Well-resolved and information-rich J-spectra are the foundation for chemical analysis based on zero-field NMR. Yet, even in relatively small molecules, the spectra may gain complexity, hindering the analysis. To address this problem, we investigate an example biomolecule characterized 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 hydrogen chemical-exchange rate in [13C, 15N2]-urea solution, the molecule, being an effective spin system XAB2A'B'2, behaves as a much simpler XA2 system (where X = 13C, A = 15N, B = 1H), manifesting through a single narrow spectral peak. Additionally, we show that introducing spin-1 nuclei into the molecule and investigating J-spectra of 1H/D isotopologues of [15N2]-urea allows to study various isolated spin subsystems: XA2, (XA)B, and XB2 (here X = 15N, A = 1H, B = D), again greatly simplifying spectra analysis. The influence of the chemical exchange process on zero-field $J$-spectra for each urea solution is elucidated by theoretical studies, demonstrating solid agreement between results and simulations. This study shows the applicability of zero-field NMR to detect complex biomolecules in aqueous solutions, and it opens the means for future in vivo/in vitro biochemical investigations, particularly in biofluids with a high concentration of water.
Supplementary Information: Zero-field J-Spectroscopy of Urea: Spin-Topology Engineering by Chemical-Exchange