Vanderbilt Institute of Chemical Biology



Discovery at the VICB






The Agony & Ecstasy of Polysaccharide Synthesis 


By: Carol A. Rouzer, VICB Communications
Published: July 31, 2019


The total synthesis of a bacterial zwitterionic capsular polysaccharide opens the door to studies of its immunological properties.


Some species of bacteria surround themselves with a capsule made predominantly of complex polysaccharides. These capsular polysaccharides (CPSs) are structurally distinct from those found in mammalian cells, so they are capable of eliciting an immune response in an infected host. In most cases, the response is triggered by binding of a CPS to multiple receptors on a B lymphocyte, cross-linking the receptors and leading to activation of the cell. These cells secrete low affinity IgM antibodies (Figure 1a). Some CPSs, however, are zwitterionic (ZPSs). These molecules can be taken up and processed by an antigen presenting cell for expression on the cell's surface in association with major histocompatability complex-II (MHC-II) proteins. This complex in turn, can interact with receptors on T helper (Th) cells, leading to their activation and cytokine secretion. The result is stimulation of cytotoxic T cells and B cells (Figure 1b). In addition, direct interaction of activated Th cells with B cells leads to conversion of the B cells to plasma cells and memory B cells. Plasma cells secrete mature, high affinity IgG antibodies, increasing the strength of the immune response, and the memory cells preserve the immune system's ability to respond to the antigen in the future (Figure 1c). Thus, ZPSs are potent activators of the immune response and have frequently been used as adjuvants that are co-administered with vaccines to increase their efficacy. This is of particular interest in the field of cancer immunotherapy, which often must depend on relatively weak tumor-associated antigens. Clearly, we would benefit from a better understanding of the structure-activity relationships of ZPSs, but this effort has been hampered by the difficulty of isolating and purifying them. Thus, Vanderbilt Institute of Chemical Biology member Steve Townsend and his graduate student Jamin Keith set out to develop a total synthesis for the repeat unit of the ZPS from Morganii morganella (MM-ZPS, Figure 2). They now report the achievement of this challenging goal [D. J. Keith and S. D. Townsend, (2019) J. Am. Chem. Soc., published online July 22,].



FIGURE 1. Immune responses to capsular polysaccharides (CPSs). (a) Most CPSs bind to the receptors of selected B cells, cross-linking the receptors and leading to production of low affinity IgM antibodies. (b) Zwitterionic polysaccharides (ZPSs) are taken up by antigen presenting cells (APCs), which process them and present them on their surface as an MHC-II complex. These complexes bind to the receptors of selected T helper (Th) cells, leading to activation and production of cytokines, which in turn activate B cells and cytotoxic lymphocytes (CTLs). (c) ZPS-activated Th cells also interact with B cells to promote their conversion to plasma cells and memory B cells. In this process, the antibodies secreted by the cells mature and switch from low affinity IgM to high affinity IgG antibodies, leading to a much more robust and long-lasting immune response. Figure reproduced with permission from D. J. Keith and S. D. Townsend, (2019) J. Am. Chem. Soc., published online July 22,



FIGURE 2. Structure of the Morganii morganella ZPS (MM-ZPS). The ZPS is a polymer of a repeating unit comprising three galactose residues. The center residue carries a phosphocholine substituent (purple). A glycerophosphate substituent (green) links the right-most residue of one repeating unit to the central residue of the next repeating unit. Note the presence of two positive and two negative charges per repeating unit.



The MM-ZPS caught the researchers' attention as a result of prior reports showing that it strongly binds to MHC-II and activates Th cells. These findings suggested that the MM-ZPS could serve as the basis for constructing better immune system boosters, but to fully exploit this possibility required the ability to manipulate, and possibly alter, the molecular structure to optimize activity. The MM-ZPS is a polymer of repeating subunits comprising three galactose residues. The central residue bears a phosphocholine substituent, and the repeating units are linked together by a glycerophosphate group. Each repeating unit bears two positive and two negative charges (Figure 2). The researchers' initial goal was to synthesize the repeating unit so that it could be used in conjunction with nanoprinting technology to create novel ZPS-based nanomaterials for assessment of biological activity.


Keith and Townsend decided to target a repeat unit in which the connecting glycerophosphate moiety was attached to the central galactose residue (Figure 3, also shown in red and purple in Figure 2). They approached the synthesis of this complex molecule by dividing the process into four major steps, in which each of the four substituents on the central galactose residue would be attached sequentially. They knew that the third step of this process (formation of a Gal-β-(1→3)-GalNAc bond) was likely to be tricky, as reactions of this nature often lead to orthoester formation. Nevertheless, they embarked on the planned synthesis. As expected, all went well until they arrived at the third step, where indeed, orthoester formation became an intractable problem. The only solution was to devise a new synthetic approach (Figure 4). In this case, they planned to execute the troublesome bond formation step first and then proceed with the other substituents. The plan was successful through step iv of the synthesis. At this point, however, they had to remove a protecting group that had been placed on the hydroxyl at carbon 2 of the central galactose residue so that they could add the phosphocholine group in the fifth step. Unfortunately, the reaction failed to remove the protecting group without damaging the molecule, so this route, also, was abandoned.




FIGURE 3. Structure of the MM-ZPS target repeat unit chosen for synthesis (left). Planned synthetic approach for the repeat unit (right). Steps are numbered in sequence from i through iv. Figure reproduced with permission from D. J. Keith and S. D. Townsend, (2019) J. Am. Chem. Soc., published online July 22,




FIGURE 4. Alternate route to the repeat unit shown in Figure 3.  This route was designed to avoid orthoester formation during establishment of the glycosidic bond in step i. Figure reproduced with permission from D. J. Keith and S. D. Townsend, (2019) J. Am. Chem. Soc., published online July 22,



At this point, the researchers hypothesized that they had, perhaps, not chosen the optimal repeat unit to make. Trying to place four large substituents on the one central galactose residue might be leading to problems with steric hindrance. So, they decided to attempt to synthesize the alternative repeat unit in which the connecting glycerophosphate group was attached to the right-most galactose in the molecule rather than the central galactose (Figure 5, also shown in blue and purple in Figure 2). Their proposed scheme for this molecule involved attaching the glycerophosphate group to the right-most galactose residue first, followed by linking together the three sugars and finally adding the phosphocholine group. This approach was highly successful until the final step, which proved tricky until they identified an appropriate phosphocholine donor. Once that obstacle was overcome, only one step remained. This was an exhaustive catalytic hydrogenation necessary to remove protecting groups and convert an azide functionality on the left-most galactose residue to an amine.





FIGURE 5. Proposed synthesis of the new target repeat unit. Figure reproduced with permission from D. J. Keith and S. D. Townsend, (2019) J. Am. Chem. Soc., published online July 22,



It is important to note that nearly every "step" in these syntheses was actually a series of steps, so this represented a daunting amount of work and is an oustanding achievement. Now, the Townsend lab has 155 mg of their repeat unit on hand, and they are wasting no time in using it to synthesize novel ZPS materials for study of their immunological properties. Stay tuned!



View JACS article: Total Synthesis of the Congested, Bis-Phosphorylated Morganella Morganii Zwitterionic Trisaccharide Repeating Unit







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