COVER STORY

PERFECT FIT Model of acetylcholinesterase inhibitor.

IN SITU CLICK CHEMISTRY

Templating strategy offers potential route to new drugs and

other functional compounds STU BORMAN, C&EN WASHINGTON

Ν THE "CLICK CHEMISTRY" STRATEGY DEVELOPED RECENT-

ly at Scripps Research Institute, reactive molecular building blocks are designed to "click" together selectively and cova-lently The Scripps researchers are now extending the idea by using protein binding sites, supramolecular complexes, or

ftinctionalized surfaces as reaction vessels to direct the in situ formation of potentially functional click chemistry products. The products might be biological inhibitors, molecular-elec­tronics components, sensor probes, non­linear optical materials, light-harvesting compounds, or compounds with any num­ber of other useful properties.

In what might be considered either a lucky break or a harbinger of the tech­nique's power, the scientists used the pro­cedure successfully on its first tryout to identify an inhibitor of the disease-associ­ated enzyme acetylcholinesterase. The in­hibitor binds at femtomolar levels—an ex­

traordinarily strong measure of binding affinity [Angew. Chem., in press}.

Click chemistry is the use of chemical building blocks with "built-in high-energy content to drive a spontaneous and irre­versible linkage reaction with appropriate complementary sites in other blocks," ex­plains Scripps chemistry professor and No­bel Laureate K. Barry Sharpless, who con­ceived the strategy [Angew. Chem. Int. Ed., 40, 2004 (2001)]. The expanded strate­

gy—in situ click chemistry—is the use of chemical and biological receptor structures as templates to guide the formation of click chemistry products.

There are powerful and plentiful prece­dents for such a templating concept, and even for the reaction Sharpless and coworkers are employing in their initial studies. But nobody has put those ele­ments together into an overall strategy for drug and materials discovery in just this way before.

In recent years, Sharpless says, there has been "an enormous emphasis on the struc­ture-guided synthesis of molecules with de­sired properties. Unfortunately, successes have been rare. For one thing, our under­standing of the systems involved is often not good enough, and the number of mol­ecules that could be considered is too vast."

In medicinal chemistry for example, the traditional approach is to lock in the struc­tures of drug candidates ahead of time— "before they are tested against a biological

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COVER STORY

target," Sharpless notes. "This traditional strategy often seems only marginally bet­ter than a shot in the dark. Wouldn't it be more productive if the biological system could somehow be directly engaged in the synthetic sequence leading to the drug candidate?"

ANY SEARCH PROCESS "that actually de pends on the biological target structure serving as the catalytic mold—for creat­ing new molecules that interfere with or change its normal function—should en­able a quantum leap in the effectiveness of drug discovery endeavors," he adds. 'When the appropriate reactive modules are pro­vided, a form-fitting small-molecule in­hibitor will be erected right in the heart of the enzyme's catalytic machinery." For bi­ological and nonbiological applications alike, "this promises to be a generic way of engaging chemical reactivity to search for and create useful functions."

Asked to comment on the in situ click chemistry strategy, bioorganic chemist Ivan Hue of the European Institute of Chemistry & Biology, Bordeaux, France, tells C&EN that the initial results by Sharpless and coworkers, in which a femtomolar in- = hibitor was found, "are tru- Ë ly remarkable in terms of ï efficiency"Althoughthein < situ part of the procedure £ is "conceptually not new," £ the findings demonstrate 5 that the strategy of enzyme § templating "can be more © successful than was ever | shown before." °

David C. Rees, head of | medicinal chemistry at As- J traZeneca, Môlndal, Swe- ο den, comments that "the | idea of using an enzyme's £ binding site to fish out two £ small organic reactants and î then force them to react with each other stereoselectively is really very elegant." Sharpless and coworkers have "applied the idea to synthesize a nov­el and very high-affinity inhibitor for a much-studied enzyme. It allows one to speculate that it might become possible to use proteins or other biopolymers as 'super reagents' or catalysts for synthesizing com­pounds with useful properties."

Ideally, click chemistry reagents are

NESTLED Model of inhibitor in the active site of acetylcholinesterase, similar to one formed experimentally using in situ click chemistry.

spring-loaded to combine pretty much on­ly with each other. At the same time, for biological applications, "these spring-loaded sites must be virtually inert toward the chemicals found in living systems," Sharpless says. This "should enable one to use them for self-assembly reactions right in the middle of functioning biosystems."

IN SITU G ROUP Sharpless (top left), coworkers (from left) Radie, Grynszpan, Green, and Lewis, and collaborator Carlier (bottom left).

In addition, "the blocks will often house structural features well known for their po­lar or hydrophobic binding affinities with biological structural motifs."

Building blocks must also be designed to combine only when they are positioned very close to one another. This makes it possible for the active site of a biomole-cule to act as a template for the combina­tion process. Only if the building blocks'

polar or hydrophobic features bind to spe­cific active-site structures do the blocks line up next to each other, permitting them to click together like Lego pieces.

For now, the reactive building blocks on which Sharpless and coworkers are focus­ing have azide and acetylene groups that combine readily with each other—when held in close proximity— to form triazoles. This type of cycloaddition reaction was one of many first studied in detail by chem­istry professor emeritus Rolf Huisgen of the University of Munich, in Germany If the polar or hydrophobic structural groups of one azide-containing and one acetylene-containing compound find adjacent bind­ing sites on a templating enzyme, the com­pounds react with each other to form a triazole product that is likely to inhibit the enzyme.

When the positioning is right, the cy­cloadditions "are strongly driven, almost inevitable transformations, depending on little else except the effective molalities of the reactants," Sharpless says.

SCRIPPS ASSOCIATE professor of chem­istry M. G. Finn, one of Sharpless' collabo­

rators, notes that a practi­cal benefit of the strategy is that detecting a hit (an in­hibitor of a protein target) "relies not upon character­izing the target protein's function in the presence of the candidate, but rather on detecting the production of a new small molecule. The analytical challenges to screening large numbers of combinations are thereby changed, sometimes for the better."

However, using an en­zyme or enzymelike mole­cule to template a bimole-cular reaction is not a novel concept and in fact has a

venerable history "Others have developed the idea of asking a target to 'template' the construction of form-fitting small-mole­cule inhibitors," Finn notes. Perhaps the first, he says, were lecturers James F. A. Chase and Philip K. Tubbs of the depart­ment of biochemistry at Cambridge Uni­versity, who reported in 1969 on the self-catalyzed inactivation of carnitine acetyltransferase. "The difference in our

"Wouldn't it be more productive if the biological system could be directly engaged in the synthetic sequence?" 3 0 C & E N / F E B R U A R Y 1 1 , 2 0 0 2 H T T P : / / P U B S . A C S . O R G / C E N

approach is not the philosophy of template assembly, but rather the chemistry that we use to accomplish ligations of blocks in the target," Finn says.

An important chemical precedent for in situ click chemistry was a series of clas­sic experiments in the 1980s by chem­istry professor William L. Mock of the University of Illinois, Chicago, and co­workers on cucurbituril—a nonadeca-cyclic cage structure that acts as a cata­lyst. The major differences are that Mock and coworkers did not use an enzyme and did not approach templating as a way to create functional compounds (such as en­zyme inhibitors).

Mock's group used cucurbituril as a tem­plating agent to guide the regiospecific for­mation of triazole products from the union of azide and alkyne substrates. In these ex­periments, only single-isomer triazole products were formed, whereas nontem-plated cycloadditions of the same compo­nents yield approximately 1:1 mixtures of isomeric syn- and anti-triazoles.

Mock and coworkers estimated that "cu­curbituril achieved a rate acceleration of 100,000 over the background thermal process," Sharpless says. "They also en­countered the same binding-constant mea­surement problem we did. Their in situ tem-plated triazole, although noncovalent and rather small, was barely able to escape from the cozy channel that fit it like a glove." Such tight binding makes it difficult to car­ry out kinetic studies and to isolate enough templated product for identification.

SHARPLESS SAYS he "cannot get over the nearly perfect match between Mock's cucurbituril experiments and our acetyl­cholinesterase experiments done 20 years later. If cucurbituril had been a small en­zyme, then the two systems would be in­distinguishable." However, the implica­tions of templating for drug discovery are more apparent today than they were at the time Mock and coworkers did their clas­sic experiments.

Researchers active more recently in tem­plating by enzymes and enzymelike com­plexes include Darryl C. Rideout of Struc­tural Bioinformatics, San Diego; chemistry professor and Nobel Prize-winner Jean-Marie Lehn and coworkers at University Louis Pasteur, Strasbourg, France; the group of chemistry professor Stephen J. Benkovic at Pennsylvania State Universi­ty, University Park; Hue's team in Bor­deaux; chemistry professor K. C. Nicolaou and coworkers at Scripps and the Univer­sity of California, San Diego; and director of chemistry Alexey V Eliseev's group at

Therascope AG, Heidelberg, Germany Their contributions include the

following: • Rideout reported in 1986 on how cy-

totoxins and drugs could self-assemble in vivo via the formation of hydrazones from aldehydes and hydrazine derivatives. The reagents reacted with one another inside living cells irreversibly but did not react "at a significant rate with biomolecules at physiological concentrations," Rideout says.

• Lehn's group developed the concept

of target-driven dynamic combinatorial chemistry, in which building blocks with properties suitable for forming a supramol-ecular entity are selected from a combina­torial library and assembled in the pres­ence of a target.

• And Eliseev and coworkers recently used neuraminidase as a template for the selective synthesis of inhibitors from high­ly diverse dynamic combinatorial libraries [Proc. Natl. Acad. Sa. USA, in press}. They found that aldehyde and amine building blocks combined by reductive animation

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CEN021102

2002 31

COVER STORY

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syn anti

TEMPLATED REACTION Generic azide-acetylene reaction in its nontemplated form (inset, top left) produces equal amounts of syn- and anti-isomeric products. But when acetylcholinesterase (blue) was used as a template for the reaction of acetylene (left) and azide (right) building blocks, the syn addition product (structure at upper right and space­filling model in enzyme active site) from the two highlighted azide and acetylene building blocks was the only compound formed. The product turns out to be the strongest noncovalent inhibitor of acetylcholinesterase yet identified.

to form inhibitors only in the presence of the enzyme.

However, Sharpless notes that many previous reports of templated reactions taking place within enzymes relied upon the reaction of a thiol with a halocarbonyl compound. One drawback of this ap­proach, he says, is that halocarbonyl com­pounds react readily with enzyme nucle-ophilic sites, which can alter the enzymes and even block the very binding sites one is trying to probe.

The reaction of azides and acetylenes to form triazoles sidesteps this problem. According to Sharpless, "The building blocks are effectively orthogonal, within limits, to the typical reactive groups and conditions encountered in enzymes and most other biological systems"—that is, they don't tend to cross-react with bio-

molecules. Hue comments that the Sharp­less group's use of the azide-acetylene re­action represents "true progress" because of its high selectivity

Some azide- and acetylene-based build­ing blocks could theoretically react with each other to a limited extent when they're free in solution, before they even en­counter an enzyme template. But by hold­ing them near each other, the enzyme speeds up the process greatly

"UNLESS THE PIECES are held close to­gether, the reaction is kinetically slow," Finn explains. "It has a reasonably high ac­tivation barrier but is thermodynamically very favorable. Most importantly, a signif­icant portion of the activation barrier is entropie—the pieces have to approach each other in precisely the right orienta­

tion. Holding the pieces close together with something else, like an enzyme bind­ing pocket, helps pay this entropie cost and thereby accelerates the process."

The selectivity of the reaction, Finn points out, is reminiscent of that of target-accelerated combinatorial synthesis, an olefin metathesis strategy developed by Nicolaou and coworkers in which the start­ing materials are similarly unreactive with biocompounds. Two years ago, Nicolaou's group used such reactions to construct highly potent vancomycin dimers.

Sharpless says his group's choice of the azide-acetylene reaction—"the most ver­satile and reliable of all the Huisgen [3+2} cycloaddition processes—was, in retro­spect, probably inevitable. Not only is it highly exergonic, but it is a pure fusion process needing only concerted reorgani-

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zation of several π bonds into two new σ bonds." Reaction yields are as high as 99% or more, although he concedes that reac­tion rates can be slow.

In their initial in situ click chemistry study Sharpless and collaborators used the active site of electric eel acetylcholinester­ase to template the formation of their fem-tomolar inhibitor. Although the nontem-plated azide-acetylene reaction forms 50-50 mixtures of syn- and anti-product isomers, the enzyme-templated reaction yielded only the syn form. This syn prod­uct was, in fact, the only triazole that formed out of 98 possible reaction prod­ucts from all combinations of the azide and acetylene building blocks used.

"This is the most potent noncovalent inhibitor ever found for the acetylcholin­esterase system, by at least two orders of magnitude," Sharpless tells C&EN—"not bad for our first effort to create an enzyme inhibitor in situ. Acetylcholinesterase is a crucial enzyme in the mammalian nervous system and a current target for drugs to al­leviate Alzheimer's disease."

Sharpless' collaborators on the acetyl­cholinesterase study were graduate student

Warren G. Lewis, research associate Luke G. Green, assistant professor of chemistry Flavio Grynszpan, and Finn at Scripps; as­sistant project scientist Zoran Radie and professor Palmer W. Taylor at the depart­ment of pharmacology of the UC San Diego School of Medicine; and associate professor of chemistry Paul R. Carlier of Virginia Polytechnic Institute & State Uni­versity. Sharpless also credits chemistry professor Daniel W Armstrong and grad­uate student Clifford R. Mitchell of Iowa State University, Ames, for chromato­graphic assistance.

THE REACTION RATE for the cycloaddi­tion system used in the acetylcholinester­ase study was extremely slow—"so slow, in fact, that we nearly failed to detect the tem-plated triazole," Sharpless says. However, "the rate of the cycloaddition process is easily 'tuned' and can be increased several hundredfold," he notes. And because of the high yield of click reactions, he believes compounds discovered this way could be produced inexpensively and thus evaluat­ed relatively easily "We could make, with­in a week, 100 g of any compound from an

initial screening library that exhibits in­teresting activity," he says.

AstraZeneca's Rees comments that ' what is exciting from a drug discovery per­spective is the prospect that in situ click chemistry could be extended to identify other small-molecule ligands or modula­tors for proteins or biopolymers, even when the structure or function of the pro­tein is unknown. This is a big challenge and often a bottleneck for medicinal chemists in the pharmaceutical industry today, es­pecially in view of the number of protein targets that have arisen from unraveling the human genome."

High-throughput biological screening of large chemical libraries can be used suc­cessfully to identify such hits, Rees says, "but there is a drive for alternative and complementary techniques, and this is just one of the areas in which in situ click chem­istry will attract attention."

However, he adds, "it is too early to be sure how generally applicable in situ click chemistry will be. Acetylcholinesterase may prove to be particularly well suited to this approach due to its lipophilic cleft and well-defined binding pockets."

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In the nonbiological area, Sharpless, Finn, and coworkers are collaborating with groups led by chemistry professors Paul S. Weiss and Raymond L. Funk of Penn State and James M. Tour of Rice University in an effort to develop surfaces with precise chemical patterns at the nanometer scale. They hope to collaborate with chemistry professor Timothy M. Swager and cowork­ers at Massachusetts Institute of Tech­nology on click chemistry-based sensors. 'And there are endless possibilities in sur­face science, chromatography, and other areas," Sharpless says.

THE APPROACH USED in these nonbio­logical studies "is basically the same as for the enzyme inhibitors," Sharpless says, "ex­cept that the molecular-scale reaction guide might now be—instead of an enzyme active site—a surface that templates azide-and acetylene-decorated pieces together if they are recruited to adjacent sites." Al­ternatively, the synthesis might now in­volve "a unique selection of polyvalent azide-acetylene blocks that generate an oligomeric structure with a defined re­peating motif, encoded by complementa­ry recognition elements in the assembling blocks. The beauty of these nonbiological projects is that a discovery can lead much more directly to real uses."

What pure chemistry offers, he says, "is constant advances in our ability to improve and understand chemical reactivity, which in turn makes it easier to make existing, useful chemical entities and to discover new ones faster. This brings me back to a quote" from former California Institute of Technology chemistry professor George S. Hammond "about 'properties,' not com­pounds, being the goal of synthesis. There are too many compounds for any one of them to be sacred, but the reactivity that connects them to each other is."

Sharpless conceived of click chemistry as a fast, high-yield form of organic syn­thesis several years ago. But he says he on­ly realized the implications of using it for in situ synthesis in biomolecular active sites in April 2000, when it "suddenly jumped out at me that Huisgen's triazole synthesis was in a class by itself. It sure has taken us a long time to prove that the in situ idea ac­tually works, but when we finally discov­ered Mock's precedent it became instant­ly clear that our present enzyme-based version had to work, too. I have always been fascinated by the way in which sim­ple and yet very important connections re­main invisible to us until their time to be seen approaches and some idiosyncrasy in our vision brings them out." •

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