Biocompatible Flash Chemiluminescent Assay Enabled by Sterically Hindered Spiro-Strained-Oxetanyl-1,2-Dioxetane.

Chemiluminescence is the emission of light that occurs as a result of a chemical reaction. Depending on the rate of chemiexcitation, light emission can occur as a long-lasting, low-intensity, glow-type reaction or a rapid, highly intense flash-type reaction. Assays using a flash-type mode of action provide enhanced detection sensitivity compared to those using a glow-type mode. Recently, our group discovered that applying spiro-strain to 1,2-dioxetanes significantly increases their chemiexcitation rate, thereby transforming glow-type chemiluminescence into flash-type chemiluminescence. However, further examination of the structure-activity relationships revealed that the spiro-strain severely compromises the chemical stability of the 1,2-dioxetanes. We hypothesized that a combination of spiro-strain, steric hindrance, and an electron-withdrawing effect, will result in a chemically stable spiro-strained dioxetane with an accelerated chemiexcitation rate. Indeed, spiro-fused tetramethyl-oxetanyl exhibited a 128-fold faster chemiexcitation rate compared to adamantyl while maintaining similar chemical stability, with a half-life of over 400 hours in PBS 7.4 buffer at room temperature. Turn-on probes composed of tetramethyl-oxetanyl spiro-dioxetane exhibited significantly improved chemical stability in bacterial and mammalian cell media compared to previously developed dioxetane probes fused to a cyclobutyl unit. The superior chemical stability enables a tetramethyl-oxetanyl dioxetane probe to detect β-Galactosidase (β-gal) activity with enhanced sensitivity in Escherichia coli (E. coli) bacterial assays and leucine aminopeptidase activity in tumoral cell lines. Overall, the development of the tetramethyl-oxetanyl dioxetane luminophore enables us to enhance the detection sensitivity of chemiluminescent probes while maintaining high chemical stability. The results obtained in this study should assist in designing improved chemiluminescent probes and underscore the significance of strain-release techniques in enhancing the detection sensitivity of chemiluminescence assays.