Automated Glycan Synthesis

OLIGOSACCHARIDES continue to defy convenient and inexpensive production through automated synthesis. Also called glycans, these assemblies of linked sugar monomers are of immense importance biologically, but the difficulty of obtaining them in pure form and constructing them from scratch has hampered their study. Applications of oligosaccharide synthesis include the development of carbohydrate-based drugs and vaccines for a range of diseases, such as malaria and AIDS.

In Division of Carbohydrate Chemistry sessions at the American Chemical Society national meeting in Salt Lake City last month, one researcher presented recent progress in simplifying and fully automating a robotic solid-phase synthesis technique his group had developed earlier. And students from another research group discussed disease-related oligosaccharides they had constructed with a newly revealed solution-phase approach.

Peter H. Seeberger of the Max Planck Institute for Colloids & Interfaces, in Potsdam, Germany, has been creating biomedically important oligosaccharides in an automated fashion since 2000, when he first reported having modified a commercial peptide synthesizer to link monosaccharides together on polystyrene beads. In Salt Lake City he reported that his team has now fully automated a solid-phase carbohydrate synthesizer and made it more amenable for use by nonspecialists.

A key change is redesigned linker chemistry, which simplifies and speeds the synthesis and for the first time permits thioglycosides to be used in the synthesizer. Thioglycosides are sugar monomers with sulfur-linked substituents; they are widely used in carbohydrate synthesis but weren't compatible with the system's earlier linker chemistry.

The new chemistry also leaves a spacer on the end of the final product's terminal sugar instead of removing it laboriously, as was done before. This terminal group can be used to attach the oligosaccharide to carbohydrate arrays, affinity beads, fluorescent markers, and quantum dots for a range of research applications.

In the past, "we showed in principle that automated synthesis works, but we made few materials that could be used by biologists. We have overcome that hurdle now," Seeberger said.

Ancora Pharmaceuticals, a Medford, Mass.-based company Seeberger cofounded, is creating beta-test models of the automated solid-phase synthesizer for academic research groups that have requested them. Seeberger's group and researchers at Ancora have been using the synthesizer to develop carbohydrate vaccine candidates active against malaria, leishmaniasis, and bacterial infections.

MEANWHILE, students in Nicola L. Pohl's group at Iowa State University, in Ames, reported promising results with an automated carbohydrate synthesis approach in which all reactions occur in solution, instead of on solid-phase beads.

With bead-based synthesizers, excess amounts of sugar monomers have to be used to drive the solid-phase reactions to completion. Some of these building blocks are wasted, and this can be quite costly because carbohydrate monomers are expensive. On the other hand, the solid-phase particles make it easy to isolate and purify the oligosaccharide products.

Pohl's group is developing a synthesizer that carries out linkage reactions in solution and thus eliminates the need for large excesses of sugar building blocks. And it purifies synthetic intermediates and final products by using affinity chromatography to hook onto fluorous tags added to the building blocks in a first step.

Monitoring solution-phase reactions is a lot easier than monitoring solid-phase reactions, Pohl and coworkers note. Another advantage, they say, is that new synthetic methods, which are generally developed in solution, are more readily translatable for use in a solution-phase automated synthesizer.

To create their automated synthesizer, the researchers modified a Chemspeed automated-synthesis workstation. A robotics platform delivers reagents to a reactor vessel, runs a linkage reaction for a set amount of time, introduces a reaction-quenching solution, delivers deprotection reagents, concentrates the product solution, loads it onto a fluorous solid-phase extraction cartridge, washes off reagents and any unused building blocks, and elutes the linkage product. The product is then returned to a reactor vessel, and the process is repeated iteratively to add additional sugars.

At the ACS meeting, grad student Heather D. Spangler reported having carried out automated solution-phase syntheses of key oligosaccharides from bacteria associated with the skin disease cellulitis. And grad student Shu-Lun (Ben) Tang reported on the first use of automated solution-phase synthesis to make β-linked mannose oligosaccharides, which play a role in fungal infections.

Pohl and coworkers have started an automated carbohydrate synthesis company, LuCella Biosciences, in Ames. Rather than selling instruments, the firm will provide bespoke sugars for scientific research, likely by the end of this year.