Expanding the scope of phosphorylase-mediated dynamic combinatorial chemistry

Carbohydrates are some of the most important biopolymers, along with proteins and polynucleotides. In nature, glycans are used for energy storage, as structural fibers, and for molecular recognition. In the pharmaceutical industry, glycans can have important applications in drug delivery systems and wound dressing. It is very difficult to produce glycans using a traditional organic synthesis approach, due to the stereochemical challenge and the need for introducing protecting groups. Enzymes could be a potential solution to this challenge. Enzymes can be used under physiological conditions and are regio- and stereoselective, therefore, protective groups are not required.
Phosphorylase can reversibly elongate and shorten linear α-1,4-glucans using glucose-1-phosphate (Glc-1-P) as a glycosyl donor. A glucose unit is attached to the non-reducing end of the linear α-1,4-glucans, releasing phosphate. Due to the reversibility of the reaction, a dynamic mixture of α-1,4-glucans is formed, called a dynamic combinatorial library (DCL). These α-1,4-glucan DCLs are under thermodynamic control. α-1,4-Glucans show binding affinities to hydrophobic rod-like guest molecules, forming inclusion complexes, and it has been shown that the product distribution in α-1,4-glucan DCLs can be influenced by the addition of a template. Until now, only Glc-1-P has been used as a glycosyl donor in such DCLs.
This thesis is divided into two parts. In the first part (Chapters 2–4), the aim was to expand the scope of phosphorylase-mediated DCC by developing a templating approach and introducing other glycosyl donors besides Glc-1-P. Herein, the concept of tethered templates is introduced, where the α-1,4-glucans are covalently linked to the template. This allows for a stronger interaction between the template and the α-1,4-glucan due to intramolecular interactions, resulting in a narrower distribution of α-1,4-glucans and better control over the DCL compared to a ‘traditional’ non-tethered template.
The introduction of phosphorylase from Aquifex aeolicus, which has a broader substrate scope than the previously used phosphorylase from rabbit muscle, allowed the inclusion of glucuronic acid-1-phosphate as a glycosyl donor in DCLs. In addition, it was shown that glucuronic acid can now be incorporated into cyclodextrins (CDs) using a double enzymatic synthesis with phosphorylase and cyclodextrin glucanotransferase. This methodology provides access to a new synthetic route toward the production of modified CDs.
In the second part of this thesis, the focus was shifted to water-soluble hydrazones and their interactions with cyclodextrins. This work was carried out in collaboration with Prof. Ivan Aprahamian at Dartmouth College, New Hampshire, during the external stay of the author. Two new water-soluble hydrazones were synthesized and their photophysical properties and their binding interaction with δ-CD were investigated. This work provides important new insight into the supramolecular chemistry of this unusual large-ring cyclodextrin.