Synthesis and bioactivity of (13Z,15E)‐octadecadienal: A sex pheromone component from Micromelalopha siversi Staudinger (Lepidoptera: Notodontidae)

Abstract BACKGROUND Micromelalopha siversi (Staudinger) (Lepidoptera: Notodontidae) is a defoliator of poplar trees, Populus spp. (Salicaceae). In our previous study, 13,15‐octadecadienal has been conformed as a female‐produced candidate sex pheromone component of M. siversi, but the Z/E stereochemistry of the 1,3‐diene system has not been identified so far. RESULTS Four unsaturated aliphatic aldehydes, Z13,E15‐18:Ald, Z13,Z15‐18:Ald, E13,E15‐18:Ald, and E13,Z15‐18:Ald, were synthesized from the commercially available 12‐bromo‐1‐decanol mainly by alkylation of lithium alkyne, normal Wittig or Wittig–Schlosser olefination, and hydroboration‐protonolysis. According to gas chromatography (GC) analysis of pheromone gland extracts, Z13,E15‐18:Ald was the main component, and a small amount of Z13,Z15‐18:Ald was also detected, with a ratio of approximately 7:3. However, the results of GC‐electroantennographic detection (GC‐EAD) showed that Z13,E15‐18:Ald was the only compound with electrophysiological activity, whereas Z13,Z15‐18:Ald elicited no activity. In the field, traps baited with only Z13,E15‐18:Ald resulted in much superior results to those with Z13,Z15‐18:Ald as well as the Z13,E15‐18:Ald and Z13,Z15‐18:Ald binary mixture. CONCLUSIONS Based on geometrically selective synthesis and bioactivity tests, the active sex pheromone component of M. siversi has been identified as Z13,E15‐18:Ald, the pheromone component that has not been identified in Lepidoptera before. The synthetic component was attractive to male moths in preliminary field traps, which provides novel technologies to monitor and control this pest.


INTRODUCTION
Poplar (Populus sp.) represents the most widely distributed and adaptable tree species in the world. An increasing amount of land is being used to plant poplars, particularly in China, South Korea, and the USA. Meanwhile, many countries with limited natural forests use poplars from plantations as an important timber source. 1 In addition, poplar plantations are also being investigated as renewable sources of energy for environmental improvement, [2][3][4] especially for important short rotation coppice (SRC) plantations, which contain and absorb vast quantities of atmospheric carbon dioxide. [5][6][7][8] Micromelalopha siversi (Staudinger) (Lepidoptera: Notodontidae), which is mainly distributed in China, is one of the defoliators that severely damage poplar plantations. 9 In addition, M. siversi larvae usually injure the mesophyll, causing balding of poplar branches, weakening the host and curtailing growth. Over recent decades, the biological characteristics of M. siversi have been extensively studied. 10,11 Commonly, M. siversi has three to four generations in northeastern China, and five to seven generations in south-central China, but the generations overlap extensively. The females have a clear circadian rhythm-related calling behavior during the scotophase, but not during the light period. 12 After mating, females will deposit their eggs on the poplar leaves. The larvae have five instar stages, and the mature larvae will pupate in the deciduous layer overwinter. 13 Outbreaks of this leaf-feeding pest across China have resulted in the wide application of natural insecticides or artificially synthesized pyrethroids, negatively affecting biodiversity as well as natural enemies within the ecosystem. 14,15 Therefore, more environmentally acceptable approaches are required to control M. siversi effectively.
At present, mass trapping or mating disruption using speciesspecific sex pheromone traps are effective approaches to control numerous Lepidoptera species. 16,17 The sex pheromone component plays an important role in contacting and promoting the chemical communication and reproductive behavior of M. siversi. 13,15-octadecadienal, an unsaturated aliphatic aldehyde, was found to be the sex pheromone component of M. siversi by our group in 2019. 18 However, the stereochemistry concerning the double bond at C-13 and C- 15 has not yet been characterized.
In this study, the total synthesis of Z13,E15-18:Ald, Z13,Z15-18: Ald, E13,E15-18:Ald, and E13,Z15-18:Ald is presented. Moreover, electrophysiological and behavior tests were carried out to characterize the active sex pheromone component of M. siversi and to prepare an effective lure to monitor and control M. siversi.

MATERIALS AND METHODS
2.1 Insects and pheromone extraction M. siversi pupae were obtained from Suiping (Henan Province, China), separated by sex and reared under the following conditions, temperature of 26 ± 1°C, light/dark cycle of 14 h/10 h, together with relative humidity (RH) of 70 ± 5%. The adults were raised with 10% honey solution that was put onto cotton. On days 1 or 2 following emergence, the female moths were adopted to extract pheromone, whereas the male counterparts were utilized in gas chromatography-electroantennographic detection (GC-EAD) analysis.
For the virgin calling female moths, their abdominal tips were cut, followed by extraction with distilled hexane for 30-40 min. Thereafter, the resultant hexane extracts were placed in 2-mL glass vials (Agilent Technologies, Palo Alto, CA, USA) and preserved in a refrigerator (Haier, Qingdao, Shandong, China) at −20°C for subsequent chemical analyses.

GC-EAD analysis
An Agilent 7890A GC containing a flame ionization detector (FID) was used to perform coupled GC-EAD. A Y splitter (5181-3397, Agilent Technologies) along with an HP-5 capillary column (inside diameter 30 m × 250 μm, thickness of film 0.25 μm; Agilent Technologies) was used in analyses. As the effluent of the GC column, the carrier gas (hydrogen and nitrogen) was separated at a ratio of 1:1 to simultaneously detect between the FID and the EAD apparatuses. A 1-μL splitless sample was added at 220°C (inlet temperature). The oven was held at 60°C for 2 min, then programmed to 250°C at a heating rate of 8°C min −1 . This final temperature was maintained for 10 min. An EAD probe with high resistance, an Intelligent Data Acquisition Controller (CS-55), type IDAC-02, along with a Signal Interface Box (Syntech, Buchenbach, Germany) were used to detect antennal depolarization. The basic segment of the moth at 1-2 days of age was cut with caution to prepare freshly resected male antenna, which was later added to a glass capillary filled with 0.9% normal saline housing 0.39mm silver wires (Sigmund Cohn Corp, Mt. Vernon, NY, USA). Electrodes were connected to a combi Probe (PRG-3, Syntech). The humid air filtered with charcoal flowed through the glass tube at a flow rate of 1 L min −1 . The glass tube was connected to the GC transfer line, which was designed to track the temperature of GC oven. A compound that was able to elicit an antennal response at least five times was considered to show electroantennographical activity.

Gas chromatography-mass spectrometry analysis
The chemicals analysis was carried out using an Agilent GC coupled with a mass spectrometry system (TRACE GC 2000). The GC system was equipped with a DB-5 ms column (30 m × 0.25 mm × 0.25 μm). Samples of 1 μL from different solutions were injected manually into the system at an injector temperature of 230°C under the splitless mode. The oven was held for 2 min at 80°C, then heated to 190°C at a heating rate of 15°C min −1 , and maintained at thei temperature for 10 min. The carrier gas (helium) was injected at a flow rate of 1.2 mL min −1 , filament bias voltage 70 eV, and ion source temperature of 250°C . The scanning mode was adopted to obtain spectra (range of mass m/z 35-500). Compound retention times (RTs) were compared with synthesized standards to identify the compounds. The NIST11 library (Scientific Instrument Services, Inc., Ringoes, NJ, USA) was used to obtain mass spectra for reference.
2.4 Gas chromatography coupled with flame ionization detection GC-FID analysis was performed on an Agilent 7890A equipped with a 30 m × 0.25 mm × 0.25-μm HP-FFAP column (Agilent Technologies). Samples of 1 μL from different solutions were injected manually into the system at the split mode (ratio 1:40) with an injector temperature of 220°C. The oven was held for 2 min at 100°C, then heated to 190°C at a heating rate of 15°C min −1 , maintained for 10 min, then further heated to 225°C at a heating rate of 8°C min −1 , and maintained for 10 min. Carrier gas (nitrogen) was injected at a flow rate of 1.0 mL min −1 . An FID operating at 230°C was used for detection. The pheromone components were quantified relative to the external standard (1 μL aliquot of 5 ng μL −1 n-tridecane).

Chemicals
All reactants used for synthesizing the four diastereomers of 13,15-octadecadienal were purchased from Sigma-Aldrich (St Louis, MO, USA). The solvents used to prepare gland extracts and to carry out chromatographic analyses were at HPLC grade and provided by Sigma-Aldrich, while those used in synthesizing compounds were at Pro analysis grade and provided by Aladdin (Shanghai, China). A Bruker NMR spectrometer (Bruker, Fällanden, Switzerland) was used to record the NMR spectra in CDCl 3 ( 1 H and 13 C at 500 and 125 MHz, respectively), with tetramethylsilane as the internal standard.
Triethyl orthoformate (44.5 g, 300 mmol) was mixed with psulphonic acid monohydrate (0.57 g, 3 mmol) and the resultant mixture was placed into 300 mL of anhydrous ethanol solution containing crude 10-bromodecanal at 0°C. Later, the resultant mixture was allowed to stand overnight at 0°C. Next, water and K 2 CO 3 solution were added to the mixture to prepare the basic solution. Diethyl ether was used to extract the mixture, followed by brine washing, MgSO 4 drying and solvent removal at reduced pressure. Finally, chromatographic analysis of the obtained residue was conducted on silica with hexane and ethyl acetate (30:1, v/v), resulting in crude 1,l-diethoxy-12-bromodecanal being obtained.

(E)-18,18-Diethoxyoctadec-3-en-5-yne (9)
Propyl triphenylphosphonium bromide (4.62 g, 12.0 mmol) was suspended in THF (100 mL), followed by 30 min of stirring with n-BuLi (4.8 mL, 2.5 M in hexane). The resulting mixture was cooled to −70°C, followed by the addition of aldehyde 8 (3.1 g, 10.0 mmol) in THF (5 mL). The mixture was stirred vigorously until the yellow coloration disappeared, and an additional amount of n-BuLi (2.5 M, 4.8 mL, 12.0 mmol) was added. The reaction mixture was stirred at −30°C for 5 min, whereupon a solution of hydrogen chloride in ethyl ether (1 M, 13.0 mmol, 13.0 mL) and then potassium tertbutoxide (2.04 g, 18.2 mmol) in 2-methyl-2-propanol (1.35 g, 18.2 mmol) were added. Later, the mixed solution was subjected to 2 h of stirring under ambient temperature, water washing till neutrality, and MgSO 4 drying. After evaporation of the solvent, the crude product (E-9:Z-9 0 = 7:1) was subjected to 10% silver nitrate SiO 2 with hexane:ethyl acetate (60:1, v/v), yielding 61% (2.65 g) of the E-9 as a colorless oil. 1  2.6.5 (13Z,15E)-Octadecadienal (1) A solution of compound 9 (2.0 g, 5.95 mmol) in THF (10 mL) was added dropwise to dicyclohexylborane solution (12.0 mmol). The suspension was stirred at −15°C for 2 h, naturally heating to ambient temperature and further stirred under ambient temperature for 2 h until no dicyclohexylborane precipitate was observed. The resulting solution was mixed with 2 mL of glacial acetic acid and stirred at 50°C for 2 h. Subsequently, 3 mL of 6 M sodium hydroxide and 3 mL of 35% hydrogen peroxide were added in succession to oxidize the resultant dicyclohexylborinate. The mixture was stirred for an additional 30 min and poured into 15 mL of ice water, extracted with hexane and dried with MgSO 4 . After evaporation, the crude product was added to oxalic acid dihydrate (3.0 g) in a solution of THF and water (60 mL, 1:1, v/v), followed by stirring and heating of the obtained mixture under 60°C in the presence of argon for 40 min. Afterwards, hexane was used to extract the mixture. Water, brine and sodium bicarbonate solution were used to wash organic solution, followed by Na 2 SO 4 drying and vacuum concentration. Column chromatography at medium pressure (hexane:ethyl acetate = 50:1, v/v) was conducted to analyze the residue, and the colorless oil 1 was produced (0.99 g, 63% yield, based on 9, isomeric purity of >95%). 1  2.6.6 (Z)-18,18-Diethoxyoctadec-3-en-5-yne (9 0 ) A solution of potassium bis(trimethylsilyl)amide (0.5 M in toluene, 62 mL, 31.0 mmol) was added to n-propyl triphenylphosphonium bromide (9.93 g, 25.8 mmol) in THF (150 mL). The resultant mixed solution was subjected to 1 h of reflux and cooled to −70°C, then a solution of the aldehyde 8 (4.0 g, 12.9 mmol) in THF (20 mL) was added dropwise. The mixture was stirred for 3 h and added to 30 mL of 10% aqueous NH 4 Cl. After separating the organic phase, hexane was used to extract the aqueous phase and Na 2 SO 4 was applied to dry the integrated organic phases. The crude product was obtained through evaporation and analyzed by column chromatography at medium pressure (hexane:ethyl acetate 60:1, v/v), and a Z-9 0 and E-9 mixture (16:1, colorless oil) was obtained (3.12 g, 72% yield). 1  2.6.7 (13Z,15Z)-Octadecadienal (2) Compound 2 was prepared from compound 9 0 (2.16 g), as described for preparing compound 1 based on compound 9, at an isomeric purity of >96% and a 62% yield based on 9 0 (1.02 g, colorless oil). 1

15,15-Diethoxypentadec-(E)-undec-2-enal (10)
A THF solution (10 mL) of compound 7 (10.0 g, 32.1 mmol) was added dropwise to a suspension of LiAlH 4 (1.22 g, 32.1 mmol) in THF (20 mL). The resultant mixed solution was subjected to 2 h of stirring under ambient temperature, and then 5 g of Celite and Na 2 SO 4 ·10H 2 O mixture (1:1, v/v) was carefully added to quench the reaction, followed by slurry filtering. Next, 20 mL of hexane was used to wash the Celite bed three times. MgSO 4 was used to dry the integrated organic phase before solvent evaporation at reduced pressure. EMD was used to oxidize the crude product. The dienal 10 was prepared according to a previous description for preparing compound 8 based on compound 7, producing a 90% yield in two steps (9.02 g, pale-yellow oil). 1  2.6.9 (13E,15E)-1,1-Diethoxy-octadecadienal (11) Compound 11 was prepared from compound 10 (3.12 g, 10 mmol) according to a previous description for preparing compound 9 from compound 8. A mixture of E-11 and Z-12 in a 10:1 ratio was obtained from this process, which was then chromatographed (10% silver nitrate SiO 2 ; hexane:ethyl acetate, 60:1) to produce the colorless oil 11 (2.03 g, 60% yield). 1  The delta-shaped traps, each with a sticky board baited with the synthetic compound (1000 μg) at different ratios and 1% butylated hydroxytoluene was injected into a polyvinyl chloride (PVC) capillary tube (inner diameter 0.1 cm, outer diameter 0.18 cm, length 10 cm) (Pherobio Technology Co. Ltd, Beijing, China), were hung from a plastic pole at a height of 3.0 m above the ground at intervals of 30-40 m. The pheromone compounds were dissolved in distilled hexane, then 10 μL of the mixed solution was added to the PVC tubes, with one PVC tube containing distilled hexane solution acting as the reference. In previous tests, no activity was elicited by the Five duplicates were set for every ratio, a total of 20 traps. All trap catches were measured on a day basis, and each trap in the field test was set according to a randomized block design.
Field experiment 2 was conducted from 1 to 14 September 2019 in Suipin, Henan, China. To illustrate bait effectiveness, all lures were baited with the synthesized compounds at 1:0, 4:1, 3:2, 0:1 and 0:0 ratios. Five duplicates were set for each treatment and the number of moths trapped was determined twice weekly.

Statistical analysis
One-way analysis of variance was adopted for data analysis, and the means were compared through Tukey's honestly significant difference test (SPSS 17.0, Inc., Chicago, IL, USA). The significance level was ⊍ = 0.05 for each test.
In field experiment 2, to further verify the attractiveness of Z13,E15-18:Ald, and the binary blends of Z13,E15-18:Ald and Z13,Z15-18:Ald were tested in another field experiment (F (4,20) = 8.866, P < 0.001) ( Fig. 3(b)). The traps baited with Z13,Z15-18: Ald alone caught fewer males among the tested lures at different ratios. Meanwhile, the numbers of catches of the binary blends were almost the same as those in traps baited with Z13,E15-18:Ald. The traps baited with Z13,E15-18:Ald alone caught the most males among the tested lures. These results indicate that Z13,E15-18:Ald is the key sex pheromone component of M. siversi. Although Z13,Z15-18:Ald exists in the pheromone extracts of M. siversi, it might make no difference to the attractiveness of Z13,E15-18:Ald.
The unsaturated aliphatic aldehyde 13,15-octadecadienal consists of a terminal formyl group and a 1,3-diene system, which act as the critical functional groups for the recognition of the sex pheromone components of M. siversi. As far as we know, there are few reports on the unsaturated dienyl type I pheromone at C-13 and C-15 in Lepidoptera. Typically, only Z13,Z15-18:Ald is suggested to be a component of the sex pheromone in Thaumetopoea solitaria. 28 The formyl group was first introduced as an acetal derivative at the early synthesis period, and this avoided isomerizing the adjoint diene system from oxidation. 29 According to our procedure, the acetal derivative 1,1-diethoxy-12-iododecane was obtained in three steps.
The presence of the 1,3-diene system is quite common in sex pheromones of Lepidoptera. For instance, the derivatives of (5Z,7E)-and (5E,7Z)-dodecadienol are the active sex pheromone components of pine caterpillars. [30][31][32][33] In addition, (11Z,13Z)-hexadecadienal, together with related derivatives, are the critical sex pheromones and attractants in Notodonfidae. 18,34 (8E,10E)dodecadienyl acetate synergizes the codlemone attraction to male Cydia pomonella, and it is also the sex pheromone of Cydia toreuta (Grote) and Melissopus latiferreanus. [35][36][37] Wittig olefination is an effective protocol to construct the 1,3-diene system of sex pheromone components. [38][39][40][41] In the normal Wittig reaction, the unstabilized ylides are produced primarily through those erythro betaine intermediates, resulting in the production of Z-alkene products. [42][43][44] By contrast, the E-alkene products are mainly produced by the Wittig-Schlosser reaction via the threo betaine intermediates. [45][46][47] Accordingly, in this study, the E configuration of the enyne and diene were constructed by the Wittig-Schlosser condition, 45 whereas the Z configurations were prepared through the normal Wittig reaction. 48,49 Although Z13,Z15-18:Ald was presented in the pheromone extracts of female M. siversi, it elicited no electrophysiological activity. In the field experiments, traps baited with the Z,Z isomer alone caught a few males, which might result from the presence of 3.6% of Z,E isomer in the bait. On the whole, the Z,Z isomer appeared to have no influence on the number of catches when it was mixed with the Z,E isomer at various ratios. More research is warranted for verification of these results.

CONCLUSION
Our results indicate that Z13,E15-18:Ald, a heretofore undescribed natural product, is the most active sex pheromone component of M. siversi. Traps baited with this synthesized sex pheromone component can be used in M. siversi monitoring or even in mating disruption. Further work will focus on the development of more convenient and efficient traps.