Synthesis of a Cytokinin Linked by a Spacer to Dexamethasone and Biotin: Conjugates to Detect Cytokinin-Binding Proteins

Abstract

Yeast cells expressing cDNA libraries have provided two new approaches to facilitate further identification of cytokinin-binding proteins and receptors. These are the yeast three hybrid (Y3H) system and fluorescence activated cell sorting (FACS). The Y3H system requires a synthetic hybrid ligand comprising an "anchor" moiety (e.g., dexamethasone) linked to a cytokinin via a spacer. In the yeast nucleus, this ligand by binding connects two fusion proteins leading to a reporter gene activation and detection and characterisation of cytokinin binding proteins. Herein is reported the first synthesis of dexamethasone-cytokinin ligands with a spacer linkage. This was attached to the purine ring of 6-benzylaminopurine (BAP) at positions 2, 8 or 9. To achieve this, dexamethasone was modified by periodate oxidation yielding a carboxylic group used for conjugation to the spacer by amide formation. Biotinyl derivatives of cytokinins for FACS included those synthesised by reaction of an activated ester of biotin with 8-(10-amino-decylamino) derivatives of BAP and BAP 9-riboside. Properties of the conjugates and some biological situations where they could be applicable are discussed briefly.

Keywords: 6-benzylaminopurine (BAP); BAP-biotin conjugates; BAP-dexamethasone conjugates; cytokinin-binding proteins; yeast three hybrid.

Figure 1

Structures of compounds used and synthesised in this study.

Figure 1

Structures of compounds used and synthesised in this study.

Figure 2

The sequence of reactions used to synthesise BAP-dexamethasone conjugates with purine ring linkage at C-8 (5b, 6b) or N-9 (4b). (A) 8-bromoadenosine; (B) 1-benzyl-8-bromoadenosine; (C) 6-benzylamino-8-bromopurine 9-riboside; (D) 6-benzylamino-9-benzyl-8-bromopurine; (E) 6-benzylaminopurin; (F) ethyl 3-(6-benzylaminopurine-9-yl)propionate. The reactions are: (i) benzylbromide in DMF at 37 °C; (ii) 0.25 M NH₄OH at 37 °C; (iii) 1,10-diaminodecan in n-PrOH at 100 °C; (iv) methanolysis with HCl at 37 °C; (v) activated ester of “dexamethasone acid” at 23 °C; (vi) ethyl acrylate/NaOEt; (vii) 1,10-diaminodecane in toluene at 110 °C.