Plant & Cell Physiol. 14: 597-606 (1973)
Isolation of a new phytoalexin-like compound,
ipomeamaronol, from black-rot fungus
infected sweet potato root tissue, and
its structural elucidation1
N. Kato, H. Imaseki2, N. Nakashima, T. Akazawa2 and I. Uritani
Department of Agricultural Chemistry, Faculty of Agriculture, Nagoya
University, Chikusa, Nagoya, Aichi 464, Japan
A new phytoalexin-like compound was isolated from sweet potato root tissue
infected by the black-rot fungus, Ceratocystisfimbriata.Its chemical structure was
similar to ipomeamarone, and the compound was identified as 14-hydroxy-ipomeamarone
and called ipomeamaronol.
In response to the infection of fungal pathogens such as Ceratocystis fimbriata,
sweet potato root tissue vigorously accumulates such terpenoids as ipomeamarone
and ipomeanine (1-4) whose chemical structures have been determined by Hiura
(1) and Kubota et al. (2). Not only the crude oily substance containing ipomeamarone and ipomeanine but also ipomeamarone and ipomeanine themselves have
been proven inhibitory to C.fimbriata(1, 5, 6).
Thin-layer chromatography indicated that the crude oily substance was
composed of ipomeamarone and ipomeanine and more than 10 kinds of terpenoids
(7), which had positive reactions toward Ehrlich's reagent (8). Some were
tentatively designated as components A and B (9), on the silica-plate chromatogram.
Devices of the solvent system for thin-layer chromatography revealed that component
B was composed of two independent substances; component Bj and B2 [10).
Several components separated by thin-layer chromatography, including
ipomeamarone and ipomeanine, had toxic effects, and component B2 also showed
a strong inhibitory effect on the sporulation of C.fimbriata(11). Thus, component
B2 can be regarded as a kind of phytoalexin, similar to ipomeamarone {12).
Component B2 was isolated on a large scale, and its chemical structure was
determined. From chemical and spectroscopic results, component B-> was shown
to be 14-hydroxy-ipomeamarone (13), and was designated as ipomeamaronol (14).
Yang et al. (75) have also isolated ipomeamaronol from sweet potato with black
1
2
This paper constitutes Part 105 of the Phytopathological Chemistry of Sweet Potato with Black
Rot and Injury.
Present address: Institute for Biochemical Regulation, Faculty of Agriculture, Nagoya University,
Nagoya.
597
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(Received November 7, 1972)
598
N. Kato, H. Imaseki, N. Nakashima, T. Akazawa and I. Uritani
rot mold infection and have independently reached the same structure for ipomeamaronol, while trying to elucidate the principal factor responsible for fatal pulmonary
edema in animals consuming moldy sweet potato roots. Both works were published
separately as short communications after mutual communication (14, 15). This
paper deals with our detailed study on the isolation of component B2, ipomeamaronol,
and its structural elucidation.
Materials and methods
Preparation of the dried powder of infected sweet potato root tissue
Isolation of component Bo
The powder (15 kg) of the infected root tissue was extracted with ether at room
temperature. This ether solution was evaporated to its residue, which was then
suspended in methanol. The suspension was filtered to remove insoluble material,
and the filtrate was concentrated in vacuo to an oily substance, whose yield was
60 g.
This oily substance (60 g) was dissolved in 60 ml of n-hexane, and the solution
was applied to a column (4.7 X 43 cm) containing 300 g of silica gel, and was eluted
by 3 liters of ethyl acetate-n-hexane (2 : 8 v/v), followed by 1 liter of ethyl acetaten-hexane ( 1 : 1 v/v), at the rate of 3 ml per min (column chromatography No. 1).
The fraction containing component B2 was concentrated to yield 7.5 g. The concentrate was dissolved in 7.5 ml of ethyl acetate-n-hexane (1 : 19 v/v), and the
solution was placed on a column (4.7x23 cm) containing aluminum oxide gel. It
was developed first with 2 liters of ethyl acetate-n-hexane (2 : 8 v/v), and successively
by 1 liter of ethyl acetate-n-hexane ( 1 : 1 v/v) at the rate of 2 ml per min (column
chromatography No. 2). The fraction containing component B2 was evaporated
to its residue (1.6 g). This residue was dissolved in 3.2 ml of chloroform-n-hexane
( 1 : 9 v/v). The solution was subjected to chromatography on an aluminum oxide
(160 g) gel column (2.8x25 cm), sequentially with 2 liters of chloroform-n-hexane
(2 : 8 v/v) and 2 liters of chloroform-n-hexane (1:1 v/v), at a rate of 2 ml per min
(column chromatography No. 3). The component B2 fraction was concentrated
by vacuum evaporation, giving 1.0 g of component B2.
Preparation and isolation of 3,5-dinitrobenzoate of component Bi
3,5-Dinitrobenzoyl chloride (1.3 g) in dried pyridine was mixed with 1.0 g
of component B2 in ether. After the mixture had stood for 3 hr at room temperature,
pyridine and 3,5-dinitrobenzoic acid were removed by successive washing with
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Roots of sweet potato (Ipomoea batatas L. cv. Norin 1) were harvested at the
Kariya Farm, Aichi Prefecture in the autumn and were stored at about 10°C until
use.
Roots were cut perpendicularly in slices 2 to 3 mm thick. Slices were inoculated with an endoconidial suspension of C.fimbriataon both surfaces, and were
incubated at 30°C under high humidity for a 55 to 60 hr period. By that time, the
fungus covered the surface and had penetrated into the tissue to a depth of 0.5 to
1.0 mm. The tissue was dried at room temperature, and pulverized.
Ipomeamaronol, a phytoalexin from diseased sweet potato
599
1 N HC1 and diluted NaHCC>3. The 3,5-dinitrobenzoate of component Bo was
extracted with ether, giving 1.5 g.
The crude 3,5-dinitrobenzoate (1.5 g) was dissolved in 3 ml of chloroform,
and the solution was applied to a column (1.6 X 15 cm) containing 15 g of silica gel.
Elution with chloroform gave a fraction containing the 3,5-dinitrobenzoate.
Removal of the solvent produced a solid material, which was recrystallized from ether
to give yellow needles (250 mg).
Preparation of the enol acetate of 3,5-dinitrobenzoate of component B-i and its ozonolysis
Thin layer chromatography
The thin layer was made with silica gel G(E. Merck, Darmstadt), and the
sample was developed on the plate by ethyl acetate- n-hexane (2 : 8 or 1:1 v/v).
Spots were shown with the Ehrlich's reagent (5 g of 0-dimethylaminobenzaldehyde
in 95% ethyl alcohol-cone. HC1 ( 1 : 1 v/v). The terpenes were colored in a wide
range of pink to violet, depending on their components. Component B-2 was colored
violet.
Determination of physico-chemical properties
The melting point was determined on a Yanagimoto micro-heating stage.
NMR spectra were recorded on Varian A-60, Varian HA-100 and JEOL 4H-100
NMR spectrometers in deuteriochloroform. The IR spectra as neat film or KBr
disc were recorded with a JASCO IR-G IR spectrometer.
Results
Isolation of component Bi
As preliminary experiments on the isolation of component B2, conditions
of thin-layer chromatography were investigated. As shown in Fig. 1, the Rf values
of the components differed, depending on the supporting material (silica gel or
aluminum oxide gel) or the composition of the developing solvent system. Silica
gel was a more active adsorbent than was aluminum oxide gel. The solvents
decreased the intensity of adsorption of the supporting material in the following
order: n-hexane > chloroform > ethyl acetate. From these results, the three
steps of preparative column chromatography were devised as shown in Materials
and methods.
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/(-Toluenesulfonic acid (40 mg) and 2 drops of dimethylsulfoxide were added
to a solution of 3,5-dinitrobenzoate (400 mg) of component B-<< in 10 ml of isopropenyl
acetate according to the method of Mofett and Weisblat (16). The solution was
refluxed for 120 hr at 110°C, then was concentrated. The residue was chromatographed over silica gel with ethyl acetate-n-hexane (3 : 17), to give a viscous liquid
(160 mg), which was composed of the 3,5-dinitrobenzoate, its enol acetate and other
compounds, based on the thin-layer chromatographic pattern. It was difficult to
purify the enol acetate by column or thin-layer chromatography.
Ozonolysis of the above mixture (45 mg) was performed with the method of
Papps et al. (17). The product was chromatographed over silica gel, and elution
with ethyl acetate-n-hexane (3 : 7), afforded an aldehyde (13 mg) as crystals.
600
N. Kato, H. Imaseki, N. Nakashima, T. Akazawa and I. Uritani
OA
C_JA.IP. P' C_jA,lp.l[
3A
—IIP
9lp
O"
• Qlp'
O
Olpn
HB,
C )'Pn
o
° N ip p
Q
o
O
8
°
o
Olpn
Q
©B,
o
o
O'Pn
0
OB,
Olpn
OBi
©B,
OB,
O Bt
III
Fig. 1.
Thin-layer chromatographic pattern of terpene components in diseased sweet potato root tissue. I, II,
Thin-layer chromatographic patterns of some of the eluted fractions after
column chromatographies No. 1, No. 2 and No. 3 are shown in Fig. 2-A, B and C.
As indicated in Fig. 2-C, component B2 was almost purified chromatographycally,
by the three sequential chromatographies.
Isolation of 3,5-dinitrobenzoate of component Bi
As shown in Materials and methods, the 3,5-dinitrobenzoate of component
B2 was purified by column chromatography. The eluted fractions were subjected
to thin-layer chromatography to check whether the 3,5-dinitrobenzoate was separated from ipomeamaronol and other contaminants. As shown in Fig 3, the
3,5-dinitrobenzoate was separated from almost all the other components.
Elucidation of the chemical structure of component Z?2
The IR spectrum of component B2 was very similar to that of ipomeamarone
(I in Fig. 4) except for a strong hydroxyl absorption at 3390 cm"1. The sample
showed the following several signals in its NMR spectrum (CDC13, 60 MHz): 8
0.87 (3H, br. d, J=6.1 Hz), 1.26 (3H, s), 2.61 (2H, s), 2.65 (1 H, m) and 7.27
(2 H, m) (see Fig. 5), and contained 60% of pure component B2, as estimated from
the area under the NMR peak at 8 0.87. Further purification by column, thinlayer or gas chromatography was almost impossible.
3,5-Dinitrobenzoate (III in Fig. 4) of component B2 (II in Fig. 4) showed
mp 81°C; vmaxKBr 1733, 1710, 1548, and 872 cm' 1 ; M+ 460. Anal. Calcd. for
C22H24N2O9: C, 57.39; H, 5.25; N, 6.08. Found: C, 57.42; H, 5.26; N, 5.96.
To determine the structure of component B2, the NMR spectrum of ipomeamarone was examined. From the spectrum (see Fig. 6), the Ci and C4 protons
on the furan ring are a doublet (J=1.8 Hz) at 8 7.29 and a singlet at 3 7.30,
respectively. The Ci proton is coupled with the neighboring C2 proton of a doublet
(J=1.8Hz) at 5 6.29. The multiplet at 3 4.84 (eight lines, J=6.0, 4.2 and
1.8 Hz) may be due to the C5 proton and is an X part of an ABMX pattern, which
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III, IV and V: silica gel and chloroform, silica gel and ethyl acetate-n-hexane (2 : 8 v/v), aluminum
oxide gel and chloroform, aluminum oxide gel and chloroform-n-hexane (3 : 7 v/v), and aluminum
oxide gel and ethyl acetate-n-hexane (2 : 8 v/v), respectively. A, Ip, Ip', Ipn, Bi and B 2 : component
A; ipomeamarone; component Ip' (shows a very similar color to ipomeamarone for Ehrlich's reagent);
ipomeanine; component Bi and component B2- O= colored strongly and Q : colored weakly.
Ipomeamaronol, a phytoalexin from diseased sweet potato
O A
O
O
601
o
IP O
oip-
o
P
o
o
e-o
Ipn Q
8
0
100
too
900
1100
1100
Bi @
2400
0
IT0O
Eluted volume (ml)
O
9
o
°
B, @
?,
Q
r,
IfOO
Eluted volume (ml)
Fig. 2.
1100
itOO
Eluted volume (ml)
Thin-layer chromatographic patterns of the eluted fractions from column chromatographies No. 1 (A),
No. 2 (B) and No. 3 (C) to isolate component Bz. A, B and C: A, column chromatography No. 1 (the
first step of chromatography using silica gel and ethyl acetate-n-hexane (2 :8, then 1 : 1 v/v) ); B,
column chromatography No. 2 (the second step of column chromatography using aluminum oxide
gel and ethyl acetate-n-hexane (2 : 8, then 1 : 1 v/v) ); and C, the third step of column chromatography using aluminum oxide gel and chloroform-n-hexane (2 : 8, then 1 : 1 v/v), (see text). A,
Ip, Ip', Ipn, and B2: see the legend to Fig. 1. Conditions for thin-layer chromatographies: silica
gel and ethyl acetate-n-hexane (2 : 8 v/v) for column chromatography No. 1, and silica gel and
ethyl acetate-n-hexane (4 : 6 v/v) for column chromatographies No. 1 and No. 2. O> O :
see the legend to Fig. 1.
is coupled with the C6 protons and one of the C7 protons (<5 1.85). A signal of
the Cio protons may be assigned to an AB-quartet signal ( J ^ 15.8 Hz) at 8 2.62,
whose chemical-shift difference between the A and B parts of an AB pattern is
close to Oppm. A doublet (J=6.2 Hz) at 8 2.29 is due to the C12 methyleneprotons coupled with the C13 methine proton. The C9 methyl protons appear as
a singlet signal at 8 1.29. Furthermore, the highest field signal (6H, d, J = 6.0
Hz) at 8 0.87 is due to the gem-dimethyl of the G14 and C15.
These assignments were applied to an analysis of the NMR spectrum of III
(see Fig. 7). Three aromatic protons appear as a multiplet of an AB» pattern
at 8 9.10. Signals at 8 7.23 (1 H, d, J=1.6 Hz), 7.19 (1 H, s), 6.24 (1 H, d , J = 1.6
Hz), 4.80 (1 H, eight lines, J=6.0, 4.2, 1.8 Hz) and 1.26 (3H, s) may correspond to
protons Ci, C4, C2, C5 and C9, respectively.
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o
N. Kato, H. Imaseki, N. Nakashima, T. Akazawa and I. Uritani
602
o
,CH3
r.
O
^CHa
3.5-ONB Bi
•
a
H
J i - n ^ C » ' .i
0
o
u
y CH3
II. R-H
<&IflR ....
([^J
0
n
OBI
0
^CHi-C=dH-CH<CHl
AcO
O=CH-CH<,-
IV. R.3,5-(NO2hCsH3CO-
3
V, R=3.5-(NOJ)2C6H3CO-
Eluled volumt(ml)
Fig. 3
Thin-layer chromatographic patterns of eluted fractions from column chromatography of the component
Bi 3,5-dinitrobenzoate product. Conditions for column chromatography: silica gel and chloroform.
3,5 DNP-B2 and B 2 : the 3,5-dinitrobenzoate of component B2 and component B2, respectively. Conditions for thin-layer chromatography: silica gel and ethyl acetate-n-hexane (3 : 7 v/v). O> O :
see the legend to Fig. 1.
Fig. 4.
Structure of ipomeamarone, component B% and its derivatives. I : ipomeamarone.
I I : component
B2 (called ipomeamaronol).
The C10 protons appear at 5 2.61 and 2.67 as an AB-quartet signal with a
14-Hz coupling. Furthermore, the spectrum shows a methyl signal (d, J=6.7
Hz) at 5 0.98 and an octet signal (2 H, J=6.0 and 10.4 Hz) centering at 5 4.25
(see part a of Fig. 8). On irradiation of a multiplet at 5 2.52, the signals vary to
a sharp singlet and a remarkable AB-quartet (J = 10.4 Hz), as shown in part b
of Fig. 8. The shape of these signals does not significantly alter in response to a
temperature change from 24 to 80°C. From these results, the somewhat strange
Fig. 5.
NMR spectrum of the component B2 sample in deuteriochbroform (60 MHz).
the sample after three sequential chromatographies.
Component B 2 sample:
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Fig. 3.
Fig. 4
Ipomeamaronol, a phytoalexin from diseased sweet potato
603
Fig. 6.
NMR spectrum of ipomeamarone in deuteriochloroform (100 MHz).
shapes of the signals in part "a" may be explained in terms of virtual spin-coupling
between the methyl protons or the C14 protons and the C13 methine proton which
is situated at about the same chemical-shift as that of the C12 protons, as reported
by Musher and Corey (18). In a pyridine-ds solution, the methyl and C14 protons
are shown as doublet signals (J=6.5 and 5.6 Hz) with a slight virtual-spin-coupling
at 5 1.00 and 4.38, respectively. Disappearance of the AB pattern of the latter
signal may be attributed to the same chemical-shifts of the two Qu-protons.
An enol acetate (IV in Fig. 4) mixture prepared from III was contaminated
by other compounds, and contained 40% of IV, as estimated from the area under
It
Fig. 7.
It
NMR spectrum of the 3,5-dinitrobenzoate of component B-i in deuteriochloroform (100 MHz).
Downloaded from http://pcp.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 17, 2016
:t
604
N. Kato, H. Imaseki, N. Nakashima, T. Akazawa and I. Uritani
•V
^
4.25
0.98
the NMR peaks at 5 1.98 and 4.95. The mixture showed umax neatfilm1755,
1736, 1685, 872cm-i; NMR (CDC13, 100 MHz): multiplets at 5 9.17 (aromatic
protons), 7.35 and 6.35 (furan ring protons) and 4.86 (C5-H), and singlets at 8
1.23 (C8-Me) and 2.07 (AcO-). In addition, the signal of the C10 protons shifts
to a higher field at 8 1.96 (2 H, s), and a doublet (J=5.0 Hz) of one proton (C12H) appears at 8 4.95 instead of the multiplet at 8 2.55 of III, as &hown by House
and Kramer (19). Signals of the geminal methyl and carbinol methylene-protons
show sharp doublets (J=6.9 and 7.5 Hz) at 8 1.07 and 4.27, respectively. The
observations indicate that the enol double bond of IV forms between carbon atoms
Cn and C12.
An aldehyde produced by ozonolysis of the mixture containing IV showed mp
125-125.5°C; [a]D21 -6.97° (C, 1.22, CHCI3); *maxKBr 1740, 1729, 1630 cm-'; M+
282. Anal. Calcd. for CuHioN207-l/3 H-ChC, 45.83; H, 3.73; N, 9.72. Found:
C, 45.76; H, 3.44; N, 9.65.
The NMR spectrum of the aldehyde in CDCI3 (100 MHz) showed the absorption of five kinds of protons. These are assigned as follows: (a) a multiplet
at 5 9.18 to the three protons of the substituted phenyl group; (b) a singlet at
8 9.80 to a proton of the aldehyde group, (c) a doublet (J = 7.6Hz) at 8 1.55
to the C15 methyl-protons, (d) a multiplet at 8 3.20 to a C13 methine proton, and (e)
eight, sharp lines centered at 8 4.89 to the substituted carbinol methylene protons.
When irradiated at 8 3.20, the sharp multiplet signal shows a typical AB pattern
with splitting (JAB = 11.3 HZ) at 5 A = 4 . 8 4 ppm and 5 B = 4 . 9 4 ppm. The principal
coupling of the methylene protons with the C13 methine-proton as an X part of the
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Fig. 8. Part of the NMR spectrum of the 3,5dinitrobenzoate of component B<> in deutcriochloroform
(100 MHz) before and after irradiation of a
multiplet at 5 2.52.
Ipomeamaronol, a phytoalexin from diseased sweet potato
605
ABX pattern affords JAX = 5.1 H Z for the A part and J B X = 6 . 6 H Z for the B part.
Similarly, the doublet signal at 8 1.55 appears as a singlet when irradiated at
the methine proton. The results confirmed the aldehyde as V.
Component B2, then, can be represented by structure II (see Fig. 4), and is
logically called ipomeamaronol.
Discussion
We express our thanks to Professor K. Munakata, of this department for his constant interest in
this work, and to the members of the Laboratory of Biochemistry for their direct or indirect helpful
association with this work.
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Elucidation of the structure of component B2 may contribute to the fields of
terpene biosynthesis and phytoalexin production. Since component B2 is produced
at almost the same time as ipomeamarone and 14C-acetate is incorporated as efficiently into component B2 as into ipomeamarone, it has been assumed that component B2 is a compound biosynthetically related to ipomeamarone, a main
constituent of terpenoids accumulated in the tissue in response to infection. This
study has demonstrated that component B2 has a very similar structure to that of
ipomeamarone, and has a hydroxyl group at the geminal methyl of ipomeamarone,
as shown in Fig. 4. This strengthens the hypothesis that component B2 is located
very close to ipomeamarone on the pathway of terpene biosynthesis. It appears
that ipomeamarone is oxidized to component B2 by a kind of oxigenase reaction.
This may be demonstrated, when 14C-labelled ipomeamarone is actively incorporated
into component B2 both in in vivo and in vitro systems.
Preliminary experiments showed that component B2 was more inhibitory on
spore germination of C.jimbriata than was ipomeamarone (11). Further detailed
investigations on the antifungal or phytoalexin-like activity of component B2,
ipomeamarone and other terpenoids and on the time-course of the content of the
components accumulated in the infected region may give a clue to the real role
of those components produced in response to the infection in a defense action or
induced resistance.
As previously indicated (20), some species of Ipomoea produce component B2
in response to infection, but others do not, but all species so far investigated produce
ipomeamarone. The lack of component B2 in the disease tissue of some species
may reflect the lack of a gene corresponding to the enzyme involved in the possible
conversion of ipomeamarone to component B2. A study of this type of species of
Ipomoea may give us some information on the biosynthetic pathway of component
B2 and the role of component B2 in the defense action.
We were unsuccessful in obtaining the semicarbazone of component B2 in a
crystalline form even though ipomeamarone gives the semicarbazone as the crystal
(1). However, the present work has made it possible to crystallize the 3,5dinitrobenzoate of component B2, and this may be useful in perform isotopic studies
on the biosynthetic mechanism of component B2, involving the oxygen-source
of its hydroxyl group which originates from oxygen molecules in air or oxygen atoms
in water.
606
N. Kato, H. Imaseki, N. Nakashima, T. Akazawa and I. Uritani
References
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