Biosynthesis of isoxazolin-5-one and 3-nitropropanoic acid containing glucosides in juvenile Chrysomelina

Article abstract of DOI:10.1039/c6ob00899b

Stable-isotope-labeled precursors were used to establish the biosynthetic pathway leading from β-alanine towards isoxazolin-5-one glucoside 1 and its 3-nitropropanoate (3-NPA) ester 2 in Chrysomelina larvae. Both structural elements originate from sequestered plant-derived β-alanine or from propanoyl-CoA that is derived from the degradation of some essential amino acids, e.g. valine. β-Alanine is converted into 3-NPA and isoxazolinone 5 by consecutive oxidations of the amino group of β-Ala. Substituting the diphospho group of α-UDP-glucose with 5 generates the isoxazolin-5-one glucoside 1, which serves in the circulating hemolymph of the larva as a platform for esterification with 3-nitropropanoyl-CoA. The pathway was validated with larvae of Phaedon cochleariae, Chrysomela populi as well as Gastrophysa viridula.

Full text of DOI:10.1039/c6ob00899b

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1 Biosynthesis of Isoxazolin-5-one and 3-Nitropropanoic acid Containing
2 Glucosides in Juvenile Chrysomelina
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Tobias Becker, Kerstin Ploss, Wilhelm Boland*
Max Planck Institute for Chemical Ecology, Bioorganic Chemistry, Hans-Knoell-Straße 8, D-07745, Jena, Germany
*boland@ice.mpg.de
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Abstract
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Stable-isotope-labeled precursors were used to establish the biosynthetic pathway leading from
-alanine towards isoxazolin-5-one glucoside 1 and its 3-nitropropanoate (3-NPA) ester 2 in
Chrysomelina larvae. Both structural elements originate from sequestered plant-derived -
alanine or from propanoyl-CoA that is derived from the degradation of some essential amino
acids, e.g., valine. -Alanine is converted into 3-NPA and the isoxazolinone 5 by two
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consecutive oxidations of the amino group of -Ala. Substituting the diphospho group of -
UDP-glucose with 5 generates the isoxazolin-5-one glucoside 1, which serves in the circulating
hemolymph of the larva as a platform for esterification with 3-nitropropanoyl-CoA. The pathway
was validated with larvae of Phaedon cochleariae, Chrysomela populi as well as Gastrophysa
viridula.
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Introduction
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Leaf beetles of the subtribe Chrysomelina1-8 and a number of legume plants9-12 produce the
glucosides 1 and 2 (Fig. 1). The latter serves as a pre-toxic storage compound for the actual
poison 3-nitropropanoic acid (3-NPA). 3-NPA derived compounds provide a second defensive
line in Chrysomelina, parallel with and independent of larval defensive secretions released from
nine paired dorsal glands.13,14
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Fig. 1 Isoxazolin-5-one glucosides in Chrysomelina larvae.
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Free 3-NPA was found in low concentration in the hemolymph of Chrysomelina, but was
observed in significant amounts in adult secretions of those beetles as well as in some plants, e.g.
Corynocarpus laevigatus, and fungi, for example in Penicillium atrovenetum.15-19 The toxic
effect of this compound is due to its isoelectronic character to succinic acid, leading to a covalent
addition product with mammalian succinate dehydrogenase.20,21 Thus, mitochondrial respiration
is inhibited in these animals. As this effect is most relevant to nerve cells22 significant economic
damage is caused to cattle feeding on 3-NPA containing food plants.23
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Aspects of the biosynthesis of 3-NPA have been characterized in Penicillium atrovenetum using
stable-isotope-labelled precursors, e.g. [2-13C,15N]-asp and 18O2.17,18,24 These studies show
incorporation when substances deriving from aspartate metabolism where applied. In plants, the
biosynthesis of isoxazolin-5-one derivatives (glucosides and non-glucosides)9,25,26 as well as of
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the 3-NPA moiety24,27 has been examined using C-labeled compounds or with in vitro assays.
In Indigofera spicata 3-NPA derives from malonate and malonyl monohydroxamate.27 The
biosynthesis might involve -alanine as an intermediate.28
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Adult leaf beetles of Chrysomela tremulae feeding on host plant leaves impregnated with [14C4]-
aspartate demonstrated incorporation of radioactivity in compounds 1 and 2.8 This experiment
indicates the ability for de novo production of compounds 1 and 2 in Chrysomelina. Since no
evidence was provided for any suggested intermediate, a contiguous metabolic pathway for the
biosynthesis of compounds 1 and 2 in Chrysomelina leaf beetles has not yet been elucidated.6,8
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Results and Discussion
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Along with previous identifications of 3-NPA in the defensive secretions of adult leaf beetles6,8
the first biosynthetic experiments with [14C4]-aspartate were published claiming aspartate as the
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ultimate precursor8. As shown in figure 2, feeding of [13C4 N]-Asp, surface-impregnated on
leaves of B. rapa pekinensis and fed to larvae of Phaedon cochleariae (see Experimental) results
in a cluster of ions in the area of the quasimolecular ion of 2 (formiate adduct; m/z = 393 to 401)
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which is composed of labelled and randomly re-assembled fragments of [13C4 N]-Asp. In
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contrast, administration of [13C3 N]--Ala displays a distinct pattern of isotopomers consistent
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with the incorporation of an intact C3-segment and the 15N of the fed [13C3 N]--Ala (m/z = 397)
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into 1. The 3-NPA ester 2 comprises two contiguous units of [13C3 N]--Ala as is obvious from
the fragment at m/z = 401. Peaks arising at m/z = 396 or 400 most likely result from loss of the
nitrogen atom during transamination or from incomplete labelling of the commercial [13C3 N]-
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-Ala. Since-alanine can be sequestered or originate from essential amino acids such as Val,
Thr, Met, or Ile,29-31 via propanoate or malonate as intermediates, its origin was further addressed
by feeding [13C3]-propanoate and [13C5 N]-valine (Table S1). In both cases the distinct fragment
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pattern from incorporation of an intact carbon skeleton of the administered precursors was
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maintained. In case of [13C3]-propanoate two fragments at m/z = 396 and 399 support
incorporation of one intact propanoate moiety into 1 and up to two units into 2. The same pattern
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is observed after feeding of [13C5 N]-valine, while the N of the precursor amino acid is lost.
This loss of the nitrogen atom together with consecutive incorporation by later transamination
can lead to either [M+4]- and [M+5]-peaks ([M+7]- and [M+8]-peaks respectively), when the
nitrogen is incorporated into 13C-labelled precursors, or [M+1]- and [M+2]-peaks, if the labelled
nitrogen is incorporated into natural unlabeled precursors.
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O
O
1.0
0.5
O
N
O
O2
N
O
HO
OH
control
OH
2
0.0
1.0
13 15
0.5
[
C
N]-Asp
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0.0
8.0
m/z 401
m/z 397
13 15
[
C
N]--Ala
4.0
3
0.0
1.0
13
[
C ]-propanoate
3
0.5
m/z 396
m/z 396
m/z 399
0.0
1.0
13 15
0.5
0.0
[
C
N]-Val
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m/z 399
5
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396
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404
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Fig. 2 Representative mass spectra of compound 2 after LC separation of larval extracts
(MeCN/H2O, 1:1) from P. cochleariae after feeding on different diets for 10 d; diets consisted of
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B. rapa pekinensis leaves, impregnated with KH2PO4/K2HPO4 buffered solutions of the
compounds given above; as a control only blank buffer was used.
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The HR-MS analysis of hydrolytically cleaved free 3-NPA from 2 confirms these findings.
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Feeding of [13C3 N]--Ala generates a molecular ion at m/z = 122,021471 (13C3H4 NO4, calc.
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for 122.021681, [M-H]-; Fig. S4). The carbon skeletons of [13C4 N]-Thr, [13C3 N]-Ala, and
[13C2]-malonate were not incorporated at all in either 1 or 2 (Table S1).
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To explore the probability of the incorporation (%, see Experimental) of plant-derived -alanine
versus the de novo synthesis of -Ala from essential precursor amino acids,29-31 defined amounts
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of [13C4 N]-Asp, [13C3]-propanoate, and [13C3 N]--Ala were injected into the hemolymph of
P. cochleariae larvae (see Experimental; Table S1) and the products 1 and 2 were analyzed by
mass spectrometry. In case of Asp no significant incorporation into 1 or 2 was observed (0.019 ±
0.241 %), while average values of 2.3 ± 1.5 and 17.4 ± 11.2 % were determined for labelled
propanoate and -Ala, respectively. Altogether, these results indicate that aspartate catabolism
plays no significant role in the biosynthesis of compounds 1 and 2 although Asp is abundantly
present in the food plants (Fig. S13). Taken together we conclude that 1 and 2 are produced from
both, sequestered and de novo produced -Ala from degradation of appropriate essential amino
acids present in the food plant.
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To investigate the later steps of the metabolic route from -alanine to the isoxazolinone
glucoside 1 and to its 3-NPA ester , potential intermediates such as [1-13C15N]-3 and [1-
13C15N]-4 were synthesized and injected into the larvae (Table S1). Accordingly, a stepwise
oxygenation at the nitrogen atom of -alanine first produces (N-hydroxyamino)propanoic acid 3
and (N-hydroxyimino)propanoic acid 4 (Fig. 3) after elimination of water from the postulated
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and unstable N-dihydroxyprecursor. Cyclization of 4 generates the isoxazolinone 5 that is con-
densed with activated -UDP-glucose to the isoxazolinone glucoside 1. Evidence for this
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reaction was provided by H NMR measurements after incubation of fat body samples of all
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three investigated species together with the substrates (Fig. S2). The isoxazolinoneglucoside 1 is
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circulating in the hemolymph and serves as a platform for alkylation with activated 3-NPA.
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Fig. 3 Proposed biosynthesis of compounds 1 and 2 in Chrysomelina larvae.
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Further oxidation of the geminal dihydroxyintermediate of -Ala generates 3-NPA that is
subsequently bound to the C(6)-hydroxy group of the isoxazolinone glucoside 1. Activation of 3-
NPA is achieved as a CoA-ester and requires ATP (Fig. S3). The increase of the glucoside ester
2 in later stages of the larval development is in line with a decrease of 1 suggesting a tight
control of the individual steps of the biosynthesis of 1 (Fig. S6-S11). A similar trend was obser-
ved for 1 and 2 in the hemolymph.1 Only very small amounts of free 3-NPA were detected in
fresh hemolymph samples.1 Furthermore, neither free -Ala nor other intermediates, such as 3 to
5 could be detected after silylation and GC-MS analysis of hemolymph samples or whole larval
extracts.
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Conclusion
Beetles produce 3-NPA- and the isoxazolinone-moiety from plant-derived -alanine or by degra-
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dation of appropriate amino acids such as L-Val via propanoate to -Ala. The extent of these
alternative routes most likely depends on the contents of the required amino acids in the food
plants (Fig. S12 and S13). Since aspartate is not a precursor of 1 or 2, this excludes a hidden
contribution from the gut microbiome since the decarboxylation of aspartate to -alanine is
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known from microbial metabolism32. It is important to note, that the oxidation products of -
alanine serve as precursors for both, 3-NPA and the isoxazolinone 1. As the production of the
isoxazolinone glucoside 1 during the very early larval stages precedes the formation of 3-NPA,
most likely different oxidases are involved that are tightly regulated. The larval enzymes are still
unknown, but show mechanistic similarities to recently described microbial enzymes28. The
isoxazolinone glucoside 1 circulates in the hemolymph of the insect and serves as a platform for
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alkylation with activated 3-NPA (as CoA ester) as was demonstrated by injection of labelled free
3-NPA into the hemolymph of the larvae which was then rapidly bound1 to the glucoside carrier
1. The steric and electronic features of the N-glucoside prevent an export of this compound from
the hemolymph into neighboured tissue or the defensive secretion of the insects, although S- and
O-glucosides, for example plant-derived salicin, are sequestered by the larvae and channeled via
the hemolymph through the whole body into the glandular system for defense production. Both,
the O- and the N-glucosides share the hemolymph as a common carrier medium, but only the
natural O-glucosides and their synthetic S-analogs are allowed to pass the separating
membranes.33 Therefore, the isoxazolinone glucoside 1 has never been found outside the
hemolymph of the larval systems. This is different in adults, which secret several 3-NPA esters
of 1 on their elytra15,34 when endangered by a predator. We assume that after ingestion of a larva
the 3-NPA esters of 1 are rapidly hydrolyzed in the gut of the predator leading to intoxication. In
a living larva, release of free 3-NPA is cured by activation as a CoA-ester and re-esterification to
the C(6) of isoxazolinone glucoside.1 The identification and cloning of the enzymes catalyzing
the transformation of the amino group of -alanine to the functionally relevant nitro group are
the next and urgent steps to understand the underlying mechanisms and the regulation of defense
production in leaf beetle larvae.
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Experimental
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Insect collection and rearing. The procedures were adapted from already described methods.1
Chrysomela populi collected near Dornburg, Germany (latitude 51.015, longitude 11.64), on
Populus canadensis. Beetles were propagated using a light/dark cycle of 16 h light and 8 h
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darkness (LD 16/8), at 18 ± 2 °C in light and 13 ± 2 °C in darkness. Gastrophysa viridula was
collected in Jena (latitude 50.929, longitude 11.597). Phaedon cochleariae (F.) was reared on
Brassica rapa subsp. pekinensis (B. rapa pekinensis) “Cantonner Witkrop” (Quedlinburger
Saatgut, Quedlinburg, Germany) and Gastrophysa viridula was reared on Rumex obtusifolius in a
Snijder chamber (Snijders Scientific, Tilburg, Netherlands) in a light/dark cycle of 16 h light and
8 h darkness (LD 16/8) and 13 °C/11 °C ± 1 °C.
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Insect sample preparation. 150 µl of MeCN/H2O 1:1 were added to individual larvae in
grinding tubes equipped with 2 steel beads (diameter/bead = 4.5 mm). The samples were ground
with a geno grinder (1210 rpm, 1 min) at rt for each larva. Then the mixtures were centrifuged at
40 °C and 13000 rcf for 30 min. 110 µl of the supernatant was transferred to a second tube. The
samples were stored at -25 °C and vortexed for 15 s directly before analysis by HPLC/MS. All in
vivo measurements are single-larva analyses. All replicates are biological replicates of individual
larvae.
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Insect dissection. The larvae were immersed in liquid nitrogen for 30 s. Then the fat body was
isolated after cutting the cuticles of the larvae with micro scissors. The tissue was stored
at -80 °C prior to use.
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Statistical evaluation. The results of the treated larvae were compared to the results of larvae of
the control groups, in order to determine the statistical significance of the difference of the mean
values. First, the data were analyzed by a Shapiro-Wilk normality test. For normally distributed
datasets, the t-test was applied. For differently distributed datasets, the Mann-Whitney rank sum
test was applied. Statistical difference is defined by a confidence level of at least 95 % (* =̂ 95 %
=̂ 0.05, ** =̂ 0.01, *** =̂ 0.001).
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HPLC/MS analysis. The analysis of compounds 1 and 2 was done by modifying the procedure
that appears in the literature.1 Measurements were carried out on an Agilent HP1100 HPLC
system equipped with an OH-endcapped RP-C18 column (RP-C18e), LiChroCART (250×4 mm,
5µm; Merck KGaA, 64271, Darmstadt, Germany) connected to a Finnigan LTQ (Thermo
Electron Corp., Dreieich, Germany) ion trap mass spectrometer operating in the APCI mode
(vaporizer temperature: 500 °C, capillary temperature 300 °C). Standard compounds for
identification were either purchased (Sigma-Aldrich (St. Louis, MO, USA) or synthesized. 2 to
5 µl of the sample volume was injected, depending on larval size (up to 20 mg larval fresh
weight: 5 µl; m > 20 mg: 2 µl). The following parameters were used: flow rate = 0.5 ml/min at rt:
90 % solvent A (H2O + 0.1 % v/v HCO2H) and 10 % solvent B (MeCN + 0.1% v/v HCO2H) for
5 minutes, linear gradient to 100 % solvent B in 5 min, then 100 % B for 2 min, linear gradient to
10 % B in 5 min and further elution with 10 % B for 5 min. For identification and quantification,
the signals of the formic acid adducts [M+HCO2H-H]- were analyzed (m/z 292 for 2-(β-D-
glucopyranosyl)-3-isoxazolin-5-one (1) and m/z 393 for 2-[6′-(3″-nitropropanoyl)-β-D-
glucopyranosyl]-3-isoxazolin-5-one (2)). The column was washed for 20 h at 40 °C with 2-
propanol/MeCN 1:1 and then equilibrated to H2O/MeCN 9:1 prior to use.
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GC/MS analysis. The measurement parameters were similar to the literature protocols.35 1 µl of
each sample was injected at a split ratio of 1:25 into a GC/MS system equipped with an A 200S
autosampler, a GC 2000 gas chromatograph, and a Voyager quadrupole mass spectrometer
including a dynode/phosphor/photomultiplier detector (all ThermoQuest, Manchester, UK).
tris(perfluorobutyl)amine (CF43) was used as reference gas for tuning. Mass spectra were
recorded from m/z 50 to 622 at 0.53 s scan-1 for trimethylsilylated samples (TMS). For the
quantifications, the parameters were as follows: An injection temperature of 230 °C was chosen,
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the interface temperature was adjusted to 250 °C, and the ion source temperature was 200 °C.
Helium flow was 1.5 mL min-1. After a 5 min solvent delay at 70 °C, the oven temperature was
increased by 5 °C min-1 to 140 °C, then by 40 °C min-1 to 310 °C; the temperature was constant
for 1 min, then cooled to 70 °C and equilibrated for 5 min. Ion trace integration was performed
manually over the complete intensities of the signals.
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NMR and HRMS analysis. NMR spectra were measured using a Bruker-Spektrospin AVANCE
400 UltraShield spectrometer operating at 400 MHz (1H) and 100 MHz (13C). Chemical shifts (δ)
are quoted in parts per million (ppm) and are referenced to the signals of residual protonated
solvents in 1H spectra (CHCl3 at  7.26 ppm; HDO at  4.79 ppm) and deuterated chloroform in
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13C spectra (CDCl3 at  77.16 ppm). Acetonitrile was added as a reference for C NMR spectra
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in D2O (H3CCN at  1.47 ppm).36 The multiplicities are given as follows: s, singlet; d, doublet; t,
triplet, dd, doublet of doublets; dt, doublet of triplets; q, quartet; dq, doublet of quartets; m,
multiplet. High-resolution mass spectra were recorded on a Bruker Maxis UHR-qTOF mass
spectrometer.
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Syntheses. Modified literature protocols were used to synthesize [1´,2´,3´,4´,5´,6´-13C6]-2-(-D-
Glucopyranosyl)-3-isoxazolin-5-one [1´,2´,3´,4´,5´,6´-13C6]-137, [1´,2´,3´,4´,5´,6´-13C6]-2-[6′-(3″-
Nitropropanoyl)-β-D-glucopyranosyl]-3-isoxazolin-5-one [1´,2´,3´,4´,5´,6´-13C6]-238, 2,2,2-
trichloroethyl 3-nitropropanoate (6)38, Isoxazolin-5(2H)-one (5)39, [1-13C15N]-3-(hydroxyamino)-
propanoate (3)18,40 and [1-13C15N]-3-(hydroxyimino)propanoate (4)39. The details of the syn-
thetic protocols as well as the analytical data are presented in the electronic supporting
information (S5-S16).
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In vitro assays. To show the incorporation of Isoxazolin-5-one 5 into compound 1, 10 mg of the
isolated fat body tissue was suspended in 400 µl H2O. In addition, one solution containing
2.6 mg of Isoxazolin-5-one 5 and one solution containing 18.7 mg of commercial -UDP-
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Glucose each in 200 µl were prepared. Solutions of compound 5 and -UDP-Glc were mixed
and split again into two solutions of 200 µl. To one solution, 200 µl of the fat body suspension
was added; to the residual solution, 200 µl of buffer (KH2PO4/K2HPO4, 100 mM) was added.
After 1 d of incubation at 30 to 40 °C, 400 µl of D2O was added to each solution, including the
1
fat body suspension, after which the mixtures were centrifuged and analyzed by 1D H NMR
experiments (512 scans each).
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The ATP/CoA-dependent incorporation of 3-NPA into compound 2 was shown according to
procedures in the literature41 Solutions of ATP (c = 125 mM), CoA (c = 5 mM), 3-NPA
(10.5 mM), compound 1 (10.5 mM) and the isolated fat body tissue (m = 30 mg) in 2 ml of
buffer (Tris-base 50 mM, sucrose 250 mM, MgCl2 2 mM, 1 µl/ml dithiotreitol solution, 10 µl/ml
protease inhibitor mix) were prepared. The fat body mixture was homogenized using a geno
grinder (1200 rpm, 30 s). Then five mixtures with an individual volume of 1 ml were prepared
from these solutions as follows: solution 1 containing ATP, CoA, 3-NPA, compound 1, fat body;
solution 2 (CoA, 3-NPA, compound 1, fat body, buffer); solution 3 (ATP, CoA, 3-NPA,
compound 1, buffer); solution 4 (ATP, 3-NPA, compound 1, fat body, buffer) and solution 5
(ATP, CoA, fat body, 2 x buffer). After 1 d of incubation at 30 °C, 200 µl was taken and
centrifuged (13000 rcf, 15 min), and the supernatant was directly analyzed by HPLC/MS.
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In vivo injection experiments. The larvae were fixed with pincers manually upon ice under a
light microscope. Then a dose of 20 to 40 nmol substance per mg larval fresh weight was
injected with a thin glass capillary attached to a microinjector. Typically injection volumes of
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200 nl containing solutions of compounds with a concentration of 0.5 mol l-1 dissolved in
potassium phosphate (KH2PO4/K2HPO4) or potassium carbonate (K2CO3) buffers were applied.
After the total volume was injected, the glass capillary was not removed from each larva for 1
min to prevent direct bleeding. Then the larvae were kept in plastic beakers covered with
cardboard with a piece of their host plant leaf (100 to 400 mg) to enable air exchange at rt unless
noted otherwise. The injected larvae were incubated for 24 h before being extracted with
MeCN/H2O 1:1.
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Feeding experiments. Whole leaves were impregnated with aqueous buffered
(KH2PO4/K2HPO4, pH 7.4, 500 mmol l-1 + 300 µl of acetone, for reduction of surface tension)
solutions of the isotopic-labeled compounds using a brush and given to P. cochleariae (L1 and
L2) as food. The larvae were extracted after 7 to 10 d of feeding. Freshly impregnated leaves
were added two or three times. The concentrations of the isotope-labeled compounds were as
15
follows: [13C4 N]-aspartate (c = 100 mmol l-1), [15N]-aspartate (c = 100 mmol l-1), [4-13C]-
15
aspartate (c = 100 mmol l-1), [13C4]-aspartate (c = 100 mmol l-1), [1,3-13C2]-malonate, [13C3 N]-
15
-L-alanine (c = 100 mmol l-1), [15N]--L-alanine (200 mmol l-1), [13C3 N]--alanine (c =
15
100 mmol l-1), [13C3]-propanoic acid (c = 100 mmol l-1), [13C5 N]-valine (200 mmol l-1) and
15
[13C4 N]-threonine (200 mmol l-1). An overview is presented in the electronic supporting
information (Table S1).
254
255
256
257
258
Quantification and identification of amino acids in B. rapa pekinensis. The samples were
analyzed according to procedures modified from the literature. 200-1000 mg FW of B. rapa
subsp. pekinensis leaves were cut, transferred into a 2 mL tube, and mixed with 100 µl of [13C3,
15N]-alanine (c = 0.6 mmol l-1). The samples were ground by a geno grinder (1200 rpm, 3 x
5min) and shaken at 40-60 °C for 3h. 1 mL of methanol was added, and the samples were
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261
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266
centrifuged for 15 min at 13000 rcf. The supernatant was pipetted into a screw-top glass tube,
and 2.5 ml of MeCN was added. The tubes were equipped with a needle, and the solvents were
removed in a desiccator under reduced pressure at rt. This procedure was repeated until the
samples were completely dried. To each sample, N-methyl-N-trimethylsilyltrifluoroacetamide
(MSTFA) was added, and the samples were shaken at 40 °C for 30 min. The clear solutions were
directly analyzed by GC/MS. The peak areas of the heavy isotope signals corresponding to the
standards and the areas of the normal isotopes were integrated. The ratio of the peak areas gave
the molar content of the amino acid cm,,leaf in nmol (mg fresh weight)-1 according to equ. (1):
푛
퐼
∙푐 ∙푉
퐴퐴 푠푡푎푛푑푎푟푑 푠푡푎푛푑푎푟푑
퐴퐴
267
푐푚,퐴퐴,푙푒푎푓
=
=
(1)
푚
푙푒푎푓
퐼
∙푚
푠푡푎푛푑푎푟푑 푙푒푎푓
268
269
270
271
272
mleaf = fresh weight of the leaf in mg, n = amount of amino acid in nmol, I = area of the non-
isotopic peak ([M+0]) of the amino acid, cstandard = molar concentration of the added solution of
the isotopically labeled amino acid standard in µmol µl-1, Vstandard = volume of added solution of
the isotopically labeled amino acid standard in µl, Istandard = area of the isotopic peak of the
isotopically labeled amino acid standard.
273
274
275
276
277
278
279
280
Quantification of compounds 1 and 2. Solutions of synthetic [1´,2´,3´,4´,5´,6´-13C6]-2-(-D-
glucopyranosyl)-3-isoxazolin-5-one [1´,2´,3´,4´,5´,6´-13C6]-1 (c = 17.33 mmol l-1, V = 10 µl) as
well as [1´,2´,3´,4´,5´,6´-13C6]-2-[6′-(3″-Nitropropanoyl)-β-D-glucopyranosyl]-3-isoxazolin-5-
one [1´,2´,3´,4´,5´,6´-13C6]-2 (c = 14.65 mmol l-1, V = 10 µl) in MeCN/H2O 1:1 were added to
each larval extract (SIL-IS42,43). The peak areas of the heavy isotope signals in the chromatogram
(m/z 298 for compound 1, m/z 399 for compound 2) were compared to the peak areas of the
normal isotopes (m/z 292 for compound 1, m/z 393 for comp. 2). The molar contents cm,analyte,larva
of compounds 1 and 2 in nmol (mg larval fresh weight)-1 were calculated using equ. (2):
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푛
퐼
∙푐 ∙푉
푎푛푎푙푦푡푒 푠푡푎푛푑푎푟푑 푠푡푎푛푑푎푟푑
푎푛푎푙푦푡푒
281
푐푚,푎푛푎푙푦푡푒,푙푎푟푣푎
=
=
(2)
푚
퐼
∙푚
푠푡푎푛푑푎푟푑 푙푎푟푣푎
푙푎푟푣푎
282
283
284
285
286
287
288
289
290
291
292
293
294
mlarva = fresh body weight of the extracted larva in mg, nanalyte = amount of compound 1 or 2 in
nmol, Ianalyte = area of the non-isotopic peak ([M+0]) of compound 1 or 2, cstandard = molar
concentration of added solution of synthetic comp. [1´,2´,3´,4´,5´,6´-13C6]-1 or [1´,2´,3´,4´,5´,6´-
13C6]-2 in µmol µl-1, Vstandard = volume of added solution of synthetic [1´,2´,3´,4´,5´,6´-13C6]-1 or
[1´,2´,3´,4´,5´,6´-13C6]-2 in µl, Istandard = area of the isotopic peak ([M+6]) of comp.
[1´,2´,3´,4´,5´,6´-13C6]-1 or [1´,2´,3´,4´,5´,6´-13C6]-2. The resulting values are presented in the
electronic supplemental information (ESI, Fig. S6-S11). Compounds containing more than one
3-NPA moiety bound to the sugar residue of an isoxazolin-5-one glucoside were detected in
secretions of adult leaf beetles.15 However, these components were not detected in whole
Chrysomelina larvae extracts; thus it is clear that compound 2 is the only 3-NPA based pre-toxic
compound found at the larval stage. Consequently, quantification of compounds 1 and 2 provides
information about the total amount of isoxazolin-5-one and 3-NPA derivatives in Chrysomelina
larvae.
295
296
297
298
299
Incorporation of injected compounds. To demonstrate the incorporation of the injected
compounds had taken place, the areas of the isotopic peaks of compounds 1 and 2 were divided
by the areas of the non-isotopic peaks. These ratios were compared to the ratios determined in
the control groups. In order to determine the percentile C of the incorporation of the injected
compounds in % equation (3) was used:
퐼
퐼 +2∙퐼
2 3
1
((
)∙푐
+(
)∙푐
)∙푚
푙푎푟푣푎,푒푛푑
푚,푐표푚푝표푢푛푑ퟏ,푙푎푟푣푎
푚,푐표푚푝표푢푛푑ퟐ,푙푎푟푣푎
퐼
퐼
푛
푀1
푀2
300
퐶 = 푑푒푡 ∙ 100 =
∙ 100
(3)
푛
푐
∙푉
푖푛푗 푖푛푗
푖푛푗
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ndet = sum of amounts of detected labeled isoxazolin-5-one and 3-NPA moieties in compound 1
and 2 in nmol, ninj = amount of injected labeled compound in nmol, I1 peak area of labeled
compound 1 (intensity of control peak is always subtracted; if 1 of n labeled atoms is
incorporated, the value of I is divided by n; if 2 of n are incorporated I is divided by n and
multiplied by 2 and so forth), IM1 = area of the non-isotopic peak of compound 1 ([Mcomp.1+0]), I2
= area of the single labeled isotopic peak of comp. 2, IM2 = area of the non-isotopic peak of
comp. 2 ([Mcomp.2+0]), I3 = area of the double labeled isotopic peak of comp. 2, mlarva,end = fresh
body weight of the larva after incubation, cinj = molar concentration of the injected compound in
nmol nl-1, Vinj = volume of the injected compound in nl. Thus, naturally occurring compounds 1
and 2 were used as internal standards within the same measurement. The ratios of isotopic versus
non-isotopic peaks determined in the control groups were subtracted from the ratios determined
in the treated groups.
313
Author contributions
314
315
316
317
318
319
T.B. and W.B. planned and designed the project and experiments. T.B. synthesized the
compounds. T.B. performed injection and feeding experiments. T.B. carried out the in vitro
experiments. T.B. prepared the samples. T.B. measured and analyzed all NMR spectra. T.B. and
K.P. performed HPLC/MS measurements. T.B. performed GC-MS measurements. T.B. analyzed
GC- and LC-mass spectra. T.B. analyzed the statistical data. T.B. and W.B. wrote the
manuscript.
320
321
322
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
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We thank Emily Wheeler for her editorial assistance. We thank Dr. Maritta Kunert and Dr. Antje
Burse for their help collecting samples. The help of Angelika Berg and Anja David for rearing
the beetles is also acknowledged. We acknowledge René R. Gretscher for artwork of the
graphical abstract. This work was supported by the Max Planck Society.
327
Notes and References
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Graphical abstract
O
O
O
N
O
O2N
O
HO
OH
15N
13C
,
labelled
compounds
OH
Biosynthesis of isoxazolin-5-one glucoside and 3-nitropropanoate esters as hemolymph
defenses in leaf beetle larvae.