N-(17-Acyloxy-acyl)-glutamines: Novel Surfactants from Oral Secretions of Lepidopteran Larvae

Article abstract of DOI:10.1021/jo0342525

N-(17-Acyloxy-acyl)-glutamine conjugates such as N-(17-linolenoyloxy-linolenoyl)-glutamine (6), N-(17-linolenoyloxy-linoleoyl)-glutamine (7), N-(17-linoleoyloxy-linolenoyl)-glutamine (8), and N-(17-linoleoyloxy-linoleoyl)-glutamine (9) were identified as novel surfactants in the oral secretion of several lepidopteran larvae (S. exigua, S. littoralis, S. frugiperda, and H. virescens) by LC-MS/MS and chemical degradation. Authentic reference compounds were synthesized via a dissymmetric bis-Wittig approach and confirmed the assigned structures.

Full text of DOI:10.1021/jo0342525

VOLUME 68, NUMBER 23
NOVEMBER 14, 2003
© Copyright 2003 by the American Chemical Society
N-(17-Acyloxy-a cyl)-glu ta m in es: Novel Su r fa cta n ts fr om Or a l
Secr etion s of Lep id op ter a n La r va e†
Dieter Spiteller and Wilhelm Boland*
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Winzerlaer Strasse 10,
D-07745 J ena, Germany
boland@ice.mpg.de
Received February 25, 2003
N-(17-Acyloxy-acyl)-glutamine conjugates such as N-(17-linolenoyloxy-linolenoyl)-glutamine (6),
N-(17-linolenoyloxy-linoleoyl)-glutamine (7), N-(17-linoleoyloxy-linolenoyl)-glutamine (8), and N-(17-
linoleoyloxy-linoleoyl)-glutamine (9) were identified as novel surfactants in the oral secretion of
several lepidopteran larvae (S. exigua, S. littoralis, S. frugiperda, and H. virescens) by LC-MS/MS
and chemical degradation. Authentic reference compounds were synthesized via a dissymmetric
bis-Wittig approach and confirmed the assigned structures.
In tr od u ction
pholipids of membranes and become major products
under phosphate-limited conditions.6 Recently, the first
hydroxylated N-acyl glutamines were found in gut secre-
tions of insects.
Conjugates of fatty acids with amino acids, in particu-
lar glutamine and glutamate, are common constituents
of gut secretions of lepidopteran larvae.1-3 The com-
pounds are also found in the digestive tract of other
arthropods, even in spiders, crabs, and crickets.4 Another
rich source of N-acyl-amino acids is microorganisms that
produce a large variety of antibacterial or antifungal
surface-active agents. Some enhance the bioavailability
of surfaces for growth, others regulate the attachment
and/or detachment of microorganisms to surfaces (quo-
rum sensing).5 Besides simple N-acyl-amino acids, there
exist more complex structures mimicking phospholipids
of membranes.6 In many cases the lipid moiety of the
amino acid conjugates comprises a 3-hydroxy core fatty
acid acylated with a second fatty acid. The first members
of such complex amphiphilic lipids were isolated from
culture media of different Gram negative bacteria, among
them are Pseudomonas, Streptomyces, and Flavobacte-
rium species.7 The zwitterionic, acylated amide lipids are
interchangeable in function with the amphiphilic phos-
N-(17-Hydroxy-linolenoyl)-L-glutamine (volicitin, 1) was
isolated from oral secretions of lepidopteran larvae
(Spodoptora spp.) and reported to elicit the biosynthesis
of volatiles after introduction into the damaged leaf by
the feeding insect.1,3,8,9 By synthesis and comparison with
the natural secretions, the chiral center in the fatty acid
moiety was shown to be always 17S with g94% ee.10
Volicitin (1) is, in some insects, accompanied by minor
amounts of another series of oxygenated conjugates such
as N-(15,16-epoxy-linoleoyl)-glutamine (4) and N-(15,16-
dihydroxy-linoleoyl)-glutamine (5).11 According to recent
biosynthetic studies, the amide bond between the fatty
acid and glutamine is apparently made by commensal
microorganisms living in the insect gut.12 Owing to their
structural analogy to microbial lipids and owing to the
involvement of bacteria in their biosynthesis, we were
encouraged to investigate the oral secretions of lepi-
dopteran larvae for the presence of O-acylated derivatives
of volicitin (1) related to the previously known 3-O-acyl
amino acids from microorganisms. Here we report on the
isolation and synthesis of a novel series of N-(17-acyloxy-
† Dedicated to Prof. Dr. W. Steglich on the occasion of his 70th
birthday.
(1) Alborn, H. T.; Turlings, T. C. J .; J ones, T. H.; Stenhagen, G.;
Loughrin, J . H.; Tumlinson, J . H. Science 1997, 276, 945-949.
(2) Pohnert, G.; J ung, V.; Haukioja, E.; Lempa, K.; Boland, W.
Tetrahedron 1999, 55, 11275-11280.
(3) Halitschke, R.; Schittko, U.; Pohnert, G.; Boland, W.; Baldwin,
I. T. Plant Physiol. 2001, 125, 711-717.
(8) Frey, M.; Stettner, C.; Pare´, P. W.; Schmelz, E. A.; Tumlinson,
J . H.; Gierl, A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14801-14806.
(9) Shen, B.; Zheng, Z.; Dooner, H. K. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 14807-14812.
(10) Spiteller, D.; Pohnert, G.; Boland, W. Tetrahedron Lett. 2001,
42, 1483-1485.
(11) Spiteller, D.; Boland, W. Tetrahedron 2003, 59, 135-139.
(12) Spiteller, D.; Dettner, K.; Boland, W. Biol. Chem. 2000, 381,
755-762.
(4) Collatz, K. G.; Mommsen, T. J . Comp. Physiol. 1974, 94, 339-
352.
(5) Ron, E. Z.; Rosenberg, E. Environ. Microbiol. 2001, 3, 229-236.
(6) Minnikin, D. E.; Abdolrah. H. FEBS Lett. 1974, 43, 257-260.
(7) Asselineau, J . In Progress in the Chemistry of Organic Natural
Products; Herz, W., Grisebach, H., Kirby, G. W., Thamm, C., Eds.;
Springer: New York, 1991; pp 1-85.
10.1021/jo0342525 CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/05/2003
J . Org. Chem. 2003, 68, 8743-8749
8743

Spiteller and Boland
ship between the two compounds. The fragment at m/z
147 is common to all conjugates of this type and indicates
the presence of glutamine. Since MS/MS experiments
with 6 established m/z 405 to be derived from m/z 683,
the difference of 278 Da suggested the presence of an
additional linolenic acid molecule in the conjugate.
This assumption was confirmed by HR-MS (C41H67N2O6
for the quasimolecular ion at m/z 683) and by metha-
nolysis of 6, which released the methyl esters of linolenic
acid and 17-hydroxy-linolenic acid from the conjugate.
Additional compounds, varying in the degree of unsat-
uration of their fatty acid building blocks, were present
in the oral secretions. The APCI mass spectrum of
compound 7 (see Figure 1 and Scheme 2) shows a
quasimolecular ion [M + H]+ at m/z 685, along with a
rather weak fragment at m/z 407, suggesting a 17-
hydroxy-linoleic acid as the central building block. This
most obvious difference between the mass spectra of 6
and 7 is due to the ease of elimination of allylic substit-
uents from the volicitin-type conjugates, yielding termi-
nally conjugated products with the most abundant ion
at m/z 405 (100%).12,13 Derivatives of 17-hydroxy-linoleic
acid are less prone to elimination reactions and, hence,
display only a weak fragment at m/z 407 (16%). Metha-
nolysis of 7 yielded the methyl esters of the fatty acid
core 17-hydroxy-linoleic acid and linolenic acid. The two
other compounds were identified as N-(17-linoleoyloxy-
linolenoyl)-glutamine (8) and N-(17-linoleoyloxy-lino-
leoyl)-glutamine (9).
F IGURE 1. N-Acyl-glutamines in oral secretions from larvae
of Spodoptera exigua (RP-18, for HPLC conditions see Experi-
mental Section).
SCHEME 1. N-Acylglu ta m in es fr om th e
Regu r gita n t of Sp od op ter a n La r va e
Information on the positions of the double bonds in the
novel conjugates was obtained by ozonolysis of purified
6 and 7. For example, oxidative cleavage of the conjugate
7 (Scheme 3) delivered the dialdehyde ester 10 (deter-
mined by GC-MS), along with the polar amino acid
conjugate 11 (determined by LC-MS), confirming the
assigned structure and its origin from common unsatur-
ated fatty acids such as linoleic and linolenic acid.
Syn th esis of N-(17-Lin olen oyloxy-lin oleoyl)-glu -
ta m in e (7). To prove the assigned structures and to
obtain material for bioassays, the conjugate 7 was
prepared following a previously established protocol for
the asymmetric synthesis of volicitin (1) (Scheme 4).13
The key step of the approach is the one-pot sequential
olefination of the bis-phosphorane 13, obtained from the
corresponding bis-phosphonium salt by deprotonation
with KN(SiMe3)2 to achieve a high (Z)-selectivity.14 The
first carbonyl component, the aldehyde 12, already car-
rying the linolenic acid moiety, was added at -78 °C, and
the reaction mixture was allowed to come to rt within 1
h. After the mixture was recooled to -78 °C, 9-oxo-
nonanoic acid (2-trimethylsilyl)-ethyl ester (14) was
introduced as the second carbonyl component. After
purification and chromatography on silica gel, the (2-
trimethylsilyl)-ethyl ester of 17-linolenoyloxy-linoleic acid
(15) was obtained in 26% overall yield. According to the
13C NMR spectra, introduction of each of the two double
bonds occurred with g90% (Z)-selectivity. Removal of the
(2-trimethylsilyl)-ethyl ester moiety with TBAF pro-
ceeded smoothly and without affecting the linolenoyl
ester moiety. Final condensation of the free acid with
acyl)glutamines that occur widespread in oral secretions
of lepidopteran larvae.
Resu lts a n d Discu ssion
Isola tion a n d Str u ctu r a l An a lysis of N-(17-Acyl-
oxy-a cyl)-glu ta m in es. Oral secretions of several lepi-
dopteran larvae were collected into glass capillaries by
gently squeezing the larva with a forceps behind the head
which caused immediate regurgitation. After collection,
the oral secretions were investigated for higher molecular
weight O-acyl conjugates by HPLC-MS on reversed phase
(RP18), using gradient elution as shown in Figure 1. The
three first eluting compounds were readily identified with
reference compounds as volicitin (1), N-linolenoyl-
glutamine (2), and N-linoleoyl-glutamine (3) (Scheme
1).1,2 The APCI-mass spectrum of the first unknown
compound 6 (40.5 min) displayed an intense quasimo-
lecular ion at m/z 683 [M + H]+ and an [M + Na]+ adduct
at m/z 705 (see Figure 2). Fragment ions at m/z 405, 259,
147, and 130 were identical with corresponding fragment
ions of volicitin (1), suggesting a close structural relation-
(13) Pohnert, G.; Koch, T.; Boland, W. Chem. Commun. 1999, 1087-
1088.
(14) Pohnert, G.; Boland, W. Eur. J . Org. Chem. 2000, 1821-1826.
8744 J . Org. Chem., Vol. 68, No. 23, 2003

N-(17-Acyloxy-acyl)-glutamines
F IGURE 2. APCI-mass spectrum of N-(17-linolenoyloxy-linolenoyl)-glutamine (6).
SCHEME 2. N-(17-Acyloxya cyl)-glu ta m in es fr om th e Regu r gita n t of Sp od op ter a n La r va e
SCHEME 3. Ozon olysis of N-(17-Lin olen oyloxy-lin oleoyl)-glu ta m in e (7)
L-glutamine was achieved as described13 and afforded
N-(17-linolenoyloxy-linoleoyl)-L-glutamine (7), identical
in all respects with the natural product.
irane yielded the alcohol 18,15 which was esterified with
linolenic acid in the presence of N,N-(dimethylamino)-
pyridine and dicyclo-hexylcarbodiimide.16
The two building blocks 12 and 14 required for the
synthesis of 15 were obtained as outlined in Scheme 5.
Grignard reaction of the dioxolane 17 with 2-methylox-
(15) Bal, S. A.; Marfat, A.; Helquist, P. J . Org. Chem. 1982, 47,
5045-5050.
J . Org. Chem, Vol. 68, No. 23, 2003 8745

Spiteller and Boland
SCHEME 4. Syn th esis of N-(17-Lin olen oyloxy-lin oleoyl)-L-glu ta m in e (7)
SCHEME 5. Syn th esis of th e Su bstr u ctu r a l Elem en ts of 12 a n d 14
Treatment of the 1,3-dioxolane 19 with aq THF/HCl
selectively removed the protecting group and afforded 12
in about 30% overall yield. The 9-oxo-nonanoic acid (2-
trimethylsilyl)-ethyl ester (14) was available from nonane-
dicarboxylic acid monomethyl ester (20) by esterification
with (2-trimethylsilyl)-ethanol17,18 and subsequent regio-
selective reduction of the less hindered methyl ester 21
with NaH2Al(OCH2CH2OMe)2.19 14 was obtained with
32% overall yield.
The synthetic concept (Scheme 4) has a high potential
and flexibility, since it allows the use and combination
of different substructural elements, tolerates a number
of functional groups, and is easily extended to highly
unsaturated or isotopically labeled fatty acids and related
compounds.14
Although direct esterification of the 17-hydroxy group
of volicitin (1) with activated derivatives of linolenic or
linoleic acid would provide a more direct route to the title
compounds, this approach suffered from competing elimi-
nation of the 17-hydroxy substitutent. Similar observa-
tions have been reported for derivatization of the mayo-
lenes.20 An attempt to acylate volicitin (1) with linolenic
acid or its vinyl ester by enzyme catalysis (porcine
(16) Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17,
522-524.
(17) Gerlach, H. Helv. Chim. Acta 1977, 60, 3039-3044.
(18) Sieber, P. Helv. Chim. Acta 1977, 60, 2711-2716.
(19) Kanazawa, R.; Tokoroyama, T. Synthesis 1976, 8, 526-527.
8746 J . Org. Chem., Vol. 68, No. 23, 2003

N-(17-Acyloxy-acyl)-glutamines
TABLE 1. N-(17-Acyloxy-a cyl)-glu ta m in es in
The compounds are potent deterrents against ants and
other enemies. Another example of a hydroxylated, but
nonconjugated pentadienyl segment is given by the ethyl
esters of 13-hydroxy-arachidonate and 13-hydroxyeisosa-
5,8,11,14,17-pentaenoic acid, both of which were isolated
from red calcareous algae from the genus Lithotham-
nion.21
Lep id op ter a n La r va ea
insects
1
2
6
7
8
9
Spodoptera exigua
Spodoptora littoralis
Spodoptera frugiperda
Heliothis virescens
++ ++
+
+(+)
+
+
+
+
+
+(+)
+
++ ++ +(+)
++ ++
++ ++
+
+
+
+
+
+(+)
a
N-acyl amino acids occur widespread in the digestive
organs of insects and beyond (spiders, crabs, etc.).4 They
may serve the organism as surfactants to emulsify food
lipids. In some cases, they may also have a defensive
function, since, for example, larvae of sawflies such as
Neodiprion lecontei discharge their enteric fluid onto
attacking enemies.22 A similar behavior is known for
some Spodopteran larvae. Unlike the very unstable
mayolenes, the conjugates 6-9 are less prone to elimina-
tion of the acyloxy substituent; even under the very
strongly basic conditions (pH ca. 9) of the insect gut the
compounds remain intact. The pronounced tendency of
compounds such as 6 to form micelles at very low
concentrations may protect the sensitive allylic acyloxy
moiety against the basic milieu. The critical micellar
concentration of, e.g., N-linolenoyl-glutamine (2) (cmc ca.
25 µM) is much lower than that of the commonly
employed detergent sodium dodecyl sulfate (SDS; cmc
8-10 mM) and competes well with that of surfactin,23
another powerful and well-studied bacterial amphiphile
(cmc ca. 9 µM). Plant feeding larvae introduce the
compounds with their regurgitant into the damaged
leaves and, owing to their strong surface activity, the
conjugates are able to depolarize plant cells and thereby
elicit defensive programs in plants. The mode of action
of the N-acyl amino acids on plant systems is currently
evaluated and will be reported soon.
Key: ++, major N-acyl-glutamines (>30%); +, minor com-
pounds (1%-30%, values in parentheses cover occasional strong
shifts in the composition of the regurgitant).
pancreas lipase in decane) was more successful and
yielded the conjugate 6, albeit in rather low yield (<5%
yield). Optimization of this approach is under investiga-
tion.
An a lysis of Or a l Secr etion s fr om Selected Lep i-
d op ter a n La r va e. Besides the known fatty acid amides
with glutamine such as 1, 2, and 3, the regurgitant from
the larvae listed in Table 1 showed different amounts of
the novel N-(17-acyloxy-acyl)-glutamines. The compounds
were not observed in oral secretions of larvae that fail to
produce N-(17-hydroxy-acyl)-glutamines [e.g., volicitin
(1)] like the tobacco hornworm (Manduca sexta).
Moreover, the quantitative composition of the amino
acid conjugates in the regurgitants shows remarkable
variations and appears to be dependent on the rearing
conditions. Occasionally, no or very low amounts of N-(17-
acyloxy-acyl)-glutamines were found. The novel conju-
gates may be either produced by acylation of volicitin (1)
or N-(17-hydroxylinoleoyl)-glutamine, or by acylation of
a 17-hydroxy acid (linolenic or linoleic acid type) by
microbial enzymes. First experiments with isolated mi-
croorganisms or even a pure enzyme isolated from a
commensal gut bacterium (unpublished results) with
simple aliphatic or hydroxylated fatty acids and natural
amino acids for conjugation revealed a very broad sub-
strate tolerance of the biocatalyst without a clear prefer-
ence for a certain fatty acid or amino acid, suggesting
that amide bond formation can occur with both types of
fatty acid substrates.
Exp er im en ta l Section
Gen er a l Met h od s. Reactions were performed under Ar.
Solvents were dried according to standard methods. 1H and
13C NMR were recorded at 400 and 500 MHz, respectively.
Chemical shifts of 1H and 13C NMR are given in ppm (δ) based
on solvent signals: DCCl3 7.26 (1H NMR) and 77.70 ppm
(13C NMR). For APCI-LC-MS/MS a microbore RP18-column
125 cm × 2 mm, 2 µm was used. High-resolution ESI-mass
spectrometry was performed by direct injection of the purified
sample. High-resolution mass spectrometry was performed on
a double-focusing magnetic sector mass spectrometer (geom-
etry EBE). For GC-MS (70 eV) a DB5 column was used
(15m × 0.25 mm, 0.25 µm); helium served as carrier gas. Silica
gel: Si 60 (0.200-0.063 mm) was used for chromatography.
Rea r in g of Lep id op ter a n La r va e a n d Collection of
Or a l Secr etion : Ar tificia l Diet. Ground white beans (500
g) were soaked overnight in water (1200 mL) and ascorbic acid
(9 g), parabene (9 g), aq formaldehyde (4 mL, 36.5%), and agar
(75 g), boiled in 1000 mL of H2O, were added. After cooling
the mixture solidified to a white waxy solid.24
Con clu sion s
Besides the previously known N-acyl glutamines, vo-
licitin (1) and N-(17-hydroxy-linoleoyl)-glutamine, a se-
ries of new N-(17-acyloxy-acyl)-glutamines is found in the
oral secretion of lepidopteran larvae. The N-(17-acyloxy-
acyl)-glutamines 6, 7, 8, and 9 are formally derived from
volicitin (1) or N-(17-hydroxy-linoleoyl)-glutamine by
additional acylation with linolenic or linoleic acid. Struc-
turally related compounds were previously only known
as microbial metabolites, and their presence in the insect
gut may be considered as another piece of evidence for
the impact of commensal microorganisms on the chemical
composition within the gut of insects. The lipid moieties
of the novel conjugates 6 to 9 exhibit similarities to the
recently isolated mayolenes from glandular hairs of the
lepidopteran larva Pieris rapae.20 Mayolenes are com-
posed of an 11-hydroxy-linolenic acid esterified to a series
of straight chain saturated fatty acids of 14 to 20 carbon
atoms, with palmitic acid and stearic acid dominating.
Rea r in g of In sect la r va e. Larvae were hatched from eggs
and reared on the agar-based artificial diet. Plastic cages with
the diet and the larvae were kept at 23-25 °C under a light-
dark regime with 16 h of illumination.
(21) Guerriero, A.; Dambrosio, M.; Pietra, F. Helv. Chim. Acta 1990,
73, 2183-2189.
(22) Costa, J . T.; Louque, R. W. Ann. Entomol. Soc. Am. 2001, 94,
480-489.
(23) Ishigami, Y.; Osman, M.; Nakahara, H.; Sano, Y.; Ishiguro, R.;
Matsumoto, M. Colloid Surf. B 1995, 4, 341-348.
(24) Bergomaz, R.; Boppre´, M. J . Lepidopterist’s Soc. 1986, 40, 131-
137.
(20) Smedley, S. R.; Schroeder, F. C.; Weibel, D. B.; Meinwald, J .;
Lafleur, K. A.; Renwick, J . A.; Rutowski, R.; Eisner, T. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 6822-6827.
J . Org. Chem, Vol. 68, No. 23, 2003 8747

Spiteller and Boland
Collection of Or a l Secr etion . Regurgitant was collected
from larvae by slight squeezing of the animals with tweezers
and collection of the discharged enteric liquid with glass
capillaries (1 mm i.d.).
(2-Tr im eth ylsilyl)-eth yl-9-oxo-n on a n a te (14). A chilled
and well-stirred solution of Al(OEtOMe)2H2 (9.5 mL, 3.5 M in
toluene) in dry THF (10 mL) was gradually treated with
N-methylpiperazine (3.9 mL, 36.6 mmol). This reagent was
then added dropwise with stirring to a cold solution (-50 to
70 °C) of the silyl ester 21 (1.5 g, 5 mmol) in THF (20 mL).19
The flask was allowed to warm to -20 °C, while the progress
of the reaction was monitored by GC-MS. The reaction was
stopped by acidification with 2 N HCl and the product was
extracted with dichloromethane (3 × 25 mL). After drying (Na2-
SO4) and removal of the solvent, the product was purified by
chromatography on silica gel, using petroleum ether:ethyl
acetate (9:1, v:v) for elution. Colorless liquid (710 mg, 47%).
1H NMR (400 MHz, CDCl3) δ 9.74 (t, J ) 1.89 Hz, 1H), 4.10-
4.19 (m, 2H), 2.40 (dt, J ) 1.89 Hz, J ) 7.55 Hz, 2H), 2.25 (t,
J ) 7.55 Hz, 2H), 1.55-1.66 (m, 4H), 1.25-1.37 (m, 6H), 0.93-
1.00 (m, 2H), 0.02 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
203.35, 174.55, 63.04, 44.50, 35.09, 29.64, 29.60, 29.56, 25.52,
22.64, 17.98, -0.84; IR (KBr, film) 2936, 2859, 2823, 2718,
1727, 1459, 1414, 1382, 1350, 1250, 1173, 1100, 1059, 1041,
936, 864, 836 cm-1; EI-MS 272 (M+•, 0.1), 257 (0.6), 243 (1),
229 (10), 211 (19), 199 (11), 173 (24), 129 (23), 117 (21), 75
(56), 73 (100); EI-HRMS calcd for C14H28SiO3 M+• 272.1808,
found 272.1815.
An a lysis of Or a l Secr etion s. The collected regurgitant
was diluted with the same volume of MeOH to precipitate
proteins. After centrifugation at 13 000 U/min (16 000 g) for
5 min, the supernatant was directly used for LC-MS analysis.
Separation was achieved by HPLC (RP18, 150 mm × 2 mm, 2
µm; Grom, Herrenberg, Germany) with programmed gradient
elution. A typical analysis was performed with a flow rate of
0.2 mL min-1, using a binary gradient of two solvent systems
(A: H2O with 0.1% AcOH; and B: MeCN with 0.1% AcOH)
starting with 100% A (3 min) programmed to 100% B in 27
min maintained for 15 min prior to equilibration to the starting
conditions. The relevant compounds eluted between 39 and
51 min and were investigated by MS/MS experiments (collision
energy: 15-25%) and HR-ESI-MS.
N-(17-Lin olen oyloxy-lin olen oyl)-glu tam in e (6): 39.5 min;
APCI-MS [M + Na]+ 705 (8), [M + H]+ 683 (28), 405 (100),
259 (6), 147 (1); HR-ESIMS [M + H]+ calcd for C41H67N2O6
683.4999, found 683.4979.
N-(17-Lin olen oyloxy-lin oleoyl)-glu ta m in e (7): 40.5 min;
APCI-MS [M + Na]+ 707, [M + H]+ 685 (100), 667 (5), 407
(16), 261 (2), 147 (1), 130 (1); HR-ESI-MS [M + H]+ calcd for
5-(1,3-Dioxola n e-2-yl)-p en ta n -2-ol (18).15 A well-stirred
suspension of magnesium turnings (1.1 g, 45 mmol) in dry THF
(10 mL) was treated at 30-35 °C with 1,2-dibromoethane (90
µL) and 2-(bromoethyl)-1,3-dioxolane (17) (2.4 mL, 20 mmol).
After completion of the reaction, the mixture was cooled to
-78 °C, and a solution of CuBr(Me2S) (0.75 g, 3.7 mmol) was
added dropwise to the well-stirred solution. After 1 h propylene
oxide (1 mL, 15 mmol) was added with a chilled syringe.
Stirring was continued for 3.5 h while the reaction was allowed
to come to -30 °C followed by another 20 h warm-up to 0 °C.
After hydrolysis with satd aq NH4Cl, extraction with dichlo-
romethane, drying (Na2SO4), and evaporation of the solvent,
the crude product was purified by chromatography on silica
gel. Elution with petroleum ether:ethyl acetate (7:3, v:v)
C
41H69N2O6 685.5156, found 685.5143.
N-(17-Lin oleoyloxy-lin olen oyl)-glu ta m in e (8): 41.5 min;
APCI-MS [M + Na]+ 707, [M + H]+ 685 (27), 405 (100), 259
(5), 147 (2).
N-(17-Lin oleoyloxy-lin oleoyl)-glu ta m in e (9): 43.0 min;
APCI-MS [M + H]+ 687 (100), 407 (18), 261 (1),147 (1).
Ozon olytic Clea va ge of th e Con ju ga tes 6 a n d 7. N-(17-
Acyloxy-acyl)-glutamine conjugates from the regurgitant (500
µL) of larvae of S. exigua were purified by HPLC (see above).
After removal of the solvent by a gentle stream of argon and
drying under high vacuum, the residue was dissolved in dry
MeOH (1 mL), and ozone (ca. 15 mg/min) was passed through
the solution for 5 min (-78 °C). The ozonides were reduced by
addition of Me2S (1 µL), and after 5 min the solvent was
removed by a stream of argon. Following an additional
treatment with diazomethane, the polar cleavage products
were identified by GC-MS. Ozonolytic degradation of conju-
gates 6 and 7 yielded the following fragments: 5-(9-oxo-
nonanoyl)-hexanal (10) [GC-MS 155 (100), 115 (24), 109 (48),
99 (88), 81 (90), 69 (35), 67 (26), 55 (81), 44 (40)] and 9-oxo-
nonanoic acid methyl ester [GC-MS 158 (7), 155 (18), 143 (26),
111 (39), 87 (67), 83 (45), 74 (100), 59 (40), 55 (75)]. N-(9-Oxo-
nonanoyl)-glutamine (11) was identified by LC-MS. APCI-MS
[M + H]+ 301 (100), 283 (37), 155 (2).
Syn th esis of N-(17-Acyloxy-a cyl)-glu ta m in es: Non a n e-
d ioic Acid (2-Tr im eth ylsilyl)-eth yl Ester Meth yl Ester
(21). To a chilled and well-stirred solution of monomethyl ester
20 (2 g, 10 mmol), N,N-(dimethylamino)-pyridine (80 mg, 0.66
mmol),16 and 2-trimethylsilylethanol17,18 (2.1 g, 15 mmol) in
dry dichloromethane (10 mL) was added, under argon, dicy-
clohexylcarbodiimide (2.3 g, 11 mmol) in 10 mL of the same
solvent. After the mixture was stirred for 5 min at 0 °C,
stirring was continued for 3 h at rt. The precipitated urea was
filtered off and the mixture was extracted with 0.5 N HCl and
satd NaHCO3 solution. After drying (Na2SO4) the solvent was
removed under reduced pressure. Purification was achieved
by chromatography on silica gel, using petroleum ether:ethyl
acetate (9:1, v:v) for elution. Colorless liquid (2.16 g, 68%). 1H
NMR (400 MHz, CDCl3) δ 4.10-4.17 (m, 2H), 3.64 (s, 3H), 2.28
(t, J ) 7.55 Hz, 2H), 2.25 (t, J ) 7.43 Hz, 2H), 1.52-1.65 (m,
4H), 1.22-1.36 (m, 6H), 0.92-0.99 (m, 2H), 0.02 (s, 9H); 13C
NMR (100 MHz, CDCl3) δ 174.86, 174.58, 63.02, 52.08, 35.10,
34.68, 29.59, 29.53, 25.53, 25.51, 17.96, -0.84; IR (KBr, film)
2951, 2861, 1735, 1437, 1355, 1250, 1172, 1062, 938, 856, 838
cm-1; EI-MS 302 (M+•, 0.5) 287 (3), 259 (45), 243 (13), 227 (9),
185 (19), 173 (45), 159 (24), 117 (29), 75 (49), 73 (100), 59 (12);
EI-HRMS calcd for C15H30O4Si M+• 302.1913, found 302.1912.
1
afforded 18 as a colorless liquid (1.39 g, 58%). H NMR (400
MHz, CDCl3) δ 4.85 (t, J ) 4.72 Hz, 1H), 3.91-3.99 (m, 2H),
3.76-3.89 (m, 3H), 1.62-1.74 (m, 2H), 1.41-1.59 (m, 4H), 1.18
(d, J ) 6.23 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 105.20,
68.53, 65.52, 39.76, 34.38, 24.10, 20.83; IR (KBr, film) 3415
(br), 2956, 2929, 2882, 1456, 1410, 1369, 1128, 1054, 1031, 943,
845 cm-1; EI-MS 159 ([M - H]+•, 1), 143 (10), 129 (8), 112 (6),
99 (6), 81 (9), 73 (100), 57 (17); EI-HRMS calcd for C8H15O3
[M - H]+• 159.1021, found 159.1019.
Lin olen ic Acid 4-(1,3-Dioxola n e-2-yl)-1-m eth yl-bu tyl
Ester (19). A solution of linolenic acid (1 g, 3.8 mmol) in dry
dichloromethane (10 mL) was treated with N,N-(dimethyl-
amino)-pyridine (100 mg, 0.82 mmol) and 5-(1,3-dioxolane-2-
yl)-pentane-2-ol (18, 450 mg, 2.8 mmol). To the chilled and
stirred solution was added dicyclohexylcarbodiimide (598 mg,
2.9 mmol). After 5 min, stirring was continued for 3 h at rt.
The urea was filtered off and the organic layer was washed
with 0.5 N HCl and satd aq NaHCO3. After drying (Na2SO4)
the solvent was removed under reduced pressure. The ester
was purified by chromatography on silica gel, using petroleum
ether:ethyl acetate (9:1, v:v) for elution. Colorless liquid (637
mg, 54%). 1H NMR (400 MHz, CDCl3) 5.26-5.43 (m, 6H), 4.90
(sext, J ) 6.23 Hz, 1H), 4.83 (t, J ) 4.77 Hz, 1H), 3.90-3.99
(m, 2H), 3.80-3.88 (m, 2H), 2.76-2.84 (pt, 4H), 2.26 (t, J )
7.48 Hz, 2H), 1.99-2.11 (m, 4H), 1.39-1.70 (m, 8H), 1.24-
1.38 (m, 8H), 1.20 (d, J ) 6.42 Hz, 3H), 0.97 (t, J ) 7.49 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ 174.16, 132.64, 130.96,
128.97, 128.94, 128.39, 127.08, 105.06, 71.19, 65.54, 36.48,
35.38, 34.34, 30.27, 29.87, 29.80, 27.89, 26.30, 26.21, 25.74,
21.23, 20.62, 14.96; IR (KBr, film) 3010, 2932, 2855, 1730,
1652, 1460, 1373, 1245, 1181, 1135, 1098, 1057, 1030, 938, 719
cm-1; EI-MS 420 (M+•, 43), 305 (25), 277 (29), 261 (20), 143
(100), 108 (57), 101 (48); EI-HRMS calcd for C26H44O4 M+•
420.3240, found 420.3239.
8748 J . Org. Chem., Vol. 68, No. 23, 2003

N-(17-Acyloxy-acyl)-glutamines
Lin olen ic Acid 1-Meth yl-5-oxo-p en tyl Ester (12). The
dioxolane was cleaved by stirring 19 (750 mg, 1.79 mmol) in
THF (50 mL) and 6 N HCl (25 mL) at 0 °C for 3 h. The mixture
was allowed to warm to rt and stirred overnight. After
extraction with dichloromethane and drying (Na2SO4), the
solvent was removed under reduced pressure. Chromatography
on silica gel, using petroleum ether:ethyl acetate (9:1, v:v) for
elution, afforded 12 a colorless liquid (562 mg, 84%). 1H NMR
(400 MHz, CDCl3) δ 9.74 (t, J ) 1.51 Hz,1H), 5.44-5.25 (m,
6H), 4.88 (qt, J ) 6.3 Hz, J ) 5.28 Hz, 1H), 2.83-2.74 (m,
4H), 2.49-2.41 (m, 2H), 2.27 (t, J ) 7.50, 2H), 2.00-2.13 (m,
4H), 1.73-1.47 (m, 6H), 1.39-1.23 (m, 8H), 1.21 (d, J ) 6.36
Hz, 3H), 0.97 (t, J ) 7.50 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 202.41, 174.05, 132.61, 130.91, 128.97, 128.94, 128.43,
127.81, 70.69, 44.15, 35.95, 35.32, 30.23, 29.81, 29.77, 27.87,
26.30, 26.21, 25.70, 21.21, 20.58, 18.63, 14.89; IR (KBr, film)
3010, 2934, 2853, 2717, 1733, 1457, 1370, 1246, 1181, 1132,
715 cm-1; EI-MS 376 (M+•, 10), 277 (11), 261 (20), 135 (5), 129
(25), 121 (7), 108 (15), 99 (100), 81 (64), 69 (15), 67 (14), 55
(35); EI-HRMS calcd for C24H40O3 M+• 376.2978, found 376.2978.
17-Lin olen oyloxy-lin oleic Acid (2-tr im eth ylsilyl)-eth yl
Ester (15). A suspension of propane-1,3-bistriphenylphospho-
nium bromide (13) (661 mg, 0.91 mmol) in dry THF (15 mL)
was stirred at -78 °C, and a solution of KN(SiMe3)2 (3.8 mL,
0.5 M in hexane) was slowly added. To achieve complete
deprotonation, the solution was allowed to warm to 0 °C for
10 min and was then recooled to -78 °C. The first carbonyl
component, (1S)-linolenic acid 1-methyl-5-oxo-pentyl ester (12)
(338 mg, 0.9 mmol) in 3 mL of dry THF, was added dropwise
via a precooled (-78 °C) syringe. After 1 h the mixture was
allowed to warm and was stirred for another 1 h at rt. After
the mixture was recooled to -78 °C, the second carbonyl
component, 14 (245 mg, 1 mmol) in dry THF (2 mL), was also
slowly added via a precooled syringe. The reaction was allowed
to warm and was stirred at rt for an additional 2 h. After
hydrolysis with NH4Cl, extraction with dichloromethane, and
drying (Na2SO4), solvents were removed under reduced pres-
sure. The pure ester 15 was obtained as a colorless liquid by
chromatography on silica gel, using petroleum ether:ether (95:
of the solvent under reduced pressure, the acid was purified
by RP18 chromatography, using a binary gradient from H2O:
MeOH (30:70, v:v), to 100% MeOH (52 mg, 62%). 1H NMR (500
MHz, CDCl3) δ 5.26-5.46 (m, 10H), 4.91 (sext, J ) 6.38 Hz,
1H), 2.85-2.73 (m, 6H), 2.35 (t, J ) 7.55, 2H), 2.26 (t, J )
7.43 Hz, 2H), 2.00-2.13 (m, 8H), 1.23-1.68 (m, 24H), 1.20 (d,
J ) 6.30 Hz, 3H), 0.97 (t, J ) 7.55 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 177.58, 174.27, 132.68, 130.99, 130.89, 130.19,
129.29, 129.03, 128.99, 128.62, 128.47, 127.87, 71.32, 36.28,
35.45, 34.24, 30.30, 30.24, 29.88, 29.84, 29.84, 29.75, 29.72,
27.93, 27.90, 27.68, 26.36, 26.26, 26.14, 25.79, 25.42, 21.26,
20.72, 14.94; IR (KBr, film) 3010, 2928, 2855, 1734, 1711, 1460,
1373, 1245, 1181, 1130, 1085, 719 cm-1; EI-MS 556 (M+•, 28),
294 (10), 278 (100), 277 (55), 236 (18), 222 (12), 135 (20), 113
(39); EI-HRMS calcd for C36H60O4 M+• 556.4492, found 556.4481.
N-(17-Lin olen oyloxy-lin oleoyl)-L-glu ta m in e (7). Free
acid 16 (50 mg, 0.10 mmol) and triethylamine (11 mg, 0.11
mmol) were stirred at -10 °C under argon in dry THF (4 mL),
and ethyl chloroformate (12 mg, 0.11 mmol) was added. After
5 min a solution of L-glutamine (30 mg, 0.20 mmol) in NaOH
(2.8 mL, 0.3 N) was added, and the mixture was stirred at rt
for 30 min. The solution was acidified with 2 N HCl and
extracted with dichloromethane. The combined organic layers
were dried (Na2SO4) and the solvent removed under reduced
pressure. The conjugate product was purified by HPLC on RP-
18, using a binary gradient of acetonitrile:water (70:30, v:v),
to pure acetonitrile (49.6 mg, 72%). 1H NMR (500 MHz, CDCl3)
δ 7.09 (s, br, 1H), 7.07 (s, br, 1H), 6.33 (s, br, 1H), 5.98 (s, br,
1H), 5.27-5.45 (m, 10H), 4.91 (sext, J ) 6.32 Hz, 1H), 4.47 (q,
J ) 6.09 Hz, 1H), 2.74-2.87 (m, 6H), 2.55-2.64 (m, 1H), 2.39-
2.48 (m, 1H), 2.26 (t, J ) 7.41 Hz, 2H), 2.25 (t, J ) 7.41 Hz,
2H), 2.15-2.24 (m, 2H), 1.99-2.13 (m, 8H), 1.24-1.68 (m,
24H), 1.20 (d, J ) 6.17 Hz, 3H), 0.98 (t, J ) 7.53 Hz, 3H); 13
C
NMR (125 MHz, CDCl3) δ 177.23, 175.42, 174.33, 173.85,
132.68, 130.99, 130.90, 130.20, 129.29, 129.04, 129.00, 128.62,
128.48, 127.87, 71.37, 53.22, 37.09, 36.26, 35.47, 32.63, 30.30,
29.90, 29.88, 29.84, 29.82, 28.34, 27.93, 27.68, 26.37, 26.36,
26.27, 26.20, 26.12, 25.80, 21.26, 20.72, 14.93; IR (KBr, film)
3429, 3327, 3210, 3013, 2959, 2927, 2858, 1731, 1657, 1542,
1456, 1423, 1260, 1188, 1098, 1026, 801 cm-1; APCI-MS 685
(100), 407 (16), 261 (2), 147 (1), 130 (1); ESI-HRMS calcd for
1
5, v:v) for elution (172 mg, 26%). H NMR (400 MHz, CDCl3)
δ 5.24-5.45 (m, 10H), 4.85-4.95 (m, 1H), 4.11-4.19 (m, 2H),
2.69-2.86 (m, 6H), 2.27 (t, J ) 7.55 Hz, 2H), 2.26 (t, J ) 7.55
Hz, 2H), 1.95-2.13 (m, 8H), 1.23-1.67 (m, 24H), 1.20 (d, J )
6.04 Hz, 3H), 0.97 (t, J ) 7.43 Hz, 3H), 0.66-1.00 (m, 2H),
0.04 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 174.71, 174.19,
132.65, 130.96, 130.91, 130.79, 130.29, 130.17, 129.23, 129.05,
128.97, 128.94, 128.51, 128.41, 127.81, 71.24, 63.05, 36.24,
35.41, 35.22, 30.28, 29.88, 29.82, 27.90, 27.64, 26.30, 26.22,
26.12, 25.76, 25.67, 21.24, 20.74, 18.01, 14.97, 1.70, -0.80; IR
(KBr, film) 3010, 2928, 2855, 1734, 1455, 1377, 1245, 1172,
1135, 856, 833 cm-1; EI-MS 656 (M+•, 2), 629 (26), 614 (6), 351
(83), 350 (100), 349 (42), 335 (55), 321 (7), 308 (11), 294 (7),
278 (67), 277 (51), 261 (46), 243 (9), 236 (14), 196 (25), 163
(16), 135 (48), 121 (44); EI-HRMS calcd for C41H72O4Si M+•
656.5200 found 656.5176.
C
41H69N2O6 [M + H]+ 685.5155, found 685.5143.
Ack n ow led gm en t. We thank Dr. Neil Oldham and
Janine Rattke for HR-MS measurements and Dr. Renate
Ellinger and Dr. Bernd Schneider for NMR measure-
ments. We are indebted to Dr. A. Elbert (Bayer AG,
D-40789 Monheim) for supplying us with egg clutches
of lepidopteran larvae and Angelika Berg for caterpillar
rearing. Financial support by the Fonds der Chemischen
Industrie, Frankfurt a.M., is gratefully acknowledged.
We thank Dr. Sabine Thiessen for the color picture of
the feeding larva of Spodoptora littoralis.
17-Lin olen oyloxy-lin oleic Acid (16). A solution of the (2-
trimethylsilyl)-ethyl ester 15 (100 mg, 0.15 mmol) in THF (2.5
mL) was stirred at rt with a solution of tetrabutylammonium
fluoride (2 mL, 1 M solution in THF) overnight. After acidifica-
tion with HCl (10 mL, 2 N), the free acid 16 was extracted
with dichloromethane. After drying (Na2SO4) and evaporation
Su p p or tin g In for m a tion Ava ila ble: Mass spectra and
1H and 13C NMR spectra of compounds 7, 12, 14, 15, 16, 18,
19, and 21. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O0342525
J . Org. Chem, Vol. 68, No. 23, 2003 8749