Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for elucidating the structures of small to medium size (MW=200 – 700 amu) organic compounds typically under development in the pharmaceutical industry.
Mass spectrometry (MS) is also commonly used by medicinal chemists to characterize the fruits of their synthetic efforts throughout the compound development process. Historically, these two analytical techniques have had separate camps of supporters and rare indeed is the analyst with a high level of proficiency and experience in both fields. However, advances in both NMR and MS made in the past decade have made it clear that the two techniques are very complementary. A good rule of thumb is that MS will answer the question “what?” and NMR will provide the answer to “where?”. In other words, MS is great for identifying the functional groups present in a molecule, either by (1) accurate mass measurements that provide a molecular formula, (2) fragmentation, or (3) isotope patterns arising from halides, for example. NMR's strength in the structure elucidation field is its ability to link pieces of the molecule together, either through bonds (scalar couplings) or through space (nuclear Overhauser effects or NOEs). The synergistic nature of NMR and MS is emphasized in early computer- aided structure elucidation (CASE) software tools, which often require NMR data and MS data as input. The success of recent NMR-based CASE systems at elucidating structures of some very large natural products containing C, H, O, and N has been quite remarkable1; however, confirming the presence of additional heteroatoms such as S, Cl, Br in a molecule generally still requires MS information.
Two of the instruments currently offered by AST Scientific could bring the type of synergism described above to your laboratory. The offered 500 MHZ Varian INOVA is a workhorse of an NMR spectrometer common in many academic and industry laboratories. With reliable hardware and standard software, information rich heteronuclear 2D NMR experiments can be conducted routinely. The Thermo Finnigan TSQ Quantum Classic can provide nominal mass and MSn capabilities for identifying fragments and functional groups. The two instruments together provide the analytical resources to tackle a wide array of structural problems that arise in many different industries.
References
1Elyashberg et al. (2009) Journal of Chemoinformatics I:3
Evolving Applications of Nuclear Magnetic Resonance
The nuclear magnetic resonance (NMR) phenomenon is an extremely versatile analytical tool. NMR Spectroscopy provides probably the most information-rich data available on molecular structures. However, while molecular structure determination is the most familiar application of magnetic resonance for many scientists , developments within the past 10-15 years have lead to broader applications for NMR. Below, I’ll briefly summarize the evolving applications of NMR with particular emphasis on the pharmaceutical industry today.
Chemical Structure Connectivity As mentioned above, the use of NMR spectroscopy to elucidate the structures of chemical compounds is the application of magnetic resonance most familiar to many scientists. Synthetic chemists rely on NMR data daily to characterize the compounds that they generate. Using 1H, 13C and 19F 1D NMR spectra, one can determine, for example, the number of different kinds of each atom that are present in a sample, as well information about how these atoms are linked together. Two-dimensional homo- and heteronuclear correlation experiments (such as COSY, HSQC and HMBC) provide further information on the chemical bonding arrangement of the atoms. On all modern NMR spectrometers, these experiments can be acquired in an automated fashion. Little or no knowledge of the experimental NMR parameters involved in acquiring and processing the data is required. Such rapid and automated acquisition of analytical data allows chemists to focus on chemistry.
3D Structure Determination Determining the chemical structure of a compound at the level of
atom-to- atom connectivity is insufficient to define the full three dimensional structure of a compound. Stereochemistry, or the relative positions of atoms in space, plays a critical role in how molecules interact with one another. NMR can be used to determine the three dimensional structure of a compound through nuclear Overhauser effects (NOEs). These short range interactions occur across space, rather than through chemical bonds, and reveal which atoms are close to one another regardless of how the atoms of the molecule are connected together. In special cases, transferred NOEs, can even be used to identify atoms that are close in space but belong to different molecules, such as an enzyme and its substrate or inhibitors. Within the last decade another type of NMR data known as residual dipolar couplings (RDC) has revolutionized the determination of the 3D structures of molecules, particularly biological macromolecules. Unlike NOEs, RDCs provide information on the orientation of bond vectors (C-H or N-H, for example) relative to the magnetic field of the NMR magnet. Such information is useful in determining the relative orientations of molecular substructures or domains located anywhere within the molecule.
Dynamics The use of NMR methods to determine the 3D structures of proteins within the pharmaceutical industry has largely been replaced by more rapid X-ray crystallographic methods. The advantage that NMR does enjoy over X-ray crystallography is its ability to characterize the structure and dynamic behavior of molecules in solution1. Several NMR parameters including relaxation rates and heteronuclear NOEs can be used to study the relative motions of different molecular domains within a compound.
Metabolomics Metabolomics is the study of the collection of small molecules involved in the biological reactions that maintain life, viewed at the level of the entire organism. The concentrations of these small molecules in bodily fluids rise and fall with changes in age, disease state, and diet, among other things. The concentrations of particular small molecules can be characteristic of gender or species. NMR has emerged as one of the main tools used for these metabolomic studies, with mass spectrometry being the other. The samples used in these experiments consist of fractions of a bodily fluid such as blood plasma , urine, or cerebrospinal fluid. The actual NMR methods used are relatively simple. One dimensional NMR spectra (usually 1H spectra) are acquired, typically with some form of solvent suppression to reduce the signal from the large amount of water present in the sample. Careful comparison of spectra acquired for diseased vs. healthy animals can be of diagnostic value and reveal information on the nature of the disease for basic research2. The adaptation of appropriate data processing and statistical analysis techniques has been a primary focus in the field. It is quite interesting to note that the same techniques applied in metabolomics studies are being used to characterize many other complex solutions, such as honey, beer3, wine4, plant extracts, dietary supplements and fruit juices.5,6 © André LeBlanc 2010
References
1Ishima, R. & Torchia, D. Nature Structure Biology 2000, 7(9):740-743
2Lindon JC et al. FEBS J. 2007 Mar;274(5):1140-51.
3Almeida C, et al. J Agric Food Chem. 2006 Feb 8;54(3):700-6.
4Anastasiadi M et al. J Agric Food Chem. 2009 Dec 9;57(23):11067-74.
5Cuny M et al. Anal Bioanal Chem. 2008 Jan;390(1):419-27.