An international team has developed NMR methodology that allows them to measure Residual Dipolar Couplings in small molecules and so determine their absolute configuration where conventional techniques fail. The work allowed the team to unambiguously assign a 3D structure to the anticancer lead compound jaborosalactol 24, which was confirmed by X-ray powder diffraction.

Roberto Gil and his postdoctoral researcher Chakicherla Gayathri at Carnegie Mellon University in Pittsburgh, USA, and colleagues are working on a new NMR methodology, that could help natural product chemists define ambiguous stereochemistry in their molecules. The technique measures Residual Dipolar Couplings (RDCs) in small molecules, the absolute configurations of which are frequently difficult and sometimes impossible to determine using conventional NMR.

Gil explains that RDCs are readouts of the interaction between atomic nuclei that reveal structural information about the molecule as well as the global orientation of all of the atoms in the molecule, even if the atoms are far away from each other.

"When molecules are forced to adopt a minor degree of alignment in solution and no longer tumble isotropically, a measurable fraction of the dipolar coupling (0.01-0.1%) can be observed in the NMR spectrum," he explains, "These RDCs contain important structural information of non-local character, since their values depend not only on the internuclear distances but also on the angles between the internuclear vectors and the external magnetic field."

Over the last ten years or so, protein chemists and researchers studying nucleic acids have used RDCs to obtain structures for their large biological molecules. Unfortunately, the approach was not amenable to small molecules. Small molecules need to be dissolved in an organic solvent prior to analysis in a typical NMR experiment, but to obtain RDCs, researchers must first partially align the molecules.

To overcome this problem scientists, including Gil, have been developing polymer gels that act as sponges to absorb a small molecule solution. Working with Nicolay Tsarevsky and Krzysztof Matyjaszewski, the Carnegie Mellon team has now used poly(methyl methacrylate) (PMMA) gel, which they explain can be compressed or stretched, in order to induce partial alignment in any small molecule sample absorbed in the gel.

Although there are other gels available that might do the same job, the NMR background signal from PMMA can be removed from the spectrum more readily than that of other gels. Additionally, PMMA is easy to prepare and relatively inexpensive.

In order to demonstrate just how powerful the RDC approach can be even for complex natural compounds, such as withanolides, which have been shown to slow the growth of breast cancer cells, Gil and colleagues demonstrated how they could unambiguously determine the structure of one example, jaborosalactol 24, a naturally occurring steroid found in the Jaborosa parviflora plant. The Carnegie Mellon team worked with natural product chemists Manuela Garcia and Viviana Nicotra at the Universidad Nacional de Córdoba in Argentina, and Armando Navarro-Vázquez from the Universidade de Universidade de Vigo in Spain, who performed DFT calculations on the structure of jaborasalactol 24 and developed the software MSpin to analyse RDCs.
Previous efforts to elucidate the molecule's absolute structure using NMR had failed. Co-workers Silvina Pagola and Peter Stephens from from the National Synchrotron Light Source and the College of William and Mary, respectively, used powder diffraction to independently confirm the structure of jaborosalactol 24, a first for such a complex natural product. Henry Krakauer of William and Mary performed DFT calculations that allowed the determination of hydrogen coordinates around the hydration water molecules.
"It was impossible to figure out the compound's structure without RDCs," Gil explains, "Conventional NMR techniques yielded three possible structural configurations for the compound. But RDCs revealed only one structure."

The researchers are now working to make their methodology easier still and spreading the word among natural product and small molecule chemists. Gil is one of only a handful of scientists around the world currently using RDCs to determine the structure of small molecules. "Our technique helped a group of natural product chemists to unambiguously determine the correct configuration of a natural compound that has since shown anti-proliferative properties in a breast cancer cell line. Our technique can help anyone that has small molecules with difficult configurations," he says.

Related links:

• Angew Chem, Int Edn, 2009, 48, 5670-5674
• Roberto Gil
• Jaborosa parviflora 

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.