The interaction of a series of phosphate diesters and triesters (1=diphenyl phosphate,2=dimethyl phosphate,3=bis(2-ethylhexyl) phosphate,4=trimethyl phosphate,5=methyldiphenyl phosphate,6=triphenyl phosphate) with [Mg(15-crown-5)](2+) (15-crown-5=1,4,7,10,13-pentaoxocyclopentadecane) was studied as a simplified model for the interaction of aqueous Mg(2+) ion with phosphate-containing biomolecules such as RNA. Using electrospray mass spectrometry, we confirm the formation of 1:1 adducts in the gas phase. Proton and (31)P NMR titration data were used to construct binding isotherms, and a 1:1 binding equilibrium was fit to the isotherms at room temperature to estimate the binding affinities. The binding affinity data are consistent with ditopic coordination of neutral dialkyl phosphate ligands to the [Mg(15-crown-5)](2+) unit. This involves inner-sphere coordination to the Mg(2+) via an oxygen atom, which is complemented by a weak hydrogen-bonding interaction with the crown ether ligand. Ditopic interaction is consistent with low-temperature NMR spectra showing four different configurations for1 coordinated to [Mg(15-crown-5)](2+), which are interpreted in terms of hindered rotation around the Mg-O(phos) bond. Thermochemical analysis of the binding affinity data suggests that the second-shell interaction contributes only about 1 kcal/mol to the binding free energy, so additional factors, such as steric constraints, must be operative to give a preferred phosphate orientation in this system. However, the experimental data do suggest that second-shell interactions contribute as much as 40% of the total binding energy, consistent with the pronounced ability of aqueous Mg(2+) to form salt-bridges linking secondary and tertiary elements of RNA structure.