Cytokinins : Synthesis and Biological Activity of Geometric and Position Isomers of

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 transand 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 3to the 2-position in goillg from dihydrozeatin to dihydroisozeatin [6(4hydroxy-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 ciszeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3, 6-dihydro-1, 2-oxazin2-yl)purine] was about /l4oo 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. cis-zeatin2 (II, III) (9) both of which have been found as their 9ribosyl derivatives in tRNA (13, 18). Because of the variety of modification of terpenoid structures in nature and in order to extend our knowledge of the influence of side chain geometry on biological activity, we have determined the structure-activity relationship of transand cis-isozeatin (V, VI) and their 9-ribosyl derivatives (XIV, XV), together with dihydrozeatin (IV) and its 9-ribonucleoside (XIII), dihydroisozeatin (VII), cis-norzeatin (VIII), and cyclic-norzeatin (IX), all compared in the tobacco callus bioassay with the naturally occurring 6-(3-methyl-2-butenylamino)purine, 2iP (I), and its 9-ribonucleoside, 2iPA (X). MATERIALS AND METHODS Bioassay Procedure. The tobacco bioassay (12) was used to determine the cytokinin activity. The medium contained the specified mineral salts (Table 6, part A, of ref. 12) and the following organic constituents: 30 g/liter of sucrose, 10 g/liter of Difco agar, 100 mg/liter of myoinositol, 2 mg/liter of indole-3-acetic acid, and 0.4 mg/liter of thiamine hydrochloride. The chemicals to be tested were dissolved in dimethylsulfoxide and added to the cooling autoclaved media at the uniform rate of 0.05% (v/v), (0.025 ml of (CH3)2SO solution to each flask with 50 ml of medium). The use of (CH3)2SO as specified does not affect growth of the tobacco tissue, and permits the addition of the test substances in sterile solutions after autoclaving, thus protecting the compounds from possible degradation by heat (14). Stock callus tissue (Nicotiana tabacum var. Wisconsin No. 38), was maintained on the above medium supplemented with 300 ,ug/liter of kinetin, and put through two 3-week passages on medium supplemented with 30 .g/liter of kinetin before use for bioassays. For each treatment 12 pieces of callus, about 40 mg each, were planted three apiece in 125-ml Erlenmeyer flasks containing 50 ml of medium. The cultures were kept at 28 C and were ocThere is a marked influence of side chain substitution and configuration on the cytokinin activity of compounds related to 6-(3methyl-2-butenylamino)purine (I),2iP, (1, 2, 5-7, 16), which is especially evident in the difference in activity between transand 'This work was supported at the University of Wisconsin by National Science Foundation Research Grant GB-25812 and by the Research Committee of the Graduate School with funds from the Wisconsin Alumni Research Foundation. Supported at the University of Illinois by National Institutes of Health Research Grant GM-05829. A. J. P. held the Eli Lilly and Company Fellowship at the University of Illinois during 1970 to 1971. 2Abbreviations: Zeatin or trans-zeatin: 6-(4-hydroxy-3-methyltrans-2-butenylamino)-9-,8-D-ribofuranosylpurine; ribosyl-cis-zeatin: 2-butenylamino)purine; ribosyl-trans-zeatin: 6-(4-hydroxy-3-methyltranis-2-butenylamino)9-3-D-ribofuranosylpurine; ribosyl-cis-zeatin: 6-(4-hydroxy -3 methyl-cis2-butenylamino)-9-,-D-ribofuranosylpu rine; tratns-isozeatin: 6-(4-hydroxy-2-methyl-trans-2-butenylamino) purine; cis-isozeatin: 6-(4-hydroxy-2-methyl-cis-2-butenylamino)purine; dihydroisozeatin: 6(4-hydroxy2-methylbutylamino)purine; cis-norzeatin: 6-(4-hydroxy-cis-2-butenylamino)purine; cyclic-norzeatin: 6-(3, 6-dihydro-1, 2-oxazin-2-yl)purine; 2iP: 6-(3-methyl-2butenylamino)purine or N8-(A2-isopentenyl)adenine; 2iPA or, in shortened form, PA: 6-(3-methyl-2-butenylamino)-9-,8-D-ribofurano sylpurine or N'-(\2-isopentenyl)adenosine. 702 www.plantphysiol.org on August 15, 2017 Published by Downloaded from Copyright © 1972 American Society of Plant Biologists. All rights reserved. GEOMETRIC AND POSITION ISOMERS OF ZEATIN