Optimised Synthesis of the Bacterial Magic Spot (p)ppGpp Chemosensor PyDPA

Abstract

Guanosine penta- or tetraphosphate (pppGpp or ppGpp, respectively) is a nucleotide signalling molecule with a marked effect on bacterial physiology during stress. Its accumulation slows down cell metabolism and replication, supposedly leading to the formation of the antibiotic-tolerant persister phenotype. A specifically tailored fluorescent chemosensor, PyDPA, allows the detection of (p)ppGpp in solution with high selectivity, relative to that of other nucleotides. Herein, an optimised synthetic approach is presented that improves the overall yield from 9 to 67 % over 7 steps. The simplicity and robustness of this approach will allow groups investigating the many facets of (p)ppGpp easy access to this probe.

Keywords: fluorescence; guanosine phosphates; nucleotides; sensors; synthesis design.

Figure 1

Left) Structure of the nucleotide signalling molecule (p)ppGpp and of its specific chemosensor PyDPA. Right) Structure of the 2:1 PyDPA/ppGpp complex.

Scheme 1

Reported synthesis of PyDPA, according to ref. 11.

Scheme 2

Palladium‐mediated coupling of 1‐iodopyrene (6) with isophthalate 5. TEA: triethylamine.

Scheme 3

Optimised approach for the preparation of 1‐(3‐bromopropyl)pyrene (3). MW: microwave.

Scheme 4

Preparation of compound 13 through the alkylation of phenol 11 with compound 3 and the final steps of the synthesis of PyDPA.

Figure 2

Fluorescence emission spectra of PyDPA (20 μm in 1 mm HEPES pH 7.5) alone and upon the addition of each nucleotide and ppGpp (7 μm). λ _ex=344 nm.