2017 Volume 65 Issue 12 Pages 1113-1116
Development of a novel, tau-selective near-infrared fluorescence (NIRF) probe was attempted by combining the 3,5-dimethoxy-N,N-dimethylaniline-4-yl moiety with an α-cyanoacetophenone via hexatrienyl π-linker. In particular, for structure–activity relationship study of the α-cyanoacetophenones, a chlorine substituent was introduced to the aromatic ring to give a series of compounds (2a–2d). Among those, compound 2c with meta-chloro aryl substituent was identified as a tau-selective NIRF probe: selectivity for tau over amyloid β (Aβ) and bovine serum albumin (BSA) was estimated to be 10.3 and 19.5 fold, respectively. The mechanism for tau-selectivity of 2c was found to be based on the specific recognition of the microenviroment of tau fibrils, which was endowed by its molecular rotor-like properties. The tau-selective NIRF probe 2c was also able to stain tau fibrils in tau-green fluorescent protein (GFP)-transgenic human neuroblastoma cells (SH-SY5Y cells).
Alzheimer’s disease (AD) is the most common cause of dementia characterized by a progressive cognitive decline and short-term memory loss.1) Due to the enormous socioeconomic costs, this devastating disease has gained significant scientific attention.2) Two kinds of abnormal proteins, senile plaques (SPs) and neurofibrillary tangles (NFTs),3) characteristically observed in AD patient’s brain have become recognized as the major hallmarks of AD.4) Misfolded amyloid β (Aβ) in SPs have long been studied as the potential target for AD treatment,5) but anti-Aβ drugs such as bapineuzumab (Pfizer, U.S.A.) and solanezumab (Eli Lilly, U.S.A.) suffered from repeated clinical failure.6) On the other hand, numerous reports support that NFTs composed of tau fibrils is highly associated with cognitive impairment.7,8) Accordingly, aggregated tau has served as another target for anti-AD therapy.9) By the same token, tau-based diagnosis of AD has also received considerable attention and various imaging modalities have been examined for staining of tau fibrils. In particular, near-infrared fluorescence (NIRF) imaging (wavelength=650–900 nm), characterized by sensitivity as well as ease of operation,10) have been utilized for detecting tau fibrils.11–14) Previously, we reported an NIRF tau tracer (1, Fig. 1) with a donor-π-acceptor architecture: a 3,5-dimethoxy-N,N-dimethylanilin-4-yl donor moiety condensed with an α-cyanoester via a hexadienyl π-linker, which showed fluorescence emission at NIR range (λem=650 nm) and, more intriguingly, tau-selective fluorescence emission (5.7 times of tau-over-Aβ selectivity).15) Nevertheless, for potential practical application as a tau-selective NIRF probe, unfavorable properties of 1 including suboptimal fluorescence emission wavelength and tau selectivity need to be further optimized. In this context, it is highly anticipated to develop a tau-selective NIRF probe with improved photochemical properties through structural modification of 1. In this study, we focused on the ester functionality of 1 and replaced it with a more stable aromatic ketone (2, Fig. 1). In particular, in order to mimic the furanyl oxygen atom of 1, a chlorine substituent was introduced into the aromatic ring of 2 (Fig. 1). Herein, we report fluorescence properties of a series of novel probes (2a–2d; Fig. 1) upon binding to tau aggregates.
Synthesis of the designed compounds is outlined in Chart 1. First, 4-(N,N-dimethylamino)-2,6-dimethoxybenzaldehyde (4) and its vinyl homologue (5) were synthesized according to the methods used to prepare 1.15) The commercially available α-cyanoacetophenones were then condensed with the key intermediate 5 to give the title compounds 2a–2d (42–65% yields).16)
The title compounds 2a–2d were tested for their stability in comparison with the reference compound 1. Thus, in basic hydrolysis conditions (pH 9.0 buffer), the title compounds 2a–2d were observed intact even after 24 h, while 1 rapidly underwent hydrolysis with a half-life of 1.5 h. Also, on continued exposure to light, the title compounds 2a–2d did not undergo decomposition (data not shown).
Fluorescence properties of the prepared compounds (2a–2d) were evaluated before and after mixing with preaggregated tau15) in phosphate-buffered saline (PBS). To evaluate the tau-selectivity of the prepared compounds, the same experiment was also performed in the presence of Aβ fibrils or bovine serum albumin (BSA) (Fig. 2). None of the newly prepared compounds was fluorescent by themselves but, as expected, the title compounds 2a–2d showed fluorescence emission at NIR region (λem=690–700 nm) with Stokes shifts of 80–120 nm upon binding to the tau fibrils (Fig. 2). Interestingly, the turn-on fluorescence was specific to tau fibrils and, in the presence of other targets such as Aβ fibrils or BSA, a markedly low fluorescence was observed. In particular, 2c with a meta-chloro phenyl group showed almost exclusive selectivity for tau fibrils (Fig. 2c), and its selectivity over Aβ fibrils and BSA reached up to 10.3 and 19.5 fold, respectively. Considering that the previously reported compound 1 exhibited emission wavelength at 650 nm and selectivity index against Aβ fibrils of 5.7,15) the newly synthesized compound 2c showed longer emission wavelength (λem=690 nm) and higher tau selectivity over Aβ fibrils (SIAβ=10.3), which demonstrates significantly enhanced property of 2c as a tau-selective NIRF probe. Fluorescence properties and selectivity of the prepared compounds as “turn-on” NIRF probes are summarized in Table 1.
| Cmpd | εa) (M−1 cm−1) | λexb) (nm) | λemc) (nm) | FId) | SIe) | |||
|---|---|---|---|---|---|---|---|---|
| FItau | FIAβ | FIBSA | SIAβ | SIBSA | ||||
| 2a | 14487 | 580 | 700 | 65.1 | 7.5 | 18.3 | 8.6 | 3.5 |
| 2b | 13923 | 610 | 690 | 84.8 | 30.5 | 18.5 | 2.7 | 4.7 |
| 2c | 11138 | 610 | 690 | 84.8 | 8.3 | 4.4 | 10.3 | 19.5 |
| 2d | 5831 | 610 | 700 | 65.7 | 11.3 | 4.7 | 5.8 | 14 |
a) Molar extinction coefficient (in dimethylsulfoxide). b) Maximum excitation wavelength of the probe (in assay buffer). c) Maximum emission wavelength of the probe (in assay buffer). d) Fold increase=fluorescence intensity of the probe bound to tau aggregates/fluorescence intensity of the probe (unbound, free). e) Selectivity index (SI)=fluorescence intensity of the probe upon binding to tau aggregates/fluorescence intensity of the probe upon binding to Aβ aggregates (or BSA).
As shown above, selective “turn-on” fluorescence upon binding to tau aggregate is the most characteristic feature of the probes discovered in this study, but the molecular mechanism behind this phenomenon remains unclear. Nevertheless, the “turn-on” fluorescence of the probes are reminiscent of that of many other molecular rotor-type probes17,18): molecular rotors with conformational flexibility do not show fluorescence at their free states, but engagement to the target and the resulting restriction of the intramolecular rotational relaxation drives the “turn-on” fluorescence. The conformation-dependent fluorescence of the molecular rotors is usually demonstrated by their fluorescence changes in solvents with different viscosities.19) Thus, fluorescence from the probes 2a–2d were observed in several solvent systems with different viscosities. The results summarized in Fig. 3 demonstrate a linear increase of the fluorescence emission intensity of the probes 2a–2d with increasing solvent viscosity, which supports that these compounds behave as a molecular rotor. Thus, the “turn-on” fluorescence of the title probes upon binding to tau aggregates might be attributed to their environment-sensitive molecular rotor-like properties, which enable specific recognition of the tau aggregates.
Fluorescence emission was measured (λex=600 nm/λem=700 nm) in various solvents at 25°C: (1) H2O, (2) glycerol, (3) ethylene glycol (EG), and (4) EG/glycerol (1 : 1, v/v).
Tau-selective fluorescence by 2c was further demonstrated in cellular milieu. Thus, human neuroblastoma cells (SH-SY5Y) expressing green fluorescent protein (GFP)-tagged tau proteins were generated by transfecting a vector containing tau-GFP fusion protein (pCMV6-htau40-GFP).15) After treatment of the cells with 2c, fluorescence emitted from GFP (λem=420 nm) and 2c (λem=700 nm) was observed by confocal microscopy, which showed green (Fig. 4a) and red (Fig. 4b) fluorescence, respectively. Interestingly, fluorescence of 2c was co-localized with the fluorescence of tau-GFP presenting in yellow fluorescence (Fig. 4c), which demonstrates specific detection of tau aggregates inside cells by 2c. In contrast, no GFP fluorescence was observed in non-transfected cells (Fig. 4e), which indicates that the green fluorescence in Fig. 4a specifically reflects cellular tau aggregates.
The fluorescence observed from (a) GFP (λem=420 nm) and (b) 2c (λem=700 nm), respectively, and the (c) merged image shows co-localization of fluorescence of GFP-tau and fluorescence of 2c as gray fluorescence. (d) Bright field image of the GFP-tau transfected SH-SY5Y cells. White arrowheads indicate co-localization foci of tau-GFP with 2c. In non-transfected SH-SY5Y cells, no fluorescence was observed (e: GFP channel image, f: bright field image).
In summary, previously reported tau-selective NIRF probe with a donor-π-acceptor architecture (1) was modified to improve its fluorescence properties. In particular, the 2-furanylmethyl ester functionality of 1 was carefully mimicked to more stable chlorinated aromatic ketones, and a series of novel probes (2a–2d) were prepared. Among those, 2c with a meta-chloro aryl substituent showed the most promising properties as a tau-selective NIRF probe; turn-on fluorescence emission at NIR range (λem=690 nm) with a large Stokes’ shift (80 nm), a large fluorescence fold increase upon binding to tau-fibrils (84.4-fold), a significant tau-selectivity over Aβ (10.3-fold) and over BSA (19.5-fold). The tau-selective turn-on fluorescence of 2c was attributed to its microenvironment-dependent molecular rotor-like properties, which enables specific recognition of the tau fibrils. The tau-selective fluorescence by 2c was further confirmed in cellular milieu. Taken together, in comparison with the previously reported compound 1, the newly discovered probe 2c exhibited longer emission wavelength (λem=690 nm) and higher tau selectivity over Aβ fibrils (SIAβ=10.3), which demonstrates significantly enhanced property of 2c as a tau-selective NIRF probe.
This study was supported by a Grant of the Korean Health Technology R&D project, Ministry of Health & Welfare, Republic of Korea (HI14C2341) and a Grant from the Priority Research Centers Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science and Technology (2009-0093824).
The authors declare no conflict of interest.
