Analyses, extensions and comparison of three experimental schemes for measuring ((n)J(CH)+D(CH))-couplings at natural abundance

Three types of experiments for measuring (n)J(CH) heteronuclear long-range coupling constants are examined and extended with state-of-the-art pulse sequence building-blocks: The use of a HMBC with corresponding reference-HSQC for accurate coupling determination is combined with the constant time technique and the conversion of antiphase magnetization into ZQ/DQ-coherences; CPMG-based LR-CAHSQC and BIRD(r,X)-HSQMBC experiments are examined in detail with respect to their coherence transfer properties; finally, the HSQC-TOCSY-IPAP experiment is introduced, a sequence derived from previously published alpha and beta selective HSQC-TOCSYs using a different spin state selection technique and a recently developed ZQ-suppression method. The experiments are characterized with their advantages and disadvantages and compared using strychnine and menthol as standard molecules.     

Sensitivity improvement and new acquisition scheme of heteronuclear active-coupling-pattern-tilting spectroscopy

A simplified phase-cycling scheme for heteronuclear active-coupling-pattern tilting (ACT) spectroscopy is presented. It is demonstrated that the theoretically expected twofold sensitivity gain over earlier implementations can be experimentally realized. A further intensity increase by a factor of 2 is obtained with standard sensitivity-enhancement pulse-sequence elements. The HSQC-HECADE sequence presented is designed for an accurate determination of heteronuclear one-bond and, with subsequent I-spin isotropic mixing, long-range coupling constants. As an exemplary application, the determination of the (3)J(N,Hbeta) coupling constants in a peptide at natural isotope abundance is demonstrated. Additionally, a new polarization-transfer step for the long-range HSQC-HECADE experiment is proposed which avoids a fixed delay tuned to a specific coupling-constant value. Thus, the long-range correlation experiment does not require prior knowledge of the coupling constants to be measured and yields more uniform cross-peak intensity for a broad range of active coupling constants.     

Optimizing delays in the MBOB,broadband HMBC,and broadband XLOC NMR pulse sequences

The MBOB, broadband HMBC, and broadband XLOC NMR pulse sequences (A. Meissner and O. W. S?rensen (2000, Magn. Reson. Chem. 38, 981-984; 2001, 39, 49-52)) were introduced as a means of obtaining heteronuclear long-range correlation spectra with broadband excitation over an interval of heteronuclear long-range J coupling constants. However, it is not trivial what combination of delays to choose for a given purpose, particularly if one-bond and long-range correlation spectra are obtained simultaneously as in MBOB. This paper presents a way to determine sets of delays for MBOB, broadband HMBC, and broadband XLOC resolving the problem. The results tabulated suit various ranges of J coupling constants and transverse relaxation times.     

Sensitivity- and Gradient-Enhanced Hetero (ω1) Half-Filtered TOCSY Experiment for Measuring Long-Range Heteronuclear Coupling Constants

An enhanced version of the X(ω₁) half-filtered TOCSY experiment for measurement of long-range heteronuclear coupling constants is proposed which yields high-quality spectra with substantially increased sensitivity and resolution. The modified method features gradient-enhanced X filtering sequences, broadband homonuclear decoupling duringt₁, optional¹J_XHscaling in theF₁domain, and gradient coherence selection in combination with the sensitivity-enhanced protocol for the TOCSY transfer. These modifications extend the applicability of the method—coupling constants can be measured accurately for natural abundance samples at low concentrations and for compounds yielding complex spectra. Computer-aided analysis of E.COSY-type multiplets is applied for the determination of heteronuclear long-range coupling constants.     

Simplified multiplet pattern HSQC-TOCSY experiment for accurate determination of long-range heteronuclear coupling constants

A new two-dimensional pulse sequence for accurately determining heteronuclear coupling constants is presented. It is derived from HSQC and HECADE techniques with B0 gradient coherence selection. The main feature of the proposed method is spectra with only one component of the IS doublet; i.e., the final result is equivalent to a selective broadband excitation of either Salpha or Sbeta spin states and a preservation of these states during the entire experiment. The effect is obtained by an appropriate combination of in- and antiphase coherences. It is demonstrated that heteronuclear single-bond as well as long-range coupling constants and their relative signs are readily evaluated. The proposed sequence is equally or less sensitive to a variation of heteronuclear one-bond couplings than previously published, closely related sequences. The new method is applied to a peptide sample for determination of 3JN, Hbeta.     

Sensitivity- and Gradient-Enhanced Hetero (ω1) Half-Filtered TOCSY Experiment for Measuring Long-Range Heteronuclear Coupling Constants

An enhanced version of the X(ω₁) half-filtered TOCSY experiment for measurement of long-range heteronuclear coupling constants is proposed which yields high-quality spectra with substantially increased sensitivity and resolution. The modified method features gradient-enhanced X filtering sequences, broadband homonuclear decoupling duringt₁, optional¹J_XHscaling in theF₁domain, and gradient coherence selection in combination with the sensitivity-enhanced protocol for the TOCSY transfer. These modifications extend the applicability of the method—coupling constants can be measured accurately for natural abundance samples at low concentrations and for compounds yielding complex spectra. Computer-aided analysis of E.COSY-type multiplets is applied for the determination of heteronuclear long-range coupling constants.     

Sensitivity- and Gradient-Enhanced Heteronuclear Coupled/Decoupled HSQC–TOCSY Experiments for Measuring Long-Range Heteronuclear Coupling Constants

A pulsed field gradient version of the sensitivity-enhanced 2D HSQC–TOCSY experiment is proposed for measurement of long-range heteronuclear coupling constants. The coupling constants are obtained by computer-aided analysis of mixed-phase multiplets with and without the heteronuclear splitting. Generation of pure phase data is not required. Since large¹J_XHandJ_HHcouplings are used for coherence transfer, smallⁿJ_XHcan be measured accurately, which could be difficult to obtain from purely heteronuclear polarization transfer experiments.     

3D HSQC-HSQMBC--increasing the resolution of long-range proton-carbon correlation experiments

A 3D HSQC-HSQMBC experiment is proposed for increasing the separation of proton-carbon long-range correlation cross peaks, the lack of which is occasionally seen in corresponding 2D experiments. It is aimed at complex molecules with many protonated carbons exhibiting a narrow spread of 13C chemical shifts e.g., complex carbohydrates. It does not yield long-range correlation of quaternary carbons. An extra indirectly detected 1H dimension of this experiment provides additional separation of long-range correlation cross peaks by utilising the chemical shifts of protons directly attached to 13C. Evolution of single-quantum coherences throughout the entire pulse sequence ensures that the cross peaks are inphase pure absorption singlets in both indirectly detected dimensions, thus maximising the resolution and sensitivity of the experiment. Partial signal cancellation can be expected due to the antiphase character of peaks in the directly detected dimension. The intensity of cross peaks depends on the length of a single long-range evolution interval and values of both active and passive long-range coupling constants of each carbon. The 3D HSQC-HSQMBC experiment provided high quality long-range correlation spectra of a 2 mg pentasaccharide sample in 27 h. The technique can also be used for measurement of long-range heteronuclear coupling constants from pure antiphase multiplets in the directly detected dimension.     

3D HSQC-HSQMBC—increasing the resolution of long-range proton–carbon correlation experiments

A 3D HSQC-HSQMBC experiment is proposed for increasing the separation of proton–carbon long-range correlation cross peaks, the lack of which is occasionally seen in corresponding 2D experiments. It is aimed at complex molecules with many protonated carbons exhibiting a narrow spread of ¹³C chemical shifts e.g., complex carbohydrates. It does not yield long-range correlation of quaternary carbons. An extra indirectly detected ¹H dimension of this experiment provides additional separation of long-range correlation cross peaks by utilising the chemical shifts of protons directly attached to ¹³C. Evolution of single-quantum coherences throughout the entire pulse sequence ensures that the cross peaks are inphase pure absorption singlets in both indirectly detected dimensions, thus maximising the resolution and sensitivity of the experiment. Partial signal cancellation can be expected due to the antiphase character of peaks in the directly detected dimension. The intensity of cross peaks depends on the length of a single long-range evolution interval and values of both active and passive long-range coupling constants of each carbon. The 3D HSQC-HSQMBC experiment provided high quality long-range correlation spectra of a 2 mg pentasaccharide sample in 27 h. The technique can also be used for measurement of long-range heteronuclear coupling constants from pure antiphase multiplets in the directly detected dimension.     

HECADE: HMQC- and HSQC-Based 2D NMR Experiments for Accurate and Sensitive Determination of Heteronuclear Coupling Constants from E.COSY-Type Cross Peaks

Two-dimensional pulse sequences for the determination of heteronuclear long-range coupling constants are presented. The sequences are based on the HMQC/HMBC or HSQC technique with subsequent optional homonuclear I-spin transfer. However, they yield tilted cross-peak patterns displaying antiphase heteronuclear coupling constants in the projections of both dimensions, which allow accurate determination of the couplings even in cases where the linewidth is of comparable magnitude. Two characteristic pulse-sequence elements were implemented to shape theF₁domain: the first element allows an arbitrary scaling of the heteronuclear coupling splittings relative to S-spin chemical-shift differences, whereas the second element achieves homonuclear broadband decoupling among the I spins in the HMQC/HMBC experiments and thus allows purely absorptive representations of such spectra. In comparison with established (ω₁) X-half-filtered TOCSY spectra, the signal dispersion inF₁is significantly improved and largely under experimental control. Furthermore, heteronuclear couplings of (I₁, S) pairs where S is either quaternary or carries one or more I spins that do not belong to the same I-coupling network as I₁can also be measured. The implementation of pulsed field gradients results in good suppression of spectral artifacts.     

Accurate measurements of multiple-bond 13C-1H coupling constants from phase-sensitive 2D INEPT spectra.

Measurements of multiple-bond 13C-)H coupling constants are of great interest for the assignment of nonprotonated 13C resonances and the elucidation of molecular conformation in solution. Usually, the heteronuclear multiple-bond coupling constants were measured either by the J(CH) splittings mostly in selective 2D spectra or in 3D spectra, which are time consuming, or by the cross peak intensity analysis in 2D quantitative heteronuclear J correlation spectra (1994, G. Zhu, A. Renwick, and A. Bax, J. Magn. Reson. A 110, 257; 1994, A. Bax, G. W. Vuister, S. Grzesiek, F. Delaglio, A. C. Wang, R. Tschudin, and G. Zhu, Methods Enzymol. 239, 79.), which suffer from the accuracy problem caused by the signal-to-noise ratio and the nonpure absorptive peak patterns. Concerted incrementation of the duration for developing proton antiphase magnetization with respect to carbon-13 and the evolution time for proton chemical shift in different steps in a modified INEPT pulse sequence provides a new method for accurate measurements of heteronuclear multiple-bond coupling constants in a single 2D experiment.     

Accurate Measurements of Multiple-Bond C–H Coupling Constants from Phase-Sensitive 2D INEPT Spectra

Measurements of multiple-bond ¹³C–¹H coupling constants are of great interest for the assignment of nonprotonated ¹³C resonances and the elucidation of molecular conformation in solution. Usually, the heteronuclear multiple-bond coupling constants were measured either by the J_CH splittings mostly in selective 2D spectra or in 3D spectra, which are time consuming, or by the cross peak intensity analysis in 2D quantitative heteronuclear J correlation spectra (1994, G. Zhu, A. Renwick, and A. Bax, J. Magn. Reson. A 110, 257; 1994, A. Bax, G. W. Vuister, S. Grzesiek, F. Delaglio, A. C. Wang, R. Tschudin, and G. Zhu, Methods Enzymol. 239, 79.), which suffer from the accuracy problem caused by the signal-to-noise ratio and the nonpure absorptive peak patterns. Concerted incrementation of the duration for developing proton antiphase magnetization with respect to carbon-13 and the evolution time for proton chemical shift in different steps in a modified INEPT pulse sequence provides a new method for accurate measurements of heteronuclear multiple-bond coupling constants in a single 2D experiment.     

Sensitivity enhancement of a two-dimensional experiment for the measurement of heteronuclear long-range coupling constants, by a new scheme of coherence selection by gradients

In this work we present a new pulse sequence for the measurement of long-range heteronuclear coupling constants in which the optimization of coherence selection by pulsed field gradients offers a net increase in sensitivity. This type of experiments is extremely valuable for conformational studies of molecules in natural abundance and in this context the use of gradients is essential for an efficient suppression of (12)C bound proton signals. A comparative analysis of the different gradient schemes available is presented with a conclusive elucidation of the relative sensitivities. Our gradient scheme could be advantageous as a building block for other related experiments.     

Development of a method for the measurement of long-range 13C-1H coupling constants from HMBC spectra

This paper describes a number of improvements to a method, developed in this laboratory and described in J. Magn. Reson. 85 (1989) 111-113, which makes it possible to determine values of long-range 13C-1H coupling constants from heteronuclear multiple bond correlation (HMBC) spectra. First, it is shown how pulsed-field gradients can be introduced into the HMBC experiment without perturbing the form of the cross-peak multiplets; a one-dimensional version of the experiment is also described which permits the rapid measurement of a small number of couplings. Second, the experiment is modified so that one-bond and long-range cross-peaks can be separated, and so that the one-bond cross-peaks have more reliable intensities. Finally, it is shown how these one-bond cross-peaks can be used to advantage in the fitting procedure.     

Improved performance accordion heteronuclear multiple-bond correlation spectroscopy-IMPEACH-MBC.

A modification of the recently reported ACCORD-HMBC long-range heteronuclear shift correlation experiment is described. The new experiment, IMPEACH-MBC (improved performance accordion heteronuclear multiple-bond correlation), introduces a new pulse sequence element, a constant time variable delay. The incorporation of the constant time variable delay into the IMPEACH-MBC sequence suppresses (1)H-(1)H coupling modulation inherent to the utilization of the accordion principle to sample a broad range of potential long-range heteronuclear couplings. (1)H-(1)H coupling modulation, which introduces an F(1) modulation or a "skew" of responses in the second frequency domain of the ACCORD-HMBC experiment, is suppressed in the IMPEACH-MBC experiment. Results of identically optimized IMPEACH-MBC and ACCORD-HMBC experiments performed on a sample of strychnine are compared.     

应用E.COSY型的13C-1H相关谱准确测定果糖-1,6-二磷酸根离子中异核31P-1H和31P-13C偶合常数

建议了一种E.COSY型的¹³C-¹H相关实验.在相应的¹³C-¹H相关谱中,³¹P核对¹³C,¹H核的被动偶合给出E.COSY型的谱峰裂分,可用于准确测定含磷化合物中的³¹P-¹H和³¹P-¹³C偶合常数及其相对符号.测定了果糖-1,6-二磷酸根离子的³¹P-¹H和³¹P-¹³C偶合常数。     

应用E.COSY型的13C-1H相关谱准确测定果糖-1,6-二磷酸根离子中异核31P-1H和31P-13C偶合常数

建议了一种E .COSY型的1 3C 1 H相关实验 .在相应的1 3C 1 H相关谱中 ,31 P核对1 3C ,1 H核的被动偶合给出E .COSY型的谱峰裂分 ,可用于准确测定含磷化合物中的31 P 1 H和31 P 1 3C偶合常数及其相对符号 .测定了果糖 1 ,6 二磷酸根离子的31 P 1 H和31 P 1 3C偶合常数     

Accurate determination of small one-bond heteronuclear residual dipolar couplings by F1 coupled HSQC modified with a G-BIRD(r) module

We report a G-BIRD(r) modified coupled HSQC experiment for the accurate determination of one-bond heteronuclear residual dipolar couplings. The G-BIRD(r) module has been employed to refocus the long-range coupling evolution of the heteronucleus during the t1 frequency labeling period. As a result, the crosspeaks obtained are split by only the direct one-bond coupling that can be extracted by measuring simple frequency differences between singlet maxima. Additionally the decoupling of long-range multiple bond splittings leads to considerable sensitivity enhancement. The modification also has been applied in a TROSY sequence resulting in a significant sensitivity and resolution improvement.     

J-modulated ADEQUATE (JM-ADEQUATE) experiment for accurate measurement of carbon-carbon coupling constants

A new method for the accurate determination of carbon-carbon coupling constants is described. The method is based on a modified ADEQUATE experiment, where a J-modulated spin-echo sequence precedes the ADEQUATE pulse scheme. The J-modulation and scaling of carbon-carbon couplings is based on simultaneous incrementation of 13C chemical shift and coupling evolution periods. The time increment for the homonuclear carbon-carbon coupling evolution can be suitably scaled with respect to the corresponding increment for the chemical shift evolution. Typically a scaling factor of 2 to 3 is employed for the measurement of one-bond coupling constants, while multiplication by a factor of 10 to 15 is applied when small long-range couplings are determined. The same pulse scheme with coupling evolution period optimized for one-bond or long-range couplings allows the determination of the corresponding carbon-carbon coupling constants. The splittings of the ADEQUATE crosspeaks in the F1 dimension yield the appropriately multiplied coupling constants.     

Measurement of one-bond heteronuclear dipolar coupling contributions for amine and diastereotopic methylene protons

One-bond heteronuclear and two-bond homonuclear residual dipolar couplings measured at methylene or amine sites can be utilized as long-range constraints in structure determination of molecules as well as to facilitate characterization of local conformation by stereospecific assignment of diastereotopic protons. We present two J-modulated HMQC type experiments to measure the one-bond heteronuclear dipolar coupling contributions of geminal protons individually. In addition two-bond homonuclear residual dipolar couplings between the diastereotopic protons are also obtained.