The position of hydroxyl groups can often be inferred from chemical shifts, but for complex, unknown molecules chemical shift arguments may not be conclusive. Chemical reaction of the alcohol is one method often used to identify the location of the group, but it is also possible to use the change in chemical shift induced by exchanging protons for deuterons. This can be as simple as changing the solvent as shown in the example below.
The nature of the hydrogen on the hydroxyl affects the chemical shift of the carbon that it is attached to. Substituting a proton for a deuteron changes the chemical shift of the attached carbon because it reduces the length of the C-H bond. The difference is normally quite small and to detect it a high resolution NMR spectrum must be obtained. A 1D 13C spectrum would probably suffice, but in many cases this experiment is not sensitive enough. A 1H-13C HSQC spectrum can also provide the information as long as the resolution in the carbon dimension is increased above that typically used.
For this comparison two samples were used - one in methanol-d4, and the other in methanol-d3. In methanol-d4 all the hydrogen atoms are deuterons. In methanol-d3 the methyl group is deuterated but the hydroxyl is not. The rapid solvent exchange of hydroxyls in methanol ensures that in methanol-d4 all solute hydroxyls will be deuterated, while in methanol-d3 the hydroxyls will be protonated. The molecule chosen for the comparison was glucose-diacetonide whose structure is shown below. It contains one hydroxyl group.
To measure the deuterium induced shifts 1H-13C HSQC spectra with 1024 increments in the 13C dimension and a 13C sweep width of 90 ppm were recorded. This gave a digital resolution of 0.176 ppm per point. Four-fold linear prediction during the processing increased the resolution further. An expansion of the overlaid spectra is shown below. The spectrum in methanol-d4 is shown in blue, and the spectrum in methanol-d3 is shown in red.
The peaks at top-left are due to the methine adjacent to the methylene on the five membered ring. The peaks at bottom-right are due to the hydroxyl bearing methine. Both pairs of peaks show a similar, slight difference in the 1H dimension, but the signals from the hydroxyl-bearing methine show a much larger difference in the 13C dimension. Bar charts for the chemical shift differences between methanol-d4 and methanol-d3 for all the peaks are shown below. Peaks 1-4 are the methyl groups, peak 8 the hydroxyl-bearing methine, peak 9 the methylene, and peak 11 is the methine between two oxygens. Clearly, the hydroxyl bearing methine shows the biggest difference between the two solvents.
To accurately measure these small chemical shift changes precise referencing of the spectra was essential. Indirect referencing of both dimensions was used. Referencing the spectra by using the residual protonated methanol signal gave poor quality data that obscured the effect, likely because the position of the residual methanol signal is also influenced by the deuterium. Nevertheless, with careful referencing this deteurium-induced change in chemical shift can be used to identify carbon-bearing hydroxyls without resorting to chemical modification of the compound or arguments based on "typical" chemical shifts.
Acknowledgments
Thanks to Prof. Ted Molinski for suggesting this experiment and to Mariam Salib for preparing the samples.
Nicely presented, Brendan. The observation of isotope shifts in -CH-OH(D) is not ours, originally: it’s been used many times to assign -CH-OH by recording 13C NMR in those two solvents. Nicely done if you have the amount of sample needed. Impossible if you have mere ‘nanomoles’ (sound familiar, Mariam & Alex? ;-). This nice implementation of hi-res HSQC, using the SSPPS 600 MHz 1.7 mm microcryoprobe, solves the problem.
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