Polymerizable heptamethine cyanine dyes and their polymers, synthesis methods and applications

Abstract

This invention discloses a polymerizable heptamethine dye, the structural formula of which is shown in general formula I: This invention introduces cyclohexene structural units into the methine chain, and uses indole and benzoindole as raw materials to synthesize a polymerizable heptamethine dye with high fluorescence quantum yield and good biocompatibility, which can be used in the biological and pharmaceutical fields.

Description

Technical Field

[0001] This invention relates to the field of organic dye technology, and more particularly to a class of polymerizable heptamethine dyes substituted with acrylates and / or alkynes, their polymers, polymerization methods, and applications. Background Technology

[0002] Cancer is a leading cause of death worldwide, and its prevalence is exacerbated by a growing and aging global population, as well as the increasing prevalence of risk factors such as smoking, obesity, lack of physical activity, and altered reproductive patterns. Early detection of cancer is crucial for preventing or delaying cancer-related deaths.

[0003] Studies have shown that lung cancer patients diagnosed early within five years have a 49% higher survival rate than those diagnosed late. Imaging techniques such as ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) can provide non-invasive anatomical and functional information about tumors, making them increasingly important for early cancer detection and recurrence monitoring. However, all these technologies have limited spatial resolution and contrast in observing the boundaries of invasive tumors or detecting metastases, resulting in a high rate of repeat surgeries due to incomplete tumor removal during the initial procedure.

[0004] Compared to other imaging methods, fluorescence imaging excels in sensitivity and resolution. Fluorescent dyes can respond to biomarkers within cancer cells at the molecular scale, labeling cancer cells using their own or their products' fluorescence signals. Fluorescent dyes offer numerous advantages, including ease of operation, good stability, high sensitivity, safety, and low cost. Compared to other fluorescent dye parent compounds, cyanine dyes possess advantages such as a large molar extinction coefficient, good biocompatibility, and near-infrared emission, making them a focus of research and application in recent years. Currently, the popular near-infrared (NIR) heptamethylcyanine dye ICG is mainly studied for hepatobiliary cancer because it is only excreted into bile and then passively accumulates in the tumor; however, when applied to intraoperative imaging of other types of tumors, unexpected false-positive results have occurred, raising concerns about the specificity of ICG. Therefore, developing a fluorescent dye that can target tumor cells is necessary in chemistry, biology, medicine, and other interdisciplinary fields.

[0005] Over the past decade, various novel bioorthogonal reactions have been developed in living cells, showing great potential for labeling or tracking biomolecules. Intracellular polymerization is a direct method for the in situ synthesis of functional macromolecules, but available protocols are limited by the cytotoxicity of the reactants and a lack of coordination with the cellular environment. Some intracellular reactions, such as azide-alkyne cycloaddition, transfer hydrogenation, and especially reactions using transition metal catalysts, provide practical methods for the in situ synthesis of small molecule fluorescent probes or therapeutic drugs. However, examples of synthesizing polymers in living cells are rare. The approach of fabricating polymerizable cyanine dyes and attempting polymerization using the tumor cell microenvironment has not yet been explored.

[0006] In conclusion, developing a fluorescent dye that can target tumor cells is necessary in chemistry, biology, medicine, and other interdisciplinary fields. However, current tumor cell imaging still faces many problems, such as poor optical performance, short response time of dye molecules within tumor cells, insufficient tissue penetration depth, weak targeting, low sensitivity, inability to identify early-stage tumors, excessively large dye molecule size, complex synthesis, low yield, and significant toxic side effects. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a class of polymerizable heptamethine cyanine dyes substituted with acrylates and / or alkynes, their polymerization methods and applications, as well as polymers. By introducing cyclohexene structural units into the methine chain and using indole and benzoindole as raw materials, polymerizable heptamethine cyanine dyes are synthesized, exhibiting high fluorescence quantum yield and good biocompatibility, and can be used in the biological and pharmaceutical fields.

[0008] To achieve the above objectives, the technical solution of the present invention is: a polymerizable heptamethylcyanine dye, the structural formula of which is shown in general formula I:

[0009]

[0010] In general formula I,

[0011] R1 and R2 can be the same or different. R1 and R2 are each independently selected from any one of hydrogen, halogen, alkyl group having 1-18 carbons, carboxyl alkyl group having 1-18 carbons, aryl group, aryl carboxylic acid group, alkyl sulfonate group, aryl sulfonate group, alkyl sulfonate or aryl sulfonate.

[0012] R3 is selected from any one of chlorine, malondicyano, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, arylcarboxylic acid, alkylsulfonate, and arylsulfonate.

[0013] R4 and R5 can be the same or different. R4 and R5 are each independently selected from acrylates with 1-18 carbon atoms or alkynes with 1-18 carbon atoms.

[0014] Furthermore, its structural formula is as follows:

[0015]

[0016] R1 and R2 are the same, and R1 and R2 are selected from any one of hydrogen, halogen, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, aryl, arylcarboxylic acid, alkylsulfonate, arylsulfonate, alkyl sulfonate or aryl sulfonate.

[0017] R3 is selected from any one of chlorine, malondicyano, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, arylcarboxylic acid, alkylsulfonate, and arylsulfonate.

[0018] Furthermore, R1 and R2 are each independently selected from any one of hydrogen, halogen, carboxyl groups having 1-8 carbons, and alkyl sulfonates;

[0019] R3 is selected from at least one of chlorine, malondicyano, alkyl groups having 1-8 carbons, and carboxyl groups having 1-8 carbons.

[0020] The synthesis method for polymerizable heptamethylcyanine dyes, where R4 and R5 are the same, and when acrylates with 1-18 carbon atoms are selected, is as follows:

[0021] (1) At 80-100℃, 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution is reacted with 2-iodoethanol in organic solvent I for 10-14h, and excess cold organic solvent II is added. After filtration, the intermediate containing R1 and R2 substitution is obtained. The molar ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution to 2-iodoethanol is 1:1.5-5. The preferred molar ratio is 1:2, and the preferred reaction temperature is 85-95℃. The volume ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution to organic solvent II exceeds 1:1.

[0022] (2) At 110-120℃, the R1 and R2 substituted intermediates and the R3 substituted 1-cyclohexene-1,3-dicarboxaldehyde were dissolved in organic solvent III. After slurrying in organic solvent III and purification by column chromatography (alumina, DCM:MeOH=10:1), intermediates containing R1, R2 and R3 substituted products were obtained.

[0023] (3) At room temperature, the intermediate containing R1, R2, and R3 substitutions is dissolved in organic solvent IV. Under ice-water bath conditions, an esterification reaction is carried out under the catalysis of an organic base. Acryloyl chloride is slowly added dropwise under nitrogen protection for 7-9 hours. After slurrying and column chromatography (alumina, DCM:MeOH = 10-15:1), a polymerizable indole parent near-infrared fluorescent dye is obtained. The preferred reaction temperature is 20℃ and the reaction time is no more than 12 hours.

[0024] Its synthesis process is as follows:

[0025]

[0026] Furthermore, in step (1) above, the organic solvent I is selected from at least one of acetonitrile, benzene, toluene, and o-dichlorobenzene; the organic solvent II used for pulping is selected from at least one of diethyl ether, tert-butyl methyl ether, and ethyl acetate.

[0027] In step (2), the organic solvent III is selected from at least one of n-butanol (boiling point 117-118): toluene (boiling point 110.6) (7:3, volume / volume) and anhydrous ethanol (with sodium ethoxide);

[0028] In step (3), the organic base is selected from at least one of triethylamine, pyridine, and DIPEA; the organic solvent IV is selected from at least one of o-dichlorobenzene and dichloromethane.

[0029] The synthesis method for polymerizable heptamethylcyanine dyes is the same for R4 and R5. When the dyes are selected from alkynes with 1-18 carbons, the synthesis method is as follows:

[0030] (1) At 100-140℃, 2,3,3-trimethyl-3H-indole containing R1 substitution is dissolved in organic solvent I and reacted for 0.5-2h. After purification by silica gel column chromatography, the intermediate containing R1 and R3 substitution is obtained. The molar ratio of 2,3,3-trimethyl-3H-indole containing R1 substitution to 6-iodine-1-hexyne is 1:1.5-3. The preferred molar ratio is 1:2.

[0031] (2) In organic solvent III, at 110-120℃, 1-cyclohexene-1,3-dicarboxaldehyde containing the intermediate R2 substituted with R1 and R3 is dissolved in organic solvent III. After slurrying in organic solvent III and purification by column chromatography (alumina, DCM:MeOH = 10:1), the indole parent near-infrared fluorescent dye containing R1, R2 and R3 substituted is obtained.

[0032] Its synthesis process is as follows:

[0033]

[0034] Furthermore, in step (1) above, the organic solvent I is selected from at least one of acetonitrile, benzene, toluene, and o-dichlorobenzene;

[0035] The solvent III used for pulping in step (2) is selected from at least one of diethyl ether, tert-butyl methyl ether, and ethyl acetate; the organic solvent III is selected from at least one of n-butanol (boiling point 117-118): toluene (boiling point 110.6) (7:3, volume / volume), and anhydrous ethanol (with sodium ethoxide);

[0036] The synthesis method for polymerizable heptamethylcyanine dyes differs for R4 and R5. When R4 and R5 are each independently selected from acrylates or alkynes with 1-18 carbon atoms, the synthesis method is as follows:

[0037] (1) 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution reacts with 6-iodo-1-hexyne in organic solvent I for 0.5-2 h, and is purified by silica gel column chromatography to obtain intermediate 1 containing R1 and R2 substitution; wherein the molar ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution to 6-iodo-1-hexyne is 1:1.5-3;

[0038] (2) At 80-100℃, 2,3,3-trimethyl-3H-indole containing R1 or R2 substitution is reacted with 2-iodoethanol in organic solvent I for 10-14h, and excess cold organic solvent II is added. After filtration, intermediate 2 containing R1 or R2 substitution is obtained. The molar ratio of 2,3,3-trimethyl-3H-indole containing R1 or R2 substitution to 2-iodoethanol is 1:1.5-5; the preferred molar ratio is 1:2, and the preferred reaction temperature is 85-95℃.

[0039] (3) At 110-120℃, the R1 and R2 substituted intermediates and the R2 substituted 1-cyclohexene-1,3-dicarboxaldehyde were dissolved in organic solvent III (n = 1:1:1). After slurrying in organic solvent III and purification by column chromatography (alumina, DCM:MeOH = 10:1), intermediate 3 containing R1, R2 and R3 substitutions was obtained.

[0040] (4) At room temperature, intermediate product 3 containing R1, R2, and R3 substitutions is dissolved in organic solvent IV. Under ice-water bath conditions, esterification reaction is carried out under the catalysis of organic base. Acryloyl chloride is slowly added dropwise under nitrogen protection for 7-9 hours. After slurrying and column chromatography (alumina, DCM:MeOH = 10-15:1), polymerizable indole parent near-infrared fluorescent dye is obtained. The preferred reaction temperature is 20℃ and the reaction time is no more than 12 hours.

[0041] Its synthesis process is as follows:

[0042]

[0043] Furthermore, in step (1) above, the organic solvent I is selected from at least one of acetonitrile, benzene, toluene, and o-dichlorobenzene; the organic solvent II used for pulping is selected from at least one of diethyl ether, tert-butyl methyl ether, and ethyl acetate.

[0044] In step (2), the organic solvent III is selected from at least one of n-butanol (boiling point 117-118): toluene (boiling point 110.6) (7:3, volume / volume) and anhydrous ethanol (with sodium ethoxide);

[0045] In step (3), the organic base is selected from at least one of triethylamine, pyridine, and DIPEA; the organic solvent IV is selected from at least one of o-dichlorobenzene and dichloromethane.

[0046] A polymerizable heptamethine dye, said polymer being a mixture of polymerizable heptamethine dye, an alkyl Br initiator, a copper-histidine complex, and sodium ascorbate in a mass ratio of 10:1:40:80.

[0047] The polymerization method for polymerizable heptamethine dye in living cells is as follows: using polymerizable heptamethine dye and N-hydroxyethyl acrylamide as monomers, Cu(II) histidine complex as catalyst, ethyl 5-bromoisobutyrate as free radical initiator, and utilizing the hypoxic microenvironment of cancer cells and the glutathione content in cancer cells that is higher than that in normal cells as reducing agent to initiate intracellular free radical polymerization.

[0048] Due to metabolic abnormalities, tumor tissues create a strong reducing environment within tumor cells. The concentration of glutathione (GSH) (2–10 mmol / L) within tumor cells is more than 1,000 times higher than the extracellular GSH concentration (2–20 μmol / L), and is also 4 times higher than the concentration in normal cells.

[0049] The polymerization mechanism of acrylate substitution is shown below:

[0050]

[0051] The polymerization mechanism of alkynyl substitution is shown below:

[0052]

[0053]

[0054] Furthermore, the excitation wavelength is 500-950nm and the fluorescence detection wavelength is 650-1000nm.

[0055] Furthermore, the polymerizable heptamethine dye can be applied to cell imaging, protein labeling, antibody-specific recognition, nucleic acid labeling, DNA sequencing, and tumor photodynamic therapy.

[0056] In summary, the present invention has the following beneficial effects:

[0057] 1. The dye described in this application improves the retention time of the dye in tumor cells: Heptamethrin dye molecules can induce strong fluorescence at near-infrared wavelengths of 700-900 nm and have high molar absorptivity and fluorescence quantum efficiency. Moreover, its near-infrared fluorescence has strong tissue penetration and is not affected by tissue autofluorescence. This application utilizes an esterification reaction to combine the dye molecules with acryloyl chloride to synthesize a polymerizable dye. After polymerization in tumor cells, the molecules are not easily expelled from the cells, thereby improving the retention time and enhancing the time for tumor cells to be fluorescently labeled. Experiments have shown that the dye under polymerization conditions not only has a longer retention time but also a significantly increased confocal brightness.

[0058] 2. The dye described in this application enhances targeting of tumor cells: Glutathione overexpression and hypoxia are two major characteristics of tumor cells. Glutathione (GSH) is reducing, so this application uses low-toxicity amino acid ligands and Cu(II) complexes to obtain Cu(I) in situ within tumor cells with GSH. The biotoxicity of the Cu(II) histidine complex has also been shown to be very low. Cu(I) activates alkyl bromide initiators, initiating the polymerization of acrylic acid monomers (or alkyne-substituted monomers) with customized functions. Furthermore, the intracellular hypoxic environment is conducive to the survival of polymerizable reducing probes. The polymerized dye molecules have a longer fluorescence labeling time than the monomer molecules and are less prone to efflux, thus possessing a certain degree of targeting. In the future, it can also be linked to recognize molecules such as folic acid on the surface of tumor cells to further explore their functions.

[0059] 3. The stability of the dye probe described in this application is further improved: the traditional Cy7 has poor stability. Although Cy3 and Cy5 have better stability, their emission wavelength is short and their fluorescence penetration is weak. Therefore, this application introduces cyclohexene structural units into the methenylene chain and uses indole and benzoindole as raw materials to synthesize an alkyne-substituted heptamethine dye with polymerization ability.

[0060] 4. The dye described in this application has good biocompatibility compared to traditional heptamethrin dyes. In the embodiments of this application, after culturing MCF-7 cells with different concentrations of dye for 24 hours, the cells still showed good survival rates. Even when the concentration was increased to 15 μmol / L, the cell survival rate remained high, indicating that this type of polymerizable heptamethrin dye has very good biocompatibility and will not produce toxic side effects on cells within the working concentration range. It can be applied to biometric imaging, nucleic acid labeling, DNA sequencing, and photodynamic therapy for tumors. Attached Figure Description

[0061] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0062] Figure 1 The high-resolution mass spectrum and proton nuclear magnetic resonance spectrum of Acr-Cy7 disclosed in this invention are shown below.

[0063] Figure 2 The high-resolution mass spectrum and proton NMR spectrum of AcrB-Cy7 disclosed in this invention are shown below.

[0064] Figure 3 The high-resolution mass spectrum and proton nuclear magnetic resonance spectrum of Cy7alk disclosed in this invention;

[0065] Figure 4 The images show the UV-Vis absorption and fluorescence spectra of Cy7alk disclosed in this invention, where a is the absorption spectrum of Cy7alk in different solvents; b is the fluorescence spectrum of Cy7alk in different solvents; c is the photostability test of Cy7alk before and after polymerization; and d is the absorption spectrum of Cy7alk in PBS under different pH conditions from 2 to 13.

[0066] Figure 5 The images show the UV-Vis absorption spectra of Acr-Cy7 and AcrB-Cy7 disclosed in this invention, where a is the absorption spectrum of Acr-Cy7 dye in different solvents; and b is the absorption spectrum of AcrB-Cy7 dye in different solvents.

[0067] Figure 6 The fluorescence spectra of Acr-Cy7 and AcrB-Cy7 disclosed in this invention are shown, where a is the fluorescence spectrum of Acr-Cy7 dye in different solvents; and b is the fluorescence spectrum of AcrB-Cy7 dye in different solvents.

[0068] Figure 7 The images show a comparison of the spectra of poly-Cy7alk and Cy7alk disclosed in this invention, where a is a comparison of absorption spectra and b is a comparison of fluorescence spectra.

[0069] Figure 8 The above are photostability test results of Cy7alk before and after polymerization as disclosed in this invention.

[0070] Figure 9The images show the MTT assay results of the polymerizable heptamethine dye disclosed in this invention in three types of tumor cells, where a is the MTT assay result of Acr-Cy7 in three types of tumor cells, b is the MTT assay result of AcrB-Cy7 in three types of tumor cells, and c is the MTT assay result of Cy7alk in three types of tumor cells.

[0071] Figure 10 MTT assay results for the Cy7alk dye monomer, poly-Cy7alk, and poly-Cy7alk with added sodium ascorbate disclosed in this invention in three types of tumor cells;

[0072] Figure 11 The graph shows the biotoxicity test results of Cu(II) amino acid complexes and Cu(I).

[0073] Figure 12 A comparison of confocal uptake of Cy7alk under monomeric and polymeric conditions in three types of tumor cells;

[0074] Figure 13 This is a comparison of confocal uptake of AcrB-Cy7 under monomeric and polymeric conditions in three types of tumor cells.

[0075] Figure 14 Comparison of confocal uptake under monomer and polymerization conditions for Acr-Cy7 and AcrB-Cy7;

[0076] Figure 15 A comparison of fluorescence intensity histograms of Acr-Cy7, AcrB-Cy7, and Cy7alk in MCF-7 from 0 to 60 min;

[0077] Figure 16 Comparison of infrared spectra before and after polymerization of Acr-Cy7 and Cy7alk;

[0078] Figure 17 The NMR changes of Acr-Cy7 and Cy7alk from 0 to 10 min;

[0079] Figure 18 The NMR changes of Acr-Cy7 from 0 to 120 min;

[0080] Figure 19 Electron micrograph of Cy7alk polymerized dialysis. Detailed Implementation

[0081] The present invention will now be described in further detail.

[0082] Unless otherwise stated, the terms used herein have the following meanings.

[0083] The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine.

[0084] The term "alkyl" as used in this invention includes straight-chain alkyl and branched-chain alkyl.

[0085] The term "MTT" as used in this invention refers to a method for detecting cell viability and growth.

[0086] The instruments and equipment used in the embodiments are as follows:

[0087] During column chromatography, 200-300 mesh and 100-200 mesh silica gel columns were purchased from Qingdao Meigao Group Co., Ltd., and 20-40 mesh analytical grade quartz sand was purchased from Tianda Chemical Reagent Factory. Neutral alumina was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0088] During the compound detection process, the mass spectrometer used was the Synapt G2-Si HDMS high-resolution mass spectrometer from Waters Corporation, USA, employing a dual-needle electrospray ionization source to detect the compounds in both positive and negative modes.

[0089] The nuclear magnetic resonance hydrogen spectrometer used was an AVANCE NEO 400.

[0090] The absorption and emission spectra of the dyes were measured using an Agilent Cary 60 UV-Vis spectrophotometer and a CaryEclipse fluorescence spectrophotometer.

[0091] Cytotoxicity assays were performed using the Varioskan LUX Multimode Microplate Reader instrument from Thermofisher, Inc.

[0092] The confocal microscopy experiment was conducted at Leica Microsystems CMC GmbH, Am Friedensplatz 368165 Mannheim, Germany.

[0093]

[0094] In general formula I,

[0095] R1 and R2 can be the same or different. R1 and R2 are each independently selected from at least one of hydrogen, halogen, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, aryl, arylcarboxylic acid, alkylsulfonate, arylsulfonate, alkyl sulfonate or aryl sulfonate.

[0096] R3 is selected from at least one of chlorine, malondicyano, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, arylcarboxylic acid, alkyl sulfonate, and aryl sulfonate.

[0097] R4 and R5 can be the same or different. R4 and R5 are each independently selected from acrylates with 1-18 carbon atoms or alkynes with 1-18 carbon atoms.

[0098] The dyes represented by general formula I will be described in detail below with reference to the embodiments.

[0099] Example 1

[0100] An acrylate-substituted polymerizable heptamethine cyanine dye, Acr-Cy7, has identical R4 and R5 values ​​selected from acrylates with one carbon atom, and its preparation method includes the following steps:

[0101] (1) Synthesis of intermediate 1a

[0102] Iodoethanol (1.2 mL, 30.0 mmol) was added to a solution of 2,3,3-trimethyl-3H-indole (4.0 g, 25.0 mmol) in 20 mL of acetonitrile and mixed thoroughly. The mixture was then refluxed at 90 °C for 12 h with vigorous stirring. After cooling to room temperature, excess chilled tert-butyl methyl ether was added, and the mixture was filtered to obtain intermediate 1a containing R1 and R3 substitutions.

[0103]

[0104] (2) Synthesis of intermediate 1b

[0105] In a round-bottom flask, the purified intermediate 1a from the previous step and the condensing agent quaternary ammonium salt were added sequentially. 2-chloro-1-cyclohexene-1,3-dicarboxaldehyde (1.7 g, 10.0 mmol) and intermediate 1a (4.1 g, 20.0 mmol) were added to a solution of n-butanol (boiling point 117-118 °C):toluene (boiling point 110.6 °C) (7:3, v / v). The mixture was stirred and refluxed overnight at 120 °C. After cooling to room temperature, excess ice-cold tert-butyl methyl ether was added, and the mixture was filtered. Column chromatography yielded 0.685 g of intermediate 1b containing R1, R2, and R3 substitutions; yield 40.3%.

[0106]

[0107] (3) Synthesis of Acr-Cy7 molecule

[0108] Under nitrogen atmosphere, intermediate 1b (65 mg, 0.1 mmol) was dissolved in o-dichlorobenzene (10 mL) with triethylamine TEA (404.8 mg, 4 mmol). Acryloyl chloride (181 mg, 2 mmol) dissolved in o-dichlorobenzene (5 mL) was added dropwise to the solution. The mixture was stirred at 0 °C for 30 min. The solution was stirred overnight at 25 °C. The crude product was precipitated with excess n-hexane, filtered, and extracted with dichloromethane and water. The organic layer was dried over anhydrous sodium sulfate, and the dichloromethane was evaporated. The residue was precipitated twice in excess n-hexane and dried under vacuum. The solid was purified by silica gel chromatography (DCM / MeOH, 10:1) to give Acr-Cy7 dye, which was pure green. 26 mg of Acr-Cy7 dye was obtained after purification, with a yield of 40% for this step. The overall yield was 14.4%.

[0109]

[0110] The structure of the product, Acr-Cy7 dye, was confirmed to be correct by nuclear magnetic resonance spectroscopy.

[0111] The 1H NMR data are as follows:

[0112] 1 H NMR(400MHz,Methanol-d4)δ8.53(s,1H),8.28(s,1H),8.04(s,2H),7.72(s,1H),7.65(s,1H),7.53(s,1H),6.46(s,1H), 6.19(s,1H),5.98(s,1H),5.83(s,1H),4.72(s,2H),3.85(s,1H),3.59(s,1H),2.80(s,2H),2.08(s,1H),2.03(s,6H).See Figure 1 ;

[0113] ESI-HRMS(C 40 H 44 ClN2O4 + m / z:[MH]-calcd651.2984,found930.21130651.3050, see also Figure 1 .

[0114] Example 2

[0115] An acrylate-substituted polymerizable heptamethylcyanine dye, AcrB-Cy7, has identical R4 and R5 values, selected from acrylates with one carbon atom. Its preparation method differs from Example 1 only in step (1), specifically: (1) Iodoethanol (1.2 mL, 30.0 mmol) is added to a solution of 2,3,3-trimethyl-3H-indole (5.23 g, 25.0 mmol) in 20 mL of acetonitrile and mixed thoroughly. The mixture is then refluxed at 90 °C for 12 h under vigorous stirring. After cooling to room temperature, excess chilled tert-butyl methyl ether is added, and the mixture is filtered to obtain intermediate 1a containing R1 and R3 substitutions.

[0116] AcrB-Cy7 dye was prepared from 5g of indole raw material, yielding 0.64g, with a yield of 12.8%. The structure was identified by nuclear magnetic resonance spectroscopy, confirming the correct structure of the Acr-Cy7 dye product.

[0117] The 1H NMR data are as follows:

[0118] 1 H NMR(400MHz,Methanol-d4)δ8.43(s,1H),7.51(s,1H),7.39(s,3H),7.29(s,1H),6.40(s,1H),6.21( s,1H),6.00(s,1H),5.84(s,1H),4.64(s,2H),3.63(s,2H),2.73(s,2H),1.78(s,1H),1.72(s,6H).See Figure 2 ;

[0119] ESI-HRMS(C 48 H 48 ClN2O4 + m / z:[MH]-calcd751.3297,found751.3363, see m / z:[MH]-calcd751.3297,found751.3363. Figure 2 .

[0120] Example 3

[0121] An acrylate-substituted polymerizable heptamethylcyanine dye, Cy7alk, has identical R4 and R5 values ​​selected from a single-carbon alkyne, and its preparation method includes the following steps:

[0122] (1) Synthesis of intermediate K2

[0123] 6-Iodo-1-hexyne (3.1 g, 15.0 mmol) was added to a solution of 2,3,3-trimethyl-3H-indole (1.0 g, 6.25 mmol) in 20 mL of acetonitrile (yellow). The mixture was refluxed at 130 °C for 1 h with vigorous stirring. After cooling to room temperature, the product was obtained by column chromatography.

[0124]

[0125] (2) Synthesis of the Cy7alk Molecular

[0126] In a round-bottom flask, the purified intermediate K2 from step (1) and the condensing agent quaternary ammonium salt were added sequentially. 2-chloro-1-cyclohexene-1,3-dicarboxaldehyde (1.7 g, 10.0 mmol) and intermediate K2 (4.1 g, 20.0 mmol) were added to a solution of n-butanol (boiling point 117-118°C):toluene (boiling point 110.6°C) (7:3, volume / volume). The mixture was stirred and refluxed overnight at 120°C. After cooling to room temperature, excess ice-cold tert-butyl methyl ether was added and the mixture was filtered. The solid was purified by silica gel chromatography (DCM / MeOH, 10:1). The dye was a pure green structure and named Cy7alk. 1.64 g of the Cy7alk dye was obtained, with a yield of 36.6%.

[0127]

[0128] The structure was identified by nuclear magnetic resonance spectroscopy, and the Cy7alk dye structure was confirmed to be correct. (See [link]). Figure 3 ;

[0129] The 1H NMR data are as follows:

[0130] 1H NMR(400MHz,Methanol-d4)δ8.34(s,1H),7.42(s,1H),7.34(s,1H),7.26(s,1H),7.20(s,1H),6.23(s,1H) ,4.11(s,2H),2.65(s,2H),2.22(s,2H),2.18(s,1H),1.93(s,1H),1.87(s,2H),1.64(s,6H),1.59(s,2H).

[0131] The polymerizable heptamethine dye described in this application can polymerize in living cells. The polymerization of the dye is described in detail below with reference to examples.

[0132] Example 5

[0133] Polymerization method of compound poly-AcrCy7:

[0134] Add 1 mL each of Acr-Cy7 (1 mM, 1 mL), alkyl Br initiator (10 mM), copper histidine complex (400 mM), and sodium ascorbate [NaAsc] (800 mM) dropwise to a 10 mL centrifuge tube containing DMSO solution, and sonicate for 30 min. Rotate the mixture to dryness, dissolve in a small amount of methanol, and dialyze the mixture against ultrapure water using a 1600 mol / L dialysis membrane for 24 h. Rotate to dryness again to obtain the polymerization product.

[0135] Example 6

[0136] Polymerization method of compound poly-AcrB-Cy7:

[0137] Add 1 mL of each of AcrB-Cy7 (1 mM, 1 mL), alkyl Br initiator (10 mM), copper histidine complex (400 mM), and sodium ascorbate [NaAsc] (800 mM) dropwise to a 10 mL centrifuge tube containing DMSO solution, and sonicate for 30 min. Rotate the mixture to dryness, dissolve in a small amount of methanol, and dialyze the mixture in ultrapure water using a 1600 mol / L dialysis membrane for 24 h. Rotate to dryness again to obtain the polymerization product.

[0138] Example 7

[0139] Polymerization method of compound poly-Cy7alk:

[0140] Add 1 mL each of Cy7alk (1 mM 1 mL), alkyl Br initiator (10 mM), copper histidine complex (400 mM), and sodium ascorbate [NaAsc] (800 mM) dropwise to a 10 mL centrifuge tube containing DMSO solution, and sonicate for 30 min. Dry the mixture by rotary evaporation, dissolve in a small amount of methanol, and dialyze the mixture against ultrapure water using a 1600 mol / L dialysis membrane for 24 h. Dry the mixture by rotary evaporation to obtain the polymerization product.

[0141] Test case

[0142] Test Example 1: Determination of UV-Vis absorption and fluorescence spectroscopy

[0143] Test method: Weigh the vacuum-dried dye precisely using a 0.01 g balance, prepare a 1 mmol / L DMSO dye stock solution in a brown sample bottle, and store it in a refrigerator at 4°C for later use.

[0144] For testing UV-Vis absorption and fluorescence spectra, 20 μL of the dye stock solution was measured using a micropipette and dissolved in a quartz cuvette containing 2 mL of the test solvent. The mixture was thoroughly mixed to obtain a dye concentration of 20.0 μmol / L, which was used for testing the absorption and fluorescence emission spectra. All tests were performed at 25°C. The test results are shown below. Figure 4-7 .

[0145] Figure 4 a shows the absorption spectra of Cy7alk dye in different solvents;

[0146] Figure 4 b shows the fluorescence spectra of Cy7alk dye in different solvents;

[0147] Figure 4 c shows the absorption spectra of Cy7alk dye in PBS under different pH conditions (2-13).

[0148] from Figure 4 It can be seen that, in different solvents, the maximum absorption wavelength of the traditional heptamethrin dye compound is 780 nm, while the maximum absorption wavelength of the compound Cy7alk in this application is 810 nm, and absorption is also observed up to 900 nm. The maximum emission wavelength of the compound Cy7alk is 821 nm, and emission is still observed up to 950 nm.

[0149] To investigate the effect of pH on absorption and emission, an aqueous solution with pH = 2.13 was prepared using hydrochloric acid and sodium hydroxide. Tests were then conducted using the method described above. The test results are shown below. Figure 4 d;

[0150] from Figure 4 As can be seen from d, within the pH range of 2-13, pH=7 exhibits better absorption and emission.

[0151] Figure 5 a shows the absorption spectra of Acr-Cy7 dye in different solvents;

[0152] Figure 5 b shows the absorption spectra of AcrB-Cy7 dye in different solvents;

[0153] like Figure 5 As shown, in different solvents, the maximum absorption wavelength of traditional heptamethrin dye compounds is 780 nm, while the maximum absorption wavelength of the compound Acr-Cy7 in this application is 790 nm, and the maximum absorption wavelength of the compound AcrB-Cy7 is 830 nm.

[0154] Figure 6 a shows the fluorescence spectra of AcrCy7 dye in different solvents;

[0155] Figure 6 b shows the fluorescence spectra of AcrBCy7 dye in different solvents;

[0156] like Figure 6As shown, the maximum emission wavelengths of compounds Acr-Cy7 and AcrB-Cy7 in different solvents are 810 nm and 850 nm, respectively, which are similar to those of conventional cyanine dyes. This indicates that the maximum absorption wavelength of the cyanine dyes of this invention is not significantly different from that of conventional cyanine dyes, but the absorption and emission ranges are greatly increased.

[0157] Figure 7 a is a comparison of the absorption spectra of poly-Cy7alk polymer and Cy7alk dye;

[0158] Figure 7 b is a comparison of the fluorescence spectra of poly-Cy7alk polymer and Cy7alk dye;

[0159] from Figure 7 It can be seen that there is still absorption at 780-800nm ​​and emission at 810-850nm. Therefore, the absorption and fluorescence spectra of the polymerized molecules have not destroyed the structure of the monomer molecules.

[0160] Test Example 2: Light Stability Test

[0161] The testing method was as follows: 100 μM aqueous solutions of Acr-Cy7, AcrB-Cy7, ICG, and Et-Cy7 were prepared respectively, and irradiated under a laser with an excitation wavelength of 808 nm and a laser power of 30 mW / cm² (voltage 11.47 V, current 0.917 A). The absorbance of each dye solution at the maximum absorption wavelength was measured every 5 minutes using a UV-Vis spectrophotometer. The test results are shown below. Figure 8 .

[0162] from Figure 8 It can be seen that the monomer molecules are more stable than traditional ICG dyes. The monomer molecules show a significantly faster decrease in absorption spectrum, proving that the monomer molecules are less stable than the polymerized molecules. After four laser irradiations over 20 minutes, 50% of the polymerized molecules were destroyed, while 60% of the monomer molecules were destroyed, demonstrating that the photostability of the polymerized molecules is increased.

[0163] Test Example 3: Cytotoxicity Experiment

[0164] The cytotoxicity of dye molecules is assessed using the MTT assay. The principle is as follows: succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet formazan crystals, which are then deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. The absorbance values ​​are measured at wavelengths of 490 nm and 570 nm using a microplate reader, indirectly reflecting the number of living cells.

[0165] Test method: MCF-7, 4T1, and HepG2 cells were seeded in 96-well plates. After a period of culture, specific concentrations of Acr-Cy7, AcrB-Cy7, and Cy7alk were added to different wells, with compound concentrations ranging from 0 to 20 μmol / L. Cells were incubated for another 24 hours, and cell viability was assessed using an MTT assay. Test results are as follows: Figure 9 .

[0166] Figure 9 a is a graph of the MTT assay of Acr-Cy7 in three types of tumor cells;

[0167] Figure 9 b is the MTT assay of AcrB-Cy7 in three types of tumor cells;

[0168] Figure 9 c is a graph from the MTT assay of Cy7alk in three types of tumor cells.

[0169] from Figure 9 It can be seen that after culturing MCF-7, 4T1, and HepG2 cells with different concentrations of Acr-Cy7, AcrB-Cy7, and Cy7alk for 24 hours, the cells still showed good survival rates, and even when the concentration was increased to 15 μmol / L, the cell survival rate remained high.

[0170] The above method was used to conduct cytotoxicity experiments on Acr-Cy7, Cy7alk dye monomers, poly-Cy7alk, poly-Acr-Cy7, and poly-Cy7alk and poly-Acr-Cy7 with added sodium ascorbate. The test results are shown in [link to results]. Figure 10 AcrB-Cy7 was not tested due to its similar structure;

[0171] from Figure 10 The low cytotoxicity indicates the presence of conditions for in-situ polymerization. These cyanine dyes exhibit excellent biocompatibility and do not produce toxic side effects on cells within the working concentration range, thus making them suitable for applications in the biological and pharmaceutical fields.

[0172] MCF-7 cells were seeded in 96-well plates. After a period of culture, Cu(II) histidine complex, copper sulfate, and Cu(I) were added to different wells to achieve a compound concentration of 400 μmol / L. After incubation for another 24 hours, cell viability was assessed using an MTT assay. The results are shown below. Figure 11 .

[0173] from Figure 11 As can be seen, copper histidine complexes have lower cytotoxicity and biocompatibility; therefore, copper histidine complexes were selected as catalysts in this application.

[0174] Test Example 3: Cell Confocal Experiment

[0175] Test method: Weigh the vacuum-dried dye precisely using a 0.01 g balance, prepare a 1 mmol / L DMSO dye stock solution in a brown sample bottle, and store it in a refrigerator at 4°C for later use.

[0176] Human breast cancer cells (MCF-7), transplantable mouse breast cancer cell line (4T1), and human liver cancer cells (HepG2) were used for in vitro imaging studies in this experiment. The cells were cultured in DMEM medium containing 10% serum and 1% penicillin / streptomycin at 37°C and 5% CO2 until the logarithmic growth phase. After trypsin digestion, 1×10⁶ cells were prepared. 5 Cell suspensions of cells / mL were used for subsequent experimental protocols.

[0177] 1 μL of compounds Acr-Cy7, AcrB-Cy7, and Cy7alk were added to confocal culture dishes 1 and 2, respectively, for cell uptake experiments. At an excitation wavelength of 638 nm, culture dish 2, 5 min after the addition of the dye, provided Cu(II) histidine complex (400 μmol / L), ethyl bromoisobutyrate (10 μmol / L), N-hydroxyethylacrylamide (10 μmol / L), and sodium ascorbate (800 μmol / L). The results of the MCF-7 uptake experiment are shown below. Figure 12-14 .

[0178] Combination Figure 12-14 It can be seen that, at the same time, under the same polymerization conditions, the dye produced stronger fluorescence in the cells, and the dye molecules under polymerization conditions remained in the cells for a longer time. At 30 min, the fluorescence of the monomer molecules was significantly weakened. The effects on 4T1 and HepG2 cells were the same as those on MCF-7 cells.

[0179] The fluorescence intensity of Acr-Cy7, AcrB-Cy7, and Cy7alk in MCF-7 from 0 to 60 min was organized as follows: Figure 15 The bar chart shown, from Figure 15 It can be seen that the fluorescence brightness under polymerization conditions is significantly higher than that under monomeric conditions, indicating that the fluorescence effect under polymerization conditions is better and lasts longer, while the fluorescence brightness of monomeric molecules is not obvious after 30 minutes.

[0180] Test Example 4: Infrared Spectroscopy Experiment

[0181] Test method: Take 1 mL each of Cy7alk (100 mM) / Acr-Cy7 (100 mM), alkyl Br initiator (10 mM), copper histidine complex (400 mM), and sodium ascorbate [NaAsc] (800 mM) and gradually add them dropwise to 1 mL of DMSO in a 10 mL centrifuge tube. Add Cy7alk / Acr-Cy7 dropwise until a visible precipitate appears in the solution. Centrifuge and dry the solution. Dialyze the solution using a 3000 molecular weight dialysis membrane to remove the solvent. Perform infrared spectroscopy experiments. See [link to results]. Figure 16 .

[0182] from Figure 16 It can be seen that the Cy7alk monomer molecule is located at 3300 cm⁻¹. -1 The terminal alkyne peak disappears and is located at 1640 cm⁻¹. -1 The appearance of the double bond characteristic peak indicates that the carbon-carbon triple bond has reacted. The Acr-Cy7 monomer molecule is located at 1640 cm⁻¹. -1 The disappearance of the characteristic peaks of the double bond indicates that the carbon-carbon double bond has undergone a reaction.

[0183] Test Example 5: 1H NMR Spectroscopy

[0184] Cy7alk was dissolved in deuterated methanol, and copper was gradually added to the NMR tube in the following proportions: alkyl Br initiator = 10 mM, copper histidine complex = 400 mM, sodium ascorbate [NaAsc] = 800 mM. 1H NMR spectra were measured at 0 min, 5 min, and 10 min. The results are shown in [reference needed]. Figure 17 .

[0185] from Figure 17 It can be seen that obvious polymerization characteristics were observed at 0 min and 10 min, and the peaks at shifts of 7.2, 7.3, 7.5 and 8.3 in the benzene ring region became wider, proving that polymerization had occurred.

[0186] Acr-Cy7 was dissolved in deuterated methanol, and copper was gradually added to the NMR tube in the following proportions: alkyl Br initiator = 10 mM, copper histidine complex = 400 mM, sodium ascorbate [NaAsc] = 800 mM. 1H NMR spectra were measured at 0 min, 5 min, 30 min, 60 min, and 120 min. The results are shown in [reference needed]. Figure 18 .

[0187] from Figure 18 It can be seen that obvious polymerization characteristics were observed at 0 min, 30 min, 60 min and 120 min, and the peaks at shifts of 7.3, 7.5 and 8.0 in the benzene ring region became wider, proving that polymerization had occurred.

[0188] Test Example 6: Transmission Electron Microscopy Test

[0189] Cy7alk (1 mM) was dissolved in methanol. Then, the following components were added incrementally to a 1 mL centrifuge tube: alkyl Br initiator = 10 mM, copper histidine complex = 400 mM, and sodium ascorbate [NaAsc] = 800 mM. The mixture was ultrasonically stirred for 4 h, the solvent was evaporated, and the solution was dissolved in methanol. The solution was dialyzed through a 3000 molecular weight dialysis membrane for 24 h. After dialysis, the solution was evaporated again to obtain a solid sample. The solid sample was then dissolved in methanol again and subjected to transmission electron microscopy (TEM). The results are shown in [reference needed]. Figure 19 .

[0190] from Figure 19 The image shows several black spherical particles of similar size and shape, indicating that the particles were obtained through polymerization.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polymerizable heptamethylcyanine dye, characterized in that, Its structural formula is shown in general formula I: In general formula I, R1 and R2 can be the same or different. R1 and R2 are each independently selected from any one of hydrogen, halogen, alkyl group having 1-18 carbons, carboxyl alkyl group having 1-18 carbons, aryl group, aryl carboxylic acid group, alkyl sulfonate group, aryl sulfonate group, alkyl sulfonate or aryl sulfonate. R3 is selected from any one of chlorine, malondicyano, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, arylcarboxylic acid, alkylsulfonate, and arylsulfonate. R4 and R5 can be the same or different. When R4 and R5 are the same, R4 and R5 are selected from acrylates with 1 to 18 carbons. When R4 and R5 are different, one of R4 and R5 is selected from acrylates with 1 to 18 carbons, and the other is selected from acrylates with 1 to 18 carbons or alkynes with 1 to 18 carbons.

2. The polymerizable heptamethine cyanine dye according to claim 1, characterized in that, Its structural formula is shown below: R1 and R2 are the same, and R1 and R2 are selected from any one of hydrogen, halogen, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, aryl, arylcarboxylic acid, alkylsulfonate, arylsulfonate, alkyl sulfonate or aryl sulfonate. R3 is selected from any one of chlorine, malondicyano, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, arylcarboxylic acid, alkylsulfonate, and arylsulfonate.

3. The polymerizable heptamethine cyanine dye according to claim 1 or 2, characterized in that, R1 and R2 are each independently selected from any one of hydrogen, halogen, carboxyl alkyl group having 1-8 carbons, and alkyl sulfonate group; R3 is selected from any one of chlorine, malondicyano, alkyl groups having 1-8 carbons, and carboxyl alkyl groups having 1-8 carbons.

4. The method for synthesizing polymerizable heptamethine cyanine dye according to claim 1, characterized in that, When R4 and R5 are the same, and the acrylates are selected from 1-18 carbon atoms, the synthesis method is as follows: (1) 2,3,3-trimethyl-3H-indole containing R1 and R2 substitutions reacted with 2-iodoethanol in organic solvent I for 10-14 h, and then ice-cold organic solvent II was added. After filtration, the intermediate containing R1 and R2 substitutions was obtained. The molar ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitutions to 2-iodoethanol was 1:1.5-5, and the volume ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitutions to organic solvent II exceeded 1:

1. (2) At 110-120℃, the R1 and R2 substituted intermediates and the R3 substituted 1-cyclohexene-1,3-dicarboxaldehyde were dissolved in organic solvent III. After slurrying and purification by column chromatography in organic solvent III, intermediates containing R1, R2 and R3 substituted products were obtained. (3) At room temperature, the intermediate containing R1, R2, and R3 substitutions is dissolved in organic solvent IV. Under ice-water bath conditions, an esterification reaction is carried out under the catalysis of an organic base. Acryloyl chloride is slowly added dropwise under nitrogen protection for 7-9 hours. After pulping and column chromatography, a polymerizable indole parent near-infrared fluorescent dye is obtained.

5. The method for synthesizing polymerizable heptamethine cyanine dye according to claim 1, characterized in that, R4 and R5 are different. When R4 and R5 are independently selected from acrylates or alkynes with 1-18 carbon atoms, their synthesis methods are as follows: (1) 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution reacts with 2-iodoethanol in organic solvent I for 0-14 h, and then excess cold organic solvent II is added. After filtration, intermediate 1 containing R1 and R2 substitution is obtained, wherein the molar ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution to 2-iodoethanol is 1:1.5-5. (2) 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution reacts with 6-iodo-1-hexyne in organic solvent I for 0.5-2 h, and is purified by silica gel column chromatography to obtain intermediate 2 containing R1 and R2 substitution; wherein the molar ratio of 2,3,3-trimethyl-3H-indole containing R1 and R2 substitution to 6-iodo-1-hexyne is 1:1.5-3; (3) At 110-120℃, 1-cyclohexene-1,3-dicarboxaldehyde containing R1, R2 substituted intermediate 1, R1, R2 substituted intermediate 2 and R3 substituted intermediate 3 were dissolved in organic solvent III. After slurrying and column chromatography purification by organic solvent III, intermediate 3 containing R1, R2, R3 substituted intermediate 3 was obtained. (4) At room temperature, intermediate product 3 containing R1, R2, and R3 substitutions was dissolved in organic solvent IV. Under ice-water bath conditions, esterification reaction was carried out under the catalysis of organic base. Acryloyl chloride was slowly added dropwise under nitrogen protection for 7-9 hours. After pulping and column chromatography, polymerizable indole parent near-infrared fluorescent dye was obtained.

6. A polymer comprising the polymerizable heptamethine cyanine dye according to any one of claims 1-3, characterized in that, The polymer is composed of a polymerizable heptamethine dye, an alkyl bromide initiator, a Cu(II) histidine complex, and sodium ascorbate in a mass ratio of 10:1:40:

80.

7. The polymerization method of the polymer according to claim 6, characterized in that, Using polymerizable heptamethine dye and N-hydroxyethylacrylamide as monomers, Cu(II) histidine complex as catalyst, alkyl bromide initiator as free radical initiator, and glutathione as reducing agent, intracellular free radical polymerization of N-hydroxyethylacrylamide and polymerizable heptamethine dye was initiated.

8. The application of the polymerizable heptamethine dye according to any one of claims 1-3 in biological and pharmaceutical fields, characterized in that, The polymerizable heptamethine dye is used in cell imaging, protein labeling, antibody-specific recognition, nucleic acid labeling, DNA sequencing, and tumor photodynamic therapy formulations.

9. The application according to claim 8, characterized in that, When applied, the excitation wavelength is 500-950nm, and the fluorescence detection wavelength is 650-1000nm.

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