild conditions was described by Sinha et al. Hence, this synthetic pathway was adopted to
produce 4ꢀvinylꢀguaiacol 2 from vanillin 1.
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Subsequently, the 4ꢀvinylꢀguaiacol hydroxyl group was temporary masked to avoid the
competitive oxidation and polymerisation sideꢀreactions. Among several protecting groups,
tertꢀbutyldimethylsilyl (TBDMS) was selected because of its stability in a variety of organic
reactions and the facility by which it can be introduced and removed under mild
conditions.26, 27 The treatment of Compound 2 with tertꢀbutyldimethylsilyl chloride and
imidazole at room temperature led to the silylꢀether derivative 3 with 85% yield (figure 1).
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Lipophilization of 3 by electrophilic esterification
As reported in the literature, various reaction parameters can influence the lipase activity and
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thus the peracids formation. Among several organic solvent tested the highest yields of
peracids were observed using toluene or hexane.28 Törnvall et al.29 and OrellanaꢀCoca et al.24
investigations have demonstrated that elevated temperatures (above 60°C) together with
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hydrogen peroxide at high concentrations (6Mꢀ12M) resulted in a rapid loss of enzyme
activity. Taking into account these different data, the vinylic derivative 3 was reacted with
caprylic peracid (octanoic peracid) in a twoꢀphase reaction. First, the CALꢀB catalyzed
perhydrolysis of caprylic acid was initiated at 40 °C during 30 min in toluene. Then, the
solution of compound 3, in the same solvent, was added to the previous mixture over 2 hours.
During the whole reaction, the hydrogen peroxide concentration did not exceed 1M.
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After 6 hours of reaction and the usual work up (described in the experimental part), the crude
product was treated with tetraꢀNꢀbutylammonium fluoride (TBAF) in THF at 0 °C, allowing
the silyl ether cleavage and the hydroxyl group recovery within 3 hours.
244
245
Instead of the expected epoxidized products, hydroxyester derivatives 4 and 5 were produced
in 76% overall yield (figure 2).
246
247
248
249
250
251
The addition of decanoic peracid to compound 3 was proved by NMR. Indeed, the
characteristic proton signals of the vinylic double bond, at 5.07, 5.64 and 6.67 ppm
disappeared and were replaced by αꢀhydroxyl (3.78ꢀ3.86 ppm for 4 and 4.89 ppm for 5) and
αꢀester (5.79 ppm for 4 and 4.16ꢀ4.25 ppm for 5) proton signals. The appearance of shielded
1
aliphatic proton signals on H NMR spectrum and the ester carboxyl signal on 13C NMR
spectrum indicated the grafting of the carboxylic acid moiety on 3.
252
253
254
255
Based on these results, other carboxylic acids (ranging from C2 to C18) were used to assess
the reactivity of compound 3 with the in situ generated corresponding peracids and to produce
phenolic hydroxyesters with different side chain lengths. The reaction products are depicted in
both figure 3 and table 1.
256
257
Considering the results displayed in table 1, two main aspects should be discussed. (i) the
peracid addition to the vinylic double bond instead of its epoxidation. (ii) the arising of two
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addition isomers with the predominance of the derivatives bearing a primary alcohol function
(4aꢀ4h derivatives).
260
261
The peracid addition to the double bond of compound 3
Generally, in the chemoꢀenzymatic oxidation of olefins, the lipase catalyzes the formation of
262
263
264
265
266
267
268
peracids, from the perhydrolysis reaction between carboxylic acids and an oxidizing agent
such as H2O2, which then donate an oxygen atom to the double bond affording the
corresponding epoxide and regenerating the carboxylic acids. The epoxidation step occurs
through a concerted transition state, also known as a symmetry butterfly transition state. The
bond between the oxygen and the alkene is being formed at the same time that the peroxy
bond is breaking and the peracid proton is transferred from the hydroxyl group to the carbonyl
oxygen.
269
270
271
272
273
274
275
276
277
278
279
280
However, in the present instance, the generated peracids did not achieve their expected task as
oxygen carrier, but an electrophilic addition, rather occurred to the vinyl double bond of 3. As
previously mentioned, the presence of the electronꢀreleasing groups OTBDMS and Oꢀmethyl
on the aromatic ring of 3 contribute, by mesomeric effect to enhance the electron density on
the vinylic double bond. The electrophilic peroxide oxygen of peracid attacks the double bond
and form a three membered cyclic transition state I19 (figure 4). Then, the electron rich
carboxylate group of peracid attacks either C7 or C8 carbons leading to the simultaneous
cleavage of the bonds between these carbons and the peroxide oxygen and the formation of
compounds 4 and 5. The intervention of the electronꢀreleasing groups to orientate the
reaction mechanism towards peracids addition is confirmed by Sarma et al30 who
demonstrated that styrene derivatives, with electronꢀwithdrawing substituents, are rather
epoxidized in the presence of peracids.
281
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283
The peracids addition products
As displayed in table 1, excluding compounds 4a, 4b and 5b, the addition products of
peracids on the vinylic double bond of 3 after hydroxyls deprotection were obtained with fair
yields ranging from 68% to 77%. The reaction of both ethanoic and butanoic peracids with 3
required a longer time and a higher amount of enzyme to afford addition products with
relatively lower yields (entry 1 and 2). This is likely due to the peracids stability which is
highly dependent on their molecular weight. Indeed, the smaller are the peracids, the faster is
their decomposition; in particular, methanoic and ethanoic peracids are highly unstable31.
284
285
286
287
288
289
290
291
292
293
294
295
296
Despite the lower reaction yield of butanoic peracid, this latter led to the formation of two
addition products 4b and 5b with a slight predominance of the derivative bearing a primary
alcohol function 4b. The same trend was observed for hexanoic, octanoic and decanoic
peracids, indicating that from C4 to C8, peracids exhibit the same reactivity towards
compound 3. Although the predominance of 4-type products is not very significant; it results
from the addition of the peracid electrophilic oxygen on the most nucleophilic double bond
carbon.
297
298
299
300
From dodecanoic to octadecanoic peracids (entries 6 to 8), only the 4ꢀtype products were
formed. The presence of a long alkyl chain in the reaction intermediate I seems to further
polarize the C7ꢀC8 bond, localizing the negative charge on C8. Hence, only the electron
deficient C7 was attacked by the carboxylate group.
301
302
Owing the good enzyme affinity to dodecanoic acid,28 its corresponding peracid afforded the
addition product (4f) with the highest yield (77%) in a relatively short time (entry 6).
303
304
305
This particular reactivity of peracids allowed the production of a set of phenolic
hydroxyesters with side chain lengths ranging from C2 to C18. To obtain equivalent reaction
yields, the other strategies based on the lipase B catalyzed esterification and
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307
transesterification; generally require harsher operational conditions, like a longer reaction
time (7 to 30 days) and higher temperatures (50 to 60°C).32
308
309
After synthesis, the antioxidant potency of these lipophilized phenolics was assessed by CAT
assay.
310
311
Determination of the antioxidant capacity of lipophilized derivatives of 4-vinyl guaiacol
In this study, the CAT assay was used to evaluate the antioxidant capacity of the synthesized
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
phenolic hydroxyesters. The triacylglycerols of tung oil are mainly (ca. 85 %) comprised of
alphaꢀeleostearic acid ((9Z,11E,13E)ꢀ9,11,13ꢀoctadecatrienoic acid). Owing to the presence of
a conjugated triene, this fatty acid strongly absorbs at 273 nm and is highly prone to
oxidation. Consequently, under oxidizing conditions, a degradation of the conjugated triene
may be simply observed upon the signal loss at 273 nm. These properties allow for an
efficient, easy, rapid and direct assessment of lipid oxidation in heterophasic system. Thus,
the CAT assay provides relevant screening of antioxidant abilities of molecules, since it is
representative of unsaturated fatty acids oxidation pathways that occur in foods and biological
systems 33. As depicted in both table 1 and figure 5, the antioxidant ability of 4ꢀvinyl guaiacol
was significantly affected by the grafting of the lipophilic moiety (alkyl chain of variable
length). Moreover, the isomer nature (phenolic ester bearing a primary or a secondary
hydroxyl) may significantly affect the antioxidant activities in such heterogeneous medium.
Indeed, isomers exhibiting the secondary alcohol function presented lower antioxidant
activities, with only a slight enhancement for the butylꢀester (CAT value = 0.49 ± 0.01) in
comparison with 4ꢀvinylguaiacol (CAT value = 0.40 ± 0.03). One may argue that the
difference between the two regioisomers might be explained by the negative inductive effect
(ꢀI) of the free aliphatic hydroxyl group. Thus, the one closest to the benzene ring would
higher disrupt the resonance stabilization of the phenoxyl radicals, resulting in lower
antioxidant ability and CAT value. As far as the phenolic esters with primary free hydroxyl
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332
333
334
335
336
337
338
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340
341
342
343
344
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346
347
(obtained with higher yields), significant improvement in the antioxidant activity was
observed with the addition of 2, 4, 6 and 8 carbons in the ester alkyl chain (4a, 4b, 4c and 4d
respectively) when compared to 4ꢀvinylguaiacol. The nonꢀlipophilized molecule 2 presented a
CAT value of 0.40 ± 0.03, while compounds 4a to 4d had CAT values of 0.80 ± 0.05, 0.49 ±
0.04, 0.55 ± 0.01 and 0.57 ± 0.03, respectively. Conversely, their counterparts with 10, 12, 16
and 18 carbons in the alkyl chain (4f, 4g and 4h respectively) showed lower antioxidant
ability in comparison with 4ꢀvinylguaiacol. The order of antioxidant capacity was: C4 > C8 ≈
C6 > C2 >> C10 ≥ C12 ≈ C16 > C18, and a clear pattern with respect to the effect of alkyl
chain length on the antioxidant ability was observed. Indeed, the effectiveness of antioxidants
has been shown to increase as a function of increasing alkyl chain length to a critical point (4
carbons), followed by steady decrease beyond that point when more hydrophobic moieties are
linked. This nonlinear effect named “cutꢀoff effect” has already been reported in literature
with similar experiments performed with series of alkyl chlorogenates, rosmarinates, ferulates
and coumarates 34-37. In these studies, the antioxidant activities were drastically improved with
increasing alkyl chain lengths until an optimized activity ranging between 8 to 12 carbon
atoms. In both series of esters synthesized from 4ꢀvinylguaiacol, the highest antioxidant
capacity was obtained with an alkyl chain of 4 carbons.
39
348
349
350
351
352
353
354
355
As previously observed,38,
the intrinsic chemical reactivity of an antioxidant toward
oxidizing species is not the only factor that governs its activity, this feature being in fact
multifactorial. Suitable location, position, mobility, and concentration of antioxidant at the
interface, but also its reactivity towards free radical can drastically change antioxidant
efficiency. It is now accepted that lipophilization mostly affects antioxidant properties by
changing the molecule’s partitioning. In such lipidꢀbased dispersions, the antioxidant
molecule may distribute in the aqueous or in the lipid phase as well as in the intermediate
pseudophase (interface) as monomer, micelle or aggregate forms, which results in a different
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358
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362
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365
366
367
368
369
370
371
372
373
374
375
reactivity. Thus, the antioxidant activity of the lipophilized molecules synthesized from 4ꢀ
vinylguaiacol can be explained by their polymorphism and partitioning in the present
environment. The first group, composed by the ethyl ester (4a), tends to partition within the
aqueous phase with limited interaction with the interface of the lipid droplets where oxidation
occurs. Consequently, it displays CAT value equal to the native molecule because of their
equivalent polarity (table 1).The second group formed by the butyl ester (4b) showed the
highest CAT values. This may be explained by a better location of this molecule at the
oil/water interface, promoting oil protection against the oxidizing species (RꢀCꢁ, RꢀCOOꢁ,
etc…) generated by the azo initiator. When the chain length was increased beyond this limit,
the amount of esters in the intermediate phase is expected to decrease while the amount in the
lipid phase is supposed to increase, which partly explain the diminution in the CAT values in
the third group composed with medium alkyl chains (C6 and C8). Also, these molecules could
be found in different polymorphisms: monomers, which may protect oil droplets against
oxidation when located at the interface and micelles, which will be only sparsely involved
against the lipid droplet oxidation. Finally, the last group composed of esters with 10 or more
carbon in the alkyl chain, exhibited very low antioxidant activities because of their high
lipophilic character. These compounds are mainly located within the lipid phase, with a
limited activity at the interface. Moreover, they might be also physically isolated from
emulsion interface through the formation of aggregates (micelles or precipitates) that reduce
their concentration in the reactive interphase.
376
377
378
379
380
Generally, phenolic compounds lipophilization catalysed by lipase B of Candida antarctica is
performed through esterification or transesterification reactions. However, it has been shown
in this study, that the perhydrolysis activity of lipase B was an efficient way to graft alkyl
chains with different lengths on 4ꢀvinyl guaiacol. The chemical structure of this vinylphenol
derivative, and particularly its nucleophilic vinylic double bond, contributed to modify the
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382
383
384
385
386
387
388
389
usual reactivity of peracids leading to the oneꢀpot production of different hydroxyesters by
electrophilic addition. Within this addition, the formation of hydroxyesters bearing primary
hydroxyl groups was promoted especially when the chain length of the peracid exceeded eight
carbons. The assessment of the antioxidant potency of the resulting hydroxyesters
demonstrated that the 4ꢀvinyl guaiacol antioxidant activity was enhanced by the grafting of
alkyl chains with 2 to 8 carbons. Therefore, this new route of phenolipids synthesis through
lipophilization of vinylphenols represents a good strategy to promote and optimize the
effectiveness of their natural antioxidant activity in heterophasic system encountered in food
emulsions, cosmetics and biological systems.
390
391
392
Supporting information including NMR spectra and HRMS data of the synthesized products
as well as the statistical analysis of CAT values is available.
393
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References
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507
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Figure captions
Figure 1: the 4ꢀvinylꢀguaiacol synthesis and its phenolic hydroxyl etherification.
528
529
Figure 2: the electrophilic esterification of compound 3 followed by the hydroxyl group
deprotection.
530
531
Figure 3: the chemical structures of hydroxyesters 4a to 4h and 5b to 5e.
Figure 4: The 1,2ꢀaddition mechanism of compound 3 with peracids.
532
533
534
535
Figure 5: CAT values of 4ꢀvinylguaiacol (ꢀ) and the lypophilized molecules: phenolic
hydroxyesters bearing primary free hydroxyl (ꢁ) and phenolic hydroxyl esters bearing
secondary free hydroxyl (ꢁ).Values followed by the same superscript letters are not
significantly different (p ≤ 0.05). Values are mean ± SD (n=3).
536
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541
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Tables
Table 1: The electrophilic addition products (after the phenolic hydroxyl deprotection) of
peracids on compound 3.
Reaction time
(h)b
Products Yield
Entry
Acid
products
CAT valuec
(%)
33
ꢀ
0.46 ± 0.05
ꢀ
4a
5a
4b
5b
4c
5c
4d
5d
4e
5e
4f
1
2
3
4
5
6
7
8
Ethanoic acid (n=0)a
Butanoic acid (n=2)a
Hexanoic acid(n=4)
Octanoic acid (n=6)
Decanoic acid (n=8)
Dodecanoic acid (n=10)
Hexadecanoic acid (n=14)
Octadecanoic acid (n=16)
126
102
4
26
22
43
32
46
30
43
31
77
ꢀ
0.80 ± 0.04
0.49 ± 0.01
0.55 ± 0.01
0.19 ± 0.05
0.57 ± 0.03
0.24 ± 0.02
0.24 ±0.04
0.22 ± 0.07
0.19 ± 0.04
ꢀ
6
7
6.5
24
20
5f
68
ꢀ
72
ꢀ
0.19 ± 0.02
ꢀ
0.12 ± 0.02
ꢀ
4g
5g
4h
5h
a Reactions of compound 3 with hexanoic to octadecanoic acids were performed by loading 10 wt% of
enzyme (relative to 3 weight). However, the addition of both ethanoic an butanoic peracids to the
compound 3 double bond required 30 wt% and 20 wt% of enzyme respectively.
b Reactions were monitored by TLC and stopped after the complete conversion of compound 3.
c CAT value of 4ꢀvinyl guaiacol is 0.40 ± 0.03.
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Figure 3
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Figure 4
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