I SCIENCE/TECHNOLOGYI
Chemical-Enzymatic Technique Used To Make Carbohydrates, Glycopeptides • Its creators believe the process could potentially provide the basis for an automated synthesizersome observers disagree Stu Borman, C&EN Washington esearchers at Scripps Research Institute in La Jolla, Calif., and Cytel Corp. in San Diego have developed 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.
R
Wong: can make larger glycopeptides
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 deprotection steps, making automation difficult. Glycopeptide synthesis is even more complicated. Efforts to overcome such problems go back to work in the early 1970s by Paulson: technique has not been optimizedchemistry professors Jean M. J. Frechet (now at Cornell University) and Conrad Automated peptide and oligonucle- Schuerch (now retired) at the State Uniotide synthesizers have been widely versity of New York College of Environavailable for years, but automated oligo- mental Science & Forestry, Syracuse. saccharide and glycopeptide synthesis Frechet and Schuerch were able to make has been elusive, largely owing to the trisaccharides by solid-phase synthesis, extraordinary complexity of carbohy- but the technique was laborious. This field has languished for many drate chemistry. An automated oligosaccharide sequencer was recently intro- years because of the seemingly overduced commercially (C&EN, Oct. 18, whelming difficulties. But the pace has 1993, page 30), but an automated synthe- picked up. sizer has yet to appear. Jiri J. Krepinsky of the department of Now, a solid-phase technique for molecular and medical genetics at the synthesis of these molecules has been University of Toronto, and coworkers, developed by postdoctoral fellows recently developed a polymer-supportMatthias Schuster and Peng Wang and ed solution technique for rapid synthechemistry professor Chi-Huey Wong at sis of reasonably pure oligosaccharides Scripps Research Institute and bio- (C&EN, July 8, 1991, page 5). Chemischemist James C. Paulson at Cytel [/. try professor Jacques H. van Boom and Am. Chan. Soc, 116,1135 (1994)]. The re- coworkers at the University of Leiden, searchers say the technique has poten- the Netherlands, have advanced the tial to be the foundation for automated use of solid-phase techniques for synsynthesis of laboratory-scale quantities thesizing oligosaccharide-based vacof carbohydrates and glycopeptides— cines. And researchers at Neose Pharwhich could be screened for biological maceuticals, Horsham, Pa., have develactivity or used in assays and affinity oped an enzyme-based carbohydrate synthesis based on the company's patchromatography. A major difficulty in carbohydrate ented technology for isolating glycosylFEBRUARY 28,1994 C&EN
37
SCIENCE/TECHNOLOGY
Glycopeptide synthesis combines chemical and enzymatic steps HoN Hexaglycine ~ — - — I addition
Chemical addition of phenylalanine
Boc-(Gly) 6 - N
with ester linkage
H?N
H?N
H
?\ H H I? H H ^ \ Boc-N^jJ^N^0^N-(Gly)6-N^^J Galactose, galactosyltransferase
95% yield)
Boc = ferf-butyloxycarbonyl protecting group Gly = glycine
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). 38
FEBRUARY 28, 1994 C&EN
The rest of the peptide and a monosaccharide residue are constructed by solid-phase chemical synthesis. Then the oligosaccharide is built up enzymatically (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 tetrasaccharide 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 deprotection in the oligosaccharide synthesis steps. Glycosyltransferases catalyze the connection of sugars in a regioselective,
substrate-selective, and stereoselective manner. However, the yields of glycosyltransferase reactions can vary widely. In the ]ACS paper, one of the glycosyltransferase 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 labscale 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-
Ester linkage H ° / H Boc-N^Q/v.N-(Gly) 6 -
Chemical addition of glycine and A/-acetylglucosamine
X) H
?\ H
H
?\
H
H
Boc-N^fi^N^0^N-(Gly)6- N ™. \ / 0 o V ^ i o
OH
Y
Sialic acid, sialyltransferase
NH
(65% yield)
NHCOCH3
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 nonnatural 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 WongPaulson 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 glycosylation steps, which means that you'll have separation problems once you're finished. The whole reason that solidphase 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
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