Characterisation of phenylethanoid glycosides by multiple-stage mass spectrometry.

RATIONALE: Although phenylethanoid glycosides (PhGs) occur widely in plants, their characterisation by liquid chromatography/mass spectrometry (LC/MS) is less well studied than other phenolic glycosides such as flavonoid glycosides. The multiple-stage mass spectrometry (MSn ) experiments required to improve the annotation of common verbascoside-type PhGs are described here. METHODS: Deprotonated, ammoniated and sodiated molecules of nine PhGs were subjected to low-energy collision-induced dissociation (CID) in a hybrid ion trap/orbitrap mass spectrometer. Most experiments were recorded at nominal mass using the linear ion trap analyser for wider applicability in the plant metabolomics community. Data interpretation was supported by high-resolution orbitrap scanning of product ions. Comparative data was acquired on the same instrument by performing higher-energy collisional dissociation (HCD) in the C-trap. RESULTS: Low-energy CID-MS2 of the deprotonated and ammoniated molecules generated diagnostic product ions from which the molecular masses of the phenolic acid and phenylethanoid moieties, respectively, could be determined. The sugar at C-3' of the core glucose was preferentially lost from the sodiated molecule following CID-MS2 , while CID-MSn produced a sodiated product ion from ring cleavage of the core glucose bearing the sugar at C-6'. Evidence of a disaccharide substitution came from a sodiated disaccharide residue in CID-MSn spectra. CONCLUSIONS: The consistency of PhG dissociation following low-energy CID-MSn of various ions is sufficient to enable annotation of verbascoside-type PhGs in LC/MS analyses of crude plant extracts. This can be achieved on a low-resolution instrument capable of MSn .

Mechanisms in mutualisms: a chemically mediated thrips pollination strategy in common elder.

MAIN CONCLUSION: This study provides first evidence of a thrips species pollinating Sambucus nigra and describes how interactions are driven by plant biochemical signalling and moderated by temporal changes in floral chemistry. The concept of flower-feeding thrips as pollinating insects in temperate regions is rarely considered as they are more frequently regarded to be destructive florivores feeding on pollen and surrounding plant tissue. Combining laboratory and field-based studies we examined interactions between Sambucus nigra (elderflower) and Thrips major within their native range to ascertain the role of thrips in the pollination of this species and to determine if floral chemicals mediated flower visits. If thrips provide a pollination service to S. nigra, then this will likely manifest in traits that attract the pollinating taxa at temporally critical points in floral development. T. major were highly abundant in inflorescences of S. nigra, entering flowers when stigmas were pollen-receptive and anthers were immature. When thrips were excluded from the inflorescences, fruit-set failed. Linalool was the major component of the inflorescence headspace with peak abundance coinciding with the highest number of adult thrips visiting flowers. Thrips were absent in buds and their numbers declined again in senescing flowers inversely correlating with the concentration of cyanogenic glycosides recorded in the floral tissue. Our data show that S. nigra floral chemistry mediates the behaviour of pollen-feeding thrips by attracting adults in high numbers to the flowers at pre-anthesis stage, while producing deterrent compounds prior to fruit development. Taking an integrative approach to studying thrips behaviour and floral biology we provide a new insight into the previously ambiguously defined pollination strategies of S. nigra and provide evidence suggesting that the relationship between T. major and S. nigra is mutualistic.

Protoilludane, Illudane, Illudalane, and Norilludane Sesquiterpenoids from Granulobasidium vellereum.

Two new and seven known sesquiterpene compounds were isolated from an agar plate culture of Granulobasidium vellereum, isolated from a log of Ulmus sp. The two new structures were elucidated with spectroscopic methods as an illudalane derivative, granulolactone (1), and a 15-norilludane, granulodione (9). The acaricidal and insecticidal activities of the isolated compounds were examined in vitro against two major horticultural pests, the two-spotted spider mite Tetranychus urticae and the glasshouse thrips Heliothrips haemorrhoidalis, respectively.

LC-MS- and (1)H NMR-Based Metabolomic Analysis and in Vitro Toxicological Assessment of 43 Aristolochia Species.

Species of Aristolochia are used as herbal medicines worldwide. They cause aristolochic acid nephropathy (AAN), a devastating disease associated with kidney failure and renal cancer. Aristolochic acids I and II (1 and 2) are considered to be responsible for these nephrotoxic and carcinogenic effects. A wide range of other aristolochic acid analogues (AAAs) exist, and their implication in AAN may have been overlooked. An LC-MS- and (1)H NMR-based metabolomic analysis was carried out on 43 medicinally used Aristolochia species. The cytotoxicity and genotoxicity of 28 Aristolochia extracts were measured in human kidney (HK-2) cells. Compounds 1 and 2 were found to be the most common AAAs. However, AA IV (3), aristolactam I (4), and aristolactam BI (5) were also widespread. No correlation was found between the amounts of 1 or 2 and extract cytotoxicity against HK-2 cells. The genotoxicity and cytotoxicity of the extracts could be linked to their contents of 5, AA D (8), and AA IIIa (10). These results undermine the assumption that 1 and 2 are exclusively responsible for the toxicity of Aristolochia species. Other analogues are likely to contribute to their toxicity and need to be considered as nephrotoxic agents. These findings facilitate understanding of the nephrotoxic mechanisms of Aristolochia and have significance for the regulation of herbal medicines.

Identification of common glycosyl groups of flavonoid O-glycosides by serial mass spectrometry of sodiated species.

Flavonoid O-glycosides are a ubiquitous and important group of plant natural products in which a wide variety of sugars are O-linked to an aglycone. Determining the identity of the sugars, and the manner in which they are linked, by mass spectrometry alone is challenging. To improve the identification of common O-linked di- and trisaccharides when analysing mixtures of flavonoid O-glycosides by liquid chromatography/mass spectrometry (LC/MS), the fragmentation of electrosprayed sodium adducts in an ion trap mass spectrometer was investigated. The sodium adducts [M + Na](+) of kaempferol 3-O-glycosides generated sodiated glycosyl groups by the neutral loss of kaempferol. The product ion spectra of these sodiated glycosyl groups differed between four isomeric kaempferol 3-O-rhamnosylhexosides and four isomeric kaempferol 3-O-glucosylhexosides in which the primary hexose was either glucose or galactose and bore the terminal glucose or rhamnose at either C-2 or C-6. Fragmentation of sodiated glycosyl groups from linear O-triglucosides and branched O-glucosyl-(1 → 2)-[rhamnosyl-(1 → 6)]-hexosides produced sodiated disaccharide residues, and the product ion spectra of these ions assisted the identification of the complete sugar. The product ion spectra of the sodiated glycosyl groups were consistent among flavonoid O-glycosides differing in the position at which the sugar was O-linked to the aglycone, and the nature of the aglycone. The abundance of sodiated species was enhanced by application of a pre-trap collision voltage, without the need to dope with salt, allowing automated LC/MS methods to be used to identify the glycosyl groups of common flavonoid O-glycosides, such as rutinosides, robinobiosides, neohesperidosides, gentiobiosides and sophorosides.

Applications of phytochemical and in vitro techniques for reducing over-harvesting of medicinal and pesticidal plants and generating income for the rural poor.

Plants provide medicine and pest control resources for millions of poor people world-wide. Widespread harvesting of medicinal and pesticidal plants puts pressure on natural populations, thus severely compromising their contribution to the income and well-being of traders and consumers. The development of in vitro propagation techniques appropriate for developing countries will provide a robust platform for effective propagation and cultivation of endangered plants. This review focuses on advances in the application of phytochemical and in vitro tools to identify and rapidly propagate medicinal and pesticidal plants. Problems of over-harvesting can be alleviated and ex situ cultivation in agroforestry systems can be facilitated through improving seed germination, in vitro cloning and the use of mycorrhizal fungi. We also present a case for effective use of phytochemical analyses for the accurate identification of elite materials from wild stands and validation of the desired quality in order to counter loss of efficacy in the long run through selection, propagation or ex situ management in agroforestry systems. Future prospects are discussed in the context of medicinal activity screening, sustainable propagation, on-farm planting, management and utilization.

Glycosylated constituents of iris fulva and Iris brevicaulis.

The major constituents of leaf extracts of Iris fulva KER GAWL. comprised a known flavone C-glycoside, 5,4'-dihydroxy-7-methoxyflavone-6-C-(6‴-O-(E)-p-coumaroyl-ß-glucopyranosyl)(1‴→2″)-ß-glucopyranoside (1) and the new monoterpene glycoside, linalyl-6'-O-(3″-hydroxy-3″-methylglutaroyl)-ß-D-glucopyranoside (2), both of which were prominent components of Iris brevicaulis RAF. leaf extracts. The structure of a new polyacylated sucrose derivative (3a) obtained from the rhizomes of I. fulva was elucidated as 3-O-(E)-p-coumaroyl-ß-D-fructofuranosyl-(2↔1')-[2″,4″,6″-tri-O-acetyl-ß-D-glucopyranosyl-(1″→3')-(2',6'-di-O-acetyl-4'-O-(E)-p-coumaroyl-α-D-glucopyranoside)]. Selective hydrolysis of the 4″-O-acetyl moiety of the terminal ß-glucopyranosyl residue of 3a occurred after several hours in solution giving 3-O-(E)-p-coumaroyl-ß-D-fructofuranosyl-(2↔1')-[2″,6″-di-O-acetyl-ß-D-glucopyranosyl-(1″→3')-(2',6'-di-O-acetyl-4'-O-(E)-p-coumaroyl-α-D-glucopyranoside)] (3b), which subsequently underwent further deacetylation.

Assigning glucose or galactose as the primary glycosidic sugar in 3-O-mono-, di- and triglycosides of kaempferol using negative ion electrospray and serial mass spectrometry.

Kaempferol 3-O-beta-glucopyranoside, kaempferol 3-O-beta-galactopyranoside and higher glycosides of these two flavonoids with alpha-rhamnose at C-2 and/or C-6 of the primary sugar were studied by negative ion electrospray ionisation and serial mass spectrometry in a three-dimensional (3D) ion trap mass spectrometer. Kaempferol 3-O-beta-glucopyranoside and kaempferol 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside could be distinguished from their respective galactose analogues by differences in the ratio of the radical aglycone ion [Y(0) - H](*-) to the rearrangement aglycone ion Y(0) (-) following MS/MS of the deprotonated molecules. Kaempferol 3-O-rhamnopyranosyl(1-->2)-beta-glucopyranoside and kaempferol 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside could be distinguished from their respective galactose analogues by differences in the product ion spectra of the [(M - H) - rhamnose](-) ion following serial mass spectrometry. In the triglycoside, it was deduced that this ion resulted from the loss of the rhamnose substituted at 2-OH of the primary sugar by observing that MS/MS of deprotonated kaempferol 3-O-beta-glucopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside showed the loss of glucose and not rhamnose. Thus the class of sugar (hexose, deoxyhexose, pentose) at C-2 and C-6 of the primary sugar can be determined. These observations aid the assignment of kaempferol 3-O-glycosides, having glucose or galactose as the primary glycosidic sugar, in LC/MS analyses of plant extracts, and this can be done with reference to only a few standards.

Flavonol pentaglycosides of Cordyla (Leguminosae: Papilionoideae: Swartzieae): distribution and taxonomic implications.

A survey of foliar flavonoids in the swartzioid legume genus Cordyla s.l. revealed that three species, C. haraka, C. pinnata and C. richardii, were rich in flavonol pentaglycosides. Their structures were elucidated by spectroscopic and chemical methods as the 3-O-alpha-L-rhamnopyranosyl(1-->3)-alpha-L-rhamnopyranosyl(1-->2)[alpha-L-rhamnopyranosyl(1-->6)]-beta-D-galactopyranoside-7-O-alpha-L-rhamnopyranosides of quercetin and kaempferol (cordylasins A and B, respectively). These compounds were not found in the remaining species, C. africana, C. densiflora, C. madagascariensis (two subspecies) and C. somalensis, which exhibited different profiles of flavonoid glycosides. The distribution of flavonol pentaglycosides in Cordyla s.l. does not support a recent proposal to place both C. haraka and C. madagascariensis in the genus Dupuya [Kirkbride, J.H., 2005. Dupuya, a new genus of Malagasy legumes (Fabaceae). Novon 15, 305-314]. The generic relationship between Cordyla s.l. and Mildbraediodendron is also reassessed on the basis of chemical characters, as the O-linked tetrasaccharide that characterises cordylasins A and B is the same as that found in mildbraedin (kaempferol 3-O-alpha-l-rhamnopyranosyl(1-->3)-alpha-l-rhamnopyranosyl(1-->2)[alpha-l-rhamnopyranosyl(1-->6)]-beta-D-galactopyranoside), the main foliar flavonoid of Mildbraediodendron excelsum. Mildbraedin itself was found to be a minor constituent of leaflet extracts of C. haraka, C. pinnata and C. richardii, and a major constituent of C. somalensis.

Flavonol tetraglycosides and other constituents from leaves of Styphnolobium japonicum (Leguminosae) and related taxa.

Two flavonol tetraglycosides comprising a trisaccharide at C-3 and a monosaccharide at C-7 were isolated from the leaves of Styphnolobium japonicum (L.) Schott and characterised as the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranosides of quercetin and kaempferol. The 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside of kaempferol, the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranosides of kaempferol and quercetin and the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside of kaempferol were also obtained from this species for the first time. Some or all of these flavonol tetra- and triglycosides were detected in 17 of 18 specimens of S. japonicum examined from living and herbarium material, although the most abundant flavonoid in the leaves was generally quercetin 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside (rutin). The triglycosides, but not the tetraglycosides, were detected in herbarium specimens of Styphnolobium burseroides M. Sousa, Rudd & Medrano and Styphnolobium monteviridis M. Sousa & Rudd, but specimens of Styphnolobium affine (Torrey & A. Gray) Walp. contained a different profile of flavonol glycosides. The flavonol tetra- and triglycosides of S. japonicum were also present in leaves of Cladrastis kentukea (Dum. Cours.) Rudd, a representative of a genus placed close to Styphnolobium in current molecular phylogenies. An additional constituent obtained from leaves of Styphnolobium japonicum was identified as the maltol derivative, 3-hydroxy-2-methyl-4H-pyran-4-one 3-O-(4'-O-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-beta-glucopyranoside.

Chromatographic behaviour of steroidal saponins studied by high-performance liquid chromatography-mass spectrometry.

The chromatographic behaviour of steroidal saponins found in Anemarrhena asphodeloides, Asparagus officinalis, Convallaria majalis, Digitalis purpurea and Ruscus aculeatus was studied by HPLC-MS using a C-18 reversed-phase column and aqueous acetonitrile or aqueous methanol mobile phase gradients, with or without the addition of 1% acetic acid. The behaviour was compared to that of triterpene saponins found in Aesculus hippocastanum, Centella asiatica, Panax notoginseng and Potentilla tormentilla. Inclusion of methanol in the mobile phase under acidic conditions was found to cause furostanol saponins hydroxylated at C-22 to chromatograph as broad peaks, whereas the peak shapes of the spirostanol saponins and triterpene saponins studied remained acceptable. In aqueous methanol mobile phases without the addition of acid, furostanol saponins chromatographed with good peak shape, but each C-22 hydroxylated furostanol saponin was accompanied by a second chromatographic peak identified as its C-22 methyl ether. Methanolic extracts analysed in non-acidified aqueous acetonitrile mobile phases also resolved pairs of C-22 hydroxy and C-22 methoxy furostanol saponins. The C-22 methyl ether of deglucoruscoside was found to convert to deglucoruscoside during chromatography in acidified aqueous acetonitrile, or by dissolving in water. Poor chromatography of furostanol saponins in acidified aqueous methanol is due to the interconversion of the C-22 hydroxy and C-22 methoxy forms. It is recommended that initial analysis of saponins by HPLC-MS using a C-18 stationary phase is performed using acidified aqueous acetonitrile mobile phase gradients. The existence of naturally-occurring furostanol saponins methoxylated at C-22 can be investigated with aqueous acetonitrile mobile phases and avoiding methanol in the extraction solvent.

Phylogenetic trends in the evolution of inflorescence odours in Amorphophallus.

The chemical composition of inflorescence odours of 80 species of Amorphophallus (Araceae) were determined by headspace-thermal desorption GC-MS. When compared to published molecular phylogenies of the genus, the data reveal evidence both of phylogenetic constraint and plasticity of odours. Dimethyl oligosulphides were found as common constituents of Amorphophallus odours and were the most abundant components in almost half of the species studied. Odours composed mainly of dimethyl oligosulphides, and perceived as being 'gaseous', were only found among Asian species, and some of these species clustered in certain clades in molecular phylogenies; e.g. in two clades in Amorphophallus subgenus Metandrium. However, some species with gaseous odours were found to be closely related to species producing odours more reminiscent of rotting meat in which various minor components accompany the dominant dimethyl oligosulphides. These two broad types of odours have co-evolved with other inflorescence characteristics such as colour, with species with rotting meat odours having darker inflorescences. Species producing pleasant odours characterised by benzenoid compounds constitute two broad groups that are not related in published phylogenies. Species having fruity odours containing 1-phenylethanol derivatives mainly occur in a clade in subgenus Metandrium while those with anise odours composed almost solely of the 2-phenylethanol derivative 4-methoxyphenethyl alcohol are restricted to a clade in subgenus Scutandrium. Phylogenetic mapping of odours also indicates that the evolution of some odour types is likely to have been influenced by ecological factors. For example, species producing fishy odours dominated by trimethylamine and occurring in N and NE Borneo are not all closely related. Conversely, two sister species, A. mossambicensis and A. abyssinicus, which are morphologically very similar and have overlapping geographical distribution, produce odours which are very different chemically. The pressure of pollinator resource has therefore been a factor influencing the evolution of odours in Amorphophallus, driving both the divergence of odour types in some taxa and the convergence of odour types in others.

Medicinally Used Asarum Species: High-Resolution LC-MS Analysis of Aristolochic Acid Analogs and In vitro Toxicity Screening in HK-2 Cells.

Species of Asarum are used in traditional Chinese medicine and, similar to members of the genus Aristolochia, they contain aristolochic acid analogs (AAAs). These compounds are known for their nephrotoxic and carcinogenic effects. So far, the phytochemistry and nephrotoxicity of species of Asarum is not well studied. A high-resolution LC-MS-based metabolomic approach was used to study the phytochemical variation in medicinally used Asarum species. The cytotoxicity of the samples was assessed using human kidney (HK-2) cells. The majority of samples contained potentially nephrotoxic AAAs, including 9-methoxy aristolactam (AL) IV, AL I, and AL IV. These compounds were present in methanol as well as water extracts. AAAs were detected in all parts of the plant. The majority of the extracts were not cytotoxic to HK-2 cells at the doses tested. However, other mechanisms relating to aristolochic acid nephropathy and cancer development, such as DNA adduct formation may occur. The results of this study provide a model for assessing lesser-known plant species for toxicity.

Variation of theanine, phenolic, and methylxanthine compounds in 21 cultivars of Camellia sinensis harvested in different seasons.

This is the first study to use chemometric methods to differentiate among 21 cultivars of Camellia sinensis from China and between leaves harvested at different times of the year using 30 compounds implicated in the taste and quality of tea. Unique patterns of catechin derivatives were observed among cultivars and across harvest seasons. C. sinensis var. pubilimba (You 510) differed from the cultivars of C. sinensis var. sinensis, with higher levels of theobromine, (+)-catechin, gallocatechin, gallocatechin gallate and theasinensin B, and lower levels of (-)-epicatechin, (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), respectively. Three cultivars of C. sinensis var. sinensis, Fuyun 7, Qiancha 7 and Zijuan contained significantly more caffeoylquinic acids than others cultivars. A Linear Discriminant Analysis model based on the abundance of 12 compounds was able to discriminate amongst all 21 tea cultivars. Harvest time impacted the abundance of EGC, theanine and afzelechin gallate.

Insect-antifeedant and antibacterial activity of diterpenoids from species of Plectranthus.

Bio-assay guided fractionation of an acetone extract of leaf material from Plectranthus saccatus Benth. resulted in the isolation of a beyerane diterpenoid. This compound, characterised by spectroscopic methods as ent-3beta-(3-methyl-2-butenoyl)oxy-15-beyeren-19-oic acid, showed insect antifeedant activity against Spodoptera littoralis. Known quinonoid abietane diterpenoids obtained from new sources included a mixture of the (4R,19R) and (4R,19S) diastereoisomers of coleon A from P. aff. puberulentus J.K. Morton, coleon A lactone from P. puberulentus J.K. Morton, and coleon U and coleon U quinone from P. forsteri 'Marginatus' Benth. These compounds, and the crude acetone extracts from the leaf surfaces of 11 species of Plectranthus, were tested for antifeedant activity against S. littoralis, antibacterial activity against Bacillus subtilis and Pseudomonas syringae and antifungal activity against Cladosporium herbarum. The coleon A mixture showed potent antifeedant activity against S. littoralis, whereas coleon U showed the greatest antimicrobial activity.

Data-directed scan sequence for the general assignment of C-glycosylflavone O-glycosides in plant extracts by liquid chromatography-ion trap mass spectrometry.

An ion trap LC-MS/MS method is described for the analysis of C-glycosylflavone O-glycosides in crude methanolic extracts of plants. The method employs survey scans with and without the application of up-front collision induced dissociation (CID) to generate diagnostic ions for data-directed MS/MS. The spectra acquired allow assignment of the C-linked sugar to either the C-6 or C-8 position of the aglycone and provide data on the molecular mass of the compound, the number and type of O-linked sugars and the molecular mass of the flavone aglycone. These data for the majority of C-glycosylflavone O-glycosides in an extract are obtained automatically in one LC-MS/MS analysis without manual pre-programming. Key to the assignment of the C-6 or C-8 site of C-glycosylation is the generation, by up-front CID, of the (0,1)X+ product ion formed by internal cleavage of the C-linked sugar. MS/MS of this ion is found to have diagnostic value in addition to the (0,2)X+ product ion described by other authors. Ion trap MS/MS spectra of [M+H]+ of the 6,8-di-C-glycosylflavones schaftoside and isoschaftoside show an additional and previously unreported diagnostic product ion that is useful in determining the type of sugar at the C-6 position. The product ion spectra of protonated kaempferol 3-O-glucosylrhamnosides show similarities to the spectra of C-glycosylflavone O-glycosides; this is a potential source of confusion if the analysis of such glycosides is limited solely to MS/MS of [M+H]+.

Application of liquid chromatography-mass spectrometry to the investigation of poisoning by Oenanthe crocata.

Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.

Monomethyl ethers of 4,5-dihydroxypipecolic acid from Petaladenium urceoliferum: Enigmatic chemistry of an enigmatic legume.

Leaves of Petaladenium (Leguminosae), an Amazonian monospecific genus recently revealed as a member of the Amburaneae clade among the earliest-diverging papilionoid legumes, were found to accumulate three monomethyl ethers of 4,5-dihydroxypipecolic acids. These were characterised by spectroscopic means as the (2S,4S,5R) and (2S,4R,5S) epimers of 5-hydroxy-4-methoxypipecolic acid and (2S,4R,5R)-4-hydroxy-5-methoxypipecolic acid. These compounds were not detected in any other genera in the Amburaneae clade or the wider Angylocalyceae-Dipterygeae-Amburaneae (ADA) clade of papilionoid legumes. Hydroxypipecolic acids, however, were detected in leaves of Myrocarpus and Myroxylon (sister genera in the Amburaneae clade), Angylocalyx and Xanthocercis (sister genera in the Angylocalyceae clade) and Monopteryx (Dipterygeae clade), and were also present in Petaladenium. Iminosugars, known to be accumulated by all four genera in the Angylocalyceae clade (Alexa, Angylocalyx, Castanospermum and Xanthocercis), were found to be characteristic of this group within the ADA clade.