Synthesis of a Phosphonate‐Linked Aminoglycoside–Coenzyme A Bisubstrate and Use in Mechanistic Studies of an Enzyme Involved in Aminoglycoside Resistance

Abstract

Aminoglycoside N-6′-acetyltransferases (AAC(6′)s) are important determinants of antibiotic resistance. A good mechanistic understanding of these enzymes is essential to overcome aminoglycoside resistance. We have previously reported the synthesis of amide-linked and sulfonamide-linked aminoglycoside-coenzyme A conjugates which were useful mechanistic and structural probes of AAC(6′)s. We report here the synthesis of a phosphonate-linked aminoglycoside-coenzyme A variant, which is expected to be a superior mimic of the tetrahedral intermediate proposed for catalysis by AAC(6′)s. This synthetic target is especially challenging for a number of reasons including the presence of multiple functional groups, the water solubility of both starting materials, and incompatibility of P(III) chemistry with water. We have overcome these challenges by adding the expensive coenzyme A in the last step via an elegant Michael-type addition onto a vinylphosphonate in water. Overall, a single protection step was needed. The decreased inhibitory potency of this bisubstrate compared to that of the amide-linked analog suggests that Enterococcus faecium AAC(6′)-Ii may not stabilize the proposed tetrahedral intermediate, and may act mainly via proximity catalysis.

A review of patents (2011–2015) towards combating resistance to and toxicity of aminoglycosides

“…115 A similar trend was observed for phosphonate-linked AGs and the decreased inhibitory potency was explained by proximity catalysis by enzymes. 116 AG-CoA bisubstrates are highly potent AAC(6′) inhibitors, however their inability to penetrate cells preclude in vivo studies.…”

Cysteine‐Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations

“…[22] Since we envisioned ethynylphosphonamidates to be more reactive,wecarried out model reactions with the N-phenyl derivative 1 and glutathione under varying pH conditions and monitored the progress by UPLC-UV.I tw as observed that the conversion rate increases from pH 7.4 to 9.0 with only aslight increase from pH 8.5 to 9.0 ( Figure S2 in the Supporting Information). [22] Since we envisioned ethynylphosphonamidates to be more reactive,wecarried out model reactions with the N-phenyl derivative 1 and glutathione under varying pH conditions and monitored the progress by UPLC-UV.I tw as observed that the conversion rate increases from pH 7.4 to 9.0 with only aslight increase from pH 8.5 to 9.0 ( Figure S2 in the Supporting Information).…”

Cysteinselektive phosphonamidatbasierte Elektrophile für modulare Biokonjugationen

“…[22] Basierend auf der Überlegung,d ass Ethinylphosphonamidate reaktiver sein kçnnten, haben wir Modelreaktionen zwischen dem N-Phenyl-Derivat 1 und Glutathion bei verschiedenen pH-Werten durchgeführt und den Verlauf der Reaktion mithilfe von UPLC-UV verfolgt. [22] Basierend auf der Überlegung,d ass Ethinylphosphonamidate reaktiver sein kçnnten, haben wir Modelreaktionen zwischen dem N-Phenyl-Derivat 1 und Glutathion bei verschiedenen pH-Werten durchgeführt und den Verlauf der Reaktion mithilfe von UPLC-UV verfolgt.…”