I SCIENCE/TECHNOLOGYI

Chemical-Enzymatic Technique Used To Make Carbohydrates, Glycopeptides

• Its creators believe the process could potentially provide the basis for an automated synthesizer-some observers disagree

Stu Borman, C&EN Washington

R esearchers at Scripps Research In­stitute in La Jolla, Calif., and Cytel Corp. in San Diego have devel­

oped a technique for solid-phase synthesis of oligosaccharides and glycopeptides—a combined chemical-enzymatic procedure that could potentially provide the basis for an automated oligosaccharide and glyco­peptide synthesizer. However, some car­bohydrate researchers express doubts about near-term prospects for automating the technique.

Carbohydrates and glycopeptides are receiving increased attention today, both as subjects of basic research and as po­tential drugs, because of the key roles they play in cell signaling, molecular recognition, and many other biological processes.

Wong: can make larger glycopeptides

Paulson: technique has not been optimized

Automated peptide and oligonucle­otide synthesizers have been widely available for years, but automated oligo­saccharide and glycopeptide synthesis has been elusive, largely owing to the extraordinary complexity of carbohy­drate chemistry. An automated oligosac­charide sequencer was recently intro­duced commercially (C&EN, Oct. 18, 1993, page 30), but an automated synthe­sizer has yet to appear.

Now, a solid-phase technique for synthesis of these molecules has been developed by postdoctoral fellows Matthias Schuster and Peng Wang and chemistry professor Chi-Huey Wong at Scripps Research Institute and bio­chemist James C. Paulson at Cytel [/. Am. Chan. Soc, 116,1135 (1994)]. The re­searchers say the technique has poten­tial to be the foundation for automated synthesis of laboratory-scale quantities of carbohydrates and glycopeptides— which could be screened for biological activity or used in assays and affinity chromatography.

A major difficulty in carbohydrate

synthesis is that each sugar residue contains several hydroxyl groups that are nearly equivalent chemically. To prevent them from reacting at random when linking the sugar to an oligosac­charide chain, they must be protected chemically, except for the one unique hydroxyl that will react to form the de­sired glycosidic bond.

Hydroxyls on the growing chain must be similarly protected, again ex­cept for one hydroxyl at the desired at­tachment point. After the addition is made, a single hydroxyl group must be selectively deprotected for the next step, and so on. The synthesis thus involves a series of tedious protection and depro-tection steps, making automation diffi­cult. Glycopeptide synthesis is even more complicated.

Efforts to overcome such problems go back to work in the early 1970s by chemistry professors Jean M. J. Frechet (now at Cornell University) and Conrad Schuerch (now retired) at the State Uni­versity of New York College of Environ­mental Science & Forestry, Syracuse. Frechet and Schuerch were able to make trisaccharides by solid-phase synthesis, but the technique was laborious.

This field has languished for many years because of the seemingly over­whelming difficulties. But the pace has picked up.

Jiri J. Krepinsky of the department of molecular and medical genetics at the University of Toronto, and coworkers, recently developed a polymer-support­ed solution technique for rapid synthe­sis of reasonably pure oligosaccharides (C&EN, July 8, 1991, page 5). Chemis­try professor Jacques H. van Boom and coworkers at the University of Leiden, the Netherlands, have advanced the use of solid-phase techniques for syn­thesizing oligosaccharide-based vac­cines. And researchers at Neose Phar­maceuticals, Horsham, Pa., have devel­oped an enzyme-based carbohydrate synthesis based on the company's pat­ented technology for isolating glycosyl-

FEBRUARY 28,1994 C&EN 37

SCIENCE/TECHNOLOGY

Glycopeptide synthesis combines chemical and enzymatic steps

HoN

H?N

Hexaglycine ~ — - — I

addition Boc-(Gly)6- N Chemical addition of phenylalanine

with ester linkage

H?N

H ?\ H H I? H H ^ \ Boc-N^jJ^N^0^N-(Gly)6-N^^J

<OHo ^ ^J \ ^ u \ NH

Galactose, galactosyltransferase

(55% yield)

H 0 NHCOCHo

H Boc-N

O H 0

HO OH

u n n M H

K-yNJV^N-(GI»)6-N

«„ f ° X)° HO

H3COCHN

Boc = ferf-butyloxycarbonyl protecting group Gly = glycine

NHCOCH. Chymotrypsin

Fucose, fucosyltransferase (>95% yield)

transferases (C&EN, March 29, 1993, page 24). Glycosyltransferases catalyze the addition of specific sugars to oli­gosaccharides.

Also last year, organic chemistry pro­fessor Samuel J. Danishefsky (now at Memorial Sloan-Kettering Cancer Center and Columbia University, New York City) and coworkers at Yale University developed a novel solid-phase synthesis of oligosaccharides based on glycal chemistry (C&EN, June 7,1993, page 30). Danishefsky has since used the tech­nique to synthesize glycopeptides as well as oligosaccharides, but this work has not yet been published.

In the alternative solid-phase tech­nique for synthesizing both oligosac­charides and glycopeptides devised by Wong, Paulson, and coworkers, silica support particles are first derivatized with a hexaglycine spacer group. An amino acid (the C-terminal residue of the peptide) is attached to the spacer by a cleavable ester linkage (or another cleavable bond).

The rest of the peptide and a mono­saccharide residue are constructed by solid-phase chemical synthesis. Then the oligosaccharide is built up enzy-matically (using glycosyltransferases). The finished glycopeptide is released from the solid support by enzymatic cleavage of the ester or other cleavable linkage.

Wong, Paulson, and coworkers dem­onstrate the technique by making a glycopeptide in which the tetrasaccha-ride sialyl Lewisx is connected to a dipeptide of phenylalanine and gly­cine. Wong says the method can also be used to make larger glycopeptides. Scripps Research Institute has filed a patent application on the technology, which has been licensed to Cytel.

A major advantage of this technique over previous solid-phase methods is that use of glycosyltransferases elimi­nates any need for protection and depro-tection in the oligosaccharide synthesis steps. Glycosyltransferases catalyze the connection of sugars in a regioselective,

substrate-selective, and stereoselective manner.

However, the yields of glycosyltrans-ferase reactions can vary widely. In the ]ACS paper, one of the glycosyltrans-ferase reactions had a yield of more than 95%, but two others had yields of only 55% and 65%.

Wong believes the technique could be adapted for automated synthesis of lab-scale quantities of oligosaccharides and glycopeptides. In fact, the glycopeptide described in the paper was made using a semiautomated solid-phase peptide syn­thesizer compatible with oligosaccharide reagents.

Would it be difficult to adapt the tech­nique for automated synthesis? "I don't think so," says Wong. "It's already worked out. The problem will be the source of the glycosyltransferase en­zymes," which are very expensive and hard to obtain. "But with recombinant DNA technology," he notes, "they're becoming more and more available." Wong concedes that the technique gen-

38 FEBRUARY 28, 1994 C&EN

Ester linkage H ° / H

Boc-N^Q/v.N-(Gly)6-

X) Chemical addition of glycine and

A/-acetylglucosamine

H ?\ H H ?\ H H B o c - N ^ f i ^ N ^ 0 ^ N - ( G l y ) 6 - N ™. \ / 0 o V ^ i o OH Y

NH

NHCOCH3

Sialic acid, sialyltransferase

(65% yield)

HO OH 0 Y° ° X)

HO H3COCHN

Glycopeptide product

erally cannot be used to make oligosac­charides containing modified sugars be­cause glycosyltransferases are specific for natural sugars—although some non-natural sugars do act as weak substrates of the enzymes.

But some researchers asked to com­ment on the technology are less enthu­siastic than Wong about its prospects. Mostly, their concerns focus on the technique's relatively low yields.

Krepinsky says the key contribution of the paper is the use of a cleavable bond and an enzyme to release the gly­copeptide from the solid support—a step that has represented a long-stand­ing problem in previous work. And he says the technique could be useful for synthesis of relatively small glycopep­tides, such as the structure reported in the paper.

But he doesn't believe the technique will provide the basis for a commercial, automated synthesizer—in part because its low yields could lead to complex product mixtures that would become in­

creasingly intractable in syntheses of larger glycopeptides. Wong replies that the oligosaccharide yields are ^satisfac­tory/' that "most bioactive oligosaccha­rides contain only four to six sugar units," and that "the only by-products are truncated sequences, which can be easily separated."

Krepinsky agrees this is true for oli­gosaccharide synthesis. However, for the synthesis of large glycopeptides, he says, impurities generated in the pep­tide and oligosaccharide parts of the synthesis would combine to yield com­plex glycopeptide mixtures that would be impossible to purify.

In addition, Krepinsky says the time saved by the elimination of protection and deprotection steps is offset by the inconvenience of glycosyltransferase reactions, which can take 10 hours to go to completion. "The work shows possibilities," says Krepinsky, "but I think practical use of this would be rather limited."

Another carbohydrate researcher,

chemistry professor Horst Kunz of Jo­hannes Gutenberg University, Mainz, Germany, believes the combined chem­ical-enzymatic approach is the most promising strategy for constructing carbohydrates and glycopeptides. But Kunz says the idea that the Wong-Paulson technique "is the key to a real­ly automated synthesis may be a little bit overstressed because the yields you can reach using glycosyltransferase re­actions do not fulfill these require­ments. You will end up in every case with a mixture."

Chemistry professor Eric J. Toone of Duke University, who specializes in enzyme-based carbohydrate synthesis, agrees with Kunz, saying: "The obvi­ous drawback is that there are very modest yields in some of the glycosyla-tion steps, which means that you'll have separation problems once you're finished. The whole reason that solid-phase peptide synthesis works is that you can get 99.9% yields in each cou­pling step."

Paulson readily concedes that "it re­mains to be demonstrated if the reac­tions can be optimized to the point where development of a useful auto­mated machine could in fact be real­ized." However, he says, "This is the first demonstration of the technique. It hasn't been optimized yet. My own ex­perience with enzymatic reactions is that an initial 50% yield is not a limita­tion. In other applications, we've taken Chi-Huey's 50% yields and moved them to 100%."

Chemistry professor Ole Hindsgaul with the University of Alberta, who works at the interface between carbo­hydrate chemistry and cell biology, says: "Even as it stands now, with rela­tively poor yields of the enzymatic re­actions, this represents a quantum leap in our ability to rapidly—in a few days instead of a few months—prepare a se­ries of related glycopeptides differing in amino acid sequence or sugar se­quence. These could then be screened for protein binding, either before or af­ter cleavage from the resin and chro­matographic purification."

Toone also believes that despite the yield problem, the work by Wong, Paul­son, and coworkers represents "an im­portant advance. It doesn't get you there yet, but it's a large step in the right di­rection. They've eliminated some fun­damental concerns that existed before they did this work." •

FEBRUARY 28, 1994 C&EN 39