In vitro runoff transcription from a double-stranded DNA template by T7 RNA polymerase is an elegant way to obtain highly pure and uniform RNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides. Here we describe the different strategies applied and optimized in our laboratory to enzymatically synthesize RNAs as necessary when working at the interface of bioinorganic chemistry, coordination chemistry, RNA biochemistry and structural biology.

A lipophilic pocket at the tRNA‐guanine transglycosylase (TGT) enzyme active site was discovered and a substantial contribution to the substrate binding free energy was observed when this pocket was filled by apolar side chains. A family of new inhibitors of TGT was developed by employing the principles of molecular recognition and structure‐based de novo design, and shown to display up to submicromolar binding affinity. Two X‐ray structures of TGT–inhibitor complexes confirmed the binding mode predicted in the design stage. An exceptional ”point mutation” effect on binding affinity was observed upon substitution of X=CH2 in 1 with O or S.

AbstracttRNA‐Guanine transglycosylase (TGT) plays a key role in the post‐transcriptional modification of tRNA. It has been linked with the pathogenicity of shigellae, the causative agents of bacillary dysentery (shigellosis). Here, we report structureactivity relationships (SARs) for a new series of 2‐aminoquinazolin‐4(3H)‐one‐based inhibitors of TGT, resulting from structure‐based design (Fig. 2). Versatile synthetic protocols allow selective functionalization of the 2‐aminoquinazolin‐4(3H)‐one core (Schemes 1–6) with H‐bond‐donor groups in position 6 (for H‐bonding to the C=O group of Leu231) and lipophilic residues in position 8 for reaching into a shallow, newly discovered lipophilic pocket lined by Val282, Val45, and Leu68. The binding mode of several of these ligands in the active site of TGT was established by crystal structure analyses (Figs. 4 and 6). A dramatic S effect was observed, with the replacement of the S‐atom in the (phenylsulfanyl)methyl residue in position 8 of inhibitor 1c (Ki=100 nM) by the O‐atom (in 1h, Ki=5.6 μM) or CH2 (in 1i, Ki=3.6 μM), resulting in a massive loss of activity (Fig. 3). Crystal structure analysis showed that the lipophilic Me group points into a highly polar region of the active site encompassed by the side chains of Asp280 and Asp102 and collides directly (d(C⋅⋅⋅O)=3.1 Å) with one of the O‐atoms of the carboxylate of Asp102. Similarly, lipophilic linkers departing from position 8 and orienting residues in the shallow hydrophobic pocket presumably encounter analogous unfavorable contacts, accounting for the modest contribution to the binding free enthalpy upon introduction of these residues. These findings provide a valuable starting point for future structure‐based lead optimization cycles leading to TGT inhibitors with increased in vitro potency.

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