Synthesis of 2-methylthio-cis- and trans-ribosylzeatin and their isolation from Pisum tRNA

The cis isomer of 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthiopurine and its 9-β- and 9-α-d-ribofuranosyl derivatives have been synthesized and their physical and spectroscopic properties are described. The biological activities of these compounds have been determined in the tobacco bioassay and are compared with those of 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-2-methylthiopurine and its β-ribofuranoside. The 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d-ribofuranosylpurine (ms-ribosylzeatin) isolated from a Pisum tRNA preparation was shown to consist of both isomers, which were separated by TLC and identified by comparisons of UV and MS with those of the synthetic compounds.     

Comparison of cytokinin activities of naturally occurring ribonucleosides and corresponding bases

Cytokinin activities in the tobacco bioassay have been determined for four adenosine derivatives known to be components of wheat germ tRNA: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino)- 2-methylthio-9-β-d-ribofuranosylpurine, and 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-d-ribofuranosylpurine. Also determined and compared with the four natural components of tRNA were the activities of the four 3-methylbutylamino analogs of the naturally occurring species and the eight substituted purines corresponding to both sets of ribonucleosides. The systematic structural modifications within this group of sixteen compounds were reflected in the variations in cytokinin activity with the level of modification.     

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).     

Cytokinins in cut carnation flowers. VII. The effect of zeatin and dihydrozeatin derivatives on flower longevity

Dihydrozeatin, at 4×10^–5 M, delayed the senescence of carnation flowers while tZ, at the same concentration, accelerated it. cis-Zeatin was ineffective. The DHZ derivatives as well as the Z derivatives gave responses very similar to those observed for the parent free bases. While additional experimentation with radiolabelled derivatives is clearly called for, the similarity between the responses observed for the respective derivatives and the free bases, suggests that in the carnation flower there is a great deal of metabolic interconversion.Abbreviations DHZ dihydrozeatin - DHZR ribosyldihydrozeatin - DHZOG glucosyl-O-dihydrozeatin - DHZ9G glucosyl-9-dihydrozeatin - DHZROG glucosyl-O-ribosyldihydrozeatin - cZ cis-zeatin - tZ trans-zeatin - ZR ribosylzeatin - Z9G glucosyl-9-zeatin - ZOG glucosyl-O-zeatin - ZROG glucosyl-O-ribosylzeatin     

Cytokinins: synthesis and biological activity of geometric and position isomers of zeatin

Geometric and position isomers of zeatin and of ribosylzeatin and other compounds closely related to zeatin have been tested in the tobacco (Nicotiana tabacum var. Wisconsin No. 38) bioassay. None was more active than zeatin itself. There was a much greater difference in activity (> 50-fold) between trans- and cis-zeatin than between trans-isozeatin [6-(4-hydroxy-2-methyl-trans-2-butenylamino) purine] and cis-isozeatin [6-(4-hydroxy-2-methyl-cis-2-butenylamino) purine], the latter being less active than cis-zeatin and trans-isozeatin. Higher concentrations were required for equivalent callus growth stimulated by the 9-ribosyl derivatives, which followed an order of decreasing activity: ribosyl-trans-zeatin > ribosyl-cis-zeatin > ribosyl-trans-isozeatin > ribosyl-cis-isozeatin, corresponding roughly to that of the bases. The effect of side chain, double bond saturation was to diminish the activity, and in the dihydro series the shift of the methyl group from the 3- to the 2-position in going from dihydrozeatin to dihydroisozeatin [6-(4-hydroxy-2-methylbutylamino) purine] resulted in a 70-fold decrease in activity. cis-Norzeatin [6-(4-hydroxy-cis-2-butenylamino) purine], which was less than one-fifth as active as cis-zeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3,6-dihydro-1,2-oxazin-2-yl) purine] was about 1/100 as active as cis-norzeatin. These findings delineate completely the effect on the cytokinin activity of zeatin of variation in side chain geometry, presence and position of the methyl substituent, presence and geometry of hydroxyl substitution, presence of the double bond, and of side chain cyclization.     

Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts

Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation.     

Cytokinins in pisum transfer ribonucleic Acid

Five cytokinin-active ribonucleosides have been isolated from the transfer RNA of 7-day-old green pea shoots (Pisum sativum L. var. Alaska). Ultraviolet spectroscopy and mass spectrometry have been used to identify 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β- d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine. The latter was separated into the cis- and trans-isomers by thin layer chromatography. The fifth cytokinin is indicated to be 6-(3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine on the basis of its chromatographic properties.     

Isolation and identification of cytokinins from Euglena gracilis transfer ribonucleic acid.

Three ribonucleosides responsible for cytokinin activity in Euglena gracilis var Bacillaris tRNA have been isolated and identified as 6-(3-methyl-2-butenylamino)-9-beta-D-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-D-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine. The structures of these compounds were assigned on the basis of their chromatographic properties and ultraviolet and mass spectra which were identical with those of the corresponding synthetic compounds. The elution profiles of cytokinin bioassay activity and of 35S radioactivity suggest the presence of a trace amount of 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine.     

Cytokinins in immature seeds of Dolichos lablab

Five cytokinins, trans-zeatin, 9-β-d-ribofuranosyl-trans-zeatin, 9-β-d-ribofuranosyl-cis-zeatin, 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)purine and 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine were identified from immature seeds of Dolichos lablab.     

Studies on avian heart pyruvate kinase during development.

The cytokinin-active nucleoside 6-(4-hydroxy-3-methyl-$\text{cis}$-2-butenylamino)-9-β-$\text{D}$-ribofuranosylpurine, i.e. ribosyl-$\text{cis}$-zeatin, has been isolated from an hydrolysate of tRNA from $\text{Corynebacterium fascians}$. The identification of ribosyl-$\text{cis}$-zeatin is based on biological activity, liquid chromatographic mobility and uv spectrum of the purified material as well as the mass spectrum and gas chromatographic mobility of its trimethylsilylated derivative.     

Mode of action of N,N′-diphenylurea: The isolation and identification of the cytokinins in the transfer RNA from tobacco callus grown in the presence of N,N′-diphenylurea

Summary The tRNA from cytokinin-dependent tobacco callus (Nicotiana tabacum) grown on mineral medium containing N,N-diphenylurea as the source of cytokinin was found to contain 3 cytokinin-active ribonucleosides. The 2 ribonucleosides present in the largest amounts were identified conclusively by their chromatographic properties, ultra-violet and low-resolution mass spectra as the naturally-occurring cytokinins 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine and 6-(3-methyl-2-butenylamino)-9--ribofuranosylpurine. A third ribonucleoside, present in smaller amounts, was identified as another naturally-occurring cytokinin 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9--D-ribofuranosylpurine on the basis of its chromatographic behaviour. No evidence was found to associate the mode of action of the non-purine cytokinin, N,N-diphenylurea, with tRNA.Abbreviation DPU N,N-diphenylurea     

The effect of cytokinins on growth and sexual organ development in the gametophyte of <Emphasis Type="Italic">Blechnum spicant</Emphasis> L.

In this study, we report the role of exogenous and endogenous cytokinins on growth and sexual organ development in the fern Blechnum spicant L. Spore-derived gametophytes (SG) were cultured in full-strength Murashige and Skoog (1962) liquid medium supplemented with (a) 4.44 μMN⁶-benzyladenine (BAP), (b) a crude extract from mature female gametophytes, and (c) 4.44 μM BAP in combination with the crude extract from mature gametophytes, respectively. Both BAP and the crude extract delayed the gametophyte development, and this effect was increased when they were added together. With respect to sexual organ development, BAP inhibited the sexual organ formation, while the crude extract favored antheridia formation; however, when added together, the percentage of antheridia decreased. The endogenous level of the cytokinins cis-zeatin (cZ), cis-zeatin-riboside (cZR), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), isopentenyl-9-glucoside (iP9G), trans-zeatin (tZ), and trans-zeatin riboside (tZR) were analyzed in female and male gametophytes of B. spicant L. The endogenous levels of cytokinins tZ, cZ, DHZ, cZR, iP, and iPR were higher in female gametophytes than in male gametophytes, with the endogenous iP and iPR content being increased more than 300 and 400 times, respectively.     

Effect of stereospecific hydroxylation of N6-(Δ 2-Isopentenyl)adenosine on cytokinin activity

The cytokinin activities of cis and trans ribosylzeatin isomers and that of N⁶-(²-isopentenyl)adenosine were compared in four bioassays. The trans isomer was found to be more active than the cis isomer in stimulation of cucumber cotyledon expansion (100x), retention of chlorophyll in detached leaf pieces (7x), induction and stimulation of chlorophyll synthesis in cucumber cotyledons (20x) and of betacyanin synthesis in Amaranthus caudatus seedlings grown in the dark (60x). The N⁶-(²-isopentenyl)adenosine adenosine was less active than the trans ribosylzeatin in all four bioassays and more active than the cis ribosylzeatin in induction and stimulation of betacyanin and chlorophyll synthesis. These results show that the hydroxylation of the trans methyl group in the N⁶ side chain of N⁶-(²-isopentenyl)adenosine increases the biological activity and that this activity is either decreased or not significantly changed when the cis methyl group is hydroxylated.Abbreviations i⁶Ado N⁶-(²-isopentenyl)adenosine or 6-(3-methyl-2-butenylamino)-9--D-ribofuranosylpurine - t-to⁶Ado trans-ribosylzeatin or 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9--D-ribofuranosylpurine - c-io⁶Ado cis-ribosylzeatin or 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine - HPLC high pressure liquid chromatography     

Identification of plant hormones from cotton ovules

An extract from 8-day-old cotton ovules (Gossypium hirsutum L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A(1), A(3), A(4), A(7), A(9), and A(13) in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2-trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine(2iP), 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine(2iPA), and 6-(4-hydroxy-3-methyl-2-trans-butenylamino)-9-beta-d- ribofuranosylpurine (zeatin riboside) in the third fraction.     

Subcellular localization of cytokinins in transfer ribonucleic Acid

Evidence on the localization of cytokinins in chloroplast tRNA was obtained by comparison of Euglena gracilis var. bacillaris light-grown and dark-grown wild type cultures and chloroplast-bleached mutant strains. The several cytokinins characteristic of tRNA were separated by Sephadex LH-20 column chromatography of the hydrolysates and were quantitatively determined by tobacco bioassays of the eluates. The results indicate that 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine (i(6) A) is formed in both the cytoplasmic and chloroplast tRNA, whereas 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-d- ribofurano-sylpurine (c-io(6)A) is produced mainly in the cytoplasmic tRNA and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-d- ribofurano-sylpurine (ms(2)io(6)A) is localized exclusively in chloroplast tRNA. The restriction of the methiolation reaction to the chloroplast is supported by results of radioisotope experiments showing that (35)S-labeled MgSO(4) is incorporated into ms(2)io(6)A in the wild type cultures, but not in the chloroplast-bleached mutant strains.     

Cytokinins from Zea mays

A number of adenine derivatives with cytokinin activity were isolated from immature sweet corn (Zea mays) kernels. The following structures were assigned: 9-β-d-ribofuranosylzeatin, 9-β-d-ribofuranosylzeatin 5′-monophosphate, 6-(1-carboxy-2-hydroxypropylamino)-9-ribofuranosylpurine, 6-(2,3,4-trihydroxy-3-methylbutylamino)purine, 2-hydroxy-6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine, 6-(3,4-dihydroxy-3-methylbutylamino)purine, a 9-glycoside of zeatin(identity of sugar moiety not established), and 6-(1,2-dicarboxyethylamino)-9-β-d-ribofuranosylpurine.     

Cytokinin biosynthesis in cultured rootless tobacco plants

Biosynthesis of cytokinin in shoots was examined by growing rootless tobacco (Nicotiana tabacum) plants in vitro. The rootless plants were originated by culturing tobacco callus on a high cytokinin-low auxin medium to induce the formation of plantlets which were then grown on medium without exogenous cytokinin and auxin. The rootless plants supplied with [(14)C]adenine synthesized ethanol-ethyl acetate-water-soluble radioactive components, portions of which had the same chromatographic and electrophoretic mobilities as N(6)-(Delta(2)-isopentenyl)adenine, N(6)-(Delta(2)-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine. The total amount of these four major cytokinins was estimated to be present at a concentration of 14 to 23 nanomoles per kilogram of rootless plant. These data indicate that adenine serves as a precursor of the purine moiety of cytokinin molecules and that the cytokinin biosynthetic sites are also located in the shoot in addition to the presumed root sites.     

Cytokinin Activity of cis-Zeatin and Phenotypic Alterations Induced by Overexpression of Putative cis-Zeatin-O-glucosyltransferase in Rice

cis-Zeatin (cZ) is generally regarded as a cytokinin with little or no activity, compared with the highly active trans-zeatin (tZ). Although recent studies suggested possible roles for cZ, its physiological significance remains unclear. In our studies with rice (Oryza sativa), cZ inhibited seminal root elongation and up-regulated cytokinin-inducible genes, and its activities were comparable to those of tZ. Tracer experiments showed that exogenously supplied cZ-riboside was mainly converted into cZ derivatives but scarcely into tZ derivatives, indicating that isomerizations of cZ derivatives into tZ derivatives are a minor pathway in rice cytokinin metabolism. We identified three putative cZ-O-glucosyltransferases (cZOGT1, cZOGT2, and cZOGT3) in rice. The cZOGTs preferentially catalyzed O-glucosylation of cZ and cZ-riboside rather than tZ and tZ-riboside in vitro. Transgenic rice lines ectopically overexpressing the cZOGT1 and cZOGT2 genes exhibited short-shoot phenotypes, delay of leaf senescence, and decrease in crown root number, while cZOGT3 overexpressor lines did not show shortened shoots. These results propose that cZ activity has a physiological impact on the growth and development of rice.     

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.     

Influence of alterations in the purine ring on biological activity of cytokinins

The 1-deaza-, 3-deaza-, 8-aza-1-deaza- and 8-aza-3-deaza-analogs of kinetin and 6-(3-methyl-2-butenylamino)purine and some of their ribosides were synthesized and their growth-promoting activities in the tobacco bioassay were determined and compared with those of the parent compounds. The replacement of nitrogen by carbon in the 1 -position of the purine ring decreases cytokinin activity 15-fold for kinetin and 2-fold for 6-(3-methyl-2-butenylamino)purine (IPA); however, the replacement of nitrogen by carbon in the 3-position decreases the activity 2000 times for kinetin and 1000 times for 6-(3-methyl-2-butenylamino)-purine. The activity of 8-aza-1-deaza-analogs appears to be of the same order of somewhat lower than the corresponding 1-deaza-analogs. The corresponding 8-aza-3-deaza-analogs are less active than kinetin (400 times and 6-(3-methyl-2-butenylamino)purine (40 times). However, they are more active than the corresponding 3-deaza-analogs. The concentration range in which the ribosides show activity is nearly the same as for the corresponding free bases, but the maximum yield of tobacco-callus for the riboside of the 3-deaza-analog of 6-(3-methyl-2-butenylamino)purine is very low.