Bruchins are long-chain a,o-diols, esterified at one or both oxygens with 3-hydroxypropionic acid. They were identified from both cowpea weevils,Callo- sobruchus maculates, and pea weevils, Bruchus pisorum, with the authors reporting these compounds to be the first natural products to induce neoplasm formation applied to intact plants (Doss et al.2000). Extraction and isolation of bruchins was accomplished through bioassay-guided normal and reverse-phase low pressure liquid chromatography. Final separation from inactive fatty acids was achieved through reaction with 2-bromoacetophenone. Alternatively, HPLC was also used instead of low pressure liquid chromatography. Prior to analysis, further micro- chemistry was applied, with compounds being hydrolysed and converted to tri- methylsilylethers using BSTFA, and subjected to ozonolysis. Synthesis of bruchins was accomplished by standard routes involving acetylene alkylations and semihy- drogenations and/or Wittig condensations. The (3-hydroxypropyl) esters were initially prepared by oxidative desilylation of 3-(phenyldimethylsilyl) propionates as described for bruchin A ((Z)-9-docosene-1,22-diol, 1-(3-hydroxypropanoate) ester) (Oliver et al. 2000). Initially, 9-decyn-1-ol was deprotonated with butyl- lithium in THF and alkylated with 12-bromododecanol THP ether. The product was hydrogenated using Lindlar catalyst and the olefinic alcohol esterified with the acid chloride obtained by treating 3-(phenyldimethylsilyl) propanoic acid with oxalyl chloride. Removal of the THP group and treatment of the resulting monoester with fluoroboric acid etherate in dichloromethane, followed by flash chromatography yielded the mono 3-(fluorodimethylsilyl)propanoate. Stirring in methanol–THF containing sodium bicarbonate, potassium fluoride and hydrogen peroxide, fol- lowed by flash chromatography, yielded the desired Bruchin A.
3.2.2 Volicitin and Related Compounds
The oral secretion of beet armyworm caterpillars (BAW), Spodoptera exigua, when applied to damaged tissues of maize, induces the production of VOCs that attract the natural enemies of the caterpillars. Alborn et al. (1997,2000) and Turlings et al. (2000) reported the identification of the key elicitor present in BAW oral secretions asN-[17-hydroxylinolenoyl]-Lglutamine (volicitin). Analysis of the oral secretion showed that it also containedN-[17-hydroxyolinoleoyl]-L-glutamine, free
17-hydroxylinolenic and 17-hydroxylinoleic acids, the glutamine conjugates of linolenic and linoleic acid as well as free linolenic and linoleic acid. Isolation of the active components included initial centrifugation, filtration of the supernatant and precipitation of proteinaceous material by treatment with citric acid, fol- lowed by SPE and further fractionation using reverse-phase HPLC. At each stage, extracts and fractions were tested for biological activity by addition to Z. mays plants in water and monitoring VOC production and parasitoid wind tunnel bioassays. Final purification was achieved using further SPE. Characterisa- tion of volicitin was achieved through fast atom bombardment mass spectroscopy (FABMS) and FABMSMS, giving information on the molecular weight, and revealing the possible presence of a glutamine unit. Acid methanolysis followed by GC-CI-MS confirmed the presence of glutamine. GC-EI-MS suggested a straight-chain unsaturated hydrocarbon, consistent with a methyl ester of an 18- carbon hydroxy acid.
Microhydrogenation of the methyl ester over PdO/H2, followed by GC-MS
indicated that more than 1 double bond was present in the side chain. GC-FTIR confirmed the presence of a hydroxyl group, indicated non-conjugation in the unsaturated side chain, and no presence oftrans double bonds. The methyl ester of the hydroxy C18 acids was subjected to further microdegradative analysis to
determine the positions of the double bonds and the hydroxyl group. Partial reduction resulted in both cases in a mixture of monoand diunsaturated products as established by GC-MS analysis. The mixtures were then ozonised, with GC-CI- MS analysis showing the presence of three diagnostic GC peaks, which was similar to that for methyl linolenate. EI mass spectra of a pyrrolidide derivative of the reduced products confirmed the C-17 location of the hydroxyl group. Alborn et al. (2000) synthesised racemic 17-hydroxylinolenic acid starting from the ethoxyethyl ester of 3,6-heptadiyn-1-ol, followed by coupling with thep-nitrobenzyl ester of L-glutamine using a method developed for peptide synthesis. Since the initial identification of volicitin, synthesis has enabled the elucidation of the absolute stereochemistry of volicitin (Sawada et al.2006; Pohnert et al.1999b).
Following the initial reports of volicitin as an insect-derived elicitor from S. exigua (Alborn et al. 1997), further fatty acid – amino acid conjugates were identified from the oral secretions of other freshly harvested Lepidopteran species by Pohnert et al. (1999a) using an APCI LC-MS method to analyse oral secretions. The compounds present in regurgitates were identified as a structurally diverse group of conjugates of glutamine and glutamic acid linkedvia an amide bond to saturated and unsaturated C14, C16 and C18 fatty acids, with proportions being
species specific. Dihydroxy and epoxy fatty acid – glutamine conjugates were later isolated from the regurgitant ofS. exigua and S. frugiperda, using LC-MS, in conjunction with methanolysis and derivatisation with MSTFA to determine the positions of the hydroxy groups by GC–MS (Spiteller and Boland 2003). The synthesis of volicitin and analogues has since been published in a number of studies (see for example Hansen and Stenstrom2000; Itoh et al.2002; Wei et al.2003; Krishnamachari et al.2007), which highlights its suitability as a natural product target for synthesis chemists.
3.2.3 Caeliferins
Caeliferins were isolated from the regurgitant of the grasshopper, Schistocerca americana (Alborn et al.2007). These novel natural products comprise saturated and unsaturated sulphated a-hydroxy fatty acids in which the o carbon is functio- nalised with either a sulphated hydroxyl group or a carboxyl conjugated with a glycine unit via an amide bond. The predominant compound possessed a 16-carbon chain and appeared to have most biological activity. Isolation and identification were achieved in an analogous manner to that of volicitin, but with negative and positive ion electrospray LC/MS and LC-MSMS used as the soft ionisation tech- nique rather than FABMS to provide initial information of the molecular mass. GC-CI-MS analyses of methanolysed material suggested methyl esters, and GC-EI- MS indicated two alcohols and a carboxylic acid methyl ester. On the basis of MS data and NMR analyses of intact molecules, compounds were shown to be 2, 16- dihydroxy C16fatty acids with the addition of two unknown 80-amu groups. The
only difference between the two compounds was the presence of a double bond, explaining its weak UV absorption. To test the one double-bond hypothesis, the methyl ester was subjected to hydrogenation, which, as expected, gave a GC/MS peak identical to that of the other methyl ester. The presence of two alcohols was confirmed by acetylation that resulted in the expected increase in molecular weight. Finally, the presence of only one carboxylic acid was confirmed by ethanolysis and GC-CI-MS that, for both compounds, gave an ethyl ester with Mþ 1 ions 14 amu higher than for the corresponding methyl esters. GC-CI-MS analyses of ozonised acetylated methyl ester confirmed the double bond was located between carbon 6 and 7 in the 16-carbon chain. GC-FTIR confirmed two non-identical alcohols and the presence of atrans double bond. NMR analysis of the original intact material did not indicate the presence of any other organic structure than a di-O(H) substituted C16fatty acid. The consecutive loss of 80 amu in LC/MS analyses was explained by
the loss of sulphate esters, which also explained the high water solubility of the natural products. Thus, two compounds were identified as 2, 16-disulfooxy-(E)-6- hexadecenoic acid and 2, 16-disulfooxyhexadecanoic acid, which were named caeliferin A16:1and caeliferin A16:0, respectively. Both proposed (racemic forms of) dihydroxy acids were synthesised and transformed to disulfate esters.