A Continuous Flow-Based Approach to Automated Microbial Oligosaccharide Synthesis.

Project Summary
The need for efficient methods for the production of well-defined oligosaccharides continues to present a major
bottleneck in the field of microbial glycobiology. Although automated oligosaccharide synthesizers have been
developed, most rely on solid-phase synthesis, which can limit the chemistry and scale of synthesis available
to them. Furthermore, existing automated approaches to oligosaccharide synthesis have focused almost
exclusively on glycosylation reactions, and do not address the time-consuming and tedious process of
converting monosaccharide feedstocks into fully-substituted glycosyl donors ready for coupling. The incredible
number of building blocks required for microbial glycan synthesis also makes keeping every possible block in
stock impossible. All these issues could be addressed by the development of automated continuous flow
platforms. Continuous flow reactions can be more easily automated than multi-step batch processes and
thereby provide greater batch-to-batch reproducibility. Through proper selection of conditions it is also possible
to telescope several reactions into a single run. The objective of this proposal is to generate platform
technologies for automated continuous flow-based oligosaccharide that is capable of automating every step of
oligosaccharide synthesis, from on-demand donor/acceptor production to assembly of these larger molecules
into target structures. We will achieve this by pursuing the following Specific Aims. Specific Aim 1 will
examine the automated production of glycosylation ready monosaccharides. By telescoping multiple reactions
into a single run and designing and controlling the system with open-source MechWolf software, this approach
will allow for the construction of these important intermediates from commercial feedstock in much more rapid
timescales than is currently possible. This will include developing rapid chemo-enzymatic syntheses of
otherwise difficult to access nonulosonic (9-carbon) acid carbohydrate building blocks commonly associated
with several pathogenic microbes. In addition, the MechWolf program will provide an open-source chemical
repository for optimal conditions for the production of any protected monosaccharide to ensure batch-to-batch
reproducibility and on-demand access of these building blocks. Specific Aim 2 will extend this technology to the
automated production of oligosaccharides. The flexible and modular nature of continuous flow synthesis will
allow for the construction of glycosidic linkages that are not trivial to make on existing platforms and for which
few if any enzymes are available. As proof of principle, the system will be used to construct several capsular
polysaccharides associated with the ESKAPE pathogen Acinetobacter baumannii; however, these
technologies and concepts could be used for the construction of any oligosaccharide. Taken together, the
technologies developed through this research will lead to a rapid, robust, reproducible, and affordable method
for automated oligosaccharide production with minimal need for human optimization and intervention.

Grant Number: 5R01GM138784-04
NIH Institute/Center: NIH
Principal Investigator: Clay Bennett