No 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.

The majority of known chemiluminescent compounds produce light through oxidation-dependent mechanisms. The unique notion of triggering chemiluminescence by a chemical reaction other than oxidation was first introduced by Schaap in 1987 with the development of chemically and enzymatically activated phenoxy-dioxetanes. Such dioxetanes are distinctive among chemiluminescent molecules since the oxidized high-energy species, the dioxetane, is stable for years at room temperature. Light emission is selectively activated by deprotection of the phenol-protecting group. The chemiluminescence quantum yields of such dioxetanes are relatively high in organic solvents like DMSO. In aqueous solution, however, light emission efficiency drops by approximately 10 000-fold due to energy loss to water molecules. As we sought to understand the low light emission efficiency in water, we realized that the dioxetane chemiexcitation leads to the release of an excited state benzoate molecule, which is a very weak emitter under aqueous conditions. Thus, we reasoned that emission in aqueous solution could be enhanced, if the emissive nature of the excited benzoate formed in water is improved. Introduction of an electron-withdrawing acrylic group at the ortho position of the phenol donor resulted in an excited benzoate species that emits light with high efficiency in aqueous solutions. A striking 3000-fold increase in chemiluminescence emission was observed by simply using an acrylonitrile substituent on the dioxetane probe. For the first time, scientists now have an effective single-entity chemiluminescent probe that can be used to evaluate biological processes. This discovery promoted us to develop numerous highly efficient chemiluminescent probes for detection of different enzymes and analytes in aqueous solution. We anticipate that further studies in this direction will lead to even better chemiluminescence probes with quantum yield emissions that are even higher than that of the luciferin/luciferase system. In this Feature Article, we describe the insights that led us to develop these unprecedented luminophores and the historical perspective that led to the current generation of chemiluminescent phenoxy-dioxetane probes.

Over the last 30 years, the quinone-methide elimination has served as a valuable tool for achieving various important molecular functions. Molecular adaptors based on quinone-methide or aza-quinone-methide reactivity have been designed, synthesized, and used in diagnostic probes, molecular amplifiers, drug delivery systems, and self-immolative dendritic/polymeric molecular systems. These unique adaptors function as stable spacers between an enzyme- or reagent-responsive group and a reporter moiety and can undergo 1,4-, 1,6-, or 1,8-type elimination reactions upon cleavage of the triggering group. Such reactivity results in the release of the reporter group through formation of a quinone-methide species. This type of elimination was applied to design distinct molecular adaptors capable of multiple quinone-methide eliminations. Using this chemistry, we have developed unique molecular structures that are known today as self-immolative dendrimers. These dendrimers disassemble upon a single triggering event in a domino-like manner from the focal point to their periphery with the consequent release of multiple end-groups. Such molecular structures are used in self-immolative dendritic prodrugs and in diagnostic probes to obtain a significant amplification effect. To further enhance amplification, we have developed the dendritic chain reaction, which uses simple molecules to achieve functionality of high-generation virtual self-immolative dendrimers. In addition, we harnessed the quinone-methide elimination reactivity to design polymers that disassemble from head-to-tail initiated by an analyte-responsive event. Following this example, other chemical reactivities were demonstrated by scientists to design such polymeric molecules. In a manner analogous to the quinone-methide elimination, electron rearrangement can lead to formation of conjugated quinone-methide-type dyes with long-wavelength emission of fluorescence. We have recently applied an intramolecular charge transfer to form a unique kind of quinone-methide type derivative based on a donor-two-acceptors molecular structure. This intramolecular charge transfer produces a new fluorochrome with an extended conjugation of π-electron system that is used for the design of long-wavelength fluorogenic probes with a turn-ON option. The rapidly expanding use of quinone-methide species, reflected in the increased number of examples reported in the literature, indicates the importance of this tool in chemistry. These species provide a useful gateway to functional molecular structures with distinct reactivities and spectroscopic characteristics.