7. Comments

The pulse sequence is divided into five sections a–e separated by dotted vertical lines. In section a we have the INEPT transfer from proton to ¹⁵N, which includes in addition a water flip-back pulse p4 and a dephasing gradient pulse (g1). At the time of the gradient pulse g1, the amide protons are described by the term 2I_HzI_Nz, and thus the gradient pulse g1 only dephases coherences that are not bonded to ¹⁵N and thus are not in the z-direction due to pulse imperfections. The final pulse p14 on ¹⁵N creates −2I_HzI_Ny for the amide protons.

In section b the magnetization is transfered further from ¹⁵N to CO by another INEPT step comprised of the two pulses p15 and p16 on ¹⁵N and the two band-selective CO pulses p24 and p25. The total length of this INEPT period is d3 + d4 + d5 = 1/[2J(N,CO)]. Furthermore, proton decoupling by the DIPSI spin-lock scheme is prepared by pulse p5 and started after delay d3 = 1/[2J(N,H)].

The section c has three purposes. First, the magnetization must be transfered from CO to C_α, then the chemical shifts of the C_α ¹³C nuclei have to be sampled, and finally, the magnetization must be transfered back to CO. Therefore, we find two more INEPT steps comprised of pulses p26, p27, p34 and p35 for going from CO to C_α, and p36, p37, p29 and p30 for going back from C_α to CO. Accordingly the delays d6 are adjusted to 1/[4J(C_α,CO)]. Pulses p33 and p39 correct for Bloch–Siegert phase shifts.

During sampling of the C_α chemical shifts, proton decoupling by DIPSI, ¹⁵N decoupling by p17, and CO decoupling by p28 removes any unwanted dephasing.

The purpose of section d is twofold. First the magnetization has to be transfered back to ¹⁵N and secondly, the ¹⁵N chemical shifts have to be sampled. The back-transfer is achieved by the pulses p31 and p18, and the sampling is performed in the constant-time scheme by decrementing the delay d8 at the same time as incrementing t₂. The total length of the period is therefore set to 1/[2J(N,CO)] ≈ 24 ms. Pulse p40 decouples C_α from ¹⁵N. Gradient pulse g2 dephases the transverse magnetization, and at the end of this period the proton decoupling is turned off.

The final back-transfer in section e from ¹⁵N to protons uses the PEP principle, and gradient g3 selects for protons bonded to ¹⁵N. GARP decoupling yields singlets for all these signals.

8. Own Observations