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Fluorine NMR: An Overlooked Nucleotide Comes into Its Own

Fluorine NMR: An Overlooked Nucleotide Comes into Its Own

The “democratization” of nuclear magnetic resonance (NMR) spectrometers has been accompanied by multiprobe, multi-nuclide capability.

Angelo DePalma, PhD

Angelo DePalma is a freelance writer living in Newton, New Jersey. You can reach him at

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A chemist waking from a 30-year nap would notice many changes in laboratory equipment, most notably the migration of instrumentation from centralized or core locations to the benchtop. The “democratization” of nuclear magnetic resonance (NMR) spectrometers has been accompanied by multiprobe, multi-nuclide capability.

Fluorine-19 (19F) is in many ways an ideal NMR nuclide. It has the same sensitivity as protons, has a wide range of chemical shifts, displays easily recognizable coupling with itself and other NMRactive nuclei, and is capable of multidimensional experiments familiar to spectroscopists who work with 13C or 1H. And fortuitously, every fluorine atom in the universe, save trace levels of 18F generated for medical imaging, is NMR-susceptible.

Although fluorine is approximately the same size as hydrogen, its electronegativity—the highest of any element—affects the reactivity of almost any molecule in which it is found. This property has been used to full effect by pharmaceutical companies.

Fluorine in pharma

Approximately 15 percent of all drugs approved in the United States contain at least one fluorine atom. Common attachment modes are fluorinated aromatics, trifluoromethyl, and aliphatic monofluorides. Drug discovery scientists use fluorination strategically to improve a drug’s pKa, lipophilicity, stability, binding affinity, permeability, metabolic oxidation potential, and more subtle activity features such as molecular recognition and conformation. A strategically positioned fluorine atom can alter the reactivity or metabolism of a drug by pulling electrons away from reactive carbon molecules.

Given the prevalence of hetero (noncarbon) elements in small-molecule pharmaceuticals, today’s NMR spectrometers can perform orthogonal analyses in one experiment by focusing on fluorine and two or three other nuclides.

“Fluorine is a billion-dollar segment of the pharmaceutical business,” says Clemens Anklin, PhD, VP of NMR applications at Bruker (Billerica, MA).

Since every fluorine in a molecule has a unique magnetic resonance signal, in just a few minutes fluorine NMR reveals the purity of a fluorinated compound and the identities of side products, contaminants, or unused starting materials. Detection down to ppm levels is not extraordinary.

Fluorine is also exquisitely sensitive to its environment. A fluorinated drug, for example, will show a different signal when bound to a target than when unbound, even if the fluorine did not participate in the binding.

This raises interesting possibilities for drug screening. It is possible to compare the well-resolved NMR spectrum of, say, 30 fluorinated drug candidates in the presence and absence of a receptor or protein. Fluorines in unbound compounds will retain their original resonance, while those attached to molecules that bind will experience a shift and broadening.

“In proton-based or ligand-based drug discovery based on NMR, six compounds is the limit for one experiment,” Anklin notes. “If you set up the experiment correctly and binding is strong, the fluorine peaks may apparently disappear. Determinations of drug-receptor dissociation constants may be calculated in this manner.”

Multidimensional analysis

Coupling between fluorine and carbon or hydrogen is predictably clean and confirmatory with respect to molecular structure and fluorine location in the molecule. Higher-level two-dimensional NMR experiments are also possible using fluorine NMR. Possibilities include fluorine-fluorine interactions and coupling among fluorine, hydrogen, carbon, and other active NMR nuclei.

As with proton NMR, interactions between fluorine and other active nuclei fall off rapidly as the distance between the two atoms increases.

Flourine-19 can serve as both a structural probe and a means of characterizing a molecule’s interactions with proteins or other targets simply by observing changes in fluorine resonance when its parent molecule binds to proteins.

Additionally, fluorine may be incorporated directly into proteins to observe such events as conformational changes, binding, and aggregation. Fluorine is introduced into intact proteins chemically through sitespecific incorporation of fluorinated ligands or during biosynthesis where the only version of a particular amino acid is fluorinated.

In similar fashion, the combination of fluorine labeling and NMR may be used to probe the behavior of oligonucleotides, polymers, and sugars.

Overcoming proton limitations

Some may argue that all the benefits of fluorine NMR mentioned above are within the realm of proton or 13C NMR. While it is true that modern NMR, particularly experiments run under two-dimensional protocols, facilitates structure elucidation from proton spectra alone, the capabilities of mononuclear spectrometry are limited.

“As the molecular weight increases, the spectral complexity and peak overlap begin to be limiting conditions in proton NMR,” says Bruce Lix, director of business development at Nanalysis Corp. (Calgary, Alberta). “At that point, your options include moving to higher dimensionality in the NMR experiment and/or analyzing additional nuclides in the detection scheme.”

Fluorine happens to be one of those “additional nuclides” that can help clear the cobwebs from NMR spectra.

“The main advantage of 19F NMR over 1H NMR alone is the much broader spectral range for fluorine, more than 20 times that of hydrogen,” Lix says, “so the chance that any two resonances will overlap is very low. Fluorine NMR is also very sensitive to its molecular or chemical environment, which is reflected by spectral changes.”

Today’s benchtop, multiprobe NMR instruments typically focus on elements of interest to the life sciences, typically nuclides of hydrogen, carbon, nitrogen, and phosphorous. Purchasers can specify a fifth element, for example, fluorine. “It is also possible to get hydrogen/fluorine combinations only,” Lix says. “Many of these probes are swappable, but at a lower field, the desired nuclides must be specified at the time of purchase.” Ordering a fluorine probe as an add-on to an instrument lacking the capability for that nuclide is possible, but expensive.

Lix describes the workflow for using fluorine NMR to determine the structure of a large, complex, fluorinated molecule. “You would start by running homonuclear proton two-dimensional correlation experiments such as COSY [correlation spectroscopy] and TOCSY [total correlation spectroscopy] to determine connectivity.”

The position of the fluorine will jump right out, since the coupling constants between fluorine and hydrogen are very large compared with hydrogen-hydrogen couplings.

“You can then determine the carbon backbone by using C-H correlation experiments and then determining the structure by measuring through-space proximity using the nuclear Overhauser effect [NOESY]. If the material is labeled with 13C or 15N, it is possible to collect even more information with three-dimensional experiments, although this probably would not be necessary. Since fluorine is strongly susceptible to changes in its chemical environment—for example, if it were absorbed into a cell or passed the blood-brain barrier—the fluorine spectrum can be used further to determine the fate of the molecule in a biological setting,” Lix says.

For additional resources on fluorine NMR, including useful articles and a list of manufacturers, visit