CN114671813B - Photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities as well as preparation method and application thereof - Google Patents
Photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biochemical materials, and provides a photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities, and a preparation method and application thereof. The photosensitizer provided by the invention has good luminous capacity, quaternary ammonium salt groups in the molecular structure enable molecules to have electropositivity, and the surface of bacteria is electronegative, and due to electrostatic interaction, the photosensitizer can be effectively combined with gram-positive bacteria, so that fluorescence imaging of the gram-positive bacteria is realized; in addition, the photosensitizer provided by the invention has strong active oxygen species generation capability, 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
Technical Field
The invention relates to the technical field of biochemical materials, in particular to a photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities, a preparation method and application thereof.
Background
Bacterial infections are one of the most serious health problems worldwide, causing millions of people to become ill each year, which poses a serious threat to global public health. Antibiotics have been widely used to treat bacterial infections since the discovery in 1928 of penicillins useful to treat bacterial infections. However, due to the clinical abuse of antibiotics in recent decades, resistant bacteria and even multi-resistant strains of bacteria have emerged and spread widely, which impair the therapeutic efficacy 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 era that common infections may lead to significant morbidity and mortality. Therefore, the development of the efficient drug-resistant bacterial infection treatment drug has extremely important significance and application value.
Photodynamic therapy (hotodynamic therapy, PDT) is a clinical treatment method that uses Photosensitizers (PSs) and appropriate wavelengths of light-generated reactive oxygen species (Reactive oxygen species, ROS) to oxidize biomolecules surrounding the lesion, thereby killing cancer cells or pathogenic microorganisms. At present, the research of photodynamic therapy is mainly focused on the aspect of tumor treatment, and the research of photodynamic antibacterial is less, but the photodynamic therapy is gradually attracting attention because the photodynamic therapy 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, and the like, and can be used for living body imaging. In the main progress of photodynamic therapy, fluorescence-mediated photodynamic therapy can simultaneously carry out real-time monitoring and efficient killing, and has wide application prospect. Therefore, it is of great importance to develop photosensitizers that have both fluorescence imaging and killing capabilities. At present, some photosensitizers which can be used for photodynamic antibiosis are reported in the literature, but few photosensitizers which have fluorescence imaging and sterilization capabilities are available.
Disclosure of Invention
In view of the above, the invention provides a photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities, and a preparation method and application thereof. The photosensitizer provided by the invention has good luminous capacity and can carry out fluorescence imaging on gram-positive bacteria; meanwhile, the photosensitizer has strong capability of generating active oxygen species, 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 fluorescence imaging and photodynamic gram-positive bacteria killing activities, which has a structure shown in a formula I or a formula II:
in the formulas I-II, R is hydrogen, alkyl, alkoxy, N-dimethyl, N-diethyl or N, N-diphenyl.
Preferably, the alkyl and alkoxy groups independently have 1 to 6 carbon atoms.
The invention also provides a preparation method of the photosensitizer, which is characterized in that when the photosensitizer has a structure shown in a 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 for carrying out a Suzuki reaction to obtain a compound with a structure shown in a formula IV;
(1-2) mixing the compound having the structure shown in the formula IV with N, N-dimethylethylenediamine to perform substitution reaction, thereby obtaining the compound having the structure shown in the formula V.
(1-3) mixing the compound with the structure shown in the formula V with methyl iodide to carry out salt forming reaction to obtain a photosensitizer with the structure shown in the formula I;
when the photosensitizer has a structure shown in a formula II, the preparation method comprises the following steps:
(2-1) mixing a compound having a structure represented by formula III with 3, 6-dichloropyridazine-1-oxide, a palladium catalyst and an inorganic base, and carrying out a Suzuki reaction in a protective atmosphere to obtain a compound having a structure represented by formula VI.
(2-2) mixing the compound having the structure shown in the formula VI with N, N-dimethylethylenediamine to perform substitution reaction, thereby obtaining a compound having the structure shown in the formula VII.
(2-3) mixing the compound with the structure shown in the formula VII with methyl iodide to carry out salt forming reaction, thus obtaining the photosensitizer with the structure shown in the formula II.
Preferably, the molar ratio of the compound with the structure shown in the formula III, 3, 6-dichloropyridazine, palladium catalyst and inorganic base in the step (1-1) is (1.1-1.5): 1 (0.03-0.08): 2-4);
the molar ratio of the compound with the structure shown in the formula III in the step (2-1), 3, 6-dichloropyridazine-1-oxygen, palladium catalyst and inorganic base is (1.1-1.5): 1 (0.03-0.08): 2-4).
Preferably, the palladium catalyst in step (1-1) and step (2-1) is independently [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride 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 in the step (1-2) to N, N-dimethyl ethylenediamine is 1 (10-20);
the molar ratio of the compound with the structure shown in the formula VI in the step (2-2) to N, N-dimethyl ethylenediamine 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 in the step (1-3) to methyl iodide is 1 (0.7-1.2);
the molar ratio of the compound with the structure shown in the formula VII in the step (2-3) to methyl iodide is 1 (3-5);
the temperature of the salification reaction in the step (1-3) and the step (2-3) is 80-130 ℃.
The invention also provides application of the photosensitizer in preparation of diagnosis and treatment reagents for killing gram-positive bacteria by photodynamic and non-therapeutic purposes and in fluorescence imaging of gram-positive bacteria.
The invention provides a photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities, which has a structure shown in a formula I or a formula II. The photosensitizer provided by the invention has good luminous capacity, quaternary ammonium salt groups in the molecular structure enable molecules to have electropositivity, and the surface of bacteria is electronegative, and due to electrostatic interaction, the photosensitizer can be effectively combined with gram-positive bacteria, so that fluorescence imaging of the gram-positive bacteria is realized; in addition, the photosensitizer provided by the invention has strong active oxygen species generation capability, 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, which 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 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-hatching with Staphylococcus aureus;
FIG. 5 is an inverted fluorescence micrograph of DAPD-O co-hatched with methicillin-resistant Staphylococcus aureus;
FIG. 6 shows the fluorescence spectrum of a mixed solution of DAPD-O and an active oxygen species scavenger DCFH as a function of illumination time;
FIG. 7 shows the change of fluorescence intensity at 530nm with time of illumination of a mixed solution of DAPD-O and active oxygen species scavenger DCFH;
FIG. 8 is a graph showing photodynamic killing of Staphylococcus aureus by DAPD-O at various concentrations;
FIG. 9 is an inverted fluorescence microscopy image of live and dead bacteria when DAPD-O (10. Mu.M) is photodynamic killed against Staphylococcus aureus with the commercial live bacterial probe Calcein-AM and commercial dead bacterial probe PI;
FIG. 10 is a graph showing photodynamic killing of methicillin-resistant Staphylococcus aureus by different concentrations of DAPD-O;
FIG. 11 is an inverted fluorescence microscopy image of live and dead bacteria when DAPD-O (10. Mu.M) was photodynamic killed against methicillin-resistant Staphylococcus aureus with the commercial live bacterial probe Calcein-AM and commercial dead bacterial probe PI;
FIG. 12 shows cytotoxicity after 40min incubation of DAPD-O with NIH3T3 cells at different concentrations.
Detailed Description
The invention provides a photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities, which has a structure shown in a formula I or a formula II:
in the formulas I-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, and specifically, the alkyl group is preferably methyl or ethyl, and the alkoxy group is preferably methoxy or ethoxy.
The invention also provides a preparation method of the photosensitizer, which is specifically provided by the scheme, and specifically comprises a preparation method of the photosensitizer shown in a formula I and a preparation method of the photosensitizer shown in a formula II, and the preparation methods are respectively described below.
In the present invention, when the photosensitizer has a structure represented by formula I, the preparation method comprises 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 for carrying out a Suzuki reaction to obtain a compound with a structure shown in a formula IV;
(1-2) mixing the compound having the structure shown in the formula IV with N, N-dimethylethylenediamine to perform substitution reaction, thereby obtaining the compound having the structure shown in the formula V.
(1-3) mixing the compound with the structure shown in the formula V with methyl iodide to carry out salt forming reaction, thus obtaining the photosensitizer with the structure shown in the formula I.
The method mixes the compound with the structure shown in the formula III with 3, 6-dichloropyridazine, a palladium catalyst, inorganic base and an organic solvent, and carries out Suzuki reaction in 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 is prepared by methods 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 (WO 2016/136425 A1).
In the present invention, the palladium catalyst is preferably [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex or tetrakis (triphenylphosphine) palladium; the inorganic base is preferably potassium phosphate or potassium carbonate; the molar 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): 1 (0.03-0.08): 2-4, 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, and an organic solvent suitable for carrying out the Suzuki reaction, such as tetrahydrofuran, which is well known to those skilled in the art, may be used. The invention has no special requirement on the dosage of the organic solvent, and can lead the reaction to be carried out smoothly.
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 particularly limited, and the Suzuki reaction can be uniformly stirred; the protective atmosphere is not particularly limited, and the protective atmosphere can be performed in a conventional protective atmosphere, such as a nitrogen atmosphere or an inert gas atmosphere; the time of the Suzuki reaction is not particularly limited, and the reaction is preferably monitored by a TLC plate (i.e., thin layer chromatography spot plate) well known in the art until 3, 6-dichloropyridazine or 3, 6-dichloropyridazine-1-oxygen completely disappears, and in the specific embodiment of the present invention, the time of the Suzuki reaction is preferably 16 hours.
After the completion of the Suzuki reaction, the present invention preferably further comprises post-treatment of the obtained Suzuki product liquid, said post-treatment preferably comprising the steps of:
mixing the product feed 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;
subjecting the concentrate to column chromatography to obtain a compound having a structure shown in formula IV.
In the present invention, the extractant for extraction is preferably methylene chloride, the number of times of extraction is preferably 3, and organic phases obtained by the 3 times of extraction are combined; the volume ratio of dichloromethane to saturated ammonium chloride aqueous solution used in 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 petroleum ether to ethyl acetate in the mixed solution is preferably 5:1. After completion of the column chromatography, the present invention preferably removes the solvent from the column chromatography product to obtain a compound having a structure represented by formula IV. The solvent removal method is not particularly limited, and a conventional solvent removal method, such as spin evaporation, may be used.
After obtaining the compound with the structure shown in the formula IV, the invention mixes the compound with the structure shown in the formula IV with N, N-dimethyl ethylenediamine to carry out substitution reaction, thus obtaining the compound with the structure shown in the formula V. In the present invention, the molar ratio of the compound having the structure represented by formula IV to N, N-dimethylethylenediamine is preferably 1 (10 to 20), more preferably 1:15.
In the present invention, the temperature of the substitution reaction is preferably 50 to 80 ℃, more preferably 50 to 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 with the structure shown in the formula IV completely disappears; in a specific embodiment of the present invention, the time for the substitution reaction is preferably 8 hours.
After the substitution reaction, the present invention preferably further comprises subjecting the product feed liquid obtained by the substitution reaction to column chromatography to obtain a compound having a structure represented by 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 completion of the column chromatography, the present invention preferably removes the solvent from the column chromatography product by a rotary evaporator to obtain a compound having a structure represented by formula V.
After obtaining the compound with the structure shown in the formula V, the invention mixes the compound with the structure shown in the formula V, methyl iodide and an organic solvent for salifying reaction to obtain the photosensitizer with the structure shown in the formula I. In the present invention, the molar ratio of the compound having the structure represented by formula V to methyl iodide is preferably 1 (0.7 to 1.2), more preferably 1:1; the temperature of the salification reaction is preferably 80-130 ℃, more preferably 90-110 ℃, and most preferably 90 ℃; the salt formation reaction is preferably carried out in a protective atmosphere, and the type 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 present invention, the salt-forming reaction is preferably carried out in an organic solvent, preferably acetonitrile, and the amount of the organic solvent is not particularly limited, so that the reaction can be smoothly carried out.
After the salification reaction is completed, the product liquid obtained by the salification reaction is preferably cooled to room temperature, then mixed liquid of methanol and ethyl acetate is added for recrystallization, and then the photosensitizer with the structure of formula I is obtained after filtration. In the invention, the volume ratio of the methanol to the ethyl acetate in the mixed liquid of the methanol and the ethyl acetate is preferably 1:5; the filtration is preferably suction filtration. The invention preferably dries the recrystallized product obtained by suction filtration 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 comprises the steps of:
(2-1) mixing a compound having a structure represented by formula III with 3, 6-dichloropyridazine-1-oxide, a palladium catalyst and an inorganic base to perform a Suzuki reaction, thereby obtaining a compound having a structure represented by formula VI.
(2-2) mixing the compound having the structure shown in the formula VI with N, N-dimethylethylenediamine to perform substitution reaction, thereby obtaining a compound having the structure shown in the formula VII.
(2-3) mixing the compound with the structure shown in the formula VII with methyl iodide to carry out salt forming reaction, thus obtaining the photosensitizer with the structure shown in the formula II.
In the invention, in the step (2-1), the reaction conditions, the post-treatment conditions, the types, the amounts and the like of the palladium catalyst and the inorganic base of the Suzuki reaction are the same as those in the step (1-1), and only the 3, 6-dichloropyridazine in the step (1-1) is replaced by 3, 6-dichloropyridazine-1-oxygen; the source of the 3, 6-dichloropyridazine-1-oxide is not particularly limited, and the 3, 6-dichloropyridazine-1-oxide may be prepared by a method known to those skilled in the art, and the 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, the post-treatment conditions and the amount of N, N-dimethylethylenediamine used in the substitution reaction are the same as those in the step (1-2), and the compound having the structure represented by the formula IV in the step (1-2) may be replaced with the compound having the structure represented by the formula VI.
In the invention, in the step (2-3), except the dosage of potassium iodide, the other conditions are the same as those in the step (1-3), and only the compound with the structure shown in the formula V in the step (1-3) is replaced by the compound with the structure shown in the formula VII; in the step (2-3), the molar ratio of the compound having the structure represented by formula VII to methyl iodide is 1 (3-5), more preferably 1:4.
In the above technical scheme, the R groups in the formulas III to VII are the same as those in the formulas I to II, and are not described in detail herein.
The invention also provides application of the photosensitizer in fluorescence imaging of gram-positive bacteria for non-therapeutic purposes and preparation of diagnosis and treatment reagents for photodynamic killing of gram-positive bacteria. In summary of the invention, the gram positive bacteria are preferably staphylococcus aureus and methicillin-resistant staphylococcus aureus; the diagnosis and treatment reagent refers to a reagent with diagnosis and treatment functions.
The photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities is of a donor-acceptor structure, and forms a push-pull electronic effect so that the photosensitizer has good luminous capacity; the quaternary ammonium salt group contained in the photosensitizer structure makes the whole compound positive and the surface of the bacteria negative, 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 the photosensitizer and gram-positive bacteria are hatched together, the bacteria are lightened under the irradiation of a certain excitation source, so that fluorescence imaging of the bacteria is realized; meanwhile, the photosensitizer has strong active oxygen species generation capability, and the signal change of the active oxygen species capturing agent is obvious when the photosensitizer is mixed with the active oxygen species capturing agent under the irradiation of white light, so 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 be used for carrying out high-efficiency photodynamic killing on 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 to normal cells, and can be used for constructing a bacterial infection diagnosis and treatment reagent with diagnosis and treatment functions.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The photosensitizer with the structure shown in the formula II, wherein R is hydrogen, is prepared by the following synthetic route, the raw materials are respectively marked as a compound 1 to a compound 4 according to marks in the synthetic route, and the products are marked as DAPO-O:
(1) Compound 1 (1 g,6 mmol), compound 2 (2.67 g,7.2 mmol), [1,1' -bis (diphenylphosphorus) ferrocene ] palladium dichloride dichloromethane complex (245 mg,0.3 mmol), potassium phosphate (3.82 g,18 mmol) and tetrahydrofuran were mixed, refluxed at 65 ℃ under the protection of nitrogen, the progress of the reaction was monitored by TLC plate until compound 1 completely disappeared, the obtained reaction liquid was mixed with saturated ammonium chloride aqueous solution dichloromethane for extraction, the organic phase was separated, the obtained aqueous phase was extracted twice with dichloromethane, the obtained organic phases were combined, purified by silica gel column chromatography using petroleum ether and ethyl acetate (volume ratio of petroleum ether and ethyl acetate was 5:1), then the solvent in the obtained column chromatography product was dried by spin to obtain yellow solid 1.5g, the calculated yield was 67%;
the yellow solid obtained was characterized and the specific data are as follows:
1 HNMR(600MHz,CDCl 3 ),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).
from the above characterization data, the yellow solid obtained is the structure shown in compound 3.
(2) Compound 3 (375 mg,1 mmol) and N, N-dimethylethylenediamine (560 mg,15 mmol) were mixed, refluxed at 60 ℃, monitored by TLC plate until compound 3 disappeared, the reaction system was purified by silica gel column chromatography using dichloromethane and methanol (volume ratio of dichloromethane to methanol: 15:1), and then the solvent in the resulting column chromatography product was spin-dried to give 320mg of yellow-green solid, calculated yield was 75%;
the resulting yellowish green solid was characterized as follows:
1 HNMR(500MHz,CDCl 3 )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). 13 CNMR(126MHz,CDCl 3 )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[C 26 H 27 N 5 O+H] + :426.22884；found:426.22925.
from the above characterization data, the resulting yellowish green solid was the structure shown in compound 4.
(3) Compound 4 (108 mg,0.25 mmol), methyl iodide (142 mg,1 mmol) and acetonitrile were mixed, refluxed at 90 ℃ under the protection of nitrogen, monitored by TLC plate until compound 4 disappeared, and the salification reaction was completed; 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, and the calculated yield is 78%;
the resulting yellowish green solid was characterized as follows:
1 HNMR(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). 13 CNMR(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[C 27 H 30 N 5 O] + :440.24449；found:440.24315.
from the above characterization data, the resulting yellowish green solid was DAPD-O.
Performance test:
(1) Light-emitting capability test of photosensitizer
To 2mL of dimethyl sulfoxide was added 20. Mu.L of APD-O in dimethyl sulfoxide (2 mM), 20. Mu.M of DAPD-O solution was obtained, 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 is a fluorescence spectrum of 20. Mu.M DAPD-O in dimethyl sulfoxide solution. As can be seen from FIGS. 1 and 2, DAPD-O has a maximum absorption at 400nm, and under the irradiation of 400nm excitation light, DAPD-O has fluorescence emission at 490nm, and has a Stokes shift of 90nm, which indicates that DAPD-O has a good luminescence capability.
(2) Aggregate particle size testing of photosensitizers
To a 2mM LPBS solution (5 mM, pH 7.4) was added a DMSO solution (2 mM) of 20. Mu.L APD-O to obtain a 20. Mu.M DAPD-O solution, and the particle size distribution of aggregates in the obtained DAPD-O solution was measured.
FIG. 3 is a particle size distribution of DAPD-O aggregates in a dimethylsulfoxide/water mixed solution having a water volume fraction of 99%. As is clear from FIG. 3, the particle size distribution of DAPD-O aggregates in the mixed solution was concentrated around 160 nm.
(3) Fluorescent imaging capability test of photosensitizer on staphylococcus aureus
Culturing Staphylococcus aureus strain in LB liquid medium after resuscitating and inoculating, at 37deg.C, in a shaking table of 220r/min for 16 hr to obtain bacterial suspension with concentration of Staphylococcus aureus of about 2×10 9 CFU/mL; 1mL of the cultured staphylococcus aureus was washed with PBS (5 mM, pH 7.4) for 2 times (1 mL each); 100. Mu.L of the bacterial suspension was added to 900. Mu.LPBS (at which time the concentration of Staphylococcus aureus was 2X 10) 8 CFU/mL), 10 mu L of DMSO solution (2 mM) of LDAPD-O was added, incubated at 37℃for 2 hours in a shaker at 220r/min, and 2 mu L of the bacterial suspension was pelletedObserving under a fluorescence microscope, and taking pictures under a bright field and a fluorescence field by using 405nm as an excitation light source to serve as an experimental group. According to the above method, only the difference is that DAPD-O is not added, and then photographing is performed under the bright field and the fluorescent field as a blank group.
FIG. 4 is an inverted fluorescence micrograph of DAPD-O after co-hatching with Staphylococcus aureus. As can be seen from FIG. 4, staphylococcus aureus was "lit" after co-hatching with DAPD-O, exhibiting a strong fluorescent signal, while no fluorescent signal was present in the blank, indicating that DAPD-O could image the Staphylococcus aureus.
(4) Fluorescent imaging capability test of photosensitizer on methicillin-resistant staphylococcus aureus
Inoculating methicillin-resistant Staphylococcus aureus strain into LB liquid medium, and culturing at 37deg.C in 220r/min shaking table for 16 hr to obtain bacterial suspension with methicillin-resistant Staphylococcus aureus concentration of about 2×10 9 CFU/mL; taking 1mL of methicillin-resistant staphylococcus aureus obtained by culture, and washing with PBS solution (5 mM, pH value is 7.4) for 2 times (1 mL each time); 100. Mu.L of the bacterial suspension was added to 900. Mu.LPBS (at which time the methicillin-resistant Staphylococcus aureus concentration was 2X 10) 8 CFU/mL), 10 μl of DMSO solution (2 mM) of LDAPD-O was added, incubated in a shaker at 37 ℃ for 2 hours at 220r/min, 2 μl of the bacterial suspension was taken for observation under an inverted fluorescence microscope, and the resultant was photographed under bright field and fluorescent field using 405nm as an excitation light source, as an experimental group. According to the above method, only the difference is that DAPD-O is not added, and then photographing is performed under the bright field and the fluorescent field as a blank group.
FIG. 5 is an inverted fluorescence micrograph of DAPD-O co-hatched with methicillin-resistant Staphylococcus aureus. As can be seen from fig. 5, methicillin-resistant staphylococcus aureus was "lit" after co-hatching with DAPD-O, exhibiting a strong fluorescent signal, whereas no fluorescent signal was present in the blank group, indicating that DAPD-O can perform fluorescent imaging on methicillin-resistant staphylococcus aureus.
(5) Reactive oxygen species production capability test of photosensitizer
A dimethyl sulfoxide solution (2 mM) of 20 mu LDAPD-O was added to a 2mLPBS solution (pH 7.4,5 mM), active oxygen species scavenger 2',7' -Dichlorofluorescein (DCFH) was added to the above solution, the fluorescence spectrum of the obtained mixed solution at 500 to 600nm was measured using light of 489nm wavelength as an excitation light source, then the mixed solution was irradiated with light using a 400nm filter mounted on a solar simulator, and the change of the fluorescence spectrum of the mixed solution at 500 to 600nm with the irradiation time was measured.
FIG. 6 shows the fluorescence spectrum of a mixed solution of DAPD-O and an active oxygen species scavenger DCFH as a function of illumination time; FIG. 7 shows the change of fluorescence intensity at 530nm with time of illumination of a mixed solution of DAPD-O and active oxygen species scavenger DCFH. As can be seen from fig. 6 and 7, the fluorescence intensity of the active oxygen species capturing agent DCFH gradually increases and eventually becomes stable as the illumination time increases, indicating that DAPD-O has the active oxygen species generating capability and is a potential photodynamic antimicrobial photosensitizer.
(6) DAPD-O photodynamic killing ability test for staphylococcus aureus
Culturing Staphylococcus aureus strain in LB liquid medium after resuscitating and inoculating, at 37deg.C, in a shaking table of 220r/min for 16 hr to obtain bacterial suspension with concentration of Staphylococcus aureus of about 2×10 9 CFU/mL; 1mL of the cultured staphylococcus aureus was taken, the medium was removed, and diluted to 2X 10 with PBS 7 CFU/mL; adding DMSO solutions (2 mM) of DAPD-O with different volumes to make the DAPD-O concentration be 0 mu M,2 mu M,4 mu M,6 mu M,8 mu M and 10 mu M in sequence, incubating the bacterial suspension in a shaking table at 37 ℃ and 220r/min for 30min, and carrying out light irradiation on the bacterial suspension by using a solar simulator and carrying out 400nm optical filter for 40min; after the illumination is finished, the concentration of the bacterial suspension is diluted by 1X 10 in a gradient way 5 Doubling and coating on LB agar solid medium; colony counts were performed after 16h incubation in 37℃incubator and survival was calculated as phototoxic group. The above procedure was followed except that no light treatment was performed, and then culture was performed and counted as a dark toxicity group.
FIG. 8 shows photodynamic killing of Staphylococcus aureus by different concentrations of DAPD-O. As can be seen from FIG. 8, the survival rate of the staphylococcus aureus in the phototoxic group is rapidly reduced with the increase of the DAPD-O concentration, the survival rate is less than 1% at 10 mu M, and the survival rate of the staphylococcus aureus in the dark toxic group is reduced, but the survival rate is still maintained above 60%, which shows that the photodynamic killing effect of the DAPD-O on the staphylococcus aureus is obvious.
Live and dead bacteria when 10 mu MDAPD-O was photodynamically killed against Staphylococcus aureus were stained with a commercial live bacterial probe Calcein-AM (green fluorescence) and a commercial dead bacterial probe PI (red fluorescence), and then subjected to inverted fluorescence microscopy imaging as follows: 1mL of Staphylococcus aureus obtained by culturing in the above manner was washed with PBS (5 mM, pH 7.4) for 2 times (1 mL each), and diluted with PBS to 2X 10 7 CFU/mL, adding 5 mu L of DAPD-O DMSO solution (2 mM), incubating the bacterial suspension in a shaker at 37 ℃ and 220r/min for 30min, and carrying out light irradiation on the bacterial suspension for 40min by using a solar simulator carrying a 400nm optical filter; different commercial probes were added separately and then observed under an inverted fluorescence microscope, which was photographed under a fluorescent field as an experimental group. According to the above method, the difference is that DAPD-O is not added, and then observation and photographing are performed as a blank group.
FIG. 9 is an inverted fluorescence microscopy image of live and dead bacteria when DAPD-O (10. Mu.M) is photodynamic killed against Staphylococcus aureus with the commercial live bacterial probe Calcein-AM and commercial dead bacterial probe PI; as can be seen from fig. 9, the fluorescent signals of the commercial live bacterial probe Calcein-AM in the photo-toxicity pictures of the experimental group are almost none, and the fluorescent signals of the commercial dead bacterial probe PI, that is, the bacteria at this time are all in a dead state, which indicates that DAPD-O photodynamic action can kill staphylococcus aureus with high efficiency, and is consistent with the result of fig. 8.
(7) DAPD-O photodynamic killing ability test for methicillin-resistant staphylococcus aureus
Inoculating methicillin-resistant Staphylococcus aureus strain into LB liquid medium, and culturing at 37deg.C in 220r/min shaking table for 16 hr to obtain bacterial suspension with methicillin-resistant Staphylococcus aureus concentration of about 2×10 9 CFU/mL; taking 1mL of methicillin-resistant staphylococcus aureus obtained by culture, removing the culture medium, and diluting with PBS2×10 7 CFU/mL; adding DMSO solutions (2 mM) of DAPD-O with different volumes to make the DAPD-O concentration be 0 mu M,2 mu M,4 mu M,6 mu M,8 mu M and 10 mu M in sequence, incubating the bacterial suspension in a shaking table at 37 ℃ and 220r/min for 30min, and carrying out light irradiation on the bacterial suspension by using a solar simulator and carrying out 400nm optical filter for 40min; after the illumination is finished, the concentration of the bacterial suspension is diluted by 1X 10 in a gradient way 5 Doubling and coating on LB agar solid medium; colony counts were performed after 16h incubation in 37℃incubator and survival was calculated as phototoxic group. The above procedure was followed except that no light treatment was performed, and then culture was performed and counted as a dark toxicity group.
FIG. 10 shows photodynamic killing of methicillin-resistant Staphylococcus aureus by different concentrations of DAPD-O. As can be seen from fig. 10, as the DAPD-O concentration increases, the survival rate of methicillin-resistant staphylococcus aureus in the phototoxic group rapidly decreases, the survival rate is less than 1% at 10 μm, and the survival rate of methicillin-resistant staphylococcus aureus in the dark toxic group decreases, but the survival rate remains above 60%, which indicates that the photodynamic killing effect of DAPD-O on methicillin-resistant staphylococcus aureus is obvious.
Live and dead bacteria when 10 mu MDAPD-O was photodynamically killed against Staphylococcus aureus were stained with a commercial live bacterial probe Calcein-AM (green fluorescence) and a commercial dead bacterial probe PI (red fluorescence), and then subjected to inverted fluorescence microscopy imaging as follows: taking 1mL of methicillin-resistant Staphylococcus aureus obtained by culturing according to the method, washing with PBS solution (5 mM, pH 7.4) for 2 times (1 mL each time), diluting with PBS to 2×10 7 CFU/mL, adding 5 mu L of DAPD-O DMSO solution (2 mM), incubating the bacterial suspension in a shaker at 37 ℃ and 220r/min for 30min, and carrying out light irradiation on the bacterial suspension for 40min by using a solar simulator carrying a 400nm optical filter; different commercial probes were added separately and then observed under an inverted fluorescence microscope, which was photographed under a fluorescent field as an experimental group. According to the above method, the difference is that DAPD-O is not added, and then observation and photographing are performed as a blank group.
FIG. 11 is an inverted fluorescence microscopy image of live and dead bacteria when 10. Mu. MDAPD-O photodynamic killing of methicillin-resistant Staphylococcus aureus was stained with a commercial live bacterial probe Calcein-AM (green fluorescence) and a commercial dead bacterial probe PI (red fluorescence); as can be seen from fig. 11, the fluorescent signals of the commercial live bacterial probe Calcein-AM in the photo-toxicity pictures of the experimental group are almost none, and are the fluorescent signals of the commercial dead bacterial probe PI, that is, the bacteria at the moment are all in a dead state, which indicates that the DAPD-O photodynamic action can kill methicillin-resistant staphylococcus aureus with high efficiency, and the result is consistent with that of fig. 10.
(8) Cytotoxicity test of DAPD-O
NIH3T3 cells are adherent cells obtained by culturing with DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37deg.C and 5% CO 2 Is cultured in a constant temperature incubator. Culturing the obtained NIH3T3 cells at a ratio of 5×10 per well 3 The density of individual cells was seeded on 96-well plates and incubated for 12h. Then incubating the cells with DAPD-O with different concentrations in a fresh culture medium for 30min, irradiating the cells with a 400nm optical filter carried by a solar simulator for 40min, and treating the cells in dark toxicity groups in dark. After further incubation for 24h, the medium was removed and washed 3 times with PBS. Cells were then incubated with fresh serum-free medium containing 10% cck-8 for 1h in the dark and finally absorbance detection was performed with a microplate reader at a wavelength of 450 nm.
FIG. 12 shows cytotoxicity after 40min incubation of DAPD-O with NIH3T3 cells at different concentrations. As can be seen from FIG. 12, even at the maximum concentration, DAPD-O had no significant dark and phototoxicity to NIH3T3 cells, indicating that the photosensitizer DAPD-O was well biocompatible.
Example 2
A photosensitizer having a structure represented by formula II, wherein R is methoxy, was prepared under the same conditions as in example 1, except that compound 2 was replaced with the following compound:
the nuclear magnetism of the final product shows that the structure of the final product is as follows:
example 3
A photosensitizer having a structure represented by formula II, wherein R is N, N-dimethyl, was prepared, except that the same conditions as in example 1 were used, except that compound 2 was replaced with the following compound:
the nuclear magnetism of the final product shows that the structure of the final product is as follows:
example 4
A photosensitizer having a structure represented by formula II, wherein R is N, N-diphenyl, was prepared, except that the same conditions as in example 1 were used, except that compound 2 was replaced with the following compound:
the nuclear magnetism of the final product shows that the structure of the final product is as follows:
the photosensitizers obtained in examples 2 to 4 were tested for their luminescence ability, particle size distribution, fluorescence imaging ability, active oxygen species generating ability, photodynamic killing ability against staphylococcus aureus and methicillin-resistant staphylococcus aureus and cytotoxicity according to the method of example 1, and the obtained results were similar to example 1, showing that the photosensitizers prepared in examples 2 to 4 had good luminescence ability and photodynamic killing ability and good biocompatibility.
As can be seen from the above examples, the photosensitizer with fluorescence imaging and photodynamic killing gram-positive bacteria activity provided by the invention has the advantages of simple synthesis steps, easy operation and good luminous capacity, and can perform fluorescence imaging on staphylococcus aureus and methicillin-resistant staphylococcus aureus in gram-positive bacteria; the photosensitizer also has strong active oxygen species generating capability, and can carry out high-efficiency photodynamic killing on staphylococcus aureus and methicillin-resistant staphylococcus aureus; meanwhile, the kit 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 merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A photosensitizer with fluorescence imaging and photodynamic gram-positive bacteria killing activities, which is characterized by having a structure shown in a formula II:
in the formula II, R is hydrogen, alkyl, alkoxy, N-dimethyl, N-diethyl or N, N-diphenyl.
2. The photosensitizer according to claim 1, characterized in that the number of carbon atoms of said alkyl and alkoxy groups is independently 1 to 6.
3. A method of preparing a photosensitizer according to claim 1 or 2, characterized by comprising the steps of:
(2-1) mixing a compound having a structure shown in formula III with 3, 6-dichloropyridazine-1-oxygen, a palladium catalyst and an inorganic base, and performing a Suzuki reaction in a protective atmosphere to obtain a compound having a structure shown in formula VI;
(2-2) mixing the compound with the structure shown in the formula VI with N, N-dimethyl ethylenediamine to perform substitution reaction to obtain a compound with the structure shown in the formula VII;
(2-3) mixing the compound with the structure shown in the formula VII with methyl iodide to carry out salt forming reaction, thus obtaining the photosensitizer with the structure shown in the formula II.
4. The process according to claim 3, wherein the molar ratio of the compound having the structure represented by formula III, 3, 6-dichloropyridazine-1-oxide, palladium catalyst and inorganic base in step (2-1) is 1.1 to 1.5:1 (0.03 to 0.08) to 2 to 4.
5. The process according to claim 3, wherein the palladium catalyst in the step (2-1) is independently [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex or tetrakis (triphenylphosphine) palladium, and the inorganic base is independently potassium phosphate or potassium carbonate.
6. The process according to claim 3, 4 or 5, wherein the temperature of the Suzuki reaction in step (2-1) is independently 50 to 100 ℃.
7. The process according to claim 3, wherein the molar ratio of the compound having the structure represented by the formula VI in the step (2-2) to N, N-dimethylethylenediamine is 1 (10 to 20).
8. The process according to claim 3 or 7, wherein the temperature of the substitution reaction in step (2-2) is independently 50 to 80 ℃.
9. The process according to claim 3 or 7, wherein the molar ratio of the compound having the structure represented by formula VII to methyl iodide in the step (2-3) is 1 (3-5);
the temperature of the salification reaction in the step (2-3) is 80-130 ℃.
10. Use of a photosensitizer according to claim 1 or 2 for the preparation of a diagnostic reagent for photodynamic killing of gram-positive bacteria and for fluorescence imaging of gram-positive bacteria for non-therapeutic purposes.
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