Metabolism of [8-14]6-(benzylamino)purine by individual carnation flower components

Isolated stem, receptacle, ovary and petal tissues of the carnation flower (Dianthus caryophyllus L. cv. White Sim) all metabolized [8-¹⁴C]6-(benzylamino) purine. Ribosyl 6-(benzylamino)purine was the major metabolite formed in all flower components. The extent of metabolism and the ratios of the various metabolites of 6-(benzylamino)purine detected within each flower component varied. The receptacle, in particular, was distinct from other flower components with respect to the extent of 6-(benzylamino)purine metabolism.Abbreviations ADE adenine - ADO adenosine - BA 6-(Benzylamino)purine - [9R]BA ribosyl 6-(benzylamino)purine - [9R-MP]BA ribosyl 6-(benzylamino)purine-5-monophosphate - [7G]BA/[9G]BA 7/9--D-glucopyranosyl 6-(benzylamino)purine     

Cytokinins in cut carnations. X. The effect of stem length and holding time on the transport and metabolism of [8-14C]6-(benzylamino)purine

Stem length and to a lesser extent holding time influenced the transport of [8-¹⁴C]6-(benzylamino)purine (BA) within the carnation flower ( Dianthus caryophyllus L.). A stem length of 40 cm as compared to one of 10 cm resulted in a lower amount of BA reaching the carnation flower head when the flowers were held in solution for 3 to 24 h. There was also a slight delay in cytokinin movement from, as well as increased metabolism within, the stem tissues. Consequently levels of radioactivity and the nature of metabolites produced in the upper floral components were influenced by movement through the stem. In the flower head the receptacle was the strongest sink for flowers with 10 cm stem lengths. A metabolite tentatively identified as ribosyl-benzylaminopurine [9R]BA was the major compound formed in all flower components. This metabolic step appeared to be reversible. Various minor stable metabolites and possible breakdown products were also produced.     

Cytokinins in Corynebacterium fascians Cultures: Isolation and Identification of 6-(4-Hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine

In addition to the four cytokinins, 6-(3-methyl-2-butenylamino)purine, 6-methylaminopurine and the cis and trans isomers of 6-(4-hydroxy-3-methyl-2-butenylamino)purine, reported earlier from our laboratories, three cytokinin-active fractions have been obtained from the aqueous medium of 6-day-old Corynebacterium fascians cultures. One of these has been identified as 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthiopurine (2-methylthio-cis-zeatin, c-ms²io⁶ Ade).     

O6-(4-Nitrophenyl)inosine and -Guanosine as Chromogenic Substrates for Adenosine Deaminase

Abstract

O⁶-(4-Nitrophenyl)inosine (la), O⁶ -(4-nitrophenyl)guanosine (1c) and O⁶ -(4-methylumbelliferonyl)inosine (2) were obtained by reaction of 6-chloro-9-(β-D-ribofuranosyl)purine (3a) or 2-amino-6-chloro-9-(β-D-ribofuranosyl)purine (3c) with sodium salts of 4-nitrophenol or 4-methylumbelliferone in N,N-dimethylformamide. Similarly, 6-chloro-9-(β-D-2,3-isopropylideneribofuranosyl)purine (3b) was transformed to 2′,3′-O-isopropylidene-O⁶-(4-nitrophenyl)inosine (1b). Deprotection of 1b with CF₃COOH gave compound la and O⁶ -(4-nitrophenyl)hypoxanthine (4). Compounds 1a and 1c are substrates for adenosine deaminase releasing 4-nitrophenol which is readily detected visually or spectrophotomemcally. Rate and extent of hydrolysis of la are significantly increased in the presence of purine nucleoside phosphorylase but xanthine oxidase has no influence. A potential fluorogenic analogue 2 is not a substrate for adenosine deaminase.

     

Synthesis and evaluation of in vitro anticancer activity of some novel isopentenyladenosine derivatives

The present study describes the synthesis, the characterization and the evaluation of some derivatives of N⁶-isopentenyladenosine on T24 human bladder carcinoma cells. In particular we have modified the hydroxyl groups in the ribose moiety, the position of the isopentenyl chain in the purine ring and the base moiety. The structures of the compounds were confirmed by standard studies of NMR, MS and elemental analysis. We here show that only two derivatives, 1-(3-methyl-2-butenylamino)-9-(3′-deoxy-β-d-ribofuranosyl)-purine hydrobromide and 2-amino-6-(3-methyl-2- butenylamino)-9-(β-d-ribofuranosyl)-purine, inhibit the growth of T24 cells, although to a lower extent than N⁶-isopentenyladenosine. We conclude that the integrity of ribosidic and purine moiety and the N⁶ position of the chain are essential for maintaining the antiproliferative activity.     

Degradation of the novel herbicide ZJ0273 by Amycolatopsis sp. M3-1 isolated from soil

ZJ0273, propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoate, is a novel and broad-spectrum herbicide. In this study, 15 bacteria capable of utilizing ZJ0273 as the sole carbon source were isolated from soil. One of the isolates belonged to the family Amycolatopsis and was designated to Amycolatopsis sp. M3-1; at 30°C and pH 7.0, degradation rate of ZJ0273 could reach at 59.3% and 68.5% in 25 days and 60 days, respectively. Furthermore, six metabolites (M1–M6) during the degradation of ZJ0273 by Amycolatopsis sp. M3-1 were identified by a combination with multi-position ¹⁴C-labeled compounds (B-ZJ0273 and C-ZJ0273), chromatography, liquid scintillation spectrometer, and LC–MS, a novel pathway of ZJ0273 degradation by Amycolatopsis sp. M3-1 was proposed based on the identified metabolites and their biodegradation courses. ZJ0273 was initially hydrolyzed into M1 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzylamino) benzoic acid), then further oxidized into M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid). M1 also could undergo a carbonylation into M2 (4-(2-(4,6-dimethoxypyrimidin-2-yloxy) benzamido) benzoic acid), and then its C–N and C–O bonds were cleaved to yield M3 (2-(4,6-dimethoxypyrimidin-2-yloxy) benzoic acid) and M4 (4,6-dimethoxypyrimidin-2-ol), respectively. Moreover, another two new metabolites, M5 (2-(4-hydroxy, 6-methoxypyrimidin-2-yloxy) benzoic acid) and M6 (2, 4-dihydroxy-pyrimidine) were found. M5 was formed through de-methyl of M3 and then hydrolyzed into M6.     

Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa

Mesophyll protoplasts obtained from leaves of shoot cultures of Rehmannia glutinosa were cultured in Murashige and Skoog (1962) liquid or liquid-over-agar medium containing 2.0 mg L^?1 naphthaleneacetic acid and 0.5 mg L^?1 benzylamino purine. An amino acid mixture of glutamine, arginine, glycine, and aspartic acid promoted sustained protoplast division, with an average plating efficiency of 27%. Protoplast-derived colonies formed callus which readily regenerated shoots on fransfer to Murashige and Skoog based agar medium with 2.0 mg L^?1 indoleacetic acid and 1.0 mg L^?1 benzylamino purine. Leaf explants also showed a marked capacity for shoot regeneration in culture.     

Cytokinin Biochemistry in Relation to Leaf Senescence : II. The Metabolism of 6-Benzylaminopurine in Soybean Leaves and the Inhibition of Its Conjugation

The metabolism of [³H]6-benzylamino purine was studied in presenescent and early senescent soybean (Glycine max [L.] Merr.) leaves. In both types of leaves, the metabolism was essentially the same. The principal metabolite was identified as β-(6-benzylaminopurin-9-yl)alanine by mass spectral studies, which included discharge ionization-secondary ion mass spectrometry and pulsed positive ion-negative ion-chemical ionization mass spectrometry. Conversion to this alanine conjugate was found to be inhibited 2,4-dichlorophenoxyacetic acid and 5,7-dichloroindoleacetic acid.     

Rapid repression of vacuolar invertase in mungbean hypocotyl segments and regulation by sucrose, auxin and light

Two previously unrecorded endogenous cytokinin metabolites,6-[2-(-D-glucopyranosyloxy)benzylamino]purine and6-[2-(-D-glucopyranosyloxy)benzylamino]-2-methylthiopurine, wereidentified, together with 6-benzylamino-9--D-glucopyranosylpurine(BAP9G),from Chenopodium rubrum cells, grown autotrophically insuspension culture. The new metabolites belong to the aromatic class ofcytokinins, in which an aromatic side chain is attached at theN⁶-position of the adenine species. The identification was performedby capillary-liquid chromatography/frit-fast atom bombardment - massspectrometry (LC/FAB MS) after pre-column derivatisation and the structuralelucidation was confirmed by organic synthesis. Cytokinin activity of thecompounds was tested in an Amaranthus bioassay. Theendogenous synthesis of the identified compounds was verified byin vivo deuterium labelling of the analysed cytokininspecies, thereby for the first time providing absolute evidence for theendogenous origin of these compounds.     

Glucosylation of cytokinin analogues

Glucosylation of adenine and 6-methylaminopurine was not detected in derooted 10-day-old radish seedlings. However, 4-(purin-6-ylamino)butanoic amide and 6-(3,4-dimethoxybenzylamino)purine (N⁶-substituted adenines with negligible cytokinin activity), like the highly active cytokinin 6-benzylaminopurine, were converted to 7-glucopyranosides. The N²-substituted guanine, 2-benzylaminopurin-6-one, and 6-benzylamino-2-(2-hydroxy-ethylamino)purine were also metabolized to glucosides which were probably 7-glucopyranosides. Hence glucosylation of purines is not restricted to N⁶-substituted adenines with strong cytokinin activity. Although only ca 1.6% of 6-benzylamino-9-(4-chlorobutyl)purine taken up by the derooted seedlings could be accounted for as 7- and 9-glucosides, a considerable proportion was metabolized to these glucosides in cotyledons excised from 2-day-old radish seedlings. The high cytokinin activity of this 9-substituted compound may be a consequence of cleavage of the 4-chlorobutyl groud at N-9.     

6-(gamma,gamma-Dimethylallylamino)Purine As A Precursor of Zeatin

A 6-(γ,γ-dimethylallylamino) purine-like compound was found in the culture medium of Rhizopogon roseolus, which had been shown earlier to synthesize zeatin. The role of 6-(γ,γ-dimethylallylamino) purine as a precursor of zeatin was studied. Rhizopogon was furnished with 6-(γ,γ-dimethylallylamino) purine-8-¹⁴C. Cochromatography, oxidation studies with potassium permanganate, and bromination indicated that labeled zeatin ribonucleoside was isolated from the medium. The fungus also incorporated labeled adenine, hypoxanthine, and 4-amino-5-imidazole carboxamide into zeatin ribonucleoside.     

Temperature-dependent Response to Indoleacetic Acid Is Altered by NH(4) in Cultured Cotton Ovules

Cut carnations (Dianthus caryophyllus L. cv. `Improved White Sim') were exposed to ultra high purity ¹⁴C₂H₄ (20 μl/1) during flower opening and senescence to study its incorporation and metabolism. During treatment precautions were taken to exclude inhibitory volatiles from rubber serum stoppers which were identified as CS₂ and COS. As with the pea seedling (Nature 1975, 255:144-147), cut carnations incorporated ¹⁴C₂H₄ into ethanol-soluble tissue metabolites and oxidized the hormone to ¹⁴CO₂. Oxidation increased from 0.5 to 3 dpm · mg dry wt⁻¹·6 hr⁻¹ during the period of flower opening and early petal wilt. As severe petal wilt set in, and the ovary increased in size and dry weight, oxidation increased to a peak of nearly 29 dpm · mg dry wt⁻¹·6 hr⁻¹. Concomitant with this peak was a similar rise in the rate of ¹⁴C₂H₄ incorporation into the petals, peduncle, bracts, and sepals. Much higher rates of incorporation were found for the reproductive and receptacle tissues. Incorporation into these tissues steadily increased during flower opening reaching a peak of over 160 dpm · mg dry wt⁻¹ · 6 hr⁻¹ just before full bloom. This peak preceded a peak of endogenous ethylene production while the ¹⁴C₂H₄ oxidation peak followed it.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Dinuclear copper(II) complexes containing 6-(benzylamino)purines as bridging ligands: Synthesis, characterization, and in vitro and in vivo antioxidant activities

A series of dinuclear copper(II) complexes involving 6-(benzylamino)purine derivatives, (HL_n), as bridging ligands were synthesized, characterized and tested for both their in vitro and in vivo antioxidant activities. Based on results of elemental analyses, temperature dependence of magnetic susceptibility measurements, UV-vis, FTIR, EPR, NMR and MALDI-TOF mass spectroscopy, conductivity measurements and thermal analyses, the complexes with general compositions of [Cu₂(μ-HL_n)₄Cl₂]Cl₂ · 2H₂O (1-4) and [Cu₂(μ-HL_n)₂(μ-Cl)₂Cl₂] (5-7) were prepared {where n = 1-4; HL₁ = 6-[(2-methoxybenzyl)amino]purine, HL₂ = 6-[(4-methoxybenzyl)amino]purine, HL₃ = 6-[(2,3-dimethoxybenzyl)amino]purine and HL₄ = 6-[(3,4-dimethoxybenzyl)amino]purine}. In the case of complexes 2, 3, 5 and 7, the antioxidant activities were studied by both in vitro {superoxide dismutase-mimic (SOD-mimic) activity} and in vivo {cytoprotective effect against the alloxan-induced diabetes (antidiabetic activity)} methods. The obtained IC₅₀ value of the SOD-mimic activity for the complex 5 (IC₅₀ = 0.253 μM) was shown to be even better than that of the native bovine Cu,Zn-SOD enzyme (IC₅₀ = 0.480 μM), used as a standard. As for the antidiabetic activity, the pretreatment of mice with complexes 3 and 7 led to the complete elimination of cytotoxic attack of alloxan and its free radical metabolites, used as a diabetogenic agent. The cytoprotective effect of these compounds was proved by the preservation of the initial blood glucose levels of the pretreated animals, as against the untreated control group.     

Activity and metabolism of C-(+/-)-abscisic Acid derivatives

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-¹⁴C-abscisic acid and the cis- and trans-1′,4′-diols of 2-¹⁴C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.     

Protoplast-derived streptomycin resistant plants of the forage legume Onobrychis viciifolia Scop. (sainfoin)

Approximately 10⁶ protoplast-derived cell colonies of sainfoin were stressed with streptomycin and two resistant colonies were recovered. Plants regenerated from these colonies could be recallused on streptomycin-containing medium three years after growth in the absence of the antibiotic.Ultrastructural studies showed cells of resistant callus grown in the presence of streptomycin to contain chloroplasts with internal thykaloids and grana. Such mutant plants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.Abbreviations BAP 6 - benzylamino purine - NAA - napthaleneacetic acid     

6-(2, 3, 4-Trihydroxy-3-methylbutylamino)purine,a cytokinin with high biological activity

6-(2, 3, 4-Trihydroxy-3-methylbutylamino)purine, isolated from the oxidation of cis- zeatin with potassium permanganate, has been identified by ¹H NMR and high resolution mass spectrometry. Its activity as a cell division factor, when examined by the soybean callus assay in the concentration range 10^?11–10^?5 M, equalled that of the parent compound.     

Dark fixation of CO2 by flowers of cut roses

Summary Complete flower heads of cut roses (cv. Baccara) were exposed to ¹⁴CO₂ for 1–4 h. The flower tissue was able to fix CO₂ via PEP carboxylase (E.C. 4.1.1.31) in the dark; various TCA products were identified in petals, ovary and anthers, including malate, aspartate, citrate, serine/glycine, glutamate and asparagine. The concentrations of these labelled products were similar in the petals and anthers, but lower in the ovary. After removal of the petals the amounts of these components were reduced in the anthers to a relatively high extent (to 1/6), whereas the amounts in the ovary increased slightly. It is suggested that the petals are necessary for supplying the anthers with the described components.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - MDH malate dehydrogenase - NADH nicotinamideade-ninedinucleotide - PEP phosphoenolpyruvate - PVP polyvinylpyrrolidone - TCA tricarboxylic acid     

Synthesis,characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives

In an attempt to improve specific biological functions of cytokinins routinely used in plant micropropagation, 33 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine (THFP) derivatives, with variously positioned hydroxy and methoxy functional groups on the benzyl ring, were prepared. The new derivatives were prepared by condensation of 6-chloropurine with 3,4-dihydro-2H-pyran or 2,3-dihydrofuran and then by the condensation of these intermediates with the corresponding benzylamines. The prepared compounds were characterized by elemental analyses, TLC, HPLC, melting point determinations, CI+ MS and ¹H NMR spectroscopy. The cytokinin activity of all the prepared derivatives was assessed in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The derivatives 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (3) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23) were selected, because of the high affinity of their parent compound meta-topolin (mT, 6-(3-hydroxybenzylamino)purine) to cytokinin receptors, as model compounds for studying their perception by the receptors CRE1/AHK4 and AHK3 in a bacterial assay. Both receptors perceived these two derivatives less well than they perceived the parent compound. Subsequently, the susceptibility of several new derivatives to enzyme degradation by cytokinin oxidase/dehydrogenase was studied. Substitution of tetrahydropyran-2-yl (THP) at the N⁹ position decreased the turnover rates of all new derivatives to some extent. To provide a practical perspective, the cytotoxicity of the prepared compounds against human diploid fibroblasts (BJ) and the human cancer cell lines K-562 and MCF-7 was also assayed in vitro. The prepared compounds showed none or marginal cytotoxicity compared to the corresponding N⁹-ribosides. Finally, the pH stability of the two model compounds was assessed in acidic and neutral water solutions (pH 3–7) by high-performance liquid chromatography (HPLC).     

Non-targeted analysis of spatial metabolite composition in strawberry (Fragariaxananassa) flowers

Formation of flower organs and the subsequent pollination process require a coordinated spatial and temporal regulation of particular metabolic pathways. In this study a comparison has been made between the metabolite composition of individual flower organs of strawberry (Fragaria × ananassa) including the petal, sepal, stamen, pistil and the receptacle that gives rise to the strawberry fruit. Non-targeted metabolomics analysis of the semi-polar secondary metabolites by the use of UPLC-qTOF-MS was utilized in order to localize metabolites belonging to various chemical classes (e.g. ellagitannins, proanthocyanidins, flavonols, terpenoids, and spermidine derivatives) to the different flower organs. The vast majority of the tentatively identified metabolites were ellagitannins that accumulated in all five parts of the flower. Several metabolite classes were detected predominantly in certain flower organs, as for example spermidine derivatives were present uniquely in the stamen and pistil, and the proanthocyanidins were almost exclusively detected in the receptacle and sepals. The latter organ was also rich in terpenoids (i.e. triterpenoid and sesquiterpenoid derivatives) whereas phenolic acids and flavonols were the predominant classes of compounds detected in the petals. Furthermore, we observed extensive variation in the accumulation of metabolites from the same class in a single organ, particularly in the case of ellagitannins, and the flavonols quercetin, kaempferol and isorhamnetin. These results allude to spatially-restricted production of secondary metabolite classes and specialized derivatives in flowers that take part in implementing the unique program of individual organs in the floral life cycle.