Formation of water-soluble vitamin derivatives from lipophilic vitamins by cultured plant cells

Article abstract of DOI:10.1016/j.tetlet.2006.02.082

Glycosylation of vitamin E, its homologues, and vitamin A by cultured plant cells of Phytolacca americana and Catharanthus roseus was investigated to produce water-soluble vitamin derivatives. Two new compounds, that is, 2,5,7,8-tetramethyl-2-(4-methylpen

Full text of DOI:10.1016/j.tetlet.2006.02.082

Tetrahedron Letters 47 (2006) 2695–2698
Formation of water-soluble vitamin derivatives from
lipophilic vitamins by cultured plant cells
Kei Shimoda,a Yoko Kondo,b Koichi Abe,c Hatsuyuki Hamadad and Hiroki Hamadae,*
aDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
bDepartment of Applied Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
cEisai Co. Ltd, 4-20-22 Koishikawa, Bunkyo-ku, Tokyo 112-8088, Japan
dNational Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390, Japan
eDepartment of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Received 17 January 2006; revised 13 February 2006; accepted 16 February 2006
Available online 3 March 2006
Abstract—Glycosylation of vitamin E, its homologues, and vitamin A by cultured plant cells of Phytolacca americana and Catha-
ranthus roseus was investigated to produce water-soluble vitamin derivatives. Two new compounds, that is, 2,5,7,8-tetramethyl-2-(4-
methylpentyl)chroman-6-yl b-D-glucopyranoside and 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6-yl b-D-glucopyranoside,
together with 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl b-D-glucopyranoside were isolated from the cultured cells
of P. americana following administration of vitamin E and its homologues, that is, 2,5,7,8-tetramethyl-2-(4-methylpentyl)-6-chro-
manol, 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)-6-chromanol and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-6-chromanol
(vitamin E). On the other hand, glycosylation by C. roseus gave two new compounds, that is, 2,5,7,8-tetramethyl-2-(4-methylpent-
yl)chroman-6-yl 6-O-b-D-glucopyranosyl-b-D-glucopyranoside and 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6-yl 6-O-b-
D-glucopyranosyl-b-D-glucopyranoside, as well. Furthermore, conversion of vitamin A (retinol) by these cultured cells afforded
retinyl b-D-glucopyranoside.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Vitamins control nutrition in humans and are involved
in various physiological phenomena in the living body.
Since the 1970s, vitamin E has attracted clinical atten-
tion because of its potential to be a very useful medicine
having effects on gynecological internal secretion control
against sterility, heart circulation, liver diseases, aging,
atherosclerosis, thrombosis, and carcinogenesis.1 On
the other hand, clinical information of vitamin A in-
cludes its effects on night blindness, coronary heart dis-
eases, certain kinds of cancer, and age-related macular
degeneration.2 Despite such specific physiological and
pharmacological activities, water-insolubility, instabil-
ity, and light decomposition of these vitamins have been
problems responsible for the poor absorption following
oral administration and for the limit of their use as medi-
cines. Recently, several attempts have been made to in-
crease the bioavailability of vitamin E and vitamin A,
that is, their amphiphilic glycosides such as b-glucoside
and b-galactoside have been synthesized by chemical
glycosylation.3–6 These glycosides would act as prodrugs
of vitamin E and vitamin A, which are expected to be
hydrolyzed by glycosidases in the living body to display
the physiological activities of the corresponding vita-
mins.7 On the other hand, glycosylation with plant cells
has been the subject of increasing attention,8,9 because
one-step enzymatic glycosylation is useful for prepara-
tion of glycosides rather than chemical glycosylation
which requires long protection–deprotection procedure.
However, there are no reports on the enzymatic glyco-
sylation of vitamin E and vitamin A with cultured plant
cells. We report, herein, the enzymatic glycosylation of
vitamin E, its homologues, and vitamin A into the cor-
responding glycosides, water-soluble vitamin deriva-
tives, by cultured plant cells of Phytolacca americana
and Catharanthus roseus.
Each callus strains, P. americana10 and C. roseus,11 were
prepared as described previously. Just prior to use for
this work, 50 g of cultured cells was transplanted to a
300 mL conical flask containing 100 mL of freshly pre-
pared SH medium (pH 5.7) containing 3% sucrose and
grown with continuous shaking for 1 week at 25 ꢁC
*
Corresponding author. Tel.: +81 86 256 9473; fax: +81 86 256
8468; e-mail: hamada@das.ous.ac.jp
0040-4039/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.02.082

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K. Shimoda et al. / Tetrahedron Letters 47 (2006) 2695–2698
under illumination (4000 lux). A total of 90 mg of each
vitamin substrates was administered to six flasks
(15 mg/flask) containing the suspension cultured cells
and the cultures were incubated at 25 ꢁC for 7 days on
a rotary shaker (120 rpm). After incubation, the cells
were harvested and extracted (·3) by homogenization
with MeOH. The yield of the products was calculated
on the basis of the peak area from HPLC using the cali-
bration curves prepared by the HPLC analyses of
authentic glycosides. The MeOH extract was concen-
trated and the residue was partitioned between H2O
and EtOAc. The H2O layer was applied to a Diaion
HP-20 column and the column was washed with H2O
followed by elution with MeOH. The MeOH eluate
was subjected to HPLC (column: 150 · 20 mm) to
give products. No products were observed in the
medium. The structures of the products were identified
using HRFABMS, 1H and 13C NMR, H–H COSY, and
C–H COSY. On administration of 2,5,7,8-tetramethyl-
2-(4-methylpentyl)-6-chromanol (1)12,13 to the cultured
to be 2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-
yl 6-O-b-D-glucopyranosyl-b-D-glucopyranoside (b-gen-
tiobioside), which was a new compound.14 On adminis-
tration of 2, two products were also isolated and
identified as b-glucoside 6 (32%) and b-gentiobioside 9
(5%), that is, 2,5,7,8-tetramethyl-2-(4,8-dimethylnon-
yl)chroman-6-yl
6-O-b-D-glucopyranosyl-b-D-gluco-
pyranoside. The product 9 was a new compound.14
When 3 was used as the substrate, only b-glucoside prod-
uct 7 (8%) was obtained, suggesting that no further glyco-
sylation products such as b-gentiobioside were produced
due to low yield of the product 7.
These demonstrate that the cultured plant cells of
P. americana are able to convert vitamin E and its
homologues into the corresponding b-glucosides,
whereas C. roseus cells are capable of further glucosyl-
ation to give b-gentiobiosides as well.
Furthermore, retinol (vitamin A, 4) was subjected to
these glycosylation systems. After incubation with the
cultured cells of P. americana, b-glucoside product, ret-
inyl b-D-glucopyranoside (10, 22%),3 was isolated. Inter-
estingly, conversion by the cultured cells of C. roseus
gave only 10 (31%) and no further glycosylation prod-
ucts such as b-gentiobioside were obtained, suggesting
that the enzymes responsible for b-gentiobioside pro-
duction from phenolic compounds are not efficient for
the formation of b-gentiobioside of primary alcohol.
These demonstrate that both of these cultured cells are
able to catalyze mono-glucosylation of vitamin A to give
the corresponding b-glucoside.
cells of P. americana,
a product 5 (63%) was
obtained (Fig. 1). The product 5 was identified as
2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yl b-
D-glucopyranoside, which was a new compound.14 Next,
2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)-6-chromanol
(2)12,13 and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-
decyl)-6-chromanol (vitamin E, 3)12 with longer side
chains were tested for the same biotransformation sys-
tem. The structures of the isolated products 6 (35%)
and 7 (7%) were determined as b-glucosides, that is,
2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6-yl
b-D-glucopyranoside and 2,5,7,8-tetramethyl-2-(4,8,12-
trimethyltridecyl)chroman-6-yl b-D-glucopyranoside.4–6
The product 6 was a new compound.14 These suggest
that the long side chains of the substrates drastically
decrease the yield of the products.
Glycosylation of organic compounds often improves
their bio- and pharmacological properties, for example,
glycosides of terpene alcohols have been widely used in
folk medicines.15 Therefore, vitamin glycosides are
expected to possess new physiological activities which
can be of pharmacological interest. The suppressive
action of the glycosides 5–10 on IgE antibody formation
On the other hand, two products, 5 (56%) and 8 (14%),
were isolated from the cultured cells of C. roseus following
administration of 1. The structure of 8 was determined
10
HO
O
HO
3
5
O
HO
6
4
9
HO
13
2
1
OH
P. americana
7
O
O
11
8
n
n
12
1: n=1; 2: n=2; 3: n=3
5: n=1; 6: n=2; 7: n=3
HO
O
HO
O
HO
HO
OH
C. roseus
m
O
O
n
n
1: n=1; 2: n=2; 3: n=3
5: m=1, n=1; 6: m=1, n=2; 7: m=1, n=3;
8: m=2, n=1; 9: m=2, n=2
HO
HO
HO
P. americana
C. roseus
O
O
HO
OH
2
2
4
10
Figure 1. Glycosylation of lipophilic vitamins 1–4 by the cultured cells of P. americana and C. roseus.

K. Shimoda et al. / Tetrahedron Letters 47 (2006) 2695–2698
2697
was examined according to the reported procedure.16,17
As a result, 6 exerted the strongest action among the gly-
cosides tested, whereas no actions were observed in the
cases of 8–10.18 This shows that the b-glucosides of vita-
min E and its homologues would be useful antiallergic
drugs.
22.4 (C-16), 22.9 (C-18, C-19), 24.1 (C-13), 29.0 (C-17),
32.6 (C-2), 40.6 (C-14), 40.7 (C-15), 62.8 (C-60), 71.7 (C-
40), 75.6 (C-1), 75.7 (C-20), 77.6 (C-50), 77.8 (C-30), 105.8
(C-10), 118.3 (C-4), 123.2 (C-5), 127.7 (C-7), 129.6 (C-8),
147.1 (C-6), 149.1 (C-9). Product 6: HRFABMS: m/z
545.2085 [M+Na]+; 1H NMR (CD3OD): d 0.88 (9H,
apparent d, J = 6.8 Hz, H-22, 23, 24), 1.07–1.55 (14H, m,
H-14, 15, 16, 17, 18, 19, 20, 21), 1.22 (3H, s, H-13), 1.77
(2H, t, J = 7.2 Hz, H-2), 2.04 (3H, s, H-10), 2.19 (3H, s, H-
12), 2.22 (3H, s, H-11), 2.57 (2H, t, J = 6.8 Hz, H-3), 3.06–
3.53 (4H, m, H-20, 30, 40, 50), 3.64 (1H, dd, J = 11.6,
5.2 Hz, H-6a0), 3.75 (1H, dd, J = 12.0, 2.4 Hz, H-6b0), 4.52
(1H, d, J = 7.6 Hz, H-10); 13C NMR (CD3OD): d 12.1 (C-
10), 13.2 (C-12), 14.1 (C-11), 20.1 (C-23), 21.6 (C-3), 22.0
(C-16), 23.1 (C-22, C-24), 24.1 (C-13), 25.8 (C-21), 29.1
(C-17), 32.6 (C-2), 33.8, 38.2, 38.6 (C-14, C-18, C-19, C-
20), 40.5 (C-15), 62.8 (C-60), 71.7 (C-40), 75.6 (C-1), 75.7
(C-20), 77.6 (C-50), 77.9 (C-30), 105.8 (C-10), 118.3 (C-4),
123.2 (C-5), 127.7 (C-7), 131.0 (C-8), 147.1 (C-6), 149.1 (C-
9). Product 8: HRFABMS: m/z 637.2171 [M+Na]+; 1H
NMR (CD3OD): d 0.88 (6H, d, J = 6.8 Hz, H-18, 19), 1.17
(2H, m, H-15), 1.20 (3H, s, H-13), 1.40–1.58 (5H, m, H-14,
16, 17), 1.79 (2H, t, J = 6.8 Hz, H-2), 2.04 (3H, s, H-10),
2.18 (3H, s, H-12), 2.21 (3H, s, H-11), 2.57 (2H, t,
J = 6.8 Hz, H-3), 3.10–3.53 (8H, m, H-20, 200, 30, 300, 40, 400,
50, 500), 3.65 (1H, dd, J = 11.6, 5.6 Hz, H-6a00), 3.73 (1H,
dd, J = 12.0, 5.2 Hz, H-6a0), 3.85 (1H, dd, J = 11.6,
2.0 Hz, H-6b00), 4.04 (1H, dd, J = 11.6, 2.4 Hz, H-6b0),
4.24 (1H, d, J = 7.6 Hz, H-100), 4.52 (1H, d, J = 7.6 Hz, H-
10); 13C NMR (CD3OD): d 12.1 (C-10), 13.2 (C-12), 14.1
(C-11), 21.6 (C-3), 22.3 (C-16), 22.9 (C-18, C-19), 24.0 (C-
13), 28.9 (C-17), 32.6 (C-2), 40.5 (C-14), 40.7 (C-15), 62.6
(C-600), 70.0 (C-60), 71.4 (C-400), 71.5 (C-40), 74.9 (C-200),
75.6 (C-1, C-20), 76.6, 77.6, 77.7 (C-30, C-300, C-50, C-500),
104.4 (C-100), 105.7 (C-10), 118.3 (C-4), 123.2 (C-5), 127.8
(C-7), 129.5 (C-8), 147.0 (C-6), 149.0 (C-9). Product 9:
HRFABMS: m/z 707.2206 [M+Na]+; 1H NMR
(CD3OD): d 0.87 (9H, apparent d, J = 6.8 Hz, H-22, 23,
24), 1.09–1.61 (14H, m, H-14, 15, 16, 17, 18, 19, 20, 21),
1.29 (3H, s, H-13), 1.78 (2H, t, J = 6.8 Hz, H-2), 2.05 (3H,
s, H-10), 2.18 (3H, s, H-12), 2.21 (3H, s, H-11), 2.59 (2H, t,
J = 6.8 Hz, H-3), 3.12–3.54 (8H, m, H-20, 200, 30, 300, 40, 400,
50, 500), 3.65 (1H, dd, J = 11.6, 5.2 Hz, H-6a00), 3.73 (1H,
dd, J = 11.6, 4.4 Hz, H-6a0), 3.85 (1H, dd, J = 11.6,
2.0 Hz, H-6b00), 4.04 (1H, dd, J = 10.8, 3.2 Hz, H-6b0),
4.24 (1H, d, J = 8.4 Hz, H-100), 4.52 (1H, d, J = 7.6 Hz, H-
10); 13C NMR (CD3OD): d 12.1 (C-10), 13.2 (C-12), 14.1
(C-11), 20.1 (C-23), 21.6 (C-3), 22.0 (C-16), 23.0, 23.1 (C-
22, C-24), 24.1 (C-13), 25.8 (C-21), 29.1 (C-17), 30.7 (C-2),
33.8, 38.2, 38.6, 40.5, 40.8 (C-14, C-15, C-18, C-19, C-20),
6020 .7 (C-600), 70.2 (C-60), 71.5 (C-400), 71.6 (C-40), 74.9 (C-
2 ), 75.7 (C-1, C-20), 76.8, 77.7, 77.8 (C-30, C-300, C-50, C-
500), 104.4 (C-100), 105.7 (C-10), 118.4 (C-4), 123.3 (C-5),
127.7 (C-7), 129.7 (C-8), 147.0 (C-6), 149.1 (C-9).
Thus, the formation of water-soluble vitamin derivatives
from lipophilic vitamins has been achieved by glycosyl-
ation with cultured plant cells of P. americana and C.
roseus. It should be emphasized that the glycosides of
vitamin E and vitamin A have been produced, for the
first time, by whole cell-mediated process. Further stud-
ies on pharmacological activities and therapeutic effects
of the glycosides are currently in progress.
Acknowledgements
This work was supported in part by a Grant-in-Aid for
Scientific Research (No. 16790014) from the Ministry of
Education, Culture, Sports, Science, and Technology,
Japan.
References and notes
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6. Witkowski, S.; Walejko, P. Z. Naturforsch. 2002, 57b, 571.
7. Barua, A. B.; Olson, J. A. Int. J. Vitam. Nutr. Res. 1992,
62, 298.
8. Hamada, H.; Tomi, R.; Asada, Y.; Furuya, T. Tetrahe-
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9. Kondo, Y.; Shimoda, K.; Takimura, J.; Hamada, H.;
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10. Hamada, H.; Nishida, K.; Furuya, T.; Ishihara, K.;
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11. Hamada, H.; Fuchikami, Y.; Ikematsu, Y.; Hirata, T.;
Williams, H.; Scott, A. I. Phytochemistry 1994, 37, 1037.
12. Substrate 1 (all racemic form) was prepared from tri-
methylhydroquinone and 3-methenyl-7-methylocta-1,6-
diene, and 2 (all racemic form) was from trimethylhydro-
quinone and 3-methenyl-7,11-dimethyldodeca-1,6-diene,
according to the reported procedure.13 Substrate 3 (RRR
form) was purchased from Sigma–Aldrich Co.
15. Mastelic, J.; Jerkovic, I.; Vinkovic, M.; Dzolic, Z.; Vikic-
Topic, D. Croat. Chem. Acta 2004, 77, 491.
13. Matsui, M.; Yamamoto, T. Jpn. Patent 292495, 1994;
Chem. Abstr. 1996, 125(13), 168389g.
16. The inhibitory action of the glycosides on IgE production
was examined as follows. Ovalbumin was used as the
antigen (1 mg/rat), and Al(OH)3 and pertussis vaccine
were used as the adjuvants (20 mg and 0.6 mL/rat,
respectively). Sensitization was made by injection of a
mixture (0.6 mL) of the antigen and the adjuvant into the
paws of each rat (male, ca. 200 g). Paw edema was
measured 24 h after injection and the treated rats were
divided in to groups with an equal average swelling
volume. Hydrocortisone was used as the positive control.
Each test glycoside was dissolved in physiological saline
containing 10% Nikkol and the solution was injected daily
into the rat for 11 d starting on the day of grouping. The
14. Spectral data for selected products; product 5:
HRFABMS: m/z 475.1924 [M+Na]+; 1H NMR
(400 MHz, CD3OD,
d in ppm): d 0.88 (6H, d,
J = 6.8 Hz, H-18, 19), 1.17 (2H, m, H-15), 1.21 (3H, s,
H-13), 1.41–1.58 (5H, m, H-14, 16, 17), 1.78 (2H, t,
J = 6.8 Hz, H-2), 2.04 (3H, s, H-10), 2.19 (3H, s, H-12),
2.22 (3H, s, H-11), 2.58 (2H, t, J = 6.8 Hz, H-3), 3.10–3.51
(4H, m, H-20, 30, 40, 50), 3.63 (1H, dd, J = 12.0, 5.2 Hz, H-
6a0), 3.76 (1H, dd, J = 12.0, 2.4 Hz, H-6b0), 4.52 (1H, d,
J = 7.6 Hz, H-10); 13C NMR (100 MHz, CD3OD, d in
ppm): d 12.0 (C-10), 13.2 (C-12), 14.1 (C-11), 21.6 (C-3),

2698
K. Shimoda et al. / Tetrahedron Letters 47 (2006) 2695–2698
amount of IgE was measured by the passive cutaneous
18. The suppressive action of b-glucosides of vitamin E and its
homologues on IgE antibody formation, that is, 5 (184 of
average of plasma IgE level), 6 (170), and 7 (195), was
stronger than that of hydrocortisone (341). The results for
b-gentiobiosides of vitamin E homologues 8 and 9,
and retinyl b-glucoside 10 were 366, 353, and 372, respec-
tively.
anaphylaxis (PCA)17 method on the 15th day. The results
were expressed as average of plasma IgE level of five rats
administered a total of 10 mg/kg of each glycoside.
17. Koda, A.; Miura, T.; Inagaki, N.; Sakamoto, O.; Ari-
mura, A.; Nagai, H.; Mori, H. Int. Arch. Allergy. Appl.
Immunol. 1990, 92, 209.