Novel oxocyanine-based compound, preparation method therefor, and use thereof

Abstract

The present invention relates to a novel oxocyanine-based compound, a preparation method therefor, and use thereof, particularly, use thereof for staining biomolecules. The compound of the present invention has excellent photostability. Thus, by using the compound of the present invention, the behavior of biomolecules can be observed in real time over a long period of time, and single-molecule imaging or high-resolution imaging that requires a strong light source can also be stably performed for a long period of time. In addition, the compound of the present invention has a small molecular size, thus not interfering with the dynamic movement of proteins, and has little blinking and is hardly affected by oxygen, thus being observable without adding an additive or artificially lowering the concentration of oxygen, and thus enables analysis under conditions closer to the actual cellular environment.

Description

Novel oxo cyanine compound, method for preparing the same, and use thereof

[0001] The present invention relates to a novel oxocyanine compound, a method for producing the same, and a use thereof.

[0002] Fluorescent dyes are key components in bioimaging and displays, widely used in research and industry. The development of superior fluorescent dyes has led to innovative technological advancements in bioimaging and displays. Organic dyes, in particular, have the greatest applicability and potential due to their small size, diverse wavelengths, ease of chemical modification, and biocompatibility. These characteristics serve as significant advantages for organic dyes, enabling their application in a wide range of fields.

[0003] However, conventional organic dyes are prone to photobleaching, making long-term observation difficult. To overcome this, inorganic fluorescent materials like quantum dots are used. However, their irregular and frequent blinking limits real-time tracking analysis. Furthermore, quantum dots are much larger than proteins, raising the possibility of multiple proteins being labeled on a single quantum dot. This can affect protein movement, potentially affecting the interpretation of results. Therefore, the small size of dyes is essential for single-molecule and super-resolution imaging.

[0004] Methodologies and technologies are continuously being developed to overcome the limitations of existing dyes. Oxygen is a key factor in photodegradation, and methods have been reported to extend observation time by artificially reducing its concentration or treating the dye with oxygen scavengers. However, these approaches have negative effects on the cells under observation. Furthermore, atmospheric composition can alter protein expression and other factors, limiting observations to limited atmospheric conditions.

[0005] Additionally, most organic dyes have a non-hydrophilic structure, which can lead to aggregation in aqueous solutions. This can lead to false positive results. Furthermore, when labeling membrane proteins, some dyes can enter cells, reducing labeling efficiency. Furthermore, organic dyes are known to exist in a triplet state in some cases, which can induce blinking or photodegradation. Using a triplet quencher to prevent this state can increase photostability. However, treatment with an oxygen scavenger is essential to achieve a dramatic increase, and moving from the in vitro to the cellular level significantly reduces the observation time.

[0006] This limitation can also be overcome by using dyes with high quantum yield, a key factor influencing fluorescence brightness. High quantum yield allows for maintaining a certain level of brightness even at relatively low light intensities, and the low energy of the light source increases the time required for photodecomposition. However, there are limits to lowering light intensity or increasing quantum yield, and since the photostability of the molecule itself is not fundamentally improved, extending the observation time is also limited.

[0007] Therefore, organic dyes with high photostability are currently absent, and their development is considered essential.

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] [Tsunoyama, TA et al., "Super-long single-molecule tracking reveals dynamic-anchorage-induced integrin function," Nat. Chem. Biol. 2018 May;14:497-506]

[0011] [Altman, RB et al., “Cyanine fluorophore derivatives with enhanced photostability,” Nat. Methods. 2012 Jan;9(1):68-71]

[0012] [Zheng, Q. et al., “On the Mechanisms of Cyanine Fluorophore Photostabilization,” J. Phys. Chem. Lett. 2012 Jul;3(16):2200-2203]

[0013] [Grimm, JB et al., “A general method to fine-tune fluorophores for live-cell and in vivo imaging,” Nat. Methods. 2017 Oct;14(10):987-994]

[0014] One object of the present invention is to provide an organic dye with high photostability that can overcome the limitations of the above-mentioned prior art, such as photodecomposition.

[0015] The present inventors have found that one of the two nitrogen moieties of a cyanine dye is oxo (Oxo, ) structure was introduced, and an asymmetric compound was synthesized for the first time, and the purpose of the present invention could be achieved through the compound.

[0016] The compound of the present invention overcomes the limitations of conventional organic dyes, and in particular, has the advantage of not being easily photodegraded, i.e., having extremely high photostability. This characteristic allows for an observation time to be extended to minutes, rather than the seconds of conventional techniques. Accordingly, the behavior of biomolecules can be observed in real time over an extended period of time, thereby enabling the observation of behaviors that were previously impossible with conventional dyes. In particular, long-term observation was nearly impossible in single-molecule imaging or high-resolution imaging, which require a strong light source. However, by using the compound of the present invention, even single-molecule imaging and high-resolution imaging can be performed stably.

[0017] Furthermore, the compound of the present invention has the advantage of being small in molecular size, allowing for results close to actual behavior without interfering with the dynamic movement of proteins. Furthermore, the compound of the present invention exhibits minimal flickering and is virtually unaffected by oxygen, enabling observation without the need for additives or artificially lowering oxygen concentrations, enabling analysis under a variety of atmospheric conditions.

[0018] The compound of the present invention is expected to enable analyses previously difficult with conventional dyes, thanks to its high photostability, which allows for extended observation time, and its ability to be utilized without significant constraints. Furthermore, it is expected to find application in various fields requiring highly photostable materials.

[0019] FIG. 1 is a diagram illustrating the structure of the ultra-photostable chemical species of the present invention. In FIG. 1(a) is a schematic diagram illustrating the photoblueing reaction setup for TSCy5. 0.13 M TSCy5 in 100 μL DMSO was placed in a rod-shaped quartz cuvette, and irradiated with a 1 W 638-nm laser while continuously flowing oxygen gas into the cuvette. As a result, the navy-colored TSCy5 solution changed to red after overnight irradiation, and the reaction mixture was analyzed by HPLC. In FIG. 1(b) is an HPLC chromatography comparing the TSCy5 photoblueing reaction before (dotted line) and after (solid line). Different colors in the chromatography diagram represent different absorption wavelengths, and the tested compounds were in the range of 254 to 630 nm. In Fig. 1(c) shows the results of in vitro photodegradation lifetime analysis of TSCy3 and PF555 derived from photoblued TSCy5, where the upper panel shows fluorescence images of PF555 at various time points (250 s intervals) during 1000 s imaging in the red channel (572-624 nm) (scale bar = 20 μm). The lower panel shows an exponential decay fitting curve plotted along the normalized fluorescence intensity data (PF555: points in the upper curve, TSCy3: points in the lower curve). In Fig. 1(d) is a single-molecule intensity profile of PF555, where the upper panel shows single-molecule images of PF555 at various time points (75 s intervals) during 300 s imaging in the red channel (572-624 nm) (scale bar = 1 μm). The lower panel shows the fluorescence intensity profile of a single PF555 molecule over time.

[0020] Figure 2 is a diagram showing that PF555 has a significantly superior photolysis lifetime compared to other organic dyes in living cells. In Figure 2(a), the fluorescence image during 320 s imaging shows HALO-EGFR overexpressed in living COS7 cells labeled with PF555, AF555, JFX549, and TMR via chloroalkane conjugation (scale bar = 20 μm). Figure 2(b) shows the photolysis lifetimes of CA-PF555, CA-AF555, CA-JFX549, and CA-TMR. The exponential decay fitting curves (colored lines) are plotted as normalized fluorescence intensity data (colored dots) for CA-PF555 (top curve), CA-AF555 (second curve from the top), CA-JFX549 (third curve from the top), and CA-TMR (bottom curve). Figure 2(c) shows the luminance of CA-PF555 (second curve from the top), CA-AF555 (bottommost curve), CA-JFX549 (topmost curve), and CA-TMR (third curve from the top) at 561 nm in the range of 100 to 32,000 mW / cm. 2 The results were measured in vitro at various laser power intensities.

[0021] Figures 3 and 4 demonstrate that previously unobserved long-term single-molecule dynamics of EGFR in live COS7 cells were revealed by MSD analysis. Figure 3(a) compares the diffusion coefficient distributions between PF555 and AF647, directly comparing the diffusivity of PF555 with that of AF647 in the same COS7 cells. The trajectory of PF555 was divided into 10 frames per trajectory, and the diffusion coefficient was calculated from the divided trajectories of PF555 and compared with that of AF647 (n > 5,000 trajectories for each dye). The distribution of the above coefficients is presented as mean ± SEM, and statistical comparisons between the two dye groups were performed using the t-test (p-value > 0.5). Figure 3(b) shows the number of fluorescent spots of PF555 detected in each 50-ms frame across 16 image streams, each lasting 80 s. PF555 was photoactivated between streams by exposure to a 405-nm laser. In Figures 4(d) to (i), the left panel is a single trajectory map using a color-coded time scale, the middle panel is a time-dependent MSD plot of the corresponding trajectory, where the red line indicates the MSD value, the blue dashed line is the MSD fitting curve, and the light red area indicates the standard deviation. The right panel is a displacement angle map.

[0022] Figure 5 shows the HR-MS negative mode spectra of PF555 monoprotonated form (left) and monosodium form (right). [M] = C 30 H 36 N2O 13 S4.

[0023] Figure 6 is an LC-MS spectrum of DM-PF595 prepared in Example 7.

[0024] Figure 7 is the LC-MS spectrum of PF555-COOH.

[0025] Figure 8 is the LC-MS spectrum of PF555-COOH_2.

[0026] Figure 9 shows the photophysical properties of PF555.

[0027] Figure 10 is an LC-MS spectrum according to Experimental Example 2, which is the spectrum of Cy5-Mal_PF555 (top) and the reaction product of Cy5-Mal_PF555 and an amino acid (cysteine) (bottom), respectively.

[0028] Figure 11 is for evaluating the photodegradation lifetime of PF555 under PCA / PCD conditions in living COS7 cells. Figure 11a is representative single molecule images of AF647, PF555, and TMR measured in living COS7 cells with or without PCA / PCD addition, and the images were captured with an EM-CCD camera at a frame rate of 20 Hz and a power of 30 W / cm. 2 Acquired using a 561 nm laser at a high intensity (scale bar = 10 μm). Figure 11b shows the time-dependent decrease in the number of fluorescent spots detected from each 50 ms frame for AF647, PF555, and TMR in the PM of live COS7 cells. The black line represents the best single exponential decay function for each dye, and the derived exponential decay time constants are indicated. Figure 11c shows the calculated photolysis lifetimes, expressed as exponential decay time constants, for AF647, PF555, and TMR under control and PCA / PCD supplemented conditions. The lifetimes were measured using a homemade MATLAB program, and the data are presented as mean ± standard deviation (SD).

[0029] Figure 12 shows a comparative analysis of the nonspecific binding levels of PF555, AF647, and ATTO647N in living COS7 cells. Figure 12 (a) is a fluorescence image of living COS7 cells cultured with PF555, AF647, and ATTO647N at concentrations ranging from 100 pM to 100 nM. (Scale bar = 20 μm). Figure 12 (b) is the average fluorescence intensity of the acquired images normalized to the intensity of the untreated sample, and the data are expressed as the mean ± standard deviation (SEM).

[0030] Figure 13 shows the photoactivity of PF555 under 405 nm irradiation, which is a fluorescence image of live COS7 cells labeled with CA-PF555 before and after 405 nm laser irradiation.

[0031] Hereinafter, the present invention will be described in detail.

[0032] The terms used in this specification are used only to describe specific embodiments and are not intended to limit the present invention, and unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0033] Throughout the specification, when it is said that a part “includes,” “contains,” or “has” a component, this means that it may further include other components, unless specifically stated otherwise.

[0034] As used herein, "alkyl(rene) having 1 to 20 carbon atoms" means a straight or branched chain alkyl(rene) having 1 to 20 carbon atoms, wherein the carbon atoms may be 1 to 15, 1 to 10, 1 to 6, or 1 to 5. Specific examples of the alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0035] In this specification, "aromatic ring having 5 to 20 carbon atoms" means a monocyclic or polycyclic unsaturated aromatic hydrocarbon ring having 5 to 20 ring skeletal carbon atoms, wherein the number of carbon atoms may be 5 to 18, 6 to 12, or 6 to 10, and examples of the aromatic ring include benzene, phenylbenzene, naphthalene, phenyl-naphthalene, naphthyl-benzene, anthracene, and the like.

[0036] As used herein, “halogen atom” includes F, Cl, Br, and I atoms.

[0037]

[0038] According to one aspect of the present invention, a compound represented by the following chemical formula 1 is provided:

[0039] [Chemical Formula 1]

[0040]

[0041] In the above chemical formula 1,

[0042] Rings A and B are each independently an aromatic ring having 5 to 20 carbon atoms, wherein ring A may be substituted with one or more R3s which are the same or different from each other, and ring B may be substituted with one or more R6s which are the same or different from each other,

[0043] R1 and R2 are each independently a hydrogen atom, an alkyl having 1 to 20 carbon atoms, -L1-R 10 , -L2-COO-L3-R 11 , -L4-CO-L5-R 12 , -L6-O-L7-R 13 , -L8-CO-NH-L9-R 14 , -L 10 -NH-L 11 -R 15 , -L 12 -SL 13 -R 16 , -L 14 -SO2-L 15 -R 17 , -L 16 -SO2-NH-L17 -R 18 , or -L 18 -PO2-L 19 -R 19 and;

[0044] R3 and R6 are each independently a hydrogen atom, a halogen atom, an alkyl having 1 to 20 carbon atoms, -L 21 -R 20 , -L 22 -COO-L 23 -R 21 , -L 24 -CO-L 25 -R 22 , -L 26 -OL 27 -R 23 , -L 28 -CO-NH-L 29 -R 24 , -L 30 -NH-L 31 -R 25 , -L 32 -SL 33 -R 26 , -L 34 -SO2-L 35 -R 27 , -L 36 -SO2-NH-L 37 -R 28 , or -L 38 -PO2-L 39 -R 29 and;

[0045] R4, R5, R7, and R8 are each independently a hydrogen atom, an alkyl having 1 to 20 carbon atoms, -L 41 -R 30 , -L 42 -COO-L 43 -R 31 , -L 44 -CO-L 45 -R 32 , -L 46 -OL 47 -R 33 , -L 48 -CO-NH-L 49 -R 34 , -L 50 -NH-L 51 -R35 , -L 52 -SL 53 -R 36 , -L 54 -SO2-L 55 -R 37 , -L 56 -SO2-NH-L 57 -R 38 , or -L 58 -PO2-L 59 -R 39 and;

[0046] L1 to L 19 , L 21 Inland L 39 , and L 41 Inland L 59 are each independently a single bond or an alkylene having 1 to 20 carbon atoms, wherein the alkylene may further include at least one linking group selected from the group consisting of -COO-, -CO-, -O-, -CO-NH-, -NH-, -S-, -SO2-, -SO2-NH-, and -PO2- within the chain,

[0047] R 10 Inland R 19 , R 20 Inland R 29 , and R 30 Inland R 39 are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 20 carbon atoms, a hydroxyl group, or a reactive substituent capable of reacting and bonding with a biomolecule,

[0048] n is 0 or an integer from 1 to 5,

[0049] However, at least one of R1 to R8 is a group having a reactive substituent capable of reacting and bonding with a biomolecule.

[0050]

[0051] According to one embodiment, the ring A may be a monocyclic or polycyclic aromatic ring having 5 to 12 carbon atoms. Here, the ring A may be substituted with 1 to 3, or 1 to 2 R3, which are the same or different from each other.

[0052] According to another embodiment, the ring A may be selected from the group consisting of the following chemical formulas a-1 to a-4:

[0053]

[0054] In the above chemical formulas a-1 to a-4, * indicates a linking moiety, and R3 is as defined above. The above chemical formulas a-1 to a-4 may be substituted with 1 to 3, or 1 to 2, R3, which are the same or different from each other.

[0055] According to one embodiment, the ring B may be a monocyclic or polycyclic aromatic ring having 5 to 12 carbon atoms. Here, the ring B may be substituted with 1 to 3, or 1 to 2 R6, which are the same or different from each other.

[0056] According to another embodiment, the ring B may be selected from the group consisting of the following chemical formulas b-1 to b-4:

[0057]

[0058] In the above chemical formulas b-1 to b-4, * indicates a linking moiety, and R6 is as defined above. The above chemical formulas b-1 to b-4 may be substituted with 1 to 3, or 1 to 2, R6, which are the same or different from each other.

[0059]

[0060] According to one embodiment, the compound of the present invention may be a compound selected from the group consisting of the following chemical formulae 2, 3a, 3b, and 3c:

[0061] [Chemical Formula 2]

[0062]

[0063] [Chemical Formula 3a]

[0064]

[0065] [Chemical Formula 3b]

[0066]

[0067] [Chemical formula 3c]

[0068]

[0069] In the above chemical formulas 2, 3a, 3b, and 3c, R1 to R8 and n are as defined above.

[0070]

[0071] According to one embodiment, the compound of the present invention may be a compound represented by the above chemical formula 2.

[0072]

[0073] According to one embodiment, R3 and R6 are each independently a hydrogen atom, an alkyl having 1 to 20 carbon atoms, or -L 21 -R 20 and the above L 21 is a single bond or alkylene having 1 to 20 carbon atoms, and R 20 may be a halogen atom, a hydroxyl group, a carboxyl group, or a sulfonic acid group.

[0074] According to one embodiment, at least one, for example 1 to 4, of R1, R2, R4, R5, R7, and R8 is a group having a reactive substituent capable of reacting and bonding with a biomolecule, and the rest are hydrogen atoms, alkyl having 1 to 20 carbon atoms, or -L 60 -R 40 and the above L 60 is a single bond or alkylene having 1 to 20 carbon atoms, and R 40 may be a halogen atom, a hydroxyl group, a carboxyl group, or a sulfonic acid group.

[0075] According to one implementation example, one of R1 and R2 is -L1-R 10 , -L2-COO-L3-R 11 , -L4-CO-L5-R 12 , -L6-O-L7-R 13 , -L8-CO-NH-L9-R 14 , -L 10 -NH-L 11 -R 15 , -L 12 -SL 13 -R 16 , -L 14 -SO2-L 15 -R 17 , -L 16 -SO2-NH-L 17 -R 18 , or -L 18 -PO2-L 19 -R 19 And here L1 to L 19 is as defined above, and R 10 Inland R 19 is a reactive substituent capable of reacting and bonding with a biomolecule; the remaining one of R1 and R2, R4, R5, R7, and R8 is a hydrogen atom, an alkyl having 1 to 20 carbon atoms, or -L 60 -R 40 and the above L 60 is a single bond or alkylene having 1 to 20 carbon atoms, and R 40 may be a halogen atom, a hydroxyl group, a carboxyl group, or a sulfonic acid group.

[0076] In one implementation, one of R4, R5, R7, and R8 is -L 41 -R 30 , -L 42 -COO-L 43 -R 31 , -L 44 -CO-L 45 -R 32 , -L 46 -OL 47 -R 33 , -L 48 -CO-NH-L 49 -R34 , -L 50 -NH-L 51 -R 35 , -L 52 -SL 53 -R 36 , -L 54 -SO2-L 55 -R 37 , -L 56 -SO2-NH-L 57 -R 38 , or -L 58 -PO2-L 59 -R 39 and here L 41 Inland L 59 is as defined above, and R 30 Inland R 39 is a reactive substituent capable of reacting and bonding with a biomolecule; the remainder of R4, R5, R7, and R8, R1, and R2 are hydrogen atoms, alkyl having 1 to 20 carbon atoms, or -L 60 -R 40 and the above L 60 is a single bond or alkylene having 1 to 20 carbon atoms, and R 40 may be a halogen atom, a hydroxyl group, a carboxyl group, or a sulfonic acid group.

[0077] According to one embodiment, n is an integer from 0 to 5, specifically an integer from 0 to 4, an integer from 0 to 1, an integer from 1 to 3, an integer from 1 to 4, or an integer from 1 to 3, for example, 0, 1, 2, 3, 4, or 5.

[0078]

[0079] A "reactive substituent capable of reacting and binding with a biomolecule" refers to any substituent having a group known in the art to be capable of reacting and binding with a biomolecule. The reactive substituents include, for example, alkenyl having 2 to 20 carbon atoms, alkynyl having 2 to 20 carbon atoms, sulfonic acid group, sulfonimide group, amine group, succinimide group, phosphoryl group, guanine group, cytosine group, thiol group, thiosulfone group, vinylsulfone group, isocyanate group, isothiocyanate group, maleimide group, carboxyl group, phenyl-isocyanate group, phenyl-isothiocyanate group, 1,2,4,5-tetrazine group, 3-methyl-6-phenyl-1,2,4,5-tetrazine group, trans-cyclooctene group, azide group, phenyl-azide group, dibenzocyclooctyne group, endo-bicyclononyne group (endo-BCN), sulfonylfluoride group, phenyl-sulfonylfluoride group, Benzophenone group, 3-phenyl-3H-diazirine group, 3-phenyl-3-(trifluoromethyl)-3H-diazirine group, 3-phenyl-3-methyl-3H-diazirine group, 3-butynyl-3H-diazirine group, tetrazole group, phenyl-tetrazole group, biotin group, hydrazine group, , , , , or a peptide probe (e.g., an oligoglycine probe, a K3 probe, a CCK probe, an R3CL probe, etc.). "A group having a reactive substituent capable of reacting and binding to a biomolecule" refers to a group including at least one selected from the reactive substituents described above.

[0080]

[0081] The reaction of labeling a target substance through a reactive substituent capable of reacting and binding to a biomolecule in the compound of the present invention is exemplified in the following reaction schemes 1 to 5 (the part indicated by a circle in the reaction schemes below is the target substance).

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] According to one embodiment, the compound of the present invention may be selected from the group consisting of the following compounds:

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] According to another aspect of the present invention, a method for preparing a compound of the present invention is provided, comprising the step of irradiating light to a compound of the following chemical formula 4 under oxygen (O2):

[0108] [Chemical Formula 4]

[0109]

[0110] In the above chemical formula 4

[0111] m is n+1, and ring A, ring B, R1 to R8, and n are as defined above.

[0112]

[0113] According to one embodiment, the compound of the above formula 4 may be in a solution state, and the light may be irradiated using a laser. Here, the wavelength of the laser may be set according to the absorption wavelength of the compound of the above formula 7, and a person skilled in the art will be able to easily determine the absorption wavelength according to the dye compound. In addition, the laser may be operated under, for example, 0.5 to 2.0 W. Here, the solution may be a solution of the compound of the above formula 7 dissolved in water or an appropriate organic solvent such as dimethylsulfoxide, dimethylformamide, ethanol, methanol, and acetonitrile, or in water.

[0114] The above manufacturing method may further include a step of purifying the reactant after the light irradiation step. The purification may be performed by a person skilled in the art by appropriately selecting from among conventional methods known in the art, such as column chromatography.

[0115]

[0116] The compound of the present invention can exhibit color in the near-infrared region, specifically in the 700 to 900 nm region. The compound of the present invention can stain a target substance, wherein the target substance may be a biomolecule. Therefore, the compound of the present invention can be used to label a biomolecule, and through such labeling, it can be used for visualization or quantification of the biomolecule.

[0117] Furthermore, the compounds of the present invention exhibit extremely long photodegradation lifetimes. For example, the compounds of the present invention may exhibit photodegradation times of approximately 300 to 350 s. Due to this excellent photostability, the compounds of the present invention can be used for imaging target substances, particularly for high-resolution imaging, both at the bulk level and at the single-molecule level. Furthermore, they can be used for observing target substances in real time over extended periods of time.

[0118] According to one aspect of the present invention, a dye composition for staining a target substance, comprising a compound of the present invention, is provided. Here, the target substance may be a biomolecule selected from the group consisting of a biomolecule, specifically a protein (e.g., a tumor marker, a signaling molecule), a peptide, a carbohydrate, a sugar, a fat, an antibody, an antigen, a lipid, a phospholipid, a lipopolysaccharide, a fatty acid, a nucleic acid (e.g., DNA, RNA), a cell membrane, a cell (e.g., a nerve cell, a tumor cell), and a microorganism, but is not necessarily limited thereto. According to one embodiment, the staining may be for obtaining an image of the target substance. According to another embodiment, the staining may be for confirming light emitted from the target substance. In addition, the staining may be for observing the behavior of the target substance in real time. The method for staining a target substance such as a biomolecule using the compound of the present invention is not particularly limited and may be appropriately selected from known methods.

[0119] According to another aspect of the present invention, a reagent, fluorescent probe, or contrast agent containing a compound of the present invention is provided. The reagent or fluorescent probe may be used to detect biomolecules or tissues. Furthermore, the contrast agent may be used to diagnose diseases.

[0120] According to another aspect of the present invention, an optical filter comprising a compound of the present invention is provided. Specifically, the optical filter may be required to have the function of absorbing light of a predetermined wavelength, and thus, by including the compound of the present invention having high photostability, a high-quality optical filter can be provided.

[0121] According to another aspect of the present invention, an image display device, such as an organic light emitting display (OLED), comprising the compound of the present invention is provided. Specifically, by including the compound of the present invention, the image display device can absorb light of unnecessary wavelengths, thereby improving image quality.

[0122] According to another aspect of the present invention, a composition for an electronic material comprising the compound of the present invention is provided. The electronic material may refer to a material used in the aforementioned optical filter or image display device.

[0123] According to another aspect of the present invention, a method for detecting a target substance is provided, comprising the step of identifying an image of the target substance or light emitted from the target substance when the target substance is brought into contact with a compound of the present invention. The target substance may be a biomolecule as described above. The contact may be performed by treating the compound with a buffer and adding it to the target substance, and a specific staining method may be appropriately selected from known methods by those skilled in the art. The detection may be performed by means known in the art, such as a fluorometer.

[0124] According to another aspect of the present invention, a method for imaging a biomolecule using a compound of the present invention is provided. The imaging may be performed at the single-molecule level. The imaging may be performed using high-resolution imaging. The imaging may be for observing the behavior of the biomolecule in real time.

[0125] According to another aspect of the present invention, a kit for detecting a target substance, for example, a biomolecule, comprising a compound of the present invention is provided.

[0126] Hereinafter, the present invention will be described in more detail with reference to examples to aid understanding. However, the following examples are provided solely to facilitate understanding of the present invention and are not intended to limit the scope of the present invention.

[0127]

[0128] [Materials and Methods for Synthesis and Photoblowing]

[0129] All compounds and solvents were purchased from Alfa Aesar (Haverhill, MA, USA), Combi-Blocks (San Diego, CA, USA), Cambridge Isotope Laboratories (Tewksbury, MA, USA), Sigma Aldrich (St. Louis, MO, USA), and Samchun chemicals (Seoul, Republic of Korea). Quartz cuvettes (Hellma, 110-1-40) were used for the photoblueing reaction. 1 H and 13C NMR spectra were obtained on a Brucker AVANCE III HD 500 and AVANCE III HD 850. HPLC chromatograms and mass spectra were recorded on a diode array detector (DAD) and HPLC (Agilent, 1260 series) with a single quadrupole mass analyzer (Agilent, 6100 series). Prep-HPLC (Shimadzu, LC-20AR) was used with a DAD (Shimadzu, SPD-M20A). HR-MS analysis was performed on a quadrupole time-of-flight instrument equipped with a nano-electrospray ionization (n-ESI) source (6560 IM-Q-TOF, Agilent, Agilent Technologies Inc., Santa Clara, CA, USA) in negative mode. The following conditions were used: gas temperature, 325 °C; dry gas, 7 L / min; Capillary voltage 2500 V. The mass value error (△m) was calculated using the following equation:

[0130] △m = (observed m / z - theoretical m / z) x 10 6 / Theoretical m / z(ppm) (1)

[0131] Observed m / z refers to the mass value data measured by mass spectrum, and theoretical m / z refers to the calculated mass value obtained from ChemDraw Professional 15.1 (PerkinElmer (Waltham, MA, USA)).

[0132]

[0133] [Example 1] Synthesis of compound PF555

[0134] (1) Synthesis of starting material TSCy5

[0135]

[0136] (1)-① Synthesis of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (compound 3)

[0137] 3-Methylbutan-2-one (Compound 1) (8.5 ml, 79.9 mmol) was added to 4-hydrazinylbenzenesulfonic acid (5 g, 26.5 mmol) in acetic acid (AcOH) (26 ml) to form a mixture, and the mixture was refluxed for 4 hours. The mixture was cooled to room temperature, filtered, and the precipitate was collected and washed with ethyl acetate (EA). Drying under reduced pressure gave a pink solid (Compound 2), which was used in the next step without further purification. KOH (1.8 g, 31.9 mmol) in isopropyl alcohol (IPA) (44 ml) was added to Compound 2 in methanol (MeOH) (44 ml) to form a mixture, and the mixture was stirred at room temperature for 24 hours. Filtering gave the precipitate, which was washed with EA. An ivory-colored solid (compound 3) was obtained by drying under reduced pressure (4.77 g, 65.0%).

[0138] 1 H NMR (500 MHz, MeOD): δ (ppm) 7.87 (sd, 1H,J= 1.2 Hz), 7.85 (dd, 1H,J= 1.7, 8 Hz), 7.50 (d, 1H,J= 7.95 Hz), 2.34 (s, 3H), 1.38 (s, 6H). 13 C NMR (125 MHz, MeOD): δ (ppm) 191.98, 153.83, 145.55, 142.49, 125.70, 119.46, 118.29, 53.90, 21.71, 13.98. ESI-MS [MK] - :m / zcalcd for C 11 H 14 NO3S 238.1, found 238.0.

[0139] (1)-② Synthesis of potassium 2,3,3-trimethyl-1-(3_sulfonatopropyl)-3H-indole-1-ium-5-sulfonate (compound 4)

[0140] 1,2-Oxathiolane 2,2-dioxide (882 mg, 7.22 mmol) was added to compound 3 (500 mg, 1.8 mmol) in 1,2-dichlorobenzene (3.6 ml) to form a mixture, and the mixture was stirred at 120°C for 12 hours. The mixture was cooled to room temperature, and the solvent was decanted. The precipitate was dissolved in a small amount of methanol (MeOH) and triturated with EA. The solid was obtained by filtration and washed with EA and IPA. Drying under reduced pressure gave a pink solid (compound 4) (683 mg, 95.1%).

[0141] 1 H NMR (850 MHz, DO): δ(ppm) 8.06 (s, 1H), 7.98 (d, 1H,J= 8.415 Hz), 7.88 (d, 1H,J= 8.5 Hz), 4.62 (t, 2H,J= 8.33 Hz), 3.05 (t, 2H,J= 6.97 Hz), 2.34 (p, 2H, J = 7.055 Hz), 1.55 (s, 6H). 13 C NMR (214 MHz, DO): δ (ppm) 199.95, 144.15, 142.74, 142.54, 127.01, 120.94, 115.73, 55.03, 47.37, 46.64, 22.64, 21.53, 21.52. ESI-MS [MK] - :m / zcalcd for C 14 H 18 NO6S2360.1, found 360.0.

[0142] (1)-③ Synthesis of compound TSCy5

[0143] Compound 4 (1 g, 2.5 mmol) and 3-Anilinoacraldehyde anil hydrochloride (324 mg, 1.25 mmol) were dissolved in H2O / EtOH = 1 / 2 (v / v) (83 ml), and sodium acetate (NaOAc) (257 mg, 3.13 mmol) and acetic anhydride (Ac2O) (296 μl, 3.13 mmol) were added. The mixture was stirred at 50 °C for 24 h and cooled to room temperature. The volatiles were removed under reduced pressure, and the residue was dissolved in a small amount of MeOH and triturated with EA. Reprecipitation using ethanol (EtOH) gave a blue solid (compound TSCy5) (754 mg, 75.1%). The obtained blue solid was used for conversion to the target compound without further purification, and only a portion of it was purified by prep-HPLC with a C18 column (Phenomenex, 5 μm, 100 Å, 21.2 × 250 mm) using H2O with 0.1% trifluoroacetic acid (TFA) added (solvent A) and acetonitrile (ACN) with 0.1% TFA added (solvent B) to obtain NMR spectral data.

[0144] Mobile phase gradient for HPLC: t=0-10 min, 10%B hold, t=10-60 min, 10%B to 99%B, t=60-65 min, 99%B hold, t=65-67 min, 99%B to 10%B, t=67-70 min, 10%B hold; flow rate: 8.0 ml / min.

[0145] 1H NMR (500 MHz, D2O):δ(ppm) 7.82 (t, 2H,J= 12.95 Hz), 7.65 (sd, 2H,J= 1.6 Hz), 7.63 (dd, 2H,J= 1.65, 8.25 Hz), 7.28 (d, 2H,J= 8.4 Hz), 6.46 (d, 1H,J= 12.45 Hz), 6.13 (d, 2H,J= 13.65 Hz), 4.14 (t, 2H,J= 7.5 Hz), 3.00 (t, 4H,J= 7.4 Hz), 2.14 (qu, 4H,J= 7.9 Hz), 1.43 (s, 12H). 13 C NMR (214 MHz, D2O):δ(ppm) 174.22, 154.87, 144.27, 141.84, 138.91, 126.45, 119.75, 110.84, 104.17, 49.11, 47.89, 42.58, 26.73, 22.22. LC-MS (ESI) [M-3Na+H] 2- :m / zcalcd for C 31 H 36 N2O 12 S4378.1, found 378.0; [M-3Na+2H] - :m / zcalcd for C 31 H 37 N2O 12 S4757.1, found 757.0. HR-MS (ESI) [M-3Na+H] 2- :m / zcalcd for C 31 H 36 N2O 12 S4378.0581, found 378.0571 (△m (ppm) = -2.645); [M-3Na+2H] - :m / zcalcd for C 31 H 37 N2O 12 S4757.1235, found 757.1201 (△m (ppm) = -4.491).

[0146] (2) 화합물 TSCy5 로부터 화합물 PF555의 합성

[0147]

[0148] 10 mg of compound TSCy5 was dissolved in 100 μl of DMSO, and the solution was transferred to a rod-shaped quartz cuvette. It was irradiated overnight with a 1.0 W 638 nm laser under an O2 flow, and the red solution was lyophilized to remove DMSO. The residue was purified by HPLC equipped with a C18 column (Phenomenex, 5 μm, 100 Å, 4.6 × 250 mm for small scale and 21.2 × 250 mm for large scale) using H2O with 0.1% TFA (solvent A) and MeOH with 0.1% TFA (solvent B), to obtain compound PF555. The average yield of compound PF555 was approximately 0.007%, and the average yield of compound TSCy3 as a by-product was approximately 1.1%.

[0149] Mobile phase gradient for prep-HPLC: t=0-5 min, 10%B hold, t=5-65 min, 10%B to 60%B, t=65-67 min, 60%B to 99%B, t=67-70 min, 99%B hold, t=70-72 min, 99%B to 10%B, t=72-75 min, 10%B hold; flow rate: 8.0 ml / min.

[0150] 1H NMR (500 MHz, D2O):δ(ppm) 8.40 (br, 1H), 7.89 (s, 1H), 7.81 (d, 1H,J= 8.5 Hz), 7.67-7.68 (m, 2H), 7.55 (d, 2H,J= 8.6 Hz), 7.39 (d, 1H,J= 7.8 Hz), 6.82 (d, 1H,J= 14.2 Hz), 6.49 (d, 1H,J= 10.5 Hz), 4.39 (t, 2H,J= 7.65 Hz), 4.17 (br, 2H), 3.02 (t, 2H,J= 6.9 Hz), 2.99 (t, 2H,J= 7.3 Hz), 2.18-2.21 (m, 4H), 1.60 (s, 6H), 1.42 (s, 6H). 13 C NMR (214 MHz, D2O):δ(ppm) 200.76, 181.31, 154.53, 147.75, 143.22, 142.94, 141.74, 138.87, 138.78, 132.24, 126.84, 126.08, 122.89, 120.18, 117.28, 113.45, 111.50, 109.39, 51.16, 47.85, 47.69, 47.18, 46.77, 44.25, 26.33, 22.77, 22.27, 21.09. LC-MS (ESI) [M-2H] 2- :m / zcalcd for C 30 H 34 N2O 13 S4379.0, found 379.2; [M-H] - :m / zcalcd for C 30 H 35 N2O 13 S4759.1, found 759.0. HR-MS (ESI) [M-2H] 2- :m / zcalcd for C 30 H 34 N2O 13 S4379.0477, found 379.0471 (△m (ppm) = -1.583); [M-3H+Na] 2- :m / zcalcd for C 30 H 33N2NaO 13 S4390.0387, found 390.0382 (△m (ppm) = -1.282).

[0151] TSCy3특성분석: 1 H NMR (500 MHz, D2O):δ(ppm) 8.47 (t, 1H,J= 13.4 Hz), 7.77 (sd, 2H,J= 1.7 Hz), 7.72 (dd, 2H,J= 1.75, 8.35 Hz), 7.29 (d, 2H,J= 8.4 Hz), 6.33 (d, 2H,J= 13.45 Hz), 4.16 (t, 4H,J= 7.5 Hz), 2.94 (t, 4H,J= 7.25 Hz), 2.15 (p, 4H,J= 7.75 Hz), 1.63 (s, 12H). 13 C NMR (125 MHz, D2O):δ(ppm) 176.21, 152.67, 144.02, 141.63, 139.45, 126.64, 119.83, 111.28, 103.71, 49.38, 47.86, 42.73, 27.09, 22.23. LC-MS (ESI) [M-2H] 2- :m / zcalcd for C 29 H 34 N2O 12 S4365.1, found 365.0; [M-3H+Na] 2- :m / zcalcd for C 29 H 33 N2NaO 12 S4376.0, found 376.0; [M-H] - :m / zcalcd for C 29 H 35 N2O 12 S4731.1, found 731.0; [M-2H+Na] - :m / zcalcd for C 29 H 34 N2NaO 12 S4753.1, found 753.0. HR-MS (ESI) [M-2H] 2- :m / zcalcd for C 29 H 34N2O 12 S4365.0503, found 365.0497 (△m (ppm) = -1.644).

[0152]

[0153] [Example 2] Synthesis of compound PF555-COOH

[0154]

[0155] Alexa Fluor TM 647 carboxylic acid (Invitrogen, A33084) (5 mg, 4.3 μmol) was dissolved in dimethyl sulfoxide (DMSO), and the solution was transferred to a rod-shaped quartz cuvette. It was irradiated with a 1.0 W 638 nm laser under an O2 flow for 6 h, and the red solution was lyophilized to remove DMSO. The residue was purified by HPLC equipped with a C18 column (Phenomenex, 5 μm, 100 Å, 4.6 × 250 mm) using H2O with 0.1% TFA (solvent A) and MeOH with 0.1% TFA (solvent B), which resulted in the yield of compound PF555-COOH (0.82 nmol, 1.9 × 10 -2 %) and A647_Cy3 (84 nmol, 1.9%) was obtained as a pink solid.

[0156] Mobile phase gradient for HPLC: t=0-5 min, 10%B hold, t=5-55 min, 10%B to 50%B, t=55-57 min, 50%B to 99%B, t=57-60 min, 99%B hold, t=60-62 min, 99%B to 10%B, t=62-65 min, 10%B hold; flow rate: 1.2 ml / min.

[0157] LC-MS (ESI) [M-2H] 2- :m / zcalcd for C 35 H 42 N2O 15 S4429.1, found 429.0; [MH]- :m / zcalcd for C 35 H 43 N2O 15 S4859.2, found 859.0. HR-MS (ESI) [M-3H] 3- :m / zcalcd for C 35 H 41 N2O 15 S4285.7135, found 285.7137 (△m (ppm) = 0.7000); [M-2H] 2- :m / zcalcd for C 35 H 42 N2O 15 S4429.0739, found 429.0730 (△m (ppm) = -2.0975).

[0158] A647_Cy3 Characterization: LC-MS (ESI) [M-2H] 2- :m / zcalcd for C 34 H 42 N2O 14 S4415.1, found 415.2; [MH] - :m / zcalcd for C 34 H 43 N2O 14 S4831.2, found 831.2. HR-MS (ESI) [M-3H] 3- :m / zcalcd for C 34 H 41 N2O 14 S4276.3819, found 276.3824 (△m (ppm) = 1.809).

[0159]

[0160] [Example 3] Synthesis of compound CA-PF555-O2

[0161] (1) Synthesis of starting material CA-A647-O2

[0162]

[0163] Alexa Fluor TM647 carboxylic acid (Invitrogen, A33084) (230 mg, 0.2 μmol), hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) (228 mg, 0.6 μmol), and N,N-diisopropylethylamine (DIPEA) (104 μL, 0.6 μmol) were dissolved in anhydrous dimethylformamide (DMF) (3 ml). The mixture was stirred at room temperature for 10 minutes, and 2-(2-(6-chlorohexyloxy)ethoxy)ethanamine (157 mg, 0.6 μmol) in anhydrous DMF (1 ml) was added, and the mixture was stirred at room temperature overnight. The volatiles were then removed under reduced pressure. The residue was purified by prep-HPLC equipped with a Phenyl-Hexyl column (Phenomenex, 5 μm, 100 Å, 21.2 x 250 mm) using H2O with 0.1% TFA (solvent A) and ACN with 0.1% TFA (solvent B) to obtain a blue solid (130 mg, 61.0%).

[0164] Mobile phase gradient for HPLC: t=0-10 min, 10%B hold, t=10-60 min, 10%B to 99%B, t=60-65 min, 99%B hold, t=65-67 min, 99%B to 10%B, t=67-70 min, 10%B hold; flow rate: 8.0 ml / min.

[0165] LC-MS (ESI) [M-2H] 2- :m / zcalcd for C 46 H 64 ClN3O 15 S4530.6, found 530.8; [MH] - :m / zcalcd for C 46 H 65 ClN3O 15 S41062.3, found 1062.0.

[0166] HR-MS (ESI) [M-3H] 3-:m / zcalcd for C 46 H 63 ClN3O 15 S4353.4282, found 353.4287 (△m (ppm) = 1.415); [M-2H] 2- :m / zcalcd for C 46 H 64 ClN3O 15 S4530.6460, found 530.6463 (△m (ppm) = 0.565).

[0167] (2) Synthesis of compound CA-PF555-O2

[0168]

[0169] Compound CA-A647-O2 (10 mg, 9.40 μmol) was dissolved in 100 μL of DMSO, and the solution was transferred to a rod-shaped quartz cuvette. The solution was irradiated overnight with a 1.0 W 638 nm laser under an O2 flow, and the red solution was lyophilized to remove DMSO. The above procedure was repeated using compound CA-A647-O2 (280 mg, 0.26 mmol). The residue was purified for the first time by HPLC equipped with a Phenyl-Hexyl column (5 μm, 100 Å, 4.6 x 250 mm) using H2O (solvent A) with 0.1% TFA and MeOH (solvent B) with 0.1% TFA, which resulted in the yield of compound CA-PF555-O2 as a purple solid and byproduct Cy3 as a pink solid (338 nmol, 0.13%). In addition, the purple solid was purified for the second time by HPLC equipped with a Phenyl-Hexyl column (5 μm, 100 Å, 4.6 x 250 mm) using H2O (solvent A) with 0.1% TFA and IPA (solvent B) with 0.1% TFA, which resulted in the yield of a purple solid (0.13 nmol, 5.4 x 10 -3 %).

[0170] Mobile phase gradient for the first HPLC purification: t=0-10 min, 20%B hold, t=10-65 min, 20%B to 50%B, t=65-67 min, 50%B to 99%B, t=67-70 min, 99%B hold, t=70-72 min, 99%B to 20%B, t=72-75 min, 20%B hold; flow rate: 10.0 ml / min. Mobile phase gradient for the second HPLC purification: t=0-5 min, 20%B hold, t=5-47 min, 20%B to 40%B, t=47-50 min, 40%B to 99%B, t=50-55 min, 99%B hold, t=55-58 min, 99%B to 20%B, t=58-60 min, 20%B hold; flow rate: 8.0 ml / min.

[0171] LC-MS (ESI) [M-2H] 2- :m / zcalcd for C 45 H 62 ClN3O 16 S4531.6, found 531.6; [MH] - :m / zcalcd for C 45 H 63 ClN3O 16 S41064.3, found 1064.2. HR-MS (ESI) [M-2H] 2- :m / zcalcd for C 45 H 62 ClN3O 16 S4531.6356, found 531.6338 6339 (△m (ppm) = -3.38581977); [M-3H] 3- :m / zcalcd for C 45 H 61 ClN3O 16 S4354.0880, found 354.0874 (△m (ppm) = -1.6945).

[0172] CA-A647_Cy3 Characterization: LC-MS (ESI) [M-2H] 2-:m / zcalcd for C 44 H 62 ClN3O 15 S4517.76, found 517.6; [MH] - :m / zcalcd for C 44 H 63 ClN3O 15 S41036.3, found 1036.2. HR-MS (ESI) [M-2H] 2- :m / zcalcd for C 44 H 62 ClN3O 15 S4517.6382, found 517.6373 (△m (ppm) = -1.7387); [M-3H] 3- :m / zcalcd for C 44 H 62 ClN3O 15 S4C 44 H 61 ClN3O 15 S4344.7563, found 344.7563 (△m (ppm) = 0).

[0173]

[0174] [Example 4] Synthesis of compound CA-PF555

[0175] (1) Synthesis of starting material CA-A647-O4

[0176]

[0177] Alexa Fluor TM647 carboxylic acid (Invitrogen, A33084) (380 mg, 0.33 mmol), N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) (464 mg, 0.49 mmol), and triethanolamine (TEA) (228 μl, 1.63 mmol) were dissolved in anhydrous DMF (6.6 ml). The mixture was stirred at 70°C overnight to prepare the NHS ester form, and 18-chloro-3,6,9,12-tetraoxaoctadecan-1-aminum chloride (137 mg, 0.39 mmol) and TEA (228 μl, 1.63 mmol) in anhydrous DMF (1 ml) were added, and the mixture was continuously stirred at 70°C overnight. The volatiles were then removed under reduced pressure. The residue was purified by prep-HPLC equipped with a C18 column (Phenomenex, 5 μm, 100 Å, 21.2 x 250 mm) using H2O with 0.1% TFA (solvent A) and ACN with 0.1% TFA (solvent B) to obtain a blue solid (compound CA-A647-O4) (207 mg, 43.0%).

[0178] HPLC를 위한 이동상 구배: t=0-5 min, 20%B hold, t=5-65 min, 20%B to 50%B, t=65-67 min, 50%B to 99%B, t=67-70 min, 99%B hold, t=70-72 min, 99%B to 20%B, t=72-75 min, 20%B hold; flow rate: 9.0 ml / min. LC-MS (ESI) [M+H]+: m / z calcd for C50H75ClN3O17S4 1152.4, found 1152.4; [M+2H]2+: m / z calcd for C50H76ClN3O17S4 576.7, found 576.8. HR-MS (ESI) [M-H]-: m / z calcd for C50H73ClN3O17S4 1150.3517, found 1150.3523 (△m (ppm) = 0.5215)

[0179] (2) 화합물 CA-PF555의 합성

[0180]

[0181] Compound CA-A647-O4 (10 mg) was dissolved in 100 μL of DMSO, and the solution was transferred to a rod-shaped quartz cuvette. It was irradiated with a 1.0 W 638 nm laser under an O2 flow overnight, and the red solution was lyophilized to remove DMSO. The above procedure was repeated using compound CA-A647-O4 (207 mg, 0.18 mmol). The residue was first purified by HPLC equipped with a Phenyl-Hexyl column (5 μm, 100 Å, 4.6 × 250 mm) using H2O (solvent A) supplemented with 0.1% TFA and MeOH (solvent B) supplemented with 0.1% TFA, resulting in compound CA-PF555 as a purple solid and the byproduct Cy3 as a pink solid (978 nmol, 0.54%). Additionally, the purple solid was purified a second time through HPLC equipped with a C18 column (5 μm, 100 Å, 4.6 x 250 mm) using H2O (solvent A) with 0.1% TFA added and IPA (solvent B) with 0.1% TFA added, and as a result, a purple solid (compound CA-PF555) was obtained (9.7 nmol, 5.4 x 10 -3 %).

[0182] Mobile phase gradient for the first HPLC purification: t=0-10 min, 35%B hold, t=10-55 min, 35%B to 60%B, t=55-57 min, 60%B to 99%B, t=57-60 min, 99%B hold, t=60-63 min, 99%B to 35%B, t=63-65 min, 35%B hold; flow rate: 10.0 ml / min.

[0183] Mobile phase gradient for the second HPLC purification: t=0-5 min, 10%B hold; t=5-55 min, 10%B to 25%B; t=55-57 min, 25%B to 99%B; t=57-60 min, 99%B hold; t=60-62 min, 99%B to 10%B; t=62-65 min, 10%B hold; flow rate: 10.0 ml / min. LC-MS (ESI) [M+H] + :m / zcalcd for C 49 H 73 ClN3O 18 S41154.3, found 1154.0; [M+2H] 2+ :m / zcalcd for C 49 H 74 ClN3O 18 S4577.7, found 577.6.

[0184] CA-A647_Cy3 Characterization: LC-MS (ESI) [M+H] + :m / zcalcd for C 48 H 73 ClN3O 17 S41126.4, found 1126.4; [M+2H] 2+ :m / zcalcd for C 48 H 74 ClN3O 17 S4563.7, found 563.8. HR-MS (ESI) [MH] - :m / zcalcd for C 48 H 71 ClN3O 17 S41124.3360, found 1124.3340 (△m (ppm) = 1.7788).

[0185]

[0186] [Example 5] Synthesis of compound DMCy5-PF555

[0187] (1) Synthesis of starting material DMCy5

[0188]

[0189] (1)-① Synthesis of 1,2,3,3-tetramethyl-3H-indole-1-ium iodide (compound 6)

[0190] Compound iodomethane (2.3 ml, 38.0 mmol) was added to compound 5 (3 g, 19.0 mmol) in acetonitrile (38 ml) to form a mixture, and the mixture was refluxed overnight. The mixture was then cooled to room temperature. The formed solid was washed with EA and ether, filtered, and a pink solid (compound 6) was obtained. The collected solid was dried under reduced pressure (5.45 g, 95.2%).

[0191] 1 H NMR (850 MHz, DMSO-d6): δ(ppm) 7.90 (dd, 1H,J= 1.36, 6.8 Hz), 7.82 (dd, 1H,J= 1.62, 6.21 Hz), 7.64 (pd, 2H,J= 1.45, 7.40 Hz), 3.95 (s, 3H), 2.75 (s, 3H), 1.52 (s, 6H). 13 C NMR (214 MHz, DMSO-d6): δ (ppm) 195.98, 142.07, 141.57, 129.29, 128.78, 123.24, 115.07, 53.88, 34.57, 21.65, 13.96.

[0192] ESI-MS [M] + :m / zcalcd for C 12 H 16 N 174.13, found 174.08.

[0193] (1)-② Synthesis of starting material DMCy5

[0194] Compound 6 (1 g, 3.32 mmol) and 3-anilinoacrylaldehyde aniline hydrochloride (344 mg, 1.33 mmol) were dissolved in EtOH (16.6 ml), and NaOAc (300 mg, 3.65 mmol) and Ac2O (0.34 ml, 3.65 mmol) were added. The mixture was stirred at 50°C for 1 h and cooled to room temperature. The mixture was filtered through a Celite plug, and the filtrate was dried in vacuo. The residue was purified by silica gel chromatography using DCM:EA=1:5 to remove non-polar impurities, and the product was eluted with a gradient of DCM:MeOH=10:1 to 5:1. Purification afforded a navy solid (compound DMCy5) (615 mg, 90.6%).

[0195] 1 H NMR (850 MHz, CDCl3):δ(ppm) 7.96 (t, 2H,J= 13.09 Hz), 7.11 (t, 4H,J= 7.57 Hz), 6.95 (t, 4H,J= 7.82 Hz), 6.54 (t, 1H,J= 12.5 Hz), 6.02 (d, 2H,J= 13.77 Hz), 3.43 (s, 6H), 1.50 (s, 12H). 13 C NMR (214 MHz, CDCl3):δ(ppm) 172.42, 152.68, 141.60, 140.05, 127.65, 125.06, 124.17, 121.25, 109.73, 102.77, 48.39, 31.47, 27.05. LC-MS (ESI) [M] + :m / zcalcd for C 27 H 31 N2383.2, found 383.2. HR-MS (ESI) [M] + :m / zcalcd for C 27 H 31 N2 + 383.2482, found 383.2482 (△m (ppm) = 0).

[0196] (2) Synthesis of compound DMCy5-PF555

[0197]

[0198] 10 mg of compound DMCy5 was dissolved in 100 μl DMSO, and the solution was transferred to a rod-shaped quartz cuvette. It was irradiated overnight with a 1.0 W 638 nm laser under an O2 flow. After that, the red solution was diluted with DCM and washed twice with H2O and brine respectively to remove DMSO. The organic layer was dried over Na2SO4, filtered, and dried in vacuo. The residue was purified by HPLC using a C18 column (Phenomenex, 5 μm, 100 Å, 4.6 × 250 mm for small scale, 21.2 × 250 mm for large scale) using 0.1% TFA (solvent A) and H2O and 0.1% TFA (solvent B) and MeOH. The average yield of compound DMCy5-PF555 was approximately 0.01%, and the average yield of byproduct DMCy3 was approximately 1.05%.

[0199] Mobile phase gradient for prep-HPLC: t=0-5 min, 20%B hold; t=5-55 min, 20%B to 70%B; t=55-60 min, 70%B to 99%B; t=60-70 min, 99%B hold; t=70-72 min, 99%B to 20%B; t=72-75 min, 20%B hold; flow rate: 8.0 ml / min. LC-MS (ESI) [M] + :m / zcalcd for C 26 H 29 N2O 385.2, found 385.2. HR-MS (ESI) [M] + :m / zcalcd for C 26 H 29 N2O 385.2274, found 385.2269 (△m (ppm) = -1.298).

[0200] DMCy3 characterization: LC-MS (ESI) [M] + :m / zcalcd for C 25 H 29 N2357.2, found 357.2. HR-MS (ESI) [M] + :m / zcalcd for C 25 H 29 N2357.2325, found 357.2324 (△m (ppm) = -0.280).

[0201]

[0202] [Example 6] Synthesis of compound Cy5-Mal_PF555

[0203] (1) Synthesis of starting material Cy5-Mal

[0204]

[0205] (1)-① Synthesis of 2,3,3-trimethyl-1-propyl-3H-indole-1-ium iodide (compound 7)

[0206] 1-Iodopropane (6.1 ml, 62.8 mmol) was added to compound 5 (2 g, 12.5 mmol) in ACN (25 ml), resulting in a mixture that was refluxed overnight. The mixture was cooled to room temperature and poured into Et2O. The bright violet crystals were collected and dried in vacuo to give compound 7 (3.60 g, 87.5%).

[0207] 1 H NMR (850 MHz, CDCl3):δ(ppm) 7.65-7.66 (m, 1H), 7.53-7.54 (m, 1H), 7.48-7.49 (m, 2H), 4.55 (t, 2H,J= 7.57 Hz), 3.02 (s, 3H), 1.97 (sext, 2H,J= 7.40 Hz), 1.56 (s, 6H), 0.99 (t, 2H,J= 7.57 Hz). 13C NMR (214 MHz, CDCl3):δ(ppm) 195.51, 141.28, 140.57, 129.87, 129.22, 123.17, 115.26, 54.40, 50.90, 22.95, 21.27, 16.84, 11.09.

[0208] (1)-② Synthesis of 1-(4-carboxybutyl)-2,3,3-trimethyl-3H-indole-1-ium bromide (compound 8)

[0209] 5-Bromovaleric acid (6.76 g, 37.3 mmol) was added to compound 5 (6 ml, 37.3 mmol) to form a mixture, which was placed in a sealed tube and stirred at 120°C for 10 hours. The mixture was cooled to room temperature, and the formed solid was washed with Et2O and Et2O:CHCl3 = 1:1 to obtain a solid. The solid was dissolved in a small amount of MeOH and triturated with EA. The solid was filtered to obtain a pale pink solid (compound 8), which was dried in vacuo (3.44 g, 27.2%).

[0210] 1 H NMR (850 MHz, MeOD-d4):δ(ppm) 7.88-7.90 (m, 1H), 7.76-7.78 (m, 1H), 7.64-7.66 (m, 2H), 4.56 (t, 2H,J= 7.74 Hz), 2.43 (t, 2H,J= 7.14 Hz), 2.03 (qui, 2H,J= 7.82 Hz), 1.79 (qui, 2H,J= 7.14 Hz), 1.61 (s, 6H). 13 C NMR (214 MHz, MeOD-d4):δ(ppm) 198.03, 176.62, 143.38, 142.54, 131.21, 130.51, 124.64, 116.50, 55.96, 33.81, 28.15, 22.84, 22.78. ESI-MS[M] + :m / zcalcd for C 12 H 16 N 260.16, found 260.25.

[0211]

[0212] (1)-③ Synthesis of compound Cy5-670

[0213] Compound 8 (100 mg, 0.29 mmol) and 3-anilinoacrylaldehyde aniline hydrochloride (91 mg, 0.35 mmol) were dissolved in Ac2O:AcOH=1:1 (v / v) (1.2 ml), and the mixture was stirred at 120°C for 4 h. The mixture was cooled to room temperature and diluted with DCM. The organic layer was washed twice with saturated aqueous NaHCO3 solution, and the aqueous layer was acidified with 1 M HCl. The acidified aqueous layer was extracted twice with DCM, and the combined organic layers were dried over Na2SO4, filtered, and dried in vacuo. The residue was used in the next step without further purification. The residue was dissolved in MeOH (5.8 ml), and compound 7 (96 mg, 0.29 mmol) and NaOAc (143 mg, 1.74 mmol) were added. The mixture was refluxed for 24 h and cooled to room temperature. The volatiles were removed under vacuum. The residue was purified by silica gel chromatography using DCM:MeOH = 20:1 to 5:1, resulting in the yield of a navy solid (compound Cy5-670) (70 mg, 38.7%).

[0214] LC-MS (ESI) [M] + :m / zcalcd for C 33 H 41 N2O2497.3, found 497.6.

[0215] (1)-④ Synthesis of compound Cy5-Mal

[0216] To Cy5-670 (152 mg, 0.27 mmol) in DMF (5.4 ml) were added PyBOP (286 mg, 0.55 mmol) and DIPEA (95 μl, 0.55 mmol), and the mixture was bubbled with nitrogen gas for 10 min. 2-Maleimidoethylamine hydrochloride (97 mg, 0.55 mmol) was then added, and the mixture was stirred at room temperature overnight. The mixture was diluted with DCM and washed twice with 1 M HCl and brine, respectively. The organic layer was dried over Na2SO4, filtered, and dried in vacuo. The residue was purified by prep-HPLC equipped with a C18 column (Phenomenex, 5 μm, 100 Å, 21.2 x 250 mm) using H2O with 0.1% TFA (solvent A) and ACN with 0.1% TFA (solvent B) to obtain a blue solid (compound Cy5-Mal) (110 mg, 65.8%).

[0217] Mobile phase gradient for HPLC: t=0-5 min, 45%B hold, t=5-45 min, 45%B to 80%B, t=45-50 min, 80%B to 99%B, t=50-60 min, 99%B hold, t=60-62 min, 99%B to 45%B, t=62-65 min, 45%B hold; flow rate: 8.0 ml / min.

[0218] 1H NMR (850 MHz, Me2CO-d6):δ(ppm) 9.22 (t, 1H,J= 5.78 Hz), 8.48-8.53 (m, 2H), 7.52 (d, 2H,J= 7.40 Hz), 7.50 (d-5 Hz), 1.3,J (m, 3H), 7.20-7.22 (m, 2H), 6.99 (t, 1H,J= 12.4 Hz), 6.86 (d, 1H,J= 13.8 Hz), 6.77 (s, 2H), 6.54 (d, 1.2,J= 1), 3 6.72 Hz), 4.20 (t, 2H,J= 7.48 Hz), 3.61 (t, 2H,J= 5.95 Hz), 3.39 (q, 2H,J= 5.95 Hz), 2.40 (t, 2H,J= 6.5.J= 8 Hz), Hz), 1.77–1.80 (m, 4H), 1.71 (s, 6H), 1.70 (s, 6H), 1.05 (t, 3H,J= 7.40 Hz). 13 C NMR (214 MHz, Me2CO-d6):δ(ppm) 174.52, 173.44, 173.26, 171.86, 155.91, 154.98, 143.55, 143.20, 142,1235.2, 129.39, 129.26, 127.49, 125.73, 125.31, 123.11, 123.07, 112.36, 111.62, 105.69, 104.17, 50.85, 49.85, 4 38.55, 38.02, 35.28, 27.97, 27.85, 26.96, 23.02, 21.51, 11.49. LC–MS (ESI) [M] + :m / zcalcd for C 39 H 47 N4O3619.4, found 619.4.

[0219]

[0220] (2) See more of Cy5-Mal_PF555

[0221]

[0222] 10 mg of compound Cy5-Mal was dissolved in 100 μl of DMSO, and the solution was transferred to a rod-shaped quartz cuvette. It was irradiated overnight with a 1.0 W 638 nm laser under an O2 flow. After that, the red solution was diluted with DCM and washed twice with H2O and brine respectively to remove DMSO. The organic layer was dried over Na2SO4, filtered, and dried in vacuo. The residue was purified by HPLC equipped with a C18 column (Phenomenex, 5 μm, 100 Å, 4.6 × 250 mm for small scale, 21.2 × 250 mm for large scale) using 0.1% TFA (solvent A) and H2O and 0.1% TFA (solvent B) and MeOH, to obtain compound Cy5-Mal_PF555.

[0223] Mobile phase gradient for prep-HPLC: t=0-5 min, 20%B hold; t=5-55 min, 20%B to 70%B; t=55-60 min, 70%B to 99%B; t=60-70 min, 99%B hold; t=70-72 min, 99%B to 20%B; t=72-75 min, 20%B hold; flow rate: 8.0 ml / min. LC-MS (ESI) [M] + :m / zcalcd for C 38 H 45 N4O4621.3, found 621.4.

[0224]

[0225] [Example 7] Synthesis of compound DM-PF595

[0226] (1) Synthesis of starting material DMCy5.5

[0227]

[0228] 1,1,2,3-Tetramethyl-1H-benzo[e]indole-3-ium iodide (100 mg, 0.28 mmol) and 3-anilinoacrylaldehyde anil hydrochloride (29 mg, 0.11 mmol) were dissolved in EtOH (2.8 ml), and NaOAc (26 mg, 0.31 mmol) and Ac2O (30 μl, 0.31 mmol) were added. The mixture was stirred at 50 °C for 24 h and cooled to room temperature. The volatiles were removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient (EA:DCM=5:1 to 1:1 until nonpolar impurities were removed, then DCM:MeOH=20:1 until the product was obtained) to obtain a blue solid (83 mg, 48.5%).

[0229] 1 H NMR (500 MHz, MeOD-d4):δ(ppm) 8.39 (t, 2H,J= 13.1 Hz), 8.26 (d, 2H,J= 8.5 Hz), 8.04 (dd, 4H,J= 8.5, 17.7 Hz), 7.67 (q, 4H,J= 8.9 Hz), 7.50 (t, 2H,J= 7.5 Hz), 6.70 (t, 1H,J= 12.4 Hz), 6.35 (d, 2H,J= 13.8 Hz), 3.76 (s, 6H), 2.02 (s, 12H). 13 C NMR (125 MHz, MeOD-d4):δ(ppm) 175.08, 152.99, 140.26, 133.47, 132.04, 130.25, 129.71, 128.02, 127.32, 124.63, 121.94, 110.53, 102.61, 50.94, 29.28, 26.13. LC-MS (ESI) [M] + :m / zcalcd for C 35 H 35 N2483.3, found 483.4.

[0230] (2) Synthesis of compound DM-PF595

[0231]

[0232] 1 mg DMCy5.5 was dissolved in 100 μl of DMSO, and the solution was transferred to a rod-shaped quartz cuvette. A 1.0 W 638 nm laser was irradiated under an O2 flow, and a 1.0 W 638 nm laser was irradiated under an O2 flow. The solution was then diluted with DCM and washed twice with H2O and brine, respectively, to remove DMSO. The organic layer was dried over Na2SO4, filtered, and vacuum-dried. The residue was purified and confirmed through an LC-MS spectrum, which is shown in Figure 6.

[0233]

[0234] [Experimental Example 1] Analysis of the chemical structure of the compound of the present invention

[0235] Among the compounds of the present invention, PF555 was selected as a representative compound and its chemical structure was analyzed. For comparison, a cyanine compound with a simple structure, TSCy5, was used (Fig. 1a). As a result of HPLC analysis, a new peak (t R = 20.05 min) was observed (Fig. 1b). This is the in vitro photolytic lifetime (t) of TSCy3 R = 18.36 min) is remarkable. The same trend was observed at the single molecule level (for 250 s) (Fig. 1d). In HR-MS, the chemical formula of PF555 was C 31 H 38 N2O 13 It was found to be S4, which is TSCy5 (C 31 H 38 N2O 12S4) had a different structure in that it had one CH2 group missing and one oxygen atom added (Fig. 5). Comparing the 1H-NMR spectra of TSCy5, TSCy3, and PF555, it was confirmed that the PF555 was asymmetric, and confirming the overlapping HMBC spectra of TSCy5, TSCy3, and PF555, it was confirmed that the asymmetric structure had one oxo-quinoline structure and the other an indole ring. The structure was different from that of conventional cyanine dyes, and it was named "3-oxo cyanine dyes" as a novel cyanine-based dye containing a ketone. In addition, when confirming the absorption spectrum and emission spectrum of PF555 in distilled water, the absorption was maximum at 555 nm, and the emission was maximum at 567 nm. Based on the above results, the structure of PF555 was confirmed as an ultra-photostable compound.

[0236] Additionally, chloroalkane (CA) was introduced into PF555 (CA-PF555) to label arbitrary HaloTag-fused proteins for bioimaging applications.

[0237]

[0238] [Experimental Example 2] Binding Experiment with Biomolecules (Amino Acids)

[0239]

[0240] To determine whether the compounds of the present invention can react and bind to biomolecules, we tested their reactivity with amino acids (cysteine) using Cy5-Mal_PF555 among the compounds of the present invention. The LC-MS spectrum of the product bound to cysteine ​​was measured and is shown in Figure 10. From Figure 10, it can be confirmed that the compounds of the present invention bind to biomolecules (amino acids).

[0241]

[0242] [Experimental Example 3] Analysis of in vivo photolysis lifetime characteristics of the compound of the present invention (bulk-level imaging, single-molecule imaging, characteristics after addition of additives)

[0243] To evaluate the photostability of CA-PF555 in a biological environment, comparative studies were performed using three conventional organic dyes with similar absorbance and emission spectra: CA-AF555, CA-JFX549, and CA-TMR. HALO-EGFR, a genetically encoded protein tag that can be labeled with chloroalkane derivatives, was conjugated to these dyes. The dye-labeled HALO-EGFR in living cells was then imaged using total internal reflection fluorescence (TIRF) microscopy using a 561 nm laser at the same laser power for all dyes. At the bulk labeling level, CA-PF555 exhibited a photolysis lifetime of 314.4 s, which was 13.3 times that of CA-AF555 (23.6 s), 34.5 times that of CA-JFX549 (9.1 s), and 56.1 times that of CA-TMR (5.6 s) (Fig. 2b). The average photodegradation lifetime of CA-PF555 measured in vivo was consistent with the in vitro value of 333.1 s (Fig. 1c).

[0244] Also, 100 to 32,000 mW / cm 2 A comparative analysis was performed to evaluate the brightness of CA-PF555 compared to CA-AF555, CA-JF549, and CA-TMR at 561 nm laser power intensity. CA-PF555 exhibited significant brightness compared to CA-TMR and CA-AF555, which provides an advantage in single-molecule imaging applications (Fig. 2c).

[0245] Single-molecule imaging of CA-PF555 was further performed and compared to the same set of dyes conjugated to HALO-EGFR in live cells cultured under identical conditions. Single-molecule signals were monitored over time using a 561 nm laser power setting (Fig. 11a). Consistent with the photolysis lifetime when labeled at the bulk level (Fig. 2b), CA-PF555 exhibited an excellent photolysis lifetime of 331.4 s at the single-molecule level compared to CA-AF555 (26.6 s), CA-JFX549 (15.1 s), and CA-TMR (5.9 s) (Fig. 11b,c). The consistency of the photolysis lifetimes at both the bulk and single-molecule levels suggests that the low blinking observed for PF555 does not affect the scale of the average photolysis lifetime. Additionally, the average photodegradation lifetime of CA-TMR calculated using the dot counting method was consistent with the values ​​reported in previous reports using the same method.

[0246] One common strategy for reducing the photodegradation of organic dyes is the use of reducing-plus-oxidizing systems (ROXS). We used PCA / PCD as a ROXS component to enhance the photodegradation lifetime of CA-PF555 and compared it with the lifetimes of AF647 and TMR (Fig. 11a). As expected, PCA / PCD increased the photodegradation lifetimes of AF647 and TMR to 10.4 s and 29.2 s, respectively (Fig. 11b-c). However, PCA / PCD did not affect the lifetime of PF555, which remained at 334.6 s. This suggests that PF555 is not sensitive to oxidation and can maintain its photodegradation lifetime under various media conditions.

[0247] PF555 exhibits minimal non-specific binding even at 100 nM (Figure 12), comparable to AF647. Atto647, on the other hand, exhibits 100-fold higher non-specific binding (Figure 12). Therefore, PF555 is ideal for accurate, low-background single-molecule imaging.

[0248]

[0249] [Experimental Example 4] Application to single-molecule tracking and behavior analysis

[0250] Considering its excellent photostability and sufficient brightness, PF555 was used for single-molecule imaging of EGFR in living cells without the use of additives. During imaging at a wavelength of 561 nm, PF555 exhibited photoactivity under 405 nm laser irradiation (Fig. 13). This was confirmed by repeatedly reactivating PF555 using the 405 nm laser for several seconds between streams (Fig. 3 a). This property was confirmed by single-particle tracking photoactivated localization microscopy (sptPALM) within individual cells. Specifically, single molecules could be tracked for over 37 minutes, capturing a large number of 116,246 single-molecule PF555 trajectories in a single cell.

[0251] To verify whether the trajectories obtained with PF555 actually provide extended long-term single-molecule information beyond the typical short-term single-molecule information, the long PF555 trajectories were divided into 10 short frames. These were then compared with the single-molecule trajectories obtained with AF647 in the same cells. The diffusion coefficient distributions of the divided frames of PF555 were consistent with those of AF647, confirming the accuracy and reliability of the long-term single-molecule data collected using PF555 (Fig. 3b).

[0252] In live COS7 cells, we observed previously unobserved long-term single-molecule dynamic motion of EGFR. Mean-squared displacement (MSD) analysis, fitting the first 30% of the MSD curve, revealed distinct diffusion patterns in the trajectories. For example, long-term moving-stationary trajectories (Fig. 4d, left panel) exhibited a downward slope in the MSD plot (Fig. 4d, middle panel) after the first 40 s, indicating sub-diffusion. After that, fluctuations or periodic patterns were observed, suggesting complex behavior during extended sptPALM. In contrast, filopodia trajectories (Fig. 4e, left panel) exhibited more directed motion, as evidenced by the displacement angle map (Fig. 4e, right panel). Furthermore, we observed a slow drift phenomenon (Fig. 4f, left panel), characterized by a linear MSD plot (Fig. 4f, middle panel) for nearly 60 seconds, after which it transitioned to a super-diffusion pattern. Abnormal diffusion was also observed in other trajectories (Fig. 4g-h, left panels), as indicated by the upward curves in the MSD plots (Fig. 4g-h, middle panels). Interestingly, both trajectories showed a limited diffusion pattern after 40 seconds of trajectory tracking (Fig. 4h, left panel) and more directional movement (Fig. 4h, right panel). Furthermore, we confirmed that the EGFR molecular population exhibited a static movement throughout the entire 80-second stream (Fig. 4i). While conventional MSD fitting well describes short-time scales, it cannot account for long-time scales exceeding 40 seconds, indicating that molecular processes are involved in the plasma membrane (PM) of living cells on time scales of tens of seconds.

[0253]

[0254] [Experimental Conclusion]

[0255] PF555 exhibits excellent photostability in biological environments, with a photolysis lifetime of 314.4 seconds, which is significantly superior to that of other commonly used dyes. In comparison, the photolysis lifetime is 13.3 times longer than that of CA-AF555 (23.6 seconds), 34.5 times longer than that of CA-JFX549 (9.1 seconds), and 56.1 times longer than that of CA-TMR (5.6 seconds). This extended photolysis lifetime is consistent with in vitro measurements, making PF555 particularly useful for long-term biological studies. Furthermore, the excellent photostability of PF555 is particularly important for single-molecule imaging applications, where the dye fluorescence lifetime is critical to data quality.

[0256] The high extinction coefficient and relatively bright properties of PF555 offer several advantages in bioimaging and molecular biology. PF555 has a maximum extinction coefficient (ε max ) is 4,416,000 M -1 cm -1 , which is relatively brighter than TMR, etc. measured under the same conditions. The brightness of organic dyes can significantly improve spatial resolution and signal-to-noise ratio and minimize photodamage to biological samples.

[0257] The quantum yield of PF555 is 0.013, which may seem low compared to other commonly used dyes. Quantum yield is a measure of how efficiently an absorbed photon is converted into emitted fluorescence. A higher quantum yield generally means the dye is more efficient at converting absorbed light into emitted light, which is often desirable in imaging applications. However, this must be considered in the context of other exceptional photophysical properties, such as its remarkable photostability. Despite its low quantum yield, PF555's high extinction coefficient compensates for this, resulting in a dye that remains bright, making it particularly useful in long-duration imaging applications where photostability is crucial. Therefore, while quantum yield is an important factor, it is not the only one that determines a dye's utility, especially when considering other properties, such as photostability.

[0258] PF555 exhibits an excitation maximum at 555 nm and an emission maximum at 567 nm, placing it in the typical near-infrared spectrum. This spectral positioning is particularly advantageous for several reasons. First, it allows seamless integration with standard fluorescence microscopy setups equipped with filter sets optimized for this wavelength range. PF555 can be adopted without specialized equipment or modifications to existing systems. Second, the near-infrared spectrum is less likely to overlap with autofluorescence of biological samples, improving the signal-to-noise ratio. Finally, its spectral characteristics allow multiplexing with other fluorophores, enabling simultaneous imaging of multiple targets. Overall, the excitation and emission maxima of PF555 not only ensure compatibility with existing fluorescence microscopy setups, but also enhance its utility in a variety of bioimaging applications.

[0259] The outstanding photostability of PF555 may be attributed to its unique molecular structure and photophysical properties. Unlike conventional cyanine dyes, PF555 belongs to a new type of ketone-containing cyanine, termed 3-oxo cyanine dyes. Its structure is asymmetric, with an oxo-quinoline structure on one end and an indole ring on the other. This asymmetric structure, combined with the carbonyl structure and unique pi-conjugation, potentially enhances oxidation resistance and contributes to its excellent photostability. Experimental data presented herein show that PCA / PCD did not affect the lifetime of PF555 (Figure 12). This suggests that PF555 is insensitive to oxidation and can maintain its photodegradation lifetime under various media conditions. Furthermore, the fluorescence lifetime (τ) of PF555 is only 0.22 nanoseconds. This short fluorescence lifetime may play a role in reducing photodegradation by minimizing the time the molecule spends in the excited state, thereby reducing the possibility of oxygen attack.

[0260] The use of PF555 for long-term sptPALM provided unprecedented insight into the complex behavior of EGFR on the PM of living COS7 cells. Due to the photoactivatable properties of PF555, multiple cycles of single-molecule imaging over a period exceeding 37 minutes could be performed, capturing up to 116,246 single-molecule PF555 trajectories in a single cell. To analyze the dynamic behavior of EGFR, Mean Squared Displacement (MSD) analysis was used. The initial 30% of the MSD curves were specifically fitted to identify the various diffusion patterns of the trajectories. While effective on short time scales, conventional MSD fitting is inadequate for capturing long-term dynamics lasting more than a minute. To overcome this limitation, we utilized the "rolling method" to dynamically evaluate the diffusion coefficient over time. This approach utilizes a temporal sliding window to calculate the diffusion coefficient using the MSD method. The window is then incrementally moved along the trajectory, and the process is repeated until the entire trajectory is analyzed. This results in time-resolved diffusion coefficient profiles, providing a more nuanced understanding of molecular dynamics. Specifically, a unique diffusion pattern was observed in the extended sptPALM of EGFR, exhibiting complex behavior on timescales of tens of seconds. Furthermore, the diffusion coefficient distribution of the segmented PF555 trajectories was found to be consistent with that obtained using AF647. This consistency suggests that PF555 does not induce dye aggregation, which may affect the diffusivity of EGFR across the plasma membrane. Therefore, the photostability and unique properties of PF555 not only enable long-term imaging but also ensure the reliability and accuracy of the collected data.

[0261]

[0262] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. A compound represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, Rings A and B are each independently an aromatic ring having 5 to 20 carbon atoms, wherein ring A may be substituted with one or more R3s which are the same or different from each other, and ring B may be substituted with one or more R6s which are the same or different from each other, R1 and R2 are each independently a hydrogen atom, an alkyl having 1 to 20 carbon atoms, -L1-R 10 , -L2-COO-L3-R 11 , -L4-CO-L5-R 12 , -L6-O-L7-R 13 , -L8-CO-NH-L9-R 14 , -L 10 -NH-L 11 -R 15 , -L 12 -SL 13 -R 16 , -L 14 -SO2-L 15 -R 17 , -L 16 -SO2-NH-L 17 -R 18 , or -L 18 -PO2-L 19 -R 19 and; R3 and R6 are each independently a hydrogen atom, a halogen atom, an alkyl having 1 to 20 carbon atoms, -L 21 -R 20 , -L 22 -COO-L 23 -R 21 , -L 24 -CO-L 25 -R 22 , -L 26 -OL 27 -R 23 , -L 28 -CO-NH-L 29 -R 24 , -L 30 -NH-L 31 -R 25 , -L 32 -SL 33 -R 26 , -L 34 -SO2-L 35 -R 27 , -L 36 -SO2-NH-L 37 -R 28 , or -L 38 -PO2-L 39 -R 29 and; R4, R5, R7, and R8 are each independently a hydrogen atom, an alkyl having 1 to 20 carbon atoms, -L 41 -R 30 , -L 42 -COO-L 43 -R 31 , -L 44 -CO-L 45 -R 32 , -L 46 -OL 47 -R 33 , -L 48 -CO-NH-L 49 -R 34 , -L 50 -NH-L 51 -R 35 , -L 52 -SL 53 -R 36 , -L 54 -SO2-L 55 -R 37 , -L 56 -SO2-NH-L 57 -R 38 , or -L 58 -PO2-L 59 -R 39 and; L1 to L 19 , L 21 Inland L 39 , and L 41 Inland L 59 are each independently a single bond or an alkylene having 1 to 20 carbon atoms, wherein the alkylene may further include at least one linking group selected from the group consisting of -COO-, -CO-, -O-, -CO-NH-, -NH-, -S-, -SO2-, -SO2-NH-, and -PO2- within the chain, R 10 Inland R 19 , R 20 Inland R 29 , and R 30 Inland R 39 are each independently a hydrogen atom, a halogen atom, an alkyl group having 1 to 20 carbon atoms, a hydroxyl group, or a reactive substituent capable of reacting and bonding with a biomolecule, n is 0 or an integer from 1 to 5, However, at least one of R1 to R8 is a group having a reactive substituent capable of reacting and bonding with a biomolecule.

2. In paragraph 1, The above ring A is selected from the group consisting of the following chemical formulas a-1 to a-4, The above ring B is a compound selected from the group consisting of the following chemical formulas b-1 to b-4: In chemical formulas a-1 to a-4 and b-1 to b-4, * indicates a connecting portion, and R3 and R6 are as defined in paragraph 1.

3. In paragraph 1, a compound represented by the following chemical formula 2: [Chemical Formula 2] In the above chemical formula 2 R1 to R8 and n are as defined in paragraph 1.

4. In paragraph 1, R3 and R6 are each independently a hydrogen atom, an alkyl having 1 to 20 carbon atoms, or -L 21 -R 20 and the above L 21 is a single bond or alkylene having 1 to 20 carbon atoms, and R 20 is a halogen atom, a hydroxyl group, a carboxyl group, or a sulfonic acid group, At least one of R1, R2, R4, R5, R7, and R8 is a group having a reactive substituent capable of reacting and bonding with a biomolecule, and the others are hydrogen atoms, alkyl having 1 to 20 carbon atoms, or -L 60 -R 40 and the above L 60 is a single bond or alkylene having 1 to 20 carbon atoms, and R 40 A compound having a halogen atom, a hydroxyl group, a carboxyl group, or a sulfonic acid group.

5. In paragraph 1, The reactive substituent capable of reacting and bonding with the above biomolecule is an alkenyl having 2 to 20 carbon atoms, an alkynyl having 2 to 20 carbon atoms, a sulfonic acid group, a sulfonimide group, an amine group, a succinimide group, a phosphoryl group, a guanine group, a cytosine group, a thiol group, a thiosulfone group, a vinylsulfone group, an isocyanate group, an isothiocyanate group, a maleimide group, a carboxyl group, a phenyl-isocyanate group, a phenyl-isothiocyanate group, a 1,2,4,5-tetrazine group, a 3-methyl-6-phenyl-1,2,4,5-tetrazine group, a trans-cyclooctene group, an azide group, a phenyl-azide group, a dibenzocyclooctyne group, an endo-bicyclononyne group (endo-BCN), a sulfonyl fluoride group, Phenyl-sulfonyl fluoride group, benzophenone group, 3-phenyl-3H-diazirine group, 3-phenyl-3-(trifluoromethyl)-3H-diazirine group, 3-phenyl-3-methyl-3H-diazirine group, 3-butynyl-3H-diazirine group, tetrazole group, phenyl-tetrazole group, biotin group, hydrazine group, , , , , or a compound that is a peptide probe.

6. In paragraph 1, a compound selected from the group consisting of the following compounds:

7. A method for producing a compound described in claim 1, comprising the step of irradiating light to a compound of the following chemical formula 4 under oxygen (O2): [Chemical Formula 4] In the above chemical formula 4 m is n+1, Ring A, ring B, R1 to R8, and n are as defined in paragraph 1.

8. A dye composition for dyeing a target material, containing the compound described in paragraph 1.

9. In the 8th paragraph, the target material is a dye composition which is a biomolecule selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, antigens, lipids, phospholipids, lipopolysaccharides, fatty acids, nucleic acids, cell membranes, cells, and microorganisms.

10. A dye composition according to claim 8, wherein the dyeing is for visualizing an image of a target material.

11. A method for detecting a target substance, comprising a step of confirming an image of a target substance contacted with a compound described in paragraph 1 or light emitted from the target substance.

Images