Adventures in Organic Chemistry

Photos: All photos courtesy Phillip Crews

In some ways, animal life doesn't get much simpler than a sponge, a creature with no mouth, gut, muscles, nerve cells, or sensory organs. A chopped up sponge can regrow from as little as a single cell. Surprising complexities, however, lie beneath the apparent simplicity of sponges. Organic chemists, for example, have been astonished by the unusual structures of some of the chemicals found in these "simple" organisms.

Sponges have been around for more than 500 million years, and genetic evidence suggests spongelike organisms were the ancestors of all animal life. Today, they are found throughout the world's oceans--and even in fresh water--and come in an amazing variety of shapes and colors. Mostly, they sit in one place and feed on bacteria and other plankton by filtering enormous volumes of water through an intricate system of canals and chambers.

Chemistry professor Phil Crews and his research team are exploring the pharmacological potential of compounds derived from marine sponges. Facing page: Crews and his research team at work in the South Pacific. Photo: r. r. jones

To hold their own in a sea teeming with predators and competitors, sponges have evolved a diverse array of chemical defenses. As stationary, soft-bodied creatures, their primary defensive strategy is to make themselves unpalatable or downright toxic. The potent chemicals that sponges use for protection have attracted intense interest from medical researchers and pharmaceutical companies seeking to develop new drugs.

Phillip Crews, a professor of chemistry at UCSC since 1970, is among the pioneers in the field of sponge chemistry. His Marine Natural Products Laboratory now holds an unparalleled collection of nearly 800 pure compounds--complex chemicals isolated from sponges and other marine organisms--as well as thousands of extracts containing mixtures of chemicals the lab has yet to separate and analyze.

In the 1980s, Crews published some of the first papers on the chemistry of sponges. Now, his research lab involves some 20 graduate students, postdoctoral researchers, undergraduates, and technical staff. Several major grants from the National Institutes of Health support the group's ongoing projects and collaborations.

Their research takes Crews and other members of his laboratory on annual expeditions to remote tropical islands, where they explore the waters around coral reefs and other habitats, collecting sponges for chemical analysis.

"It's neat to be a chemist and get to do scuba diving as part of the job," says graduate student Chris Wegerski. "I was afraid of the ocean until I joined the lab, and now I love to dive."

The researchers are careful to avoid overharvesting any particular sponge, and always leave behind part of each specimen so the sponge can regrow, says Karen Tenney, the lab's research coordinator. "We know these are fragile ecosystems, so the collecting is carefully targeted," she says.

Crews still remembers the day in 1974 when he decided to explore the chemistry of sponges. Thumbing through a book entitled Poisonous and Venomous Marine Animals of the World, he read that extracts from sponges had shown antibiotic and antiparasitic properties. Turning to the section on the chemistry of sponges, he saw just one word: "Unknown."

"That was the moment when it struck me that this is what I should do," Crews says.

Crews set about methodically acquiring the knowledge, skills, and equipment he would need to pursue this new path. Through the campus's recreation department, he learned scuba diving so he could collect specimens. Later, he took sailing lessons so he could rent boats on his expeditions to the tropics.

"There was a 10-year period when we learned how to organize ourselves to explore these very remote areas," Crews says.

Each extract contains hundreds of chemicals, one of which may yield a new treatment for a disease like cancer or arthritis. The challenge is to find the potentially useful compounds.

As an organic chemist with little background in biology or ocean sciences, he had to spend a lot of time learning about the natural history of sponges. Now, when Crews goes on an expedition, he knows what to look for.

"We have a sense of what kinds of sponges have been important in terms of chemical leads, based on our experience in previous years," he says. "We also know which ones have already been well studied, and we try to avoid those."

Over the years, Crews has focused much of his collecting effort around the South Pacific islands of Fiji, the Solomon Islands, and Papua New Guinea. The variety of coral reef habitats in this region has given rise to great biological and chemical diversity in the sponges. Crews has found that even within the same species of sponge, the chemistry can differ from one locale to another.

The samples Crews and his colleagues collect go through an extensive extraction procedure designed to separate interesting chemicals from the tissues of the sponge. It starts on the boat with a good soaking in a 50 percent alcohol solution (100 proof vodka will do in a pinch), and concludes in the laboratory with a series of separation procedures, yielding a half-dozen crude liquid extracts from each specimen. Then the really interesting work begins.

Each extract contains hundreds of chemicals, one of which may yield a new treatment for a disease like cancer or arthritis. The challenge is to find the potentially useful compounds. Biological assays or tests can be used to identify extracts with valuable properties. The National Cancer Institute, for example, has a standard battery of assays for antitumor activity. If an extract shows activity in one of these assays-- stopping the growth of breast cancer cells, for example--the Crews lab can further refine it and try to isolate and characterize a specific compound that can serve as the basis for the development of a new drug.

Crews's lab also searches the extracts for compounds that are interesting in their own right, either because of a unique chemical structure or a similarity to a compound already known to have useful properties. This involves the use of sophisticated equipment and techniques for separating and analyzing the individual compounds in an extract.

"With instruments like the mass spectrometer, we're able to get a sense early on of any novel chemistry in the organism," Crews says. "That's buttressed by the biological assays performed by our various collaborators and partners."

Determining the exact chemical structure of a new compound can be a lengthy and complicated process. For Crews, however, it is the most interesting part of his research.

Although much of his work has a very practical orientation, Crews remains at heart a theoretical organic chemist. What really gets him excited is the discovery of a new compound with a unique chemical structure.

"Chemists can do combinatorial chemistry to create novel synthetic structures, but they're never going to envision what nature can do. I look at nature as the ultimate chemist," he says.

The most promising drug lead to come out of the program so far is a group of compounds called bengamides, which Crews first isolated from sponges collected in the Benga Lagoon in the Fiji Islands. The bengamides have shown potent antitumor activity, and the pharmaceutical company Novartis has been investigating them for clinical use. A bengamide-derived drug is currently in clinical trials to test its safety and effectiveness as a treatment for breast cancer.

Sometimes the lab discovers compounds that are not entirely new but are interesting variants within a class of known compounds. The value of such discoveries often lies in the added complexity of the chemical structure or slight differences in biological activity, Crews says.

Wegerski, for example, was screening a library of extracts from sponges collected in Papua New Guinea when he found some new types of manzamines, compounds previously shown to have antimalarial properties.

"Papua New Guinea has a bad malaria problem, so it would be nice if something we discovered there could be used to help them out," he says.

Drug leads are not the only useful products to come out of Crews's library of chemical compounds and extracts. Some of the compounds his lab has isolated have proved to be very useful tools for cell biologists because they bind to and inhibit specific cellular proteins. These inhibitors can be used as molecular probes to tease apart complex biological processes. The Crews lab doles out precious samples of these compounds to other scientists around the world.

Crews is constantly moving forward into fresh territory--adopting new technologies, establishing new collaborations, and pursuing new areas of investigation. A few years ago, he began exploring the chemistry of marine fungi. "The microorganisms --both fungi and bacteria-- may be the next place where we're going to see a lot of new chemistry," he says.

Crews decided to study the microorganisms from sponges in part because he thought they might explain why completely different sponges sometimes contain identical chemical compounds. Perhaps, he thought, the compound is produced by a microorganism associated with both sponges. So far, he hasn't found a case like that, but he has found a lot of interesting chemistry produced by the fungi associated with sponges.

Four Ph.D. theses have now come out of his lab's research on the chemistry of microorganisms associated with sponges. Some of the compounds derived from sponge fungi have shown anticancer activity in the National Cancer Institute's panel of screening assays and are being evaluated further.

The vast library of extracts and pure compounds Crews has amassed from 20 years of expeditions is becoming increasingly valuable as new technologies emerge for analyzing the extracts and finding useful compounds.

Phil Crews and his crew: (l-r) Itchung Cheung, Paul Ralifo, Akiko Amagata, Phil Crews, Jocelyn Flanary, Chris Wegerski, Karen Tenney, Nate Segraves, Claudia Meents, and Robert Cichewicz. not pictured: Taro Amagata, Laura Clifford, Dan Kuhn, Tyler Johnson, Jeff Gautschi, Rachel Sonnenschein, and Phil Wenzel. Photo: r. r. jones

Crews works with an impressive array of collaborators to make the most of this remarkable collection. Novartis's Institute for Biomedical Research and the Josephine Ford Cancer Center are both involved in ongoing partnerships with Crews, screening his library to find drug leads. And Crews is working with a growing number of colleagues at UCSC to find new ways of exploring his collection.

"The way he has run his operation is a model for all natural products researchers," says Theodore Holman, an associate professor of chemistry and biochemistry at UCSC and one of Crews's collaborators. "He loves collaborations where his compounds are used in other people's assays, and he is truly a fun person to work with. His personality really helps the collaborative endeavor work and makes the repository even more valuable."

Holman studies enzymes called lipoxygenases that have been implicated in a broad range of human diseases, including cancer and heart disease. He found 20 different lipoxygenase inhibitors by screening just part of Crews's collection of pure compounds and crude extracts.

"To find 20 novel inhibitors so quickly from this one repository is phenomenal, and that's just with my one little assay," Holman says. "When you look at the quantity of extracts he has and the biodiversity that it comes from, the potential is amazing."

New "high-throughput" technologies may speed up the pace of discovery. For example, assistant professor of chemistry and biochemistry Scott Lokey is setting up a robotic system that can screen 10,000 compounds in one day in an assay for selective destruction of cancer cells. New instruments also make it possible to purify large numbers of compounds in a short time.

In the past, standard screening procedures have involved running a relatively small set of crude extracts through an assay to see, for example, if an extract kills cancer cells. If activity is detected in one of the extracts, it is then further purified. The partially purified fractions are run through the assay again, and the process is repeated until, with a bit of luck, it yields a pure, active compound.

Recently, the Crews lab took some of their crude extracts to the laboratory of molecular pharmacologist Kip Guy at UC San Francisco, where they used a sophisticated new instrument to quickly generate hundreds of purified fractions from each extract. Each fraction contains a pure compound or perhaps a mixture of two compounds. Next, they will screen these fractions using Lokey's robotic assay system.

"Our technology continues to evolve," Crews says. "We just bought a high-pressure extractor that can do in less than an hour what used to take us a week. And if you were to come back to our lab in a few months, you would see new pieces of equipment."

Even with the latest technology, the path from detecting biological activity in an extract to developing a useful drug is long and tortuous, and there are many pitfalls along the way. One of the drawbacks of sponges as a source of pharmaceuticals is the limited availability of the raw materials. Many of the most interesting compounds make up less than 1 percent of the sponge by weight. It would be impractical, not to mention environmentally irresponsible, to harvest large quantities of wild sponges for drug production.

Synthesizing the compounds in the laboratory is one alternative. Although that can be quite challenging, chemists may not need to synthesize the entire compound as it occurs in nature, according to Holman. Having found 20 different lipoxygenase inhibitors, he is now trying to identify the common feature that accounts for their potency.

"If we can determine what that core feature is, then maybe we can synthesize a compound with that basic scaffold and develop it into a drug. So the natural product can help us design the drug," Holman says.

Crews calls this "inspirational chemistry."

Another approach is to identify the genes involved in making the compound in the sponge, then try to transfer the genes into a microorganism that can be cultured on an industrial scale. Crews is pursuing this approach in collaboration with David Sherman, a microbiologist at the University of Minnesota. As an undergraduate at UCSC in the 1970s, Sherman did his senior-thesis research in Crews's lab.

"We're trying to understand the machinery that nature uses to put these molecules together," Crews says. "So this has sent us in yet another direction."

Despite the diverse ways in which his research program continues to evolve, Crews is never likely to stray far from his first love, organic chemistry. In fact, he is currently deeply engaged in writing a revised second edition of his textbook, Organic Structure Analysis.

By staying at the forefront of organic chemistry, while developing a broad range of interdisciplinary collaborations, Crews has managed to develop a program that effectively combines basic and applied research. Biomedical researchers are already exploiting the novel chemistry Crews has found in sponges and other obscure organisms, but they have only begun to tap into the full potential of his lab's repository.

"There are millions of compounds in his repository," Holman says. "We have just begun to scratch the surface in terms of the potential benefit to mankind."

-Tim Stephens