Synthesis and characterization of 6-O-acyl-2-O-α-D-glucopyranosyl-L- ascorbic acids with a branched-acyl chain

Article abstract of DOI:10.1248/cpb.51.175

We previously reported the chemical synthesis of a series of novel monoacylated vitamin C derivatives, 6-O-acyl-2-O-α-D-glucopyranosyl-L- ascorbic acids (6-Acyl-AA-2G) possessing a straight-acyl chain of varying length from C₄ to C₁₈, as effective skin antioxidants. In this paper, we describe branched type of 6-Acyl-AA-2G derivatives (6-bAcyl-AA-2G) synthesized by use of a 2-branched-chain fatty acid anhydride as an acyl donor. The stability of 6-bAcyl-AA-2G in neutral solution was much higher than that of 6-Acyl-AA-2G, while they were susceptible to enzymatic hydrolysis for exerting vitamin C effect. These branched derivatives as well as 6-Acyl-AA-2G increased the radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl and the lipophilicity in octanol/water-partitioning systems with increasing length of their acyl group. In addition, the 6-bAcyl-AA-2G derivative with an acyl chain of C₁₂, 6-bDode-AA-2G had the excellent solubility to various solvents, suggesting easy handling in cosmetic use. These characteristics of 6-bAcyl-AA-2G may be available for skin care application as an effective antioxidant.

Full text of DOI:10.1248/cpb.51.175

February 2003
Chem. Pharm. Bull. 51(2) 175—180 (2003)
175
Synthesis and Characterization of 6-O-Acyl-2-O-a-D-glucopyranosyl-L-
ascorbic Acids with a Branched-acyl Chain
a
a
b
a
,a
Akihiro TAI, Daisuke KAWASAKI, Kenji SASAKI, Eiichi GOHDA, and Itaru YAMAMOTO*
a
b
Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University; and Department of
Pharmaceutical Fundamental Science, Faculty of Pharmaceutical Sciences, Okayama University; Okayama 700–8530,
Japan. Received October 24, 2002; accepted November 22, 2002
We previously reported the chemical synthesis of a series of novel monoacylated vitamin C derivatives, 6-O-
acyl-2-O-a-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) possessing a straight-acyl chain of varying length
from C to C , as effective skin antioxidants. In this paper, we describe branched type of 6-Acyl-AA-2G deriva-
4
18
tives (6-bAcyl-AA-2G) synthesized by use of a 2-branched-chain fatty acid anhydride as an acyl donor. The sta-
bility of 6-bAcyl-AA-2G in neutral solution was much higher than that of 6-Acyl-AA-2G, while they were suscep-
tible to enzymatic hydrolysis for exerting vitamin C effect. These branched derivatives as well as 6-Acyl-AA-2G
increased the radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl and the lipophilicity in
octanol/water-partitioning systems with increasing length of their acyl group. In addition, the 6-bAcyl-AA-2G
derivative with an acyl chain of C , 6-bDode-AA-2G had the excellent solubility to various solvents, suggesting
1
2
easy handling in cosmetic use. These characteristics of 6-bAcyl-AA-2G may be available for skin care application
as an effective antioxidant.
Key words 6-acyl ascorbic acid 2-glucoside; lipophilic ascorbate; stable ascorbate; branched-acyl chain; solubility; skin appli-
cation
A stable ascorbate derivative, 2-O-a-D-glucopyranosyl-L- tion, although its stability was less superior to that of AA-
1
—4)
10)
ascorbic acid (AA-2G), developed in our laboratory
has 2G. The insufficient stability may be ascribed to the hy-
already been utilized as a medical additive in the cosmetic drolysis of the ester bond at the C-6 hydroxyl group of AA.
field. This stable hydrophilic vitamin C derivative exhibits vi- Branched fatty acid esters such as cetyl 2-ethylhexanoate,
tamin C activity in vitro and in vivo after enzymatic hydroly- glyceryl tri-2-ethylhexanoate and glyceryl triisopalmitate,
5
—9)
sis to ascorbic acid (AA) by a-glucosidase.
have succeeded in the chemical synthesis of a series of lized as cosmetic materials.
monoacylated AA-2G with an efficient transdermal activity
In this paper, we describe the synthesis and characteriza-
Moreover, we which are excellent in the hydrolytic stability, are widely uti-
1
0)
in relatively good yields. The monoacyl AA-2G derivatives tion of the improved monoacyl vitamin C derivatives, 6-O-
were identified as 6-O-acyl-2-O-a-D-glucopyranosyl-L-ascor- acyl-2-O-a-D-glucopyranosyl-L-ascorbic acids possessing a
bic acids (6-Acyl-AA-2G) possessing a straight-acyl chain of 2-branched-acyl chain. The properties of the derivatives are
varying length from C to C . 6-Acyl-AA-2G was also syn- evaluated by the stability in neutral solution, radical scaveng-
4
18
thesized by protease-catalyzed regioselective acylation in ing activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH),
1
1)
pyridine. Some of them with an appropriate length acyl susceptibility to enzymatic hydrolysis, partitioning behavior
1
0)
chain group exhibited satisfactory stability and radical in n-octanol/buffer systems, and solubility in a wide variety
1
2,13)
scavenging activity.
6-Acyl-AA-2G was susceptible to of solvents.
enzymatic hydrolysis by mammalian tissue esterase and/or
1
0,14)
a-glucosidase to produce AA-2G and AA.
Extremely high stability in the long-term storage is re-
Results and Discussion
Synthesis of 6-bAcyl-AA-2G Monoacylated ascorbic
quired in the case of the application of a cosmetic material. acid derivatives with high stability in the long-term storage
6
-Acyl-AA-2G was shown to be very stable in a neutral solu- were developed from a requirement for cosmetic application.
Chart 1
*
To whom correspondence should be addressed. e-mail: iyamamoto@pheasant.pharm.okayama-u.ac.jp
© 2003 Pharmaceutical Society of Japan

1
76
Vol. 51, No. 2
Fig. 1. Structure of 6-O-Acyl-2-O-a-D-glucopyranosyl-L-ascorbic Acids
with a Straight- or Branched-Acyl Chain
We expected that increasing the substitution at the a position
to the carbonyl in the acyl moiety might inhibit the deacyla-
tion. 2-O-a-D-Glucopyranosyl-L-ascorbic acid (AA-2G) was
coupled with acid anhydrides with various branched-chain
lengths in pyridine to give 6-O-acyl-2-O-a-D-glucopyra-
nosyl-L-ascorbic acid having a 2-branched-acyl chain (6-
bAcyl-AA-2G) as shown in a Chart. They were identified
as 6-O-(2-ethylbutyryl)-2-O-a-D-glucopyranosyl-, 2-O-a-D-
glucopyranosyl-6-O-(2-propylpentanoyl)- and 2-O-a-D-glu-
copyranosyl-6-O-(2-pentylheptanoyl)-L-ascorbic acid (abbre-
viations: 6-bHexa-AA-2G, 6-bOcta-AA-2G, and 6-bDode-
AA-2G, respectively) by UV spectra, elemental analyses and
nuclear magnetic resonance spectroscopy (Fig. 1). In order to
avoid the possibility of different biological effects by the
enantiomer, an acyl group that has the same length of
aliphatic groups attached to the C-2 was employed as a
model for a 2-branched-acyl chain. 6-bAcyl-AA-2G had su-
perior yields when the molar ratio of AA-2G to acid anhy-
drides was essentially a 1 : 2 ratio, while the synthesis of 6-
O-acyl-2-O-a-D-glucopyranosyl-L-ascorbic acid having a
Fig. 2. Stability of 6-bAcyl-AA-2G (a) and 6-Acyl-AA-2G (b) in Aque-
ous Solution
Ascorbic acid (c, ᭛), AA-2G (c, ᭜), 6-Hexa-AA-2G (᭝), 6-bHexa-AA-2G (᭡), 6-
Octa-AA-2G (ᮀ), 6-bOcta-AA-2G (᭿), 6-Dode-AA-2G (᭺) and 6-bDode-AA-2G (᭹)
were dissolved in 100 mM potassium phosphate buffer (pH 6.5) to give 10 mM solution,
and then incubated at 50 °C for the indicated time. The concentration of the test com-
straight-acyl chain (6-Acyl-AA-2G, Fig. 1) needed only 1.2 pounds was analyzed by HPLC. Each value represents the meanϮS.D. (nϭ6).
1
0)
equivalent of acid anhydrides. The detailed examination of
molar ratio permitted producing 6-bAcyl-AA-2G as a major degradation product of 6-Acyl-AA-2G was found to be AA-
product, although monoacylation without the use of any pro- 2G (Fig. 3). AA-2G was scarcely degraded during the tested
tective group was seemed to be difficult. However, the reason period, while AA disappeared after 21 d (Fig. 2c). These re-
that one hydroxyl group at the C-6 of AA was regioselec- sults indicate that 6-bAcyl-AA-2G was more stable than 6-
tively acylated from two primary hydroxyl groups of the Acyl-AA-2G. However, the deacylation (AA-2G release)
whole molecule is unclear.
profile of 6-bAcyl-AA-2G as shown in Fig. 3 was in conflict
Stability of 6-bAcyl-AA-2G in Aqueous Solution The with the initial remarkable decrease of the intact form as
stability of 6-bAcyl-AA-2G in a neutral aqueous solution shown in Fig. 2a. The similar results were observed in 6-
(
10 mM) at 50 °C was evaluated on the basis of remaining Acyl-AA-2G (Figs. 2b, 3). A new peak, with the remarkable
ratio measured by HPLC (Fig. 2). AA-2G, the deacylation decrease, was observed by HPLC analysis, and the peak area
form from 6-bAcyl-AA-2G was also analyzed by HPLC. It ratio of 6-bAcyl-AA-2G (or 6-Acyl-AA-2G) to the new com-
was found that 82—84% of 6-bAcyl-AA-2G remained intact pound was kept approximately constant through the experi-
after 28 d (Fig. 2a). 6-bAcyl-AA-2G sharply decreased dur- ment (data not shown). It is difficult to suppose the unidenti-
ing the first one day, after which was kept approximately con- fied compound as 6-O-acyl AA, because the remaining
stant. The difference in stability among their derivatives was amount of AA-2G revealed the high stability of the ether
not observed. The very small releasing amounts (1—2%) of linkage at the C-2 of AA. Intramolecular acyl migrations
AA-2G indicated that 6-bAcyl-AA-2G avoided the hy- have been previously reported to occur for ascorbic acid es-
1
5,16)
7)
drolytic cleavage of the ester bond at the C-6 of AA (Fig. 3). ters,1
and they are well-known reactions of glycerol es-
On the other hand, 61—68% of 6-Acyl-AA-2G was found to ters. Thus it seems likely that 6-bAcyl-AA-2G underwent
remain intact after 28 days (Fig. 2b). Although the significant an intramolecular acyl migration to yield the isomer and was
difference in stability among their derivatives was not ob- in equilibrium with the isomer within one day. Therefore,
served, the remaining amount of 6-Acyl-AA-2G tended to in- these findings lead to a conclusion that 6-bAcyl-AA-2G had
crease with increasing length of their acyl group. A major a good ability of avoiding the hydrolysis comparable to that

February 2003
177
Fig. 3. Release of AA-2G from 6-bAcyl-AA-2G and 6-Acyl-AA-2G by
the Hydrolytic Cleavage of the Ester Bond
Fig. 4. Hydrolysis of 6-bAcyl-AA-2G and 6-Acyl-AA-2G by Rice Seed
a-Glucosidase
Each sample (1.0 mM) of AA-2G (᭜), 6-Hexa-AA-2G (᭝), 6-bHexa-AA-2G (᭡), 6-
Octa-AA-2G (ᮀ), 6-bOcta-AA-2G (᭿), 6-Dode-AA-2G (᭺) and 6-bDode-AA-2G (᭹)
was incubated with rice seed a-glucosidase (1.0 unit/ml) at 37 °C for the indicated
time. Remaining amount of the derivatives was determined by HPLC analysis. Each
value represents the meanϮS.D. (nϭ3).
The deacylation of 6-Hexa-AA-2G (᭝), 6-bHexa-AA-2G (᭡), 6-Octa-AA-2G (ᮀ),
6
-bOcta-AA-2G (᭿), 6-Dode-AA-2G (᭺) and 6-bDode-AA-2G (᭹) was determined
by measurement of AA-2G release. This experiment was carried out with the experi-
ment on Fig. 2. The concentration of AA-2G was analyzed by HPLC. Each value repre-
sents the meanϮS.D. (nϭ6).
a)
Table 1. Radical Scavenging Activity of 6-bAcyl-AA-2G against DPPH
enging.
Ϫ5
Susceptibility of 6-bAcyl-AA-2G to Enzymatic Hydro-
lysis Ascorbic acid 2-phosphate (AA-2P) and ascorbic acid
2-sulfate (AA-2S) are well known as stable derivatives. AA-
Sample
EC50 (ϫ10 M)
AA
AA-2G
2.4
4.5
3.7
3.6
3.3
3.7
3.6
3.1
2
P was demonstrated to exert an antiscorbutic activity in
6
6
6
6
6
6
-Hexa-AA-2G
-Octa-AA-2G
-Dode-AA-2G
-bHexa-AA-2G
-bOcta-AA-2G
-bDode-AA-2G
guinea pigs and rhesus monkeys, and the activity is explained
2
1,22)
by phosphatase-catalyzed hydrolysis to AA.
However,
2
3,24)
AA-2S was devoid of a substantial vitamin C activity.
The oral administration of AA-2P in rats increased the serum
AA levels, but the treatment of AA-2S caused only a slight
5
)
increase. In addition, ascorbic acid 2-O-methyl ether with-
a) After the reaction was carried out at 25 °C for 20 min, the decolorization of
2
5)
DPPH was measured at 516 nm. The EC50 value was determined as the concentration of out enzymatic degradation had no vitamin C activity. In
each sample required to give 50% of the absorbance shown by a blank. Each EC50 value
contrast, 6-bromo-6-deoxy-L-ascorbic acid possessed an anti-
represents the mean of three separate experiments.
2
6)
scorbutic activity in guinea pigs. These facts demonstrate
that the free 2- and 3-enolic hydroxyl groups of AA are es-
sential to exert the antiscorbutic activity.
of AA-2G under the nonenzymatic condition.
Radical Scavenging Activity of 6-bAcyl-AA-2G
A
In order to get the free 2- and 3-enolic hydroxyl groups of
model for scavenging the stable radical 1,1-diphenyl-2- AA, the hydrolysis by a-glucosidase of 6-bAcyl-AA-2G was
picrylhydrazyl (DPPH) has been used extensively to predict investigated. Rice seed a-glucosidase, which effectively re-
2
)
the antioxidant activities. The radical scavenging activity of leased AA from AA-2G in our previous research, was used
AA derivatives has been estimated by the colorimetric in this experiment. Hydrolysis of 6-bAcyl-AA-2G to 6-O-
1
8—20)
method using DPPH.
We previously showed that 6- acyl AA was determined by HPLC analysis. The remaining
Acyl-AA-2G was slightly superior as a radical scavenger to amounts of 6-bAcyl-AA-2G were the same levels as those of
AA-2G, although the scavenging activity was lower than that 6-Acyl-AA-2G (Fig. 4). These results indicate that the con-
1
0,12)
of AA.
quantity of radicals quenched by AA-2G and 6-Acyl-AA-2G had no effect on the hydrolysis by a-glucosidase. The hy-
Stoichiometric evaluation also indicated that the version of a straight-acyl chain into a branched-acyl chain
1
3)
was superior to that of AA in a long-term reaction.
drolytic cleavage rate of AA-2G was less than that of 6-Acyl-
Whether the branched-acyl group affects the reactivity and 6-bAcyl-AA-2G. In addition, the susceptibility to a-glu-
with DPPH radical was investigated. Table 1 shows the 50% cosidase of 6-Acyl- and 6-bAcyl-AA-2G tended to increase
effective concentration (EC ) calculated from the dose re- with increasing length of their acyl group. The increased sus-
5
0
sponse curve of radical scavenging activity. The EC of each ceptibility seems to result from the high affinity of a-glucosi-
5
0
Ϫ5
6
-bAcyl-AA-2G (3.1—3.7ϫ10 M) was smaller than that of dase for their derivatives with increasing length of their acyl
Ϫ5
AA-2G (4.5ϫ10 M), indicating that 6-bAcyl-AA-2G is group. 6-Acyl-AA-2G was hydrolyzed with mammalian tis-
more efficient than AA-2G in terms of the radical scavenging sue esterase and/or a-glucosidase to produce AA-2G and
potency. The scavenging activity of both 6-Acyl-AA-2G and AA.
1
0,14)
In contrast, 6-bAcyl-AA-2G was predominantly hy-
6
-bAcyl-AA-2G tended to increase with increasing length of drolyzed to 6-O-acyl AA with tissue homogenates from
their acyl group. In addition, the values of 6-bHexa-, 6- guinea pigs (data not shown), suggesting that the prevented
bOcta-, and 6-bDode-AA-2G were nearly identical to those enzymatic degradation of the ester bonds was attributed to
of 6-Hexa-, 6-Octa-, and 6-Dode-AA-2G, respectively. These the steric hindrance by the 2-alkyl groups of their acyl moi-
results indicate that the conversion of a straight-acyl chain eties. It was reported that 6-O-palmitoyl ascorbate stimulated
into a branched-acyl chain had no effect on the radical scav- collagen synthesis in cultured human fibroblasts at lower

1
78
Vol. 51, No. 2
2
7)
10)
doses than does AA. Uesato et al. reported that some of 6- ity in a human skin model. The lipophilicity of 6-bAcyl-
O-acylated ascorbic acids possessing a straight- and a AA-2G was investigated with an n-octanol/10 mM sodium ac-
branched-acyl chain displayed the marked anti-tumor pro- etate buffer (pH 5.0) system at 37 °C, because the acidity of
2
8)
moting activities. It appears that the 6-O-acylated AA de- human skin surface varies within the pH range of about 4—
rivatives efficiently penetrated into the hydrophobic region of 6.
3
0—32)
The distribution percent of 6-bAcyl-AA-2G in oc-
lipid bilayer to exert the activity. Thus, various physiological tanol increased with increasing length of their acyl group
and pharmacological actions of 6-bAcyl-AA-2G could be ef- (Table 2). 6-bHexa-, 6-bOcta-, and 6-bDode-AA-2G were
fectively elicited by the hydrolysis not to AA but to 6-O-acyl found to be 12.6, 53.2, and 99.3% in octanol, respectively.
AA.
The distribution percent of 6-Acyl-AA-2G also increased
In addition, the intact form of 6-bAcyl-AA-2G possessed with increasing length of their acyl group. The lipophilicity
the radical scavenging activity as described above, while the of 6-bDode-AA-2G was equivalent to that of 6-Dode-AA-
radical scavenging activity of ascorbyl 2,6-dipalmitate and 2G, while the lipophilicity was slightly decreased by the sub-
1
0)
AA-2P is extremely weak. Ascorbyl 2,6-dipalmitate and stitution from the straight-acyl chain to the branched-acyl
AA-2P are relatively stable in vitro and exhibit antioxidative chain of C or C carbon atoms. In contrast, AA and AA-2G
6
8
activity after enzymatic hydrolysis to AA by esterase, or were not distributed into octanol.
phosphatase in vivo. Therefore, the prominent effectiveness
The lipophilicity of 6-bAcyl-AA-2G was also investigated
of 6-bAcyl-AA-2G as an antioxidant may be shown not only with an n-octanol/10 mM potassium phosphate buffer (pH
by the fact that the intact form possessed the radical scaveng- 7.0) system in consideration of the penetration into the cell
ing activity, but also by the possibility that 6-bAcyl-AA-2G membrane after skin permeation (Table 2). The same ten-
seems to be enzymatically metabolized and to play an impor- dency as the above results was observed in this system, al-
tant part in antioxidation as AA or 6-O-acyl AA in vivo. though the distribution percent of 6-bHexa-, 6-Hexa-, 6-
These results indicate that 6-bAcyl-AA-2G may be usable as bOcta-, and 6-Octa-AA-2G was found to decrease slightly
an effective pharmacological agent and as a promising an- with increase in pH. The decrease was assumed to be caused
tioxidant in vivo.
by deprotonation at the C-3 hydroxyl group of AA. In con-
Partitioning Behavior of 6-bAcyl-AA-2G in n-Octanol/ trast, the major portion of 6-bDode- and 6-Dode-AA-2G was
Buffer Systems The partition between n-octanol and water distributed into octanol under both conditions, suggesting
is generally accepted to be a suitable model for studying that these compounds have high affinity for cell membranes
affinity on cell membranes or on the corneal layer of epider- and for the corneal layer of epidermis. These results indi-
2
9)
mis. Our previous research showed that partitioning behav- cated that the modification of the acyl group exerted little or
ior in an n-octanol/buffer system agreed with skin permeabil- no effect on the partitioning behavior in the n-octanol/buffer
systems. We previously reported that 6-Dode-AA-2G of 6-
Acyl-AA-2G having a straight-acyl chain was effectively per-
meated into a human skin model. Therefore, it is expected
Table 2. Partitioning Behavior of 6-bAcyl-AA-2G in n-Octanol/Buffer
Systems
a)
10)
that 6-bDode-AA-2G may be applicable to skin care field as
a cosmetic material.
Distribution percent in octanol
Sample
Solubility of 6-bDode-AA-2G in a Wide Variety of Sol-
vents Considering the application to a cosmetic material, it
is desired to easily dissolve in solvents generally employed as
a cosmetic base. Table 3 shows the solubility of the promis-
ing 6-bDode-AA-2G in various solvents including cosmetic
bases such as water, ethanol, glyceryl tri-2-ethylhexanoate
and squalane. 6-bDode-AA-2G was readily dissolved in
water and ethanol at a concentration of above 250 mM, al-
though the partitioning behavior in Table 2 showed the high
lipophilicity. The solubility in acetone, ethyl acetate and
pH 5.0
pH 7.0
AA
AA-2G
0
0
0
0
6-Hexa-AA-2G
6-Octa-AA-2G
6-Dode-AA-2G
6-bHexa-AA-2G
6-bOcta-AA-2G
6-bDode-AA-2G
21.0Ϯ0.6
73.4Ϯ0.7
96.1Ϯ1.8
12.6Ϯ0.6
53.2Ϯ0.5
99.3Ϯ0.5
7.3Ϯ0.6
43.1Ϯ0.7
98.8Ϯ0.2
5.1Ϯ0.9
25.2Ϯ0.2
95.0Ϯ0.2
a) 6-bAcyl-AA-2G was dissolved in sodium acetate buffer (pH 5.0) or potassium
phosphate buffer (pH 7.0) to give 5 ml of 1 mM solution. Five ml of n-octanol was glyceryl tri-2-ethylhexanoate was 46.0, 2.40 and 0.090 mM,
added to the resulting solution, vigorously mixed and partitioned at 37 °C for 30 min.
respectively. 6-bDode-AA-2G was dissolved in a wide vari-
The amount of 6-bAcyl-AA-2G in octanol was analyzed by HPLC. Each value repre-
sents the meanϮS.D. (nϭ3).
ety of solvents, but not in squalane. In contrast, 6-Dode-AA-
a)
Table 3. Solubility of 6-bDode-AA-2G in Various Solvents
Solubility (mM)
Sample
Glyceryl tri-2-
ethylhexanoate
Water
Ethanol
Acetone
Ethyl acetate
Squalane
AA-2G
Ͼ250
0.102Ϯ0.013
Ͼ250
ND
14.8Ϯ1.1
Ͼ250
ND
1.27Ϯ0.12
46.0Ϯ4.5
ND
0.073Ϯ0.004
2.40Ϯ0.08
ND
0.001Ϯ0.001
0.090Ϯ0.004
ND
ND
ND
6-Dode-AA-2G
-bDode-AA-2G
b)
b)
b)
6
a) Sample (10—30 mg) added 200 ml of each solvent was stirred for 6 h at 25 °C. The resulting mixture was transferred into a centifugal ultrafiltration tube and centrifuged.
The amount of AA derivative in the filtrate was determined by HPLC. Each value represents the meanϮS.D. (nϭ3). ND, not detectable. The statistical significance of differences in
the mean of each data was calculated with Student’s t-test. b) pϽ0.001 as compared with 6-Dode-AA-2G.

February 2003
179
1
0)
2
G that had the same partitioning behavior in Table 2 as 6- scribed previously.
Synthesis of 6-O-acyl-2-O-a-D-glucopyranosyl-L-ascorbic Acids with a
Branched-acyl Chain (6-bAcyl-AA-2G). 6-O-(2-Ethylbutyryl)-2-O-a-D-
glucopyranosyl-L-ascorbic Acid (6-bHexa-AA-2G) The preparation of
acid anhydride was carried out according to the procedure of Mestres and
bDode-AA-2G was difficult to be dissolved in the all tested
solvents. The solubility was about 15 mM even in ethanol in
which 6-Dode-AA-2G was most well dissolved. AA-2G, a
3
3)
stable hydrophilic vitamin C derivative was dissolved only in Palomo. Diphenyl phosphorochloridate (10.3 ml, 50 mmol) was added to a
water at a concentration of above 250 mM.
solution of 2-ethylbutyric acid (12.6 ml, 100 mmol) and triethylamine
14.0 ml, 100 mmol) in dichloromethane (30 ml) and the mixture was stirred
at room temperature for 15 min. The solution was washed with cold water
3ϫ200 ml), the organic layer was separated and dried over anhydrous
sodium sulfate. Evaporation of the solvent gave 2-ethylbutyric anhydride
(
It is noteworthy that 6-bDode-AA-2G, as well as AA-2G,
was easily dissolved in water utilized as a main solvent in
cosmetic field. The characteristic that 6-bDode-AA-2G with
(
1
the possibility of high skin permeability showed the high sol- (10.5 g, 98.2%): H-NMR (500 MHz, CDCl ) d 0.97 (12H, t, Jϭ7.5 Hz,
3
ubility in water seemed to be advantageous for cosmetic ma- CH
terials. These findings indicated that the conversion of a
straight-acyl chain into a branched-acyl chain gave excellent
CH
ϫ4), 1.54—1.64 and 1.64—1.74 (each 4H, each m,
3
2
CH CHCH ϫ2), 2.31 (2H, tt, Jϭ5.5, 8.2 Hz, CHCOϫ2). The acid anhy-
2
2
dride was used for the following reaction without further purification.
A mixture of AA-2G (4.0 g, 11.8 mmol) and 2-ethylbutyric anhydride
results in the solubility. 6-bDode-AA-2G could be dissolved
(
5.5 ml, 24.1 mmol) in pyridine (40 ml) was stirred for 2 h at 60 °C. The re-
in a wide variety of solvents, suggesting easy handling in action mixture was concentrated in vacuo. The oil residue was dissolved in
water (80 ml), and then partitioned with EtOAc (4ϫ80 ml). The water layer
cosmetic use. Therefore, 6-bDode-AA-2G seems to be supe-
rior for practical use to AA-2G which has been widely uti-
lized as a cosmetic material.
was concentrated and chromatographed on a Sephadex LH-20 column (f
4
.0ϫ31 cm) eluted with MeOH–H O–AcOH (40 : 59 : 1, v/v). Recrystalliza-
2
tion of the product from benzene–isopropanol (4 : 1, v/v) gave 6-bHexa-AA-
G (1.28 g, 24.9%). UV lmax (MeOHϩHCl) nm (e): 234 (9700); UV l
2
max
1
Conclusion
(MeOHϩNaOH) nm (e): 261 (14800). H-NMR (500 MHz, CD OD) d:
0
3
.91 (6H, t, Jϭ7.5 Hz), 1.51—1.59 (2H, m), 1.59—1.68 (2H, m), 2.28 (1H,
We synthesized 6-O-acyl-2-O-a-D-glucopyranosyl-L-
ascorbic acids having a 2-branched-acyl chain, 6-bAcyl-AA-
tt, Jϭ5.3, 8.7 Hz), 3.40 (1H, dd, Jϭ9.4, 10.1 Hz, 4Ј-H), 3.53 (1H, dd, Jϭ3.7,
9
3
.4 Hz, 2Ј-H), 3.71 (1H, dd, Jϭ4.9, 11.9 Hz, 6Ј-Ha), 3.78 (1H, t, Jϭ9.4 Hz,
Ј-H), 3.79 (1H, dd, Jϭ2.4, 11.9 Hz, 6Ј-Hb), 4.03 (1H, ddd, Jϭ2.4, 4.9,
2
G. The acyl moiety that has the same length of aliphatic
groups attached to the C-2 was employed as a model for a 2- 10.1 Hz, 5Ј-H), 4.14 (1H, ddd, Jϭ1.8, 6.1, 7.0 Hz, 5-H), 4.20 (1H, dd,
branched-acyl chain. 6-bAcyl-AA-2G exhibited a higher sta- Jϭ6.1, 11.0 Hz, 6-Ha), 4.26 (1H, dd, Jϭ7.0, 11.0 Hz, 6-Hb), 4.82 (1H, d,
1
3
Jϭ1.8 Hz, 4-H), 5.37 (1H, d, Jϭ3.7 Hz, 1Ј-H). C-NMR (125 MHz,
bility than 6-Acyl-AA-2G possessing a straight-acyl chain
under a nonenzymatic condition, and the stability was com-
parable to that of AA-2G. The high stability in the long-term
CD OD) d: 12.11 (ϫ2), 26.03, 26.08, 50.09, 62.06 (6Ј-C), 65.40 (6-C),
3
6
7.88 (5-C), 71.00 (4Ј-C), 73.32 (2Ј-C), 74.40 (3Ј-C), 74.77 (5Ј-C), 77.28
(
4-C), 101.68 (1Ј-C), 120.32 (2-C), 161.99 (3-C), 172.07, 177.39 (1-C).
storage satisfied a requirement for the application to a cos- Anal. Calcd for C H O : C, 49.540; H, 6.467. Found: C, 49.561; H, 6.396.
1
8
28 12
metic material. The derivatives showed the radical scaveng- mp 159—161 °C.
2
-O-a-D-Glucopyranosyl-6-O-(2-propylpentanoyl)-L-ascorbic Acid (6-
ing activity, and the hydrolysate by a-glucosidase was also
considered to exhibit antioxidative activity in vivo. In addi-
tion, 6-bAcyl-AA-2G increased the lipophilicity in skin per-
bOcta-AA-2G) From 2-propylpentanoic acid (15.9 ml, 100 mmol) and
diphenyl phosphorochloridate (10.3 ml, 50 mmol), 2-propylpentanoic anhy-
dride (13.1 g, 97.0%) was prepared in a similar manner as 2-ethylbutyric
1
meation model with increasing length of their acyl group. Of anhydride. H-NMR (500 MHz, CDCl ) d: 0.92 (12H, t, Jϭ7.3 Hz,
3
CH CH CH ϫ4), 1.31—1.43 (8H, m, CH CH CH ϫ4), 1.44—1.51 and
6
-bAcyl-AA-2G derivatives, the promising 6-bDode-AA-2G
3
2
2
3
2
2
1
.61—1.69 (each 4H, each m, CH CHCH ϫ2), 2.45 (2H, tt, Jϭ5.2, 8.5 Hz,
2 2
could be dissolved in a wide variety of solvents, suggesting
easy handling in cosmetic use. These findings indicate that 6-
bAcyl-AA-2G may be an effective antioxidant in skin care.
CHCOϫ2).
The synthetic reaction of 6-bOcta-AA-2G was essentially the same as that
of 6-bHexa-AA-2G. From AA-2G (4.0 g, 11.8 mmol) and 2-propylpentanoic
In closing, we expect that 6-bAcyl-AA-2G with the charac- anhydride (7.0 ml, 23.6 mmol), 6-bOcta-AA-2G was obtained as a crystal
(
(
0
1
1.17 g, 21.4% yield). UV lmax (MeOHϩHCl) nm (e): 234 (9500); UV l
max
1
teristic properties is utilized in not only cosmetic field but
also various fields.
MeOHϩNaOH) nm (e): 261 (14500). H-NMR (500 MHz, CD OD) d:
3
.91 (6H, t, Jϭ7.3 Hz), 1.27—1.36 (4H, m), 1.41—1.49 (2H, m), 1.56—
.64 (2H, m), 2.44 (1H, tt, Jϭ5.2, 9.0 Hz), 3.40 (1H, dd, Jϭ9.4, 10.4 Hz, 4Ј-
Experimental
H), 3.53 (1H, dd, Jϭ3.7, 9.4 Hz, 2Ј-H), 3.71 (1H, dd, Jϭ4.9, 11.9 Hz, 6Ј-
1
13
General Experimental Procedure H- and C-NMR spectra were Ha), 3.78 (1H, t, Jϭ9.4 Hz, 3Ј-H), 3.80 (1H, dd, Jϭ2.3, 11.9 Hz, 6Ј-Hb),
recorded on a Varian VXR-500 Instrument with TMS. Melting points were 4.03 (1H, ddd, Jϭ2.3, 4.9, 10.4 Hz, 5Ј-H), 4.12 (1H, ddd, Jϭ1.8, 6.1, 7.0 Hz,
determined on a Yanagimoto micro-melting point apparatus, and are uncor- 5-H), 4.18 (1H, dd, Jϭ6.1, 11.0 Hz, 6-Ha), 4.25 (1H, dd, Jϭ7.0, 11.0 Hz, 6-
13
rected. Elemental analyses were performed on a Yanagimoto MT-5 CHN Hb), 4.80 (1H, d, Jϭ1.8 Hz, 4-H), 5.37 (1H, d, Jϭ3.7 Hz, 1Ј-H). C-NMR
Corder elemental analyzer. UV spectra were obtained on a Shimadzu UV- (125 MHz, CD OD) d: 14.32 (ϫ2), 21.61 (ϫ2), 35.74, 35.77, 46.43, 62.07
3
1
200 spectrophotometer. The HPLC analyses were carried out with a system (6Ј-C), 65.41 (6-C), 67.85 (5-C), 71.02 (4Ј-C), 73.33 (2Ј-C), 74.41 (3Ј-C),
consisting of a Shimadzu SCL-10A system controller, LC-10AD pump, 74.78 (5Ј-C), 77.27 (4-C), 101.68 (1Ј-C), 120.33 (2-C), 161.96 (3-C),
SPD-10AV UV-Vis spectrophotometric detector, SIL-10AD auto injector, 172.07, 177.69 (1-C). Anal. Calcd for C H O ·1/5H O: C, 51.321; H,
2
0
32 12
2
CTO-6A column oven, and C-R7A chromatopac.
Chemicals 2-O-a-D-Glucopyranosyl-L-ascorbic acid (AA-2G) was ob-
tained from Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan). bDode-AA-2G) 2-Pentylheptanoic acid was synthesized according to the
6.977. Found: C, 51.167; H, 6.936. mp 98—101 °C.
2-O-a-D-Glucopyranosyl-6-O-(2-pentylheptanoyl)-L-ascorbic Acid (6-
1
,1-Diphenyl-2-picrylhydrazyl and diphenyl phosphorochloridate were pur- procedure of the malonic ester synthesis, because it was not commercially
chased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Ascorbic acid available. Diethyl malonate was alkylated by n-amyl bromide in EtONa/
was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). a- EtOH, and the product was alkylated again. The dialkylmalonic ester was
Glucosidase from rice seed and 2-propylpentanoic acid (Sigma) were com- hydrolyzed to 2,2-dipentylmalonic acid. Decarboxylation of the substituted
1
mercially available. 2-Ethylbutyric acid and Sephadex LH-20 were pur- malonic acid gave 2-pentylheptanoic acid: H-NMR (500 MHz, CDCl ) d:
3
chased from Nacalai Tesque, Inc. (Kyoto, Japan) and Amersham Bio- 0.88 (6H, br t, Jϭ7.0 Hz, pentyl-5-Hϫ2), 1.22—1.36 (12H, m, pentyl-2,3,4-
sciences, respectively. 2-O-a-D-Glucopyranosyl-6-O-hexanoyl-L-ascorbic
acid (6-Hexa-AA-2G), 2-O-a-D-glucopyranosyl-6-O-octanoyl-L-ascorbic
acid (6-Octa-AA-2G) and 6-O-dodecanoyl-2-O-a-D-glucopyranosyl-L-
ascorbic acid (6-Dode-AA-2G) were synthesized with AA-2G and each acid
Hϫ2), 1.42—1.51 and 1.58—1.67 (each 2H, each m, CH CHCH ), 2.31—
2 2
2.37 (1H, m, CHCO H), 10.36 (1H, br, D O exchangeable, CO H); FAB-MS
2
2
2
ϩ
ϩ
m/z 401 (2MH ), 201 (MH ).
From 2-pentylheptanoic acid (11.4 ml, 50 mmol) and diphenyl phospho-
anhydride in pyridine. The products were purified by recrystallization as de- rochloridate (5.2 ml, 25 mmol), 2-pentylheptanoic anhydride (8.9 g, 93.7%)

1
80
Vol. 51, No. 2
1
was prepared in a similar manner as 2-ethylbutyric anhydride. H-NMR area of the samples with reference to the calibration of authentic AA, AA-
(
(
500 MHz, CDCl ) d: 0.88 (12H, br t, Jϭ7.0 Hz, pentyl-5-Hϫ4), 1.22—1.38 2G, 6-bAcyl-AA-2G and 6-Acyl-AA-2G, respectively.
3
24H, m, pentyl-2,3,4-Hϫ4), 1.45—1.53 and 1.61—1.69 (each 4H, each m,
CH CHCH ϫ2), 2.41 (2H, tt, Jϭ5.5, 8.5 Hz, CHCOϫ2).
Acknowledgment The authors thank Mr. J. Takebayashi for his techni-
2
2
The synthetic reaction of 6-bDode-AA-2G was essentially the same as cal assistance in part of this work.
that of 6-bHexa-AA-2G. From AA-2G (4.0 g, 11.8 mmol) and 2-pentylhep-
tanoic anhydride (10.0 ml, 22.5 mmol), 6-bDode-AA-2G was obtained as a References
crystal (0.88 g, 14.3% yield). UV lmax (MeOHϩHCl) nm (e): 233 (9900);
1) Yamamoto I., Muto N., Nagata E., Nakamura T., Suzuki Y., Biochim.
1
UV lmax (MeOHϩNaOH) nm (e): 261 (15000). H-NMR (500 MHz,
Biophys. Acta, 1035, 44—50 (1990).
2) Yamamoto I., Muto N., Murakami K., Suga S., Yamaguchi H., Chem.
Pharm. Bull., 38, 3020—3023 (1990).
CD OD) d: 0.89 (6H, t, Jϭ7.0 Hz), 1.21—1.37 (12H, m), 1.43—1.52 (2H,
3
m), 1.56—1.65 (2H, m), 2.40 (1H, tt, Jϭ5.2, 9.0 Hz), 3.41 (1H, dd, Jϭ9.4,
1
1
6
6
1
0.1 Hz, 4Ј-H), 3.53 (1H, dd, Jϭ3.7, 9.4 Hz, 2Ј-H), 3.71 (1H, dd, Jϭ4.9,
1.9 Hz, 6Ј-Ha), 3.78 (1H, t, Jϭ9.4 Hz, 3Ј-H), 3.79 (1H, dd, Jϭ2.4, 11.9 Hz,
Ј-Hb), 4.03 (1H, ddd, Jϭ2.4, 4.9, 10.1 Hz, 5Ј-H), 4.12 (1H, dt, Jϭ1.8,
.6 Hz, 5-H), 4.18 (1H, dd, Jϭ6.6, 11.0 Hz, 6-Ha), 4.26 (1H, dd, Jϭ6.6,
1.0 Hz, 6-Hb), 4.80 (1H, d, Jϭ1.8 Hz, 4-H), 5.37 (1H, d, Jϭ3.7 Hz, 1Ј-H).
3) Aga H., Yoneyama M., Sakai S., Yamamoto I., Agric. Biol. Chem., 55,
1751—1756 (1991).
4) Mandai T., Yoneyama M., Sakai S., Muto N., Yamamoto I., Carbohydr.
Res., 232, 197—205 (1992).
5) Yamamoto I., Suga S., Mitoh Y., Tanaka M., Muto N., J. Pharmaco-
bio-Dyn., 13, 688—695 (1990).
6) Wakamiya H., Suzuki E., Yamamoto I., Akiba M., Otsuka M.,
Arakawa N., J. Nutr. Sci. Vitaminol., 38, 235—245 (1992).
7) Yamamoto I., Muto N., Murakami K., Akiyama J., J. Nutr., 122, 871—
877 (1992).
13
C-NMR (125 MHz, CD OD) d: 14.36 (ϫ2), 23.50 (ϫ2), 28.16 (ϫ2),
3
3
7
2.85 (ϫ2), 33.52, 33.56, 46.89, 62.05 (6Ј-C), 65.29 (6-C), 67.81 (5-C),
0.99 (4Ј-C), 73.34 (2Ј-C), 74.41 (3Ј-C), 74.77 (5Ј-C), 77.19 (4-C), 101.71
(
1Ј-C), 120.32 (2-C), 161.99 (3-C), 172.05, 177.66 (1-C). Anal. Calcd for
C H O : C, 55.374; H, 7.745. Found: C, 55.176; H, 7.695. mp 158—
24
40 12
1
60 °C.
8) Kumano Y., Sakamoto T., Egawa M., Iwai I., Tanaka M., Yamamoto I.,
J. Nutr. Sci. Vitaminol., 44, 345—359 (1998).
Stability of 6-bAcyl-AA-2G in Aqueous Solution The test compounds
were dissolved in 100 mM potassium phosphate buffer (pH 6.5) to give 10 ml
of 10 mM solution. The resulting solution was stored at 50 °C for periods up
9) Kumano Y., Sakamoto T., Egawa M., Tanaka M., Yamamoto I., Biol.
Pharm. Bull., 21, 662—666 (1998).
to 28 d, and 100 ml samples were periodically taken. The concentration of the 10) Yamamoto I., Tai A., Fujinami Y., Sasaki K., Okazaki S., J. Med.
test compounds was analyzed by HPLC. The difference from initial concen-
Chem., 45, 462—468 (2002).
tration was taken as the remaining ratio. AA-2G, the deacylation form was 11) Tai A., Okazaki S., Tsubosaka N., Yamamoto I., Chem. Pharm. Bull.,
also analyzed by HPLC.
49, 1047—1049 (2001).
Free-Radical Scavenging Activity of 6-bAcyl-AA-2G Free-radical 12) Fujinami Y., Tai A., Yamamoto I., Chem. Pharm. Bull., 49, 642—644
scavenging activity of 6-bAcyl-AA-2G was assayed using a relatively stable
(2001).
free radical, DPPH, according to the method of Blois. The reaction mix- 13) Takebayashi J., Tai A., Yamamoto I., Biol. Pharm. Bull., 25, 1503—
3
4)
Ϫ6
ture contained 1 ml of 0.5 mM DPPH in ethanol and 4 ml of 1.25ϫ10
,
1505 (2002).
Ϫ5
Ϫ5
Ϫ4
1
.25ϫ10 , 6.25ϫ10 , and 1.25ϫ10 M antioxidant in ethanol/H O (1 : 1,
v/v). After the reaction was carried out at 25 °C for 20 min, the decoloriza-
14) Tai A., Fujinami Y., Matsumoto K., Kawasaki D., Yamamoto I., Biosci.
Biotechnol. Biochem., 66, 1628—1634 (2002).
2
tion of DPPH was measured at 516 nm. The EC50 value was determined as 15) Nomura H., Sugimoto K., Chem. Pharm. Bull., 14, 1039—1044
the concentration of each sample required to give 50% of the absorbance
shown by a blank.
Hydrolysis of 6-bAcyl-AA-2G by Rice Seed a-Glucosidase Reaction
mixtures were composed of 800 ml of 1.25 mM sample dissolved in 100 mM
potassium phosphate buffer (pH 7.0) and 200 ml of enzyme solution (1.0
(1966).
16) Paulssen R. B., Chatterji D., Higuchi T., Pitman I. H., J. Pharm. Sci.,
64, 1300—1305 (1975).
17) Crossley A., Freeman I. P., Hudson B. J. F., Pierce J. H., J. Chem. Soc.,
1959, 760—764.
unit/ml final concentration). The hydrolytic reaction was carried out at 18) Kato K., Terao S., Shimamoto N., Hirata M., J. Med. Chem., 31, 793—
7 °C, and 50 ml of sample was periodically taken. The reaction mixture was
798 (1988).
diluted 10 times with 75% MeOH–H O containing 1% acetic acid, and then 19) Nihro Y., Miyataka H., Sudo T., Matsumoto H., Satoh T., J. Med.
3
2
centrifuged at 8000ϫg for 10 min. Remaining amount of 6-bAcyl-AA-2G in
Chem., 34, 2152—2157 (1991).
the supernatant was determined by HPLC analysis.
20) Morisaki K., Ozaki S., Chem. Pharm. Bull., 44, 1647—1655 (1996).
Partition of 6-bAcyl-AA-2G between n-Octanol and Water 6-bAcyl- 21) Imai Y., Usui T., Matsuzaki T., Yokotani H., Miwa H., Araki Y., Jpn. J.
AA-2G was dissolved in 10 mM sodium acetate buffer (pH 5.0) or 10 mM
potassium phosphate buffer (pH 7.0) to give 5 ml of 1 mM solution. Five ml
of n-octanol was added to the resulting solution, vigorously mixed and parti-
Pharmacol., 17, 317—324 (1967).
22) Machlin L. J., Garcia F., Kuenzig W., Brin M., Am. J. Clin. Nutr., 32,
325—331 (1979).
tioned at 37 °C for 30 min. The amount of 6-bAcyl-AA-2G in octanol was 23) Kuenzig W., Avenia R., Kamm J. J., J. Nutr., 104, 952—956 (1974).
analyzed by HPLC. The difference from the total amount was taken as distri- 24) Machlin L. J., Garcia F., Kuenzig W., Richter C. B., Spiegel H. E., Brin
bution % of 6-bAcyl-AA-2G in octanol.
Solubility of 6-bDode-AA-2G Sample (10—30 mg) was put into a 25) Lu P.-W., Lillard D. W. Jr., Seib P. A., Kramer K. J., Liang Y.-T., J.
.5 ml Eppendorf tube, after which 200 ml of each solvent was added. After
Agric. Food Chem., 32, 21—28 (1984).
sonication for 5 min and incubation at 50 °C for 5 min, the mixture was 26) Kasai T., Ishikawa Y., Inoue K., Tsujimura M., Hasegawa T., Int. J.
placed in a thermoregulated shaker (M·BR-022, TAITEC, Saitama, Japan),
Vitam. Nutr. Res., 65, 36—39 (1995).
and was stirred at 500 r/min for 6 h at 25 °C. The resulting mixture was 27) Rosenblat G., Perelman N., Katzir E., Gal-Or S., Jonas A., Nimni M.
M., Am. J. Clin. Nutr., 29, 825—831 (1976).
1
transferred into a centifugal ultrafiltration tube and centrifuged at 2000ϫg
for 10 min. The amount of AA derivative in the filtrate was determined by
HPLC.
E., Sorgente N., Neeman I., Connect. Tissue Res., 37, 303—311
(1998).
28) Uesato S., Kitagawa Y., Kaijima T., Tokuda H., Okuda M., Mou X. Y.,
Mukainaka T., Nishino H., Cancer Lett., 166, 143—146 (2001).
HPLC Conditions The separation of AA and AA-2G was achieved by
isocratic elution from an Inertsil ODS-3 column (f 4.6ϫ250 mm, 5 mm, GL 29) Kitagawa S., Li H., Sato S., Chem. Pharm. Bull., 45, 1354—1357
Sciences Inc., Tokyo, Japan) kept at 40 °C with 0.1 M potassium phosphate-
phosphoric acid buffer (pH 2.1, containing 10 mg/l of EDTA) at a flow rate
(1997).
30) Zlotogorski A., Arch. Dermatol. Res., 279, 398—401 (1987).
of 0.7 ml/min. The absorbance at 240 nm was monitored. The separation of 31) Korting H. C., Hübner K., Greiner K., Hamm G., Braun-Falco O., Acta
6
-bAcyl-AA-2G and 6-Acyl-AA-2G was carried out by isocratic elution
Derm. Venereol., 70, 429—431 (1990).
from an Inertsil Ph column (f 4.6ϫ250 mm, 5 mm, GL Sciences Inc.) kept at
32) Yosipovitch G., Xiong G. L., Haus E., Sackett-Lundeen L., Ashkenazi
I., Maibach H. I., J. Invest. Dermatol., 110, 20—23 (1998).
4
0 °C with 75% MeOH–H O containing 1% acetic acid at a flow rate of
2
0
.7 ml/min. The absorbance at 240 nm was monitored. AA, AA-2G, 6- 33) Mestres R., Palomo C., Synthesis, 1981, 218—220.
bAcyl-AA-2G and 6-Acyl-AA-2G contents were determined from the peak 34) Blois M. S., Nature (London), 181, 1199—1200 (1958).