CN114671813A - Photosensitizer with activities of fluorescence imaging and photodynamic killing of gram-positive bacteria and preparation method and application thereof - Google Patents

Photosensitizer with activities of fluorescence imaging and photodynamic killing of gram-positive bacteria and preparation method and application thereof Download PDF

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CN114671813A
CN114671813A CN202210400915.6A CN202210400915A CN114671813A CN 114671813 A CN114671813 A CN 114671813A CN 202210400915 A CN202210400915 A CN 202210400915A CN 114671813 A CN114671813 A CN 114671813A
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王建国
姜国玉
龚建业
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Abstract

The invention relates to the technical field of biochemical materials, and provides a photosensitizer with activities of fluorescence imaging and photodynamic killing of gram-positive bacteria, and a preparation method and application thereof. The photosensitizer provided by the invention has good luminous capacity, the quaternary ammonium salt group in the molecular structure of the photosensitizer enables molecules to have electropositivity, the surface of bacteria is electronegativity, and the photosensitizer can be effectively combined with gram-positive bacteria due to electrostatic interaction, so that fluorescence imaging of the gram-positive bacteria is realized; in addition, the photosensitizer provided by the invention also has strong active oxygen species generation capacity, and can carry out high-efficiency photodynamic killing on gram-positive bacteria; meanwhile, the photosensitizer provided by the invention has no toxicity to normal cells, and can be used for constructing a bacterial infection diagnosis and treatment reagent with diagnosis and treatment functions. The preparation method of the photosensitizer provided by the invention has simple steps and is easy to operate.

Description

Photosensitizer with activities of fluorescence imaging and photodynamic killing of gram-positive bacteria and preparation method and application thereof
Technical Field
The invention relates to the technical field of biochemical materials, in particular to a photosensitizer with the activities of fluorescence imaging and photodynamic gram-positive bacteria killing and a preparation method and application thereof.
Background
Bacterial infections are one of the most serious health problems worldwide, causing millions of people to become sick each year, which poses a serious threat to global public health. Antibiotics have been widely used to treat bacterial infections since 1928 where penicillin was found to be useful. However, due to the clinical abuse of antibiotics in recent decades, drug-resistant bacteria and even multiple drug-resistant strains have emerged and spread widely, which impair the therapeutic effect of antibiotics, resulting in high mortality and serious waste of medical resources. The world health organization warns that in the near future, we will enter a post-antibiotic age where common infections can lead to a significant number of morbidity and mortality. Therefore, the development of efficient drug-resistant bacterial infection treatment drugs has extremely important significance and application value.
Photodynamic therapy (PDT) is a clinical treatment that uses Photosensitizers (PSs) and light of appropriate wavelength to generate Reactive Oxygen Species (ROS) that oxidize and damage surrounding biomolecules, thereby killing cancer cells or pathogenic microorganisms. At present, the research on photodynamic therapy mainly focuses on tumor treatment, and the research on photodynamic antibacterial is less, but the photodynamic therapy gradually draws attention because it is not easy to induce bacteria to generate drug resistance. The fluorescence imaging technology has the advantages of simple operation, high sensitivity, low detection limit, application to in vivo imaging and the like. In the main development of the photodynamic therapy, the fluorescence-mediated photodynamic therapy can simultaneously carry out real-time monitoring and efficient killing, and has wide application prospect. Therefore, the development of the photosensitizer with fluorescence imaging capability and killing capability is of great significance. The literature reports some photosensitizers which can be used for photodynamic antibacterial, but few photosensitizers have the capability of fluorescence imaging and sterilization.
Disclosure of Invention
In view of the above, the present invention provides a photosensitizer with both fluorescence imaging and photodynamic gram-positive bacteria killing activity, and a preparation method and an application thereof. The photosensitizer provided by the invention has good luminous capacity and can perform fluorescence imaging on gram-positive bacteria; meanwhile, the photosensitizer has strong active oxygen species generation capacity and can carry out high-efficiency photodynamic killing on gram-positive bacteria.
In order to achieve the above object, the present invention provides the following technical solutions:
a photosensitizer with both fluorescence imaging and photodynamic gram-positive bacteria killing activities has a structure shown in a formula I or a formula II:
Figure BDA0003600137440000021
in the formulas I to II, R is hydrogen, alkyl, alkoxy, N-dimethyl, N-diethyl or N, N-diphenyl.
Preferably, the number of carbon atoms of the alkyl group and the alkoxy group is independently 1 to 6.
The invention also provides a preparation method of the photosensitizer in the scheme, and when the photosensitizer has a structure shown in the formula I, the preparation method comprises the following steps:
(1-1) mixing a compound with a structure shown in a formula III with 3, 6-dichloropyridazine, a palladium catalyst and inorganic base to perform a Suzuki reaction to obtain a compound with a structure shown in a formula IV;
Figure BDA0003600137440000022
(1-2) mixing the compound with the structure shown in the formula IV and N, N-dimethylethylenediamine for substitution reaction to obtain the compound with the structure shown in the formula V.
Figure BDA0003600137440000023
(1-3) mixing the compound with the structure shown in the formula V and methyl iodide for salt forming reaction to obtain a photosensitizer with the structure shown in the formula I;
when the photosensitizer has a structure shown in formula II, the preparation method comprises the following steps:
(2-1) mixing the compound with the structure shown in the formula III, 3, 6-dichloropyridazine-1-oxygen, a palladium catalyst and an inorganic base, and carrying out a Suzuki reaction under a protective atmosphere to obtain the compound with the structure shown in the formula VI.
Figure BDA0003600137440000031
(2-2) mixing the compound with the structure shown in the formula VI and N, N-dimethylethylenediamine for substitution reaction to obtain a compound with a structure shown in the formula VII.
Figure BDA0003600137440000032
(2-3) mixing the compound with the structure shown in the formula VII and methyl iodide for salt forming reaction to obtain the photosensitizer with the structure shown in the formula II.
Preferably, the mol ratio of the compound with the structure shown in the formula III, the 3, 6-dichloropyridazine, the palladium catalyst and the inorganic base in the step (1-1) is (1.1-1.5): 1, (0.03-0.08): 2-4;
the mol ratio of the compound with the structure shown in the formula III, 3, 6-dichloropyridazine-1-oxygen, the palladium catalyst and the inorganic base in the step (2-1) is (1.1-1.5): 1, (0.03-0.08): 2-4.
Preferably, the palladium catalyst in the step (1-1) and the step (2-1) is independently [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex or tetrakis (triphenylphosphine) palladium, and the inorganic base is independently potassium phosphate or potassium carbonate.
Preferably, the temperature of the Suzuki reaction in the step (1-1) and the step (2-1) is independently 50 to 100 ℃.
Preferably, the molar ratio of the compound with the structure shown in the formula IV to the N, N-dimethylethylenediamine in the step (1-2) is 1 (10-20);
the molar ratio of the compound with the structure shown in the formula VI in the step (2-2) to the N, N-dimethylethylenediamine is 1 (10-20).
Preferably, the temperature of the substitution reaction in the step (1-2) and the step (2-2) is independently 50 to 80 ℃.
Preferably, the molar ratio of the compound with the structure shown in the formula V to the methyl iodide in the step (1-3) is 1 (0.7-1.2);
the molar ratio of the compound with the structure shown in the formula VII to the methyl iodide in the step (2-3) is 1 (3-5);
the temperature of the salt forming reaction in the step (1-3) and the step (2-3) is 80-130 ℃.
The invention also provides application of the photosensitizer in the scheme in preparation of diagnosis and treatment reagents for killing gram-positive bacteria by photodynamic and non-treatment-purpose fluorescence imaging of the gram-positive bacteria.
The invention provides a photosensitizer with the activities of fluorescence imaging and photodynamic gram-positive bacteria killing, which has a structure shown in a formula I or a formula II. The photosensitizer provided by the invention has good luminous capacity, the quaternary ammonium salt group in the molecular structure of the photosensitizer enables molecules to have electropositivity, the surface of bacteria is electronegativity, and the photosensitizer can be effectively combined with gram-positive bacteria due to electrostatic interaction, so that fluorescence imaging of the gram-positive bacteria is realized; in addition, the photosensitizer provided by the invention also has strong active oxygen species generation capacity, and can carry out high-efficiency photodynamic killing on gram-positive bacteria; meanwhile, the photosensitizer provided by the invention has no toxicity to normal cells, and can be used for constructing a bacterial infection diagnosis and treatment reagent with diagnosis and treatment functions.
The invention also provides a preparation method of the photosensitizer in the scheme, and the preparation method provided by the invention is simple in steps and easy to operate.
Drawings
FIG. 1 is an absorption spectrum of 20. mu.M DAPD-O in dimethyl sulfoxide solution;
FIG. 2 is a fluorescence spectrum of 20. mu.M DAPD-O in dimethyl sulfoxide solution;
FIG. 3 is a graph showing the particle size distribution of DAPD-O aggregates in a dimethylsulfoxide/water mixed solution having a water volume fraction of 99%;
FIG. 4 is an inverted fluorescence micrograph of DAPD-O after co-incubation with Staphylococcus aureus;
FIG. 5 is an inverted fluorescence micrograph of DAPD-O after co-incubation with methicillin-resistant Staphylococcus aureus;
FIG. 6 shows the change of fluorescence spectrum of mixed solution of DAPD-O and active oxygen species scavenger DCFH with illumination time;
FIG. 7 shows the change of fluorescence intensity at 530nm with illumination time of a mixed solution of DAPD-O and a reactive oxygen species scavenger DCFH;
FIG. 8 shows the photodynamic killing of Staphylococcus aureus by different concentrations of DAPD-O;
FIG. 9 is an inverted fluorescence micrograph of live and dead bacteria when DAPD-O (10. mu.M) is stained with a commercial live bacteria probe Calcein-AM and a commercial dead bacteria probe PI for photodynamic killing of Staphylococcus aureus;
FIG. 10 is a graph of photodynamic killing of methicillin-resistant Staphylococcus aureus with different concentrations of DAPD-O;
FIG. 11 is an inverted fluorescence micrograph of live and dead bacteria when DAPD-O (10. mu.M) is stained with a commercial live bacteria probe Calcein-AM and a commercial dead bacteria probe PI for photodynamic killing of methicillin-resistant Staphylococcus aureus;
FIG. 12 shows cytotoxicity of different concentrations of DAPD-O after 40min incubation with NIH3T3 cells.
Detailed Description
The invention provides a photosensitizer with both fluorescence imaging and photodynamic gram-positive bacteria killing activity, which has a structure shown in a formula I or a formula II:
Figure BDA0003600137440000051
in the formulas I to II, R is hydrogen, alkyl, alkoxy, N-dimethyl, N-diethyl or N, N-diphenyl.
In the present invention, the number of carbon atoms of the alkyl group and the alkoxy group is independently preferably 1 to 6, more preferably 2 to 5, specifically, the alkyl group is preferably a methyl group or an ethyl group, and the alkoxy group is preferably a methoxy group or an ethoxy group.
The invention also provides a preparation method of the photosensitizer in the scheme, which specifically comprises a preparation method of the photosensitizer shown in the formula I and a preparation method of the photosensitizer shown in the formula II, and the preparation methods are respectively explained below.
In the present invention, when the photosensitizer has a structure represented by formula I, the preparation method includes the steps of:
(1-1) mixing a compound with a structure shown in a formula III with 3, 6-dichloropyridazine, a palladium catalyst and inorganic base to perform a Suzuki reaction to obtain a compound with a structure shown in a formula IV;
Figure BDA0003600137440000061
(1-2) mixing the compound with the structure shown in the formula IV and N, N-dimethylethylenediamine for substitution reaction to obtain the compound with the structure shown in the formula V.
Figure BDA0003600137440000062
(1-3) mixing the compound with the structure shown in the formula V and methyl iodide for salt forming reaction to obtain the photosensitizer with the structure shown in the formula I.
The compound with the structure shown in the formula III is mixed with 3, 6-dichloropyridazine, a palladium catalyst, inorganic base and an organic solvent, and the Suzuki reaction is carried out in a protective atmosphere to obtain the compound with the structure shown in the formula IV. In the present invention, the source of the compound having the structure shown in formula III is not particularly limited, and the compound having the structure shown in formula III can be prepared by a method known to those skilled in the art, and specifically, the compound having the structure shown in formula III can be prepared by referring to international patent application (WO2016/136425a 1).
In the present invention, the palladium catalyst is preferably [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex or tetrakis (triphenylphosphine) palladium; the inorganic base is preferably potassium phosphate or potassium carbonate; the mol ratio of the compound with the structure shown in the formula III to the 3, 6-dichloropyridazine, the palladium catalyst and the inorganic base is preferably (1.1-1.5) to 1 (0.03-0.08) to (2-4), and more preferably 1.2:1:0.05: 3.
In the present invention, the Suzuki reaction is preferably carried out in an organic solvent, and the kind of the organic solvent is not particularly limited in the present invention, and an organic solvent suitable for the Suzuki reaction, which is well known to those skilled in the art, may be used, such as tetrahydrofuran. The invention has no special requirements on the dosage of the organic solvent, and can ensure that the reaction is smoothly carried out.
In the invention, the temperature of the Suzuki reaction is preferably 50-100 ℃, more preferably 50-80 ℃, and most preferably 65 ℃; the Suzuki reaction is preferably carried out under stirring conditions and protective atmosphere, the stirring speed is not specially limited, and the Suzuki reaction can be uniformly stirred; the protective atmosphere is not particularly limited, and the method can be carried out in a conventional protective atmosphere, such as a nitrogen atmosphere or an inert gas atmosphere; the Suzuki reaction time is not particularly limited in the present invention, and is preferably monitored by TLC plate (i.e. thin-layer chromatography dot plate) known in the art until 3, 6-dichloropyridazine or 3, 6-dichloropyridazine-1-oxygen disappears, and in the specific embodiment of the present invention, the Suzuki reaction time is preferably 16 h.
After the Suzuki reaction is completed, the invention preferably further comprises post-treatment of the Suzuki product liquid, and the post-treatment preferably comprises the following steps:
mixing the product liquid obtained by the Suzuki reaction with a saturated ammonium chloride aqueous solution for extraction, and concentrating the obtained organic phase to obtain a concentrate;
and carrying out column chromatography on the concentrate to obtain the compound with the structure shown in the formula IV.
In the invention, the extracting agent for extraction is preferably dichloromethane, the extraction times are preferably 3 times, and organic phases obtained by 3 times of extraction are combined; the volume ratio of dichloromethane to saturated aqueous ammonium chloride solution used for each extraction is preferably 1: 1; the eluent for column chromatography is preferably a mixed solution of petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate in the mixed solution is preferably 5: 1. After the column chromatography is finished, the solvent in the column chromatography product is preferably removed, so that the compound with the structure shown in the formula IV is obtained. The solvent removal method is not particularly limited, and a conventional solvent removal method, such as rotary evaporation, can be adopted.
After the compound with the structure shown in the formula IV is obtained, the compound with the structure shown in the formula IV and N, N-dimethylethylenediamine are mixed for substitution reaction to obtain the compound with the structure shown in the formula V. In the invention, the molar ratio of the compound with the structure shown in the formula IV to N, N-dimethylethylenediamine is preferably 1 (10-20), and more preferably 1: 15.
In the invention, the temperature of the substitution reaction is preferably 50-80 ℃, more preferably 50-70 ℃, and most preferably 60 ℃. The time of the substitution reaction is not particularly limited, and the reaction is preferably monitored by a TLC plate until the compound having the structure shown in formula IV completely disappears; in a specific embodiment of the present invention, the time of the substitution reaction is preferably 8 h.
After the substitution reaction, the method preferably further comprises performing column chromatography on a product liquid obtained by the substitution reaction to obtain the compound with the structure shown in the formula V. In the present invention, the eluent for column chromatography is preferably a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixed solution is preferably 15: 1. After the column chromatography is finished, the solvent in the column chromatography product is preferably removed by using a rotary evaporator to obtain the compound with the structure shown in the formula V.
After the compound with the structure shown in the formula V is obtained, the compound with the structure shown in the formula V, methyl iodide and an organic solvent are mixed for salt forming reaction, and the photosensitizer with the structure shown in the formula I is obtained. In the invention, the molar ratio of the compound with the structure shown in the formula V to methyl iodide is preferably 1 (0.7-1.2), and more preferably 1: 1; the temperature of the salt forming reaction is preferably 80-130 ℃, more preferably 90-110 ℃, and most preferably 90 ℃; the salt-forming reaction is preferably carried out in a protective atmosphere, the kind of the protective atmosphere is not particularly limited, and a conventional protective atmosphere, such as a nitrogen atmosphere or an inert gas atmosphere, may be used. In the invention, the salt forming reaction is preferably carried out in an organic solvent, the organic solvent is preferably acetonitrile, and the invention has no special requirement on the using amount of the organic solvent and can ensure that the reaction can be smoothly carried out.
After the salt-forming reaction is completed, the invention preferably cools the product liquid obtained by the salt-forming reaction to room temperature, then adds the mixed solution of methanol and ethyl acetate for recrystallization, and then filters to obtain the photosensitizer with the structure of formula I. In the present invention, the volume ratio of methanol to ethyl acetate in the mixed solution of methanol and ethyl acetate is preferably 1: 5; the filtration is preferably suction filtration. According to the invention, the recrystallized product obtained by suction filtration is preferably dried to obtain the pure photosensitizer with the structure shown in the formula I.
In the present invention, when the photosensitizer has a structure represented by formula II, the preparation method includes the steps of:
(2-1) mixing the compound with the structure shown in the formula III, 3, 6-dichloropyridazine-1-oxygen, a palladium catalyst and an inorganic base for carrying out Suzuki reaction to obtain the compound with the structure shown in the formula VI.
Figure BDA0003600137440000091
(2-2) mixing the compound with the structure shown in the formula VI and N, N-dimethylethylenediamine for substitution reaction to obtain a compound with a structure shown in the formula VII.
Figure BDA0003600137440000092
(2-3) mixing the compound with the structure shown in the formula VII and methyl iodide for salt forming reaction to obtain the photosensitizer with the structure shown in the formula II.
In the present invention, in the step (2-1), the reaction conditions and post-treatment conditions of the Suzuki reaction, and the kinds, amounts, etc. of the palladium catalyst and the inorganic base are the same as those in the step (1-1), and only 3, 6-dichloropyridazine in the step (1-1) is replaced with 3, 6-dichloropyridazine-1-oxygen; the source of the 3, 6-dichloropyridazine-1-oxygen is not particularly limited, and the preparation method is adopted by a method known by a person skilled in the art, and the preparation method can be specifically prepared by referring to Org.Lett.2016,18, 5142-5145-.
In the present invention, in the step (2-2), the reaction conditions, post-treatment conditions and the amount of N, N-dimethylethylenediamine used in the substitution reaction may be the same as those in the step (1-2), and the compound having the structure represented by formula IV in the step (1-2) may be replaced with the compound having the structure represented by formula VI.
In the present invention, in the step (2-3), the conditions other than the amount of potassium iodide are the same as those in the step (1-3), and only the compound having the structure represented by the formula V in the step (1-3) is replaced with the compound having the structure represented by the formula VII; in the step (2-3), the molar ratio of the compound having the structure shown in the formula VII to methyl iodide is 1 (3-5), and more preferably 1: 4.
In the above technical solutions, the types of R groups in formulas III to VII are the same as those in formulas I to II, and are not described herein again.
The invention also provides application of the photosensitizer in the scheme in non-treatment-purpose fluorescence imaging of gram-positive bacteria and preparation of diagnosis and treatment reagents for killing gram-positive bacteria through photodynamic. In summary of the present invention, the gram-positive bacteria are preferably staphylococcus aureus and methicillin-resistant staphylococcus aureus; the diagnosis and treatment reagent is a reagent with diagnosis and treatment functions.
The photosensitizer with the activities of fluorescence imaging and photodynamic gram-positive bacteria killing is of a donor-receptor structure, and forms a push-pull electron effect so that the photosensitizer has good luminous capacity; the quaternary ammonium salt group contained in the photosensitizer structure enables the whole compound to be electropositive, the surface of bacteria to be electronegative, and the photosensitizer can be effectively combined with gram-positive bacteria due to electrostatic interaction, so that a good foundation is laid for subsequent fluorescence imaging and photodynamic killing of the bacteria; after a photosensitizer and gram-positive bacteria are incubated together, the bacteria are lightened under the irradiation of a certain excitation source, and the fluorescence imaging of the bacteria is realized; meanwhile, the photosensitizer has strong active oxygen species generation capacity, the photosensitizer is mixed with the active oxygen species capture agent, and the signal change of the active oxygen species capture agent is obvious under the irradiation of white light, which indicates that a large amount of active oxygen species are generated; the photosensitizer is used for photodynamic antibiosis, and experimental results show that the photosensitizer can efficiently kill staphylococcus aureus and methicillin-resistant staphylococcus aureus; in addition, the photosensitizer provided by the invention has good biocompatibility, has no obvious phototoxicity and dark toxicity on normal cells, and can be used for constructing a bacterial infection diagnosis and treatment reagent integrating diagnosis and treatment functions.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Preparing a photosensitizer with a structure shown in a formula II, wherein R is hydrogen, wherein the synthesis route is as follows, raw materials are respectively marked as compounds 1 to 4 according to marks in the synthesis route, and products are marked as DAPO-O:
Figure BDA0003600137440000111
(1) compound 1(1g, 6mmol), compound 2(2.67g, 7.2mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (245mg, 0.3mmol), potassium phosphate (3.82g, 18mmol) and tetrahydrofuran were mixed, refluxing at 65 ℃ under the protection of nitrogen, monitoring the reaction process by using a TLC plate until the compound 1 completely disappears, mixing the obtained reaction solution with saturated ammonium chloride aqueous solution dichloromethane for extraction, separating an organic phase, extracting the obtained water phase twice with dichloromethane, combining the obtained organic phases, purifying with silica gel column chromatography, eluting with petroleum ether and ethyl acetate (volume ratio of petroleum ether to ethyl acetate is 5:1), then the solvent in the obtained column chromatography product is dried in a spinning mode to obtain 1.5g of yellow solid, and the calculated yield is 67%;
the obtained yellow solid was characterized, and the specific data are as follows:
1HNMR(600MHz,CDCl3),δ(ppm):7.83(dt,J＝13.4,4.1Hz,2H),7.78(d,J＝12.8Hz,1H),7.38–7.31(m,5H),7.18–7.08(m,8H).
according to the above characterization data, the obtained yellow solid has the structure shown in compound 3.
(2) Mixing compound 3(375mg, 1mmol) and N, N-dimethylethylenediamine (1320mg, 15mmol), refluxing at 60 ℃, monitoring by TLC plate until compound 3 disappears, purifying the reaction system by silica gel column chromatography using dichloromethane and methanol (the volume ratio of dichloromethane to methanol is 15:1) as eluent, and then spin-drying the solvent in the obtained column chromatography product to obtain 320mg of yellow-green solid with a calculated yield of 75%;
the obtained yellow-green solid was characterized, and the specific data are as follows:
1HNMR(500MHz,CDCl3)δ7.78(d,J＝8.8Hz,2H),7.43(d,J＝8.8Hz,1H),7.30(d,J＝8.1Hz,3H),7.15–7.06(m,9H),7.01(d,J＝8.9Hz,1H),6.85(t,J＝5.0Hz,1H),3.38(dd,J＝11.1,5.5Hz,2H),2.68(t,J＝5.4Hz,2H),2.34(s,6H).13CNMR(126MHz,CDCl3)δ148.92,147.28,146.16,144.74,129.38,128.10,127.00,124.96,123.50,122.70,116.37,113.02,57.55,45.28,39.95.HRMS(ESI):m/z:[M+H]+calcdfor[C26H27N5O+H]+:426.22884；found:426.22925.
according to the above characterization data, the obtained yellowish green solid has a structure shown in compound 4.
(3) Mixing compound 4(108mg, 0.25mmol), methyl iodide (142mg, 1mmol) and acetonitrile, refluxing at 90 ℃ under the protection of nitrogen, and monitoring by TLC plate until compound 4 disappears to complete salt-forming reaction; then adding a mixed solution of methanol and ethyl acetate for recrystallization, wherein the volume ratio of the added mixed solution of methanol and ethyl acetate is 1:5, and then carrying out suction filtration to obtain 112mg of yellow-green solid, wherein the calculated yield is 78%;
the obtained yellow-green solid was characterized by the following specific data:
1HNMR(500MHz,MeOD)δ7.85(t,J＝9.3Hz,3H),7.57(d,J＝8.9Hz,1H),7.33(t,J＝7.8Hz,4H),7.12(d,J＝8.3Hz,6H),7.07(d,J＝8.7Hz,2H),3.95(t,J＝5.8Hz,2H),3.68(t,J＝6.5Hz,2H),3.28(s,9H).13CNMR(126MHz,MeOD)δ149.57,147.48,147.16,144.05,129.20,126.74,124.97,124.89,123.67,121.79,119.00,115.68,63.92,52.83,35.85.HRMS(ESI):m/z:[M-I]+calcdfor[C27H30N5O]+:440.24449；found:440.24315.
according to the above characterization data, the obtained yellow-green solid is DAPD-O.
And (3) performance testing:
(1) luminous power test of photosensitizers
A20. mu.M solution of DAPD-O was prepared by adding a 20. mu.M solution of LDAPD-O in dimethyl sulfoxide (2mM) to 2mL of dimethyl sulfoxide, and the absorption spectrum and fluorescence spectrum of the obtained DAPD-O solution were measured.
FIG. 1 is an absorption spectrum of 20. mu.M DAPD-O in dimethyl sulfoxide solution; FIG. 2 shows the fluorescence spectrum of 20. mu.M DAPD-O in dimethyl sulfoxide solution. As can be seen from FIG. 1 and FIG. 2, DAPD-O has the maximum absorption at 400nm, and under the irradiation of the excitation light at 400nm, DAPD-O has the fluorescence emission at 490nm and has the Stokes shift of 90nm, which indicates that DAPD-O has good luminous capacity.
(2) Aggregate size testing of photosensitizers
A20. mu.M DAPD-O solution was obtained by adding a 20. mu.M DMSO solution (2mM) of LDAPD-O to a 2mM PBS solution (5mM, pH 7.4), and the particle size distribution of the aggregates in the obtained DAPD-O solution was determined.
FIG. 3 is a graph showing the particle size distribution of DAPD-O aggregates in a mixed dimethyl sulfoxide/water solution having a water volume fraction of 99%. As can be seen from FIG. 3, the particle size distribution of the DAPD-O aggregates in the mixed solution was concentrated around 160 nm.
(3) Fluorescence imaging ability test of photosensitizer for staphylococcus aureus
The staphylococcus aureus strain is revived, inoculated into LB liquid culture medium and cultured for 16h in a shaking table at 37 ℃ and 220r/min, and the concentration of the staphylococcus aureus in the obtained bacterial suspension is about 2 multiplied by 109CFU/mL; 1mL of cultured Staphylococcus aureus was washed 2 times (1 mL each) with PBS (5mM, pH 7.4); 100 μ L of the bacterial suspension was added to 900 μ L of LPBS (in this case, the concentration of Staphylococcus aureus was 2X 10)8CFU/mL), adding 10 mu L of DMSO solution (2mM) of LDAPD-O, incubating for 2h in a shaking table at 37 ℃ and 220r/min, taking 2 mu L of bacterial suspension slices, observing under an inverted fluorescence microscope, taking pictures under bright field and fluorescence field by using 405nm as an excitation light source, and taking pictures as experimental groups. Following the above procedure, the only difference was that no DAPD-O was added, and then photographs were taken in bright and fluorescent fields as blanks.
FIG. 4 is an inverted fluorescence micrograph of DAPD-O after co-incubation with Staphylococcus aureus. As can be seen from FIG. 4, Staphylococcus aureus was "lighted" after co-incubation with DAPD-O, and exhibited a strong fluorescence signal, but no fluorescence signal was observed in the blank group, indicating that DAPD-O can perform fluorescence imaging on Staphylococcus aureus.
(4) Fluorescence imaging capability test of photosensitizer to methicillin-resistant staphylococcus aureus
Inoculating methicillin-resistant Staphylococcus aureus strain into LB liquid culture medium after resuscitation, and culturing in shaking table at 37 deg.C and 220r/min for 16h to obtain bacterial suspension with methicillin-resistant Staphylococcus aureus concentration of 2 × 109CFU/mL; 1mL of methicillin-resistant Staphylococcus aureus obtained by culturing was washed 2 times (1 mL each time) with a PBS solution (5mM, pH 7.4); 100 μ L of the bacterial suspension was added to 900 μ L of LPBS (at a concentration of 2X 10 methicillin-resistant Staphylococcus aureus)8CFU/mL), 10. mu.L of LDAPD-O in DMSO (2mM) at 37 ℃ at 220r/minAfter incubation for 2h in a shaking table, 2 μ L of the bacterial suspension is sliced and observed under an inverted fluorescence microscope, and pictures are taken under a bright field and a fluorescence field by using 405nm as an excitation light source to serve as an experimental group. Following the above procedure, the only difference was that no DAPD-O was added, and then photographs were taken in bright and fluorescent fields as blanks.
FIG. 5 is an inverted fluorescence micrograph of DAPD-O after co-incubation with methicillin-resistant Staphylococcus aureus. As can be seen from FIG. 5, methicillin-resistant Staphylococcus aureus was "lighted up" after co-incubation with DAPD-O, and showed a strong fluorescence signal, but no fluorescence signal was present in the blank group, indicating that DAPD-O can perform fluorescence imaging on methicillin-resistant Staphylococcus aureus.
(5) Active oxygen species generating capability test of photosensitizer
A20 mu LDAPD-O dimethyl sulfoxide solution (2mM) was added to a 2mM PBS solution (pH7.4, 5mM), an active oxygen species-capturing agent 2',7' -Dichlorodihydrofluorescein (DCFH) was added to the solution, the fluorescence spectrum of the resulting mixed solution at 500 to 600nm was measured using 489nm wavelength light as an excitation light source, and then the mixed solution was illuminated with light using a solar simulator equipped with a 400nm filter, and the change in fluorescence spectrum of the mixed solution at 500 to 600nm with the illumination time was measured.
FIG. 6 shows the change of fluorescence spectrum of mixed solution of DAPD-O and active oxygen species scavenger DCFH with illumination time; FIG. 7 shows the change of fluorescence intensity at 530nm of the mixed solution of DAPD-O and active oxygen species scavenger DCFH with the time of illumination. As can be seen from FIGS. 6 and 7, the fluorescence intensity of the active oxygen species scavenger DCFH gradually increases and eventually stabilizes with the increase of the illumination time, indicating that DAPD-O has the active oxygen species generating ability and is a potential photodynamic antibacterial photosensitizer.
(6) Test of photodynamic killing capability of DAPD-O to staphylococcus aureus
Reviving staphylococcus aureus strain, inoculating into LB liquid culture medium, culturing at 37 deg.C and 220r/min for 16h in shaking table to obtain bacterial suspension with staphylococcus aureus concentration of about 2 × 109CFU/mL; collecting 1mL of cultured golden yellow grape ballBacteria, remove medium, dilute to 2X 10 with PBS7CFU/mL; adding DMSO solutions (2mM) of DAPD-O with different volumes to ensure that the concentration of DAPD-O is 0 muM, 2 muM, 4 muM, 6 muM, 8 muM and 10 muM in sequence, incubating the bacterial suspension in a shaking table with the temperature of 37 ℃ and the speed of 220r/min for 30min, and then carrying a 400nm optical filter by using a solar simulator to illuminate the bacterial suspension for 40 min; after the illumination is finished, the bacterial suspension is diluted by 1 multiplied by 10 in a concentration gradient way5Doubling and spreading on LB agar solid culture medium; colony counts were performed after 16h of incubation in an incubator at 37 ℃ and the survival rate was calculated as a phototoxic group. The dark toxicity group was obtained by the above method except that the light treatment was not performed, and then the culture and counting were performed.
FIG. 8 shows the photodynamic killing of Staphylococcus aureus by different concentrations of DAPD-O. As can be seen from figure 8, the survival rate of staphylococcus aureus in phototoxic group is rapidly reduced with the increase of DAPD-O concentration, the survival rate is less than 1% at 10 mu M, although the survival rate of staphylococcus aureus in dark toxic group is reduced, the survival rate is still maintained above 60%, and the photodynamic killing effect of DAPD-O on staphylococcus aureus is obvious.
The method comprises the following steps of staining live and dead bacteria when 10 mu MDAPD-O (Methylococcus aureus) is subjected to photodynamic killing by using a commercial live bacteria probe Calcein-AM (green fluorescence) and a commercial dead bacteria probe PI (red fluorescence), and then carrying out inverted fluorescence microscopic imaging, wherein the steps are as follows: 1mL of Staphylococcus aureus cultured as described above was washed 2 times (1 mL each) with PBS (5mM, pH 7.4), and diluted to 2X 10 with PBS7Adding 5 mu L of DAPD-O DMSO solution (2mM) into CFU/mL, incubating the bacterial suspension in a shaking table at 37 ℃ and 220r/min for 30min, and carrying a 400nm optical filter by using a solar simulator to illuminate the bacterial suspension for 40 min; different commercial probes were added, and observed under an inverted fluorescence microscope, and photographed under a fluorescence field as an experimental group. Following the above procedure, except that no DAPD-O was added, observations were made and photographed as a blank.
FIG. 9 is an inverted fluorescence micrograph of live and dead bacteria when DAPD-O (10. mu.M) is stained with a commercial live bacteria probe Calcein-AM and a commercial dead bacteria probe PI for photodynamic killing of Staphylococcus aureus; as can be seen from FIG. 9, the fluorescence signals of the commercially viable bacterial probe Calcein-AM in the phototoxicity pictures of the experimental group are almost none, and are the fluorescence signals of the commercially dead bacterial probe PI, that is, the bacteria are all dead, which indicates that the DAPD-O photodynamic action can kill Staphylococcus aureus with high efficiency, and is consistent with the results of FIG. 8.
(7) Test of photodynamic killing ability of DAPD-O to methicillin-resistant staphylococcus aureus
Inoculating methicillin-resistant Staphylococcus aureus strain into LB liquid culture medium after resuscitation, and culturing in shaking table at 37 deg.C and 220r/min for 16h to obtain bacterial suspension with methicillin-resistant Staphylococcus aureus concentration of 2 × 109CFU/mL; taking 1mL of methicillin-resistant Staphylococcus aureus obtained by culture, removing the culture medium, and diluting to 2 × 10 with PBS7CFU/mL; adding DMSO solutions (2mM) of DAPD-O with different volumes to ensure that the concentration of DAPD-O is 0 muM, 2 muM, 4 muM, 6 muM, 8 muM and 10 muM in sequence, incubating the bacterial suspension in a shaking table with the temperature of 37 ℃ and the speed of 220r/min for 30min, and then carrying a 400nm optical filter by using a solar simulator to illuminate the bacterial suspension for 40 min; after the illumination is finished, the bacterial suspension is diluted by 1 multiplied by 10 in a concentration gradient way5Doubling and spreading on LB agar solid culture medium; colony counts were performed after 16h of incubation in an incubator at 37 ℃ and the survival rate was calculated as a phototoxic group. The dark toxicity group was obtained by the above method except that the light treatment was not performed, and then the culture and counting were performed.
FIG. 10 is a graph of the photodynamic killing of methicillin-resistant Staphylococcus aureus by various concentrations of DAPD-O. As can be seen from FIG. 10, with the increase of the concentration of DAPD-O, the survival rate of methicillin-resistant Staphylococcus aureus in phototoxic group is rapidly reduced, the survival rate is less than 1% at 10 μ M, the survival rate of methicillin-resistant Staphylococcus aureus in dark toxic group is reduced, but the survival rate is still maintained above 60%, which shows that the photodynamic killing effect of DAPD-O on methicillin-resistant Staphylococcus aureus is obvious.
10 μ MDAPD-O photodynamic killing of Staphylococcus aureus was stained with commercial live bacterial probe Calcein-AM (Green fluorescence) and commercial dead bacterial probe PI (Red fluorescence)Live and dead bacteria, and then inverted fluorescence microscopy imaging is carried out, and the specific steps are as follows: collecting 1mL of methicillin-resistant Staphylococcus aureus cultured as above, washing with PBS solution (5mM, pH 7.4) for 2 times (1 mL each time), and diluting to 2 × 10 with PBS7Adding 5 mu L of DAPD-O DMSO solution (2mM) into CFU/mL, incubating the bacterial suspension in a shaking table at 37 ℃ and 220r/min for 30min, and carrying a 400nm optical filter by using a solar simulator to illuminate the bacterial suspension for 40 min; different commercial probes were added, and observed under an inverted fluorescence microscope, and photographed under a fluorescence field as an experimental group. Following the above procedure, except that no DAPD-O was added, observations were made and photographed as a blank.
FIG. 11 is an inverted fluorescence micrographs of live and dead bacteria when 10 μ M DPD-O is stained with a commercial live bacteria probe Calcein-AM (Green fluorescence) and a commercial dead bacteria probe PI (Red fluorescence) for photodynamic killing of methicillin-resistant Staphylococcus aureus; as can be seen from FIG. 11, the fluorescence signals of the commercially viable bacterial probe Calcein-AM in the phototoxicity pictures of the experimental group are almost none, and are the fluorescence signals of the commercially dead bacterial probe PI, that is, the bacteria are all dead, which indicates that the DAPD-O photodynamic action can efficiently kill methicillin-resistant Staphylococcus aureus, and the results are consistent with those in FIG. 10.
(8) Cytotoxicity assay of DAPD-O
NIH3T3 cells were adherent cells, cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37 deg.C and 5% CO2The constant temperature incubator is used for culture. Cultured NIH3T3 cells at 5X 10/well3The density of individual cells was seeded in 96-well plates and incubated for 12 h. Then incubating the cells and DAPD-O with different concentrations in a fresh culture medium for 30min, carrying the cells on a 400nm optical filter by using a solar simulator, irradiating for 40min, and simultaneously carrying out dark toxicity group dark protection treatment. After further incubation for 24h, the medium was removed and washed 3 times with PBS. Then, the cells were incubated with fresh serum-free medium containing 10% CCK-8 in the dark for 1h, and finally, absorbance detection was performed with a microplate reader at a wavelength of 450 nm.
FIG. 12 shows cytotoxicity of various concentrations of DAPD-O after 40min incubation with NIH3T3 cells. As can be seen from FIG. 12, even at the maximum concentration, DAPD-O had no significant dark toxicity or phototoxicity to NIH3T3 cells, indicating that the photosensitizer DAPD-O was good in biocompatibility.
Example 2
Preparing a photosensitizer having a structure shown in formula II, wherein R is methoxy, under the same conditions as in example 1, except for replacing compound 2 with the following compound:
Figure BDA0003600137440000171
the nuclear magnetism of the final product showed that the structure of the final product was as follows:
Figure BDA0003600137440000172
example 3
A photosensitizer having a structure represented by formula II, in which R is N, N-dimethyl, was prepared under the same conditions as in example 1 except that compound 2 was replaced with the following compound:
Figure BDA0003600137440000181
the nuclear magnetism of the final product shows that the structure of the final product is as follows:
Figure BDA0003600137440000182
example 4
Preparing a photosensitizer having a structure shown in formula II, wherein R is N, N-diphenyl, under the same conditions as in example 1, except for replacing compound 2 with the following compound:
Figure BDA0003600137440000183
the nuclear magnetism of the final product shows that the structure of the final product is as follows:
Figure BDA0003600137440000191
the photosensitizer obtained in the embodiment 2 to 4 is tested for luminous capacity, particle size distribution, fluorescence imaging capacity, active oxygen species generation capacity, staphylococcus aureus and methicillin-resistant staphylococcus aureus photodynamic killing capacity and cytotoxicity according to the method of the embodiment 1, and the obtained result is similar to that of the embodiment 1, which indicates that the photosensitizer prepared in the embodiment 2 to 4 has good luminous capacity and photodynamic sterilization capacity and good biocompatibility.
According to the embodiment, the photosensitizer with the fluorescence imaging and the photodynamic gram positive bacteria killing activity has the advantages of simple synthesis steps, easiness in operation, good luminous capacity and capability of performing fluorescence imaging on staphylococcus aureus and methicillin-resistant staphylococcus aureus in gram positive bacteria; the photosensitizer also has strong active oxygen species generating capacity, and can carry out high-efficiency photodynamic killing on staphylococcus aureus and methicillin-resistant staphylococcus aureus; meanwhile, the reagent has no toxicity to normal cells, and can be used for constructing a bacterial infection diagnosis and treatment reagent with diagnosis and treatment functions.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A photosensitizer with both fluorescence imaging and photodynamic gram-positive bacteria killing activities is characterized by having a structure shown in a formula I or a formula II:
Figure FDA0003600137430000011
in the formulas I to II, R is hydrogen, alkyl, alkoxy, N-dimethyl, N-diethyl or N, N-diphenyl.
2. The photosensitizer according to claim 1, wherein the alkyl group and the alkoxy group independently have 1 to 6 carbon atoms.
3. The process for preparing the photosensitizer of claim 1 or 2, wherein when the photosensitizer has the structure represented by formula I, the process comprises the steps of:
(1-1) mixing a compound with a structure shown in a formula III, 3, 6-dichloropyridazine, a palladium catalyst and an inorganic base for a Suzuki reaction to obtain a compound with a structure shown in a formula IV;
Figure FDA0003600137430000012
(1-2) mixing the compound with the structure shown in the formula IV and N, N-dimethylethylenediamine for substitution reaction to obtain the compound with the structure shown in the formula V.
Figure FDA0003600137430000013
(1-3) mixing the compound with the structure shown in the formula V and methyl iodide for salt forming reaction to obtain a photosensitizer with the structure shown in the formula I;
when the photosensitizer has a structure shown in formula II, the preparation method comprises the following steps:
(2-1) mixing the compound with the structure shown in the formula III, 3, 6-dichloropyridazine-1-oxygen, a palladium catalyst and an inorganic base, and carrying out a Suzuki reaction under a protective atmosphere to obtain the compound with the structure shown in the formula VI.
Figure FDA0003600137430000021
(2-2) mixing the compound with the structure shown in the formula VI and N, N-dimethylethylenediamine for substitution reaction to obtain a compound with a structure shown in the formula VII.
Figure FDA0003600137430000022
(2-3) mixing the compound with the structure shown in the formula VII and methyl iodide for salt forming reaction to obtain the photosensitizer with the structure shown in the formula II.
4. The method according to claim 3, wherein the molar ratio of the compound having the structure represented by formula III, 3, 6-dichloropyridazine, the palladium catalyst and the inorganic base in the step (1-1) is (1.1-1.5): 1, (0.03-0.08): 2-4;
the mol ratio of the compound with the structure shown in the formula III, 3, 6-dichloropyridazine-1-oxygen, the palladium catalyst and the inorganic base in the step (2-1) is (1.1-1.5): 1, (0.03-0.08): 2-4.
5. The production method according to claim 3, wherein the palladium catalyst in the step (1-1) and the step (2-1) is independently [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex or tetrakis (triphenylphosphine) palladium, and the inorganic base is independently potassium phosphate or potassium carbonate.
6. The method according to claim 3, 4 or 5, wherein the temperature of the Suzuki reaction in the step (1-1) and the step (2-1) is independently 50 to 100 ℃.
7. The method according to claim 3, wherein the molar ratio of the compound having the structure represented by formula IV to N, N-dimethylethylenediamine in the step (1-2) is 1 (10-20);
the molar ratio of the compound with the structure shown in the formula VI in the step (2-2) to the N, N-dimethylethylenediamine is 1 (10-20).
8. The method according to claim 3 or 7, wherein the temperature of the substitution reaction in the step (1-2) and the step (2-2) is independently 50 to 80 ℃.
9. The preparation method according to claim 3 or 7, wherein the molar ratio of the compound having the structure represented by the formula V to methyl iodide in the step (1-3) is 1 (0.7-1.2);
the molar ratio of the compound with the structure shown in the formula VII to the methyl iodide in the step (2-3) is 1 (3-5);
the temperature of the salt forming reaction in the step (1-3) and the step (2-3) is 80-130 ℃.
10. The use of the photosensitizer of claim 1 or 2 for the preparation of diagnostic reagents for photodynamic killing of gram-positive bacteria and for non-therapeutic purposes of fluorescence imaging of gram-positive bacteria.
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CN116715636A (en) * 2023-08-04 2023-09-08 内蒙古大学 Anion-pi type fluorescent probe and preparation method and application thereof
CN116715636B (en) * 2023-08-04 2023-12-05 内蒙古大学 Anion-pi type fluorescent probe and preparation method and application thereof

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