Fig. 3.

Pulse sequences in top panels are for diagonal-free Aro-Methyl 3D SOFAST HMQC-NOESY-HMQC (a) and reference 3D HSQC-NOESY-HMQC sequence (b). Frequency labeling is C_Aro(F₁)-C_M(F₂)H_M(F₃). Top a): the smaller and larger ¹H shaped pulses are 1.69 ms long 90° PC9_4_90 and 1.15 ms long 180° REBURP, respectively. The first and second vertical arrows on ¹H and ¹³C channels indicate the ¹H(¹³C) carrier frequency setting to 8.5 ppm (122 ppm) and 4.7 ppm (17.5 ppm), respectively. The offsets of ¹H shaped pulses before and after τ_mix are 0 (at 4.7 ppm) and −3,230 Hz (at 0.9 ppm), respectively. The narrow bars represent 90° hard pulses. The spectral centers of ¹³C(F₁), ¹³C(F₂), ¹H(F₃) dimensions are at 122.0, 16.5, and 4.7 ppm, respectively. The delays are: d1 = 200 ms, Δ1 = 3.1 ms, Δ2 = 4.0 ms, τ_mix = 300 ms. The phase cycling are: φ1 = (x, −x), φ2 = (x, −x, −x, x), φ3 = 4(x), 4(y), φ4 = 8(x), 8(−x), φ_rec = (x, −x, −x, x, −x, x, x, −x, −x, x, x, −x, x, −x, −x, x). Bruker decoupling scheme bi_garp_2pl is used. The quadrature detections in t₁ and t₂ dimensions are acquired via States-TPPI of φ1 and φ2, respectively. The durations and strengths of the gradients are G1 = (1 ms, 15 G/cm), G2 = (1 ms, 5 G/cm). Top (b): reference WATERGATE (W5’) 3D HSQC-NOESY-HMQC pulse sequence used for the S/N comparison. The filled narrow and wide bars represent 90° and 180° hard pulses, respectively. The open narrow (41.6 µs) and wide (37.2 µs) bars on ¹³C channel represent 90° and 180° soft pulses, respectively, that have null excitation at an offset of 109 ppm. ¹³C shaped pulses are 423 µs IBURP2.1000 180° pulses (Geen and Freeman 1991). The first and second vertical arrows indicate the 13C carrier frequency setting to 122.0 ppm and 17.5 ppm (centers of aromatic and methyl ¹³C chemical shifts), respectively. The distances between 3-9-19 W5’ pulses are 148 µs. The delays are: d1=1.0 sec, Δ1 = 3.1 ms, Δ2 = 4.0 ms, δ = t₁(0) + 2*pw (initial t₁ value and ¹H 180° pulse width), τ_mix = 0.3 sec. The phase cycling is: φ1 = (x, −x, −x, x), φ2 = (x, −x), φ3 = 4(x), 4(y), φ_rec = (x, −x, −x, x, −x, x, x, −x). Bruker decoupling scheme bi_garp_2pl is used. The quadrature detections in t1 and t2 dimensions are acquired via States-TPPI of φ1 and φ2, respectively. The durations and strengths of the gradients are G1 = (1 ms, 10 G/cm), G2 = (1 ms, 5 G/cm), G3 = (2 ms, 10 G/cm). Bottom panels show comparison of pure NOE F2(C_M)-F3(H_M) planes run using the FliT-FliJ complex sample (see main text Materials and Methods) that contained both ¹H-¹³C methyl and aromatic labels. Mixing time was 0.3 s, d1 = 0.2 s, and 90 degrees variable angle pulse (α) for SOFAST version.