A simple efficient synthesis and biological evaluation of 3-O-ethylascorbic acid

Article abstract of DOI:10.1080/09168451.2014.946396

A single-step synthesis of 3-O-ethyl-L-ascorbic acid was performed without the induction of protecting groups. Sodium L-ascorbate reacted with ethyl bromide in DMSO to give 3-O-ethylascorbic acid in a yield of 51.0%. 3- O-Ethylascorbic acid enhanced dibutyryl cyclic AMP-induced neurite outgrowth in PC12 cells.

Full text of DOI:10.1080/09168451.2014.946396

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A simple efficient synthesis and biological evaluation
of 3-O-ethylascorbic acid
Akihiro Taia, Mei Aburadaa & Hideyuki Itob
a Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima,
Shobara, Japan
b Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan
Published online: 15 Aug 2014.
To cite this article: Akihiro Tai, Mei Aburada & Hideyuki Ito (2014): A simple efficient synthesis and biological evaluation
of 3-O-ethylascorbic acid, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2014.946396
To link to this article: http://dx.doi.org/10.1080/09168451.2014.946396
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Bioscience, Biotechnology, and Biochemistry, 2014
Note
A simple efficient synthesis and biological evaluation of 3-O-ethylascorbic acid
Akihiro Tai1,*, Mei Aburada1 and Hideyuki Ito2
2
1Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Japan; Faculty of
Health and Welfare Science, Okayama Prefectural University, Soja, Japan
Received May 28, 2014; accepted June 25, 2014
http://dx.doi.org/10.1080/09168451.2014.946396
A single-step synthesis of 3-O-ethyl-L-ascorbic acid
was performed without the induction of protecting
groups. Sodium L-ascorbate reacted with ethyl
bromide in DMSO to give 3-O-ethylascorbic acid in
a yield of 51.0%. 3-O-Ethylascorbic acid enhanced
dibutyryl cyclic AMP-induced neurite outgrowth in
PC12 cells.
we succeeded in a single-step synthesis of 3-O-ethy-
lascorbic acid without the induction of protecting
groups, and we examined its ability as an enhancer of
neurite outgrowth-promoting activity of dibutyryl cyclic
AMP (Bt2cAMP) in PC12 cells.
Some reports have appeared in the literature recently
regarding the direct 3-O-alkylation of L-ascorbic acid
without protecting groups. Ascorbic acid directly reacts
with alkyl bromides or with alkyl mesylates using
sodium hydrogen carbonate as a base in DMSO.15,16)
Tahir and Hindsgaul have developed a novel methodol-
ogy for the preparation of 3-O-alkyl derivatives of
ascorbic acid under Mitsunobu conditions.17) We
considered a simple reaction system consisting of
sodium L-ascorbate as a starting material and an alkyl-
ation reagent in DMSO for the synthesis of 3-O-ethy-
lascorbic acid (Scheme 1 (B)). This one-step procedure
further simplified the direct 3-O-alkylation method15) of
Beifuss and co-workers. By replacing ascorbic acid
with sodium ascorbate as a starting material, there was
no need to add sodium hydrogen carbonate as a base
to the reaction system, thereby eliminating the neutral-
ization step in the work-up process.
Sodium ascorbate (39.6 mg, 0.2 mmol) and 1.0 mL
of solvent were put into a 2.0-mL screw-capped micro-
tube, and the tube was then placed in an M·BR-022
thermo-regulated shaker (Taitec, Saitama, Japan) and
the mixture was stirred at 1300 r/min for 10 min at
40–60 °C. After adding an ethylation agent (0.8–1.6
equiv.), the mixture was further stirred at 1300 r/min at
40–60 °C. Aliquots of 50 μL were withdrawn at the
indicated times and were diluted 400-fold with 15%
MeOH-H2O containing 0.5% formic acid. The
methanolic solution was subjected to HPLC analysis.
3-O-Ethylascorbic acid was separated by isocratic elu-
tion in an Inertsil ODS-3 column (ϕ 4.6 × 100 mm,
3 μm; GL Sciences, Tokyo, Japan) kept at 40 °C with
15% MeOH-H2O containing 0.5% formic acid at a
flow rate of 0.7 mL/min. The absorbance at 245 nm
was monitored.
Key words: 3-O-ethylascorbic
acid;
single-step
synthesis; PC12 cells; neurite outgrowth
2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G), a
stable ascorbic acid derivative developed by Yamamoto
et al.,1–4) has been approved by the Japanese Govern-
ment as a quasi-drug principal ingredient for skin care
and as a food additive, and it is now widely used as a
medical additive in commercial cosmetics. AA-2G
exhibits vitamin C activity in vitro and in vivo after
enzymatic hydrolysis to ascorbic acid by α-glucosi-
dase.5–7) We have recently chemically synthesized the
monoacylated derivative of AA-2G, 6-O-dodecanoyl-2-
O-α-D-glucopyranosyl-L-ascorbic acid (6-sDode-AA-
2G), in order to improve the bioavailability of
AA-2G.8,9) 6-sDode-AA-2G had outstanding permeabil-
ity in a human living skin equivalent model. The ascor-
bic acid derivative was susceptible to enzymatic
hydrolysis by tissue esterase and α-glucosidase to
produce ascorbic acid.
More recently, 3-O-ethyl-L-ascorbic acid, an ascorbic
acid derivative with a simple structure and efficient
transdermal activity, has been developed as an additive
in commercial cosmetics. The derivative is synthesized
by a three-step procedure as shown in Scheme 1(A).10)
A series of 3-O-alkylascorbic acids has been synthe-
sized in the three-step reaction to act as radical scav-
engers for active oxygen species and free radicals.10–12)
These derivatives inhibited lipid peroxidation, and
some of them also reduced coronary reperfusion-
induced arrhythmias in anesthetized rats. In addition,
3-O-ethylascorbic acid has been reported to effectively
inhibit the induction of nephroblastomas, an uncommon
tumor mostly found in childhood in humans,13) and to
reduce rat mammary tumor induction.14) In this study,
Effects of the ethylation agent and reaction tempera-
ture on 3-O-ethylascorbic acid formation are shown in
Fig. 1(A). Generally, it is not easy to mix carbohy-
drates and alkyl donors in a reaction medium because
*Corresponding author. Email: atai@pu-hiroshima.ac.jp
© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
A. Tai et al.
6
5
O
O
OH
OH
(A)
OH
OH
O
O
1) NaHCO3
2) C2H5Br
O
O
O
O
1
O
O
O
O
4
H+/H2O
AcCl/acetone
DMSO
3
2
C2H5O
OH
C2H5O
OH
HO
OH
HO
OH
3-O-Ethylascorbic acid
L-Ascorbic acid
OH
OH
O
OH
OH
O
(B)
Ethylation agent
DMSO
O
O
C2H5O
OH
NaO
OH
Sodium L-ascorbate
3-O-Ethylascorbic acid
Scheme 1. Synthesis of 3-O-ethylascorbic acid: (A) conventional method; (B) simple method.
of their different solubilities. As a result of screening
for solvents that can dissolve sodium ascorbate and an
ethylation agent, DMSO was chosen as a solvent to
obtain a good yield of 3-O-ethylascorbic acid (data not
shown). The reaction of sodium ascorbate and each
ethylation agent (1.0 equiv.) was carried out in DMSO
for 12 h at 40, 50, and 60 °C. At all the reaction tem-
peratures, the reaction with ethyl bromide gave high
yields. The yield was best when sodium ascorbate and
ethyl bromide reacted at 50 °C. In order to improve the
yield, the reaction of sodium ascorbate and ethyl bro-
mide (0.8, 1.0, 1.2, 1.4, and 1.6 equiv.) was carried out
in DMSO at 50 °C for 24 h (Fig. 1(B)). With 0.8 and
1.0 equivalents of ethyl bromide, the reaction yield
gradually increased and reached a plateau (54% and
60%, respectively) at 6 h and then remained fairly con-
stant until the experiment ended in 24 h. With 1.2
equivalents of ethyl bromide, the reaction yield reached
a maximum (61%) at 3 h and only slightly decreased
by the subsequent reaction. With 1.4 and 1.6 equiva-
lents of ethyl bromide, the reaction yield reached a
maximum (62%) at 3 h and rapidly decreased by the
subsequent reaction. The maximum reaction yield was
slightly better than that with 1.2 equivalents of ethyl
bromide, but the periods for good yields of 3-O-ethy-
lascorbic acid were far shorter than that with 1.2 equiv-
alents of ethyl bromide. These results indicated that the
optimal reaction conditions for simple synthesis of
3-O-ethylascorbic acid were a molar ratio of sodium
ascorbate to ethyl bromide of 1:1.2, a reaction tempera-
ture of 50 °C, a reaction time of 3 h, and use of DMSO
as the reaction solvent.
(A)
60
40
20
0
Ethyl bromide Ethyl iodide Diethyl sulfate
(B)
60
40
20
0
0
6
12
18
24
Time (h)
Fig. 1. Effect of ethylation agent and reaction temperature (A) and
effect of ethyl bromide content and reaction time (B) on 3-O-ethy-
lascorbic acid formation.
The optimal reaction conditions were applied to
scale-up the 3-O-ethylation of ascorbic acid. Sodium
ascorbate (1.98 g, 10.0 mmol) was dissolved in DMSO
(50 mL) and was stirred for 10 min at 50 °C. After add-
ing ethyl bromide (896 μL, 12.0 mmol), the mixture
was further stirred for 3.5 h at 50 °C. The reaction mix-
ture was diluted twofold with water and was subjected
to column chromatography to remove DMSO and unre-
acted starting materials. The diluted reaction mixture
was chromatographed in a DIAION HP20 column
(ϕ 3.0 × 33 cm; Mitsubishi Chemical Co., Tokyo) eluted
Notes: (A) The reaction mixture contained sodium ascorbate
(39.6 mg, 0.2 mmol) and each ethylation agent (1.0 equiv.) in 1.0 mL
of DMSO. The reaction was carried out at 40, 50, and 60 °C for
12 h. The reaction mixture was analyzed by HPLC. Each value is the
mean SD of three independent experiments. (B) The reaction
mixture contained sodium ascorbate (39.6 mg, 0.2 mmol) and ethyl
bromide in 1.0 mL of DMSO: ethyl bromide contents of 0.8 equiv.
(
(
), 1.0 equiv. (Δ), 1.2 equiv. ( ), 1.4 equiv. (□), and 1.6 equiv.
). The reaction was carried out at 50 °C for 24 h. Aliquots were
periodically withdrawn and analyzed by HPLC. Each value is the
mean SD of three independent experiments. The absence of an SD
bar means that the SD bar is within the symbol.

A simple synthesis and bioactivity of 3-O-ethylascorbic acid
3
by the total number of cells, which amounted to
300–400 cells per well. 3-O-Ethylascorbic acid showed
neurite outgrowth-promoting activity at 3–30 μM
(Fig. 2). Ascorbic acid had neurite outgrowth-promot-
ing activity at 0.75–7.5 μM. The percentage of cells
with neurites (25.9%) at 10 μM of 3-O-ethylascorbic
acid was higher than the maximum level (23.9% at
2.5 μM) of neurite outgrowth induced by ascorbic acid,
although a higher concentration of 3-O-ethylascorbic
acid than that of ascorbic acid was required for neurite
outgrowth promotion. The stability of 3-O-ethylascor-
bic acid (1.0 mM) in a medium at 37 °C was evaluated
on the basis of the remaining ratio determined
by HPLC. It was found that 97.1 1.9% (n = 4) of
3-O-ethylascorbic acid remained intact after 24 h
(unpublished data). These results suggested that 3-O-
ethylascorbic acid per se, not ascorbic acid released
from 3-O-ethylascorbic acid, might exhibit neurite out-
growth-promoting activity. Therefore, a new activity of
3-O-ethylascorbic acid for enhancement of Bt2cAMP-
induced neurite outgrowth in PC12 cells was revealed.
In conclusion, we succeeded in synthesizing 3-O-eth-
ylascorbic acid from sodium ascorbate and ethyl bro-
mide in DMSO by a single-step procedure and found
that 3-O-ethylascorbic acid enhanced Bt2cAMP-induced
neurite outgrowth in PC12 cells.
30
20
10
0
**
**
**
*
*
*
1
10
100
1000
0
Concentration (µM)
Fig. 2. Effects of 3-O-ethylascorbic acid on Bt2cAMP-induced
neurite outgrowth in PC12 cells.
Notes: PC12 cells were plated at 4.0 × 103 cells/well and cultured
with indicated concentrations of 3-O-ethylascorbic acid (
) and
ascorbic acid (○) in the presence of 0.5 mM Bt2cAMP. The extent of
neurite outgrowth was measured after 24 h and expressed as the mean
percentage of 300–400 cells from three independent experiments. The
bars represent SD. *p < 0.05, **p < 0.01 (Dunnett’s test) compared
with the control (Bt2cAMP 0.5 mM only).
with a stepwise gradient of MeOH-H2O solvent system
(0, 10, 20, and 30%, v/v). The 3-O-ethylascorbic acid-
containing fraction (30% MeOH eluate) was concen-
trated to dryness. The residue was recrystallized from
EtOAc to obtain 1.04 g (51.0% yield) of 3-O-ethylasc-
orbic acid. The results of analysis of the purified com-
pound by 1HNMR, HRMS, and optical rotation are
shown as follows. 1HNMR (600 MHz, CD3OD) δΗ:
1.41 (3H, t, J = 7.2 Hz), 3.69 (2H, d, J = 6.6 Hz), 3.88
(1H, dt, J = 1.8, 6.6 Hz), 4.59 (2H, q, J = 7.2 Hz), 4.81
(1H, d, J = 1.8 Hz). ESI-HRMS m/z [M−H]−: calcd for
C8H11O6: 203.0561, found: 203.0562. [α]25D + 48.1°
(c 1.0, MeOH). The product was determined to be
3-O-ethylascorbic acid by its 1HNMR spectrum and
mass spectrum, and by comparing the 1HNMR spec-
trum with that in a previous report.10) The results indi-
cate that the described method here enables preparation
of 3-O-ethylascorbic acid very easily and quickly com-
pared with the conventional method,10) and suggest that
the simple single-step synthesis of 3-O-ethylascorbic
acid can be applied to the procedure of a large scale.
The ability of 3-O-ethylascorbic acid as an enhancer
of neurite outgrowth-promoting activity of Bt2cAMP
was investigated in PC12 cells. Evaluation of neurite
outgrowth-promoting activity was carried out basically
according to our previous procedure.18) Briefly, PC12
cells (RIKEN Cell Bank, Tsukuba, Japan) were grown
in RPMI 1640 medium supplemented with 5% heat-
inactivated fetal bovine serum, 10% heat-inactivated
horse serum, 100 units/mL penicillin G sodium salt,
and 100 μg/mL streptomycin sulfate, and then incu-
bated in a humidified atmosphere containing 95% air
and 5% CO2 at 37 °C. The suspended PC12 cells in
the medium were seeded in 96-well plates coated with
porcine tendon collagen at a density of 4.0 × 103 cells/
90 μL/well. After 24 h, 10 μL of a medium containing
each sample and Bt2cAMP was added to the cultured
cells. Neurite formation was examined 24 h after the
treatment. The number of cells bearing neurites longer
than one cell body diameter after treatment was divided
Acknowledgments
The authors are grateful to the SC-NMR Laboratory
of Okayama University and the MS Laboratory of the
Faculty of Agriculture at Okayama University. We
thank Prof. Yoshiaki Amakura at College of Pharma-
ceutical Sciences, Matsuyama University for the mea-
surement of optical rotation.
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