Fig. 1.
Pulse sequence scheme for (a) C(aliph)HMQC-NOESY-C(aro)HSQC and (b) C(aro)HMQC-NOESY-C(aliph)HSQC. 90° and 180° ‘hard’ pulses are represented by filled and open bars, respectively. Shaped pulses are represented as follows: (a) 40 ppm selective pulse of the iburp-2 profile (Geen and Freeman 1991) (659 μs duration, 7.6 kHz peak r.f. field) is shown as wide open bell-shaped pulse (denoted ‘E’). Wide grey rectangular pulses (‘A’ and ‘C’) are of duration of 61 μs and peak r.f. 4.1 kHz (calibrated to give null excitation 90 ppm off -resonance). (b) Wide grey bell-shaped pulse denotes 80 ppm-selective hyperbolic secant adiabatic pulse (Silver et al. 1984) of duration of 1 ms (r.f. peak 6.9 kHz). Narrow grey bell-shaped pulses (‘F’ and ‘H’) utilize Gaussian profile (Bauer et al. 1984) (truncated at 1 %, duration of 178 μs, peak r.f. 3.4 kHz, ca. 60 ppm bandwidth). Note that for some hardware the shaped 90° pulses (‘A’, ‘C’, ‘F’ and ‘H’) may require small angle phase adjustment to compensate 0th order phase shift with respect to refocusing pulses (‘B’ and ‘G’) which are applied at full power. 15N refocusing pulse enclosed in dashed-line box (sequence b) is optional and may be used if delay complementing to half-dwell time, ξ = [(sw3)-1 − pw180(N)]/2, is positive. Otherwise ξ = [(sw3)-1 − pw180(H)]/2. Similar rules apply to the delay ζ for sequence (a). Refocusing of Caliph-C’ couplings in t 2 (a) or t 3 (b) may be considered if max. evolution time of aliphatic 13C spins exceeds 9 ms. The composite 34.2°−x123°x197.6°−x288.8°x pulse (‘D’ and ‘I’) was used in HMQC for broadband inversion of 13C spins (Shaka 1985). WATERGATE (Piotto et al. 1992) with 3-9-19 pulses separated by (3.2 kHz)−1 was employed for experiment (a). 13C composite pulse decoupling was performed employing WURST scheme (Kupče and Freeman 1995). The durations of ‘hard’ π/2 pulses were 7.1, 14.3 and 31 μs for 1H, 13C and 15N, respectively. ϕ1, ϕ2 and ϕ3 are incremented for quadrature detection in t 1, t 2 and t 3 using States (ω1) or States-TPPI (ω2, ω3) method. Four-step phase cycle is as follows: ϕ1 = 45°; ϕ2 = x, −x; ϕ3 = 2(x), 2(−x); ϕ4 = 2(x), 2(−x); ϕ5 = 135°; ϕrec = x, −x, x, −x. Delays are set as follows: τa = 1.79 ms ≈ (4 JCHaliph)−1, τb = 1.56 ms ≈ (4 JCHarom)−1. NOESY mixing time τm = 150 ms was used. For the semi-constant time evolution in t 1 (1H) the delays τ1, τ2 and τ3 are t 1/2, t 1(1 − 2Δ/t 1,max)/2 and Δ(1 − t 1/t 1,max), where Δ = 2τa, or Δ = 2τb for sequences (a) and (b), respectively (Stanek et al. 2012). Gradient levels and durations are: G1 (2 ms, 6.5 Gs/cm), G2 (2 ms, 14.2 Gs/cm), G3 (0.5 ms, 1.77 Gs/cm), G4 (2 ms, 11.3 Gs/cm), G5 (2 ms, −12.9 Gs/cm), G6 (0.5 ms, 5.4 Gs/cm). Proton carrier frequency was set on resonance with water (4.68 ppm), carbon carrier was set to 35 ppm and switched to 125 ppm as indicated by the vertical arrow; 15N carrier was set to 117 ppm and shifted to 162 ppm at the beginning of NOESY mixing period. For the aliphatic-to-aromatic NOESY (a) 4,400 sampling points (t1,t2,t3) were randomly chosen from 120 × 84 × 30 Cartesian grid according to Gaussian probability distribution, p(t) = exp[−(t/t max)2/2σ2], σ = 0.5, with Poisson disk restrictions (Kazimierczuk et al. 2008). Maximum evolution times of 15 (t 1), 6 (t 2) and 5 ms (t 3) were achieved in the indirectly detected dimensions. Spectral widths of 8, 14, 6 and 12 kHz were set in ω1, ω2, ω3 and ω4 dimensions, respectively. In the full 4D spectrum any residual diagonal peaks can be folded in 13C (ω2 and ω3) dimensions without the risk of overlap and misinterpretation with genuine peaks. The only requirement is to ensure proper 1H (ω1) spectral width to avoid aliasing in this indirect dimension. The restriction of 13C spectral widths is very practical as it saves vast amounts of disk space. Inter-scan delay of 1.2 ms was used. The total experimental time was 57 h