Insect Pheromone Biosynthesis: Stereochemical Pathway Of

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6834-6838, July 1993 Chemistry Insect pheromone biosynthesis: Stereochemical pathway of hydroxydanaidal production from alkaloidal precursors in Creatonotos transiens (Lepidoptera, Arctiidae)* (pyrrolizidine alkaloids/biosynthetic mechanisms/stereochemistry/deuteriated alkaloids/sexual selection) S. SCHULZt, W. FRANCKEt, M. BOPPREt, T. EISNER§, AND J. MEINWALD¶ tInstitut fur Organische Chemie, Universitat Hamburg, Martin-Luther-King-Platz 6, D-2000 Hamburg 13, Germany; *Forstzoologisches Institut der Universitait Freiburg im Breisgau, Bertoldstrasse 17, D-7800 Freiburg im Breisgau, Germany; fSection of Neurobiology and Behavior, Seeley G. Mudd Hall, Cornell University, Ithaca, NY 14853-2702; and lDepartment of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301 Contributed by J. Meinwald, April 6, 1993 AESTRACT The mechanism by which the moth Creatono- HO. HO. tos transiens produces its male pheromone, (7R)-hydroxydanaidal, from heliotrine, an alkaloidal precursor of opposite (7S) stereochemistry, was investigated. Specifically deuteriated ,,, 00 HO CHO 54 samples of heliotrine and epiheliotrine were prepared and fed * 2 81 to C. transiens larvae, and the steps in the biosynthetic process 0A 1 were monitored by gas chromatography/mass spectrometry. 5 4 3 to These analyses indicate that heliotrine is initially epimerized 2 3 R,=OH, R2=H (7S)-epiheliotrine by oxidation to the corresponding ketone followed by stereospecific reduction. The order of the subse- 4 R1=D, R2=OH quent steps is (i) aromatization of the dihydropyrrole ring, (u) 5 R,=OH, R2=D ester hydrolysis, and (iii) oxidation of the resulting primary 7 R,=H, R2=OH alcohol to thermal aldehyde. The ecological implications ofthis insect's ability (and the inability of another moth, Utetheisa R, OH ornatrix) to, use representatives of two stereochemical families R2 H O of alkaloids as pheromone precursors are discussed. That some insect species require plant alkaloids as biosyn- 6 8 R,=H, R2=OH thetic precursors for their male courtship pheromones is well 11 R,=OH, R2-H known (2, 3). Although these transformations appear unre- OH :OH markable from an organic chemical viewpoint, few details of 0 the underlying biosynthetic pathways have been elucidated HO (4-7). The conversion of monocrotaline (1, see Fig. 1) into hydroxydanaidal (2) in the Asian arctiid moth Creatonotos N/ transiens, for example, requires only aromatization of a 9 10 R,=H, R2=OH dihydropyrrole ring, ester hydrolysis, and oxidation of a 12 R,=OH, R2=H primary benzylic alcohol to the corresponding aldehyde (8, 9). The observation that this process occurs with retention of the R confi'guration at the one asymmetric center (C-7) HOH HO OH common to the alkaloidal precursor and the pheromonal 0 product, although not at all surprising, led us to wonder how OM Creatonotos would process a related pyrrolizidine alkaloid with the opposite configuration (S) at C-7. Would the insect 13 14 15 fail to recognize and metabolize a pyrrolizidine with the "incorrect" stereochemistry, would it utilize the new pre- FIG. 1. Structures of compounds mentioned in the text. D, cursor to make the mirror image form of its pheromone deuterium. (7S-2), or would it invert the configuration at C-7 to produce the original 7R-2? In an experiment in which Creatonotos (ii) There might be selective oxidation of only the 7S larvae were fed heliotrine (3, a 7S alkaloid) as the only precursor, followed by reduction to a 7R product. pyrrolizidine source, 7R-2 was in fact produced, demonstrat- (iii) There might be an SNi solvolysis of both precursors, ing that this insect can take up and metabolize alkaloids with followed by collapse of the cationic intermediate to a 7R either configuration at C-7 to make the same final product (8). product. We now ask how this stereochemical result is achieved. (iv) There might be selective SNi solvolysis of only the 7S Five mechanistic possibilities come quickly to mind, as precursor, followed by formation of the 7R product. summarized in Scheme I. (v) There might be selective inversion of the 7S precursor (i) There might be an oxidation of both the 7R and 75 by an SN2 substitution mechanism to give a 7R product. precursors to a common C-7 ketone, which could then To study these mechanistic possibilities, we prepared two undergo stereospecific reduction to a 7R product. deuterium-labeled pheromone precursors: (7R)-7-deuterio- The publication costs of this article were defrayed in part by page charge Abbreviation: El, electron ionization. payment. This article must therefore be hereby marked "advertisement" *This is paper no. 117 in the series Defense Mechanisms ofArthro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. pods; no. 116 is LaMunyon and Eisner, ref. 1. 6834 Downloaded by guest on September 29, 2021 Chemistry: Schulz et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6835 Ho H Chrompack) fused silica column was used with helium as carrier gas. Column chromatography was performed on silica HOH gel (230-400 mesh, Merck). Feeding Experiments. Creatonotos transiens Walker larvae from an established laboratory culture originating from fe- males collected in Indonesia and maintained on an artificial diet (10) were used. Alkaloids were fed to final instar larvae II as their first meal after molting; 100 Al of a 1 ,ug/,ul alkaloid solution in dichloromethane or methanol was applied to a Ho H3 5-cm2 piece of dandelion (Taraxacum officinale) leaf and HO HH offered after the solvent had evaporated. After the larvae had consumed the entire sample, they were fed with artificial diet iii gN until pupation. Extraction of Whole Insects. Bodies of 12-h-old Creatono- )i tos males were macerated in methanol/water (9:1, vol/vol), HO the solvent was removed, and the residue was taken up in 1 /it M sulfuric acid. Zinc was added to reduce any N-oxides, and L HO the' solution was extracted with chloroform, made alkaline, H OH and extracted with chloroform as described by L'Empereur tv<N et al. (11). The' chloroform solution was analyzed by gas chromatography/mass spectrometry (GC-MS). Pharate males (male pupae <12 h to eclosion) were macerated in 2 ml of dry dichloromethane. The mixture was filtered and the ivN filtrate was immediately analyzed by GC-MS. Extraction of Coremata. Hydroxydanaidal is produced in f the Creatonotos male by two abdominal scent brushes called E S ~~~~~~HO coremata. Coremata were removed from males 12 h after eclosion. They were macerated in 50 ,ul of dichloromethane and left for 30 min. After filtration through cotton, the eHPR R'O H extracts were analyzed by GC-MS or silylated at room temperature by the addition of 50 ,ul of N,O-bis-(trimethyl- silyl)trifluoroacetamide (BSTFA). After 2 h, the mixture was concentrated under a stream of nitrogen, and the residue was Scheme I taken up in dichloromethane for analysis. Biosynthetic Experiments with Utetheisa ornatrix. For com- epiheliotrine (4) and (7S)-7-deuterioheliotrine (5). Table 1 parative purposes, we examined hydroxydanaidal biosynthe- summarizes the results of biosynthetic experiments to be sis in another arctiid moth, Utetheisa ornatrix, originally anticipated from feeding of Creatonotos larvae with these from Florida, which we maintain in culture in our Cornell labeled alkaloids, according to each of these five mecha- laboratories. In this moth also, hydroxydanaidal is produced nisms. While no discrimination among pathways (iii, iv, and by the male, from a pair of coremata, as a courtship phero- v) involving any of the nucleophilic substitution (SN) mech- mone (4). The moth presumably derives hydroxydanaidal anisms could be revealed, this set of pathways should be from pyrrolizidine alkaloid, since it fails to produce the clearly distinguished from either of the oxidation/reduction pheromone when raised on an alkaloid-free diet (4). Three mechanisms, i and 11. groups of Utetheisa larvae (n = 20-30 per group), raised on an alkaloid-free diet [PB diet (4)], were injected with monocrotaline, monocrotaline hydrochloride, or heliotrine in in- MATERIALS AND METHODS sect saline solution (12) (=350-500 ,g of alkaloid per larva). Instrumentation. 'H and 13C NMR spectra were obtained Larvae were held for pupation and adult emergence. Three with Bruker WM 400, AC250P, and Varian XL-200 instru- days after emergence, the coremata of males were removed ments, and tetramethylsilane or residual solvent protons and placed in 0.5 ml ofdichloromethane. These samples were were used as standards. Mass spectra (70 eV) were obtained analyzed for hydroxydanaidal by GC. with a VG 70/250 S mass spectrometer coupled to a Hewlett- Synthesis of (R)- and (S)-Hydroxydanaidal. These enantio- Packard HP 5890 A gas chromatograph. Gas chromato- mers were prepared as previously described (9). graphic analyses were carried out on a Carlo-Erba Fractovap Synthesis of (8R)-5,6,7,8-Tetrahydro-3H-pyrrolizin-7-on-1- 2101 gas chromatograph equipped with a flame ionization yl)methyl (2S,3R)-2-Hydroxy-2-(1-methoxyethyl)-3-methylbu- detector, using on-column injection and a 30-m CP-Sil-8-CB tanoate (6). A solution of216 Al ofdry dimethyl sulfoxide (2.8 (internal diameter 0.32 mm, film thickness 0.25 ,um, mmol) in 0.75 ml of dichloromethane was added under a Chrompack, Middelburg, The Netherlands) fused silica col- nitrogen atmosphere to 128 ,4 of oxalyl chloride (1.4 mmol). umn with hydrogen as carrier gas. For enantiomer separa- The resulting mixture was stirred for 10 min and cooled to tions, a 50-m Chirasil-L-Val -10°C. A solution of 200 mg of heliotrine (3) (0.62 mmol) in (internal diameter 0.25 mm, 3 ml of dimethyl sulfoxide was added, and the mixture was Table 1. Labeling patterns expected in hydroxydanaidal (2) after stirred for 15 min and cooled to -55°C.

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