NMR Method Downsizes

RESIDUAL DIPOLAR couplings, or RDCs, have become an invaluable tool for scientists trying to decipher the structure of large biological molecules with nuclear magnetic resonance spectroscopy. Chemists are now finding that the NMR parameter is just as useful for pinning down the stereochemical details of small molecules, providing structural information that has been impossible to obtain from other methods.

"I think RDCs are very useful for all purposes in organic structure determination—relative configuration determination, diastereotopic assignments, and, hopefully soon, detailed investigation of dynamics and absolute configuration determination," says Christina M. Thiele of Germany's Darmstadt Technical University. "I think that it is a real alternative" to other parameters, she says, because RDCs give information over the length of the entire molecule and aren't limited to the short-distance relationships that are observed in standard NMR parameters such as nuclear Overhauser effects (NOEs).

To understand how RDCs work, it helps to think of a molecule as a series of magnets strung together. Each nucleus has its own magnetic field, and these fields influence one another, giving rise to dipolar couplings. The strength of these dipolar couplings depends on the distance between the nuclei and the direction of their internuclear vectors with respect to the NMR's external magnetic field. In cyclohexane, for example, the vectors of all the axial protons are parallel to one another, but the vectors of the equatorial protons are oriented at angles that are different from the axial protons.

Dipolar couplings are well-known in solid-state NMR spectroscopy, but they're not observed in most conventional high-resolution NMR experiments because, as the molecules tumble in solution, their vectors average out with respect to the external magnetic field. Dipolar couplings are quite large—on the order of kilohertz—and they're responsible for the strong signal broadening that's observed in solid-state NMR.

Decades ago, scientists figured out that if you take an NMR spectrum in some sort of ordered media, such as a liquid crystal, you can coax a small percentage of the molecules to align. From that alignment you can obtain a residual echo of the dipolar couplings. These RDCs contain the same structural information about the relative orientations of bond vectors, but they are about three orders of magnitude smaller than dipolar couplings and their line broadening isn't nearly as pronounced.

"The beauty of RDCs is that they provide information about the relative orientations between internuclear vectors regardless of the distance between them," says Roberto R. Gil, a research professor and NMR spectroscopist at Carnegie Mellon University.

When trying to solve the structure of a small molecule with NMR, most chemists use NOEs and J-couplings. But these parameters are sensitive only to what's happening with their nearest neighbors, so they can't unravel some structural problems.

Those NMR parameters "just don't give you answers when things are far apart from each other," says Michael Shapiro, a professor at the University of Maryland School of Pharmacy, Baltimore. For example, he says, "two stereocenters more than four bonds away might as well be in two different molecules using NOEs and J-couplings. With the RDCs they could be on two different planets; as long as you can measure those vectors relative to one another in the magnetic field, the distance between them no longer matters, and it doesn't really matter what the intervening bonds are."

Christian Griesinger, of the Max Planck Institute for Biophysical Chemistry in Güttingen, Germany, and Masaru Hashimoto, of Hirosaki University in Japan, recently used the technique to determine the stereochemistry of four stereocenters in the novel glycoside sucro-neolambertellin (Angew. Chem. Int. Ed. 2008, 47, 2032). Quantitative NOE analysis was only able to narrow the structure to four relative configurations, but RDCs were able to differentiate the correct structure from the other three. Without the benefit of the parameter, it might have been necessary to synthesize four different compounds to verify the structure.

RDCs have been popular for studying large biological molecules since the 1990s, but until recently the technique hadn't been practical for small-molecule studies. That's because the available alignment media could be used only with aqueous solutions.

Now, however, alignment media that are compatible with organic solvents are coming onto the scene. It's possible to use chloroform and other organic solutions with liquid crystalline poly-γ-benzyl-L-glutamate or poly-γ-ethyl-L-glutamate.

Stretched polymer gels can also induce RDCs in organic solutions. To use these materials, an NMR tube is loaded with a small cylindrical plug of a polymer gel, such as polystyrene, poly(acrylonitrile), poly(dimethylsiloxane), or poly(methyl methacrylate). Adding an organic solvent prompts the polymer to swell in an anisotropic fashion along the glass wall of the tube. Molecules dissolved inside these gels will align, and the degree of that alignment can be adjusted by changing the cross-linking within the polymer or the diameter of the unswollen polymer plug.

CHEMISTS FAMILIAR with RDCs tell C&EN that the technique is quite easy to use. "I could teach this in a day," Gil says. It's simply a matter of taking a couple of NMRs, usually proton-coupled heteronuclear single-quantum coherence experiments in which one sample is in regular NMR solvent and the other sample is in the aligning medium. After that, you just need to do some simple arithmetic and then enter some values and some structural information into a computer program.

"RDCs have many applications in small-molecule NMR spectroscopy," says Burkhard Luy, an assistant professor at Germany's Technical University of Munich. "They are very well suited to verify or falsify a given structural model. For example, RDCs are tools to determine the relative configuration of a molecule. It is also possible to distinguish enantiomers and determine enantiomeric excess using chiral alignment media. Another large field of applications is the conformational analysis of molecules of practically any size, which can be significantly refined by RDCs."

So far, RDCs have mostly been used on conformationally rigid small molecules. "The big challenge," Griesinger says, "lies in the elucidation of conformationally flexible molecules." He and Thiele have had some preliminary success working with flexible systems.

RDCs may be easy to use and provide a wealth of structural information, but the technique really hasn't caught on among organic chemists. Only a dozen or so researchers around the world are using RDCs for structural determination of small molecules.

Jacques Courtieu, a pioneer in small-molecule RDCs at France's University of South Paris, has some ideas why this is so. Conventional NMR techniques give the right answer in 80 to 90% of cases, he says, so NMR in aligning media is useful only 10 to 20% of the time. He also points to software issues and the fact that a general strategy isn't yet available to solve all possible stereochemical problems. "The strategy has to be adapted to each case," he says.

Courtieu adds that "time is needed to solve a given problem. This technique is not routine, and the solution may take a month or two to be reached. It depends how strongly chemists want the solution."

Luy notes, however, that the current limitations are mainly technical details that will work themselves out as using RDCs for small molecules becomes more commonplace. "It will advance from a parameter only for specialists to a standard NMR parameter," he tells C&EN. "It is already clear that many problems can be solved with RDCs as an additional source of structural information that cannot be solved with classical NMR parameters alone."