Still On the Case

By Eric Sauter

Chi-Huey Wong's laboratory has several new papers out this spring, all of which push the horizon of his pioneering carbohydrate research and enzymatic synthesis even further out.

These latest accomplishments notwithstanding, this hasn't been easy. From a research point of view, carbohydrates are not user-friendly. So dizzyingly ubiquitous that they have been called the body's third functional language after DNA and proteins, carbohydrates are complex, hard to synthesize, and difficult to handle in the laboratory.

A Tale of Two Hurdles

"There are two major hurdles in the field of carbohydrates," says Wong, an investigator at The Scripps Research Institute who is the Ernest W. Hahn Professor and Chair in Chemistry and member of Scripps Research's Skaggs Institute for Chemical Biology. "One is homogenous glycoprotein synthesis—producing an abundance of pure, molecularly well defined glycoproteins with each batch— which we are pretty close to solving. The problem now is making the process more general, efficient, and economical.

He continues, "The other [problem] is the development of the glycan array, to see how many sugars we can put on the chip—the highest number is still unknown—and how to characterize and study the binding properties and functions of those same sugars. There are nine monosaccahrides and theoretically you can make 15 million different structures out of those nine, but nature probably only has use for a certain number of them."

Wong was one of the pioneers in the development of glycan or complex carbohydrate microarrays, which got their start in 2003 at Scripps Research with The Consortium for Functional Glycomics, an international group led by principal investigator Jim Paulson and supported by a $37 million grant from the National Institute of General Medical Sciences (NIGMS).

The microarray, a glass slide or other solid surface on which hundreds of different glycan chains are printed robotically, gives scientists the power to assess specific properties of glycan binding proteins. Recently, a collaboration led by Scripps Research scientists used a glycan microarray to identify the mutations that could enable adaptationof H5N1 strain of avian influenza into the human population in the laboratory.

For Wong and his colleagues, the two major challenges in the field tend to run together because without high-speed synthesis you won't have the ready structures for the kind of large-scale glycan array that Wong and his colleagues have been working to achieve. What is needed, he says, is some real breakthrough chemistry to attack the problem.

They have come very close in a paper published in a recent issue of Angewandte Chemie International Edition (2006, 45, 2753-2757). That study involves the one-pot synthesis of a tumor associated antigen octasaccharide and a new strategy for putting sugars on a silicon array which, the study notes "should be useful for preparation and characterization of a large glycan array for high throughput analysis."

The One-Pot Solution

The one-pot solution is Wong's method for the mass production of whatever carbohydrate structure you might want in a matter of hours or minutes. Basically, this is Carbs To Go.

Wong's work in the field has been a critically important development because the complexity of carbohydrates has until now made them extremely difficult to synthesize. By placing large numbers of chemical building blocks into a reaction vessel and then inducing sequential chemical reactions in sequence—entering the sequence into a computer—researchers can create very specific final products based on the reaction sequence. Before this, there was no automatic or programmable way to do that.

Wong and his colleagues began working on this several yeas ago and by now it's close to where he wants it to be.

"With the one-pot method you can basically make any saccharide you need," Wong says. "Now we have to optimize the process and consider using enzymes together to make sure that we have a minimum set of building blocks available to make any type of structure we want."

The one-pot technology has been licensed to Optimer Pharmaceuticals, a company co-founded by Wong, which is using it to develop new antibiotic candidates and cancer vaccines. So far, two products are in Phase III clinical trials, while a new vaccine for breast cancer is about to go in to a combined II/III trial.

"But that is the commercialization part of science," he says. "My main interest is still to solve the problem of carbohydrates in biology, something that could lead to new discoveries. I'm still dealing with basic research in the field."

Avian Influenza and HIV

Because carbohydrates are found on the surface of all cells, they play critical roles in intercellular interactions. Right now, Wong is focused on two specific viral interactions, those involving avian influenza and HIV. He and his laboratory colleagues are working on a small-molecule vaccine approach to the avian influenza virus, one that would provide protection even in the face of the virus's known ability to mutate. Another project is focused on cancer and cancer stem cell biology.

When asked if he's concerned about the prospect of a pandemic, Wong says, "Yes, I'm very concerned about it, we have to prepare for the worst. What happened in 1918, the virus went through a simple mutation that made human transmission possible. It hasn't happened with this new virus so far. We are trying to understand how this virus evolves, and its mutation trend, so we may be able to develop a general strategy to attack it. "

He looks to future success with HIV as well, hoping to create a workable vaccine that is still under development. His breast cancer vaccine is already on the development track.

The other paper of note describes a new method for the synthesis of glycopeptides and glycoproteins from readily available materials, an "attractive route to homogeneous products for structural and functional studies," one of the lab's primary goals. The paper—published on line by the Journal of the American Chemical Society – describes a "sugar-assisted" ligation method of synthesis. With the assistance of a Glycopeptide sugar moiety—a specific segment of the molecule—Wong and his colleagues were able to complete the ligation of a cysteine-free glycopeptide to another peptide. Cysteine, an amino acid that is relatively scarce in proteins, is usually required for this ligation, something which restricts the number of proteins available for synthesis.

The new method, which produced the desired product rapidly and in significant quantities, offers an "attractive potential in glycopeptides and glycoprotein synthesis," the study noted, and researchers are studying various applications of sugar-assisted ligation.

A third study, published in March by the Proceedings of the National Academy of Sciences (PNAS 2006, 103, 3972-3977) focused on defining the structure of dendritic cell surface CD1 receptor proteins binding to a bacterial glycolipid to activate natural killer T (NKT) cells. Once activated by antigens on receptors that play an important role in our innate immune system, NKT cells are vital in defending against infection or cancer. These structural findings could help produce more powerful NKT cell agonists that could have significant potential in future development of vaccines or immunomodulators.

No More Awards to Win

Wong, of course, is well known as the leading pioneer in the field of carbohydrates, their synthesis, and how these complex sugars are involved in various cellular interactions. Last spring he won the 2005 American Chemical Society Award for Creative Work In Synthetic Organic Chemistry and with that, he's won basically all the awards available in the field of carbohydrates. He told the crowd gathered for the award ceremony that uncovering the functions of carbohydrates is going to be the future.

His two major accomplishments over the past year—the further development of new tools for the synthesis of glycoproteins and advances in the size and scope of glycan arrays—bring that future a bit closer.

Now, it can no longer be the awards that he's after but something a bit more intangible.

"I will stay with carbohydrates because it's getting more and more interesting," Wong says, forgetting, perhaps, that it's because of his own work that it got that way.

Send comments to: mikaono[at]scripps.edu

"My main interest is still to solve the problem of carbohydrates in biology," says Professor Chi-Huey Wong, "something that could lead to new discoveries."