Preface

Nuclear magnetic resonance (NMR) is a technique for determining the structure of organic molecules and biomolecules in solution. The covalent structure (what atoms are bonded to what), the stereochemistry (relative orientation of groups in space), and the conformation (preferred bond rotations or folding in three dimensions) are available by techniques that measure direct distances (between hydrogens) and bond dihedral angles. Specific NMR signals can be identified and assigned to each hydrogen (and/or carbon, nitrogen) in the molecule.

You may have seen or been inside an MRI (magnetic resonance imaging) instrument, a medical tool that creates detailed images (or "slices") of the patient without ionizing radiation. The NMR spectroscopy magnet is just a scaled-down version of this huge clinical magnet, rotated by 90° so that the "bore" (the hole that the patient gets into) is vertical and typically only 5 cm (2 in.) in diameter. Another technique, solid-state NMR, deals with solid (powdered) samples and gives information similar to solution NMR. This book is limited to solution-state NMR and will not cover the fields of NMR imaging and solid-state NMR, even though the theoretical tools developed here can be applied to these fields.

NMR takes advantage of the magnetic properties of the nucleus to sense the proximity of electronegative atoms, double bonds, and other magnetic nuclei nearby in the molecular structure. About one half of a micromole of a pure molecule in 0.5 mL of solvent is required for this nondestructive test. Precise structural information down to each atom and bond in the molecule can be obtained, information rivaled only by X-ray crystallography. Because the measurement can be made in aqueous solution, we can also study the effects of temperature, pH, and interactions with ligands and other biomolecules. Uniform labeling (13C, 15N) permits the study of large biomolecules, such as proteins and nucleic acids, up to 30 kD and beyond.

Compared to other analytical techniques, NMR is quite insensitive. For molecules of the size of most drugs and natural products (100-600 Da), about a milligram of pure material is required, compared to less than 1 /g for mass spectrometry. The intensity of NMR signals is directly proportional to concentration, so NMR "sees all" and "tells all," even giving multiple signals for stereoisomers or slowly interconverting conformations. This complexity is very rich in information, but it makes mixtures very difficult to analyze. Finally, the NMR instrument is quite expensive (from US $200,000 to more than $5 million depending on the magnet strength) and can only analyze one sample at a time, with some experiments requiring a few minutes and the most complex ones requiring up to 4 days to acquire the data. But used in concert with complementary analytical techniques, such as light spectroscopy and mass spectrometry, NMR is the most powerful tool by far for the determination of organic structure. Only X-ray crystallography can give a comparable kind of detailed information on the precise location of atoms and bonds within a molecule.

The kind of information NMR gives is always "local": The world is viewed from the point of view of one atom in a molecule, and it is a very myopic view indeed: This atom can "see" only about 5 A or three bonds away (a typical C-H bond is about 1 A or 0.1 nm long). But the point of view can be moved around so that we "see" the world from each atom in the molecule in turn, as if we could carry a weak flashlight around in a dark room and try to put together a picture of the whole room. The information obtained is always coded and requires a complex (but very satisfying) puzzle-solving exercise to decode it and produce a three-dimensional model of a molecule. In this sense, NMR does not produce a direct "picture" of the molecule like an electron microscope or an electron density map obtained from X-ray crystallography. The NMR data are a set of relationships among the atoms of the molecule, relationships of proximity either directly through space or along the bonding network of the molecule. With a knowledge of these relationships, we can construct an unambiguous model of the molecular structure. To an organic chemist trained in the interpretation of NMR data, this process of inference can be so rapid and unconscious that the researcher really "sees" the molecule in the NMR spectrum. For a biochemist or molecular biologist, the data are much more complex and the structural information emerges slowly through a process of computer-aided data analysis.

The goal of this book is to develop in the reader a real understanding of NMR and how it works. Many people who use NMR have no idea what the instrument does or how the experiments manipulate the nuclei of the molecule to reveal structural information. Because NMR is a technique involving the physics of magnetism and superconductivity, radio frequency electronics, digital data processing, and quantum mechanics of nuclear spins, many researchers are understandably intimidated and wish only to know "which button to push." Although a simple list of instructions and an understanding of data interpretation are enough for many people, this book attempts to go deeper without getting buried in technical details and physical and mathematical formalism. It is my belief that with a relatively simple set of theoretical tools, learned by hands-on problem solving and experience, the organic chemist or biologist can master all of the modern NMR techniques with a solid understanding of how they work and what needs to be adjusted or optimized to get the most out of these techniques.

In this book we will start with a very primitive model of the NMR experiment, and explain the simplest NMR techniques using this model. As the techniques become more complex and powerful, we will need to expand this model one step at a time, each time avoiding formal physics and quantum mechanics as much as possible and instead relying on analogy and common sense. Necessarily, as the model becomes more sophisticated, the comfortable physical analogies become fewer, and we have to rely more on symbols and math. With lots of examples and frequent reminders of what the practical result (NMR spectrum) would be at each stage of the process, these symbols become familiar and useful tools. To understand NMR one only needs to look at the interaction of at most two nearby nuclei in a molecule, so the theory will not be developed beyond this simplest of relationships. By the end of this book, you should be able to read the literature of new NMR experiments and be able to understand even the most complex biological NMR techniques. My goal is to make this rich literature accessible to the "masses" of researchers who are not experts in physics or physical chemistry. My hope is that this understanding, like all deep understanding of science, will be satisfying and rewarding and, in a research environment, empowering.

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