Bioscience, Biotechnology, and Biochemistry

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A simple efficient synthesis and biological

evaluation of 3-O-ethylascorbic acid

Akihiro Tai, Mei Aburada & Hideyuki Ito

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,
78:12, 1984-1987, DOI: 10.1080/09168451.2014.946396

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Bioscience, Biotechnology, and Biochemistry, 2014
Vol. 78, No. 12, 1984–1987

Note
A simple efficient synthesis and biological evaluation of 3-O-ethylascorbic acid
Akihiro Tai1,*, Mei Aburada1 and Hideyuki Ito2
1
Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Japan; 2Faculty 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 we succeeded in a single-step synthesis of 3-O-ethy-

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

*Corresponding author. Email: [email protected]

© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

 A simple synthesis and bioactivity of 3-O-ethylascorbic acid 1985

(A) 6 OH O O OH
1) NaHCO3
5 OH O 2) C2H5Br O OH
O 1 O O O O O O
4 O
AcCl/acetone DMSO H+/H2O
3 2
HO OH HO OH C2H5O OH C2H5O OH

L-Ascorbic acid 3-O-Ethylascorbic acid

(B) OH OH
OH Ethylation agent OH
O O O
O
DMSO

NaO OH C2H5O 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 ethylation agent, DMSO was chosen as a solvent to
for solvents that can dissolve sodium ascorbate and an 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
(A) for 12 h at 40, 50, and 60 °C. At all the reaction tem-
60 peratures, the reaction with ethyl bromide gave high
Reaction yield (%)

yields. The yield was best when sodium ascorbate and

ethyl bromide reacted at 50 °C. In order to improve the
40
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
20 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
0
Ethyl bromide Ethyl iodide Diethyl sulfate 60%, respectively) at 6 h and then remained fairly con-
stant until the experiment ended in 24 h. With 1.2
(B) equivalents of ethyl bromide, the reaction yield reached
60
a maximum (61%) at 3 h and only slightly decreased
Reaction yield (%)

by the subsequent reaction. With 1.4 and 1.6 equiva-

lents of ethyl bromide, the reaction yield reached a
40 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
20 bromide, but the periods for good yields of 3-O-ethy-
lascorbic acid were far shorter than that with 1.2 equiv-
0
alents of ethyl bromide. These results indicated that the
0 6 12 18 24 optimal reaction conditions for simple synthesis of
Time (h) 3-O-ethylascorbic acid were a molar ratio of sodium
ascorbate to ethyl bromide of 1:1.2, a reaction tempera-
Fig. 1. Effect of ethylation agent and reaction temperature (A) and ture of 50 °C, a reaction time of 3 h, and use of DMSO
effect of ethyl bromide content and reaction time (B) on 3-O-ethy- as the reaction solvent.
lascorbic acid formation. The optimal reaction conditions were applied to
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
scale-up the 3-O-ethylation of ascorbic acid. Sodium
of DMSO. The reaction was carried out at 40, 50, and 60 °C for ascorbate (1.98 g, 10.0 mmol) was dissolved in DMSO
12 h. The reaction mixture was analyzed by HPLC. Each value is the (50 mL) and was stirred for 10 min at 50 °C. After add-
mean ± SD of three independent experiments. (B) The reaction ing ethyl bromide (896 μL, 12.0 mmol), the mixture
mixture contained sodium ascorbate (39.6 mg, 0.2 mmol) and ethyl was further stirred for 3.5 h at 50 °C. The reaction mix-
bromide in 1.0 mL of DMSO: ethyl bromide contents of 0.8 equiv. ture was diluted twofold with water and was subjected
( ), 1.0 equiv. (Δ), 1.2 equiv. ( ), 1.4 equiv. (□), and 1.6 equiv.
to column chromatography to remove DMSO and unre-
( ). The reaction was carried out at 50 °C for 24 h. Aliquots were
periodically withdrawn and analyzed by HPLC. Each value is the
acted starting materials. The diluted reaction mixture
mean ± SD of three independent experiments. The absence of an SD was chromatographed in a DIAION HP20 column
bar means that the SD bar is within the symbol. (ϕ 3.0 × 33 cm; Mitsubishi Chemical Co., Tokyo) eluted
1986 A. Tai et al.
by the total number of cells, which amounted to
30 ** ** 300–400 cells per well. 3-O-Ethylascorbic acid showed
% of cells with neurites

** neurite outgrowth-promoting activity at 3–30 μM

* (Fig. 2). Ascorbic acid had neurite outgrowth-promot-
20 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
10
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-
0
0 1 10 100 1000 bic acid (1.0 mM) in a medium at 37 °C was evaluated
on the basis of the remaining ratio determined
Concentration (µM)
by HPLC. It was found that 97.1 ± 1.9% (n = 4) of
Fig. 2. Effects of 3-O-ethylascorbic acid on Bt2cAMP-induced 3-O-ethylascorbic acid remained intact after 24 h
neurite outgrowth in PC12 cells. (unpublished data). These results suggested that 3-O-
Notes: PC12 cells were plated at 4.0 × 103 cells/well and cultured ethylascorbic acid per se, not ascorbic acid released
with indicated concentrations of 3-O-ethylascorbic acid ( ) and from 3-O-ethylascorbic acid, might exhibit neurite out-
ascorbic acid (○) in the presence of 0.5 mM Bt2cAMP. The extent of growth-promoting activity. Therefore, a new activity of
neurite outgrowth was measured after 24 h and expressed as the mean 3-O-ethylascorbic acid for enhancement of Bt2cAMP-
percentage of 300–400 cells from three independent experiments. The
bars represent SD. *p < 0.05, **p < 0.01 (Dunnett’s test) compared
induced neurite outgrowth in PC12 cells was revealed.
with the control (Bt2cAMP 0.5 mM only). 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
with a stepwise gradient of MeOH-H2O solvent system that 3-O-ethylascorbic acid enhanced Bt2cAMP-induced
(0, 10, 20, and 30%, v/v). The 3-O-ethylascorbic acid- neurite outgrowth in PC12 cells.
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- Acknowledgments
orbic acid. The results of analysis of the purified com-
pound by 1HNMR, HRMS, and optical rotation are The authors are grateful to the SC-NMR Laboratory
shown as follows. 1HNMR (600 MHz, CD3OD) δΗ: of Okayama University and the MS Laboratory of the
1.41 (3H, t, J = 7.2 Hz), 3.69 (2H, d, J = 6.6 Hz), 3.88 Faculty of Agriculture at Okayama University. We
(1H, dt, J = 1.8, 6.6 Hz), 4.59 (2H, q, J = 7.2 Hz), 4.81 thank Prof. Yoshiaki Amakura at College of Pharma-
(1H, d, J = 1.8 Hz). ESI-HRMS m/z [M−H]−: calcd for ceutical Sciences, Matsuyama University for the mea-
C8H11O6: 203.0561, found: 203.0562. [α]25D + 48.1° surement of optical rotation.
(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- References
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