Aminolysis of linoleic and salicylic acid derivatives with Candida antarctica lipase B: A solvent-free process to obtain amphiphilic amides for cosmetic application

Article abstract of DOI:10.1016/j.molcatb.2016.01.002

In this biotechnological process, the fatty amides (9Z,12Z)-N-dodecyloctadeca-9,12-dienamide (3) and N-dodecyl-2-hydroxybenzamide (5), respectively derived from linoleic acid and salicylic acid were synthesized through aminolysis reactions catalyzed by Ca

Full text of DOI:10.1016/j.molcatb.2016.01.002

Accepted Manuscript
Title: Aminolysis of linoleic and salicylic acid derivatives with
Candida antarctica lipase B: A solvent-free process to obtain
amphiphilic amides for cosmetic application
Author: Ana Maria Mouad Delphine Taupin Laurent Lehr
´
Florent Yvergnaux Andre Luiz Meleiro Porto
PII:
S1381-1177(16)30002-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.01.002
MOLCAB 3305
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
23-6-2015
7-1-2016
8-1-2016
Please cite this article as: Ana Maria Mouad, Delphine Taupin, Laurent Lehr,
´
Florent Yvergnaux, Andre Luiz Meleiro Porto, Aminolysis of linoleic and salicylic
acid derivatives with Candida antarctica lipase B: A solvent-free process to obtain
amphiphilic amides for cosmetic application, Journal of Molecular Catalysis B:
Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.01.002
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Aminolysis of linoleic and salicylic acid derivatives with Candida
antarctica lipase B: A solvent-free process to obtain amphiphilic
amides for cosmetic application
Ana Maria Mouada,b,*, Delphine Taupinb, Laurent Lehrb, Florent Yvergnauxb, André
Luiz Meleiro Portoa
aUniversidade de São Paulo, Instituto de Química de São Carlos, Av. João Dagnone,
1100, 13563-120, São Carlos-São Paulo, Brazil
bBioEurope-Groupe Solabia, Route d’Oulins-28260, Anet, Eure-et-Loir, France
*ana.m.mouad@gmail.com
Graphical abstract:
O
O
H2N
H2N
O
O
+
+
OH
Vacuum
-EtOH
65\ C
Vacuum
-EtOH
65\C
20 h
20 h
CALB
CALB
d eri vati ve
O
O
O
Ô m ega 6
N
H
2N
NH
O
+
H
N - do d ecy l am in e ( 2)
e th yl lino leate ( 1)
OH
N-dodecyl-2-hydroxybenzamide
(9Z,12Z)-N-dodecyloctadeca-9,12-dienamide
6 5°C , vacuu m
2 0
h
C AL B
O
N H
3
( )
(9 Z ,1 2Z )- N -do de cy lo ctad eca -9 ,1 2- d ien a mi de
Skin care
1

Highlights
 In this work, an enzymatic and solvent-free process was developed
aiming the production of amphiphilic molecules for cosmetic
application. The fatty amides (9Z,12Z)-N-dodecyloctadeca-9,12-
dienamide (3) and N-dodecyl-2-hydroxybenzamide (5), were synthesized
through aminolysis reactions catalyzed by Candida antarctica lipase B.
The reactions were carried out in a solvent-free process, at 65°C and
reduced pressure (50 mbar). The products were monitored by HPLC
analysis. The conversion rates were measured through disappearance of
ethyl linoleate and ethyl salicylate. The best conversion rates (up to
95%) were obtained by adding an enzyme amount of 5.0 g/mol of
acyl donor group substrate and an equimolar substrates ratio (1:1). This
work reveals that enzymatic synthesis provides an attractive way for the
cosmetic industrial production of fatty amides.
Abstract
In this biotechnological process, the fatty amides (9Z,12Z)-N-dodecyloctadeca-
9,12-dienamide (3) and N-dodecyl-2-hydroxybenzamide (5), respectively derived from
linoleic acid and salicylic acid were synthesized through aminolysis reactions catalyzed
by Candida antarctica lipase B. These amphiphilic compounds receive great attention
from cosmetic industry due to a range of beneficial properties for skin. The aminolysis
reactions were performed with the esters ethyl linoleate (1) and ethyl salicylate (4) as
acyl group donors and the fatty compound N-dodecylamine (2) as the nucleophilic
substrate. The aminolysis reactions were carried out in a solvent-free process, which is
beneficial from an environmental and economical perspective, at 65°C and reduced
pressure (50 mbar). Parameters as enzyme amount and substrates molar ratios were
investigated and the products were monitored by HPLC analysis. The conversion rates
were measured through disappearance of ethyl linoleate and ethyl salicylate. The best
conversion rates (up to 95%) were obtained by adding an enzyme amount of 5.0
g/mol of acyl donor group substrate and an equimolar substrates ratio (1:1). The
products characterization was performed by High Resolution Mass Spectrometry,
Infrared spectroscopy and Nuclear Magnetic Ressonance. This work reveals that
enzymatic synthesis provides an attractive way for the cosmetic industrial production of
2

fatty amides, wich may represent key ingredients to maintain and/or restore a healthy
skin.
Keywords: Biocatalysis, Solvent-free process, Lipase, Skin care, Antioxidants, Green
chemistry..
3

1. Introduction
Over the last decades, the application of enzymes as catalysts has expanded
horizons in several fields of biotechnology. This expansion may be related to a ‘greener’
way to produce fine chemicals with high degree of selectivity, none or minimal side
reactions and relatively milder conditions compared to the chemical counterparts [1].
In this scenario, lipases (triacylglyceride hydrolases; EC 3.1.1.3) constitute the
most important enzymes for biotechnological applications [2]. They present commercial
availability, stability and usually do not require expensive cofactors [3,4]. These
biocatalysts are capable of reacting with several substrates, catalyzing not only the
hydrolysis and synthesis of long-chain acylglycerols but also alcoholysis, acidolysis,
interesterification and the transfer of acyl groups from esters to amines (nucleophiles),
promoting aminolysis reactions [5,6]. The use of lipases in aminolysis of esters in
anhydrous media has been successfully described for the synthesis of peptides, fatty
acid amides, polymers, surfactants and low cost detergents [7, 8].
From the cosmetic sector standpoint, lipases represent great biocatalysts in the
synthesis of fine chemicals such as specialty esters, aroma compounds and active
ingredients [9]. Notedly, the lipase B from Candida antarctica (CALB) has proven to
be a versatile catalyst to synthesize cosmetic products for skin care, such as retinol
(vitamin A) and ascorbic acid (vitamin C) derivatives, widely used as natural
antioxidants in the treatment of photoaging and skin disorders [10].
In a previous work, CALB catalyzed aminolysis reactions in a solvent-free
process, leading to the production of ceramides analogous molecules, which are
essential for epidermal permeability barrier function and overall condition of the skin
[11-13]. In this work, we proposed the employment of CALB as catalyst in aminolysis
reactions between the esters ethyl linoleate (1) and ethyl salicylate (4), respectively
derived from linoleic and salicylic acids, and the fatty compound N-dodecylamine (2).
The interest in the application of these compounds as cosmetic ingredients can
be described as follows; firstly, the presence of a linoleic acid (LA) derivative is
interesting in cosmetic formulations since it represents an important skin constituent,
with specific functions in the production and maintance of epidermal permeability
barrier [14-16]. LA has received increased attention in cosmetic market due to its anti-
inflammatory, acne reduction and moisture retention properties [12]. Furthermore, this
polyunsaturaded fatty acid (PUFA) is featured with 18 carbon atoms and two cis double
bonds at position 6 (omega-6 family), representing an essential fatty acid. Whereas the
4

human body can not synthesize double bonds at position 6, LA must be necessarily
obtained from the diet or topical applications [14].
With respect to ethyl salicylate (4), salicylic acid (SA) derivatives composes
the phenolic acid family, recognized by the antioxidant activity against the highly
reactive free radicals, which are involved in adverses skin effects such as photoaging,
wrinkling, general loss of elasticity and cancer [17-20]. SA is frequently used in the
treatment of Acne vulgaris due to its keratolytic propertie and is also known as an
ultraviolet B sunscreen, reducing sunburn and melasma production [21-23].
Lipophillic derivatives of salicylic acid also have been proven to present anti-
aging, photoprotective, keratolytic and antibacterial properties. In fact, it has been
shown that the addition of fatty chains to SA molecule confers superior efficacy on the
original molecule [24]. The greater efficacy is related to the skin permeability, which is
lower for hydrophilic compounds, since the outermost layer of skin represents a highly
lipophilic barrier constituted of ceramides, fatty acids and cholesterol [25]. In this
context, the addiction of fatty chains can be extended to LA derivatives, where the
increasing of fatty chains in unsaturated fatty acids, particularly those of cis
configuration, confers greater protective effect against cutaneous dehydration and
xenobiotics [26]. Thus, this structural mofication represents an attractive way to
improve the skin-product compatibility.
Additionally, this reactions may lead to the production of amphiphilic
molecules, which presenting an hydrophobic region (soluble in lipophilic medium) and
a hydrophilic head (solubility in aqueous environment), represent ideal stabilizers for
emulsion formulations, being recognized as emulsifiers or surfactants [27]; once there is
a concern about the classical synthesis of surfactants derived from petroleum [28, 29],
biosurfactants containing amide bonds are potential substitutes, presenting skin
tolerance, good biological degradability and significantly lower toxicological effects
[30].
Therefore, the structural modification of LA and SA esters (1 and 4) via
aminolysis reactions catalyzed by CALB with the fatty N-dodecylamine (2), in a
solvent-free process, can be used as a tool to produce amphiphilic amides, which may
atract great interest from cosmetic industry and differentiated applications for skin
cosmetic formulations.
5

2. Materials and methods
2.1 Materials
The lipase B from C. antarctica (Novozym 435, immobilized) was kindly
provided by Novozymes A/S, Bagsvaerd, Denmark. Ethyl linoleate (1) was purchased
from Stearinerie Dubois (Ciron, France), while N-dodecylamine (2) and ethyl salicylate
(4) were purchase from Sigma-Aldrich (St. Louis, MO).
2.2 The reaction parameters
It is important to consider that to run enzymatic reactions under solvent-free
conditions, at least one of the reactants has to be kept in the liquid state; thus, the
reaction temperature has to be high enough (65°C) to shift the equilibrium in the right
sense. Also, to limite the possible reverse reaction (hydrolysis), the reactions were
carried out under vacuum, evaporating thus, the ethanol (by-product) formed during the
reaction.
2.3 Enzymatic reactions
In order to obtain the amphiphilic amides 3 and 5 in great yields, two studies
were perfomed to evaluate the effect of substrates molar ratio and the biocatalyst
amount. In the effect of substrates molar ratio study, the reactants acyl donor groups 1
or 4 and the nucleophile N-dodecylamine (2) were added in 50 mL flasks in the
following values: 0.8: 1.0; 1.0:1.0 and 1.0:2.0 (acyl donor 1 or 4: nucleophile 2). Then,
after 5 min under vacuum (50 mbar) for gas removal of the system, 5.0 g of Candida
antarctica B/ mol of ethyl esters (1 or 4) were added to the reaction mixtures.
Also, one sequence of reactions was performed by changing the biocatalyst
amount (CALB) in order to find the higher yields. This study was carried out in 50 mL
glass reactors, wherein equimolar substrates concentrations [1 acyl donor groups (1 or
4) : 1 nucleophile (2)] were added. After 5 min under vacuum (50 mbar) for gas
removal of the system, different CALB amounts were added to the reaction mixtures
(0.6; 1.2; 2.5; 5.0; 7.5; 10 g/mol acyl donor reactants 1 or 4).
6

All the reactions were conducted under stirring (250-300 rpm) using an overhead
stirrer IKA RW 16 Basic, Staufen, Germany, equipped with a plastic propeller, at 65°C.
After 20 h, the products were purified through flash cromatography and the rates of
conversion of the reactions were monitored through the disappearance of ethyl linoleate
(1) or/and ethyl salicylate (4) using HPLC analysis.
3. Analytical and characterization methods
3.1 High Performance Liquid Cromatography (HPLC)
The aminolysis reactions were monitored by High-Performance Liquid
Chromatography (HPLC analysis) carried out in a system (Alliance-Waters) composed
of a column (Xterra MS C18 5M, 150 mm × 2.1 mm), a column oven (temperature 40
°C), an autoinjector and UV/vis detector (PDA, W2996= 210 nm). The compounds were
evaluated with an eluent system of methanol (A) and water (B), both containing 0.1% of
trifluoroacetic acid, as shown in Table 1.
3.2 Nuclear Magnetic Resonance (NMR)
The NMR specters of proton and carbon (1H and 13C NMR) of the chemical
structure of the products were obtained using a Bruker AM 500 at the Analytical Center
of the Institute of Chemistry of São Carlos (IQSC/USP), operating in 500 MHz (1H
NMR) and 125 MHz (13C NMR), using CDCl3 as the deutered solvent and TMS as the
internal standart unless otherwise noted. The chemical shifts are given in ppm and the
coupling constants (J) in Hz.
3.3 High Resolution Mass Spectrometry (HRMS)
The analyses were performed on a High Resolution Mass Spectrometer
quadrupole / flight time, operating in the MS mode in the unit: microTOF-QII, detector
Daltonis Bruker (Bremen Germany). In these analyzes, the samples were ionized in the
positive mode using atmospheric pressure chemical ionization (APCI). The analyses
7

were performed at the Institute of Chemistry of São Carlos, in the Chromatography
Laboratory (Prof. Dr. Fernando Mauro Lanças, FAPESP Grant n°2004/09498-2).
4.Results and Discussion
4.1 Effect of molar reactants concentrations in the yield reactions
The study concerning the effect of substrates molar ratio was performed with the
addition of different values of the substrates (0.8: 1.0; 1.0:1.0 and 1.0:2.0 – wherein acyl
donor: nucleophile) and 5.0 g CALB/mol of the acyl donor group 1 or 4 in all reactions
(Scheme 1).
The reactions were carried out following the described procedures and the
synthesized products were monitored by HPLC analysis. The results are summarized in
Table 2.
The reactions 1-6 were performed as a preview study, in order to find the proper
molar ratios of the reactants. As can be seen in the analysed reactions 1-3, by adding a
equal molar ratio of the substrates 1 and 2 and 5.0 g CALB/mol of acyl donor group, it
was possible to obtain the high yield of 99% of the fatty amide 3. The results were
similar in the reactions 4-6, where a yield of 96% of the fatty amide 5 was obtained by
adding a equimolar ratio of the reactants 4 and 2. These results opened the way for the
standardization of the equal molar ratio of reactants for the subsequent reactions.
4.2 The effect of the biocatalyst amount in the yield reactions
In sequence, the aminolysis reactions among ethyl linoleate (1) and N-
dodecylamine (2) with different enzyme amount catalyzed by CALB were carried out
following the described procedures and the synthesized product was monitored by
HPLC analysis. The results are summarized in Table 3.
8

The results showed that the fatty amide 3 was obtained with 30% yield by
adding the lower enzyme amount selected (0.6 g/ mol ethyl ester 1). The HPLC analysis
exhibited that this yield was raised to 73% after duplicating the added enzyme amount
(1.2 g/mol ethyl ester 1). The addiction of 2.5 g CALB/mol ethyl linoleate (1) to the
mixture reaction led to a 88% yield after 20 h.
A great yield of 99% was obtained by adding 5.0 g CALB/mol ethyl linoleate (1)
and therefore, this enzyme amount was standarzided for further reactions. The reactions
carried out with 7.5 g and 10.0 g CALB/mol ethyl linoleate (1) led to the production of
the fatty amide 3 with similar yields (99-100%).
Sequentially, aminolysis reactions were carried out with the reagents ethyl
salicylate (4) and N-dodecylamine (2) in order to produce the amphiphilic N-dodecyl-2-
hydroxybenzamide (5). The aminolysis reactions of ethyl salicylate (4) and N-
dodecylamine (2) catalyzed by CALB were carried out following the described
procedures and the synthesized products were monitored by HPLC analysis (Scheme
2). The results are summarized in Table 4.
In this case, the fatty amide 5 was obtained with 25% yield by adding the lower
enzyme amount selected (0.6 g/ mol ethyl ester 4). The HPLC analysis exhibited that
this yield was raised to 68% after duplicating the added enzyme amount (1.2 g/mol
ethyl ester 4). The reaction 3 achieved a 85% yield of the fatty amide 5 with the
addiction of 2.5 g CALB/mol ethyl salicylate (4). The results showed that by adding 5.0
g CALB/mol ethyl salicylate (4) it was possible to obtain a 96% yield of the fatty amide
5 and the results with the addiction of 7.5 and 10 g CALB/ mol ethyl ester 4 were
approximated, with 97% yield. Considering this proximity between the reaction yields,
the experimental conditions of the reaction 4 (5.0 g CALB/mol ester) may be
standardized for the production of N-dodecyl-2-hydroxybenzamide (5).
Amide compounds are of great value in different fields, including cosmetic,
pharmaceutical and agrochemical [31, 32]. Due to this interest, the syntheses of these
compounds has been conducted either by chemical and enzymatic pathways.
Conventional chemical syntheses of amides may be carried out under high temperatures
and extended reaction times, involving for example alkoxides, anhydrides, alkyllithium,
acyl chlorides, metallic catalysts or Grignard reagents [33-36]. Amides syntheses have
also been reported via microwave radiation [31, 37].
9

Related to enzymatic syntheses, CALB has proven to be the most efficient
catalyst for aminolysis reactions and its environmentally benign character is desirable
for large scale industrial aplications [38]. Kaushik et al., 2015 performed studies were a
serie of N-alkyl-substituted amides have been synthesized in the presence of CALB at
60-90°, in a solvent-free system with yields between 75-83% [39].
Studies have demonstrated that CALB can be useful for direct formation of
ethanolamides of various long-chain fatty acids, optically pure amides and lactams
through aminolysis [33, 38, 40]. Phenolic compounds such as caffeic acid amide can
also be obtained by CALB aminolysis reactions, as demonstrated in the study performed
by Xiao et al., 2013 [41].
CALB also catalyzed the direct amidation of fatty and hydroxy fatty acids (RA:
ricinoleic acid, LQA: lesquerolic acid and TOD: 7,10,12-trihydroxy-8-(E)-octadecenoic
acid) to their primary amides with ammonia in organic solvent. At 55 °C and a 2:1
ammonia to fatty acid substrate ratio, the transformation reactions of LQA, RA and
TOD approached completion (over 95%) within 24 h, while a decreased in the ratio to
1:1 reduced the transformation to approximately 80% [42]. Studies investigated the
direct amidation of the carboxylic acid 7,10-dihydroxy-8(E)-octadecenoic acid with N-
methylethanol amine catalyzed by CALB. After 72 h, in organic solvent medium,
CALB mediated the production of a novel secondary amide N-methyl-7,10-dihydroxy-
8-(E)-octadecenoylethanolamide with 95% yield, which displayed antimicrobial and
antioxidative activities [43]. Furthermore, CALB catalyzed the synthesis of optically
enriched α-haloamides and N-alkyl substituted amides with potential antinemic activity
[44, 45].
In this work, the fatty amides (9Z,12Z)-N-dodecyloctadeca-9,12-dienamide (3)
and N-dodecyl-2-hydroxybenzamide (5) were synthesized through a solvent-free
biotechnological process, where products yields over 96% were obtained after 20 h in
reactions carried out in equimolar substrates ratio and 5.0 g CALB/mol of acyl group
donors substrates. The amphiphilic compounds 3 and 5 are currently passing through
studies, exhibiting efficacy in their biological activities so far. Since amides derived
from fatty acids are important for the maintenance a healthy skin and their long
hydrophobic chains constitute a barrier against evaporation of water and the resulting
dry skin [46], these amphiphilic compounds represent potential candidates for skincare
cosmetic formulations, agregatting as well the beneficial properties previous described.
10

5. Conclusion
The cosmetic market is renowned for moving a huge financial capital, attracting
the attention of academic and research centers to the development of new skin care
chemicals. In this sense, compounds which aggregate cutaneous beneficial properties
with economic and environmentally friendly routes are of major interest.
Fatty amides are of great interest due to their ranging of industrial applications
not only in cosmetics, but also as surfactants and detergents formulations. In this way,
enzymatic synthesis of fatty amides in milder conditions are interesting from an
economical and envirommental aspect. In this work, two amphiphilic amides (3 and 5)
with potential for skin care application were synthesized through an enzymatic
biotechnological process. The syntheses of the cosmetic chemicals were achieved due to
the application of CALB enzyme as biocatalyst, which provided high yields (96-99%),
under mild conditions and in a solvent-free system. This work shows that CALB and its
acylating activity exhibits high catalytic efficiency for implementation in the production
of cosmetic ingredients.
Acknowledgements
A.M.M. express gratitude to Capes (Brazil) for the scholarship and to all the
BioEurope-Solabia Group (Anet, France), especially to Dr. Florent Yvergnaux and Dr.
Gerard Josset for the support. The authors are grateful to FAPESP and the
Chromatography Group of the Instituto de Química de São Carlos-USP (including
Guilherme M. Titano) for the HRSM analysis (FAPESP Grant n° 2004/09498-2).
11

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15

O
H2N
O
+
vacuum
-EtOH
CALB
65°C, 20 h
O
NH
(9Z,12Z)-N-dodecyloctadeca-9,12-dienamide
Scheme 1. Aminolysis reaction catalyzed by CALB for the synthesis of (9Z,12Z)-N-dodecyloctadeca-
9,12-dienamide (3).
Scheme 2. Aminolysis reaction catalyzed by CALB for the synthesis of N-dodecyl-2-hydroxybenzamide
(5).
16

Table 1. Chromatography conditions for analysis of aminolysis reactions by HPLC
Fatty
amides
Time
(min)
Flow
(mL/min)
(A)%
MeOH
(B)%
H2O
tr
tr
(reactant)
(fatty
amide)
22.2
3
5
40
30
0.27
0.27
80
50
20
50
14.5
9.2
21.0
tr: retention time, column: X terra MS C18 5M.
Table 2. Aminolysis reactions performed with different molar ratios of reactants for the synthesis of
(9Z,12Z) -N-dodecyloctadeca-9,12-dienamide (3) and N-dodecyl-2-hydroxybenzamide (5).
Reaction
Ethyl linoleate (1): N-dodecylamine (2)
Remaining ester 1
Fatty amide 3a
(Mols)
(%)
27
(%)
73
1
0.8: 1.0
2
3
1.0:1.0
1.0:2.0
1
1
99
99
Reaction
Ethyl salicylate (4): N-dodecylamine (2)
Remaining ester 4
Fatty amide 5a
(Mols)
0.8:1.0
(%)
34
(%)
66
4
5
6
1.0:1.0
1.0:2.0
4
1
96
99
Experimental conditions: T=65 °C, p=50 mbar, CALB: 5 g/mol of esters 2 or 4, time: 20 hours, a: yields
values obtained by HPLC analysis.
Table 3. Aminolysis reactions of ethyl linoleate (1) and N-dodecylamine (2) for the synthesis of (9Z,12Z)
-N-dodecyloctadeca-9,12-dienamide (3) catalyzed by CALB.
Reaction
CALB
Remaining ethyl
(9Z,12Z) -N-
(g/mol ethyl ester 1)
linoleate (1) (%)
dodecyloctadeca-9,12-
dienamide (3)a (%)
1
2
3
4
5
6
0.6
1.2
70
27
12
1
30
73
2.5
88
5.0
99
7.5
1
99
10.0
0
100
17

Experimental conditions: T=65 °C, p=50 mbar, CALB: g/mol of ethyl linoleate (1), equimolar
concentration of reactants (1:1), time: 20 h, a: yields obtained by HPLC analysis.
Table 4. Aminolysis reactions among ethyl salicylate (4) and N-dodecylamine (2) for the synthesis of N-
dodecyl-2-hydroxybenzamide (5).
Reaction
CALB
(g/mol ethyl ester 4)
Remaining ethyl salicylate
N-dodecyl-2-
hydroxybenzamide (5)a(%)
(4) (%)
1
2
3
4
5
6
0.6
1.2
2.5
5.0
7.5
10
75
32
15
4
25
68
85
96
97
97
3
3
Experimental conditions: T=65 °C, p=50 mbar, CALB: g/mol of ethyl salicylate (4), equimolar
concentration of reactants (1:1), time: 20 hours, a: yields obtained by HPLC analysis.
18