br ( E )-4-Oxo-2-hexenal Dimers in the Scent Glands of the Bark Bug Phloea subquadrata ( Heteroptera , Phloeidae )

Bark bugs belonging to the family Phloeidae are known for their camouflage on tree trunks. The nymphs store in dorsal abdominal glands defensive secretions with a pungent odor mainly constituted of (E)-2-hexenal, (E)-2-octenal, and (E)-4-oxo-2-hexenal. The metathoracic glands of adults (male and female) store (E)-2-hexen-1-ol and (E)-2-hexenyl acetate, which are less irritating than their corresponding aldehydes. Additional compounds of m/z 224 were detected in the scent glands of these insects and were previously suggested to be dimers of the (E)-4-oxo-2-hexenal. Thus, the aim of this study was to elucidate the details of the chemical structure of the dimers found in the scent glands of Phloea subquadrata. These dimers were obtained by synthesis and were compared with the natural products, confirming the dimeric structures of the latter. These (E)-4-oxo-2-hexenal dimeric compounds are novel and have not been reported before.


Introduction
Bark bugs belonging to the family Phloeidae are known for their camouflage on tree trunks. 1 This family comprises three genus: Phloea, with two species (Phloea subquadrata and Phloea corticata); Phloeophana (Phloeophana longirostris) and Serbana (Serbana borneensis), both with one species each. 2 P. subquadrata occurs exclusively in Brazil, from Southern Bahia to Rio Grande do Sul. 3All four species have cryptic coloration, with a flattened body with broad foliated expansions. 2 This insect is phytophagous, feeding on the sap of certain species of Myrtaceae trees, but also Combretaceae and Phyllanthaceae. 4It is subsocial and females with eggs or early nymphs display parental care. 3oth nymphs and adults release an odoriferous yellow liquid when disturbed.
The defensive secretion stored in the dorsal abdominal glands of nymphs has a pungent odor characteristic of aldehydes and the metathoracic glands of adults (male and female) store similar derivatives that are less aggressive.
Additional compounds were detected in this insect's scent glands, many of which are still unknown.
Among these compounds, our attention was particularly attracted to the (E)-4-oxo-2-hexenal dimers, which were detected in the metathoracic and the dorsal abdominal glands of P. subquadrata individuals.The occurrence of these dimers has been mentioned in the literature, but no structure or biosynthesis has ever been proposed for these molecules. 5imeric natural products are ubiquitous and arise from aldol reactions, Michael-type reactions, Mannich-type reactions, etherification, and so forth, consequently, the dimerization attribute does not give a clue on the structure. 6he aim of this study was, therefore, to elucidate the chemical structure of the dimers (m/z 224) found in the scent glands of P. subquadrata.

Chemical analysis
P. subquadrata individuals (males, females, and second to fifth instar nymphs) and exuviae were collected from Plinia caulifora (Myrtaceae) at the Serra do Japi Biological Reserve (Jundiaí, São Paulo State, Brazil) and Itapetinga State Park (Atibaia, São Paulo State, Brazil) from 2010 to 2012.Fresh exuviae were collected wherever newly moulted nymphs were found.All adult bugs were dissected within 48 h of capture under a stereoscopic magnifying microscope.The contents of the metathoracic glands of adults were sampled by piercing the gland with a microsyringe and analyzed (10 μL, see Figure 1).Exuviae and the insects (second, third, fourth and fifth instars) were macerated in bidistilled ethyl acetate (500 μL) and filtrated.Solvent evaporation yielded exuviae and insect extracts.The nymphal dorsal abdominal glands were analyzed using the exuviae extracts or insect gland content.All samples were stored at -20 ºC in the dark.The gas chromatography-mass spectrometry (GC-MS) analyses were performed with an Agilent 6890 chromatograph and a (70-eV) Hewlett Packard 5975 MSD equipped with a fused capillary column (HP-5MS, 30 m × 0.25 mm × 0.25 μm).Helium (1 mL min -1 ) was the carrier gas.The analysis conditions consisted of a splitless mode (1 μL), mass range m/z 40-600, and temperatures of 250 and 280 ºC for the injector and detector, respectively.The column temperature program was 40 ºC (3 min) increasing to 290 °C at 10 °C min -1 .Samples of the glandular content of male and female individuals were injected (1 μL) in the splitless mode, without solvent.The exuvial glandular content was extracted in ethyl acetate (1 mg mL -1 ).The retention indices of all compounds were calculated using the retention times of a standard mixture of n-alkane mixture (C8-C32, Sigma, USA), at 20 ppm with a split ratio of 1:100.

NMR analysis
The nuclear magnetic resonance (NMR) analyses were conducted with either a Varian Inova 500 operating at 499.89 and at 125.71 MHz for 1 H and 13 C, respectively, a Bruker Avance III-400 operating at 400.13 and 100.61MHz for 1 H and 13 C, respectively, or a Bruker Avance 250 operating at 250.13 and 62.90 MHz for 1 H and 13 C, respectively.Deuterated chloroform was used as the solvent and the samples were analyzed in 5 mm diameter NMR tubes.The chemical shifts (d) are measured in parts per million, with the tetramethylsilane (TMS) signal at 0.0 ppm or the residual solvent signal (7.26 ppm) as the internal reference.All spectra were processed using the VNMRJ or TopSpin 2.1 programs and 13 C NMR, with decoupling, distortionless enhancement by polarization transfer (DEPT) 135º, DEPT 90º, and 2D NMR ( 1 H, 13 C heteronuclear single quantum correlation (HSQC), 1 H, 13 C heteronuclear multiple-bond correlation (HMBC), 1 H-1 H correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY)) were applied in the structural elucidation of the compounds.

Results and Discussion
The GC-MS analyses of the glandular contents of P. subquadrata individuals (glands of nymphs and adults and exuviae, see Figure 1) revealed the presence of lowmolecular weight compounds and dimers of (E)-4-oxo-2hexenal (2) (see Figure 2 and Table 1).Most of the known glandular components were characterized by their relative retention indices, which were compared with data in the literature, and their respective mass spectra matched those in the Wiley spectral data. 11(E)-4-Oxo-2-hexenal (2) was one of the most abundant constituents in these mixtures, characterized by 1 H NMR (P.subquadrata males), and possesses E stereochemistry, displaying the characteristic vicinal coupling constants between H-2 and H-3 (J 16.4 Hz).Joint analysis of the spectral data produced a more detailed data set of the major male metathoracic gland constituents (Figure 2).

(E)-4-Oxo-2-hexenal dimmers
Minor constituents of m/z 224 were present in the glandular content of P. subquadrata nymphs and adults, which suggested the dimerization of two 4-oxo-2-hexenals (twice m/z 112).The synthetic dimers were obtained from 4-oxo-2-hexenal catalyzed by silica gel.The GC-MS analysis revealed the presence of four isomeric dimers.These dimers were compared to the natural compounds present in the glandular content of males, revealing perfect co-elution of all the constituents (see Figure 3).
The rationale of the 4-oxo-hexenal dimerization is depicted in Figure 4 and implies an aldol dimerization as a first step followed by an intramolecular Michael addition leading to a cyclic derivative of 5 or 6 member.This could be explained applying the 5-exo-trig and 6-endo-trig pathways, both of which are favored by Baldwin's rules for ring closure (see Figure 4). 12he relative configurations of the three chiral centers was achieved by NMR employing a sample containing a major dimer, as shown in the chromatogram in Figure 3.The 13 C NMR chemical shifts of the two isomers were discriminated by their relative signal intensities, taking care to compare carbons bonded to equal numbers of hydrogens (CH 3 , CH 2 and CH) and with similar relaxation times. 12hus, the relative abundance and signal intensities were compatible.
Assignment of the 13 C NMR signals was based on the comparison of the carbon chemical shifts of the monomer and dimer (see Table 2).The 2D NMR data ( 1 H, 13 C HMBC and HSQC) were used to confirm the suggested structures.
This set of chemical shifts led to structures possessing either a five-or a six-member ring (Figure 4).However, the substantial limitation of these rules was supplanted by the careful mass fragmentation analysis that revealed the presence of fragment m/z 180, which could only arise from the five-membered ring (see Figure 5).This analysis revealed that both dimers displayed similar hydrogen and carbon chemical shifts, with major differences in the chemical shifts of carbon 4 and 2 (Δd ca.2).The NOESY and selective NOE experiments with the most abundant isomer indicated that the saturation of methyl 8 (1.30 ppm) enhanced signals at 2.50 ppm (H-6, 0.2%) and 4.56 ppm (H-5, 0.7%), consistent with the five-membered ring substituent relative stereochemistry of 5,2-trans, 4,2-cis, and 4,5-trans (13).

Conclusions
These dimeric structures were never reported before, Peak numbers refer to the compounds listed in Table 1.therefore, the present report contributes novel data on the chemistry of bark bugs.Concerns about whether these dimers were naturally occurring or not were mitigated by the detection of these compounds in gland liquid that was directly injected in the GC-MS but not when the pure synthetic monomer was analyzed under the same conditions, ruling out dimerization during GC-MS analysis.Therefore, these dimeric structures do occur naturally in the bark bugs P. subquadrata and could be a storage strategy, since one molecule of dimer yields two monomers.Additionally, one cannot dismiss that the monomer is probably more toxic than the dimer, which is an advantage for the insects, since monomer toxicity is present when needed to fight enemies and produced from the dimer by retro-Michael and retro-aldol reactions.

Supplementary Information
data (1D and 2D NMR, IR and MS spectra) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Figure 3 .
Figure 3. Chromatograms of a synthetic dimer mixture, natural dimers from P. subquadrata males, and co-elution of both natural synthetic dimers and natural dimers.

Figure 4 .
Figure 4. Dimerization of 4-oxo-hexenal.Pathways A and B are possible, but only the products produced by pathway A were observed, allowing for the appearance of four dimers (13, 14, 15, 16).

Figure 5 .
Figure 5. Diagnostic fragment of m/z 180 observed in the four dimers' mass spectra.

Table 1 .
Detected volatile compounds of P. subquadrata