Sharpless Clicks at Informex 2004

Nobel Laureate K. Barry Sharpless drew a big crowd at an educational workshop on homogeneous catalysis at Informex 2004, held last month in Las Vegas. However, Sharpless talked little of conventional asymmetric catalysis and focused instead on his new passion, "click chemistry," where reagents fuse easily, irreversibly.

According to the click chemistry manifesto written by Sharpless and his Scripps Research Institute colleagues Hartmuth C. Kolb and M. G. Finn three years ago, reactions that qualify as click chemistry "are modular, [are] wide in scope, give very high yields, generate only inoffensive by-products that can be removed by nonchromatographic methods, and [are] stereospecific (but not necessarily enantioselective)" [Angew. Chem. Int. Ed., 40, 2004 (2001)]. In addition, the reaction must consistently give high yields with various starting materials, be easy to carry out, be insensitive to oxygen and water, and use only readily available reagents [Drug Discovery Today, 8, 1128 (2003)].

The goal, Sharpless said, is to emulate nature's modular approach to synthesis to improve the speed and reach of synthetic organic chemistry. "Click chemistry is about picking the best reactions in the world," he said. "Those reactions usually are fusions between two energetically self-contained reactants. Hence, the best click reactions are all highly exergonic, with thermodynamic driving forces from 20 to 50 kcal per mole."

Sharpless' chemistry has always been inspired by nature. The catalytic asymmetric oxidations for which he won the 2001 Nobel Prize in Chemistry grew out of the conviction that nature's enantioselectivity can be duplicated by human hands. With click chemistry, Sharpless hopes to emulate nature to quickly discover and/or form functional molecules from a few building blocks.

Sharpless pointed out that nature is the original combinatorial chemist, producing "staggering diversity" from only about 35 building blocks, the supreme example being proteins. All are polyamides based on 20 l-amino acids. "Life is completely modular," he said. "Nature has a few building blocks, and she makes most compounds by uniting the blocks with heteroatom links." Carbon-heteroatom links are the key, he explained, because click-chemistry-type reactions with a net yield of new carbon-carbon bonds are rare.

In exploring click chemistry, Sharpless received an unexpected "gift"--realizing the importance of reactions on water. Reactions that take days to occur in organic solvent can be complete in hours when reactants are floating on water; reactions that would explode when carried out with neat reactants are tamed.

One example involves quadricyclane, a potentially inexpensive hydrocarbon that the military has been evaluating as a possible fuel. When floating on ice water, it reacts with dimethyl azodicarboxylate quantitatively in about one hour, he pointed out. The reaction occurs at least 1,000 times faster than when the neat liquids are mixed at 0 °C.

In another example, β-pinene and diethyl azodicarboxylate on water react within 30 minutes, and the pure product drops underneath the water phase. In process chemistry, "the ideal reaction is one where a chemist with one arm pours ingredients into a bathtub and collects the product pure from the drain hole," Sharpless pointed out, rewording a famous statement by Nobel Laureate Sir John W. Cornforth. "This reaction could be easily developed," he added.

So far, the fittest click chemistry reaction is the Huisgen 1,3-dipolar cycloaddition of alkynes and azides to form triazoles, Sharpless said. "It is not used much, even though it goes downhill by about 50 kcal per mole." He described how he has used this chemistry to let enzymes reveal their "deepest, darkest secrets"--that is, to let "enzymes discover their own best inhibitors."

Proof of principle was established with acetylcholinesterases, which are involved in neurotransmission. The enzyme's active site is located at the bottom of a deep gorge. Known inhibitors bind either to the active site or to a secondary site, the rim of the gorge. Those that bind the active site have long been used to treat Alzheimer's dementia.

Sharpless and coworkers used acetylcholinesterase as a reaction vessel for the triazole-forming reaction. The reactants consisted of dozens of azides and acetylenes attached to building blocks made up of known inhibitors that are selective for either the enzyme's active site or the secondary site. The hope was that click chemistry occurring within the enzyme would produce more potent and more selective inhibitors, binding both the active and the secondary sites.

The cycloaddition reaction between azides and acetylenes usually yields a 1:1 mixture of syn and anti triazoles. It is very slow at room temperature, but in certain cases it is dramatically accelerated by acetylcholinesterase. By using the enzyme's active site as a template, Sharpless and coworkers found one pair of building blocks that form the most potent noncovalent inhibitors of acetylcholinesterase to date. Furthermore, the enzyme-mediated reaction gives only the syn product, which binds the enzyme more strongly than does the anti product [Angew. Chem. Int. Ed., 41, 1053 (2002)].

TO UNDERSTAND why the inhibitors generated by click chemistry are so powerful--inhibiting at picomolar levels--the Sharpless group turned to several acetylcholinesterase experts: biochemist Pascale Marchot and structural biologist Yves Bourne, working in Marseille for the French National Center for Scientific Research (CNRS), and pharmacologists Zoran Radic and Palmer W. Taylor at the University of California, San Diego.

Their recently published structural studies of the inhibitors complexed to acetylcholinesterase explain not only why the click-chemistry inhibitors are so good but also why the enzyme-synthesized syn product is so much better than the chemically synthesized anti product [Proc. Natl. Acad. Sci. USA, 101, 1449 (2004)]. The findings suggest a different approach to drug discovery: harnessing a protein's flexible template to reveal vulnerable conformations that can be accessed by specific chemistries.

The structural studies confirm that the click-chemistry inhibitors bind both the enzyme's active and secondary sites. They also show that the triazoles formed are not simply passive linkers but actually interact with the gorge. "Binding interactions within the gorge itself have not been observed with other inhibitors," Marchot tells C&EN. "These interactions may contribute to the exquisite potency of the click-chemistry products."

The difference in the potency of the syn and anti products is explained by their binding at the secondary site. A part of the syn product inserts itself between certain enzyme residues near the gorge's rim, whereas the anti product does not. The insertion makes it difficult for the enzyme to make the usual motions for shuttling substrates or inhibitors in and out of the active site. The enzyme is forced into a conformation from which it cannot easily free itself.

According to Marchot, the insertion is made possible by a newly found, more open conformation of the enzyme, in which a tryptophan residue at the gorge's rim flips out to the solvent, creating the space that makes insertion possible. Such a conformation had not been seen in other structures--of the free enzyme or its complexes with inhibitors bound at the secondary site--that she and Bourne have analyzed [EMBO J., 22, 1 (2003)]. The new conformation "may have important implications for the enzyme's functioning," she says.

"I'm not sure how many people really understand the significance of these results," Sharpless told C&EN. "For the triazole to form in the enzyme, the two pieces--the azide and the acetylene--and the enzyme danced together up the energy surface to a point that people don't see normally," Sharpless explained. "When they got there, the two pieces felt at home and formed the bond. And the product got stuck." Ironically, such simple triazoles are poorly represented in the literature, he noted.

"Triazoles could become the best pharmacophores in drug discovery," Sharpless continued. "They are not harmful. They are stable, but unlike benzene, they are water soluble and they have a big dipole. I think we ought to go through all the drugs that have benzene rings, especially those that are meta-substituted, and just put triazoles in them. That would be almost an industry in its own right."

THE AZIDE-ALKYNE click chemistry has been further enriched by copper catalysis, which makes the reaction almost unstoppable. The effect of copper was recognized by Valery Fokin, an assistant professor at Scripps, when he filled a scintillation vial with copper wire, water, tert-butyl alcohol, a diazide, and an acetylene. After about eight hours, Sharpless recalled, all that was left was the triazole product, water, tert-butyl alcohol, and part-per-million traces of copper in solution. However, a few days after this experiment, Fokin carried out the reaction in fresh human plasma--"gooey yellow stuff," as Sharpless described it--and the reaction went even faster.

Next, Fokin examined whether the reaction could work just as well with copper sulfate, which is plentiful and inexpensive, in the presence of a reducing agent. "Valery went to Trader Joe's and got some vitamin C, which he thought was the best reagent for reducing copper sulfate," Sharpless recalled. The next day, Fokin worked up a 50-g-scale reaction solely in pure water with copper sulfate and vitamin C. "The products just crashed out," he said.

Fokin has since used the reaction to prepare nonnatural amino acids. He has collaborated with Scripps colleague Chi-Huey Wong to apply the reaction to the discovery of inhibitors of fucosyltransferase, a target for controlling inflammation and cancer metastasis [J. Am. Chem. Soc., 125, 9588 (2003)]. Another Scripps colleague, Benjamin F. Cravatt III, has used the reaction for activity-based profiling of whole proteomes. And together with Craig J. Hawker's group at IBM Almaden Research Center, Fokin has applied it to the synthesis of dendrimers with polyazide cores. "Valery is a genius. He has made this reaction roar," Sharpless said.

"This catalytic reaction is so robust that it gives the impression of alien reactivity principles at work," Sharpless noted. Guanidines, thiols, and phosphines in the mixture don't have any effect. Temperature, pH, solvent, and the presence of other functional groups on the reactants don't matter. And overall yields are greater than 99.9%--even for a 60-step linear sequence. "I don't know what will stop this reaction besides H₂S and HCN," he said.

At the beginning of his lecture, Sharpless said his early attempts to explore click chemistry had been rebuffed by funding agencies, which he described as being "under the sway of people who don't care about efficiency and [who] just want elegance." Yet, he emphasized, it is the simplicity of click chemistry that has led to the discovery of unusual reactivity. The copper-catalyzed cycloaddition of azides and acetylenes, he added, may "prove to be the most powerful metal-catalyzed organic reaction yet found."

Toward the end of his lecture, Sharpless showed photographs taken last December by one of his sons during a family vacation in Antarctica. One showed Antarctica's top predator--a leopard seal--lying lazily on a piece of sea ice, looking happy and content. A few feet away stood a penguin, the seal's prey. The seal and the penguin, he said, are metaphors for big pharma and a fast click chemist.

"The best trained synthetic chemists today are proud of achieving difficult syntheses, which are the antithesis of the survival-of-the-fittest, fast, and broad synthetic approach that is click chemistry," Sharpless said. "But you just can't let go of these simple findings" of click chemistry. "If you don't believe that the penguin has a chance, you might end up being part of the food chain.

"If the penguin gets results a hundred or a thousand times faster, then the seal might be in trouble. The little penguins might be able to jump everywhere, find everything; when they swim, the seal can't get them. The seal is slow. It just waits for the penguin chicks to walk off the ice at the end of summer and gobbles them up. Not much talent there."