Biosynthesis of iridoid glucosides in Galium mollugo,G. spurium var. Echinospermon and Deutzia crenata. Intermediacy of deoxyloganic acid,loganin and iridodial glucoside

Administration of ²H-labelled compounds to Galium mollugo, G. spurium var. echinospermon and Deutzia crenata established that deoxyloganic acid is a precursor of asperuloside, geniposidic acid and secogalioside in G. mollugo as well as asperuloside in G. spurium, while iridodial glucoside is a precursor of deutzioside in D. crenata. Additionally, the intermediacy of loganic acid in the biosynthesis of the iridoid and secoiridoid glucosides in the Galium plants was reconfirmed.     

Age-dependent variations of the efficiency of iridoid biosynthesis in Verbena officinalis

²H Labelled deoxyloganin gave ca a 30 % incorporation into each of dihydrocornin and cornin in Verbena officinalis plants before their flow     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Implication of tyramine in the biosynthesis of morphinan alkaloids in Papaver

Doubly-labeled [³H, ¹⁴C]tyrosines, [1-¹³C-]tyramine or [2-¹⁴C]tyramine, administered to the stems of intact Papaver somniferum L. plants, were found to be incorporated into the morphinan alkaloids of the plant with comparable efficiency. ³H/¹⁴C ratios of alkaloids from plants fed the tyrosines were consistent with an almost equal conversion of this amino acid into the tetrahydroisoquinoline (TIQ) and benzyl-derived segments. Nuclear magnetic resonance (NMR) analyses of morphine isolated after administration of [1-¹³C]tyramine demonstrated selective labeling of C-16 of the alkaloid, indicating the conversion of this amine primarily into the TIQ-derived moiety. Morphine and thebaine labeled by [2-¹⁴C]tyramine were degraded to phenanthridines and N,N-dimethyl ethylamines. Of the total radioactivity in the alkaloids 97% was found to be associated with the ethylamines, a distribution consistent with the NMR data. This preferential utilization of tyramine in the biosynthesis of morphinan alkaloids can be explained by the compartmentalization of intermediates and enzymes of the pathway.Abbreviations L-dopa L-3,4-dihydroxyphenylalanine - HPLC high-pressure liquid chromatography - NMR nuelear magnetic resonance - TIQ tetrahydroisoquinoline     

Intermediacy of 8-epiiridodial in the biosynthesis of iridoid glucosides by Gardenia jasminoides cell cultures

By administration of ²H-labelled iridodial, its congeners and ¹³C-labelled 10-hydroxygeraniol to Gardenia jasminoides cell suspension cultures it was demonstrated that tarennoside and gardenoside were biosynthesized, after iridodial cation formation, via 8-epiiridodial, 8-epiiridotrial, 8-epiiridotrial glucoside and 7,8-dehydroiridotrial glucoside. However, the coexistence of a pathway via the iridodial cation, 7,8-dehydroiridodial, 7,8-dehydroiridotrial and 7,8-dehydroiridotrial glucoside could not be excluded.     

Intermediacy of iridodial in the biosynthesis of some iridoid glucosides

Administration of MVA-[2-¹⁴C] to Lamium applexicaule and Deutzia crenata as well as of 11-hydroxyiridodial glucoside-[10-³H] and 7-deoxyloganic acid-[10-³H] to the former plant suggested that, in contrast to secoiridoid-indole alkaloids, iridoid glucosides such as ipolamiide, lamiide, lamioside, deutzioside and scabroside in these plants are biosynthesized via iridodial. Iridodial as a precursor for the biosynthesis of asperuloside was also suggested from the results of the administration of MVA-[2-¹⁴C] to Galium spurium var. echinospermon.     

Biosynthetic preparation of cardenolides from [1-14C]acetic acid by stem discs of the milkweed Asclepias curassavica

[¹⁴C]Calotropin (11.2 μCi/mmol) and uscharidin (14.1 μCi/mmol) were biosynthesized by stem discs of Asclepias curassavica incubated in a medium containing [1-¹⁴C]acetic acid. Relative isotope enrichment sites determined by ¹³C NMR spectroscopy of [¹³C]calotropin prepared by the same method were at C-23 (0.71 %), C-2′ (0.28 %) and C-4′ (0.21 %).     

Biosynthesis of dioscorine : Incorporation of nicotinic acid into the isoquinuclidine moiety

The administration of nicotinic-[2-¹⁴C] acid to Dioscorea hispida plants afforded radioactive dioscorine (1.9% absolute incorporation) and a systematic degradation of the alkaloid indicated that essentially all the activity was located at C-3. Dioscorine derived from nicotinic-[5,6-¹⁴C, ¹³C₂] acid was also labelled. Its proton noise decoupled ¹³C NMR spectrum contained satellites at C-1 and C-7 due to spin-spin coupling of contiguous ¹³C atoms arising from direct incorporation of the labelled nicotinic acid. A biosynthetic scheme representing a novel utilization of nicotinic acid is proposed.     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

Investigation of Uña De Gato I. 7-Deoxyloganic acid and 15N NMR spectroscopic studies on pentacyclic oxindole alkaloids from Uncaria tomentosa

The C-8-(S) isomer of deoxyloganic acid (7-deoxyloganic acid), together with beta-sitosteryl glucoside, five known stereoisomeric pentacyclic oxindole alkaloids and the tetracyclic oxindole isorhyncophylline, were isolated from the inner bark of Uncaria tomentosa. Structures of the isolated compounds were based on 1H and 13C NMR data, mainly 2D NMR experiments, including 1H-13C HMBC and 1H-1H NOESY correlation. Furthermore, the hitherto unreported 15N chemical shifts of the isomeric oxindole alkaloids, using 1H-15N HMBC experiments, were utilized to facilitate their characterization. Uncarine D showed weak cytotoxic activity against SK-MEL, KB, BT-549 and SK-OV-3 cell lines with IC(50) values between 30 and 40 microg/ml, while uncarine C exhibited weak cytotoxicity only against ovarian carcinoma (IC(50) at 37 microg/ml).     

5-deoxystansioside,an iridoid glucoside from Tecoma stans

In addition to plantarenaloside and stansioside, a new iridoid glucoside with a formyl group at C-4 has been isolated from Tecoma stans. The new glucoside was shown to be 5-deoxystansioside by ¹³C NMR and ¹H NMR spectroscopy.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Biosynthesis of salicylic acid in fungus elicited Catharanthus roseus cells

Feeding experiments using [1-¹³C]-d-glucose to Catharanthus roseus (L.) G.Don cell suspension cultures followed by elicitation with Pythium aphanidermatum extract were performed in order to study the salicylic acid (SA) biosynthetic pathway and that of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a comparison. A strongly labeled C-7 and a symmetrical partitioning of the label between C-2 and C-6 would occur if SA was synthesized from phenylalanine. In case of the isochorismate pathway, a relatively lower incorporation at C-7 and a non-symmetrical incorporation at C-2 and C-6 would be obtained. Relatively, high- and non-symmetrical enrichment ratios at C-2 and C-6, and a lower enrichment ratio at C-7 were observed in both SA and 2,3-DHBA detected by ¹³C NMR inverse gated spectrometry leading to the conclusion that the isochorismate pathway is responsible for the biosynthesis of both compounds. However, different enrichment ratios of the labeled carbons in SA and 2,3-DHBA indicate the use of different isochorismate pools, which means that their biosynthesis is separated in time and/or space.     

Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR spectroscopy

Plants have been shown to use the mevalonate pathway for the biosynthesis of sterols and triterpenes in the cytoplasm and the recently discovered deoxyxylulose phosphate pathway for the biosynthesis of a variety of hemiterpenes, monoterpenes, diterpenes, as well as for the biosynthesis of carotenoids and the phytol side chain of chlorophyll in plastids. Despite the compartmental separation, at least one terpene precursor can be exchanged between the two pathways. In order to assess quantitatively the crosstalk between the two isoprenoid pathways, [2-¹³C₁]mevalonolactone or [U-¹³C₆]glucose were supplied to cell cultures of Catharanthus roseus grown under illumination or in darkness. Sitosterol, lutein and phytol were isolated and analysed by NMR spectroscopy. The incorporations of exogenous [2-¹³C₁]mevalonolactone were 48% and 7% into the DMAPP and IPP precursors of sitosterol and lutein, respectively. With [U-¹³C₆]glucose as precursor, at least 95% of sitosterol precursors were obtained via the mevalonate pathway, whereas phytol appeared to be biosynthesised via the deoxyxylulose phosphate pathway (approximately 60%) as well via the mevalonate pathway (approximately 40%). The apparent ratios for the contribution of the two pathways depend on the nature of the precursor supplied as well as the nature of the target compound. Thus, crosstalk between the two terpenoid pathways cannot be explained in detail by a simple two compartment model and requires an additional in depth study of complex regulatory mechanisms.     

Structure of renifolin and reconfirmation of the structure of pirolatin

The ¹H and ¹³C NMR spectral analysis of the glucoside renifolin, isolated from Pyrola renifolia, demonstrated the so far unknown binding site of the glucose moiety to be at C-8 and hence its structure as 8-β-D-glucosyloxy-2,7-dimethyl-1,4-dihydronaphthalen-5-ol. The earlier reported structure for the glucoside pirolatin, isolated from Pyrola japonica, was also reconfirmed by the ¹³C NMR spectral analysis.     

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-¹³C]- and [1,4-¹³C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-¹³C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson     

Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the β-glucuronidase gene (uidA) drives β-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [²H]₅-L-tryptophan, they converted this precusor to [²H]₅-indole-3-acetonitrile and [²H]₅-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [²H]₅-indole-3-acetic acid extracted from roots. When [1-¹³C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-¹³C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan. Received: 3 February 1998 / Accepted: 17 April 1998     

Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase.

Particulate enzyme preparations of cotton fibers catalyze the acylation of exogenous steryl glucoside to form acylated steryl glucoside. The acyl transferase involved in this reaction was solubilized by treatment of the membrane fractions with Triton X-100 and was partially purified by chromatography on DEAE-cellulose and gel filtration. This solubilized enzyme had an absolute requirement for Triton X-100 and phospholipid in order to catalyze the acylation of the steryl glucoside. The best phospholipid substrate was phosphatidylethanolamine but egg and soybean phosphatidylcholine were also active. The phospholipid was shown to function as an acyl donor by demonstrating that [¹⁴C]fatty acid from ¹⁴C-labeled phospholipid could be transferred to steryl-[³H]glucoside to form [¹⁴C,³H]acylated steryl glucoside. Saponification of this compound yielded [¹⁴C]fatty acid and steryl-[⁷H]glucoside.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.