Sex pheromone biosynthetic pathway for disparlure in the gypsy moth, Lymantria dispar

Abstract

The pheromone biosynthetic pathway for production of the sex pheromone disparlure, 2-methyl-7R,8S-epoxy-octadecane, was determined for the gypsy moth. Each step in the pathway was followed by using deuterium-labeled compounds that could be identified by using GCMS. This approach provides unequivocal determination of specific reactions in the pathway. It was shown that the alkene precursor, 2-methyl-Z7-octadecene, is most likely made in oenocyte cells associated with abdominal epidermal cells. The pathway begins with valine contributing carbons for chain initiation, including the methyl-branched carbon, followed by chain elongation to 19 carbons. The double bond is introduced with an unusual Delta12 desaturase that utilizes a methyl-branched substrate. The resulting 18-methyl-Z12-nonadecenoate is decarboxylated to the hydrocarbon, 2-methyl-Z7-octadecene. The alkene is then transported to the pheromone gland through the hemolymph, most probably by lipophorin. At the pheromone gland, the alkene is unloaded and transformed into the epoxide disparlure for release into the environment. A chiral HPLC column was used to demonstrate that the (R,S)-stereoisomer of the epoxide, (+)-disparlure is found in pheromone glands.

Figure 1

Synthesis of labeled compounds. Reagents and conditions: (a) Trityl chloride/pyridine/25°C/24 h (95%); (b) n-BuLi/THF/0°C, then 1-iodo-3-methylbutane/HMPA/0–25°C/2 h (92%); (c) D₂/RhCl(PPh₃)₃/benzene/25°C/36 h (99%); (d) MeOH/CHCl₂COOH/25°C/48 h (90%); (e) NBS/DMF/Ph₃P/25°C/1 h, then MeOH/25°C/5 min (75%); (f) 1, Ph₃P/CH₃CN/reflux/24 h; 2, n-BuLi/THF/HMPA/–50°C/30 min, then 13,15-dioxahexadecanal/THF/–78°C/1 h, then 25°C/3 h (60%); (g) H₂/Pd/CmeOH/25°C/48 h (90%); (h) PDC/DMF/25°C/48 h (70–75%); (i) 1, Ph₃P/CH₃CN/reflux/24 h; 2, n-BuLi/THF/HMPA/–50°C/30 min, then undecanal/THF/–78°C/1 h, then 25°C/3 h (69%).

Figure 2

Valine incorporation into the alkene 2me-7-18:Hc (A) and disparlure (B). D₈-Valine (20 μg per 20 μl of saline) was injected into 1-day-old females once daily until 3 days old (three injections total). One hour after the last injection, hemolymph and pheromone glands were extracted and hydrocarbons were purified and analyzed by GC/MS. MS analysis was performed in the SIM mode, analyzing for specific ions plus 7, because one deuterium is lost during the conversion of valine to isobutyryl-CoA. The ions that were monitored were 43 and 266 for the alkene. These were selected because 266 is the molecular ion and 43 can come from the methyl branch and both are of significant intensity. The ions 43, 141, and 183 were monitored for disparlure. These ions were selected because of their intensity, and the latter two are fragments on either side of the epoxide. Control females were injected with unlabeled valine, and the peaks for ions 50 and 273 were absent in the alkene (data not shown). The labeled ion peaks elute just before the natural unlabeled peaks, which is indicative of deuterium labeling.

Figure 3

Incorporation of D₄-18me-19:COOH (A) and D₄-18me-12-19:COOH (B) into the alkene, 2me-7-18:Hc. Isolated cuticular abdominal tissue was incubated with the deuterium-labeled compound (25 μg per 100 μl of Grace's medium) for 6 h, and then the tissue was extracted and hydrocarbons were purified and analyzed by GC/MS. MS was performed in the SIM mode, monitoring for the molecular ion 266 and ion 270 for label incorporation. (C) Control tissues were incubated with Grace's medium without labeled compounds.

Figure 4

Incorporation of D₄-2me-7-18:Hc into disparlure after injection into females. One-day-old females were injected with 5 μg of D₄-2me-7-18:Hc in 5 μl of Grace's medium once per day for 3 days. Two hours after the last injection, pheromone glands were removed, extracted, and analyzed for incorporation by complete scan GC/MS. (A) Ions 141 and 145 were selected from the scan for presentation. (B) Complete ion scan under the ion 145 peak. (C) Complete ion scan under the ion 141 peak.

Figure 5

Incorporation of D₄-2me-7-18:Hc into disparlure in isolated pheromone glands. Pheromone glands were incubated with 2.5 μg of D₄-2me-7-18:Hc in 5 μl of Grace's medium for 3 h; then the glands were extracted and analyzed by SIM GC/MS.

Figure 6

Partial HPLC/MS chromatograms of racemic mixture of disparlure (A), (+)-disparlure (B), and disparlure isolated from four pheromone glands (C). HPLC/MS conditions are described in Materials and Methods. The chromatograms represent SIM of ion 300 (M + 18).

Figure 7

Proposed biosynthetic pathway for producing the alkene (A) and disparlure (B) by oenocyte and pheromone gland cells, respectively.