Key result
Phosphoramidates of proline hydrolyzed rapidly, with a half-life as short as 2.4 hours for Pro-AMP at 37 °C, 45-fold faster than Ala-AMP and 120-fold faster than Phe-AMP.
Why the study?
Enzymatic hydrolysis of prodrug phosphoramidate metabolites into nucleotides has been shown, but whether a spontaneous chemical process may also occur was unclear.
The rate of spontaneous chemical hydrolysis of phosphoramidate prodrugs depends strongly on the amino acid, with proline derivatives hydrolyzing exceptionally fast, which is significant for antiviral prodrug design.
Proline phosphoramidates hydrolyze rapidly without enzymes; leaves open whether amino acid choice improves prodrug delivery or cardiac safety profiles.
Phosphoramidates composed of an amino acid and a nucleotide analogue are critical metabolites of prodrugs, such as remdesivir. Hydrolysis of the phosphoramidate liberates the nucleotide, which can then be phosphorylated to become the pharmacologically active triphosphate. Enzymatic hydrolysis has been demonstrated, but a spontaneous chemical process may also occur. We measured 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 a half‐life time as short as 2.4 h for Pro‐AMP in ethylimidazole‐containing buffer at 37 °C; 45‐fold faster than Ala‐AMP and 120‐fold faster than Phe‐AMP. Crystal structures of Gly‐AMP, Pro‐AMP, βPro‐AMP and Phe‐AMP bound to RNase A as crystallization chaperone showed how well the carboxylate is poised to attack the phosphoramidate, helping to explain this reactivity. Our results are significant for the design of new antiviral prodrugs.
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Jovanovic et al. (2020) studied this question. Phosphoramidates of ribonucleotides with amino acids was evaluated on Rate of enzyme-free hydrolysis at pH 7.5. Phosphoramidates of proline hydrolyzed rapidly, with a half-life as short as 2.4 hours for Pro-AMP at 37 °C, 45-fold faster than Ala-AMP and 120-fold faster than Phe-AMP.
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