Thursday, September 1, 2016

Measuring long range heteronuclear couplings

Three bond coupling constants are particularly useful for determining stereochemistry as the Karplus relationship relates them to torsion angles. Typically, however, only a qualitative analysis to define rotamers is used, as measuring the couplings accurately is not easy. Homonuclear 3JHH couplings are normally obtained from 1D 1H spectra, and are fairly straightforward to measure, but heteronuclear 3JCH couplings are more difficult to obtain. There are a variety of methods available for measuring long range heteronuclear couplings, each with their own advantages and disadvantages. Three of them, that have been implemented at the Skaggs NMR Facility, will be described below.


The HETLOC1 experiment produces a 1H-1H spectrum where each correlation is split in the indirect dimension by the 1JCH coupling. In the directly-detected dimension the two peaks are offset by the long range nJCH coupling. This experiment gives both the magnitude and the sign of the coupling constant. In the example shown below the coupling is positive. If the upper peak were to the left of the lower then the coupling would be negative.

Another advantage of the HETLOC is that it records all couplings that it is capable of recording in a single experiment. One of the experiment's disadvantages, due to its use of a TOCSY transfer step, is that it can only measure couplings between a protonated heteronucleus and protons that correlate with it by TOCSY. Other disadvantages are, the doubling of peaks that increases the chances of overlap and peak distortion making measurements less accurate, and the fact that the chemical shift in the indirect dimension is not that of the heteronucleus involved in the coupling but of the proton attached to it. This latter disadvantage makes it easy to get confused as to what coupling is actually being measured. Nevertheless the HETLOC is a good way of collecting a lot of information quickly and is quite sensitive.


The J-HMBC2 experiment produces a 1H-13C spectrum where the correlations are split in the indirect heteronuclear dimension. The size of the peak separation is proportional to the long range heteronuclear coupling but is scaled by a factor (typically 50-100) determined by the experimental parameters. The scaling factor reduces the need for high resolution in the indirect dimension and so reduces experimental time.

The big advantage of the J-HMBC is its ability to measure coupling constants between nuclei that cannot be connected by a TOCSY transfer, thus providing information that the HETLOC cannot. Also, the chemical shift of the correlations are those of the nuclei involved, simplifying analysis. Like the HETLOC, it too records all couplings its capable of measuring in a single experiment. Disadvantages are its doubling of peaks, leading to a greater chance of overlap, reduced sensitivity relative to the HETLOC, and inability to determine the sign of the coupling constant.


The In-Phase Anti-Phase (IPAP) HSQMBC3 uses the spin state selective methodology to separate the α and β lines of a doublet into separate 1H-13C spectra. Correlations appear as single peaks in each spectrum offset from their natural chemical shift. Overlaying the spectra and measuring the distance between the α and β lines gives the magnitude of the coupling constant, while the direction of the offset gives the sign. In the spectrum below, the β line of the doublet in green is upfield of the α line in blue, indicating that the coupling is positive.

The main advantage of the IPAP-HSQMBC is its separation of the doublet lines into separate subspectra, thereby reducing the chance of overlap and peak distortion and enabling more accurate measurements. Measurement of the coupling in the directly detected 1H dimension also means that high resolution does not require excessive acquisition times. The IPAP HSQMBC can measure couplings between nuclei that cannot be connected by a TOCSY transfer, and it provides the sign of the coupling as well as it magnitude. The main drawback of this experiment is that the resonances in the directly detected 1H dimension cannot be coupled, requiring selective or band selective excitation. To measure all the couplings requires multiple experiments, or more specialized multi site selective excitation. An extension of this experiment, the IPAP HSQMBC-TOCSY4, adds a TOCSY transfer to generate more correlations and overcome this limitation somewhat.


1. Uhrín, Dusan, Batta, Gyula, Hruby, Victor J., Barlow, Paul N., Kover, Katalin E., 'Sensitivity- and Gradient-Enhanced Hetero (ω1) Half-Filtered TOCSY Experiment for Measuring Long-Range Heteronuclear Coupling Constants', Journal of Magnetic Resonance, vol. 130, no. 2, 155-161 (1998).

2. Meissner, Axel, Sørensen, Ole W., 'Measurement of J(H,H) and long-range J(X,H) coupling constants in small molecules. Broadband XLOC and J-HMBC', Magn. Reson. Chem., vol. 39, no. 1, 49-52 (2001)

3. Gil, Sergi, Espinosa, Juan F., Parella, Teodor, 'Accurate measurement of small heteronuclear coupling constants from pure-phase α/β HSQMBC cross-peaks', Journal of Magnetic Resonance, vol. 213, no. 1, 145-150 (2011).

4. Saurí, Josep, Espinosa, Juan F., Parella, Teodor, 'A Definitive NMR Solution for a Simple and Accurate Measurement of the Magnitude and the Sign of Small Heteronuclear Coupling Constants on Protonated and Non-Protonated Carbon Atoms', Angew. Chem. Int. Ed., vol. 51, no. 16, 3919-3922 (2012).

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