A promiscuous fatty acid ω-hydroxylase CYP94A90 is likely to be involved in biosynthesis of a floral nitro compound in loquat (Eriobotrya japonica).

Nitro groups are often associated with synthetically manufactured compounds such as medicines and explosives, and rarely with natural products. Loquat emits a nitro compound, (2-nitroethyl)benzene, as a flower scent. The nitro compound exhibits fungistatic activity and is biosynthesised from l-phenylalanine via (E/Z)-phenylacetaldoxime. Although aldoxime-producing CYP79s have been intensively studied, it is unclear what enzymes form nitro groups from aldoximes either in plants or in other organisms. Here, we report the identification of two cytochrome P450s that are likely to be involved in (2-nitroethyl)benzene biosynthesis in loquat through differential gene expression analysis using RNA-seq and functional identification using yeast and tobacco. CYP79D80 and CYP94A90 catalysed the formation of (E/Z)-phenylacetaldoxime from l-phenylalanine and (2-nitroethyl)benzene from the aldoxime, respectively. Expression profiles of CYP79D80 and CYP94A90 were correlated with the emission of (2-nitroethyl)benzene from loquat flowers. CYP94A90 also functioned as a fatty acid ω-hydroxylase as do other CYP94A fatty acid ω-hydroxylases. The CYP94As tested from other plants were all found to catalyse the formation of (2-nitroethyl)benzene from (E/Z)-phenylacetaldoxime. CYP79D80 and CYP94A90 are likely to operate in concert to biosynthesise (2-nitroethyl)benzene in loquat. CYP94A90 and other CYP94As are 'promiscuous fatty acid ω-hydroxylases', catalysing the formation of nitro groups from aldoximes, and are widely distributed in dicot plants.

Biosynthesis of (2-nitroethyl)benzene and (Z)- and (E)-(2-nitroethenyl)benzenes from (Z)- and (E)-phenylacetaldoximes and phenylacetonitrile; defense allomone of Eutrichodesmus elegans and Eutrichodesmus armatus (Polydesmida: Haplodesmidae).

The defense allomones of two haplodesmid millipedes, Eutrichodesmus elegans and E. armatus (Polydesmida: Haplodesmidae), are known as a mixture of the following three nitro compounds: (2-nitroethyl)benzene and (Z)- and (E)-(2-nitroethenyl)benzenes. Administrations of a mixture of 2H-labeled (Z)- and (E)-phenylacetaldoximes and of 2H-labeled phenylacetonitrile as precursors resulted in the same production of three 2H-labeled nitro compounds, [2'-nitroethyl][2,3,4,5,6-2H5]benzene and [(Z)- and (E)-2'-nitroethenyl][2,3,4,5,6-2H5]benzenes, in both species. Oxime administration at an appropriate dose resulted in the production of three nitro compounds with similar natural ratios more effectively than nitrile administration. Conversion from oximes to nitrile and vice versa was evidenced during administration. Occurrences of three precursors (Z- and E-oximes and nitrile) were detected sporadically in millipede extracts by selected ion chromatography.

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.

Generation of (2-Nitroethyl)benzene and related benzenoids from L-Phenylalanine; flower scents of the Japanese Loquat Eriobotrya japonica [Rosales: Rosaceae].

(2-Nitroethyl)benzene, methyl 4-methoxybenzoate and 4-methoxybenzaldehyde have been known as major scent components in flowers of the Japanese loquat Eriobotrya japonica [Rosales: rosaceae], together with 13 related benzenoids, including Z- and E-2-phenylacetaldoxime and benzyl alcohol. The scents air-trapped from a flowering panicle during 24 h incubation with d8-L-phenylalanine were composed of 15 deuterium labeled compounds {d6-styrene, d5-benzaldehyde, d7-2-phenylacetaldehyde, methyl d5-benzoate, d7 -2-phenylethanol, d7-2-phenylacetonitrile, d4-1,4-dimethoxybenzene, d7-Z-2-phenylacetaldoxime, d4-4-methoxybenzaldehyde, d7-E-2-phenylacetaldoxime, d4-4-methoxybenzyl alcohol, d7-(2-nitroethyl)benzene, methyl d4-4-methoxybenzoate, methyl d6-cinnamate and ethyl d4-4-methoxybenzoate}. On the other hand, hexane extracts of the flower petal incubate with a mixture of d5-Z- and d5-E-2-phenylacetaldoxime after 24 h indicated generation of six d5-labeld components {d5-benzaldehyde, d5-benzyl alcohol, d5-2-phenylacetaldehyde, methyl d5-benzoate, d5-2-phenylethanol, and d5-(2-nitroethyl)benzene}. By comparing those results, (2-nitroethyl)benzene was concluded as a product directly generated from a mixture of Z- and E-2-phenylacetaldoxime together with six minor benzenoids, while two major compounds (4-methoxybenzaldehyde and methyl 4-methoxybenzoate) together with three minors from L-phenylalanine, presumably via L-tyrosine. The other two minor components were derived from L-phenylalanine.

Hydrogen peroxide as a new defensive compound in "benzoyl cyanide" producing polydesmid millipedes.

Hydrogen peroxide was newly and simultaneously demonstrated with well-known hydrogen cyanide as a component of defensive secretions of "benzoyl cyanide" producing polydesmid millipedes. Presence of hydrogen peroxide was successively evidenced by Trinder reagent's spray with colorless as well as oily smears of defensive secretions containing benzoyl cyanide and hydrogen cyanide by alkaline picrate paper treatment. Linear correlation was demonstrated between quantities of hydrogen peroxide and benzoyl cyanide. By qualitative assay, seven benzoyl cyanide containing polydesmidans (six species of adults and one species of a nymph at stadium I) tested positive to Trinder reagent, indicative of the presence of hydrogen peroxide (together with hydrogen cyanide), while two cyanogenic species without benzoyl cyanide exhibited negative responses to the reagent. Two types of millipedes were elucidated as species of cyanogenic Polydesmida.

A novel cytochrome P450, CYP3201B1, is involved in (R)-mandelonitrile biosynthesis in a cyanogenic millipede.

Specialized arthropods and more than 2500 plant species biosynthesize hydroxynitriles and release hydrogen cyanide as a defensive mechanism. The millipede Chamberlinius hualienensis accumulates (R)-mandelonitrile as a cyanide precursor. Although biosynthesis of hydroxynitriles in cyanogenic plants and in an insect are extensively studied, (R)-mandelonitrile biosynthesis in cyanogenic millipedes has remained unclear. In this study, we identified the biosynthetic precursors of (R)-mandelonitrile and an enzyme involved in (R)-mandelonitrile biosynthesis. Using deuterium-labelled compounds, we revealed that (E/Z)-phenylacetaldoxime and phenylacetonitrile are the biosynthetic precursors of (R)-mandelonitrile in the millipede as well as other cyanogenic organisms. To identify the enzymes involved in (R)-mandelonitrile biosynthesis, 50 cDNAs encoding cytochrome P450s were cloned and coexpressed with yeast cytochrome P450 reductase in yeast, as cytochrome P450s are involved in the biosynthesis of hydroxynitriles in other cyanogenic organisms. Among the 50 cytochrome P450s from the millipede, CYP3201B1 produced (R)-mandelonitrile from phenylacetonitrile but not from (E/Z)-phenylacetaldoxime, whereas plant and insect cytochrome P450s catalysed the dehydration of aldoximes and hydroxylation of nitriles. CYP3201B1 is not phylogenetically related to cytochrome P450s from other cyanogenic organisms, indicating that hydroxynitrile biosynthetic cytochrome P450s have independently evolved in distant species. Our study will shed light on the evolution of cyanogenesis among plants, insects and millipedes. DATABASE: Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers LC125356-LC125405.

Biosynthetic pathway of aliphatic formates via a Baeyer-Villiger oxidation in mechanism present in astigmatid mites.

Astigmatid mites depend on bioactive glandular secretions, pheromones, and defensive agents to mediate intra- and interspecies interactions. Aliphatic formates, such as (Z,Z)-8,11-heptadecadienyl formate (8,11-F17) and (Z)-8-heptadecenyl formate (8-F17), are rarely encountered natural products that are abundant in Sancassania sp. Sasagawa (Acari: Acaridae) mite secretions. Linoleic acid and oleic acid are predicted as key intermediates in the synthesis of the closely related aliphatic formates. To gain insight in this biosynthetic pathway, acarid mite feeding experiments were conducted using 13C-labeled precursors to precisely track incorporation. Analyses using 13C NMR spectroscopy demonstrated that the 13C-labeling pattern of the precursors was detectable on formates in exocrine secretions and likewise on fatty acids in total lipid pools. Curiously, the results demonstrated that the formates were biosynthesized without the dehomologation of corresponding fatty acids. Careful examination of the mass spectra from labeling experiments revealed that the carbonyl carbon of the formates is originally derived from the C-1 position of the fatty acids. Consistent with a Baeyer-Villiger oxidation reaction, labeling studies support the insertion of an oxygen atom between the carbonyl group and carbon chain. Empirical data support the existence of a Baeyer-Villiger monooxygenase responsible for the catalyzation of the Baeyer-Villiger oxidation. The predicted existence of a Baeyer-Villiger monooxygenase capable of converting aliphatic aldehydes to formates represents an exciting opportunity to expand the enzymatic toolbox available for controlled biochemical synthesis.

A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase.

Soldiers of some eusocial insects exhibit an altruistic self-destructive defense behavior in emergency situations when attacked by large enemies. The swarm-forming invasive millipede, Chamberlinius hualienensis, which is not classified as eusocial animal, exudes irritant chemicals such as benzoyl cyanide as a defensive secretion. Although it has been thought that this defensive chemical was converted from mandelonitrile, identification of the biocatalyst has remained unidentified for 40 years. Here, we identify the novel blood enzyme, mandelonitrile oxidase (ChuaMOX), which stoichiometrically catalyzes oxygen consumption and synthesis of benzoyl cyanide and hydrogen peroxide from mandelonitrile. Interestingly the enzymatic activity is suppressed at a blood pH of 7, and the enzyme is segregated by membranes of defensive sacs from mandelonitrile which has a pH of 4.6, the optimum pH for ChuaMOX activity. In addition, strong body muscle contractions are necessary for de novo synthesis of benzoyl cyanide. We propose that, to protect its swarm, the sacrificial millipede also applies a self-destructive defense strategy-the endogenous rupturing of the defensive sacs to mix ChuaMOX and mandelonitrile at an optimum pH. Further study of defensive systems in primitive arthropods will pave the way to elucidate the evolution of altruistic defenses in the animal kingdom.

Identification and Synthesis of (Z,Z)-8,11-Heptadecadienyl Formate and (Z)-8-Heptadecenyl Formate: Unsaturated Aliphatic Formates Found in the Unidentified Astigmatid Mite, Sancassania sp. Sasagawa (Acari: Acaridae).

We identified two aliphatic formates, (Z,Z)-8,11-heptadecadienyl formate and (Z)-8-heptadecenyl formate in the opisthonotal gland secretions of an unidentified acarid species, namely Sancassania sp. Sasagawa. Both compounds were isolated using silica gel column chromatography and the structures were elucidated by ¹H-NMR and GC/FT-IR. Further information on the double bond positions was obtained by GC-MS analysis of the corresponding dimethyl disulfide derivatives. Based on the estimated structures of the two formates and using linoleic and oleic acids as the respective starting materials, a simple four-step synthesis was achieved via Barton decarboxylation as the key step. The aliphatic formates identified in acarids thus far are neryl formate ((Z)-3,7-dimethylocta-2,6-dienyl formate) and lardolure (1,3,5,7-tetramethyldecyl formate), and both have been reported to have pheromone functions. The biological function of the two formates isolated in this study is currently being investigated. Although we can speculate that the two compounds were biosynthesized from linoleic and oleic acid, there is a possibility that the synthetic processes featured a novel chain shortening and formic acid esterification mechanism.

Chemical polymorphism in defense secretions during ontogenetic development of the millipede Niponia nodulosa.

A mixture of defense compounds (benzaldehyde, benzoyl cyanide, benzoic acid, mandelonitrile, and mandelonitrile benzoate), found commonly in cyanogenic polydesmid millipedes, was identified in the non-cyanogenic millipede Niponia nodulosa. These compounds were major components in 1st-4th instars, but were absent in older instars and adults. Extracts of older instars and adults contained 1-octen-3-ol, 2-methyl-2-bornene, E-2-octen-1-ol, 2-methyl-isoborneol, and geosmin; these compounds were minor components in 1st-4th instars. This ontogenetic allomone shift may be explained by the high cost of biosynthesis of polydesmid compounds from L-phenylalanine being offset by their potency in protecting the insect during fragile and sensitive growth stages. However, as the cuticle hardens in older juveniles (5th, 6th, 7th instars) and adults, this allows for a switch in defense to using less effective and less costly volatile organic compounds (presumably microbial in origin) that are ubiquitous in the millipede's habitat or are produced by symbiotic microbes and may be readily available through food intake or aspiration.

(2-Nitroethyl)benzene: a major flower scent from the Japanese loquat Eriobotrya japonica [Rosales: Rosaceae].

(2-Nitroethyl)benzene was identified as a major component of the flower scent of the Japanese loquat Eriobotrya japonica [Rosales: Rosaceae], together with p-methoxybenzaldehyde and methyl p-methoxybenzoate. The corresponding volatiles from chopped leaves did not contain these three compounds. This is the first time that 1-nitro-2-phenyl-ethane has been demonstrated to be a natural product among Japanese plants, although two Japanese millipedes are known to possess the same aromatics.

De novo biosynthesis of linoleic acid and its conversion to the hydrocarbon (Z,Z)-6,9-heptadecadiene in the astigmatid mite, Carpoglyphus lactis: incorporation experiments with 13C-labeled glucose.

De novo biosynthesis of linoleic acid (LA) and its conversion to (Z,Z)-6,9-heptadecadiene were examined in Carpoglyphus lactis (Acarina, Carpoglyphidae). Experiments involving (13)C-administration using [1-(13)C]-d-glucose revealed that (13)C atoms were incorporated into LA of total lipid extracted from the mite, resulting in labeling of all even-numbered carbons. This result demonstrated that LA was produced from (13)C-labeled acetyl-CoA, which is indicative of direct de novo biosynthesis. In these feeding experiments involving [1-(13)C]-D-glucose, (13)C atoms were also incorporated into (Z,Z)-6,9-heptadecadiene, which is one of the major secretory components in the mite. The labeling pattern of (Z,Z)-6,9-heptadecadiene at odd-numbered carbons agreed well with that of LA after loss of the carboxyl carbon. It was concluded that the mites could stably convert LA into (Z,Z)-6,9-heptadecadiene without the dietary requirement of this essential fatty acid.

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae).

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d31-hexadecanoic acid (16:0), d27-octadecadienoic acid (18:2), produced from d31-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d27-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.

Chemical ecology of astigmatid mites LXXXVII. S-(+)-isopiperitenone: re-identification of the alarm pheromone as the female sex pheromone in Tyrophagus similis (Acari: Acaridae).

Behavioral analysis revealed that S-(+)-isopiperitenone [(S)-3-methyl-6-isopropenyl-2-cyclohexen-1-one], previously identified as an alarm pheromone, is also the female sex pheromone of Tyrophagus similis (Astigmata: Acaridae), showing maximum male attraction at a dose of 0.1 female equivalent. Although the antipode, R-(-)-isopiperitenone, was not detectable in the mite extract, this synthetic optical isomer (80% e.e.) also induced activity at a dose of 100 ng, a response indicative of S-(+)-isopiperitenone being the active compound. The average content was determined to be 38.5 ng per female and 19.8 ng per male. This is the first example of an astigmatid mite species possessing a compound that functions as an alarm as well as a sex pheromone.

The absolute configuration of chrysomelidial: a widely distributed defensive component among oribotririid mites (Acari: Oribatida).

The absolute configuration of the iridoid monoterpene chrysomelidial from the oribatid mite, Austrotritia dentate Aoki, was elucidated by the GC-MS and GC comparisons with four synthetic stereoisomers of this well-known natural product. This identification was made possible by asymmetric synthesis of the known alcohol, (5S,8S)-chrysomelidiol. The GC retention time of diol derived from the natural oribatid dial agreed with that of the synthetic (5S,8S)-chrysomelidiol, confirming that the absolute configurations at C5 and C8 positions of the natural chrysomelidial are both S. Chrysomelidial was detected as a single or a major component in nine oribatid mites examined; thus, this compound is considered to be commonly distributed in Oribotririidae where it serves a defensive role.

n-Hexyl laurate and fourteen related fatty acid esters: new secretory compounds from the julid millipede, Anaulaciulus sp.

A total of fifteen saturated fatty acid esters were newly identified from the secretions of an unidentified Anaulaciulus sp. (Julida: Julidae). The fatty acid components of the esters were composed of normal chain acids (from C(10) to C(14)) and of branched chain acids (from iso-C(12) to iso-C(15) and anteiso-C(15)). The alcohol moieties were all composed of normal chain alcohols varying from n-butanol to n-octanol. The most abundant component found in the total esters was n-hexyl laurate (64.7%). Novel compounds identified from the millipede secretion extracts include six branched iso- and anteiso-fatty esters, an odd-numbered C(11)-fatty acid ester, a C(13)-fatty acid ester, and a C(7)-alcohol ester, all of which were previously undescribed natural products. In addition, a characteristic mixture of benzoquinones, such as 2-methyl-1,4-benzoquinone, 2-methoxy-3-methyl-1,4-benzoquinone, 2,3-dimethoxy-1,4-benzoquinone, 2-methoxy-6-methyl-1,4-benzoquinone, and 2,3-dimethoxy-5-methyl-1,4-benzoquinone were identified from the secretions, together with trace amounts of 1,4-benzoquinone.

Geraniol, E-3,7-dimethyl-2,6-octadien-1-ol, as the alarm pheromone of the sycamore lace bug Corythucha ciliata (Say).

Although adult sycamore lace bugs Corythucha ciliata (Say) show no sign of aggregation, nymphs at all developing stages are gregarious. When an individual nymph in the center of a colony was squashed with a needlepoint, proximate nymphs showed evasive behavior. The same evasive reaction was produced by exposing aggregated nymphs to nymph hexane extract. The active component, E-3,7-dimethyl-2,6-octadien-1-ol, geraniol, was responsible for the evasive behavior, and identified as the alarm pheromone. One nanogram of the alarm pheromone elicited activity similar to that in a third instar nymph. Presence of 2-acylcyclohexane-1,3-diones and their 4-hydroxy analogues are reconfirmed as nymph-specific components, though their biological significance remains unknown.

Release of hydrogen cyanide via a post-secretion Schotten-Baumann reaction in defensive fluids of polydesmoid millipedes.

Mandelonitrile benzoate, a minor defense component produced by polydesmoid millipedes, is produced in large amounts together with hydrogen cyanide following shake-disturbances administered to individuals of Nedyopus tambanus tambanus, Parafontaria tonominea, Epanerchodus sp., and Epanerchodus fulvus. These species commonly produce mandelonitrile and benzoyl cyanide (the oxidized product after discharge). The newly generated mandelonitrile benzoate was identified as a product of post secretion Schotten-Baumann reaction under basic conditions of bled bodily fluids (pH ca. 9.0), and was not an enzymatic reaction product. The reaction occurred in vitro even under less basic conditions [1M Tris-HCl buffer (pH 8.0)], and could be defined as a new mechanism of hydrogen cyanide release occurring in roughly half of polydesmoid millipedes. Species possessing no benzoyl cyanide, such as Oxidus gracilis and Cryptocorypha sp., could also produce mandelonitrile benzoate under conditions in which benzoyl cyanide was exogenously provided.

Stereochemistry of female-specific normonoterpenes, sex pheromone candidates from the acarid mite, Tyreophagus sp. (Astigmata: Acaridae).

Two normonoterpenes were detected from an unidentified Tyreophagus sp. as new female-specific components. Both planar structures were identified to be 2,6-dimethyl-5-heptenal (1) and 2,6-dimethyl-5-hepten-1-ol (2) by GC/MS co-chromatography with synthetic 1 and 2. The stereochemistry of 2 was determined to be R by a GC analysis with a chiral column, while that of 1 was presumed to be similar to 2 based on the biosynthetic aspects.

Syntheses of bioactive bisabolane-type Cryptomeria japonica sesquiterpenes.

The first diastereoselective synthesis of (1S,6R)-1-hydroxy-2,7(14),10-bisabolatrien-4-one, an antifeedant against Acusta despesta and Locusta migratoria, was produced from Cryptomeria japonica (commonly known as Japanese cedar), starting from (R)-(-)-carvone via (R)-(-)-cryptomerione. The enantiomer was transformed into (1S,3R,6R)-1-hydroxy-7(14),10-bisaboladien-4-one, a novel antifeedant against L. migratoria from the same tree, by 1,4-selective reduction of the enone moiety.