The Synthesis of 3H-Labelled 8-Azido-N6-Benzyladenine and Related Compounds for Photoaffinity Labelling of Cytokinin-Binding Proteins

Abstract

The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology. Photoaffinity labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present experimental details for two methods for synthesis of 8-azido-N6-benzyladenine, which has the azido affinity group in the preferred position of the purine ring. Synthesis from [2-3H]adenosine yielded the above-mentioned PAL reagent with 3H in the purine ring and also gave labelled 9-riboside and 8-azido-N6,9-dibenzyladenine. 8-Azido-N6-benzyladenine was also prepared from 6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin analogues in assessing the significance of PAL is discussed.

The Staudinger Ligation

“…Over the past decade, a large number of photobioconjugation methods have been emerged with organic azides. Besides classical 357 photoaffinity labeling 358 with azides, 359 also quite complex constructs have been used to attach probes to biological compartments.…”

Zeatin: The 60th anniversary of its identification

“…Their presence is an absolute requirement for leaf growth and, as such, cytokinins are essential for life on earth. However, as Letham et al (2019) noted, the function of cytokinins extends far beyond plants. For example, Z is produced by certain human microbial pathogens that also respond to applied cytokinin.…”

“…This was shown with an obligate protozoan pathogen ( Andrabi et al 2018 ) and with Mycobacterium tuberculosis which occurs naturally only in humans ( Samanovic et al 2015 , 2018 ). M. tuberculosis, several other human pathogens, other bacteria, and even archaea have been shown to contain homologs of LOG, the plant cytokinin activating enzyme (see references in Letham et al 2019 ; Chen et al 2022 ). Much remains to be elucidated regarding the role of cytokinins in such organisms.…”