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Some pairs of click chemistry components will fit together snugly inside the acetylcholinesterase and some will not. The pairs that do fit snugly together are much more likely to snap together in the presence of acetylcholinesterase. In orienting and initiating the reaction, and cutting the reaction time from years to minutes, the enzyme functions as a chemical catalyst.

Sharpless calls this variation of click chemistry "in situ," which is Latin for "in the natural position." In this case, the reaction is in situ because the enzyme directs which way the pieces come together. "Once together in the correct orientation, they will click," he says.

More than the sum of the two parts, the triazole acetylcholinesterase inhibitor the team found has powerful "femtomolar" " (10-15) activity against the enzyme. This exceeds by several hundred times the potency of the hundreds of previously known acetylcholinesterase inhibitors.

"I think it is one of the most fascinating ideas I have ever heard," says Professor Samuel J. Danishefsky of Memorial Sloan-Kettering Cancer Center and Columbia University, who heard Sharpless lecture on this research at Columbia University about a week after the announcement of his Nobel Prize. "The very enzyme that you are trying to inhibit was used to assemble the inhibitor."

"It works a lot better than we ever anticipated," says Sharpless.

A Simple and Fascinating New Approach

The reaction is an example of what Sharpless calls "click chemistry," a methodology for chemical synthesis he invented a few years ago.

"The idea [of click chemistry] is a very simple one," says TSRI Associate Professor M.G. Finn of the Department of Chemistry and The Skaggs Institute for Chemical Biology, who is an author on the report. "If you are going to make a drug (or anything), why do it with techniques that are difficult when you can do it with techniques that are easy?"

In click chemistry, chemicals (like acetylcholinesterase inhibitors) are made from modular chemical "blocks" that can be joined together in various combinations in very few steps. Reactions are chosen from readily available starting materials that react with high reliability and form easily isolated products in high yield without additional reagents.

Sharpless calls the reactions that join these blocks together "spring-loaded" because the blocks are designed to have a higher energy content than the product, which enables them to react together and form larger structures reliably.

The azides and acetylenes that were used to make the acetylcholinesterase inhibitors are, according to Sharpless, "cream of the crop" building blocks for click chemistry, because they will not react with other molecules but instead fuse irreversibly into various product structures (triazoles) when brought together.

Selecting the one triazole that is the best inhibitor of acetylcholinesterase was the job of the acetylcholinesterase enzyme itself.

This enzyme has a large binding pocket with separate places for the azides and acetlyenes to bind. When the two separate building blocks both bind to the acetylcholinesterase, they can react and form a triazole—the one that fits best inside acetylcholinesterase.

The best inhibitors thus formed will be those that bind tighter than the azides and acetylenes from which they are formed.

The research article "Click Chemistry In Situ: Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of a Femtomolar Inhibitor from an Array of Building Blocks" is authored by Warren G. Lewis, Luke G. Green, Flavio Grynszpan, Zoran Radic, Paul R.Carlier, Palmer Taylor, M.G.Finn, and K. Barry Sharpless and appears in the March 15, 2002 issue of Angewandte Chemie.

The research was funded by the National Institute for General Medical Sciences, the National Institutes of Health, the National Science Foundation, The Skaggs Institute for Chemical Biology, the W.M. Keck Foundation, and the J.S. Guggenheim Memorial Foundation.


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The enzyme acetylcholinesterase preferentially assembles one pair of the reactants, each bearing a group that binds to adjacent positions on the protein structure (the choline binding site of the active center and the peripheral site), into a triazole adduct that is the most potent noncovalent inhibitor of the enzyme yet developed. Courtesy Flavio Grynszpan, TSRI.