Biotransformation of benzaldehyde-type and acetophenone-type derivatives by Pharbitis nil hairy roots

Article abstract of DOI:10.1248/cpb.53.361

The glucosylation of some coumarin and flavone derivatives on incubation with the hairy roots of morning glory (Pharbitis nil) was previously reported. We further studied the biotransformation of benzaldehyde- and acetophenone-type derivatives. Vanillin a

Full text of DOI:10.1248/cpb.53.361

April 2005
Chem. Pharm. Bull. 53(4) 361—365 (2005)
361
Biotransformation of Benzaldehyde-Type and Acetophenone-Type
Derivatives by Pharbitis nil Hairy Roots
Hideki KANHO,a Sayaka YAOYA,a Nobuo KAWAHARA,b Takahisa NAKANE,c Yoichi TAKASE,c
c
,a
*
Kazuo MASUDA, and Masanori KUROYANAGI
a School of Bioresources, Hiroshima Prefectural University; 562 Nanatsuka, Shobara, Hiroshima 727–0023, Japan:
b National Institute of Health Sciences; 1–18–1 Kamiyoga, Setagaya, Tokyo 158–8501, Japan: and c Showa Pharmaceutical
University; 3–3165 Higashi-Tamagawagakuen, Machida, Tokyo 194–8543, Japan.
Received October 13, 2004; accepted December 28, 2004
The glucosylation of some coumarin and flavone derivatives on incubation with the hairy roots of morning
glory (Pharbitis nil) was previously reported. We further studied the biotransformation of benzaldehyde- and
acetophenone-type derivatives. Vanillin and isovanillin were reduced to alcoholic derivatives and glucosylated at
the phenolic and the alcoholic hydroxyl groups. In the case of 3,4-dihydroxybenzaldehyde, the formyl group was
reduced and the 3-hydroxyl or 4-hydroxyl groups were glucosylated to give monoglucosides. The 3-hydroxyl
group was predominantly glucosylated to the 4-hydroxyl group. 4-b-D-Glucopyranosyloxy-3-methoxybenzylalco-
hol was obtained in low yield. In time-course experiments with vanillin, it was found that the high-level reduction
of the formyl group and glucosylation of the phenolic hydroxyl group occurred, and finally 4-O-b-D-glucopyra-
nosylvanillylalcohol was obtained as the main product. In the case of 3,4-dimethoxybenzaldehyde, 3,4,5-
trimethoxybenzaldehyde, and salicylaldehyde, the formyl groups were reduced, and then the hydroxyl groups at
the benyl position were glucosylated to give alcoholic glucosides in relatively high yields. In 4-hydroxy-3-
methoxyacetophenone, the 4-hydroxyl group was glucosylated and two dimerized glucosides, biphenyl and
biphenylether types, were obtained in low yields. In acetophenone, 1-b-D-glucopyranosyloxy-1-phenylethane and
2-b-D-glucopyranosyloxyacetophenone were obtained. As mentioned above P. nil hairy roots showed various bio-
transformative activities including glucosylation of phenolic and benzylic hydroxyl groups, reduction of the
formyl group near the benzene ring, and phenol oxidation dimerization. The glucosylation reaction was espe-
cially interesting for the production of valuable glucosides.
Key words Pharbitis nil hairy root; glucosylation; reduction; benzaldehyde derivative; acetophenone deivative
Hairy roots induced by infection with Agrobacterium rhi- group of these compounds.
zogenes are differentiated cells capable of the biosynthesis of
secondary metabolites.1—3) Hairy roots also have a high rate Results and Discussion
of proliferation. They are therefore expected to become
Benzaldehyde-Type Compounds One of the most pop-
widely used culture systems for the production of useful sec- ular benzaldehyde derivatives, vanillin (1) (100 mg), was in-
ondary metabolites4) and for studies on the production of bio- cubated with the hairy root culture (5 flasks with 200 ml of
logically active compounds and transformation of organic 1/2 MS medium) for 5 d and gave the products 2 (3 mg), 3
substrates.5) We reported the production of a stress com- (25 mg), and 5 (8 mg) (obtained as pure compounds after pu-
pound, umbelliferone, upon treatment of morning glory rification with preparative TLC and HPLC). Vanilyl alcohol
(Pharbitis nil) hairy roots with CuSO4 and that umbellifer- (4) (100 mg) was also incubated with the hairy root culture to
one was highly glucosylated to a glucoside, skimmin, by P. give 3 (28 mg) and 5 (6 mg). Isovanillin (6) gave 7 (23 mg).
nil hairy roots.6) We further reported the glucosylation of Isovanillyl alcohol (8) gave 7 (28 mg) and 9 (5 mg). 3,4-Di-
phenolic compounds, such as coumarin and flavone deriva- hydroxybenzaldehyde (10) gave 11 (26 mg), 12 (3 mg), and 3
tives by the hairy roots.7) Many types of secondary metabo- (2 mg). 3,4-Dimethoxybenzaldehyde (13) gave 14 (22 mg)
lites exist as glycosides such as saponines,8) terpene glyco- and 15 (67 mg). 3,4,6-Trimehoxybenzaldehyde (16) gave 17
sides,9) flavone glycosides,10) and other phenolic glycosides. (56 mg). Salicylaldehyde (18) gave 19 (35 mg) and 20
Some glycosides exist as prebioactive specimens,11) detoxi- (50 mg).
cated specimens,12) and bioactive specimens. Thus the bio-
The fast-atom bombardment (FAB)-MS, 1H- and 13C-
transformation of various organic substrates to glycosides by NMR spectral data (see Experimental) showed that 2, 3, 5, 7,
plant cell cultures should be useful for supplying glucosides. and 9 were monoglucosides, an that 2 had a formyl group but
We are also interested in the glucosylation of other simple 3, 5, 7 and 9 had oxymethyl groups. These results indicated
phenolic compounds. Vanillin is a common natural product that 2 was vanillin-4-O-b-D-glucopyranoside and 3, 5, 7, and
with a benzaldehyde skeleton. The 3- and 4-positions of ben- 9 were glucosides of which the formyl groups were reduced.
zaldehyde and acetophenone derivatives correspond to the 6- The position of the glucopyranosyl groups was determined
and 7-positions of coumarin derivatives. Thus the biotrans- from the heteronuclear multiple-bond correlation (HMBC)
formation of benzaldehyde- and acetophenone-type deriva- spectra. The structures of 3, 5, 7, and 9 were therefore deter-
tives were carried out using P. nil hairy roots.
mined to be vanillyl alcohol-4-O-b-D-glucopyranoside, vanil-
This report deals with the glucosylation of benzaldehyde- lyl alcohol-7-O-b-D-glucopyraniside, isovanillyl alcohol-3-
and acetophenone-type derivatives having phenolic hydroxyl O-b-D-glucopyranoside, and isovanillyl alcohol-7-O-b-D-glu-
groups or not, and the reduction of a formyl and a carbonyl copyranoside, respectively.
∗ To whom correspondence should be addressed. e-mail: kuroyang@bio.hiroshima-pu.ac.jp
© 2005 Pharmaceutical Society of Japan

362
Vol. 53, No. 4
The FAB-MS, 1H- and 13C-NMR spectral data (see the Ex-
perimental) indicated that 11, 12, 15, 17, 19, and 20 were
monoglucosides with oxymethyl groups. Their glucopyranoyl
group positions were determined from the HMBC spectrum.
The structures of 11, 12, 15, 17, 19, and 20 were therefore
determined to be 3,4-dihydroxybenzyl alcohol-3-O-b-D-gluco-
pyranoside, 3,4-dihydroxybenzyl alcohol-4-O-b-D-glucopyrano-
side, 3,4-dimethoxybenzyl alcohol-7-O-b-D-glucopyranoside,
3,4,5-trimethoxybenzyl alcohol-7-O-b-D-glucopyranoside,
salicyl alcohol-2-O-b-D-glucopyranoside, and salicyl alcohol-
7-O-b-D-glucopyrsnoside, respectively. The structure of 14
was determined to be 3,4-dimethoxybenzyl alcohol based on
the spectral data (see Experimental).
The hairy roots had good ability to reduce a formyl group
of benzaldehyde derivatives to a hydroxymethyl group. The
glucosylation of phenolic hydroxyl groups of benzaldehyde
derivatives and reduced benzyl alcohol derivatives was also
easily carried out, but the primary alcoholic hydroxyl groups
were only slightly glucosylated by the hairy roots. Thus, in
the case of benzaldehyde with phenolic hydroxyl groups, the
final main product is benzyl alcohol derivatives with O-b-D-
glucopyranosyl groups at a phenolic hydroxyl group. But in
the case of benzaldehyde derivatives lacking a phenolic hy-
droxyl group, the main product is a benzyl alcohol derivative
with O-b-D-glucopyranosyl groups at the alcoholic hydroxyl
groups. Transformation of benzaldehyde-type derivatives and
isolation yields of products are shown in Fig. 1.
Acetophenone Derivatives 4-Hydroxy-3-methoxyace-
tophenone (21) gave 22 (12 mg), 23 (3 mg), and 24 (3 mg).
Acetophenone (25) gave 26 (9 mg) and 27 (5 mg). The spec-
tral data of 22 showed that it is a monoglucoside of 21. Thus
the structure of 22 was determined to be 4-hydroxy-3-
methoxyacetophenone-4-O-b-D-glucopyranoside.
Fig. 1. Transformation of Benzaldehyde-Type Derivatives by Pharbitis nil
Hairy Roots and Isolation Yields of Products
The FAB-MS of 23 showed a pseudomolecular ion at m/z
515 [MꢀNa]ꢀ for C24H28O11Na, and m/z 493 [MꢀH]ꢀ for
1
C24H29O11. The H-NMR spectrum of 23 showed the pres-
ence of two acetyl groups [d 2.47 (3H, s), 2.54 (3H, s)], two
methoxyl groups [d 3.89 (3H, s), 3.92 (3H, s)], five aromatic
H [d 6.90 (1H, d, Jꢁ8.5 Hz), 7.16 (1H, d, Jꢁ2.0 Hz), 7.44
(1H, d, Jꢁ2.0 Hz), 7.57 (1H, dd, Jꢁ8.5, 2.0 Hz), 7.63 (1H, d,
Jꢁ2.0 Hz)], and an b-D-glucopyranosyl moiety [d 3.55 (1H,
dd, Jꢁ11.5, 5.5 Hz, H-6ꢂ), 3.67 (1H, dd, Jꢁ11.5, 2.5 Hz, H-
6ꢂ), 5.17 (1H, d, Jꢁ7.5 Hz, H-1ꢂ)]. The 13C-NMR data of 23
also showed the presence of two acetyl groups (d 26.4, 26.5,
198.6, 199.2), two methoxyl groups (d 56.6, 57.0), 12 aro-
matic carbons (d 109.6, 113.0, 114.4, 118.9, 124.0, 134.3,
134.4, 142.4, 150.2, 151.4, 151.8, 154.9), and a glucopyra-
nosyl moiety (d 62.4, 71.2, 75.6, 77.7, 78.3, 103.9). These
data indicated that 23 was a dimeric glucoside derivative of
21. The coupling patterns of the aromatic H showed the pres-
ence of 1,2,4-trisubstituted and 1,2,3,5-tetrasubstituted ben-
zene rings and a biphenyl ether structure. Dimeric and gluco-
sylated positions of 23 were determined from the HMBC ex-
periments of 23 (see Fig. 2). The structure of 23 was there-
fore determined to be 5,4ꢃ-diacetyl-3,2ꢃ-dimethoxy-2-b-D-
glucopyranosyloxybiphenyl ether.
Fig. 2. Significant HMBC Correlations of Dimeric Derivatives 23 and 24
pled aromatic H at d 7.49 (1H, d, Jꢁ2.0 Hz), 7.53 (1H, d,
Jꢁ2.0 Hz), 7.41 (1H, d, Jꢁ2.0 Hz), and 7.71 (1H, d,
Jꢁ2.0 Hz). This indicated that 24 had a biphenyl structure
from the dimerization of 21. The dimeric and glucosy posi-
tions of 24 were determined from the HMBC experiments
(see Fig. 2). The structure of 24 was therefore determined to
be 5,5ꢃ-diacetyl-3,3ꢃ-dimethoxy-2-b-D-glucopuranosyloxy-
2ꢃ-hydroxybiphenyl.
The dimeric products 23 and 24 should be produced from
21 through a phenol oxidative coupling reaction13) followed
by glucosylation. The dimeric positions are consistent with a
phenol oxidative coupling reaction. The phenol oxidative
coupling reaction is important for the formation of some
types of natural products,14) such as lignins, lignans, tannins,
stilbene polymers, etc. These reactions are catalyzed by ox-
idative enzymes, such as catalase, peroxidase, laccase, and
tyrosinase, and some types of nonenzymatic oxidation. Com-
pound 21 was not dimerized and recovered from the medium
after incubation of 21 with the medium without the hairy
The FAB-MS of 24 showed a pseudo molecular ion at m/z
515 [MꢀNa]ꢀ C24H28O11Na, and m/z 493 [MꢀH]ꢀ for
1
C24H29O11. The H- and 13C-NMR spectral data (see Experi-
mental) for 24 showed that 24 is also a dimeric glucoside of
21. Product 24 showed the presence of two pairs of m-cou-

April 2005
363
Fig. 3. Transformation of Acetophenone-Type Derivatives by Pharbitis nil Hairy Roots and Isolation Yields of Products
roots. Thus the formation of 23 and 24 from 21 should be
carried out through a biologic process via dimerization in the
presence of hairy roots in the culture medium.
FAB-MS, 1H-, and 13C-NMR data for 26 showed that 26 is
a glucopyranoside of the reduced product of 25. The struc-
ture of 26 was therefore determined to be 1-b-D-glucopyra-
nosyloxy-1-phenylethane. The structure of 27 was assumed
to be 2-b-D-glucopyranosyloxyacetophenone based on the
1H- and 13C-NMR data and comparison with the authentic
data.7) Transformation of acetophenone-type derivatives and
isolation yields of products are shown in Fig. 3.
Time Course of Vanillin (1) Transformatioin by P. nil
Hairy Roots The amount of vanillin (1) incubated with the
hairy root culture decreased to zero in a short time (in 6 h) in
the medium and vanillyl alcohol (4) appeared a few hours
after the addition of 1 to the hairy root culture. The level of 4
in the medium reached a maximum at 12 h and decreased
thereafter. In the hairy roots, the level of vanillin-4-O-b-D-
glucopyranoside (2) increased, peaking 6 h after the addition,
and then decreased. On the other hand, the level of vanillyl
alcohol-4-O-b-D-glucopyranoside (3) in the hairy roots in-
creased with the the decrease in the level of 2 and 4. The
level of vanillyl alcohol-7-O-b-D-glucopyranoside (5) also
changed the same as that of 3 (see Fig. 4). Glucosides, 2, 3,
and 5 were not identified in the medium. These results indi-
cate that the reduction of the formyl group and glucosylation
at C-4 of 1 were carried out to give 4 and 2 first. O-Glucosy-
lation of 4 and reduction of 2 continuously gave 3 as the
main product. Some of 4 was glucosylated at C-7 to give 5.
Aglycons 1 and 4 were transferred through the cell mem-
brane but glucosides 2, 3, and 5 were not transferred through
the cell membrane from the hairy roots to the medium. The
reduction of a formyl group of 1 was carried out at the same
level as glucosylation at C-4 of 1 (see Fig. 5). In the time-
course experiment, production of 4 was observed, but 4 could
not be isolated from the incubation experiment on 1 because
of overlaping with constituents of the hairy roots in a prepar-
ative TLC.
Fig. 4. Time-Course of Transformation of Vanillin (1) by Pharbitis nil
Hairy Root Culture
After incubation of 1 with the hairy root culture, amounts of transformed products,
vanillyl alcohol (4), vanillin 4-O-b-D-glucopyranoside (2), vanillyl alcohol 4-O-b-D-
glucopyranoside (3), vanillyl alcohol 7-O-b-D-glucopyranoside (5), and
1 in the
medium (MD) and the hairy roots (HR) were determined. Glucosides, 2, 3, and 5, were
not identified in the medium and 1 was not identified in the hairy roots on TLC detec-
tion. Trace 2 was identified in the medium 120 h after incubation.
Fig. 5. Biotransformation of Vanillin (1) by Pharbits nil Hairy Roots via
Vanillyl Alcohol (4) and Vanillin 4-O-b-D-Glucopyranoside (2)
Reduction and glucosilation reactions of 1 were took place in similar level by incuba-
tion with the hairy roots. Aglycones 1 and 4 were transferred through the cell mem-
brane but glucosides 2, 3, and 5 were not.
volved, glucosylation was especially interesting for the bio-
transformation of phenolic compounds to new types of glu-
cosides. Isolation yields of the glucosides should be im-
proved by optimization of the incubation conditions and iso-
lation procedurs. The substrate specificity of the biotransfor-
mation of further phenolic compounds must be investigated
for actual application. We are studying cloning of glucosyl-
transferase genes and their expression to clarify the substrate
specificity of the enzyme.
P. nil hairy roots showed not only a potent ability to gluco-
sylate the phenolic hydroxyl group but also to reduce the
formyl group on the aromatic ring along with an ability to
glucosylate the alcoholic hydroxyl group at the benzyl posi-
tion, methylation of the phenolic hydroxyl group, and an ox-
idative phenol coupling reaction. In the case of acetophe-
none, reduction of the carbonyl group and hydroxylation at
the neighboring position of the acetyl group were observed.
Of these various reactions in which the hairy roots were in-

364
Vol. 53, No. 4
Experimental
135.8 (C-1), 147.9 (C-3), 150.1 (C-4).
General Experimental Procedures FAB-MS was measured on a JEOL
JMS-AX505F mass spectrometer using m-nitrobenzyl alcohol as a matrix.
1H- and 13C-NMR spectra were measured on a JEOL a-500 (500 MHz for
1H and 125 MHz for 13C-NMR spectrometer including NOE, HMQC, and
HMBC experiments. Chemical shifts are give as d-values (ppm) with 3.24 (1H, overlap, H-5ꢃ), 3.28 (1H, t, Jꢁ8.0 Hz, H-3ꢃ), 3.32 (1H, t,
tetramethylsilane (TMS) as an internal standard. Analytical and semiprepar- Jꢁ9.0 Hz, H-2ꢃ), 3.68 (1H, dd, Jꢁ12.0, 5.5 Hz, H-6ꢃ), 3.83 (3H, s, MeO),
Incubation of Isovanillyl Alcohol (8) The MeOH eluate from the P. nil
hairy roots incubated with 100 mg of 8 gave 7 (28 mg) and 9 (5 mg). FAB-
MS: m/z, 339 [MꢀNa]ꢀ C14H20O8Na, m/z 317 [MꢀH]ꢀ C14H21O8, m/z 316
1
[M]ꢀ C14H20O8. H-NMR (CD3OD) d: 3.21 (1H, dd, Jꢁ9.0, 8.5 Hz, H-4ꢃ),
ative HPLC was carried out using a reverse-phase (ODS, Mightysil RP-18
GP packed column, Kanto Chemical Co.) column. TLC was carried out
using precoated silica gel 60F254 (Merck) 200ꢄ200ꢄ0.25-mm plates for
analysis and 200ꢄ200ꢄ0.5-mm plates for the preparation of products.
Chemicals Vanillin was purchased from Kanto Chemical Co. iso-
3.88 (1H, dd, Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.31 (1H, d, Jꢁ8.0 Hz, H-1ꢃ), 4.54 (1H,
d, Jꢁ11.0 Hz, H-7), 4.78 (1H, d, Jꢁ11.0 Hz, H-7), 6.84 (1H, dd, Jꢁ8.0,
1.5 Hz, H-6), 6.87 (1H, d, Jꢁ8.0 Hz, H-5), 6.88 (1H, d, Jꢁ1.5 Hz, H-2). 13C-
NMR (CD3OD) d: 56.3 (MeO), 62.8 (C-6ꢃ), 71.5 (C-4ꢃ), 71.7 (C-7), 75.1
(C-2ꢃ), 77.9 (C-3ꢃ), 78.1 (C-5ꢃ), 102.8 (C-1ꢃ), 112.3 (C-5), 116.6 (C-2),
vanillin, vanillyl alcohol, isovanillyl alcohol, and salicylaldehyde were 121.0 (C-6), 131.7 (C-1), 147.4 (C-3), 148.7 (C-4).
obtained from Aldrich Chemical. 3,4,5-Trimethoxybenzaldehyde, 3,4-
Incubation of 3,4-Dihydroxybenzaldehyde (10) The MeOH eluate
dimethoxybenzaldehyde, acetophenone, and 4-hydroxy-3-methoxyacetophe- from the P. nil hairy roots incubated with 100 mg of 10 gave 11 (26 mg), 12
none were purchased from Tokyo Kasei Kogyo Co.
(3 mg), and 3 (2 mg). 11: FAB-MS: m/z 325 [MꢀNa]ꢀ C13H18O8Na. 1H-
Hairy Root Culture, Incubation of Substrates, and Purification of NMR (CD3OD) d: 3.40 (1H, t, Jꢁ9.5 Hz, H-4ꢃ), 3.41 (1H, over lap, H-5ꢃ),
Products The induction of hairy roots of P. nil by inoculation of A. rhizo- 3.46 (1H, t, Jꢁ9.0 Hz, H-3ꢃ), 3.49 (1H, t, Jꢁ9.0 Hz, H-2ꢃ), 3.72 (1H, dd,
genes (MAFF 03-1725) was described in a previous paper.6) The hairy roots
were subcultured every 4 weeks in 1/2 MS medium, supplemented with 3%
sucrose. A 100-mg aliquot of each substrate was incubated with the 3-week-
Jꢁ2.5, 5.0 Hz, H-6ꢃ), 3.90 (1H, dd, Jꢁ12.5, 2.0 Hz, H-6ꢃ), 4.48 (2H, s, H-7),
4.77 (1H, d, Jꢁ7.5 Hz, H-1ꢃ), 6.81 (1H, d, Jꢁ7.5 Hz, H-5), 6.91 (1H, dd,
Jꢁ7.5, 2.0 Hz, H-6), 7.20 (1H, d, Jꢁ2.0 Hz, H-2). 13C-NMR (CD3OD) d:
old hairy root culture (five flasks including 200 ml of 1/2 MS medium and 62.6 (C-6ꢃ), 65.1 (C-7), 71.4 (C-4ꢃ), 75.0 (C-2ꢃ), 77.7 (C-3ꢃ), 78.4 (C-5ꢃ),
20 mg of substrate/flask) for 5 d. After incubation, the culture medium was
filtered to give the hairy roots and medium. The hairy roots were lyophilized,
mashed, and extracted with boiling water. The extract was passed through a
104.5 (C-1ꢃ), 117.0 (C-5), 118.2 (C-2), 123.8 (C-6), 134.5 (C-1), 146.7 (C-
3), 147.9 (C-4). 12: FAB-MS: m/z 325 [MꢀNa]ꢀ C13H18O8Na, m/z 303
[MꢀH]ꢀ C13H19O8. 1H-NMR (CD3OD) d: 3.39 (2H, overlap, H-4ꢃ, 5ꢃ), 3.45
HP 20 (Mitsubishi Chemical) column, washed thoroughly with water, and (1H, t, Jꢁ9.0 Hz, H-3ꢃ), 3.48 (1H, Jꢁ9.0 Hz, H-2ꢃ), 3.72 (1H, dd, Jꢁ12.0,
eluted with MeOH to give a MeOH eluate. The MeOH eluate was analyzed
using TLC and HPLC and purified by means of semipreparative HPLC and
TLC to give glucosylated products.
5.0 Hz, H-6ꢃ), 3.89 (1H, dd, Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.73 (1H, d, Jꢁ8.5 Hz,
H-1ꢃ), 4.48 (2H, s, H-7), 6.75 (1H, dd, Jꢁ8.5, 2.0 Hz, H-6), 6.85 (1H, d,
Jꢁ2.0 Hz, H-2), 7.14 (1H, d, Jꢁ8.5 Hz, H-5). 13C-NMR (CD3OD) d: 62.5
(C-6ꢃ), 65.0 (C-7), 71.4 (C-4ꢃ), 75.0 (C-2ꢃ), 77.7 (C-3ꢃ), 78.4 (C-5ꢃ), 104.7
The culture medium was passed through an HP 20 column, washed thor-
oughly with water, and eluted with MeOH to give the MeOH eluate. The (C-1ꢃ), 116.1 (C-2), 118.9 (C-5), 119.4 (C-6), 138.6 (C-1), 146.2 (C-4),
MeOH eluate was analyzed for the presence of residual substrate and prod-
ucts.
Incubation of Vanillin (1) The MeOH eluate from the hairy roots incu-
bated with 100 mg of 1 gave pure products 2 (3 mg), 3 (25 mg), and 5
148.8 (C-3).
Incubation of 3,4-Dimethoxybenzaldehyde (13) The MeOH eluate
from the P. nil hairy roots incubated with 100 mg of 13 gave 14 (22 mg) and
15 (67 mg). 14: FAB-MS: m/z 169 [MꢀH]ꢀ C9H13O5, m/z 168 [M]ꢀ
(8 mg). 2: FAB-MS: m/z 337 [MꢀNa]ꢀ C14H18O9Na, m/z 315 [MꢀH]ꢀ C9H12O3. 1H-NMR (CD3OD) d: 3.80 (3H, s, MeO), 3.83 (3H, s, MeO), 4.52
C14H19O8. 1H-NMR (CD3OD) d: 3.40 (1H, dd, Jꢁ9.5, 9.0 Hz, H-4ꢃ), 3.48 (2H, s, H-7), 6.87 (1H, dd, Jꢁ8.0, 1.5 Hz, H-6), 6.90 (1H, d, Jꢁ8.0 Hz, H-
(1H, t, Jꢁ9.5 Hz, H-3ꢃ), 3.48 (1H, ddd, Jꢁ9.0, 6.0, 2.0 Hz, H-5ꢃ), 3.54 (1H, 5), 6.97 (1H, d, Jꢁ1.5 Hz, H-2). 13C-NMR (CD3OD) d: 56.4 (MeO), 56.5
dd, Jꢁ9.5, 8.0 Hz, H-2ꢃ), 3.69 (1H, dd, Jꢁ11.5, 6.0 Hz, H-6ꢃ), 3.88 (1H, dd, (MeO), 65.1 (C-7), 112.2 (C-5), 112.8 (C-2), 120.7 (C-6), 135.6 (C-1),
Jꢁ11.5, 2.0 Hz, H-6ꢃ), 3.91 (3H, s, MeO), 5.06 (1H, d, Jꢁ7.5 Hz, H-1ꢃ), 149.8 (C-4), 150.5 (C-3). 15: FAB-MS: m/z 330 [M]ꢀ C15H22O8. H-NMR
1
7.31 (1H, d, Jꢁ8.5 Hz, H-5), 7.50 (1H, d, Jꢁ1.5 Hz, H-2), 7.52 (1H, dd, (CD3OD) d: 3.22 (1H, dd, Jꢁ9.0, 8.0 Hz, H-4ꢃ), 3.24 (1H, over lap, H-5ꢃ),
Jꢁ8.5, 1.5 Hz, H-6), 9.83 (1H, s, H-7). 13C-NMR (CD3OD) d: 56.6 (MeO),
62.4 (C-6ꢃ), 71.2 (C-4ꢃ), 74.7 (C-2ꢃ), 77.8 (C-3ꢃ), 78.3 (C-5ꢃ), 101.7 (C-1ꢃ),
116.5 (C-5), 111.7 (C-2), 126.9 (C-6), 132.8 (C-1), 151.2 (C-4), 153.5 (C-3),
3.27 (1H, t, Jꢁ8.5 Hz, H-3ꢃ), 3.32 (1H, t, Jꢁ8.0 Hz, H-2ꢃ), 3.67 (1H, dd,
Jꢁ12.0, 6.0 Hz, H-6ꢃ), 3.81 (3H, s, MeO), 3.83 (3H, s, MeO), 3.89 (1H, dd,
Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.30 (1H, d, Jꢁ7.5 Hz, H-1ꢃ), 4.61 (1H, d,
Jꢁ11.5 Hz, H-7), 4.84 (1H, d, Jꢁ11.5 Hz, H-7), 6.89 (1H, d, Jꢁ8.0 Hz, H-
192.9 (C-7). 3: FAB-MS: m/z 339 [MꢀNa]ꢀ C14H20O8Na, m/z 317 [MꢀH]ꢀ
1
C14H21O8. H-NMR (CD3OD) d: 3.38 (2H, overlap, H-2ꢃ, 5ꢃ), 3.45 (1H, t, 6), 6.93 (1H, dd, Jꢁ8.0, 2.0 Hz, H-6), 7.08 (1H, d, Jꢁ2.0 Hz, H-2). 13C-
Jꢁ9.0 Hz, H-3ꢃ), 3.48 (1H, t, Jꢁ9.0 Hz, H-2ꢃ), 3.68 (1H, dd, Jꢁ12.0, NMR (CD3OD) d: 56.4 (6H, s, MeOꢄ2), 62.8 (C-6ꢃ), 71.5 (C-7), 71.7 (C-
4.0 Hz, H-6ꢃ), 3.86 (3H, s, MeO), 3.86 (1H, dd, Jꢁ12.0, 1.5 Hz, H-6ꢃ), 4.53 4ꢃ), 75.1 (C-2ꢃ), 78.0 (C-3ꢃ), 78.1 (C-5ꢃ), 102.8 (C-1ꢃ), 112.5 (C-5), 113.3
(2H, s, H-7), 4.86 (1H, d, Jꢁ7.5 Hz, H-1ꢃ), 6.87 (1H, dd, Jꢁ8.0, 2.0 Hz, H- (C-2), 122.0 (C-6), 131.7 (C-1), 150.1 (C-4), 150.4 (C-3).
6), 7.02 (1H, d, Jꢁ2.0 Hz, H-2), 7.12 (1H, d, Jꢁ8.0 Hz, H-5). 13C-NMR
(CD3OD) d: 56.6 (MeO), 62.5 (C-6ꢃ), 64.9 (C-7), 71.3 (C-4ꢃ), 74.9 (C-2ꢃ),
77.8 (C-3ꢃ), 78.1 (C-5ꢃ), 102.9 (C-1ꢃ), 112.6 (C-2), 117.9 (C-5), 120.7 (C-6),
Incubation of 3,4,5-Trimethoxybenzaldehyde (16) The MeOH from
the hairy roots incubated with 100 mg of 16 gave 17 (56 mg). FAB-MS: m/z
361 [MꢀH]ꢀ C16H15O9, m/z 360 [M]ꢀ C16H14O9. 1H-NMR (CD3OD) d: 3.25
137.7 (C-1), 147.2 (C-4), 150.8 (C-3). 5: FAB-MS: m/z 339 [MꢀNa]ꢀ (1H, dd, Jꢁ9.0, 8.0 Hz, H-4ꢃ), 3.28 (1H, t, Jꢁ9.0 Hz, H-3ꢃ), 3.33 (1H, t,
1
C14H20O8Na, m/z 317 [MꢀH]ꢀ C14H21O8. H-NMR (CD3OD) d: 3.22 (1H, Jꢁ9.0 Hz, H-2ꢃ), 3.67 (1H, dd, Jꢁ11.5, 6.0 Hz, H-6ꢃ), 3.74 (3H, s, MeO at
dd, Jꢁ9.0, 8.0 Hz, H-4ꢃ), 3.25 (1H, overlap, H-5ꢃ), 3.27 (1H, t, Jꢁ9.0 Hz, H-
3ꢃ), 3.30 (1H, overlap, H-2ꢃ), 3.68 (1H, dd, Jꢁ12.0, 5.5 Hz, H-6ꢃ), 3.85 (3H,
C-4), 3.83 (6H, s, MeO at C-3, 5), 3.89 (1H, dd, Jꢁ11.5, 2.0 Hz, H-6ꢃ), 6.75
(2H, s, H-2, 6). 13C-NMR (CD3OD) d: 56.6 (MeO at C-3, 5), 61.1 (MeO at
s, MeO), 3.89 (1H, dd, Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.30 (1H, d, Jꢁ8.0 Hz, H-1ꢃ), C-4), 62.8 (C-6ꢃ), 71.5 (C-7), 71.8 (C-4ꢃ), 75.2 (C-2ꢃ), 78.1 (C-3ꢃ, 5ꢃ), 103.0
4.58 (1H, d, Jꢁ11.3 Hz, H-7), 4.81 (1H, d, Jꢁ11.3 Hz, H-7), 6.74 (1H, d,
Jꢁ8.0 Hz, H-5), 6.82 (1H, dd, Jꢁ8.0, 2.0 Hz, H-6), 7.04 (1H, d, Jꢁ2.0 Hz,
H-2). 13C-NMR (CD3OD) d: 56.4 (MeO), 62.9 (C-6ꢃ), 71.7 (C-7 or C-4ꢃ),
(C-1ꢃ), 106.4 (C-2, 6), 135.1 (C-1), 138.4 (C-4), 154.5 (C-3, 5).
Incubation of Salicylaldehyde (18) The MeOH eluate from the hairy
roots incubated with 100 mg of 18 gave 19 (35 mg) and 20 (50 mg). 19:
1
71.8 (C-4ꢃ or C-7), 75.1 (C-2ꢃ), 78.0 (C-3ꢃ), 78.1 (C-5ꢃ), 102.7 (C-1ꢃ), 113.3 FAB-MS: m/z 309 [MꢀNa]ꢀ C13H18O7Na. H-NMR (CD3OD) d: 3.38 (1H,
(C-2), 115.8 (C-5), 122.5 (C-6), 130.3 (C-1), 147.4 (C-4), 148.9 (C-3).
Incubation of Vanillyl Alcohol (4) The MeOH eluate from the P. nil
hairy roots incubated with 100 mg of 4 gave 3 (28 mg) and 5 (6 mg).
Incubation of Isovanillin (6) The MeOH eluate from the hairy roots in-
cubated with 100 mg of 6 gave 7 (23 mg). FAB-MS: m/z 339 [MꢀNa]ꢀ
dd, Jꢁ9.5, 8.3 Hz, H-4ꢃ), 3.42 (1H, ddd, Jꢁ9.5, 5.2, 2.0 Hz, H-5ꢃ), 3.45 (1H,
t, Jꢁ8.3 Hz, H-3ꢃ), 3.50 (1H, dd, Jꢁ9.2, 7.5 Hz, H-2ꢃ), 3.70 (1H, dd,
Jꢁ12.0, 5.4 Hz, H-6ꢃ), 3.89 (1H, dd, Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.56 (1H, d,
Jꢁ12.9 Hz, H-7), 4.77 (1H, d, Jꢁ12.9 Hz, H-7), 4.86 (1H, overlap, H-1ꢃ),
7.02 (1H, dt, Jꢁ7.5, 1.1 Hz, H-5), 7.20 (1H, dd, Jꢁ7.2, 1.1 Hz, H-3), 7.25
(1H, dt, Jꢁ7.2, 1.5 Hz, H-4), 7.32 (1H, dd, Jꢁ7.5, 1.5 Hz, H-6). 13C-NMR
1
C14H20O8Na, m/z 317 [MꢀH]ꢀ C14H21O8. H-NMR (CD3OD) d: 3.36 (1H,
d, Jꢁ9.5, 9.0 Hz, H-4ꢃ), 3.41 (1H, ddd, Jꢁ9.0, 5.5, 2.0 Hz, H-5ꢃ), 3.46 (1H, (CD3OD) d: 61.0 (C-7), 62.5 (C-6ꢃ), 71.4 (C-4ꢃ), 75.1 (C-2ꢃ), 78.0 (C-3ꢃ),
t, Jꢁ9.0 Hz, H-3ꢃ), 3.49 (1H, t, Jꢁ9.5 Hz, H-2ꢃ), 3.68 (1H, dd, Jꢁ12.0, 78.3 (C-5ꢃ), 103.4 (C-1ꢃ), 117.0 (C-3), 123.7 (C-5), 129.9 (C-4 or 6), 130.0
5.5 Hz, H-6ꢃ), 3.84 (3H, s, MeO), 3.88 (1H, dd, Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.51 (C-6 or 4), 167.2 (C-2). 20: FAB-MS: m/z 309 [MꢀNa]ꢀ C13H18O7Na, m/z
(2H, s, H-7), 4.90 (1H, d, Jꢁ7.5 Hz, H-1ꢃ), 6.96 (1H, d, J=8.0 Hz, H-5), 6.98
(1H, dd, Jꢁ8.0, 2.0 Hz, H-6), 7.17 (1H, d, Jꢁ2.0 Hz, H-2). 13C-NMR
(CD3OD) d: 56.8 (MeO), 62.6 (C-6ꢃ), 65.0 (C-7), 71.4 (C-4ꢃ), 74.9 (C-2ꢃ),
77.8 (C-3ꢃ), 78.3 (C-5ꢃ), 102.8 (C-1ꢃ), 113.5 (C-5), 117.2 (C-2), 122.7 (C-6),
287 [MꢀH]ꢀ C13H19O7. 1H-NMR (CD3OD) d: 3.23 (1H, dd, Jꢁ8.6, 8.0 Hz,
H-4ꢃ), 3.29 (2H, overlap, H-3ꢃ, 5ꢃ), 3.34 (1H, t, Jꢁ7.7 Hz, H-2ꢃ), 3.68 (1H,
dd, Jꢁ12.0, 5.4 Hz, H-6ꢃ), 3.88 (1H, dd, Jꢁ12.0, 2.0 Hz, H-6ꢃ), 4.38 (1H, d,
Jꢁ7.7 Hz, H-1ꢃ), 4.72 (1H, d, Jꢁ12.0 Hz, H-7), 4.92 (1H, d, Jꢁ12.0 Hz, H-

April 2005
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7), 6.77 (1H, dd, Jꢁ8.0, 1.0 Hz, H-3), 6.80 (1H, dt, Jꢁ7.4, 1.1 Hz, H-5),
7.11 (1H, dt, Jꢁ1.7, 8.0 Hz, H-4), 7.33 (1H, dd, Jꢁ7.4, 1.7 Hz, H-6). 13C-
NMR (CD3OD) d: 62.8 (C-6ꢃ), 67.8 (C-7), 71.7 (C-4ꢃ), 75.1 (C-2ꢃ), 78.1 (C-
3), 103.5 (C-1ꢃ), 116.3 (C-3), 120.5 (C-5), 125.2 (C-1), 130.1 (C-4 or 6),
131.0 (C-6 or 4), 156.6 (C-2).
2.5 Hz, H-5ꢃ), 4.12 (2H, overlap, H-3ꢃ, 4ꢃ), 4.26 (1H, t, Jꢁ8.5 Hz, H-2ꢃ),
4.40 (1H, br d, Jꢁ11.5 Hz, H-6ꢃ), 4.56 (1H, br d, Jꢁ11.5 Hz, H-6ꢃ), 4.82
(1H, d, Jꢁ7.5 Hz, H-1ꢃ), 5.35 (1H, q, Jꢁ6.5 Hz, H-7), 7.27 (1H, t,
Jꢁ7.5 Hz, H-4), 7.36 (2H, t, Jꢁ7.5 Hz, H-3, 5), 7.68 (2H, d, Jꢁ7.5 Hz, H-2,
6). 13C-NMR (pyridine-d5) d: 25.0 (C-8), 62.9 (C-6ꢃ), 71.8 (C-4ꢃ), 74.8 (C-
Incubation of 4-Hydroxy-3-methoxyacetophenone (21) The MeOH 7), 75.5 (C-2ꢃ), 78.5ꢄ2 (C-3ꢃ, 5ꢃ), 101.6 (C-1ꢃ), 127.2 (C-3, 5), 127.7 (C-4),
eluate from the hairy roots incubated with 100 mg of 21 gave 22 (12 mg), 23
128.8 (C-2, 6), 144.1 (C-1). Product 27 was in comparison with the authen-
(3 mg) and 24 (3 mg). 22: FAB-MS: m/z 351 [MꢀNa]ꢀ C15H20O8Na, m/z
tic data of 2-b-D-glucopyranosyloxyacetophenone.7)
1
329 [MꢀH]ꢀ C15H21O8. H-NMR (DMSO-d6) d: 2.53 (3H, s, Me-8), 3.27
Time-Course Experiment of Transformation of Vanillin (1) by P. nil
(3H, overlap, H-2ꢃ, 3ꢃ, 4ꢃ), 3.34 (ddd, Jꢁ9.5, 5.5, 2.0 Hz, H-5ꢂ), 3.44 (1H, Hairy Roots Vanillin (1) was added to each hairy root culture (200 ml of
dt, Jꢁ12.0, 6.0 Hz, H-6ꢃ), 3.65 (1H, ddd, Jꢁ12.0, 5.0, 2.0 Hz, H-6ꢃ), 3.82 1/2 MS liquid medium, 4 weeks old) and incubated. Sampling of the culture
(3H, s, MeO), 4.52 (1H, t, Jꢁ5.5 Hz, OH at C-6ꢃ), 5.02 (1H, br d, Jꢁ5.0 Hz, medium and hairy roots for analysis was carried out 0, 3, 6, 12, 24, 48, 72,
OH), 5.05 (1H, d, Jꢁ7.5 Hz, H-1ꢃ), 5.31 (1H, br d, Jꢁ5.5 Hz, OH), 7.17 and 144 h after adding 1 to the culture. Each hairy root obtained from the
(1H, d, Jꢁ8.5 Hz, H-5), 7.46 (1H, d, Jꢁ2.0 Hz, H-2), 7.57 (1H, dd, Jꢁ8.5, cultures was lyophilized and extracted with boiling water. The water solu-
2.0 Hz, H-6), 13C-NMR (DMSO-d6) d: 26.4 (C-8), 55.6 (MeO), 60.5 (C-6ꢃ), tions were passed through an HP 20 column, washed with water, and finally
69.5 (C-4ꢃ), 73.1 (C-2ꢃ), 76.8 (C-3ꢃ), 77.1 (C-5ꢃ), 99.4 (C-1ꢃ), 110.9 (C-5),
eluted with MeOH. The MeOH eluate was analyzed using HPLC. The cul-
ture medium was passed through a HP 20 column, washed with water and
114.1 (C-2), 122.7 (C-6), 130.8 (C-1), 148.7 (C-4), 150.6 (C-3), 196.4 (C-7).
23: FAB-MS: m/z 515 [MꢀNa]ꢀ C24H28O11Na, m/z 493 [MꢀH]ꢀ C24H29O11. eluted with MeOH. The MeOH eluate was analyzed by HPLC.
1H-NMR (CD3OD) d: 2.47 (3H, s, Me-8), 2.54 (3H, s, Me-8ꢃ), 3.14 (1H,
ddd, Jꢁ9.0, 5.0, 2.0 Hz, H-5ꢂ), 3.23 (1H, dd, Jꢁ9.5, 8.0 Hz, H-2ꢂ), 3.30 (1H, References
overlap, H-4ꢂ), 3.32 (1H, t, Jꢁ9.0 Hz, H-3ꢂ), 3.55 (1H, dd, Jꢁ11.5, 5.5 Hz,
H-6ꢂ), 3.67 (1H, dd, Jꢁ11.5, 2.5 Hz, H-6ꢂ), 3.89 (3H, s, MeO at C-2ꢃ), 3.92
(3H, s, MeO at C-3), 5.17 (1H, d, Jꢁ7.5 Hz, H-1ꢂ), 6.90 (1H, d, Jꢁ8.5 Hz,
H-6ꢃ), 7.16 (1H, d, Jꢁ2.0 Hz, H-6), 7.44 (1H, d, Jꢁ2.0 Hz, H-4), 7.57 (1H,
dd, Jꢁ8.5, 2.0 Hz, H-5ꢃ), 7.63 (1H, d, Jꢁ2.0 Hz, H-3ꢃ). 13C-NMR (CD3OD):
d: 26.4 (C-8), 26.5 (C-8ꢃ), 56.6 (MeO at C-2ꢃ), 57.0 (MeO at C-3), 62,4 (C-
6ꢂ), 71.2 (C-4ꢂ), 75.6 (C-2ꢂ), 77.7 (C-3ꢂ), 78.3 (C-5ꢂ), 103.9 (C-1ꢂ), 109.6
(C-4), 113.0 (C-3ꢃ), 114.4 (C-6), 118.9 (C-6ꢃ), 124.0 (C-5ꢃ), 134.3 (C-4ꢃ),
134.4 (C-5), 142.4 (C-2), 150.2 (C-1), 151.4 (C-2ꢃ), 151.8 (C-1ꢃ), 154.9
(C-3), 198.6 (C-7), 199.2 (C-7ꢃ). 24: FAB-MS: m/z 515 [MꢀNa]ꢀ
C24H28O11Na, m/z 493 [MꢀH]ꢀ C24H29O11. 1H-NMR (DMSO-d6) d: 2.50
(3H, s, Me-8ꢃ), 2.53 (3H, s, Me-8), 2.91 (1H, dd, Jꢁ8.5, 8.0 Hz, H-2ꢂ), 3.01
(1H, ddd, Jꢁ9.0, 4.5, 1.5 Hz, H-5ꢂ), 3.05 (1H, t, Jꢁ9.5 Hz, H-4ꢂ), 3.12 (1H,
br t, Jꢁ8.0 Hz, H-3ꢂ), 3.37 (1H, dd, Jꢁ12.0, 5.0 Hz, H-6ꢂ), 3.55 (1H, br dd,
Jꢁ12.0, 1.5 Hz, H-6ꢂ), 3.88 (3H, s, MeO at C-3ꢃ), 3.90 (MeO at C-3), 5.27
(1H, d, Jꢁ7.5 Hz, H-1ꢂ), 7.41 (1H, d, Jꢁ2.0 Hz, H-4ꢃ), 7.49 (1H, d,
1) Kamada H., Okamura N., Satake M., Harada H., Shimomura K., Plant
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101.1 (C-1ꢂ), 108.8 (C-4ꢃ), 111.6 (C-4), 124.2 (C-1ꢃ), 125.1 (C-6), 126.8 (C-
5ꢃ), 126.8 (C-6ꢃ), 131.5 (C-1), 131.8 (C-5), 146.5 (C-2), 147.8 (C-3ꢃ), 149.5
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Incubation of Acetophenone (25) The MeOH eluate from the hairy
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1
MS: m/z 307 [MꢀNa]ꢀ C14H20O6Na, m/z 285 [MꢀH]ꢀ C14H21O6. H-NMR
2587—2595 (2004).
(pyridine-d5) d: 1.50 (3H, d, Jꢁ6.5 Hz, Me-8), 3.78 (1H, ddd, Jꢁ9.0, 5.0,