NMR Relaxation Measurements

The transverse relaxation rates (R₂ = 1/T₂) serve as a valuable tool to interrogate molecular interactions. The T₂ data was collected using the standard Bruker pulse program library sequence CPMGESGP2D. This is a pseudo-2D NMR experiment (a series of 1D ¹H NMR spectra are recorded in an interleaved manner) and involves the CPMG block: 90–[τ–180−τ–τ–180−τ−]_n– where 2τ is the spin-echo time between two 180° (inversion) pulses of length 25 μs and n is the loop counter used to vary the CPMG-based T₂ time. For decay of transverse magnetization, the CPMG time was varied as 3.3, 6.6, 13.2, 19.8, 26.4, 52.8, 79.2, 105.6, 118.8, 132.0, and 158.4 ms, and for this, we used spin-echo time (2τ) equal to 800 μs, 180° RF hard pulse of 25 μs, and n was varied as 2, 4, 8, 12, 16, 32, 48, 64, 72, 80, and 96. The T₂ data sets were acquired on both free and bound states of the ligand. Each dataset was averaged over 512 transients using recycle delay equal to 6.0 s, whereas other acquisition parameters used were similar to those used for ZGESGP experiment. The acquired FID signals in the 2D array format were then processed using Bruker command ≪xf2≫ (other processing parameters were identical to those used for processing ZGESGP spectra). The resultant spectra were processed and analyzed in software TopSpin v3.6 using its T₁/T₂ analysis application tool. The resultant peak intensity profiles were plotted as a function of T₂ time and then fitted to a two-parameter exponential decay equation given below

where I(t) is the NMR peak intensity at CPMG-based T₂ time equal to t and I₀ is the reference signal intensity at t = 0.