Design and Synthesis of Carbohydrate-Based lin-Benzoguanines as Inhibitors of tRNA–Guanine Transglycosylase

Shigellosis is a severe inflammatory intestinal disease mainly occurring in developing countries. The urgent need for the development of innovative therapeutics has been emphasized by the World Health Organization (WHO). Structural similarity of the enzyme active sites of tRNA–guanine transglycosylase (TGT) in Shigella (S.) bacteria and Zymomonas mobilis (Z. mobilis) promotes the latter as ideal drug target for the structure-based design of potent inhibitors. This enzyme catalyzes the base exchange of G34 at the wobble position of the anticodon of tRNA in prokaryotes for preQ1 ultimately leading to queuine (Q). In contrast, TGT of humans directly catalyzes the exchange for Q, which is available by food uptake. Based on X-ray crystallography studies, the collaboration of Diederich and Klebe developed suitable ligands targeting the guanine/preQ1 binding site. They identified the lin-benzoguanine core structure and derivatization at C(2) and C(4) to enable selective examination of the ribose-33 and ribose-34 pocket. Herein, this guanine benzo-extended scaffold was used for the design and synthesis of new inhibitors investigating the molecular recognition properties of the ribose binding sites independently by means of carbohydrate substituents. First, a series of C(2) substituted carbohydrate-based inhibitors was designed resembling the natural structure of tRNA. Preparation of these non-classical bioisosteres containing an acetal/acetonide motif was successfully achieved by an optimized synthetic procedure. In this context, tert-butyloxycarbonyl (Boc) and [2-(trimethylsilyl)ethoxy]methyl (SEM) were identified as new imidazole protecting groups and the crucial guanylation step was improved by a one-pot thiourea formation, followed by an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI)-promoted intramolecular cyclization. This ligand series provides a multi-faceted structure–activity relationship (SAR), with affinities ranging from low nanomolar to micromolar. X-ray co-crystal structures showed the progressive filling of the ribose-33 binding site and contributed to the elucidation of the role of water solvation in this pocket. An energetically favorable five-water cluster seems to solvate the complex with the properly-sized ribose-33 substituents. The last chapter investigates the impact of small molecules on the protein–protein interaction (PPI) in TGT. Previous investigations observed the dislocation of the Leu68 residue to induce the displacement of the 11-loop helix motif, which is assumed to be crucial for the dimer stability. Therefore, furanoside–substituted lin-benzoguanines were designed and synthesized matching the spatial constraint and hydrophobic character of the ribose-34 pocket. Examination of the X-ray co-crystal structures as well as nanoESI-MS measurements confirmed the ligand-induced modulation of the enzyme interface. To enable future studies with carbohydrate-substituents with increased lipophilicity, an O-trifluormethoxy derivatization method was established