The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum information out of challenging biomolecules. Here we investigate the source of and propose solutions for abnormal peak splitting ranging from 152 to 80 Hz and below that were found in three popular TOCSY-based experiment types: H(F1)-C(F2)-DIPSI-H(F3), C(F1)-DIPSI-C(F2)-H(F3), and C(F1)-DIPSI-N(F2)-HN(F3). Peak splitting occurs in the indirect C(F1) or C(F2) dimension before DIPSI and analyses indicate that the artifacts are resulted mainly from the DIPSI transforming a double spin order [Formula: see text] from 13C-13C scalar 1JCC coupling during t1 into observable megnetization. The splitting is recapitulated by numerical simulation and approaches are proposed to remove it. Adding a pure delay of 3.7 ms immediately before DIPSI is a simple and effective strategy to achieve 3D HCCH-TOCSY and HCC(CO)NH-TOCSY spectra free of splitting with full crosspeak intensity.
Keywords: Artifacts; DIPSI; HCC(CO)NH-TOCSY; HCCH-TOCSY; J-coupling; PFG; Splitting.