Biotransformation of low-molecular-weight alcohols by Coleus forskohlii hairy root cultures

Article abstract of DOI:10.1016/S0008-6215(03)00011-9

Coleus forskohlii hairy root cultures were shown to biotransform methanol and ethanol to the corresponding β-D-glucopyranosides and β-D-ribo-hex-3-ulopyranosides, and 2-propanol to its β-D-glucopyranoside.

Full text of DOI:10.1016/S0008-6215(03)00011-9

Carbohydrate Research 338 (2003) 729–731
www.elsevier.com/locate/carres
Biotransformation of low-molecular-weight alcohols by Coleus
forskohlii hairy root cultures
Wei Li,a Kazuo Koike,a Yoshihisa Asada,b Takafumi Yoshikawa,b Tamotsu Nikaidoa,*
aFaculty of Pharmaceutical Sciences, Toho Uni6ersity, 2-2-1 Miyama, Funabashi-City, Chiba 274-8510, Japan
bSchool of Pharmaceutical Sciences, Kitasato Uni6ersity, 5-9-1, Minato-ku, Tokyo 108-8641, Japan
Received 29 October 2002; accepted 3 December 2002
Abstract
Coleus forskohlii hairy root cultures were shown to biotransform methanol and ethanol to the corresponding b-
D
-glucopyran-
osides and b-
D
-ribo-hex-3-ulopyranosides, and 2-propanol to its b-
D-glucopyranoside. © 2003 Elsevier Science Ltd. All rights
reserved.
Keywords: Biotransformation; Coleus forskohlii; Hairy root
Plant cell, root or hairy root cultures are considered
to be an important source of useful secondary metabo-
lites. Furthermore, the ability of some of the cultures to
specifically convert administered organic compounds
into useful analogues has been the subject of increasing
attention. The reactions involved in the biotransforma-
tion include oxidation, reduction, hydroxylation, ester-
ification, methylation, isomerization, and also
glycosylation, which occurs readily in plant cells but
with difficulty in microorganisms. Cultures with the
ability of glycosylation have been reported in some
plant cell, root or hairy root cultures, such as that of
Panax ginseng,1,2 Eucalyptus perriniana,3 Artemisia an-
nua,4 Coffea arabica,5 etc.
3 weeks in the same medium (120 mL), to a final
concentration of 1% (v/v). The cultures not adminis-
tered any alcohol were used as negative controls. The
hairy roots were further cultured for 1 week. Each
culture was separated into roots and medium by filtra-
tion through a filter paper. The methanol extracts of
the roots and the medium were analyzed by TLC (6:4:1
CHCl3–MeOH–H2O). Two biotransformation prod-
ucts (1, Rf 0.25 and 2, Rf 0.35) were found in the
methanol extracts of the roots administered methanol,
but none were found in the medium or negative con-
trols. Further separation of the extract by silica gel
open column (solvent as 60:29:6 CHCl3–MeOH–H2O)
gave products 1 (30.0 mg) and 2 (11.5 mg).
Coleus forskohlii hairy roots induced from Agrobac-
terium rhizogenes are reported to be a producer of
forskolin.6 Herein, we report the C. forskohlii hairy
roots also have the ability to biotransform low-molecu-
Product 1 revealed an [M+Na]+ ion peak at m/z
217.1 in the positive-ion ESIMS spectrum. The 1H
NMR spectrum showed a methoxy signal and a set of
b-glucopyranose signals, with the resonance for the
anomeric proton at l 4.16 (1 H, d, J 7.8 Hz). Further
comparison of the [h]D of 1 with that of the standard
lar-weight alcohols into the corresponding b-
D-glucopy-
ranosides or b- -ribo-hex-3-ulopyranosides.
D
The C. forskohlii hairy root cultures induced from A.
rhizogenes MAFF 03-01724 were subcultured on Gam-
borg B5 basal liquid medium7 at 4-week intervals.
Methanol was administered to hairy roots cultured for
compound showed that it is methyl b-D-
glucopyranoside.
* Corresponding author. Tel.: +81-47-4721161; fax: +81-
47-4721404
E-mail address: nikaido@phar.toho-u.ac.jp (T. Nikaido).
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00011-9

730
W. Li et al. / Carbohydrate Research 338 (2003) 729–731
tometer detector, was used. Column chromatography
was carried out using Kieselgel 60 (E. Merck). TLC was
conducted in Kieselgel 60 F254 plates (E. Merck).
Product 2 revealed an [M+Na]+ ion peak at m/z
215.2 in the positive-ion ESIMS spectrum, which was 2
1
mass units smaller than that of 1. Comparing the H
NMR spectrum of 2 with 1, H-3 of 2 disappeared, and
H-2 (l 4.10) and H-4 (l 4.22) showed a lower field shift
of about 1 ppm. The 13C NMR spectrum of 2 showed
a ketone signal at l 207.0. Further 2D NMR analysis
1.2. Culture methods and feeding experiments
The root cultures were established as described in a
previous paper.6 The root cultures were subcultured on
Gamborg B5 basal liquid medium7 containing 2% su-
crose at 25 °C in the dark at 60 rpm on a rotary shaker
at 4-week intervals. Methanol, EtOH or 2-propanol
(each 1.2 mL) was administered to hairy roots (approx
6 g, Fr. Wt.) cultured for 3 weeks in B5 liquid medium
(120 mL per flask), respectively. The cultures devoid of
any alcohol were used as negative controls. The hairy
roots were further cultured for 1 week.
suggested that 2 is a methyl D-ribo-hex-3-ulopyranoside
(the 3-keto derivative of 1). Reduction of 2 with NaBH4
afforded two products, of which one product (2a) was
identical with 1 by its NMR spectrum and [h]D data.
The other product (2b) was characterized as the methyl
b-
D
-allopyranoside. All these data show 2 to be methyl
-ribo-hex-3-ulopyranoside.
b-
D
Although the a-form is known to be thermodynami-
cally more stable than the b-form of methyl -glucopy-
D
ranoside, we could not find the a-glucoside as a
product. The result shows that the biotransformation
was an enzyme-catalyzed reaction.
1.3. Extraction and separation procedures of biotrans-
formation products
With the same experimental method, ethanol was
administered to C. forskohlii hairy root cultures, similar
Each culture was separated into roots and medium by
filtration through a filter paper. The roots were ex-
tracted with MeOH under ultrasonic treatment three
times for 1 h each at room temperature. The MeOH
extracts and the media were analyzed by TLC (6:4:1
CHCl3–MeOH–H2O). Two biotransformation prod-
ucts were found in the methanolic extract of roots
administered MeOH and EtOH, whereas only one
product was found in the methanolic extract of roots
administered 2-propanol. No product was found in the
media or negative controls. Further separation of each
extract by silica gel open-column chromatography gave
products 1 (30.0 mg, Rf 0.25 on TLC) and 2 (11.5 mg,
Rf 0.35) from the MeOH extract of roots administered
MeOH, products 3 (23.4 mg, Rf 0.32) and 4 (4.6 mg, Rf
0.43) from the methanolic extract of roots administered
EtOH, and product 5 (15.0 mg, Rf 0.38) from the
methanolic extract of roots administered 2-propanol.
to the case of methanol, and ethyl b-
(3, 23.4 mg) and ethyl b- -ribo-hex-3-ulopyranoside (4,
4.6 mg) were isolated as biotransformation products.
D-glucopyranoside
D
When 2-propanol was administered, 2-propyl b-D-
glucopyranoside (5, 15.0 mg) was isolated as the bio-
transformation product. The lack of 2-propyl
b-D-ribo-hex-3-ulopyranoside as a product is probably
due to the higher toxicity of 2-propanol to the hairy
roots.
b-D-ribo-Hex-3-ulopyranosides rarely exist in the
plant kingdom and have only been reported as
iridoid8–10 and cardenolide11,12 glycosides. Interestingly,
it has been reported that b-D-glucopyranose-3-oxidase
was present in Agrobacterium tumeffaciens, which be-
longs to the same genus with A. rhizogenes used to
induce the hairy roots.13 We are now working to deter-
mine whether the b-D-glucopyranose-3-oxidase activity
present in the hairy roots is due to the fact that the
genome of this enzyme is transferred into C. forskohlii
hairy roots from A. rhizogenes.
1.4. Methyl b-D-glucopyranoside (1)
Powder, [h]2D4 −33.7° (c 1.03, MeOH). ESIMS (posi-
1
tive-ion) m/z: 217.1 [(M+Na)+]. H NMR (500 MHz,
MEOH-d4): l 3.15 (1 H, dd, J 9.1, 7.8 Hz, H-2), 3.26 (1
H, m, H-5), 3.27 (1 H, dd, J 9.1, 9.1 Hz, H-4), 3.34 (1
H, dd, J 9.1, 9.1 Hz, H-3), 3.52 (3 H, s, CH3), 3.66 (1
H, dd, J 11.9, 5.2 Hz, Ha-6), 3.86 (1 H, dd, J 11.9, 2.1
Hz, Hb-6), 4.16 (1 H, d, J 7.8 Hz, H-1). 13C NMR (125
MHz, MeOH-d4): l 57.3 (CH3), 62.8 (C-6), 71.7 (C-4),
75.0 (C-2), 77.9 (C-5), 78.1 (C-3), 105.4 (C-1).
1. Experimental
1.1. General methods
Optical rotations were measured with a JASCO DIP-
370 digital polarimeter in a 0.5-dm cell. The ESIMSs
were taken on an LCQ mass analyzer. The H and 13C
1
NMR spectra were measured with a JEOL ECP-500
spectrometer in MeOH-d4 solution with Me4Si as the
internal reference, and chemical shifts are expressed in
l (ppm). For HPLC, a JASCO PU-2080 HPLC system,
equipped with a Shodex RI-101 Differential Refrac-
1.5. Methyl b-D-ribo-hex-3-ulopyranoside (2)
Powder, [h]2D4 −27.8° (c 0.76, MeOH). ESIMS (posi-
1
tive-ion) m/z: 215.2 [(M+Na)+]. H NMR (500 MHz,
MeOH-d4): l 3.31 (1 H, ddd, J 10.1, 4.9, 2.1 Hz, H-5),

W. Li et al. / Carbohydrate Research 338 (2003) 729–731
731
3.58 (3 H, s, CH3), 3.79 (1 H, dd, J 12.1, 4.9 Hz, Ha-6),
3.94 (1 H, dd, J 12.1, 2.1 Hz, Hb-6), 4.10 (1 H, dd, J
7.9, 1.8 Hz, H-2), 4.22 (1 H, dd, J 10.1, 1.8 Hz, H-4),
4.27 (1 H, d, J 7.9 Hz, H-1). 13C NMR (125 MHz,
MeOH-d4): l 57.5 (CH3), 62.5 (C-6), 73.6 (C-4), 78.2
(C-5), 78.3 (C-2), 106.8 (C-1), 207.0 (C-3).
13C NMR (125 MHz, MeOH-d4): l 15.4 (CH3), 62.6
(C-6), 66.5 (CH2), 73.7 (C-4), 78.4 (C-2 and C-5), 105.6
(C-1), 207.2 (C-3).
1.9. 2-Propyl b-D-glucopyranoside (5)
NaBH4 (5.0 mg) was added to a solution of 2 (4.8
mg) in EtOH (1 mL), and the mixture was stirred at
room temperature for 30 min. The reaction solution
was neutralized by adding Dowex 50W-X8 (H+) and
then filtered through a filter paper to remove the
Dowex. Further separation by HPLC (H2O as solvent)
gave two products (2a, 0.8 mg and 2b, 0.8 mg). Com-
pound 2a showed the same Rf value on TLC and tR on
HPLC and was identical with 1 by its NMR spectrum
and [h]D data.
Powder, [h]2D4 −34.4° (c 1.00, MeOH). ESIMS (posi-
1
tive-ion) m/z: 245.2 [(M+Na)+]. H NMR (500 MHz,
MeOH-d4): l 1.18 (3 H, d, J 6.2 Hz, CH3), 1.22 (3 H,
d, J 6.2 Hz, CH3), 3.13 (1 H, dd, J 9.0, 7.8 Hz, H-2),
3.25 (1 H, m, H-5), 3.27 (1 H, dd, J 9.2, 9.2 Hz, H-4),
3.34 (1 H, dd, J 9.0, 9.2 Hz, H-3), 3.65 (1 H, dd, J 11.9,
5.5 Hz, Ha-6), 3.84 (1 H, dd, J 11.9, 2.3 Hz, Hb-6), 4.03
(1 H, m, CH), 4.25 (1 H, d, J 7.9 Hz, H-1). 13C NMR
(125 MHz, MeOH-d4): l 22.1 (CH3), 23.8 (CH3), 62.9
(C-6), 71.8 (C-4), 72.6 (CH), 75.2 (C-2), 77.9 (C-5), 78.2
(C-3), 102.6 (C-1).
1.6. Methyl b-D-allopyranoside (2b)
Powder, [h]2D4 −40.0° (c 0.07, MeOH). ESIMS (posi-
Acknowledgements
1
tive-ion) m/z: 217.1 [(M+Na)+]. H NMR (500 MHz,
MeOH-d4): l 3.27 (1 H, dd, J 8.0, 3.0 Hz, H-2), 3.47 (1
H, dd, J 9.6, 3.0 Hz, H-4), 3.51 (3 H, s, CH3), 3.65 (1
H, dd, J 11.3, 5.7 Hz, Ha-6), 3.68 (1 H, ddd, J 9.6, 5.7,
2.1 Hz, H-5), 3.84 (1 H, dd, J 11.3, 2.1 Hz, Hb-6), 4.51
(1 H, d, J 8.0 Hz, H-1). 13C NMR (125 MHz, MeOH-
d4): l 57.2 (CH3), 63.2 (C-6), 69.1 (C-4), 72.4 (C-3), 72.9
(C-2), 75.5 (C-5), 103.0 (C-1).
The authors wish to thank Dr Koichiro Shimomura
(Tsukuba Medicinal Plant Research Station, National
Institute of Health Sciences) for C. forskohlii hairy root
strain.
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1.7. Ethyl b-D-glucopyranoside (3)
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1
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Hb-6), 3.95 (1 H, dd, J 9.5, 7.1 Hz, CH2), 4.25 (1 H, d,
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1.8. Ethyl b- -ribo-hex-3-ulopyranoside (4)
D
Powder, [h]2D4 −55.8° (c 0.45, MeOH). ESIMS (posi-
1
tive-ion) m/z: 229.2 [(M+Na)+]. H NMR (500 MHz,
MeOH-d4): l 1.25 (3 H, dd, J 7.3, 7.3 Hz, CH3), 3.30
(overlapped by MeOH-d4, H-5), 3.67 (1 H, dd, J 9.6,
7.3 Hz, CH2), 3.78 (1 H, dd, J 12.4, 5.0 Hz, Ha-6), 3.92
(1 H, dd, J 12.4, 2.3 Hz, Hb-6), 3.99 (1 H, dd, J 9.6, 7.3
Hz, CH2), 4.10 (1 H, dd, J 7.8, 1.8 Hz, H-2), 4.21 (1 H,
dd, J 10.1, 1.8 Hz, H-4), 4.36 (1 H, d, J 7.8 Hz, H-1).