No.151

The High Functionality of Glycosylation Using Plant Suspension Cells
Hiroki Hamada1)* and Kei Shimoda2)
1) Department of Life Science, Faculty of Science, Okayama University of Science,
1-1 Rida-cho Kita-ku Okayama 700-0005, Japan*Corresponding author: e-mail:hamada@dls.ous.ac.jp
2) Department of Chemistry, Faculty of Medicine, Oita University,
1-1 Hasama-machi, Oita 879-5593, Japan
E-mail: shimoda@med.oita-u.ac.jp

1. Introduction
Because prodrugs and prosupplements (precursors of drugs
and dietary supplements that are metabolized within the bodyto form the corresponding bioactive materials) have recently
attracted a great deal of attention, there is a growing need fortechniques that are capable of selective chemical modificationof functional compounds. We have focused on glycosylation,
particularly glucosylation, as one such type of chemical
modification.
There have been many studies on the production of useful
substances by organic syntheses through routes that incorporate
reactions induced by biocatalysts, such as cultured cells or
enzymes, and the results of such studies have been appliedin the production of a range of fine chemicals, including
pharmaceuticals, aroma chemicals, and food additives. Amongthe biocatalysts that have been used are microorganisms, fungi,
yeasts, animal cells, and enzymes extracted from these sources.
In addition, biotransformations effected by using cultured plant
cells as biocatalysts have recently attracted attention. Plants,
which live on land and are generally incapable of movement,
produce various secondary metabolites for the purposes of selfdefense and signal transduction. As a result, plant cells containa range of enzymes that have inherent abilities to transform or
to synthesize organic substances. With the aim of utilizing the
intrinsic ability of plant enzymes to effect biotransformations,
we have studied biotransformations of exogenous substances bycultured plant cells and we have succeeded in effecting a rangeof reactions, including reduction, hydrolysis, isomerization,
glycosylation, esterification, and hydroxylation reactions, byusing cultured plant cells as biocatalysts. Glycosylation byplant cells is a particularly important reaction that is involvedin the activation of metabolites in cells and might, therefore, be
useful in stabilizing various biologically active compounds or in
activating various physiological functions.
A major advantage of using biocatalysts in organic
synthesis is their high selectivity. Furthermore, glycosides, thechemical synthesis of which generally involves a complicatedrange of procedures, can be obtained by means of a one-step
enzymatic reaction with a biocatalyst. For these reasons, theapplication of cultured plant cells that are capable of highly
stereoselective glycosylation reactions in organic synthesis has

2been eagerly anticipated.
We have attempted to apply glycosylation reactions
catalyzed by cultured plant cells to the transformation of
bioactive compounds into synthetic compounds with improved
stabilities and new bioactivities. This review introduces theresults of our studies to date on the transformation and activationof bioactive compounds by means of cultured plant cells.

2. Glycosylation of tocophenols
Tocopherols have attracted clinical attention 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. Despite of such specific physiological andpharmacological activities, water-insolubility, instability, andlight decomposition of these vitamins have been problems
responsible for the poor absorption following oral administrationand for the limit of their use as medicines. To synthesize watersoluble tocopherol glycosides, glycosylation of tocopherols with
cultured plant cells was investigated.
Just prior to use for this work, 50 g of cultured cells wastransplanted to a 300 mL conical flask containing 100 mL offreshly prepared SH medium (pH 5.7) containing 3% sucroseand grown with continuous shaking for 1 week at 25 °C underillumination (4000 lux). A total of 90 mg of each vitamin
substrates was administered to the 6 flasks (15 mg/flask)
containing the suspension cultured cells and the cultures wereincubated 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 wascalculated on the basis of the peak area from HPLC using thecalibration curves prepared by the HPLC analyses of authentic
glycosides. The MeOH extract was concentrated and the residue
was partitioned between H2O and EtOAc. The H2O layer wasapplied to a Diaion HP-20 column and the column was washedwith 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.

No.151

products were also isolated and identified as β-glucoside 5(32%) and β-gentiobioside 8 (5%), i.e., 2,5,7,8-tetramethyl-2(4,8-dimethylnonyl)chroman-6-yl 6-O-β- D-glucopyranosylβ-D-glucopyranoside. When 3 was used as the substrate, only
β-glucoside product 6 (8%) was obtained.
Glycosylation of organic compounds often improves theirbio- and pharmacological properties, e.g., glycosides of terpene
alcohols have been widely used in folk medicines. Therefore,
vitamin glycosides are expected to possess new physiologicalactivities which can be of pharmacological interest. The
suppressive action of the glycosides 4-8 on IgE antibody
formation was examined according to the reported procedure(Table 1).2,3 As a result, 5 exerted the strongest action among
the glycosides tested, whereas no actions were observed in thecases of 7 and 8. This shows that the β-glucosides of vitamin E
and its homologues would be useful antiallergic drugs.

On administration of 2,5,7,8-tetramethyl-2-(4methylpentyl)-6-chromanol (tocopherol derivative, 1) tothe cultured cells of Phytolacca americana, a product 4(63%) was obtained (Fig. 1). 1 The product 4 was identified
as 2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yl
β- D -glucopyranoside, which was a new compound. Next,
2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)-6-chromanol (2) and
2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-6-chromanol(vitamin E, 3) with longer side chains were tested for thesame biotransformation system. The structures of the isolated
products 5 (35%) and 6 (7%) were determined as β-glucosides,
i.e., 2,5,7,8-tetramethyl-2-(4,8-dimethylnonyl)chroman-6yl β- D -glucopyranoside and 2,5,7,8-tetramethyl-2-(4,8,12trimethyltridecyl)chroman-6-yl β-D-glucopyranoside.
On the other hand, two products, 4 (56%) and 7 (14%),were isolated from the cultured cells of C. roseus following
administration of 1. The structure of 7 was determined to be
2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yl 6-O-βD -glucopyranosyl-β- D -glucopyranoside (β-gentiobioside),which was a new compound. On administration of 2, two

HO
HO
HO

10

HO

6

5

4

7
11

9

8
12

3

O

2
1

13

P. americana

O

O

OH
O

n

1: n=1; 2: n=2; 3: n=3n

4: n=1; 5: n=2; 6: n=3

HOHO
C. roseus

O

O

HO
HO

OH

O
m

O

n

1: n=1; 2: n=2; 3: n=3n4: m=1, n=1; 5: m=1, n=2; 6: m=1, n=3;
7: m=2, n=1; 8: m=2, n=2

Figure 1. Glycosylation of tocopherols by cultured plant cells.

Table 1. Anti-allergic activity of tocopheryl glycosides.

Compounds

IgE level

4

184

5

170

6

195

7

366

8

353

Hydrocortisone341

3

No.151

3. Glycosylation of flavones

4. Glycosylation of 4-phenylbutan-2-ones

Natural polyphenols such as quercetin, epicatechin, andcatechin occur in plants and have diverse biological activities.
Quercetin is a principal flavonoid in onion Allium cepa, andhas anti-oxidative, anticarcinogenic, and anticancer activities.
Epicatechin and catechin are main flavonoids found in tea
Camellia seinensis, and have been reported on their antioxidative, anti-angiogenesis, anti-inflammatory, and anticancer
effects. Irrespective of such physiological and pharmacologicalactivities, the use of these polyphenols as drugs and foodadditives is limited because of their water-insolubility and low
absorbability after oral administration.
Quercetin 3-O-β- D-glucoside (10) was obtained as the
major product in 51% yield together with 3-O-(6-O-malonyl)β- D-glucoside (11, 10%), 3-O-[6-O-(α- L-rhamnosyl)]-β- Dglucoside (β-rutinoside, 12, 3%), 3,4'-O-β-D-diglucoside (13,1%), and 3,7-O-β- D -diglucoside (14, 1%) after incubationof Nicotiana tabacum cells with quercetin (9) (Fig. 2).4 On
the other hand, three products were isolated from the MeOH
extracts of N. tabacum cells treated with epicatechin ((2R,3R)15). The products were identified as 3'-O-β- D -glucoside((2R,3R)-16, 38%), 5-O-β-D-glucoside ((2R,3R)-17, 7%), and7-O-β-D-glucoside ((2R,3R)-18, 15%). No further glycosylation
and acylation products were obtained. Similar tendency was
found in the biotransformation of catechin ((2R,3S)-15) with
N. tabacum cells. Three products, 3'-O-β-D-glucoside ((2R,3S)16, 46%), 5-O-β-D-glucoside ((2R,3S)-17, 10%), and 7-O-βD-glucoside ((2R,3S)-18, 17%), were isolated, and no further
products were detected despite careful HPLC analyses.

4-(4-Hydroxyphenyl)butan-2-one (raspberry ketone) and4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone) aremajor aromatic compounds of red raspberry Rubus idaeus
and zinger Zingiber officinale, respectively, and have beenused worldwide as food additives and spices. Recently, it
has been reported that these compounds, the structures ofwhich are similar to those of capsaicinoids, showed stronger
anti-obesity activity than capsaicin (N-[(4-hydroxy-3methoxyphenyl)methyl]-8-methyl-6-nonamide) and synephrine(1-(4-hydroxyphenyl)-2-methylaminoethanol). Despite suchbio- and physiological activities, their use as lipid degradation
ingredients and medicines has been limited, due to theirinsolubility in water and decomposition under light.
After cultured cells of P. americana were incubated with
raspberry ketone (19) for three days, the glycosylated products22-26 were isolated from the cells by extraction with MeOH. On
the other hand, none were detected in the medium. On the basisof their HRFABMS, 1H and 13C NMR (Table 1), H-H COSY,
C-H COSY, and NOE-spectroscopic analyses, the products were
determined to be (2S)-4-(4-hydroxyphenyl)-2-butanol (20, 2%),(2S)-4-(3,4-dihydroxyphenyl)-2-butanol (21, 5%), 4-[4-(β-Dglucopyranosyloxy)phenyl]-2-butanone (22, 19%), 4-[(3S)-3hydroxybutyl]phenyl-β-D-glucopyranoside (23, 23%), (2S)-4(4-hydroxyphenyl)but-2-yl-β-D-glucopyranoside (24, 20%),2-hydroxy-4-[(3S)-3-hydroxybutyl]phenyl-β-D-glucopyranoside(25, 12%), and 2-hydroxy-5-[(3S)-3-hydroxybutyl]phenyl-β-Dglucopyranoside (26, 11%) (Fig. 3).5

OH

OH
1

8

HO 7
6
5

2'

9 O 2

10

OH

4

3'

3

OR1

OH
4'
N. tabacum

5'

1'

R3 O

O

6'

OR2

OH
OH

O

O

10: R1,R3 = H, R2 = Glc;
11: R1,R3 = H, R2 = MalonylGlc;
12: R1,R3 = H, R2 = GlcRham;
13: R1,R2 = Glc, R3 = H;
14: R1 = H, R2,R3 = Glc

9

OR1

OH
8

HO 7
6
5

1

2'

9 O 2

10

4

3'

3

OH

OH

4'
1'

5'

N. tabacum

3

R O

O

6'

OH

OH
OR2

OH
15

16: R1 = Glc, R2,R3 = H;
17: R1,R3 = H, R2 = Glc;
18: R1,R2 = H, R3 = Glc

Figure 2. Glycosylation of flavones by cultured plant cells.

4No.151

Glycoside products 29-32 were obtained from the
MeOH extracts of the cells treated with zingerone (27),whereas product 28 was isolated from EtOAc extract ofthe medium. The products were identified as (2S)-4-(4hydroxy-3-methoxyphenyl)-2-butanol (28, 15%), 4-[4-(β-Dglucopyranosyloxy)-3-methoxyphenyl]-2-butanone (29, 21%),4-[(3S)-3-hydroxybutyl]-2-methoxyphenyl-β-D-glucopyranoside

HO 8(30, 24%), (2S)-4-(4-hydroxy-3-methoxyphenyl)but2-yl-β- D -glucopyranoside (31, 17%), and (2S)-2-(β- D glucopyranosyloxy)-4-[4-(β- D -glucopyranosyloxy)-3methoxyphenyl]butane (32, 16%).

9
10

7

4

5

6

3

P. americana

1

2

GlcO

O

22

O

23

OH

24

OGlc

25

OH

26

OH

29

O

30

OH

31

OGlc

32

OGlc

19

GlcO
HO
HO
OH
20

GlcO
HO

HO

HO
HO

OH
21

HO 8

GlcO

9
10

H3CO 7 6 5

4

3

2

1

P. americana

GlcO
H3CO

O

27

GlcO
H3CO
HO

HO

H3CO
OH
28

H3CO
GlcO
H3CO

Figure 3. Glycosylation of 4-phenylbutan-2-ones by cultured plant cells.

5No.151

5. Glycosylation of curcumin

6. Glycosylation of capsaicin

Curcuma longa Linn. has been used as a spice for centuries
worldwide. Also it has been used in folk medicines for thetreatment of a variety of inflammatory conditions. Its intake
reduces the risk of certain kinds of cancers and renders otherprotective pharmacological effects in human. These medicinal
properties have been attributed mainly to the curcuminoids,
and the main component present in C. longa L. is curcumin[1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5dione] that has been widely studied for its anticancer, antiinflammatory, antiaging, antiangiogenic, wound healing, andantioxidant effects. Irrespective of such pharmacologicalactivities, its use as a medicine has been limited, because of its
water insolubility and poor absorption after oral administration.
Incubation of Strophanthus gratus suspension cells withcurcumin (33) gave glucoside 34 as the sole product in 68%
yield.6 Cultured cells of P. americana converted 33 into 34(27%) and doubly glucosylated product (5%). On the other
hand, no glycosylation products were isolated from the cultured
N. tabacum cells which had been treated with 33. The differencein the ability of glucosylation of curcumin between the threeplant species is probably due to the substrate specificity of
glucosyltransferases in these plant cells. Glucosylation of
curcumin with S. gratus described here is considerably efficientmethod to give curcumin β-D-glucoside rather than chemical
glucosylation. Biocatalytic glycosylation of curcumin β- Dglucoside with CGTase was attempted to synthesize curcumin
β-maltooligosides. As a result, products 35 (28%), 36 (20%),37 (14%), and 38 (10%) were obtained after 24 h incubationof curcumin β-D-glucoside (34), which had been prepared by
glucosylation of curcumin (33) with S. gratus, with CGTase in
the presence of soluble starch (Fig. 4).

Capsaicin, N-[(4-hydroxy-3-methoxyphenyl)methyl]8-methyl-(E)-6-nonenamide, is the most pungent principle
among naturally occurring capsaicinoids. Capsaicin has also
been reported to decrease adipose tissue weight and serum
triacylglycerol content in rats by enhancing energy metabolism.
Capsaicin has shown a wide range of pharmacologicalproperties, such as analgesic, antigenotoxic, antimutagenic,
and anticarcinogenic effects, and has been used to treat various
peripheral painful conditions, including rheumatoid arthritis anddiabetic neuropathy. However, capsaicinoids possess extensive
neurological toxicity, and direct irritant effects on skin andmucous membrane. Furthermore, capsaicinoids are scarcely
soluble in water and poorly absorbed after oral administration.
These disadvantages prevent capsaicinoids from being used asfood additives and medicines.
Glycoside product 40 was isolated from cultured cells of
P. americana that had been previously treated with capsaicin(39) by extraction with MeOH.7 The substrate, capsaicin (39),
was detected in both the media and cell extracts. The glycosideproduct was purified and isolated by preparative HPLC for
structure identification by spectroscopic methods. The product40 was identified as capsaicin 4-O-β-D-glucopyranoside bycomparison of its 1 H and 13 C NMR data with previously
reported data (Fig. 5).

H3CO 3''HO 4''

2''

O
1''

1

2

3

OH
4

5

6

6''

7

1'

2'

6'

5''

5'

O
3' OCH3

S. gratus

6a

HO
HO 4a
HO

4' OH

CGTase

HO
HO

5a O

O
O
HO

1a O

OH
34

O

H3CO
O

OCH3

3a 2aOH

33

HO

OH

H3CO

HO
n HO

O

OCH3

O

OH

OH
35: n=1; 36: n=2; 37: n=3; 38: n=4

Figure 4. Glycosylation of curcumin by cultured plant cells and CGTase.

HO
H3CO

H
N

P. americana

O

HO
HO

OH
O

O

OH
H3CO

O

39

40

Figure 5. Glycosylation of capsaicin by cultured plant cells.

6HNNo.151

7. Summary

References

Biocatalytic glycosylation using plant cultured cells andenzymes is a convenient and useful method to prepare costly
and scarce glycosides from inexpensive and plentiful organic
compounds. It was demonstrated that cultured plant cells andenzymes such as CGTase can glycosylate exogenously added
compounds. The diverse glycosylation of exogenous compounds
was observed between the cultured cells. Purification of the
glucosyltransferases which catalyze the biotransformation ofexogenous compounds and studies on the pharmacologicalactivities of the glycosides are now in progress in our laboratory.
In the future we strongly believe that this process andmethod will play an important role in pharmaceutical andsupplement industry.

1)	K. Shimoda, Y. Kondo, K. Abe, H. Hamada, H. Hamada,
Tetrahedron Lett. 2006, 47, 2695-2698.
2)	R. K. Uhrig, M. A. Picard, K. Beyreuther, M. Wiessler,
Carbohydr. Res. 2000, 325, 72-80.
3)	T. Satoh, H. Miyataka, K. Yamamoto, T. Hirano, Chem.
Pharm. Bull. 2001, 49, 948-953.
4)	K. Shimoda, T. Otsuka, Y. Morimoto, H. Hamada, H.
Hamada, Chem. Lett. 2007, 36, 1292-1293.
5)	K. Shimoda, T. Harada, H. Hamada, H. Hamada,
Phytochemistry 2007, 68, 487-492.
6)	K. Shimoda, T. Hara, H. Hamada, H. Hamada, Tetrahedron
Lett. 2007, 48, 4029-4032.
7)	H. Hamada, S. Ohiwa, T. Nishida, H. Katsuragi, T. Takeda,
H. Hamada, N. Nakajima, K. Ishihara, Plant Biotechnol.
2003, 20, 253-255.

Introduction of the authors :Hiroki Hamada

Professor, Department of Life Science, Faculty of Science, Okayama University of Science, Japan

He was born in August 3, 1952. He completed his master course in 1982 and joined to Faculty of Science inOkayama University of Science in 1983. He received his Ph.D. degree from Hiroshima University in 1987. From1988 to 1990, he worked as a postdoctoral fellow in Oklahoma State University and Texas A&M University.
In 1992, he was promoted to an assistant professor in Okayama University of Science and in 1998, he wasappointed as a professor.
His research interests include bioorganic chemistry, biotechnology, and synthetic organic chemistry. Specifically,
his current research involves development of novel biocatalysts including microbes, plants, and algae. Recently
he is interested in glycosylation of biological active compounds using the living cells (plant and glycosyltransferase
so on).

Kei Shimoda

Associate Professor, Department of Chemistry, Faculty of Medicine, Oita University, JapanHe finished his master course in Hiroshima University in 1994. He received his Ph.D. degree from Hiroshima
University in 1999. He worked as a postdoctoral fellow at Liverpool University, UK from 1999 to 2000. In 2002,
he was promoted as an associate professor of Oita University.

7No.151

TCI Related Compounds: Contribution (pp. 2-7)

CH3
O

CH3

CH3

HO

OH

2

CH3

HO

CH3
CH3

CH3

DL-a-Tocopherol

25g[T2309]25g, 250g[T0251]

OOH. xH2O

OH
HO

OH

O

CH3

2

CH3

D-a-Tocopherol

OH
HO

CH3

CH3

CH3
O

CH3

CH3

O
CH
O HO
HO 3
HO
HO OH

O
OH

OH

OH

Quercetin Hydrate
25g[P0042]

O
HO

O
O

O OH

. 3H2O. xH2O
Rutin Trihydrate
25g[R0035](+)-Catechin Hydrate
1g, 10g[C0705]

O

O
CH3O

OH

HO

CH3

CH3

HO

HO

4-(4-Hydroxy-3-methoxyphenyl)-2-butanone(= Zingerone)
25g[H1314]4-(4-Hydroxyphenyl)-2-butanone(= Raspberry Ketone)
25g, 250g[H0604]OCH3O
C CH CH

O

OH

CH3O

CH2
C CH CH

OH

N
H

HO

O
OCH3

Capsaicin (Natural)
1g[M1149]Curcumin (Synthetic)
5g, 25g[C2302]

HO
HO

O

OH
O
O
HO
CH3O

CH3O
CH3

H
N
O

Capsaicin b-D-Glucopyranoside
200mg[C2548]

8

CH3

HO

N
H
N-Vanillylnonanamide[= Capsaicin (Synthetic)]
10g[M0900]

CH3OH