title: Constant time INEPT CT-HSQC (CTi-CT-HSQC) - A new NMR method to measure accurate one-bond J and RDCs with strong 1H-1H couplings in natural abundance.
abstract: Strong (1)H-(1)H coupling can significantly reduce the accuracy of (1)J(CH) measured from frequency differences in coupled HSQC spectra. Although accurate (1)J(CH) values can be extracted from spectral simulation, it would be more convenient if the same accurate (1)J(CH) values can be obtained experimentally. Furthermore, simulations reach their limit for residual dipolar coupling (RDC) measurement, as many significant, but immeasurable RDCs are introduced into the spin system when a molecule is weakly aligned, thus it is impossible to have a model spin system that truly represents the real spin system. Here we report a new J modulated method, constant-time INEPT CT-HSQC (CTi-CT-HSQC), to accurately measure one-bond scalar coupling constant and RDCs without strong coupling interference. In this method, changing the spacing between the two 180° pulses during a constant time INEPT period selectively modulates heteronuclear coupling in quantitative J fashion. Since the INEPT delays for measuring one-bond carbon-proton spectra are short compared to (3)J(HH), evolution due to (strong) (1)H-(1)H coupling is marginal. The resulting curve shape is practically independent of (1)H-(1)H coupling and only correlated to the heteronuclear coupling evolution. Consequently, an accurate (1)J(CH) can be measured even in the presence of strong coupling. We tested this method on N-acetyl-glucosamine and mannose whose apparent isotropic (1)J(CH) values are significantly affected by strong coupling with other methods. Agreement to within 0.5Hz or better is found between (1)J(CH) measured by this method and previously published simulation data. We further examined the strong coupling effects on RDC measurements and observed an error up to 100% for one bond RDCs using coupled HSQC in carbohydrates. We demonstrate that RDCs can be obtained with higher accuracy by CTi-CT-HSQC, which compensates the limitation of simulation method.
authors.affiliation: Laboratory of Bacterial Polysaccharides, CBER/FDA, Bethesda, MD 20892, United States.
authors.forename: Bingwu
authors.initials: B
authors.lastname: Yu
authors.forename: Hugo
authors.initials: H
authors.lastname: van Ingen
authors.forename: Darón I
authors.initials: DI
authors.lastname: Freedberg
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry*; D000465:Algorithms; D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008358:Mannose / Q000737:chemistry*; D059629:Signal-To-Noise Ratio; D013329:Structure-Activity Relationship
chemical_list: D002247:Carbon Isotopes; D002244:Carbon; D008358:Mannose; D000117:Acetylglucosamine