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.

Keywords: 8-azido-N6-benzyladenine; cytokinin-binding proteins; cytokinins; photoaffinity labelling; receptors; synthesis of cytokinin analogues.

Figure 1

The sequence of reactions used to prepare photoaffinity compounds from [2-³H]adenosine. The compounds and intermediates are numbered 1 to 9 as in the text. The 1-substituted adenosine derivatives, 3 and 4, were not preparatively separated but the mixture was subjected to conditions to induce a Dimroth rearrangement yielding the N⁶-derivatives 5 and 6. The reactions were (i) bromination; (ii) reaction with benzylbromide in DMF; (iii) rearrangement in 0.25 M NH₄OH; (iv) NaN₃ at 70 ^oC; and (v) 0.5 M HCl at 80 ^oC.

Figure 2

The distribution of radioactivity over the TLC chromatograms show the effects of temperature and solvent on bromination of [2-³H]adenosine. (A) water at 26 ^oC (conditions often used to brominate nucleotides); (B) 50% methanol at 4 ^oC (conditions used herein). The quantities used in the reactions are those given in Section 3.3.1. The letters a and b denote adenosine and 8-bromoadenosine, respectively. For both graphs, the reaction time was 0.75 h and solvent A was used for TLC.

Figure 3

Reaction sequences for synthesis of 8-azido-N⁶-benzyladenine from 2,6-dichloropurine (DCP). A. Synthesis of the unlabelled compound (AZ) as described herein. B. Proposed synthesis of ³H-labelled compound of high specific activity, i.e., 8-azido-N⁶-[3-³H]benzyladenine (13). Intermediates shown in the figure are: N⁶-benzyl-8-chloro-adenine (10), 8-chloro-N⁶-(3-iodobenzyl)adenine (11), N⁶-[3-³H]benzyl-8-chloro-adenine (12), 8-azido-N⁶-[3-³H]benzyladenine (13). The reactions were with: (i) benzylamine at 100 ^oC; (ii) NaN₃ in propan-1-ol containing acetic acid; (iii) 3-iodobenzylamine; (iv) tritium gas over palladium catalyst.