plied to chemical synthesis, suggests they might also want to try Œoss-linking isomerase, an enzyme used commercially for isomerizing glucose to fructose. "High-fructose corn syrup has to go through a purification step to increase the fructose concentration," says Frost. 'It's known that if you were to run the isomerization at higher temperature you would get that percentage of fructose without purification, but the problem has been that the catalyst doesn't last long enough. Any kind of technology that can potentially stabilize enzymes like isomerase has incredible ramifications." In previous research, some lyophilized enzymes have been found to be both stable and active when suspended in near-anhydrous organic solvents. However, in such systems, even a tiny amount of added water often leads to rapid loss of activity. What's unique about CLECs is that they're stable over the whole range of water concentrations in aqueous-organic mixed solvents. "If you read the literature on use of enzymes in organic solvents," says Navia, "it always refers to near-anhydrous organic solvents. If the water level goes up too high, it facilitates the breaking of bonds and the protein will unravel. We've demonstrated activity in 50% aqueous organic solvents, including 50% aqueous THF—a very harsh condition for an enzyme to be in." Chemistry professor Alexander M. Klibanov of Massachusetts Institute of Technology, who specializes in studies of biochemistry in nonaqueous media, comments that cross-linking of enzyme crystals "may turn out to be an improvement compared to other methods of putting enzymes in organic solvents, but if s really hard to say at this point because simply not enough is known. However, I would certainly view it as a significant advance and something that potentially may have very substantial implications." Because CLECs are self-supporting assemblies, they could be attractive for biosensor applications, where the largest possible signal per unit volume is often desired. The Vertex group has formulated jackbean urease CLECs for use in clinical biosensors to measure urea as an early indication of renal disease. The ability of CLECs to resist high temperatures and proteases also may make them valuable as therapeutic agents. Free enzymes used as drugs— such as streptokinase, used to treat myocardial infarctions—are often very short-
acting because they are attacked by proteases in the blood. CLECs, which are relatively stable to proteolysis, might be administered less frequently or in reduced amounts, and might even provide an alternative to gene therapy for enzyme replacement in patients with enzyme deficiency diseases. In addition, CLEC proteins could potentially be taken orally as drugs without being broken down by the digestive system. Vertex researchers are currently working on a proprietary strategy to get such drugs into the bloodstream once they have passed safely through the stomach. Still another potential application would be catalytic antibody CLECs. According to chemistry professor Stephen J. Benkovic of Pennsylvania State University, who studies catalytic antibodies, cross-linking of enzyme crystals "looks very promising. If it can be generalized to other enzymes—and catalytic antibodies fall into that category—it may very well prove to be quite useful." •
Techniques facilitate fullerene separations A significant obstacle to research on fullerenes, the carbon-cage molecules of which buckminsterfullerene (C^) is the prototype, has been obtaining pure samples of C60, C70, and their relatives. Producing soot rich in fullerenes is easy enough, but separating the fullerenes from each other has been time consuming and expensive. Recent research in two independent laboratories may change that. Chemists at the University of South Carolina, Columbia, have discovered that activated charcoal is an efficient stationary phase for chromatographic separations of gram quantities of Cœ. And chemists at Regis Chemical Co., Morton Grove, Dl., using a stationary phase developed at the University of Illinois at Urbana-Champaign, have developed a high-performance liquid chromatography column that will be useful for separations of fullerenes.
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SCIENCE/TECHNOLOGY At the University of South Carolina, associate chemistry professor James M. Tour and graduate students Walter A. Scrivens and Peter V. Bedworth obtain as much as a gram of pure C60 in less than two hours using activated char coal as the stationary phase in a flash chromatography column [/. Am. Chem. Soc., 114, 7917 (1992)]. Tour is a synthetic organic chemist whose chromatography discovery was born out of frustration with the standard technique for preparative separations of fullerenes—liquid chromatography us ing alumina as the stationary phase. Tour says that obtaining 1 g of C^ using this technique requires 10 kg of alumina, which is expensive, and 50 L of solvent. "We became interested in reactions in volving fullerenes/' Tour says. 'To carry out reactions involving compounds that are sensitive to moisture or air, you have to work on a larger scale. To run milli gram-quantity reactions with organolithium reagents, for example, is very diffi cult. "So we had to produce larger amounts
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Stationary phase selector envelops spherical fullerene molecules of Qo," he continues. "We were separating it on nor mal alumina columns, and it was just killing us in terms of cost and time re quired for the separa tions/' The problem with using alumina columns to sepa rate fullerenes is that pure toluene, in which fullerenes are reasonably soluble, cannot be used as the mo bile phase because separa tion of the fullerenes is not achieved. Instead, 5% tolu ene/95% hexane solutions commonly have been used as the mobile phase with these columns. Although separations are accomplished with this mobile phase, large amounts of solvent are required because fullerenes have a very low solu bility in hexane. The South Carolina chemists discov ered in "The Chemist's Companion: A Handbook of Practical Data, Techniques, and References" [John Wiley & Sons, New York, 1973] that activated charcoal has been used as a stationary phase for molecular size selection-based chroma tography. Since the main difference among C^, C70, and the higher ful lerenes is their size, the researchers de veloped a column based on activated charcoal and a silica gel support. Tour says that producing a gram of C 60 using an alumina column costs about $200 and takes 16 to 20 hours. By contrast, producing a gram of C^ using the activated charcoal column costs about $2.00 and takes less than two hours. The separation is achieved with a column containing 36 g of activated charcoal and 72 g of silica gel and re quires only 600 mL of pure toluene. At Regis Chemical, Christopher J. Welch, a senior research chemist, has continued research he began as a grad uate student at the University of Illi nois, in the laboratory of chemistry professor William H. Pirkle. Welch and Pirkle investigated the chromatograph ic behaviors of C60, C70, and eight polycyclic aromatic hydrocarbons using 10 π-electron-deficient or π-electron-rich aromatic HPLC stationary phases [/. Chromatography, 609, 89 (1992)]. The Il linois chemists conducted their initial studies using hexane as the mobile phase because, despite the poor solu bility of C60 in this solvent, it gives suit able retention on a (dinitrobenzoyl)-
phenylglycine-derived stationary phase developed in Pirkle's lab. To develop an improved stationary phase, Welch says, "we used a molecular recognition approach to design a concave host for the spherical fullerene molecules and utilized π-π aromatic forces as attrac tive interactions." This approach led to a stationary phase selector that consists of three dinitrophenyl groups linked to a common tether. The idea is that the three electron-deficient aromatic groups will preferentially "wrap around" the spheri cal Qo molecules in a "ball-and-socket" fashion. The idea works. "The selector pro vides enhanced retention for the spher ical fullerenes, permitting larger appli cations of crude fullerene mixture per chromatographic run," Welch says. "Retention of the fullerenes is suffi ciently high that toluene-based mobile phase can now be used." Additionally, he notes, the high de gree of selectivity demonstrated by this stationary phase permits resolution of a number of as yet unidentified minor components of the crude fullerene mix ture. "The selectivity seen with this sta tionary phase will almost certainly prove useful in the separation of the various products of fullerene derivatization reactions, where the number of possible isomers produced can be quite large," Welch says. Welch says he and coworkers at Regis have worked to improve the loading of the selector onto the silica support. The company specializes in chromatography products and, Welch says, "has some very good techniques worked out for stationary phase production." The com pany has recently released a commercial version of the fullerene column which they call the ''Buckyclutcher I." Rudy Baum
