A Nile blue derivative compound and its preparation method and application

Abstract

The present invention discloses a Nile blue derivative compound, its preparation method, and application. The Nile blue derivative compound provided has the following general structural formula (I). The Nile blue derivative compound exhibits good structural stability, excellent membrane permeability, and strong antioxidant properties. It can be used as a mitochondrial single-molecule localization imaging probe. It not only has good mitochondrial targeting, but also can specifically label mitochondria through simple co-incubation without the need for transfection, resulting in high labeling efficiency and no damage to cells or mitochondria. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the technical field of biological imaging, and particularly relates to a Nile blue derivative compound and a preparation method and application thereof. Background Art

[0002] Mitochondria are not only the cell's "energy source" but also a key platform for numerous cellular signaling processes, participating in a wide range of eukaryotic metabolic processes. This functional diversity is closely linked to the continuous remodeling of the mitochondrial network, involving fusion, fission, and membrane ultrastructural remodeling, processes collectively known as mitochondrial dynamics. It not only influences and coordinates metabolism but also influences complex cellular signaling events, such as those involved in regulating cell division, differentiation, aging, and death. Therefore, studying mitochondrial dynamics is crucial for understanding diseases associated with mitochondrial dysfunction.

[0003] Fluorescence microscopy has been used for many years to study the morphology of whole mitochondria. Due to the extremely small size of individual mitochondria, traditional fluorescence microscopy, constrained by the diffraction limit of light, is insufficient for observing the distribution, fine structure (cristae) and dynamics of individual mitochondrial proteins. Therefore, super-resolution fluorescence microscopy (SRFM) is crucial as a tool for capturing spatiotemporal dynamics. SRFM improves the resolution of optical microscopes to a few nanometers and is widely used to observe the fine structure and dynamics of various organelles. SRFM usually requires high radiation intensity to produce a sufficient number of effective photons, but high radiation intensity can cause damage to living cells and organelles. Compared to organelles such as the nucleus, microtubules and endoplasmic reticulum, mitochondria are constantly dividing, fusing and moving under physiological conditions and are more sensitive to radiation intensity. Single-molecule localization microscopy (SMLM), a form of SRFM, relies on the ability of individual organic dye molecules or fluorescent proteins to cyclically switch between a fluorescent bright state and a non-fluorescent dark state under certain conditions. By precisely locating individual fluorescent dye molecules that are spatially distant from each other, SMLM overcomes the diffraction limitation and achieves ultra-high resolution. SMLM uses the laser only for the excitation transitions of the dye molecules, allowing its intensity to be significantly reduced, making it ideal for imaging living cells.

[0004] Over the past decade, the use of chemical methods to develop small-molecule fluorescent probes with novel structures and excellent properties for mitochondrial SMLM imaging has been a cutting-edge hot topic in the field of bioimaging and has promoted the cross-integration of chemistry and biology. Fluorophores with an overall negative charge, such as cyanine-based Alexa 647, Cy5, and rhodamine-based fluorescein, Alexa 488, show good water solubility, but their cell membrane permeability is poor, so they are limited to cell membrane imaging or can only be used for fixed cell imaging. Although electroporation can allow them to enter the cell interior, this technique often causes certain damage or death to the cells. The double-membrane structure of mitochondria can prevent most foreign molecules from entering, making the use of small-molecule fluorescent probes for SMLM imaging of mitochondria in living cells more challenging.

[0005] In 2012, Zhuang et al. applied the commercial mitochondrial labeling probes MitoTracker Red (MTR, whose structure is shown below, derived from rhodamine) and MitoTracker Deep Red (MTDR, whose structure is shown below, derived from anthocyanin) to live cell SMLM imaging and successfully captured the dynamic fusion and fission of mitochondria (Shim, S.-H. et al. Super-Resolution Fluorescence Imaging of Organelles in Live Cells with Photoswitchable Membrane Probes. Proc. Natl. Acad. Sci. 2012, 109(35), 13978–13983. https: / / doi.org / 10.1073 / pnas.1201882109). This technology requires an oxygen scavenging system and the applied laser intensity is relatively high, causing significant damage to mitochondria.

[0006]

[0007] The small molecule fluorophores currently used for mitochondrial SMLM imaging are mostly structurally derived from rhodamine and anthocyanin. Although these two types of fluorophores have been fully applied in cell imaging, they both have inherent disadvantages. The excitation wavelength of rhodamine is about 540nm, and the damage to the mitochondria of living cells caused by strong lasers cannot be ignored. The excitation wavelength of anthocyanin is about 650nm, which belongs to the near-infrared region. The phototoxicity is significantly reduced at the same intensity, but photobleaching (that is, the molecules in the excited state are oxidatively destroyed and the fluorescence ability is permanently lost) is a key obstacle that limits its spatiotemporal resolution. Methods to improve the stability of anthocyanin include the use of reducing additives (thiols, cyclooctatetraene, etc.) and oxygen scavenging systems in imaging buffers, but they have potential toxic side effects in living cells. In addition, the "phototruncation effect" of anthocyanin, that is, it is oxidized by singlet oxygen ( 1 The double carbon truncation and shortened absorption wavelength in the polymethylene bridge chain caused by the oxidative cleavage of O2 have also caused concerns about the artifacts caused by photoinduced blue shift in multicolor imaging.

[0008] Unlike cyanine and rhodamine dyes, Nile blue is an oxazine fluorophore, characterized by a longer excitation wavelength (approximately 650 nm), strong fluorescence, good membrane permeability, and solvatochromism. Solvatochromism refers to the fact that Nile blue's fluorescence brightness is higher in lipid-soluble solvents and lipophilic environments, but weaker in aqueous environments, which helps reduce background fluorescence signals. Another notable property of oxazine fluorophores is their high electron affinity. In their excited state, they readily accept electrons for reduction, resulting in a dark state, which can then be oxidized back to the ground state by oxygen and other substances. This reversible cycling between the dark and bright states makes them suitable for SMLM imaging. Their high electron affinity also imparts them with strong resistance to oxidation and photobleaching. A successful example is the use of the oxazine dye ATTO 655 (structure shown below) for SMLM imaging of the nucleus in living cells. However, the application of this dye for mitochondrial imaging has not been reported because its overall negative charge makes it difficult for it to enter mitochondria.

[0009]

[0010] Current modifications to the structure of Nile blue have primarily focused on altering the substituents at the 5 / 9 amino groups. When the total number of substituents is three, it is called trisubstituted Nile blue (NBA; its structure is shown below). However, its 5-amino group is susceptible to deprotonation, resulting in a blue-shift in absorption wavelength. When used for live-cell imaging, trisubstituted Nile blue can passively diffuse into lysosomes. This is attributed to the formation of a neutral imine upon deprotonation of the 5-amino group. This imine has high membrane permeability, allowing it to enter the acidic interior of the lysosome (pH 4-5) and become protonated there. However, this protonation reduces membrane permeability, trapping it within the lysosome. High concentrations of trisubstituted Nile blue can diffuse into other cellular organelles, including the nucleus, cell membrane, and cytoplasm. Therefore, trisubstituted Nile blue itself is not suitable for biological imaging. Tetrasubstituted Nile blue (TENB) (its structure is shown below) is a dye with four substituents on the 5 / 9 amino groups. Because mitochondria have an outside-to-inside potential difference, they readily absorb lipophilic cations. Tetrasubstituted Nile blue, with its positive charge delocalized throughout the conjugated system, naturally possesses excellent mitochondrial targeting. However, few reports have yet reported this type of cationic Nile blue. This is due to the significant steric repulsion between the 5-amino substituents and the naphthalene ring, making it sensitive to nucleophiles and unstable in living cells. Furthermore, characterization of this type of dye is currently limited to melting point and mass spectrometry, with no evaluation in biological systems.

[0011]

[0012] At present, there is an urgent need to develop small molecule fluorescent probes suitable for mitochondrial SMLM imaging, which need to have stable structure, can specifically label mitochondria through simple co-incubation without transfection, and do not cause damage to cells and mitochondria. Summary of the Invention

[0013] In response to one or more problems existing in the prior art, the present invention provides a Nile blue derivative compound, which can be used as a mitochondrial single-molecule localization imaging probe. The provided Nile blue derivative compound has the following structural formula (I):

[0014]

[0015] in:

[0016] R1 and R2 are each independently selected from C 1-5 alkyl;

[0017] X is selected from H, OH, NH2, halogen, C 1-4 Haloalkyl, COOR3, and COO - , wherein R3 is selected from H and C 1-5alkyl.

[0018] Since the Nile blue derivative compound of the present invention contains a tetravalent amine cation, which is usually present in the form of a salt, it also contains a compensating anion; or, when the X substituent is COO - In the case of, forming an inner salt. In the present application, the compensating anion used to form the salt is not limited, and any anion commonly used in the art can be selected to form the salt. In the structural formula (I) shown in the present application, the group used as the compensating anion is not shown.

[0019] In some embodiments, the halogen is selected from F, Cl, Br and I, and the C 1-4 Haloalkyl is CH2-halogen selected from chloromethyl and bromomethyl.

[0020] In some embodiments, the X substituent is COO - In the case of forming an inner salt, the inner salt can be formed by reacting the Nile blue derivative compound of formula (I) containing CO2H with an organic base or an inorganic base; optionally, the organic base can be selected from methylamine, ethylamine, ethylenediamine, propylenediamine, propylamine, ethanolamine, diethanolamine, triethanolamine, trimethylamine, triethylamine, tributylamine, N-methylglucamine, N,N-dimethylaminopyridine, diisopropylethylamine, potassium tert-butoxide, tetrapropylammonium hydroxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetraisopropylammonium hydroxide , tetraisobutylammonium hydroxide, tetrabutylammonium hydroxide, sodium methoxide, sodium ethoxide, potassium ethoxide and potassium tert-butoxide; the inorganic base can be selected from ammonia, alkali metal, alkaline earth metal or transition metal hydroxide, oxide, sulfide, carbonate, phosphate, carboxylate, or sulfonate, for example, the inorganic base can be selected from ammonia, potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, calcium oxide, calcium hydroxide, lithium hydroxide, magnesium hydroxide, strontium hydroxide, copper hydroxide, zinc hydroxide, aluminum hydroxide, iron hydroxide and cobalt hydroxide.

[0021] In some embodiments, the Nile blue derivative compound is selected from the following compounds:

[0022]

[0023] Another aspect of the present invention provides a method for preparing a Nile blue derivative compound, wherein in the general structural formula (I), X is H, and the preparation method comprises the following steps:

[0024] S1: 1-amino-5-bromonaphthalene is reacted with 1,4-dibromobutane to prepare compound 2;

[0025]

[0026] S2: reacting compound 2 with phenylboronic acid to obtain compound 3;

[0027]

[0028] S3: reacting compound 3 with compound 4' to obtain the Nile blue derivative compound;

[0029] wherein R1 and R2 are each independently selected from C 1-5 alkyl.

[0030] Another aspect of the present invention provides a method for preparing a Nile blue derivative compound, wherein in the general structural formula (I), X is CH2-halogen, and the preparation method comprises the following steps:

[0031] T1: reacting 1-amino-5-bromonaphthalene with 1,4-dibromobutane to prepare compound 2;

[0032]

[0033] T2: reacting compound 2 with 4-methoxycarbonylphenylboronic acid to obtain compound 5;

[0034]

[0035] T3: Compound 5 is subjected to a reduction reaction and then reacted with CR4, wherein R is selected from Cl and Br, to obtain compound 6';

[0036]

[0037] T4: reacting compound 6' with compound 4' to obtain the Nile blue derivative compound;

[0038] wherein R1 and R2 are each independently selected from C 1-5 alkyl.

[0039] Another aspect of the present invention provides a method for preparing a Nile blue derivative compound, wherein in the general structural formula (I), X is CO2H, and the preparation method comprises the following steps:

[0040] M1: 1-amino-5-bromonaphthalene reacts with 1,4-dibromobutane to prepare compound 2;

[0041]

[0042] M2: Compound 2 is reacted with 4-methoxycarbonylphenylboronic acid to prepare compound 5;

[0043]

[0044] M3: hydrolyzing compound 5 and reacting it with compound 4' to obtain the Nile blue derivative compound;

[0045] wherein R1 and R2 are each independently selected from C 1-5 alkyl.

[0046] In another aspect, the present invention provides a method for preparing a Nile blue derivative compound, wherein in the general structural formula (I), X is COO - , the preparation method further comprises the following steps based on step M3:

[0047] M4: The Nile blue derivative prepared in step M3 is mixed with a base (organic base or inorganic base) in DCM, methanol or DMF to obtain a compound of the general formula (I) wherein X is COO - Nile blue derivatives.

[0048] In some embodiments, the reaction of step S2, T2 or M2 is carried out in the presence of Pd(dppf)Cl2 under an inert atmosphere; and / or, the reaction of step S3, T4 or M3 is carried out in the presence of an organic acid, preferably selected from HOAc, CF3CO2H and CH3SO3H; and / or, step T3 comprises a reduction reaction in an inert atmosphere under alkaline conditions with a LiAlH4 catalyst, followed by reaction with CR4 in the presence of PPh3.

[0049] In another aspect, the present invention also provides the use of the Nile blue derivative compound in mitochondrial single-molecule localization imaging, and the use of the Nile blue derivative compound in preparing a probe for mitochondrial single-molecule localization imaging or in preparing a biological sample labeling reagent.

[0050] In some embodiments, the biological sample is mitochondria in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figures 1A-1D The absorption and emission curves of compound B and MTDR as well as the solvatochromic phenomenon are depicted, where Figure 1A represents the absorption and emission curves of compound B and MTDR in PBS pH 7.4, Figure 1B represents the absorption and emission curves of compound B and MTDR in the lipophilic solvent n-octanol, Figure 1C Indicates the solvatochromic phenomenon of compound B, Figure 1D It represents the solvatochromic phenomenon of MTDR.

[0052] Figures 2A-2D The performance of Compound B or MTDR in live cell confocal microscopy without washing is depicted, where Figures 2A-2CRespectively represent the performance of 50nM, 250nM, and 500nM compound B in live cell confocal microscopy without washing. Figure 2D Figure 5 shows the performance of 500 nM MTDR in live cell confocal microscopy without washing; scale bar, 10 μm.

[0053] Figures 3A-3C Confocal microscopy of live cells without washing is shown for 50 nM, 250 nM, and 500 nM of Compound A, respectively; scale bar, 10 μm.

[0054] Figures 4A-4D is the curve of HO· and HOCl oxidative quenching of MTDR and compound B fluorescence, where Figures 4A-4B The curves showing the fluorescence quenching of MTDR and compound B by HO· oxidation, respectively, Figures 4C-4D The curves represent the fluorescence quenching of MTDR and compound B by HOCl oxidation, respectively.

[0055] Figure 5 Mitochondrial staining images of HeLa cells after incubation with compound B and MTDR for 24 hours. Scale bar, 5 μm.

[0056] Figures 6A-6J Describe the application of compound B in mitochondrial SMLM imaging, where Figure 6A Depicts the reduction of compound B by sodium dithionite (Na2S2O4) and its reoxidation by exposure to air; Figures 6B-6C The absorption (6B) and emission spectra (6C) of the oxidized and reduced forms of compound B, respectively; Figures 6D-6E Depicts ultra-high-resolution SMLM images of mitochondria reconstituted in fixed cells using compound B; Figures 6F-6G Depicts a diffraction-limited wide-field image captured prior to SMLM imaging; Figure 6H Transverse profiles of five individual mitochondria marked with red dashed lines in the reconstructed super-resolution image are depicted; Figure 6I The SMLM images of compound B reveal mitochondrial dynamics in living cells. Each image was reconstructed using 1000 consecutive frames at a rate of 100 frames per second. The red arrows indicate multiple mitochondrial fission and fusion events. Figure 6J Depicted are SMLM imaging of mitochondria in living cells using MTDR, which exhibits rapid photobleaching and high background. Scale bars: 2 μm in (D) / (F) / (I) / (J), 500 nm in (E) / (G). DETAILED DESCRIPTION

[0057] Given that the small molecule fluorophores used for mitochondrial SMLM imaging in the existing technology have the following disadvantages. (1) Small molecule fluorophores derived from rhodamine and anthocyanin in structure have inherent disadvantages. For example, when the concentration of MTDR is high, it not only stains mitochondria, but also non-specifically labels the endoplasmic reticulum and cell nucleus, and causes a large number of mitochondrial fragmentation and rounding during the staining process, which is highly toxic. In addition, due to the influence of its polymethylen bridge chain structure, MTDR has weak antioxidant capacity, and some common reactive oxygen species in mitochondria can easily damage its structure. The frontier orbital energy of MTDR in the ground state is high and the electron affinity is weak. In the excited state, the frontier orbital electrons transition to higher energy levels, making the structure more unstable and more susceptible to oxidation. This determines that it is very easy to be photobleached when conducting SMLM experiments under physiological conditions. (2) Trisubstituted Nile blue is not suitable for biological imaging because it is easily deprotonated by the 5-amino group and the absorption wavelength shifts blue. (3) Tetrasubstituted Nile blue is sensitive to nucleophilic substances and is unstable in living cells. Given that many small molecule probes (such as (1)-(3) above) currently require transfection experiments before being used for ultra-high resolution imaging of mitochondria, each cell has a different response to a specific transfection reagent or method, and any transfection method will inevitably cause some cell death, the labeling of mitochondria by plasmid transfection has certain limitations. Based on the structural characteristics of Nile blue dye, the applicant has creatively developed a class of cationic tetrasubstituted Nile blue fluorophores with a novel and unique structure, stable structure (strong antioxidant capacity), which can specifically label mitochondria by simple co-incubation without transfection, and does not cause damage to cells and mitochondria.

[0058] Specifically, the applicant replaced the 5-amino group of trisubstituted Nile Blue (NBA) or tetrasubstituted Nile Blue (TENB) with a pyrrolidine ring and introduced a substituted or unsubstituted benzene ring at the C1 position, and used the thus modified Nile Blue derivative compounds as small molecule fluorophores for mitochondrial SMLM imaging.

[0059] Therefore, the general structural formula of the cationic tetrasubstituted Nile blue fluorophore (also known as a Nile blue derivative compound) provided by the present invention, which can be used as a mitochondrial single-molecule localization imaging probe, can be shown as the following formula (I):

[0060]

[0061] in:

[0062] R1 and R2 are each independently selected from C 1-5 alkyl;

[0063] X is selected from H, OH, NH2, halogen, C 1-4 Haloalkyl, -COOR3, and COO - , wherein R3 is selected from H or C 1-5alkyl.

[0064] Since the Nile blue derivative compound of the present invention contains a tetravalent amine cation, which is usually present in the form of a salt, it also contains a compensating anion; or, when the X substituent is COO - In this case, an inner salt is formed.

[0065] In this application, the compensating anion used to form the salt is not limited, and any anion commonly used in the art can be selected to form the salt, including but not limited to: CF3COO - HCOO - 、CH3COO - 、C2F5COO - 、C3F7COO - 、C4F9COO - 、CH3SO3 - 、Cl3CO2 - 、F - 、Cl - Br - , I - PO4 3- 、SO4 2- 、ClO4 - BrO4 - 、BF4 - In the structural formula (I) shown in the present application, the group serving as the compensating anion is not shown.

[0066] In a specific embodiment, R1 and R2 are each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl and neopentyl; preferably, R1 and R2 are each independently ethyl.

[0067] In a specific embodiment, R3 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl and neopentyl; preferably, R3 is H.

[0068] In a specific embodiment, the halogen is selected from F, Cl, Br and I; preferably, the halogen is Cl.

[0069] In a specific embodiment, C 1-4 Haloalkyl is CH2-halogen selected from chloromethyl and bromomethyl.

[0070] In a specific embodiment, when the X substituent is COO -In the case of forming an inner salt, the inner salt can be formed by reacting the Nile blue derivative compound of formula (I) containing CO2H with an organic base or an inorganic base; optionally, the organic base can be selected from methylamine, ethylamine, ethylenediamine, propylenediamine, propylamine, ethanolamine, diethanolamine, triethanolamine, trimethylamine, triethylamine, tributylamine, N-methylglucamine, N,N-dimethylaminopyridine, diisopropylethylamine, potassium tert-butoxide, tetrapropylammonium hydroxide, tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetraisopropylammonium hydroxide , tetraisobutylammonium hydroxide, tetrabutylammonium hydroxide, sodium methoxide, sodium ethoxide, potassium ethoxide and potassium tert-butoxide; the inorganic base can be selected from ammonia, alkali metal, alkaline earth metal or transition metal hydroxide, oxide, sulfide, carbonate, phosphate, carboxylate, or sulfonate, for example, the inorganic base can be selected from ammonia, potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, calcium oxide, calcium hydroxide, lithium hydroxide, magnesium hydroxide, strontium hydroxide, copper hydroxide, zinc hydroxide, aluminum hydroxide, iron hydroxide and cobalt hydroxide.

[0071] The applicant chose to modify the pyrrolidine ring because the pyrrolidine ring is less polar and more lipophilic than NH2, and compared with tetraethyl Nile blue (TENB), the ring strain and conformational constraints of the pyrrolidine ring in the modified structure greatly reduce the steric repulsion between the CH group at the C4 position and the CH2 group of the pyrrolidine ring. And unlike the trisubstituted Nile blue with an extra proton in the 5-amino group, the structure after the pyrrolidine ring modification is less sensitive to the environmental pH value because it does not undergo a deprotonation-protonation process. In addition, the applicant found that the use of the pyrrolidine ring is the best choice for preparing this type of tetrasubstituted Nile blue derivative compound, and attempts to replace the pyrrolidine ring in the modified structure with azetidine, aziridine, or piperidine were unsuccessful. Without being bound by any theory, the applicant speculates that this may be due to changes in ring strain or steric hindrance, and therefore it is necessary to select an appropriate nitrogen monocyclic structure for modification to obtain suitable ring strain and steric hindrance.

[0072] In addition, the benzene ring at the C1 position can be substituted or unsubstituted, which can further increase the lipophilicity of the molecule, thereby improving the transmembrane ability. The Nile blue derivative compound modified with the unsubstituted benzene ring is named compound A in this application. In addition, a group selected from OH, NH2, halogen, C 1-4 A haloalkyl group (preferably CH2Cl, the Nile blue derivative compound thus modified is named compound B), COOR3 (wherein R3 may be H, the Nile blue derivative compound thus modified is named compound C), and COO -(forming an inner salt, and the Nile blue derivative compound thus transformed is named as compound D) a substituent X. The carboxylic acid (COOR3, wherein R3 is H) introduced on the phenyl ring can serve as a linking group, and by covalently binding with drug molecules, protein inhibitors, etc., mitochondrial-specific delivery of small molecules is achieved, and the direct interaction between drugs and organelles is studied. The CH2 connected to the halogen acts as an electrophilic reaction site, and can undergo a nucleophilic substitution reaction with the nucleophilic residues on the mitochondrial protein, so that the Nile blue derivative compound is covalently attached to the protein, and the fluorescent signal is not attenuated due to mitochondrial damage and membrane potential changes. In addition, the alkyl group connected to the C9 amino group can be a C1-C5 alkyl group, preferably each an ethyl group.

[0073]

[0074] In a more specific embodiment, the structure of the cationic tetrasubstituted Nile blue fluorophore provided by the present invention, which can be used as a mitochondrial single-molecule localization imaging probe, is selected from the following compounds A, B, and C (omitting their counterions), or compound D (inner salt):

[0075]

[0076] Unless otherwise specified, the terms used herein have the following meanings.

[0077] The term "alkyl" as used herein includes straight chain alkyl and branched chain alkyl, for example, "C 1-5 "Alkyl" includes C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 Alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl and neopentyl, etc.

[0078] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0079] As used herein, the term "zwitterion" refers to a salt formed by neutralization of a positively charged group and a negatively charged group in the same molecule.

[0080] The present invention will be further described below with reference to specific examples. It should be understood that the specific examples are only used to further illustrate the present invention, and are not intended to limit the scope of the present invention.

[0081] The methods used in the following examples are conventional methods unless otherwise specified.

[0082] The methods for obtaining the various biological materials described in the examples are intended merely to provide a means for experimental acquisition to achieve full disclosure and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0083] English abbreviations and their meanings:

[0084] ACN: acetonitrile

[0085] CCl4: Tetrachloromethane

[0086] DCM: dichloromethane

[0087] DMF: N,N-dimethylformamide

[0088] Et2O: ethyl ether

[0089] EtOAc: ethyl acetate

[0090] HOAc: acetic acid

[0091] HPLC: High Performance Liquid Chromatography

[0092] MeOH: methanol

[0093] TFA: trifluoroacetic acid

[0094] THF: Tetrahydrofuran

[0095] PPh3: triphenylphosphine

[0096] LiAlH4: lithium aluminum hydride

[0097] Pd(dppf)Cl2:[1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride

[0098] Example 1 :Synthesis of a Nile Blue-based Single-molecule Localization Imaging Probe for Mitochondria

[0099] In this example, the synthesis of compound A, compound B, compound C, and compound D is described in detail, and the synthesis route is as follows:

[0100]

[0101] 1) Synthesis of Compound A

[0102] 1.1) Argon was bubbled through a mixture of 1-amino-5-bromonaphthalene (compound 1, 2.22 g, 10 mmol, 1 equiv), KCO (5.53 g, 40 mmol, 4 equiv), and KI (332 mg, 2 mmol, 0.2 equiv) in anhydrous DMF (25 mL) for 5 minutes, followed by the addition of 1,4-dibromobutane (2.38 mL, 20 mmol, 2 equiv). The resulting mixture was stirred and heated to 100°C under argon for 12 hours, then cooled to room temperature and diluted with 250 mL of water. The mixture was extracted with EtOAc (50 mL x 3), and the combined organic phases were washed with saturated NaCl, dried over NaSO, filtered, concentrated, and purified by silica gel column chromatography (eluent: hexane to hexane / EtOAc = 100 / 1) to afford compound 2 as a brown oil (2.24 g, 81%). 1 H NMR (400MHz, CDCl3) δ8.26(d,J=8.6Hz,1H),7.94(d,J=8.4Hz,1H),7.81(d,J=7.2Hz,1H),7.52(d ,J=8.2,1H),7.30(t,J=7.9Hz,1H),7.07(d,J=7.6Hz,1H),3.44–3.28(m,4H),2.12–1.94(m,4H); 13 CNMR(101MHz, CDCl3)δ148.2,133.5,129.9,129.6,127.4,124.9,124.4,123.2,120.3,112.5,53.0,24.9; LRMS(ESI) for C 14 H 15 BrN([M+H] + ):Calcd.276.04,278.04,found276.32,278.35; HRMS(ESI)for C 14 H 15 BrN([M+H] + ):Calcd.276.0388,278.0367,found276.0378,278.0358.

[0103]

[0104] 1.2) Argon was bubbled through a solution of compound 2 (192 mg, 0.695 mmol, 1 eq), phenylboronic acid (93 mg, 0.765 mmol, 1.1 eq), Pd(dppf)Cl2 (25 mg, 0.034 mmol, 0.05 eq), and NaHCO3 (117 mg, 1.39 mmol, 2 eq) in dioxane (4 mL) and H2O (1 mL) for 5 minutes, then stirred and heated to 75°C under argon. After reacting for 3 hours, the mixture was cooled to room temperature and filtered through silica gel, washed with EtOAc, and concentrated to dryness. The residue was purified by silica gel column chromatography (eluent: hexane / EtOAc = 100 / 1) to give compound 3 as a brown oily liquid (187 mg, 98%). 1 H NMR(400MHz, CDCl3)δ8.32(d,J=8.5Hz,1H),7.59–7.50(m,6H),7.49–7.42(m,2H), 7.36(t,J=8.0Hz,1H),7.06(d,J=7.5Hz,1H),3.48–3.37(m,4H),2.14–2.04(m,4H); 13 C NMR (101MHz, CDCl3) δ148.1,141.6,140.5,133.2,130.3,128.9,128.2,127.1,126.8,126.0,124.5,123.9,119.7,111.7,53.0,24.8; LRMS(ESI) for C 20 H 20 N([M+H] + ):Calcd.274.16,found 274.51.HRMS(ESI)for C 20 H 20 N([M+H] + ):Calcd.274.1596,found274.1590.

[0105]

[0106] 1.3) Compound 3 (27 mg, 0.1 mmol, 1 eq) and compound 4 (5-diethylamino-2-nitrosophenol hydrochloride, 23 mg, 0.1 mmol, 1 eq) were stirred in HOAc (2 mL) and heated to 115°C. After 4 hours, another portion of compound 4 (23 mg, 0.1 mmol, 1 eq) was added, and the reaction was continued for 4 hours before cooling to room temperature. The mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH = 30 / 1 to 15 / 1) to yield a dark blue solid. If necessary, it could be purified by preparative HPLC on a C18 column (mobile phase: ACN / H2O (containing 0.1% TFA) = 70:30) to yield a dark blue solid, the TFA salt of compound A (28 mg, 50%). 1 H NMR (500MHz, CD3OD) δ8.40(d,J=8.2Hz,1H),7.75(t,J=7.8Hz,1H),7.66(d,J=7.2Hz,1H),7.45–7.37(m ,3H),7.28–7.21(m,2H),6.95(dd,J=9.4,2.7Hz,1H),6.93(s,1H),6.74–6.66(m,2H),4.17–4.04(broad s,4H),3.59(q,J=7.1Hz,4H),2.23–2.12(broad s,4H),1.26(t,J=7.1Hz,6H); 13 C NMR (126MHz, CD3OD) δ160.9,155.3,151.8,148.4,145.6,143.8,136.2,136.0,133.8,131.2,12 9.6,128.96,128.94,128.7,128.5,127.61,127.58,115.5,98.7,96.5,56.6,46.8,26.6,12.8; 19 F NMR(471MHz,CD3OD)δ–77.2; LRMS(ESI)for C 30 H 30 N3O(M + ):Calcd.448.24,found 448.55; HRMS(ESI)for C 30 H 30 N3O(M + ):Calcd.448.2383, found 448.2386.

[0107]

[0108] 2) Synthesis of Compound B

[0109] 2.1) Argon was bubbled through a mixed solution of compound 2 (1.15 g, 4.04 mmol, 1 equiv), 4-methoxycarbonylphenylboronic acid (763 mg, 4.24 mmol, 1.05 equiv), Pd(dppf)Cl (147 mg, 0.2 mmol, 0.05 equiv), and NaHCO (679 mg, 8.08 mmol, 2 equiv) in dioxane (8 mL) and H O (2 mL) for 5 minutes, then heated to 75° C. under argon with stirring. After 6 hours, the mixture was cooled to room temperature and filtered through silica gel, washed with EtOAc, and concentrated. The residue was purified by silica gel column chromatography (eluent: hexane / EtOAc = 100 / 1 to 40 / 1) to provide compound 5 as a brown oily liquid (1.23 g, 92%). 1 H NMR (400MHz, CDCl3) δ8.37(d,J=8.6Hz,1H),8.24(d,J=8.2Hz,2H),7.63(d,J=8.1Hz,2H),7.55–7.46(m,2H),7.41 (d,J=6.9Hz,1H),7.35(t,J=8.0Hz,1H),7.04(d,J=7.5Hz,1H),4.02(s,3H),3.47–3.31(m,4H),2.13–1.96(m,4H); 13 C NMR (101MHz, CDCl3) δ166.8,148.0,146.2,139.1,132.6,130.1,129.4,128.7,1 28.6,126.6,126.2,124.9,123.5,118.8,111.6,52.7,51.9,24.6; LRMS(ESI)for C 22 H 22 NO2([M+H] + ):Calcd.332.17,found 332.52; HRMS(ESI)for C 22 H 22 NO2([M+H] + ):Calcd.332.1651,found332.1649.

[0110]

[0111] 2.2) To a solution of compound 5 (331 mg, 1 mmol, 1 eq) in anhydrous Et2O (10 mL) was slowly added LiAlH4 (38 mg, 1 mmol, 1 eq) at room temperature. The resulting mixture was stirred under argon for 1 hour, followed by the addition of H2O (40 μL), 15% aqueous NaOH (80 μL), and H2O (0.12 mL). After drying over Na2SO4, the mixture was filtered through celite, washed with EtOAc, and concentrated to afford a light yellow oil, which was dissolved in CCl4 (10 mL), followed by the addition of PPh3 (262 mg, 1 mmol). The resulting mixture was heated to reflux under argon for 28 hours, then cooled to room temperature, concentrated and purified by silica gel column chromatography (eluent: hexane to hexane / EtOAc = 50 / 1) to obtain compound 6 (Cl in the structure is derived from CCl4; to synthesize a compound in which the substituent Cl in compound 6 is replaced by Br, CCl4 can be replaced by CBr4 (1 equivalent), and dry DCM (1-10 mL) is used as solvent) as a light yellow oily liquid (210 mg, 65%). 1 H NMR(400MHz, CDCl3) δ8.29(d,J=8.6Hz,1H),7.56–7.44(m,6H),7.40(dd,J=7.0,1.3Hz,1H),7. 33(t,J=8.0Hz,1H),7.04(d,J=7.5Hz,1H),4.71(s,2H),3.46–3.31(m,4H),2.12–2.00(m,4H); 13 C NMR (101MHz, CDCl3) δ148.2,141.8,139.8,136.3,133.1,130.6,128.8,128. 5,126.8,126.1,124.7,123.9,119.4,111.8,53.0,46.3,24.8; LRMS(ESI)for C 21 H 21 ClN([M+H] + ):Calcd.322.14,324.13,found 322.44,324.47; HRMS(ESI)for C 21 H 21 ClN([M+H] + ):Calcd.322.1363,324.1333,found 322.1357,324.1335.

[0112]

[0113] 2.3) A solution of compound 6 (32 mg, 0.1 mmol, 1 eq) and compound 4 (23 mg, 0.1 mmol, 1 eq) in HOAc (3 mL) was heated to 115°C under air. After 3 hours, another portion of compound 4 (23 mg, 0.1 mmol, 1 eq) was added, and the reaction was continued for 3 hours before cooling to room temperature. The mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH = 30 / 1 to 12 / 1) to yield a dark blue solid. This was further purified by preparative HPLC on a C18 column (mobile phase: ACN / H2O (containing 0.1% TFA) = 70:30) to yield a dark blue solid, the TFA salt of compound B (19 mg, 31%). 1 H NMR(500MHz,CD3OD)δ8.47(dd,J=8.4,1.3Hz,1H),7.80(dd,J=8.5,7.5Hz,1H),7.7 1(dd,J=7.5,1.0Hz,1H),7.51–7.45(m,2H),7.30–7.25(m,2H),7.03(s,1H),6.97( dd,J=9.4,2.7Hz,1H),6.83(d,J=9.5Hz,1H),6.78(d,J=2.5Hz,1H),4.80(s,2H),4 .23–4.10(m,4H),3.62(q,J=7.1Hz,4H),2.26–2.12(m,4H),1.26(t,J=7.2Hz,6H); 13 C NMR (126MHz, CD3OD) δ161.1,155.4,152.1,148.6,145.8,143.2,138.1,136.08,136.06,133.9,1 31.3,130.0,129.5,129.1,128.9,128.6,127.7,115.4,98.8,96.5,56.5,47.2,46.8,26.7,12.8; 19 F NMR(376MHz,CD3OD)δ–77.3; LRMS(ESI)for C 31 H 31 ClN3O(M + ):Calcd.496.22,found 496.26; HRMS(ESI)for C 31 H 31 ClN3O(M + ):Calcd.496.2150, found 496.2165.

[0114]

[0115] 3) Synthesis of Compound C

[0116] 3.1) A mixture of compound 5 (193 mg, 0.582 mmol, 1 eq) in THF (6 mL) and 6N NaOH solution (1 mL) was heated to reflux for 18 hours. The mixture was cooled to room temperature, and the THF was then removed by rotary evaporation. The residue was diluted with 3 mL of water and adjusted to pH 6 with 3N HCl, forming a yellow precipitate. This precipitate was collected by filtration, washed with water, and then dried under high vacuum (182 mg, quant.). A solution of this yellow solid (32 mg, 0.1 mmol, 1 eq) and compound 4 (23 mg, 0.1 mmol, 1 eq) in HOAc (2 mL) was heated to 115°C under air. After 4 hours, another portion of compound 4 (23 mg, 0.1 mmol, 1 eq) was added, and the reaction was continued for another 4 hours before cooling to room temperature. The mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH (containing 1% HOAc) = 50 / 1 to 10 / 1) to yield a dark blue solid. If necessary, the product can be further purified by preparative HPLC on a C18 column (mobile phase: ACN / H2O (containing 0.1% TFA) = 47 / 53) to give a dark blue solid, which is the TFA salt of compound C (25 mg, 41%). 1 HNMR (400MHz, CD3OD) δ8.43(d,J=8.3Hz,1H),8.03(d,J=7.8Hz,2H),7.77(t,J=7.9Hz,1H),7.63(d,J=7. 2Hz,1H),7.32(d,J=8.0Hz,2H),6.97–6.85(m,2H),6.69(s,1H),6.57(d,J=9.2Hz,1H),4.18–4.04(broad s,4H),3.58(q,J=7.1Hz,4H),2.25–2.10(broad s,4H),1.25(t,J=7.0Hz,6H); 13 C NMR (126MHz, CD3OD) δ169.8,160.6,155.4,151.7,150.5,148.5,142.4,135.8,135.5,133.4,130 .9,130.4,130.1,129.8,129.2,129.1,128.6,127.5,115.8,98.8,96.6,56.6,46.9,26.7,12.8; 19 FNMR(376MHz,CD3OD)δ–77.2; LRMS(ESI)for C 31 H 30 N3O3([M]+ ):Calcd.492.23,found 492.34; HRMS(ESI)for C 31 H 30 N3O3([M] + ):Calcd.492.2282, found 492.2290.

[0117]

[0118] 4) Synthesis of Compound D (Inner Salt)

[0119] 4.1) To a solution of the TFA salt of compound C (12 mg, 0.02 mmol, 1 eq) in MeOH (2 mL) was added 0.1 N NaOH solution (0.2 mL, 0.02 mmol, 1 eq). After stirring at room temperature for 10 minutes, the solvent was removed by rotary evaporation, and the residue was purified by preparative HPLC on a C18 column (mobile phase: ACN / H2O = 47 / 53) to give a dark blue solid, compound D (9 mg, 92%). 1 H NMR (400MHz, CD3OD) δ8.43(d,J=8.3Hz,1H),8.03(d,J=7.8Hz,2H),7.77(t,J=7.9Hz,1H),7.63(d,J=7.2 Hz,1H),7.32(d,J=8.0Hz,2H),6.97–6.85(m,2H),6.69(s,1H),6.57(d,J=9.2Hz,1H),4.18–4.04(broad s,4H),3.58(q,J=7.1Hz,4H),2.25–2.10(broad s,4H),1.25(t,J=7.0Hz,6H); 13 C NMR (126MHz, CD3OD) δ169.8,160.6,155.4,151.7,150.5,148.5,142.4,135.8,135.5,133.4,130.9,130 .4,130.1,129.8,129.2,129.1,128.6,127.5,115.8,98.8,96.6,56.6,46.9,26.7,12.8; LRMS(ESI)forC 31 H 30 N3O3([M+H] + ):Calcd.492.23,found 492.34; HRMS(ESI)for C 31 H 30 N3O3([M+H] +):Calcd.492.2282, found 492.2290.

[0120]

[0121] Example 2: Lipophilicity and membrane permeability evaluation of Nile blue derivative compounds

[0122] 2.1) n-Octanol is often used to evaluate the lipophilicity of organic small molecule drugs. The distribution of drugs between water and n-octanol can be used to calculate their partition coefficient, which is a good approximation of their affinity for the cytoplasm (water) or lipid membranes of living systems. In this example, the lipophilicity and solvatochromic phenomena of compound B synthesized in Example 1 were evaluated and compared with MTDR (MitoTracker TM Deep Red FM (purchased from ThermoFisher Scientific) was compared to determine its lipophilicity and solvatochromic properties. The following steps were included.

[0123] 2.1.1) Compound B was dissolved in DMSO (Sigma-Aldrich) to prepare a 10 mM stock solution. The solution was diluted to 10 μM using n-octanol (Sigma-Aldrich) or phosphate buffer (PBS pH 7.4, ThermoFisher Scientific). 3 mL was placed in a 1 cm × 1 cm quartz cuvette and the UV-visible absorption spectrum of the sample solution was measured using a Shimadzu UV-3150 spectrometer. Fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer. The results are shown in Figure 2. Figures 1A-1C shown.

[0124] 2.1.2) The fluorescent dye MTDR was dissolved in DMSO to prepare a 1 mM stock solution. The solution was diluted to 1 μM using n-octanol or phosphate buffer (PBS pH 7.4) and placed in a 1 cm × 1 cm quartz cuvette. The UV-visible absorption spectrum of the sample solution was measured using a Shimadzu UV-3150 spectrometer. Fluorescence measurements were performed at room temperature on a Hitachi F-7000 fluorescence spectrophotometer. The results are shown in Figure 2. Figures 1A-1B , as shown in 1D.

[0125] Figures 1A-1D The absorption and emission curves of compound B and MTDR as well as the solvatochromic phenomenon are shown, where Figure 1A represents the absorption and emission curves of compound B and MTDR in PBS pH 7.4, Figure 1B represents the absorption and emission curves of compound B and MTDR in the lipophilic solvent n-octanol, Figure 1C Indicates the solvatochromic phenomenon of compound B, Figure 1D Indicates the solvatochromic phenomenon of MTDR. Figures 1A-1B It can be seen that compound B exhibits a large Stokes shift (40-50 nm) in both the lipophilic solvent n-octanol and the aqueous buffer, which is caused by the red shift of its fluorescence emission (~700 nm). In contrast, the absorption / emission curve of MTDR in PBS or n-octanol remains essentially unchanged, which leads to a smaller Stokes shift in PBS and n-octanol. Figure 1C and Figure 1D As shown, unlike the cyanine dye MTDR, the cationic Nile blue type dye (Compound B) exhibits a unique solvatochromic phenomenon, and its fluorescence in the lipophilic medium n-octanol is enhanced by about 8 times compared with the aqueous medium, while the fluorescence of MTDR in the lipophilic medium n-octanol is only enhanced by about 2-3 times compared with the aqueous medium.

[0126] 2.2) In this example, the membrane permeability of Compound B synthesized in Example 1 was evaluated and compared with that of MTDR. Specifically, the following steps were included.

[0127] 2.2.1) HeLa cell line (ATCC) culture and inoculation: HeLa cells were grown in complete medium (i.e., DMEM (ThermoFisher Scientific) medium containing 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific) and 1% penicillin-streptomycin (ThermoFisher Scientific) by volume). The culture flasks were placed in a vented culture incubator maintained at 37°C with 5% carbon dioxide. When the cell density at the bottom of the flask reached 70-80%, the culture medium was aspirated and discarded. The cells were washed twice with 1 mL of PBS buffer. Then, 1 mL of trypsin (ThermoFisher Scientific) was added and the flask was placed in the incubator for digestion for 1 minute. The digestion was quickly terminated by adding 2 mL of complete medium to the flask. The flask was tapped to dislodge the cells. The entire cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded and 2 mL of complete medium was added. The cells were resuspended by pipetting, counted, and diluted to 1 × 10 with complete medium. 5 Pipette 2 mL of this suspension into a glass-bottom confocal imaging dish and incubate in an incubator for 24 hours before incubating with the probe.

[0128] 2.2.2) Probe Incubation and Imaging: The culture medium in the glass-bottomed confocal imaging dish was discarded, and the cells were washed with 1 mL of HBSS solution (Thermofisher Scientific). Phenol red-free DMEM solution containing the specified concentration of probe (Compound B or MTDR) was added to the dish. The dish was incubated in an incubator for 40 minutes, and confocal fluorescence microscopy was performed directly. The microscope used was a Zeiss LMS 780 or 880 laser scanning confocal microscope equipped with a 64× or 40× / 1.3 oil-immersion objective. The cell environment was maintained at 37°C and 5% CO2. The excitation wavelength was 633 nm, and fluorescence was collected at a wavelength of 650-750 nm.

[0129] Figures 2A-2D The performance of compound B or MTDR in live cell confocal microscopy without washing is shown, wherein Figures 2A-2C The results of 50nM, 250nM and 500nM compound B in live cell confocal microscopy without washing are shown in Figure 2. Figures 2A-2B The blue fluorescence in the sample comes from Hoechst dye (ThermoFisher Scientific), which is used to label cell nuclei at a working concentration of 0.5 μg / mL. Cells were co-incubated with compound B. Figure 2D The figure shows the performance of 500nM MTDR in live cell confocal microscopy without washing. The scale bar is 10μm. Figures 2A-2D As shown, it can be seen that compound B exhibits extremely low background fluorescence at different concentrations. At a lower concentration (50nM), it can penetrate from the outside of the cell membrane to the inside of the cell membrane, showing excellent membrane permeability, and is not easy to diffuse under the three concentration conditions of the experiment, and can specifically stain and mark mitochondria. In contrast, it can be clearly observed that MTDR at a concentration of 500nM easily diffuses into the cytoplasm and the nucleus, and the specificity for mitochondrial staining is not high. This result shows that compound B has excellent membrane permeability, is not easy to diffuse, and has a high specificity for mitochondrial staining. Similar to compound B, compound A also has excellent membrane permeability, and its imaging results according to the above steps 2.2.1) and 2.2.2) are as follows Figures 3A-3C Shown (scale bars are 10 μm) are the performance of 50 nM, 250 nM and 500 nM compound A in live cell confocal microscopy without washing.

[0130] In summary, the results of 2.1 and 2.2 above indicate that the Nile blue derivative compound provided by the present invention exhibits a unique solvatochromic phenomenon and has excellent membrane permeability, which makes it more suitable for non-washing live cell imaging (i.e., direct imaging without washing after incubation of the probe with the cells).

[0131] Example 3: Evaluation of the antioxidant activity of a Nile blue-based mitochondrial single-molecule localization imaging probe

[0132] Mitochondrial respiratory metabolism produces reactive oxygen species (ROS), including hypochlorous acid (HOCl), hydroxyl radicals (HO ·), etc., which are highly oxidizing. The interaction between them and dyes in different excited states is a common chemical process that can lead to irreversible photobleaching. Therefore, the antioxidant properties of the probe are crucial for mitochondrial imaging. In this example, the antioxidant capacity of compound B synthesized in Example 1 was evaluated as an example, and compared with the antioxidant capacity of the commercial cyanine-based mitochondrial dye MTDR, specifically including the following steps.

[0133] 3.1) Dissolve the fluorescent dye MTDR in DMF (Sigma-Aldrich) to prepare a 1 mM stock solution. Dilute to 1.67 μM using phosphate buffer (PBS pH 7.4, ThermoFisher Scientific), take 3 mL and place in a 1 cm × 1 cm quartz cuvette, add 40 equivalents of H2O2 (30% aqueous solution, Sigma-Aldrich) and then add 4 equivalents of FeSO4 (Sigma-Aldrich) solution to generate 4 equivalents of HO· in situ and oxidize MTDR, and the volume of the added test solution does not exceed 1% of the total volume. After the solution is thoroughly mixed, the fluorescence intensity curve is measured on a fluorescence measurement Hitachi F-7000 fluorescence spectrophotometer with an excitation wavelength of 633 nm and a photomultiplier voltage of 950 V. The preparation method of 10 to 50 equivalents of HO· is similar. The results are shown in the figure. Figure 4A shown.

[0134] 3.2) Compound B was dissolved in DMF to prepare a 10 mM stock solution. Dilute to 16.67 μM using PBS pH 7.4, take 3 mL and place in a 1 cm × 1 cm quartz cuvette, add 100 equivalents of H2O2 (30% aqueous solution) and mix well, then add 10 equivalents of FeSO4 solution to generate 10 equivalents of HO· in situ and oxidize compound B, and the volume of the added test solution should not exceed 1% of the total volume. After the solution is thoroughly mixed, the fluorescence intensity curve is measured on a fluorescence measurement Hitachi F-7000 fluorescence spectrophotometer, with an excitation wavelength of 647 nm and a photomultiplier voltage of 950 V. The preparation method of 30 and 50 equivalents of HO· is similar. The results are shown in the figure below. Figure 4B shown.

[0135] 3.3) Dissolve the fluorescent dye MTDR in DMF to prepare a 1 mM stock solution. Dilute to 2 μM using PBS pH 7.4, take 3 mL and place in a 1 cm × 1 cm quartz cuvette, add Figure 4CIndicated equivalents of HOCl (5% aqueous solution, bleach), and the volume of the test solution added does not exceed 1% of the total volume. After the solution is thoroughly mixed, the fluorescence intensity curve is measured on a Hitachi F-7000 fluorescence spectrophotometer with an excitation wavelength of 633 nm and a photomultiplier voltage of 700 V. The results are shown in Figure 2. Figure 4C shown.

[0136] 3.4) Dissolve compound B in DMF to prepare a 10 mM stock solution. Dilute to 16.67 μM using PBS pH 7.4, take 3 mL and place in a 1 cm × 1 cm quartz cuvette, add Figure 4D The indicated equivalents of HOCl (5% aqueous solution, derived from bleach), and the volume of the test solution added does not exceed 1% of the total volume. After the solution is thoroughly mixed, the fluorescence intensity curve is measured on a Hitachi F-7000 fluorescence spectrophotometer with an excitation wavelength of 647nm and a photomultiplier voltage of 950V. The results are shown in Figure 2. Figure 4D shown.

[0137] Figures 4A-4D The curves showing the oxidation quenching of MTDR and compound B by HO· and HOCl. Figures 4A-4D As can be seen, HO· can oxidatively quench the fluorescence of the cyanine-based mitochondrial dye MTDR in a concentration-dependent manner, but it has little effect on the fluorescence of compound B. In addition, three equivalents of HOCl can completely bleach MTDR but only cause a small loss of fluorescence for compound B. This result indicates that compound B has strong antioxidant capacity, which contributes to its good stability in living cells.

[0138] Example 4: Evaluation of mitochondrial damage using a Nile blue-based mitochondrial single-molecule localization imaging probe

[0139] The state of mitochondria is highly sensitive to external stimuli. In this example, the potential damage caused by compound B synthesized in Example 1 to this organelle was evaluated and compared with MTDR, which specifically includes the following steps.

[0140] Follow the above step 2.2.1. When the HeLa cells reach a density of 70-80% at the bottom of the culture flask, aspirate and discard the culture medium in the flask. Wash the cells twice with 1 mL of PBS buffer, add 1 mL of trypsin, and place the culture flask in the incubator for digestion for 1 minute. Quickly add 2 mL of complete culture medium to the flask to terminate the digestion. Tap the flask wall to remove the cells. Transfer the entire cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, and add 2 mL of complete culture medium. Resuspend the cells by pipetting, count, and dilute them with complete culture medium to a density of 5×104 Cells were suspended at a concentration of 10 cells / mL, and 2 mL of this cell suspension was transferred to a glass-bottomed confocal imaging dish. After incubation for 24 hours in an incubator, the culture medium in the dish was discarded, and the cells were washed with 1 mL of HBSS. 2 mL of phenol red-free DMEM solution containing the indicated concentration of dye (Compound B or MTDR) was added to the dish. The dish was incubated in an incubator for 24 hours. The DMEM solution in the dish was discarded, and the cells were washed twice with HBSS before adding 1 mL of phenol red-free DMEM solution for confocal fluorescence microscopy. The microscope was a Zeiss LMS 780 or 880 laser scanning confocal microscope equipped with a 64× or 40× / 1.3 oil immersion objective. The cell environment was maintained at 37°C and 5% CO2. The excitation wavelength was 633 nm, and fluorescence was collected at a wavelength of 650-750 nm.

[0141] The results are as follows Figure 5 As shown (scale bar, 5 μm), mitochondria in HeLa cells treated with MTDR swell and enlarge. In contrast, mitochondria in HeLa cells treated with compound B largely maintain their elongated, filamentous morphology. This result suggests that compound B causes less damage to mitochondria, maintaining their health.

[0142] Example 5: Application of Nile Blue-based Mitochondrial Single-Molecule Localization Imaging Probe in SMLM Imaging of Mitochondria in Living Cells

[0143] 5.1) Evaluation of the redox properties of a Nile Blue-based mitochondrial single-molecule localization imaging probe in the ground state

[0144] The redox properties of small molecule fluorophores in the ground state determine the ease with which they switch from a fluorescent bright state to a non-fluorescent dark state. If the transition between the bright and dark states is easier to achieve, the conditions required for photoswitching under physiological conditions will be milder. In this example, the redox properties of compound B synthesized in Example 1 were evaluated in the ground state and compared with other commercial mitochondrial imaging probes (MTR) (MitoTracker TM The redox characteristics of Red CMXRos (purchased from ThermoFisher Scientific) were compared with those of MTDR in the ground state, which specifically included the following steps.

[0145] 5.1.1) Dissolve Compound B in DMSO (Sigma-Aldrich) to prepare a 10 mM stock solution. Dilute to 10 μM with DCM (Sigma-Aldrich). Place 3 mL of this solution in a 1 cm × 1 cm quartz cuvette and measure the UV-Vis absorption spectrum of the sample solution using a Shimadzu UV-3150 spectrometer. Mix the DCM solution of Compound B with a freshly prepared concentrated solution of Na2S2O4 (Sigma-Aldrich). Vortex thoroughly and allow to stand until the DCM solution fades from blue to colorless or light yellow. Remove the lower organic phase and place it in a 1 cm × 1 cm quartz cuvette. Measure the UV-Vis absorption and fluorescence spectra. Air is introduced into the quartz cuvette until the solution returns to a blue color with no further deepening. Measure the fluorescence spectrum.

[0146] 5.1.2) Dissolve the fluorescent dye MTDR or MTR in DMSO to prepare a 1 mM stock solution. Dilute to 1 μM in DCM. Take 3 mL of this solution and mix it with the freshly prepared concentrated Na2S2O4 solution. Vortex thoroughly and let it stand until no color fading is observed in the DCM phase.

[0147] The results are as follows Figures 6A-6C As shown, Figure 6A This represents a reaction in which compound B is reduced by sodium dithionite (Na2S2O4) and then reoxidized by exposure to air; Figures 6B-6C are the absorption of oxidized and reduced forms of compound B ( Figure 6B ) and launch ( Figure 6C ) spectrum. It can be seen that after thoroughly mixing a 10 μM dichloromethane solution of compound B with a freshly prepared sodium dithionite solution, the characteristic blue-green color of compound B and its near-infrared absorption / emission completely disappear, indicating that the reduced state of the dye is formed; when air is bubbled into the light yellow dichloromethane solution, the blue-green color and absorption / emission energy gradually recover. This result shows that the redox conversion exhibited by compound B is reversible, and this conversion can be repeated more than 10 times before the dye is significantly degraded. In contrast, rhodamine-based MTR or cyanine-based MTDR dyes cannot achieve this redox switching. This is because their reduction potentials are low and they are less likely to accept electrons, so extremely strong reducing agents (such as sodium borohydride) or high laser irradiation are required to put these probes into the dark state.

[0148] 5.2) Evaluation of the applicability of Nile Blue-based mitochondrial single-molecule localization imaging probes for mitochondrial SMLM imaging in fixed cells

[0149] In this experiment, compound B was used as an example to evaluate its applicability in SMLM imaging of mitochondria in fixed cells. A redox buffer (PBS solution containing 1 mM ascorbyl palmitate and 1 mM methyl amethystine) was used to assist the photoswitching process of the dye. The specific steps included were as follows.

[0150] 5.2.1) Experimental materials and instruments

[0151] The cells used for SMLM imaging were U-2OS cell line (ATCC), and the complete culture medium required for cell growth was Mccoy's 5A (ThermoFisher Scientific) medium containing 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific) and 1% penicillin-streptomycin (ThermoFisher Scientific).

[0152] SMLM experiments were performed using the NBI SRiS STROM and Ningbo Lixian iSTORM instruments. The microscope objective was a Nikon CFI Apochromat 100× NA1.49 oil-immersed total internal reflection fluorescence (TIRF) microscope, and the detector was an Oxford Instruments Andor iXon Ultra 897 EMCCD camera. The excitation wavelength was 647 nm.

[0153] 5.2.2) SMLM experiment of fixed cells: U-2OS cells were grown in a breathable culture flask containing complete culture medium and placed in an incubator with a constant temperature of 37°C and 5% carbon dioxide. When the cell growth density at the bottom of the culture flask reached 70-80%, the culture medium in the cell culture flask was aspirated and discarded. After adding 1 mL of PBS buffer to wash the cells twice, 1 mL of trypsin was added and the culture flask was placed in the incubator for digestion for 1 minute. 2 mL of complete culture medium was quickly added to the flask to terminate the digestion. The flask wall was knocked to make it fall off. The entire cell suspension was transferred to a 15 mL centrifuge tube. After centrifugation at 1000 rpm for 3 minutes, the supernatant was discarded and 2 mL of complete culture medium was added. The cells were resuspended by pipetting and counted, and diluted to 1×10 with complete culture medium. 5Cells / mL cell suspension. 2 mL of this suspension was transferred to a 35 mm φ cell culture dish containing a 18 mm φ glass slide and incubated in an incubator for 24 hours. The culture medium in the dish was discarded. The cells were washed with 1 mL of PBS solution. 2 mL of phenol red-free DMEM solution containing 250 nM compound B was added to the dish and incubated in an incubator for 1 hour. The liquid in the dish was then discarded, the cells were washed twice with PBS, and 2 mL of 4% paraformaldehyde solution (ThermoFisher Scientific) was added. After incubation at room temperature for 20 minutes, the liquid in the dish was discarded and the cells were washed twice with PBS. The 18 mm φ glass slide containing the cells was removed and placed in the imaging chamber of the SMLM instrument. 0.38 mL of Tris-HCl 8.0 (ThermoFisher Scientific) buffer containing 1 mM vitamin C (Sigma-Aldrich) and 1 mM methyl amethyst (Sigma-Aldrich) was added, and a 28 mm × 28 mm coverslip was placed above the liquid level. Turn on the camera, select 647nm laser, adjust the laser intensity to WF (wide field imaging), and capture the wide field image of the cell mitochondria (i.e., the diffraction-limited image, such as Figure 6F Gradually increase the laser intensity (<3kW / cm 2 ) When you see a flicker of fluorescence in the mitochondria, click "Start Capture" to continuously capture the fluorescence signal. The camera automatically records the fluorescence intensity and position at each pixel. The number of raw image frames captured is 9000.

[0154] 5.2.3) Reconstruction of super-resolution images: Reconstruction was performed using ImageJ software equipped with ThunderSTORM (Ovesny, M.; Krizek, P.; Borkovec, J.; Svindrych, Z.; Hagen, GM, ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 2014, 30(16), 2389-90.) and HAWK (Marsh, RJ; Pfisterer, K.; Bennett, P.; Hirvonen, LM; Gautel, M.; Jones, GE; Cox, S., Artifact-free high-density localization microscopy analysis. Nat. Methods 2018, 15(9), 689-692.) plug-ins. 9000 frames of original images were imported into ImageJ and preprocessed using the HAWK plug-in. The "number of levels" input was 5. The processed images were further processed using the ThunderSTORM plug-in with the default parameters. The output data contained key information such as the coordinates, intensity (photon number), background intensity, and positioning uncertainty of each fluorescent point. From this, the average photon number and positioning uncertainty of all fluorescent points can be calculated. Abnormal points with too high or too low fluorescence intensity were discarded, and the remaining fluorescent points were overlapped in one image to obtain a super-high-resolution image of the mitochondria (such as Figure 6D shown).

[0155] 5.2.4) Plotting FWHM data: Select the region of interest in the widefield image or super-high resolution image and first draw a straight line (e.g. Figure 6E and 6G According to Analyze->Plotprofile in Fiji software, the image profile is obtained (as shown in the red dotted line in the figure). Figure 6H shown).

[0156] In the reconstructed ultra-high resolution image ( Figure 6D ) with red dashed lines marking the transverse profiles of five individual mitochondria, showing an average full width at half maximum (FWHM) of approximately 250 nm, which is significantly smaller than that obtained from diffraction-limited total internal reflection fluorescence (TIRF) imaging ( Figure 6H) (approximately 1 μm). Data output from reconstructing super-high-resolution images showed that, on average, each probe (Compound B) emitted 680 photons per imaging frame, achieving a spatial resolution of approximately 13 nm, an improvement of approximately one order of magnitude compared to traditional optical microscopy techniques.

[0157] 5.3) Evaluation of the applicability of Nile Blue-based mitochondrial single-molecule localization imaging probes for mitochondrial SMLM imaging in living cells

[0158] In living cells, the photoreduction efficiency of organic fluorophores is determined by the local thiol concentration and pH, because thiolates (RS – ) acts as a reducing substance. Studies on intracellular glutathione (GSH) have shown that mitochondria have the highest concentration of reduced GSH / GSSG (oxidized glutathione) pool among various organelles (Jones, DP Redox Sensing: Orthogonal Control in Cell Cycle and Apoptosis Signalling: Symposium: Redox Sensing in Cellular Control. J. Intern. Med. 2010, 268 (5), 432–448. https: / / doi.org / 10.1111 / j.1365-2796.2010.02268.x), and its interior is alkaline with a pH value of about 7.8, so it has a strong photoreduction ability. This unique feature makes it possible to use the Nile blue-based mitochondrial single-molecule localization imaging probe provided by the present invention to perform SMLM imaging of mitochondria in living cells. In this experiment, compound B synthesized in Example 1 was used to perform SMLM imaging evaluation of mitochondria in living cells, which specifically includes the following steps.

[0159] 5.3.1) SMLM experiment of living cells: Use the same operation as the fixed cell experiment above (step 5.2.2) to seed U-2OS into a φ35mm cell culture dish with a φ18mm glass slide. After culturing for 24 hours, discard the culture medium in the culture dish and wash the cells with 1mL PBS solution. Add 2mL of phenol red-free DMEM solution containing 250nM compound B or MTDR to the culture dish, place it in the incubator and incubate for 1 hour, then discard the liquid in the culture dish and wash the cells twice with PBS. Take out the φ18mm glass slide containing the cells and place it in the imaging chamber equipped with the SMLM instrument, add 0.38mL of phenol red-free DMEM solution to it, and cover it with a 28mm×28mm coverslip above the liquid surface. Turn on the camera, select 647nm for the laser, adjust the laser intensity to WF (widefield imaging), and capture the widefield image of the cell mitochondria. Gradually increase the laser intensity (<3kW / cm2 ) When you see a flicker of fluorescence in the mitochondria, click "Start Capture" to continuously capture the fluorescence signal. The camera automatically records the fluorescence intensity and position at each pixel. The number of raw image frames captured is 20,000.

[0160] 5.3.2) Reconstruction of ultra-high resolution images: The method is the same as step 5.2.3 above.

[0161] The results obtained from the above experiments can be used to study mitochondrial dynamics under physiological conditions, and multiple fission and fusion events have been successfully observed, such as Figure 6I (SMLM images of compound B reveal mitochondrial dynamics in living cells. Scale bar is 2 μm. Each image was reconstructed using 1000 consecutive frames at a rate of 100 frames per second) indicated by the red arrow. The data output when reconstructing super-high-resolution images shows that each dye molecule (compound B) emits an average of 800 photons per imaging frame, resulting in a localization accuracy of approximately 18 nm for a single compound B probe. Other oxazine dyes (such as Atto 655) have also been found to achieve redox conversion under physiological conditions. However, their limited membrane permeability and tendency to nonspecifically stain hinder their effectiveness in penetrating mitochondria and imaging.

[0162] In this experiment, SMLM imaging of mitochondria in living cells was performed using cyanine-based MTDR under the same imaging conditions as compound B. Super-resolution images could be reconstructed using the initial 3000 frames of MTDR. However, it was observed that MTDR experienced rapid photobleaching during imaging ( Figure 6J , scale bar is 2μm), which ultimately produces a reconstructed image dominated by the background. In contrast, the blinking behavior of compound B lasts significantly longer ( Figure 6I The mechanism behind photobleaching is complex, but there is evidence that in the presence of molecular oxygen, the excited triplet state of the dye is easily oxidized to form free radicals, leading to photobleaching. This mechanism is particularly applicable to cyanine dyes (Altman, RB et al. Cyanine Fluorophore Derivatives with Enhanced Photostability. Nat. Methods 2012, 9(1), 68–71. https: / / doi.org / 10.1038 / nmeth.1774), because Figure 4 shows that it is easily oxidized in the ground state and combined with Figures 1A-1B Figure 4 Figures 6A-6CThe result of the experiment 5.1.2 above that MTDR cannot be reduced to the dark state by Na2S2O4 indicates that the ground state frontier orbital energy of compound B is lower than that of MTDR, and the frontier orbital electrons are not easily oxidized. Figures 1A-1B This shows that the absorption wavelengths of compound B and MTDR are not much different, indicating that the energy required for the two to enter the excited state is not much different. This proves that in the excited state, compound B has lower frontier orbital electron energy than MTDR and is relatively less susceptible to oxidation, while MTDR is more easily oxidized in the excited state.

[0163] Based on the results of the above examples, it can be seen that the mitochondrial single-molecule localization imaging probe based on a Nile blue derivative compound provided by the present invention can replace the commonly used commercial MTDR probe. Compared with MTDR, the mitochondrial single-molecule localization imaging probe based on Nile blue provided by the present invention is a more cost-effective mitochondrial-specific probe and has potential commercial value.

[0164] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to substitute equivalent features for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A Nile blue derivative compound having the following general structural formula (I): in: R1 and R2 are each independently selected from C 1-5 alkyl; X is selected from H, OH, NH2, halogen, C 1-4 Haloalkyl, COOR3, and COO - , wherein R3 is selected from H and C 1-5 alkyl.

2. The Nile blue derivative compound according to claim 1, wherein the halogen is selected from F, Cl, Br and I, and / or the C 1-4 Haloalkyl is CH2-halogen selected from chloromethyl and bromomethyl.

3. The Nile blue derivative compound according to claim 1, which is selected from the following compounds:

4. The method for preparing the Nile blue derivative compound according to claim 1, wherein in the general structural formula (I), X is H, and the preparation method comprises the following steps: S1: 1-amino-5-bromonaphthalene is reacted with 1,4-dibromobutane to prepare compound 2; S2: reacting compound 2 with phenylboronic acid to obtain compound 3; S3: reacting compound 3 with compound 4' to obtain the Nile blue derivative compound; wherein R1 and R2 are each independently selected from C 1-5 alkyl.

5. The method for preparing a Nile blue derivative compound according to claim 1, wherein in the general structural formula (I), X is CH2-halogen, and the method comprises the following steps: T1: reacting 1-amino-5-bromonaphthalene with 1,4-dibromobutane to prepare compound 2; T2: reacting compound 2 with 4-methoxycarbonylphenylboronic acid to obtain compound 5; T3: Compound 5 is subjected to a reduction reaction and then reacted with CR4, wherein R is selected from Cl and Br, to obtain compound 6'; T4: reacting compound 6' with compound 4' to obtain the Nile blue derivative compound; wherein R1 and R2 are each independently selected from C 1-5 alkyl.

6. The method for preparing the Nile blue derivative compound according to claim 1, wherein in the general structural formula (I), X is CO2H, and the preparation method comprises the following steps: M1: 1-amino-5-bromonaphthalene reacts with 1,4-dibromobutane to prepare compound 2; M2: Compound 2 is reacted with 4-methoxycarbonylphenylboronic acid to prepare compound 5; M3: hydrolyzing compound 5 and reacting it with compound 4' to obtain the Nile blue derivative compound; wherein R1 and R2 are each independently selected from C 1-5 alkyl.

7. The method according to any one of claims 4 to 6, wherein the reaction of step S2, T2 or M2 is carried out in the presence of Pd(dppf)Cl2 in an inert atmosphere.

8. The method according to any one of claims 4 to 6, wherein the reaction of step S3, T4 or M3 is carried out in the presence of an organic acid.

9. The method of claim 8, wherein the organic acid is selected from the group consisting of HOAc, CF3CO2H, and CH3SO3H.

10. The method according to any one of claims 4 to 6, wherein step T3 comprises a reduction reaction in an inert atmosphere under a LiAlH4 catalyst and alkaline conditions, followed by a reaction with CR4 in the presence of PPh3.

11. Use of the Nile blue derivative compound according to any one of claims 1 to 3 in mitochondrial single-molecule localization imaging.

12. Use of the Nile blue derivative compound according to any one of claims 1 to 3 in the preparation of a biological sample labeling reagent.

13. The use according to claim 12, wherein the biological sample is mitochondria in living cells.

14. Use of the Nile blue derivative compound according to any one of claims 1 to 3 in preparing a probe for mitochondrial single-molecule localization imaging.

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