Gijs van der Marel and Jeroen Codée received an NWO/ECHO grant for their proposal entitled "Automated solid phase synthesis of teichoic acids".
This proposal aims at the development of automated solid phase synthesis techniques to generate a diverse library of structurally well defined teichoic acid (TA) structures. TAs are an important class of glycopolymers, abundantly present in the cell wall of Gram-positive bacteria. They form a polyanionic network surrounding the bacteria and perform a vital role in cell-wall functioning and the interaction of the bacterial cell with its environment. As such TAs also interact with our immune system. Although the immunological properties of TAs, in particular lipoteichoic acids, are widely accepted their exact role at a molecular level is not definitively established, and there is ongoing debate regarding the precise nature of the active substance in TA preparations. To increase our insight into the immunomodulatory activity of TAs, with the ultimate goal to use TA structures in future vaccine applications, structurally well-defined TA fragments are required. However, the availability of these is hampered by the structural diversity of TAs, which differ between bacterial species but also between different strains of the same bacterium. Furthermore, the isolation of sufficient amounts of well-defined TAs from natural sources is hampered by the intrinsic lability of TAs, in particular the unstable D-alanine esters in their structures. Organic synthesis has the potential to deliver well-defined, single compounds and derivatives thereof, with, for example, increased stability. In addition, through the aid of organic synthesis, chemoselective ligation handles can be incorporated, allowing the attachment to proteins or surfaces. TAs are polyanionic glycopolymers, of which the backbone is composed of repeating alditol (glycerol, ribitol, mannitol) phosphates. The constituting monomers can be (randomly) decorated with (oligo)saccharides or D-alanine esters. TAs can be subdivided into lipoteichoic acids (LTAs), noncovalently attached to the bacterial cell membrane via a glycolipid anchor and wall teichoic acids (WTAs) that are covalently linked to the peptidoglycan layer. Up to now the synthesis of teichoic acids is investigated using solution phase approaches. In particular the assembly of LTA fragments of Staphylococcus aureus and Streptococcus pneumoniae has been pursued. To tackle the large structural diversity presented by TAs, the present proposal aims at the development of automated solid phase synthesis procedures to construct a library of TAs. The presence of interresidual phosphodiester linkages in the common structure of TAs indicates that an automated synthesis can be attained by the adaptation of phosphoramidite chemistry and solid supports, as developed for DNA/RNA synthesis. The viability of this approach will be explored by the generation of a library of TA structures of the Gram-positive bacteria Enterococcus faecalis. This choice is made on the basis of a collaboration we started with the group of Huebner and ongoing collaborations with the groups of Huizinga and Ossendorp on the immunological evaluation of bacterial constituents.
An operative automated solid phase approach of TAs relies on an efficient synthesis of the phosphoramidites of otherwise suitably protected repeating units of the TAs. As can be deduced from a general structure (shown in the figure above) four types of repeating units can be discerned. Besides, three types of glycolipid anchors (not shown) are known for the LTA of Enterococcus faecalis. Further structural diversity of the projected LTAs comes from the nature and the length of the lipid tail (R2), the length of the LTA chain as well as the nature, the number (n,m,p) and positions of the repeating units.
Moreover the projected TA fragments will be provided with a suitable handle (X = amino or carbonyl function) for further funtionalization. Having the phosphoramidites in hand the solid phase synthesis will be explored with the aid of an automated DNA synthesizer, available in our laboratory. Each step of the elongation cycle comprising phosphitylation, oxidation, capping and removal of the temporary dimethoxytrityl protective group will be optimized. At the end of the automated synthesis the immobilized fragments will be (partially) deprotected, cleaved from the solid support. Different purification techniques of partially and completely deprotected TA fragments will be investigated. The immunological properties of the prepared TAs fragments will be evaluated in both innate and adaptive immunesystem settings in the laboratories of our collaborators.