Identification and synthesis of volicitin and related components from beet armyworm oral secretions

Article abstract of DOI:10.1023/A:1005401814122

Oral secretion of beet armyworm caterpillars (BAW), when applied to damaged tissues of corn seedlings, induces the seedlings to emit volatile compounds that attract the natural enemies of the caterpillars. The key elicitor present in BAW oral secretions is N-[17-hydroxylinolenoyl]-L- glutamine (volicitin). Analysis of the oral secretion showed that it also contained N-[17-hydroxyolinoleoyl]-L-glutamine, free 17-hydroxylinolenic, and 17-hydroxylinoleic acid, the glutamine conjugates of linolenic and linoleic acid as well as free linolenic and linoleic acid. Here we present the identification and synthesis of the hydroxy acids and of glutamine conjugates.

Full text of DOI:10.1023/A:1005401814122

Journal of Chemical Ecology, Vol. 26, No. 1, 2000
IDENTIFICATION AND SYNTHESIS OF VOLICITIN AND
RELATED COMPONENTS FROM BEET ARMYWORM
ORAL SECRETIONS
H. T. ALBORN,1 T. H. JONES,2 G. S. STENHAGEN,3
and J. H. TUMLINSON1,*
1 Center for Medical, Agricultural, and Veterinary Entomology
United States Department of Agriculture, Agricultural Research Service
Gainesville, Florida 32608
2 Department of Chemistry
Virginia Military Institute
Lexington, Virginia 24450
3 Department of Organic Chemistry
Chalmers University of Technology
S-412 96 Go¨teborg, Sweden
(Received February 10, 1999; accepted September 3, 1999)
Abstract—Oral secretion of beet armyworm caterpillars (BAW), when
applied to damaged tissues of corn seedlings, induces the seedlings to emit
volatile compounds that attract the natural enemies of the caterpillars. The
key elicitor present in BAW oral secretions is N-[17-hydroxylinolenoyl]-L-
glutamine (volicitin). Analysis of the oral secretion showed that it also
contained N-[17-hydroxyolinoleoyl]-L-glutamine, free 17-hydroxylinolenic,
and 17-hydroxylinoleic acid, the glutamine conjugates of linolenic and
linoleic acid as well as free linolenic and linoleic acid. Here we present the
identification and synthesis of the hydroxy acids and of glutamine conjugates.
Key Words—Spodoptera exigua Hubner, Zea mays L., elicitors, isolation,
¨
identification, synthesis, volicitin, 17-hydroxylinolenic acid, 17-hydroxylinoleic
acid, linolenic acid, linoleic acid, glutamine conjugates.
INTRODUCTION
Injury by insect herbivores induces plants to release a blend of volatile ter-
penoids and indole that attract natural enemies of the herbivores (Turlings et
al., 1990, 1991). This intriguing defensive reaction is triggered when substances
*To whom correspondence should be addressed.
203
0098-0331 00 0100-0203$18.00 0  2000 Plenum Publishing Corporation
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ALBORN, JONES, STENHAGEN, AND TUMLINSON
in the oral secretion of the insect herbivores contact damaged plant tissue. Isola-
tion of the inducing factors in the oral secretion of beet armyworm (Spodoptera
¨
exigua Hubner) caterpillars resulted in a single fraction that contained all the
biological activity of the crude secretion. This fraction contained two compo-
nents in about equal concentrations (Turlings et al., 2000). Both components
had almost identical retention times on HPLC and identical UV spectra; how-
ever, only one of them induced corn (Zea mays L.) seedlings to release the
same blend of volatile compounds as are released when they are damaged by
caterpillar feeding (Turlings et al., 2000). The identification and synthesis of
the active component, N-[17-hydroxylinolenoyl]-L-glutamine, named volicitin,
was reported by Alborn et al. (1997). Here we present an improved synthesis
of volicitin and the synthesis of the second component, which was identified
as N-[17-hydroxylinoleoyl]-L-glutamine by mass and infrared spectroscopy and
by chemical transformations. We also describe an improved analysis of BAW
oral secretion that revealed the presence of related components including free
linolenic, linoleic, 17-hydroxylinolenic, and 17-hydroxylinoleic acid, as well as
the glutamine conjugates of linolenic and linoleic acid.
METHODS AND MATERIALS
Isolation of Biologically Active Natural Product. Following procedures
described in Turlings et al. (1993), 50 ml of oral secretion were collected from
third to fifth instar beet armyworms by gentle squeezing, causing them to regur-
gitate. Each purification step was monitored by analyzing the induced release of
terpenoids and indole from corn seedlings (Zea mays, variety Ioana and LG11
sweet corn) that had been allowed to take up a test solution through the cut
stem overnight (Turlings et al., 1993). The purification is described in detail in
Alborn et al. (1997) and Turlings et al. (2000). The crude oral secretion was cen-
trifuged at 1600g for 30 min and filtered through a sterilizing membrane (0.22
mm Millex GV, Millipore, Bedford, Massachusetts). An equal amount of 50 mM
pH 3 sodium phosphate buffer was added, and the precipitated proteins were
removed by centrifugation as before. The active material was purified with a
series of water–acetonitrile gradient reverse-phase HPLC separations (LDC 4100
pump, SM 5000 diode array detector, LDC Analytical, Riviera Beach, Florida)
with a Nova Pac C18 reverse phase column, 4-mm particle size, 4 mm ID × 150
mm long (Waters, Millford, Massachusetts) and with a reverse-phase ODS-AQ
S-5 column, 4.6 mm ID × 250 mm long (YMC, Kyoto, Japan). The UV absorp-
tion was monitored at 200 nm. Two closely related and difficult to separate com-
ponents were isolated and designated B and C. Component B was as biologically
active as the crude oral secretion while C showed no activity (Turlings et al.,
2000).

BEET ARMYWORM ORAL SECRETIONS
205
Spectrometric Instrumentation. A VG Analytical Zab-spec (Fison Instru-
ments, Manchester, England) high-resolution mass spectrometer was used for
fast atom bombardment (FAB) analysis of the natural products. Up to 4 ml of 10
ng ml solutions in 50% acetonitrile–water were added to a 10-ml glycerol matrix.
/
Samples were protonated by addition of 1 ml trifluoroacetic acid. Sodium adducts
were obtained by adding 1 ml of 1 M aqueous sodium chloride. Polyethylene gly-
col (1 ml) with an average mass of 400 Da (PET 400) was added to the FAB
glycerol matrix to give reference ions of known mass for an exact calibration of
the mass scale and subsequent estimation of the elemental composition.
Daughter ion FAB-MS MS spectra were obtained from samples in the same
/
matrix as above, and analyzed on a tandem four sector mass spectrometer (Jeol
HX HX110A, Tokyo, Japan). The nitrogen collision gas was adjusted to give
/
60% reduction in intensity of the parent ion.
Electron impact (EI) and chemical ionization (CI) mass spectrometric anal-
yses were obtained on a Finnigan TSQ 700, quadrupole spectrometer (Finnigan
MAT, San Jose, California) interfaced to a Hewlett-Packard 5890 gas chromato-
graph (Palo Alto, California), equipped with either a polar OV 351 column (25
m long × 0.25 mm ID; Scandinaviska Genetec, Kungsbacka, Sweden), or a non-
polar DB-1 column (25 m × 0.25 mm ID; J&W Scientific, Folsom, California).
Methane was used as the reaction gas for CI. Synthesis products were analyzed
with a Shimadzu QP-5000, quadrupole GC-MS (Kyoto, Japan), in the EI mode,
equipped with an RTX-5 column, (30 m × 0.25 mm ID, SGE Austin, Texas),
or with a Jeol SX102 instrument equipped with an HP-5 column (15 m × 0.20
mm ID).
GC-FTIR analysis of natural products were performed on a Hewlett-
Packard model 5965B Fourier transform infrared spectrometer interfaced to a
model 5890 GC equipped with a DB-1 column (25 m long × 0.32 mm ID; J&W
scientific). Synthesis products were analyzed with the same type of instrumen-
tation fitted with an RTX-5 amine column (30 m × 0.32 mm). FTIR spectra of
neat liquids were obtained with a Perkin-Elmer 1600 series FTIR instrument. In
all GC analyses injections were made in the splitless mode at 225 C with the
column held at 60 C for 3 min, then programmed at 10 C min to 250 C.
/
Acid Methanolysis. Following procedures modified from Mee et al. (1977),
we evaporated an amount of each purified compound equivalent to 100 ml oral
secretion to dryness and added 50 ml of dry methanol and 10 ml acetic anhy-
dride. In a sealed 1-ml glass ampule filled with nitrogen, the sample was then
heated to 100 C for 10 min, allowed to cool, and evaporated to dryness with
a gentle stream of N2. Fifty microliters of CH2Cl2 were added, and the sample
was analyzed by GC-MS.
Location of Double Bonds and Hydroxyl Groups. Following procedures in
Attygalle et al. (1995), we evaporated 50-ml oral secretion equivalents of each
fatty acid methyl ester obtained by methanolysis (above) to dryness and redis-

206
ALBORN, JONES, STENHAGEN, AND TUMLINSON
solved in 10 ml ethanol. After the addition of 30 ml of 10% hydrazine in ethanol
and 30 ml of 0.6% hydrogen peroxide in ethanol, the solution was heated at 60 C
for 1 hr. The sample was cooled to room temperature and 35 ml of 5% aqueous
HCl was added. The solution was extracted with two 40-ml aliquots of GC2 Hex-
ane (Burdick and Jackson, Muskegon, Michigan), and the hexane solution was
washed with four 50-ml aliquots of water. The partially reduced hydroxy acid
methyl esters were ozonized following standard procedures (Beroza and Bierl,
1966, 1967) and the products analyzed by GC-MS to determine the locations of
the double bonds.
To determine the location of the hydroxyl groups, the double bonds of the
methyl esters were reduced by adding 5 mg of PdO to 50 ml of oral secretion
equivalent sample solution in 50 ml of ethyl acetate, and H2 was gently bubbled
through the solution for 18 hr. The products were analyzed by GC-MS.
Pyrrolidide derivatives of the fatty acid methyl esters also were prepared
by dissolving a sample in 10 ml of 1% glacial acetic acid in freshly distilled
pyrrolidine and heating to 100 C for 30 min in a sealed tube (Anderson, 1978).
The product was cooled to room temperature, 10 ml of CH2Cl2 were added,
and the product analyzed by GC-MS. This derivative significantly reduces the
characteristic hydrocarbon MS fragmentation pattern of long chain fatty acids
and was originally developed to determine the location of double bonds in the
chain. It also resulted in increased intensity of diagnostic ions for the location
of hydroxyl groups.
HPLC Analysis of BAW Oral Secretion. Following determination of the
structure of volicitin, we developed an HPLC method to analyze the acidic com-
ponents of the BAW oral secretion. To 1 ml of filter sterilized oral secretion
100 ml acetic acid and 2 ml of CH2Cl2 were added. The solution was shaken
for 5 min, and the organic phase was evaporated to dryness under vacuum. One
milliliter of 50 mM pH 8 sodium phosphate buffer was added and 10 ml of the
solution was analyzed on HPLC with the ODS-AQ S-5 column described above
(flow, 1 ml min, gradient 40 to 100% of solvent B in A in 10 min, 100% B for 15
/
min, A 0.05% aqueous acetic acid and B 0.05% acetic acid in acetonitrile).
The column eluent was monitored by UV detection at 200 nm.
Synthesis of 17-Hydroxylinolenic Acid (Figure 1). Under an argon atmo-
sphere, 1 M solution of ethylmagnesium bromide in 65 ml tetrahydrofuran
(THF) was added dropwise to 10.7 g (59 mmol) of the ethoxyethyl ether of
3,6-heptadiyn-1-ol (Brandsma, 1988; Huang et al., 1983) in 20 ml of THF at
0 C. After 1 hr, 4 ml of freshly distilled acetaldehyde in 10 ml of THF were
added dropwise, and the mixture was stirred for 1 hr. Five milliliters of freshly
distilled pyridine and 7.6 ml of benzoyl chloride were then added, and after 3 hr,
20 ml of saturated aqueous NH4Cl were added, and the mixture was extracted
with ether (3 × 50 ml). The combined extracts were dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was chromatographed on silica gel

BEET ARMYWORM ORAL SECRETIONS
207
FIG. 1. Synthetic scheme for the synthesis of 17-hydroxylinolenic acid.
(100 g; hexane eluent) to provide 19.0 g of 1 [(8-benzoyloxy)-3,6-nonadiyn-1-ol
ethoxyethyl ether, >95% pure by GC analysis]. GC-FT-IR: 3074, 2981, 2943,
2890, 1735(s), 1602, 1453, 1382, 1357, 1310, 1271(s), 1176, 1106, 1025, 891,
1
and 857 cm ; MS, m z (rel %) 255 (3), 115 (10), 105 (85), 91 (12), 77 (25), 73
/
(100), 51 (10), and 45 (80). HR-MS, calc. for C20H24O4: 328.1675; observed,
328.1674 and calc. for C19H21O4 (M-CH3): 313.1440: observed 313.1450.
A solution of 18.2 g of 1 and 100 mg of p-toluenesulfonic acid in 100 ml of
MeOH was stirred for 2 hr. GC-MS analysis indicated that the ethoxyethyl ether
group had been removed to provide the corresponding alcohol. After the addition
of 5 ml of saturated NaHCO3, the solvent was removed in vacuo. The residue
was taken up in ether and dried over anhydrous MgSO4 and the solvent removed
to provide 13 g of crude diyne alcohol. A solution containing 8.0 g of this alcohol
and 0.6 ml of quinoline in 100 ml ethanol was hydrogenated over 2.0 g of Lindlar
catalyst (Aldrich Chemical Co.). As the uptake of H2 slowed, additional portions
of catalyst were added (total of 6.0 g). After the uptake of 1450 ml of hydrogen,
the mixture was filtered through Celite (Fisher Chemical Co.) and the solvent
was removed in vacuo. The residue was taken up in ether, washed with dilute
HCl and saturated NaHCO3, and dried over MgSO4. Filtration and removal of
the solvent provided 7.8 g of 2 [(Z,Z)-(8-benzoyloxy)-3,6-nonadien-1-ol, >90%
pure by GC analysis, less than 1% of other double bond isomers]. GC-FT-IR,
3667, 3023, 2964, 2938, 1733(s), 1452, 1314, 1267(s), 1178, 1102, 1046, 905,
1
and 859 cm ; MS, m z (rel %), 260 (1, M+), 229 (2), 175 (3), 138 (12), 123 (4),
/

208
ALBORN, JONES, STENHAGEN, AND TUMLINSON
105 (100), 93 (16), 91 (14), 79 (45), and 77 (55); HR-MS, calc. for C16H20O3:
260.1412; observed, 260.1407, and calc. for C15H17O5 (M-CH2OH): 229.1229;
observed, 229.1231.
A solution of 6.0 g (23 mmol) of 2 and 2 ml of triethylamine in 50 ml of
CH2Cl2 was cooled and treated with 2 ml of methanesulfonyl chloride. After 5
hr the mixture was diluted with 200 ml diethyl ether and washed sequentially
with dilute HCl, water, and saturated NaHCO3. The organic solution was dried
over anhydrous MgSO4 and after filtration the solvent was removed to provide
the unstable mesylate. This product was immediately dissolved in 50 ml of dry
acetone containing 5.7 g of NaI and refluxed under Ar for 1.5 hr. The solution
was cooled, and diluted with 100 ml of diethylether, and filtered through 60 g of
neutral alumina with excess diethyl ether. Concentration in vacuo provided the
pale yellow iodide. Analysis by GC-MS of the extremely unstable mesylate and
iodide derivatives showed that they were ca. 90% pure. The iodide was imme-
diately taken up in 70 ml acetonitrile containing 5.25 g of triphenylphosphine
and refluxed 6 hr. Most of the solvent was removed in vacuo and the residue
was triturated with diethylether. The ether layer was decanted and the residue
was washed with 2 × 50 ml of ether. The solvent was removed from the residue
to provide 8.0 g of the phosphonium salt 3 as a brittle foam. A 1 M solution
of sodium hexamethyldisilazide in hexane (14 ml) was added dropwise to 8 g
(12.7 mmol) of 3 in 50 ml of THF under Ar at 78 C. After 15 min, 2.45 g of
methyl 9-oxononanoate (Pennington et al., 1953; Burgstahler et al., 1976) in 5
ml of THF were added dropwise, and the resulting mixture was stirred at 78 C
for 1 hr and warmed to room temperature for 8 hr. After addition of 10 ml sat
aq. NH4Cl and 30 ml diethyl ether, the organic phase was separated, dried over
anhydrous MgSO4 and the solvent was removed in vacuo. The residue was trit-
urated with 70 ml of 1 : 1 ether–petroleum ether. After filtration and removal
of the solvent, the organic layer provided 1.3 g of residue that was approxi-
mately a 1 : 1 mixture (by GC-MS) of triphenylphosphine oxide and 4 (methyl
17-benzoyloxylinolenate); MS, m z (rel %) 290 (7, M-PhCO2H), 175 (2), 161
/
(2), 147 (5), 133 (14), 119 (16), 105 (100), 94 (20), 93 (16), 91 (28), 81 (11),
80 (28), 79 (34), 77 (27), 67 (20), 55 (28), 41 (30); HR-MS, calc. for C19H30O2
(M-PhCO2H), 290.2246; observed, 290.2246. The mixture was dissolved in 20
ml THF and cooled to 20 C, which resulted in a slow precipitation of the tri-
phenylphosphine oxide over several days, which then was removed by filtration.
The solvent was removed by a gentle stream of N2. The protecting group was
removed by stirring a 500-mg aliquot of neat material with 2 ml of 1 M LiOH
overnight at room temperature under N2. The solution was extracted 3× with 3
ml CH2Cl2 and 3× with 3 ml hexane. The pH of the water solution was then
adjusted to <7 with 1 M HCl and the product extracted three times with 2 ml
of CH2Cl2. While gently heating and stirring the combined solution, we added
approximately 2 ml of hexane, and the solution was then allowed to cool, result-

BEET ARMYWORM ORAL SECRETIONS
209
FIG. 2. Synthetic scheme for the synthesis of 17-hydroxylinoleic acid.
ing in precipitation of most of the benzoic acid. The final solution was concen-
trated under vacuum to thick yellow liquid. HPLC analysis with UV detection
at 200 nM showed two major components, benzoic acid (45% of total area) and
hydroxylinolenic acid (50% of total area). GC analysis after treatment with 4 M
HCl in MeOH showed the hydroxy acid to be 95% pure, with less than 1% of
trans isomers and approximately 3% of benzoic acid.
Synthesis of 17-Hydroxylinoleic Acid (Figure 2). Under Ar, 1.6 M solution
of n-butyllithium in hexane (65 ml) was added dropwise to a solution contain-
ing 5.1 g (40 mmol) of the ethoxyethyl ether of propargyl alcohol in 20 ml THF
at 0 C. After 30 min, 8.79 g (35 mmol) of freshly distilled 2-(7-bromohept-1-
yl)-1,3,-dioxolane (Gil et al., 1996) were added dropwise followed by 15 ml of
dimethylsulfoxide (DMSO). The mixture was warmed to room temperature over
8 hr. After addition of 150 ml hexane, the mixture was washed four times with
50 ml of water, dried over anhydrous MgSO4, and after filtration the solvent
was removed in vacuo. The residue was taken up in 100 ml of benzene con-
taining 5 ml of ethylene glycol and 100 mg p-toluenesulfonic acid and heated
to reflux for 2 hr to selectively remove the ethoxyethyl protecting group. The
cooled mixture was washed with saturated NaHCO3, dried over MgSO4, filtered,
and concentrated in vacuo and the residue was chromatographed over silica gel
(100 g; hexane ether 9 : 1 eluent) to provide 5.2 g (66%) of 6 [2-(10-hydroxy-8-
/
decyn-1-yl)-1,3-dioxolane]. GC-FTIR: 2939, 2873, 1397, 1139, 1014, and 943

210
ALBORN, JONES, STENHAGEN, AND TUMLINSON
1
cm ; MS, m z (rel %) 225 (1), 209 (1), 195 (1), 73 (100), 55 (12), and 45 (50).
/
HR-MS, calc. for C23H21O3 (M-1): 225.1491; observed, 225.1498.
Compound 7 {2[16-(1,3-dioxolan-2-yl)-4,7,-hexadecadiyn-1-yl]-2methyl-
1,3-dioxolane} was prepared by dropwise addition of 12.6 g (60 mmol) of 2-
(3-bromoprop-1-yl)-2-methyl-1,3-dioxolane (Bellas et al., 1969) to a suspension
of 8.3 g of lithium acetylide–ethylene diamine complex in 20 ml of THF, fol-
lowed by the addition of 15 ml of DMPU. The mixture was stirred overnight and
worked up in the usual manner to provide 6.9 g of 2-(4-pentyl-1-yl)-2-methyl-
1,3-dioxolane (74% yield), bp 77–82 C (22 mm Hg). GC-FTIR: 3328, 2989,
1
2958, 2885, 2118, 1447, 1378, 1307, 1245, 1217, 1132, 1063, 943 and 883 cm ;
MS, m z (rel %) 153 (1), 139 (25), 99 (20), 87 (100), 55 (10), and 43 (90); HR-
/
MS calc. for C9H14O2: 154.0994; observed, 154.0994.
Methanesulfonyl chloride (0.5 ml) was added to a solution containing 1.18
g (5.22 mmol) of 6 and 0.6 ml of triethylamine in 15 ml of CH2Cl2 cooled to
0 C. The mixture was stirred 4 hr (completion of reaction confirmed by TLC),
washed with saturated NaHCO3, dried over MgSO4, filtered, and concentrated
in vacuo. Analysis of the product showed one major component (the unstable
mesylate of 6): MS, m z (rel %) 303 (1, M-1), 225 (1, M-MeSO2), 207 (1),
/
73 (100, dioxolanyl). This product was passed through a short silica gel col-
umn, eluted with hexane–diethyl ether (2 : 1), and the solvent was removed in
vacuo.
A solution containing 1.23 g (8 mmol) of 2-(4-pentyn-1-yl)-2-methyl-1,3-
dioxolane in 10 ml of THF was treated with 9 ml of 1 M ethylmagnesium bro-
mide in THF. After 1.5 hr the mixture was cooled to 0 C and 85 mg of CuCl were
added. After 20 min, the above mesylate in 3 ml of THF was added dropwise,
and the mixture was stirred overnight and allowed to warm to room tempera-
ture. The mixture was diluted with diethyl ether, and washed sequentially with
saturated NH4Cl and brine, dried over MgSO4, and filtered. After removal of the
solvent, the residue contained only the ethylene ketal of 6-heptyn-2-one and the
dine 7 (70%). MS, m z (rel %) 362 (1, M+), 347 (4), 99 (10), 87 (100), 73 (83),
/
55 (12), and 45 (11); HR-MS scale, calc. for C22H34O4: 362.2457; observed,
362.2444.
The above mixture was taken up in 10 ml of acetone containing 2.0 ml
of 5% aqueous HCl at room temperature. After 6 hr the reaction was neutral-
ized with saturated NaHCO3 and the solvent removed in vacuo. The mixture
was extracted three times with ether and the combined ether extracts were dried
(MgSO4), filtered, and the solvent was removed in vacuo. The residue was taken
up in 40 ml of acetone and added dropwise to a cold (0 C) solution of 1.5 g
of CrO3 in 15 ml of 3.5 M H2SO4. After 5 hr, the usual work up (Oehlslager
et al., 1986) provided 0.6 g of 17-oxo-9,12-octadecadiynoic acid (40% from 6).
Methyl ester (CH2N2) GC-FT-IR: 2939, 2865, 1754, 1439, 1360, 1313, 1167
1
and 1022 cm ; MS, m z (rel %) 303 (M-1, 1), 289 (2), 273 (1), 162 (8), 161
/

BEET ARMYWORM ORAL SECRETIONS
211
(10), 147 (10), 145 (10), 131 (11), 119 (16), 117 (18), 105 (23), 91 (25), 87
(2), 81 (9), 79 (16), 74 (6), 55 (20), and 43 (100); HR-MS, calc. for C19H28O3:
304.2038; observed, 304.2046.
A solution of the 17-oxo-9,12-octadecadiynoic acid in 5 ml of ethanol was
added to P-2 nickel freshly prepared from 0.75 g of nickel(II) acetate under a
hydrogen atmosphere. After 4 hr, the usual work up (Millar and Oehlschlager,
1984) provided (Z,Z)-17-oxo-9,12-octadecadienoic acid. Methyl ester (CH2N2)
1
GC-FTIR: 3016, 2935, 2865, 1756, 1440, 1360, 1170 and 1023 cm ; MS, m z
/
(rel %) 308(1, M+), 377 (2), 376 (3), 250 (5), 150 (6), 135 (7), 133 (6), 121
(8), 108 (15), 107 (16), 95 (12), 94 (20), 93 (21), 91 (14), 87 (10), 81 (22), 80
(32), 79 (42), 74 (7), 67 (41), 55 (25), 43 (100), and 41 (38); HR-MS, calc. for
C19H32O3: 308.2351; observed, 308.2361.
The (Z,Z)-17-oxo-9,12-octadecadienoic acid prepared above was reduced
with NaBH4 as described in the literature (Oehlschlager et al., 1986) to pro-
vide 0.35 g of 17-hydroxylinoleic acid (8) (58% yield from 17-oxo-9,12-
octadecadiynoic acid). Methyl ester (CH2N2) GC-FTIR: 3016, 2935, 2865, 1758,
1
1441, 1358, 1241, and 1173 cm ; MS, m z (rel %) 292 (1, M-18), 250 (2), 178
/
(5), 164 (5), 163 (4), 150 (7), 149 (8), 135 (16), 122 (12), 121 (16), 101 (10), 108
(25), 107 (24), 95 (32), 94 (50), 93 (30), 91 (15), 87 (15), 81 (39), 80 (37), 79
(61), 74 (14), 67 (64), 59 (100), 55 (50), 45 (12), 43 (30), and 41 (65); HR-MS,
calc. for C19H34O3: 310.2508; observed, 310.2512.
Synthesis of Glutamine Conjugates of 17-Hydroxylinolenic and 17-Hy-
droxylinoleic Acids. Glutamine conjugates of the hydroxyacids were prepared
by procedures modified after Sheehan et al. (1965). Five hundred milligrams
of L-glutamine-p-nitrobenzyl ester (Bachem Bioscience Inc. King of Prussia,
PA) were dissolved in 3 ml of dimethylformamide (DMF), and then 200 ml of
water and 250 ml of triethylamine (TEA) were added. In a second flask, 400
mg of the hydroxyacid was dissolved in 3 ml DMF and then 500 mg of 1-
hydroxybenzotriazole (HOBT) (Sigma) were added and allowed to totally dis-
solve. Subsequently 300 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDAC) (Sigma) were added. After stirring for 2 min, the contents of the first
flask were added to the second and the mixture was stirred overnight at ambient
temperature under N2. The following day, 5 ml of CH2Cl2 were added, and the
mixture extracted with 5 ml of 100 mM aqueous ammonia, 5 ml of water, 5
ml of 100 mM acetic acid, and finally three additional 5-ml aliquots of water
before the CH2Cl2 fraction was concentrated under vacuum to a thick yellow
liquid (approximately 300 mg). HPLC analysis revealed for each coupling one
major component [N-(17 hydroxylinolenoyl)-p-nitrobenzyl-glutamine and N-(17
hydroxylinoleoyl) p-nitrobenzyl-glutamine].
The nitrobenzyl ester was dissolved in 2 ml THF, 2 ml of 1 M Na2S in
50% water–THF was added, and the mixture stirred over night to remove the
protective p-nitrobenzyl group (Lammert et al., 1978). The mixture was con-

212
ALBORN, JONES, STENHAGEN, AND TUMLINSON
centrated to near dryness and redissolved in 5 ml water. The aqueous solution
was extracted with 4 × 5 ml CH2Cl2 to remove most of the p-nitrobenzyl alco-
hol. HPLC analysis of the aqueous fraction revealed a totally deprotected final
product (70%) and the presence of a closely eluting by-product (25%) with iden-
tical UV absorption. There were also traces of p-nitrobenzyl alcohol (5%). The
crude product in water was chromatographed on a solid-phase extraction col-
umn (Mega bondelut C18, Varian, Harbor City, California, 25 mm ID × 25
mm long). The column was rinsed with 40 ml water to remove sulfide. Twenty
milliliters of 20% acetonitrile–water removed remaining traces of p-nitroben-
zoyl alcohol. The partially purified product was recovered with 40 ml of 60%
acetonitrile–water followed by 20 ml of 100% acetonitrile (200 mg). HPLC anal-
ysis by the improved procedures for analysis of BAW oral secretion described
above revealed the fraction to contain only the expected product and the by-prod-
uct (70 30%). Pure conjugates were obtained by HPLC on the YMC ODS-AQ
/
column eluted with 45% acetonitrile and 0.5% acetic acid in water at a flow of
1.0 ml min, monitored by UV detection at 200 nm.
/
Synthesis of Glutamine Conjugates of Linolenic and Linoleic Acids. L-Glu-
tamine t-butyl ester was coupled to 500 mg of linolenic and linoleic acids
(Aldrich, Milwaukee, Wisconsin) by the same procedures as described above.
The protective t-butyl group was removed by dissolving and stirring the product
overnight in 2 ml of 88% formic acid. Five milliliters of CH2Cl2 was then added
and the solution was extracted three times with 5 ml of water. The organic phase
was concentrated to dryness under vacuum to give 600 mg of the product (>95%
pure). The main impurity was noncoupled free fatty acid.
RESULTS
HPLC separations of BAW oral secretion monitored by the corn seedling
bioassay described in Turlings et al. (1993) showed consistently that all activ-
ity appeared to be located in one fraction that contained two very similar com-
ponents (Turlings et al., 2000). However, a final separation revealed that only
one of these components, B, identified as N-(17-hydroxylinolenoyl)-L-glutamine
(volicitin) had biological activity. Fraction C was identified by the same mass
spectrometric and micro-degradative analysis as described for volicitin (fraction
B) in Alborn et al. (1997). Fast atom bombardment mass spectroscopy (FAB-
MS) analysis gave exact mass m z 425.3050 (M+H)+ in the positive ion mode
/
and subsequently a molecular weight of 424.297 Da for the neutral molecule.
There were also traces of m z 407 (M+H-H2O)+. This component was not ana-
/
lyzed in the negative ion mode. The addition of sodium chloride to the FAB
matrix gave the same result as for B, with reduced intensity of the M+1 ion at
m s 425 and subsequent strong m z 447 (MH+Na)+ and 469 (MNa+Na)+. These
/
/

BEET ARMYWORM ORAL SECRETIONS
213
results indicated that the molecular weights of B and C only differed by the mass
of two hydrogens.
FAB-MS MS daughter ions of the M+1 ions at m z 423 for B and m z 425
/
/
/
for C gave very strong ions representing the loss of water [B: m z 405 (423
/
18), C: m z 407 (425 18)]. The lower mass region of both components showed
characteristic hydrocarbon fragmentation combined with an identical pattern of
/
m z 147, 130, 101, 84, 67, which resembled the electron impact (EI) mass spec-
/
tra of glutamine (NIST, 1995). A daughter ion spectrum of pure glutamine (M+1
147) strongly supported the presence of glutamine by showing the same char-
acteristic fragments (m z 147, 130, 101, 84, 67). Subtraction of glutamine, linked
/
via an ester or amide bond would give C18H30O3 as the elemental composition
of the second part of component B and similarly C18H32O3 for C.
GC analysis of both components after acid methanolysis showed two promi-
nent peaks. Chemical ionization (CI) MS analysis of the first of thees peaks,
common to both components, revealed a prominent (M+1)+ ion at m z 144 and
/
electron impact (EI) MS analysis revealed a molecular ion at m z 143 and di-
/
agnostic ions at m z 84 (base peak), 56 and 41, identifying it as the methyl ester
/
of pyroglutamate (NIST, 1995), which by congruence in GC retention time and
mass spectrum with the product of glutamine treated in the same way confirmed
the presence of glutamine in both components. The CI mass spectrum of the
second GC peak for B showed no molecular ion but a predominant fragment at
m z 291 due to a loss of water (M+1 18)+. Loss of methanol gave an ion at m z
/
/
277 (M+1 32)+, and the loss of both water and methanol gave an ion at m z 259
/
(M+1 18 32)+. The EI spectrum of the same peak showed a very weak molec-
ular ion at m z 309 and a strong m z 290 due to the loss of water (M 18)+.
/
/
The lower mass region of the spectrum also showed a characteristic fragmenta-
tion pattern of ions (14 and 12 Da apart), strongly suggesting a straight-chain
unsaturated hydrocarbon. These results were consistent with the methyl ester of
an 18-carbon hydroxy acid. The GC trace from C showed a second peak with
a slightly longer GC retention time than the second peak from B. The CI mass
spectrum of this peak showed a dominating m z 293 (M+1 18) and no molec-
/
ular ion. Loss of methanol gave an ion at m z 279 (M+1 32)+, and the loss of
/
both water and a methanol gave an ion at m z 261 (M+1 18). The EI spectra
/
showed a smaller m z 292 and no molecular ion but otherwise a fragmentation
/
very comparable to that of B.
Both methyl esters were reduced by treatment with PdO H2 over night. GC-
/
MS (CI) analysis showed an identical product with a dominating ion at m z 296
/
(M+1 18). These results indicated that component B and C contained an unsat-
urated 18-carbon hydroxy acid with three and two double bonds, respectively,
and that both contained glutamine.
Fourier transform infrared analysis of the two hydroxy acid methyl esters
1
produced diagnostic absorption at 3646 cm , confirming a hydroxyl group and

214
ALBORN, JONES, STENHAGEN, AND TUMLINSON
1
absorption bands at 3019, 2935, 2865 cm and indicating straight-chain non-
conjugated unsaturated hydrocarbons. The intensity of the 3019 cm peak rel-
1
ative to the others indicated that B contained one more double bond than C and
1
the absence of a peak in the area of 980 cm indicated that no trans double
1
bonds were present. Both molecules showed intense absorptions at 1758 cm
as expected for methyl esters.
These results confirmed that both molecules consisted of two subunits, glu-
tamine and a C18 hydroxyacid with two or three cis double bonds. These two
components could be attached in three ways to give the right elemental compo-
sition. However, only an amide bond between glutamine and the acid moiety of
the hydroxy acid would result in a free hydroxyl as indicated by the FAB-MS
experiments, without also giving a free amine, for which there was no evidence.
The methyl esters of the hydroxy C18 acids were subjected to further micro-
degradative analysis to determine the positions of the double bonds and the
hydroxyl group. Partial reduction resulted in both cases in a mixture of mono-
and diunsaturated products as established by GC-MS analysis. The mixtures
were then ozonized and CI-GC-MS analysis showed the presence of three diag-
nostic GC peaks for B with (M+1)+ ions at m z 187, 229, and 271, corresponding
/
to H(CO)(CH2)nCOOCH3 with n 7, 10, and 13, respectively. Methyl linolenate
treated in the same way gave identical products. Thus, the olefinic bonds in the
chain are located on carbons 9, 12, and 15, and the alcohol group on either the
17th or 18th carbon. CI-GC-MS analysis of the C sample showed the presence
of two diagnostic GC peaks with (M+1)+ ions at m z 187 and 229, correspond-
/
ing to HCO(CH2)nCOOCH3 with n 7 and 10, respectively. Thus the olefinic
bonds were located on carbons 9 and 12, and C was confirmed as containing
hydroxylated linoleic acid but with the position of the hydroxyl still unclear.
EI-GC-MS analysis of the reduced methyl esters showed the identical prod-
uct with no visible molecular ion but a strong ion at m z 296 (60) (M+1 18).
/
Weak m z 299 (5) (M 15)+ and strong m z 270 (100) 271 (35) (M 44)+ was
/
/
/
indicative of a hydroxyl group on C-17. EI mass spectra of a pyrrolidide deriva-
tive of the reduced products produced the expected diagnostic ions at m z 309
/
(20) (M 44)+ and m z 338 (30) (M 15)+ confirming the C-17 location of the
hydroxyl group.
/
These data proved that the components isolated from the regurgitant
of BAW larvae were N-(17-hydroxylinolenoyl)-L-glutamine, which we named
volicitin (component B), and N-(17-hydroxylinoleoyl)-L-glutamine (component
C). We could find no previous reports of these compounds in the literature.
Analysis of Oral Secretion. After the structure of volicitin was determined,
a new method was developed for analysis of volicitin and similar compounds
in oral secretions of BAW and other insects. Reverse-phase HPLC analysis of
the methylene chloride extract of acidified BAW oral secretion revealed several
related acidic components with similar UV absorption (Figure 3). The most polar

BEET ARMYWORM ORAL SECRETIONS
215
FIG. 3. HPLC separation of acidic components in BAW oral secretion. The YMC ODS-
AQ S-5 column (4.6 mm ID × 250 mm long) was eluted at a flow of 1 ml min with
/
a gradient 40–100% solvent B in A in 10 min and then 100% B for 15 min. A
0.05% aqueous acid, B 0.05% acetic acid in acetonitrile. The column eluent was mon-
itored by UV detection at 200 nm. B: Volicitin, (N-[17-hydroxylinolenoyl]-L-glutamine),
C: N-[17-hydroxylinoleoyl]-L-glutamine, D: 17-hydroxylinolenic acid, E: N-linolenoyl-
L-glutamine, F: 17-hydroxylinoleic acid, G: N-linoleoyl-L-glutamine, H: linolenic acid,
I: linoleic acid, J: unknown unsaturated C18 acid, K: oleic acid.
components were volicitin (B) and C. The presence of free 17-hydroxylinolenic
and 17-hydroxylinoleic acids (peaks D and F) was confirmed by GC-MS analysis
of their methyl esters and by coinjection of synthetic components on HPLC and
GC. The free linolenic and linoleic acid were identified as major components by
coinjection of commercial standards (peaks H and I) and by GC-MS analysis of
the methyl esters of the collected peaks and of the standards. Two components
with retention times similar to the free hydroxy acids (peaks E and G), and UV
absorption identical to that of volicitin, were collected and analyzed. FAB-MS
gave a molecular weight of 406 Da for E and 408 Da for G. GC-MS analysis
after acid methanolysis gave in both cases a peak corresponding to pyroglutamate
methyl ester. Peak E also gave a methyl linolenate and G gave methyl linoleate.
This suggested that these two components were N-linolenoyl-L-glutamine (E)
and N-linoleoyl-L-glutamine (G).

216
ALBORN, JONES, STENHAGEN, AND TUMLINSON
Synthesis of Glutamine Conjugates. We have earlier shown that only the
N-[17-hydroxylinolenoyl]-L-glutamine (volicitin) was biologically active in elic-
iting volatile production and that the D-glutamine form had no biological activity
(Alborn et al., 1997). We therefore chose to use only L-glutamine in developing
an improved synthesis of volicitin and in the synthesis of component C. The
earlier described procedures (Alborn et al., 1997) resulted in less than 10% final
product.
Racemic 17-hydroxylinolenic acid (5) and 17-hydroxylinoleic acid (8) were
synthesized by the routes illustrated in Figures 1 and 2. The synthesis of 5 was
based on the lithium salt-free Wittig method to form the final Z double bond
(Bestmann et al., 1976, 1987), based on standard methodology to prepare the
required aldehyde ester and (Z,Z)-dienephosphonium salt. The synthesis of 8
also was based on previously described methodology (Oelschlager et al., 1986)
wherein the preparation of a (Z,Z)-dienoic acid containing a secondary alco-
hol includes oxidation of a ketoaldehyde to the keto acid, reduction of a diyne
with P-2 nickel to the (Z,Z)-diene, and subsequent reduction of the ketone to a
secondary alcohol.
Both 5 and 8 were coupled with p-nitrobenzyl ester of L-glutamine by a
method developed for peptide synthesis. This resulted in a significantly higher
efficiency than earlier (approximately 75% compared to 10%), but required sub-
sequent deprotection and purification steps. HPLC analysis of the de-protected
product showed the presence of an unidentified substance that comprised about
25% of the mixture. This impurity could not be removed by recrystallization or
flash chromatography and had to be removed by HPLC. Thus, it still seriously
limited the amount of material that could be produced. An alternative protect-
ing group, t-butyl ester, could not be used when glutamine was coupled with
the hydroxy acids. The necessary acid deprotecting conditions caused the alco-
hol to shift position on the fatty acid. However, following the procedures above,
this protecting group could be used when L-glutamine was coupled with linolenic
and linoleic acid and resulted in products that were considerably purer than when
the p-nitrobenzyl group was used. The synthetic N-linolenoyl-L-glutamine and
N-linoleoyl-L-glutamine coeluted on HPLC with component E and G (Figure 3).
All the synthetic conjugates showed separation characteristics and UV spectra
identical to the natural products. FAB-MS analysis gave molecular ions identi-
cal to those of the natural products. GC-MS and GC-FTIR analysis of the prod-
ucts after acid methanolysis also produced identical results when compared to
methanolysis of the natural products.
DISCUSSION
The structure of volicitin shows an intriguing similarity to substrates of
the lipoxygenase pathway (Farmer and Ryan, 1992a; Krumm et al., 1995). In

BEET ARMYWORM ORAL SECRETIONS
217
plant systems studied thus far, biosynthesis and release of volatile compounds
appear to be induced by jasmonic acid, which is produced from linolenic acid
by the octadecanoid signaling pathway (Krumm et al., 1995). Jasmonates also
have been reported to stimulate numerous physiological and defensive processes
in plants (Farmer and Ryan, 1992a,b; Krumm et al., 1995). Furthermore, amino
acid conjugates of jasmonic acid were suggested to be involved in physiological
and developmental processes in many plants (Kramell et al., 1995). Therefore,
the presence of an elicitor that is an octadecatrienoate conjugated to an amino
acid suggests that the elicitor molecule in some way interacts with or amplifies
the octadecanoid pathway in herbivore-damaged plants. This association with
the lipoxygenase pathway and jasmonic acid is further strengthened by the fact
that the inactive compound C is based on linoleic acid, which is not a jasmonic
acid precursor.
We have established that the biological activity of BAW oral secretion is not
diet related and thus volicitin is not plant produced (Turlings et al., 1993). How-
ever, both linolenic and linoleic acids are plant-produced essential fatty acids for
Lepidoptera and are regularly added to artificial diet for these insects. We have
recently shown (Pare et al., 1998) that beet armyworm larvae synthesize volic-
´
itin by hydroxylating linolenic acid obtained from the plant and conjugate the
hydroxylinolenic acid to glutamine.
Of the linolenic and linoleic acid related components present in BAW oral
secretion, only volicitin induced production of volatiles in corn seedlings in
bioassays, and volicitin was shown to be as active as the crude oral secretion.
We now know that N-linolenoyl-L-glutamine also can induce corn seedlings in
a similar way but that it is considerably less active than volicitin (about 30%,
Alborn unpublished data), which, combined with the relatively low concentration
of this component in the oral secretion, explains why only volicitin was isolated
initially. We do not yet know how widespread or uniform the response of plants
to this type of molecule will be and if other plants might respond differently to
some of the other components found in the BAW regurgitant. We have found
that both corn and cotton respond to BAW damage and to the oral secretions
of BAW applied to damaged leaves (Pare and Tumlinson, 1997). While some
´
compounds, such as indole, ocimene, and farnesene are released by both plants,
others are unique to each plant, undoubtedly as a result of differences in plant
chemistry. In addition, both plants respond systemically to BAW oral secretion
¨
by releasing induced volatiles from undamaged leaves of injured plants Rose et
al., 1996; Turlings and Tumlinson, 1992). Furthermore, it was shown recently
that in cotton the induced volatile compounds are synthesized de novo, rather
than being released from storage (Pare and Tumlinson, 1997, 1998).
´
It is possible that different species of herbivorous insects may produce
different types of compounds that induce plants to release volatiles. The oral
secretions of at least five species of lepidopterous larvae and of grasshoppers

218
ALBORN, JONES, STENHAGEN, AND TUMLINSON
induce corn seedlings to release the same volatile components (Turlings et al.,
1993). However, corn seedlings release greater quantities of volatiles when
treated with grasshopper oral secretion than when treated with caterpillar secre-
tion (Turlings et al., 1993). Furthermore, we found that feeding damage from
Heliothis virescens induces the release of different ratios of volatile compounds
than does feeding damage by Helicoverpa zea on cotton, corn, and tobacco plants
(De Moraes et al., 1998). At this point, it seems reasonable to speculate that
closely related insect species may have plant volatile elicitors with the same
or very similar structures as volicitin and the other components found in beet
armyworm, but that these components might not have the same effect on all
plants. We also do not know what effect the isolated components may have on
other defense systems in plants, such as the systemic induction of anti feedants.
For example, beet armyworm feeding induces a strong systemic reaction in cot-
ton plants that results in feeding deterrence and toxicity (Alborn et al., 1996;
McAuslane et al., 1997; McAuslane and Alborn, 1998).
It is still unknown what benefit insects gain from these compounds that
elicit strong defensive reactions in plants. One possibility is that they are in
some way related to or involved in the insects’ defense against bacteria and other
pathogens. As noted by Farmer and Ryan (1992a), the octadecanoid signaling
pathway in plants is similar in many ways, particularly in the chemical struc-
tures of the active molecules, to the eicosanoid pathways in animals that produce
prostaglandins and leukotrienes. In insects it has been reported that eicosanoids
may be involved in mediating cellular responses to bacterial infections as well as
regulating several other physiological functions (Stanley-Samuelson, 1994a,b).
The production of volicitin and its related components also might be a way for
the insect to improve the uptake of two essential fatty acids from the diet. Both
volicitin and C are much more water soluble than linolenic and linoleic acid, and
the attached glutamine might improve absorption and transport by the digestive
system.
Acknowledgments—This project was funded in part by a grant from the Swedish Natural Sci-
ence Research Council. We are grateful to A. T. Proveaux, M. Appelgren, and H. Karlsson for assis-
tance with mass spectrometric analysis of the natural product. We are also grateful to T. Spande and
L. Pannell, NIDDK, NIH, for GC-FTIR and high-resolution mass spectral analysis of synthetic com-
pounds. Special thanks to J. Lockerman and S. Sharp for oral secretion collection; to M. Brennan
for technical assistance, and to J. G. Millar, A. Renwick, and K. Slessor for valuable comments on
the manuscript.
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