Figure 4.

¹³C NMR spectra of [¹³C-¹⁵N₄]-4 and cathepsin K at 125.76 MHz. (A) Compound 4 (15 mM) in DMSO-d₆ at 20°C, 3,000 scans, 1.5-s repetition period, ¹H broadband decoupling. The triplet structure of the ¹³C-labeled carbonyl resonance peak is caused by the one-bond ¹³C-¹⁵N spin–spin coupling with two adjacent ¹⁵N-labeled amines. (B) Cathepsin K (0.45 mM) complexed with 4 in 90% water/10%D₂O, 50 mM acetate-d₃, 250 mM NaCl, and 2 mM l-Cys, pH 4.0, 5°C, 123,283 scans, 1.5-s repetition period, ¹H broadband decoupling. The doublet structure is caused by the ¹³C-¹⁵N spin–spin coupling with only one adjacent ¹⁵N-labeled amine. (C) Same sample and experimental conditions as in B but with simultaneous ¹H and ¹⁵N broadband decoupling, 240,000 scans, 1.5-s repetition period. The doublet seen in B coalesces into a singlet because of ¹⁵N decoupling.