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July 8, 2002
Volume 80, Number 27
CENEAR 80 27 p. 7
ISSN 0009-2347

Combination technique facilitates structure and mechanism studies


For decades, the highly charged biological polysaccharides heparin and heparan sulfate have been used therapeutically as commercial anticoagulants. It's been difficult to study the structure, mechanism, and structure-activity relationships of these molecules because they are hard to purify. But a new analytical method should help make the job easier.

MIT researchers (from left) Beeler, Rosenberg, Lech, Kuberan, Wu, and Zhang developed new approach for analyzing large oligosaccharides.

The technique can separate heparin and heparan sulfate oligosaccharides that are three to four times larger than was previously possible. It was developed by biology professor Robert D. Rosenberg and coworkers Balagurunathan Kuberan, Miroslaw Lech, Lijuan Zhang, Zhengliang L. Wu, and David L. Beeler at Massachusetts Institute of Technology [J. Am. Chem. Soc., published online June 25, http://dx.].

The researchers use a hyphenated technique--reversed-phase capillary high-performance liquid chromatography (RP-HPLC) with microelectrospray ionization mass spectrometry (ESI-MS)--to separate large heparan sulfate precursors (heparosans).

"There's a lot of interest in understanding the structure of heparin and heparan sulfate," says University of Iowa chemistry professor Robert J. Linhardt, a specialist in polysaccharide synthesis, sequencing, and interactions. "Up to now, most people have focused on single techniques--MS, electrophoresis, or HPLC methods. This is the first real application of a hyphenated technique to do this sort of separation."

HARD TO ANALYZE Heparan sulfate polysac-charides are heterogeneous linear polymers that can contain 100 or more copies of the type of highly variable disaccharide repeating unit shown here.
Rosenberg and coworkers "have extended the size range to a pretty high level--up to 40 sugar units," Linhardt says. "That's a pretty impressive result and a massive improvement. It's about three to four times what people have been able to do before, in terms of chain length. The biggest we've done in our lab is 12. I think they have a technique that's going to be used by a lot of people."

However, there's still more work to be done, he notes. Heparosans are nonsulfated polysaccharides that are less highly charged than heparin or heparan sulfate, making them easier to analyze. "Now the approach needs to be applied to more complicated polymers," Linhardt says.

Until now, polysaccharides have been analyzed mostly by matrix-assisted laser desorption/ionization MS, which can't accept HPLC samples directly. Rosenberg and coworkers used volatile aliphatic amine ion-pairing re- agents to separate the oligosaccharides by RP-HPLC, followed by direct ESI-MS analysis.

The ion-pairing reagents neutralize the highly charged oligo-saccharides, making them sufficiently hydrophobic to be separated on an RP column. The volatile reagents evaporate harmlessly when they enter the MS chamber, making direct ESI-MS analysis of the oligo-saccharides possible as well.

A major motivation for the JACS study, Rosenberg says, was that "no one has been able to figure out the critical structural parameters responsible for the biological activity" of heparin and heparan sulfate. The new analytical approach may now make this possible. In a soon-to-be-published study, Rosenberg and coworkers will report having used isotopic labeling and their new analytical approach to identify critical groups on polysaccharides responsible for biological function and to determine their distance apart on the polymers. "That turns out to be sufficient to establish structure-function relationships," Rosenberg says.

Rosenberg's group has also developed energy minimization techniques for calculating the three-dimensional structure of polysaccharides. "You are beginning to be able to study polysaccharides in the same way as proteins and DNA," he says. "It's only taken 30 years, but the time is here."


Chemical & Engineering News
Copyright © 2002 American Chemical Society

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