Zero-Field NMR of Urea: Spin-Topology Engineering by Chemical Exchange

Abstract

Well-resolved and information-rich J-spectra are the foundation for chemical detection in zero-field NMR. However, even for relatively small molecules, spectra exhibit complexity, hindering the analysis. To address this problem, we investigate an example biomolecule with a complex J-coupling network─urea, a key metabolite in protein catabolism─and demonstrate ways of simplifying its zero-field spectra by modifying spin topology. This goal is achieved by controlling pH-dependent chemical exchange rates of ¹H nuclei and varying the composition of the D₂O/H₂O mixture used as a solvent. Specifically, we demonstrate that by increasing the proton exchange rate in the [¹³C,¹⁵N₂]-urea solution, the spin system simplifies, manifesting through a single narrow spectral peak. Additionally, we show that the spectra of ¹H/D isotopologues of [¹⁵N₂]-urea can be understood easily by analyzing isolated spin subsystems. This study paves the way for zero-field NMR detection of complex biomolecules, particularly in biofluids with a high concentration of water.

Figure 1

Simulated and experimental ZULF NMR spectra of [¹⁵N₂]-urea and [¹³C,¹⁵N₂]-urea in aprotic (DMSO) and protic (H₂O) solvents. The structural formulas are shown with J-coupling values used in the simulation.

Figure 2

Experimental (top) and simulated (bottom) zero-field J-spectra of [¹⁵N₂]-urea and [¹³C,¹⁵N₂]-urea in aqueous solutions at various pH values. The peaks arising from one-bond, strong J-coupling interaction between ¹⁵N and ¹H are green shaded (120–150 Hz), while the narrow peaks (around 30 Hz), originating from one-bond J-coupling between ¹³C and ¹⁵N, are highlighted in red.

Figure 3

Experimental and simulated zero-field J-spectra of [¹⁵N₂]-urea in aqueous solutions with various ¹H/D ratios. J-coupling values used in simulations are shown with chemical structures of an example ¹H/D isotopologue of urea. All isotopologues’ structures and corresponding simulated zero-field J-spectra are shown in the Supporting Information.

Figure 4

Left and right: Energy-level structures for XA, (XA)B, and XA₂ spin subsystems. High and low-frequency transitions in (XA)B spin system are denoted by v_1–3 and , respectively. The transition in XA₂ spin system is represented as v₄ which corresponds to 3/2J_XA. The manifolds are grouped by the quantum numbers I_A and I_B that denote the spin number of A nuclei and B nuclei, respectively. Each manifold is labeled by its total spin quantum number F or F_T (see Supporting Information for detailed energy level analysis).^, Only a single sublevel in each manifold and a single transition at each frequency are shown for clarity. Middle: Experimental spectra of [¹⁵N₂]-urea in the mixture of D₂O/H₂O (1:1). For all peaks in the spectrum, the corresponding transitions (v_1–4, ) are determined by the first-order perturbation theory.