The Enzyme‐Free Release of Nucleotides from Phosphoramidates Depends Strongly on the Amino Acid

Abstract

Phosphoramidates composed of an amino acid and anucleotide analogue are critical metabolites of prodrugs,such as remdesivir.Hydrolysis of the phosphoramidate liberates the nucleotide,w hichc an then be phosphorylated to become the pharmacologically active triphosphate.E nzymatic hydrolysis has been demonstrated, but as pontaneous chemical process may also occur.W em easured the rate of enzyme-free hydrolysis for 17 phosphoramidates of ribonucleotides with amino acids or related compounds at pH 7.5. Phosphoramidates of proline hydrolyzed fast, with ahalf-life time as short as 2.4 hf or Pro-AMP in ethylimidazole-containing buffer at 37 8 8C; 45-fold faster than Ala-AMP and 120-fold faster than Phe-AMP.C rystal structures of Gly-AMP,P ro-AMP, bPro-AMP and Phe-AMP bound to RNase Aa sc rystallization chaperone showed how well the carboxylate is poised to attack the phosphoramidate,h elping to explain this reactivity.O ur results are significant for the design of new antiviral prodrugs.

Determining the Diastereoselectivity of the Formation of Dipeptidonucleotides by NMR Spectroscopy

“…The aminoacidyl nucleotides of Scheme 1 were prepared from the 2‐methylimidazolide of AMP and free amino acids in aqueous solution, following a synthetic approach described recently [30] . Details of the synthetic protocol and spectroscopic data are given in the Supporting Information (SI).…”

“…The aminoacidyl nucleotides of Scheme 1 were prepared from the 2‐methylimidazolide of AMP and free amino acids in aqueous solution, following a synthetic approach described recently. [30] Details of the synthetic protocol and spectroscopic data are given in the Supporting Information (SI). The group of amino acids linked to the ribonucleotide included aliphatic, cationic and aromatic amino acids to sample a certain breadth of amino acid structure space.…”

“… Conformations of aminoacidyl nucleotides found in X‐ray crystal structures, with A) phenylalanine and B) proline as amino acid residue and AMP as nucleotide. Graphics were generated in VMD, using coordinates of PDB entries 6XHF and 6XHD [30] …”

“… [24] The chemistry producing the covalently linked ′peptido RNAs′ in the condensation mixture is compatible with all proteinogenic amino acids, [25] and also induces the formation of cofactors from precursor molecules. [22] The phosphoramidates of amino acids and nucleoside 5′‐monophosphates are called ‘aminoacidyl nucleotides’ or ‘amino acidyl nucleotides’ [30] to avoid confusing them with 2′/3′‐aminoacyl nucleotides that feature an ester link between the carboxy group of the amino acid moiety and the ribose ring of the nucleotide. Alaninyl nucleotides are long‐known intermediates of the metabolism of antiviral prodrugs, [26] and are structurally different from the alanyl products of aminoacylation.…”

Co-crystallization of an organic solid and a tetraaryladamantane at room temperature

“…There are several approaches to overcome the reluctance of some organic compounds to crystallize. Among them are the crystallization of small molecules at very low temperatures [5][6][7], and the diffusion into a crystal lattice set up by a larger compound or crystalline complex [8,9]. For some medium-size molecules, supercritical fluids have been used to obtain co-crys- tals [10].…”