Patent Application
20230333019
The present disclosure relates to a two-photon fluorescent probe (marker) capable of targeting specific organelles and labeling ions to be detected, and more particularly, to a blue emission two-photon fluorescent probe for detecting calcium ions by targeting the cytoplasm, mitochondria, and plasma membrane, respectively, and a green emission two-photon fluorescent probe for detecting hydrogen ions (proton ions) by targeting the lysosome, a manufacturing method of these two-photon probes, and a method for imaging calcium ions in specific organelles and lysosomal hydrogen ions separately or simultaneously using these two-photon probes.
Ca2+ is known to perform several important functions in vivo, such as signaling, exocytosis, apoptosis, transcription, muscle contraction, and bone formation. Defects in Ca2+ signaling may cause diseases such as neurodegeneration, muscle defects, heart disease, and skin diseases. Calcium (Ca) ions enter the cytoplasm from an extracellular space or internal Ca2+ storage such as the endoplasmic reticulum and the mitochondria.
The concentration of free Ca2+ is about 1 mM in the extracellular space, whereas the concentrations in the cytoplasm, the endoplasmic reticulum, and the mitochondria are 0.1 to 1 μM, 0.1 to 1 mM, and 0.1 to 20 μM, respectively. The signal is triggered when Ca2+ is released from the intracellular storage or introduced into the cell through ion channels.
Fluorescence imaging using fluorescent probes such as Fluo-4, Fura-2, Rhod-2, or Calcium Green-1 is the most common method to study Ca2+ biology in the cell. However, these probes require relatively short excitation wavelengths of 350 to 500 nm, which is unsuitable for tissue imaging due to limited penetration depth (<80 μm) and autofluorescence. Two-photon (TP) microscopy (TPM) may enable long-term imaging of live tissue at depths of 500 μm or more to overcome these drawbacks.
The two-photon microscopy has advantages of higher spatial resolution, less photobleaching, and less phototoxicity than one-photon microscopy currently widely used. These advantages are much remarkable when a two-photon fluorescent probe (two-photon marker) with high two-photon absorption and high quantum yield is used together, and it is advantageous for obtaining 3D real-time images of life phenomena occurring in live cells and tissues. However, due to the small number of two-photon fluorescent probes (two-photon markers) which have been currently developed, it was impossible to observe various life phenomena using the two-photon microscopy.
In particular, there is an urgent need to develop an organelle-specific Ca2+ TP probe suitable for multicolor imaging for monitoring Ca2+ transport from one organelle to the other organelle in live tissue. Accordingly, several TP probes for Ca2+ have been developed in the related art, but there is no attempt to develop a Ca2+ TP probe suitable for multicolor imaging to realize the purpose. There are also few blue emission TP probes for Ca2+.
Therefore, the present inventors synthesized a Ca2+ two-photon probe by binding a benzoxazole derivative and a calcium ion receptor suitable for measuring changes in calcium concentration as a result of research to develop a blue light two-photon probe capable of detecting Ca2+ in vivo, confirmed that two-photon excitation fluorescence was increased by binding the blue emission two-photon fluorescent probe with the calcium ions in the specific organelles to measure changes in calcium ions specifically in organelles in real time, and then completed the present disclosure.
The present disclosure has been made in an effort to provide a blue emission two-photon fluorescent probe compound capable of detecting calcium through fluorescent signals by targeting specific organelles, a preparing method thereof, a two-photon fluorescent probe composition for detecting calcium ions in live cells or tissues containing the compound, and a method for imaging calcium ions in live cells or tissues using the compound.
In order to achieve the above purpose, an embodiment of the present disclosure provides a two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 below:
In Chemical Formulas 1 to 3, Y is each independently K, Na, CH3, or CH2OC(O)CH3, and in Chemical Formula 3, X is H, OH, or O(CH2)5CH3.
Another embodiment of the present disclosure provides a two-photon fluorescent probe composition for detecting calcium ions in live cells or tissues including at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3.
Yet another embodiment of the present disclosure provides a method for imaging calcium ions including injecting at least one compound selected from two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3 into cells or tissues isolated from a living body; irradiating an excitation source to the cells or tissues isolated from the living body; and observing fluorescence generated from the two-photon fluorescent probe compound by two-photon microscopy.
Still another embodiment of the present disclosure provides a method for preparing a two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3, including reacting a compound represented by Chemical Formula 5 below with a compound represented by Chemical Formula 6 or 7 below:
In Chemical Formula 5, 6, or 7, Y is each independently K, Na, CH3, or CH2OC(O)CH3, X is H, OH, O(CH2CH2O)2CH3, O(CH2)3Br, O(CH2)3PPh3+Br−, or O(CH2)5CH3, R1 is CO2C(CH3)3 or CO2H, Z is H or F, and L is H or NH2.
Still yet another embodiment of the present disclosure provides a two-photon fluorescent probe compound represented by Chemical Formula 4 below:
In Chemical Formula 4, W is NH(CH2)2N(CH3)2 or OCH3.
Still yet another embodiment of the present disclosure provides a two-photon fluorescent probe composition for detecting hydrogen ions in live cells or tissues, including the two-photon fluorescent probe compound represented by Chemical Formula 4.
Still yet another embodiment of the present disclosure provides a method for imaging hydrogen ions including injecting the two-photon fluorescent probe compound represented by Chemical Formula 4 into cells or tissues isolated from a living body; irradiating an excitation source to the cells or tissues isolated from the living body; and observing fluorescence generated from the two-photon fluorescent probe compound by two-photon microscopy.
Still yet another embodiment of the present disclosure provides a method for preparing a two-photon fluorescent probe compound represented by Chemical Formula 4, including reacting a compound represented by Chemical Formula 8 below with a compound represented by Chemical Formula 9 below:
In Chemical Formula 8, R2 is NO2 or NH2.
Still yet another embodiment of the present disclosure provides a two-photon fluorescent probe composition for simultaneously detecting calcium ions and hydrogen ions including at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3 and the compound represented by Chemical Formula 4.
Still yet another embodiment of the present disclosure provides a method for simultaneously imaging calcium ions and hydrogen ions including injecting at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3 and the two-photon fluorescent probe compound represented by Chemical Formula 4 into cells or tissues isolated from a living body; irradiating an excitation source to the cells or tissues isolated from the living body; and observing fluorescence generated from the two two-photon fluorescent probe compounds by two-photon microscopy.
According to the embodiments of the present disclosure, a blue emission two-photon fluorescent probe for detecting calcium ions is a two-photon fluorescence probe (marker) capable of selectively imaging ions to be detected by targeting specific organelles, that is, the cytoplasm, mitochondria, or plasma membrane, respectively, so that it is possible to provide two-photon probes with low cytotoxicity, high photostability, and pH independence.
In addition, a blue emission two-photon fluorescent probe for detecting calcium ions by targeting the cytoplasm, mitochondria, and plasma membrane, respectively, and a green emission two-photon fluorescent probe for detecting hydrogen ions by targeting the lysosome can simultaneously image calcium ions in the cytoplasm, mitochondria, or plasma membrane and hydrogen ions in the lysosome of live cells and tissues, thereby simultaneously monitoring a distribution of calcium ions and a change in acidity (pH) in vivo.
The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
In the following detailed description, reference is made to the accompanying drawing, which forms a part hereof. The illustrative embodiments described in the detailed description, drawing, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.
Hereinafter, the present disclosure will be described in detail.
In order to design a two-photon (TP) probe (marker) for detecting Ca2+ for multicolor imaging, the present inventors developed organelles-specific blue emission TP probes BCa-1-AM, BCa-2mito-AM, and BCa-3mem for Ca2+ by using 6-(benzo[d]oxazol-2-yl)-2-naphthalylamine (compound B) as a fluorophore and 0,0′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid (BAPTA) and 2-(2′-morpholino-2′-oxoethoxy)-N,N-bis(hydroxycarbonylmethyl)aniline (MOBHA) derivatives as Ca2+ receptors. Meanwhile, since BCa-3mem has too low aqueous solubility and its photophysical properties cannot be accurately measured in an aqueous buffer, the present inventors further designed BCa-3°mem to mimic BCa-3mem. In addition, the present inventors developed a TP probe FHEt-1lyso for detecting lysosomal H+ by using 2-acetyl-7-aminofluorene (Compound F) as a green emission TP fluorophore and 4-diethylaminophenyl group as an H+ receptor. The present inventors confirmed that the prepared TP probes for detecting Ca2+ had high selectivity for specific organelles (cytoplasm, mitochondria, plasma membrane, respectively) and calcium ions, confirmed that the TP probe for detecting Ca2+ and the TP probe for detecting lysosomal H+ could be used together to simultaneously monitor changes in cytoplasmic Ca2+ and lysosomal 1-1+ concentrations in live cells and tissues in real time by dual-color TP microscopy, and then completed the present disclosure.
At this time, the compounds B and F were prepared by deriving from BLT-blue targeting the lysosome and FMT-green targeting mitochondria, respectively. The BLT-blue and FMT-green trackers have maximum TP excited fluorescence (TPEF) at 451 and 523 nm, respectively, and have high TP action cross-section values (Φδeff) of 2120×and 1480×10−50 cm4s/photon (GM). In previous studies, it is confirmed that these trackers enable dual-color imaging of two organelles in probe-labeled cells.
In one aspect, the present disclosure provides a two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 below:
In Chemical Formulas 1 to 3, Y is each independently K, Na, CH3, or CH2OC(O)CH3, and in Chemical Formula 3, X is H, OH, or O(CH2)5CH3.
Preferably, in Chemical Formula 1, Y may be K or CH2OC(O)CH3.
Preferably, in Chemical Formula 2, the Y may be K or CH2OC(O)CH3.
Preferably, in Chemical Formula 3, Y may be K or CH3, most preferably K, and X may be H or O(CH2)5CH3.
According to one embodiment of the present disclosure, the compound represented by Chemical Formula 1 may be a compound represented by [BCa-1] or [BCa-1-AM] below:
According to one embodiment of the present disclosure, the compound represented by Chemical Formula 2 may be a compound represented by [BCa-2-mito] or [BCa-2-mito-AM] below:
According to one embodiment of the present disclosure, the compound represented by Chemical Formula 3 may be a compound represented by [BCa-3mem] or [BCa-3°mem] below:
The two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 according to the present disclosure may specifically bind to calcium ions (Ca2+), may react with calcium ions to form a complex, and may emit blue fluorescence with a fluorescence reaction to calcium ions.
The two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 according to the present disclosure may be for selectively imaging calcium ions in the cytoplasm, mitochondria, or plasma membrane.
According to one embodiment of the present disclosure, the two-photon fluorescent probe compound represented by Chemical Formula 1, the two-photon fluorescent probe compound represented by Chemical Formula 2, and the two-photon fluorescent probe compound represented by Chemical Formula 3 may be for selectively imaging calcium ions in the cytoplasm, calcium ions in the mitochondria, and calcium ions in the plasma membrane, respectively.
In addition, the present disclosure provides a two-photon fluorescent probe composition for detecting calcium ions in live cells or tissues including at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3.
The two-photon fluorescent probe composition for detecting calcium ions according to the present disclosure may be selectively imaging calcium ions in the cytoplasm, mitochondria, and/or plasma membrane. The depth of the imaging may be 90 to 140 μm.
The two-photon fluorescent probe composition for detecting calcium ions according to the present disclosure may also include only one compound among the compounds represented by Chemical Formula 1, 2, or 3, or a mixture of two compounds, and may also include all three compounds represented by Chemical Formulas 1, 2, and 3, respectively.
In one embodiment, the composition may be a two-photon fluorescent probe composition for detecting calcium ions in live cells or tissues including a compound represented by Chemical Formula 1 (preferably, [BCa-1] and/or [BCa-1-AM]), a compound represented by Chemical Formula 2 (preferably [BCa-2mito] and/or [BCa-2mito-AM]), and a compound represented by Chemical Formula 3 (preferably [BCa-3mem] and/or [BCa−3°mem]). The composition may be for simultaneously imaging calcium ions located in organelles, preferably the cytoplasm, mitochondria, and plasma membrane, respectively, and for simultaneously monitoring changes in distribution or concentration of calcium ions in the cytoplasm, mitochondria, and plasma membrane, respectively.
The two-photon fluorescent probe composition for detecting calcium ions according to the present disclosure may further include at least one fluorescent probe compound selected from the group consisting of MitoTracker Red, BLT-blue, and FMT-green.
Further, the present disclosure provides a method for imaging calcium ions including injecting at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3 into cells or tissues isolated from a living body; irradiating an excitation source to the cells or tissues isolated from the living body; and observing fluorescence generated from the two-photon fluorescent probe compound by two-photon microscopy.
The method for imaging calcium ions according to the present disclosure may target the cytoplasm, mitochondria, and/or plasma membrane to selectively image calcium ions in the cytoplasm, mitochondria, and/or plasma membrane. The depth of the imaging may be 90 to 140 μm.
In the method for imaging calcium ions according to the present disclosure, the injecting step may be injecting at least one compound itself selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3, and may be injecting the composition containing at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3, for example, the two-photon fluorescent probe composition for detecting calcium ions.
In addition, it is possible to monitor changes in calcium distribution or concentration in live cells or tissues using the method for imaging calcium ions according to the present disclosure, and it is also possible to quantitatively measure the changes as well as qualitative analysis.
In addition, in the method for imaging calcium ions according to the present disclosure, the injecting step may be further injecting at least one fluorescent probe compound selected from the group consisting of MitoTracker Red, BLT-blue, and FMT-green, and it is possible to simultaneously monitor changes in calcium distribution or concentration in each organelle using the same.
In addition, the present disclosure provides a method for preparing a two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 below, including reacting a compound represented by Chemical Formula 5 below with a compound represented by Chemical Formula 6 or 7 below:
In Chemical Formula 1, 2, 3, 5, 6, or 7, independently of each other, Y is Na, K, Na, CH3, or CH2OC(O)CH3, X is H, OH, O(CH2CH2O)2CH3, O(CH2)3Br, O(CH2)3PPh3+Br−, or O(CH2)5CH3, R1 is CO2C(CH3)3 or CO2H, Z is H or F, and L is H or NH2.
Preferably, the method for preparing the compounds represented by Chemical Formulas 1 and 2 may include reacting the compound represented by Chemical Formula 5 with the compound represented by Chemical Formula 6, and the method for preparing the compound represented by Chemical Formula 3 may include reacting the compound represented by Chemical Formula 5 with the compound represented by Chemical Formula 7.
In one embodiment, in Chemical Formula 1, Y is K or CH2OC(O)CH3, in Chemical Formula 5, X is OH, R1 is CO2H, and in Chemical Formula 6, Y is CH3, Z is H, and L may be NH2. At this time, the method for preparing the two-photon fluorescent probe compound represented by Chemical Formula 1 may further include substituting X in Chemical Formula 5 with O(CH2CH2O)2CH3 and substituting CH3 corresponding to Y in Chemical Formula 6 with K or CH2OC(O)CH3 corresponding to Y in Chemical Formula 1.
In one embodiment, in Chemical Formula 2, Y is K or CH2OC(O)CH3, in Chemical Formula 5, X is OH and R1 is CO2H, and in Chemical Formula 6, Y is CH3, Z is F, and L may be NH2. At this time, the method for preparing the two-photon fluorescent probe compound represented by Chemical Formula 2 may further include substituting the OH with O(CH2)3Br or O(CH2)3PPh3+Br− when X in Chemical Formula 5 is OH, and further include substituting the CH3 with K or CH2OC(O)CH3 when Y in Chemical Formula 6 is CH3.
In one embodiment, in Chemical Formula 3, Y is K or CH3 and X is O(CH2)5CH3 or H, in Chemical Formula 5, X is H or OH and R1 is CO2H, and in Chemical Formula 7, Y is CH3 and L may be NH2. At this time, the method for preparing the two-photon fluorescent probe compound represented by Chemical Formula 3 may further include substituting the OH with O(CH2)5CH3 when X in Chemical Formula 5 is OH, and further include substituting the CH3 with K or CH2OC(O)CH3 when Y in Chemical Formula 7 is CH3.
In one embodiment, the two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 may be prepared by a method including Reaction Formula 1 below, but is not limited thereto.
In Reaction Formula 1, reaction conditions may be (a) Compound 2, Pd(OAc)2, CuI, Cs2CO3, Xphos, dimethylformamide (DMF), 120° C.; (b) (i) trifluoroacetic acid, CH2Cl2, RT; (ii) HATU, (i-Pr)2NEt, H2N-BAPTA-Me, DMF, RT; (c) (i) Me(OCH2CH2)2OTs, K2CO3, DMF, 120° C.; (ii) KOH, EtOH/dioxane, 0° C.; (d) BrCH2OC(0)Me, (i-Pr)2NEt, MeCN/DMF, RT; (e) (i) BrCH2CH2CH2Br, K2CO3, DMF, 75° C.; (ii) PPh3, MeCN, 90° C.; (iii) KOH, EtOH/dioxane, RT; (f) n-C6H13Br, K2CO3, DMF, 80° C.; (ii) KOH, EtOH/dioxane, 0° C.; and (g) KOH, EtOH/dioxane, 0° C. The two-photon fluorescent probe compound represented by Chemical Formula 1 prepared by the method including Reaction Formula 1 may be BCa-1 or BCa-1-AM, the two-photon fluorescent probe compound represented by Chemical Formula 2 prepared by the method including Reaction Formula 1 may be the BCa-2mito or BCa-2mito-AM, and the two-photon fluorescent probe compound represented by Chemical Formula 3 prepared by the method including Reaction Formula 1 may be the BCa-3mem or BCa-3°mem.
In one aspect, the present disclosure provides a two-photon fluorescent probe compound represented by Chemical Formula 4 below:
In Chemical Formula 4, W is NH(CH2)2N(CH3)2 or OCH3. Preferably, W may be NH(CH2)2N(CH3)2.
According to one embodiment of the present disclosure, the compound represented by Chemical Formula 4 may be a compound represented by [FHEt-1lyso]:
The two-photon fluorescent probe compound represented by Chemical Formula 4 according to the present disclosure may specifically bind to hydrogen ions (H+) and emit green fluorescence by a fluorescence reaction with hydrogen ions.
The two-photon fluorescent probe compound represented by Chemical Formula 4 according to the present disclosure may be for selectively imaging hydrogen ions in the lysosome or for imaging changes in acidity (pH) in the lysosome.
In addition, the present disclosure provides a two-photon fluorescent probe composition for detecting hydrogen ions in live cells or tissues, including the two-photon fluorescent probe compound represented by Chemical Formula 4.
The two-photon fluorescent probe composition for detecting hydrogen ions according to the present disclosure may selectively image hydrogen ions in the lysosome, and the depth of the imaging may be 90 to 140 μm.
Further, the present disclosure provides a method for imaging hydrogen ions including injecting the two-photon fluorescent probe compound represented by Chemical Formula 4 into cells or tissues isolated from a living body; irradiating an excitation source to the cells or tissues isolated from the living body; and observing fluorescence generated from the two-photon fluorescent probe compound by two-photon microscopy.
The method of imaging hydrogen ions according to the present disclosure may be selectively imaging hydrogen ions in the lysosome by targeting the lysosome.
In addition, it is possible to monitor changes in distribution or concentration of hydrogen ions in live cells or tissues using the method for imaging the hydrogen ions according to the present disclosure, and it is also possible to quantitatively measure the changes as well as qualitative analysis and to monitor a change in pH.
In addition, the present disclosure provides a method for preparing a two-photon fluorescent probe compound represented by Chemical Formula 4 below, including reacting a compound represented by Chemical Formula 8 below with a compound represented by Chemical Formula 9 below:
In Chemical Formula 4, W is NH(CH2)2N(CH3)2 or OCH3, and in Chemical Formula 8, R2 is NO2 or NH2.
Preferably, in Chemical Formula 4, W may be NH(CH2)2N(CH3)2, and in Chemical Formula 8, R2 may be NH2. At this time, the method for preparing the two-photon fluorescent probe compound represented by Chemical Formula 4 according to the present disclosure may further include substituting CO2Me in Chemical Formula 8 with CO2NH(CH2)2N(CH3)2.
In one embodiment, the two-photon fluorescent probe compound represented by Chemical Formula 4 may be prepared by a method including Reaction Formula 2 below, but is not limited thereto.
In Reaction Formula 2, the reaction conditions may be (a) (i) BrCH2CO2Me, NaOtBu, dimethyl sulfoxide; (ii) HNO3, CH2Cl2, 0° C.; (iii) MeC(O)Cl, AlCl3, nitrobenzene, 60° C.; (b) Fe, NH4Cl(aq), EtOH/dioxane, reflux; (c) Compound A (compound represented by Chemical Formula 9), (i-Pr)2NEt, DMF; and (d) (i) KOH, EtOH, RT; (ii) N,N-dimethylethylenediamine, HATU, (i-Pr)2NEt, DMF, RT.
In one aspect, the present disclosure provides a two-photon fluorescent probe composition for simultaneously detecting calcium ions and hydrogen ions including at least one compound selected from two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3 below and a compound represented by Chemical Formula 4 below:
In Chemical Formulas 1 to 3, Y is each independently K, Na, CH3, or CH2OC(O)CH3, in Chemical Formula 3, X is H, OH or O(CH2)5CH3, and in Chemical Formula 4, W is NH(CH2)2N(CH3)2 or OCH3.
Preferably, in Chemical Formula 1, Y is K or CH2OC(O)CH3, in Chemical Formula 2, Y is K or CH2OC(O)CH3, in Chemical Formula 3, Y is K, and X is H or O(CH2)5CH3, and in Chemical Formula 4, W may be NH(CH2)2N(CH3)2.
According to one embodiment of the present disclosure, the compound represented by Chemical Formula 1 may be the compound represented by [BCa-1] or [BCa-1-AM], the compound represented by Chemical Formula 2 may be the compound represented by [BCa-2-mito] or [BCa-2-mito-AM], the compound represented by Chemical Formula 3 may be the compound represented by [BCa-3mem] or [BCa-3°mem], and the compound represented by Chemical Formula 4 may be the compound represented by [FHEt-1lyso].
In the two-photon fluorescent probe composition for simultaneously detecting calcium ions and hydrogen ions according to the present disclosure, the two-photon fluorescent probe compound represented by any one of Chemical Formulas 1 to 3 may target the cytoplasm, mitochondria, or plasma membrane to selectively image calcium ions in the cytoplasm, mitochondria, or plasma membrane, and the compound represented by Chemical Formula 4 may target the lysosome to selectively image hydrogen ions in the lysosome.
Preferably, the compound of Chemical Formula 1 may target the cytoplasm to selectively image calcium ions in the cytoplasm, the compound of Chemical Formula 2 may target the mitochondria to selectively image calcium ions in the mitochondria, the compound of Chemical Formula 3 may target the plasma membrane to selectively image calcium ions in the plasma membrane, and the compound of Chemical Formula 4 may target the lysosome to selectively image hydrogen ions in the lysosome.
Further, the present disclosure provides a method for simultaneously imaging calcium ions and hydrogen ions including injecting at least one compound selected from the two-photon fluorescent probe compounds represented by any one of Chemical Formulas 1 to 3 and the two-photon fluorescent probe compound represented by Chemical Formula 4 into cells or tissues isolated from a living body; irradiating an excitation source to the cells or tissues isolated from the living body; and observing fluorescence generated from the two-photon fluorescent probe compounds by two-photon microscopy.
In addition, by using the method of simultaneously imaging calcium ions and hydrogen ions according to the present disclosure, it is possible to monitor changes in distribution or concentration of calcium ions in the cytoplasm, mitochondria, and/or plasma membrane and hydrogen ions in the lysosome in live cells or tissues and also to measure quantitatively the changes as well as qualitative analysis.
In the method of simultaneously imaging calcium ions and hydrogen ions according to the present disclosure, the imaging may be simultaneously imaging changes in distribution of calcium ions in the cytoplasm, mitochondria, and/or plasma membrane and pH of the lysosome.
In one embodiment of the present disclosure, it was confirmed that organelles-specific blue emission TP probes BCa-1-AM, BCa-2mito-AM, and BCa-3mem for Ca2+ prepared by the method according to the present disclosure had a maximum absorption (λmax) of 350 to 358 nm, a maximum emission (λfl) of 464 to 466 nm, and a TP action cross-section (Φδmax) of 55 to 70×10−50 cm4s/photon at 750 nm in the presence of excess Ca2+. It was confirmed that the TP probes had dissociation constants of 0.18, 2.7, and 100 μM, which were appropriate values for detecting Ca2+ in the cytoplasm, mitochondria, and plasma membrane (cell membrane), respectively, and it was confirmed through TP microscopic analysis that the TP probes facilitated real-time detection of Ca2+ in the cytoplasm, mitochondria, and plasma membrane of live cells and tissues. In addition, it was confirmed that the green emission TP probe FHEt-1lyso for lysosomal H+ prepared by the method according to the present disclosure had λmax of 359 nm, λfl of 571 nm, and Φδmax of 70×10−50 cm4s/photon in a universal buffer at pH 4.3, and it was confirmed through TP microscopy analysis that the TP probe facilitated the detection of H+ in the lysosome. Specifically, it was confirmed that by using BCa-1-AM (or BCa-1) and FHEt-1lyso, it is possible to simultaneously monitor changes in cytoplasmic Ca2+ and lysosomal H+ concentrations in live cells and tissues (or Hela cells) in real time by dual-color TP microscopy.
Hereinafter, preferred Examples will be proposed in order to help in understanding of the present disclosure. However, the following Examples are just provided to more easily understand the present disclosure and the contents of the present disclosure are not limited by Examples.
Preparation Example 1. Devices and Materials
1H NMR and 13C NMR spectra were measured by using CDCl3, (CD3)250, CD3OD, or CD3CN as a solvent and internal reference [CDCl3: δ H 7.26, δ C 77.0; (CD3)2SO: δ H 2.50, δ C 39.51; CD3CN: δ H 1.94, δ C 1.39] in Varian 300 (300 MHz for 1H NMR and 75 MHz for 13C NMR) and Bruker Avance III 500 and 600 (500 and 600 MHz for 1H NMR and 151 MHz for 13C NMR) spectrometers.
High-resolution mass spectra (HRMS) were analyzed using AB SCIEX Triple TOF® 5600 plus (for BCa-1, BCa-2 onto-AM, BCa-3mem, BCa−3°mem and FHEt-1lyso) and Thermo Scientific Q Exactive (for BCa-1-AM and BCa-2 onto) mass spectrometry.
All chemical reagents were purchased from commercial suppliers and used without further purification unless otherwise specified.
Compound 3°, Compound C2, BAPTA-Me, BAPTA-F-Me, and MOBHA-Me were synthesized according to the related art [Analytical Chemistry (2012) 84:8110-8113, Angewandte Chemie International Edition (2013) 52:3874-3877, Nature Chemical Biology (2007) 3:423-431]. Synthesis of other compounds was shown in Reaction Formulas 1-1 and 1-2 below.
First, BCa-1, BCa-1-AM, BCa-2mito, BCa-2 onto-AM, BCa-3mem, and BCa-3°mem were synthesized by a method of Reaction Formula 1-1 below.
Briefly, Compound 3 was prepared by combining Compound 1 and Compound 2 in 70% yield. Compound 3 was hydrolyzed and then combined with an receptor moiety (BAPTA-Me, BAPTA-F-Me, or MOBHA-Me) to synthesize Compounds 4 to 7 in yields of 65% to 89%. Compounds 4 to 7 prepared BCa-1, BCa-3mem, BCa−3°mem, and BCa-2 mito in yield of 47% to 72% through introduction of one targeting moiety of the organelles and hydrolysis of ester groups. BCa-1 and BCa-2 onto reacted with BrCH2OC(0)Me to prepare BCa-1-AM and BCa-2mito-AM in yield of 28% and 29%, respectively. A crude product (97% purity according to high-performance liquid chromatography) was used for additional experiments because the product was degraded slowly during the work-up (
Meanwhile, FHEt-1lyso was prepared from 9-methylfluorene by the method of Reaction Formula 2-1 below.
Briefly, Compound 8 was obtained in 41% yield of the total by reacting 9-methylfluorene with BuLi and then through an alkylation process using methyl bromoacetate, nitration, and acylation. Compound 9 was obtained in 46% yield through a reduction reaction of a nitro group and a reaction between an amino group and Compound A. After hydrolysis of the ester group, FHEt-1lyso was prepared in yield of 40% through binding to 4-diethylaminoaniline.
Structures and specific synthetic processes of each compound of Reaction Formulas 1-1 and 1-2 were shown below.
1-1. Preparation of Compound 1
A solution of 6-bromo-2-aminonaphthalene (6.7 g, 30 mmol), K2CO3 (12.4 g, 90 mmol), and BrCH2CO2Bu-t (8.8 g, 45 mmol) in MeCN (60 mL) was refluxed for 12 hours. The mixture was cooled to room temperature (RT), diluted with distilled water, and then extracted with ethyl acetate. The organic layer was washed with brine, dried with anhydrous Na2SO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using hexane/EtOAc (3: 1) as a mobile phase to obtain Compound 1 in a white solid state (yield 7.8 g, 78%).
1H NMR (500 MHz, CDCl3): δ 7.82 (1H, d, J=2.0 Hz), 7.54 (1H, d, J=8.8 Hz), 7.47 (1H, d, J=8.7 Hz), 7.42 (1H, dd, J=8.7, 2.0 Hz), 6.94 (1H, dd, J=8.8, 2.4 Hz), 6.67 (1H, d, J=2.4 Hz), 4.56 (1 H, br s), 3.89 (2 H, s), 1.51 (9 H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 169.9, 145.0, 133.5, 129.6, 129.5, 128.7, 128.1, 127.6, 118.8, 115.3, 104.4, 82.2, 46.3, 28.0(3) ppm.
1-2. Preparation of Compound 2
A solution containing 5-hydroxybenzoxazole (9.0 g, 67 mmol), t-butyldimethylsily chloride (TBDMS-Cl; 12.1 g, 80 mmol), imidazole (0.45 g, 6.7 mmol), and Et3N (8.1 g, 80 mmol) in CH2Cl−2 (200 mL) was stirred at room temperature for 1 hour. The mixture was added with distilled water and a product was extracted with CH2Cl−2. The organic layer was washed with brine and dried with anhydrous MgSO4 and then the solvent was evaporated. The residue was purified by silica gel column chromatography using hexane/EtOAc (3: 1) as a mobile phase to obtain Compound 2 in a pale yellow oil state (yield 15.7 g, 94%).
1H NMR (500 MHz, CDCl3): δ 8.03 (1 H, s), 7.39 (1H, d, J=8.8 Hz), 7.22 (1 H, d, J=2.4 Hz), 6.89 (1H, dd, J=8.8, 2.4 Hz), 0.99 (9 H, s), 0.20 (6 H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 153.2, 152.9, 145.0, 140.8, 118.7, 110.7, 110.7, 25.6(3), 18.2,-4.6(2) ppm.
1-3. Preparation of Compound 3
Compound 1 (1.1 g, 3.3 mmol), Compound 2 (1.3 g, 5.0 mmol), Cs2CO3 (1.3 g, 5.0 mmol), CuI (0.13 g, 0.67 mmol), Xphos (0.16 mg, 0.33 mmol), and Pd(OAc)2 (0.036 g, 0.16 mmol) were added sequentially to DMF (10 mL) in an argon (Ar) atmosphere. The mixture was stirred at 120° C. for 3 hours, cooled to room temperature, diluted with CH2Cl−2, and stirred for 10 minutes. Thereafter, the mixture was filtered through a Celite pad and washed with CH2Cl−2. The filtrate was evaporated, and the residue was purified by silica gel column chromatography using hexane/EtOAc (1: 1) as a mobile phase to obtain Compound 3 in a pale yellow solid state (yield 0.90 g, 70%).
1H NMR (500 MHz, CD3OD/CDCl3 at 1:4):δ 8.48 (1H, d, J=1.7 Hz), 8.04 (1H, dd, J=8.8, 1.7 Hz), 7.73 (1H, d, J=8.8 Hz), 7.66 (1H, d, J=8.8 Hz), 7.37 (1H, dd, J=8.8 Hz), 7.09 (1H, d, J=2.4 Hz), 6.99 (1 H, dt, J=8.8, 2.4 Hz), 6.83 (1 H, dd, J=8.8, 2.4 Hz), 6.69 (1H, d, J=2.4 Hz), 3.91 (2 H, s), 1.47 (9 H, s) ppm; 13C NMR (75 MHz, CD3OD/CDCl3 at 1/4):δ 170.1, 164.4, 154.3, 146.5, 144.4, 142.1, 136.6, 130.0, 127.8, 126.6, 126.4, 123.9, 119.7, 118.6, 113.2, 110.3, 104.0, 103.8, 82.3, 45.8, 27.7(3) ppm.
1-4. Preparation of Compound C1
A mixture of TFA and CH2Cl−2 (1: 1, 20 mL) was dropped to a solution of Compound 3 (3.0 g, 7.7 mmol) in CH2Cl−2 (10 mL) at 0° C., stirred at room temperature overnight, and added with diethyl ether (30 mL), and then the solvent was evaporated. The formed precipitate was resuspended in diethyl ether, filtered, and washed with diethyl ether. The product (Compound C1) was obtained in the form of a dark yellow solid (yield 2.3 g, 89%).
1H NMR (500 MHz, DMSO-d6): δ 9.51 (1 H, br s), 8.50 (1H, d, J=1.7 Hz), 8.01 (1H, dd, J=8.7, 1.7 Hz), 7.85 (1H, d, J=8.8 Hz), 7.71 (1H, d, J=8.7 Hz), 7.55 (1H, d, J=8.8 Hz), 7.15 (1H, dd, J=8.8, 2.4 Hz), 7.07 (1H, d, J=2.4 Hz), 6.81 (1H, dd, J=8.8, 2.4 Hz), 6.74 (1H, d, J=2.4 Hz), 3.97 (2 H, s) ppm; 13C NMR (75 MHz, DMSO-d6): δ 172.3, 163.7, 155.0, 148.2, 144.0, 142.9, 136.7, 129.9, 127.5, 126.4, 125.9, 123.9, 119.2, 119.1, 113.2, 110.6, 104.6, 102.6, 44.5 ppm.
1-5. Preparation of Compound 4
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU; 0.57 g, 1.5 mmol) was added to a solution containing Compound C1 (0.42 g, 1.3 mmol), BAPTA-Me (0.65 g, 1.2 mmol), and (i-Pr)2NH (0.43 mL, 2.5 mmol) in DMF (3.8 mL). The mixture was stirred at room temperature for 1 hour, then added with distilled water and the product was extracted with CH2Cl2. The organic layer was washed with brine and dried with anhydrous Na2SO4, and then the solvent was evaporated. The residue was purified by silica gel column chromatography using CH2Cl2/EtOAc (1: 2) as a mobile phase to obtain Compound C4 in a pale yellow solid state (yield 1.0 g, 89%).
1H NMR (500 MHz, CD3CN): δ 8.60 (1 H, br s), 8.55 (1H, s), 8.09 (1 H, dd, J=8.8, 1.8 Hz), 7.87 (1 H, d, J=8.9 Hz), 7.75 (1H, d, J=8.8 Hz), 7.46 (1H, d, J=8.8 Hz), 7.26 (1H, d, J=2.3 Hz), 7.15 (1H, dd, J=8.9, 2.4 Hz), 7.12 (1H, d, J=2.4 Hz), 7.05 (1H, dd, J=8.7, 2.3 Hz), 7.03 (1H, s), 6.82-6.92 (5H, m), 6.74-6.79 (2H, m), 5.52 (1H, t, J=5.9 Hz), 4.16-4.21 (4H, m), 4.05 (4H, s), 4.04 (4H, s), 4.00 (2H, d, J=5.6 Hz), 3.50 (6 H, s), 3.48 (6 H, s) ppm; 13C NMR (75 MHz, CD3CN): δ 172.7(2), 172.6(2), 169.4, 165.2, 155.3, 151.0, 150.8, 148.6, 145.9, 144.2, 139.9, 137.8, 136.2, 133.9, 130.9, 128.4, 127.8, 127.6, 125.2, 122.6, 122.0, 121.5, 120.2, 119.6, 119.2, 113.9, 113.8, 113.1, 111.4, 106.4, 105.7, 104.8, 68.0, 67.8, 54.0(4), 52.1(4), 48.7 ppm.
1-6. Preparation of Compound C3
A solution of Compound 4 (0.57 g, 0.66 mmol), 2-(2-methoxyethoxy)ethyl 4-methyl benzenesulfonate (0.24 g, 0.86 mmol) and K2CO3 (0.14 g, 0.99 mmol) in DMF (2.2 mL) was stirred at 90° C. for 10 hours, and then the mixture was diluted with distilled water, and extracted with CH2Cl−2. The organic layer was washed with brine and dried with anhydrous MgSO4 and then the solvent was evaporated. The residue was purified by silica gel column chromatography using EtOAc as the mobile phase to obtain Compound C3 in a yellow solid state (yield 0.56 g, 84%).
1H NMR (500 MHz, CDCl3): δ 8.60 (1H, s), 8.35 (1H, s), 8.18 (1H, dd, J=8.7, 1.8 Hz), 7.83 (1 H, d, J=8.9 Hz), 7.73 (1 H, d, J=8.7 Hz), 7.45 (1 H, d, J=8.8 Hz), 7.29 (1H, d, J=2.0 Hz), 7.25 (1H, d, J=1.8 Hz), 7.05 (1H, dd, J=8.9, 1.8 Hz), 6.96 (2H, dd, J=8.8, 2.4 Hz), 6.81-6.92 (6H, m), 6.77 (1H, d, J=8.7 Hz), 4.24-4.29 (4H, m), 4.20--4.22 (2H, m), 4.14 (4H, s), 4.10 (4H, s), 4.05 (2 H, s), 3.90-3.93 (2H, m), 3.74-3.77 (2H, m), 3.60-3.62 (2H, m), 3.57 (6H, s), 3.54 (6H, s), 3.41 (3 H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 171.9(2), 171.8(2), 168.2, 164.2, 156.1, 150.3, 150.1, 146.3, 145.2, 142.7, 139.0, 136.3, 135.6, 132.4, 130.2, 127.6, 127.1, 126.7, 124.4, 122.2, 121.3, 120.8, 118.9, 118.8, 118.4, 113.6, 112.7, 112.4, 110.3, 105.5, 105.0, 103.1, 71.8, 70.6, 69.7, 67.9, 67.0, 66.7, 59.0, 53.2(4), 51.6(4), 48.8 ppm.
1-7. Preparation of BCa-1
A solution of Compound C3 (0.024 g, 0.025 mmol) in dioxane (0.20 mL) and EtOH (0.30 mL) was dropped with KOH (aq) (1 N, 0.20 mL, 0.20 mmol) while stirring at 0° C. The mixture was stirred at room temperature for 5 hours, and then dropped with HCl (aq) (1 N, 0.10 mL, 0.10 mmol). After 10 minutes, the mixture was dropped to a vial containing MeCN (2 mL) while stirring. The formed precipitate was obtained by filtration and washed with diethyl ether to obtain BCa-1 in a yellow solid state (yield 0.018 g, 67%).
1H NMR (500 MHz, DMSO-d6): δ 8.46 (1H, s), 7.96 (1H, d, J=8.7 Hz), 7.82 (1H, d, J=8.9 Hz), 7.72 (1H, d, J=8.7 Hz), 7.55 (1H, d, J=8.7 Hz), 7.20 (1H, d, J=2.3 Hz), 7.09 (1H, d, J=8.9 Hz), 6.99 (1 H, br s), 6.95 (2H, dd, J=9.0, 2.3 Hz), 6.76-6.87 (7H, m), 4.15-4.23 (4H, m), 4.08-4.12 (2H, m), 3.99 (2 H, s), 3.72-3.75 (2H, m), 3.56-3.59 (2H, m), 3.44-3.47 (2H, m), 3.42 (4H, s), 3.39 (4H, s), 3.21 (3 H, s) ppm; 13C NMR (151 MHz, DMSO-d6): δ 178.0(2), 177.9(2), 171.7, 165.6, 157.4, 151.3, 151.3, 149.1, 146.1, 143.0, 141.5, 138.6, 138.0, 133.7, 131.6, 129.1, 128.1, 127.6, 125.2, 123.9, 122.4, 120.5, 120.1, 119.6(2), 115.3, 114.7, 113.2, 112.6, 106.8, 104.7, 104.3, 72.3, 70.8, 70.2, 69.2, 67.4, 67.1, 59.4(4), 58.8(2) ppm. HRMS (electrospray ionization; ESI) m/z [M+K]+ calculated for C46H47KN5O15: 948.2701, found: 948.2706.
1-8. Preparation of BCa-1-AM
A solution of BCa-1 (0.045 g, 0.049 mmol), bromomethyl acetate (0.075 g, 0.49 mmol), and DIPEA (0.050 g, 0.39 mmol) in MeCN (0.99 mL) was stirred at room temperature for 40 hours under an Ar atmosphere. Thereafter, the product was extracted with CH2Cl−2, washed with brine, and dried with anhydrous Na2SO4 and then the solvent was evaporated. The residue was purified by semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC; YMC-PACK ODS-A, 5 μm, 250×20 mm). At this time, RP-HPLC was performed on a YMC HPLC system (YMC LC-Forte/R 100) using MeCN/H2O (1: 1) containing 0.1% formic acid at a flow rate of 10 mL/min for 20 minutes. The obtained product, BCa-1AM was a yellow solid, and the yield was 0.016 g (28%).
1H NMR (500 MHz, CDCl3): δ 8.59 (1H, s), 8.39 (1H, s), 8.17 (1H, d, J=9.0 Hz), 7.82 (1H, d, J=9.0 Hz), 7.73 (1H, d, J=9.0 Hz), 7.44 (1H, d, J=9.0 Hz), 7.31 (1H, d, J=2.0 Hz), 7.25 (1H, d, J=2.1 Hz), 7.04 (1 H, dd, J=9.0, 2.1 Hz), 7.00 (1H, d, J=8.5 Hz), 6.96 (1H, d, J=8.7 Hz), 6.93 (1H, d, J=8.5 Hz), 6.84-6.90 (4H, m), 6.82 (1H, d, J=8.7 Hz), 5.64 (4H, s), 5.60 (4H, s), 4.82 (1H, t, J=5.4 Hz), 4.25-4.32 (4H, m), 4.19-4.22 (2H, m), 4.18 (4H, s), 4.15 (4H, s), 4.05 (2H, d, J=5.2 Hz), 3.89-3.91 (2H, m), 3.74-3.77 (2H, m), 3.59-3.62 (2H, m), 3.41 (3H, s), 2.05 (12 H, s) ppm; 13C NMR (151 MHz, CDCl3): δ 170.2(2), 170.0(2), 169.6(2), 169.5(2), 168.0, 164.3, 156.4, 150.8, 150.5, 146.2, 145.5, 143.0, 138.6, 136.4, 135.4, 132.9, 130.5, 127.8, 127.6, 126.9, 124.8, 123.0, 121.7, 121.5, 120.3, 119.8, 118.4, 113.9, 113.6, 112.9, 110.5, 106.4, 105.6, 103.7, 79.3(2), 79.2(2), 71.9, 70.8, 69.8, 68.2, 67.4, 67.0, 59.1, 53.4(2), 53.4, 53.3, 49.2, 20.7(2), 20.7(2) ppm. HRMS (ESI) m/z [M+Na]+ calcd for C58H63N5NaO23: 1220.3806, found: 1220.3807.
1-9. Preparation of Compound 5
HATU (0.55 g, 1.4 mmol) was added to a solution of Compound C1 (0.40 g, 1.2 mmol), BAPTA-F-Me (0.68 g, 1.2 mmol), and (i-Pr)2NH (0.41 mL, 2.4 mmol) in DMF (4 mL). The mixture was stirred at room temperature for 2 hours, then added with distilled water and the product was extracted with CH2Cl2. The organic layer was washed with brine and dried with anhydrous Na2SO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using CH2Cl2/EtOAc (1: 2) as a mobile phase to obtain Compound 5 (0.68 g, 65%) in a pale yellow solid state.
1H NMR (500 MHz, CDCl3): δ 8.60 (d, J=1.8 Hz, 1H), 8.18 (dd, J=8.8, 1.8 Hz, 1H), 8.18 (br s, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.71 (d, J=8.7 Hz, 1H), 7.41 (d, J=8.7 Hz, 1H), 7.26 (br s, 1H), 7.20 (d, J=2.5 Hz, 1H), 7.05-6.97 (m, 2H), 6.92-6.83 (m, 4H), 6.72-6.65 (m, 2H), 4.39-4.35 (m, 2H), 4.30-4.26 (m, 2H), 4.17 (s, 4H), 4.14 (s, 4H), 4.03 (s, 2H), 3.66 (s, 6H), 3.63 (s, 6H) ppm; 13C NMR (75 MHz, CD3OD/CDCl3 at 1/9): δ 171.9(2), 171.5(2), 168.4, 164.2, 156.4 (d, J=245.1 Hz), 154.4, 150.6, 146.6, 144.6, 143.7 (d, J=3.8 Hz), 142.3, 138.3 (d, J=12.8 Hz), 136.5, 135.6, 132.3, 130.3, 127.8, 127.2, 126.8, 124.2, 123.6 (d, J=9.7 Hz), 120.6, 119.8, 118.5, 114.7, 113.4, 112.5, 110.4, 109.9 (J=19.8 Hz), 106.1, 104.9, 104.3, 71.3, 67.6, 53.4(4), 51.7(2), 51.6(2), 48.4 ppm.
1-10. Preparation of Compound C4
A solution of Compound 5 (0.55 g, 0.62 mmol), 1,3-dibromopropane (0.31 mL, 3.1 mmol), and K2CO3 (0.34 g, 2.5 mmol) in DMF (2.5 mL) was stirred for 6 hours at 72° C. Then, distilled water was added and the product was extracted using CH2Cl2. The organic layer was washed with brine and dried with anhydrous Na2SO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using EtOAc as a mobile phase to obtain Compound C4 (0.37 g, 60%) in a pale yellow solid state.
1H NMR (500 MHz, CDCl3): δ 8.58 (d, J=1.8 Hz, 1H), 8.35 (s, 1H), 8.17 (dd, J=8.8, 1.8 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 7.71 (d, J=8.8 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.26 (br s, 1H), 7.26 (s, 1H), 7.02 (dd, J=8.8, 2.4 Hz, 1H), 7.00-6.81 (m, 5H), 6.71-6.62 (m, 2H), 4.38-4.34 (m, 2H), 4.25-4.21 (m, 2H), 4.18-4.14 (m, 2H), 4.17 (s, 4H), 4.16 (s, 4H), 4.02 (s, 2H), 3.65 (s, 6H), 3.64 (t, J=6.1 Hz, 2H), 3.61 (s, 6H), 2.36 (p, J=6.1 Hz, 2H) ppm; 13C NMR (75 MHz, CDCl3): δ 171.7(2), 171.3(2), 167.9, 164.4, 156.5 (d, J=245.4 Hz), 156.3, 150.7, 146.4, 145.5, 143.8 (d, J=3.7 Hz), 143.0, 138.4 (d, J=12.6 Hz), 136.4, 135.6, 132.5, 130.4, 127.8, 127.4, 126.9, 124.6, 123.7 (d, J=9.4 Hz), 121.2, 119.8, 118.4, 114.8, 113.6, 112.5, 110.5, 110.0 (d, J=19.8 Hz), 106.2, 105.4, 103.6, 71.3, 67.7, 66.1, 53.4(4), 51.7(2), 51.6(2), 49.0, 32.3, 30.0 ppm.
1-11. Preparation of Compound C5
A solution of Compound C4 (0.21 g, 0.21 mmol) and Ph3P (0.55 g, 2.1 mmol) in MeCN (2 mL) was stirred at 90° C. for 12 hours. The solvent was evaporated and the residue was purified through silica gel column chromatography using CH2Cl2/MeOH (10: 1) as a mobile phase to obtain Compound C5 (0.11 g, 40%) in a yellow solid state.
1H NMR (500 MHz, CDCl3): δ 9.46 (br s, 1H), 8.16 (d, J=1.7 Hz, 1H), 7.80 (dd, J=8.9, 1.7 Hz, 1H), 7.77-7.66 (m, 9H), 7.67-7.58 (m, 6H), 7.47 (d, J=2.3 Hz, 1H), 7.44 (d, J=8.9 Hz, 1H), 7.37 (d, J=8.7 Hz, 1H), 7.15 (dd, J=8.7, 2.2 Hz, 1H), 7.12 (d, J=8.8 Hz, 1H), 7.08 (dd, J=8.7, 2.3 Hz, 1H), 6.91-6.81 (m, 2H), 6.72 (d, J=8.7 Hz, 1H), 6.69 (dd, J=8.8, 2.4 Hz, 1H), 6.66-6.60 (m, 3H), 6.23 (s, 1H), 4.34-4.29 (m, 2H), 4.21-4.11 (m, 8H), 4.11 (s, 4H), 4.01 (s, 2H), 3.86-3.76 (m, 2H), 3.60 (s, 6H), 3.59 (s, 6H), 2.08-2.02 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3): δ 171.8(2), 171.3(2), 169.0, 164.2, 156.5 (d, J=245.5 Hz), 155.3, 150.5, 147.1, 145.2, 143.8 (d, J=3.9 Hz), 142.7, 138.4 (d, J=12.9 Hz), 136.4, 135.2(3) (d, J=3.0 Hz), 134.9, 133.6, 133.4(6) (d, J=10.0 Hz), 130.5 (6) (d, J=12.6 Hz), 129.7, 127.5, 126.6, 126.4, 123.9, 123.6 (d, J=9.3 Hz), 119.8, 119.7, 119.0, 117.7 (3) (d, J=86.4 Hz), 114.9, 112.4, 112.2, 110.2, 109.9 (d, J=19.4 Hz), 105.8, 104.3, 103.6, 71.4, 67.5, 66.9 (d, J=16.1 Hz), 53.4(4), 51.7(2), 51.5(2), 48.6, 22.8, 19.6 (d, J=53.4 Hz) ppm.
1-12. Preparation of BCa-2mito
A solution of Compound C5 (0.022 g, 0.017 mmol) in dioxane (0.070 mL) and MeOH (0.21 mL) was dropped with KOH (aq) (1 N, 0.14 mL, 0.14 mmol) while stirring at 0° C. The mixture was stirred at room temperature for 24 hours, and then dropped with HCl (aq) (1 N, 0.070 mL, 0.070 mmol). The crude product (97% purity according to HPLC) was used in the next step without additional purification.
HRMS (ESI) m/z [M]+ calcd for C62H56FN5O13P+: 1128.3591, found: 1128.3587.
1-13. Preparation of BCa-2mito-AM
A solution of BCa-2 mite (0.089 g, 0.074 mmol), bromomethyl acetate (0.28 g, 1.9 mmol), and DIPEA (0.19 g, 1.5 mmol) in MeCN (1.4 mL) and DMF (0.90 mL) was stirred at room temperature for 72 hours under an Ar atmosphere. The product was extracted with CH2Cl−2, washed with brine, and dried with anhydrous Na2SO4 and then the solvent was evaporated. The residue was purified by performing medium-pressure liquid chromatography (Yamazen ODS-SM, 50 μm, 2.3×12.3 cm) in a Yamazen MPLC system (Yamazen W-Prep 2XY) using MeCN/H2O (1: 1) containing 0.1% formic acid as a mobile phase at a flow rate of 10 mL/min for 20 minutes. The obtained product, BCa-2mito-AM was a pale yellow solid, and the yield was 0.032 g (29%).
1H NMR (500 MHz, CD3CN): δ9.15 (br s, 1H), 8.50 (d, J=1.8 Hz, 1H), 8.05 (dd, J=8.7, 1.8 Hz, 1H), 7.88-7.81 (m, 4H), 7.77-7.67 (m, 13H), 7.51 (d, J=8.9 Hz, 1H), 7.34 (d, J=2.2 Hz, 1H), 7.21 (d, J=2.4 Hz, 1H), 7.17 (dd, J=8.9, 2.2 Hz, 1H), 7.12 (dd, J=8.5, 2.4 Hz, 1H), 6.97-6.91 (m, 2H), 6.86 (d, J=2.0 Hz, 1H), 6.80 (d, J=8.5 Hz, 1H), 6.77-6.70 (m, 2H), 5.89 (t, J=5.6 Hz, 1H), 5.62 (s, 4H), 5.60 (s, 4H), 4.33-4.29 (m, 2H), 4.22-4.11 (m, 12H), 4.02 (d, J=5.6 Hz, 2H), 3.49-3.42 (m, 2H), 2.13-2.06 (m, 2H), 1.97 (s, 6H), 1.96 (s, 6H) ppm; 13C NMR (75 MHz, CDCl3): δ170.0(2), 169.5(2), 169.4(2), 169.0(2), 166.9, 164.4, 156.6 (d, J=245.3 Hz), 155.2, 150.8, 147.2, 145.3, 143.3 (d, J=3.3 Hz), 142.9, 138.7 (d, J=12.7 Hz), 136.6, 135.3(3) (d, J=2.8 Hz), 134.3, 134.2, 133.2(6) (d, J=9.9 Hz), 130.5(6) (d, J=12.5 Hz), 129.8, 127.6, 126.5, 126.4, 124.0, 123.7 (d, J=9.4 Hz), 120.4, 119.8, 118.9, 117.5(3) (d, J=86.5 Hz), 115.3, 115.3, 112.4, 110.4 (d, J=19.3 Hz), 110.3, 106.0, 104.1, 103.6, 79.0(4), 71.4, 67.6, 66.7 (d, J=16.3 Hz), 53.3(4), 48.4, 22.6, 20.6(4), 19.3 (d, J=53.8 Hz) ppm. HRMS (ESI) m/z [M]+ calcd for C74H72PN5O21P+: 1416.4436, found: 1416.4442.
1-14. Preparation of Compound 6
HATU (0.89 g, 2.3 mmol) was added to a solution of Compound C1 (0.68 g, 2.0 mmol), MOBHA-Me (0.76 g, 1.9 mmol), and (i-Pr)2NH (0.69 mL, 2.0 mmol) in DMF (6 mL), and the mixture was stirred at room temperature for 30 minutes. The mixture was added with distilled water and extracted with EtOAc, and then the organic layer was washed with brine and dried with anhydrous Na2SO4 and the solvent was evaporated. The product was purified through silica gel column chromatography using EtOAc/i-PrOH (98: 2) as a mobile phase to obtain Compound 6 in a pale yellow solid state (yield 1.09 g, 78%).
1H NMR (500 MHz, DMSO-d6/CD3CN at 1:5):δ 9.36 (s, 1H), 9.08 (s, 1H), 8.56 (d, J=1.7 Hz, 1H), 8.10 (dd, J=8.8, 1.7 Hz, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.76 (d, J=8.8 Hz, 1H), 7.48 (d, J=8.6 Hz, 1H), 7.26 (d, J=2.3 Hz, 1H), 7.20 (dd, J=8.8, 2.4 Hz, 1H), 7.13 (d, J=2.4 Hz, 1H), 7.09 (dd, J=8.6, 2.3 Hz, 1H), 6.88 (dd, J=8.6, 2.4 Hz, 1H), 6.86 (d, J=2.4 Hz, 1H), 6.83 (d, J=8.6 Hz, 1H), 6.08 (t, J=5.9 Hz, 1H), 4.77 (s, 2H), 4.15 (s, 4H), 4.03 (d, J=5.9 Hz, 2H), 3.66-3.58 (m, 4H), 3.65 (s, 6H), 3.52-3.44 (m, 4H) ppm; 13C NMR (75 MHz, DMSO-d6/CD3CN at 1:5):δ 171.5(2), 168.5, 166.0, 164.0, 155.2, 149.6, 148.2, 144.4, 143.2, 136.9, 134.8, 133.6, 130.0, 127.5, 126.5, 126.5, 124.2, 120.0(2), 119.4, 113.2, 112.6, 110.4, 105.9, 104.7, 103.3, 66.3, 66.2, 66.0, 53.1(2), 51.3(2), 47.3, 45.0, 41.8 ppm.
1-15. Preparation of Compound C6
A solution of Compound 6 (0.15 g, 0.21 mmol), 1,3-dibromopropane (31 μL, 0.23 mmol), and K2CO3 (37 mg, 0.27 mmol) dissolved in DMF (0.7 mL) was stirred at 80° C. for 16 hours. Next, distilled water was added and the product was extracted with EtOAc, and then the organic layer was washed with brine and dried with anhydrous Na2SO4 and the solvent was evaporated. The residue was purified through silica gel column chromatography using CH2Cl2/EtOAc (1: 10) as a mobile phase to obtain Compound C6 in a white solid state (yield 0.14 g, 85%).
1H NMR (500 MHz, CDCl3): δ 8.61 (d, J=1.7 Hz, 1H), 8.35 (br s, 1H), 8.19 (dd, J=8.7, 1.7 Hz, 1H), 7.83 (d, J=8.7 Hz, 1H), 7.73 (d, J=8.7 Hz, 1H), 7.45 (d, J=8.7 Hz, 1H), 7.35 (d, J=2.3 Hz, 1H), 7.25 (d, J=2.5 Hz, 1H), 7.05 (dd, J=8.7, 2.3 Hz, 1H), 6.95 (dd, J=8.7, 2.3 Hz, 1H), 6.94 (dd, J=8.9, 2.5 Hz, 1H), 6.87 (d, J=8.9 Hz, 1H), 6.87 (d, J=2.3 Hz, 1H), 4.74 (s, 2H), 4.12 (s, 4H), 4.05-3.99 (m, 4H), 3.67-3.64 (m, 4H), 3.66 (s, 6H), 3.62-3.58 (m, 2H), 3.53-3.49 (m, 2H), 1.82 (m, 2H), 1.49 (m, 2H), 1.36 (m, 4H), 0.95-0.87 (m, 3H) ppm; 13C NMR (75 MHz, CDCl3): δ 171.5(2), 168.1, 166.2, 164.2, 156.8, 150.0, 146.3, 145.2, 143.0, 136.4, 135.7, 132.8, 130.4, 127.7, 127.4, 126.8, 124.6, 121.3, 120.3, 118.4, 113.8, 113.6, 110.4, 107.2, 105.4, 103.4, 68.8, 67.3, 66.7, 66.6, 53.5(2), 51.7(2), 49.0, 45.4, 42.2, 31.6, 29.2, 25.7, 22.6, 14.0 ppm.
1-1.6 Preparation of BCa-3mem
KOH (aq) (1 N, 0.087 mL, 0.087 mmol) was dropped to a solution of Compound C6 (0.034 g, 0.043 mmol) in dioxane (0.20 mL) and EtOH (0.52 mL) while stirring at 0° C. After stirring at room temperature for 48 hours, the mixture was dropped to a vial containing MeCN (2.5 mL). The formed precipitate was collected by filtration and washed with diethyl ether. The obtained product, BCa-3mem was a yellow solid and the yield was 0.024 g (72%).
1H NMR (500 MHz, DMSO-d6/CD3CN at 9:1):δ 10.51 (s, 1H), 8.50 (d, J=1.8 Hz, 1H), 8.01 (dd, J=8.8, 1.8 Hz, 1H), 7.84 (d, J=8.8 Hz, 1H), 7.70 (d, J=8.8 Hz, 1H), 7.62 (d, J=8.8 Hz, 1H), 7.39 (s, 1H), 7.30 (d, J=2.5 Hz, 1H), 7.19 (dd, J=8.8, 2.3 Hz, 1H), 7.09 (br s, 1H), 6.95 (dd, J=8.8, 2.5 Hz, 1H), 6.85 (d, J=8.8 Hz, 1H), 6.79 (s, 1H), 4.79 (s, 2H), 4.05-3.98 (m, 4H), 3.65-3.61 (m, 2H), 3.57-3.53 (m, 2H), 3.48-3.42 (m, 4H), 3.21 (br s, 4H), 1.78-1.71 (m, 2H), 1.49-1.42 (m, 2H), 1.36-1.30 (m, 4H), 0.92-0.87 (m, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): δ174.2(2), 168.7, 166.9, 164.2, 156.8, 150.0, 148.6, 145.0, 143.0, 137.3, 137.1, 133.5, 130.3, 127.9, 126.9, 126.3, 124.3, 119.6, 119.2, 119.0, 113.8, 112.2, 111.3, 104.6, 103.6, 103.1, 68.6, 66.3, 66.2, 64.9, 59.1(2), 47.0, 44.8, 42.0, 31.4, 29.0, 25.6, 22.5, 14.4 ppm. HRMS (ESI) m/z [M+K]+ calcd for C41H45KN5O10: 806.2798, found: 806.2800.
1-17. Preparation of Compound 7
HATU (0.99 g, 2.6 mmol) was added to a solution of Compound C2 (0.76 g, 2.4 mmol), MOBHA-Me (0.89 g, 2.3 mmol), and (i-Pr)2NH (0.81 mL, 4.7 mmol) in DMF (7.5 mL), and the mixture was stirred at room temperature for 30 minutes. Distilled water was then added and the product was extracted with EtOAc. The organic layer was washed with brine and dried with anhydrous Na2SO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using CH2Cl2/EtOAc (1: 3) as a mobile phase to obtain Compound 7 in a pale yellow solid state (yield 1.17 g, 70%).
1H NMR (500 MHz, CDCl3): δ 8.59 (s, 1H), 8.50 (br s, 1H), 8.17 (d, J=8.7, 1H), 7.81-7.73 (m, 2H), 7.70 (d, J=8.7 Hz, 1H), 7.61-7.54 (m, 1H), 7.38-7.31 (m, 3H), 7.03 (br d, J=8.6 Hz, 1H), 6.98 (br d, J=8.6 Hz, 1H), 6.88-6.81 (m, 2H), 4.71 (s, 2H), 4.11 (s, 4H), 4.00 (s, 2H), 3.66 (s, 6H), 3.66-3.62 (m, 4H), 3.60-3.55 (m, 2H), 3.50-3.45 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3): δ 171.5(2), 168.2, 166.3, 163.5, 150.7, 150.0, 146.4, 142.2, 136.5, 135.7, 132.9, 130.4, 127.9, 127.4, 126.9, 124.8, 124.7, 124.4 121.1, 120.4, 119.6, 118.5, 113.6, 110.4, 107.2, 105.3, 67.3, 66.6, 66.5, 53.5(2), 51.7(2), 48.9, 45.4, 42.2 ppm.
1-18. Preparation of BCa−3°mem
KOH (aq) (1 N, 0.18 mL, 0.18 mmol) was dropped to a solution of Compound 7 (0.050 g, 0.072 mmol) in dioxane (0.50 mL) and MeOH (0.50 mL) while stirring at 15° C., and then the mixture was stirred at room temperature for 15 hours. The reaction mixture was dropped to a round bottom flask containing MeCN (3 mL) while stirring. The formed precipitate was collected by filtration and washed with diethyl ether. The obtained product, BCa−3°mem was a yellow solid and the yield was 0.024 g (47%).
1H NMR (500 MHz, DMSO-d6): δ 8.52 (d, J=1.7 Hz, 1H), 8.02 (dd, J=8.8, 1.7 Hz, 1H), 7.84 (d, J=8.9 Hz, 1H), 7.76-7.72 (m, 2H), 7.70 (d, J=8.8 Hz, 1H), 7.40-7.36 (m, 2H), 7.27 (d, J=2.2 Hz, 1H), 7.16 (dd, J=8.9, 2.2 Hz, 1H), 6.95 (dd, J=8.7, 2.0 Hz, 1H), 6.86 (d, J=8.7 Hz, 1H), 6.76 (d, J=2.0 Hz, 1H), 4.78 (br s, 2H), 3.99 (br s, 2H), 3.61-3.55 (m, 2H), 3.53-3.47 (m, 2H), 3.44-3.37 (m, 4H), 3.34-3.26 (m, 4H) ppm; 13C NMR (151 MHz, DMSO-d6): δ 174.3(2), 168.7, 166.9, 163.5, 150.5, 150.1, 148.6, 142.0, 137.2, 137.1, 133.6, 130.2, 128.0, 126.8, 126.2, 125.4, 125.2, 124.3, 119.7, 119.6, 119.1, 119.0, 112.2, 111.0, 104.6, 103.1, 66.2, 66.1, 64.8, 59.2(2), 47.0, 44.7, 42.0 ppm. HRMS (ESI) m/z [M+K]+ calcd for C35H33KN5O9+: 706.1910, found: 706.1928.
1-19. Preparation of Compound C7
t-BuONa (5.3 g, 55 mmol) was added to a solution of 9-methyl-9H-fluorene (4.0 g, 22 mmol) in DMSO (100 mL) while stirring at 10° C. Next, the solution was stirred at room temperature for 2 hours, and added with distilled water. The product was extracted with EtOAc, washed with brine, dried with anhydrous Na2SO4 and evaporated.
The residue was purified through silica gel column chromatography using hexane/EtOAc (5: 1) as a mobile phase to obtain Compound C7 in a white solid state (yield 3.6 g, 65%).
1H NMR (500 MHz, CDCl3): δ 7.77-7.74 (2H, m), 7.50-7.47 (2H, m), 7.38 (2H, td, J=7.3, 1.2 Hz), 7.34 (2H, td, J=7.3, 1.2 Hz), 3.48 (3H, s), 2.88 (2H, s), 1.63 (3H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 171.1, 150.7(2), 139.5(2), 127.4(2), 127.2(2), 122.9(2), 119.9(2), 51.1, 48.3, 43.9, 25.3 ppm.
1-20. Preparation of Compound C8
HNO3 (1.4 mL, 33 mmol) was dropped to a solution of Compound C7 (2.4 g, 8.1 mmol) in CH2Cl2 (16 mL) while stirring at 0° C. After stirring at 0° C. for 20 minutes, the reaction mixture was poured into a beaker containing ice and a saturated NaHCO3(aq) solution. The product was extracted with CH2Cl2, washed with brine, dried with anhydrous MgSO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using hexane/EtOAc (4: 1) as a mobile phase to obtain Compound C8 in a white solid state (yield 1.8 g, 75%).
1H NMR (500 MHz, CDCl3): δ 8.35 (1H, d, J=2.2 Hz), 8.28 (1H, dd, J=8.3, 2.2 Hz), 7.83 (1H, d, J=8.3 Hz), 7.82 (1H, dd, 8.3, 0.7 Hz), 7.53-7.50 (1H, m), 7.46-7.41 (2H, m), 3.43 (3H, s), 3.02 (1H, d, J=15.2 Hz), 2.94 (1H, d, J=15.2 Hz), 1.62 (3H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 170.3, 152.1, 151.7, 146.9, 146.2, 137.2, 129.4, 128.0, 123.7, 123.1, 121.4, 120.1, 118.6, 51.4, 48.5, 43.2, 25.5 ppm.
1-21. Preparation of Compound 8
A solution of Compound C8 (3.3 g, 11 mmol) and acetyl chloride (3.8 mL, 54 mmol) in nitrobenzene (15 mL) was stirred at 40° C. for 1 hour. AlCl3 (7.2 g, 54 mmol) was added slowly and the mixture was stirred at 60° C. for 5 hours. The mixture was cooled on an ice bath and the reaction was quenched using 1 N HCl (aq). The product was extracted with CH2Cl2, washed with brine, dried with anhydrous MgSO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using hexane/EtOAc (3: 1) as a mobile phase to obtain Compound 8 in a pale yellow solid state (yield 3.2 g, 85%).
1H NMR (500 MHz, CDCl3): δ 8.35 (1H, d, J=2.0 Hz), 8.30 (1H, dd, J=8.4, 2.0 Hz), 8.11 (1H, d, 1.6 Hz), 8.04 (1H, dd, J=7.9, 1.6 Hz), 7.90 (1H, d, J=6.6 Hz), 7.89 (1H, d, J=6.6 Hz), 3.40 (3H, s), 3.09 (1H, d, J=15.6 Hz), 3.04 (1H, d, J=15.6 Hz), 2.67 (3H, s), 1.63 (3H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 197.6, 170.0, 153.0, 152.6, 147.9, 144.9, 142.0, 137.6, 128.9, 123.9, 122.7, 121.4, 121.2, 118.7, 51.6, 48.8, 43.0, 26.9, 25.8 ppm.
1-22. Preparation of Compound C9
A solution of Compound 8 (2.2 g, 6.4 mmol), Fe powder (1.1 g, 19 mmol), and NH4C1 (0.69 g, 13 mmol) in EtOH (10 mL) and 1,4-dioxane (10 mL) was refluxed for 2 hours. The mixture was cooled, neutralized by adding Na2CO3 (aq) and then filtered through Celite. The product was extracted with CH2Cl2, washed with brine, dried with anhydrous MgSO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using hexane/EtOAc (1: 1) as a mobile phase to obtain Compound C9 in a pale yellow solid state (yield 1.7 g, 80%).
1H NMR (500 MHz, CDCl3): δ 7.99 (1H, d, J=1.5 Hz), 7.93 (1H, dd, J=8.0, 1.5 Hz), 7.61 (1H, d, J=8.0 Hz), 7.57 (1H, d, J=8.1 Hz), 6.82 (1H, d, J=2.1 Hz), 6.72 (1H, dd, J=8.1, 2.1 Hz), 3.46 (3H, s), 2.89 (1H, d, J=15.0 Hz), 2.84 (1H, d, J=15.0 Hz), 2.63 (3H, s), 1.56 (3H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 197.7, 170.8, 154.1, 150.1, 147.6, 145.2, 134.2, 128.9, 128.8, 122.4, 122.0, 118.1, 114.5, 109.4, 51.2, 47.9, 43.6, 26.6, 25.6 ppm.
1-23. Preparation of Compound 9
A solution of Compound C9 (3.0 g, 8.8 mmol), 2-bromo-N-(4-(diethylamino)phenyl)acetamide (5.1 g, 18 mmol), and (i-Pr)2NH (3.1 mL, 18 mmol) in DMF (20 mL) was stirred at 80° C. for 12 hours. Distilled water was added to the mixture and the product was extracted with EtOAc. The organic layer was washed with brine and dried with anhydrous MgSO4 and then the solvent was evaporated. The residue was purified through silica gel column chromatography using hexane/EtOAc (1:2) as a mobile phase to obtain Compound 9 in a pale yellow solid state (yield 2.6 g, 58%).
1H NMR (500 MHz, CDCl3): δ 8.20 (1H, s), 8.00 (1H, d, J=1.6 Hz), 7.94 (1H, dd, J=7.8, 1.6 Hz), 7.63 (1H, d, J=7.8 Hz), 7.62 (1H, d, J=8.2 Hz), 7.30 (2H, d, J=9.0 Hz), 6.79 (1H, d, J=2.2 Hz), 6.70 (1H, dd, J=8.2, 2.2 Hz), 6.62 (2H, d, J=9.0 Hz), 4.62 (1H, t, J=5.2 Hz), 3.97 (2H, d, J=5.2 Hz), 3.42 (3H, s), 3.31 (4H, q, J=6.9 Hz), 2.90 (1H, d, J=15 Hz), 2.87 (1H, d, J=15 Hz), 2.63 (3H, s), 1.56 (3H, s), 1.12 (6H, t, J=6.9 Hz) ppm; 13C NMR (75 MHz, CDCl3): δ 197.8, 170.8, 167.9, 154.3, 150.3, 148.1, 145.4, 144.9, 134.7, 130.1, 128.9, 125.6, 122.4, 122.3(3), 118.5, 113.1, 112.1(2), 108.1, 53.4, 51.4, 49.5, 48.2, 44.5, 43.6, 26.7, 25.9, 12.5(2) ppm.
1-24. Preparation of FHEt-1lyso
KOH (aq) (1 N, 5 mL, 5 mmol) was dropped to a solution of Compound 9 (0.26 g, 0.5 mmol) in EtOH (1 mL) while stirring at room temperature for 12 hours. The mixture was stirred at room temperature for 48 hours, and then dropped with HCl (aq) (1 N, 5 mL, 5 mmol). The formed precipitate was collected by filtration, washed with distilled water, dried in vacuum, and used in the next step without additional purification. HATU (0.13 g, 0.34 mmol) was added to a solution of the precipitate (0.13 g, 0.26 mmol), N,N-dimethylethylenediamine (0.034 g, 0.39 mmol), and (i-Pr)2NH (0.090 mL, 0.52 mmol) in DMF (1 mL), and the mixture was stirred at room temperature for 12 hours. Distilled water was added, the product was extracted with EtOAc, and then the organic layer was washed with brine and dried with anhydrous Na2SO4 and the solvent was evaporated. The residue was purified through silica gel column chromatography using CH2Cl2/MeOH (1: 1) as a mobile phase to obtain FHEt-1lyso in a yellow solid state (total yield 0.11 g, 40%).
1H NMR (600 MHz, CDCl3): δ 8.86 (1H, s), 8.55 (1H, s), 8.03 (1H, d, J=1.6 Hz), 7.92 (1H, dd, J=7.9 Hz, 1.6 Hz), 7.90 (1H, br s), 7.59 (1H, d, J=7.9 Hz), 7.57 (1H, d, J=8.3 Hz), 7.34 (2H, d, J=9.1 Hz), 6.84 (1H, d, J=2.2 Hz), 6.64 (1H, dd, J=8.3, 2.2 Hz), 6.62 (2H, d, J=9.1 Hz), 4.02 (1H, d, J=17 Hz), 3.98 (1H, d, J=17 Hz), 3.36-3.29 (1H, m), 3.31 (4H, q, J=7.0 Hz), 3.11-3.04 (1H, m), 2.94 (1H, d, J=14 Hz), 2.83 (1H, d, J=14 Hz), 2.68-2.63 (1H, m), 2.63 (3H, s), 2.57-2.52 (1H, m), 2.37 (6H, s), 1.53 (3H, s), 1.12 (6H, t, J=7.0 Hz) ppm; 13C NMR (75 MHz, CDCl3): δ 198.0, 170.3, 168.4, 153.8, 150.5, 148.2, 145.2, 144.9, 134.5, 129.7, 128.6, 126.2, 122.7, 122.4(2), 122.2, 118.3, 112.2(2), 111.9, 108.8, 57.4, 48.9, 48.9, 45.4, 44.5(2), 43.0(2), 34.2, 27.2, 26.8, 12.4(2) ppm. HRMS (ESI) m/z [M+H]+ calcd for C34H43N5O3+: 570.3439, found: 570.3445.
2-1. Spectroscopy
Absorption and emission spectra of a two-photon probe (TP probe) were measured using an Agilent 8453 diode array UV-Vis spectrophotometer and a Hitachi fluorescence spectrophotometer F-7000 system using a previously reported method (Chem. Asian J. 2015, 10(10):2240-2249). Fluorescence quantum yield (Φ) was measured by a method used in another study (Anal. Chem. 2011, 83(4):1232-1242) using Coumarin 540A. Two-photon (TP) excitation was implemented using a mode-locked Ti:sapphire laser (Chameleon, 90 MHz, 200 fs; Coherent Inc.).
2-2. Water Solubility
The water solubility of BCa-1, BCa-2mito, BCa−3°mem, and FHEt-1lyso in an aqueous buffer (pH 7.4) was evaluated using a conventional fluorescence technique (Chem. Asian J. 2015, 10(10):2240-2249).
2-3. Measurement of TP Action Cross-Section (Φδ) and Effective TP Action Cross-Section (Φδeff)
Φδ and Φδeff for the probes were evaluated using the fluorescence method as reported in other studies (Chem. Asian J. 2015, 10(10):2240-2249).
2-4. Measurement of Apparent Dissociation Constants
A dissociation constant (Kd) for binding the probe to Ca2+ was measured using a conventionally known method. Calcium calibration buffers (CBs; 10 mM 3-[N-morpholino]propanesulfonic acid [MOPS], 100 mM KCl, pH 7.2) containing Ca2+ at concentrations of 0 to 40 μM were prepared using a calcium calibration buffer kit (Biotium, CA, USA). A buffer containing 401.1M or more of Ca2+ was prepared by adding an appropriate amount of CaCl2 to a MOPS buffer. Probes (BCa-3°mem at a concentration of 0.3 μM and BCa-1 and BCa-2mito at concentrations of 0.1 μM) were added to calcium CB and fluorescence intensity was measured. Each Kd was calculated through a resultant titration curve. In addition, in order to evaluate a fluorescence titration of FHEt-1lyso, the fluorescence intensity was measured by adding FHEt-1lyso probe at a concentration of 0.6 μM in a universal buffer (UB; 0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M tris[hydroxymethyl]aminomethane, 0.1 M KCl) at pH 4 to 11. The pKa value was calculated through a titration curve.
2-5. Preparation of Cells
HeLa cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured by a method described in previous studies (Chem. Asian J. 2015, 10(10):2240-2249). Before two days of imaging, HeLa cells (5×104/mL) were plated on a confocal glass bottom dish (#200350, SPL Life Sciences, Gyonggi-do, Korea). The cells were washed with an aqueous buffer (pH 7.4), a Hank's balanced salt solution (HBSS; LB003-02, WelGene Inc., Gyeongsangbuk-do, Korea), or a Ringer's solution (RB; 124 mM NaCl, 26 mM NaHCO3, 10 mM d-glucose, 1.3 mM MgSO4, 3 mM KCl, 1.25 mM NaH2PO4) unless otherwise specified. Thereafter, the probe (0.5 to 3 μM) and Pluronic® F-127 (0.03%, Sigma-Aldrich, St. Louis, MO, USA) were added to the cells in one of the three aqueous buffers (1.0 mL). The mixture was cultured (incubated) at 37° C. (BCa-1-AM and BCa-2mito-AM) for 40 minutes or at room temperature (RT; BCa-3mem) for 10 minutes. The cells labeled with the probe were washed 3 times with each corresponding buffer before imaging.
2-6. Preparation of Tissues
Ex vivo brain slices were obtained from the hippocampus of 14-day-old Sprague-Dawley rats (Orient Bio Inc., Korea) according to a protocol approved by the institutional review board (IRB) of the Korea University. Fresh hippocampal slices (thickness: 400 μm) were prepared using a previously reported technique on an oscillating blade microtome (Chem. Asian J. 2015, 10(10):2240-2249).
2-7. Two-Photon Imaging (TP Imaging)
TP microscopy (TPM) images were obtained using spectral confocal and multiphoton microscopy (Leica TCS SP2; Leica). TPM images of probe-labeled HeLa cells were obtained using a Leica TCS SP2 using a conventional method (Chem. Asian J. 2015, 10(10):2240-2249). Time-dependent changes in TPEF intensity were monitored and measured at intervals of 1.6 to 2.0 seconds in the xyt mode upon excitation at 750 nm. To monitor changes in [Ca2+ ] in the plasma membrane, the cells were washed successively with RB containing 5, 0.1, and 0 mM of EGTA and then incubated with BCa-3mem (2 μM). The TPEF intensity was measured before and after adding CaCl2 (2 mM) to RB and EGTA (2 mM) in distilled water.
Before and after adding histamine dihydrochloride (100 μM, Sigma-Aldrich) to HBSS, Ca2+ oscillation was evaluated by TPEF intensity monitoring in HeLa cells labeled with BCa-1-AM (2 μM).
Before and after adding carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 0.5 M, cat. #15218, Cayman, USA) to HBSS, changes in mitochondrial Ca2+ were analyzed by monitoring the TPEF intensity in HeLa cells labeled with BCa-2mito-AM (2 μM).
To simultaneously monitor changes in lysosomal pH and cytoplasmic [Ca2+], HeLa cells were co-labeled with a mixture of BCa-1-AM (3 μM) and FHEt-1lyso (1 μM). The cells were washed twice with RB, once with RB containing 5 mM EGTA, and twice with RB containing 0.1 mM EGTA. The TPEF intensity of the cells was measured before and after adding monensin (20 μM, Sigma-Aldrich) to EtOH.
For tissue imaging, brain slices were labeled with one of the following: BCa-1-AM (7 mM) and FHEt-1lyso (3 mM) for 60 minutes at 37° C. for dual-color imaging, BCa-2mito-AM (7 μM) for 60 minutes at 37° C. for mitochondrial imaging, or BCa-3mem (2 μM) for 10 minutes at RT for plasma membrane imaging. TPM images were obtained through the method described above except that six cross-sectional images were captured at 10 μm intervals at 90 to 140 μm depths from the tissue surface along a z-direction to minimize errors due to surface damage occurring during tissue preparation.
2-8. Cytotoxicity
The cytotoxicity of BCa-1-AM, BCa-2mito-AM, BCa-3mem, and FHEt-1lyso was measured using Cell Counting Kit-8 (Dojindo, Japan).
2-9. Photostability
Photostability was measured by monitoring a time-dependent decrease in TPEF intensity under imaging conditions for HeLa cells labeled with BCa-1-AM, BCa-2mito-AM, BCa-3mem, or FHEt-1lyso.
Table 1 showed results of analyzing the physical properties of the two-photon (TP) probes BCa-1-AM, BCa-2mito-AM, BCa-3mem, and FHEt-1lyso prepared in Example of the present disclosure.
2λmaxf
a λmax of one-photon absorption spectra in nanometer unit.
bλfl of one-photon absorption spectra in nanometer unit.
cMolar extinction coefficient.
d,eFluorescence quantum yield (d) in the absence or presence of excess Ca2+ (in parentheses) and fluorescence quantum yields (e) at pH 11 and pH 4.3 (in parentheses).
fλmax of TPEF spectra in nanometer unit in aqueous buffer or Hela cells (in parentheses).
g,hDissociation constant (g) in micrometer unit and negative log (h) of acid dissociation constant.
iFluorescence enhancement factor.
jMaximum TP action cross-section at 10−50 cm4s/photon (GM), error range: ±15%.
kEfficient TP action cross-section in GM.
3-1. Spectroscopic Characteristics
BCa-1, BCa-2 onto, and BCa-3°mem showed a molar extinction coefficient (c) of 30,000 to 31,000 cm-1M-1 and the maximum absorbance (λmax) of 350 to 358 nm, as a result of a spectral study performed in phosphate buffered saline (pH 7.4;
The water solubility measured by the absorption method for each probe compound was found to be 0.4 to 2.0 μM, which was sufficient to stain (color) cells (
HeLa cells (cervical cancer cell line) labeled with BCa-3mem, BCa-1-AM, BCa-2mito-AM, or FHEt-1lyso emitted broad TPEF spectra based on 427, 461, 462, and 550 nm, respectively, when excited at 750 nm in a scanning lambda mode (
3-2. Fluorescence Titration
When Ca2+ was added little by little to BCa-1, BCa-2mito, and BCa−3°mem in CB, the fluorescence intensity was increased rapidly (
The dissociation constants (Kd) of BCa-1, BCa-2mito, and BCa−3°mem calculated from the titration curve were 0.18, 2.7, and 100 μM, respectively. A similar value (KdTP=95 μM) for BCa-3°mem was determined in the TP process. The KdTP values for BCa-1 and BCa-2mito were hardly calculated because the Fmin value of the TP process was too low to make accurate measurements (
The BCa-1, BCa-2mito, and BCa-3°mem probes of the present disclosure all showed weak responses to Mg2+ at 2 mM and Fe2+, Cu2+, and Co2+ at 100 μM, showed moderate responses to Zn2+ and Mn2+ at 100 μM, whereas showed high selectivity for Ca2+ (
Meanwhile, the fluorescence titration of FHEt-1lyso was performed in UB at pH 4 to 11. The pKa value of FHEt-1lyso was 6.5, which was within the pH range of the cell (
3-3. Brightness of Two-Photon Probe
To evaluate the brightness of the TPM image, a TP action cross-section (Φδ) was measured by a conventionally known method. In the presence of excess Ca2+, the Φδ maximum values (Φδmax) of BCa-1, BCa-2mito, and BCa−3°mem were 55, 70, and 60 GM at 750 nm, respectively, whereas the Φδ maximum value of FHEt-1lyso measured at pH 4.3 was 70 GM at 750 nm (
In addition, the Φδeff value of the probe was determined in probe-labeled cells by comparing the TPEF intensity in MeOH with the intensity of 5.0 μM rhodamine 6 G. The Φδeff values of BCa-1, BCa-2mito, BCa-3mem, and FHEt-1lyso in HeLa cells were 1400, 2000, 4800, and 21000 GM at 750 nm, respectively, which were sufficient values to obtain bright TPM images. As a result of measuring the effective concentrations (ceff) of BCa-1, BCa-2mito, BCa-3mem, and FHEt-1lyso using Equation of ceff=Φδeff/Φδmax, the bright spots were 25, 29, and 80, and 300 μM, respectively. These ceff values were 17 to 600-fold higher than the probe concentrations measured in a staining medium, 1.5 μM for BCa-1, 1.0 μM for BCa-2mito, and 0.5 μM for BCa-3mem and FHEt-1lyso. These results indicate that the microenvironment of the probes is more favorable to the cells than the staining medium.
To evaluate the photostability of the probes, the probe was continuously irradiated with femtosecond pulses while monitoring the TPEF intensity of regions of interest (ROIs) in the TPM image of the probe-labeled cells (
TPEF of HeLa cells labeled with BCa-3mem was measured at wavelengths of 380 to 660 nm. For colocalization experiments on HeLa cells co-labeled with BCa-2mito-AM/MitoTracker Red′ (cat. #M22425, Invitrogen, Waltham, MA, USA), BLT-blue/FHEt-1lyso−, and BCa-1-AM/FHEt-1lyso, respectively, the detection window was measured using the TPEF spectra of the two probes to be compared. It was confirmed that the detection windows of the probes had well-separated emission bands and similar levels of emission intensities from the two probes to be compared.
The detection windows for BCa-2mito-AM, MitoTracker Red, BLT-blue, FHEt-1lyso, and BCa-1-AM were 380 to 540, 600 to 680, 380 to 480, 550 to 660, and 380 to 480 nm, respectively (
6-1. Detection of Ca2+ in Plasma Membrane
First, whether BCa-3mem may detect Ca2+ in the plasma membrane was evaluated. TPM images of HeLa cells labeled with BCa-3mem contained bright spots on the plasma membrane, and these signals persisted for at least 1 hour (
Meanwhile, in contrast to the results of BCa-3mem, the images were much darker and blurry when the cells were labeled with BCa−3°mem (
6-2. Detection of Ca2+ in Cytoplasm
Whether BCa-1 may detect Ca2+ in the cytoplasm was evaluated. TPM images of HeLa cells labeled with BCa-1-AM showed bright spots in the cytoplasm. When adding histamine, a reagent that induces Ca oscillation, the TPEF intensity increased and then decreased. The increase and decrease of the TPEF intensity were repeated for 800 seconds, which was consistent with the results of previous histamine-induced Ca oscillation studies. Accordingly, it was confirmed that BCa-1 may target the cytoplasm and detect Ca2+ in the cytoplasm (
6-3. Detection of Ca2+ in Mitochondria
Whether BCa-2mito-AM may detect Ca2+ in the mitochondria was evaluated. TPM images of HeLa cells labeled with BCa-2mito-AM showed well-colocalized bright spots with MitoTracker Red having a Pearson's colocalization coefficient (A) of 0.84 (
When the cells were treated with FCCP, a reagent that depolarizes the mitochondrial membrane, the TPEF intensity rapidly decreased in the mitochondria (FIG. 17F, black curve), and then the TPEF intensity increased temporarily in the cytoplasm and then gradually decreased to a baseline (
6-4. Detection of H+ in lysosome
Whether FHEt-1lyso may detect H+ in the lysosome was evaluated. TPM images of HeLa cells labeled with FHEt-1lyso showed well-colocalized bright spots with BLT-blue having a Pearson's colocalization coefficient (A) of 0.86 as a TP probe for the lysosome (
6-5. Dual-Color Imaging
Whether BCa-1 and FHEt-1lyso could be used to simultaneously monitor changes in cytoplasmic [Ca2+ ] and lysosomal pH during dual-color TPM was evaluated. TPM images of HeLa cells co-labeled with BCa-1-AM and FHEt-1lyso showed Ca2+ in the cytoplasm (green pixels) and H+ in the lysosome (red pixels,
The BCa-1-AM and FHEt-1lyso probes of the present disclosure were evaluated for usefulness as TP probes for tissue imaging. Hippocampal tissue slices from 14-day-old Sprague-Dawley rats were incubated with 7 M BCa-1-AM and 3 M FHEt-1lyso at 37° C. for 60 minutes. Six TPM images were obtained at depths of 90 to 140 μm to visualize the distribution of cytoplasmic Ca2+ and lysosomal H+ ions at different depths on pyramidal neuron layers in CA1 and dentate gyrus regions (
In addition, cross-sectional TPM images (depths of 90 to 140 μm) of hippocampal slices labeled with BCa-2mito-AM and BCa-3mem were obtained (
Therefore, it was confirmed that BCa-2mito-AM and BCa-3mem could detect Ca ions in the mitochondria and plasma membrane in live tissues during TPM, respectively.
The present inventors have prepared blue emission TP probes (BCa-1-AM, BCa-2mito-AM, BCa-3mem) for Ca2+ and a green emission TP probe (FHEt-1lyso) for H+ capable of organelles-specific detection in the cytoplasm, mitochondria, plasma membrane, and the lysosome, respectively. These probes showed significant TP cross-section, high selectivity and sensitivity for Ca2+ to be detected, high photostability, low pH dependence, and negligible cytotoxicity. BCa-1-AM, BCa-2mito-AM, and BCa-3mem may selectively detect Ca2+ in real time in the cytoplasm, mitochondria, and plasma membrane of live cells and tissues, respectively, and FHEt-1lyso may detect lysosomal H+ in live cells and tissues. In addition, BCa-1-AM and FHEt-1lyso may simultaneously detect cytoplasmic Ca2+ and lysosomal H+ in live cells and tissues through dual-color TPM images. Through these results, it was confirmed that the organelles-specific blue emission TP probe derived from 6-(benzoxazol-2-yl)-2-naphthalylamine of the present disclosure was useful as a TP Ca2+ probe capable of detecting Ca ions in the cytoplasm, mitochondria, and plasma membrane of live cells and tissues. In addition, the probe of the present disclosure can be used to examine crosstalk between metal ions through multicolor TPM imaging. Accordingly, the probe of the present disclosure may be useful for biomedical research including physiological research on living biological tissues.
From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0025902 | Feb 2022 | KR | national |
| 10-2022-0025903 | Feb 2022 | KR | national |
| 10-2022-0025904 | Feb 2022 | KR | national |
