NORD: NO Relaxation Delay NMR Spectroscopy

Abstract

The novel concept of NORD (NO relaxation delay) NMR spectroscopy is introduced. The idea is to design concatenated experiments in a way that the magnetization used in the first relaxes toward equilibrium during the second and vice versa, thus saving instrument time. Applications include complete well-resolved ¹ H-¹ H and ¹ H-¹³ C one-bond and long-range correlation maps of an 80 mM solution of a trisaccharide recorded in less than two minutes and hydrocortisone with extensive spectral overlap.

Keywords: H2OBC/2BOB; NMR spectroscopy; NORD; SEA XLOC/HMBC; heteronuclear correlation.

Figure 1

700 MHz ¹H NMR spectra illustrating the $v$ ‐angle dependence of the applicable BIG‐BIRD element using a 2:1 mixture of ¹³CH₃COONa and ¹²CH₃COONa dissolved in D₂O and representing the IS (¹³CH₃) and I (¹²CH₃) pools of magnetization, respectively. The spectrum at the bottom is acquired with a single 90°_y excitation pulse. All the spectra were processed with identical parameters and phase correction.

Figure 2

Excerpts of H2OBC (left) and HMBC spectra of a trisaccharide (1) (45 mg in D₂O) recorded in 1 min 48 s on a Bruker 700 MHz Avance NEO spectrometer equipped with a TCI z‐gradient prodigy probe using the NORD HMBC‐H2OBC experiment with 25 % NUS. The assignment walks of D, E, and F residues are labeled by colored dotted lines in the H2OBC spectrum. Peaks framed by a red box in the HMBC spectrum verify the sequential connectivities of D–E and E–F residues. The spectra were acquired with the parameters: Δ=83 ms, T=23 ms, spectral widths of 5.1 ppm (¹H) and 190.0 ppm (¹³C), using 64 NUS points in t₁ with a single scan per increment and 1024 data points in t₂. In the BANGO and BIG‐BIRD elements a CAWURST‐20(240 ppm, 1.92 ms; H2L) adiabatic ¹³C inversion pulse was used. β ^I was set to 120° for the BANGO pulse and the $v$ angle of BIG‐BIRD to 20°. Before standard processing the obtained combined data was separated into HMBC and H2OBC blocks using the Bruker au‐program splitx. Then the non‐uniformly sampled data were reconstructed with the compressed sensing (CS) approach implemented in TopSpin and processed as in the corresponding stand‐alone experiments. The pulse sequence code for Bruker spectrometers can be found in the Supporting Information.

Figure 3

Overlay of excerpts of edited 2BOB spectra of hydrocortisone (45.7 mg in 550 μL [D₆]DMSO) acquired using the NORD SEA XLOC‐HMBC‐2BOB experiment (outlined in Figure S1) on a Bruker Avance NEO 700 MHz spectrometer equipped with a TCI z‐gradient prodigy probe. One‐bond (1B) correlations of CH2 and CH carbons are plotted in green and blue, respectively, whilst the corresponding two‐bond (2B) correlations are plotted in red and brown, respectively. The experiment was performed with the following parameters: Δ _{SEA XLOC/HMBC}=83 ms, T _H2OBC=23 ms, spectral widths of 6.5 ppm (¹H) and 190.0 ppm (¹³C), 512 points in t₁ with a single scan per increment in the SEA XLOC and HMBC modules and 128 points with 4 scans in the 2BOB module due to the four‐step editing cycle. For all three modules 2048 data points were acquired in t₂ amounting to 42 minutes measurement time. In the BANGO and BIG‐BIRD elements a CAWURST‐20 (240 ppm, 1.92 ms; H2L) adiabatic ¹³C inversion pulse was used. β ^I was set to 110° for the first and to 120° for the second BANGO, respectively, and the $v$ angle of BIG‐BIRD to 20°. Before standard processing the obtained combined data set is separated into three blocks, corresponding to SEA XLOC, HMBC and 2BOB data, using the Bruker au‐program splitx. The data block of SEA XLOC is then divided further with the au‐program split to separate ZQ and 2Q data. The 2BOB block is divided into four data sets with the same au‐program split for subsequent linear combinations. Excerpts from the SEA XLOC and HMBC spectra can be found in the Supporting Information (Figure S7).