Quantification of cytokinins by selected ion monitoring using15N labelled internal standards

Abstract

A procedure has been developed for the synthesis of 6-chloropurine labelled with four * ' N atoms. This compound can be used in the synthesis of a range of labelled cytokinin bases, which can be ribosylated by an enzymatic procedure. 15N labelled zeatin riboside was synthesized and used as an internal standard in the measurement by selected ion monitoring of the levels of this cytokinin in cultured crown gall and normal tissues of Vinca rosea, which were found to contain 402 and 2.5 ng per g fresh weight respectively.

Synthesis of [15N4] purine labeled cytokinin glycosides derived from zeatins and topolins with 9‐β‐d, 7‐β‐d‐glucopyranosyl, or 9‐β‐d‐ribofuranosyl group

“…Therefore, we decided to prove direct enzymatic transglycosylation reaction of 7a‐e to give desired ribosides 8a‐e . Non‐ribosylated labeled derivatives 7a‐e were already reported, and only labeled [ 15 N 4 ]‐ trans ‐zeatin‐9‐β‐ d ‐ribofuranoside 8b was provided according to the method described by Sivadjian, where ribose‐1‐phosphate was used. To transform cytokinins 7 to their β‐ d ‐ribofuranosides 8, we adapted and modified enzymatic ribosylating method employing 7‐methylguanosine as a ribose‐1‐phosphate donor .…”

“…For this reason, the high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS) method is the most frequently used . An addition of a known quantity of the isotopically labeled cytokinin standard into the plant tissue extract enables to monitor possible loses during the purification and determine the amount of the natural cytokinin by the ratio of the area of the MS peaks of the natural and labeled cytokinin …”

Hormones in wheat kernels during embryony

“…Lyophilized samples were homogenized in 80 % methanol (Brinkmann, Polytron) and spiked with DL-cis,trans-[G-3 H]abscisic acid (TRK.644, Amersham), 4.25 TBq × mmol -1 specific activity; 3-[5(n)-3 H]indolylacetic acid (TRK.781, Amersham), 1.04 TBq × mmol -1 specific activity; and 3 H-trans-[9R]Z, 3 H-diH[9R]Z and 3 H-[9R]iP, each of which were synthesized using nucleoside phosphorylase (Scott and Horgan 1980). Extracted hormones were purified by C 18 octadecyl silica solid phase chromatography and HPLC (MacDonald and Morris 1985) using an ODS C 18 , 250 × 4.6 mm, 5 mm column (Alltech Associates).…”

Total synthesis of [ 15 N]-labelled C6-substituted purines from [ 15 N]-formamide—easy preparation of isotopically labelled cytokinins and derivatives

“…These and other considerations as well as the purine-core structure make use of the 15 N nuclide a reasonable choice. Despite the existence of previous studies describing [ 15 N 5 ] adenine and trans -zeatin preparation [22,34] as well as formamide cyclization to adenine [35], a significant improvement of continuous and fully functional synthesis of 15 N-core-labelled CKs from [ 15 N]-formamide is presented herein. Furthermore, the yields are strongly improved compared to the less effective procedures described above.…”

“…Alternatively, 2 H nuclide can be introduced to the t Z side chain as well to produce [ 2 H 5 ]- t Z [21]. A few years later, alongside the first attempt to prepare 15 N 4 trans- zeatin [22], another complex approach has been developed, which gradually combines side chain catalytic constructions [23]. By using deuterated reduction systems and solvents, isotopically labelled t Z side chains can be produced for reaction with 6-chloropurine [23].…”