Pennelliisides A-C, 2,3,4-Trisubstituted Acyl Glucoses Isolated from Solanum pennellii

Article abstract of DOI:10.1021/acs.jnatprod.9b01234

Solanum species accumulate a variety of secondary metabolites in their trichomes, and it is well known that acyl sugars are specialized metabolites secreted by the trichomes. However, very few reports provide detailed information on the chemical structure of polyacylated glucose derivatives, due to the α and β isomerization that can occur at the C-1 position. In this study, a strategy was established to isolate polyacylated glucose derivatives. According to the developed strategy, hydroxy groups were derivatized to a benzyloxy group using TriBOT. After isolation of the compounds in pure form and deprotection of the benzyloxy group, the chemical structures of pennelliisides A-C were determined as 2,3,4-O-triisobutyryl-d-glucose, 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-d-glucose, and 3-O-decanoyl-2,4-O-diisobutyryl-d-glucose, respectively. Structural elucidation was performed using spectroscopic techniques, including 1D and 2D NMR, FD-MS, and GC-MS. It was also found that the fatty acid moiety contributes to the allelopathic properties of the isolated compounds.

Full text of DOI:10.1021/acs.jnatprod.9b01234

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Pennelliisides A−C, 2,3,4-Trisubstituted Acyl Glucoses Isolated from
Solanum pennellii
Tenki Nakashima,† Yurika Nambu,† Yutaka Inoue, Rishni Masimbula, and Hideyuki Matsuura*
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ABSTRACT: Solanum species accumulate a variety of secondary
metabolites in their trichomes, and it is well known that acyl sugars
are specialized metabolites secreted by the trichomes. However,
very few reports provide detailed information on the chemical
structure of polyacylated glucose derivatives, due to the α and β
isomerization that can occur at the C-1 position. In this study, a
strategy was established to isolate polyacylated glucose derivatives.
According to the developed strategy, hydroxy groups were
derivatized to a benzyloxy group using TriBOT. After isolation
of the compounds in pure form and deprotection of the benzyloxy group, the chemical structures of pennelliisides A−C were
determined as 2,3,4-O-triisobutyryl-D-glucose, 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-D-glucose, and 3-O-decanoyl-2,4-O-
diisobutyryl-D-glucose, respectively. Structural elucidation was performed using spectroscopic techniques, including 1D and 2D
NMR, FD-MS, and GC-MS. It was also found that the fatty acid moiety contributes to the allelopathic properties of the isolated
compounds.
lants produce a series of specialized metabolites, some of
which are known to be important for subsistence, such as
isomerization that can occur at the C-1 position. In this
study, a strategy was established to isolate the acylglucoses of S.
pennellii as follows: (1) protection of the hydroxy group of
acylglucose using a reagent to make benzyl ether derivatives,
(2) isolation and evaluation of the chemical structures of the
isolated derivatives, and (3) deprotection of the benzyl group
to give naturally derived compounds. Following this strategy,
herein the isolation and structural determination of one known
compound, 2,3,4-O-triisobutyryl D-glucose, and two new
acylglucoses, 2,4-O-diisobutyryl 3-O-(8-methylnonanoyl)-D-
glucose and 3-O-decanoyl 2,4-O-diisobutyryl-D-glucose,
named, in turn, pennelliisides A−C (1−3), respectively, were
accomplished, and the results are presented in this report.
Furthermore, the allelopathic properties of these compounds
were investigated.
P
for protection against pathogens and herbivory.1,2 A tomato
species, Solanum pennellii Correll (Solanaceae), is categorized
as a wild-type tomato, for which the full genomic information
was investigated by the 100 Tomato Genome Sequencing
Consortium in 2014.3 Solanum species accumulate a variety of
secondary metabolites in their trichomes, which, in the case of
S. pennellii, account for more than 25% of the total dry weight.4
It is well known that acyl sugars are specialized metabolites
secreted by the trichomes.4 For S. pennellii, it was reported that
its acyl sugars have insecticidal,5 pest repellant,6 and weed
growth inhibitory7 activities. Although the fruits of the plant
contain toxic glycoalkaloids,8,9 S. pennellii is regarded as an
agriculturally useful species due to its ability to hybridize with
cultivar tomatoes.10 Recently, it was revealed that insects can
modulate not only the trichome density but also the contents
of allelochemicals, for which the degree of response is thought
to depend on the magnitude and/or type of induction.11 Thus,
it was hypothesized that the biosynthesis of acyl sugars might
be affected by fluctuations in plant hormones. One research
group has studied the biosynthetic pathway of those acyl
sugars.12 However, their biosynthesis remains to be inves-
tigated, especially due to the lack of chemical structural
information.
RESULTS AND DISCUSSION
■
The upper parts of S. pennellii (1.7 kg) were rinsed with
ethanol, and the volatile components of the organic solvent
were removed under reduced pressure to give an epicuticular
lipophilic extract (Figure S1, Supporting Information). The
crude extract was roughly purified using column chromatog-
Received: December 15, 2019
From the chemical structural viewpoint, the structures of
acyl sugars can be classified into two main groups: polyacylated
sucrose and polyacylated glucose.2 However, very few reports
provide detailed information on the chemical structure13 of
polyacylated glucose derivatives, due to the α and β
© XXXX American Chemical Society and
American Society of Pharmacognosy
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raphy, which was followed by benzylation to give a crude
extract containing benzyl derivatives of acylglucose. The benzyl
derivatives were purified to give compounds 4 (47 mg), 5 (33
mg), and 6 (17 mg).
5.29)/C-C-1 (δC 174.6) were indicative of the linkage of the
fatty acid moiety with the basic glucose structure. Furthermore,
the HMBC correlations (Figures 2 and S5, Supporting
Information) of H-A-2 (δH 2.41, methine proton)/C-A-1 (δC
174.4), H-B-2 (δH 2.39, methine proton)/C-B-1 (δC 175.5),
and H-C-2 (δH 2.30, methine proton)/C-C-1 (δC 174.6),
together with the COSY correlations (Figures 2 and S4,
Supporting Information) of δH 1.07 (H-A-3 and A4) to 2.41
(H-A-2), δH 1.06 (H-B-3 and B-4) to 2.39 (H-B-2), and δH
1.01 (H-C-3) and 0.97 (H−C4) to 2.30 (H−C-2), suggested
that three isobutyryl ester moieties are attached to the basic
glucose structure. In addition, the HMBC correlations between
the phenylmethylene protons of δH 4.74/4.73 (H-1a′ and -1b′)
and anomeric carbon C-1 (δC 99.5) and between the
phenylmethylene protons of δH 4.33/4.30 (H-1a″ and 1b″)
and C-6 (δC 69.2) indicated that two substituted benzyloxy
groups are attached at the C-1 and C-6 positions. Therefore, it
was determined that the structure of 4 is 1,6-O-dibenzyl-2,3,4-
O-triisobutyryl-β-D-glucose. Analysis of the 1H NMR, 13C
NMR, DEPT, HMQC, and HMBC spectroscopic data gave
the total 1H and 13C NMR assignments for 4, as listed in Table
S1 (Supporting Information).
Compound 4 was isolated as a colorless oil. The molecular
formula, C32H42O9, was deduced from the HRFDMS data (m/
z 570.2823 [M]+, calcd 570.2829) (Figure S2, Supporting
Information), and the index of hydrogen deficiency was
1
calculated to be 12. The H NMR resonances (Figure S3 and
Table S1, Supporting Information) and the correlations
observed in the COSY spectrum (Figure 2 and Figure S4,
Compound 5 was purified as a colorless oil. The molecular
formula, C38H54O9, was determined from the HRFDMS data
(m/z 654.3750 [M]+, calcd 654.3767) (Figure S9, Supporting
Information) with the index of hydrogen deficiency again
calculated to be 12. The 1H NMR resonances (Figure S10 and
Table S2, Supporting Information) and the correlations
observed in the COSY spectrum (Figures 3 and S11,
Supporting Information) between the signals at δH 4.39 (H-
1, J1,2 = 7.7 Hz), 5.46 (H-2, J2,3 = 9.0 Hz), 5.49 (H-3, J3,4 = 9.2
Hz), 5.31 (H-4, J4,5 = 9.1 Hz), 3.43 (H-5), and 3.48 (H-6b),
together with their coupling constants, suggested that the basic
skeleton of 5 is also a glucopyranose backbone and again was
supported by the HMBC correlations shown in Figures 3 and
S12 (Supporting Information). The NOESY correlations in
Figures 3 and S13 (Supporting Information) showing H-1/H-
3/H-5 cross-peaks substantiated this determination, and the δC
99.4 resonance in the 13C NMR spectrum (Figure S14, Table
S2, Supporting Information) indicated a β anomeric structure.
The correlations in the HMBC spectrum (Figures 3 and S12,
Supporting Information) of H-2 (δH 5.46)/C-A-1(δC 174.5),
H-3 (δH 5.49)/C-B-1 (δC 172.3), and H-4 (δH 5.31)/C-C-
1(δC 174.6) were indicative of the linkage of the fatty acid
moiety with the basic glucose structure. Furthermore, the
correlations in the HMBC spectrum (Figures 3 and S12,
Supporting Information) of H-A-2 (δH 2.45, methine proton)/
C-A-1 (δC 174.5) and H-C-2 (δH 2.34, methine proton)/C-C-
1(δC 174.6) together with the correlations in the COSY
spectrum (Figures 3 and S11, Supporting Information) of δH
1.12 (H-A-3 and H-A-4) to 2.45 (H-A-2) and δH 1.05 (H−C-
3) and 1.00 (H−C-4) to 2.34 (H−C-2) suggested that two
isobutyryl ester moieties are attached to the basic glucose
structure. In addition, the correlations in the COSY spectrum
(Figures 3 and S11, Supporting Information) of δH 0.88 (H-B-
9 and B-10, each 3H) and δH 1.47 (H-B-8, 1H) suggested the
occurrence of other types of fatty acid moieties with isopropyl
terminal branching structures. Furthermore, the molecular
formula and index of hydrogen deficiency indicated that the
fatty acid moiety is an 8-methylnonanoyl ester at the C-4
position of glucopyranose. To verify the structure, methyl 8-
methylnonanoate (7) was synthesized (Scheme 1, Figure S16
in Supporting Information). Compound 5 was treated with
Figure 1. Structures of pennelliisides A−C and their derivatives.
Supporting Information) between the signals at δH 4.36 (H-1,
J1,2 = 6.3 Hz), 5.45 (H-2, J2,3 = 8.6 Hz), 5.43 (H-3, J3,4 = 9.3
Hz), 5.29 (H-4, J4,5 = 9.3 Hz), 3.39 (H-5), and 3.47 (H-6),
together with their coupling constants (Figure 2A), suggested
that the basic skeleton of 4 is a glucopyranose backbone, as
supported by the HMBC correlations (Figures 2B and S5,
Supporting Information). The NOESY correlations shown in
Figures 2C and S6 (Supporting Information) showing H-1/H-
3/H-5 cross-peaks substantiated this determination, and the δC
99.5 resonance in the 13C NMR spectrum (Figure S7 and
Table S1, Supporting Information) indicated a β anomeric
structure. The HMBC correlations (Figures 2 and S5,
Supporting Information) of H-2 (δH 5.45)/C-A-1 (δC
174.4), H-3 (δH 5.43)/C-B-1 (δC 175.5), and H-4 (δH
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Figure 2. Key 1H NMR resonances and COSY (blue), HMBC (red), and NOESY correlations of compound 4. NOESY interactions were observed
between each proton filled with the same color.
Figure 3. Key 1H NMR resonances and COSY (blue), HMBC (red), and NOESY correlations of compound 5. NOESY interactions were observed
between each proton filled with the same color.
MeONa in anhydrous MeOH to give a crude mixture
containing methyl esters, which was subjected to GC-MS
analysis. The GC-MS chromatogram of the synthesized methyl
8-methylnonanoate (7) is given in Figure S17 (Supporting
Information), which showed that the retention time of the
target compound was at 12.2 min, and GC-MS features are
given in Figure S18 (Supporting Information). The GC-MS
chromatogram of the crude sample derived from compound 5
C
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4.39 (H-1, J1,2 = 7.3 Hz), 5.47 (H-2, J2,3 = 9.7 Hz), 5.49 (H-3,
J3,4 = 9.6 Hz), 5.32 (H-4, J4,5 = 9.6 Hz), 3.43 (H-5), and 3.48
(H-6) together with their coupling constants suggested that
the basic skeleton of 6 has a glucopyranose backbone, which
was supported by HMBC correlations (Figures 4 and S24,
Supporting Information). The NOESY correlations in Figures
4 and S25 showing H-1/H-3/H-5 cross-peaks substantiated
this determination, and the δC 99.4 resonance in the 13C NMR
spectrum (Figure S26 and Table S3, Supporting Information)
indicated a β anomeric structure. The correlations in the
HMBC spectrum (Figures 4 and S24, Supporting Information)
of H-2 (δH 5.47)/C-A-1(δC 174.5), H-3 (δH 5.49)/C-B-1 (δC
172.3), and H-4 (δH 5.32)/C-C-1(δC 174.6) were indicative of
the linkage of the fatty acid moiety with the basic glucose
structure. Furthermore, the correlations in the HMBC
spectrum (Figures 4 and S24, Supporting Information) of H-
A-2 (δH 2.45, methine proton)/C-A-1 (δC 174.5) and H-C-2
(δH 2.34, methine proton)/C-C-1(δC 174.6) together with the
correlations in the COSY spectrum (Figures 4 and S23,
Supporting Information) of δH 1.14−0.98 (H-A-3 and H-A-4)
to 2.45 (H-A-2) and δH 1.14−0.98 (H-C-3 and H-C-4) to 2.34
(H−C-2) suggested that two isobutyryl esters are attached to
the basic glucose structure. In addition, the molecular formula,
the index of hydrogen deficiency, and the correlations in the
HMBC spectrum (Figures 4 and S24, Supporting Information)
of δH 2.22 (H-3, 2H)/δC 172.3 (C-B-1) together with the δH
0.91 (H-B-10, 3H, triplet) resonance in the 1H NMR spectrum
(Figure S22, Supporting Information) indicated the decanoyl
ester moiety to be attached at the 3-position of the basic
glucopyranose structure. Finally, the HMBC correlations
between the phenylmethylene protons of δH 4.75/4.45 (H-
Scheme 1. Synthesis of Compound 7
is given in Figures S19 and S20 (Supporting Information), in
which a peak having almost the same GC-MS features as that
of synthesized 7 was observed at 12.4 min. Thus, it was found
that 8-methylnonanoate is attached to the basic glucopyranose
structure at the C-3 position to form an ester. Finally, the
HMBC correlations between the phenylmethylene protons of
δH 4.75/4.46 (H-1a′ and -1b′) and anomeric carbon C-1 (δC
99.4) and between the phenylmethylene protons of δH 4.35/
4.31 (H-1a″ and -1b″) and C-6 (δC 69.1) indicated that two
benzyloxy groups are attached to the C-1 and C-6 positions.
Therefore, it could be proposed that the chemical structure of
5 is 1,6-O-dibenzyl-3-O-(8-methylnonanoyl)-2,4-O-diisobutyr-
1
yl-β-D-glucose. Analysis of the H NMR, 13C NMR, DEPT,
HMQC, and HMBC spectroscopic data gave the complete 1H
and 13C NMR assignments for 5, as listed in Table S2
(Supporting Information).
Compound 6 was isolated as a colorless oil. The molecular
formula, C38H54O9, was assigned from the HRFDMS data (m/
z 654.3769 [M]+, calcd 654.3767) (Figure S21, Supporting
Information), based on which the index of hydrogen deficiency
1
was once more calculated to be 12. The H NMR resonances
(Figure S22 and Table S3, Supporting Information) and the
correlations observed in the COSY spectrum (Figure 4 and
Figure S23, Supporting Information) between the signals at δH
Figure 4. Key 1H NMR resonances and COSY (blue), HMBC (red), and NOESY correlations of compound 6. NOESY interactions were observed
between each proton filled with the same color.
D
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Table 1. NMR Spectroscopic Data for Compound 1 in CDCl3 (500 MHz)
α anomer
β anomer
position
δC
90.2
71.2
68.6
68.5
69.4
61.0
176.2
34.1−33.6
19.4−18.5
19.0−18.5
175.8
34.1−33.6
19.4−18.5
19.0−18.5
176.8
type
δH, mult. (J in Hz)
position
δC
95.7
73.3
72.3
68.3
74.4
61.1
177.2
34.1−33.6
19.4−18.5
19.0−18.5
175.9
34.1−33.6
19.4−18.5
19.0−18.5
176.4
type
δH, mult. (J in Hz)
1
2
3
4
5
6
A-1
A-2
A-3
A-4
B-1
B-2
B-3
B-4
C-1
C-2
C-3
C-4
CH
CH
CH
CH
CH
CH2
C
CH
CH3
CH3
C
CH
CH3
CH3
C
CH
CH3
CH3
5.41 d (3.6)
4.86, dd (10.9, 3.6)
5.59, dd (11.1, 10.9)
4.97, dd (11.1, 9.9)
4.00, br d (10.0)
1
2
3
4
5
6
A1
A2
A3
A4
B1
B2
B3
B4
C1
C2
C3
C4
CH
CH
CH
CH
CH
CH2
C
CH
CH3
CH3
C
CH
CH3
CH3
C
CH
CH3
CH3
4.68, d (8.0)
4.82, dd, (10.2, 8.0)
5.32, dd (10.2 9.4)
5.00, dd, (9.7, 9.4)
3.50, m
3.70−3.60, m, 3.53−3.45, m
3.70−3.60 m, 3.53−3.45, m
2.51−2.38, m
1.11−0.99, m
1.11−1.01, m
2.51−2.38, m
1.11−0.99, m
1.11−1.01, m
2.51−2.38, m
1.11−0.99, m
1.11−1.01, m
2.51−2.38, m
1.11−0.99, m
1.11−1.01, m
34.1−33.6
19.4−18.5
19.0−18.5
2.51−2.38, m
1.11−0.99, m
1.11−1.01, m
34.1−33.6
19.4−18.5
19.0−18.5
2.51−2.38, m
1.11−0.99, m
1.11−1.01, m
Table 2. NMR Spectroscopic Data for Compound 2 in CDCl3 (500 MHz)
α anomer
β anomer
position
δC
90.2
71.2
68.9
68.8
70.4
61.1
177.2
type
δH, mult. (J in Hz)
position
δC
type
δH, mult. (J in Hz)
1
2
3
4
5
6
CH
CH
CH
CH
CH
CH2
C
5.42 d (3.2)
4.80, dd (13.7, 3.2)
5.61, dd (13.7, 10.3)
4.93, dd (10.3, 9.5)
4.00, br d (9.0)
1
2
3
4
5
6
95.7
71.2
71.3
73.5
74.5
70.5
176.3
CH
CH
CH
CH
CH
CH2
C
4.68 d (7.7)
4.78, dd (10.2, 7.7)
5.32, dd (10.2 9.7)
4.99, dd (9.7, 8.8)
3.48, m
3.71−3.57, m, 3.54−3.45, m
3.71−3.57, m, 3.54−3.45, m
A-1
A-2
A-3
A-4
B-1
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
C-1
C-2
C-3
C-4
A1
A2
A3
A4
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
C1
C2
C3
C4
34.1−33.6
19.0−18.5
19.0−18.5
172.7
34.4−33.5
29.1
24.8
29.4
27.1
38.9
27.9
22.4
22.4
176.8
34.1−33.6
19.0−18.5
19.0−18.5
CH
CH3
CH3
C
2.53, m
1.11−1.01, m
1.11−1.01, m
34.1−33.6
19.0−18.5
19.0−18.5
172.7
34.4−33.5
29.1
24.8
29.4
27.1
38.9
27.9
22.4
22.4
176.5
34.1−33.6
19.0−18.5
19.0−18.5
CH
CH3
CH3
C
2.53, m
1.11−1.01, m
1.11−1.01, m
CH2
CH2
CH2
CH2
CH2
CH2
CH
CH3
CH3
C
2.15, 7 (7.0)
1.46, m
CH2
CH2
CH2
CH2
CH2
CH2
CH
CH3
CH3
C
2.15, 7 (7.0)
1.46, m
1.23−1.10, m
1.23−1.10, m
1.23−1.10, m
1.23−1.10, m
1.43, m
1.23−1.10, m
1.23−1.10, m
1.23−1.10, m
1.23−1.10, m
1.43, m
0.78, d (6.5)
0.78, d (6.5)
0.78, d (6.5)
0.78, d (6.5)
CH
CH3
CH3
2.53, m
1.11−1.01, m
1.11−1.01, m
CH
CH3
CH3
2.53, m
1.11−1.01, m
1.11−1.01, m
1a′ and -1b′) and anomeric carbon C-1 (δC 99.4) and between
the phenylmethylene protons of δH 4.35/4.31 (H-1a″ and
-1b″) and C-6 (δC 69.1) correspond with two substituted
benzyloxy groups being attached at the C-1 and C-6 positions.
Therefore, it was determined that the chemical structure of 6 is
1,6-O-dibenzyl-3-O-decanoyl-2,4-O-diisobutyryl-β-D-glucose.
Compounds 4 (25 mg), 5 (13 mg), and 6 (11 mg) were
dissolved in ethyl acetate and mixed with palladium black
under a hydrogen atmosphere with balloon pressure to give
pennelliisides A (1, 7 mg), B (2, 11 mg), and C (3, 4 mg),
respectively.
Pennelliiside A (1) was obtained as a colorless oil. The
molecular formula, C18H30O9, was determined from the
HRFDMS data (m/z 391.1983 [M]+, calcd 391.1968) (Figure
S28, Supporting Information). Since the chemical structure of
4 was determined to be 1,6-O-dibenzyl-2,3,4-O-triisobutyryl-D-
glucose, that of 1 should be 2,3,4-O-triisobutyryl-D-glucose.
1
Analysis of the H NMR, 13C NMR, DEPT, HMQC, and
1
HMBC spectroscopic data gave the complete H and 13C
NMR assignments for 6, as listed in Table S3 (Supporting
Information).
E
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Table 3. NMR Spectroscopic Data for Compound 3 in CDCl3 (500 MHz)
α anomer
β anomer
type
position
δC
91.1
72.0
69.8
69.5
type
δH, mult. (J in Hz)
position
δC
96.6
74.3
72.1
69.3
75.3
62.0
178.1
34.6−34.9
20.2−19.1
20.2−19.1
173.5
35.0
25.6
29.7−30.7
29.7−30.7
32.7
23.5
14.9
δH, mult. (J in Hz)
1
2
3
4
CH
CH
CH
CH
CH
CH2
C
CH
CH3
CH3
C
CH2
CH2
CH2
CH2
CH2
CH2
CH3
C
5.41 d (3.6)
4.83, dd (10.6, 3.6)
5.65, t (11.0)
4.97, br. t (10.0)
4.05, br d (10.6)
3.66, m, 3.55, m
1
2
3
4
5
6
CH
CH
CH
CH
CH
CH2
C
CH
CH3
CH3
C
CH2
CH2
CH2
CH2
CH2
CH2
CH3
C
4.68 d (8.0)
4.83, br t (10.0)
5.38, t (10.2)
5.00, br t, (9.3)
3.54, m
5
70.4
6
61.9
3.66, m, 3.55, m
A-1
A-2
A-3
A-4
B-1
B-2
B-3
B-4 - B-6
B-7
B-8
B-9
B-10
C-1
C-2
C-3
C-4
177.1
A1
A2
A3
A4
B1
B2
B3
B4 - B-6
B7
B8
B9
B10
C1
C2
C3
C4
34.6−34.9
20.2−19.1
20.2−19.1
173.5
35.0
25.6
29.7−30.7
29.7−30.7
32.7
23.5
14.9
2.53, m
1.15−1.08, m
1.15−1.08, m
2.53, m
1.15−1.08, m
1.15−1.08, m
2.20, dd (7.8, 7.5)
1.51, m
1.30−1.20, m
1.26, m
1.28, m
1.25, m
0.85, t (7.3)
2.20, dd (7.8, 7.5)
1.51, m
1.30−1.20, m
1.26, m
1.28, m
1.25, m
0.85, t (7.3)
177.7
177.3
34.6−34.9
20.2−19.1
20.2−19.1
CH
CH3
CH3
2.53, m
1.15−1.08, m
1.15−1.08, m
34.6−34.9
20.2−19.1
20.2−19.1
CH
CH3
CH3
2.53, m
1.15−1.08, m
1.15−1.08, m
Burke et al. reported the isolation of 2,3,4-O-triisobutyryl-D-
glucose from S. pennellii.14 For convenience, compound 1 was
named pennelliiside A, although the compound was previously
reported. The reported 1H and 13C NMR spectra showed good
accordance with those recorded in this study (Figures S29 and
S30, Supporting Information). The COSY, HMQC, and
HMBC spectra of 1 are given in Figures S31−S34, Supporting
Information. Compound 1 contained α and β anomers, and it
was revealed that the ratio of α and β anomers was ca. 2:1 from
the peak area ratio of the resonances of the H-1 position in α
and β anomers. The individual assignments of α and β
anomers were clarified by the following procedure. The
resonances of H-1 of α and β anomers were determined to
be δH 5.41 and 4.68, respectively, due to the 13C NMR
chemical shifts of directly attached carbon with the protons,
such as δC 90.2 for H-1α and δC 95.7 for H-1β together with
their coupling constant values as 3.6 Hz of H-1α and 8.0 Hz of
H-1β. Partial COSY spectra for 1 are given in Figure S32,
which revealed the networks as H-1α (δH 5.41)/H-2α (δH
4.86)/H-3α (δH 5.59)/H-4α (δH 4.97)/H-5α (δH 4.00)/H-6α
(δH 3.70−3.60 and 3.53−3.45) for the α anomer and H-1β (δH
4.68)/H-2β (δH 4.82)/H-3β (δH 5.32)/H-4β (δH 5.00)/H-5β
(δH 3.50)/H-6β (δH 3.70−3.60 and 3.53−3.45) for the β
anomer. Since there were overlapping resonances of acyl
moieties, complete total assignments of the α and β anomers
for each were not able to be accomplished. However, partial
1H and 13C NMR assignments for 1 are listed in Table 1.
Pennelliiside B (2) was obtained as a colorless oil. The
molecular formula, C24H42O9, was determined from the
HRFDMS data (m/z 475.2889 [M + H]+, calcd 475.2907)
B-10) (Figure S35, Supporting Information). Since the
chemical structure of 5 was determined to be 1,6-O-dibenzyl-
3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-β-D-glucose, that
of 2 should be 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-D-
1
glucose. Analysis of the H NMR, 13C NMR, DEPT, HMQC,
HMBC, and HSQC-TQCSY spectroscopic data provided only
partial 1H and 13C NMR assignments for 2 due to the
overlapping resonance of acyl moieties as listed in Table 2.
However, assignments of α and β anomers attributed to the D-
glucose structure were accomplished according to the
procedure employed for compound 1. Partial COSY spectra
for 2 are given in Figure S39 (Supporting Information). The
ratio of α and β anomers was ca. 2:1, which was deduced from
the peak area ratio of the resonances of the H-1 position in the
α and β anomers.
Pennelliiside C (3) was isolated as a colorless oil. The
molecular formula, C24H42O9, was determined from the
HRFDMS data (m/z 475.2913 [M + H]+, calcd 475.2907)
(Figure S43, Supporting Information). Similar to pennelliiside
A (1) and pennelliiside B (2), compound 3 showed complex
resonances and cross-peaks in the 1H NMR, 13C NMR, COSY,
HMBC, HSQC, and HSQC-TQCSYspectra (Figures S44−50,
Supporting Information), but the signals were evident for 2H
protons for each at δH 2.53 (m, H-A-2 and H-C-2) and the 3H
triplet terminal methyl protons at δH 0.85 (H-B-10) (Figure
S44, Supporting Information). Since the chemical structure of
6 was determined as 1,6-O-dibenzyl-3-O-decanoyl-2,4-O-
diisobutyryl-β-D-glucose, that of 3 should be 3-O-decanoyl-
1
2,4-O-diisobutyryl-D-glucose. Analysis of the H NMR, 13C
NMR, DEPT, HMQC, HMBC, and HSQC-TQCSY spectro-
1
(Figure S35, Supporting Information). The H NMR, 13C
scopic data yielded the partial H and 13C NMR assignments
1
NMR, COSY, HMBC, HSQC, and HSQC-TQCSY spectra
(Figures S36−S42, Supporting Information) were complex
features, but they also gave distinctive signals attributed to 2H
multiplet methine protons at δH 2.53 (H-A-2 and H-C-2) and
the 6H doublet terminal methyl protons at δH 0.78 (H-B-9/H-
for 3 due to the overlapping resonance of acyl moieties as
listed in Table 3. However, assignments of α and β anomers
attributed to the D-glucose structure were accomplished
according to the procedure employed for compound 1. Partial
COSY spectra for 3 are given in Figure S47 (Supporting
F
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Figure 5. Root elongation assessment of acylglucose and their constituted fatty acids. Assays were performed by preparing MS medium (1/40
concentration) and mixing each test compound, (A) penelliside A (1), (B) penelliside B (2), (C) and penelliside C (3), and their constituent fatty
acids, (D) isobutyric acid (8), (E) 8-methylnonanoic acid (9), and (F) decanoic acid (10). Error bars indicate SE (n = 8; results are from one
representative experiment) (*: p < 0.05, **: p < 0.01, ***: p < 0.001 are significantly different by Welch’s t-test).
Information). The ratio of α and β anomers was ca. 2:1, which
was deduced from the peak area ratio of the resonances of the
H-1 position in α and β anomers.
reported method,16 except that ethyl 5-bromopentanoate and
methyl 6-bromohexanoate, respectively, were used, followed by
treatment with a solution of NaOH in EtOH in each case, and
the concentrations of the compounds were set to 50 μM. As a
result, Figure 6, 8-methylnonanoic acid (9), 4-methylpentanoic
acid (12), 5-methylhexanoic acid (13), 6-methylheptanoic acid
(14), and 7-methyloctanoic acid (15) showed more potent
activity than those of isobutyric acid (8) and isovaleric acid
(11), even though isobutyric acid (8) showed a significant
inhibitory effect on seed germination (Figure 5D). Figure 6
In a previous paper, Peterson et al. reported in 1997 that
acyl sucrose acts as a phytoalexin to inhibit the seed
germination of velvetleaf.15 Thus, the biological activities of
penelliiside A (1), penelliiside B (2), and penelliiside C (3)
were evaluated. Root elongation was assessed for 1−3 and
their constituent fatty acids, isobutyric acid (8), 8-methyl-
nonanoic acid (9), and decanoic acid (10). Compound 9 was
synthesized according to a reported method,16 except that
methyl 7-bromoheptanoate was used, followed by treatment
with a solution of NaOH in EtOH. The compounds tested
were dissolved in MeOH to final concentrations of 10, 50, and
100 μM, and the seeds of Arabidopsis thaliana were used for
the assay. The results are given in Figure 5; no activity was
observed for penelliiside A (1), penelliiside B (2), penelliiside
C (3), and decanoic acid (10), although isobutyric acid (8)
and 8-methylnonanoic acid (9) did show activity.
Since a fatty acid with a longer carbon chain with an
isopropyl terminal branching structure showed more potent
activity than that with a shorter carbon chain, an activity
correlation test focusing on carbon chain length was
performed. For the evaluation, fatty acids with isopropyl
terminal branching structures, namely, isobutyric acid (8),
isovaleric acid (11), 4-methylpentanoic acid (12), 5-methyl-
hexanoic acid (13), 6-methylheptanoic acid (14), 7-methyl-
octanoic acid (15), and 8-methylnonanoic acid (9), were used.
Compounds 14 and 15 were synthesized according to a
Figure 6. Activity correlation test between carbon chain length and
root elongation inhibitory activity. Arabidopsis thaliana incubated for
2 weeks was used. MS medium (1/40 concentration) was prepared
mixed with each saturated terminal branched fatty acids 8, 9, and 11−
15. Error bars indicate SE (n = 8; results are from one representative
experiment). The plots that do not share the same letter differ
significantly between the treatments (p < 0.05, Tukey−Kramer’s test).
G
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Figure 7. Herbicidal activity test of 8-methylnonanoic acid (9) and nonanoic acid (16). The spray solution was adjusted to a sample concentration
of 5 mg/mL, ethanol 2%, Tween 20 0.3% and sprayed on A. thaliana grown for 30 days under long-day conditions.
FDMS and FIMS analyses were performed on a JMS-T100GCV
(JEOL) instrument. GC-MS analysis was completed on a Varian CP-
3800 gas chromatograph with a Varian 1200L quadrupole MS/MS in
electron ionization mode. Compounds 8, 10−13, and 16 and all
reagents were purchased from Kanto Chemical Co., Inc.
Plant Material. Seeds of Solanum pennellii were obtained from the
National Bioresource Project (NBRP, Tsukuba). The plants were
grown in an artificial weather room (16 h light, 8 h dark, room
temperature 25 °C) of the Faculty of Agriculture, Hokkaido
University, Hokkaido, Japan, in 2018 and 2019. The plants were
used for experiments approximately 80 days after sowing.
Extraction and Isolation. The aerial parts of the plants (1.7 kg)
were rinsed by dipping the shoots in EtOH (5 L) for 30 s, and the
epicuticular lipophilic extract was filtered and concentrated using a
rotary evaporator, yielding a crude material. This crude material was
then partitioned with EtOAc (500 mL) and saturated aqueous
NaHCO3 (500 mL). The organic layer was dried over MgSO4 and
concentrated, yielding a crude material, which was subjected to silica
gel column chromatography (MeOH−CHCl3−CH3COOH,
5:95:0.1) to give a subfraction containing acylglucose (27 g). To a
stirred mixture of the material containing acylglucose (4.5 g) in 1,4-
dioxane (100 mL) were added TriBOT (300 mg) and TfOH (0.035
mL) under a N2 atmosphere, and the reaction mixture was stirred for
16 h at room temperature. The volatile components of the reaction
mixture were removed under reduced pressure to give an oil, which
was subjected to silica gel column chromatography (100 g, EtOAc−n-
hexane−CH3COOH, 1:4:0.1) to give subfractions Fr-1 (1.0 g) and
Fr-2 (4.1 g). Fr-1 and Fr-2 were subjected to HPLC (InertSustain C18,
10 × 250 mm, 2.5 mL/min, A210 nm, MeOH−H2O−CH3COOH,
91:1:0.1) to give 4 (47 mg), 5 (33 mg), and 6 (17 mg).
indicates that the root length decreased as the length of the
carbon chain increased up to six carbon atoms and remained
almost the same for carbon chains of six and 10 carbon atoms,
and 8-methylnonanoic acid (9) showed the most potent
activity of the test compounds.
A weed-withering herbicide containing nonanoic acid (16)
as a key ingredient is on the market. Therefore, it was assumed
that 8-methylnonanoic acid (9) also has an ability to be used as
an herbicide. An herbicidal activity test was performed on the
basis of a previous report.17 Solutions of the test samples (5
mg/mL), ethanol (2%), and Tween 20 (0.3%) were sprayed
once on the leaves of A. thaliana. As a result, 8-methylnonanoic
acid (9) showed approximately the same level of activity as
nonanoic acid (16) (Figure 7).
In this investigation, one reported and two new acylglucoses
named pennelliisides A (1), B (2), and C (3) were isolated
from a wild tomato species, S. pennellii, although derivatization
to a benzyl ether was needed to isolate the compounds in their
pure forms. Since a reaction to remove the benzyl ether group
using palladium black under a H2 atmosphere was required to
isolate the native compounds, the strategy adopted in this
study cannot be used for isolation of compounds that react
under the conditions of palladium black under a H2
atmosphere. Although the adopted strategy had drawbacks, it
was possible to isolate the compounds, discern the chemical
nature of the compounds, and measure their physical and
spectroscopic parameters. Based on the obtained information,
synthesis of internal standards of the isolated compounds
could be possible, which would enable endogenous amounts of
the compounds to be evaluated in the observed biological
events, such as whether the biosynthesis of acylglucose is
affected by fluctuations of plant hormones or not. Further-
more, it was found that pennelliisides A (1), B (2), and C (3)
did not have an inhibitory effect on root elongation itself, but
isobutyric acid (8) and 8-methylnonanoic acid (9) exhibited
such activity, which led to the hypothesis that their biological
activity might be achieved when the plants withered. Nonanoic
acid (16) is commercially used as an herbicide component,
and it was very interesting to have discovered that S. pennellii
uses a compound with a similar skeleton to combat other
plants.
1,6-O-Dibenzyl-2,3,4-O-triisobutyryl-β-D-glucose (4). Yellow oil;
1
[α]20 +31 (c 0.5, CHCl3); H and 13C NMR see Table S1, Figures
D
S3 and S7 (Supporting Information); HRFDMS m/z 570.28225 [M]+
(calcd for C32H42O9 570.28288).
1,6-O-Dibenzyl-2,4-O-diisobutyryl-3-O-(8-methylnonanoyl)-β-D-
1
glucose (5). Oil; [α]20 −19 (c 0.6, CHCl3); H and 13C NMR see
D
Table S2, Figures S10 and S14 (Supporting Information); HRFDMS
m/z 654.37501 [M]+ (calcd for C38H54O9 654.37679).
1,6-O-Dibenzyl-3-O-decanoyl-2,4-O-diisobutyryl-β-D-glucose (6).
1
Oil; [α]20 −16 (c 0.6, CHCl3); H and 13C NMR see Table S3,
D
Figures S22 and S26 (Supporting Information); HRFDMS m/z
654.37678 [M]+ (calcd for C38H54O9 654.37679).
Synthesis of Methyl 8-Methylnonanoate (7). Compound 7
(560 mg, 3 mmol, 13%) was synthesized according to a reported
method except for using methyl 7-bromoheptanoate.16
Methyl 8-methylnonanoate (7). 1H NMR (CDCl3, 270 MHz,
Figure S16, Supporting Information) δ 3.53 (3H, s), 2.20 (2H, t, J =
7.1 Hz), 1.64−1.35 (3H, m), 1.30−1.00 (8H, m), 1.10−0.96 (2H, m),
0.76 (6H, d, J = 6.4 Hz); GC-MS found, m/z 186 (Figures S17 and
S18, Supporting Information).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
obtained with a JASCO P-2200 polarimeter. NMR spectra were
recorded in CDCl3 and benzene-d6 using a JNM-EX 270 FT-NMR
spectrometer (JEOL, 1H NMR: 270 MHz, 13C NMR: 67.5 MHz) and
AMX 500 (Bruker, 1H NMR: 500 MHz, 13C NMR: 126 MHz).
Analysis of Methyl 8-Methylnonanoyl Moiety in Compound
2. To a stirred solution of 5 (10 mg) in anhydrous MeOH (1 mL)
was added a powder of NaOMe (10 mg), and the reaction mixture
H
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was further stirred for 24 h. The usual workup was employed to give a
solution containing methyl ester in hexane. A portion (1 μL) of the
solution was analyzed using GC-MS in the following manner. For the
analysis of the methyl ester using GC-MS, the injection temperature
was 200 °C, and a fused-silica capillary column (BetaDEX120; 30 m ×
0.25 mm i.d., 0.25 μm film thickness; GL Sciences) was used. The
temperature program started at 80 °C for 1 min and subsequently
increased at 5 °C/min to 220 °C, which was maintained for 1 min.
Helium was used as the carrier gas at a linear velocity of 1.2 mL/min,
and all spectra were scanned within the range m/z 10−600. A GC-MS
chromatogram and a chart of the MS pattern are given in Figures S19
and S20, Supporting Information, respectively.
AUTHOR INFORMATION
Corresponding Author
■
Hideyuki Matsuura − Research Faculty of Agriculture,
Hokkaido University, Sapporo 060-8589, Japan; orcid.org/
0000-0002-9605-7188; Phone: +81-11-706-2495;
Email: matsuura@chem.agr.hokudai.ac.jp
Authors
Tenki Nakashima − Research Faculty of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan
Yurika Nambu − Research Faculty of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan
Yutaka Inoue − Research Faculty of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan
Rishni Masimbula − Research Faculty of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan
Replacements of Benzyl Ether Groups to Give Compounds
1−3. To a stirred mixture of 4 (25 mg, 44 mmol) in EtOAc (1 mL)
was added palladium black (4 mg) under a H2 atmosphere with
balloon pressure, and the reaction mixture was further stirred for 3 h
at room temperature. The reaction mixture was filtered using Celite
and concentrated under reduced pressure to give an oil, which was
subjected to silica gel chromatography to give compound 1 (2,3,4-O-
triisobutyryl-D-glucose, 7 mg, 19 mmol, 43%). Compounds 2 (2,4-O-
diisobutyryl-3-O-(8-methylnonanoyl)-D-glucose, 11 mg, 23 mmol,
49%) and 3 (3-O-decanoyl-2,4-O-diisobutyryl-D-glucose, 4 mg, 8
mmol, 54%) were obtained following the same method, except that 5
(13 mg, 20 mmol) and 6 (11 mg, 17 mmol) were used, respectively.
Synthesis of Compounds 9, 14, and 15. Compounds 9, 14,
and 15 were synthesized according to a reported method16 except for
using ethyl 6-bromohexanoate, ethyl 5-bromobutanoate, and ethyl 6-
bromopentanoate followed by treatment with a solution of NaOH in
EtOH to give 9 (78 mg, 4.5 × 10−1 mmol, 71%), 14 (1.1 g, 6.1 mmol,
54%), and 15 (2.0 g, 12 mmol, 59%), respectively.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b01234
Author Contributions
†T. Nakashima and Y. Namubu contributed equally to this
work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Mr. Y. Takata and Dr. E.
Fukushi (Faculty of Agriculture, Hokkaido University) for
assistance in obtaining the spectroscopic data.
1
8-Methylnonanoic Acid (9). H NMR (CDCl3, 270 MHz, Figure
S51, Supporting Information) δ 2.34 (2H, t, J = 7.1 Hz), 1.74−1.42
(3H, m), 1.40−1.00 (8H, m), 0.86 (6H, d, J = 6.6 Hz).
6-Methylheptanoic Acid (14). 1H NMR (CDCl3, 270 MHz, Figure
S52, Supporting Information) δ 2.28 (2H, t, J = 7.8 Hz), 1.67−1.37
(3H, m), 1.35−1.00 (4H, m), 0.80 (6H, d, J = 7.62 Hz).
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