Pentaphosphorylated Magic Spot Nucleotides: Chemoenzymatic Synthesis and Disassembly-Based Sensing

Abstract

The magic spot nucleotides (MSNs), (p)ppGpp and (p)ppApp, play central roles in bacterial stress signaling, yet their selective detection and chemical accessibility remain limited. This work presents a scalable chemo-enzymatic synthesis of natural and functionalized pentaphosphorylated MSNs based on a cyclic pyrophosphoryl phosphoramidite (cPyPA) mediated phosphorylation and RNase T2-catalyzed hydrolysis. This approach enables preparative access to defined 3′monophosphates (ppAp, ppGp) and the pentaphosphorylated products pppApp and pppGpp. In parallel, a metal-ligand disassembly-based fluorescence probe that operates in water was developed for the selective detection of MSNs. Coordination of the alarmone to an Fe(III)-salen complex induces its demetallation and fluorescence activation through salicylaldehyde release, supported by theoretical and spectroscopic studies. The probe displays high selectivity for (p)ppGpp and (p)ppApp over other nucleotides and responds most strongly to MSNs bearing 3′-and 5′-pyrophosphate groups. The probe also detects enzymatically generated ppGpp from Staphylococcus aureus RelP reactions in vitro. This work combines a robust synthesis route for pentaphosphorylated MSNs with a readily accessible fluorescence sensor, thereby laying the foundation for future investigations into bacterial stress signaling.