Search

Therapy of molybdenum cofactor (Moco) deficiency has received US Food and Drug Administration (FDA) approval in 2021. Whereas urothione, the urinary excreted catabolite of Moco, is used as diagnostic biomarker for Moco-deficiency, its catabolic pathway remains unknown. Here, we identified the urothione-synthesizing methyltransferase using mouse liver tissue by anion exchange/size exclusion chromatography and peptide mass fingerprinting. We show that the catabolic Moco S-methylating enzyme corresponds to thiopurine S-methyltransferase (TPMT), a highly polymorphic drug-metabolizing enzyme associated with drug-related hematotoxicity but unknown physiological role. Urothione synthesis was investigated in vitro using recombinantly expressed human TPMT protein, liver lysates from Tpmt wild-type and knock-out (Tpmt

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.

A comprehensive analysis of low molecular weight compounds in biological samples by the hyphenated method of GC/MS in general detects a large number of peaks which can not be identified by searching of commercially available mass spectral libraries. Therefore, more information is required for a successful identification of these compounds. Some structural features like molecular weight and number of derivatization groups present in the molecule can be determined by variation of the derivatization prior to GC/MS. The use of deuterated derivatizing reagents and the newly developed N-methyl-N-ethyldimethylsilyl-trifluoracetamide for this purpose is described. The knowledge of these structural properties alone undoubtedly will not lead to the structure elucidation of novel metabolites. However, it may be helpful in identifying derivatives of known metabolites or artifacts. Thus, by use of the molecular weight as search criterion it is possible to find plausible candidates in metabolic pathway databases. It is shown that the application of this method to a hydrophilic extract of C. glutamicum lead to the identification of 31 in previous analyses unidentified peaks, in their majority representing minor derivatives of common metabolites.

Hydroxyiridals − monocyclic triterpenoids − are found abundantly in sword lilies. They feature a homofarnesyl side chain that is hydroxylated at one of the allylic methylene groups, which gives rise to a chiral carbon atom. The absolute configuration of six hydroxyiridals from different sources was determined by the exciton chirality method and by NMR spectroscopy of the diastereoisomeric MTPA and MPA esters. All three measurements gave consistent results, establishing the (R) configuration for all of the hydroxyiridals. The reliability of these methods for the stereochemical analysis of acyclic secondary allylic alcohols was confirmed by applying them to a synthetic model compound of known configuration. In the course of this study, two new iridals − 16,26-dihydroxyiridal 7 and 18,19-epoxyiridal 8 − were isolated from extracts of I. versicolor and the iridal fraction of I. missouriensis was analyzed thoroughly. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Fractionation of the lipid extract from rhizomes of Iris hoogianaDykes resulted in the isolation of one new and several known iridals. The latter were identified by comparison with authentic standards as 1 – 5. The structure of the new natural product, hoogianal (11), was elucidated by spectroscopic analysis. Oxidative degradation yielded β-irone (10), identified by GC- and LC-MS. The (−)-(2S)-configuration of this oxidation product was determined by enantioselective GC on a chiral cyclodextrin phase and by comparison with the corresponding ketones in laevo- and dextrorotatory commercial Iris oils.

Three new iridals were isolated from rhizomes of Iris cristata. After spectral analysis their structures were respectively established as 22,23-epoxy-21-hydroxyiridal 5, 22,23-epoxyiridal 6 and 22,23-epoxy-10-deoxy-21-hydroxyiridal 7.

Besides numerous known iridals, which are unusual mono- or bicyclic triterpenoids of Iridaceae, lipid extracts of Tigridia pavonia bulbs contain tigridial (17), a new spirobicyclic hemiacetal with a conjugated pentaene moiety in the terpenoid side chain. The isolated natural product decomposes rapidly despite exclusion of light and oxygen. In contrast, derivatives of 17 formed by Diels–Alder reaction with 4-phenyl-3H-1,2,4-triazole-3,5-(4H)-dione (13) are stable, as are the products of a subsequent reduction with NaBH4. The products 15a,b and 16a,b of this reaction sequence were isolated and their structures elucidated by spectroscopic techniques. From these results structure 17 was inferred for the new natural product, which was confirmed by 1D and 2D NMR spectroscopy of a mixture of 11 and 17, in which the latter had been stabilized by addition of hydroquinone. The stereochemistry of 17 was deduced from 2D NOE experiments and comparison with known compounds.

The metabolism of iridals, unusual triterpenoids found in sword lilies, was studied in a cell culture of Iris pseudacorus Linn. The cells contain mainly spiroiridals, esterified with capric acid, which are hydrolyzed by esterases upon cell disruption. In ether extracts of these cell homogenates 10-desoxyiridal, a hitherto unknown precursor of the iridals, was identified by comparison with a semisynthetic standard. The compound was isolated and its structure verified by spectroscopic means. Upon incubation of the cells with 3-amino-1,2,4-triazole, a bleaching agent, the amounts of iridal and 10-desoxyiridal in the cells increase, whereas the level of the conjugated iridotrienes is diminished. This proves that 10-desoxyiridal plays a central role in iridal biosynthesis and that the conjugated triene moiety of the iridotrienes is formed by the action of a desaturase on iridal.

Corticrocin is synthesized by mycelia of Piloderma croceum in contrast to mycorradicin, which is deposited into the vacuoles of maize root cells upon colonization with arbuscular mycorrhizal fungi. Corticrocin is formed only when P. croceum is grown in darkness. This indicates that the compound is unlikely of a carotenoid origin which also follows from the chemical structure. Corticrocin is deposited at the surface of the fungal hyphae. The all-E- geometry of the polyunsaturated chain is confirmed by comparing the spectral and chrom atographic properties of the natural product with a synthetic reference.

Numerous 2-methoxy-6-n-alkyl-1,4-benzoquinones with interesting biological activities have been found as constituents of plants and plant seeds. The most prominent is primin, the n-pentyl derivative, which is stored in trichomes on leaves and stems of Primula obconica and is responsible for primrose dermatitis. The biosynthesis of primin was studied by feeding experiments with radioactively labelled precursors and unlabelled homologues.

Besides primin 1 and its hydroquinone, miconidin 2, surface extracts of Primula obconica contain their longer chain homologues 1a and 2a. In addition to several derivatives of 2, namely the methyl ethers 3 and 4 and the acetate 5, the resorcinol 6 and its methyl ether 7 are found. The structures of the compounds were elucidated by spectroscopic methods and - if necessary - by comparison with synthetic references. The possible biosynthesis of these natural products is discussed.

Dehydration of slices of rhizomes led to an increase of iridal esters (IE) with a concomitant drop of free iridals (FI), as well as phospholipids (PL), in the drought-resistant species, Iris germanica . By contrast, in the wetland species, I. pseudacorus , lysis of IE is observed, while the PL content remains around the control level. The origin of fatty acids incorporated into IE and the possible role of iridals against stress injury within Iris species are discussed.

The metabolism of iridals, unusual triterpenoids found in sword lilies, was studied in a cell culture of Iris pallida . By incubation of the cells with radioactively labelled and unlabelled compounds the biogenetic pathway from iridal, the parent compound of the family, to the spiroiridals was shown to lead via 26-hydroxyiridal, which is dehydrogenated to form the corresponding 26-hydroxytriene. A dehydration of the 16- or 17-hydroxyiridals, respectively, was not observed. All triterpenoids are readily taken up by the cells from the medium and are esterified at C3, predominantly with medium-chain fatty acids.

Viele Eigenschaften, mit deren Hilfe sich Pflanzen dagegen wehren, gefressen zu werden, sind offensichtlich und uns bestens bekannt. Pflanzen mit Stacheln oder Dornen haben eine gute Chance, von groseren Tieren in Ruhe gelassen zu werden. Brennesseln beruhrt niemand gern, und spatestens die Erfahrung lehrt, das ubel riechende, unangenehm schmeckende oder giftige Pflanzen fu den Verzehr nicht geeignet sind. Trotzdem werden Pflanzen, die solche physikalischen oder chemischen Abwehrstrukturen entwickelt haben, haufig von Insekten befallen und in erheblichem Mas geschadigt. Andere Pflanzen dagegen, die ganz wehrlos aussehen, sind gegen Frasschadlinge sehr resistent und werden nur auserst selen in groserem Ausmas von Insekten heimgesucht. Deshalb stellt sich die Frage: „Wie wehren sich diese scheinbar wehrlosen Pflanzen eigentlich, wenn sie von Insekten gebissen oder gestochen werden?”

The four novel iridals 8–10 and 18 were isolated from rhizome extracts of Iris pallida and Iris foetidissima, and their structures were established by spectroscopic methods and oxidative degradation. The compounds 8–10 bearing a conjugated triene moiety are extremely labile and decompose rapidly. Thus, the monocyclic triene 8 was only isolated after its conversion to a Diels-Alder adduct with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (14), The tricyclic iridal 18 is a hitherto unknown precursor of α-irone. The possible biogenesis of these unusual triterpenoids is discussed.

Four novel iridals (compounds 8, 11, 12, and 13), representing new examples of these unusual monocyclic triterpenoids from sword lilies, have been isolated from rhizome extracts of I. sibirica and I. versicolor. Their structures have been elucidated by spectroscopic methods combined with oxidative degradation. Based on the stereochemistry of the 10-deoxy-17-hydroxyiridal 13, the possible biosynthesis of these compounds is discussed.

A new iridal has been isolated from rhizomes of Iris germanica var. “Rococo”. On the basis of spectral data, its structure was established as 18,19-epoxy-10-deoxyiridal (5). This compound is the first epoxidized iridal discovered so far.

Besides numerous known iridals, which are unusual mono- or bicyclic triterpenoids of Iridaceae, lipid extracts of Tigridia pavonia bulbs contain tigridial (17), a new spirobicyclic hemiacetal with a conjugated pentaene moiety in the terpenoid side chain. The isolated natural product decomposes rapidly despite exclusion of light and oxygen. In contrast, derivatives of 17 formed by Diels–Alder reaction with 4-phenyl-3H-1,2,4-triazole-3,5-(4H)-dione (13) are stable, as are the products of a subsequent reduction with NaBH4. The products 15a,b and 16a,b of this reaction sequence were isolated and their structures elucidated by spectroscopic techniques. From these results structure 17 was inferred for the new natural product, which was confirmed by 1D and 2D NMR spectroscopy of a mixture of 11 and 17, in which the latter had been stabilized by addition of hydroquinone. The stereochemistry of 17 was deduced from 2D NOE experiments and comparison with known compounds.

We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 +/- 0.5 and 6 +/- 1 mug/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus.

The seed oils of I.pseudacorus LINN., I.sibirica LINN., and I.missouriensis NUTTAL. contain appreciable amounts of phenols, quinones, and resorcinols, substituted with homologous alkyl or alkenyl side chains. The structure elucidation of these compounds is described, and their possible biosynthesis is discussed.

Epicuticular leaf waxes of Ginkgo biloba, Magnolia grandiflora and Liriodendron tulipifera contain homologous series of hydrocarbons, wax esters, benzyl acyl esters, aldehydes, primary alcohols, and fatty acids. None of these lipid classes is found to contain any main component dominating. In addition to these usual wax lipids, in G. biloba leaf wax a secondary alcohol namely nonacosan-10-ol (15.0%), γ-tocopherol (1.7%) and acetates (0.3%) is also present. The wax of L. tulipifera, however, contains hentriacontan-16-one (23%) and several triterpenols (10%), additionally. On G. biloba leaves a dense arrangement of tubular wax crystalloids are found on the lower as well as on the upper leaf surface. The openings of the tubules can be seen very well in the SEM figures at a magnification of 20000. The small tubules are a clear indication for the presence of nonacosan-10-ol as also reported previously for conyferyl waxes. Leaves of M . grandiflora have an abaxial epidermis with a continuous wax layer without any crystalloids or sculptures. The adaxial epidermis also shows a continuous wax layer but with little irregular granular sculptures. L. tulipifera leaves show an abaxial epidermis with a continuous wax layer superimposed by a dense arrangement of crystalloids in shape of angular rodlets which are composed of several piled up layers. The adaxial leaf surface is also superimposed with wax crystalloids, the rodlets of which, however, are not sculptured in such definite way. They usually appear melted up and also form granular sculptures. The wax crystalloids in shape of angular rodlets on the abaxial surface are formed by hentriacontan-16-one. The abaxial and adaxial leaf surfaces show most different micromorphological wax ultrastructures, as shown for all trees studied.

Four novel iridals (compounds 8, 11, 12, and 13), representing new examples of these unusual monocyclic triterpenoids from sword lilies, have been isolated from rhizome extracts of I. sibirica and I. versicolor. Their structures have been elucidated by spectroscopic methods combined with oxidative degradation. Based on the stereochemistry of the 10-deoxy-17-hydroxyiridal 13, the possible biosynthesis of these compounds is discussed.

The composition of iridals, new triterpenoids from sword-lilies, and their fatty acid esters in roots, rhizomes and leaves of I. germanica Linn., I. pseudacorus Linn, and two different varieties of I. pallida Lam. was determined. Quantitative analysis of their total content in the various parts of the plants in the course of the year showed specific seasonal changes in rhizomes and leaves. Based on these results the possible significance of these compounds for the plants is discussed.

The four novel iridals 8–10 and 18 were isolated from rhizome extracts of Iris pallida and Iris foetidissima, and their structures were established by spectroscopic methods and oxidative degradation. The compounds 8–10 bearing a conjugated triene moiety are extremely labile and decompose rapidly. Thus, the monocyclic triene 8 was only isolated after its conversion to a Diels-Alder adduct with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (14), The tricyclic iridal 18 is a hitherto unknown precursor of α-irone. The possible biogenesis of these unusual triterpenoids is discussed.

Isolation of intact photoreceptive organelles (eyespot apparatuses) involved in blue-light mediated photoresponses in a flagellate green alga (Spermatozopsis similis) allowed for the first time the identification of both 11-cis- and all-trans-retinal in a plant cell. Both isomers were identified by HPLC analysis in conjunction with UV spectra. Additionally, reconstitution of a distinct absorption band, centered around 540 nm, was achieved by addition of exogenous 9-cis-retinal to bleached, isolated eyespot apparatuses.

Two novel nortriterpenoids and two, hitherto unknown, representatives of the rare malabaricane and polypodane type, respectively, were found in the neutral fraction of gum mastic, the resin ofPistacia lentiscus. In addition, several tri-, tetra- and pentacyclic triterpenes were identified. The significance of these results for the geometry of the epoxyqualene cyclase/substrate interaction inPistacia lentiscus is discussed.

Natural irones found in the essential oil of Iris rhizomes often are mixtures of enantiomers. The separation of all optical isomers of the irones by GLC on modified cyclodextrins and the determination of their composition within different Iris oils is described. The significance for the biosynthesis of the cycloiridals, C31-triterpenoids, which serve as precursors of the irones, is discussed.

Four novel types of iridals (compounds 8, 9, 15 and 16) have been isolated from lipid extracts of roots and rhizomes of I. pseudacorus and their structures (except the stereochemistry of 15 at C-13 and C-26 and of 16 at C-13) elucidated by oxidative degradation combined with spectroscopic methods. They are up to now unknown triterpenoids with unusual spiro-bicyclic structures. The possible biogenesis of these compounds is descussed.

The novel iridal 10 has been isolated from rhizome extracts of Iris foetidissima. Its structure was established by spectroscopic methods and oxidative degradation. Final proof of the spirobicyclic nature of the compound – a new feature in the triterpenoid field – was afforded by the correlations observed in the 2D-HMBC- (1H-detected multiple-bond heteronuclear multiple-quantum coherence) spectrum. The possible biogenesis of this unusual compound is discussed.

Of the hyphenated techniques used for metabolic profiling of cell and tissue extracts, GC/MS is in some ways advantageous as it allows the simultaneous fingerprinting of chemically very different metabolites, and the electron impact mass spectra recorded in many cases lead to unambiguous identification of the compounds. However, prior to chromatography, the hydrophilic substances of the cell extracts have to be converted to vaporizable derivatives, the mass spectra of which often are not known or not listed in the available spectral libraries, even if they are derived from simple biochemicals. Thus, numerous chromatographic peaks remain as yet unidentified. Attempts to identify these peaks afford the acquisition of more data on these compounds. The value of in vivo labeling of metabolites with 13C and 15N for this purpose is described.

Linear hydroxyproline-bound O-glycans of the unicellular green alga Chlamydomonas reinhardtii were isolated from outer cell wall glycoproteins and their structure elucidated by chemical and spectroscopic methods. They consist exclusively of arabinose and galactose, the latter in the furanose form, unusual for plants. The first two arabinoses linked to hydroxyproline have the same anomeric configuration and linkage to each other as do the arabinosides isolated from land plants, suggesting that these two steps of hydroxyproline-O-glycosylation have been conserved during evolution. In a concomitant analysis of Hyp-bound O-tri- and tetraarabinans isolated from Arabidopsis thaliana, we were able to unambiguously identify the natural trans-4-l-Hyp-bound O-glycans and the artificially produced d-allo forms and to identify diagnostic signals in NMR spectra of these compounds for the first time.

A new iridal has been isolated from rhizomes of Iris germanica var. “Rococo”. On the basis of spectral data, its structure was established as 18,19-epoxy-10-deoxyiridal (5). This compound is the first epoxidized iridal discovered so far.

Fractionation of the lipid extract from rhizomes of Iris variegata Linn. resulted in the isolation of one new and four known iridals. The latter were identified by comparison with authentic standards and NMR-spectroscopic analyses as 21-hydroxyiridal 1, 21-hydroxy-10-deoxyiridal 2, 16-hydroxyiridal 3 and 22-methyl-gamma-cycloiridal 4. The (R) configuration of C21 in compound 2 was determined by the exciton chirality method. The same stereochemistry has previously been found for the appropriate moieties in 1 and 3. Based on its spectroscopic properties the new triterpenoid was shown to be the 23-hydroxyiridal 5.

The pheromone produced by the female gametes of the green flagellate Chlamydomonas allensworthii to lure the male gametes for sexual fusion is the plastohydroquinone derivative lurlene, (all-E)-14-[2′-hydroxy-3′,4′-dimethyl-5′-(1″;-β-D-xylopyranosyloxy)phenyl]-4,8,12-trimethyltetradeca-4,8,12-trienoic acid (1).

Roots of gramineous plants turn deep yellow upon colonization with arbuscular mycorrhizal fungi. At the same time methanolic extracts of maize roots show a polyene absorption with maxima at 375 and 395 nm that is not found in the extracts of controls. After esterification with diazomethane the compound responsible for this absorption was isolated by column chromatography and shown to be all- E -4,9-dimethyldodeca-2,4,6,8,10-pentaenedioic acid by NMR and mass spectrometry. The compound presumably is derived from oxidative degradation of a carotenoid.

Fractionation of a methanol extract obtained from rhizomes of I. spuria (Zeal) resulted in the isolation of seven iridalglycosides 5a,b, 6a-c, 7, 8. The structures of the aglycons and the position of the glycosidic bonds were elucidated by spectroscopic analyses. The nature of the sugar moieties was determined as glucose from capillary GC and GC/MS analyses after hydrolysis of the compounds, reduction and derivatization of the resulting hexitols in comparison to authentic standards. Methylation analysis revealed the glycosyl-linkage composition. The number and position of the glucose residues was determined by ozonolysis.

Since 1990, the National Cancer Institute has performed extensive in vitro screening of compounds for anticancer activity. To date, more than 70 000 compounds have been screened for their antiproliferation activities against a panel of 60 human cancer cell lines. We probed this database to identify novel structural classes with a pattern of biological activity on these cell lines similar to that of the phorbol esters. The iridals form such a structural class. Using the program Autodock, we show that the iridals dock to the same position on the C1b domain of protein kinase C delta as do the phorbol esters, with the primary hydroxyl group of the iridal at the C3 position forming two hydrogen bonds with the amide group of Thr12 and with the carbonyl group of Leu 21 and the aldehyde oxygen of the iridal forming a hydrogen bond with the amide group of Gly23. Biological analysis of two iridals, NSC 631939 and NSC 631941, revealed that they bound to protein kinase C alpha with K(i) values of 75.6 +/- 1.3 and 83.6 +/- 1.5 nM, respectively. Protein kinase C is now recognized to represent only one of five families of proteins with C1 domains capable of high-affinity binding of diacylglycerol and the phorbol esters. NSC 631939 and NSC 631941 bound to RasGRP3, a phorbol ester receptor that directly links diacylglycerol/phorbol ester signaling with Ras activation, with K(i) values of 15.5 +/- 2.3 and 41.7 +/- 6.5 nM, respectively. Relative to phorbol 12,13-dibutyrate, they showed 15- and 6-fold selectivity for RasGRP3. Both compounds caused translocation of green fluorescent protein tagged RasGRP3 expressed in HEK293 cells, and both compounds induced phosphorylation of ERK1/2, a downstream indicator of Ras activation, in a RasGRP3-dependent fashion. We conclude that the iridals represent a promising structural motif for design of ligands for phorbol ester receptor family members.

Four novel types of iridals (compounds 8, 9, 15 and 16) have been isolated from lipid extracts of roots and rhizomes of I. pseudacorus and their structures (except the stereochemistry of 15 at C-13 and C-26 and of 16 at C-13) elucidated by oxidative degradation combined with spectroscopic methods. They are up to now unknown triterpenoids with unusual spiro-bicyclic structures. The possible biogenesis of these compounds is descussed.

The novel iridal 10 has been isolated from rhizome extracts of Iris foetidissima. Its structure was established by spectroscopic methods and oxidative degradation. Final proof of the spirobicyclic nature of the compound – a new feature in the triterpenoid field – was afforded by the correlations observed in the 2D-HMBC- (1H-detected multiple-bond heteronuclear multiple-quantum coherence) spectrum. The possible biogenesis of this unusual compound is discussed.

The incorporation of [2-14C]acetate, [2-14C]mevalonate, and [3H]squalene proved the squalenoid nature of the iridals, novel triterpenoids from rhizomes of swordlilies. Methionine is readily incorporated into cycloiridalls of Iris pallida dalmatica, thus indicating that the methylation of iridals via S-adenosyl-L-methionine leads to the formation of the irone moiety of the bicyclic compounds. The 3H/14C ratio of the transferred labelled methyl group remained unchanged. Therefore, the methylation/cyclization of the terpenoid side chain of iridals must proceed by a concerted mechanism without formation of a cyclopropanoid intermediate.

Rhizomes of Iris pallida and Iris florentina contain - increasing on storage - violet-like smelling C14-ketones (irones 1 a-c) which develop by oxidative degradation of C31-triterpenoids. The structure of these precursors is reported as well as the structure of respective C30-homologues. The unusual C31-structures may derive biogenetically from the respective C30 compounds by a methylation which initiates the formation of the ring-closed irone-moiety

Quantity and composition of epicuticular leaf wax of Euphorbia dendroides L. was examined. The wax contained rt-alkanes, wax esters, aldehydes, fatty acids and primary alcohols. In addition to these common epicuticular wax constituents several triterpenoids and benzoic acid esters were found. The triterpenols β-amyrin and lupeol occurred free as well as esterified with long chain fatty acids. The ketones Δ 12-oleanen-3-one and lupen-3-one have also been identified.

The epicuticular leaf waxes from E. characias, E. nicaeensis and E. peplus were found to contain pentacyclic triterpenoids in great amounts in addition to the common lipid wax constituents: alkanes, wax esters, aldehydes, primary alcohols and fatty acids. The gross amount of triterpenoids were triterpenols, however, their acetates and fatty acid esters as well as the corresponding ketones were found, too. Distinctive variations in the occurrence of 13 triterpenoids α- and β-amyrin, lupeol, germanicol, α-fernenol, simiarenol, β-amyrinone, lupenone, lupeol acetate, lupeol esters, α- and β-amyrin esters and α-fernenol esters were observed. Therefore all Euphorbia species showed very species specific compositions concerning these triterpenoids.

Gene 4 from the T-region of Ti plasmids is responsible for cytokinin effects in crown gall cells; we investigated whether it codes for an enzyme of hormone biosynthesis. In a first set of experiments, gene 4 from octopine plasmid pTiAch5 and nopaline plasmid pTiC58 was expressed in Escherichia coli, and the gene products were identified by reaction with antiserum raised against a decapeptide derived from the DNA sequence of the gene. Extracts from cells expressing the gene contained high isopentenyl-transferase activity catalyzing the formation of N6-(delta2-isopentenyl)adenosine from 5'-AMP and delta2-isopentenylpyrophosphate. The cytokinin was identified by sequential h.p.l.c. chromatography and mass spectrometry. In a second set of experiments it was shown that crown gall cells contained isopentenyltransferase activity and a protein of mol. wt. 27 000 which was identified as the product of gene 4 by reaction with the antiserum. Isopentenyltransferase activity was specifically inhibited by the antiserum. No comparable enzyme activity or immunoreactive protein was detected in cytokinin-autotrophic, T-DNA free tobacco cells. The results establish that gene 4 from the T-region of octopine and nopaline Ti plasmids codes for an enzyme of cytokinin biosynthesis.

Esters of very long chain fatty acids with benzyl alcohol and the homologue 2-phenyl-ethanol-1 were found in the epicuticular waxes from Jojoba (Simmondsia chinensis) leaves. The isolation of these esters by chromatographic methods, the structural elucidation by GC-MS and their chemical reactions and synthesis are described.

Majusculamides A and B are major lipophilic constituents of the blue-green alga Lyngbya majuscula Gomont. Detailed spectral analysis, chemical degradation, and x-ray crystallographic studies show that majusculamide A is N-((2R)-2-methyl-3-oxodecanoyl)-D-N,O-dimethyltyrosyl-L-N-methylvalinamide (6a) and that majusculamide B is N-((2S)-2-methyl-3-oxodecanoyl)-D-N,O-dimethyltyrosyl-L-N-methylvalinamide (6b). Majusculamide B is epimerized into a mixture of majusculamides A and B and then degraded into D-N,O-dimethyltyrosyl-L-N-methylvaline lactam (2) and racemic 2-methyl-3-oxodecanoic amide (1) at 140/sup 0/C in anhydrous dimethyl sulfoxide.

A monocyclic C31-triterpenoid was isolated from rhizomes of Iris versicolor L. Its structure was elucidated as 12 by spectroscopy combined with oxidative degradation. The identity of the degradation-products was proven by comparison with synthetic references.