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International Journal of
Molecular Sciences
Article
Chemo-Enzymatic Synthesis of Renewable
Sterically-Hindered Phenolic Antioxidants with
Tunable Polarity from Lignocellulose and
Vegetal Oil Components
Louis Hollande 1,2, Sandra Domenek 2and Florent Allais 1, *
1Chaire ABI, AgroParisTech, CEBB 3 rue des Rouges Terres 51110 Pomacle, France;
louis.hollande@agroparistech.fr
2UMR GENIAL, AgroParisTech, INRA, UniversitéParis-Saclay, Avenue des Olympiades,
91300 Massy, France; sandra.domenek@agroparistech.fr
*Correspondence: florent.allais@agroparistech.fr
Received: 28 September 2018; Accepted: 24 October 2018; Published: 26 October 2018
Abstract:
Despite their great antioxidant activities, the use of natural phenols as antioxidant additives
for polyolefins is limited owing to their weak thermal stability and hydrophilic character. Herein,
we report a sustainable chemo-enzymatic synthesis of renewable lipophilic antioxidants specifically
designed to overcome these restrictions using naturally occurring ferulic acid (found in lignocellulose)
and vegetal oils (i.e., lauric, palmitic, stearic acids, and glycerol) as starting materials. A predictive
Hansen and Hildebrand parameters-based approach was used to tailor the polarity of newly
designed structures. A specific affinity of Candida antarctica lipase B (CAL-B) towards glycerol
was demonstrated and exploited to efficiently synthesized the target compounds in yields ranging
from 81 to 87%. Antiradical activity as well as radical scavenging behavior (H atom-donation, kinetics)
of these new fully biobased additives were found superior to that of well-established, commercially
available fossil-based antioxidants such as Irganox 1010
®
and Irganox 1076
®
. Finally, their greater
thermal stabilities (302 < T
d
5% < 311
◦
C), established using thermal gravimetric analysis, combined
with their high solubilities and antioxidant activities, make these novel sustainable phenolics a very
attractive alternative to current fossil-based antioxidant additives in polyolefins.
Keywords: ferulic acid; fatty acid ethyl esters; CAL-B; antioxidant; DPPH
1. Introduction
In contact with atmospheric oxygen, polymers undergo oxidative degradation reactions during
fabrication processes, storage, and throughout their use [
1
–
4
]. Involving undesirable radical species
(R
•
) and mechanisms, these oxidative degradation reactions drastically impact the initial aesthetic
and mechanical properties of the polymers [
1
,
5
,
6
]. This ineluctable phenomenon is known as
thermo-oxidative ageing of polymers. Mixing polymeric materials with a complex blend of additives
able to inhibit or delay their deterioration is the best way to prevent premature ageing [
7
]. These
additives are named stabilizers, or more commonly antioxidants. They are classified according to their
mechanisms of action: primary (AO-I) and secondary (AO-II) antioxidants.
Sterically-hindered phenols (SHP) such as Irganox 1010
®
and Irganox 1076
®
, derived from
the controversial butylated hydroxytoluene moiety, are the best benchmark antioxidant additives
belonging to the AO-I group [
7
]. They bring oxidative degradation reactions to a close by transferring
hydrogen atoms from their phenols to R
•
, resulting in nonreactive phenoxyl radicals. Precisely, SHP act
Int. J. Mol. Sci. 2018,19, 3358; doi:10.3390/ijms19113358 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2018,19, 3358 2 of 12
like radical scavengers thanks to chain-breaking reactions, and thus prevent the formation of R
•
, well
known to be responsible for polymer degradation [8,9].
Recently, the additive industry has shown increasing interest in naturally occurring
p-hydroxycinnamic acids (HCAs), such as ferulic, caffeic, sinapic, and p-coumaric acids, because
of their nontoxic nature yet powerful chain-breaking antioxidative properties, acting through radical
scavenging [
10
–
14
]. Consequently, we previously reported a library of bisphenolic AO-I derived
from HCAs found in lignocellulose (p-coumaric, ferulic, and sinapic acids) and biobased diols
(1,3-propanediaol, 1,4-butanediol, and isosorbide) [
15
]. To optimize their antiradical activities,
we assessed the structure–activity relationships (aka SAR) of these phenolics [
16
]. The best candidates,
obtained from the reaction between ferulic acid and 1,4-butanediol (BDF), 1,3-propane-diol (PDF),
or isosorbide (IDF), exhibited potent antioxidant activities competing with commercially available
molecules such as Irganox 1010
®
. Their high thermal stabilities (>200
◦
C) render them compatible
with harsh polymer production processes. Furthermore, their molar mass greater than 500 g.mol
−1
prevents leaching and other volatility issues. Finally, their eco-friendly preparation makes them an
attractive choice as sustainable additives and therefore increases their market value even further.
Unfortunately, the polar character of this series of biobased antioxidants leads to a very poor solubility
in nonpolar media, such as polyolefins [
15
]. This major drawback thus limits their reactivity with
radicals responsible for oxidation and subsequently reduces their protective role against premature
polymer ageing [
17
]. Indeed, good solubility and mobility have an important positive impact on the
stabilization properties of antioxidants [3].
A promising approach to increase compatibility with nonpolar matrix, namely lipophilization,
consists in the covalent grafting of lipophilic moieties to phenolics in order to improve both miscibility
and incorporation of a given antioxidant in nonpolar media. Lecomte et al. have reported that
the grafting of a medium chain-length is the best strategy to design potent lipophilized antioxidants [
18
].
To date, many of these antioxidants, called phenolipids, have been synthesized from phenolic acid,
flavonoids or tocopherols [
19
–
21
]. However, the design of these molecules is usually not optimized
to provide sustainable alternatives able to challenge commercial additives for polymeric materials in
terms of activity, solubility and thermal stability.
This work reports on the design and preparation of new potent sustainable bisphenolics AO-I
from lignocellulosic and oleaginous biomasses as we dedicate ourselves to integrated biorefinery
concepts. To insure a high antiradical activity, the targeted bisphenolic structures were based on
our previously reported structure–activity relationship study (SAR) [
15
,
16
] and their solubilities into
common polymers were evaluated using Hansen and Hildebrand parameters [
22
]. Once the structural
designs were validated, the chemo-enzymatic synthesis of the target structures was optimized through
sustainable chemo-enzymatic processes involving a lipase-catalyzed transesterification strategy [
23
].
Finally, their activities and thermal stabilities were benchmarked against that of Irganox 1010
®
and
lipophilic Irganox 1076®, two widely used fossil-based antioxidant additives.
2. Experimental
2.1. Materials
Ferulic acid, lauric acid, palmitic acid, stearic acid, glycerol, benzyl bromide,
N,N-dimethyl-4-aminopyridine (DMAP), and N,N-diispropylcarbodiimide (DIC) were purchased
from Sigma-Aldrich (Saint-Louis, MO, USA). Candida antarctica Lipase B immobilized on resin
(LC200291, 10,000 propyl laurate units g
−1
) was obtained from Novozyme. Reagents were used as
received. All solvents were bought either from ThermoFisher Scientific (Waltham, MA, USA) or VWR
France (Fontenay-sous-Bois, France). Deuterated chloroform (CDCl
3
) was purchased from Euriso-top
(Saint-Aubin, France).
Int. J. Mol. Sci. 2018,19, 3358 3 of 12
2.2. Analytical Methods
Column chromatography was carried out with an automated flash chromatograph (PuriFlash
4100, Interchim (Montluçon, France), prepacked with INTERCHIM PF-30SI-HP (30
µ
m silica gel)
columns using a gradient of cyclohexane and ethyl acetate for elution. NMR analyses were recorded
on a Bruker (Billerica, MA, USA) Fourier 300.
1
H NMR spectra of samples were determined in CDCl
3
at 300 MHz and chemical shifts were reported in parts per million (CDCl
3
, CHCl
3
residual signal
at
δ
= 7.26 ppm).
13
C NMR spectra of samples were recorded at 75 MHz (CDCl
3
signal at
δ
= 77.16
ppm). High-resolution mass spectroscopy (HRMS) analyses were carried out by the PLANET platform
at URCA using a Micromass GC-TOF. Thermogravimetric analyses (TGA) were executed on a Q500
(TA Instruments (
Milford, MA, USA
)). About 10 mg of each sample was heated from 30 to 500
◦
C at a
rate of 10 ◦C min−1under constant nitrogen flow (60 mL min−1).
2.3. Synthesis of Benzylated Ethyl Ferulate
Ethyl ferulate
23
(25 g, 0.1 mol, 1 eq), benzyl bromide (15 mL, 0.12 mol, 1.2 eq), and K
2
CO
3
(27 g, 0.2 mol, 2 eq) were dissolved in N,N-dimethylformamide (DMF) (0.5 M) and heated to 85
◦
C.
The reaction was monitored by TLC and let run until complete conversion of the starting material (3 h).
After cooling to room temperature (r.t.), the mixture was concentrated and filtered to remove K
2
CO
3
.
The resulting phase was evaporated under reduced pressure and the crude product was purified by
flash chromatography on silica gel using cyclohexane and ethyl acetate (90:10) as eluent, providing the
desired product as a white powder (32 g, 92%, Figure 1). M.p.: 70.4
◦
C,
1
H (300 MHz, CDCl
3
)
δ
: 3.90
(3H, s, H
10
), 5.18 (2H, s, H
CH2OBn
), 6.30 (2H, d, J = 15.9 Hz, H
2
), 6.83 to 6.87 (3H, m, H
5, 8 and 9
), 7.30 to
7.35 (5H, m, H
ArOBn
), 7.62 (2H, d, J = 15.9 Hz, H
3
),
13
C (75 MHz, CDCl
3
)
δ
: 14.4 (C
12
), 56.0 (C
10
), 60.4
(C
11
), 70.8 (C
(CH2)OBn
), 110.2 (C
9
), 113.3 (C
8
), 115.0 (C
2
), 122.8 (C
5
), 128.1 (C
4
), 127.3 to 136. 6 (C
(ArOBn)
),
145.8 (C7), 149.8 (C6), 150.5 (C3), 167.4 (C1).
Int. J. Mol. Sci. 2018, 19, x 3 of 13
Column chromatography was carried out with an automated flash chromatograph (PuriFlash
4100, Interchim (Montluçon, France), prepacked with INTERCHIM PF-30SI-HP (30 µm silica gel)
columns using a gradient of cyclohexane and ethyl acetate for elution. NMR analyses were recorded
on a Bruker (Billerica, MA, USA) Fourier 300. 1H NMR spectra of samples were determined in CDCl3
at 300 MHz and chemical shifts were reported in parts per million (CDCl3, CHCl3 residual signal at δ
= 7.26 ppm). 13C NMR spectra of samples were recorded at 75 MHz (CDCl3 signal at δ = 77.16 ppm).
High-resolution mass spectroscopy (HRMS) analyses were carried out by the PLANET platform at
URCA using a Micromass GC-TOF. Thermogravimetric analyses (TGA) were executed on a Q500
(TA Instruments (Milford, MA, USA)). About 10 mg of each sample was heated from 30 to 500 °C at
a rate of 10 °C min−1 under constant nitrogen flow (60 mL min−1).
2.3. Synthesis of Benzylated Ethyl Ferulate
Ethyl ferulate23 (25 g, 0.1 mol, 1 eq), benzyl bromide (15 mL, 0.12 mol, 1.2 eq), and K2CO3 (27 g,
0.2 mol, 2 eq) were dissolved in N,N-dimethylformamide (DMF) (0.5 M) and heated to 85 °C. The
reaction was monitored by TLC and let run until complete conversion of the starting material (3 h).
After cooling to room temperature (r.t.), the mixture was concentrated and filtered to remove K2CO3.
The resulting phase was evaporated under reduced pressure and the crude product was purified by
flash chromatography on silica gel using cyclohexane and ethyl acetate (90:10) as eluent, providing
the desired product as a white powder (32 g, 92%, Figure 1). M.p.: 70.4 °C, 1H (300 MHz, CDCl3) δ:
3.90 (3H, s, H10), 5.18 (2H, s, HCH2OBn), 6.30 (2H, d, J = 15.9 Hz, H2), 6.83 to 6.87 (3H, m, H5, 8 and 9), 7.30 to
7.35 (5H, m, HArOBn), 7.62 (2H, d, J = 15.9 Hz, H3), 13C (75 MHz, CDCl3) δ: 14.4 (C12), 56.0 (C10), 60.4
(C11), 70.8 (C(CH2)OBn), 110.2 (C9), 113.3 (C8), 115.0 (C2), 122.8 (C5), 128.1 (C4), 127.3 to 136. 6 (C(ArOBn)),
145.8 (C7), 149.8 (C6), 150.5 (C3), 167.4 (C1).
Figure 1. Benzylated ethyl ferulate.
2.4. Lipase-Catalyzed Transesterification of Benzylated Ethyl Ferulate into Glycerol Dibenzyl Ferulate
(GDFoBn)
Selective lipase-catalyzed transesterification was performed in presence of glycerol (5 g, 54.3
mmol, 1 eq), benzylated ethyl ferulate (42 g, 135.7 mmol, 2.5 eq) and CAL-B (10% w/w relative to the
total weight of batch). The reaction mixture was heated to 75 °C, kept under reduced pressure and
magnetically stirred for three days. It was then dissolved in acetone and filtered to remove CAL-B
beads. The solvent was evaporated under vacuum and the crude product was purified by flash
chromatography on silica gel eluted with cyclohexane/ethyl acetate to provide GDFoBn as a highly
viscous oil (32 g, 93%, Figure 2). 1H (300 MHz, CDCl3) δ: 3.89 (6H, s, H10), 4.22 to 4.39 (4H, m, H11),
4.28 (1H, s, HOH), 5.17 (4H, s, H(CH2)OBn), 6.30 (2H, d, J = 15.9 Hz, H2), 6.83 to 7.05 (6H, m, H5, 8 and 9), 7.25
to 7.40 (10H, m, HArOBn), 7.61 (2H, d, J = 15.9 Hz, H3), 13C (75 MHz, CDCl3) δ: 56.0 (C10), 65.4 (C11), 68.6
(C12), 70.8 (C(CH2)OBn), 110.2 (C9), 113.3 (C8), 115.0 (C2), 122.8 (C5), 128.14 (C4), 127.3 to 136. 6 (C(ArOBn)),
145.8 (C7), 149.8 (C6), 150.5 (C3), 167.4 (C1).
Figure 2. Glycerol dibenzyl ferulate.
2.5. Lipophilization: Synthesis of GDFx
Figure 1. Benzylated ethyl ferulate.
2.4. Lipase-Catalyzed Transesterification of Benzylated Ethyl Ferulate into Glycerol Dibenzyl Ferulate (GDFoBn)
Selective lipase-catalyzed transesterification was performed in presence of glycerol (5 g, 54.3 mmol,
1 eq), benzylated ethyl ferulate (42 g, 135.7 mmol, 2.5 eq) and CAL-B (10% w/wrelative to the total
weight of batch). The reaction mixture was heated to 75
◦
C, kept under reduced pressure and
magnetically stirred for three days. It was then dissolved in acetone and filtered to remove CAL-B
beads. The solvent was evaporated under vacuum and the crude product was purified by flash
chromatography on silica gel eluted with cyclohexane/ethyl acetate to provide GDFoBn as a highly
viscous oil (32 g, 93%, Figure 2).
1
H (300 MHz, CDCl
3
)
δ
: 3.89 (6H, s, H
10
), 4.22 to 4.39 (4H, m, H
11
),
4.28 (1H, s, H
OH
), 5.17 (4H, s, H
(CH2)OBn
), 6.30 (2H, d, J = 15.9 Hz, H
2
), 6.83 to 7.05 (6H, m, H
5, 8 and 9
),
7.25 to 7.40 (10H, m, H
ArOBn
), 7.61 (2H, d, J = 15.9 Hz, H
3
),
13
C (75 MHz, CDCl
3
)
δ
: 56.0 (C
10
), 65.4
(C
11
), 68.6 (C
12
), 70.8 (C
(CH2)OBn
), 110.2 (C
9
), 113.3 (C
8
), 115.0 (C
2
), 122.8 (C
5
), 128.14 (C
4
), 127.3 to 136.
6 (C(ArOBn)), 145.8 (C7), 149.8 (C6), 150.5 (C3), 167.4 (C1).
Int. J. Mol. Sci. 2018, 19, x 3 of 13
Column chromatography was carried out with an automated flash chromatograph (PuriFlash
4100, Interchim (Montluçon, France), prepacked with INTERCHIM PF-30SI-HP (30 µm silica gel)
columns using a gradient of cyclohexane and ethyl acetate for elution. NMR analyses were recorded
on a Bruker (Billerica, MA, USA) Fourier 300. 1H NMR spectra of samples were determined in CDCl3
at 300 MHz and chemical shifts were reported in parts per million (CDCl3, CHCl3 residual signal at δ
= 7.26 ppm). 13C NMR spectra of samples were recorded at 75 MHz (CDCl3 signal at δ = 77.16 ppm).
High-resolution mass spectroscopy (HRMS) analyses were carried out by the PLANET platform at
URCA using a Micromass GC-TOF. Thermogravimetric analyses (TGA) were executed on a Q500
(TA Instruments (Milford, MA, USA)). About 10 mg of each sample was heated from 30 to 500 °C at
a rate of 10 °C min−1 under constant nitrogen flow (60 mL min−1).
2.3. Synthesis of Benzylated Ethyl Ferulate
Ethyl ferulate23 (25 g, 0.1 mol, 1 eq), benzyl bromide (15 mL, 0.12 mol, 1.2 eq), and K2CO3 (27 g,
0.2 mol, 2 eq) were dissolved in N,N-dimethylformamide (DMF) (0.5 M) and heated to 85 °C. The
reaction was monitored by TLC and let run until complete conversion of the starting material (3 h).
After cooling to room temperature (r.t.), the mixture was concentrated and filtered to remove K2CO3.
The resulting phase was evaporated under reduced pressure and the crude product was purified by
flash chromatography on silica gel using cyclohexane and ethyl acetate (90:10) as eluent, providing
the desired product as a white powder (32 g, 92%, Figure 1). M.p.: 70.4 °C, 1H (300 MHz, CDCl3) δ:
3.90 (3H, s, H10), 5.18 (2H, s, HCH2OBn), 6.30 (2H, d, J = 15.9 Hz, H2), 6.83 to 6.87 (3H, m, H5, 8 and 9), 7.30 to
7.35 (5H, m, HArOBn), 7.62 (2H, d, J = 15.9 Hz, H3), 13C (75 MHz, CDCl3) δ: 14.4 (C12), 56.0 (C10), 60.4
(C11), 70.8 (C(CH2)OBn), 110.2 (C9), 113.3 (C8), 115.0 (C2), 122.8 (C5), 128.1 (C4), 127.3 to 136. 6 (C(ArOBn)),
145.8 (C7), 149.8 (C6), 150.5 (C3), 167.4 (C1).
Figure 1. Benzylated ethyl ferulate.
2.4. Lipase-Catalyzed Transesterification of Benzylated Ethyl Ferulate into Glycerol Dibenzyl Ferulate
(GDFoBn)
Selective lipase-catalyzed transesterification was performed in presence of glycerol (5 g, 54.3
mmol, 1 eq), benzylated ethyl ferulate (42 g, 135.7 mmol, 2.5 eq) and CAL-B (10% w/w relative to the
total weight of batch). The reaction mixture was heated to 75 °C, kept under reduced pressure and
magnetically stirred for three days. It was then dissolved in acetone and filtered to remove CAL-B
beads. The solvent was evaporated under vacuum and the crude product was purified by flash
chromatography on silica gel eluted with cyclohexane/ethyl acetate to provide GDFoBn as a highly
viscous oil (32 g, 93%, Figure 2). 1H (300 MHz, CDCl3) δ: 3.89 (6H, s, H10), 4.22 to 4.39 (4H, m, H11),
4.28 (1H, s, HOH), 5.17 (4H, s, H(CH2)OBn), 6.30 (2H, d, J = 15.9 Hz, H2), 6.83 to 7.05 (6H, m, H5, 8 and 9), 7.25
to 7.40 (10H, m, HArOBn), 7.61 (2H, d, J = 15.9 Hz, H3), 13C (75 MHz, CDCl3) δ: 56.0 (C10), 65.4 (C11), 68.6
(C12), 70.8 (C(CH2)OBn), 110.2 (C9), 113.3 (C8), 115.0 (C2), 122.8 (C5), 128.14 (C4), 127.3 to 136. 6 (C(ArOBn)),
145.8 (C7), 149.8 (C6), 150.5 (C3), 167.4 (C1).
Figure 2. Glycerol dibenzyl ferulate.
2.5. Lipophilization: Synthesis of GDFx
Figure 2. Glycerol dibenzyl ferulate.
Int. J. Mol. Sci. 2018,19, 3358 4 of 12
2.5. Lipophilization: Synthesis of GDFx
GDFoBn (20 g, 32.1 mmol, 1 eq) and fatty acid (lauric, palmitic or stearic, 1 eq) were dissolved
in dichloromethane (DCM) (0.25 M) with a catalytic amount of DMAP, (1.1 g, 9.6 mmol, 0.3 eq).
Subsequently, DIC (5.45 mL, 35.2 mmol, 1.1 eq) was added to the mixture and the reaction was
magnetically stirred at r.t. overnight. The precipitate urea was removed via filtration and the filtrate
concentrated under vacuum. The crude product was dissolved in THF and stirred under N
2
flow at
room temperature. After 10 min, palladium on activated charcoal (Pd/C, 10% w/w) was added and the
solution was stirred under N
2
for another 10 min, before being submitted to H
2
flow to simultaneous
reduce the C=C double bond and the benzyl protecting group (Bn). The solution was finally filtered
using Celite
®
pads and evaporated under reduced pressure. Target bisphenol was purified by flash
chromatography on silica gel eluted with cyclohexane/ethyl acetate. Structures were named GDF
x
,
for Glycerol Diferulate, where the incrementation “x” indicates the alkyl chain length.
GDF
10
(84%, Figure 3).
1
H (300 MHz, CDCl
3
)
δ
: 0.87 (3H, t
app
, J = 6.8 Hz, H
24
), 1.24 (16H, m,
H
16 to 23
), 1.61 (2H, m, H
15
), 2.28 (2H, t, J = 7.5 Hz, H
14
), 2.60 (4H, t, J = 7.6 Hz, H
3
), 2.86 (4H, t, J = 7.6
Hz, H
2
), 3.79 (6H, s, H
10
), 4.18 (4H, dd, J = 4.2, 11.7 Hz, H
11, 110
), 5.22 (1H, m, H
12
), 5.53 (2H, s, H
OH
),
6.65 (2H, s, H
5
), 6.68 (2H, s, H
9
), 6.81 (2H, d, J = 7.8 Hz, H
8
),
13
C (75 MHz, CDCl
3
)
δ
: 14.2 (C
24
), 24.9
(C
15
), 29.1–29.7 (C
16 to 23
), 30.6 (C
3
), 34.2 (C
14
), 36.1 (C
2
), 55.9 (C
10
), 62.3 (C
11
), 68.8 (C
12
), 110.9 (C
9
),
114.5 (C8), 120.9 (C5), 132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3456
(ArOH), 2922 and 2851 (aliphatic chain), 1733 (C=O), Td
5%-Loss
: 302
◦
C, HRMS (TOF MS, ES+): m/z
calcd for C35H50 O10Na: 653.3295; found 653.3302.
Int. J. Mol. Sci. 2018, 19, x 4 of 13
GDFoBn (20 g, 32.1 mmol, 1 eq) and fatty acid (lauric, palmitic or stearic, 1 eq) were dissolved in
dichloromethane (DCM) (0.25 M) with a catalytic amount of DMAP, (1.1 g, 9.6 mmol, 0.3 eq).
Subsequently, DIC (5.45 mL, 35.2 mmol, 1.1 eq) was added to the mixture and the reaction was
magnetically stirred at r.t. overnight. The precipitate urea was removed via filtration and the filtrate
concentrated under vacuum. The crude product was dissolved in THF and stirred under N2 flow at
room temperature. After 10 min, palladium on activated charcoal (Pd/C, 10% w/w) was added and
the solution was stirred under N2 for another 10 min, before being submitted to H2 flow to
simultaneous reduce the C=C double bond and the benzyl protecting group (Bn). The solution was
finally filtered using Celite® pads and evaporated under reduced pressure. Target bisphenol was
purified by flash chromatography on silica gel eluted with cyclohexane/ethyl acetate. Structures
were named GDFx, for Glycerol Diferulate, where the incrementation “x” indicates the alkyl chain
length.
GDF10 (84%, Figure 3). 1H (300 MHz, CDCl3) δ: 0.87 (3H, tapp, J = 6.8 Hz, H24), 1.24 (16H, m, H16 to
23), 1.61 (2H, m, H15), 2.28 (2H, t, J = 7.5 Hz, H14), 2.60 (4H, t, J = 7.6 Hz, H3), 2.86 (4H, t, J = 7.6 Hz, H2),
3.79 (6H, s, H10), 4.18 (4H, dd, J = 4.2, 11.7 Hz, H11, 11′), 5.22 (1H, m, H12), 5.53 (2H, s, HOH), 6.65 (2H, s,
H5), 6.68 (2H, s, H9), 6.81 (2H, d, J = 7.8 Hz, H8), 13C (75 MHz, CDCl3) δ: 14.2 (C24), 24.9 (C15), 29.1–29.7
(C16 to 23), 30.6 (C3), 34.2 (C14), 36.1 (C2), 55.9 (C10), 62.3 (C11), 68.8 (C12), 110.9 (C9), 114.5 (C8), 120.9 (C5),
132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3456 (ArOH), 2922 and 2851
(aliphatic chain), 1733 (C=O), Td5%-Loss: 302 °C, HRMS (TOF MS, ES+): m/z calcd for C35H50O10Na:
653.3295; found 653.3302.
Figure 3. GDF10.
GDF14 (81%, Figure 4). 1H (300 MHz, CDCl3) δ: 0.87 (3H, tapp, J = 6.3 Hz, H28), 1.23 (24H, m, H16 to
27), 1.59 (2H, m, H15), 2.28 (2H, t, J = 7.5 Hz, H14), 2.61 (4H, t, J = 5.1 Hz, H3), 2.88 (4H, t, J = 7.5 Hz, H2),
3.78 (6H, s, H10), 4.17 (4H, dd, J = 4.5, 12.0 Hz, H11, 11′), 5.23 (1H, m, H12), 5.51 (2H, s, HOH), 6.65 (2H, s,
H5), 6.68 (2H, s, H9), 6.81 (2H, d, J = 7.8 Hz, H8), 13C (75 MHz, CDCl3) δ: 14.2 (C28), 24.9 (C15), 29.1–29.7
(C16 to 27), 30.6 (C3), 34.2 (C14), 36.0 (C2), 55.9 (C10), 62.3 (C11), 68.8 (C12), 110.9 (C9), 114.5 (C8), 120.9 (C5),
132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3460 (ArOH), 2921 and 2850
(aliphatic chain), 1734 (C=O), Td5%-Loss: 311 °C, HRMS (TOF MS, ES+): m/z calcd for C39H58O10Na:
709.3937; found 709.3928.
Figure 4. GDF14.
Figure 3. GDF10 .
GDF
14
(81%, Figure 4).
1
H (300 MHz, CDCl
3
)
δ
: 0.87 (3H, t
app
, J = 6.3 Hz, H
28
), 1.23 (24H, m,
H
16 to 27
), 1.59 (2H, m, H
15
), 2.28 (2H, t, J = 7.5 Hz, H
14
), 2.61 (4H, t, J = 5.1 Hz, H
3
), 2.88 (4H, t, J = 7.5
Hz, H
2
), 3.78 (6H, s, H
10
), 4.17 (4H, dd, J = 4.5, 12.0 Hz, H
11, 110
), 5.23 (1H, m, H
12
), 5.51 (2H, s, H
OH
),
6.65 (2H, s, H
5
), 6.68 (2H, s, H
9
), 6.81 (2H, d, J = 7.8 Hz, H
8
),
13
C (75 MHz, CDCl
3
)
δ
: 14.2 (C
28
), 24.9
(C
15
), 29.1–29.7 (C
16 to 27
), 30.6 (C
3
), 34.2 (C
14
), 36.0 (C
2
), 55.9 (C
10
), 62.3 (C
11
), 68.8 (C
12
), 110.9 (C
9
),
114.5 (C8), 120.9 (C5), 132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3460
(ArOH), 2921 and 2850 (aliphatic chain), 1734 (C=O), Td
5%-Loss
: 311
◦
C, HRMS (TOF MS, ES+): m/z
calcd for C39H58 O10Na: 709.3937; found 709.3928.
Int. J. Mol. Sci. 2018, 19, x 4 of 13
GDFoBn (20 g, 32.1 mmol, 1 eq) and fatty acid (lauric, palmitic or stearic, 1 eq) were dissolved in
dichloromethane (DCM) (0.25 M) with a catalytic amount of DMAP, (1.1 g, 9.6 mmol, 0.3 eq).
Subsequently, DIC (5.45 mL, 35.2 mmol, 1.1 eq) was added to the mixture and the reaction was
magnetically stirred at r.t. overnight. The precipitate urea was removed via filtration and the filtrate
concentrated under vacuum. The crude product was dissolved in THF and stirred under N2 flow at
room temperature. After 10 min, palladium on activated charcoal (Pd/C, 10% w/w) was added and
the solution was stirred under N2 for another 10 min, before being submitted to H2 flow to
simultaneous reduce the C=C double bond and the benzyl protecting group (Bn). The solution was
finally filtered using Celite® pads and evaporated under reduced pressure. Target bisphenol was
purified by flash chromatography on silica gel eluted with cyclohexane/ethyl acetate. Structures
were named GDFx, for Glycerol Diferulate, where the incrementation “x” indicates the alkyl chain
length.
GDF10 (84%, Figure 3). 1H (300 MHz, CDCl3) δ: 0.87 (3H, tapp, J = 6.8 Hz, H24), 1.24 (16H, m, H16 to
23), 1.61 (2H, m, H15), 2.28 (2H, t, J = 7.5 Hz, H14), 2.60 (4H, t, J = 7.6 Hz, H3), 2.86 (4H, t, J = 7.6 Hz, H2),
3.79 (6H, s, H10), 4.18 (4H, dd, J = 4.2, 11.7 Hz, H11, 11′), 5.22 (1H, m, H12), 5.53 (2H, s, HOH), 6.65 (2H, s,
H5), 6.68 (2H, s, H9), 6.81 (2H, d, J = 7.8 Hz, H8), 13C (75 MHz, CDCl3) δ: 14.2 (C24), 24.9 (C15), 29.1–29.7
(C16 to 23), 30.6 (C3), 34.2 (C14), 36.1 (C2), 55.9 (C10), 62.3 (C11), 68.8 (C12), 110.9 (C9), 114.5 (C8), 120.9 (C5),
132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3456 (ArOH), 2922 and 2851
(aliphatic chain), 1733 (C=O), Td5%-Loss: 302 °C, HRMS (TOF MS, ES+): m/z calcd for C35H50O10Na:
653.3295; found 653.3302.
Figure 3. GDF10.
GDF14 (81%, Figure 4). 1H (300 MHz, CDCl3) δ: 0.87 (3H, tapp, J = 6.3 Hz, H28), 1.23 (24H, m, H16 to
27), 1.59 (2H, m, H15), 2.28 (2H, t, J = 7.5 Hz, H14), 2.61 (4H, t, J = 5.1 Hz, H3), 2.88 (4H, t, J = 7.5 Hz, H2),
3.78 (6H, s, H10), 4.17 (4H, dd, J = 4.5, 12.0 Hz, H11, 11′), 5.23 (1H, m, H12), 5.51 (2H, s, HOH), 6.65 (2H, s,
H5), 6.68 (2H, s, H9), 6.81 (2H, d, J = 7.8 Hz, H8), 13C (75 MHz, CDCl3) δ: 14.2 (C28), 24.9 (C15), 29.1–29.7
(C16 to 27), 30.6 (C3), 34.2 (C14), 36.0 (C2), 55.9 (C10), 62.3 (C11), 68.8 (C12), 110.9 (C9), 114.5 (C8), 120.9 (C5),
132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3460 (ArOH), 2921 and 2850
(aliphatic chain), 1734 (C=O), Td5%-Loss: 311 °C, HRMS (TOF MS, ES+): m/z calcd for C39H58O10Na:
709.3937; found 709.3928.
Figure 4. GDF14.
Figure 4. GDF14 .
Int. J. Mol. Sci. 2018,19, 3358 5 of 12
GDF
16
(87%, Figure 5).
1
H (300 MHz, CDCl
3
)
δ
: 0.87 (3H, t
app
, J = 6.3 Hz, H
30
), 1.24 (28H, m,
H
16 to 30
), 1.58 (2H, m, H
15
), 2.27 (2H, t, J = 7.5 Hz, H
14
), 2.60 (4H, t, J = 8.1 Hz, H
3
), 2.85 (4H, t, J = 7.5
Hz, H
2
), 3.86 (6H, s, H
10
), 4.17 (4H, dd, J = 4.5, 12.0 Hz, H
11, 110
), 5.23 (1H, m, H
12
), 5.48 (2H, s, H
OH
),
6.65 (2H, s, H
5
), 6.68 (2H, s, H
9
), 6.81 (2H, d, J = 7.5 Hz, H
8
),
13
C (75 MHz, CDCl
3
)
δ
: 14.2 (C
30
), 24.9
(C
15
), 27.1–29.7 (C
16 to 29
), 30.6 (C
3
), 34.2 (C
14
), 36.0 (C
2
), 55.9 (C
10
), 62.3 (C
11
), 68.8 (C
12
), 110.9 (C
9
),
114.5 (C8), 120.9 (C5), 132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3454
(ArOH), 2921 and 2850 (aliphatic chain), 1735 (C=O), Td
5%-Loss
: 308
◦
C, HRMS (TOF MS, ES+): m/z
calcd for C41H62 O10Na: 737.4252; found 737.4241.
Int. J. Mol. Sci. 2018, 19, x 5 of 13
GDF16 (87%, Figure 5). 1H (300 MHz, CDCl3) δ: 0.87 (3H, tapp, J = 6.3 Hz, H30), 1.24 (28H, m, H16 to
30), 1.58 (2H, m, H15), 2.27 (2H, t, J = 7.5 Hz, H14), 2.60 (4H, t, J = 8.1 Hz, H3), 2.85 (4H, t, J = 7.5 Hz, H2),
3.86 (6H, s, H10), 4.17 (4H, dd, J = 4.5, 12.0 Hz, H11, 11′), 5.23 (1H, m, H12), 5.48 (2H, s, HOH), 6.65 (2H, s,
H5), 6.68 (2H, s, H9), 6.81 (2H, d, J = 7.5 Hz, H8), 13C (75 MHz, CDCl3) δ: 14.2 (C30), 24.9 (C15), 27.1–29.7
(C16 to 29), 30.6 (C3), 34.2 (C14), 36.0 (C2), 55.9 (C10), 62.3 (C11), 68.8 (C12), 110.9 (C9), 114.5 (C8), 120.9 (C5),
132.2 (C4), 144.2 (C7), 146.5 (C6), 172.5 (C1), 173.0 (C13), FT-IR (neat), νmax: 3454 (ArOH), 2921 and 2850
(aliphatic chain), 1735 (C=O), Td5%-Loss: 308 °C, HRMS (TOF MS, ES+): m/z calcd for C41H62O10Na:
737.4252; found 737.4241.
Figure 5. GDF16.
2.6. Calculation of Solubility Parameters
The theoretical solubility and incorporation of additives into polymers were estimated by using
the van Krevelen and Hoftyzer atomic group contribution method [22]. The Hansen solubility
parameters of compounds were calculated using following equation and the database supplied in
electronic supplementary information (ESI).
δd = ΣFdi/ΣVi; Dispersion component (J1/2/cm−3/2) (1)
δp = ΣF²pi/ΣVi; Polar component (J1/2/cm−3/2) (2)
δh = ΣEhi/ΣVi; Hydrogen-bonding component; (J1/2/cm−3/2) (3)
with
Fdi: Dispersion contribution of the molar attraction constant [(J1/2 cm−3/2)/mol−1]
Fpi: Polar contribution of the molar attraction constant [(J1/2 cm−3/2)/mol−1]
Ehi: Hydrogen-bonding energy contribution of the molar attraction constant (J/mol)
V: Molar volume contribution of the chemical group involved (cm3/mol).
The Hildebrand solubility parameters (HiSP) were then calculated in J1/2/cm−3/2 with the
simplified following equation:
HiSP = √ (δ²d + δ²p + δ²h) (4)
2.7. Analysis of the Radical Scavenging Power of Antioxidants
The radical scavenging power was measured with a method derived from Berset et al. [24]. A
total amount of 190 µL homogenate DPPH (2,2-diphenyl-1-picrylhydrazyl) ethanol solution (200
µM) was added to a 96-well plate containing 10 µL of potential antiradical molecule ethanol
solutions at different concentrations, ranging from 300 µM to 9.3 µM. The experiments were
performed in an invariable excess of the DPPH radical (200 µM, 40 nmol). The phenolic
concentrations were selected to get linear dose-response intervals. The reaction was followed by a
microplate Multiskan FC 1 scan every 5 min for 7.5 h at 515 nm. The use of different amounts of the
Figure 5. GDF16 .
2.6. Calculation of Solubility Parameters
The theoretical solubility and incorporation of additives into polymers were estimated by using the
van Krevelen and Hoftyzer atomic group contribution method [
22
]. The Hansen solubility parameters
of compounds were calculated using following equation and the database supplied in electronic
supplementary information (ESI).
δd=ΣFdi/ΣVi; Dispersion component (J1/2 /cm−3/2) (1)
δp=ΣF2pi/ΣVi; Polar component (J1/2 /cm−3/2) (2)
δh=ΣEhi/ΣVi; Hydrogen-bonding component; (J1/2/cm−3/2) (3)
with
Fdi: Dispersion contribution of the molar attraction constant [(J1/2 cm−3/2)/mol−1]
Fpi: Polar contribution of the molar attraction constant [(J1/2 cm−3/2)/mol−1]
Ehi: Hydrogen-bonding energy contribution of the molar attraction constant (J/mol)
V: Molar volume contribution of the chemical group involved (cm3/mol).
The Hildebrand solubility parameters (HiSP) were then calculated in J
1/2
/cm
−3/2
with the
simplified following equation:
HiSP = √(δ2d+δ2p+δ2h) (4)
2.7. Analysis of the Radical Scavenging Power of Antioxidants
The radical scavenging power was measured with a method derived from Berset et al. [
24
].
A total amount of 190
µ
L homogenate DPPH (2,2-diphenyl-1-picrylhydrazyl) ethanol solution (200
µ
M)
was added to a 96-well plate containing 10
µ
L of potential antiradical molecule ethanol solutions
at different concentrations, ranging from 300
µ
M to 9.3
µ
M. The experiments were performed in an
invariable excess of the DPPH radical (200
µ
M, 40 nmol). The phenolic concentrations were selected
to get linear dose-response intervals. The reaction was followed by a microplate Multiskan FC 1 scan
every 5 min for 7.5 h at 515 nm. The use of different amounts of the potential antioxidant gave rise
Int. J. Mol. Sci. 2018,19, 3358 6 of 12
to the EC
50
value, which is defined as the concentration needed to reduce half the initial amount of
DPPH. Each analysis was performed four times. The total stoichiometries (n) is the number of DPPH
moles reduced by 1 mol of antioxidant (n= DPPHtot/EC50 ×2).
3. Results and Discussions
3.1. Design of Lipophilic Antioxidants: Predictive Approaches
The design of the new bisphenolic antioxidants was based on our earlier studies on the
Structure–Activity Relationships (SAR) of ferulic acid-based diphenolic antioxidants [
15
]. Ferulic
acid is a well-known natural antioxidant because of its hydrogen-donating ability [
13
,
25
]. It was
shown that removing the
α
,
β
-unsaturation was beneficial for the radical-scavenger ability of bisphenol
structures [
16
]. Therefore,
α
,
β
-saturated ferulic acid moieties were selected to confer optimal radical
scavenging ability. A previously published SAR study [
16
] showed that the nature of the linker
between the antiradical moieties slightly impacts the antioxidant activity. In this specific study,
the linker had to be a triol able to carry the two phenolic moieties as well as an alkyl chain allowing
the fine tuning of the lipophilic character of the molecule. Glycerol that carries two primary
alcohols and one secondary alcohol was selected. Glycerol is a byproduct during the production
of biodiesel, therefore available in high amounts, the use of which helps reduce the environmental
impact of this industry [
26
]. The lipophilic part was based on recent advances, which show that
the grafting of a medium chain-length fatty acid can be used to design potent custom-made lipophilic
antioxidants [
18
,
21
]. Herein, three linear fatty acids were tested. The theoretical miscibility of additives
used in the formulation with PP was calculated with the help of the Hildebrand solubility parameters
(HiSP, Equation (4)). The lower the difference of HiSP between two compounds, the higher their mutual
solubility. In the literature HiSP values in the range between 16.8 and 19.0 J
1/2
cm
−3/2
are reported
for PP. The bisphenolics previously developed in our laboratory (i.e., BDF, PDF, and IDF) exhibited
HiSP values greater than 25.0 J
1/2
/cm
−3/2
(Figure 6, Table 1). Among them, IDF has the highest
polarity contribution (
δp
7.2 vs. 3.5–3.7 J
1/2
/cm
−3/2
) linked to the polarity of the isosorbide linker.
In comparison, benchmark antioxidant Irganox 1010
®
exhibits a low HiSP value of 21.4 J
1/2
/cm
−3/2
.
The commercial lipophilic antioxidant Irganox 1076
®
exhibits a HiSP value much more comparable
to that of PP, thanks to a lower contribution of the aliphatic chain to polarity (
δp
) and the other
hydrogen bonding (
δh
) factors [
27
]. The increase of the polar contribution of the target antioxidants
was calculated for esters with lauric (C
12
), palmitic (C
16
), and stearic (C
18
) acid. This group of lipophilic
antioxidants was named GDF
x,
for Glycerol Diferulate, where the incrementation “x” indicates the alkyl
chain length. The HiSP values of the GDFxfamily were lowered compared to the initial antioxidants,
therefore better solubility in PP might be expected (Table 1). The HiSP values showed the importance
of alkyl chain length. Indeed, data show that an increase of the chain length by six carbon atoms
(GDF10 vs. GDF16) leads to a HiSP value drop of 1.00 J1/2/cm−3/2 (Table 1).
Int. J. Mol. Sci. 2018, 19, x 7 of 13
Figure 6. Developed structures of phenolic additives.
Table 1. Hildebrand solubility parameters (HiSP) of phenolic antioxidants.
Compound
δd
(J1/2 cm−3/2)
δp
(J1/2 cm−3/2)
δh
(J1/2 cm−3/2)
HiSP
(J1/2 cm−3/2)
BDF 21.3 3.5 13.6 25.5
PDF 21.5 3.7 13.9 25.9
IDF 23.3 7.2 14.9 28.5
GDF10 19.8 2.5 11.7 23.2
GDF14 19.5 2.2 10.9 22.5
GDF16 19.3 2.1 10.7 22.2
Irganox 1010® 18.9 1.3 10.0 21.4
Irganox 1076® 17.4 1.3 7.0 18.8
3.2. Synthesis of the Targets (GDFx)
As we committed ourselves to the use of sustainable processes for the production of the
targeted bisphenols, the use of the previously reported lipase-catalyzed synthesis of IDF, PDF, and
BDF was envisaged [23]. Indeed, the use of immobilized lipase CAL-B as a biocatalyst is a great tool
for the development of sustainable processes. Not only does CAL-B not require a solvent, it is
inactive toward phenolic hydroxyl groups, but can also be reused for further reaction cycles. In
addition, CAL-B has been proven to promote the easier (trans)esterification on esters rather than
acids moieties. Hence, α,β-saturated ethyl ferulate (aka ethyl dihydroferulate) and fatty acid ethyl
ester (FAEE) were selected as starting materials rather than the corresponding acids. Several
strategies were investigated to determine the best synthetic pathway to GDFx. The different
pathways (Figure 7) are the
(I) Stoichiometric one pot-one step enzymatic strategy,
(II) One pot-two step strategy, and
(III) Chemo-enzymatic strategy.
Figure 6. Developed structures of phenolic additives.
Int. J. Mol. Sci. 2018,19, 3358 7 of 12
Table 1. Hildebrand solubility parameters (HiSP) of phenolic antioxidants.
Compound δd
(J1/2 cm−3/2)
δp
(J1/2 cm−3/2)
δh
(J1/2 cm−3/2)
HiSP
(J1/2 cm−3/2)
BDF 21.3 3.5 13.6 25.5
PDF 21.5 3.7 13.9 25.9
IDF 23.3 7.2 14.9 28.5
GDF10 19.8 2.5 11.7 23.2
GDF14 19.5 2.2 10.9 22.5
GDF16 19.3 2.1 10.7 22.2
Irganox 1010®18.9 1.3 10.0 21.4
Irganox 1076®17.4 1.3 7.0 18.8
3.2. Synthesis of the Targets (GDFx)
As we committed ourselves to the use of sustainable processes for the production of the targeted
bisphenols, the use of the previously reported lipase-catalyzed synthesis of IDF, PDF, and BDF was
envisaged [
23
]. Indeed, the use of immobilized lipase CAL-B as a biocatalyst is a great tool for the
development of sustainable processes. Not only does CAL-B not require a solvent, it is inactive toward
phenolic hydroxyl groups, but can also be reused for further reaction cycles. In addition, CAL-B has
been proven to promote the easier (trans)esterification on esters rather than acids moieties. Hence,
α
,
β
-saturated ethyl ferulate (aka ethyl dihydroferulate) and fatty acid ethyl ester (FAEE) were selected
as starting materials rather than the corresponding acids. Several strategies were investigated to
determine the best synthetic pathway to GDFx. The different pathways (Figure 7) are the
(I)
Stoichiometric one pot-one step enzymatic strategy,
(II)
One pot-two step strategy, and
(III)
Chemo-enzymatic strategy.
Int. J. Mol. Sci. 2018, 19, x 8 of 13
Figure 7. Stoichiometric one pot-one step enzymatic strategy (I), one pot-two step strategy (II),
chemo-enzymatic strategy (III), and corresponding yields (I.a: 34%, II.a: 91%, II.b: 40%, III.a: 93%,
III.b GDF10: 84%, GDF14: 81%, GDF16: 87%).
3.3. Stochiometric One Pot-One Step Enzymatic Strategy (Pathway I, Figure 7)
The first strategy (I) involved a one pot-one step lipase-mediated transesterification of glycerol
with a mixture of ethyl dihydroferulate (1) (2 eq) and FAEE (1 eq) (I.a—Figure 7). It is worth
mentioning that ethyl dihydroferulate can be easily obtained from ethyl ferulate through a simple
palladium-catalyzed hydrogenation in yield greater than 98% [23]. Under such stoichiometric
conditions, Candida antarctica lipase B was not regioselective toward glycerol primary and secondary
alcohols. Indeed, despite the total conversion of ethyl dihydroferulate, the enzyme functionalized
primary and secondary alcohols randomly with ethyl dihydroferulate or FAEE, leading to a complex
mixture of the target structure and corresponding regioisomers. After purification, the targeted
GDFx were isolated in 34% yield.
3.4. One Pot-Two Step Strategy
The transesterification was then performed on α,β-unsaturated ethyl ferulate (2). In that case,
Candida antarctica lipase B proved unable to esterify the secondary alcohol of glycerol, giving access
to the symmetric bisphenol (3) in high yield after purification (II.a, 91%, Figure 7). The
α,β-unsaturation of ethyl ferulate (2) conferred probably more rigidity to the molecule and
decreased the accessibility to the active site of the lipase. Furthermore, it also decreased significantly
the electronic density on the C from the carbonyl, thus limiting its reactivity. With the aim to access
GDFx, a second step involving a lipase-catalyzed transesterification followed by a
palladium-catalyzed hydrogenation was then performed on intermediate bisphenol (3) (II.b, Figure
7). This second biocatalytic step induced however transesterification issues due to CAL-B esterase
activity, resulting in a complex mixture of regioisomers and therefore in the decrease in yield down
to 40% after purification.
3.5. Chemo-Enzymatic Strategy
A protecting group-based chemo-enzymatic strategy was finally developed to produce
lipophilic antioxidants in high yields. The first step involved the lipase-mediated transesterification
of benzylated ethyl ferulate (4) with the two primary alcohols of glycerol leading to an intermediate
bisphenol (5) isolated in 93% yield (III.a, Figure 7). Then, fatty acids were grafted onto the available
secondary alcohol through a conventional Steglich esterification, immediately followed by a
OEtO
MeO
OH CAL-B, 75 °C, vaccum
Solvent free, 3 days
OHHO
OH
x = 10, 14, 16
O
OEt
O
O
OH
MeO
OH
OMe
OO O
O
x
x
+
OH
O
OBn
MeO
OBn
OMe
OO O
13
OEtO
MeO
OBn
III.b
Lipophilization
I.a
No selectivity
III.a
High selectivity on position
1 and 3 of glycerol
Target strucutre
GDF
x
OH
O
OH
MeO
OH
OMe
OO O
13
OEtO
MeO
OH
CAL-B, 75 °C, vaccum
Solvent free, 3 days
OHHO
OH
x = 10, 14, 16
O
OEt
x
1) CAL-B, 75 °C, vaccum
Solvent free, 3 days
2 )H
2
, Pd/C, 3h
II.a
High selectivity on p osition
1 and 3 of glycerol
II.b
Transesterifications
x = 10, 14, 16
O
OH
x
1) DCI, DMAP, r.t, 5h
2) H
2
, Pd/C, 3h
CAL-B, 75 °C, vaccum
Solvent free, 3 days
OHHO
OH
(III)
(II)
(I)
x = 10, 14, 16
(1)
(2) (3)
(4) (5)
Figure 7.
Stoichiometric one pot-one step enzymatic strategy (
I
), one pot-two step strategy (
II
),
chemo-enzymatic strategy (
III
), and corresponding yields (I.a: 34%, II.a: 91%, II.b: 40%, III.a: 93%, III.b
GDF10: 84%, GDF14: 81%, GDF16: 87%).
Int. J. Mol. Sci. 2018,19, 3358 8 of 12
3.3. Stochiometric One Pot-One Step Enzymatic Strategy (Pathway I, Figure 7)
The first strategy (I) involved a one pot-one step lipase-mediated transesterification of glycerol
with a mixture of ethyl dihydroferulate (1) (2 eq) and FAEE (1 eq) (I.a—Figure 7). It is worth
mentioning that ethyl dihydroferulate can be easily obtained from ethyl ferulate through a simple
palladium-catalyzed hydrogenation in yield greater than 98% [
23
]. Under such stoichiometric
conditions, Candida antarctica lipase B was not regioselective toward glycerol primary and secondary
alcohols. Indeed, despite the total conversion of ethyl dihydroferulate, the enzyme functionalized
primary and secondary alcohols randomly with ethyl dihydroferulate or FAEE, leading to a complex
mixture of the target structure and corresponding regioisomers. After purification, the targeted GDF
x
were isolated in 34% yield.
3.4. One Pot-Two Step Strategy
The transesterification was then performed on
α
,
β
-unsaturated ethyl ferulate (2). In that case,
Candida antarctica lipase B proved unable to esterify the secondary alcohol of glycerol, giving access to
the symmetric bisphenol (3) in high yield after purification (II.a, 91%, Figure 7). The
α
,
β
-unsaturation
of ethyl ferulate (2) conferred probably more rigidity to the molecule and decreased the accessibility
to the active site of the lipase. Furthermore, it also decreased significantly the electronic density on
the C from the carbonyl, thus limiting its reactivity. With the aim to access GDF
x
, a second step
involving a lipase-catalyzed transesterification followed by a palladium-catalyzed hydrogenation was
then performed on intermediate bisphenol (3) (II.b, Figure 7). This second biocatalytic step induced
however transesterification issues due to CAL-B esterase activity, resulting in a complex mixture of
regioisomers and therefore in the decrease in yield down to 40% after purification.
3.5. Chemo-Enzymatic Strategy
A protecting group-based chemo-enzymatic strategy was finally developed to produce lipophilic
antioxidants in high yields. The first step involved the lipase-mediated transesterification of benzylated
ethyl ferulate (4) with the two primary alcohols of glycerol leading to an intermediate bisphenol (5)
isolated in 93% yield (III.a, Figure 7). Then, fatty acids were grafted onto the available secondary
alcohol through a conventional Steglich esterification, immediately followed by a palladium-catalyzed
hydrogenation to simultaneously reduce the
α
,
β
-unsaturation and cleave the benzyl protecting group
(III.b, Figure 7). Using this third strategy, targeted bisphenols were obtained as single regioisomers in
high yields (GDF
10
: 84%, GDF
14
: 81%, GDF
16
: 87%). It is worth mentioning that this synthesis was
then successfully implemented on a large scale (≈20 g).
The thermal properties of the new additives described in Table 2were investigated by
thermogravimetric analysis (TGA).
Table 2. Thermostability (Td5%) of GDFx.
Compound Thermostability (Td5%, ◦C)
GDF10 302
GDF14 311
GDF16 308
Irganox1076®236
Irganox1010®347
Thermogravimetric analyses (TGA) of the bisphenols revealed thermostability (T
d
5%) in the range
of 302 to 311
◦
C. Furthermore, the alkyl chain length does not significantly impact the degradation
temperature. Indeed, replacing lauric (C
12
) with palmitic (C
16
) or stearic (C
18
) moieties shift their
thermostability by less than 10 ◦C.
Int. J. Mol. Sci. 2018,19, 3358 9 of 12
3.6. Analysis of the Antiradical Activity of Lipophilic Bisphenols
The DPPH method is an easy and rapid analytical tool to determine the free radical scavenging
activity of phenolic compounds and their abilities to quench radicals. [
24
] The total H atom donation
capacities can be evaluated with the EC
50
value, defined as the quantity of antioxidant (nmol) needed
to protonate/quench 50% of the initial population of DPPH. The lower the EC
50
, the higher the
antioxidant activity of a compound is. Because the EC
50
is intrinsically linked to the structure of
the antioxidant, in particular to the number of free phenols, a compound can be also characterized
by its antioxidant stoichiometry (n), i.e., the number of DPPH radicals reduced by one molecule of
antioxidant [28]:
n = DPPHtot/EC50 ×2 (5)
where DPPHtot is the initial amount of DPPH in nmol.
The principal kinetic parameters of the DPPH reaction are the time needed to reach the steady state
at the concentration EC
50
, i.e., T
EC50
, and the rate of reaction towards free radicals [
29
]. By combining H
atom-donation abilities and kinetic parameters, the Antioxidant Efficiency (AE) [
29
,
30
] can be defined
in order to characterize the behavior of a substance as antioxidant after:
AE = (1/EC50)×|m(Ec50 )| (6)
with m(EC
50
) the slope in the first minutes of the absorbance-versus-time plots at EC
50
of
each compound.
The reaction kinetics of all compounds with DPPH are shown in ESI, and an example of EC
50
determination can been seen in Figure 8. The reaction proceeds in two stages, with a fast decay in
absorbance during the first minutes, followed by a slower one until equilibrium is reached. EC
50
values and total stoichiometries (n) obtained with the newly synthesized and commercial phenolic
additives classified by chemical structure are shown in Table 3.
Int. J. Mol. Sci. 2018, 19, x 10 of 13
Figure 8. (A) Reaction kinetics of GDF16 solution at given concentrations: 300 µM (brown), 150 µM
(red), 75 µM (orange), 37.5 µM (yellow), 18.8 µM (light green), and 9.4 µM (dark green), (B) EC50
determination for GDF16. Blue line: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and Green line:
RPPH reduced.
As shown in Table 3, the EC50 values for GDFx are homogenous around 5 nmol (mean value:
4.95 nmol). It is noteworthy to mention that no correlation between the alkyl chains size and the
antiradical activity was observed. Irganox 1010® is the most potent scavenging compound, whereas
Irganox 1076® is the least one; GDFx have intermediate EC50 values. The number of phenols available
for DPPH quenching is an important feature for the antioxidant activity. Irganox 1010® and 1076®
contains four and one free phenol groups, respectively, while the GDFx family has only two. The
stoichiometries value n takes into account this factor. As expected, with its four phenols, Irganox
1010® exhibited an antiradical activity twice as high than that found for the GDFx family that only
bears two phenols. Similarly, the monophenolic compound Irganox 1076® displayed an activity
twice as low than the bisphenolic compound GDFx.
Table 3. Radical scavenging parameters of phenolic antioxidants.
Compound Free Phenols EC50 (nmol) Stoichiometries (n) ׀m(EC50)׀ AE
GDF10 2 4.81 ± 0.17 4.17 ±0.15 2.19 0.45
GDF14 2 5.38 ± 0.12 3.72 ± 0.09 2.30 0.42
GDF16 2 4.66 ± 0.15 4.30 ± 0.14 2.16 0.46
Irganox 1010® 4 2.52 ± 0.16 7.98 ± 0.47 0.76 0.30
Irganox 1076® 1 11.48 ± 0.17 1.74 ± 0.03 0.68 0.06
The stoichiometry value n does not coincide with the number of hydroxyl groups available.
Indeed, the direct abstraction of the phenol H-atom and the electron transfer process from ArOH to
DPPH radical is not the only mechanism involved in the reaction between DPPH and phenols.
Various studies [31,32] have already proposed potential phenol regeneration pathways leading to
reaction products such as dimers or quinone methides, able to further react with DPPH radicals and
thus leading to n values higher than 2 (Figure 9).
Figure 8.
(
A
) Reaction kinetics of GDF
16
solution at given concentrations: 300
µ
M (dark green),
150
µ
M (light green), 75
µ
M (yellow), 37.5
µ
M (orange), 18.8
µ
M (red), and 9.4
µ
M (brown), (
B
) EC
50
determination for GDF
16
. Blue line: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and Green line:
RPPH reduced.
Int. J. Mol. Sci. 2018,19, 3358 10 of 12
As shown in Table 3, the EC
50
values for GDF
x
are homogenous around 5 nmol (mean value:
4.95 nmol). It is noteworthy to mention that no correlation between the alkyl chains size and
the antiradical activity was observed. Irganox 1010
®
is the most potent scavenging compound,
whereas Irganox 1076
®
is the least one; GDF
x
have intermediate EC
50
values. The number of phenols
available for DPPH quenching is an important feature for the antioxidant activity. Irganox 1010
®
and
1076
®
contains four and one free phenol groups, respectively, while the GDF
x
family has only two.
The stoichiometries value n takes into account this factor. As expected, with its four phenols, Irganox
1010
®
exhibited an antiradical activity twice as high than that found for the GDF
x
family that only
bears two phenols. Similarly, the monophenolic compound Irganox 1076
®
displayed an activity twice
as low than the bisphenolic compound GDFx.
Table 3. Radical scavenging parameters of phenolic antioxidants.
Compound Free Phenols EC50 (nmol) Stoichiometries (n) |m(EC50)| AE
GDF10 2 4.81 ±0.17 4.17 ±0.15 2.19 0.45
GDF14 2 5.38 ±0.12 3.72 ±0.09 2.30 0.42
GDF16 2 4.66 ±0.15 4.30 ±0.14 2.16 0.46
Irganox 1010®4 2.52 ±0.16 7.98 ±0.47 0.76 0.30
Irganox 1076®1 11.48 ±0.17 1.74 ±0.03 0.68 0.06
The stoichiometry value n does not coincide with the number of hydroxyl groups available.
Indeed, the direct abstraction of the phenol H-atom and the electron transfer process from ArOH to
DPPH radical is not the only mechanism involved in the reaction between DPPH and phenols. Various
studies [
31
,
32
] have already proposed potential phenol regeneration pathways leading to reaction
products such as dimers or quinone methides, able to further react with DPPH radicals and thus
leading to n values higher than 2 (Figure 9).
Int. J. Mol. Sci. 2018, 19, x 11 of 13
Figure 9. Chemical behavior of phenolic antioxidants.
Finally, in order to determine the antioxidant efficiencies (AE), the absorbance-vs-time plots at
the EC50 concentration of each phenolic compound were established. When concentration of DPPH
was in large excess, the slope in the first minutes of these plots (mEC50 = Δy/Δx 0→10 min) could be
assimilated to the rate constants of fast reaction of proton abstraction (a) in Figure 9. [29]
Consequently, antioxidant efficiency values (AE) calculated using equation (6) (Table 3 and ESI),
take into consideration both H atom-donation capacity and kinetic aspects of the phenolic
compounds tested. Here, the higher the AE, the higher the antioxidant activity of a compound is.
Based on these results, the antioxidant Irganox 1076® was the least efficient of all tested phenolic
compounds. It is interesting to note that a compound such as Irganox 1010®, with an EC50 value of
2.50, was found to be the best antioxidant of all phenolic compounds, but instead showed a
moderate antioxidant efficiency (AE = 0.30), due to its relative slow rate of reaction (Table 3). Finally,
the newly created GDFx family appeared to be competitive showing the highest AE values, ranging
from 0.42 to 0.46, due to the combination of high H atom-donation capacities and fast kinetics.
4. Conclusions
A novel class of renewable bisphenol (GDFx) was successfully designed from ferulic acid and
vegetal oil (glycerol and fatty acids) and proved to be potent antioxidant additives for polyolefins.
By playing with the chemical structure of the starting materials, their polarities can be easily tuned
so to display theoretical solubilities into polypropylene similar to that of Irganox 1010®. The efficient
synthesis of these novel bisphenols has been achieved through a chemo-enzymatic process involving
a highly regioselective lipase-mediated transesterification, allowing the preparation of well
structurally defined targets in very good yields (81–87%). The H atom-donation capacities and
kinetic aspects of these novel bisphenols were evaluated using the DPPH free radical technique and
benchmarked against commercially available antioxidant compounds. All of the results
demonstrated the high radical scavenging capacity of these renewable bisphenols and their potential
as promising renewable alternatives. The study of their antioxidant capacity in polyolefins matrices
will be reported in due course.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1.
O
OO
R
O
O
OO
R
O
HDPPH
DPPH-H
O
OO
R
O
O
OO
R
O
O
O
O
R
O
O
O
OR
O
OH
O
O
R
O
HO
O
OR
O
H
H
DPPH
O
OO
R
O
DPPH-H
N
NO
2
O
2
N
NO
2
N
O
OO
R
ON
NO
2
O
2
N
NO
2
N
(a)
Dimer formation Quinone methide formation
Figure 9. Chemical behavior of phenolic antioxidants.
Finally, in order to determine the antioxidant efficiencies (AE), the absorbance-vs-time plots
at the EC
50
concentration of each phenolic compound were established. When concentration of
DPPH was in large excess, the slope in the first minutes of these plots (m
EC50
=
∆
y/
∆
x 0
→
10 min)
Int. J. Mol. Sci. 2018,19, 3358 11 of 12
could be assimilated to the rate constants of fast reaction of proton abstraction (a) in Figure 9[
29
].
Consequently, antioxidant efficiency values (AE) calculated using equation (6) (Table 3and ESI), take
into consideration both H atom-donation capacity and kinetic aspects of the phenolic compounds
tested. Here, the higher the AE, the higher the antioxidant activity of a compound is.
Based on these results, the antioxidant Irganox 1076
®
was the least efficient of all tested phenolic
compounds. It is interesting to note that a compound such as Irganox 1010
®
, with an EC
50
value of
2.50, was found to be the best antioxidant of all phenolic compounds, but instead showed a moderate
antioxidant efficiency (AE = 0.30), due to its relative slow rate of reaction (Table 3). Finally, the newly
created GDF
x
family appeared to be competitive showing the highest AE values, ranging from 0.42 to
0.46, due to the combination of high H atom-donation capacities and fast kinetics.
4. Conclusions
A novel class of renewable bisphenol (GDF
x
) was successfully designed from ferulic acid and
vegetal oil (glycerol and fatty acids) and proved to be potent antioxidant additives for polyolefins.
By playing with the chemical structure of the starting materials, their polarities can be easily tuned so to
display theoretical solubilities into polypropylene similar to that of Irganox 1010
®
. The efficient synthesis
of these novel bisphenols has been achieved through a chemo-enzymatic process involving a highly
regioselective lipase-mediated transesterification, allowing the preparation of well structurally defined
targets in very good yields (81–87%). The H atom-donation capacities and kinetic aspects of these novel
bisphenols were evaluated using the DPPH free radical technique and benchmarked against commercially
available antioxidant compounds. All of the results demonstrated the high radical scavenging capacity of
these renewable bisphenols and their potential as promising renewable alternatives. The study of their
antioxidant capacity in polyolefins matrices will be reported in due course.
Supplementary Materials:
The following are available online at http://www.mdpi.com/1422-0067/19/11/3358/
s1.
Author Contributions:
Conceptualization, F.A.; Methodology, L.H., F.A.; Validation, S.D. and F.A.; Formal
Analysis, L.H.; Investigation, L.H. and F.A.; Resources, F.A.; Data Curation, L.H.; Writing-Original Draft
Preparation, L.H.; Writing-Review & Editing, S.D. and F.A.; Supervision, S.D. and F.A.; Project Administration,
F.A.; Funding Acquisition, F.A.
Funding: This research was funded by Grand Reims, Conseil Général de la Marne and Région Grand Est.
Acknowledgments:
The authors are grateful to Grand Reims, the Conseil Général de la Marne and Région Grand
Est for their financial support.
Conflicts of Interest: The authors declare no conflict of interest.
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