Unprecedented Photoinduced‐Electron‐Transfer Probe with a Turn‐ON Chemiluminescence Mode‐of‐Action

Abstract

PeT‐based fluorescent probes were demonstrated to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. While numerous examples of fluorescence‐based PeT have been frequently reported, there is not even a single example of a PeT probe that operates via a chemiluminescence mode. Here we report the first PeT‐based turn‐on probe that acts via a chemiluminescent operation mode. We designed, synthesized, and evaluated a novel chemiluminescent probe, featuring a PeT‐based turn‐on mechanism. The probe consists of a phenoxy‐1,2‐dioxetane, linked to an azide unit that acts as a PeT quencher. Upon cycloaddition of a strained cycloalkyne with the azide, a triazole‐dioxetane is formed, which undergoes relatively slow chemiexcitation, resulting in a measurement window with an exceptionally high signal‐to‐noise ratio (over 5000‐fold). The PeT‐dioxetane probe could effectively detect and image two model proteins labeled with strained cycloalkyne units (Myc‐DBCO and Max‐DBCO) through either NHS or maleimide conjugations. Comparative analysis shows that our PeT‐based chemiluminescent probe significantly outperforms a commercially available fluorescent analog. We anticipate that the insights gained from this study will facilitate the development of additional chemiluminescent probes utilizing various PeT‐quenching pathways.

The first PeT‐based turn‐on probe utilizing a chemiluminescent mechanism is presented. The probe is activated through a reaction between a quenched dioxetane‐azide and a strained cycloalkyne, resulting in high signal‐to‐noise protein detection and outperforming a fluorescent analog.

Introduction

Photoinduced electron transfer (PeT) is a fundamental electron transfer mechanism, which is widely employed in the design of fluorescent probes. ^[1] A typical fluorescent PeT probe consists of a fluorophore linked to a quencher unit. Broad studies have shown that the fluorescent signal of the fluorophore is usually quenched through an electron transfer from the HOMO energy level of the quencher to the HOMO energy level of the fluorophore. Interaction of the analyte of interest with the quencher unit significantly diminishes the PeT quenching effect and turns on the signal of the fluorophore upon its excitation. ^[2]

Chemiluminescence is a highly effective technique for developing molecular probes,[ ³ , ⁴ , ⁵ , ⁶ , ⁷ , ⁸ ] and its combination with PeT mechanism could offer significant potential. While numerous examples of fluorescence‐based PeT have been frequently reported, there is not even a single example of a PeT probe that operates in a chemiluminescence mode. This fact can be possibly explained by the different operation modes of fluorescence and chemiluminescence. The emission signal of a fluorogenic dye is initiated through excitation by an external light source. The signal can be generated at any given time and its intensity depends on the number of active fluorescent molecules accumulated over time. Unlike fluorescence dyes, chemiluminogenic molecules emit light through a single chemiexcitation event, resulting in a kinetic light emission profile that occurs within a specific time interval. Light emission is initiated by the chemiexcitation of a chemiluminophore, and the intensity and duration depend on the rate of the chemiexcitation and the chemiluminescent quantum yield. Once all the chemically excited molecules have decayed to their ground state, the time interval that enables measurement has ended and the light emission signal ceases to exist.

This limitation poses a major challenge for designing a PeT‐based probe that operates via chemiluminescence. To meet this challenge, one should design a chemiluminophore‐quencher adduct that undergoes rapid chemiexcitation when standing alone. However, after interacting with the analyte of interest, the chemiluminophore chemiexcitation rate should significantly slow down. Such a design generates a light emission signal that provides a sufficiently wide time interval to enable measurement that facilitates a high S/N ratio.

Phenoxy‐adamantyl‐1,2‐dioxetane chemiluminescent luminophores undergo efficient chemiexcitation upon generation of their phenolate ion. ^[9] This chemiexcitation occurs through electron transfer from the phenolate ion to the sigma star orbital of the dioxetane‐peroxide bond. This event is followed by a C−C bond cleavage and the formation of an excited benzoate, which decays to its ground state through the emission of visible light.[ ¹⁰ , ¹¹ , ¹² ] While the light emission of phenoxy‐1,2‐dioxetanes is highly efficient in polar organic solvents, the presence of water results in nearly complete quenching of the emitted light. Several years ago, we discovered that 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 and increases the light‐emission intensity of the chemiluminescent luminophore by up to 3000‐fold (Figure 1A). ^[13] This groundbreaking development enabled the use of the new chemiluminescent luminophores as single‐component probes with no required additives.[ ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² , ²³ , ²⁴ , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² ] Numerous research groups worldwide, took advantage of the ortho‐substituted phenoxy‐adamantyl‐1,2‐dioxetane luminophore to develop a wide range of useful chemiluminescent probes for detection and imaging for in vitro and in vivo applications.[ ¹⁷ , ³³ , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ ]

Figure 1.

A) Activation and chemiexcitation pathway of ortho‐acrylate‐adamantyl‐phenoxy‐1,2‐dioxetanes. B) Design and activation pathway of a PeT‐based phenoxy‐dioxetane chemiluminescent probe through a click azide‐cycloalkyne cycloaddition reaction.

The ability of the ortho‐acrylate phenoxy‐1,2‐dioxetane to efficiently emit light under physiological conditions has stimulated us to evaluate, if a PeT‐based turn‐on mechanism, can be effectively incorporated in such a chemiluminophore. We sought to integrate on the phenoxide unit an azide functional group that should quench the light emission signal of the dioxetane chemiluminophore via a PeT pathway. A click reaction of the azide with an acetylene unit to form a triazole should diminish the PeT quenching effect and turn on the chemiluminescent light emission signal (Figure 1B). Here we report the first PeT‐based turn‐on probe with a chemiluminescent operation mode.

Results and Discussion

Nearly 20 years ago, it was first demonstrated that the azide functional group could quench the fluorescent emission of a fluorogenic dye through a PeT mechanism, using 7‐hydroxycoumarin as the model compound. ^[42] The fluorescent emission of the azido‐coumarin could be recovered after a Cu‐click reaction with an acetylene derivative to form a triazole unit. The Bertozzi group used this concept to develop a Cu‐free variant of click chemistry that can label biomolecules rapidly and selectively in living systems. ^[43] Later on, the same group was able to identify potential PeT‐quenched azido‐fluorescein molecules that undergo an increase in fluorescence quantum yield upon copper‐free cycloaddition with cyclic alkynes. ^[44] A specific azido‐fluorescein derivative has enabled cell imaging under no‐wash conditions upon the formation of its corresponding triazole unit.[ ⁴⁵ , ⁴⁶ ]

To evaluate the ability of the azide functional group to quench the chemiluminescence light emission of a phenoxy‐1,2‐dioxetane luminophore we initially sought to test this quenching effect on the fluorescence emission of the acrylate‐benzoate species, generated during the chemiexcitation. We have previously shown that the chemiluminescence light emission intensity of acrylate‐substituted phenoxy‐1,2‐dioxetanes, under physiological conditions, can be adequately predicted by the fluorescent properties of the corresponding acrylate‐benzoate in water. ^[13] Indeed, the fluorescence emission of Azido‐MA‐benzoate (Figure 2A) was significantly quenched compared to its non‐azide derivative (see Figure S2). The cycloaddition reaction of DBCO‐amine (dibenzocyclooctyne‐amine) with Azido‐MA‐benzoate to form the triazole derivative has resulted in up to 26‐fold enhancement of the fluorescent intensity (Figure 2B). The observed quenching outcome of the azide group on the fluorescent of the benzoate species and the obtained turn‐on effect as a result of the click reaction with DBCO‐amine suggest that this PeT mode‐of‐action can be incorporated in the chemiluminescence turn‐on pathway of phenoxy‐dioxetanes.

Figure 2.

Copper‐free click reaction of Azido‐MA‐benzoate with DBCO‐amine. B) (1) Fluorescence spectra at different time points and (2) fluorescence intensity vs time of Azido‐MA‐benzoate [100 μM] in the presence of DBCO‐amine [200 μM], in PBS, pH 7.4, with 10 % DMSO at room temperature (λ_ex=398 nm, λ_em=530 nm). Measurements were performed in triplicate using independent samples.

We next synthesized the corresponding azido‐phenoxy‐dioxetane, Azido‐MA‐dioxetane (see Supporting Information) and evaluated its chemiluminescent light emission properties before and after a click reaction with DBCO‐amine in PBS 7.4 (Figure 3A). Upon the addition of Azido‐MA‐dioxetane to the PBS solution, the chemiexcitation process is initiated immediately. However, in the presence of DBCO‐amine, the click reaction occurs simultaneously to give the triazole‐dioxetane product. The total light emission of Azido‐MA‐dioxetane in PBS 7.4, without DBCO‐amine, is relatively low. Additionally, its chemiexcitation rate is fast, leading to complete signal decay within 2 hours. The cycloaddition reaction of DBCO‐amine with Azido‐MA‐dioxetane to form the triazole derivative enhances total light emission by 20‐fold and slows the chemiexcitation rate, enabling the signal to last for more than 16 hours (Figure 3B1). The enhancement in total light emission and the significant difference in chemiexcitation rates between Azido‐MA‐dioxetane and triazole‐dioxetane enables the sensitive detection of the triazole‐dioxetane as the background signal becomes negligible upon full decomposition of the Azido‐MA‐dioxetane. The light emission signal produced by the triazole‐dioxetane after 2 hours was 1,142 times higher than the signal produced by Azido‐MA‐dioxetane. Remarkably, after 8 hours, this ratio increased to 5,050‐fold, generating a noticeably very high signal‐to‐noise (Figure 3B2). Since the light emission profile of Azido‐MA‐dioxetane was significantly different from that of the in situ generated triazole‐dioxetane, the chemiluminescent properties of the latter were studied to rule out the influence of other variables, such as the click reaction rate (see Triazole‐MA‐dioxetane and Figure S8 in the Supporting Information). The chemiexcitation rate of Azido‐MA‐dioxetane was also measured and compared to the click reaction rate (see Figure S7). The results indicate that the dioxetane decomposes faster than the click reaction, suggesting that increasing the click reaction rate could enhance light emission by even more than 20‐fold since only a portion of the dioxetane has reacted with the DBCO‐amine.

Figure 3.

A) Chemiluminescence enhancement of Azido‐MA‐dioxetane in the presence of DBCO‐amine. B) (1) Chemiluminescence kinetic profile and (2) total light emission ratio at different times of Azido‐MA‐dioxetane [10 μM] in the presence and absence of DBCO‐amine [20 μM], in PBS, pH 7.4, with 10 % DMSO at 30 °C. Measurements were performed in triplicate using independent samples.

With these results in hand, we decided to extend our strategy to more complex molecules and to evaluate whether the PeT‐based chemiluminescence mode could be applied to detect and image proteins labelled with DBCO. Two essential transcription factor (TF) proteins were selected to exemplify this new technique: Myc and Max. ^[47] These TFs regulate the rate of gene expression by interacting with specific DNA sequences to either initiate or suppress gene transcription. ^[48] This network governs the expression of ~15 % of the human genome, with its dysregulation linked to over 50 % of human cancers. ^[49]

We initially conjugated the DNA binding domain of Myc (10 kDa) to DBCO through its amino‐lysine residues and the N‐terminus by a reaction with DBCO‐NHS‐ester (See Supporting Information). Since the polypeptide backbone of Myc contains several lysine residues, conjugation with DBCO‐NHS‐ester resulted in an average of 4.5 DBCO molecules per Myc protein. The Max analog (10 kDa) was synthesized with an additional cysteine unit in its C‐terminus and the thiol functional group of this cysteine was used for conjugation with DBCO by a reaction with a DBCO‐maleimide derivative (See Supporting Information). ^[50] Conversely, since only one cysteine residue was engineered into the Max protein, its conjugation with DBCO‐maleimide resulted in a single DBCO attachment.

The two DBCO‐labeled proteins (Figure 4A) were then incubated with Azido‐AA‐dioxetane (AA is an abbreviation for Acrylic Acid). The use of an acrylic acid substituent, instead of a methyl‐acrylate one, increases the water solubility of the dioxetane probe and also induces a faster chemiexcitation rate. ^[51] The light emission profile of Azido‐AA‐dioxetane, in the absence of DBCO‐labeled proteins, rapidly decays and almost completely diminishes after 20 min. However, intense, long‐lasting light emission profiles are generated upon copper‐free click reaction of Myc‐DBCO and Max‐DBCO with Azido‐AA‐dioxetane (Figure 4B1). The signal intensities produced by Myc‐DBCO and the Max‐DBCO after 35 min, are about 1,034‐fold and 203‐fold higher than the signal of Azido‐AA‐dioxetane (Figure 4B2). As expected, Myc‐DBCO produced a stronger chemiluminescent signal than Max‐DBCO, likely due to the higher degree of labeling with DBCO units.

Figure 4.

A) Chemiluminescence enhancement of Azido‐AA‐dioxetane in the presence of Myc‐DBCO or Max‐DBCO B) (1) Chemiluminescence kinetic profiles, (2) light emission intensity after 35 min, and (3) IVIS images of Azido‐AA‐dioxetane [0.1 μM] in the presence and absence of Myc‐DBCO and Max‐DBCO [1 μM], in PBS, pH 7.4, with 10 % DMSO at 30 °C (IVIS images were taken at 37 °C). Measurements were performed in triplicate using independent samples.

The successful attempt to detect Myc‐DBCO and Max‐DBCO with probe Azido‐AA‐dioxetane, promoted us to compare the detection sensitivity of our PeT‐based chemiluminescent probe to that of a known analog fluorescent probe. 3‐Azido‐7‐hydroxycoumarin is a commercially available quenched derivative which upon click reaction with alkynes generates a triazole with an increased fluorescent emission (Figure 5A). The probes Azido‐AA‐dioxetane and 3‐Azido‐7‐hydroxycoumarin were incubated with varying concentrations of Myc‐DBCO and Max‐DBCO and the turn‐on luminescence response was measured. The resulting signal‐to‐noise ratios were plotted against the corresponding concentrations to extrapolate the limit of detection (LOD) values (Figures 5B1 and 5B2). Notably, the chemiluminescent probe Azido‐AA‐dioxetane exhibited a LOD value that is 157‐fold and 63‐fold more sensitive than the fluorescent probe 3‐Azido‐7‐hydroxycoumarin for Myc‐DBCO and Max‐DBCO, respectively. Under the present conditions, these results effectively demonstrate the superior performance of a chemiluminescence‐based PeT probe compared to a fluorescence‐based PeT probe.

Figure 5.

A) Light emission enhancement of Azido‐AA‐dioxetane or 3‐Azido‐7‐hydroxycoumarin in the presence of Myc‐DBCO or Max‐DBCO B) Lowest detected concentration of (1) Myc‐DBCO and (2) Max‐DBCO in the presence of Azido‐AA‐dioxetane [0.1 μM] or 3‐Azido‐7‐hydroxycoumarin [10 μM] in PBS, pH 7.4, with 10 % DMSO at room temperature. Measurements were performed in triplicate using independent samples.

In 1985, de Silva introduced the first PeT‐based fluorescent probe, which utilized an aminomethyl‐anthracene derivative as a fluorescent pH indicator. ^[52] Since then, PeT‐based fluorescent probes have proven to be powerful tools for detection and imaging, owing to their significant fluorescence enhancement in response to specific targets. In this work, we present the first chemiluminescent probes with a turn‐on mechanism, utilizing a PeT operation mode. Given that the light emission mechanism of chemiluminescent molecules involves a chemiexcitation step with a limited time interval, we aimed to design a probe where chemiexcitation occurs rapidly before interaction with the analyte of interest and slowly after the probe encounters the analyte. In this process, the remaining low‐intensity light emission from the quenched dioxetane will quickly diminish, resulting in an almost negligible background signal after a short period.

Incorporating an azide group into phenoxy‐1,2‐dioxetane (probes Azido‐MA‐dioxetane and Azido‐AA‐dioxetane) produced chemiluminescent molecules with relatively fast chemiexcitation. Appealingly, the cycloaddition reaction between the strained cycloalkyne and the azide to form a triazole yielded a phenoxy‐1,2‐dioxetanes with a slower chemiexcitation mode, likely due to the electron‐withdrawing effect of the triazole unit. Previous studies by our group have demonstrated that the chemiexcitation rate of 1,2‐dioxetanes can be influenced by altering the electronic properties on the phenol side of the dioxetane or, more recently, by introducing spiro strain on the opposite side.[ ³² , ⁵¹ ] Focusing on the first mechanism, our group showed that electron‐withdrawing substituents decrease the electron density on the phenol, thereby reducing the probability of electron transfer and resulting in a slower chemiexcitation rate.

This difference in chemiexcitation rates between the phenoxy‐1,2‐dioxetane‐azide derivatives and the phenoxy‐1,2‐dioxetane‐triazole derivatives provides a sufficiently wide time interval, allowing for measurements that facilitate a high S/N ratio after the interaction with the analyte of interest.

Conclusions

In summary, we designed, synthesized, and evaluated a new chemiluminescent probe with a PeT‐based turn‐on operation mode. The probe is comprised of a phenoxy‐1,2‐dioxetane attached to an azide unit, which acts as a PeT quencher. A cycloaddition reaction between the strained cycloalkyne and the azide forms a triazole‐dioxetane, which undergoes relatively slow chemiexcitation, resulting in a measuring window with a very high signal‐to‐noise ratio (over 5000‐fold). Two model proteins labeled with strained cycloalkyne unit (Myc‐DBCO and Max‐DBCO) through either NHS or maleimide conjugations, could be effectively detected and imaged by probe Azido‐AA‐dioxetane. Comparison analysis of our PeT‐based chemiluminescent probe against commercially available fluorescent analog demonstrated the substantially superior performance of the chemiluminescent probe. We anticipate that the insights gained from this study will open a door to the design of further new chemiluminescent probes, based on various PeT‐quenching pathways.

Conflict of Interests

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

David M., Gutkin S., Nithun R. V., Jbara M., Shabat D., Angew. Chem. Int. Ed. 2025, 64, e202417924. 10.1002/anie.202417924

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.