Figure 2.

Pulse sequence for the quantitative J-correlation ²J_HN HNN–COSY experiment. Carrier positions in the present work were 189.1 p.p.m. for ¹⁵N, and 4.75 p.p.m. for ¹H^N, respectively. All ¹H pulses were given on resonance for the water signal. High power proton pulses were applied with a field strength of 26.2 kHz. ¹⁵N decoupling during acquisition employed a 1.26 kHz GARP field (40), while high power ¹⁵N pulses were applied with a field strength of 7.0 kHz. The inversion of nitrogen magnetization during the first and the final INEPT transfer step is achieved using composite 90_x180_y90_x 180° (N) pulses. The phase cycling is φ₁ = x, –x, φ₂ = x, φ₃ = 2(y), 2(–y), receiver = x, –x. Quadrature detection was obtained in the t₂ dimension by altering φ₁, φ₂ and φ₃ according to States–TPPI (41). The delays for the INEPT and N,N transfers were Δ = 28 ms and τ = 45 ms, respectively (see text). Sine-shaped gradient durations and amplitudes were: G₁ 0.32 ms (5 G/cm); G₂ 0.64 ms (3.5 G/cm); G₃ 0.64 ms (–8.5 G/cm); G₄ 0.32 ms (7.5 G/cm); G₅ 1.28 ms (15 G/cm). For experiments carried out in 90% H₂O, 10% D₂O the last non-selective 180° (H) pulse in the reverse INEPT transfer was replaced by the binomial WATERGATE sequence in order to achieve adequate water suppression (42).