Green enzymatic synthesis of L-ascorbyl fatty acid ester: An antioxidant

Article abstract of DOI:10.1080/00397910802513045

In green chemistry, biocatalysis using microwaves is a very attractive tool for various regioselective syntheses. L-Ascorbyl fatty acid esters were synthesized by an immobilized lipase from Bulkholderia multivorans using microwaves, with a dynamically enh

Full text of DOI:10.1080/00397910802513045

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Green Enzymatic Synthesis of
L-Ascorbyl Fatty Acid Ester: An
Antioxidant
a
a
Mazaahir Kidwai , Poonam Mothsra , Namita
b
b
b
Gupta , S. Suresh Kumar & Rani Gupta
a
Green Chemistry Research Laboratory, Department
of Chemistry , University of Delhi , Delhi, India
b
Department of Microbiology , University of Delhi ,
South Campus, India
Published online: 04 Mar 2009.
To cite this article: Mazaahir Kidwai , Poonam Mothsra , Namita Gupta , S. Suresh
Kumar & Rani Gupta (2009) Green Enzymatic Synthesis of L-Ascorbyl Fatty Acid
Ester: An Antioxidant, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 39:7, 1143-1151
To link to this article: http://dx.doi.org/10.1080/00397910802513045
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1
Synthetic Communications , 39: 1143–1151, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910802513045
Green Enzymatic Synthesis of L-Ascorbyl Fatty
Acid Ester: An Antioxidant
1
Mazaahir Kidwai, Poonam Mothsra, Namita Gupta,
1
2
2
S. Suresh Kumar, and Rani Gupta
2
1
Green Chemistry Research Laboratory, Department of Chemistry,
University of Delhi, Delhi, India
Department of Microbiology, University of Delhi, South Campus, India
2
Abstract: In green chemistry, biocatalysis using microwaves is a very attractive
tool for various regioselective syntheses. L-Ascorbyl fatty acid esters were synthe-
sized by an immobilized lipase from Bulkholderia multivorans using microwaves,
with a dynamically enhanced rate of reaction and appreciable yield of around
80%. Microwave radiation had no effect on enzymic inactivation, however, syner-
gism between microwaves and biocatalyst was observed.
Keywords: Antioxidants, ascorbic acid, fatty acid ester, microwaves, regio-
selective
INTRODUCTION
Antioxidants are widely used in foods and cosmetics to prevent oxidation
of lipids. Synthetic antioxidants, such as butylatedhydroxy toluene
(
additives, but their application has been reassessed because of toxic or
BHT) and butylated hydroxyanisole (BHA), are widely used as food
[
1]
carcinogenic components formed during degradation. L-Ascorbyl fatty
acid esters (3a–d) are oil-soluble derivatives of L-ascorbic acid (1),
which is a widely used natural antioxidant. The hydrophilic nature of
Received July 19, 2008.
Address correspondence to Mazaahir Kidwai, Green Chemistry Research
Laboratory, Department of Chemistry, University of Delhi, Delhi 110007, India.
E-mail: kidwai.chemistry@gmail.com
1
143

1144
M. Kidwai et al.
L-ascorbic acid (1) limits its application in cosmetics or in the presence of
fats and oils, whereas fatty acid esters of L-ascorbic acid are oil-soluble
antioxidants extensively used in medical hygiene, cosmetics, and food
industries. They are also used for inhibition of enzymic browning in apple
[
2]
[3]
juice and for treating sunburn damage to skin, and they act as carriers
[4]
of ascorbate into neural tissue. They can even inhibit the development
[
of cancer.
L-Ascorbyl fatty acid esters are produced commercially by chemical
5]
[
6]
means. This has a number of disadvantages, including the use of less
biocompatible chemicals and solvents, the formation of by-products
due to instability of vitamin C, and low yields. As an alternative, the
use of enzymes to catalyze these synthetic reactions recently has become
a much more promising method. Lipases are the most sought-after
catalysts for the synthesis of fatty acid esters of L-ascorbic acid because
of their specificity, regioselectivity, and high effectiveness under mild
reaction conditions, which prevent oxidation of both L-ascorbic acid
and its esters during reaction. Though the lipase-catalyzed syntheses of
[
7–9]
L-ascorbyl fatty acid esters (3a–d) have been already described,
they
are so far hampered by long reaction times or the use of solvents, which
are not permitted for the manufacture of food-related products. In the
present communication, our aim was to study the enzyme in dry media
under microwaves and how microwaves provide new inputs for the synth-
esis of L-ascorbyl fatty acid esters (3a–d).
Microwave usage to promote reaction rates of chemical reactions has
[
10–13]
become fairly routine as reported by our laboratory.
in enzyme-catalyzed reactions is relatively limited, and moreover literature
Their application
[
14–17]
is contradictory on the stability of enzymes under microwaves.
RESULTS AND DISCUSSION
Stability of Immobilized Lipase Under Microwaves
Supported enzymes were placed under microwave irradiation for 30 min
ꢀ
ꢀ
at the temperature range of 70 C to 100 C and subsequently tested in the
esterification reaction of benzoic acid. Within experimental error, their
activities were found to be same as those of freshly prepared, nonheated,
supported enzymes. This confirms the stability of the enzyme in micro-
waves. Keeping in view these experimental results, we tried the synthesis
of ascorbyl fatty acid esters under microwaves. As discussed in Table 1, it
was observed that immobilized enzyme over neutral aluminium oxide
could be reused two or three more times in the reaction under study
without loss of activity, thus confirming the good stability and the

Green Enzymatic Synthesis
1145
Table 1. Lipase-catalyzed transesterification under microwaves and reusability of
catalyst
a
Esters
Mode of activation
Time (min)
Yield (%)
b
Methyl laurate
Methyl laurate
MW
MW
35
35
45
45
360
40
40
40
40
40
78
78
75
76
39
83
82
81
80
78
c
b
Methyl myristate
Methyl myristate
Methyl palmitate
Methyl palmitate
Methyl palmitate
Methyl palmitate
Methyl stearate
Methyl stearate
MW
MW
c
d
D
MW
b
c
MW
MW
MW
MW
c
b
c
a
Isolated and unoptimized yield.
Fresh biocatalysts (support) used.
Reused biocatalysts (support).
b
c
d
ꢀ
D: Conventional thermal heating at 80 C.
Lyophilised enzyme.
e
possibility of recycling such systems. This can be attributed to the fact
that mineral support avoids high-temperature enhancement, as only weak
interaction of microwaves could occur, and this thus shows improvement
of enzyme stability. In addition to an increase in enzyme stability, immo-
bilized enzymes offer enzyme reuse and ease of product separation,
whereas lyophilized enzymes do not show such promising results.
Influence of Substrate Ratio
Theoretically, an equimolar ratio of L-ascorbic acid (1) to methyl fatty
acid ester (2a–d) would be sufficient; however, to increase the rate of
reaction and conversion, higher proportions of methyl fatty acid esters
(
2a–d) were used. In practice under microwaves, we observed 65% pro-
duct with an equimolar ratio but considerably higher yields, up to 83%,
when the ratio of methyl palmitate (2c) to L-ascorbic acid (1) was
increased from 1:1 to 1:2 (Fig. 1). Further increase in the ratio does
not reveal promising results. In contrast to the conventional synthesis,
[
7–9]
where five- to nine-fold excess substrate was required,
only two-fold
of fatty acid ester works well in microwaves because the reaction equili-
brium is favorable under microwaves, which allows the reaction to go
to completion.

1146
M. Kidwai et al.
Figure 1. Influence of substrate ratio.
Effect of Microwave on L-Ascorbyl Fatty Acid Ester (3a–d) Synthesis
In this article, we describe the solventless synthesis of L-ascorbyl fatty
acid esters (3a–d) using an enzymatic protocol under microwaves. This
is the first report on enzymatic synthesis of L-ascorbyl fatty acid ester
(3a–d) under microwaves using dry media. To work under dry-media
conditions, the enzyme was immobilized on solid support. L-Ascorbic
acid (1) and fatty acid esters (2a–d) were solubilized in organic solvents
and then impregnated on the enzyme-loaded support by subsequent eva-
poration of these solutions with continuous stirring (Scheme 1).
The loaded alumina was then irradiated under microwave irradia-
tion. Yields and rate of reactions were significantly enhanced (75–80%).
An advantage of microwaves in terms of reactivity lies in the fact that
the associated rate enhancements may allow the reaction to go to com-
pletion in cases where it is barely attainable under classical heating.
Microwaves allow better energy usage with increases in reaction rates
Scheme 1. 2a) Methyl laurate; 2b) methyl myristate; 2c) methyl palmitate; and
2d) methyl stearate.

Green Enzymatic Synthesis
1147
and induce the equilibrium in a forward direction by evaporation of
secondary reaction products such as water or methanol, which strongly
[
18]
interact with the electromagnetic radiation.
side reactions such as hydrolysis of fatty acid esters (2a–d).
This also prevents other
[
19]
CONCLUSIONS
The environmentally benign nature of the reaction is maintained because
we take the added advantage of two ecofriendly technologies, which are
complementary in nature. Enzymatic catalysis using immobilized enzyme
in dry media and microwave activation in solvent free conditions enhance
reactivity of the enzymatic reaction. All the transesterifications per-
formed in dry media under microwave activation were found to be faster
than those described in the literature. Because of the reusability of the
enzyme, we concluded that the enzyme does not get deactivated or dena-
tured as a result of microwave irradiation. Microwave-assisted enzyme
catalysis can become an attractive procedure for rapid characterization
of a large number of enzyme samples and substrates, which otherwise
is a cumbersome and time-consuming exercise.
EXPERIMENTAL
1 13
H NMR and C NMR spectra were recorded on a Bruker Avance
Spectrospin 300 (300 MHz) using tetramethylsilane (TMS) as internal
standard. Infrared (IR) spectra were obtained on Perkin-Elmer Fourier
transform Infrared (FTIR) 1710 spectrophotometer using Nujol film.
Mass spectra were recorded on a Jeol JMS-DX 303. The temperature
of the reaction mixture was measured through a noncontact IR thermo-
meter (AZ, Mini Gun type, model 8868). A Kenstar microwave oven at
2
were purchased from Sigma Aldrich. Aluminium oxide (Aldrich Chem
450 MHz was used for microwave irradiation. All chemical compounds
2
˚
Co., ꢁ150 mesh, 58 A, surface area 155 m =g) and silica gel (Aldrich
2
˚
Chem Co., mesh 130–270, 60 A, BET surface area ꢁ500 m =g, pore
2
volume 0.75 cm =g) were also from Aldrich.
General Procedure for the Synthesis of L-Ascorbyl Fatty Acid Esters
(
3a–3d)
Ascorbic acid (100 mmol) and methyl fatty acid esters (200 mmol) were
dissolved in a minimum amount of methanol (saturated solution) and

1148
M. Kidwai et al.
impregnated on 500 mg enzyme-loaded alumina by subsequent evapora-
tion of these solutions under vacuum. Loaded alumina was taken in a
beaker with a dummy load, and the reactions were performed in an open
vessel. The dry media were irradiated under microwaves (Kenstar model
no. OM-9925E) for a sufficient interval of time as indicated in Table 1.
ꢀ
Temperature of the system raises up to 80 ꢂ 2 C and was checked by
a noncontact IR thermometer (AZ, Mini Gun type, model 8868).
Formation of product was checked by thin-layer chromatography,
TLC (silica-gel-coated Merck aluminium plates). A solution of 2,6-
dichlorophenol-indophenol (1 mg=ml) in ethanol was used as a spray to
observe TLC. After TLC analysis indicated high or complete conversion,
the reaction mixture was extracted three times with 15 ml of methanol,
followed by filtration to remove enzyme-loaded alumina for further
use. The solvent was removed by evaporation in vacuum. After concen-
tration under reduced pressure, the product was purified by silica-gel
chromatography (60–120 mesh) using CHCl –methanol system (80:20
3
by vol). For the sake of comparison and to check specific microwave
(purely nonthermal) effects, synthesis of L-ascorbyl palmitate was also
performed conventionally. Ascorbic acid (100 mmol) and methyl palmi-
tate (200 mmol) were taken in 2-methyl-2-butanol (as solvent) with
500 mg of immobilized enzyme in a thermostatted oil bath at the same
temperature with as close as possible heating profiles.
General Procedure for Enzyme Production and Adsorption
Lipase from Burkholderia multivorans (Gen Bank accession no.
A4597545) was produced under the same culture conditions, with slight
[20]
modifications, as reported for B. cepacia. After incubation, the culture
ꢀ
broth was centrifuged at 10,000 g for 20 min at 4 C in a centrifuge (Sigma
3
K30), and the cell-free supernatant was used as the lipase enzyme.
The crude enzyme was lyophilized to a powdered form. Lipase activity
was determined spectrophotometrically, using p-nitrophnenyl palmitate
as the substrate. One unit of lipase activity was defined as the amount
ꢃ1
ꢃ1
of enzyme required to release 1 mmol p-nitrophenol ml
5
min
For adsorption, 200 mg neutral alumina was wetted with 2 ml
ethanol. To this, 17 ml double-distilled water and 3 ml phosphate buffer
0.05 M, pH 7.0) were added. Lyophilized enzyme (500 mg, containing
at
ꢀ
0 C.
[21]
(
about 93 lipase units, 4 mg protein) was then added to the support.
ꢀ
The suspension was shaken at 100 rpm and 25 ꢂ 2 C for 2 h for adsorp-
tion to occur. The support was filtered off, washed twice with phosphate
buffer (0.05 M, pH 7.0) to remove unbounded protein, and air dried at
room temperature for 10–15 h.

Green Enzymatic Synthesis
1149
Spectroscopic Data of Products (3a–d)
6
-O-Lauroyl-L-ascorbic Acid (3a)
ꢃ1
1
0
IR n (cm ): 1737 (C¼O). H NMR (DMSO-d , TMS) d 4.72 (d, H-4 ,
6
0
0
1H), 4.39 (m, H-5 1H), 4.06 (d, H-6 , 3H), 2.32 (t, H-2, 2H), 1.56 (m,
H-3, 2H), 1.28 (H-4 to H-11, 16H), 0.91 (t, H-12, 3H). C NMR
1
3
0
0
(
1
DMSO-d , TMS): d 171.9 (C-1 ¼O), 170.2 (C-1¼O), 152.1 (C-2 ),
6
0
0
0
0
18.0 (C-3 ), 74.6 (C-4 ), 65.5 (C-5 ), 64.0 (C-6 ), 33.2 (C-2), 31.2 (C-
0), 28.9 (C-6,7), 28.7 (C-5,8), 28.6 (C-9), 28.1 (C-4), 24.2 (C-3), 21.9
1
(
C-11), 13.8 (C-12).
6
-O-Myristoyl-L-ascorbic acid (3b)
ꢃ1
1
0
IR n (cm ): 1752 (C¼O). H NMR (DMSO-d , TMS): d 4.69 (d, H-4 ,
6
0 0
1
2
H), 4.13-4.03 (m, H-5 and H-6 , 3H), 2.31 (t, H-2, 2H), 1.61 (m, H-3,
1
H), 1.22 (H-4 to H-13, 20H), 0.89 (t, H-14, 3H). C NMR (DMSO-
3
0
0
d , TMS): d 171.9 (C-1 ¼O), 170.4 (C-1¼O), 152.1 (C-2 ), 118.1
6
0
0
0
0
(
(
C-3 ), 74.6 (C-4 ), 65.5 (C-5 ), 64.1 (C-6 ), 33.1 (C-2), 31.2 (C12), 28.9
C-8,9), 28.8 (C-7), 28.7 (C-6,10), 28.6 (C-11), 28.5 (C-5), 28.3 (C-4),
2
4.2 (C-3), 21.9 (C-13), 13.9 (C-14).
6
-O-Palmitoyl-L-ascorbic Acid (3c)
ꢃ1
1
0
IR n (cm ): 1731 (C¼O). H NMR (DMSO-d , TMS): d 4.66 (d, H-4 ,
6
0 0
1
2
H), 4.05-3.95 (m, H-5 and H-6 , 3H), 2.31 (t, H-2, 2H), 1.52 (m, H-3,
1
3
H), 1.23 (H-4 to H-15,24H), 0.85 (t, H-16, 3H). C NMR (DMSO-
0
0
d , TMS): d 171.9 (C-1 ¼O), 170.4 (C-1¼O), 152.1 (C-2 ), 118.1
6
0
0
0
0
(
(
C-3 ), 74.6 (C-4 ), 65.5 (C-5 ), 64.1 (C-6 ), 33.2 (C-2), 31.2 (C14), 28.9
C-8,9,10), 28.6 (C-7,11), 28.5 (C-6,12) 28.3 (C-13), 28.2 (C-5), 28.1 (C-
4
), 24.3 (C-3), 21.9 (C-15), 13.9 (C-16).
6
-O-Stearoyl-L-ascorbic Acid (3d)
ꢃ1
1
0
IR n (cm ): 1754 (C¼O). H NMR (DMSO-d , TMS): d 4.66 (d, H-4 ,
6
0
0
1
2
3
1
3
H), 4.12-3.97 (m, H-5 and H-6 , 3H), 2.33 (t, H-2, 2H), 1.54 (m, H-3,
H), 1.48 (m, H-4,5, 4H), 1.31 (m, H-6 to H17, 24H), 0.91 (t, H-18,
1
3
0
H). C NMR (DMSO-d , TMS): d 172 (C-1 ¼O), 170.4 (C-1¼O),
6
0
0
0
0
0
52.1 (C-2 ), 118.1 (C-3 ), 74.6 (C-4 ), 65.5 (C-5 ), 64.1 (C-6 ), 33.2 (C-2),
1.2 (C-16), 28.9 (C-10,11), 28.8 (C-9,12), 28.7 (C-8), 28.6 (C-7,13), 28.5
(
C-5,6,14), 28.4 (C-15), 28.3 (C-4), 24.2 (C-3), 22.0 (C-17), 13.9 (C-18).

1150
M. Kidwai et al.
ACKNOWLEDGMENTS
The authors are thankful to the Council of Scientific and Industrial
Research, New Delhi, India, for financial assistance.
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