Preparation, biological activity and endogenous occurrence of N6-benzyladenosines.

Cytokinin activity of forty-eight 6-benzyladenosine derivatives at both the receptor and cellular levels as well as their anticancer properties were compared in various in vitro assays. The compounds were prepared by the condensation of 6-chloropurine riboside with corresponding substituted benzylamines and characterized by standard collection of physico-chemical methods. The majority of synthesized derivatives exhibited high activity in all three of the cytokinin bioassays used (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The highest activities were observed in the senescence bioassay. For several of the compounds tested, significant differences in activity were found between the bioassays used, indicating that diverse recognition systems may operate. This suggests that it may be possible to modulate particular cytokinin-dependent processes with specific compounds. In contrast to their high activity in bioassays, the tested compounds were recognized with only very low sensitivity in both Arabidopsis thaliana AHK3 and AHK4 receptor assays. The prepared derivatives were also investigated for their antiproliferative properties on cancer and normal cell lines. Several of them showed very strong cytotoxic activity against various cancer cell lines. On the other hand, they were not cytotoxic for normal murine fibroblast (NIH/3T3) cell line. This anticancer activity of cytokinin ribosides may be important, given that several of them occur as endogenous compounds in different organisms.

Batch immunoextraction method for efficient purification of aromatic cytokinins.

A range of benzylaminopurines naturally occur in plants and exhibit high biological activity. Others have been synthesized, such as 6-(2-hydroxy-3-methoxybenzylamino)purine riboside (2OH3MeOBAPR), which has shown interesting anti-cancer activity under in vitro conditions. In order to study the biological activity of this interesting compound in more detail, a rapid and highly efficient method for its purification from complex samples (e.g. blood and plant extracts) is needed. Therefore, we prepared monoclonal antibodies against 2OH3MeOBAPR. The antibody had undetectable cross-reactivity with all natural isoprenoid cytokinins, but relatively high cross-reactivity with aromatic cytokinins as well as some synthetic di- and tri-substituted 6-benzylaminopurines and the corresponding ribosides. The antibody also showed strong responses and specificity in enzyme-linked immunoassays (ELISAs). In addition, it was used to prepare, for the first time, an immunoaffinity sorbent with high specificity and capacity for aromatic cytokinins. A batch immunoextraction method was then developed and optimized for the purification of 2OH3MeOBAPR from murine blood samples. The high efficacy and simplicity of this method (in off-line combination with HPLC-MS) for the isolation of target analytes from biological material is demonstrated in this study.