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

Abstract

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

“…Syntheses of all desired target [ 15 N 4 ] purine labeled cytokinin glycosides 5, 6, and 8 started with completely 15 N‐labeled 6‐chloro[ 15 N 4 ]purine ( 1 ), which was prepared by improved procedure . However, all pilot studies were performed with unlabeled chloropurine 1 .…”

“…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 .…”

“…All commercially available reagents were used without further purification and purchased from standard chemical suppliers. Isotopically labeled 6‐chloro[ 15 N 4 ]purine ( 1 ) (98% 15 N) was provided by authors of publication where the improved preparation is described. Purine nucleotide phosphorylase 120 Unit/mL was purchased from Sigma‐Aldrich (Prague, Czech Republic).…”

“…7‐Methylguanosine (30 mg, 0.1 mmol) was added to a solution of corresponding labeled [ 15 N 4 ] zeatin derivative 7a or 7b (10.0 mg, 0.045 mmol) in a mixture of aqueous 0.02 M K 2 HPO 4 (1 mL) and pure 96% ethanol (1 mL). This solution reached pH = 7.8 and was not further modified.…”

Endocytic Trafficking Promotes Vacuolar Enlargements for Fast Cell Expansion Rates in Plants

“…Plant extracts (2 µL) were injected onto a Kinetex C18 column and separated using chromatographic conditions as described above. The high-resoluion mass spectrometry (HRMS) analysis was achieved by a hybrid Q-TOF tandem mass spectrometer Synapt G2-Si (Waters MS Technologies) as described previously (Buček et al, 2018). Briefly, the effluents were introduced into the HRMS instrument (ESI-; Capillary Voltage, 0.75 kV; Source Offset, 30 V; Desolvation/Source Temperature, 550/120°C; Desolvation/Cone Gas Flow, 1000/50 l hr−1; LM/HM Resolution, 2.8/14.75; Ion Energy 1/2, 0.5/1.0 V; Entrance/Exit Voltages, 0.5 V; Collision energy, 6 eV).…”