Biosynthesis of avenanthramides in suspension cultures of oat (<Emphasis Type="Italic">Avena sativa</Emphasis>)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.     

Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.     

Identification of antioxidant phenolic compounds in feverfew (Tanacetum parthenium) by HPLC-ESI-MS/MS and NMR

Antioxidant polyphenolic acids in the medicinal herb feverfew (Tanacetum parthenium) were isolated through in vitro bioassay-orientated antioxidant tests in response to 1,1-diphenyl-2-picrylhydrazyl (DPPH*) free radical scavenging and Fe(2+)-chelating activities. Purification of the active compounds and their structural elucidation involved a variety of techniques including open-column chromatography, HPLC, GC-MS, LC-MS and NMR. Major compounds with potent DPPH* scavenging activities were characterised as 3,5-, 4,5- and 3,4-di-O-caffeoylquinic acids (DCQAs). This is the first report of DCQAs found in feverfew.     

Induction of biosynthetic enzymes for avenanthramides in elicitor-treated oat leaves

The accumulation of oat (Avena sativa L.) phytoalexins, avenanthramides, occurred in leaf segments treated with oligo-N-acetylchitooligosaccharides. The amount of avenanthramide A, the major oat phytoalexin, reached a maximum 36–48 h after elicitor treatment. This accumulation was preceded by a marked increase in enzyme activities of phenylpropanoid pathway members, including phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamate 4-hydroxylase (EC 1.14.13.11) and 4-coumarate:CoA ligase (EC 6.2.1.12). These enzyme activities reached a maximum 6–12 h after elicitor treatment, when the avenanthramides were produced most rapidly. Both phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities decreased thereafter to undetectable levels 72 h after treatment, while cinnamate 4-hydroxylase activity showed a second increase 48 h after treatment. Among the chitooligosaccharides tested, tetra- and pentasaccharides most effectively induced these enzyme activities in a dose-dependent manner. The elicitor-induced 4-coumarate: CoA ligase accepted all hydroxycinnamic acids occurring in the avenanthramides as substrates, with the exception of avenalumic acid. These findings indicate that accumulation of the avenanthramides results from de-novo synthesis through the general phenylpropanoid pathway and that early biosynthetic enzymes function as regulatory points of carbon flow to the avenanthramides. Received: 3 December 1998 / Accepted: 27 January 1999     

Metabolism of avenanthramide phytoalexins in oats

Oat leaves produce phytoalexins, avenanthramides, in response to infection by pathogens or treatment with elicitors. The metabolism of avenanthramides was investigated using low molecular weight, partially deacetylated chitin as an elicitor. When oat leaf segments are floated on the elicitor solution, avenanthramides accumulate in the solution. The transfer of elicited oat leaves to solutions containing stable-isotope-labeled avenanthramides resulted in a rapid decrease in the labeled avenanthramides, suggesting the metabolism of avenanthramides. The rate of decrease was enhanced by elicitor treatment, and was dependent on the species of avenanthramides, with avenanthramide B decreasing most rapidly. The rates of biosynthesis and metabolism of avenanthramides A and B were measured using a model of isotope-labeling dynamics. Avenanthramide B was found to be more actively biosynthesized and metabolized than avenanthramide A. Radiolabeled avenanthramide B was incorporated into the cell wall fraction and 99% of incorporated activity was released by alkaline treatment. Gel filtration indicated that high-molecular-weight compounds derived from avenanthramide B were released by alkaline treatment. The decrease in stable-isotope-labeled avenanthramides was suppressed by catalase, salicylhydroxamic acid, and sodium ascorbate, suggesting the involvement of peroxidase in the metabolism. Consistent with this, peroxidase activity that accepts avenanthramide B as a substrate was induced in apoplastic fractions by elicitor treatment. The appearance of multiple basic isoperoxidases was observed by activity staining with 3-amino-9-ethylcarbazole coupled with isoelectric focusing of proteins from elicitor-treated leaves. These findings suggest that accumulated avenanthramides are further metabolized in apoplasts in oat leaves by inducible isoperoxidases.     

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

QSAR study on inhibition of brain 3-hydroxy-anthranilic acid dioxygenase (3-HAO): A molecular connectivity approach

The ability of 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3-hydroxyanthranilic acid derivatives to reduce the production of the excitotoxin quinolinic acid (QUIN) by inhibition of brain 3-hydroxyanthranilic acid dioxygenase (3-HAO) has been investigated using molecular connectivity indices (⁰χ^v, ¹χ^v, ²χ^v). The in-vivo inhibition of 3-HAO in rat cortex (pIC₅₀, nM) is used for this purpose. The regression models obtained suggest that the degree of branching of the compounds under study have a dominant role in the observed inhibition potency. The data were used to generate quantitative structure–activity relationship (QSAR) models for estimating the potency of 3-HAO. The information obtained from the correlation should be useful in designing more potent analogues.     

Purification and identification of antiviral components from Laggera pterodonta by high-speed counter-current chromatography

Although Laggera pterodonta, a folk medicine has been widely used for several centuries as an antiviral agent, there have been no studies to identify its antiviral components. A bioassay-guided phytochemical examination of L. pterodonta disclosed that its aqueous extract, which was made up of three dicaffeoylquinic acids showed significant inhibitory activity of virus replication. Then a simple and efficient preparative high-speed counter-current chromatography (HSCCC) method was successfully established by using ethyl acetate-n-butanol-water (3:2:5, v/v) as the two-phase solvent system to isolate and purify three bioactive dicaffeoylquinic acids, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid. The flow rate was 1.5 ml/min and revolution speed was 800 rpm. The isolation was done in less than 6h, and 34.6 mg of 3,5-O-dicaffeoylquinic acid, 29.4 mg of 3,4-O-dicaffeoylquinic acid and 27.1mg of 4,5-O-dicaffeoylquinic acid was yielded from 600 mg of the crude sample in one-step separation with the purity of 98.3%, 96.7% and 96.2%, respectively, as determined by high-performance liquid chromatography (HPLC). The structures of these three bioactive dicaffeoylquinic acids were identified by ultraviolet (UV), electrospray ionization mass spectrometry (ESI-MS), proton nuclear magnetic resonance ((1)H NMR) and carbon-13 nuclear magnetic resonance (13C NMR). In the antiviral experiment, three dicaffeoylquinic acids all showed significant inhibitory activity against herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2) and influenza viruses A (IVA) in vitro with high selectivity indexes. However, among the three compounds, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid were the more active than 3,4-O-dicaffeoylquinic acid against HSV-1, HSV-2 and IVA. This study demonstrated a direct link between the antiviral activity of L. pterodonta and the three dicaffeoylquinic acids.     

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.     

Variation in Phenolic Content,Profile, and Antioxidant Activity of Seeds among Different Paeonia ostii Cultivated Populations in China

The purpose of this study was to analyze the phenolic profiles of seeds from fifteen Paeonia ostii cultivated populations in China and identify their relationship with antioxidant activities and associated environmental factors. Thirteen individual phenolic compounds were quantitatively determined by HPLC, and (+)‐catechin was the most abundant phenolic compound in the seeds. Correlation analysis showed that phenolics were the most effective antioxidant compound class by evaluating DPPH, ABTS, and hydroxyl radical scavenging activities as well as ferric reducing antioxidant power. Latitude and annual rainfall had significant effects on the contents of many phenolic compounds, and elevation was only significantly correlated with gallic acid content. Within fifteen P. ostii cultivated populations, the seeds of Tongling population exhibited the highest phenolic contents and strongest antioxidant activities. These results suggest that Tongling population has a relatively high utilization value and a potential for sources of natural antioxidants.     

High-resolution spatial and temporal analysis of phytoalexin production in oats

The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antioxidant properties ofErigeron annuus extract and its three phenolic constituents

The antioxidant activity of the extract ofErigeron annuus was assessed by means of two differentin vitro tests: bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test) and the scavenging of authentic peroxynitrite in company with peroxynitrite generation from 3-morpholinosydnonimine (SIN-1). In both tests, the 85% aq. MeOH andn-BuOH soluble fractions of the crude extract showed a significant scavenging effect on peroxynitrite and DPPH radical in comparison to L-ascorbic acid. And bioassay-guided fractionation of then-BuOH soluble fraction led to the isolation of three compounds: Apigenin (1), quercetin-3-O-glucoside (2), and caffeic acid (3). The structures of the isolated compounds were elucidated on the basis of their spectroscopic data and their antioxidant activities were measured by determining their capacity to scavenge peroxynitrite and the DPPH radical.     

Synthesis,antioxidative and antiviral activity of hydroxycinnamic acid amides of thiazole containing amino acid

The synthesis and the biological (antioxidant and antiviral) activities of novel hydroxycinnamic acid amides of a thiazole containing TFA.valine-4-carboxylic acid ethyl ester are reported. The amides have been synthesized from p-coumaric, ferulic and sinapic acids with the corresponding TFA.valine-thiazole-4-carboxylic acid ethyl ester using the coupling reagent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 4-(dimethylamino) pyridine (DMAP) as a catalyst. The antioxidant properties of the newly synthesized amides have been studied for then antioxidative activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH)* test. The newly synthesized compounds have been tested against the replication in vitro of influenza virus A (H3N2) and human herpes virus 1 and 2 (HSV-1 and HSV-2).     

Seed Polyphenolic Profile,Antioxidative Activity,and Fatty Acids Composition of Wild and Cultivated Carthamus Species

Total flavonoid content (TFC) and cyanidin‐3‐glucoside (Cyd‐3‐glu) of seed and seed coat extract of 16 genotypes from five species of Carthamus were studied, and their major polyphenolic compounds and antioxidant activity of the seed coat extracts were determined using HPLC analysis and DPPH assay, respectively. Additionally, fatty acids composition of the seed oil was analyzed by GC. In general, TFC and Cyd‐3‐glu content of seed coat extracts were higher than those of seed extracts. A novel breeding line with black seed coat (named A82) depicted lower TFC (3.79 mg QUE/g DW) but higher Cyd‐3‐glu (24.64 mg/g DW) compared to the white and other seed‐pigmented genotypes. DPPH radical scavenging activity showed a strong association with Cyd‐3‐glu content (r = 0.84), but no correlation with TFC (r = ?0.32). HPLC analysis of seed coat extracts revealed that four compounds were dominant constituents including rutin (7.23 – 117.95 mg/100 g DW), apigenin (4.37 – 64.88 mg/100 g DW), quercetin (3.09 – 14.10 mg/100 g DW), and ferulic acid (4.49 – 30.41 mg/100 g DW). Interestingly, the genotype A82 with an appropriate polyunsaturated/saturated fatty acids index (5.46%) and a moderate linoleic fatty acid content (64.70%) had higher nutritional and pharmaceutical value than all the other genotypes.     

Aryl hydroxylation: A selective mechanism for the herbicides, diclofop-methyl and clofop-isohutyl, in gramineous species

Esters of substituted phenoxy-phenoxy propionic acid constitute a new class of herbicides that are effective against gramineous weed and crop species. Slight changes in chemical structure alter drastically the spectrum of weeds controlled by this class of herbicides. Wheat (Triticum aestivum L.) is resistant to diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy] propanoate) (DM) and clofop-isobutyl (iso-butyl 2-[4-(4′-phenoxy)phenoxy] propanoate) (CI), oat (Avena sativa L.) and wild oat (Avena fatua L.) are susceptible to DM but resistant to CI, and corn (Zea mays L.) is susceptible to both compounds. The antagonism of IAA-induced elongation in the coleoptile straight growth test was determined to measure biological activity of the herbicides. The basis for the differential responses by gramineous species was related to the metabolism and deioxication of the herbicides in coleoptiles. Growth of wheat coleaptiles was relatively unaffected by both compounds, oat coleoptile growth was inhibited by DM but not by CI. but corn coleoptile growth was inhibited equally by both compounds. Coleoptiles and excised shoots of the three species rapidly hydrolyzed both compounds to their respective acids (diclofop, clofop). The acids were conjugated to a water-soluble ester conjugate or they were hydroxylated in the chlorine-substituted phenyl ring and conjugated as a phenolic conjugate. Aryl hydroxylation is a detoxication mechanism in resistant plants. Plants resistant to DM or CI contained low concentrations of the parent ester and the free or bound (ester conjugate) acid and a high concentration of free or bound (phenolic conjugate) aryl hydroxylated acid in coleoptile and shoot tissues, Differential responses by the three gramineous species to DM and CI axe due apparently to differences in their detoxication mechanism. The enzyme for aryl hydroxylation in oat appears to have a higher affinity for the 4-chloro- than for the 2,4-dichloro-substituted moiety. Therefore, oat hydroxylated clofop rapidly and was tolerant to CI but the limited ability of oat to hydroxylate diclofop resulted in oat being extremely susceptible to DM.     

A peroxygenase pathway involved in the biosynthesis of epoxy fatty acids in oat

While oat (Avena sativa) has long been known to produce epoxy fatty acids in seeds, synthesized by a peroxygenase pathway, the gene encoding the peroxygenase remains to be determined. Here we report identification of a peroxygenase cDNA AsPXG1 from developing seeds of oat. AsPXG1 is a small protein with 249 amino acids in length and contains conserved heme-binding residues and a calcium-binding motif. When expressed in Pichia pastoris and Escherichia coli, AsPXG1 catalyzes the strictly hydroperoxide-dependent epoxidation of unsaturated fatty acids. It prefers hydroperoxy-trienoic acids over hydroperoxy-dienoic acids as oxygen donors to oxidize a wide range of unsaturated fatty acids with cis double bonds. Oleic acid is the most preferred substrate. The acyl carrier substrate specificity assay showed phospholipid and acyl-CoA were not effective substrate forms for AsPXG1 and it could only use free fatty acid or fatty acid methyl esters as substrates. A second gene, AsLOX2, cloned from oat codes for a 9-lipoxygenase catalyzing the synthesis of 9-hydroperoxy-dienoic and 9-hydroperoxy-trienoic acids, respectively, when linoleic (18:2-9c,12c) and linolenic (18:3-9c,12c,15c) acids were used as substrates. The peroxygenase pathway was reconstituted in vitro using a mixture of AsPXG1 and AsLOX2 extracts from E. coli. Incubation of methyl oleate and linoleic acid or linolenic acid with the enzyme mixture produced methyl 9,10-epoxy stearate. Incubation of linoleic acid alone with a mixture of AsPXG1 and AsLOX2 produced two major epoxy fatty acids, 9,10-epoxy-12-cis-octadecenoic acid and 12,13-epoxy-9-cis-octadecenoic acid, and a minor epoxy fatty acid, probably 12,13-epoxy-9-hydroxy-10-transoctadecenoic acid. AsPXG1 predominately catalyzes intermolecular peroxygenation.     

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.     

Changes in composition of phenolic compounds and antioxidant properties of <Emphasis Type="Italic">Vitis amurensis</Emphasis> seeds germinated under osmotic stress

The research focused on the changes of phenolic compounds as well as their antiradical activity and reducing power isolated from Amur grape (Vitis amurensis) seeds during germination under optimal conditions and under osmotic stress. The seeds were found to contain tannins, (+) catechin, (−) epicatechin, and gallic acid (in free, ester- and glycoside-bound forms). Extracts from the seeds were also shown to contain two other phenolic acids: caffeic and p-coumaric acids, in very low levels. During a 3-day seed germination test under osmotic stress (−0.5 MPa), the content of total phenolics, tannins and phenolic acids declined as compared to the control. However, seed germination under stress conditions led to a significant increase in the amount of catechins. Because catechin is the one of the units in condensed tannins, its dynamic increase during seed germination may be involved in metabolism of tannins under osmotic stress. It is also likely that the synthesis of catechins is greater under stress conditions and these compounds may be engaged in the process of acclimatization of grapevines to stress conditions. The content of total phenolic compounds in seed extracts is positively correlated with their antioxidant properties. The extracts from seeds germinated under optimal conditions exhibited strong antiradical properties against the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical as well as reducing power. As regards the extracts from grape seeds germinated under osmotic stress, this capability was much weaker. The research demonstrated that antioxidants could interfere with the oxidation process induced by various stresses by acting as oxygen scavengers, therefore the tolerance to drought stress might be correlated with an increase in the antioxidant potential.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.