Preparation method and application of cyanine dye variants and cyanine protein composite fluorophores

Abstract

The present invention is applicable to the technical field of near-infrared zone II imaging, provides a preparation method of a cyanine dye variant and a cyanine protein composite fluorophore, successfully prepares a series of cyanine dye-protein composite probes by using a protein encapsulation strategy, and forms a covalently bound complex by protein encapsulation and its application, wherein the covalently bound complex is formed by covalently linking a cyanine dye molecule and a protein; compared with the existing near-infrared zone II fluorescent probe, the composite of the present invention has better biocompatibility and luminescent behavior, a longer wavelength, good optical stability, high brightness, and superior imaging effect in fluorescent imaging applications.

Description

Technical Field

[0001] The invention belongs to the technical field of near-infrared second-zone imaging, and in particular relates to a preparation method of a cyanine dye variant and a cyanine protein composite fluorophore. Background Art

[0002] Fluorescence imaging technology is convenient for medical observation and tracking of living organisms due to its non-invasiveness, high sensitivity, and high signal-to-noise ratio. Compared with the near-infrared region I and visible light regions, near-infrared region II imaging has a longer wavelength, which can greatly reduce the absorption, scattering and autofluorescence of light by tissues, thereby achieving deep tissue imaging with excellent contrast and resolution. At present, only two near-infrared region I fluorescent dyes, namely methylene blue (MB) and indocyanine green (ICG), have been approved for clinical use by the US Food and Drug Administration (FDA). With the continuous exploration of the field of long-wavelength imaging, near-infrared region II fluorescent materials have gradually been developed. However, most of the current near-infrared fluorescent materials, such as inorganic materials such as single-walled nanotubes, quantum dots, and rare earth nanoparticles, usually have high quantum yields and emission peaks exceeding 1000nm, which have unique advantages in deep tissue imaging of organisms. However, because of the low level of biosafety and uncontrollable pharmacokinetic characteristics of heavy atoms contained in their skeletons, they remain in the body for a long time after imaging and accumulate, limiting their clinical transformation. In addition, there are some organic small molecule dyes, such as DAD small molecules with donor-acceptor-donor structure, cyanine dyes, etc. DAD molecules often show large Stokes shift, but have low molar extinction coefficients, and large conjugated structures are more susceptible to attack by water molecules and then quenched, which leads to low quantum yield. Unlike DAD dyes, cyanine dyes are usually composed of two heterocyclic end groups connected by polymethylene chains of different lengths, and have higher absorption coefficients, which makes them have great potential for imaging applications. However, the current near-infrared zone II cyanine dyes such as IR-26, IR-1061, and IR-1048 have problems such as poor molecular stability and short cycle life, so there are still many difficulties in developing near-infrared zone II dyes that can be put into clinical use. At the same time, in recent years, researchers have used the fluorescence emission tailing (>1000nm) of commercial clinically approved near-infrared zone I cyanine dyes and successfully obtained a series of near-infrared zone II fluorescent probes with good biocompatibility and bright near-infrared zone II luminescence ability by combining them with albumin, but rarely involve near-infrared zone II cyanine dyes. Therefore, there is an urgent need to develop near-infrared zone II fluorophores that have high quantum efficiency, excellent optical properties and good biosafety, which will effectively promote the clinical application of near-infrared zone II fluorescence imaging technology. Summary of the invention

[0003] The purpose of the embodiments of the present invention is to provide a method for preparing a cyanine dye variant and a cyanine protein composite fluorophore, aiming to solve the problems in the background technology.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] A cyanine dye variant, the structure of the cyanine dye variant is as follows:

[0006]

[0007] Wherein, X=CI, Br; R1=Et; R2=n-Pr; R3=(CH2)5COOH; R4=(CH2)4SO3H.

[0008] The present invention provides a synthetic route for preparing 1080 series fluorescent probes as follows:

[0009]

[0010] A method for preparing the cyanine dye variant as described above, the method for preparing the cyanine dye variant comprises the following steps:

[0011] Step 1, using 2-(2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-propenoic acid), alkyl chain, carbonate chain and sulfonic acid chain as reaction raw materials, and conducting reflux reaction in an organic solvent under normal pressure;

[0012] Step 2, placing the product obtained in step 1 in a concentrated hydrochloric acid solution for reflux reaction, the reaction time is 10min to 2h, cooling to room temperature after the reaction is completed, filtering the crude product, washing with water and ethyl acetate, and drying;

[0013] Step 3: placing the product obtained in step 2 and a ketone compound under alkaline conditions, heating under reflux in a toluene and n-butanol system, and reacting for 3 to 12 hours to obtain the final product.

[0014] As a further scheme of the embodiment of the present invention, the organic solvent described in step 1 is N,N-dimethylformamide, dimethyl sulfoxide ethanol, methanol, acetonitrile, dichloromethane or anhydrous toluene; the reaction temperature is 60-120° C., and the reaction time is 6h-36h; the solvent used for silica gel chromatography purification is ethyl acetate, dichloromethane, methanol or a mixture of acetone and petroleum ether, and the volume ratio of dichloromethane to methanol is 30:1-10:1.

[0015] As a further scheme of the embodiment of the present invention, the strong acid solution described in step 2 is concentrated sulfuric acid, concentrated hydrochloric acid, acetic acid or formic acid; the reaction temperature is 60-120°C, and the reaction time is 0.5h-6h; and after the reaction is completed, the obtained product is cooled to room temperature, the crude product is filtered, washed with any solvent of toluene, ether or ethyl acetate, and dried after washing.

[0016] As a further scheme of the embodiment of the present invention, the alkaline solution described in step 3 is triethylamine, 2,6-di-tert-butyl-4-methylpyridine, pyridine, sodium acetate and piperidine; the reaction solution described in step 3 is methanol, ethanol, toluene-n-butanol, acetic anhydride and n-butanol; the reaction temperature is 60-120°C, and the reaction time is 4h-16h; and after the reaction is completed, the obtained product is cooled to room temperature, the crude product is filtered, washed with any solvent selected from toluene, ether or ethyl acetate, and dried after washing.

[0017] In addition, the 1080 series fluorescent probes provided by the present invention are probes in the near-infrared region II. These fluorescent molecules show relatively low fluorescence intensity in aqueous solution and lack good biocompatibility in vivo, which limits their practical application to a certain extent. Therefore, the present invention also provides a preparation method and application of a covalently bound complex of albumin and cyanine dye molecules, and obtains a series of near-infrared region II fluorescent probes with good biocompatibility and bright luminescence ability in the near-infrared region II. The covalently bound complex of the present invention has good biocompatibility and low cytotoxicity in vivo, high quantum yield in the near-infrared region II, good photostability, high brightness, and superior imaging effect and imaging ability in the field of fluorescence imaging.

[0018] A cyanine protein composite fluorophore is formed by controllable self-assembly of protein and cyanine dye molecules. The preparation method of the cyanine protein composite fluorophore comprises the following steps:

[0019] Step 1: mixing a solution containing albumin and a solution containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of cyanine dye molecules to albumin in the reaction solution is 0.5:1, 1:1, 2:1, 4:1, 8:1 and 16:1, respectively, and the concentration of albumin in the reaction solution is 0-600 μM;

[0020] Step 2: The reaction solution is placed in an environment of 30 to 90° C. and heated for 0 to 5 hours to obtain a cyanine protein complex fluorophore.

[0021] As a further embodiment of the present invention, the protein is any one of lipocalin such as fetal bovine serum albumin, human serum albumin and human serum albumin domain III;

[0022] The cyanine dye molecule has any one of fluorine, chlorine, bromine or iodine atomic structures.

[0023] As a further solution of an embodiment of the present invention, the cyanine protein complex fluorophore is albumin, which reacts with the four-membered, five-membered and six-membered rings of chlorine, bromine and iodine atoms in the middle of the cyanine dye molecule according to its own specific site to obtain a covalently bound complex; the outer layer of the covalently bound complex is albumin, and the cyanine dye molecule is wrapped inside the albumin.

[0024] The cyanine dye molecule is one of Et-1080, St-1080, CO-1080, FD-1080, Er-1080, Sr-1080, Cr-1080, and Fr-1080. It is worth noting that this method can be extended to other cyanine dyes with chlorine, bromine or iodine atoms.

[0025] The cyanine protein composite fluorophore as described above is used as a fluorescent imaging developer in the near-infrared second window for biological imaging.

[0026] As a further solution of an embodiment of the present invention, the fluorescent probe is applied to imaging in living organisms.

[0027] The method for preparing a cyanine dye variant and a cyanine protein composite fluorophore provided in an embodiment of the present invention has the following beneficial effects:

[0028] The present invention provides a cyanine dye and a preparation method thereof. The cyanine dye is a series of dyes with long absorption and emission wavelengths, and the fluorescence wavelength reaches 1078nm. At the same time, CO-1080 and Cr-1080 introduce active group carboxyl groups, which can react with amino groups, provide target sites, and are easy to modify and modify. The synthesis method is simple, the conditions are mild, and the yield is high, which provides good application prospects in the fields of near-infrared second-zone fluorescence imaging.

[0029] The covalently bonded composite fluorophore of the present invention has good biocompatibility and low biotoxicity in vivo, exhibits high quantum yield, bright luminescence ability and excellent photostability in the near-infrared region II, and has excellent imaging potential in fluorescence imaging applications;

[0030] Inspired by albumin coating technology, the present invention uses albumin to perform external modification on near-infrared zone II cyanine dye molecules, and obtains near-infrared zone II molecular probes with bright luminescence ability, which effectively reduces the aggregation quenching of cyanine dye molecules in aqueous solution, limits the spatial structure of cyanine dye molecules, reduces vibration relaxation, promotes charge transfer within twisted molecules, and greatly increases the quantum yield. In addition, the present invention maintains the small size and biocompatibility of a single albumin or protein domain, and will not cause oversizing and changes in in vivo circulation behavior due to cross-linking between protein molecules. The composite method can be extended to other protein systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of Et-1080 of the present invention;

[0032] Figure 2 The nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of St-1080 of the present invention;

[0033] Figure 3 The nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of CO-1080 of the present invention;

[0034] Figure 4 It is the nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of FD-1080 of the present invention;

[0035] Figure 5 It is the nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of Er-1080 of the present invention;

[0036] Figure 6 It is the nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of Sr-1080 of the present invention;

[0037] Figure 7 It is the nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of Cr-1080 of the present invention;

[0038] Figure 8 It is the nuclear magnetic characterization, 1H-NMR and mass spectrometry characterization and liquid phase high-resolution mass spectrometry of Fr-1080 of the present invention;

[0039] Fig. 9 Characterization of absorption spectra of the 1080 series dyes of the present invention in various solvents;

[0040] Fig.10 To characterize the fluorescence spectra of the 1080 series dyes of the present invention in various solvents;

[0041] Fig.11 This is a statistical diagram of the brightness of 1080 series dyes in DMSO according to the present invention;

[0042] Fig.12 It is a schematic diagram of the binding of cyanine dye and albumin according to the present invention;

[0043] Fig.13 This is a statistical diagram of brightness of 1080 series dyes in PBS according to the present invention;

[0044] Fig.14 This is a statistical graph of the brightness of the cyanine dye@albumin composite fluorescent probe of the present invention;

[0045] Fig.15 The present invention characterizes the absorption and fluorescence spectra of the composite of cyanine dye and albumin;

[0046] Fig.16 This is a statistical diagram of the photostability of free dyes and cyanine dyes@albumin according to the present invention;

[0047] Fig.17 The biocompatibility evaluation table of the present invention for free CO-1080 and CO-1080@albumin composite fluorophore;

[0048] Fig.18 This is a diagram for exploring the in vivo metabolic behavior of the free CO-1080 and CO-1080@albumin composite fluorophore of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0050] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0051] Combination Figures 1 to 18 : Example 1

[0052]

[0053] (1) Compound 1 (500 mg, 1.6 mmol) was dissolved in anhydrous DMF (5 mL), iodoethane (316 mg, 1.6 mmol) was added, and then potassium carbonate (701 mg, 5.1 mmol) was added; the resulting solution was stirred under reflux overnight; the solvent was then concentrated in vacuo, and the residue was purified by silica gel chromatography (PE / EtOAc, 10:1, v / v) to obtain compound 1a as an orange solid (300 mg, 55%). 1H-NMR (400MHz, CDCl3, ppm) δ9.01 (d, J=7.4hz, 1H), 8.17 (d, J=8.0hz, 1H), 7.87–7.78 (m, 2H), 7.62 (t, J=7.8hz, 1H ), 7.52 (d, J = 7.2hz, 1H), 4.41 (q, J = 7.2hz, 2H), 1.83 (s, 6H), 1.56 (t, J = 7.2hz, 3H).LC-HRMS (ESI-TOF): calcd.for C19H18NO4[M+h]+324.1236; found324.1280.

[0054] (2) A mixture of compound 1a (200 mg, 1 mmol) in concentrated hydrochloric acid (2 mL) was stirred under reflux for 2 h; cooled to room temperature, then saturated potassium iodide solution was added, filtered, and washed with ethyl acetate to obtain compound 1b as a red solid (130 mg, 75%). 1H-NMR (400MHz, CD3OD, ppm) δ8.91 (d, J=7.2hz, 1H), 8.78 (d, J=8.2hz, 1H), 8.47-8.44 (m, 2H), 8.19 (t, J=7. 2hz, 1H), 8.03 (t, J=7.2hz, 1H), 4.80 (q, J=7.4hz, 2H), 1.70 (t, J=7.4hz, 3H).LC-HRMS (ESI-TOF): calcd.for C14H14N+[M]+196.1121; found1961170.

[0055] (3) Compound 1b (50 mg, 0.15 mmol) and compound 2 (11.9 mg, 0.070 mmol) were dissolved in acetic acid (1 mL), triethylamine (0.50 mL) and acetic anhydride (0.50 mL), and then heated to 60°C for overnight reaction. After cooling to room temperature, ethyl acetate was added, and the mixture was filtered and recrystallized in ethanol to obtain compound 1c, dark green solid Et-1080 (45 mg, 45%). 1H-NMR (400MHz, DMSO, ppm) δ8.71 (d, J=14.6hz, 2H), 8.43 (d, J=6.9hz, 2H) , 8.28 (d, J = 8.0hz, 2H), 7.97 (t, J = 7.6hz, 2H), 7.81 (d, J = 7.8hz, 2H), 7.72– 7.62 (m, 4H), 6.90 (d, J = 13.9hz, 2H), 4.45 (q, J = 7.2hz, 4H), 2.91 (m, 4H), 1.96 (p, J = 6.4hz, 4H), 1.39 (t, J = 7.2hz, 6H). LC-HRMS (ESI-TOF): calcd.for C36H32ClN2+[M]+527.2249; found 527.2248.

[0056] Example 2

[0057]

[0058] (1) Compound 1 (500 mg, 1.6 mmol) was dissolved in anhydrous DMF (5 mL), iodopropane (346 mg, 2.0 mmol) was added, and then potassium carbonate (701 mg, 5.1 mmol) was added; the resulting solution was stirred under reflux overnight; the solvent was then concentrated in vacuo, and the residue was purified by silica gel chromatography (PE / EtOAc, 10:1, v / v) to obtain compound 2a as an orange solid (300 mg, 55%). 1H-NMR (400MHz, CDCl3, ppm) δ9.01 (d, J=7.4hz, 1H), 8.16 (d, J=8.0hz, 1H), 7.85–7.77 (m, 2H), 7.60 (t, J=7.7hz, 1H), 7.49 (d, J=7.3hz, 1H), 4. found 338.1386.

[0059] (2) A mixture of compound 2a (200 mg, 1 mmol) in concentrated hydrochloric acid (2 mL) was stirred under reflux for 2 h; cooled to room temperature, then saturated potassium iodide solution was added, filtered, and washed with ethyl acetate to obtain compound 2b as a red solid (130 mg, 75%). 1H-NMR (400MHz, CD3OD, ppm) δ9.01 (d, J=7.4hz, 1H), 8.16 (d, J=8.0hz, 1H), 7.85–7.77 (m, 2H), 7.60 (t, J=7.7hz, 1H), 7.49 (d, J=7.3hz, 1H), 4.36 (t, J=7.5hz, 2H), 1.95 (h, J=7.2hz, 2H), 1.83 (s, 6H), 0.94 (t, J=7.4hz, 3H).LC-HRMS (ESI-TOF): calcd.for C15H18N+[M]+210.1277; found210.1297.

[0060] (3) Compound 2b (50 mg, 0.15 mmol) and compound 4 (11.9 mg, 0.070 mmol) were dissolved in acetic acid (1 mL), triethylamine (0.50 mL) and acetic anhydride (0.50 mL), and then heated to 60 °C for overnight reaction. After cooling to room temperature, ethyl acetate was added, filtered and recrystallized in ethanol to obtain compound 2c, dark green solid St-1080 (45 mg, 45%). 1H-NMR (400 MHz, DMSO, ppm) δ8.69 (d, J = 13.7 Hz, 2H), 8.42 (d, J = 7.5 Hz, 2H), 8.27 (d, J = 8.7 Hz, 2H), 7.96 (t, J = 7.7 Hz, 2H), 7.79 (d, J = 7.5 Hz, 2H), 7.71–7.61 (m, 4H), 6 .89 (d, J=14.1hz, 2H), 4.37 (t, J=7.2hz, 4H), 2.90 (t, J=7.2hz, 4H), 1.97 (p, J=5. 4hz, 2H), 1.89–1.80 (m, 4H), 0.99 (t, J=7.3hz, 6H).LC-HRMS (ESI-TOF): calcd.for C38H36ClN2+[M]+555.2562; found 555.2562.

[0061] Example 3

[0062]

[0063] (1) Compound 1 (500 mg, 1.4 mmol) was dissolved in anhydrous DMF (5 mL), 6-bromohexyl acetate (363 mg, 1.6 mmol) was added, and then potassium carbonate (562 mg, 4.1 mmol) was added; the resulting solution was stirred under reflux overnight; the solvent was then concentrated in vacuo, and the residue was purified using silica gel chromatography (DCM / MeOH, 10:1, v / v) to obtain compound 3a as an orange solid (332 mg, 56%). 1H-NMR (400MHz, CDCl3, ppm) δ8.99 (d, J=7.3hz, 1H), 8.17 (d, J=8.0hz, 1H), 7.86–7.7 8 (m, 2H), 7.61 (t, J=7.8hz, 1H), 7.49 (d, J=7.3hz, 1H), 4.37 (t, J=7.6hz, 2H), 4.09 (q, J=7.2hz, 2H), 2.26 (t, J=7.4hz, 2H), 1.96 (p, J=7.4hz, 2H), 1.83 (s, 6H), 1.64 (p, J=7. 4hz, 2H), 1.38 (p, J=7.4hz, 2H), 1.22 (t, J=7.2hz, 3H).LC-HRMS (ESI-TOF):calcd.For C21H18NO6[M]+380.1134; found 380.1462.

[0064] (2) A mixture of compound 3a (200 mg, 0.46 mmol) in concentrated hydrochloric acid (2 mL) was stirred under reflux for 2 h; cooled to room temperature, then saturated potassium iodide solution was added, filtered, and washed with ethyl acetate to obtain compound 3b as a red solid (135 mg, 72%). 1H-NMR (400MHz, CD3OD, ppm) δ8.88 (d, J=7.2hz, 1H), 8.75 (d, J=8.0hz, 1H), 8.45–8.39 (m, 2H), 8.21–8.13 (m, 1H), 8.04–7.97 (m, 1H), 4.7 For C18H20NO2+[M]+282.1489, found 282.1455.

[0065] (3) Compound 3b (50 mg, 0.12 mmol), compound 4 (9.4 mg, 0.055 mmol) and 2,6-di-tert-butyl-4-methylpyridine (75 mg, 0.37 mmol) were dissolved in toluene (1.6 mL) and n-butanol (0.40 mL), and then heated under reflux overnight. After cooling to room temperature, ethyl acetate was added, and the mixture was filtered and recrystallized in ethanol to obtain compound 3c as a dark green solid CO-1080 (45 mg, 45%). 1H-NMR (400MHz, DMSO, ppm) δ8.54 (d, J=14.0hz, 2H), 8.25 (d, J=7.8hz, 2H), 8.14 (d, J=8.1hz, 2H), 7 .88 (t, J=7.7hz, 2H), 7.63 (d, J=8.1hz, 2H), 7.54 (t, J=7.7hz, 2H), 7.48 (d, J=7.3hz, 2H), 6.70 (d, J= 14.1hz, 2H), 4.24 (t, J=7.8hz, 4H), 2.83 (t, J=6.4hz, 4H), 2.21 (t, J=7.3hz, 4H), 1.96-1.93 (m, 2H) , 1.76 (p, J = 7.3hz, 4H), 1.57 (p, J = 7.4hz, 4H), 1.40 (p, J = 7.6hz, 4H).LC-HRMS (ESI-TOF): calcd.For C44H44ClN2O4+[M]+699.2984; found 699.2980.

[0066] Example 4

[0067]

[0068] (1) Compound 1 (400 mg, 1.4 mmol) and KOH (152 mg, 2.7 mmol) were mixed in NMP (4 mL) and stirred at room temperature for 30 minutes. 1,4-Butanesultone (221 mg, 1.6 mmol) was then added and heated at 90° C. overnight. The solvent was then concentrated in vacuo and the residue was purified by silica gel chromatography (DCM / MeOH, 10:1, v / v) to give compound 4a as an orange solid (327 mg, 0.76 mmol, 56%). 1H-NMR (400MHz, D2O, ppm) δ8.99 (d, J=7.3hz, 1H), 8.17 (d, J=8.0hz, 1H), 7.86–7.78 ( m, 2H), 7.61 (t, J=7.8hz, 1H), 7.49 (d, J=7.3hz, 1H), 4.37 (t, J=7.6hz, 2H), 4.09 (q, J =7.2hz, 2H), 2.26 (t, J = 7.4hz, 2H), 1.96 (p, J = 7.4hz, 2H), 1.83 (s, 6H), 1.64 (p, J = 7. 4hz, 2H), 1.38 (p, J=7.4hz, 2H), 1.22 (t, J=7.2hz, 3H).LC-HRMS (ESI-TOF):calcd.for C21H21NO7S[M+2h]+432.1117; found432.1140.

[0069] (2) A mixture of compound 4a (150 mg, 0.19 mmol) in concentrated hydrochloric acid (2 mL) was stirred under reflux for 2 h; cooled to room temperature, then the solvent was concentrated in vacuo and washed with ethyl acetate to give compound 4b as a red solid (110 mg, 82%). 1H-NMR (400MHz, D2O, ppm) δ8.55 (d, J=7.2hz, 1H), 8.46 (d, J=8.1hz, 1H), 8.17-8.12 (m, 2H), 7.90 (t, J=7.7hz, 1H), 7.79 (t, J=7.8hz, 1H), 4.59 (t, J =7.6hz, 2H), 2.97 (t, J = 7.6hz, 2H), 2.12 (p, J = 7.9hz, 2H), 1.90 (p, J = 7.9hz, 2H). LC-HRMS (ESI-TOF): calcd.forC16H18NO3S+[M]+304.1002; found 304.1035.

[0070] (3) Compound 4b (60 mg, 0.2 mmol), compound 4 (13 mg, 0.079 mmol) and 2,6-di-tert-butyl-4-methylpyridine (120 mg, 0.59 mmol) were dissolved in toluene (1.6 mL) and n-butanol (0.40 mL), and then heated under reflux overnight. After cooling to room temperature, ethyl acetate was added, filtered and recrystallized in ethanol to obtain compound 4c, dark green solid FD-1080 (45 mg, 45%). LC-HRMS (ESI-TOF): calcd. for C40H40ClN2O6S2+[M]+743.2011; found743.2002.

[0071] Example 5

[0072]

[0073] Compound 1b (50 mg, 0.15 mmol) and compound 5 (15.1 mg, 0.070 mmol) were dissolved in acetic acid (1 mL), triethylamine (0.50 mL) and acetic anhydride (0.50 mL), and then heated to 60°C for overnight reaction. After cooling to room temperature, ethyl acetate was added, filtered and recrystallized in ethanol to obtain compound 5c, dark green solid Er-1080 (33 mg, 68%). 1H-NMR (400MHz, DMSO, ppm) δ8.56 (d, J=13.2hz, 2H), 8.29 (d, J=7.4hz, 2H), 8.14 (d, J=7.9hz, 2H), 7.88 (t, J=7.5hz, 2H), 7.62 (d, J=7.9hz, 2H), 7.56–7 .46(m, 4H), 6.73(d, J=14.0hz, 2H), 4.30(q, J=7.2hz, 4H), 2.88–2.85(m, 4H ), 1.99–1.91(m, 2H), 1.33(t, J=7.2hz, 6H).LC-HRMS(ESI-TOF):calcd.for C36H32BrN2+[M]+571.1743; found 571.1723.

[0074] Example 6

[0075]

[0076] Compound 2b (50 mg, 0.15 mmol) and compound 5 (15.9 mg, 0.07 mmol) were dissolved in acetic acid (1 mL), triethylamine (0.50 mL) and acetic anhydride (0.50 mL), and then heated to 60 ° C for overnight reaction. After cooling to room temperature, ethyl acetate was added, filtered and recrystallized in ethanol to obtain compound 6c, dark green solid Sr-1080 (40 mg, 80%). 1H-NMR (400 MHz, DMSO, ppm) δ8.54 (d, J = 13.9 Hz, 2H), 8.28 (d, J = 7.5 Hz, 2H), 8.14 (d, J = 8.0 Hz, 2H), 7.88 (t, J = 7.7 Hz, 2H), 7.62 (d, J = 7.8 Hz, 2H), 7.56–7.47 (m, 4H) , 6.71 (d, J = 14.0hz, 2H), 4.21 (t, J = 7.1hz, 4H), 2.85 (t, J = 5.6hz, 4H), 1.96-1.92 (m, 2H), 1.81-1.72 (m, 4H), 0.95 (t, J=7.3hz, 6H).LC-HRMS (ESI-TOF): calcd.for C38H36BrN2+[M]+599.2056; found599.2077.

[0077] Example 7

[0078]

[0079] Compound 3b (50 mg, 0.12 mmol), compound 5 (9.4 mg, 0.055 mmol) and 2,6-di-tert-butyl-4-methylpyridine (75 mg, 0.37 mmol) were dissolved in toluene (1.6 mL) and n-butanol (0.40 mL), and then heated under reflux overnight. After cooling to room temperature, ethyl acetate was added, filtered and recrystallized in ethanol to obtain compound 7c, dark green solid Cr-1080 (38 mg, 65%). 1H-NMR (400MHz, DMSO, ppm) δ8.53 (d, J=13.8hz, 2H), 8.26 (d, J=7.5hz, 2H), 8.12 (d, J=8.2hz, 2H), 7.87 (t, J=7.7hz, 2H), 7.61 (d, J=8.0hz, 2H), 7.52 (t, J=7.7hz, 2H), 7.45 (d, J=7.3hz, 2H), 6.68 (d, J=13.9hz, 2H), 4.20 (t, J=7.6hz, 4H), 2.88–2.80 (m, 4H), 2.21 (t, J=7.2hz, 4H), 1.99–1.90 (m, 2H), 1.73 (p, J=7.5hz, 4H), 1.56 (p, J=7.5hz, 4H), 1.40 (p, J=7.4hz, 4H).LC-HRMS (ESI-TOF): calcd.for C44H44BrN2O4+[M]+743.2479; found 743.2477.

[0080] Example 8

[0081]

[0082] Compound 4b (60 mg, 0.2 mmol), compound 5 (13 mg, 0.079 mmol) and 2,6-di-tert-butyl-4-methylpyridine (120 mg, 0.59 mmol) were dissolved in toluene (1.6 mL) and n-butanol (0.40 mL), and then heated under reflux overnight. After cooling to room temperature, ethyl acetate was added, filtered and recrystallized in ethanol to obtain compound 8c, dark green solid Fr-1080 (43 mg, 45%). LC-HRMS (ESI-TOF): calcd.for C40H40BrN2O6S2+[M]+787.1506; found 787.1533.

[0083] Example 9

[0084] This embodiment provides a cyanine dye-protein composite fluorescent probe, which is formed by the reaction of the protein through its specific site with the chlorine or bromine atom carried by the six-membered ring in the middle of the cyanine dye molecule to obtain a covalently bound complex. The outer layer of the covalently bound complex is albumin, and the cyanine dye molecules are wrapped inside the albumin. The cyanine dye molecules are coated by the hydrophobic cavity of the albumin itself to form a stable and firm fluorescent probe.

[0085] The method for preparing the above protein-cyanine dye complex comprises the following steps:

[0086] S1: mixing a solution A containing albumin and a solution B containing cyanine dye molecules to obtain a reaction solution;

[0087] The molar ratio of cyanine dye molecules to albumin in the reaction solution is 0.5:1, and the concentration of albumin in the reaction solution is 400 μM;

[0088] S2: then heating and incubating at 60°C for 2 hours to obtain a covalently bound complex, wherein the cyanine dye molecules are Et-1080 and Er-1080;

[0089] Example 10

[0090] The present embodiment provides a cyanine dye-protein composite fluorescent probe, which is formed by the reaction of albumin through its specific site with the chlorine or bromine atom carried by the six-membered ring in the middle of the cyanine dye molecule to obtain a covalently bound complex. The outer layer of the covalently bound complex is albumin, and the cyanine dye molecules are wrapped inside the albumin. The cyanine dye molecules are coated by the hydrophobic cavity of the albumin itself to form a stable and firm fluorescent probe.

[0091] The method for preparing the above protein-cyanine dye complex comprises the following steps:

[0092] S1: mixing a solution A containing albumin and a solution B containing cyanine dye molecules to obtain a reaction solution;

[0093] The molar ratio of cyanine dye molecules to albumin in the reaction solution is 1:1, and the concentration of albumin in the reaction solution is 100 μM;

[0094] S2: Then, the mixture was heated and incubated at 60° C. for 2 h to obtain a covalently bound complex, the cyanine dye molecules St-1080 and Sr-1080.

[0095] Embodiment 11

[0096] This embodiment provides a novel cyanine dye-protein composite fluorescent probe, which is formed by the reaction of albumin through its specific site with the chlorine atom carried by the six-membered ring in the middle of the cyanine dye molecule to obtain a covalently bound complex. The outer layer of the covalently bound complex is albumin, and the cyanine dye molecules are wrapped inside the albumin. The cyanine dye molecules are coated by the hydrophobic cavity of the albumin itself to form a stable and firm fluorescent probe.

[0097] The method for preparing the above-mentioned cyanine dye-albumin complex comprises the following steps:

[0098] S1: mixing a solution A containing albumin and a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the albumin in the reaction solution is 2:1, and the concentration of the albumin in the reaction solution is 20 μM;

[0099] S2: then heating and incubating at 50°C for 1 hour to obtain a covalently bound complex, wherein the cyanine dye molecules are CO-1080 and Cr-1080;

[0100] Example 12

[0101] This embodiment provides a novel cyanine dye-protein composite fluorescent probe, which is formed by the reaction of albumin through its specific site with the chlorine atom carried by the six-membered ring in the middle of the cyanine dye molecule to obtain a covalently bound complex. The outer layer of the covalently bound complex is albumin, and the cyanine dye molecules are wrapped inside the albumin. The cyanine dye molecules are coated by the hydrophobic cavity of the albumin itself to form a stable and firm fluorescent probe.

[0102] The method for preparing the above-mentioned cyanine dye-albumin complex comprises the following steps:

[0103] S1: mixing a solution A containing albumin and a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the albumin in the reaction solution is 3:1, and the concentration of the albumin in the reaction solution is 200 μM;

[0104] S2: then heating and incubating at 80°C for 5 hours to obtain a covalently bound complex, wherein the cyanine dye molecules are FD-1080, Fr-1080;

[0105] Experimental verification shows that when the albumin concentration in the reaction solution in step S1 is 30 μM, the brightness is the best. However, at this concentration, the composite fluorophore has been partially quenched and does not conform to the linear increasing trend. Therefore, an albumin concentration of 10 μM is selected as the optimal reaction condition.

[0106] In the above step S2, the heating co-incubation temperature in the above step S2 can also be 40-60°C, and the most preferred temperature is 50°C or 60°C.

[0107] In addition, by comparing the fluorescence intensity and gel running data of the complex of albumin and cyanine dye, it can be found that albumin has a fluorescence enhancement effect on chlorine / bromine-containing cyanine dye molecules, but the enhancement effect on chlorine-containing cyanine dye molecules is more obvious. In addition, the fluorescence brightness enhancement effect of the CO-1080@albumin complex is the most prominent, and an effective covalent bond is formed between the two.

[0108] Embodiment 13

[0109] The biosafety of free dye probes and composite probes was investigated. Fig.17 As shown, unlike the free dye probe which has certain cytotoxicity under high concentration conditions, the complex probe exhibits better biocompatibility and shows negligible cytotoxicity to L929 and 4T1 cells.

[0110] Embodiment 14

[0111] On the basis of the good biosafety mentioned above, the metabolic behavior of the composite probe in vivo was also explored. The prepared fluorescent probe was injected into Balb / c mice through the tail vein and monitored by a near-infrared zone II imaging device. Fig.18 As shown, the CO-1080@albumin composite probe exhibited excellent and rapid metabolic behavior - hepatobiliary metabolism, was almost completely excreted from the body 72 hours after injection, and showed negligible skin absorption, further indicating the good biosafety of the complex.

[0112] In summary, we synthesized a series of small molecule dyes in the near-infrared region II, and used a protein-encapsulated strategy to modify the molecules and optimize their optical properties, resulting in a composite probe with long wavelength, high brightness, good photostability and good biocompatibility. The CO-1080@albumin complex has excellent biological system imaging capabilities in the near-infrared region II window, and is expected to be used in subsequent in-depth research on vascular and lymphatic system imaging, accelerating the process of clinical application of near-infrared region II fluorescence imaging technology.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cyanine protein composite fluorophore, which is formed by controllable self-assembly of albumin and cyanine dye molecules, characterized in that: The preparation method of the cyanine protein composite fluorophore comprises the following steps: Step 1: mixing a solution containing albumin and a solution containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of cyanine dye molecules to albumin in the reaction solution is 0.5:1, 1:1, 2:1, 4:1, 8:1 and 16:1, respectively, and the concentration of albumin in the reaction solution is 0-600 μM; Step 2: The reaction solution is placed in an environment of 30-90°C for heating and incubation for 0-5 h to obtain a cyanine protein complex fluorophore; The albumin is any one of bovine serum albumin and human serum albumin; The cyanine dye molecule is: ; The cyanine protein complex fluorophore is a covalently bound complex obtained by the reaction of albumin with the six-membered ring of chlorine atoms in the middle of the cyanine dye molecule according to its own specific site; The outer layer of the covalently bound complex is albumin, and the albumin encapsulates cyanine dye molecules.

2. Use of the cyanine protein composite fluorophore as claimed in claim 1 in the preparation of a fluorescent imaging developer.

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