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senal and may often be used in combina tion with one another. "What everybody in this field has real ized," Alliance's Keipert observes, "is that these products are more than just a simple blood substitute. They truly are therapeu tic oxygen-delivery agents, and they can be used in a variety of clinical situations where tissues are at risk of hypoxia." A transfusion of donor blood may end up be ing a therapy a patient gets in addition to a substitute because a problem can't be fixed any other way, he says, "but here we have an immediate intervention that can be giv en to potentially correct that problem." If any one blood substitute approach is garnering more bets than another, recom binant technology is the one, many re searchers in the field agree. That is "the Baxter perspective," Farmer says. "Recombinant technology probably will be the ultimate way to go," NHLBFs Nemo predicts, "because molecules can be constructed according to need." Once a need is determined, he says, the struc ture and function relationships for a prod uct can be worked out. Biopure's Rausch adds that while re combinant technology may be the way
to go, so far only single-molecule hemo globins have been made this way. He notes that the polyhemoglobins seem to be working better than the singlemolecule hemoglobins at this point, and a recombinant route to polyhemoglobins hasn't yet been established. "Success of oxygen carriers will de pend on completion of very complicated clinical trials and then the companies' ability to scale manufacturing and to price the product competitively," UCSD's Winslow says. "They can compete only if they work, they are safe, and the cost is competitive." And the question of whether approv al of first-generation substitutes can help drive research into second- and thirdgeneration products is still wide open. "Unless the condition demands it," McGill's Chang says, "there is no need to use a more expensive and more compli cated second- and third-generation blood substitute if the first-generation blood substitute can be used." "Let the work go as it may, and we'll see who the winners are," Nemo says. "The marketplace will determine it in the end."^
Studies Probe Catalytic Antibody Mechanisms
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ey details about how catalytic anti bodies work on a molecular level are revealed in recent studies by two independent research groups. The studies suggest new strategies that might be taken to improve the activity of these artificial enzymes in the future. And one of the studies also helps confirm a hypothesis about how the immune system generates diversity as effectively as it does. One study was carried out by professor of organic and theoretical chemistry Ken dall N. Houk of the University of Califor nia, Los Angeles; chemistry professor Kim D. Janda of Scripps Research Institute, La Jolla, Calif.; molecular biology professor Ian A. Wilson of Scripps; and coworkers [Science, 279,1934 (1998)]. The research ers used crystallographic and molecular modeling techniques to identify key as pects of the mechanism of a catalytic anti body (called 13G5) that catalyzes an ener getically disfavored Diels-Alder reaction. A Diels-Alder reaction is a cycloaddition 44
MAY 18, 1998 C&EN
process in which a conjugated diene adds to an alkene or alkyne with one or more electron-withdrawing groups (a "dienophile"), yielding a six-membered cyclic product. The specific reaction accelerated by 13G5 is an exo Diels-Alder reaction in which a butadiene adds to an acrylamide dienophile to form an enantiomerically pure cyclohexene product with trans substituents. The cis adduct is the primary product of the uncatalyzed reaction, but the trans adduct is produced almost exclu sively when 13G5 is used. This type of reaction is one for which no natural enzyme catalyst is known, and the 1995 discovery of 13G5 by Janda's group was considered an important devel opment at the time. "But people won dered about the mechanism by which the protein could do this reaction and control the stereochemistry," says Janda. To learn more about this, Houk, Jan da, Wilson, and coworkers determined
the crystal structure of the antibody in complex with a ferrocene inhibitor simi lar to the ferrocene hapten used to cre ate the antibody. To produce a catalytic antibody, a hapten designed to be a tran sition-state analog is introduced into a mouse, the mouse's immune system pro duces antibodies against the hapten, and the antibodies are screened for catalytic activity. The study revealed that three of the antibody's amino acid residues play a crucial mechanistic role in the reaction. A tyro sine residue acts as a Lewis acid to activate the dienophile for nucleophilic attack by the diene, and hydrogen-bonding of an asparagine and aspartate to the diene accel erates the reaction and determines its ste reoselectivity. Altogether, there are three hydrogen-bonding interactions and 45 van der Waals (hydrophobic) interactions be tween antibody and inhibitor in the com plex—presumably representing the types of interactions that occur between anti body and transition state in the catalyzed reaction as well. The group also carried out a molecular modeling study that helps explain why 13G5 catalyzes production of the trans re action product exclusively. "We obtained a quantum mechanical transition state and then docked it into the binding site to see how the binding-site residues stabilize it," says Houk. "This had not been done be fore for a catalytic antibody." At the same time, chemistry professor Peter G. Schultz and assistant professor of chemistry Raymond C. Stevens of the Uni versity of California, Berkeley, and Law rence Berkeley National Laboratory (LBNL) and coworkers carried out structural and functional studies on another Diels-Alderase catalytic antibody [Science, 279, 1929 (1998)]. In the reaction catalyzed by their antibody (called 39-A11), a substituted bu tadiene adds to a bicyclic dienophile to form a tricyclic DielsAlder product. Schultz and coworkers generated the antibody by immunizing with a bicyclo[2.2.2]octenebased transition-state analog. By determining the structure of a com plex of 39-A11 and its hapten, Schultz, Stevens, and coworkers found that two hydrogen-bonding and 89 van der Waals interactions between antibody and hapten activate the dienophile and control the rel ative geometries of the bound substrates in the antibody-catalyzed reaction. To bet ter understand the antibody's immunologi cal origins, the researchers also deter mined the structure of the precursor anti body from which 39-A11 develops. They found the precursor to be a "promiscu-
Research teams studied similar Diels-Alder reactions Scripps—UCLA group 0 ^CH3
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