Projected Volume Method for Accurate Measurement of Cross-Peak Intensity in Two-Dimensional NMR Spectra

Abstract

Trandolapril, an angiotensin-converting enzyme (ACE) inhibitor, undergoes two-state exchange in organic solvents arising from cis–trans isomerization around a N–C bond. A previous NMR study reported different equilibrium constants depending on which ¹H nuclei were used for analysis. Such variations have been attributed to experimental error but require experimental resolution. In this study, we developed a new method for measuring cross-peak volumes based on a projection technique and applied the method to a series of two-dimensional ¹H–¹³C HSQC spectra of trandolapril, acquired using the time-zero HSQC (HSQC0) scheme. The Proj-Vol method yielded consistent equilibrium constant values across multiple ¹H nuclei, demonstrating that trandolapril has a single equilibrium constant, consistent with its single exchange mechanism. The Proj-Vol method is based on constructing 1D ¹³C projections of narrow rectangular regions around the cross-peaks. The use of 1D projection provides several advantages, including fewer fitting parameters and the elimination of the need to consider peak splitting due to ¹H homonuclear J-couplings. It also offers other useful benefits, such as a narrower projection box size to reduce the contributions of other diagonally overlapping cross-peaks in 2D HSQC spectra, the improved signal-to-noise ratio of projection spectra by slice summation, and the cancellation of dispersion components caused by spectral misphasing in the ¹H dimension. These advantages and benefits increase the accuracy of cross-peak volume determination in 2D HSQC spectra, compared with existing methods that directly fit 2D cross-peak shapes.