Studies toward biomimetic claisen condensation using nucleic acid templates and ribozyme catalysis

Abstract

Many different experimental approaches were attempted to achieve carbon-carbon bond formation by nucleic acid template-directed reactions and ribozyme catalysis as potential lipid synthesizing machineries in the RNA world. A novel biomimetic condition for decarboxylative Claisen condensation in polyketide biosynthesis was discovered. The reaction of a malonic acid half oxyester with a Nhydroxysuccinmidyl ester forming reagent resulted in self-condensation to provide the corresponding 1,3-acetonedicarboxylic acid diester in the absence of a divalent metal chelator or a coordinating solvent. The decarboxylative Claisen condensation of malonyl adenosine using a poly-U template in solution or with immobilized poly-U was attempted. Various analytical methods demonstrated that malonyl adenosine underwent an exclusive hydrolysis reaction instead of condensation in the given conditions. Similar results were observed for the reaction of malonyl-CoA with acetyl-CoA on poly-U templates. No evidence for the decarboxylative Claisen condensation was observed by a DNA-templated system although a double helical structure of DNA duplex was proven to facilitate a bimolecular reaction by offering a favorable proximity effect. Therefore, it seems that the unsuccessful condensation resulted not from the bad template effect but from the intrinsic properties of the decarboxylative Claisen condensation reaction itself. Two tRNA molecules loaded with a malonic acid were prepared by ligation of truncated tRNAs with malonylated dinucletides. Our initial attempts to probe carbon-carbon bond formation by subjecting malonylated tRNAs to the in vitro translational machinery were not successful. Novel carbon isosteres of α-amino acids are suggested as a potential source of a more stable and reactive carbanion for future experiments. Isoprenoid conjugates of nucleoside 5??-diphosphates, which were proposed as either an initiator nucleotide or substrate molecule for in vitro selection of prenyl-transferase ribozyme were prepared by one step nucleophilic displacement reactions. A random DNA pool was constructed for selection of a ketosynthase ribozyme. A substrate bearing a biotin tag was prepared by one-step conjugation. Hig-tagged T7 RNA polymerase was expressed and purified for a large scale transcription reaction. In vitro transcription of the random DNA pool with a 5??-thiol modified GMP analogue as an initiator nucleotide produced a thiol-modified random RNA library.