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. 2014 Apr 9;20(21):6332–6338. doi: 10.1002/chem.201304992

Figure 1.

Figure 1

Pulse sequence designed to measure the proton exchange rates kex of the NHδ1, NHε2, and NH3+ groups in histidine. The π and π/2 pulses are represented by narrow open and filled rectangles, respectively, whereas decoupling sequences are represented by wide rectangles. Continuous-wave (cw) proton decoupling was applied in both experiments A and B for a duration δ, but the CPMG train, also of length δ, was delayed until after the decoupling interval in experiment A, whereas both were applied at the same time in experiment B. This prevents differences in temperature induced by decoupling. All π/2 and π pulses applied to 13C in the first and last INEPT blocks were nonselective rectangular pulses, whereas the 13C refocusing pulses in the other INEPT blocks had REBURP profiles[35] with a duration of 4 ms. The 15N inversion π pulse in the INEPT block had a REBURP profile of 2 ms duration for observing NH3+ protons and Q3 profiles[36] with a duration of 30 ms for probing the NHε2 and NHδ1 protons. Continuous-wave proton decoupling was used during the blocks A and B but WALTZ-16 decoupling[37] was used during the INEPT sequences that bring about coherence transfer between 13C and 15N. The delays were set to τ1=1.69 ms≈1/(4 1J(C,H)), τ2=23.43 ms≈1/(4 1J(N,C)) for NH3+, τ1=1.22 ms≈1/(4 1J(C,H)), τ2=16.7 ms≈1/(4 1J(N,C)) for NHε2, τ1=1.12 ms≈1/(4 1J(C,H)), τ2=17.9 ms≈1/(4 1J(N,C)) for NHδ1. All phases were along the x axes unless indicated otherwise. The phases were cycled according to: ϕ1=2{y}, 2{−y}, ϕ2={x}, {−x}, ϕ3=4{x}, 4{−x}, ϕ4=y, ϕ5=8{x}, 8{−x} with a receiver phase ϕrec={x, −x, −x, x, −x, x, x, −x, −x, x, x, −x, x, −x, −x, x}. The gradients need to be carefully adjusted to avoid accidental refocusing. The value α=γC/γH. The labile Hδ1, Hε2, and NH3+ protons examined in this work are highlighted with colors and the pathways for the transfer of magnetization are indicated on the molecular structure. By way of example, spectra A and B of the Hδ2 proton are shown for indirect detection of NHε2 at pH 3.2 and 292.5 K.