Enantioselective Synthesis and Activity of All Diastereoisomers of (E)-Phytal, a Pheromone Component of the Moroccan Locust, Dociostaurus maroccanus

Article abstract of DOI:10.1021/acs.jafc.8b06346

The Moroccan locust, Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae), is a polyphagous pest capable of inflicting large losses in agriculture under favorable environmental and climatic conditions. Currently, control of the pest relies solely on the application of conventional insecticides that have negative effects on the environment and human safety. In the search for a more rational, environmentally acceptable approach for locust control, we have previously reported that (Z/E)-phytal (1) is a male-produced candidate sex pheromone of this acridid. This molecule, with two stereogenic centers at C-7 and C-11, has four different diastereomers along with the Z/E stereochemistry of the double bond at C-2. In this paper, we present for the first time the enantioselective synthesis of the four diastereomers of (E)-phytal and their electrophysiological and behavioral activity on males and females. Our results demonstrate that the (R,R)-phytal is the most active diastereomer in both assays, significantly attracting females in a double-choice Y olfactometer, and confirming the previous chromatographic assignment as component of the sex pheromone of the Moroccan locust.

Full text of DOI:10.1021/acs.jafc.8b06346

Subscriber access provided by University of Otago Library
Agricultural and Environmental Chemistry
Enantioselective Synthesis and Activity of all
Diastereoisomers of (E)-Phytal, a Pheromone Component
of the Moroccan Locust, Dociostaurus maroccanus
Angel Guerrero, Victoria Elena Ramos, Sergio López, José María Álvarez-
Calero, Aroa Dominguez, Mª Milagro Coca, Mª Pilar Bosch, and Carmen Quero
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06346 • Publication Date (Web): 17 Dec 2018
Downloaded from http://pubs.acs.org on December 18, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 34
Journal of Agricultural and Food Chemistry
1
2
3
Enantioselective Synthesis and Activity of All Diastereoisomers of (E)-
Phytal, a Pheromone Component of the Moroccan Locust, Dociostaurus
maroccanus
Angel Guerrero†,* Victoria Elena Ramos†, Sergio López†, José María Alvarez†, Aroa
Domínguez†, María Milagro Coca-Abia‡, María Pilar Bosch†, and Carmen Quero†
†Department of Biological Chemistry and Molecular Modelling, Institute of Advanced
Chemistry of Catalonia (CSIC), 08034 Barcelona, Spain
‡Plant Health Department, Centro de Investigación y Tecnología Agroalimentaria de Aragón
and Instituto Agroalimentario de Aragón-IA2 (CITA-Zaragoza University), 50059 Zaragoza,
Spain
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 34
4
5
ABSTRACT
The Moroccan locust, Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae),
is a polyphagous pest capable of inflicting large losses in agriculture under favorable
environmental and climatic conditions. Currently, control of the pest relies solely on the
application of conventional insecticides that have negative effects on the environment and
human safety. In the search for a more rational, environmentally-acceptable approach for
locust control, we have previously reported that (Z/E)-phytal (1) is a male-produced
candidate sex pheromone of this acridid. This molecule, with two stereogenic centers at C-7
and C-11, has four different diastereomers along with the Z/E stereochemistry of the double
bond at C-2. In this paper, we present for the first time the enantioselective synthesis of the
four diastereomers of (E)-phytal and their electrophysiological and behavioral activity on
males and females. Our results demonstrate that the (R,R)-phytal is the most active
diastereomer in both assays, significantly attracting females in a double-choice Y
olfactometer, and confirming the previous chromatographic assignment as component of the
sex pheromone of the Moroccan locust.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
KEYWORDS: Moroccan locust, Dociostaurus maroccanus, sex pheromone, phytal, ,
20
21
(2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-enal, enantioselective synthesis,
electrophysiology, behavioral activity.
2
ACS Paragon Plus Environment

Page 3 of 34
Journal of Agricultural and Food Chemistry
22
INTRODUCTION
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
According to data from the Food and Agricultural Organization of the United Nations (FAO)
the world food production will need to rise by 70% and to double in the developing countries
by 2050. In addition, it is likely that these countries will have to face devastating attacks
promoted by locust plagues.1 Control of outbreaks and plagues of these pests has relied
primarily on the application of conventional insecticides to hopper bands and swarms.2 These
treatments have been highly controversial for environmental, human safety and economic
reasons and therefore, alternative, environmentally-friendly strategies for locusts control are
urgently needed.2-4 First attempts have been implemented using biopesticides based on the
fungus Metarhizium anisopliae var. acridum as an alternative biological control measure,5
but chemical control nevertheless remains the most effective defence against locusts so far.4
The Moroccan locust, Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae),
is a polyphagous pest of crops and pastures, particularly in Southern Europe, North Africa
and the Middle East, but devastating outbreaks have also been seen in Afghanistan, Iran, and
adjacent countries of the former USSR.6-8 The locust is highly voracious and, as such,
considered a “major pest of many crops”.9 As for other acridid species, like Schistocerca
gregaria (Forskål, 1775) or Locusta migratoria (L., 1758),10 if environmental and climatic
conditions are favorable, the Moroccan locust can also shift from solitarious to gregarious
phase, increasing dramatically in number with formation of hopper bands and large
landscape-devouring swarms.11 This fact has led to an increasing use of insecticides in
infested zones to prevent spreading to non-infested areas. Thus, 651,600 ha were treated in
Uzbekistan in 1984, 290,000 ha in Kazakhstan in 1993, 219,700 ha in Turkmenistan in 1996,
and ca. 650,000 ha in Spain in the period 2004-2007,11,12 among others. However, in spite of
its economic incidence, no reports have been found in the literature to tackle management of
this locust through a more rational, environmentally acceptable approach. In a previous
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 34
47
48
49
50
51
52
53
54
55
56
57
58
59
60
study,13 we reported the identification, synthesis and biological activity of (Z/E)-phytal, 1
(Figure 1) as a male-specific candidate sex pheromone compound of the acridid.
Biosynthetically, it has been postulated that phytal proceeds from oxidation of phytol in
marine bacteria,14,15 and later supported by us to occur in the gut of Moroccan locust males.13
Like phytol, the alcohol moiety of the chlorophyll molecule, the phytal molecule (3,7,11,15-
tetramethylhexadec-2-enal, 1) features four methyl substituents, two of them generating a
stereogenic center at C-7 and C-11. Therefore, four different diastereoisomers are possible, in
addition to the Z/E stereochemistry of the double bond at C-2. Only the natural (R,R)-phytal
and the racemic compound have been prepared by oxidation of the corresponding
phytols13,16,17 or by reduction of the corresponding nitrile.18 In this study, we developed an
enantioselective synthesis of all diastereoisomers of (E)-phytal, 2-5 (Figure 2) from
commercially available chiral synthons, and their electrophysiological and behavioral activity
on males and females of the Moroccan locust to establish the most active diastereomer as
well as the final assignment of the natural pheromone.
61
MATERIALS AND METHODS
62
63
64
65
66
67
68
69
70
71
General. Chemicals and solvents were purchased from commercial suppliers or purified by
standard techniques. Racemic and (2E,7R,11R)-phytol were commercially available from
Sigma-Aldrich (Tres Cantos, Madrid, Spain) and TCI Europe (Zwijndrecht, Belgium),
respectively. All reactions employing organometallic compounds were carried out under inert
atmosphere. IR spectra were recorded on a Avatar 360 FT-IR spectrometer (Thermo Nicolet,
1
13
Madison, WI). NMR spectra were recorded at 400 MHz for H and 100 MHz for C on a
Mercury 400 spectrometer (Varian, Palo Alto, CA). Spectra were recorded in CDCl3 at
ambient temperature using the CHCl3 present in the solvent (7.25 ppm for 1H NMR and 77.0
ppm for 13C NMR spectra) as internal standard. Mass spectra (MS) were obtained on a Trace
MS (ThermoQuest-Finnigan, Manchester, United Kingdom) coupled to a GC2000 gas
4
ACS Paragon Plus Environment

Page 5 of 34
Journal of Agricultural and Food Chemistry
72
73
74
75
76
77
78
chromatograph (ThermoQuest-Finnigan, Milan, Italy). High resolution mass spectra (HRMS)
were run on a Acquity UPLC coupled to an LCT Premier XE mass spectrometer (Waters,
Milford, MA) using a 50 mm x 2.1 mm i.d., 1.7 µm, BEH C18 column (Waters, Milford,
MA) eluting with CH3CN/H2O (80:20, v/v). Elemental analyses were determined on a Flash
2000 Organic Elemental Analyzer (Thermo Scientific, Cambridge, United Kingdom). Optical
rotations were measured on a model 341 polarimeter (Perkin-Elmer, Waltham, MA) at 589
nm.
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
Synthesis of Diastereomers of (E)-phytal, 2-5 (Figures 3 and 4).
The general approach is represented by the synthesis of (2E,7S,11R)-phytal, 2, from the
intermediate (2S,6R)-2,6,10-trimethylundecyl alcohol, 6 (Figure 3), as follows:
(2S,6R)-2,6,10-Trimethylundecyl methanesulfonate [(2S,6R)-16]. To a solution of chiral
alcohol (2S,6R)-6 (1.20 g, 5.6 mmol) in dichloromethane (11 mL) was added triethylamine
(2.50 mL, 18 mmol) and methanesulfonyl chloride (0.78 mL, 10.1 mmol) at 0 ºC. The
reaction was stirred at 0 ºC for 1 h, and at room temperature for 3 h. The mixture was
quenched with a saturated NH4Cl solution (10 mL) and extracted with ether (3 x 40 mL). The
combined extracts were washed with brine (20 mL), dried with anhydrous Na2SO4, and
evaporated under reduced pressure. The residue was chromatographed (silica, hexane/diethyl
ether 85:15) to give mesylate (2S,6R)-16 (1.40 g, 86%) as a pale yellow oil. 1H NMR δ 4.07
(dd, J= 9.4, 5.7 Hz, 1 H), 3.98 (dd, J= 9.4, 6.7 Hz, 1 H), 2.98 (s, 3 H), 1.86 (m, 1 H), 1.50 (m,
13
1 H), 1.43–1.01 (m, 13 H), 0.97 (d, J= 6.7 Hz, 3 H), 0.89–0.80 ppm (m, 9 H). C NMR δ
76.7 (CH2), 39.3 (CH2), 37.2 (CH3), 37.1 (CH2), 37.0 (CH2), 33.0 (CH), 32.9 (CH2), 32.6
(CH), 27.9 (CH), 24.7 (CH2), 24.0 (CH2), 22.6 (CH3), 22.5 (CH3), 19.5 (CH3), 16.4 ppm
(CH3). IR (film): 2939, 2924, 2871, 1454, 1352, 1322, 1184, 1059, 974, 809 cm-1. MS (EI):
m/z (%): 291 (1) [M-1]+, 196 (25), 168 (11), 154 (9), 140 (24), 126 (81), 111 (91), 97 (77), 83
(83), 69 (100), 57 (95), 43 (94).
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 34
97
98
(3S,7R)-3,7,11-Trimethyldodecanenitrile [(3S,7R)-17]. To a solution of mesylate (2S,6R)-16
(1.40 g, 4.78 mmol) in THF/DMSO (1:1, v/v) (14 mL) was added sodium cyanide (516 mg,
10.50 mmol) and the mixture was refluxed for 3 h. The reaction mixture was cooled to room
temperature, quenched with water (10 mL) and stirred for 10 min. The organic material was
extracted with ethyl acetate (3 x 35 mL). The combined extracts were washed with brine (20
mL) and water (15 mL), dried with anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was chromatographed (silica, hexane/diethyl ether 9:1) to give nitrile
99
100
101
102
103
104
105
106
107
108
109
25
1
(3S,7R)-17 (930 mg, 87%) as a colourless oil. [α]D = +3.06 (c 1.11, hexane). H NMR δ
2.31 (dd, J= 16.7, 5.8 Hz, 1 H), 2.22 (dd, J= 16.7, 6.8 Hz, 1 H), 1.83 (m, 1 H), 1.50 (m, 1 H),
13
1.43–1.00 (m, 13 H), 1.05 (d, J= 6.7 Hz, 3 H), 0.84 ppm (t, J = 6.9 Hz, 9 H). C NMR δ
118.9 (C), 39.2 (CH2), 37.2 (CH2), 36.8 (CH2), 36.1 (CH2), 32.6 (CH), 30.4 (CH), 27.9 (CH),
24.7 (CH2), 24.5 (CH2), 24.2 (CH2), 22.6 (CH3), 22.5 (CH3), 19.5 (CH3), 19.4 ppm (CH3). IR
(film): 2954, 2925, 2868, 2248, 1461, 1383 cm-1. MS (EI): m/z (%): 224 (2) [M+1]+, 223 (1)
+

110
[M] , 222 (7) [M-1]+, 208 (69), 194 (20), 180 (70), 166 (60), 152 (82), 138 (100), 124 (63),
111
112
113
114
115
116
117
118
119
120
121
111 (81), 97 (79), 82 (68), 71 (92), 57 (98), 43 (92). HRMS calc for C15H28N: 222.2222 [M-
1]+, found 222.2223.
(3S,7R)-3,7,11-Trimethyldodecanal [(3S,7R)-18]. A 1M solution of diisobutylaluminium
hydride (DIBAH) in hexane (5.30 mL, 5.30 mmol) and nitrile (3S,7R)-17 (928 mg, 4.09
mmol) was stirred in hexane (68 mL) at room temperature under Ar for 2.5 h. Ethanol (55
mL) and water (35 mL) were added dropwise to the mixture and stirred for 1 h. The mixture
was extracted with diethyl ether (3 x 40 mL). The combined extracts were washed with 3M
HCl (25 mL), water (25 mL), saturated sodium hydrogen carbonate solution (25 mL) and
brine (25 mL). After drying with anhydrous Na2SO4, the solvent was evaporated under
reduced pressure to furnish aldehyde (3S,7R)-18 (916 mg, 97%) as a colourless oil, pure
enough for the next step without need of further purification. [α]D25 = -1.22 (c 1.14, hexane).
6
ACS Paragon Plus Environment

Page 7 of 34
Journal of Agricultural and Food Chemistry
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
1H NMR δ 9.74 (t, J = 2.3 Hz, 1 H), 2.38 (ddd, J= 16.0, 5.7, 2.0 Hz, 1 H), 2.21 (ddd, J = 16.0,
7.8, 2.6 Hz, 1 H), 2.03 (m, 1 H), 1.59–0.99 (m, 14 H), 0.94 (d, J= 6.6 Hz, 3 H), 0.84 ppm (dd,
13
J= 8.7, 6.6 Hz, 9 H). C NMR δ 203.1 (CH), 51.1 (CH2), 39.3 (CH2), 37.3 (CH2), 37.2
(CH2), 37.0 (CH2), 32.6 (CH), 28.1 (CH), 27.9 (CH), 24.7 (CH2), 24.3 (CH2), 22.6(CH3), 22.5
(CH3), 19.9 (CH3), 19.5 ppm (CH3). IR (film): 2952, 2924, 2868, 1728, 1461, 1377, 1365,
1144, 976 cm-1. MS (EI): m/z (%): 226 (1) [M]+, 182 (67), 154 (9), 137 (18), 126 (74), 123
(85), 111 (69), 97 (89), 81 (87), 71 (100), 57 (95), 43 (94). HRMS calc for C15H29O:
225.2218 [M-1]+, found 225.2224.
2,4-Dimethylpent-3-yl 3-methylbut-2-enoate (19). This compound was obtained from
dimethylacrylic acid (4.00 g, 40 mmol), 2,4-dimethylpentan-3-ol (14 mL, 100 mmol) in the
presence of 4,4’-dimethylaminopyridine (440 mg, 3.6 mmol) in dichloromethane and N,N’-
dicyclohexylcarbodiimide (1.0 M in dichloromethane, 48 mL, 48 mmol) to give ester 19 in
51% isolated yield, as previously described.19
((1-(2,4-Dimethylpent-3-yloxy)-3-methylbuta-1,3-dien-1-yl)oxy)trimethylsilane (20). This
compound was prepared by reaction of ester 19 (4.08 g, 20.6 mmol) with lithium
diisopropylamide (LDA) (22.7 mmol) in hexane followed by the addition of
chlorotrimethylsilane (TMSCl) (4.70 mL, 37.08 mmol) to provide enol ether 20 (4.96 g, 89%;
1
95% pure by H NMR), as previously described.19 The compound was used in the next step
without further purification.
2,4-Dimethylpent-3-yl
(2E,5RS,7S,11R)-5-hydroxy-3,7,11,15-tetramethylhexadec-2-enoate
[(2E,5RS,7S,11R)-21]. A solution of silyl enol ether 20 (3.60 g, 12.63 mmol) in
dichloromethane (10 mL) was added at -78 ºC to a mixture of aldehyde (3S,7R)-18 (916 mg,
4.04 mmol) in dichloromethane (25 mL) and 1.0 M solution of titanium tetrachloride in
dichloromethane (12.5 mL, 12.5 mmol) over 20 min. The mixture was stirred at -78 ºC for 1
h, left to warm to room temperature, and quenched with saturated sodium hydrogen carbonate
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 34
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
solution (80 mL). After extraction with diethyl ether (3 x 40 mL), the extracts were
combined, washed with brine (20 mL), dried with anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by column chromatography (silica gel,
hexane/diethyl ether 98:2 to 95:5) to give alcohol (2E,5RS,7S,11R)-21 (246 mg, 14%) as a
colourless oil (none of the Z isomer was detected). Stereomeric mixture: 1H NMR δ 5.76 (s, 1
H), 4.61 (t, J= 6.2 Hz, 1 H), 3.95–3.78 (m, 1 H), 2.25 (m, 2 H), 2.19 (s, 3 H), 1.89 (m, 2 H),
13
1.75–1.01 (m, 18 H), 0.95–0.81 ppm (m, 24 H). C NMR δ 166.5 (C), 155.7 (C), 118.4
(CH), 81.7 (CH), 67.3 (CH), 67.0 (CH), 49.8 (CH2), 44.8 (CH2), 39.3 (CH2), 38.1 (CH3), 37.3
(CH2), 37.2 (CH2), 37.1 (CH2), 32.7 (CH), 29.4 (CH), 29.2 (CH), 27.9 (CH), 24.7 (CH2), 24.3
(CH2), 22.7 (CH3), 22.6 (CH3), 20.0 (CH3), 19.6 (CH3), 19.5 (CH3), 19.1 (CH3), 17.3 (2CH3),
17.2 ppm (2CH3). IR (film): 3384 (b), 2956, 2926, 2867, 1715, 1644, 1462, 1220, 1129 cm-1.
MS (EI): m/z (%): 423 (1) [M-1]+, 309 (49), 198 (21), 123 (15), 111 (41), 100 (100), 83 (86),
71 (53), 57 (83), 43 (65). HRMS calc for C27H53O3: 425.3995 [M+1]+, found 425.3976.
2,4-Dimethylpent-3-yl
(2E,5RS,7S,11R)-3,7,11,15-tetramethyl-5-(methylsulfonyloxy)hexa-
dec-2-enoate [(2E,5RS,7S,11R)-22]. To a solution of alcohol (2E,5RS,7S,11R)-21 (245 mg,
0.58 mmol) in dichloromethane (2.5 mL) was added anhydrous triethylamine (0.4 mL, 2.9
mmol) and methanesulfonyl chloride (0.14 mL, 1.73 mmol) at 0 ºC. The reaction was stirred
at 0 ºC for 1 h and at room temperature for 2 h. The mixture was quenched with a saturated
NH4Cl solution (10 mL) and extracted with diethyl ether (3 x 15 mL). The combined extracts
were washed with brine (10 mL) and water (10 mL), dried with anhydrous Na2SO4, and
evaporated under reduced pressure. The residue was chromatographed (silica, hexane/ethyl
acetate, 98:2 to 96:4) to give mesylate (2E,5RS,7S,11R)-22 (181 mg, 63%) as a pale yellow
oil. This compound was used immediately for the next step. Stereomeric mixture: 1H NMR δ
5.76 (s, 1 H), 5.03–4.89 (m, 1 H), 4.61 (t, J= 6.2 Hz, 1 H), 2.95 and 2.93 (2s, 3 H), 2.66–2.37
(m, 2 H), 2.20 (s, 3 H), 1.89 (m, 2 H), 1.81–0.99 (m, 14 H), 0.93 (d, J= 6.4 Hz, 3 H), 0.90–
8
ACS Paragon Plus Environment

Page 9 of 34
Journal of Agricultural and Food Chemistry
13
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
0.79 ppm (m, 24 H). C NMR δ 166.2 (C), 153.0 (C), 119.1 (CH), 81.9 (CH), 79.6 (CH),
79.2 (CH), 46.8 (CH2), 46.1 (CH2), 42.5 (CH2), 42.2 (CH2), 39.3 (CH2), 38.7 (CH3), 37.5
(CH2), 37.2 (CH2), 36.8 (CH2), 32.7 (CH), 29.3 (CH), 29.0 (CH), 27.9 (CH), 24.7 (CH2), 24.1
(CH2), 24.0 (CH2) 22.6 (CH3), 22.5 (CH3), 19.6 (CH3), 19.5 (2CH3), 19.1 (CH3), 19.0 (CH3),
18.9 (CH3), 17.2 ppm (4CH3). IR (film): 2952, 2925, 2867, 1709, 1629, 1366, 1314, 1171,
1054, 967, 796, 750 cm-1.
(2E,5RS,7S,11R)-1-Hydroxy-3,7,11,15-tetramethylhexadec-2-en-5-yl
methanesulfonate
[(2E,5RS,7S,11R)-23]. A 1.2M solution of diisobutylaluminium hydride in toluene (1.5 mL,
1.80 mmol) was added to a cold (-78 ºC) solution of mesylate (2E,5RS,7S,11R)-22 (180 mg,
0.36 mmol) in anhydrous THF (0.7 mL). After 2 h of stirring, the reaction mixture was
warmed to -20 ºC and stirred for 2 h more. Then, saturated NH4Cl solution (1 mL) was added
and the reaction mixture allowed to reach room temperature. The mixture was extracted with
ethyl acetate (3 x 10 mL) and the combined extracts were washed with brine (2 x 4 mL),
dried with anhydrous Na2SO4 and concentrated in vacuo. The crude was passed through a pad
of silica gel eluting with ether to give alcohol (2E,5RS,7S,11R)-23 (138 mg, 96%) as a
colourless oil. This compound was used immediately for the next step without purification.
Stereomeric mixture: 1H NMR δ 5.51 (t, J= 6.5 Hz, 1 H), 4.92 (m, 1 H), 4.13 (m, 2 H), 2.97
and 2.96 (2s, 3 H), 2.50–2.21 (m, 2 H), 1.72 (s, 3 H), 1.64–0.97 (m, 17 H), 0.91 (dd, J= 6.3,
13
3.7 Hz, 3 H), 0.87–0.75 ppm (m, 9 H). C NMR δ 134.4 (C), 128.0 (CH), 80.3 (CH), 80.1
(CH), 59.0 (CH2), 45.9 (CH2), 45.1 (CH2), 42.4 (CH2), 42.2 (CH2), 39.3 (CH2), 38.8 (CH3),
37.6 (CH2), 37.3 (CH2), 37.2 (CH2), 36.8 (CH2), 32.7 (CH), 30.2 (CH), 29.4 (CH), 29.1 (CH),
27.9 (CH), 24.7 (CH2), 24.2 (CH2), 24.0 (CH2), 22.6 (CH3), 22.5 (CH3), 19.7 (CH3), 19.6
(CH3), 19.2 (CH3), 16.4 ppm (CH3). IR (film): 3380 (b), 2952, 2925, 2867, 1462, 1334, 1171,
902 cm-1.
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 34
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
(2E,7S,11R)-3,7,11,15-Tetramethylhexadec-2-en-1-ol, (2E,7S,11R)-phytol, 24. A 1.0M
solution of lithium aluminium hydride in ether (0.55 mL, 0.55 mmol) was added dropwise to
a solution of compound (2E,5RS,7S,11R)-23 (138 mg, 0.353 mmol) in anhydrous ether (2.0
mL) at room temperature, and the mixture was stirred for 4 h. Then, water (0.3 mL) was
added followed by 1M HCl (0.3 mL). The mixture was extracted with ethyl acetate (3 x 10
mL), the combined extracts were washed with brine (2 x 5 mL), dried with anhydrous
Na2SO4 and concentrated in vacuo. The residue was chromatographed (silica gel,
hexane/ethyl acetate, 95:5) to give 80 mg of a mixture of (2E,7S,11R)-phytol, 24, α,β,γ,δ-
diunsaturated alcohol (2E,4E,7S,11R)-24a, and (7S,11R)-2,3-dihydrophytol in 78:5:17 ratio
as a clear oil. 24: 1H NMR δ 5.39 (tq, J= 6.9, 1.3 Hz, 1 H), 4.25 (d, J= 7.0 Hz, 2 H), 1.97 (t,
13
J=7.9 Hz, 2 H), 1.64 (s, 3 H), 1.57–0.95 (m, 19 H), 0.90–0.77 ppm (m, 12 H). C NMR δ
140.2 (C), 123.0 (CH), 59.4 (CH2), 39.8 (CH2), 39.3 (CH2), 37.3 (CH2), 37.2 (CH2), 37.2
(CH2), 36.7 (CH2), 32.7 (CH), 32.6 (CH), 27.9 (CH), 25.1 (CH2), 24.8 (CH2), 24.4 (CH2),
22.6 (CH3), 22.5 (CH3), 19.6 (2CH3), 16.1 ppm (CH3). IR (film): 3324 (b), 2952, 2924, 2866,
1669, 1461, 1377, 1000 cm-1. MS (EI): m/z (%): 296 (1) [M]+, 196 (4), 179 (3), 151 (4), 137
25
(11), 123 (76), 111 (39), 95 (68), 81 (77), 71 (100), 57 (75), 43 (74). [α]D = -0.91 (c 0.99,
1
CHCl3). 24a (characteristic data): H NMR δ 6.05 (d, J=15.4 Hz, 1H), 5.68 (dt, J=15.0, 7.3
Hz, 1H), 5.55 (bt, J=6.9 Hz, 1H), 4.26 (d, J=7.3 Hz, 2H). 13C NMR δ 134.9 (C), 129.3 (CH),
1
127.5 (CH), 125.3 (CH), 61.35 (CH2). (7S,11R)-2,3-Dihydrophytol (characteristic data): H
NMR δ 3.72 (m, 2H). 13C NMR δ 61.31 (CH2).
216
217
218
219
220
(2E,7S,11R)-3,7,11,15-Tetramethylhexadec-2-enal, (2E,7S,11R)-phytal, 2. Activated MnO2
(470 mg, 5.40 mmol) was added at room temperature to a solution of (2E,7S,11R)-phytol, 24
(80 mg, 0.27 mmol) in dichloromethane (1.4 mL) and the reaction mixture was refluxed for
24 h. The solid was then filtered through a Celite pad and washed with dichloromethane. The
combined organic layers were concentrated in vacuo, and the residue was purified by column
10
ACS Paragon Plus Environment

Page 11 of 34
Journal of Agricultural and Food Chemistry
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
chromatography (silica, hexane/diethyl ether, 95:5) to obtain (2E,7S,11R)-phytal, 2 as a clear
oil in three batches: first batch, 22 mg, 94% pure by 1H NMR, E/Z ratio 96:4, containing 6%
1
of α,β,γ,δ-diunsaturated aldehyde (2E,4E,7S,11R)-2a; second batch, 12 mg, 86% pure by H
1
NMR; E/Z ratio 96:4, containing 14% of 2a; third batch, 20 mg, 76% pure by H NMR,
1
containing 12% of 2a. The calculated yield of pure 2 was 58%. E isomer (2E,7S,11R)-2: H
NMR δ 9.99 (d, J = 8.1 Hz, 1 H), 5.87 (dq, J= 8.1, 1.1 Hz, 1 H), 2.21-2.13 (m, 2 H), 2.15 (d,
13
J=1.2 Hz, 3H), 1.60–0.96 (br, 19 H), 0.88–0.77 ppm (m, 12 H). C NMR δ 191.4 (CHO),
164.4 (C), 127.2 (CH), 40.9 (CH2), 39.3 (CH2), 37.3 (2CH2), 37.2 (CH2), 36.5 (CH2), 32.7
(CH), 32.6 (CH), 27.9 (CH), 24.7 (CH2), 24.6 (CH2), 24.3 (CH2), 22.6 (CH3), 22.5 (CH3),
19.6 (CH3), 19.5 (CH3), 17.4 ppm (CH3). IR (film): 2952, 2924, 2867, 1675, 1632, 1461,
1379, 1193, 1116, 864, 735 cm-1. MS (EI): m/z (%): 294 (2) [M]+, 279 (3), 276 (3), 263 (7),
194 (8), 191 (10), 179 (10), 163 (25), 150 (26), 149 (64), 140 (61), 139 (56), 121 (66), 111
25
(76), 97 (84), 84 (100), 71 (75), 69 (77), 57 (79), 55 (79), 43 (78). [α]D = +0.55 (c = 0.90,
ethanol). HRMS calc for C20H39O: 295.3001 [M+1]+, found 295.2988. Z isomer (2Z,7S,11R)-
2 (characteristic data): 1H NMR δ 9.95 (d, J = 8.3 Hz, 1 H, CHO). 13C NMR δ 190.7 (CHO),
164.8 (C), 128.4 (CH). MS (EI): m/z (%): 294 (1) [M]+, 279 (1), 263 (3), 191 (3), 179 (4),
163 (9), 150 (10), 149 (26), 140 (23), 139 (23), 121 (29), 111 (47), 97 (84), 95 (42), 84 (100),
83 (56), 69 (58), 57 (67), 55 (63), 43 (71). α,β,γ,δ-Unsaturated aldehyde (2E,4E,7S,11R)-2a
(characteristic data): 1H NMR δ 10.10 (d, J = 8.1 Hz, 1 H, CHO). 13C NMR δ 191.4 (CHO),
154.8 (C), 138.6 (CH). 134.4 (CH), 128.3 (CH). MS (EI) m/z (%): 292 (4) (M+), 277 (1), 208
(2), 179 (2), 137 (6), 110 (16), 109 (40), 96 (52), 95 (100), 81 (37), 71 (38), 57 (51), 55(45),
43 (51).
Synthesis of (2E,7R,11S)-phytal, 3, and (2E,7S,11S)-phytal, 4 (Figure 2).
These compounds were obtained by a route similar to 2 from the corresponding alcohols.
Synthesis of (2E,7R,11R)-phytal, 5 (Figure 2).
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 34
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
This compound was obtained from commercially available (2E,7R,11R)-phytol in 85% yield,
as previously reported.13
Insects. Fourth- and fifth-instar nymphs (N4 and N5) and adults of D. maroccanus were
collected from the field around Alhama de Aragón (Zaragoza) (840 m altitude, Latitude (41°
20’ 58.54’’ (ºN)/Longitude (1° 55’ 39.73’’ (ºW)), Alcudia Valley (Ciudad Real) (685 m
altitude, Latitude (38° 34’ 49.08’’ (ºN)/Longitude (4° 20’ 0.60’’ (ºW)), and Tierra de
Ledesma (Salamanca) (833 m altitude, Latitude (41° 3’ 7.00’’ (ºN)/Longitude (6° 13’ 33.12’’
(ºW)) during the period May-July 2013-2018. After collection, insects were placed in
50x50x50 cm metallic cages except the ceiling and the bottom that were of polyvinyl
chloride. Next to the lateral walls and outside the cages, 60W incandescent light bulbs were
placed to facilitate thermoregulation of the insects. The cages were placed into a climatic
room (27±2 ºC, 50±10% RH) under 12:12 L:D photoperiod. Nymphs and adults were fed
once a day with fresh alfalfa (Medicago sativa) and common reed (Phragmites australis).
Before last molt, insects were sorted by sex to get virgin adults and after last molt, adults
were separated to control the age. When required, insects were sent to the Institute of
Advanced Chemistry of Catalonia (CSIC) where they were maintained in similar cages in a
climatic room at 29±3 ºC, 50±10% RH and 14:10 L:D photoperiod for electroantennographic
(EAG) recordings and behavioral activity.
264
265
266
267
268
269
270
EAG activity. The electroantennogram apparatus was commercially available from Syntech
(Kirchzarten, Germany) and the methodology used was based on standardized protocols.20
Briefly, one antenna from a non-anesthetized insect was excised, and mounted on an
electrode holder (Syntech). A flow of humidified pure air (ca. 750 mL/min) was
continuously directed over the antenna to clean the environment and prevent its desiccation.
Test stimulations were carried out by giving puffs of air (ca. 200 mL/min) for 200 ms through
a Pasteur pipette with the aid of a CS-01 stimulus controller (Syntech). The pipette contained
12
ACS Paragon Plus Environment

Page 13 of 34
Journal of Agricultural and Food Chemistry
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
a piece of Whatman filter paper (2.5 cm diameter) containing either the solvent (n-hexane, 10
µL) alone as control or the corresponding amount of the chemical, dissolved in hexane,
which was allowed to evaporate before the tests. For each compound, two consecutive
stimulations of each testing dose were applied at 60 s intervals over the antennae. Control
puffs were applied before and after each pair of stimuli. The output signals were amplified
(10×), filtered (DC to 1 kHz) with an IDAC-2 interface (Syntech), further amplified (10×),
digitized on a PC and analyzed with the EAG 2000 program (Syntech). The whole EAG
preparation was enclosed in a Faraday cage (70×65×60 cm) connected to the ground to
prevent extraneous electric signals. The net EAG responses were calculated by subtracting
the mean response to control (hexane) before and after each stimulus from the mean response
to the corresponding chemical. Two EAG tests were conducted. In the first assay, a dose-
response profile with (R,R)-phytal and the racemic mixture was obtained for males (N=4-10
antennae) and females (N=8-14 antennae) (8-10 weeks-old) using three doses (2, 20 and 200
µg) for each chemical. In the second assay, the electrophysiological activity elicited by 200
µg of the four diastereomers and the racemic mixture was evaluated. This high dose was
required according to the only significant response shown by the antennae in the first assay.
Each antenna was stimulated with all compounds in a random sequence and a total of 9-11
male antennae and 8-10 female antennae (7-8 weeks-old) were tested. Prior to each assay,
adults were individually isolated for 24 h to avoid possible interference of odors from
conspecific individuals.
291
292
293
294
295
Behavioral activity. Behavioral responses of virgin adults of controlled age (7-15 days-old
females and 9-16 days-old males to all diastereoisomers of (E)-phytal and ca 5-7 weeks to
racemic phytal at 200 µg dose were evaluated in a Y-shaped dual-choice olfactometer. The
olfactometer consisted of a main glass tube (21 cm long x 29 mm i.d.) with two 10 cm arms
separated by 90º. Each arm was connected to a glass adaptor (10.4 cm long x 21 mm i.d.)
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 34
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
containing a Whatman filter paper (25 mm diameter) with the test sample or as control.
Charcoal-filtered air (700-800 mL/min was passed through the arms to carry the stimuli to the
test insects. The system was lit by a 60W white bulb centered in the middle of the
olfactometer to ensure homogeneous illumination. The experiments were performed from
10:00 a.m. to 6:00 p.m. at 26-29 ºC and 35-50% RH. The locusts were removed from their
containers and kept individually in BugDorm round-bottomed polypropylene containers
(Entomopraxis, Barcelona, Spain) covered with mesh screen lid for 24 h at 26±2 ºC, 50±10%
RH and 14:10 L:D photoperiod. For the experiments, locusts were placed at the base of the
main arm, and individuals walking upwind and reaching the middle of one side arm without
returning to the intersection point within 5 min were recorded as positive response, and those
that failed to choose either arm in the same period were excluded from analysis. A total of 25
insects per diastereomer were tested and assays for each treatment were performed on at least
two different days. When half of the insects of an specific experiment was tested, the
olfactometer arms were reversed to avoid any possible positional bias. After testing one
compound, the Y-tube was cleaned with soap, ethanol, and acetone, allowed to dry and
heated in an oven at 200 ºC for 1 h. Then, 10 µL of a 20 µg/µL solution of the diastereomer
in nanograde hexane was deployed in the filter paper, the hexane allowed to evaporate and
the paper placed in the treatment container. The number of insects directed to either arm was
counted and the treatment response was calculated as the number of insects attracted to the
compound relative to the total number of insects moving to either arm of the olfactometer
(treatment+control).
317
318
319
320
Statistical analysis. Mean net electroantennographic responses were subjected to one-way
ANOVA followed by Fisher's least significant difference (LSD) post-hoc test. If necessary,
data were log-transformed to fulfill the assumptions of normality and heteroscedasticity. For
behavioral assays and to assess the null hypothesis that the locust adults had no preference for
14
ACS Paragon Plus Environment

Page 15 of 34
Journal of Agricultural and Food Chemistry
321
322
323
the treatment and control arms the Chi-square goodness-of-fit test was used. In both types of
assays, the significance level was set at α = 0.05 and all tests were performed using SPSS
Statistics 17.0.
324 RESULTS AND DISCUSSION
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
Synthesis of all diastereomers of (E)-phytal
It has been reported that phytal, also known as phytenal, is a degradation product of phytol,
widely present in plants.21 Therefore, the easiest way to prepare (7R,11R)-phytal, 5 is by
direct oxidation of the corresponding chiral phytol.13,16 This diterpenic alcohol had been
previously obtained by ring-opening reaction of (R)-β-methyl-β-propiolactone with (R)-4,8-
dimethylnonyl bromide,22 and by Mukaiyama reaction of (3R,7R)-3,7,11-trimethyldodecanal
with silyl enol ether 20.19 For the oxidation to phytal, active MnO2 in CH2Cl2 is the reagent of
choice since other oxidizing agents, such as Jones reagent/acetone and pyridinium
dichromate/CH2Cl2 produce variable amounts (4-10%) of hexahydrofarnesyl acetone,
resulting from the double bond oxidative cleavage.13 Pyridinium chlorochromate/CH2Cl2 has
also been successfully used as oxidizing agent apparently with no side reaction.17 Racemic
(E,Z)-phytal has also been obtained as E:Z 1.2:1 mixture from the corresponding nitrile
precursor by reduction with diisobutylaluminium hydride in CH2Cl2.18 However, no synthetic
approaches for the synthesis of the other diastereomers 7R,11S, 7S,11R, and 7S,11S of (E)-
phytal or the corresponding phytol precursors have been reported.
340
341
342
343
344
Synthesis of the diastereomers (2E,7S,11R)-phytal, 2; (2E,7R,11S)-phytal, 3; and
(2E,7S,11S)-phytal, 4 was accomplished through the corresponding building blocks (2S,6R)-
6, (2R,6S)-6 and (2S,6S)-618,23 from (R)- or (S)-citronellal, 7, and methyl (R)- and (S)-3-
hydroxy-2-methylpropionate, 11, as chiral sources (Figures 3 and 4). Key processes of our
synthesis involve: i) Li2CuCl4-mediated coupling of the differently protected alcohol groups
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 34
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
of chiral 2-methyl-1,3-propanediol, one of them as tetrahydropyranyl (THP) or tert-
butyldimethylsilyl (TBDMS) ether and the other as a suitable leaving group such as tosylate
or mesylate,24 and ii) Lewis-acid catalyzed Mukaiyama aldol reaction25 between silyl enol
ether 20 with a suitable chiral aldehyde such as (3S,7R)-18, to introduce a 5-carbon prenyl
unit.19 Reduction of (R)-citronellal, 7 to (R)-citronellol, 8 followed by hydrogenation (H2,
10% Pt/C, EtOH) provided alcohol (R)-9 in 92% overall yield, which was transformed into
the corresponding bromide (R)-10 (N-bromosuccinimide, Ph3P, CH2Cl2, 90%)
Tetrahydropyranyl-protected tosylate (R)-14 was prepared by protection of hydroxyester (R)-
11 as tetrahydropyranyl ether (R)-1226-28 followed by diisobutylaluminium hydride reduction
in toluene/THF at -20 ºC, to produce mono-protected diol (S)-13 in 81% overall yield from
(R)-11. It should be noted that the intermediate aldehyde can be isolated in good yield when
the reaction is performed in CH2Cl2 at -78 ºC.28 Tosylation of (S)-13 under standard
conditions provided the required tosylate (R)-14 in 87% yield. Coupling reaction of (R)-14
with the Grignard reagent derived from (R)-10 under Schlosser’s conditions29 followed by
acid hydrolysis afforded the intermediate (2S,6R)-6 in 66% overall yield from (R)-14 (Figure
3). The alternative protection of alcohol (R)-11 as tert-butyldimethylsilyl ether afforded
mixtures of products when the corresponding tosylate or mesylate was reacted with bromide
(R)-10 (not shown). Mesylation of (2S,6R)-6 followed by cyanation provided nitrile (3S,7R)-
17 that was reduced with diisobutylaluminium hydride in hexane to aldehyde (3S,7R)-18 in
72.6% overall yield from (2S,6R)-6 (Figure 4). Introduction of a new prenyl unit into the
chain of aldehyde 18 was achieved through Mukaiyama aldol reaction with silyl dienol ether
20.19 The protected enol ether30 was obtained by deprotonation of ester 19 followed by
silylation in 45.4% yield from dimethylacrylic acid. Coupling reaction of aldehyde (3S,7R)-
18 with vinyl ether 20 in TiCl4/CH2Cl2 afforded mixture of stereomeric alcohols 21 at C-5,
from which isomer (2E,5RS,7S,11R)-21 was obtained after careful column chromatography
16
ACS Paragon Plus Environment

Page 17 of 34
Journal of Agricultural and Food Chemistry
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
purification. None of the Z isomer was isolated (Figure 4). The Mukaiyama reaction was used
by Fleming et al.19 to transform aldehyde (3R,7R)-18 into (7R,11R)-phytol as a 3:1 E:Z
mixture after mesylation and lithium aluminium hydride reduction. In our hands, however,
direct treatment of mesylate (2E,5RS,7S,11R)-22 with lithium aluminium hydride under
different conditions gave an impractical mixture of products. Therefore, after an extensive
optimization of the reduction conditions, we found that this process is preferably
implemented in two steps: a first chemoselective reduction of the conjugated ester group of
22 with diisobutylaluminium hydride in toluene at -78 ºC to provide hydroxy mesylate
(2E,5RS,7S,11R)-23 in almost quantitative yield, followed by lithium aluminium
hydride/ether treatment at room temperature. However, the second elimination reaction was
not completely chemoselective since a small proportion (5%) of the α,β,γ,δ diunsaturated
alcohol (2E,4E,7S,11R)-24a was also obtained from the competing dehydromesylation
reaction. In addition, (7S,11R)-2,3-dihydrophytol (17%) was also obtained from
overreduction. The expected (2E,7S,11R)-phytol, 24 was obtained in 46.6% overall yield
from alcohol 21. Selective oxidation of 24 with active MnO2 in dichloromethane as
previously described,29 gave, after careful column chromatography purification, a first batch
of (2E,7S,11R)-phytal, 2, 94% pure as 96:4 E:Z mixture, containing 6% of the α,β,γ,δ
diunsaturated aldehyde [(2E,4E,7S,11R)-2a. Two more fractions from the column provided
the expected phytal 2 less pure (76-86%). The calculated yield of pure phytal was 58%
(Figure 4). Since MnO2 is a well-known selective oxidation reagent for allylic alcohols,31 the
2,3-dihydrophytol present in the alcohol mixture was not oxidized under the conditions used
and was easily removed by column chromatography. It should be noted that the E isomer of
all synthetic diastereomers partially isomerized to the Z isomer in few hours at room
temperature to reach a ca. 75:25 E:Z stereochemical mixture. Therefore, all the diastereomers
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 34
394
395
were stabilized with 20% w/w of BHT for the electroantennographic and behavioral assays.
In previous tests no activity was elicited by the antioxidant.
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
Electroantennographic activity
In the first experiment aimed to determine the electrophysiological activity elicited by
different doses of (2E,7R,11R)-phytal, 5 relative to the racemic material, female antennae
were more sensitive than male antennae, particularly at the highest doses tested (20 and 200
µg) (Figure 5). Within each sex, the depolarization responses were always higher to
stimulations with (R,R)-phytal, except for the 2 µg dose on female antennae, although
significant difference (P≤0.05) was only apparent at the intermediate dose (20 µg). In the
second experiment, aimed to assess the electroantennographic responses of each synthetic
diastereomer relative to the racemic material, female antennae responded also better to all
diastereomers than male antennae, with the depolarizations induced by the R,R diastereomer
on females being significantly higher than those displayed by any other diastereomer (Figure
6). In males, the same trend was noticed with the highest statistically significant activity
being displayed by the R,R diastereomer. In this regard, it should be noticed the R,R
configuration of natural phytol, a very common component of locust food, so it is reasonable
to argue that this configuration is maintained also in its degradation products phytal13 and
phytanic acid15. In both sexes the following more active diastereomers were R,S (42% and
58% relative to R,R in males and females, respectively) and S,R (53% and 56%, respectively),
while the S,S and the racemic mixture elicited the weakest response. In no case, the difference
was significant.
In all cases, the net electroantennographic depolarizations elicited by the different
diastereomers were quite small, probably because of the very low volatility of the
pheromone, in spite of the huge amount of the compound tested, and particularly by the small
number of chemoreceptor organs in the antenna.32,33 In this regard, the antennal sensory
18
ACS Paragon Plus Environment

Page 19 of 34
Journal of Agricultural and Food Chemistry
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
system of acridids present a phenotypic variation in the number and type of sensilla arising
from differences in food habits, crowding and previous odor environmental experience.34 In
the Moroccan locust, like in other Acrididae,35 the most abundant sensilla are basiconic
followed by coleoconic, trichoid and chaotic.36 Although knowledge of the molecular
mechanisms of olfaction in locusts is highly unknown, it appears established that odorant
receptors expressed in neurons located in sensilla coeloconica would be tuned to plant and
fruit odors,37 those expressed in basiconic sensilla would be tuned to odors related to social
behaviors,38 and receptors expressed in neurons of sensilla trichodea are probably related to
sexual recognition.39 As result of the low depolarizations elicited along with the low stability
of the signal, only a limited number of antennae were considered for analysis (e.g. in
experiment 1 from 19-20 male and female antennae only 4-10 for males and 8-14 for females,
respectively, were used). A similar situation had been found with phytol, which gave small, if
any, electroantennogram responses although this terpenic alcohol was found to be tuned to a
specific receptor cell in Spodoptera littoralis eliciting a clear response.40
Behavioral activity
In previous experiments, sexually mature 7-15 days-old virgin males and females did not
elicit a significant preferential response towards the (R,R)-phytal at the dose of 20 µg (data
not shown). When sexually mature 7-15 days-old virgin females were tested, the (R,R)-phytal
at the dose of 200 µg was clearly the most effective diastereomer in a dual-choice Y
olfactometer with 15 insects (79% response) being attracted to the chemical-containing arm
and 4 individuals (21% response) to the control arm (6 locusts did not respond) (Table 1).
The response to the treatment was significant (p=0.012, Chi-square goodness-of-fit test).
Similarly, the (R,S)- and (S,S)-phytal attracted more insects to the arm containing the
chemical than the control arm but the difference was not significant (p=0.275 and 0.180,
respectively). The (S,R)-phytal and the racemic material did not induce any preferential
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 34
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
response on the insects tested although the latter was assayed on field-collected insects of 5-7
weeks-old (Table 1). When all diastereomers were tested on males of similar age, neither one
elicited a significant preferential response towards the treatment arm (Table 1). As for
females, (R,R)- and (S,S)-phytal were more attractive than the other two diastereomers but the
difference vs control was not significant. The racemic material was also ineffective.
Previous experiments conducted with racemic phytal (20 µg) on a bioassay arena41 with
groups of 10-15 adults resulted in a preferred attraction to females over males, but attraction
to males was also significant.13 In this regard, we postulated that the attraction to males could
be the result of a male-produced sex pheromone that is eavesdropped on by male congeners
to locate possible mating partners.42 Some points can be considered to explain the apparently
contradictory results obtained in both types of assay. First, the different design of the
bioassays; second, the age of insects (7-16 days old in the Y olfactometer and 10-30 days old
in the arena bioassay); and third, the different state of the insects used, i.e. groups of 10-15
individuals in the arena experiment vs single locusts in the Y olfactometer. It may be possible
that grouped individuals produce other minor components in addition to phytal to attract
other males whereas single insects do not. This speculation will be tackled in future
investigations of our group.
461
462
463
464
In summary, we have synthesized and tested electrophysiologically and behaviorally all
diastereomers of (E)-phytal, and assessed that the R,R isomer is the most active, confirming
the initial chromatographic identification as component of the sex pheromone of the
Moroccan locust.
465
466
ASSOCIATED CONTENT
Supporting Information
20
ACS Paragon Plus Environment

Page 21 of 34
Journal of Agricultural and Food Chemistry
467
468
469
Synthesis of intermediate (2S,6R)-6, and stereoisomers (2E,7R,11S)-phytal, 3; (2E,7S,11S)-
1
phytal, 4 and (2E,7R,11R)-phytal 5. H and 13C NMR spectra of all stereoisomers. This
material is available free of charge via the Internet at http://pubs.acs.org.
470
471
472
473
474
475
476
AUTHOR INFORMATION
Corresponding Authors
*Phone: +34 93 4006120; Fax +34 93 2045904; E-mail: agpqob@cid.csic.es.
E-mail: cqlqob@cid.csic.es
ORCID
Angel Guerrero: 0000-0003-2323-2691
Sergio López: 0000-0002-8641-1622
477
478
José María Álvarez-Calero: 0000-0002-4506-0299
Carmen Quero: 0000-0003-3599-2778
479
480
481
482
483
484
Funding
We gratefully acknowledge MINECO for an FPI contract to A.D., a postdoctoral contract to
V.E.R. and J.M.A. and financial support (AGL2012-39869-C02-01, AGL2015-66469-R)
with assistance from the European Regional Development Fund. E. Beltran (Regional
Community of Aragón) provided financial support for fieldwork.
Notes
485
486
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
487
488
489
490
We gratefully acknowledge J. Coll for helpful discussions, E. Faggi for help in some EAG
experiments, G. Rosell for HRMS measurements, S. Cahill and M. Casals for help in the
bioassays. C. Muñoz and T. Ruiz provided field collected insects and I. Tenas and P. Esteban
assisted in the fieldwork.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 34
491
REFERENCES
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
(1) Steedman, A. The locust handbook. Overseas Development National Resources Institute: London,
1988
(2) Showler, A. T. A summary of control strategies for the desert locust, Schistocerca gregaria
(Forskål). Agr. Ecosyst. Environ. 2002, 90, 97-103.
(3) Cressman, K. Monitoring desert locusts in the Middle East: An overview. Yale F. ES. Bull. 1998,
103, 123-141.
(4) Enserink, M. Can the war on locusts be won? Science (Washington DC) 2004, 306, 1880-1882.
(5) Arthurs, S.; Thomas, M. B.; Langewald, J. Field observations of the effects of fenitrothion and
Metarhizium anisopliae var. acridum on non-target ground dwelling arthropods in the Sahel. Biol.
Control 2003, 26, 333-340.
(6) El Ghadraoui, L.; Petit, D.; Picaud, F.; El Yamani, J. Relationship between labrum sensilla number
in the Moroccan locust Dociostaurus maroccanus and the nature of its diet. J. Orthop. Res. 2002, 11,
11-18.
(7) Latchininsky, A. V.; Launois-Luong, M. H. Le criquet marocain, Dociostaurus maroccanus
(Thunberg, 1815), dans la partie orientale de son aire de distribution : étude monographique relative
à l'ex-URSS et aux pays proches. CIRAD-PRIFAS: Montpellier/France, VIZR, St Petersburg, Russia,
1992
(8) Uvarov, B. P. Grasshoppers and Locusts. A Handbook of General Acridology. Vol. II: Behaviour,
Ecology, Biogeography Population Dynamics. Centre for Overseas Pest Research.: London, 1977 pp
613
(9) Anonymous Locust and Grasshopper Agricultural Manual. Center for Overseas Pest Research:
London, 1982
(10) Simpson, S. J.; Sword, G. A. Locusts. Curr. Biol. 2008, 18, 364-366.
(11) Latchininsky, A. V. Moroccan locust Dociostaurus maroccanus (Thunberg, 1815): a faunistic
rarity or an important economic pest? J. Insect Conserv. 1998, 2, 167-178.
22
ACS Paragon Plus Environment

Page 23 of 34
Journal of Agricultural and Food Chemistry
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
(12) CPAGE Reuniones anuales de los grupos de trabajo fitosanitarios. Ministerio de Agricultura,
Pesca y Alimentación: Madrid, 2004-2007 ISBN: 2004, 84-491-0674-5; 2005, 84-491-0735-0; 2006,
978-84-491-0785-6; 2007, 978-84-491-0853-2.
(13) Fürstenau, B.; Muñoz, L.; Coca-Abia, M.; Rosell, G.; Guerrero, A.; Quero, C. Phytal: A
candidate sex pheromone component of the Moroccan locust Dociostaurus maroccanus.
ChemBioChem 2013, 14, 1450-1459. Corrigendum: ChemBioChem 2013, 1414, 1391-1397.
(14) Rontani, J.-F.; Bonin, P. C.; Volkman, J. K. Biodegradation of free phytol by bacterial
communities isolated from marine sediments under aerobic and denitrifying conditions. Appl.
Environ. Microbiol. 1999, 65, 5484-5492.
(15) van den Brink, D. M.; Wanders, R. J. A. Phytanic acid: production from phytol, its breakdown
and role in human disease. Cell. Mol. Life Sci. 2006, 63, 1752.
(16) Jackman, L. M.; Rüegg, R.; Ryser, G.; von Planta, C.; Gloor, U.; Mayer, H.; Kofler, M.; Isler, O.;
Schudel, P. Über die Chemie des Vitamins K. 2. Mitteilung. Totalsynthese und Stereochemie von
trans- und cis-(7′R, 11′R)-Phyllochinon und verwandten Verbindungen. Helv. Chim. Acta 1965, 48,
1332-1349.
(17) Saikia, D.; Parihar, S.; Chanda, D.; Ojha, S.; Kumar, J. K.; Chanotiya, C. S.; Shanker, K.; Negi,
A. S. Antitubercular potential of some semisynthetic analogues of phytol. Bioorg. Med. Chem. Lett.
2010, 20, 508-512.
(18) Gembus, V.; Sala-Jung, N.; Uguen, D. Efficient access to (all-rac)-alpha-tocopherol acetate by a
Crombie chromene synthesis. Bull. Chem. Soc. Jpn. 2009, 82, 843-854.
(19) Fleming, I.; Maiti, P.; Ramarao, C. Stereocontrol of 1,5-related stereocentres using an
intermediate silyl group-the diastereoselectivity of nucleophilic attack on a double bond adjacent to a
stereogenic centre carrying a silyl group. Org. Biomol. Chem. 2003, 1, 3989-4004.
(20) Acín, P.; Rosell, G.; Guerrero, A.; Quero, C. Sex pheromone of the Spanish population of the
beet armyworm Spodoptera exigua. J. Chem. Ecol. 2010, 36, 778-786.
(21) Jansen, G. A.; Wanders, R. J. Alpha-oxidation. Biochim. Biophys. Acta 2006, 1763, 1403-1412.
(22) Fujisawa, T.; Sato, T.; Kawara, T.; Ohashi, K. A stereocontrolled total synthesis of optically
active (R,R)-phytol. Tetrahedron Lett. 1981, 22, 4823-4826.
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 34
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
(23) Huo, S.; Negishi, E.-i. A convenient and asymmetric protocol for the synthesis of natural
products containing chiral alkyl chains via Zr-catalyzed asymmetric carboalumination of alkenes.
Synthesis of phytol and vitamins E and K. Org. Lett. 2001, 3, 3253-3256.
(24) Schmid, R.; Antoulas, S.; Rüttimann, A.; Schmid, M.; Vecchi, M.; Weiserb, H. Synthesis of all
four stereoisomers of (E)-vitamin KT (phylloquinone), analysis of their diastereoisomeric and
enantiomeric purities and determination of their biopotencies. Helv. Chim. Acta 1990, 73, 1276-1299.
(25) Ishida, A.; Mukaiyama, T. Total synthesis of (±)-variotin and its analogs. Bull. Chem. Soc. Jpn.
1978, 51, 2077-2081.
(26) Mori, K. Synthesis of all the stereoisomers of 6-methyl-2-octadecanone, 6,14-dimethyl-2-
octadecanone and 14-methyl-2-octadecanone, the components of the female-produced sex pheromone
of a moth, Lyclene dharma dharma. Tetrahedron 2009, 65, 2798-2805.
(27) Nakamura, Y.; Mori, K. Synthesis of (3S,7S)- and (3S,7S,15S)-stereoisomers of 3,7-dimethyl-2-
heptacosanone and 3,7,15-trimethyl-2-heptacosanone, the ketones identified from the locust
Schistocerca gregaria. Eur. J. Org. Chem. 2000, 2000, 1307-1312.
(28) Spaltenstein, A.; Carpino, P. A.; Miyake, F.; Hopkins, P. B. New approaches to the synthesis of
trans-alkene isosteres of dipeptides. J. Org. Chem. 1987, 52, 3759-3766.
(29) Fouquet, G.; Schlosser, M. Improved carbon-carbon linking by controlled copper catalysis.
Angew. Chem. Int. Ed. Eng. 1974, 13, 82-83.
(30) Fleming, I.; Goldhill, J.; Paterson, I. γ-Alkylation of unsaturated ketones and esters: zinc
bromide-catalysed alkylation of O-silylated dienolates. Tetrahedron Lett. 1979, 20, 3209-3212.
(31) Fieser, F.; Fieser, M., Reagents for Organic Synthesis. John Wiley & Sons, Inc.: New York,
1967; 637-643; 1012-1013.
(32) Chen, H. H.; Zhao, Y. X.; Kang, L. Antennal sensilla of grasshoppers (Orthoptera: Acrididae) in
relation to food preferences and habits. J. Biosci. (Bangalore) 2003, 28, 743-752.
(33) Dumas, P.; Tetreau, G.; Petit, D. Why certain male grasshoppers have clubbed antennae? C. R.
Biol. 2010, 333, 429-437.
24
ACS Paragon Plus Environment

Page 25 of 34
Journal of Agricultural and Food Chemistry
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
(34) Guerrero, A.; Coca-Abia, M.; Quero, C. The Moroccan locust Dociostaurus maroccanus
(Thunberg): Biology, economic impact and control. In Advances in Animal Science and Zoology;
Jenkins, O. P., Ed. Nova Science Pub., Inc.: New York, 2017; Vol. 10, p 260.
(35) Ochieng, S. A.; Hallberg, E.; Hansson, B. Fine structure and distribution of antennal sensilla of
the desert locust, Schistocerca gregaria (Orthoptera: Acrididae). Cell Tissue Res. 1998, 291, 252-536.
(36) Fürstenau, B. Comunicación química intraespecífica de Coroebus spp. (Coleoptera: Buprestidae)
y Dociostaurus maroccanus /Orthoptera: Acrididae), dos insectos plaga de la Península Ibérica. Ph.D.
Dissertation, University of Barcelona, Barcelona, 2011.
(37) Anton, S.; Hansson, B. S. Antennal lobe interneurons in the desert locust Schistocerca gregaria
(Forskal): Processing of aggregation pheromones in adult males and females. J. Comp. Neurol. 1996,
370, 85-96.
(38) Hansson, B. S.; Ochieng, S. A.; Grosmaitre, X.; Anton, S.; Njagi, P. G. N. Physiological
responses and central nervous projections of antennal olfactory receptor neurons in the adult desert
locust, Schistocerca gregaria (Orthoptera: Acrididae). J. Comp. Physiol. A 1996, 179, 157-167.
(39) Ochieng, S. A.; Hansson, B. S. Responses of olfactory receptor neurones to behaviourally
important odours in gregarious and solitarious desert locust, Schistocerca gregaria. Physiol. Entomol.
1999, 24, 28-36.
(40) Anderson, P.; Hilker, M.; Hansson, B. S.; Bombosch, S.; Klein, B.; Schildknecht, H. Oviposition
deterring components in larval frass of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae): A
behavioural and electrophysiological evaluation. J. Insect Physiol. 1993, 39, 129-137.
(41) Torto, B.; Njagi, P. G. N.; Hassanali, A.; Amiani, H. Aggregation pheromone system of nymphal
gregarious desert locust, Schistocerca gregaria (Forskål) (Orthoptera: Acrididae). J. Chem. Ecol.
1996, 22, 2273-2281.
(42) Wyatt, T. D., Pheromones and Animal Behaviour: Communication by Smell and Taste.
Cambridge University Press: N.Y., 2003; pp 408.
596
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 34
597
598
599
FIGURE CAPTIONS
Figure 1. Structure of (±)-(Z/E)-phytal, 1.
Figure 2. Structure of all diastereoisomers of (E)-phytal, 2-5.
600
601
602
Figure 3. Synthesis of intermediate (2S,6R)-6. (Abbreviations: NBS, N-bromosuccinimide;
DHP, 3,4-dihydro-2H-pyran; DIBAH, diisobutylaluminium hydride; TsCl, p-toluenesulfonyl
chloride; TEA, triethylamine).
603
604
605
Figure 4. Synthesis of (2E,7S,11R)-phytal, 2. (Abbreviations: MsCl, methanesulfonyl
chloride; TEA, triethylamine; DIBAH, diisobutylaluminium hydride; LDA, lithium
diisopropylamine; TMSCl, chlorotrimethylsilane).
606
607
608
609
Figure 5. Mean electroantennographic responses (mV ± SEM) from antennae of D.
maroccanus males (N=4-10) and females (N=8-14) to different doses of (R,R)- and racemic
phytal. Means with different letters within sex are statistically different (one-way ANOVA
followed by LSD post-hoc test, α=0.05).
610
611
612
613
Figure 6. Mean electroantennographic responses (mV ± SEM) from antennae of D.
maroccanus males (n=9-11) and females (N=8-10) to 200 µg of the four diastereoisomers of
(E)-phytal and the racemic mixture. Means with different letters within sex are statistically
different (one-way ANOVA followed by LSD post-hoc test, α=0.05).
614
26
ACS Paragon Plus Environment

Page 27 of 34
Journal of Agricultural and Food Chemistry
Table 1. Behavioral Response of Virgin D. maroccanus Females and Males to all Diastereomers of
(E)-Phytal and the Racemic Material in a Dual-Choice Y Olfactometer
Compound
N
Sex
Age (d)
Treatment
Control
Treatment
response (%)
78.95
Significance
(R,R)-phytal
(R,S)-phytal
(S,S)-phytal
(S,R)-phytal
(±)-phytal
25
25
25
25
24
25
25
25
25
21
F
F
7-15
7-15
7-15
7-15
15
13
13
10
10
13
9
4
8
*
61.90
65.00
50.00
47.62
56.52
50.00
59.09
34.78
44.44
NS
NS
NS
NS
NS
NS
NS
NS
NS
F
7
F
10
11
10
9
1
F
--
(R,R)-phytal
(R,S)-phytal
(S,S)-phytal
(S,R)-phytal
(±)-phytal
M
M
M
M
M
9-16
9-16
9-16
9-16
--1
13
8
9
15
10
8
*p = 0.012 (Chi-square goodness-of-fit test at α = 0.05)
NS = non significant
1Unknown age (field collected).
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 34
Table of Contents Graphic
28
ACS Paragon Plus Environment

Page 29 of 34
Journal of Agricultural and Food Chemistry
CHO
(±)-(Z/E)-phytal, 1
Figure 1
ACS Paragon Plus Environment