Chemists Make Molecules with Potential to Kill Tumors

By Cassie Ferguson

Gazette Staff

Using off-the-shelf ingredients, Harvard scientists have yielded two separate methods for synthesizing twin molecules with the potential to be potent tumor killers.

Researchers working in two independent chemistry labs constructed related ring-shaped chemicals identical to those used for self-defense by a brown sponge scraped from the bottom of the Indian Ocean.

The properties of the molecules that protect the sponge from hungry fish and marauding bacteria may also be responsible for the anticancer activity reported five years ago by researchers at Arizona State University.

However, the Harvard chemists didn't synthesize the chemicals (which differ from each other by just one atom) just for their potential biological applications.

"We're in it for the reactions," said chemistry graduate student Wes Trotter. "Someone might take our synthesis and make an analog. Those analogs could be used for biological studies, though it's nowhere near being practical to use it as a drug."

Trotter, who presented the first-time synthesis of his molecule last week at the American Chemical Society meetings in Dallas, has been working on the reactions with his colleagues in the lab of David Evans -- the Abbott and James Lawrence Professor of Chemistry -- since January 1994.

Last July, they finished the last reaction -- stripping off extraneous chemicals added to the structure to protect delicate parts of the ring that might be damaged while other parts of the chemical react. With the last step complete, all they had left to do was to compare it to a natural sample extracted from the sponge.

On the morning of Sept. 22, Trotter inserted a bit of his synthesized chemical into an NMR machine, which takes a fingerprint of the protons and carbons in the molecule. After a quick glance to compare the structures, champagne corks popped in the lab, a moment shared by those who'd worked on the project: Professor Evans and postdoctoral researchers Paul Coleman, Bernard Côté, Luiz Carlos Dias, and graduate student Hemeka Rajapakse.

The following month Rebecca Roth, a graduate student down the hall in the lab of Yoshito Kishi -- Morris Loeb Professor of Chemistry -- took her turn at the NMR machine, anxiously awaiting for a confirmation of a completely separate synthesis of a similar molecule. Riding on the printout was the result of a four-year thesis project.

She said, "I knew I'd get the Ph.D., but it was a matter of how sweet it would be."

It turned out to be pretty sweet. Roth's experiment, like Trotter's, confirmed the identity of the molecules.

"The most important part of this experiment is that we proved the structure," said Trotter.

Chemical Construction

The race to build the chemicals started soon after the Arizona researchers isolated a set of giant circular molecules from a sponge pulled out of the ocean.

In 1993, they enthusiastically reported the chemicals to be, "the most extraordinarily potent substance presently known against a subset of highly chemo-resistant tumor types."

They later found that both molecules act like a monkey wrench thrown into the protein machinery that makes cells divide. It latches onto a protein in the cell called tubulin, tweaking it out of shape so that other sections of the machinery can't mesh.

Inactivating the tubulin arrests cell division, killing cancerous growth.

"They both do the exact same thing," said Trotter. "But the one with the chlorine [Roth's] is a bit more potent."

Intrigued by the problem of constructing the large exotic molecules, the Harvard chemists, along with a dozen other groups around the country, took on the challenge of synthesizing them -- a complex problem since the exact structures were uncertain. The chemists could have come up with molecules with the same ingredients, but parts of them might have wound up out of alignment or even misplaced.

"As we built it, we proved parts of the structure," said Trotter.

In addition to not knowing the exact molecular architecture, the researchers had to invent a recipe for making the molecules from scratch. For both groups, that recipe turned out to have more than 30 steps, dozens of ingredients, and involved months of cooking at each stage. To a non-chemist, this is like baking an elaborate cake from scratch, knowing neither the ingredients, how long it will take to cook, nor the exact flavor it will be when it pops out of the oven.

They started from the most educated guess as to the structure of the molecules, then figured out how to piece together the molecule's long carbon skeleton.

"To figure out the synthesis, you go in reverse and think about the reactions you'll have at the very end," said Trotter.

Both groups first approached the problem with the strategy of building the major parts of the molecule and then gluing those chunks together. But that's where the resemblance ends.

"The overall concept for the two syntheses is very similar, but at the microscopic level, they're very different," said Roth.

For example, Roth attached a chlorine atom in a spot where the other group didn't. In Trotter's synthesis, he hooked a chain of carbons to a major part of the molecule several steps later than the other group.

"You can say certain parts are better here and some parts are better there. They're two routes to essentially the same thing. Perhaps some combination of the two might be best," said Roth.

The next move would be to make the synthesis more practical, since both groups started with a few cups worth of materials and finished with a minute pinch of the product.

In each reaction, part of the starting material transforms into an unwanted byproduct. If a reaction takes 30 steps, each losing 10 percent of the material, by the time step 29 occurs, all but a few grains of the product are gone.

In order to make the synthesis more efficient, they'd have to make it shorter.

"If you can delete four steps, the yield is much higher," said Roth.

Inventing a more efficient method to construct the chemical isn't out of the question, said Trotter. "Given any molecule, there are many ways of making it."

"Different people are going to look at the same molecule and come up with different ways to make it," echoed Roth. "Putting together something like this is a bit of an art."