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.

Derivatives of 3′‐Azidothymidine with 6‐Cyanopyridone as Base or as Phosphoramidate Ester and their Antiretroviral Activity

“…Besides known alaninyl methyl ester 23 , [24,25] which was prepared to test the synthetic method and to serve as positive control, prolinyl phosphoramidate 26 was selected as target. The latter represents a design that should also allow for non‐enzymatic release of the free monophosphate intracellularly [29] …”

“…They feature a phosphate group masked by an O ‐aryl group and an N ‐linked amino acidyl ester. The free nucleotide is released intracellularly in a series of steps, starting with the enzymatic hydrolysis of the amino acidyl ester and subsequent hydrolysis of the phosphoramidate [28,29] …”

“…Here, we report the synthesis of a more polar derivative of AZW, containing a cyano rather than an ethynyl group, together with exploratory studies on two ProTide constructs of AZT with a less lipophilic amino acid ester moiety. One of these is a new construct with a prolinyl methyl ester as amino acid component, motivated by our observation of facilitated release for such species [29] …”

“…The free nucleotide is released intracellularly in a series of steps, starting with the enzymatic hydrolysis of the amino acidyl ester and subsequent hydrolysis of the phosphoramidate. [28,29] In our previous work, we employed the alaninyl ester known from sofosbuvir [30] and remdesivir, [31] and did not test less lipophilic alternatives. Here, we report the synthesis of a more polar derivative of AZW, containing a cyano rather than an ethynyl group, together with exploratory studies on two ProTide constructs of AZT with a less lipophilic amino acid ester moiety.…”

“…One of these is a new construct with a prolinyl methyl ester as amino acid component, motivated by our observation of facilitated release for such species. [29]…”