Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g fresh weight). We have developed a simple microscale magnetic immunoaffinity-based method for selective one-step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one-step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30-80% depending on individual CKs. Immunoaffinity extraction using group-specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry-based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N -glucosides) in 1.0-mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.

Cytokinins (CKs) and their metabolites and derivatives are essential for cell division, plant growth regulation and development. They are typically found at minute concentrations in plant tissues containing very complicated biological matrices. Therefore, defined standards labelled with stable isotopes are required for precise metabolic profiling and quantification of CKs, as well as in vivo elucidation of CK biosynthesis in various plant species. In this work, 11 [15N]-labelled C6-purine derivatives were prepared, among them 5 aromatic (4, 5, 6, 7, 8) and 3 isoprenoid (9, 10, 11) CKs. Compared to current methods, optimized syntheses of 6-amino-9H-[15N5]-purine (adenine) and 6-chloro-9H-[15N4]-purine (6-chloropurine) were performed to achieve more effective, selective and generally easier approaches. The chemical identity and purity of prepared compounds were confirmed by physico-chemical analyses (TLC; HRMS; HPLC–MS; 1H, 13C, 15N NMR). The presented approach is applicable for the synthesis of any other desired [15N4]-labelled C6-substituted purine derivatives.

Synthesis of [ 15 N 4 ] purine labeled cytokinine glycosides derived from zeatinsand topolins containing a 9-β-D, 7-β-D-glucopyranosyl, or 9-β-D-ribofuranosyl group is described. These N 6 -substituted adenine derivatives are intended as internal analytic standards for phytohormone analysis. All labeled compounds were prepared from 6-chloro[ 15 N 4 ]purine (1). The equilibrium reaction of 1 with acetobromo-α-D-glucose gave isomeric 7-β-D (3) and 9-β-D (4) chloro glucosyl precursors, which were treated with the corresponding amines to get desired labeled cytokinin 7-β-D (6) and 9-β-D (5) glucopyranosides. Cytokinins containing 9-β-D-ribofuranosyl group (8) were obtained by direct enzymatic transglycosylation reaction of cytokinins (7) prepared from 6-chloro[ 15 N 4 ] purine (1).