Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins Journal Article

Author(s): Kubiasová, Karolina; Mik, Václav; Nisler, Jaroslav; Hönig, Martin; Husičková, Alexandra; Spíchal, Lukáš; Pěkná, Zuzana; Šamajová, Olga; Doležal, Karel; Plíhal, Ondřej; Benková, Eva; Strnad, Miroslav; Plíhalová, Lucie
Article Title: Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins
Affiliation IST Austria
Abstract: Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.
Keywords: Cytokinin; 6-[(3-methylbut-2-en-1-yl)amino]purine; ARR5:GUS; competitive receptor bioassay; fluorescent label; fluorescent probe; isoprenoid; linker; live cell confocal microscopy; N6-isopentenyladenine
Journal Title: Phytochemistry
Volume: 150
ISSN: 0031-9422
Publisher: Elsevier  
Date Published: 2018-06-01
Start Page: Epub ahead of print
DOI: 10.1016/j.phytochem.2018.02.015
Notes: This work was supported by the Ministry of Education Youth and Sports, Czech Republic (grant LO1204 from the National Program of Sustainability I and Agricultural Research ) and by Czech Science Foundation grants 16-04184S , 501/10/1450 and 13-39982S and by IGA projects IGA_PrF_2018_033 and IGA_PrF_2018_023 . We would like to thank Jarmila Balonová, Olga Hustáková and Miroslava Šubová for their skillful technical assistance and Mgr. Tomáš Pospíšil, Ph.D. for his measurement of 1 H NMR and analysis of some 2D NMR spectral data.
Open access: no