Article pubs.acs.org/JACS
Near-Infrared Dioxetane Luminophores with Direct Chemiluminescence Emission Mode Ori Green,†,§ Samer Gnaim,†,§ Rachel Blau,‡ Anat Eldar-Boock,‡ Ronit Satchi-Fainaro,‡ and Doron Shabat*,† †
School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, and ‡Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel S Supporting Information *
ABSTRACT: Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-tonoise 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 energytransfer 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.
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INTRODUCTION Optical imaging modalities have become powerful tools for noninvasive visualization of biomolecular processes in real time with high spatial resolution.1,2 Moreover, such imaging devices are relatively inexpensive, easy to use, portable, and adaptable to acquire physiological and functional information from microscopic to macroscopic levels.3,4 Fluorescence is a common useful modality for optical imaging that is broadly used for detection and monitoring of various biological processes in vitro and in vivo.5−8 The obtained signal-to-noise ratio in such measurements is often limited by light interference resulting from excitation and the autofluorescence of biological tissues. In bioluminescence imaging, such limitations are avoided, as no excitation light source is required and background signal is not affected by phenomena like autofluorescence.9−14 In addition, bioluminescence imaging techniques rely heavily on cells that are engineered to express the enzyme luciferase. Chemiluminescence imaging could offer some attractive advantages over bioluminescence, as light generation is initiated by a specific chemical reaction without further enzymatic dependency.15,16 So far, only a few reports have described chemiluminescence systems that were used for in vivo imaging.17−29 Such systems are usually based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Furthermore, the activation mechanism of the chemiluminescent © 2017 American Chemical Society
precursors used in most systems is limited by an oxidation step, which diminishes the generality of the platform. Schaap’s adamantylidene-1,2-dioxetane30−32 is a chemiluminescent compound that does not require a prior oxidation step, since it is already oxidized in the form of a thermally stable dioxetane (Figure 1A). Its chemiexcitation mechanism relies on a phenol deprotection and an electron transfer from a phenolate to the peroxide bond of the dioxetane. As a result, various analyteresponsive groups can be applied to trigger the chemiexcitation mechanism. Thus, different probes can be designed for detection of a variety of analytes. Unfortunately, due to a quenching effect of the emitter species by water molecules, the chemiluminescence signal generated by Schaap’s dioxetane is extremely weak under physiological conditions.33 Additionally, the blue photon released by the emitter has a great tendency to be absorbed by organic tissues. In order to use Schaap’s dioxetanes for in vivo imaging, it is essential to obtain better emitter species in water and to shift their light emission wavelength toward the near-infrared (NIR) region. This region is considerably more useful for in vivo imaging, since NIR wavelengths can better penetrate and are less scattered by living tissues.34,35 Until now, in vivo imaging assays using Received: August 9, 2017 Published: August 30, 2017 13243
DOI: 10.1021/jacs.7b08446 J. Am. Chem. Soc. 2017, 139, 13243−13248
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Journal of the American Chemical Society
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.
Figure 2. General design and activation pathway of NIR luminophores based on Schaap’s 1,2-dioxetanes.
Figure 3. Molecular structure and spectral analysis of NIR luminophores 1 and 2. (Left) Chemiluminescence kinetic profile upon incubation of luminophores 1 and 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.
Schaap’s dioxetane could not be applied without the addition of a surfactant and a fluorogenic dye.36−38
Recently, we have developed a new methodology to rationally design chemiluminescence turn-ON dioxetane probes with 13244
DOI: 10.1021/jacs.7b08446 J. Am. Chem. Soc. 2017, 139, 13243−13248
Article
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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.
whereas luminophore 2 has, in addition, an acrylic acid substituent at the ortho position of the phenol. The chemiluminescence emission spectra and light-emission kinetic profile of the luminophores were evaluated under physiological conditions. As expected, the emission spectra of luminophores 1 and 2 were found to be in the NIR region, with maximum emission wavelengths of 660 and 690 nm, respectively (Figure 3, right). Generally, NIR wavelengths are considered to be between 700 and 2500 nm. Although our luminophores’ maximum emission wavelengths are a bit below 700 nm, the emission spectrum is quite broad and reaches beyond 750 nm. As previously observed for our green luminophores, the fluorescence spectra of the released benzoate esters overlapped with the chemiluminescence emission spectra of their corresponding luminophores (see Supporting Information, Figure S1). Luminophores 1 and 2 exhibited typical chemiluminescence kinetic profile behavior with an initial signal increase to a maximum, followed by a gradual decrease to zero (Figure 3, left). Luminophore 2 showed a faster kinetic profile (T1/2 = 53 min) compared to that of luminophore 1 (T1/2 = 178 min). This difference is attributed to the reduced pKa of the phenol in luminophore 2, since the chemiexcitation of the dioxetane−luminophore is essentially dependent on the relative concentration of the phenolate species. Incubation of a phenol with lower pKa, under physiological conditions, resulted in an enriched phenolate population. The spectral properties of luminophores 1 and 2 are summarized in Table S1 (see Supporting Information). The promising results obtained so far have encouraged us to investigate whether our new NIR luminophores could serve as imaging probes for in vitro and in vivo applications. Turn-ON chemiluminescent probes can be generated by simply masking the phenol functionality of the luminophores by an analyte/enzyme-responsive group. Probe 1a was prepared with an enzyme-responsive group that is designed for activation by β-galactosidase (Figure 4). This enzyme is commonly used as a gene reporter and also has important physiological roles, such as a cancer biomarker.45−48 The chemiluminescence behavior of probe 1a, under physiological conditions, was evaluated by
remarkable efficiency of light emission under physiological conditions (Figure 1B). The chemiluminescence efficiency of Schaap’s dioxetane essentially depends on the emissive nature of the obtained excited-state benzoate, which suffers from an extremely low quantum yield emission in water (Figure 1A, benzoate I). We have drastically improved the benzoate emission ability under aqueous conditions (3000-fold) simply by installing an acceptor substituent on the phenol donor (Figure 1B, benzoate II).39 Here, we have notably extended our methodology to develop the first NIR chemiluminescence turn-ON probes with direct mode of emission under physiological conditions.
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RESULTS AND DISCUSSION
As explained above, the chemiluminescence emission wavelength of Schaap’s 1,2-dioxetane essentially depends on the fluorescence properties of the benzoate species generated during the chemiexcitation pathway. Therefore, in order to obtain a luminophore with NIR emission, we sought to design a dioxetane derivative that, upon chemiexcitation, will produce an excited-state benzoate that can emit a NIR photon. To achieve this task, we simply incorporated an appropriate acceptor substituent with an extended conjugated π-electron system at the para position of the phenolic luminophore (Figure 2), with or without an additional ortho substituent. In recent years, much attention has been given to NIR fluorescent dyes that are based on a phenol donor and a dicyanomethylchromone (DCMC) acceptor.40−44 Such DCMC-based push−pull chromophores are known to produce NIR emissive dyes with decent fluorescence quantum yield and high photostability. In order to obtain NIR luminophores with a direct mode of emission, we chose to integrate the DCMC acceptor as a substituent on Schaap’s 1,2-dioxetanes. Activation of such luminophores by deprotonation initiates a chemiexcitation process to release an excited-state benzoate with an extended donor−acceptor pair. Such an excited-state benzoate should decay to its ground state through emission of an NIR photon. Based on this design, we synthesized two new luminophores that include the DCMC acceptor (Figure 3). Luminophores 1 and 2 both have a DCMC acceptor at the para position of the phenol, 13245
DOI: 10.1021/jacs.7b08446 J. Am. Chem. Soc. 2017, 139, 13243−13248
Article
Journal of the American Chemical Society
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.
growth, proliferation, differentiation, and migration. Therefore, there is an urgent demand to develop new methods for real-time monitoring of hydrogen peroxide in living organisms.12,49−51 Probe 2a was composed with an aryl-boronate moiety as triggering substrate for hydrogen peroxide (Figure 6). Such an aryl-boronate can selectively react with hydrogen peroxide to generate the active form of luminophore 2. Also, probe 2a, like luminophore 2, has an additional acrylic acid ortho substituent. This substituent significantly increases the aqueous solubility of the probe through the ionizable carboxylate group. That is obviously important in a probe used for in vivo study, where solubility under physiological conditions is required. The ability of probe 2a to detect hydrogen peroxide was evaluated by measuring the light emitted from solutions of the probe in the presence and absence of hydrogen peroxide. Probe 2a exhibited a light-emission kinetic profile with a signal-to-noise ratio of 17-fold, with chemiluminescence emission wavelength in the NIR region (Figure 6). The selectivity and sensitivity of probe 2a toward hydrogen peroxide were evaluated as described in the Supporting Information (Figures S3 and S4). These results suggest that probe 2a could indeed be used to image hydrogen peroxide in real time. Since the overproduction of hydrogen peroxide in vivo is associated with the development of numerous inflammatory diseases,
measuring the light emission signal over time in the presence and absence of β-galactosidase (Figure 4). Probe 1a displayed a typical chemiluminescence kinetic profile with a signal-to-noise ratio of 17-fold. As expected, the NIR-emission wavelength of probe 1a overlaps with that of its parent luminophore 1. Addition of a galactosidase inhibitor substantially decreases the chemiluminescence signal (see Supporting Information, Figure S2). Next, we investigated the feasibility of probe 1a to image endogenous β-galactosidase activity in Human embryonic kidney cells, HEK 293, transfected with the Lac-Z gene. Probe 1a was incubated with HEK293-Lac-Z and HEK293-wild type (wt) cells, and its light emission was monitored. Remarkably, probe 1a generated an intense chemiluminescence signal when incubated with HEK293-Lac-Z cells, while negligible signal was observed with the HEK293-wt cells. The quantified chemiluminescence signal is presented in Figure 5. The ratio between the signal intensities observed by the transfected cells and the wild-type cells was found to be approximately 14-fold. These results clearly demonstrate the chemiluminescence ability of probe 1a to image β-galactosidase activity in living cells in real time. To demonstrate the ability of our NIR luminophores to serve as imaging probes for in vivo use, we sought to image a biologically relevant analyte. As a secondary metabolite, hydrogen peroxide is tightly linked to diverse cellular processes such as 13246
DOI: 10.1021/jacs.7b08446 J. Am. Chem. Soc. 2017, 139, 13243−13248
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Figure 7. (Left) In vivo imaging of endogenous hydrogen peroxide in the peritoneal cavity of mice during an LPS-induced inflammatory response, using probes 2a and 2b. Images of mice were recorded on BioSpace Lab PhotonIMAGER. Group A: 1 mL of 0.1 mg/mL LPS was injected into the peritoneal cavity of mice, followed 4 h later by an i.p. injection of Probe 2a [100 μM, 100 μL, PBS 7.4]. Group B: 1 mL of 0.1 mg/mL LPS was injected into the peritoneal cavity of mice, followed 4 h later by an i.p. injection of probe 2b [100 μM, 100 μL in PBS 7.4]. Group C: 1 mL of PBS 7.4 was injected into the peritoneal cavity of mice, followed 4 h later by an i.p. injection of probe 2a [100 μM, 100 μL in PBS 7.4]. (Right). Quantification of signal intensities measured from each group of mice (three mice per group).
we wanted to investigate the ability of probe 2a to image endogenously produced hydrogen peroxide in a mice model. We have used a known model of acute inflammation in mice that can be induced by lipopolysaccharide (LPS).19,23,24 Mice were injected intraperitoneally (i.p.) with LPS (1 mL of 0.1 mg/mL), followed 4 h later by an additional i.p. injection of 100 μL of probe 2a [100 μM], and thereafter imaged by non-invasive luminescence imaging system (BioSpace Lab PhotonIMAGER). Figure 7 shows that LPS-treated mice injected with probe 2a (group A) generated a significantly greater intensity of NIR light emission signal compared with non-LPS-treated mice injected with the probe (group C). The slight light emission signal observed with non-LPS-treated mice is attributed to basal levels of hydrogen peroxide produced in living animals. Practically, no light emission signal was observed in mice treated with LPS and control probe 2b (a probe that cannot be activated by hydrogen peroxide, group B). The signal-to-noise ratio of the NIR light emission intensity observed by the hydrogen peroxide imaging probe in mice treated with LPS was about 20-fold higher than that of the nontreated group. For comparison, previously reported chemiluminescence imaging probe system, which is based on energy-transfer mechanism, has produced only 2-fold signal-to-noise ratio in identical LPS-treated mice model.19 As far as we know, this is the first demonstration for an in vivo imaging, obtained by a NIRchemiluminescence probe with direct mode of emission. The key advantage of chemiluminescence probes is in their extremely low background signal. In addition, undesirable phenomena such as photobleaching and phototoxicity are avoided. For in vivo imaging, the advantage of chemiluminescence over fluorescence is even more significant, as the autofluorescence of living tissues significantly increases the background signal and thus limits the use of fluorescence imaging. Accordingly, the obtained results emphasize the uniqueness of luminophore 2 to serve as a chemiluminescent reporter for imaging of biologically relevant analytes in small animal models.
Currently, chemiluminescence signal amplification is gaining increased attention.52 Last year, we reported a new practical synthetic route for preparation of turn-ON fluorophore-tethered dioxetane chemiluminescent probes.53 The chemiluminescence emission of the fluorophore-tethered dioxetane probes was amplified in comparison to that of a classic dioxetane probe through an energy-transfer mechanism. One of the fluorophoretethered dioxetanes was composed with an NIR dye. This probe’s ability to provide effective chemiluminescence for in vivo imaging was successfully demonstrated. Nevertheless, a probe with a direct NIR emission mode is superior to a probe with an energytransfer mode in almost every possible aspect. A probe based on energy transfer is structurally based on a conjugate of two separated entities: a dioxetane moiety and a fluorescent dye. On the other hand, a probe with a direct emission mode is a single entity, smaller in size, and emits NIR light with greater efficiency (about 10-fold). In addition, such dioxetane structure exhibits greater photostability, and its chemical synthesis is simpler.
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CONCLUSION We have developed the first chemiluminescent dioxetane luminophores with a direct mode of NIR light emission, suitable for use under physiological conditions. The NIR luminophores were obtained by introducing an acceptor substituent with an extended π-electron system (DCMC) at the para position of the phenol−dioxetane donor. Masking of the luminophores with analyte-responsive groups has resulted in two different turn-ON probes for detection and imaging of β-galactosidase and hydrogen peroxide. The probes’ ability to image their corresponding analytes was effectively demonstrated in vitro for β-galactosidase activity and in vivo for an inflammation model in mice. We anticipate that our strategy for designing and preparing NIR luminophores will open new doors for further exploration of complex biomolecular processes using non-invasive chemiluminescence imaging techniques. 13247
DOI: 10.1021/jacs.7b08446 J. Am. Chem. Soc. 2017, 139, 13243−13248
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b08446. Supplementary figures, synthetic schemes and experimental procedures, NMR and MS spectra of key compounds, and details of in vitro and in vivo experiments (PDF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Doron Shabat: 0000-0003-2502-639X Author Contributions §
O.G. and S.G. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS D.S. thanks the Israel Science Foundation (ISF), the Binational Science Foundation (BSF), and the German Israeli Foundation (GIF) for financial support. This work is supported in part by a grant from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics, and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund. R.S.-F. thanks the European Research Council for the ERC Consolidator Grant Agreement No. [617445]- PolyDorm.
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