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NMR spectroscopy advances biotechnology research.

NMR is helping researchers working on new pharmaceuticals, heavy metal toxicology, and agricultural biotechnology.

THE AUTHOR:

Barbara Myers-Acosta is product marketing manager for the Unity brand NMR spectrometer line at Varian Associates, Palo Alto, CA. She received her PhD from Univ. of California, Santa Cruz, specializing in the characterization of marine natural products by NMR and other methods. Prior to joining Varian, she established an NMR laboratory at Lockheed for both liquid and solid-state NMR techniques.

Nuclear magnetic resonance spectroscopy is helping researchers in many biotechnological fields obtain data that were not available via other methods, including x-ray crystallography, circular dichroism, infrared spectroscopy, or mass spectrometry.

Many studies combine information from these methods with theoretical calculations and NMR techniques to determine the conformation, folding, and metabolism of biologically active molecules in solution.

This research could lead to the development of new pharmaceuticals, better understanding of heavy metal toxicology, and improvements in agricultural research methods.

The noninvasive, nondestructive nature of NMR technology yields benefits that are similar to those seen in studies of living cells. Drug molecules can be analyzed as they interact with receptors; this activity can be studied over time. Newer NMR techniques, such as two- and three-dimensional NMR, rely on the increasing power of computer hardware and software to attain higher resolution and rule out potential conformations that do not comply with empirical findings.

Another important benefit is a significant savings in time and costs, since NMR helps researchers make more intelligent choices leading to the correct molecular structures. Thomas Gadek, a scientist in the Medicinal & Biomolecular Chemistry Dept. at Genentech, South San Francisco, CA, says he uses NMR spectroscopy to "work smarter."

"To develop new products and bring them to market as quickly as possible, we need to focus our efforts and control research costs," says Gadek. "Research is an interactive process. We're trying to cut down the number of iterations by gaining better insight into the properties of the structure we're investigating. Through NMR techniques, we can determine parts of the molecule (or molecules) that aren't necessarily connected to each other, but are close in space. Then, based on those observations, we go into molecular modeling with a set of distance constraints."

Gadek wants to relate the biological activity to the exact substructural piece responsible for the activity. He believes that gaining an understanding of all possible chemical structures would enable researchers to simulate each individually and determine which is being recognized by the receptor or enzyme.

"Then we can make a molecule that has all of the features important for biological activity, but is conformationally constrained so it can't rotate or do anything else but look like the conformation that's recognized by the receptor or enzyme," Gadek continues. "By excluding useless conformations, we might be able to increase potency and improve selectivity, while reducing the number of side effects. The ratio of active compounds to inactive compounds we produce is much higher than it would be without NMR."

Gadek also uses his NMR findings to complement those obtained through x-ray crystallography. "While the differences between the two techniques are often not that great when dealing with large proteins, the solution-phase structure of smaller molecules can be very different," he says. "There are many more possible low-energy conformations in solution than one sees reflected in crystalline structures."

At Genentech, a network of computers unites the NMR and modeling functions. Gadek's Varian NMR spectrometer is equipped with a Sun SPARC workstation, which controls the NMR and also processes the data. The Sun workstation is connected by Ethernet to a DEC VAX system, which is used to archive all the data. The computer network at Genentech allows Gadek to perform some modeling tasks on his desktop Macintosh computer; he can offload some tasks from the Sun workstation and benefit from the convenience of data access in his office.

B.N. Ramasinga Rao, a scientist at Glycomed Inc., Alameda, CA, also uses NMR spectroscopy to study molecular conformations in solution. Where Gadek's group is working to simulate the activities of peptides, Rao's team emphasizes the role of sugar residues bound to proteins and cell surfaces.

Carbohydrates are widely distributed in nature and are seen to have significant effects on the level of activity and selectivity of pharmaceutical compounds. Unlike peptides and oligonucleotides of similar size, NMR of oligosaccharides is significantly more complex.

This requires high magnetic field strengths on the NMR spectrometer to separate overlapping resonance lines and simplify the NMR spectrum interpretation of the data. Also, since carbohydrates are isolated from natural sources, Rao often has to work with very small quantities of compounds, so spectrometer sensitivity becomes very important.

"One of the aspects of sensitivity is that it is also directly dependent on field strength," says Rao. "For example, high field strength is required if you have to work with very small quantities, say a few hundred nanomoles or less of a naturally occurring oligosaccharide."

Rao finds it advantageous to use NMR for studying molecular structure in solution, because this "gives us an idea of the conformation of the molecule as it may exist in the body and yields structural information that could be functionally important." He highlights the significance of using NMR as a complementary method to computations and other empirical techniques.

"Among physical techniques," he says, "one could get gross structural information by using an optical technique, such as fluorescence measurements-getting separation between two fluorophors, for instance. High-resolution electron microscopy also can be useful, although the results are not as detailed as is NMR spectroscopy. High-resolution x-ray crystal structure studies provide the most detailed information, not only on the structure but also the conformation of the molecule as it exists in the crystalline state. Computational techniques, such as quantum chemical calculations and empirical energy computations, provide in-depth information on molecular conformations; however, the computation methods require experimental verification." Rao concludes, "NMR spectroscopy and molecular modeling work together, with the experimental data from NMR measurements providing certain constraints as input for the molecular modeling calculations."

NMR spectroscopy also is being used by manufacturers of pharmaceuticals fermented from genetically altered cell cultures to examine and regulate the balance of nutrients and wastes in these cultures.

Maintaining homeostasis is very difficult in some of these fermentations, so NMR is extremely useful for quality control. It provides high-resolution, noninvasive, nondestructive analysis of the conditions under which these fragile cells are being cultivated.

Dallas L. Rabenstein, professor of chemistry and chairman of the Dept. of Chemistry at Univ. of California, Riverside, has been taking advantage of the benefits of NMR spectroscopy since 1966. The early research of his group included exploring the chemistry of heavy metal toxicology by studying the binding of metal ions by amino acids and peptides.

With the advent of NMR spectrometers that used supercooled superconducting magnets to generate stronger magnetic fields, Rabenstein's group began studying the activity of mercury, cadmium, and lead ions in intact red blood cells.

"That work has gone on to include NMR studies of other cells as well, particularly mast cells, which are involved in the storage of histamine in biological systems," says Rabenstein. "More recently, we have become interested in the biological chemistry of sulfur and selenium. We're using NMR techniques to study the chemistry of sulfur, for example, in peptide hormones."

Before NMR was used to study red blood cells, most red blood cell research used classical biochemical techniques, requiring that cells be lysed. Their components had to be separated and highly purified before the chemistry or biochemistry could be studied.

"That's all different now," says Rabenstein. "With the development of high-field NMR spectroscopy and some very simplified techniques, we can make measurements on intact cells and we can see individual compounds directly in the cell. There is no other tool today that lets us do this."

Different kinds of experiments take varying amounts of time by NMR. Rabenstein finds that he can measure an NMR spectrum of a red blood cell in less than a minute, while the measurement of time courses in the study of metabolic processes might involve leaving the sample in the spectrometer for 1 to 1.5 hours.

NMR also can be used to measure the rate of transport across the cell membrane. "The advantage of NMR spectroscopy is that not only can you study transport across the cell membrane, but then, if the molecule undergoes metabolic reaction, that can be studied at the same time," he says. "This makes the NMR technique unique. You get a lot of information from a single experiment."

NMR spectroscopy is able to monitor metabolic processes as well as study claudication and ischemia in muscle tissue by measuring pH. NMR might become increasingly useful in cardiology, since it can be used to visualize blood flow and heart motion, and, according to Rabenstein, "it is already in the domains of the neurologist and radiologist."

Agriculture researchers also are using NMR to study molecular conformation. "For small molecule structure identification, there is no substitute for NMR," says Geoffrey K. Cooper, principal scientist at the Plant Cell Research Institute, Dublin, CA. "Mass spectrometry is complementary in a way, but it doesn't give me anywhere near the kinds of useful information that NMR spectroscopy does. Basically, I couldn't do my research without NMR."

NMR's nondestructive properties make it especially useful to Cooper's group, which is developing an assay for analyzing genetically altered lipids in whole seeds. Often, only a minute amount of a genetically altered sample is available; gaining additional samples often requires growing another generation of plants. Being able to analyze the seeds without destroying them is a significant advantage.

Cooper's group is working on problems ranging from metabolic studies in support of licensing a fungicide, to more protein-oriented projects involved in developing pathogen resistance and nutritional improvements in crops. "Most of our work involves small molecules that are probes of a biological system, or are important in a biological interaction," he says.

One of Cooper's primary interests is affinity labeling, which he says, "involves synthesizing a small molecule that will tag an enzyme or receptor in a cell or in an extract of a cell. The molecules are generally made in radioactive form and resemble substrates or ligands of the enzyme or the receptor.

"Since they are radioactive, you can isolate the protein by isolating the radioactive band. This involves synthesizing a molecule, using NMR spectroscopy to analyze the reaction product so we know what we have structurally. Then the protein itself is analyzed by mass spectrometry after it has been labeled."

This procedure can be used in studies to support licensing of a fungicide, for example, by synthesizing radiochemically labeled samples of the fungicide and using NMR spectroscopy to examine the structure of these metabolites.

As to the future of NMR in agricultural research, Cooper says: "I think the study of proteins and their interactions with ligands is going to become more powerful. If you want to map a binding site on a protein in solution, NMR is the only way to go. It complements x-ray crystallography and protein structure determination. And it provides new information about short-range interactions in solution, which is where enzymes work.

"Any company that's in biotechnology that focuses on particular gene products, which are proteins, wants to know how they work. They will probably turn more and more to NMR.

"Biotechnology started out being mostly microbiology but, as it develops and they try to increase the number of products, chemistry is going to become more and more important. For a chemist, NMR spectroscopy is the single most useful and powerful analytical technique. So I would expect to see the biotechnology industry make increasing use of NMR."

A brief history of NMR:

In 1946, Felix Bloch of Stanford (CA) Univ. and Edward M. Purcell of Harvard Univ., Cambridge, MA, independently discovered nuclear magnetic resonance (NMR), a phenomenon in which nuclei absorb radio-frequency energy and therefore resonate at that exact frequency while in the presence of a magnetic field.

Other results were soon reported, including the discovery that characteristic and reproducible chemical shifts are apparent for different compounds. The nuclei in different compounds absorb specific radio frequencies, resulting in the characteristic chemical shifts. These significant discoveries led to the development of NMR spectroscopy and NMR imaging techniques.

In the ensuing four decades, NMR has evolved into a family of state-of-the-art techniques for chemical analysis and spectroscopic NMR imaging. Many researchers find that NMR spectroscopy provides information not available by any other technique.

Among NMR's distinguishing characteristics is its ability to do noninvasive and nondestructive analysis. Since modern NMR spectroscopy provides extremely high resolution, it is used to examine the chemistry and structure of living tissue down to the cellular level. And because this living tissue is not affected or harmed in the NMR experiments, the same tissue can be observed over time, revealing such changes as the metabolism of selected molecules.
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Title Annotation:R&D Instrumentation; nuclear magnetic resonance
Author:Myers-Acosta, Barbara
Publication:R & D
Date:Jan 1, 1991
Words:2144
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