Immunodetection and Identification of N6-(o-Hydroxybenzylamino)Purine as a Naturally Occurring Cytokinin in Populus x canadensis Moench cv Robusta Leaves

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Abstract
A highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for 9-beta-d-ribofuranosyl-N(6)-(o-hydroxybenzylamino)purine [(oOH)[9R]BAP] and structurally related cytokinins. As little as 3 femtomoles of the compound could be detected by this method. Cross-reactivity studies demonstrated the specificity of four polyclonal antibodies for (oOH)[9R] BAP and its free base in preference to a range of natural cytokinins and other purines. After evaluating the method by internal standardization employing [2-(3)H](oOH)[9R]BAP of high specific radioactivity as recovery marker by dilution analyses and by immunohistograms, it was possible to apply ELISA to quantify (oOH)[9R]BAP in plant extracts. In addition to (oOH)[9R]BAP, an unknown cytokinin reacting with the same antibody was detected in high performance liquid chromatography-fractionated extracts of mature Populus x canadensis Moench cv Robusta. The structure of the new compound was determined by gas chromatography-mass spectrometry and finally confirmed by synthesis as N(6)-(o-hydroxybenzylamino)purine.