Of Molecules and Methods

By Ulrika Kahl

Most people use tennis balls when they, well, play tennis. In addition to using them to play tennis, Professor Julius Rebek, director of The Skaggs Institute for Chemical Biology at The Scripps Research Institute (TSRI), uses tennis balls—and softballs, jelly donuts, and other items—to illustrate the shape of, and forces within, the molecular structures that he and his colleagues study in the laboratory.

"It's all about the space," says Rebek, explaining why molecules behave in the way they do. Parameters like temperature, pH, and illumination of course matter, but space is critical. Molecules want to fit into the space, and make sure that they have enough of it, although not too much of it either. And, according to Rebek, the magic number is 55. "If you have a space, and you fill 55 percent of it, then you have a happy complex," he explains. Organic solutions, for instance, are 55 percent molecules, the rest pure space. Typical crystals are 75 percent molecule, 25 percent space.

Rebek uses a tennis ball to illustrate his point. If you cut a tennis ball along the curved line, you will end up with two identical halves. Imagine that these halves are molecules. In the space between these two large host molecules, other molecules, like methane and similar gases, can be encapsulated. The tennis ball capsule and the encapsulated molecule will assemble into a complex, held together by reversible intermolecular hydrogen bonds. If the encapsulated molecule fills 55 percent of the space in the capsule, the whole complex will be in equilibrium, "a happy complex."

Molecules such as benzene, whose structure is flat, will need a different environment. Using another of Rebek's examples, benzene would be better off in the space of a jelly donut. But benzene, too, will still strive towards 55 percent occupancy.

Rebek and his colleagues presented their tennis ball capsules in 1994 in an article in Science. There, they showed that the capsule molecules they had created in a chemical reaction could form homodimers (a complex consisting of two identical molecules) that assembled spontaneously, resulting in an energy-minimized structure. In the sphere inside the dimer capsule, small molecules like methane, ethane, and ethylene could be entrapped reversibly, as was shown with nuclear magnetic resonance (NMR). The degree of occupancy was shown to be largely determined by the nature of the solvent the complex was in. The solvent molecules would compete with the guest molecules for the space inside the capsule, and the molecule coming closest to filling 55 percent of the space would be the one most likely to be found in the capsule.

From the perspective of a chemist, Rebek's findings lead to an elegant molecular assembly technique. Instead of having to rely on extreme conditions or complicated chemical syntheses, molecules recognize each other and self-assemble in solution. This process is strikingly similar to that used by living cells, which may have taken millions of years to refine to perfection. Complex cellular structures like membranes, ribosomes, and viruses are all products of self-assembling mechanisms.

From a medical perspective, this encapsulation strategy holds promise for creating a technique using molecular shells to deliver drugs to target cells—in fact, in a manner similar to that which viruses use, entering host cells and releasing their DNA inside. It also suggests guidelines for designing drugs. "If you have a structure of an enzyme and want to design an inhibitor," says Rebek, "then look for something that fills 55 percent of the binding site." In other words, you will have the greatest chance of succeeding if you give the drug an optimal amount of space to perform its action.

The self-assembly of molecules that Rebek and co-workers observed in their study has become a focus of further research in the past half-decade. Self-assembly can be used for the rapid construction of large and elaborate molecular structures, an approach that is widely used in what is referred to as combinatorial chemistry. Combinatorial chemistry is a method for rapidly making a large number of different molecules in one and the same reaction, and is currently one important part of Rebek's research.

"Seven years ago, no companies were interested in combinatorial chemistry," Rebek says. "Today, every drug company has a combinatorial chemistry section. Using combinatorial chemistry is a smart, efficient way to study molecular interactions and develop novel drugs."

Combinatorial libraries of molecules rely on a numbers game. Let's say a scientist is presented with a core molecule with four reactive corners. If he or she throws in 20 bases, for example amines, which are all able to react with each one of the four reactive groups of the core molecule, in 10 minutes the reaction will yield around 100,000 statistically possible compounds. Then, if the researcher runs this library of compounds in an activity assay—for instance one in which the inhibition of the protease trypsin is measured—and get a positive signal, it means that one or more compounds in the library are potential competitive protease inhibitors, acting on trypsin.

The most problematic step in combinatorial chemistry is the analysis of the libraries obtained. It is—even with the most advanced instruments and analytical methods available—hard to separate and characterize hundreds of thousands of molecules in a reaction mix. There are two main approaches to creating combinatorial libraries: on solid support and in solution. The latter method is the one Rebek prefers. To identify the products in a solution-created library, one can either use tagged molecules in the reaction, or employ the method of deconvolution.

Deconvolution is an iterative selection procedure, in which several smaller sublibraries are first created, each of them lacking a few of the initial building blocks. Activity measurements of the sublibraries will then reveal which of the building blocks did not contribute to the overall activity in the main library. Another set of sublibraries is then made based on the first ones, and the activity is once again measured. Eventually, after the cycle is repeated a number of times, only one or a few active compounds are left. These compounds can be separated with chromatography methods like high-performance liquid chromatography (HPLC). Finally, mass spectrophotometry and NMR analysis will reveal the composition and structure of the active compounds.

The unique efficiency and complexity of combinatorial chemistry becomes more useful as we learn more about the mechanisms that keep the cells and organs in our body running. For instance, based on the known structure of the binding site for a certain nerve transmitter in the brain, a researcher may be able to choose potential building blocks with high precision in the combinatorial synthesis of a drug targeted at this binding site.

Rebek's most important contributions to science to date are probably the many experimental methods that he together with co-workers developed in self-assembling systems and combinatorial chemistry over the years. These accomplishments have grown out of Rebek's love of designing and building molecules and methods.

Rebek has been drawn to designing and building throughout his life. Initially, he planned to become an architect. The architect dream was, at least to some extent, fulfilled by a certain Cape Cod beach house—designed by Rebek, of course.

When Rebek came to TSRI in 1996, he became director of the newly founded Skaggs Institute for Chemical Biology, which was funded by a generous gift from Aline and Sam Skaggs through the Skaggs Institute for Research and their family foundation, the ALSAM Foundation. Rebek appreciates the commitment of Skaggs family members, who frequently visit TSRI, and show an interest in the research they have made possible.

In his role as director of the Skaggs Institute and head of an active laboratory, Rebek has had the opportunity to build both molecules and a future generation of scientists. Throughout his career, over 150 students and associates have studied under Rebek's guidance. Along with his contributions to science, Rebek's investment in future scientists will have a lasting impact. As Rebek expressed it a few years ago in his overview of The Skaggs Institute, "the ultimate research identity of The Skaggs Institute will be the scientists it produces."

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Professor Julius Rebek's most important contributions to science to date are probably the many experimental methods that he together with co-workers developed in self-assembling systems and combinatorial chemistry. Photo by Mark Dastrup.

The Rebek lab's self-assembling capsule—dubbed the "tennis ball"—encapsulates methane and is held together by eight hydrogen bonds. The capsule has a lifetime of about one second. Illustration by Arash Rebek and Lubomir Sebo.

Since 1970, over 150 students and associates have studied under Rebek's guidance. Photo by Arash Rebek.