Spirostrain-Accelerated Chemiexcitation of Dioxetanes Yields Unprecedented Detection Sensitivity in Chemiluminescence Bioassays

Abstract

Chemiluminescence is a fascinating phenomenon that involves the generation of light through chemical reactions. The light emission from adamantyl-phenoxy-1,2-dioxetanes can glow from minutes to hours depending on the specific substituent present on the dioxetane molecule. In order to improve the light emission properties produced by these chemiluminescent luminophores, it is necessary to induce the chemiexcitation rate to a flash mode, wherein the bulk of light is emitted instantly rather than slowly over time. We report the realization of this goal through the incorporation of spirostrain release into the decomposition of 1,2-dioxetane luminophores. DFT computational simulations provided support for the hypothesis that the spiro-cyclobutyl substituent accelerates chemiexcitation as compared to the unstrained adamantyl substituent. Spiro-linking of cyclobutane and oxetane units led to greater than 100-fold and 1000-fold emission enhancement, respectively. This accelerated chemiexcitation rate increases the detection sensitivity for known chemiluminescent probes to the highest signal-to-noise ratio documented to date. A turn-ON probe, containing a spiro-cyclobutyl unit, for detecting the enzyme β-galactosidase exhibited a limit of detection value that is 125-fold more sensitive than that for the previously described adamantyl analogue. This probe was also able to instantly detect and image β-gal activity with enhanced sensitivity in E. coli bacterial assays.

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

“…The incorporation of an acrylate substituent at the ortho position of a phenoxy-adamantyl-1,2-dioxetane, prevents water-mediated quenching of the excited intermediate, enabling up to 3000-fold enhancement of light-emission intensity. These probes found widespread applications in bioimaging, immunoassays, and real-time monitoring of cellular events both in vitro and in vivo . − Here, we report a highly sensitive chemiluminescent probe for the detection of PYRase and its use for the rapid screening of its activity across numerous Gram-positive and Gram-negative bacterial strains.…”