Nodupetide, a potent insecticide and antimicrobial from Nodulisporium sp. associated with Riptortus pedestris

Article abstract of DOI:10.1016/j.tetlet.2017.01.009

Nodupetide (1), a new cyclodepsipeptide unique in its incorporation of a 3-hydroxy-4-methylhexanoic acid (HMHA) derived motif, was discovered from Nodulisporium sp. IFB-A163, a fungus residing in the insect (Riptortus pedestris) gut. The nodupetide structure was elucidated by its MS/MS and 2D NMR spectra, and its absolute configuration by the X-ray crystallography and modified Marfey's method. Nodupetide is insecticidal against rice brown planthopper (Nilaparvata lugens) with an LD₅₀value of 70?ng/larva, and inhibitory towards the drug-resistant human pathogenic bacterium Pseudomonas aeruginosa with its MIC value (5.0?μM) comparable to that (3.2?μM) of ciprofloxacin, a prescribed antibacterial agent co-assayed equally.

Full text of DOI:10.1016/j.tetlet.2017.01.009

Tetrahedron Letters 58 (2017) 663–665
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Tetrahedron Letters
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Nodupetide, a potent insecticide and antimicrobial from Nodulisporium
sp. associated with Riptortus pedestris
Hui Min Wu a,1, Li Ping Lin a,b,c,1, Qin Lan Xu a, Wen Bo Han a, Song Zhang d, Ze Wen Liu d, Ya Ning Mei e,
Zhu Jun Yao a, Ren Xiang Tan a,c,
⇑
a Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China
b Jiangsu Center for Research & Development of Medicinal Plants, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
c School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
d Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
e Department of Clinical Laboratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nodupetide (1), a new cyclodepsipeptide unique in its incorporation of a 3-hydroxy-4-methylhexanoic
acid (HMHA) derived motif, was discovered from Nodulisporium sp. IFB-A163, a fungus residing in the
insect (Riptortus pedestris) gut. The nodupetide structure was elucidated by its MS/MS and 2D NMR spec-
tra, and its absolute configuration by the X-ray crystallography and modified Marfey’s method.
Nodupetide is insecticidal against rice brown planthopper (Nilaparvata lugens) with an LD50 value of
70 ng/larva, and inhibitory towards the drug-resistant human pathogenic bacterium Pseudomonas
Received 28 December 2016
Accepted 3 January 2017
Available online 4 January 2017
Keywords:
Nodulisporium sp.
Riptortus pedestris
Cyclodepsipeptide
Absolute configuration
Insecticidal and antimicrobial activities
aeruginosa with its MIC value (5.0
lM) comparable to that (3.2 lM) of ciprofloxacin, a prescribed
antibacterial agent co-assayed equally.
Ó 2017 Elsevier Ltd. All rights reserved.
The newer generation of pesticides and agricultural antibiotics
is always indispensable for the continuous improvement of crop
productions to feed on the rapidly growing world’s population.1
However, the efficacy of currently applied insecticidal and antimi-
crobial agents keeps decreasing, or even vanishing, because of the
drug resistance development of crop-threatening pests and
microbes to most if not all of the marketed agrochemicals.2
Accordingly, there is a pressing need to search for novel leads for
insecticidal and antimicrobial agents, the latter being required as
well for the ‘‘war” against human microbial pathogens.3
cules to help counteract the challenges from bacterial infectors,
snatcher and predators.7 Based on these theories, we therefore
hypothesized that R. pedestris might be among the reliable host
of the microbes capable of producing insecticidal and/or antimicro-
bial chemicals. As a result, Nodulisporium sp. IFB-A163, an ana-
morph-form genus of Xylariaceae,8 was isolated from the R.
pedestris gut. Some Nodulisporium fungi were demonstrated to be
efficient producers of bioactive secondary metabolites such as
insecticidal indole terpenes,9 and viridins which inhibit amyloid
b-peptide aggregation involved in the Alzheimer’s disease inci-
dence.10 Those evidences inspired the subsequent bioassay-guided
fractionation of the extract derived from the scaled-up fermenta-
tion of the fungal strain IFB-A163. Then a structurally undescribed
cyclodepsipeptide named nodupetide (1) was afforded, rather than
indole terpenes and viridins, as the first cyclodepsipeptide member
characterized from cultures of Nodulisporium fungi.
The fungal strain IFB-A163 was cultured in the malt extract
(ME) medium (100 Â 400 mL) for 14 days at 28 °C with an agita-
tion of 200 rpm. Filtration of the culture gave the fermentation
broth which was partitioned with EtOAc with the mycelium
extracted with a methanol-dichloromethane mixture (1:1). The
obtained extracts were combined and purified by column chro-
matographies over silica gel, octadecyl silane (ODS), followed by
Sephadex LH-20 column with CH2Cl2-MeOH system (v/v 1:1)
Owing to the intimate host-microbe interaction,4 symbionts are
being recognized as versatile producers of small molecule sec-
ondary metabolites with unforeseeable architectures and diverse
bioactivities.4,5 Widely spread in China are the herbivorous insects
such as Riptortus pedestris (Hemiptera: Alydidae), which feed on
crop species such as soybean.6 From a symbiotic perspective,
insects usually harbor a particular community of microorganisms,
some of which might generate low-molecular-weight biomole-
⇑
Corresponding author at: Institute of Functional Biomolecules, State Key
Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
China; School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing
210023, China.
E-mail address: rxtan@nju.edu.cn (R.X. Tan).
H.M. Wu and L.P. Lin contributed equally.
1
http://dx.doi.org/10.1016/j.tetlet.2017.01.009
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

664
H.M. Wu et al. / Tetrahedron Letters 58 (2017) 663–665
6
O
5
Gly2
H
N
HMHA1
4
1
O
2
7
3
O
NH
Val3
O
Val6
O
HN
NH
Leu4
N
H
O
O
Ala5
COSY
HMBC
Fig. 1. Chemical and crystal structures of nodupetide (1).
purification to nodupetide (1, Fig. 1), which was evidenced to have
a molecular formula of C28H49N5O7 from the Na+-liganded molecu-
lar ion at m/z 590.3522 (calculated for C28H49N5O7Na:590.3524) in
its high resolution electrospray ionization mass spectrometry
(HR-ESI-MS).
Its depsihexapeptide nature was evidenced both from the five
characteristic amide proton signals in the range of dH 7.44–8.66
of its 1H NMR spectrum, and from the six carbonyl groups between
dC 169 and 172 in its 13C NMR spectrum (Table S1, Figs. S2–3). With
the information in mind, the assignment of its 1H and 13C NMR sig-
nals to the amino acid residues was subsequently accomplished by
interpreting its 1H-1H COSY, HSQC, and HMBC spectra (Table S1,
Figs. S5–7), indicating that 1 was likely composed of glycine
(Gly2), valine (Val3), leucine (Leu4), alanine (Ala5), and valine
(Val6) residues. Furthermore, a linear alkyl chain was indicated
by the HMBC correlations of a diastereotopic methylene (H2-2) sig-
nals at dH 2.54 and 2.25 with the carbonyl C-1 (d 169.8) and
oxymethine carbon C-3 (d 75.3), and of H-3 (dH 4.92) of
3-hydroxy-4-methylhexanol acid (HMHA) moiety to Val6-CO (d
170.7), suggesting the attachment of this oxymethine C-3 to C-2.
The absence of correlation between the CO (Val6) and an amide
proton associated with the HMBC correlation of the oxymethine
carbon C-3 (d 75.3) suggests that the ester linkage is at this posi-
tion. And the rest of this alkyl chain is easily characterized with
the COSY and HMBC correlations. Finally, these enabled the
determination of the complete cyclic sequence as cyclo [HMHA1-
Gly2-Val3-Leu4-Ala5-Val6] by the HMBC correlations of the amide
protons of amino acid residues with the corresponding carbonyls,
respectively. The sequence proposed for 1 was reinforced by its
LC-MS/MS analysis which pinpointed the successive cleavage of
amino acid moieties from its protonated molecular ion at m/z
568.3707 (Fig. 2).
Fig. 2. The nodupetide (1) sequence indicated by the pronounced fragment ions in
its ESI-MS/MS spectrum. HMHA, 3-hydroxy-4-methylhexanoic acid; Gly, glycine;
Val, valine; Leu, leucine; Ala, alanine.
crystallographic analysis with the anomalous dispersion of Cu Ka
radiation (Fig. 1). To our knowledge, 1 seems to be the first charac-
terized fungal metabolite with the 3-hydroxy-4-methylhexanoyl
motif, which is very rare in nature. Prior to the study, this substruc-
ture was only found in the secondary metabolites of the bacterium
Paenibacillus ehimensis13 and the plant Quillaja saponaria.14
Inspired by the biosynthetic pathways of emericellamide A
from Aspergillus nidulans15 and fusaristatin
A from Fusarium
The optical purity of 1 was secured by its specific rotation
graminearum,16 nodupetide (1) perhaps was biosynthetically of
mixed origins with polyketide and amino acid substructures.
According to the basic common logic of natural polyketide biosyn-
thesis,17 HMHA might be assembled under the catalysis of fungal
polyketide synthases (PKSs). The HMHA-based polyketide chain
may subsequently serve as a precursor to directly assimilated by
the downstream nonribosomal peptide synthetase,16 or be trans-
ferred to a CoA thioester by an acyl-CoA ligase and then loaded
onto an acyl-transferase, which subsequently shuttles the product
to the NRPS.15 Following the successive incorporation of the five
amide acid residues (Gly, Val, Leu, Ala, and Val), nodupetide (1)
is released through an intramolecular esterification reaction which
seems to be shared by the biosynthesis of fungal cyclodepsipep-
tides (Fig. 3).
([a
]
D
25 = À63.5°, c = 0.085, MeOH) and its CD spectrum (Fig. S1) and
the advanced Marfey’s method was subsequently applied to address
the absolute configuration of the cyclodepsipeptide.11,12 Hydrolysis
of 1 with 6 N HCl at 110 °C for 20 h gave a hydrolysate. After deriva-
tised with 1-fluoro-2,4-dinitro-5-(L)-alanine amide (FDAA), the
hydrolysate was demonstrated to contain Gly, L-Ala, L-Val, and D-
Leu by the HPLC comparison with the authentic samples in an ace-
tonitrile-water gradient (3:7 ? 7:3). However, this effort was unable
to clarify the absolute configuration of the 3-hydroxy-4-methylhex-
anoyl residue. We therefore resolved to obtain the single crystal of 1,
and fortunately succeeded. Relative to the established chirality of
L-Ala, L-Val, and D-Leu, the 3S- and 4S-configurations were assigned
for the 3-hydroxy-4-methylhexanoyl moiety of 1 through its X-ray

H.M. Wu et al. / Tetrahedron Letters 58 (2017) 663–665
NRPS
665
PKS
T
C
A
A
T
S
C
T
Cy
KS AT DH MT ER KR ACP
A
T
C
T
C
A
T
E
C
A
T
S
S
S
S
S
S
HO
CoA-S
O
O
O
O
O
O
O
O
O
NH
NH
OH
HN
HN
HN
HN
OH
OH
OH
HN
O
O
O
O
O
O
O
O
HN
NH
HN
NH
NH
OH
O
O
O
O
O
or
1
OH
HN
NH
O
HO
O
OH
HN
O
OH
OH
OH
Fig. 3. Proposed biosynthetic pathway for nodupetide (1) in Nodulisporium sp.
Using the purified sample, nodupetide (1) was examined to be
substantially insecticidal against the 3rd instar nymphae of brown
rice planthopper Nilaparvata lugens with its median lethal dose
(LD50) being 70 ng/larva.18,19 Furthermore, 1 was screened for its
antibacterial activities against Peptostreptococcus anaerobius,
Clostridium prazmowski, Pseudomonas aeruginosa, and Bacteroides
fragilis. To our surprise, 1 displayed exclusively an inhibitory action
A. Supplementary material
Supplementary data (1H, 13C NMR, DEPT, HSQC, HMBC, COSY,
NOESY, and HR-ESI-MS spectroscopic data for compound 1) associ-
ated with this article can be found in the online version.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2017.01.
009.
towards P. aeruginosa with its MIC value (5.0 lM) close to that of
ciprofloxacin (3.2 lM), a marketed antibacterial agent co-assayed
identically as a positive reference in the study.20
References
In summary, one of the major objectives concerning natural
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Acknowledgments
The work was successively financed by the National Natural
Science Foundation of China Grants (21672101, 81530089,
81421091, 21402090, 21132004, and 81503232).