Ring-substituted and side-chain amino-substituted Nile blue dye as well as synthesis method and application thereof

Abstract

The invention discloses a novel ring-substituted and side-chain amino-substituted nile blue dye and a synthesis method and application thereof, a series of dyes which can be simultaneously used for bacterial fluorescence imaging and photodynamic antibacterial treatment are designed and synthesized by taking the nile blue dye as a parent, and the structure of the novel ring-substituted and side-chain amino-substituted nile blue dye is as shown in a general formula I, the dye has high molar extinction coefficient, fluorescence imaging efficiency and active oxygen generation efficiency, and MNBS-C2-NH2 and MNBS-C6-NH2 synthesized in the embodiment show excellent absorption and fluorescence properties in PBS (Phosphate Buffer Solution) and dichloromethane, especially in a range of 640-660nm. A test result shows that the dyes have a remarkable killing effect on escherichia coli under an illumination condition, log CFU is obviously reduced, and dark toxicity is relatively low. Scanning electron microscope observation further proves that the bacteria subjected to light treatment have obvious membrane surface collapse and rupture phenomena. Through the outer membrane protein of the gram-negative bacteria, the enhanced penetrating ability and killing ability on the gram-negative bacteria can be realized, and wide application prospects are realized in the field of antibacterial treatment.

Description

Technical Field

[0001] The invention relates to the technical field of fine chemical organic dyes, in particular to a type of Nile blue dye and a preparation method and application thereof. Background Art

[0002] Bacterial infection has always been an important factor threatening human health. Due to the increasing antimicrobial resistance (AMR), the speed of new drug development is far behind the speed of drug-resistant bacteria. In addition, the existing antibiotics commonly used to treat drug-resistant bacterial infections, such as polymyxin and vancomycin, have significant side effects such as renal damage, hearing damage, neurotoxicity, liver damage, and anaphylactic shock. The problem of drug-resistant bacterial infection has become a global challenge in the field of public health. Therefore, it is particularly important to explore new treatment methods for drug-resistant bacterial infections.

[0003] Photodynamic antimicrobial therapy (PDAT) is a new type of non-antibiotic antimicrobial therapy. It has great potential to solve the increasingly serious clinical multidrug-resistant bacteria because it can overcome the antibiotic resistance of multidrug-resistant bacteria and almost does not produce resistance itself. It has received more and more attention and research in recent years. At present, photodynamic antimicrobial therapy has been clinically used in the treatment of skin and oral infections. It can effectively replace antibiotics and reduce their use in the treatment of local infections. It has been reported that a variety of resistant bacteria can be inactivated by photodynamic antimicrobial therapy. Although the antibacterial treatment of photosensitizers has made certain progress, their poor water solubility, poor bacterial penetration ability and low reactive oxygen production rate have seriously restricted their practical application, especially the unsatisfactory bactericidal effect on Gram-negative bacteria. The enhanced penetration ability for Gram-negative bacteria is also the focus and difficulty of antibacterial dye design. Therefore, it is urgent to develop a class of photosensitizers with excellent bacterial penetration performance and photosensitization ability to enhance the penetration and killing ability of Gram-negative bacteria and multidrug-resistant bacteria, which is of great significance for solving the diagnosis and treatment of bacterial infections. Summary of the invention

[0004] Aiming at the problems of poor water solubility, poor penetration ability for Gram-negative bacteria and low active oxygen yield of existing photosensitizers, the present invention provides a class of ring-substituted and side-chain amino-substituted Nile blue dyes, a synthesis method and application thereof, using a hydrophilic photosensitizer with a positive charge and an amino group to adsorb and bind to bacteria through electrostatic action, and using the transport capacity of porins in the outer membrane of Gram-negative bacteria to penetrate into the internal structure of Gram-negative bacteria, thereby enhancing the imaging and killing ability for Gram-negative bacteria, and can be used for fluorescence imaging, not only can it kill Gram-positive bacteria, but also has a good photodynamic antibacterial effect on most Gram-negative bacteria that are less effective with photosensitizers, and has important research significance for the diagnosis and treatment of bacterial infections.

[0005] In order to achieve the above object, the technical scheme of the present invention is: a method for synthesizing a ring-substituted and side-chain amino-substituted Nile blue dye, the structure of which is shown in general formula I:

[0006]

[0007] In the general formula I,

[0008] X is selected from one of oxygen, sulfur, selenium and tellurium. More preferably, in the embodiment of the present invention, X is selected from sulfur.

[0009] Y - Selected from halogen ions, ClO 4 - , BF 4 - PF 6 - In one embodiment of the present invention, further preferably, Y - Chloride ion is selected;

[0010] R 1 Selected from One of the groups, further preferably, in the embodiment of the present invention, R 1 Select

[0011] n is a positive integer selected from 1-18, which is the length of the alkyl chain of the side chain, further preferably 2-6, more preferably, in the embodiment of the present invention, n is 2 or 6;

[0012] R 2 , R 3 Each independently selected from H, methyl, ethyl, propyl, One of the groups, further preferably, in the embodiment of the present invention, R 2 , R 3 Select H for both.

[0013] The synthesis method of ring-substituted and side-chain amino-substituted Nile blue dye, the process of which is as follows:

[0014]

[0015] Its synthesis method comprises the following steps:

[0016] (1) In aqueous solution, 1 The substituted aniline was added to the aluminum sulfate aqueous solution, and NaS was added successively under stirring at low temperature. 2 O 3 and ZnCl 2 , add the oxidant dropwise, after sufficient reaction, filter out the precipitate, and then purify it by recrystallization to obtain the compound represented by formula Z-1;

[0017] Or will contain R 1 The substituted phenol is added to 5-50 wt% acid; sodium nitrite is slowly added under low temperature and stirring, and the mixture is fully reacted, allowed to stand, and a precipitate is obtained by suction filtration, and the precipitate is washed to obtain a compound represented by formula Z-2;

[0018] (2) Add 1-bromonaphthalene, an inorganic base, a catalyst and a mixture containing R in an organic solvent at 80-150°C. 2 and R 3 The amino compound of the substituent is reacted for 12-48 hours, and the compound represented by formula Z-3 is obtained by decompression, concentration and purification;

[0019] (3) in an organic solvent at 0-80°C, the compound represented by formula Z-1 or formula Z-2 and the compound represented by formula Z-3 are oxidized by an oxidant or catalyzed by a base to form a ring, and then - The compound shown in formula I is obtained by treating with acid, concentrating and purifying.

[0020] Further preferably, in step (1), the 1 Substituted aniline, aluminum sulfate, NaS 2 O 3 、ZnCl 2 The molar ratio of the oxidant is 1:1-2:1-3:1-2:0.1-2, and the preferred ratio is 1:1-1.5:2-3:1-1.5:0.3-1.

[0021] More preferably, in the above step (1), R 1 The molar ratio of the substituted phenol to sodium nitrite is 1:1-3, preferably 1:1-1.5; the acid is further preferably 10-20wt%.

[0022] Further preferably, in the above step (1), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate and potassium chlorate; and the acid is selected from at least one of hydrochloric acid, sulfuric acid, hydrobromic acid and acetic acid.

[0023] Further preferably, in step (2), the 1-bromonaphthalene, catalyst, inorganic base and R 2 and R 3 The molar ratio of the amino compound of the substituent is 1:0.05-0.6:1-3:1-5, and the preferred ratio is 1:0.05-0.2:1-1.5:2-3.

[0024] Further preferably, in the above step (2), the organic solvent is selected from one or a mixed solvent of ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, ethylene glycol monobutyl ether, propylene glycol methyl ether, butanediol, ethanol, acetonitrile, and ethyl acetate; the inorganic base is selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, and sodium ethoxide; and the catalyst is selected from at least one of cuprous iodide, cuprous bromide, cuprous chloride, cuprous oxide, cupric sulfate, cupric sulfate pentahydrate, cupric oxide, and cupric chloride.

[0025] Further preferably, in the step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the compound represented by formula Z-3 is 1:0.5-3, preferably 1:1-1.5.

[0026] Further preferably, in the step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the oxidant or base is 1:0.5-5, preferably 1:1-2.

[0027] Further preferably, in the above step (3), the organic solvent is selected from one or a combination of methanol, ethanol, isopropanol, petroleum ether, ethyl acetate, glycerol, dichloromethane, DMF;

[0028] Further preferably, in step (3), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, potassium chlorate and hydrogen peroxide.

[0029] Further preferably, in step (3), the base is selected from at least one of silver carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium ethoxide, sodium hydroxide, sodium perchlorate, potassium perchlorate, sodium hexafluorophosphate, sodium fluoroborate, and triethylamine;

[0030] Further preferably, in step (3), the Y-containing - The acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, perchloric acid, fluoroboric acid and hexafluorophosphoric acid.

[0031] Another aspect of the present invention is to protect the application of the class of ring-substituted and side-chain amino-substituted Nile blue dyes, which includes the fields of spectral testing technology, bacterial imaging technology and photodynamic therapy technology used in non-disease diagnosis and treatment, with particular emphasis on the practical application of high-definition fluorescence imaging technology of bacteria and antibacterial treatment technology.

[0032] In summary, the present invention has the following beneficial effects:

[0033] 1. The present invention discloses a novel type of Nile blue dye with ring substitution and side chain amino substitution, which retains the fluorescence properties of traditional Nile blue dye and improves the water solubility and photosensitization ability of the dye. MNBS-C2-NH 2 and MNBS-C6-NH 2 They exhibited excellent absorption and fluorescence properties in both PBS and dichloromethane, especially in the range of 640-660 nm. In addition, the solubility of these dyes in water was significantly improved, which is convenient for practical applications.

[0034] 2. Experimental tests have shown that the Nile blue dye with ring substitution and side chain amino substitution described in the present application can effectively penetrate Gram-negative bacteria represented by Escherichia coli, has good fluorescence imaging ability for negative bacteria, has strong killing ability for negative bacteria under light conditions, and retains the penetration and killing ability of traditional photosensitizers for Gram-positive bacteria. Test Example 3 of the present invention shows that MNBS-C2-NH 2 and MNBS-C6-NH 2 The fluorescence signal intensity of E. coli gradually increased from 0 to 120 minutes, indicating that it has good bacterial penetration ability. Test Example 5 further confirmed that under light conditions, MNBS-C2-NH 2 and MNBS-C6-NH 2 It has a significant killing effect on Escherichia coli, with a significant reduction in log CFU and low dark toxicity.

[0035] 3. The ring-substituted and side-chain amino-substituted Nile blue dyes described in the present application have low dark toxicity, good fluorescence properties and enhanced antibacterial ability, and have good application prospects in bacterial imaging and treatment of bacterial infections. Test Example 6 of the present invention was observed by scanning electron microscopy and found that the light-treated bacteria (L+ group) showed obvious membrane surface collapse, wrinkling and rupture, while the bacterial morphology of the light-protected group (L- group) and the control group remained intact, further verifying the efficient antibacterial ability of these dyes in photodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0037] Figure 1 Embodiment of the present invention MNBS-C2-NH 2 MNBS-C6-NH 2 Absorption and fluorescence spectra in different reagents.

[0038] Figure 2 Embodiment of the present invention MNBS-C2-NH 2 MNBS-C6-NH 2 UV absorption decay curve of active oxygen scavenger DPBF in dichloromethane.

[0039] Figure 3 Embodiment of the present invention MNBS-C2-NH 2 MNBS-C6-NH 2 Compared with the comparative example 1MNBS-C0-NH 2 Uptake experiments in different bacteria.

[0040] Figure 4 Embodiment of the present invention MNBS-C2-NH 2 MNBS-C6-NH 2 Analysis of the penetration ability of the outer membrane protein of the Gram-negative bacterium Escherichia coli.

[0041] Figure 5 Embodiment of the present invention MNBS-C2-NH 2 MNBS-C6-NH 2 Compared with Comparative Example 2, the bacterial colony counts of ampicillin killing different bacteria.

[0042] Figure 6 Embodiment of the present invention MNBS-C2-NH 2 The morphological damage of different bacteria was observed under a scanning electron microscope. DETAILED DESCRIPTION

[0043] Hereinafter, the present invention will be described in further detail.

[0044] Unless otherwise specified, the terms used herein have the following meanings.

[0045] The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.

[0046] The term "alkyl group" used in the present invention includes straight-chain alkyl groups and branched-chain alkyl groups.

[0047] The ring-substituted and side-chain amino-substituted Nile blue dyes shown in the general formula I are described in detail below with reference to the examples.

[0048] Example 1

[0049] Nile Blue Dye MNBS-C2-NH 2 Has the following structural formula:

[0050]

[0051] Its synthesis method comprises the following steps:

[0052]

[0053] (1) Synthesis of Compound 1

[0054]

[0055] Aluminum sulfate 18hydrate (3.66g, 5.5mmol) was dissolved in 10mL of water to prepare an aqueous solution of aluminum sulfate. Weigh 4-morpholinoaniline (0.89g, 5mmol) and add it to the above aqueous solution of aluminum sulfate, then add sodium thiosulfate pentahydrate (2.85g, 11.5mmol) and zinc chloride (0.72g, 5.25mmol) in sequence, and cool in an ice bath. Weigh potassium dichromate (0.44g, 1.5mmol) and prepare 4mL of aqueous solution, and slowly add it dropwise to the reaction system. Stir in an ice bath for about 2h to filter out the solid precipitate and dissolve it in 12mL of methanol. After reflux for 10min, cool, filter and dry to obtain an off-white solid, which can be directly used in the next step without further purification. The yield is 0.60g, and the yield is 41%.

[0056] (2) Synthesis of Compound 2

[0057]

[0058] Weigh 1-bromonaphthalene (2.07 g, 10.0 mmol), ethylenediamine (1.20 g, 20.0 mmol), cuprous iodide (0.095 g, 0.5 mmol) and cesium carbonate (3.26 g, 10 mmol), add 20 mL of ethylene glycol monomethyl ether, and react at 125 ° C for 24 h under nitrogen protection. After the reaction is completed, the solvent is removed by distillation under reduced pressure, and column chromatography is used for separation and purification (V 二氯甲烷 :V 甲醇 =5:1) to obtain a light yellow oily liquid with a yield of 1.12 g and a yield rate of 61%.

[0059] (3) Target compound MNBS-C2-NH 2Synthesis

[0060]

[0061] Weigh compound 1 (0.145 g, 0.50 mmol) and compound 2 (0.122 g, 0.65 mmol), add 3 mL of dimethyl sulfoxide, slowly add potassium dichromate (0.162 g, 0.55 mmol), stir at room temperature for 10 min, then add 50 mL of methanol and 6 mL of dilute hydrochloric acid solution (1.0 mol / L), and continue to react at room temperature for 40 min. After the reaction is completed, the solvent is removed by vacuum distillation on a rotary evaporator, and column chromatography is used for separation and purification (V 二氯甲烷 :V 甲醇 =100:14) to give a dark blue solid with a yield of 0.059 g and a yield rate of 30%.

[0062] 1 H NMR (500 MHz, D 2 O)δ7.72(d,J=6.1Hz,1H),7.30(d,J=8.2Hz,3H),6.84(d,J=8.5Hz,1H),6.61(d,J=8.2Hz,1H),6 .45(s,1H),6.11(s,1H),3.71(s,4H),3.56(t,J=7.0Hz,2H),3.20(t,J=7.0Hz,2H),3.15(s,4H). 13 C NMR (126MHz, D 2 O)δ152.64,152.19,140.71,136.46,134.38,132.54,132.49,131.76,131.50,130 .08,125.21,123.70,122.06,118.11,105.82,101.87,66.04,46.68,40.75,37.71.

[0063] Example 2

[0064] MNBS-C6-NH 2 The dye has the following structural formula:

[0065]

[0066] Its synthesis method comprises the following steps:

[0067]

[0068] (1) Synthesis of Compound 1

[0069] The synthesis of intermediate compound 1 in Example 2 is the same as that of intermediate compound 1 in Example 1.

[0070] (2) Synthesis of Compound 3

[0071]

[0072] Weigh 1-bromonaphthalene (2.07 g, 10.0 mmol), 1,6-hexanediamine (2.33 g, 20.0 mmol), cuprous iodide (0.095 g, 0.5 mmol) and cesium carbonate (3.26 g, 10 mmol), add 20 mL of ethylene glycol monomethyl ether, and react at 125 ° C for 24 h under nitrogen protection. After the reaction is completed, the solvent is removed by distillation under reduced pressure, and column chromatography is used for separation and purification (V 二氯甲烷 :V 甲醇 =5:1) to obtain a light yellow oily liquid with a yield of 1.45 g and a yield rate of 60%.

[0073] (3) Target compound MNBS-C6-NH 2 Synthesis

[0074]

[0075] Weigh compound 1 (0.145 g, 0.50 mmol) and compound 3 (0.157 g, 0.65 mmol), add 3 mL of dimethyl sulfoxide, slowly add potassium dichromate (0.162 g, 0.55 mmol), stir at room temperature for 10 min, then add 50 mL of methanol and 6 mL of dilute hydrochloric acid solution (1.0 mol / L), and continue to react at room temperature for 40 min. After the reaction is completed, the solvent is removed by vacuum distillation on a rotary evaporator, and column chromatography is used for separation and purification (V 二氯甲烷 :V 甲醇 =100:12) to give a dark blue solid with a yield of 0.078 g and a yield rate of 34%.

[0076] 1 H NMR (500 MHz, D 2 O)δ8.05(d,J=8.0Hz,1H),7.51(t,J=7.3Hz,1H),7.44(dt,J=14.6,7.6Hz,2H),7.08(d,J=9.1Hz,1H),6.73(d,J=9.4Hz,1H),6.53(s ,1H),6.35(s,1H),3.77(t,J=5.1Hz,4H),3.23(d,J=5.1Hz,6H),3.00–2.94(m,2H),1.65(q,J=13.0,9.9Hz,4H),1.44–1.37(m,4H). 13 C NMR (126MHz, D 2O)δ152.68,151.44,140.11,135.47,134.22,131.68,131.60,131.38,129.98,129.72,124.96,1 23.31,121.76,116.57,105.52,102.63,65.93,46.14,44.12,39.45,28.11,26.79,25.93,25.55.

[0077] Comparative Example 1

[0078] Comparative Example of Nile Blue Dye with Left Ring Substitution Only MNBS-C0-NH 2 Use and Example MNBS-C2-NH 2 MNBS-C6-NH 2 Synthesized by a similar method, the structure and synthetic route are as follows:

[0079]

[0080] 1 H-NMR (500MHz, Methanol-d 4 )δ9.09(d,J=8.1Hz,1H),8.30(d,J=8.1Hz,1H),8.04(d,J=9.2Hz,1H),7.97(t,J=7.5Hz,1H),7.87(t,J =7.5Hz,1H),7.48(d,J=9.1Hz,1H),7.37(s,1H),7.23(s,1H),3.89–3.84(m,4H),3.66(d,J=4.4Hz,4H). 13 C-NMR (126MHz, Methanol-d 4 )δ159.32,154.34,142.49,138.15,137.27,135.97,134.43,133.53,131.8 7,131.52,126.96,125.41,124.79,118.69,107.95,107.65,67.53,48.34.

[0081] Comparative Example 2

[0082] (2S,5R,6R)-3,3-dimethyl-6-[(R)-2-amino-2-phenylacetylamino]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid sodium salt (Ampicillin Sodium) is a commercial antibiotic used clinically. The structure is as follows. The product can be purchased from Bid Pharmaceutical Co., Ltd.

[0083]

[0084] Test Case

[0085] In order to prove that the Nile blue dye with ring substitution and side chain amino substitution prepared in Example 1 and Example 2 can effectively penetrate Gram-negative bacteria represented by Escherichia coli, has good fluorescence imaging ability for negative bacteria, has strong killing ability for negative bacteria under illumination conditions, and retains the penetration and killing ability of traditional photosensitizers for Gram-positive bacteria, which has important research significance for the treatment of drug-resistant bacterial infections, the Nile blue dye synthesized in the above examples was tested as follows:

[0086] Test Example 1 UV-visible absorption spectrum and fluorescence quantum yield determination

[0087] MNBS-C2-NH 2 MNBS-C6-NH 2 Prepared into 10 μM concentration test solution, tested MNBS-C2-NH 2 and MNBS-C6-NH 2 The absorption spectrum and fluorescence spectrum of the test result are shown in Figure 1 .

[0088] Figure 1 (a) is MNBS-C2-NH 2 MNBS-C6-NH 2 Absorption curve in dichloromethane; Figure 1 (a) is MNBS-C2-NH 2 MNBS-C6-NH 2 Absorption curve in phosphate buffered saline (PBS).

[0089] Table 1. MNBS-C2-NH 2 MNBS-C6-NH 2 The absolute fluorescence quantum yield.

[0090] sample Solvents Absolute fluorescence quantum yield <![CDATA[MNBS-C2-NH 2 ]]> Dichloromethane 0.216 <![CDATA[MNBS-C2-NH 2 ]]> PBS 0.020 <![CDATA[MNBS-C6-NH 2 ]]> Dichloromethane 0.377 <![CDATA[MNBS-C6-NH 2 ]]> PBS 0.026

[0091] from Figure 1 It can be seen that in the PBS and dichloromethane system, MNBS-C2-NH 2 and MNBS-C6-NH 2It exhibits excellent optical absorption at 640nm-660nm, has significant fluorescence in dichloromethane, and has a low fluorescence quantum yield in water. In practical applications, it is beneficial for in vivo imaging, can effectively reduce background fluorescence interference, and improve the signal-to-noise ratio and resolution of imaging.

[0092] Test Example 2 Singlet oxygen generation capacity test

[0093] The compound MNBS-C2-NH 2 and MNBS-C6-NH 2 They were added to a quartz dish containing 3 mL of dichloromethane, and a certain amount of DPBF was added to make the absorbance at 415 nm approximately equal to 1. Using near-infrared light (660 nm, 2 mW / cm 2 ) Irradiate the test quartz dish and test the UV absorption every 1 minute. The test results are shown in Figure 2 .

[0094] Figure 2 (a) is MNBS-C2-NH 2 Absorption curve with illumination time; Figure 2 (b) is MNBS-C6-NH 2 Absorption curves with illumination time.

[0095] Depend on Figure 2 It can be seen that under 660 nm light irradiation, the absorbance of the DPBF solution containing the photosensitizer dye at 415 nm decreased rapidly, indicating that the photosensitizer has a good ROS production ability and can be used for subsequent antibacterial treatment.

[0096] Test Example 3 Bacterial uptake test

[0097] MNBS-C2-NH 2 MNBS-C6-NH 2 And comparative example 1MNBS-C0-NH 2 , dripped onto a glass-bottomed confocal cell culture dish and imaged using a single-photon laser scanning confocal microscope, collecting bright field images and fluorescence images of bacteria at different time periods to observe the bacterial uptake of photosensitizers. For test results, see Figure 3 .

[0098] Figure 3 (a) Bacteria take up MNBS-C2-NH 2 Fluorescence signal changes over time; Figure 3 (b) Bacterial uptake of MNBS-C6-NH 2 Fluorescence signal changes over time; Figure 3(c) Bacterial uptake of MNBS-C0-NH 2 Fluorescence signal changes over time. Figure 3 Scale bar is 10 μm.

[0099] from Figure 3 It can be seen that within 0-120 min, MNBS-C2-NH 2 and MNBS-C6-NH 2 The fluorescence signal intensity of Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli gradually increased, indicating that it has good penetration ability for bacteria; while the comparative example 1MNBS-C0-NH 2 The bacterial penetration ability is poor, especially the low fluorescence intensity of Gram-negative bacteria Escherichia coli. This characteristic makes MNBS-C2-NH 2 and MNBS-C6-NH 2 It has broad application potential in bacterial imaging and antibacterial therapy, and can be used to monitor bacterial growth and distribution in real time and improve diagnostic accuracy.

[0100] Test Example 4MNBS-C2-NH 2 and MNBS-C6-NH 2 Analysis of the penetrating ability of the outer membrane protein of Gram-negative bacteria Escherichia coli

[0101] Spermine was used as a gram-negative bacterial membrane protein inhibitor to prepare two groups of gram-negative Escherichia coli bacterial suspensions. One group was pre-added with 20 μM spermine and incubated for 30 minutes, and then the dye was added and incubated for 2 hours to observe the effect of the inhibitor on the uptake of the dye by Escherichia coli.

[0102] like Figure 4 As shown in the figure, there is an obvious red fluorescence signal in the Gram-negative bacteria in the group without inhibitor, while the fluorescence signal is significantly reduced after adding the inhibitor, indicating that the dye MNBS-C2-NH 2 and MNBS-C6-NH 2 It mainly enters Gram-negative bacteria through membrane protein channels. Figure 4 Scale bar is 10 μm. This mechanism enables the dye to efficiently penetrate and accumulate within bacterial cells, enabling it to be used to precisely monitor bacterial distribution and activity, thereby enhancing the efficacy of its fluorescence imaging and antibacterial treatment.

[0103] Test Example 5 Bacterial Phototoxicity and Dark Toxicity

[0104] To evaluate the phototoxicity and dark toxicity of bacteria, 100 μL of bacterial suspension (10 8 ~10 9 CFU / mL) were added to the test sample MNBS-C2-NH 2MNBS-C6-NH 2 , Comparative Example 1MNBS-C0-NH 2 The ampicillin sodium sample solution of Comparative Example 2 was placed in a light-proof incubator for 2 hours, and then the light treatment group was irradiated (640-660nm LED, 30mW cm -2 , 10 minutes). Subsequently, 100 μL of each treated sample was taken, serially diluted 10 times, and the dilutions were plated on LB solid plates for overnight culture, the number of bacterial colonies was counted, and the log CFU of each sample was calculated. The log CFU was recorded as 0 for the colony number less than or equal to 1 on the lowest dilution plate. All experiments were repeated three times.

[0105] Figure 5 (a) is MNBS-C2-NH 2 Changes in log CFU of bacteria under light and dark conditions; Figure 5 (b)MNBS-C6-NH 2 Changes in log CFU of bacteria under light and dark conditions; Figure 5 (c)MNBS-C0-NH 2 Changes in log CFU of bacteria under light and dark conditions; Figure 5 (d)MNBS-C6-NH 2 Changes in log CFU of bacteria under light and dark conditions; Figure 5 (e)MNBS-C2-NH 2 MNBS-C6-NH 2 , Comparative Example 1MNBS-C0-NH 2 And the bacterial plate images of Comparative Example 2, ampicillin sodium, under light and light-proof conditions.

[0106] like Figure 5 As shown, under light-protected conditions, the bacteria 2 MNBS-C6-NH 2 After treatment, the log CFU was basically similar to that of the control group, indicating that the two dyes were less toxic under dark conditions, while after illumination, the log CFU decreased significantly, indicating that the two dyes had high bacterial killing ability under light conditions, while the log CFU of bacteria in the control group of ampicillin decreased less, indicating that the photosensitizer MNBS-C2-NH 2 and MNBS-C6-NH 2 It has excellent bacterial killing ability under light conditions, proving its great potential in photodynamic therapy. It can be used to kill bacteria efficiently, thereby improving the treatment effect.

[0107] Test Example 6 Observation of bacterial damage under different treatment conditions under scanning electron microscope

[0108] 15 mL of overnight cultured bacterial suspension was divided into three groups and centrifuged. The first group was resuspended in physiological saline, and the second group was resuspended in 10 μM dye MNBS-C2-NH 2 The samples were resuspended in saline and incubated at 37°C for 30 min before being illuminated at 660 nm (30 mWcm -2 ) light irradiation for 10 min (L+ group), and the second group was treated with 10 μM dye MNBS-C2-NH 2 The cells were resuspended in saline and incubated at 37°C for 30 min, then placed in the dark for 10 min (L-group). After treatment, the cells were centrifuged, fixed with glutaraldehyde solution for 4 h, gradient eluted with ethanol solutions of different contents, and then dripped onto the ultra-thin carbon film. After drying, the cells were observed by scanning electron microscopy.

[0109] Figure 6 Scale bar is 1 μm.

[0110] like Figure 6 As shown, the bacterial morphology of the saline group and the light-protected group was intact, while the overall morphology of the negative bacteria was significantly damaged after light exposure, and obvious collapse, wrinkling and rupture appeared on the membrane surface, indicating that the photosensitizer had effectively damaged the bacteria.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 Nile blue dye, characterized in that Its structure is shown in general formula I: In the general formula I, X is selected from one of oxygen, sulfur, selenium and tellurium; Y - Selected from halogen ions, ClO4 - 、BF4 - PF6 - One of; R1 is selected from One of the groups; n is a positive integer selected from 1 to 18; R2 and R3 are each independently selected from H, methyl, ethyl, propyl, One of the groups.

2. The method for synthesizing a type of Nile blue dye as claimed in claim 1, characterized in that: The process is as follows: Its synthesis method comprises the following steps: (1) In an aqueous solution, aniline containing an R1 substituent is used as a raw material, added to an aqueous solution of aluminum sulfate, NaS2O3 and ZnCl2 are added in sequence under stirring at low temperature, an oxidant is added dropwise, and after sufficient reaction, a precipitate is obtained by suction filtration, and then purified by recrystallization to obtain a compound represented by formula Z-1; Alternatively, a phenol containing an R1 substituent is added to 5-50 wt% acid, sodium nitrite is slowly added under stirring at low temperature, the mixture is fully reacted, allowed to stand, a precipitate is obtained by suction filtration, and the precipitate is washed to obtain a compound represented by formula Z-2; (2) adding 1-bromonaphthalene, an inorganic base, a catalyst and an amino compound containing R2 and R3 substituents in an organic solvent at 80-150° C., reacting sufficiently, and then reducing pressure, concentrating and purifying to obtain a compound represented by formula Z-3; (3) in an organic solvent at 0-80°C, the compound represented by formula Z-1 or formula Z-2 and the compound represented by formula Z-3 are oxidized by an oxidant or catalyzed by a base to form a ring, and then - The compound shown in Formula I is obtained by treating with an acid or salt, concentrating and purifying.

3. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that: In step (1), the molar ratio of aniline containing R1 substituent, aluminum sulfate, NaS2O3, ZnCl2 and oxidant is 1:1-2:1-3:1-2:0.1-2; in step (1), the molar ratio of phenol containing R1 substituent and sodium nitrite is 1:1-3.

4. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that: In step (1), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate and potassium chlorate; in step (1), the acid is selected from at least one of hydrochloric acid, sulfuric acid, hydrobromic acid and acetic acid.

5. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that: In step (2), the molar ratio of 1-bromonaphthalene, catalyst, inorganic base and amino compound containing R2 and R3 substituents is 1:0.05-0.6:1-3:1-5.

6. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that: In step (2), the organic solvent is selected from one or a combination of ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, ethylene glycol monobutyl ether, propylene glycol methyl ether, butanediol, ethanol, acetonitrile, and ethyl acetate; in step (2), the inorganic base is selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, cesium carbonate, sodium carbonate, and sodium ethoxide; in step (2), the catalyst is selected from at least one of cuprous iodide, cuprous bromide, cuprous chloride, cuprous oxide, cupric sulfate, cupric sulfate pentahydrate, cupric oxide, and cupric chloride.

7. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that: In step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the compound represented by formula Z-3 is 1:0.5-3; In step (3), the molar ratio of the compound represented by formula Z-1 or formula Z-2 to the oxidant or base is 1:0.5-5.

8. The method for synthesizing a type of Nile blue dye according to claim 2, characterized in that: In step (3), the organic solvent is selected from one or a combination of methanol, ethanol, isopropanol, petroleum ether, ethyl acetate, glycerol, dichloromethane, DMF and a mixed solvent; In step (3), the oxidant is selected from at least one of potassium dichromate, potassium permanganate, potassium perchlorate, potassium chlorate, and hydrogen peroxide; In step (3), the base is selected from at least one of silver carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium ethoxide, sodium hydroxide, sodium perchlorate, potassium perchlorate, sodium hexafluorophosphate, sodium fluoroborate, and triethylamine; In step (3), the Y-containing - The acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid, perchloric acid, fluoroboric acid and hexafluorophosphoric acid.

9. The use of a type of Nile blue dye according to claim 1, characterized in that: The applications include spectroscopy for diagnosis and treatment of non-diseases, bacterial imaging, and photodynamic therapy.

10. The use according to claim 9, characterized in that: The applications include fluorescent imaging of bacteria and antimicrobial treatment.

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