Biosynthesis of Quinoline by a Stick Insect

Article abstract of DOI:10.1021/acs.jnatprod.0c00945

The Peruvian stick insect Oreophoetes peruana is the only known animal source for unsubstituted quinoline in nature. When disturbed, these insects discharge a defensive secretion containing quinoline. Analysis of samples obtained from l-[2′,4′,5′,6,′7′-2H

Full text of DOI:10.1021/acs.jnatprod.0c00945

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Biosynthesis of Quinoline by a Stick Insect
Athula B. Attygalle,* Kithsiri B. Hearth, Vikram K. Iyengar, and Randy C. Morgan
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ABSTRACT: The Peruvian stick insect Oreophoetes peruana is the only known
animal source for unsubstituted quinoline in nature. When disturbed, these insects
discharge a defensive secretion containing quinoline. Analysis of samples obtained
from L-[2′,4′,5′,6,′7′-2H5]tryptophan-fed stick insects demonstrated that the
insects convert it to [5,6,7,8-2H4]quinoline by removing the 2′-CH moiety in
the indole ring of tryptophan. Analogous experiments using L-[1′-15N]tryptophan
and L-[1′-15N,15NH2]tryptophan showed that the indole-N atom is retained while
the α-amino group is eliminated during the biosynthesis. Mass spectra recorded
from quinoline derived from [2-13C1]tryptophan-fed insects indicated that the α-
carbon atom of tryptophan is incorporated as the C-2 atom of the quinoline ring.
he so-called stick insects of the order Phasmatodea are
Ttypically cryptic, armed with spines, or protected by
camouflage by resemblance to twigs or leaves. Because they are
mostly wingless and slow moving, these animals are expected
to be vulnerable to predators. In this respect, the Peruvian fire
stick, Oreophoetes peruana (Floyd, 1993),1 is atypical because
these insects are strikingly colored. The males of O. peruana
are black with conspicuous bright red patches, while females
have orange markings with longitudinal yellow stripes.
Presumably, O. peruana, can exhibit its aposematic coloration
blatantly because it is protected by the allomones present in
their defensive glands. Previously, we reported that O. peruana
when disturbed ejects a white malodorous fluid from its
defensive glands in the prothorax, and quinoline (1) was
identified as the major constituent in the secretion.2
pharmaceutical preparations.7,8 Although many quinoline-
based alkaloids are known from plants, animals, and microbial
sources,9−16 unsubstituted quinoline as an animal natural
product has been identified only from O. peruana.2
A compound that bears the quinoline core, 6-hydroxykynur-
enic acid, has been shown to be biosynthesized from L-
tryptophan (2) in Nicotiana tabacum.16 In an analogous
manner, 8-hydroxyquinoline-2-carboxylic acid found in the
foregut content of the cotton leafworm Spodoptera littoralis has
been demonstrated to be generated from tryptophan via
kynurenine, kynurenic acid, and quinaldic acid.17 Xanthurenic
acid (4,8-dihydroxyquinoline-2-carboxylic acid) found in
Drosophila melanogaster10,11 and several mosquito species13
has been shown to originate from L-tryptophan via 3-
hydroxykynurenine.18−21 As many quinolinic compounds
have been shown to be products of tryptophan metabo-
lism,22,23 we hypothesized that unsubstituted quinoline in O.
peruana could be biosynthesized by an oxidative trans-
formation of tryptophan. Herein, we report on the evaluation
of biosynthetic pathways for the formation of quinoline from
tryptophan, using several stable isotope-labeled precursors
such as L-[2′,4′,5′,6′,7′-2H5]tryptophan, L-[1′-15N]tryptophan,
L-[1′-15N,15NH2]tryptophan, and [2-13C1]tryptophan.
The analysis of an extract made from the defensive secretion
of O. peruana by GC-MS showed essentially one chromato-
Received: August 27, 2020
Published: January 26, 2021
Quinoline (1) is an aromatic compound first identified from
3
̈
coal tar by Runge in 1834. About a century later, Spath and
Pikl4 reported it as one of the trace constituents (about
0.003%) of angostura bark (Cusparia trifoliata). Many
compounds that have a quinoline-core structure such as
quinine5 and chloroquine6 are used as active ingredients in
© 2021 American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00945
J. Nat. Prod. 2021, 84, 527−530
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Figure 1. Reconstructed gas chromatogram (RGC) and ion-intensity profiles extracted from acquired data for the m/z 129 (B) and 133 (C) from a
defensive secretion extract prepared from Oreophoetes peruana maintained on a regular diet. Panels D (RGC), E (m/z 129), and F (m/z 133) show
analogous profiles recorded from a defensive secretion extract prepared from insects maintained on a diet of fern leaves laced with L-
[2′,4′,5′,6,′7′-2H5]tryptophan. Analytical conditions are described in the text.
Figure 2. Electron-ionization (70 eV) mass spectra corresponding to the quinoline peak in Figure 1A (A) and that for the deuteriated compound
that eluted immediately before the quinoline peak (B).
graphic peak, which represented quinoline (1) as reported
previously (Figure 1A).2 Data acquired from extracts made
from the defensive fluid of insects fed with L-
[2′,4′,5′,6,′7′-2H5]tryptophan showed a similar chromato-
graphic profile (Figure 1D). However, a careful scrutiny of
the mass spectra recorded from the sample obtained from L-
[2′,4′,5′,6,′7′-2H5]tryptophan-fed insects showed the presence
of an extra mass peak at m/z 133, in addition to that at m/z
129 expected for the molecular ion of quinoline. Time−
intensity profiles for m/z 129 and 133 ions, extracted from the
acquired GC-MS data, confirmed the presence of an additional
chromatographic peak for a compound that eluted slightly
before the quinoline peak (Figure 1F). In fact, it is well known
that deuteriated isotopologues elute earlier than their non-
labeled counterparts under GC conditions.24−26 Evidently, the
earlier eluting peak we observed represented a deuterio-
isotopologue of quinoline. By following a careful background
subtraction protocol, we were able to obtain a representative
mass spectrum for the deuteriated compound (Figure 2).
Because the nominal mass of the new compound was 133 u
(Figure 2B), which is four mass units higher than that of
quinoline (Figure 2A), it was evident that of the five deuterium
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atoms initially present in the L-[2′,4′,5′,6,′7′-2H5]tryptophan,
only four of them have been incorporated into the labeled
product (Figure 2B). Moreover, in the spectrum in Figure 2B,
a fragment-ion peak was observed at m/z 106 for an expulsion
of a 27 Da hydrogen cyanide molecule from the precursor m/z
133 ion. Based on rationalizations documented for the mass
spectral fragmentation of quinoline under electron-ionization
conditions, the hydrogen atom for the lost HCN originates
from the heterocyclic ring of quinoline.27 Thus, it was apparent
that all deuterium atoms on the phenylene ring of L-
[2′,4′,5′,6,′7′-2H5]tryptophan are retained in the course of its
conversion to labeled quinoline, while the 2′-2H atom is
eliminated (Supporting Information Scheme S1).
By taking similar pathways proposed for the biosynthesis of
quinoline derivatives in other systems and our experimental
data into consideration, we propose that quinoline in O.
peruana is made from tryptophan as outlined in Scheme 1.
Scheme 1. Conversion of L-Tryptophan (2) to Quinoline
(1)
The electrospray-ionization mass spectrum recorded from
the defensive secretion of O. peruana maintained on a diet
laced with L-[2′,4′,5′,6,′7′-2H5]tryptophan provided further
support to confirm the proposed biosynthetic pathway. The
positive-ion mass spectrum acquired from a sample prepared in
acidified MeCN/H2O extract showed a mass peak at m/z 134
for protonated quinoline-d4 (Figure S1 and Scheme S1).
Moreover, gas chromatograms recorded from extracts made
from the secretion obtained from insects fed with L-
[1′-15N]tryptophan and L-[1′-15N,15NH2]tryptophan also
showed a chromatographic peak at a retention time similar
to that obtained from insects on a regular diet (Figure 1A).
However, it was not straightforward to monitor the formation
of a labeled quinoline from 15N1-labled tryptophan because the
expected product [15N1]quinoline is isobaric to natural
[13C1]quinoline. Nevertheless, definitive deductions could be
ascertained by extracting ion intensity data from the acquired
GC-MS file. The mass spectrum recorded from native
quinoline showed two mass peaks at m/z 129 and 130 for
M+• and [M + 1]+• ions, respectively (Figure 2A). The area
ratio of peaks in mass chromatograms extracted for m/z 129
and 130 ions for native quinoline is about 100:10 (Figure S2).
In contrast, the mass spectrum corresponding to the composite
quinoline peak and the extracted ion chromatograms recorded
from the defensive secretion obtained after feeding L-
[1′-15N]tryptophan manifested an m/z 129 to 130 peak
intensity ratio of about 100:45 (Figures S3 and S4). Analogous
results were obtained from L-[1′-15N,15NH2]tryptophan-fed
insects as well (Figures S5 and S6). The relative increase of the
intensity of the m/z 130 peak confirmed that the indole
nitrogen atom is retained when tryptophan is transformed to
quinoline.
Further biosynthetic experiments were conducted with
[2-13C1]tryptophan. The mass spectrum corresponding to the
quinoline peak and the extracted ion chromatograms recorded
from the defensive secretion obtained after feeding [2-13C1]-
tryptophan manifested an m/z 129 to 130 peak intensity ratio
of about 100:75, which compared to a 100:10 ratio for native
quinoline, indicating a very high degree of incorporation of the
experimentally provided stable-isotope label (Figures S7 and
S8). Although the mass spectrum recorded was a composite of
[2-13C1]quinoline and all other isotopomers of natural
[13C1]quinoline, the m/z 129 to 130 peak intensity ratio of
100:75 confirmed that the [13C1]-isotopologue that contrib-
uted primarily to the spectrum was the [2-13C1]-isotopologue
of quinoline. Moreover, the intense peak at m/z 102 (Figure
S7) for H12CN or H13CN losses from m/z 129 and 130 ions,
respectively, indicated that the incorporated 13C-label was
primarily located at the C-2 position of the quinoline ring.27
Although the putative intermediates were not isolated, we
presume that the 2,3-double bond of the pyrrole ring of
tryptophan is cleaved by a tryptophan 2,3-dioxygenase to form
N-formylkynurenine.28 A pathway to remove the formyl group
by a kynurenine formylase to form kynurenine has been
reported by Paglino et al.28 A kynurenine aminotransferase can
then cyclize kynurenine to kynurenic acid (Scheme 1).
Alternatively, the ketone moiety in kynurenine can be reduced
before cyclization. In an earlier study, Slaytor et al. has
demonstrated that in Nicotiana tabacum tryptophan is
converted to 8-hydroxykynurenic acid.16 Eventually, a
deoxygenation of kynurenic acid can yield quinaldic acid.
Previously, rabbits have been shown to be able to deoxygenate
kynurenic acid or xanthurenic acid to quinaldic acid and 8-
hydroxyquinaldic acid, respectively.29 Apparently, O. peruana
has evolved to decarboxylate quinaldic acid to form quinoline
as the final product.
More than 3000 species of stick insects exist, and the
defensive chemistry of some of them has been inves-
tigated.2,30−33 However, this is the first report on the
biosynthetic origin of defensive allomones of any stick insect.
Although the intermediates participating in the conversion
process were not isolated, our data unambiguously demon-
strate that the stick insect O. peruana is able to convert
tryptophan to quinoline.
EXPERIMENTAL SECTION
Insect Samples. Oreophetes peruana specimens used in this study
originated from a colony maintained at the Cincinnati Zoo &
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Botanical Garden, OH, USA. They were caged in individual plastic
containers (10 cm × 10 cm × 10 cm) and fed routinely on a diet of
Nephrolepis exaltata (Boston fern). The defensive secretion of O.
peruana was “milked” by holding specimens by hand and collecting
the fluid that oozed from the glands into glass capillary tubes.
Chemicals. L-[2′,4′,5′,6′,7′-2H5]Tryptophan, L-[1′-15N]-
tryptophan, L-[1′-15N,15NH2]tryptophan, and [2-13C1] glycine were
purchased from Cambridge Isotope Laboratories, Inc. Indole-3-
aldehyde was obtained from Sigma-Aldrich Chemical Co. DL-
[2-13C1]Tryptophan was synthesized by adapting the procedures
described by Ellinger and Flamand34 (see Supporting Information).
Stock solutions of each test chemical were made in deionized H2O (1
mg/100 μL).
Busching from the Cincinnati Zoo, who provided the O.
peruana culture, and M. Faehr for photos of O. peruana.
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Experimental data on the synthesis of labeled com-
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Athula B. Attygalle − Department of Chemistry and Chemical
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stevens.edu
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Authors
Kithsiri B. Hearth − Department of Chemistry and Chemical
Biology, Stevens Institute of Technology, Hoboken, New Jersey
07030, United States
Vikram K. Iyengar − Department of Biology, Villanova
University, Villanova, Pennsylvania 19085, United States
Randy C. Morgan − Insectarium, Cincinnati Zoo & Botanical
Garden, Cincinnati, Ohio 45220, United States
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is respectfully dedicated to the memory of
Professors Thomas Eisner and Jerrold Meinwald, two pioneers
of chemical ecology. This project was initiated when both were
mentoring our research. We thank K. Schmidt and M.
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