Near-Infrared Dioxetane Luminophores with Direct Chemiluminescence Emission Mode

Ori Green, Samer Gnaim, Rachel Blau, Anat Eldar-Boock, Ronit Satchi-Fainaro, Doron Shabat. J. Am. Chem. Soc., 2017, 139, 13243–13248.

DOI: 10.1021/jacs.7b08446

ABSTRACT

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for in vivo use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for in vivo imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemiexcitation pathway of Schaap’s adamantylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of β-galactosidase and hydrogen peroxide. The probes’ ability to image their corresponding analyte/enzyme was effectively demonstrated in vitro for β-galactosidase activity and in vivo in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intravital chemiluminescence imaging techniques.

 

Introduction

Figure 1. (A) Chemiexcitation activation mechanism of Schaap’s dioxetane (PG, protecting group). (B) Here, direct chemiluminescence amplification and red-shifted wavelength emission were obtained by a substituent effect.

 

Details

Figure 2 shows the general design and activation pathway of NIR luminophores based on Schaap’s 1,2-dioxetanes.

 

Figure 3 shows the molecular structure and spectral analysis of NIR luminophores 1 and 2. (Left) Chemiluminescence kinetic profile upon incubation of luminophores 1and 2 [1 μM] in PBS, pH 7.4, 10% fetal bovine serum (FBS), at 37 °C. (Right) Chemiluminescence emission spectra of luminophores 1 and 2 [50 μM] in PBS, pH 7.4, 10% FBS, at 37 °C.

 

Figure 4. (Top) Molecular structure of probe 1a and its activation pathway upon reaction with β-galactosidase. (Bottom) Chemiluminescence kinetic profile and total photon count of probe 1a [1 μM] in PBS, pH 7.4 (10% DMSO), in the presence or absence of 1.5 units/mL β-galactosidase.

 

Figure 5. (Left) Chemiluminescence imaging of probe 1a [5 μM] in HEK293 cells. (Right) Quantification of signal intensities evolving from HEK293 cells.

 

Figure 6. (Top) Molecular structure of probe 2a and its activation pathway upon reaction with H₂O₂. (Bottom) Chemiluminescence kinetic profile and total photon count of probe 2a [50 μM] in serum (5% DMSO), with and without of H₂O₂ [250 μM] at temperature of 37 °C.

 

 

Reference