Figure 2. Chemical activation of the steroid analogue probe with the alkyne moiety in aqueous solutions.

(a) Alkyne formation of steroid analogue probe 1. (b–f) ¹H NMR measurement of production of alkynyl probe 1 from precursor 2. The ¹H NMR spectra of 2 were obtained after incubation with TsNHNH₂ in D₂O/DMSO for (b) 0, (c) 2.0, (d) 7.5, and (e) 31 h. (f) The ¹H NMR spectrum of 1 was obtained as positive control. The black arrow indicates the chemical shifts of the proton of 2 that is marked in red in the chemical structure shown above the spectra (2.97 and 3.02 ppm of enantiomers). The gray arrow indicates the chemical shift of the red-colored proton indicated in 1 (2.06 ppm). (g) The integral ratio of the chemical shifts of the red-colored proton indicated in 2 (indicated by the black arrow) was plotted as a function of the reaction time.