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almost identical in shape to thymine, di fluorotoluene replaces each of thymine's two carbonyl groups with afluorineatom. At first glance, Zimmerman points out, one might predict that fluorine is electro negative enough to form hydrogen bonds with adenine much the way carbonyl groups do. However, the Rochester chem ists tested for that and were unable to in duce hydrogen bonding between their mimic and adenine, even in solvents such as chloroform that stabilize hydrogenerybody go: 'Hmm, what's going on bonded complexes. Organofluorine biochemistry is full of here?' " "It's a very clever experiment and an surprises, Matteucci notes, and this ex interesting and important observation," periment may be another one. It's com says molecular biology professor Myron F. mon in medicinal chemistry to substitute Goodman of the University of Southern fluorine for a hydrogen in a promising California. Goodman, for one, doesn't find drug candidate and—for reasons that are the result surprising. He and a few others not understood—end up with a more ac have been arguing for the past several tive molecule, he says. "Nobody really years on the basis of free-energy consider seems to understand what fluorine does ations that shape is likely to be more im to a molecule. It doesn't hydrogen bond portant than hydrogen bonding in explain in the traditional sense, yet there's a diing the extreme accuracy of DNA poly pole there," he says. merase enzymes in copying DNA. Such responses don't really surprise "It's great to make suggestions and Kool. "This is going to be a controversial hypothesize things," Goodman says, result," he says, "so we need to do this "but Kool has done something very im experiment many times and in many dif portant. He's put together a system to ferent contexts to see how general the result is." He wants, for example, to per address this question." Kool, along with postdoctoral re form the experiment using more than searchers Sean Moran and Rex X.-F. Ren one DNA polymerase enzyme and to try and graduate student Squire Rumney IV, it using RNA polymerase to see if that made their mimic by substituting difluo- enzyme will make the correct RNA poly rotoluene for thymine, one of the four mer from a DNA template containing the bases normally found in DNA. Although mimic. His group is working on the re verse experiment, to see if DNA § polymerase will incorporate a μ difluorotoluene-containing deg oxynucleoside triphosphate into ο a growing DNA chain. The re| searchers are also trying to make § nonpolar shape mimics for the % other three natural bases in DNA
MIMIC TRICKS DNA POLYMERASE Shape of base, rather than H-bonding, may be key to faithful copying of DNA
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hemists at the University of Roch ester have "tricked" the enzyme that synthesizes DNA into accept ing an unnatural base in the template strand of DNA. The mimic has the same shape as the base thymine, but unlike thymine, it is unable to form strong hy drogen bonds with thymine's comple mentary base, adenine. Nevertheless, the enzyme "reads" the base as if it were thymine and inserts an adenine in the complementary strand with a specificity almost as high as that for thymine itself. "Most biochemistry textbooks cite hy drogen bonds as the primary reason that DNA is copied accurately," says chemis try professor Eric T. Kool, who headed the research effort. But in this experi ment, DNA is copied accurately even though the base completely lacks con ventional hydrogen bonds \f. Am. Chem. Soc, 119, 2056(1997)]. "It now appears that the shape of the DNA base may be the chief mechanism by which DNA-replicating enzymes se lect the right bases to insert into a growing strand," Kool says. "These results are very intrigu ing," says chemistry professor Steven C. Zimmerman of the Uni versity of Illinois, Urbana-Champaign. Because it can't form the expected hydrogen bonds, the thymine mimic destabilizes the DNA helix, Zimmerman points out. "The idea that something can destabilize the DNA helix and yet be incorporated with such selectivity is very unex pected," he says. These experiments "reverse the well-accepted dogma," says Mark Matteucci, director of bioorganic chemistry at Gilead Thymidine Difluorotoluene Sciences, a biotechnology com deoxynucleoside deoxynucleoside pany in Foster City, Calif. "The recognition that shape is the Thymidine deoxynucleoside mimic, left, has the same determining factor in this poly shape as the natural nucleoside, right, but lacks elec merase insertion of bases dur tronegative carbonyl groups (red and yellow) needed to form hydrogen bonds with its base pair, adenine. ing replication is totally unprec edented. It's going to make ev
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to see if polymerases can also ac cept them. Not all of the mimics they are trying to build contain fluorine, Kool says. Already people are beginning to speculate even more broadly about what the experiment might mean. "In what other ar eas might something like this be occurring, where we thought all along that there was a hydrogenbonding interaction and, in fact, there really isn't?" Matteucci asks. "Right now, it's fuzzy what the extensions of this work will be. But I think it will provoke people to think about this." Rebecca Rawls MARCH 3, 1997 C&EN 9
