Pure shift NMR approach for fast and accurate extraction of heteronuclear couplings

Abstract

The direct and accurate determination of heteronuclear (ⁿJ_HX, X = ¹⁹F, ³¹P) couplings from the one dimensional ¹H-NMR spectrum is severely hampered due to the simultaneous presence of large numbers of ⁿJ_HH. The present study demonstrates the utility of the pure shift NMR approach for spectral simplification, and precise and direct measurement of heteronuclear couplings. As a consequence of refocusing of homonuclear couplings (ⁿJ_HH) by the pure shift NMR, only heteronuclear couplings (ⁿJ_HX) appear as simple multiplets at the resonance position of each chemically non-equivalent proton, enabling their direct measurement from the 1D-¹H spectrum. The experiment is demonstrated on a number of molecules containing either ¹⁹F or ³¹P, where ⁿJ_HF and ⁿJ_HP could be precisely measured in a straightforward manner. The distinct advantage of the experiment is demonstrated on molecules containing more than one fluorine atom, where most of the available NMR experiments fail or have restricted utility.

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Introduction

NMR spectroscopy is one of the widely used analytical techniques for the study of small molecules, proteins and peptides.¹ Availability of higher magnetic fields, sophisticated electronics, and modern probe design permits easy spectral analyses² and quantification at low concentration levels.³ The multiplicity pattern provides valuable information about the scalar couplings (J). The ⁿJ_XH (n = 2, 3) is extremely useful in the assignment of relative configurations and conformations.⁴ The well known Karplus equation is employed to obtain the relative configurations in small molecules using the vicinal coupling constants.⁴ Homo- and hetero-nuclear J-based analysis have also been proven to be very useful for the analysis of natural products and relative stereochemistry of complex acyclic molecules.^5,6 Nevertheless the challenging problem of reducing the spectral complexity for the straightforward determination of couplings continues to persist. The direct extraction of short and long range J_HX (X = F, P, etc.) couplings from the one dimensional ¹H NMR spectrum is a challenging task due to the paradigm of homonuclear couplings. Although there exist many 2D experiments for the extraction of ⁿJ_HF, such as, 2D ¹H–¹³C,^{7 13}C–¹⁹F,^{8 1}H–¹⁹F⁹ correlation, majority of them demand either specially designed ¹⁹F probe or require more investment on the instrument time, and also suffer from problem of sensitivity when low abundant nuclei, such as, ¹³C is involved. Long back the skew projected 2D J-resolved spectrum has been reported for the measurement of heteronuclear couplings, which does not insist on more experimental time and additional equipment.¹⁰ We have also reported spin selective double quantum,¹¹ double quantum J-resolved, spin selective triple quantum,¹² and heteronuclear ¹⁵N–¹H double quantum-single quantum correlation,¹² 2D ¹H–¹⁵N¹³ experiments for the extraction of signs and magnitudes of homo- and hetero-nuclear couplings. The higher quantum detection experiments suffer from both the loss of sensitivity and the linear scaling of inhomogeneity contributions with the higher quantum order.¹² One dimensional 1D-HSQC,¹⁴ 1D-HSQMBC-TOCSY experiments¹⁵ and selective TOCSY experiment¹⁶ though available for the extraction of signs and magnitudes of the couplings, they require higher concentrations of sample and cannot be applied in a straightforward manner when more than one fluorine atom is present in the molecule. Furthermore, some of these experiments have limitations due to severe overlap of peaks posing problem for selective excitation. It is always beneficial to devise a simple and convenient one dimensional experiments for such a purpose which is free from such limitations. Thus in the present work we are exploring the utility of a simple NMR experiment, which decouples the entire proton coupled network of spins¹⁷ well known in the literature as pure shift NMR¹⁸ and a number of related experiments have been developed by the group of G.A. Morris.¹⁸ The pulse sequence thus retains only heteronuclear couplings allowing a fast and reliable method of measuring ⁿJ_HX (X = ¹⁹F, ³¹P) couplings from the complex ¹H spectrum. The usefulness of pure shift experiments is demonstrated on fluorine and phosphorous containing aliphatic and aromatic molecules which exhibit structural and spectral complexity, such as, epifluorohydrin (a), 2-fluroacetanilide (b), 2-fluoropyridine (c), 2,3,4-trifluorophenylcyanide (d), 3,4-difluoro-2-nitrophenol (e) and dimethyl vinylphosphonate (f) (Fig. 1).

Fig. 1 Chemical structures of molecules investigated for the extraction of the ⁿJ_HX (X = ¹⁹F, ³¹P) couplings using pure shift experiments.

Results and discussion

Initially the utility of pure shift approach is demonstrated on epiflurohydrin (molecule a) whose ¹H NMR spectrum is well dispersed but has complex multiplet pattern at each proton site due to both J_HH and J_HF. The direct extraction of ⁿJ_HF couplings from this spectrum is difficult. The application of pure shift sequence removes all the proton–proton couplings and retains only ⁿJ_HF giving a doublet at the chemical shift position of each chemically non-equivalent proton. The direct measure of doublet separation yields ⁿJ_HF depicting the immediate advantage of the utility of pure shift. The ¹H-NMR spectrum of epiflurohydrin with and without implementation of pure shift is given in Fig. 2a and 2b.

Fig. 2 (a) ¹H NMR spectrum of epiflurohydrin (molecule a) in CDCl₃; (b) pure shift spectrum of the same molecule, depicting only ⁿJ_HF couplings.

For wide utility, the experiments were carried out on molecules, such as, 2-fluroacetanilide (b) and 2-fluoropyridine (c), 2,3,4-trifluorophenylcyanide (d), 3,4-difluoro-2-nitrophenol, whose spectra are either broader without any coupling fine structures or complex, preventing the direct extraction of ⁿJ_HF. All the corresponding spectra are reported in the ESI† along with the magnitudes of the measured ⁿJ_HF.

The experiment is not restricted to molecules containing single fluorine atom but also can be employed for molecules containing more than one fluorine atoms. For such a purpose the molecule, 2,3,4-trifluorophenylcyanide (d) was chosen. The ¹H spectrum of this molecule comprises of two well isolated but severely overlapping multiplet pattern in the region δ 7.20–8 ppm, due to the number of proton–proton and proton–fluorine couplings, prohibiting the measurements of ⁿJ_HF (Fig. 3a). On other hand the application of pure shift sequence aids in simplifying the spectrum, the analysis of which provides coupling of ¹H with all the three fluorines (Fig. 3b). Thus the utility of pure shift is fast and reliable for deriving all ⁿJ_HF from the complex ¹H spectrum. The pure shift utility also has distinct advantages over other available methods, such as, 1D-HSQC¹⁴ and 1D-HSQMBC-TOCSY¹⁵ experiments, where the extraction of the couplings fails when the molecules contains more than one fluorine atom. The G-SERF¹⁹ experiment though provides the coupling information, requires multiple experiments to extract all ⁿJ_HF, whereas in the case of the pure shift experiment the coupling can be extracted in one shot from a single experiment. The experiment is also demonstrated on molecule e containing two fluorine atoms, the derived spectral parameters are reported in the ESI.†

Fig. 3 (a) ¹H NMR spectrum of 2,3,4-trifluoronitrobenzene in CDCl₃; (b) pure shift spectrum of the same molecule, depicting only ⁿJ_HF couplings.

The pure shift experiment also finds immediate application when other abundant heteronuclei, such as, phosphorous is present in the molecule, where we are able to extract ⁿJ_HP couplings from the simple 1D-¹H pure shift NMR spectrum. This is demonstrated on dimethyl vinylphosphonate (f) whose chemical structure is reported Fig. 1 and the corresponding ¹H spectrum is given in Fig. 4. The ¹H spectrum exhibits a complex pattern due to the presence of large number of ⁿJ_HH and ⁿJ_HP couplings. As a consequence the spectrum is severely overlapped and does not display any coupling fine structures. In such situations the extraction of ⁿJ_HP is severely hampered and peaks are overlapped posing a challenge for the assignment of peaks. Recently reported selective 1D-TOCSY experiment¹⁶ also has restricted utility in situations since the selective excitation of any of the peaks is not possible due to overlap. However, the pure shift NMR has an immediate advantage and ⁿJ_HP could easily be extracted without any ambiguity. The extracted couplings and the pure shift spectrum are given in Fig. 4.

Fig. 4 (a) ¹H NMR spectrum of dimethyl vinylphosphonate in CDCl₃; (b) pure shift spectrum of the same molecule, depicting only ⁿJ_HP couplings.

The studies clearly reveal that pure shift approach has enormous advantages as the heteronuclear coupling information between two abundant spins is directly derived from the simplified one dimensional spectrum. It is fast, effective and can be easily implemented. However, one limitation of the present approach is that the relative signs of the couplings cannot be derived. Nevertheless this problem can be circumvented by the blend of pure shift with two dimensional sequence such as HSQC [pureshift HSQC], which demands more investment on the instrument time.

Conclusions

In summary, the pure shift NMR approach offers the enormous advantages as it provides all the couplings between proton and other abundant heteronuclei for a given molecule in a single experiment there by filling the lacuna in the current available methods as far as the measurement of ⁿJ_HP and ⁿJ_HF coupling constants from conventional the 1D-NMR is concerned. The experiment can be performed in a matter of few minutes and the complex multiplets gets simplified to an easily interpretable doublet patterns in cases when single fluorine or phosphorous is present. This is the biggest advantage. Compared to 1D-HSQC, 1D-selecitve TOCSY and 1D-HSQMBC-TOCSY experiments, the utility of pure shift is faster, more sensitive, and easier to interpret and does not demand more than one experiment.

Experimental

The commercially available samples were purchased and used as received. The samples were prepared directly in the NMR sample tube with nearly 20 mM concentration in the solvent CDCl₃. All the spectra were recorded on Bruker AV-III 800 MHz NMR spectrometer. For carrying out pure shift experiments the pulse program “push1dzs” available in the public domain of the Manchester NMR methodology group website (http://nmr.chemistry.manchester.ac.uk) was used. The protocol provided on the same website was followed for carrying out the experiment. The refocusing step was carried out using rsnob (shape pulse) of duration 22 ms, combined with slice selection gradient strength of 0.6 to 0.9 G cm⁻¹. Each of the 32 increments in t₁ was acquired with 4 scans with a recycle delay of 2 s between two successive fids. The total time domain points in t₂ dimension are 8 K. The time dome data was zero filled to 1024 K points before processing and was processed with sinebell window function. Data was processed automatically with the AU program named pshift provided at the same website (http://nmr.chemistry.manchester.ac.uk). The AU program converts the raw data to pure shift FID. The FID was subsequently Fourier transformed with line broadening window function of 0.3 Hz.

Acknowledgements

We are very much grateful to Prof. G. A. Morris for permitting us to download and use the relevant pulse program and macros from their website (http://nmr.chemistry.manchester.ac.uk). SRC would like to thank IISc for RA. NS gratefully acknowledges the generous financial support by the Science and Engineering Research Board, Department of Science and Technology, New Delhi (grant no. SR/S1/PC-42/2011).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra01436g

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