Absolute Configuration of Solenopsis Piperidines is a Tool to Classify Fire Ants (Formicidae:Myrmicinae)

Piperidine alkaloids are predominant in ant venom glands of S. invicta and S. saevissima. These ants are broadly sympatric in Southeastern Brazil, and potentially diagnostic compounds could assist in ant identification. We determined the absolute configuration of 2,6-dialkyl-piperidines of S. invicta and S. saevissima workers and used these data to group Southeastern Brazil species. The monitoring of venom samples by chiral gas chromatography coupled to a flame ionization detector (GC-FID) revealed that S. saevissima produces the four stereoisomers of 2-methyl-6-undecylpiperidines, 2-methyl-6-tridecyl-piperidines and 2-methyl-6-tridecenyl-piperidines, while S. invicta venom consisted primarily of a single enantiomer cis and trans from each piperidine alkaloid. Our findings showed that the absolute configuration of piperidine alkaloids of fire ant venom may be a potential chemical tool to distinguish S. invicta and S. saevissima sympatric species in Southeastern Brazil.


Introduction
Ants of the genus Solenopsis (Hymenoptera: Formicidae), known as fire ants, produce a variety of oxygen-free polyketide alkaloids in their venom glands such as piperidine, piperideine, pyrrolidine, indolizidine, and pyrrolizidine alkaloids.][3] The most widely studied Solenopsis species are S. invicta, S. richteri, S. geminata, S. saevissima, S. xyloni, and S. aurea. 3The significant morphological and behavioral similarity of some species has prompted studies to assist in their identification, which is hindered by the existence of intermediate hybrids. 5An important tool to overcome this problem is the analysis of potentially diagnostic compounds, such as 2,6-dialkyl-piperidines and cuticular hydrocarbons. 6,7all'Aglio-Holvorcem et al. 6 analyzed two chemical classes in widely separated populations of S. invicta and S. saevissima.They found that piperidine alkaloids clustered S. invicta, but not S. saevissima, while cuticular hydrocarbons strongly clustered both species.However, this study did not take into account the absolute configuration of alkaloids.Our laboratory has developed a rapid methodology for efficient determination of the absolute configuration of 2-methyl-6-alkylpiperidine using chiral gas chromatography coupled to a flame ionization detector (GC-FID). 4Comparison of the chromatographic profiles of synthetic racemic mixtures of 2-methyl-6-undecylpiperidines with venom samples of S. saevissima workers and queens from different nests showed the natural occurrence of four stereoisomers of 2-methyl-6-undecylpiperidine in both trifluoroacetylated and acetylated samples.These results demonstrated that trans alkaloids are not always (2R,6R) and cis alkaloids are not always (2R,6S), contrasting with previously reported data. 2 In order to find chemical criteria to separate S. saevissima and S. invicta within an area of 75,000 km 2 in Southeastern Brazil, the present study aimed to determine the absolute configuration of 2,6-dialkyl-piperidines and use these data to group the Southeastern Brazil species.

Ants
The eighteen fire ant alkaloid samples were the same as those used by Dall'Aglio-Holvorcem et al. 6 These samples were stored at -20 °C for five years and the alkaloid pattern had not changed.Thirty larger worker ants of S. invicta and S. saevissima species were collected in three individual nests at six localities in São Paulo State, in Southeastern Brazil. 6he locality codes for S. invicta samples were Instituto Agronômico de Assis, Assis (AS); Companhia Agrícola Brejo das Almas, Botucatu (BT); Fazenda Experimental/ UNIMAR, Marília (MA); Bosque-Zoológico Municipal, São José do Rio Preto (RP); and for S. saevissima: Parque Pedra do Baú, Campos do Jordão (CJ); Parque Estadual Intervales, Capão Bonito, (INT).To extract the alkaloids, the large worker ants were manually homogenized for 10 min in a test tube containing sea-sand (Fluka, Geneva, Switzerland) and 2 mL of dichloromethane (residual pesticide grade, Carlo Erba, Cer Brasil, SP, Brazil). 6

Instrumentation
Gas chromatography coupled with mass spectrometry (GC-MS) analyses (70 eV) were carried out with an Agilent 6890 chromatograph coupled to a Hewlett-Packard 5973 mass detector, equipped with a DB5 fused silica capillary column (30 m × 0.25 mm × 0.25 µm i.d.).Mass spectra were recorded over the 40-600 amu range at 3.54 scans s -1 from m/z 40 to 600.Helium was the carrier gas at a flow rate of 1 mL min -1 .The injector and interface temperatures were kept at 250 and 280 °C, respectively.The oven temperature program was 50-290 °C at 25 °C min -1 , and then maintained for 20 min at 290 °C.One-microliter samples (1 mg mL -1 ) were injected using a 10:1 split ratio.
GC-FID analyses were performed using an Agilent 6890 chromatograph equipped with a Chrompack Chirasil CB chiral column (25 m × 0.25 mm × 0.25 µm).High-purity hydrogen was the carrier gas at a flow rate of 2 mL min -1 . 1 µL samples were injected, with a 1:100 split ratio.The temperature program was 130-180 °C (0.3 °C min -1 ), maintained for 30 min at 180 °C, with the detector at 250 °C and the injector at 220 °C.

Catalytic hydrogenation microderivatization reaction
Approximately 0.2 to 0.5 mg of ant venom containing a mixture of trifluoroacetylated alkaloids, ethyl acetate (1 mL), and catalytic amount of Pd/C (10%) was added to a round-bottom flask (5 mL).The flask was sealed with a rubber septum, connected to a hydrogen reservoir (1 atm), and magnetically stirred for 6 h.The reaction was filtered using a silica gel column (1 g, 1 cm diameter), eluted with ethyl acetate (10 mL) and analyzed by GC-MS and GC-FID (chiral column). 8rification of unsaturated ant venom alkaloids by silver nitrate-silica gel chromatography Silica gel impregnated with silver nitrate was prepared as previously described. 9Thus, a Pasteur pipette (1 cm diameter) was wrapped in aluminum foil and packed with silver nitrate/ silica gel in hexane.The sample was then added in the form of pastilles to silica gel.Elution was performed using a hexane/ ethyl ether gradient (100:0, 90:1 and 80:20, v/v).Fraction composition was determined by GC-MS, demonstrating a 90:1 gradient elution for saturated trifluoroacetylated alkaloids and an 80:20 (v/v) gradient elution for unsaturated alkaloids.Fractions containing saturated trifluoroacetylated alkaloids were analyzed by GC-FID, while those containing unsaturated trifluoroacetylated alkaloids were hydrogenated for GC-FID analysis.

Results and Discussion
We developed a method to separate diatereomeric and enantiomeric piperidine alkaloids in the natural venoms in two species of fire ants (S. invicta and S. saevissima) trying to use these alkaloids as a chemotaxonimic tool to separate both species.We used large worker of three nests from six localities in São Paulo state to extract the alkaloids.After the trifluoroacetylation reaction (Figure S1 in the Supplementary Information (SI) section) and GC-FID analyses using the modified method proposed by Pianaro et al., 4 natural venom containing piperidine alkaloids showed overlapping peaks in the chromatogram region where unsaturated alkaloids were eluted (Figure 1a).The peaks corresponding to the 2-methyl-6-tridecenylpiperidine stereoisomers were broad with partially overlapping peaks of 2-methyl-6-tridecylpiperidine stereoisomers, preventing good chiral discrimination and the possibility of co-injection with enantiomerically-enriched synthetic products.Thus, saturated and unsaturated alkaloids were separated using silver nitrate-silica gel chromatography (Figure S2 in the SI section).The fraction containing saturated alkaloids was directly analyzed by chiral GC-FID, and the fraction containing the unsaturated trifluoroacetylated alkaloids was submitted to catalytic hydrogenation (Scheme S1 in the SI section) to obtain the corresponding saturated derivatives.As such, compounds containing unsaturation in the side chain were converted into their saturated products without any chemical modification in the central chiral portion of the piperidine ring.Catalytic hydrogenation was extremely efficient, converting reactants to products almost quantitatively (Figure S3, in the SI section).After semi-purification and hydrogenation of unsaturated trifluoroacetylated alkaloids, chiral GC-FID analysis was fast and simple, exhibiting less overlapping (Figure 1b) and enabling coinjection with enantiomerically-enriched synthetic standards.Analysis of 2-methyl-6-tridecenylpiperidine (Figure S4 in the SI section) and 2-methyl-6-pentadecenylpiperidine (Figure S5 in the SI section) trifluoroacetamide DMDS derivatives revealed a double bond at carbon 4 and 6 of the alkyl chains, respectively.
The cluster analysis was performed using three nests at six different localities and seven diastereomeric or enantiomeric ratio of piperidine alkaloid trifluoroacetamides (see Experimental section for more details).The analysis showed that is possible to group the two species (S. saevissima and S. invicta) using the absolute configuration of the stereogenic center of 2-methyl-6-undecylpiperidine, 2-methyl-6-tridecylpiperidine and 2-methyl-6-tridecenylpiperidine (Figure 2).The nests of S. saevissima (localities CJ and INT) were separated from S. invicta (localities AS, BT and MA).However, S. invicta from RP locality clustered near S. saevissima.It is interesting to note that the RP population of S. invicta showed different alkaloid and hydrocarbon profiles from other S. invicta populations, suggesting an undescribed species. 6Due to the small nest sample in each locality (only 3), it is difficult to explain why some S. invicta populations (AS and BT) clustered together, while others did not (MA and RP).Geographical variation, hybrids, cryptic species or even unicoloniality 11 have been hypothesized, but further studies with a large number of nests and molecular genetic analysis are needed to clarify this issue.

Conclusions
S. invicta and S. saevissima venom analysis of seven different regions of São Paulo State, Brazil, revealed the presence of four stereoisomers of 2-methyl-6-undecylpiperidines, 2-methyl-6-tridecyl-piperidines and 2-methyl-6-tridecenyl-piperidines in S. saevissima, and the predominance of a single enantiomer cis and trans in each 2,6-dialkyl-piperidine in S. invicta.Our findings showed the potential of the absolute configuration of the piperidine alkaloid of fire ant venom as a chemical tool to distinguish the sympatric species S. invicta and S. saevissima in Southeastern Brazil.By integrating a natural synthetic product approach with classical taxonomic identification, we were able to improve the accuracy of Solenopsis identification.An integrative prespective combining, for example, molecular genetics with natural products, 12 may contribute to solving taxonomic problems in several organisms.