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Friday, January 10, 2020

Where is my INEPT signal? - Proton Exchange Issues

INEPT and DEPT sequences are routinely used to enhance the NMR signals for low γ nuclides such as 15N or 13C.  The enhancement relies on polarization transfer between the protons J-coupled and the low γ nuclide.  The pulse sequences incorporate delays based on the reciprocal of the J-coupling constant between the protons and the low γ nuclide.  In the case of 15N INEPT, the enhancement for each scan can be as much as γHN (~ 10) compared to that from a conventional one-pulse sequence with inverse gated decoupling.  Furthermore, the recycle delay for the INEPT sequence depends on the the 1H T1 relaxation time rather than that of 15N.  1H T1's are typically an order of magnitude (or more) less than those of 15N so the recycle delays required for 15N INEPT spectra are at least ten (and possibly 100 times) shorter than those required for one-pulse data collection.  These two factors mean that the true time saving for a 15N INEPT measurement compared to a one-pulse 15N measurement can be on the order of 100 - 1000 times.  There are, however cases where 15N INEPT signals are attenuated or entirely nonexistent.  Attenuated 15N INEPT signals are observed when the protons (with short T1) coupled to 15N exchange with those of water (longer T1) on a time scale of seconds.*  The problem arises because of saturation transfer during the inverse gated decoupling used during the acquisition time. The partially saturated protons are unable to transfer as much polarization to the 15N as they would were they fully polarized. The problem can be reduced if a recycle delay much greater than the T1 relaxation time of the water protons is employed.  If the protons bound to 15N undergo exchange with other labile protons at a rate fast with respect to the 1H-15N J coupling interaction, polarization transfer from 1H to 15N is not possible and a 15N INEPT signal cannot be observed.  This is demonstrated in the figure below.
Concentrated solutions of the methyl ester of anthranilic acid and anthranilic acid were prepared in DMSO-d6.  The 15N NMR data were collected on a 600 MHz instrument with a cryoprobe.  The left-hand panel of the figure compares the 15N one-pulse spectrum with inverse gated decoupling (bottom) to the INEPT spectrum (top) for the methyl ester.  The spectra were collected with the same number of scans. For the methyl ester, the 15N bound protons do not exchange with any other labile protons.  The enhancement in the 15N INEPT spectrum is clear.  Similar spectra for anthranilic acid are shown on the right-hand side of the figure.  In anthranilic acid, the 15N bound -NH2 protons undergo intramolecular exchange with the acid proton at a rate fast with respect to the one-bond 15N-1H coupling constant (~90 Hz).  As a result, polarization transfer is not possible and no INEPT signal is observed.  The same is true for the meta- and para- isomers (data not shown). 

Thank you to Jin Hong for sharing her experience with collecting 15N INEPT data for anthranilic acid and Mojmir Suchy for kindly providing the samples.

* G.D. Henry and B.D. Sykes, J. Magn. Reson. B, 102, 193 (1993).

Friday, January 3, 2020

1H J-Resolved Spectroscopy to Evaluate 1H-1H and 1H-19F Coupling Constants

2D 1H J-RESolved spectroscopy (JRES) is able to separate the 1H chemical shift and J coupling interactions in the F2 and F1 domains of the 2D data, respectively. The F2 projection represents the pure-shift 1H decoupled 1H NMR spectrum while the individual F1 slices at each chemical shift reveal the 1H - 1H J coupling for each resonance.  When this technique is applied to a spin system with both homonuclear 1H-1H coupling and heteronuclear coupling, it has the ability to provide both the homonuclear and heteronuclear coupling constants.  This is demonstrated in the figure below for 2,3-difluoro pyridine which has both 1H-1H and 1H-19F coupling.
The top trace in the figure is the 1H NMR spectrum showing the complex resonances due to both the homonuclear and heteronuclear coupling.  The 2D JRES spectrum is highlighted in grey.  The 1H-1H coupling is shown in the F1 slices which were summed to produce the blue, red and green vertical traces in the figure for 1H resonances A, C and B, respectively.  These traces are identical to the resonances in the separately collected 1H spectrum with 19F decoupling shown in the bottom trace of the figure.  The F2 projection of the JRES spectrum is shown in the trace directly on top of the 2D spectrum, colour coded in yellow.  The F2 projection represents the 1H decoupled 1H spectrum showing only the 1H- 19F coupling.  It can be compared to the separately collected PSYCHE pure-shift 1H spectrum, colour coded in orange which is very nearly identical.  Clearly this very simple, often overlooked, technique can provide a great deal of both homonuclear and heteronuclear coupling information.