Determination of heteronuclear long‐range H,X coupling constants from gradient‐selected HMBC spectra

Abstract

A gradient-selected J-HMBC experiment is presented. Coherence selection with magnetic field gradients results in HMBC spectra in which t , noise is very low or completely eliminated. This makes it possible to detect unequivocally very small ( < t Hz) long-range H,X coupling constants. With a slight modification, the HMBC experiment can be used for the quantitative measurement of these couplings. The signal amplitude of spectra with increasing coupling evolution time z shows the characteristic sin (zJXH t) dependence. By fitting a sine curve to the experimental data or by performing a 3D J-resolved HMBC experiment, accurate "JXH coupling constants are obtained. This method is especially useful for estimating coupling constants between protons and unprotonated heteronuclei, e.g. quaternary carbons, "N nuclei in labelled prolines or "P in organophosphates. These experiments offer the coupling information independent of the linewidth, because the coupling constant need not be extracted from the signal fine structure but rather is available from the displacement of two signal elements. All experiments of this kind have one common shortcoming: they require a proton-bound heteronucleus. Coupling constants to unprotonated heteronuclei are not accessible in this way. Furthermore, signals arising from TOCSY or NOESY transfers may appear that are not related to a long-range J coupling. Additionally, the signal splitting by the 'J,, coupling in F, reduces the intensity, and the signal separation is limited because the experiment is performed as an H,H correlation. For * Author to whom correspondence should be addressed. a sufficient separation these experiments are often extended into three dimensions. Another technique, i.e. the quantitative J correlation, measures the intensity ratio of cross peaks to reference peaks. One version has been implemented as a carbon-excited 3D experiment".' whereas another uses a refocussed HMBC experiment." The HMBC experiment was also used by Bermel et al. 13 and Titman et al.14, Bermel et al. compared the total multiplet width of an HMBC signal (including the long-range coupling) with that from a COSY-type experiment (without the long-range coupling). Titman et al. fitted the multiplet with the aid of reference multiplets.We used the HMBC experiment to measure the longrange H,X coupling constants by stepwise incrementation of the coupling evolution time in a constant-time manner. The HMBC experiment is in principle the most suitable experiment to obtain information on heteronuclear long-range couplings, because all long-range correlations are accessible, even those to unprotonated heteronuclei. However, the HMBC experiment in the phase cycled version" often suffers from unacceptably intense t , noise. This artifact can be removed almost completely by using magnetic field gradients to select the coherence pathway.I6 We use a slight modification of the previously presented gradientselected (gs) HMBC,'77'8 which might be called gs-J-HMBC (see Fig. 1). The long-range evolution delay t is varied...

Conformational Studies of the O-specific Polysaccharide of Shigella flexneri 5a and of Four Related Synthetic Pentasaccharide Fragments Using NMR and Molecular Modeling

“…Two different methods were used to measure long range heteronuclear 3 J C,H coupling constants, EXSIDE (19) and gs-J-HMBC (20). With EXSIDE, all proton band-selective pulses were Gaussian cascade Q3 pulses (37) with phase modulation to achieve off-resonance inversion.…”

“…Indeed, the intensity, s(t 1 ,t 2 ), of a particular correlation in a HMBC spectrum depends on the amplitude of the long range J C,H and can be described by the following equation (20,38): The 13 C spin-lattice relaxation times of O-SP 5a were measured at 125 MHz by means of two-dimensional double-INEPT-type inversedetected experiments with suppression of cross-correlations (39). Eight experiments were performed with relaxation delays, , ranging from 0.005 to 1 s. T 1 values were deduced by measuring the cross-peak intensities as a function of T and fitting the volumes to the equation, y ϭ A exp(Ϫ /T 1 ).…”

“…The 3 J C,H coupling constants were determined by two different techniques, EXSIDE (excitation-sculptured indirect-detection experiments) (19) and gradient-selected J-HMBC experiments (gs-J-HMBC) (20). The molecular modeling of the pentasaccharides involved a complete conformational search with the CI-CADA algorithm (21) interfaced with MM3 force field (22,23), which is very efficient for exploring the conformational space of flexible molecules (21), including oligosaccharides (24 -26).…”

Conformational studies on a unique bis-sulfated glycolipid using NMR spectroscopy and molecular dynamics simulations

“…Furthermore, 3 J C,H values in glycosidic linkages are often smaller than the digital resolution in two-dimensional spectra, making their extraction from the spectra difficult [25]. The J-HMBC 2D experiment overcomes this disadvantage by using a curve-fitting step for signal intensities of cross-peaks [26,27]. For crowded spectra, it could be more advantageous than the proton-detected 1D experiment, selective INEPT [28].…”

PFG J-HMBC 2D NMR Spectroscopic Observation of the Natural Abundance 1H-15N Long-Range Coupling

“…Recently, a new method, PFG J-HMBC 2D, using modified PFG HMBC pulse sequence has been developed for the measurement of the heteronuclear long-range coupling constants including non-protonated heteronuclei. 5 However, the measurement of 1 H-15 N long-range coupling constants without enrichment is still difficult because of the insufficient sensitivity caused by the low natural abundance of 15 N. 5 The measurement of this coupling constant was also attempted by the use of conventional HMBC with PFG. 6 But the limited coupling constant having the value of more than 7 -8 Hz was observed in this method.…”