Synthesis and Properties of N6, C-8 and C-2 Spin-Labelled Derivatives of Adenosine Cyclic 3':5'-Monophosphate

N6, C-8 and C-2 spin-labelled derivatives of adenosine cyclic 3′:5′-monophosphate (cAMP) were synthesized by reacting the respective halogen-substituted compounds with 4-amino-1-oxyl-2,2,6,6,-tetramethylpiperidine and 3-methylamino-1-oxyl-2,2,5,5,-tetramethylpyrrolidine. Circular dichroic spectra indicate that substitution at N6 does not markedly influence the conformational equilibrium in cAMP about the glycosidic bond, whereas the derivatives at C-8 from steric reasons have a syn conformation; probably the C-2 compound also prefers the syn conformation. The latter compound as the 2,6-diamino-purine derivative reveals strong fluorescence. In order to determine the mobility of the spin label at the different positions, electron spin resonance spectra were recorded and analysed in glycerol-water mixtures of increasing viscosities. The results show that the label is most strongly restricted at C-8 and most weakly at N6 position. The insertion of a CH2 link between the label and the purine significantly reduces restriction at C-8 but has no effect at the N6 position. The marked restriction of mobility at C-2 relative to N6 is consistent with a preferred syn conformation of this labelled derivative. With cAMP-dependent protein kinases from bovine brain and rabbit muscle the binding of these compounds were measured by competition with tritiated cAMP. The N6 derivatives are bound as strongly as unsubstituted cAMP, while the affinity of C-8 and C-2 labelled compounds is reduced by about 2 kcal/mol.