NMR investigation of magnesium chelation and cation-induced signal shift effect of testosterone

We have previously reported that testosterone (Tes) is able to interact with magnesium chloride dissolved in methanol. In this study, we have applied ¹H and ¹³C NMR spectroscopies to a series of Tes solutions containing Mg²⁺ at various concentrations. High-resolution ¹³C NMR spectra of Tes/Mg²⁺ revealed well-resolved ¹³C signals, and the intensities of those arising from C3, C5, C16, and C17 decreased linearly with increasing Mg²⁺ concentration. The magnitude of the chelation affinity could be deduced from the slopes of the ¹³C intensity variations; typically, the greater the slope the higher the chelation affinity. The results revealed Tes/Mg²⁺ chelation to be mediated by the oxygen atom attached to C3 in ring A, and the hydroxyl group attached to C17 in ring D. With regard to the chelation specificity, we showed that Tes chelates Mg²⁺, but not Ca²⁺ or Zn²⁺. We also explored the cation-induced signal shift effects of Tes in the presence of Mg²⁺, Ca²⁺, or Zn²⁺. We demonstrate that high-resolution ¹³C NMR spectroscopy provides a better probe than ¹H NMR for the detection of cation chelation and cation-induced signal shift effects for steroid compounds such as Tes.