N-(15,16-Dihydroxylinoleoyl)-glutamine and N-(15,16-epoxylinoleoyl)-glutamine isolated from oral secretions of lepidopteran larvae

Article abstract of DOI:10.1016/S0040-4020(02)01489-8

N-(15,16-Dihydroxylinoleoyl)-glutamine (1) and N-(15,16-epoxylinoleoyl)-glutamine (2) and were identified in the regurgitant of lepidopteran larvae (Spodoptera exigua and Spodoptera frugiperda) by LC-MS. After methanolysis and derivatisation with MSTFA, the positions of the hydroxy groups of 1 were identified by GC-MS. The structures of both conjugates were confirmed by synthesis.

Full text of DOI:10.1016/S0040-4020(02)01489-8

Tetrahedron 59 (2003) 135–139
N-(15,16-Dihydroxylinoleoyl)-glutamine and
N-(15,16-epoxylinoleoyl)-glutamine isolated from oral secretions
of lepidopteran larvae
*
Dieter Spiteller and Wilhelm Boland
¨
Max-Planck-Institut f u¨ r Chemische Okologie, Winzerlaer Straße 10, D-07745 Jena, Germany
Received 16 September 2002; accepted 18 November 2002
Abstract—N-(15,16-Dihydroxylinoleoyl)-glutamine (1) and N-(15,16-epoxylinoleoyl)-glutamine (2) and were identified in the regurgitant
of lepidopteran larvae (Spodoptera exigua and Spodoptera frugiperda) by LC–MS. After methanolysis and derivatisation with MSTFA, the
positions of the hydroxy groups of 1 were identified by GC–MS. The structures of both conjugates were confirmed by synthesis. q 2002
Elsevier Science Ltd. All rights reserved.
1
6
1
. Introduction
the family of cytochrome P450 enzymes. Since these
biocatalysts not only introduce hydroxy groups, but also
convert double bonds into epoxides,
1
,2
17,18
N-Acylamino acid conjugates, such as N-acylornithines,
3
we screened the
4
N-acylserines and N-acylisoleucines are well known
bacterial metabolites. Some of them have pharmacological
regurgitant from lepidoteran larvae (S. exigua and
Spodoptera frugiperda) for additional oxygen-containing
fatty acid metabolites, in particular epoxy- and dihydroxy
fatty acids.
5
–7
and industrial importance as antibiotics and surfactants.
The compounds are produced by many different micro-
organisms, among them Streptomycetes, Flavobacteria and
1
,5,6
Pseudomonas species.
N-acylglutamine ‘volicitin’, (17S)-(17-hydroxylinolenoyl)-
In 1997, Alborn et al. isolated the
2
. Results and discussion
L-glutamine, from oral secretions of the larvae of the beet
8
armyworm (Spodoptera exigua). After introduction of the
Compounds from freshly collected regurgitant from larvae
of S. exigua or S. frugiperda were separated by HPLC on
reverse phase under programmed conditions and analysed
by mass spectrometry using atmospheric pressure chemical
ionization (APCI). Two peaks in the midpolar region of the
chromatographic run displayed quasimolecular ions as
expected for an epoxide or a dihydroxy derivative of
compound into the damaged leaf by the feeding larvae,
volicitin may act as an elicitor of plant volatile biosynthesis.
The compound is not generally active, but triggers volatile
8
,9
biosynthesis in corn (Zea mays) or sweet potato (Ipomoea
1
0
batatas). Besides volicitin, several other N-acyl-L-gluta-
mines were found in the regurgitates of caterpillars
1
1,12
(
ing in chain length (C –C ) and in the number of double
Noctuidae, Geometridae),
containing fatty acids vary-
N-(linolenoyl)-glutamine. First eluted the polar N-(di-
þ
min; ca. 5% with respect to the major constituent volicitin
1
4
18
hydroxylinoleoyl)-glutamine 1 ([MþH] ¼441; t ¼21.1
ret
bonds. Previous work on the biosynthesis of these
conjugates has provided the first evidence that the amide
followed by the less polar N-(epoxylinoleoyl)-glutamine 2
[
1
1
bond between the plant derived fatty acid and L-glutamine
is made by microorganisms living in the fore- and midgut of
þ
volicitin).
MþH] ¼423, t ¼24.7 min; ca. 3% with respect to
ret
1
3
the insect larvae. Moreover, the introduction of the (17S)-
1
4
hydroxy group into the linolenic acid moiety of volicitin
represents another transformation which is typical for
bacterial metabolism. Well known are the v, v-1, or v-2
The APCI mass spectrum of the dihydroxy conjugate 1
(Fig. 1(a)) showed a strong quasimolecular ion at m/z¼441
followed by the loss of two molecules of water
1
5
hydroxylations which are often achieved by enzymes from
(
m/z¼441!423!405). The fragment at m/z¼147 is
characteristic for the amino acid fragment (Gln) and results
from fragmentation across the protonated amide bond. The
epoxide 2 displayed a similar spectrum with a quasi-
molecular ion at m/z¼423 and fragments at m/z¼405 and
388. For further analysis, the fraction of the dihydroxy
Keywords: N-(15,16-epoxylinoleoyl)-glutamine;
dihydroxylinoleoyl)-glutamine; 15,16-epoxylinoleate;
N-(15,16-
15,16-
dihydroxylinoleate; Spodoptera exigua.
*
Corresponding author. Tel.: þ49-36-4157-1200, fax: þ49-36-4157-1202;
e-mail: boland@ice.mpg.de
0040–4020/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01489-8

136
D. Spiteller, W. Boland / Tetrahedron 59 (2003) 135–139
Figure 1. (a) APCI-MS of N-(15,16-dihydroxylinoleoyl)-L-glutamine (1), (b) EI-MS of 15,16-bis-trimethylsilanyloxylinoleic acid methyl ester (3) and
c) O-TMS mediated fragmentation of 3. Reference compounds display identical mass spectra.
(
conjugate 1 was collected, methanolysed with Ac O/MeOH
2
to the fragments at m/z¼131 and m/z¼339 along with the
dominant allylic cleavage yielding m/z¼233 characteristic
for the EtCH(OTMS)CH(OTMS) fragment (Fig. 1(b)). The
ion at m/z¼310 (Fig. 1(c)) results from transfer of the
trimethylsiloxy group to the carboxyl group followed by
cleavage of the C(15)–C(16) bond resulting in loss of
2-trimethylsilyloxybutanal in analogy to well known
1
4
at 808C for 0.5 h, derivatised with MSTFA at 408C for 1 h
and investigated by GC–MS. The EI-MS of the bistri-
methylsilylated ester 3 showed a molecular ion at m/z¼470,
and a fragment at m/z¼439, indicating a loss of OMe. The
position of the dihydroxy moiety was proven by the
cleavage between the two vicinal O-TMS groups leading
Scheme 1. Synthesis of (15RS,16RS)-oxygenated conjugates of octa-9,12-dienoic acid and glutamine.

D. Spiteller, W. Boland / Tetrahedron 59 (2003) 135–139
137
degradation reactions of other trimethylsilyloxy fatty
1
acids.
70 eV. High resolution EI mass spectra were recorded with a
Micromass MasSpec 2, for high resolution ESI mass spectra
a Micromass Quattro II was used (Micromass, Manchester,
9
1
The identity of both conjugates 1 and 2 was additionally
proven by synthesis (Scheme 1). Linolenic acid was
converted into peroxylinolenic acid 4 following a protocol
UK). IR: Bruker Equinox 55 FTIR spectrophotometer. H
1
3
and C NMR: Bruker Avance 400 spectrometer (Bruker,
D-76287 Rheinstetten/Karlsruhe, Germany). Chemical
2
0
1
13
of Corey et al. using linolenoylimidazole in combination
2
shifts of H and C NMR are given in ppm (d) based on
1
1
with urea–hydrogen peroxide instead of anhydrous H O
2
solvent peaks: DCCl 7.26 ppm ( H NMR) and 77.70 ppm
3
2
1
3
1
(
Scheme 1).
( C NMR); CD OD 3.31 ppm ( H NMR) and 49.00 ppm
3
1
3
(
C NMR).
At low temperature (08C), intramolecular epoxidation
occurred predominantly at the terminal double bond
3.1.1. Isolation and degradation of N-(15,16-dihydroxy-
linoleoyl)-glutamine (1) from lepidopteran larvae. Com-
pounds from freshly collected regurgitant of S. exigua
larvae or S. frugiperda (ca. 0.5 ml) were separated by HPLC
on reverse phase (Grom-Sil ODS-3 CP, 120 mm£2 mm)
under programmed conditions from 100% A (3 min) to
(
with minor amounts of the 9,10- and 12,13-isomers (ca.
ca. 75%) yielding 15,16-epoxylinoleic acid 5 along
1
2
2
2
2
5%).
Formation of the glutamine conjugate 2 was achieved via
the mixed anhydride protocol using chloroformate and
L-glutamine. Hydrolysis of 2 was accomplished with aq.
100% B (27 min). Solvent A: H O, 0.1% AcOH. Solvent B:
2
1
CH CN, 0.1% AcOH. Flow: 0.2 ml min2 . Fractions
3
HClO (1.3%) and provided the dihydroxy conjugate 1 in
4
containing N-(dihydroxylinoleoyl)-glutamine (1) were com-
bined and evaporated to dryness. The crude residue was
dissolved in methanol (2.5 ml), acetic acid anhydride
(0.25 ml) was added and then heated to 808C for 30 min.
After removal of solvent, the diol was silylated for 1 h with
MSTFA at 408C. The bis-OTMS ether 3 (Fig. 1(b) or (c))
was directly analysed by GC–MS. MS (EI 70 eV) m/z (%):
59 (8), 73 (100), 75 (32), 131 (54), 143 (40), 147 (22), 217
(5), 233 (35), 299 (2), 310 (10), 339 (4), 380 (0.5), 439 (0.5),
470 (0.5).
moderate yield (30%). Both synthetic compounds proved to
be identical with the natural products. However, the
absolute configurations of the epoxide- or the diol moiety
of the conjugates 1 and 2 were left open owing to the very
low concentrations of the natural products.
Oxygenated octadecanoids generally display a wide range
of biological activities. Free 15,16-epoxyoleic acid and all
three positional isomers of epoxylinoleic acid were first
2
3,24
isolated by Kato et al. from rice plants.
The epoxy acids
induce resistance against the rice blast fungus
3.1.2. rac-14-(3-Ethyl-oxiranyl)-tetradeca-9,12-dienoic
2
0,21
(
(
(
Magnaporthe grisea) by preventing spore germination
ED50 20–30 ppm). The structurally related vernolic acid
12,13-epoxyoleic acid, ¼leukotoxin B) exhibits bacteri-
acid (5); [5(15RS,16RS)-15,16-epoxylinoleic acid].
Linolenic acid (0.278 g) and 1,1 -carbonyldiimidazole in
dry CH Cl (10 ml) were stirred for 20 min at rt. In a second
0
2
2
2
5
cidal and antifungal properties. Other epoxy acids
modulate cellular signalling by affecting hormone levels,
flask, the stable urea–H O adduct (10 g, 30%) and
2 2
Na HPO (5.0 g) were suspended in CH Cl (50 ml).
2 4 2 2
After cooling (08C) and addition of lithium imidazolide
(0.2 equiv.), the activated linolenic acid was added within
2 min. Stirring was continued for 5 min, KHSO (5.0 g),
4
2
6
2
7
28
the action of G proteins and ion channels. Dihydroxy
fatty acids, derived from vernolic and isovernolic acid by
action of an epoxide hydrolase, are associated with the acute
respiratory distress syndrome. The biological potential of
the novel glutamine conjugates 1 and 2 is, as yet,
unexplored and remains to be established with appropriate
plant and animal assay systems.
2
9
CH Cl (50 ml), and Na SO (5.0 g) were added upon
2
2
2
4
which the solution clarified within 5 min of stirring. The
reaction was allowed to stand at 08C. The progress of the
reaction was monitored by GC–MS of aliquots after
esterification with diazomethane. The reaction was termi-
nated after ca. 60% conversion (ca. 1 week) and solids were
removed by filtration and washed with 50 ml CH Cl . After
First results on the mode of action of N-acyl glutamines on
plants and the up-regulation of well-defined defense
responses will be reported in due course.
2
2
removal of solvents, the residue was purified by chroma-
tography on RP 18 with MeOH/H O (75:25, v/v) for elution.
2
1
Yield: 0.149 g (51%, ca. 75% 15,16-epoxide). H NMR
3
. Experimental
(400 MHz, CDCl
m, 8H), 1.48–1.68 (m, 4H), 2.01–2.10 (m, 2H), 2.17–2.27
(m, 1H), 2.34 (pt, J¼7.47 Hz, 2H), 2.37–2.45 (m, 1H), 2.80
pt, J¼6.89 Hz, 2H), 2.90 (dt, J¼4.24, 6.31 Hz, 1H), 2.96
3
) d 1.06 (t, J¼7.51 Hz, 3H), 1.25–1.41
(
3
.1. General
(
1
3
Reactions were performed under Ar; solvents were dried
according to standard methods. LC–MS (APCI): Hewlett–
Packard HP 1100, equipped with a Grom-Sil ODS-3 CP,
(dt, J¼4.24, 6.41 Hz, 1H), 5.26–5.55 (m, 4H). C NMR
(100 MHz, CDCl ) d 11.30, 21.72, 25.39, 26.49, 26.81,
3
27.89, 29.69, 29.73, 29.77, 30.18, 34.62, 57.33, 59.14,
124.82, 127.97, 131.24, 131.50, 179.36. IR (NaCl, neat):
3011 m, 2971, 2931, 2856, 1711, 1455, 1260, 1240, 1085,
1
20 mm£2 mm (Fa. Grom, D-71083 Herrenberg, Germany)
for LC separation, and attached to a Finnigan LCQ with
Finnigan LC/MS APCI interface (CA 95134 San Jose,
USA). Vaporizer temperature: 4508C. GC–MS: Trace MS,
910, 815, 795, 730 cm2 . MS (EI 70 eV) m/z (%) 294 (M ,
0.5), 276 (6), 236 (10), 222 (32), 135 (12), 121 (21), 107
(40), 95 (45), 93 (73), 91 (34), 81 (67), 80 (84), 79 (100), 67
1
þz
2
000 Series (Thermoquest, D-63329 Egelsbach, Germany)
equipped with an Alltech EC5 (15 m£0.25 mm, 0.25 mm).
(77), 59 (18), 55 (68); exact mass 276.2090; calcd for
þz
Mass spectra were measured in electron impact (EI) mode at
C
18
H
28
O
2
[M 2H
2
O] 276.2089.

138
D. Spiteller, W. Boland / Tetrahedron 59 (2003) 135–139
3
.1.3. (15RS,16RS)-4-Carbamoyl-2-[14-(3-ethyloxi-
ranyl)-tetradeca-9,12-dienoylamino]-butyric acid (2)
5N-(15,16-epoxylinoleoyl)-L-glutamine]. A stirred and
Acknowledgements
[
We thank Dr Ales Svatos and Janine Rattke for high
resolution mass spectra and Dr A. Elbert (Bayer AG,
Monheim) for continuous supply with egg clutches of
lepidopteran larvae. Rearing of insect cultures by Angelika
Berg is gratefully acknowledged.
chilled solution of 15,16-epoxylinoleic acid (100 mg) in dry
tetrahydrofuran (10 ml) was treated with triethylamine
(
55 ml) and ethyl chloroformate (34 ml). After 2 min,
L-glutamine (125 mg), dissolved in 0.3N NaOH (7 ml),
was added. After 5 min, the solution was allowed to come to
rt and the reaction was stopped by careful acidification with
2
N HCl. The conjugate was extracted with CH Cl2
2
(
the residue was purified by medium-pressure chroma-
3£10 ml). After drying (Na SO ) and removal of solvent,
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4
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(
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þ
C H N O [MþH] 441.2965.
2
3 41 2 6

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2