Identification of saturated and unsaturated 1-methoxyalkanes from the Thai millipede Orthomorpha communis as potential “Raincoat Compounds”

Mixtures of saturated and unsaturated 1-methoxyalkanes (alkyl methyl ethers, representing more than 45.4% of the millipede hexane extracts) were newly identified from the Thai polydesmid millipede, Orthomorpha communis, in addition to well-known polydesmid defense allomones (benzaldehyde, benzoyl cyanide, benzoic acid, mandelonitrile, and mandelonitrile benzoate) and phenolics (phenol, o- and p-cresol, 2-methoxyphenol, 2-methoxy-5-methylphenol and 3-methoxy-4-methylphenol). The major compound was 1-methoxy-n-hexadecane (32.9%), and the mixture might function as “raincoat compounds” for the species to keep off water penetration and also to prevent desiccation.

Using gas chromatography coupled with mass spectrometry (GC/MS) analysis of the species, we happened to detect a series of saturated and unsaturated wax-like components, other than the conventional mixtures derived from mandelonitrile. These wax-like components have not been reported in other millipedes, and our hypothesis is that they have a function similar to the "raincoat" found in the oribatid mite, Liacarus subterraneus (Acari: Oribatida). This mite secretes di-glycerides via an esterification reaction between fatty acids and glycerin 5 . As a result, the chemical structures of these saturated and unsaturated wax-like components was investigated. Fig. 2, the GC-profile showed a total of 31 peaks (=compounds) including non-resolved components [2-methoxyphenol (5) and benzoyl cyanide (6), as mentioned later].

GC/MS analyses of the Thai millipede. As shown in
Mass spectra of all peaks are summarized in Table 1, including identified results by co-chromatography with authentic compounds and (or) elucidated structures, as mentioned later.
Chromatographic behavior of waxy compounds on SiO 2 column. When the hexane extracts from seven adult millipedes were separated by silica gel column (300 mg, 0.5φ × 2.1 cm) chromatography, most of the waxy compounds were recovered in the fraction (3 ml) eluted with the 1% Et 2 O in hexane, indicating their non-polar nature (less polar than esters). Methyl ether mixtures prepared from palm oil were also recovered in the same 1% Et 2 O in hexane fractions, as mentioned above. Peak 1 was eluted by 2% Et 2 O in hexane (3 ml) with the remaining waxy compounds. As shown in Fig. 3, peaks 11, 18, 27, 28, and 30 derived from the palm oil were identical to those derived from millipedes using GC t R s and mass spectra (mentioned below).  the same as the spectra for the 1-methoxyalkanes found in palm oil, except for a difference in the integral on long chain methylenes. The following chemical shifts were observed for millipedes; ω-methyl (3H, t, J = 6.6 Hz at δ 0.88 ppm), long chain methylenes (26H, m at δ 1.52-1.60 ppm), -CH 2 -CH 2 -O-(2H, m, at δ 1.52-1.60 ppm), CH 3 -O-(3H, s, at δ 3.33 ppm), and -CH 2 -CH 2 -O (2H, t, J = 6.6 Hz at δ 3.36 ppm), thus, identifying the structure as 1-methoxy-hexadecane. As a result, waxy components from millipede were shown to be a mixture of saturated and unsaturated 1-methoxyalkanes.

Discussion
The present polydesmid millipede has recently revealed the possession of a large amount of waxy compounds (mentioned later) in addition to the conventional chemical defense systems represented by five polydesmid com-pounds{1, 6, 7, 10, and 26, produced by two enzymes; hydroxynitrile lyase (HNL) 11 and mandelonitrile oxidase (MOX) 12 from 10 as the common substrate with subsequent chemical and biochemical reactions} and of six phenolic compounds (2, 3, 5, 8, and 9). Then the species corresponds to one of HCN and H 2 O 2 emitter 13 . The structures of waxy compounds were elucidated and identified as saturated, unsaturated or methyl branched 1-methoxyalkanes (more than 45.4% of total hexane extracts) by NMR and GC/MS spectra. Most of components (86.6% of total ethers) were identified by co-chromatography with authentic compounds prepared from the palm oil, as follows; 11 (0.7%), 16 (2.2%), 18 (72.5%), 23 (0.7%), 27 (2.6%), 28 (7.0%), and 30 (0.9%). Structures of the other ethers (11.6% of total methyl ethers; 12, 13, 14, 17, 19, 21, 22, 24, 25, 29, and 31) were elucidated by calculation using Kovat's retention index as summarized in Table 1. The position of a double bond in 15 (1.1%) and that of methyl residue in 20 (0.9%) remained obscure. Alkyl moieties of methyl ethers are mostly identical to those of long chain fatty acids, and presumably have those fatty acids as a precursor. At present, there are no other polydesmid millipedes known to possess those waxy compounds, such as hydrocarbons and fatty acid esters, nor other related presumably functioning as "raincoat compounds".
As far as our data searches are concerned, no other millipedes including Polydesmida or other animals have been reported to produce 1-methoxyalkanes (methyl alkyl ethers) 8,9 , except for the spider Nephila clavipes [Arachnida: Araneae] 18 . The major components of the spider silk extracted by pentane or methylene chloride consist of a complex mixture of methyl-branched 1-methoxyalkanes [total 51 compounds, all of those contained up to four methyl groups in each carbon chain (chain length between C 28 and C 34 )], together with small amounts of hydrocarbons and alcohols 18 .
In conclusion, 1-methoxyalkanes (a total of 20 compounds of methyl alkyl ethers, a class of compounds) were discovered as components of hexane extracts in the present species. It is only the second example of its presence in the animal kingdom, and it is presumably present on the body surface as a raincoat to keep off water penetration and also to prevent desiccation. The present discovery of unusual 1-methoxyalkanes in this polydesmid millipede might represent evidence of a chemical evolution of the species that adapts and thrives in heavy tropical rainfalls. Further examples of similar species should be expected.

Materials and Methods
Millipedes. The   Hexane extraction of millipedes. Five adult millipedes as a group and also one adult millipede (two times) were dipped into n-hexane (1 ml, separately) for three minutes, using appropriate glass vials (5 ml volume). Then, the resultant hexane extracts after separation from residual millipede bodies by decantation were each subjected to gas chromatography coupled with mass spectrometry (GC/MS) analyses.
Analytical methods. GC/MS spectra were obtained as reported 24 using Hewlett Packard HP 5975 C Inert XL EI/CI MSD with triple-Axis Detector at 75 eV coupled with a 7890 A GC-system equipped with an HP-5 column (30 m × φ 0.25 mm; 0.25 μm in film thickness) operated in the split-less mode at 60 °C for 2 min, then programmed to increase at 10 °C/min to 290 °C, and finally held at this temperature for 5 min. Helium was used as the carrier gas at a flow rate of 1.00 ml/min. GC and GC/MS data were processed using ChemStation (Hewlett Packard Co.), with reference to an MS database (Wiley 9 th /NIST 2011 MS Library; Hewlett Packard Co.). In the case of the non-resolved peaks (5 and 6), relative amounts were calculated by selected ion chromatography (SIC) using each base ion (m/z 105 and 109, respectively). Retention indices 25 were calculated under the same GC conditions mentioned above, as described in Bodner and Raspotnig 26 , and were used for structure elucidation. 1 H-NMR spectra (400 MHz, TMS at δ00.00 as internal standard) were recorded on a Bruker Biospin AC400M spectrometer.
Preparation of standard 1-methoxyalkane mixtures from palm oil. A mixture of alcohols derived from the palm oil by reduction with lithium aluminum hydride were lead to the corresponding mixture of 1-methoxyalkanes using Williamson's ether synthesis (reaction between sodium alcoholate and methyl iodide). After addition of water to dissolve solids, the reaction products were extracted by hexane. The hexane extracts, without concentration, was poured on a silica gel column (Wako Gel C-200, 300 mg, 0.5φ × 2.1 cm in length) prepared without solvent, using a disposable glass pipette (1.5 ml, Iwaki). 1-Methoxyalkanes were obtained as an eluted fraction by 1% ether in hexane.