Biomimetic Aminoacylation of Ribonucleotides and RNA with Aminoacyl Phosphate Esters and Lanthanum Salts

Abstract

Aminoacylation of tRNA in cells involves activation of the amino acid as an aminoacyl adenylate, a mixed anhydride with AMP, which reacts with tRNA. We have now established that aminoacyl phosphate esters in the presence of lanthanide ions in water will acylate hydroxyls at the 3'-terminus of RNA or a simple nucleotide. By extension, this will permit synthetically aminoacylated tRNA to be produced in a single-step biomimetic process. The reactions of Boc-4-fluorophenylalanyl ethyl phosphate were followed by HPLC separation, MS, and 19F NMR analysis. In stoichiometric combination with lanthanum salts in aqueous buffer, Boc-4-fluorophenylalanyl ethyl phosphate rapidly produces 2'- and 3'-monoesters of cytidine and cytidine monophosphate. Reaction of the reagent with RNA in the presence of lanthanum and magnesium salts introduces a specifically detectable signal into the RNA, which is evidence of formation of the aminoacyl ester. When the same RNA is initially oxidized with periodate to convert the 3'-terminal vicinal diol to the cleaved dialdehyde, reaction with the aminoacyl phosphate no longer occurs as evidenced by the lack of a signal in the 19F NMR spectrum. The results are consistent with a requisite chelation mechanism in which lanthanum serves as a template for both the aminoacyl phosphate and the 3'-terminal diol of RNA and nucleotides. The coordinated diol will then react through specific base-catalyzed intramolecular addition of the alkoxide nucleophile to the acyl group of the aminoacyl phosphate. Assessment of the method with a single tRNA was also achieved using the fluorescent reagent N-dansyl-glycyl ethyl phosphate. Lanthanide-promoted aminoacylation at the 3'-terminus of tRNAPhe is detected by the introduction of fluorescence (detected directly and by antibody-enhanced emission). This does not occur if the 3'-terminus is converted to the dialdehyde by reaction with periodate.