Entrega Controlada y Liberación Inducida por Luz de Nucleótidos de Punto Mágico en Escherichia coli

The "magic spot" nucleotides (MSNs) ppGpp and pppGpp constitute bacterial alarmones that orchestrate the conserved stringent response, a global regulatory mechanism enabling bacteria to adapt to nutrient deprivation and other environmental stresses. Current strategies to manipulate MSN levels rely mainly on genetic or environmental approaches, which are slow and lack temporal control. Chemical tools such as photocaged MSN analogues could provide such temporal control over MSN levels. However, the high negative charge of MSNs prevents spontaneous passage through the complex bacterial cell envelope. Here, we report the synthesis of photocaged, clickable, and isotope-labeled MSN analogues and their delivery into Escherichia coli comparing different approaches. A cyclodextrin-based synthetic nucleotide transporter facilitated uptake. Upon 400 nm irradiation, these probes were photo-released inside living cells, where we tracked their conversion from pppGpp to ppGpp by capillary electrophoresis mass spectrometry and demonstrated their ability to alter growth in a (p)ppGpp⁰ mutant. These probes lay the foundation for spatially and temporally controlled studies of MSN function and of other highly negatively charged metabolites in vivo.