Hyper-Responsive Chemiluminescent Probe Reveals Distinct PYRase Activity in Pseudomonas aeruginosa

Pyrrolidone carboxyl peptidase, commonly known as PYRase, is an exopeptidase that catalytically cleaves an N-terminal pyroglutamic acid from peptides or proteins. The diverse functions of PYRases in bacterial enzymology have prompted the development of various bacterial diagnostic techniques. However, the specific physiological role and activity of this enzyme across the bacterial kingdom remain unclear. Here, we present a functional phenoxy-1,2-dioxetane chemiluminescent probe (PyrCL) that can selectively detect PYRase activity in both Gram-positive and Gram-negative bacteria. The probe activation mechanism is based on the cleavage of a pyroglutamyl substrate, followed by a release of the phenoxy-dioxetane luminophore, which then undergoes efficient chemiexcitation to emit a green photon. Probe PyrCL exhibits an effective turn-on response with superior detection capability in terms of response time and sensitivity compared to existing fluorescence probes. The superior detection sensitivity of the chemiluminescent probe enables us to reveal previously undetected PYRase activity in Streptococcus mutans. Furthermore, it enables the discrimination of Pseudomonas aeruginosa from other Gram-negative bacteria in the tested panel, based on their distinct PYRase activity. We expect that probe PyrCL will have great value for PYRase-based bacteria diagnosis with use in basic research and clinical applications.


General methods
All reactions requiring anhydrous conditions were performed under an Argon atmosphere.All reactions were carried out at room temperature unless stated otherwise.Chemicals and solvents were either A.R. grade or purified by standard techniques.Thin-layer chromatography (TLC): silica gel plates Merck 60 F254: compounds were visualized by irradiation with UV light.Column chromatography (FC): silica gel Merck 60 (particle size 0.040-0.063mm), eluent given in parentheses.Reverse-phase high-pressure liquid chromatography (RP-HPLC): C18 5u, 250x4.6mm,eluent given in parentheses.Preparative RP-HPLC: C18 5u, 250x21mm, eluent given in parentheses. 1 H-NMR spectra were measured using Bruker Avance operated at 400MHz. 13C-NMR spectra were measured using Bruker Avance operated at 100 MHz.Chemical shifts were reported in ppm on the δ scale relative to a residual solvent (CDCl3: δ = 7.26 for 1 H-NMR and 77.16 for 13 C-NMR, DMSO-d6: δ = 2.50 for 1 H-NMR and 39.52 for 13 C-NMR).Multiplicities are reported with the following abbreviations: br, broad; s, singlet; d, doublet; t, triplet; dt, doublet of triplets; dd, doublet of doublets; ddd, doublet of doublet of doublets; qd, quartet of doublets; m, multiplet; eq, equatorial; ax, axial.Coupling constants (J) are given in Hertz.Mass spectra were measured on Waters Xevo TQD.Chemiluminescence was recorded on Molecular Devices Spectramax iD3.Fluorescence was recorded on Tecan infinite 200 Pro.All chemicals, unless otherwise stated, were obtained from commercial sources.Light irradiation for photochemical reactions: LED PAR38 lamp (19W, 3000K).

Chemiluminescent PYRase probe-PyrCL
PyrCL was synthesized according to our recently reported procedure. 1

Compound 2
Compound 1 (400 mg, 1.71 mmol, 1 eq.) was dissolved in 4 mL of ACN and cooled to 0 ℃.Sodium Iodide (764 mg, 5.13 mmol, 3 eq.) was added followed by the rapid addition of TMS-Cl (647 μl, 5.13 mmol, 3 eq.).The reaction was allowed to warm up to room temperature and monitored by TLC (EtOAc: Hex mixture).Upon completion, the reaction mixture was diluted with EtOAc, and washed with saturated Na2S2O3 followed by brine.The organic layer

Compound 3
Phenol enol ether b 2 (796 mg, 2.05 mmol, 1.2 eq.) and K2CO3 (354 mg, 2.56 mmol, 1.5 eq.) were dissolved in DMF (5 mL).The solution was stirred for 5 minutes, then compound 2 was added.The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc:Hex mixture).Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 1M HCl (50 mL) and brine (50 mL).The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure.The crude product was purified by column chromatography on silica gel (EtOAc: Hex mixture) to afford compound 3 (827 mg, 80% yield) as a white solid.

Probe PyrCL
Compound 4 (10 mg, 0.02 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM.Oxygen was bubbled through the solution while irradiating with yellow light.The reaction was monitored by RP-HPLC.Upon completion, the solvent was concentrated under reduced pressure, and the product was purified by preparative RP-HPLC (mobile phase: acetonitrile in H2O containing 0.1% TFA; gradient from 70 to 100%; flow rate: 20 mL/min).Probe PyrCL was obtained as a white solid (8.8 mg, 84% yield).

List of bacterial strains and protocols for measurements of PYRase activity in bacteria
Table S1.Bacteria strains.

Protocol for limit-of-detection measurements
Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 47085 were cultured in Lysogeny broth at 37°C for 18 hours under aerobic conditions.The initial culture was rinsed with PBS (centrifuged at 5000 rpm, 10 minutes), and the resulting bacterial pellet was resuspended in 4 mL of PBS to facilitate a 1:4 dilution experiment.
For the subsequent procedure, a 96-well plate was utilized, with each well initially loaded with 50µL of the pyro- Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 47085 OD600 values were converted to CFU/mL according to reported ratios. 3,4 tocol for substrate-specificity evaluation The specificity of the pyro-glutamyl aminopeptidase chemiluminescent probe (PyrCL) was assessed in the presence of ten commercially available recombinant enzymes.

Protocol for chemiluminescent measurements of PYRase activity in bacteria
All bacterial strains were cultured in LB/BHI at either 37°C or 30°C for 18 hours under aerobic conditions.
Subsequently, the initial culture was subjected to a PBS wash (centrifuged at 5000 rpm, 10 minutes), and the bacterial pellet obtained was reconstituted in 4 mL of PBS, aiming for an OD600 of 0.8.Following this, a 96-well plate was utilized, and each well was pre-loaded with 50 µL of the chemiluminescent probe (PyrCL) [20 µM, 0.2% DMSO].
Next, 50 µL of bacterial aliquot was introduced into each well, bringing the final OD600 to 0.4.The resultant chemiluminescence signal was monitored using a Molecular Devices Spectramax iD3 over 1 hour of incubation at 37°C.B.

Figure S16 .
Figure S16.Total light emitted (A.) and linear calibration curve (B.) after 90 min of PyrCL probe [10 µM] with various bacterial optical densities of P. aeruginosa ATCC 47085 [OD600 0.4 -1.52*10 -6] in PBS 7.4, 0.1% DMSO, 37°C.The limit of detection (L.O.D) was determined using two methods: the blank + 3SD (standard deviation) method (left), and secondly, by a linear calibration curve.For the latter, the limit of detection is defined as 3 times the standard deviation of the blank divided by the slope of the linear calibration curve (L.O.D = 3σ/k) (right).
84% S was separated, dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure.The crude product was further purified by column chromatography (EtOAc: Hex mixture) to afford compound 2 in the form of a yellow solid (476 mg, 1.38 mmol, 81%).