Journal of Chemical Ecology, Vol. 32, No.

1, January 2006 ( #2006)

DOI: 10.1007/s10886-006-9359-6

IDENTIFICATION, SYNTHESIS, AND FIELD TESTING OF

THE SEX PHEROMONE OF THE CITRUS LEAFMINER,
Phyllocnistis citrella

JARDEL A. MOREIRA, J. STEVEN MCELFRESH,

and JOCELYN G. MILLAR*
Department of Entomology, University of California, Riverside, CA 92521, USA

(Received June 9, 2005; revised August 11, 2005; accepted October 3, 2005)

Abstract—The citrus leafminer is an important vector of citrus canker in

many of the major citrus production areas of the world. (7Z,11Z)-Hexadeca-
dienal was reported as a sex attractant for this insect in the 1980s, based on
trap catches during pheromone screening trials in Japan. However, attempts to
reproduce this work in other areas of the world have not been successful. We
report here that (7Z,11Z)-hexadecadienal is only one component of the
pheromone, with the other critical component being the analogous trienal,
(7Z,11Z,13E)-hexadecatrienal. Both compounds were identified in the effluvia
from live female moths by coupled gas chromatography (GC)-electro-
antennography using nonpolar and polar GC columns, and the identifications
were confirmed by comparisons of mass spectra with those of authentic
standards. Stereoisomers of the two compounds, and a number of analogs,
were synthesized to confirm the identifications. In field trials, neither
compound alone was attractive to male moths, but blends of the two were
highly attractive, with thousands of insects being caught per trial. Addition of
the isomeric (7Z,11Z,13Z)-hexadecatrienal inhibited attraction to the two-
component blend.

Key Words—Sex pheromone, (7Z,11Z)-hexadecadienal, (7Z,11Z,13E)-hexa-

decatrienal, 5,7,11-hexadecatrienal, citrus leafminer, (7Z,11Z,13Z)-hexade-
catrienal.

* To whom correspondence should be addressed. E-mail: jocelyn.millar@ucr.edu

This paper and the preceeding paper (Leal et al.) were submitted within a few days of each other.
The editors and the authors agreed that they should be published in tandem.

169
0098-0331/06/0100-0169/0 # 2006 Springer Science + Business Media, Inc.
170 MOREIRA ET AL.

INTRODUCTION

The citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepidoptera:

Gracillariidae), is a pest in most of the citrus-growing regions of the world
(Heppner, 1993). It attacks all varieties of citrus and some related plant species,
with grapefruit, tangerine, and pumello being among the most susceptible hosts
(Legaspi and French, 2003). Although mature trees can tolerate some damage,
growth of young trees in nurseries and newly planted orchards is reduced.
Damage is caused by the larvae forming serpentine mines in the leaves, in which
they are well protected from insecticide sprays, making them difficult to control.
Even more important than direct damage is the potential for the adult moths to
vector the highly contagious and lethal citrus canker bacterium, Xanthomonas
axonopodis pv citri, where this pathogen occurs (Ando et al., 1985). The
importance of this highly contagious disease to growers is reflected in the control
program in Florida, USA, which calls for the destruction of all exposed citrus
trees within 1900 ft of an infected one (Florida Department of Agriculture and
Consumer Services, http://www.doacs.state.fl.us/pi/canker/cankerflorida.pdf ).
There are currently no good methods for early detection and sampling of
this insect to track the continued spread through California and other citrus-
growing regions in the United States. Often, the first signs of its presence are the
characteristic mines in the leaves, by which time the infestation may be well
established. Although (7Z,11Z)-hexadecadienal (7Z,11Z-16:Ald) was reported
as a sex attractant for this insect from screening trials 20 yr ago (Ando et al.,
1985), in subsequent trials in other parts of the world, including China, Spain,
Florida, and Italy (Jacas and Peña, 2002), this compound was not effective. Due
to the threat of CLM to the California citrus industry, we undertook an
investigation of its pheromone chemistry, and report that the pheromone of
California populations consists of at least two components, the previously
identified (7Z,11Z)-hexadecadienal, and the corresponding (7Z,11Z,13E)-hex-
adecatrienal (7Z,11Z,13E-16:Ald).

METHODS AND MATERIALS

Insects. Insects were obtained by collecting branches with infested leaves

from lemon orchards in the Coachella Valley, Riverside Co., CA, USA and
from Escondido, San Diego Co., CA. Branches with larvae were kept in
humidified, vented plastic boxes [32  17  9 cm (L  W  H), with four 18-
mm vents covered with fine brass screening, or for larger quantities, 40  30 
14 cm with four 38-mm screen-covered vents) until the insects pupated. Pupae
were removed from cocoons and transferred, using a damp camel-hair brush if
necessary, into a Petri dish lined with a damp filter paper. The sex of pupae was
PHEROMONE OF CITRUS LEAFMINER 171

determined by examining the terminal abdominal segments (Garrido and Jacas,

1996). Pupae were then transferred to individual 1-dram glass shell vials (45 
15 mm, L  diam.) with a small disk of filter paper to prevent them from
becoming trapped in condensation on the glass. The vials, closed with a plastic
cap with a pinhole for ventilation, were held in groups in a vented, humidified
plastic box. Vials were checked daily for adult emergence. Adults were fed a
sugar–water solution (10 g sugar in 100 ml deionized water) absorbed on 2- to
3-mm cubes of cellulose sponge (ca. 10–20 ml/cube). Male moths were used for
coupled gas chromatography-electroantennogram detection (GC-EAD) studies,
and females were used for pheromone sampling (see below).
Collection of Pheromone from Live Females. Pheromone was collected
from female CLM by placing groups of 1–30 virgin females in aeration
chambers for 1–4 nights, collecting the emitted pheromone on solid phase
microextraction (SPME) fibers. The aeration chambers were constructed from
Swagelok\ fittings (two 1/8 to 1/2 in. unions and one 1/2 to 1/2 in. union)
connected to central 12-mm OD glass tubes (6.5 cm long). Activated-charcoal
purified medical air was humidified by passage through a õ5  1 cm (L 
diam.) wad of Soxhlet-extracted glass wool wetted with 1 ml of MilliQ\
purified water, then it was passed through a 5  1 cm (L  diam.) bed of 50–
200 mesh activated charcoal, and finally into the aeration chamber through a
steel frit, at a flow rate of õ8–10 ml/min. An SPME fiber (100 mm poly-
dimethylsiloxane coating, SPME portable field sampler; Supelco Inc., Belle-
fonte, PA, USA) was inserted into the 1-mm-diam. Teflon outlet tube at the
downwind end of the chamber for collection of volatiles. Insects were added to
the chamber prior to assembly, and were provided with sugar–water on a
cellulose sponge cube during aerations. When there were >3 females, a 1  9
cm piece of clean, fine brass screening was provided as a perching substrate.
Aerations were conducted in the laboratory with natural daylight, augmented
with room fluorescent lighting during the day. Room temperatures varied from
20 to 25-C and were not precisely controlled.
Coupled Gas Chromatography-Electroantennography. Female-produced
volatiles collected by SPME and synthetic standards were analyzed by GC-
EAD on Hewlett-Packard 5890 series II gas chromatographs, with the first fitted
with a DB-5 column (30 m  0.25 mm ID, 0.25 mm film, 100-C/1 min, 10-/min
to 275-C for 15 min; J&W Scientific, Folsom, CA, USA) and the second with a
DB-WAX column (30 m  0.25 mm ID, 0.25 mm film, 100-C/1 min, 10-/min to
240-). Loaded SPME fibers were desorbed in the injection port for 1 min prior
to starting the GC. Helium was used as the carrier and makeup gas, and all
injections were made splitless. The column effluent was split equally with a
glass X-cross with one branch going to the flame ionization detector (FID),
another to the EAG detector (EAD), and the final branch providing helium
makeup gas (3 ml/min). The EAD branch passed through a heated conduit
172 MOREIRA ET AL.

(250-C) and into the side of a 15-mm ID glass tube swept with humidified
medical air (770 ml/min), with the air flow directed over the antennal
preparation. Signals were recorded on either matched HP 3394 integrators or
with SRI model 202 PeakSimple chromatography data system running on a
personal computer with PeakSimple v. 2.83 software (SRI Instruments,
Torrance, CA, USA).
Males (1–2 d old) used in GC-EAD analyses were not chilled or
anesthetized prior to use. Males were held with fine forceps, the head removed
with a scalpel, and the antennal tips cut off. The head was mounted on the
ground electrode and both cut antennal tips were placed in contact with the
saline-filled glass recording electrode.
Field Trials. In all trials, moths were counted under a stereomicroscope to
ensure correct identification (based on distinctive wing patterns) because of the
insects’ small size (less than 3 mm) and the large numbers trapped. Moths were
readily identified from their distinctive wing patterns. Voucher specimens,
UCRC 92975-92982 (pinned) and 92983 (specimens in alcohol), have been
deposited in the UC Riverside Entomology Museum.
Lures consisted of 11-mm gray rubber septa (West Pharmaceutical
Services Co., Lionville, PA, USA) individually labeled with a treatment
number and loaded with heptane solutions (75 or 100 ml) of test blends. Field
trials were conducted in September 2004, in a heavily infested lemon orchard in
Escondido, CA. Green Delta traps were used [Pherocon IIID traps (Trécé Inc.,
Salinas, CA, USA) or Intercept D traps (IPM Tech Inc., Portland, OR, USA)].
In the first two trials, testing blend ratio and total dose, respectively, five
replicate blocks containing a complete set of treatments (including a solvent
control) were spaced four to seven rows apart. Treatments were randomly
placed within the rows, four trees in from the end and on every other tree within
a row. Traps were positioned just inside the canopy, 1.5–2 m above ground.
Counts were taken 1–2 d after initial setup, and then the traps were re-
randomized, with a second count made 2 d later. Traps with more than 10 moths
were changed by transferring the rubber septum to a new trap, and the moths in
the old trap counted. In the first trial, testing blend ratios, trap catches from the
two counts were summed prior to transformation (¾x + 0.5), and then subjected
to two-way analysis of variance using SigmaStat v. 1.0 (Jandel Scientific, San
Rafael, CA, USA). Significantly different treatment means were distinguished
using the Student–Neuman–Keuls test (SNK, a = 0.05). In the second trial,
testing dose response, data could not be normalized by transformation, and
so the data were analyzed by Friedman’s two-way nonparametric ANOVA on
the untransformed data, followed by Bonferroni’s tests for separation of means
(a = 0.05).
A final trial testing the effects of 7Z,11Z,13Z-16:Ald as a possible synergist
or antagonist was carried out on June 7–8 2005, using a standard blend of
PHEROMONE OF CITRUS LEAFMINER 173

7Z,11Z,13E-16Ald and 7Z,11Z-16:Ald (100:33 mg) augmented with 0 to 100 mg

7Z,11Z,13Z-16:Ald. The experiment was set up in the same orchard as used pre-
viously. After log10(x + 1) transformation to normalize the data, the transformed
counts were analyzed by two-way ANOVA followed by SNK tests (a = 0.05).
Synthesis of Pheromones and Related Compounds. Tetrahydrofuran (THF)
was distilled from sodium/benzophenone under argon atmosphere. 1H and 13C
NMR spectra were recorded with a Varian INOVA-400 (400 and 100 MHz,
respectively) spectrometer, as CDCl3 solutions. Chemical shifts are expressed
in ppm relative to CDCl3 (7.26 and 77.23 ppm for 1H and 13C NMR,
respectively). Mass spectra were obtained with an HP 5890 GC equipped with a
DB5-MS column (25 m  0.20 mm ID  0.33 mm film; J&W Scientific,
Folsom, CA, USA), interfaced to an HP 5970 mass selective detector, in EI
mode (70 eV) with helium carrier gas. Products were purified by flash or
vacuum flash chromatography using silica gel (230–400 mesh; EM Science,
Gibbstown, NJ, USA). Reactions with air- or water-sensitive reagents were
carried out in dried glassware under argon atmosphere. Worked up reaction
mixtures were dried over anhydrous Na2SO4 and concentrated by rotary
evaporation under reduced pressure.
The syntheses of the 5,7,11-hexadecatrienal isomers (Schemes 1 and 2),
and the previously known (7Z,11Z)-hexadecadienal (Scheme 5) are described in
detail in the online supplement to this manuscript (Electronic Supplementary
Material is available for this article at http://dx.doi.org/10.1007/s10886-006-
9359-6 and is accessible for authorized users).
Short, Nonstereospecific Preparation of 7,11,13-Hexadecatrienal Isomers.
(Z)-11-Acetoxyundec-4-enal (28). (Scheme 3) A solution of KMnO4 (1.70 g,
10.7 mmol) in water (12.7 ml) was added dropwise to a stirred solution of
(7Z,11E/Z)-hexadecadienyl acetate 25 (3.00 g, 10.7 mmol; Shin-Etsu Chemical
Co., Tokyo, Japan) in ethanol (35 ml) at 0-C over 2.5 hr, and the resulting
mixture was stirred at ambient temperature for 2 hr. The mixture was then
filtered through a pad of Celite and the filtrate was concentrated. The residue
was taken up in Et2O (100 ml), washed with brine, dried, and concentrated. The
crude product was filtered through a plug of silica gel (hexane/EtOAc, 9:1)
giving 0.71 g of diols 26 and 27. The diols (0.61 g, 1.94 mmol) were added to a
solution of NaIO4 (1.45 g, 6.8 mmol) in a mixture of THF/acetone/water
(8:4:13–20 ml) and stirred for 4 hr at 0-C, then diluted with ethyl acetate (50
ml), washed with water and brine, dried, and concentrated. Purification by
vacuum flash chromatography (hexane/EtOAc, 95:5 as eluent) afforded 0.21 g
of aldehyde 28 (10.1% yield over two steps). MS: 226 (M+, trace), 148 (6), 122
(14), 109 (3), 95 (11), 93 (14), 84 (19), 81 (27), 80 (17), 79 (19), 69 (13), 68
(28), 67 (51), 55 (35), 54 (20), 43 (100), 41 (56).
(7Z,11E/Z,13Z)-Hexadecatrienyl Acetate (31). LDA (1.5 M in cyclohex-
ane, 0.44 ml, 0.66 mmol) was added dropwise to a suspension of (Z)-2-pentenyl
174 MOREIRA ET AL.

SCHEME 1. Synthesis of (5,7,11Z)-hexadecatrienals.

triphenylphosphonium bromide 30 (0.273 g, 0.66 mmol) in THF (2.0 ml) at

j10-C. The mixture was stirred for 2 hr and then cooled to j20-C, and (Z)-11-
acetoxyundec-4-enal 28 (0.100 g, 0.44 mmol) dissolved in THF (1.0 ml) was
added dropwise. The resulting mixture was warmed to room temperature and
stirred overnight, then quenched with water and poured into saturated aqueous
PHEROMONE OF CITRUS LEAFMINER 175

SCHEME 2. Synthesis of (5Z,7Z,11Z)-hexadecadienal 24.

NH4Cl solution. The organic layer was separated and the aqueous phase
extracted with Et2O (3  10 ml). The combined organic layers were washed
with brine, dried, and concentrated. The residue was purified by flash chroma-
tography (hexane/ethyl acetate, 95:5) affording (7Z,11E/Z,13Z)-hexadecatrienyl

SCHEME 3. Synthesis of (7Z,11,13)-hexadecatrienals.

176 MOREIRA ET AL.

acetate 31 (0.052 g) in 42.3% yield, in a 49:51 ratio of geometric isomers. MS:

7Z,11E,13Z (minor) isomer: 278 (M+, 10), 207 (1), 149 (3), 135 (8), 119 (5), 95
(100), 81 (16), 79 (23), 67 (48), 55 (21), 43 (51), 41 (30). MS: 7Z,11Z,13Z
(major) isomer: 278 (M+, 8), 149 (2), 135 (7), 121 (7), 95 (100), 81 (17), 79
(24), 67 (58), 55 (24), 43 (54), 41 (31).
(7Z,11E/Z,13Z)-Hexadecatrien-1-ol (32). (7Z,11E/Z,13Z)-hexadecatrienyl
acetate 31 (0.040 g, 0.14 mmol) was added to a solution of NaOH (6 M in
water, 2 drops) in methanol (1.0 ml) and stirred for 4 hr at room temperature.
After concentration, the residue was diluted with EtOAc, washed with water and
brine, dried, and then concentrated. The residue was purified by flash chro-
matography (hexane/EtOAc, 5:1) affording (7Z,11E/Z,13Z)-hexadecatrien-1-ol
32 (0.030 g) in 88.2% yield. MS: 7Z,11E,13Z (minor) isomer: 236 (M+, 6), 163
(1), 149 (2), 135 (5), 121 (4), 95 (100), 81 (14), 79 (18), 67 (46), 55 (24), 41
(36). MS: 7Z,11Z,13Z (major) isomer: 236 (M+, 5), 149 (2), 135 (7), 121 (5), 95
(100), 81 (15), 79 (19), 67 (54), 55 (27), 41 (37).
(7Z,11E/Z,13Z)-Hexadecatrienal (33). (7Z,11E/Z,13Z)-Hexadecatrien-1-ol
32 (5.0 mg, 0.021 mmol) was oxidized with PCC and powdered 4 Å molecular
sieve in CH2Cl2 as described above, yielding (7Z,11E/Z,13Z)-hexadecatrienal
33 in 56.6% yield (2.8 mg). MS: 7Z,11E,13Z (minor) isomer: 234 (M+, 3), 163
(1), 149 (2), 135 (5), 121 (2), 95 (100), 79 (16), 67 (50), 55 (25), 41 (36). MS:
7Z,11Z,13Z (major) isomer: 234 (M+, 3), 163 (1), 149 (1), 135 (3), 121 (1), 95
(100), 79 (13), 67 (44), 55 (21), 41 (34).
(7Z,11E/Z,13E)-Hexadecatrienyl acetate (35). In the same manner de-
scribed for the preparation of (7Z,11E/Z,13Z)-hexadecatrienyl acetate 31, the
reaction of aldehyde 28 with the ylide from (E)-2-pentenyltriphenylphospho-
nium bromide 34 (0.273 g, 0.66 mmol) gave (7Z,11E/Z,13E)-hexadecatrienyl
acetate 35 (0.071 g, 57.7% yield), in a 41:59 ratio of geometric isomers. MS:
7Z,11Z,13E (minor) isomer: 278 (M+, 7), 189 (1), 175 (1), 161 (2), 149 (2), 135
(5), 121 (4), 95 (100), 81 (13), 79 (22), 67 (47), 55 (23), 43 (47), 41 (29). MS:
7Z,11E,13E (major) isomer: 278 (M+, 7), 175 (1), 161 (1), 149 (1), 135 (4), 121
(3), 95 (100), 81 (10), 79 (13), 67 (40), 55 (18), 43 (40), 41 (23).
(7Z,11E/Z,13E)-Hexadecatrien-1-ol (36). (7Z,11E/Z,13E)-Hexadecatrienyl
acetate 35 (0.060 g, 0.22 mmol) was hydrolyzed as described above for acetate
31, giving (7Z,11E/Z,13E)-hexadecatrien-1-ol 36 (0.041 g, 80.6% yield). MS:
7Z,11Z,13E (minor) isomer: 236 (M+, 3), 207 (traces), 163 (1), 149 (2), 135 (4),
121 (3), 95 (100), 81 (10), 79 (16), 67 (45), 55 (24), 41 (34). MS: 7Z,11E,13E
(major) isomer: 236 (M+, 4), 207 (traces), 149 (1), 135 (3), 121 (2), 95 (100), 81
(8), 79 (13), 67 (39), 55 (20), 41 (28).
(7Z,11E/Z,13E)-Hexadecatrienal (37). Alcohol 36 (5.0 mg, 0.021 mmol
was oxidized as described above for alcohol 32, yielding (7Z,11E/Z,13E)-
hexadecatrienal 37 (3.1 mg) in 62.7% yield. MS: 7Z,11Z,13E (minor) isomer:
234 (M+, 2), 149 (2), 135 (3), 121 (2), 95 (100), 79 (15), 67 (47), 55 (22), 41
PHEROMONE OF CITRUS LEAFMINER 177

SCHEME 4. Synthesis of (7Z,11Z,13E)-hexadecatrienal 49.

(32). MS: 7Z,11E,13E (major) isomer: 234 (M+, 3), 216 (traces), 149 (1), 135
(2), 121 (2), 95 (100), 79 (11), 67 (37), 55 (19), 41 (27).
Preparation of (7Z,11Z,13E)-Hexadecatrienal (49). 1-(t-Butyldimethylsily-
loxy)-3-bromopropane (39). (Scheme 4) 4-(Dimethylamino)pyridine (70 mg,
0.57 mmol) and triethylamine (6.42 g, 63.4 mmol) were added dropwise to a
j10-C, stirred mixture of 3-bromo-1-propanol 38 (8.00 g, 57.6 mmol) and
t-butyl-dimethylsilyl chloride (9.11 g, 60.4 mmol) in CH2Cl2 (65 ml). After
1 hr, the mixture was warmed to room temperature and stirred for 16 hr. Water
(50 ml) was added, and the mixture then was extracted three times with Et2O
(1  80, 2  50 ml). The combined organic phases were washed with 10%

SCHEME 5. Synthesis of (7Z,11Z)-hexadecadienal 56.

178 MOREIRA ET AL.

aqueous HCl (3  30 ml), saturated NaHCO3 solution (2  50 ml), and brine,

then dried and concentrated. The residue was Kugelrohr distilled (oven temp.
68–72-C, 10 mmHg) giving 13.66 g (93.7% yield) of bromide 39. 1H NMR: d
0.07 (s, 6H), 0.90 (s, 9H), 1.93–2.00 (m, 2H), 3.51 (t, 2H, J = 6.4 Hz), 3.73
(t, 2H, J = 5.8 Hz). 13C NMR: d j5.17, 18.51, 26.12, 30.89, 35.77, 60.64 ppm.
MS: m/z 197 (M+ j57, 48), 195 (45), 169 (61), 167 (60), 155 (5), 153 (6), 139
(100), 137 (97), 115 (65), 99 (7), 85 (8), 75 (23), 73 (28), 59 (27), 58 (16), 57
(24), 47 (19), 45 (31), 43 (20), 41 (43). The NMR spectrum matched literature
values (Kerr et al., 1990).
1-(Tetrahydropyranyloxy)-oct-7-yne (41). Dihydropyran (16.00 g, 190.2
mmol) was added dropwise to a solution of 7-octyn-1-ol 40 (16.00 g, 126.8
mmol; prepared from 2-octyn-1-ol by the acetylene zipper reaction; Abrams and
Shaw, 1988) and a few crystals of PTSA in CH2Cl2 (30 ml) at 0-C under argon.
The reaction was allowed to warm to room temperature and stirred overnight.
Most of the solvent was removed by rotary evaporation, the residue was diluted
with hexane (150 ml), washed with saturated NaHCO3 solution (2  50 ml) and
brine, dried, and concentrated. The resulting oil was purified by vacuum flash
chromatography (hexane, then hexane/ethyl acetate, 95:5) followed by
Kugelrohr distillation (bp õ94–96-C, 1.0 mmHg) yielding 25.48 g (95.6%)
of pure product. 1H NMR: d 1.32–1.48 (m, 4H), 1.48–1.65 (m, 8H), 1.67–1.75
(m, 1H), 1.78–1.88 (m, 1H), 1.93 (t, 1H, J = 2.7 Hz), 2.18 (td, 2H, J = 2.7 and
6.8 Hz), 3.38 (dt, 1H, J = 9.8 and 6.4 Hz), 3.46–3.53 (m, 1H), 3.73 (dt, 1H, J =
9.8 and 6.8 Hz), 3.83–3.90 (m, 1H), 4.57 (dd, 1H, J = 4.4 and 2.7 Hz). 13C
NMR: d 18.57, 19.92, 25.70, 25.99, 28.65, 28.79, 29.83, 31.00, 62.58, 67.75,
68.33, 84.90, 99.09 ppm. MS: m/z 209 (M+ j 1, 1), 125 (1), 109 (3), 101 (29),
85 (100), 67 (40), 55 (23), 43 (21), 41 (51).
1-(t-Butyldimethylsilyloxy)-11-(tetrahydropyranyloxy)-undec-4-yne
(42). n-BuLi (1.6M in hexanes, 12.2 ml, 19.5 mmol) was added dropwise to a
stirred solution of 1-(tetrahydropyranyloxy)-oct-7-yne 41 (3.90 g, 18.5 mmol) in
dry THF (20 ml) at j40-C. The mixture was allowed to warm to j10-C and
stirred for 4 hr. 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU,
9.0 ml, 74.2 mmol) was added over 10 min, the resulting mixture was stirred for
1 hr, and then cooled to j40-C. A solution of 1-(t-butyldimethylsilyloxy)-3-
bromopropane 39 (4.70 g, 18.5 mmol) in dry THF (10 ml) was added dropwise,
and the mixture was allowed to warm to room temperature over õ4 hr and
then stirred overnight. The reaction was quenched with saturated NH4Cl
solution (20 ml) and extracted with ethyl acetate (4  20 ml). The combined
organic phases were washed with saturated NaHCO3 solution and brine, dried,
and concentrated. The residue was purified by vacuum flash chromatography
(hexane/ethyl acetate, 95:5) affording 6.83 g of product contaminated with
unreacted 1-(t-butyldimethylsilyloxy)-3-bromopropane 39. This impure material
was used directly in the next step. 1H NMR: d 0.05 (s, 6H), 0.89 (s, 9H), 1.33–
PHEROMONE OF CITRUS LEAFMINER 179

1.88 (m, 16H), 2.18 (tt, 2H, J = 7.0 and 2.3 Hz), 2.26 (tt, 2H, J = 7.0 and 2.3
Hz), 3.38 (dt, 1H, J = 9.6 and 6.6 Hz), 3.47–2.53 (m, 1H), 3.68 (t, 2H, J = 6.0
Hz), 3.73 (dt, 1H, J = 9.6 and 6.8 Hz), 3.77–3.87 (m, 1H), 4.57 (dd, 1H, J = 4.3
and 2.7 Hz). 13C NMR: d j5.12, 15.36, 18.57, 18.91, 19.90, 25.72, 26.04,
26.16, 28.93, 29.29, 29.88, 30.99, 32.36, 61.98, 62.54, 67.80, 79.90, 80.51,
99.06 ppm. MS: m/z 325 (M+ j57, 2), 241 (5), 165 (3), 159 (12), 93 (9), 85
(100), 81 (12), 79 (10), 75 (29), 73 (13), 67 (19), 57 (10), 55 (13), 43 (12), 41 (22).
11-(Tetrahydropyranyloxy)-undec-4-yn-1-ol (43). A solution of tetrabutyl-
ammonium fluoride (1.0 M in THF, 21.4 ml, 21.4 mmol) was added to a
solution of 1-(t-butyldimethylsilyloxy)-11-(tetrahydropyranyloxy)-undec-4-yne
42 (6.82 g) in dry THF (35 ml), at room temperature. The mixture was stirred
until the starting material had been consumed (1.5 hr), then diluted with Et2O
(50 ml) and washed with water. The organic phase was separated, and the
aqueous layer was extracted with Et2O (2  20 ml). The combined organic
phases were washed with brine, dried, and concentrated. The residue was
purified by vacuum flash chromatography (hexane/EtOAc, 4:1) to yield 3.49 g
of 43 (70.1% over two steps). 1H NMR: d 1.31–1.64 (m, 10H), 1.66–1.88 (m,
4H), 1.73 (quint, 2H, J = 6.3 Hz), 2.13 (tt, 2H, J = 6.8 and 2.5 Hz), 2.27 (tt, 2H,
J = 6.8 and 2.5 Hz), 3.38 (dt, 1H, J = 9.6 and 6.6 Hz), 3.46–3.54 (m, 1H), 3.73
(dt, 1H, J = 9.6 and 6.8 Hz), 3.74 (t, 2H, J = 6.0 Hz), 3.82–3.90 (m, 1H), 4.57
(dd, 1H, J = 4.5 and 2.5 Hz). 13C NMR: d 15.64, 18.88, 19.88, 25.71, 25.95,
28.84, 29.15, 29.82, 30.98, 31.80, 62.24, 62.56, 67.76, 79.61, 81.22, 99.08 ppm.
MS: m/z 268 (M+, trace), 195 (3), 183 (3), 167 (3), 125 (2), 111 (3), 101 (18), 85
(100), 81 (13), 79 (15), 67 (21), 57 (14), 55 (26), 43 (19), 41 (44). Anal.
calculated for C16H28O3: C, 71.60; H, 10.52. Found: C, 71.85; H, 10.59. The 1H
NMR spectrum was in agreement with that reported in the literature (Yadav
et al. 1988).
(Z)-11-(Tetrahydropyranyloxy)undec-4-en-1-ol (44). Alkynol 43 (3.40 g,
12.67 mmol) was reduced to the corresponding alkenol with hydrogen and P2-
nickel catalyst as described above. The crude product was purified by vacuum
flash chromatography (hexane/ethyl acetate, 9:1), yielding 3.14 g of 44 (91.8%
yield). 1H NMR: d 1.24–1.42 (m, 6H), 1.44–1.64 (m, 8H), 1.64–1.73 (m, 1H),
1.73–1.85 (m, 1H), 1.96–2.07 (m, 2H), 2.07–2.14 (m, 2H), 3.36 (dt, 1H, J = 9.6
and 6.6 Hz), 3.44–3.51 (m, 1H), 3.62 (dt, 2H, J = 5.3 and 6.4 Hz), 3.70 (dt, 1H,
J = 9.6 and 6.8 Hz), 3.81–3.88 (m, 1H), 4.55 (dd, 1H, J = 4.3 and 2.7 Hz), 5.30–
5.41 (m, 2H). 13C NMR: d 19.89, 23.81, 25.70, 26.31, 27.30, 29.28, 29.79,
29.90, 30.97, 32.86, 62.57, 62.76, 67.87, 99.06, 129.18, 130.85 ppm. MS: m/z
270 (M+, trace), 186 (1), 168 (1), 150 (1), 109 (3), 101 (4), 95 (7), 85 (100), 81
(11), 67 (19), 57 (10), 55 (17), 43 (12), 41 (28). The 1H NMR spectrum was in
agreement with that reported in the literature (Sharma et al. 1994).
(4Z)-1-Iodo-11-(tetrahydropyranyloxy)undec-4-ene (45). Iodine (2.91 g,
11.46 mmol) was added in small portions to a j40-C solution of Ph3P (3.00 g,
180 MOREIRA ET AL.

11.46 mmol), imidazole (1.56 g, 22.92 mmol), and (Z)-11-(tetrahydropyranyl-

oxy)undec-4-en-1-ol 44 (3.10 g, 11.46 mmol) in dry THF (18 ml). The
resulting mixture was warmed to 0-C and stirred for 3 hr. The mixture was
diluted with Et2O (100 ml), washed successively with saturated sodium
thiosulfate (100 ml), water (2  50 ml), and brine, then dried and concentrated.
The residue was suspended in hexane to precipitate most of the Ph3PO and
filtered. After concentration, the crude product was purified by vacuum flash
chromatography (hexane/ethyl acetate, 9:1) yielding 2.15 g of 45 (57.6% yield)
as colorless oil and 0.201 g of unreacted starting material. 1H NMR: d 1.22–
1.42 (m, 6H), 1.45–1.64 (m, 6H), 1.64–1.75 (m, 1H), 1.77–1.87 (m, 1H), 1.87
(quint, 2H, J = 7.0 Hz), 2.05 (q, 2H, J = 6.6 Hz), 2.13 (q, 2H, J = 7.2 Hz), 3.18
(t, 2H, J = 6.8 Hz), 3.37 (dt, 1H, J = 9.6 and 6.6 Hz), 3.46–3.52 (m, 1H), 3.72 (dt,
1H, J = 9.6 and 6.8 Hz), 3.82–3.90 (m, 1H), 4.57 (dd, 1H, J = 4.3 and 2.7 Hz),
5.28 (dtt, 1H, J = 10.7, 7.2 and 1.4 Hz), 5.42 (dtt, 1H, J = 10.7, 7.4 and 1.4 Hz).
13
C NMR: d 6.87, 19.93, 25.73, 26.37, 27.56, 28.14, 29.35, 29.88, 29.95, 31.02,
33.64, 62.57, 67.86, 99.07, 127.51, 131.92 ppm. MS: m/z 183 (1), 155 (4), 109
(4), 101 (4), 95 (10), 85 (100), 81 (13), 69 (8), 67 (17), 55 (18), 43 (9), 41 (27).
Anal. calculated for C16H29IO2: C, 50.53; H, 7.69. Found: C, 50.64; H, 7.73.
(Z)-11-(Tetrahydropyranyloxy)undec-4-enyl triphenylphosphonium iodide
(46). A solution of Ph3P (1.66 g; 6.31 mmol) and (Z)-1-iodo-11-(tetrahydropyr-
anyloxy)undec-4-ene 45 (2.40 g; 6.31 mmol) in toluene (7.0 ml) was refluxed for
2 d (GC analysis showed no remaining iodide), then poured with vigorous stirring
into 50 ml hexane, to produce a viscous oil. The mixture was stirred for 1 hr, the
solvent was replaced with fresh hexane (50 ml), and then stirred for an additional
1 hr. The solvent was removed and the remaining phosphonium salt 46 was
pumped under high vacuum for 8 hr, then used without further purification.
(7Z,11Z,13E)-1-(Tetrahydropyranyloxy)-hexadecatriene (47). Lithium
hexamethyldisilazide (LiHMDS, 1.0 M in THF, 4.50 ml, 4.5 mmol) was added
to a solution of dry DMPU (2.56 g, 20.0 mmol) in THF (12 ml) at 0-C. The
resulting mixture was stirred for 30 min, then cooled to j78-C. A solution of
(Z)-11-(tetrahydropyranyloxy)undec-4-enyl triphenylphosphonium iodide 46
(3.21 g, 5.00 mmol) in THF (18.0 ml) was added dropwise. After stirring for
1 hr, (E)-2-pentenal (0.560 g, 6.50 mmol) in THF (2 ml) was added dropwise.
The resulting mixture was stirred for 2 hr at j78-C, then warmed to room
temperature and stirred an additional 2 hr. The mixture was diluted with hexane/
Et2O (3:1–50 ml) and washed with saturated NH4Cl solution and brine, dried,
and concentrated. Purification by vacuum flash chromatography (hexane/
EtOAc, 95:5) afforded a 93:7 mixture of the (7Z,11Z,13E)- and (7Z,11E,13E)-
trienes, respectively (0.664 g, 41.5% yield). (7Z,11Z,13E)-isomer: 1H NMR: d
1.01 (t, 3H, J = 7.6 Hz), 1.22–1.40 (m, 6H), 1.46–1.64 (m, 6H), 1.66–1.80 (m,
2H), 1.96–2.06 (m, 2H), 2.06–2.16 (m, 4H), 2.16–2.26 (m, 2H), 3.37 (dt, 1H, J
= 9.6 and 6.6 Hz), 3.45–3.55 (m, 1H), 3.72 (dt, 1H, J = 9.6 and 6.8 Hz), 3.84–
PHEROMONE OF CITRUS LEAFMINER 181

3.90 (m, 1H), 4.56 (dd, 1H, J = 4.3 and 2.7 Hz), 5.30 (dt, 1H, J = 10.7 and 7.4
Hz), 5.32–5.42 (m, 2H), 5.70 (dt, 1H, J = 15.2 and 6.6 Hz), 5.95 (t, 1H, J = 10.9
Hz), 6.25–6.33 (m, 1H). 13C NMR: d 13.85, 19.92, 25.73, 26.11, 26.38, 27.45,
27.58, 28.07, 29.37, 29.88, 29.95, 31.01, 62.56, 67.87, 99.06, 124.85, 129.18,
129.21, 129.51, 130.69, 136.64 ppm. MS: m/z 320 (M+, 2), 302 (trace), 236 (1),
235 (1), 175 (1), 161 (1), 135 (2), 122 (3), 95 (35), 85 (100), 79 (15), 67 (29), 55
(20), 43 (11), 41 (30).
(7Z,11Z,13E)-Hexadecatrien-1-ol (48). The THP protecting group was
removed from (7Z,11Z,13E)-1-(tetrahydropyranyloxy)-hexadecatriene 47 (0.650 g,
2.02 mmol) with PTSA in methanol, as described above. The crude product was
purified by vacuum flash chromatography (hexane/ethyl, acetate 5:1) followed by
Kugelrohr distillation (oven temp. õ 112-C, 0.03 mmHg), yielding (7Z,11Z,13E)-
hexadecatrien-1-ol (0.338 g, 70.8% yield). The isomeric purity of the product was
increased to 97% by recrystallization from hexane at j20-C. 1H NMR: d 1.01
(t, 3H, J = 7.4 Hz), 1.26–1.42 (m, 6H), 1.52–1.60 (m, 2H), 1.98–2.06 (m, 2H),
2.06–2.16 (m, 4H), 2.18–2.26 (m, 2H), 3.63 (t, 2H, J = 6.7 Hz), 5.30 (dt, 1H, J =
10.9 and 7.4 Hz), 5.35–5.42 (m, 2H), 5.70 (dt, 1H, J = 15.2 and 6.6 Hz), 5.96 (t,
1H, J = 10.9 Hz), 6.29 (ddq, 1H, J = 15.2, 10.9 and 1.6 Hz). 13C NMR: d 13.84,
25.86, 26.11, 27.41, 27.58, 28.06, 29.28, 29.87, 32.97, 63.25, 124.84, 129.18,
129.28, 129.49, 130.61, 136.67 ppm. MS: m/z 236 (M+, 3), 163 (1), 149 (2), 135
(4), 121 (3), 107 (3), 96 (10), 95 (100), 93 (12), 81 (10), 79 (18), 67 (48), 55 (24),
53 (9), 41 (37). Anal. calculated for C16H28O: C, 81.29; H, 11.94. Found: C,
80.11; H, 12.06.
(7Z,11Z,13E)-Hexadecatrienal (49). (7Z,11Z,13E)-Hexadecatrien-1-ol 48
(18.0 mg, 0.076 mmol) was oxidized to the aldehyde with PCC and powdered 4
Å molecular sieve in CH2Cl2 as described above, and the crude product was
flash chromatographed (pentane/Et2O 9:1) yielding (7Z,11Z,13E)-hexadecatrie-
nal 49 in 77.4% yield (13.8 mg). 1H NMR: d 1.01 (t, 3H, J = 7.4 Hz), 1.34–1.42
(m, 4H), 1.63 (quint, 2H, J = 7.4 Hz), 2.00–2.06 (m, 2H), 2.06–2.17 (m, 4H),
2.18–2.26 (m, 2H), 2.42 (td, 2H, J = 1.8 and 7.4 Hz), 5.30 (dt, 1H, J = 10.8 and
7.4 Hz), 5.34–5.42 (m, 2H), 5.71 (dt, 1H, J = 15.2 and 6.6 Hz), 5.96 (t, 1H, J =
10.9 Hz), 6.29 (ddq, 1H, J = 15.2, 10.9 and 1.6 Hz), 9.76 (t, 1H, J = 1.8 Hz). 13C
NMR: d 13.84, 22.21, 26.11, 27.25, 27.59, 28.02, 29.00, 29.62, 44.10, 124.82,
129.23, 129.42, 129.53, 130.27, 136.71, 203.04 ppm. MS: m/z 234 (M+, 2), 216
(trace), 187 (1), 173 (1), 149 (2), 135 (3), 121 (2), 107 (2), 95 (100), 93 (11), 79
(14), 67 (45), 55 (22), 41 (32).
Preparation of (7Z,11Z,13Z)-hexadecatrienal (62)(Scheme 6). 2-Pentynal
(58). 2-Pentyn-1-ol 57 was oxidized with PCC, as described above. The crude
product was flash chromatographed (pentane/Et2O, 9:1), and the solvent was
distilled off using a Vigreux column, giving 2-pentynal 58 (0.832g) in a ca. 15%
mixture with solvent. MS: m/z 82 (M+, 34), 81 (M+ j1, 57), 67 (1), 66 (7), 54
(57), 53 (100), 52 (12), 51 (32), 50 (40), 49 (17).
182 MOREIRA ET AL.

SCHEME 6. Synthesis of (7Z,11Z,13Z)-hexadecatrienal 62.

(Z)-1-(Tetrahydropyranyloxy)-hexadec-7,13-diyn-11-ene (59). The title

compound was prepared by a Z-selective Wittig between the anion of 11-
(tetrahydropyranyloxy)-undec-4-ynyl triphenylphosphonium iodide 52 and 2-
pentynal 58 in THF/DMPU as described above. After workup, purification by
vacuum flash chromatography (hexane/EtOAc, 95:5) gave endiyne 59 in 57.7%
yield. 1H NMR: d 1.161 (t, 3H, J = 7.6 Hz), 1.26–1.59 (m, 12H), 1.61–1.69 (m,
1H), 1.72–1.82 (m, 1H), 2.08–2.17 (m, 2H), 2.17–2.26 (m, 2H), 2.33 (dq, 2H,
J = 7.6 and 2.2 Hz), 2.45 (q, 2H, J = 7.2 Hz), 3.32 (dt, 1H, J = 9.6 and 6.4 Hz),
3.40–3.47 (m, 1H), 3.67 (dt, 1H, J = 9.6 and 6.8 Hz), 3.76–3.84 (m, 1H), 4.51–
4.59 (m, 1H), 5.37–5.48 (m, 1H), 5.83 (dt, 1H, J = 10.8 and 7.2 Hz). 13C NMR:
d 13.43, 14.26, 18.64, 18.92, 19.90, 25.72, 26.03, 28.90, 29.24, 29.87, 29.88,
31.00, 62.54, 67.80, 76.62, 79,55, 80.90, 96.50, 99.07, 110.54, 140.69. MS: m/z
287 (1), 231 (1), 215 (2), 201 (1), 185 (3), 171 (3), 157 (9), 145 (13), 143 (16),
131 (13), 129 (21), 117 (16), 91 (39), 85 (100), 77 (40), 67 (22), 57 (19), 55
(21), 43 (18), 41 (47).
(7Z,11Z,13Z)-1-(Tetrahydropyranyloxy)-hexadecatriene (60). A solution
of cyclohexene (0.549 g, 6.68 mmol) in dry THF (2.0 ml) was added dropwise
under argon to a solution of BH3IDMDS (solution 2.0 M in THF, 1.67 ml, 3.34
mmol) in dry THF (5.0 ml) at j10-C. The resulting mixture was stirred for 3 hr
between j10 and 0-C, then allowed to warm to room temperature and stirred
for 1 hr. The resulting white slurry of dicyclohexylborane was recooled to
j20-C and a solution of (Z)-1-(tetrahydropyranyloxy)-hexadec-7,13-diyn-11-
ene 59 (0.264 g, 0.834 mmol) in dry THF (2.0 ml) was added dropwise over
PHEROMONE OF CITRUS LEAFMINER 183

20 min. The resulting mixture was slowly warmed to 0-C (ca. 2.5 hr) and then
stirred for 4 hr. Glacial acetic acid (0.84 ml) was added dropwise, and the
mixture was warmed to room temperature and stirred overnight. The solution
was cooled to 0-C and treated with aqueous NaOH (20% by wt., 6.0 ml)
followed by dropwise addition of hydrogen peroxide (30% by wt., 0.40 ml),
keeping the temperature below 15-C. The mixture was warmed to room
temperature and stirred for 1 hr, diluted with water (10 ml), and extracted with
hexane (4  15 ml). The combined organic layers were washed with brine,
dried, concentrated, and purified by vacuum flash chromatography on silica gel
(hexane/EtOAc, 95:5) giving (7Z,11Z,13Z)-1-(tetrahydropyranyloxy)-hexadeca-
triene 60 (0.213 g) in 79.8% yield as a 92:8 mixture of the (7Z,11Z,13Z)- and
(7Z,11E,13Z)-trienes, respectively. 1H NMR: d 0.98 (t, 3H, J = 7.6 Hz), 1.26–
1.43 (m, 6H), 1.46–1.65 (m, 6H), 1.66–1.74 (m, 1H), 1.76–1.80 (m, 1H), 1.96–
2.06 (m, 2H), 2.06–2.26 (m, 6H), 3.36 (dt, 1H, J = 9.6 and 6.6 Hz), 3.44–3.52
(m, 1H), 3.71 (dt, 1H, J = 9.6 and 6.8 Hz), 3.81–3.89 (m, 1H), 4.53–4.58 (m,
1H), 5.20–5.30 (m, 2H), 5.30–5.39 (m, 2H), 6.06–6.20 (m, 2H). 13C NMR: d
14.40, 19.91, 21.01, 25.73, 26.37, 27.43, 27.47, 27.83, 29.34, 29.86, 29.94,
31.00, 62.54, 67.85, 99.05, 123.17, 124.03, 129.13, 130.71, 131.47, 134.09 ppm.
MS: m/z 320 (M+, 2), 236 (1), 235 (1), 175 (1), 161 (1), 135 (2), 121 (3), 95
(38), 85 (100), 79 (12), 67 (33), 55 (20), 43 (11), 41 (32).
(7Z,11Z,13Z)-Hexadecatrien-1-ol (61). The THP protecting group was re-
moved from (7Z,11Z,13Z)-1-(tetrahydropyranyloxy)-hexadecatriene 60 (0.163 g,
0.51 mmol) with PTSA in methanol as described above. The crude product was
purified by vacuum flash chromatography (hexane/ethyl acetate, 9:1), yielding
(7Z,11Z,13Z)-hexadecatrien-1-ol (0.096 g, 79.9% yield). The isomeric purity of
the product was increased to 95% by chromatography over silica gel with 10%
AgNO3 (hexane/Et2O, 9:1). 1H NMR: d 0.99 (t, 3H, J = 7.6 Hz), 1.28–1.42 (m,
6H), 1.50–1.62 (m, 2H), 1.98–2.06 (m, 2H), 2.07–2.26 (m, 6H), 3.62 (t, 2H, J =
6.6 Hz), 5.32–5.40 (m, 2H), 5.40–5.48 (m, 2H), 6.16–6.30 (m, 2H). 13C NMR: d
14.40, 21.02, 25.85, 27.39, 27.47, 27.83, 29.26, 29.85, 32.98, 63.26, 123.16,
124.03, 129.21, 130.64, 131.47, 134.14 ppm. MS: m/z 236 (M+, 7), 207 (1), 163
(1), 149 (3), 135 (8), 121 (5), 107 (5), 96 (11), 95 (100), 93 (17), 81 (19), 79
(23), 67 (65), 55 (31), 53 (13), 41 (46).
(7Z,11Z,13Z)-Hexadecatrienal (62). (7Z,11Z,13Z)-Hexadecatrien-1-ol 61
(13.0 mg, 0.055 mmol) was oxidized to the aldehyde with PCC and powdered
4 Å molecular sieve in CH2Cl2 as described above, and the crude product was
flash chromatographed (pentane/Et2O, 9:1) affording (7Z,11Z,13Z)-hexadeca-
trienal 62 in 71.5% yield (9.2 mg). 1H NMR: d 0.99 (t, 3H, J = 7.6 Hz), 1.28–
1.42 (m, 4H), 1.63 (quint, 2H, J = 7.4 Hz), 1.99–2.06 (m, 2H), 2.06–2.26 (m,
6H), 2.41 (td, 2H, J = 2.0 and 7.4 Hz), 5.34–5.39 (m, 2H), 5.39–5.49 (m, 2H),
6.16–6.30 (m, 2H), 9.76 (t, 1H, J = 2.0 Hz). 13C NMR: d 14.40, 21.02, 22.20,
27.23, 27.48, 27.79, 28.99, 29.61, 44.09, 123.13, 124.08, 129.46, 130.30,
184 MOREIRA ET AL.

131.39, 134.19, 203.01. MS: m/z 234 (M+, 6), 219 (trace), 187 (1), 173 (1), 149
(2), 135 (6), 121 (3), 107 (3), 95 (100), 93 (15), 79 (20), 67 (59), 55 (28), 41 (43).

RESULTS

The amounts of the pheromone components collected by SPME from

overnight aerations of 1–30 female moths were usually below the detection
limit of GC and GC-MS. Therefore, identification of the active compounds was
primarily guided by a combination of GC-EAD, using the retention behavior of
the compounds on GC columns of different polarity, and the relative responses
of male moth antennae to various standards with different functional groups.
Male moth antennae responded consistently to two compounds in the effluvia
from females. Sporadically, there was a third, much smaller response visible
when extracts were analyzed on the DB-5 column (Figure 1, Table 1). The
earliest response on both columns (for DB-5, Figure 1, peak 1) matched the
retention time of synthetic 7Z,11Z-16:Ald, the previously reported sex attractant

FIG. 1. Coupled gas chromatography-electroantennogram detection traces showing res-

ponses from the GC and a male moth antenna to volatiles collected from two citrus
leafminer females over 2 nights on a SPME fiber. GC responses with letters were also
present in blank runs. Peak 1: (7Z,11Z)-hexadecadienal; peak 2: (7Z,11Z,13E)-
hexadecatrienal; peak 3: tentatively identified as (7Z,11Z,13Z)-hexadecatrienal. Column:
DB-5 (30 m  0.25 mm ID, 0.25 mm film), 100-C/1 min, 10-C/min to 275-C.
PHEROMONE OF CITRUS LEAFMINER 185

TABLE 1. COMPARISON OF RETENTION INDICES ON DB-5 AND DB-WAX GC COLUMNS

OF INSECT-PRODUCED COMPOUNDS ELICITING ANTENNAL RESPONSES IN GC-EAD
EXPERIMENTS, AND OF SYNTHETIC STANDARDS

Retention indices
a
Compound DB-5 DB-WAXa

Insect-produced compounds
Leafminer first response 17.96 22.19
Leafminer second response 18.43 23.71
Leafminer third response 18.58 –b
Synthetic standards
Z11–16:Ald 18.10 21.86
Z11,E13–16:Ald 18.56 23.35
E11,Z13–16:Ald 18.65 23.47
Z11,Z13–16:Ald 18.74 23.57
Z7,Z11–16:Ald 17.95 22.20
Z7,E11–16:Ald 17.93 22.12
5Z,7E,11Z-16:Ald 18.29 23.46
5E,7Z,11Z-16:Ald 18.36 23.60
5Z,7Z,11Z-16:Ald 18.55 23.83
5E,7E,11Z-16:Ald 18.62 23.86
7Z,11Z,13E-16:Ald 18.43 23.70
7Z,11Z,13Z-16:Ald 18.57 23.89
7Z,11E,13E-16:Ald 18.56 23.97
7Z,11E,13Z-16:Ald 18.44 23.75

Matches are shown in boldface type.

a
GC conditions: DB-5 (30 m  0.25 mm ID, 0.25 mm film, 100-C/1 min, 10-C/min–275-C/15 min);
DB-WAX (30 m  0.25 mm ID, 0.25 mm film, 100-C/1 min, 10-C/min–240-C/10 min). Because
the final temperature was reached before the tetracosane standard eluted on DB-WAX, the retention
indices on this column may differ slightly from true Kovat’s indices.
b
Third response not seen on this column.

for this species (Ando et al., 1985). Furthermore, when challenged with synthetic
standards of 7Z,11Z-16:Ald, male moth antennal responses were consistently
stronger to the aldehyde than to the corresponding acetate or alcohol.
The second compound, which consistently elicited the largest antennal
responses, eluted about 50 and 150 retention units later than 7Z,11Z-16:Ald
from the DB-5 and DB-WAX columns, respectively (Table 1). Both retention
time and antennal response data ruled out the possibility of the unknown being
7Z,11Z-16:OH or 7Z,11Z-16:Ac. Retention times on both the nonpolar and polar
columns were markedly longer than those of 7Z,11Z-16:Ald, suggesting the
presence of a conjugated diene or triene in the unknown. The presence of a
conjugated triene (e.g., a 7,9,11–16:Ald) was ruled out by examination of
retention time differences of standards available from other projects
(10E,12E,14E-16:Ald, Chen and Millar, 2000; 9E,11Z,13Z-16:Ald, Millar
186 MOREIRA ET AL.

et al., 1996). On the DB-5 column, these standards eluted >100 retention units
later than the unknown, whereas the difference was even greater on the DB-
WAX column (208 and 179 retention units, respectively). Therefore, the most
likely candidates for the unknown were the 5E/Z,7Z,11Z- or 7Z,11Z,13E/Z-
16:Ald isomers. These isomers were synthesized, and only 7Z,11Z,13E-16:Ald
matched the retention times of the unknown on both columns.
The identifications of both 7Z,11Z-16:Ald and 7Z,11Z,13E-16:Ald were sub-
sequently confirmed from full-scan mass spectra of both compounds (Figure 2)

FIG. 2. EI mass spectra of (A) (7Z,11Z,13E)-hexadecatrienal and (B) (7Z,11Z)-

hexadecadienal.
PHEROMONE OF CITRUS LEAFMINER 187

obtained from an SPME sample collected from two females aerated over five
consecutive nights. In particular, the spectrum of 7Z,11Z,13E-16:Ald exhibits a
diagnostic base peak at m/z 95, corresponding to a C7H11+ ion arising from
cleavage between allylic carbons 9 and 10, with the charge stabilized by the con-
jugated diene system. An analogous cleavage has been reported for 4,6,10–16:Ac
isomers, which have a similar arrangement of double bonds (Beevor et al., 1986).
The third EAD response matched the retention time of 7Z,11Z,13Z-16:Ald
on the DB-5 column. However, it was not seen on the DB-WAX column, nor
was it possible to obtain a mass spectrum, and so the identity of this compound
could not be confirmed.
Synthesis of the Pheromone Components and Analogs. The GC-EAD data
indicated that the pheromone extracts contained 7Z,11Z-16:Ald, and we
reasoned that the putative trienal, with a conjugated diene present, must either
be a 5,7,11- or a 7,11,13-hexadecatrienal. Thus, we initially focused on short,
nonstereoselective syntheses that would provide mixtures of isomers for compar-
isons of retention times with those of the insect-produced compound. These
compounds enabled us to eliminate the 5,7,11-isomers, and confirmed that one of the
7,11,13-isomers matched the insect produced triene, so stereoselective syntheses
were developed.
The syntheses of both pairs of (5E/Z,7E,11Z)-hexadecatrienals 11 and (5E/
Z,7Z,11Z)-hexadecatrienals 16 started with the preparation of the key inter-
mediate 4 (Scheme 1). Thus, (Z)-3-octen-1-ol 1 was transformed into its cor-
responding tosylate 2 (83% yield), which was then coupled with the terminal
alkyne 3 followed by deprotection to give alcohol 4 in 31% yield in two steps.
Enyne alcohol 4 was reduced to the corresponding (E)- and (Z)-allylic alcohols
7 and 12 by reaction with lithium aluminum hydride or P-2 Ni and H2, re-
spectively. The alcohols were treated with PBr3 to give bromides 8 and 13. To
introduce the third double bond into the alkyl chain, the bromides 8 and 13 were
converted into the phosphonium salts 9 and 14, followed by Wittig reactions of
the corresponding ylides with protected hydroxyaldehyde 6. Deprotection of the
resulting trienes with PTSA in methanol gave (5E/Z,7E,11Z)-hexadecatrien-1-ol
10 (71% yield over two steps, 42:58 ratio of isomers) and (5E/Z,7Z,11Z)-
hexadecatrien-1-ol 15 (75% yield, 55:45 ratio of isomers). Oxidation of alcohols
10 and 15 with PCC in dichloromethane gave the desired aldehydes 11 and 16
as pairs of isomers.
The synthesis of the (5E/Z,7Z,11Z)-hexadecatrienal mixture 16 gave an
approximately 1:1 ratio of isomers. To determine the identities of the two
isomers, one of which eluted very close to the insect-produced trienal, a ste-
reospecific synthesis of the 5Z,7Z,11Z-isomer was carried out (Scheme 2). The
key steps were the sequential alkylation of (Z)-1,2-dichloroethene 19 with the
terminal alkyne 18 in the presence of CuI, PdCl2(PPh3)2, and diisopropylamine,
followed by a second alkylation of the resulting chloroenyne 20 with (Z)-3-
octenyl magnesium bromide with Fe(acac)3 catalysis and N-methylpyrrolidi-
188 MOREIRA ET AL.

none (NMP) as cosolvent (Cahiez and Avedissian, 1998). The latter coupling
step was inefficient, returning mainly unreacted starting material, but it did
furnish 21 in sufficient yield (13.5%) to proceed. Dienyne 21 was stereo-
selectively reduced to the corresponding triene 22 (76% yield) with dicyclohex-
ylborane, in >99% isomeric purity by GC analysis. Removal of the THP group
by acid catalysis in methanol gave trien-1-ol 23, which was oxidized to (5Z,
7Z,11Z)-hexadecatrienal 24 with PCC.
Although (5Z,7Z,11Z)-hexadecatrienal had retention times on both col-
umns that were close to those of the insect-produced compound (Table 1), none
of the four 5,7,11-isomers had retention times that exactly matched those of the
insect-produced trienal on the two columns. Thus, we embarked on the syn-
theses of the other most likely isomers, the 7,11,13-trienals.
Initially, four of the eight possible geometric isomers of 7,11,13-hexadeca-
trienal were synthesized as two pairs of isomers, that is, (7Z,11E/Z,13E)-hex-
adecatrienal and (7Z,11E/Z,13Z)-hexadecatrienal, starting from commercially
available (7Z,11E/Z)-hexadecadienyl acetate 25 (pheromone of pink bollworm)
(Scheme 3). Thus, oxidative cleavage of 25 gave aldehyde 28 (2 steps, 10.1%
yield), which was reacted separately with the ylides generated from the
phosphonium salts 30 and 34. These reactions yielded the mixtures of trienes
(7Z,11E/Z,13Z)-31 and (7Z,11E/Z,13E)-35 in 42 and 58% yield, respectively.
After hydrolysis of the acetates, the corresponding alcohols 32 and 36 were
oxidized with PCC, furnishing the desired mixtures of aldehydes 33 and 37.
With these mixtures in hand, the insect-produced trienal was identified as the
7Z,11Z,13E-16:Ald isomer. A more stereoselective route was developed to
provide material for field trials, along with a parallel route to provide the
diene component, 7Z,11Z-16:Ald, in high isomeric purity as well.
The synthetic routes to 7Z,11Z,13E-16:Ald 49 and 7Z,11Z-16:Ald 56 are
shown in Schemes 4 and 5. The key step in both routes was a stereoselective
Wittig reaction that placed a (Z) double bond in the 11 position of the desired
compounds. The synthetic route to 7Z,11Z,13E-16:Ald 49 was based on a (C8 +
C3) + C5 approach. Thus, alkyne 41 (Waanders et al., 1987), prepared from 7-
octyn-1-ol 40 by protection of the hydroxyl group as the tetrahydropyranyl
(THP) ether, was deprotonated with BuLi, followed by addition of bromide 39
to give diprotected yne-diol 42. Selective removal of the t-butyldimethylsilyl
ether with tetrabutylammonium fluoride furnished monoprotected yne-diol 43
(Yadav et al., 1988) (70% over two steps). The alkyne was reduced by P-2
nickel-catalyzed reduction of 43 with H2 (Brown and Ahuja, 1973) to produce
44 (Sharma et al., 1994) in 92% yield. Alcohol 44 was converted to the
corresponding iodide 45 by reaction with iodine, triphenylphosphine, and
imidazole (Gnädig et al., 2001) (58%), followed by reaction of 45 with
triphenylphosphine to give the corresponding phosphonium salt. Wittig reaction
of ylide 46 derived from this salt with (E)-2-pentenal in a THF–DMPU solvent
PHEROMONE OF CITRUS LEAFMINER 189

mixture, in a modification of the Z-selective olefination method of Labelle et al.

(1990), afforded triene 47 as a 93:7 mixture of the 7Z,11E/Z,13,E-trienes in
42% yield. The Labelle et al. (1990) method called for a THF–HMPA solvent
mixture, whereas our results showed that DMPU was an effective and safer
substitute for toxic and carcinogenic HMPA. Trienol 48 was obtained by
removal of the THP protecting group with PTSA in methanol, and the isomeric
purity of 48 was increased to 97:3 by recrystallization from hexane. Oxidation
of 48 with pyridinium chlorochromate (PCC) then produced the desired
aldehyde 49 in 77% yield.
The synthesis of 7Z,11Z-16:Ald was carried out by a modification of this
route (Scheme 5). Thus, alkyne 41 was treated with BuLi followed by addition
of 1-chloro-3-iodopropane to give chloride 50 (Sonnet, 1974) (64% yield). This
was converted to the corresponding iodide 51 by refluxing with sodium iodide
in acetone (95% yield). Reaction of 51 with triphenylphosphine gave the
corresponding phosphonium salt 52, which can be used as an intermediate for
the syntheses of both the diene and the triene pheromone components. Wittig
reaction of the ylide derived from 52 via treatment with lithium hexamethyldi-
silazide in THF–DMPU solvent gave the Z olefinic compound 53 (Yadav et al.,
1988) in 65% yield as a 98.8Z:1.2E isomeric mixture. After removal of the THP
group with PTSA in methanol, alcohol 54 (Yadav et al., 1988) was reduced with
P-2 nickel and hydrogen, yielding dienol 55 in 71% yield. 7Z,11Z-16:Ald 56
was obtained by oxidation of 55 with PCC as before.
The synthetic route for the preparation of 7Z,11Z,13Z-16:Ald 62 is depicted
in Scheme 6. Phosphonium salt 52, previously used to prepare 7Z,11Z-16:Ald
56, was deprotonated with lithium hexamethyldisilazide in THF–DMPU, and
reacted with 2-pentynal to give protected endiynol 59 (58%), which was then
stereoselectively reduced to the corresponding triene 60 with dicyclohexylbor-
ane (80%), yielding a 92:8 mixture of the (7Z,11Z,13Z)- and (7Z,11E,13Z)-
trienes, respectively. After removal of the THP protecting group with PTSA in
methanol (80%), the isomeric purity of trienol 61 was increased to 95:5 by
chromatography on silica gel impregnated with 10% AgNO3. Oxidation of 61
with PCC completed the synthesis, giving aldehyde 62 in 73% yield.
Field Trials. A preliminary, unreplicated field trial run for 1 d provided
evidence that both 7Z,11Z-16:Ald and 7Z,11Z,13E-16:Ald were required for
attraction of male moths, because traps baited with 100 mg of either component
alone attracted no moths, whereas traps baited with 100:10, 100:50, or 100:100
mg ratios of 7Z,11Z,13E-16:Ald to 7Z,11Z-16:Ald caught 135, 192, and 146
moths, respectively. A subsequent, replicated field trial testing different blend
ratios of the two components caught >13,000 moths, with a 3:1 ratio of the
trienal and dienal being most attractive (Figure 3). A dose-response trial of the
3:1 ratio showed that trienal doses of 100–1000 mg were more attractive than
lower doses of 1.0–33 mg/lure (Figure 4). Many thousands of moths were
190 MOREIRA ET AL.

FIG. 3. Citrus leafminer field trial to determine the optimum ratio of the trienal to the dienal,
September 8–12, 2004. Each septum was loaded with 100 mg Z7,Z11,E13–16:Ald and
variable amounts of Z7,Z11–16:Ald. Bars with different letters are significantly different
(two-way ANOVA followed by Student–Neumann–Keuls test, a = 0.05, F = 180.4, df = 4,
24, P < 0.001). Total moths trapped = 13,915. Solvent controls and the 1 mg dose of
Z7,Z11–16:Ald each caught 1 moth, and were not included in the statistical analysis.

FIG. 4. Effect of pheromone dose on trap catches of citrus leafminer, October 15–22, 2004.
The trienal/dienal ratio was fixed at 3:1. Bars with different letters are significantly different
(two-way nonparametric ANOVA followed by Bonferroni’s t-tests for means separation, a =
0.05). For treatment, F = 22.92, df = 6, 34, P < 0.001; for block effect, F = 0.0, df = 4, 34,
P = 1.00. Total number of moths trapped = 13,122. Control traps caught two moths, and
were not included in the statistical analysis.
PHEROMONE OF CITRUS LEAFMINER 191

FIG. 5. Citrus leafminer blend ratio trial, June 2005. The trienal/dienal ratio was fixed at
100:33 mg, and variable amounts of 7Z,11Z,13Z–16:Ald were added to this base blend.
Bars surmounted with different letters are significantly different (two-way ANOVA on
log10(x + 1) transformed data, followed by Student–Neumann–Keuls test, a = 0.05). For
treatment, F = 44.0, df = 5, 29, P < 0.001; for block, F = 1.93, df = 4, 29, P = 0.14. Total
moths trapped = 617. Control traps caught no moths, and were not included in the
statistical analysis.

caught, again indicating the effectiveness of the lure. The addition of increasing
amounts of 7Z,11Z,13Z-16:Ald to the two component blend of 7Z,11Z,13E-
16:Ald and 7Z,11Z-16:Ald (100:33) resulted in decreasing numbers of moths
caught, indicating that this isomer was inhibitory at higher percentages of the
blend (Figure 5).

DISCUSSION

Solvent extracts of pheromone glands were difficult to prepare because of

the tiny size of these moths, and the pheromone components were not detected
in these extracts by GC or GC-MS. In contrast, the SPME method of dynamic
collection of pheromone from the effluvia from live female moths provided
extracts in which the amounts of the pheromone components were detectable by
GC and GC-MS, and these provided an accurate representation of the
compounds released by the moths. However, even with overnight SPME
collections made from groups of as many as 30 moths, and with the efficiency
inherent in the desorption of the entire SPME sample directly into the injection
port of the GC or GC-MS, we frequently could not detect the pheromone
192 MOREIRA ET AL.

components with either of these instruments. Thus, the preliminary identifica-

tion of the two compounds was based solely on the combination of data from
relative retention times on two GC columns of different polarities determined
from EAD responses (Table 1), the relative sizes of antennal responses to
different functional groups and double bond orientations, and latterly, the
bioassay results. The sum total of these data presented a compelling case for the
identification of the pheromone components. Confirmation of the pheromone
structures was finally acquired after the field trials, when mass spectra of the
two compounds were obtained from an SPME sample obtained from aeration of
female moths for several sequential nights.
The analytical results and the field trials conclusively demonstrate that the
citrus leafminer pheromone for California populations of this moth consists of
two major synergistic components, with no attraction at all to either compound
alone. This explains why researchers in several areas of the world had not been
able to reproduce the results of Ando et al. (1985), who reported attraction of
several hundred moths of a Japanese population of this species to 7Z,11Z-
16:Ald as a single component. Furthermore, the marked difference between the
attractiveness of 7Z,11Z-16:Ald between populations in Japan and other areas of
the world suggests that the Japanese population may constitute a geographically
distinct pheromone race, as has been reported for other lepidopteran species
(e.g., turnip moth, Agrotis segetum, Tóth et al., 1992; moths in the genus
Hemileuca, McElfresh and Millar, 2002).
The GC-EAD traces from the DB-5 column showed occasional weak
antennal responses to another trace compound, tentatively identified as
7Z,11Z,13Z-16:Ald. However, addition of this isomer to the optimal two-
component blend did not increase trap catches, and at higher doses, was actually
inhibitory. Given that our field bioassays indicated that the two-component
blend of 7Z,11Z,13E-16:Ald and 7Z,11Z-16:Ald was highly attractive to male
moths, with many thousand moths being caught in each of the two bioassays in
fall of 2004, it seems unlikely that this compound would be an important
component of the pheromone blend.
To our knowledge, the citrus leafminer is the only lepidopteran species
known to use 7Z,11Z-16:Ald, although the corresponding acetate has been
reported as a sex attractant or sex pheromone component for one stathmopid,
eight gelechiid, and one noctuid species (Witzgall et al., 2004). This is the first
report of the trienal component, 7Z,11Z,13E-16:Ald, and to our knowledge, the
7Z,11Z,13E-16:X structural motif has not been reported before from any
lepidopteran pheromone or other natural source. However, this may be a
reflection of the few Phyllocnistis or related leafminer species’ pheromones
studied rather than a unique structural motif in nature.
In summary, this new pheromone blend will find immediate use in
monitoring citrus leafminer populations, particularly in areas such as California
PHEROMONE OF CITRUS LEAFMINER 193

where the insect has been introduced fairly recently, and where it is still expanding
its range.

Acknowledgments—We thank Mariana Krugner for technical assistance, and the Citrus
Research Board of California for financial support for this project.

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