Preparation and Characterization of Thermochemiluminescent Acridine-Containing 1,2-Dioxetanes as Promising Ultrasensitive Labels in Bioanalysis

Abstract

Thermochemiluminescence is the luminescence process in which a thermodynamically unstable molecule decomposes with light emission when heated above a threshold temperature. We recently reported the thermochemiluminescence properties of an acridine-containing 1,2-dioxetane, which emits at relatively low temperatures (i.e., below 100 °C). Herein, we explored the effect of the introduction of methyl substituents in the acridine system. The methyl group did not determine an excessive destabilization of 1,2-dioxetane ring nor significantly affect the general physical properties of the molecule. Monosubstituted methyl derivatives and a series of derivatives bearing several combinations of two, three, and four methyl groups were prepared. The rate of formation of 1,2-dioxetane derivatives 1b-k strongly depended on the methyl substitution pattern. All members of this library of mono-, di-, tri-, and tetramethyl-substituted derivatives were characterized in terms of photophysical and thermochemiluminescence properties. The introduction of methyl groups into the acridine ring caused a marked decrease in the activation energy of the thermochemiluminescent reaction. Tri- and tetramethyl-substituted acridones had the highest fluorescence quantum yields, in the range 0.48-0.52, and the corresponding 1,2-dioxetanes 1h and 1j showed in thermochemiluminescence imaging experiments limit of detection values more than ten times lower with respect to the unsubstituted derivative.

Synthesis of 1,2‐Dioxetanes as Thermochemiluminescent Labels for Ultrasensitive Bioassays: Rational Prediction of Olefin Photooxygenation Outcome by Using a Chemometric Approach

“…2 h , entry 2, Table ) . The observed behavior also proved to be peculiar in comparison with the fluorescence quantum yield trend previously recorded for (poly)methylated acridones . In the presence of EDGs, the φ F values showed an increase for substitution at both the 2‐ and 3‐positions.…”

“…Conversely, 3,6‐difluoro‐ ( 2 h ) and 1,6‐difluoro‐substituted ( 2 i ) acridones showed decreased fluorescence quantum yields, but the corresponding dioxetanes 4 h – i displayed significantly longer half‐life values. A comparison of these data with those recorded for methyl‐substituted acridan‐based dioxetanes confirmed that the introduction of weak EDGs increased the ketone fluorescence quantum yield and lowered the dioxetane activation energy, regardless of the position on the aromatic ring. On the contrary, the impact of fluorine atoms depended dramatically on the substituent position.…”

“…After examining the photophysical properties of ketones 2 h – l , we turned our attention to the activation parameters of the thermal decomposition of the fluorinated 1,2‐dioxetanes 4 h – l . The trend shown by acridane 1,2‐dioxetanes containing EDGs consists of lowered activation energies ( E a ) and pre‐exponential coefficients ( A ) and increased fluorescence quantum yields ( φ F ) . We reasoned that the introduction of fluorine atoms as EWGs should give more stable and easy to handle 1,2‐dioxetanes, without causing an excessive impairment of the fluorescence quantum yield.…”

“…Previously investigated acridane‐based alkenes 3 u – x and the 1‐Me, 6‐F derivative 3 y are all characterized by the presence of a methyl group at the 1‐position of the aromatic system. Such results suggest that steric crowding in this region disfavors the addition of singlet oxygen.…”

“…In a preliminary stage, we studied the effect of weak electron‐donating groups (EDGs), such as methyl substituents, on the acridan moiety of 4 a ( 4 b – 4 g ; Figure ) . For example, we found that, with acridones as the emitting species, tri‐ and tetramethyl‐substituted acridones showed the highest fluorescence quantum yields ( φ F ), in the range of 0.48–0.52.…”