Method for oxygen-resistant photooxidation reduction catalytic release of CO / NO and application

Abstract

The invention relates to the technical field of single-component CO / NO donor compounds, photocatalysis and photosensitization, and discloses a method for releasing CO / NO through oxygen-resistant photooxidation reduction catalysis and application, carbon monoxide (CO) and nitric oxide (NO) are triggered to be released from a single component under the action of a photooxidation reduction catalyst, and the photooxidation reduction catalyst also serves as a photosensitizer to play a role. The CO release motif and the NO release motif are directly coupled to prepare a single-component molecule containing the CO release motif and the NO release motif. The assembly can release CO and NO at the same time through light stimulation under the normal oxygen condition, and gram-positive and gram-negative bacterial pathogens can be effectively killed.

Description

Technical Field

[0001] The invention relates to the technical field of single-component CO / NO donor compounds, photocatalysis and photosensitization, and in particular to a method and application of releasing CO / NO by oxygen-resistant photooxidation-reduction catalysis. Background Art

[0002] The exogenous GSM systems used for research are mainly single GSM donors, which are involved in key physiological and pathological processes of treatment. However, it is currently hoped that the synergistic therapeutic effects of two or more GSMs can be studied. Some single-component dual-GSM donor molecules have been designed in previous research works, such as ultrasound-stimulated CO / H 2 S release molecules, UV triggers NO / H 2 S 2 and NO / CO releasing molecules, as well as green light triggered NO / CO releasing molecules. Physiological and pathological studies on the synergistic effects of CO and NO basically involve the use of single component GSM donors and donors that release similar ratios of two GSMs. Considering the initial CO release, it is also necessary to study its crosstalk with different ratios of NO. In addition, from the above studies, it is also necessary to use higher wavelength dual donor excitation with low biological toxicity.

[0003] Catalysis plays an important role in the release of GSM. However, it is difficult to achieve photoredox catalysis without adding any oxygen scavenging agents under normal oxygen conditions, especially under complex physiological conditions.

[0004] Therefore, we proposed a method and application of oxygen-resistant photo-redox catalytic release of CO / NO to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of oxygen-resistant photo-redox catalytic release of CO / NO, involving three CO / NO donor molecules and one NO donor molecule. The gas donor molecules can be prepared by covalent bonds and loaded into nano-components through self-assembly together with photocatalysts (photosensitizer molecules); CO / NO is generated under light conditions to kill Gram-positive and Gram-negative bacteria and drug-resistant Gram-negative bacteria.

[0006] To achieve the above object, the present invention provides the following technical solution: CO / NO donor molecules have the structures of formula VI, VII and VIII:

[0007]

[0008] The CO / NO donor molecule of the present invention has an N-nitrosoamine group in the structure as the NO releasing part and an oxygen / thioflavonol as the CO releasing part.

[0009] The present invention provides a NO donor molecule having a structure of Formula IX:

[0010]

[0011] In the NO donor molecule of the present invention, the N-nitrosoamine group in the structure is the NO releasing part, and the caged flavonol part is the non-functionalized CO releasing part.

[0012] The nanoassembly obtained by co-assembling the gas donor molecule and the photocatalyst molecule (photosensitizer molecule) is a platform for triggering CO / NO release by oxygen-resistant red light photoredox catalysis. The photocatalyst molecule (photosensitizer molecule) has the following chemical structural characteristics:

[0013]

[0014] The above-mentioned nanoassembly preparation methods include: organic water mixing, nano flash precipitation method, thin film hydration method, etc. Gas molecules can self-assemble with photocatalyst molecules (photosensitizer molecules) in the presence of block copolymers to prepare nanoassemblies with a particle size of 40-200nm.

[0015] In a preferred embodiment, nano-assemblies loaded with photocatalyst molecules (photosensitizer molecules) and gas donor molecules can be prepared by dissolving the gas donor molecules, photocatalysts (photosensitizer molecules) and block copolymers in an organic solvent (tetrahydrofuran and dimethyl sulfoxide), and adding them into water (such as ultrapure water, Wahaha water) at a certain temperature and stirring speed at one time, and dialyzing to remove the organic solvent to obtain nano-assemblies loaded with gas donors and photocatalyst molecules (photosensitizer molecules).

[0016] In the present invention, the nanoassembly loaded with gas donor and photocatalyst molecule (photosensitizer molecule) can release CO and NO simultaneously under illumination. In the embodiment of the present invention, there is no clear restriction on the illumination conditions, as long as it is within the absorption wavelength range of the photocatalyst (photosensitizer) molecule. There is no clear restriction on the intensity of light, and any illumination condition that excites the photocatalyst (photosensitizer) molecule and does not adversely affect the release performance is acceptable.

[0017] In the present invention, the photocatalyst molecule (photosensitizer molecule) can be used as a type II photosensitizer to generate O under light conditions. 1 2 , O 1 2 It can then react with the CO donor part of the dual donor molecule to release CO, and can also act as a photoredox catalyst to activate the NO donor part of the same dual donor molecule to release NO under the same lighting conditions.

[0018] In the present invention, the photocatalyst molecule (photosensitizer molecule) and the NO donor molecule are assembled to release NO as expected. However, for the non-functionalized (caged) CO donor part, the O generated by the photosensitizer molecule 1 2 Under light conditions, it does not react with the caged CO donor moiety to remove O 1 2 ; hence, it cannot be used as a photoredox catalyst to activate the NO donor part of the same molecule to release NO unless any scavenging agents are used.

[0019] The present invention also provides an application of CO / NO released by the above-mentioned oxygen-resistant light redox catalytic CO / NO release method in killing Gram-positive and Gram-negative bacterial pathogens.

[0020] Compared with the prior art, the beneficial effects of the present invention are: under the action of a photoredox catalyst, carbon monoxide (CO) and nitric oxide (NO) are triggered to be released from a single component, the photoredox catalyst also acts as a photosensitizer, and the CO release motif and the NO release motif are directly coupled to prepare a single-component molecule containing CO and NO release motifs. The assembly can simultaneously release CO and NO through light stimulation under normal oxygen conditions, and can effectively kill Gram-positive and Gram-negative bacterial pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The hydrogen and carbon nuclear magnetic resonance spectra of compound A in Example 1 of the present invention are shown in FIG.

[0022] Figure 2 The hydrogen and carbon nuclear magnetic resonance spectra of compound B in Example 1 of the present invention are shown in FIG.

[0023] Figure 3 The hydrogen and carbon nuclear magnetic resonance spectra of compound C in Example 1 of the present invention are shown in FIG.

[0024] Figure 4 The hydrogen nuclear magnetic resonance spectrum, carbon spectrum and HRMS spectrum of the double donor molecule MCNO in Example 2 of the present invention are shown;

[0025] Figure 5 The hydrogen NMR spectrum, carbon NMR spectrum and HRMS spectrum of the dual donor molecule MHCNO1-1 in Example 3 of the present invention;

[0026] Figure 6 The hydrogen NMR spectrum, carbon NMR spectrum and HRMS spectrum of the dual donor molecule MHCNO1-2 in Example 4 of the present invention are shown;

[0027] Figure 7 The hydrogen nuclear magnetic resonance spectrum, carbon spectrum and HRMS spectrum of the double donor molecule CMCNO in Example 5 of the present invention are shown;

[0028] Figure 8 The particle size characterization of the oxygen-resistant photo-redox catalytically triggered GSM release nanocarrier in Example 6 of the present invention;

[0029] Fig. 9 The morphological characterization of the oxygen-resistant photo-redox catalytically triggered GSM release nanocarrier in Example 6 of the present invention;

[0030] Fig.10 (a) CO and (b) NO released by the nanocarrier triggered by oxygen-tolerant photoredox catalysis before and after illumination in Example 7 of the present invention;

[0031] Fig.11 The antioxidant photo-redox catalytically triggered CO / NO release nanocarriers of Examples 2-6 of the present invention have anti-(a) Staphylococcus aureus (S. aureus), (b) Escherichia coli (E. col Ⅰ) and (c) methicillin-resistant Staphylococcus aureus (MRSA) activities. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The molecular structure of NO donor carboxylate is as follows:

[0034]

[0035] The structure of the CO donor molecule is as follows:

[0036]

[0037] The molecular structure of the caged CO donor is as follows:

[0038]

[0039] The dual donor molecule is a single component molecule obtained by direct conjugation of a NO donor molecule and a CO donor molecule.

[0040] A single NO donor molecule is a single-component molecule obtained by directly coupling a NO donor molecule and a caged CO donor molecule.

[0041] The double donor single component molecules have the structure of formula VI-VIII:

[0042]

[0043]

[0044] The double donor molecule has a molecular weight of;

[0045] The molecular weight of VI is 584.15

[0046] The molecular weight of Ⅶ is 600.13

[0047] The molecular weight of Ⅷ is 1120.26

[0048] The NO donor molecule has the structure of Formula IX:

[0049]

[0050] The molecular weight of NO donor molecule is;

[0051] The molecular weight of Ⅸ is 674.20

[0052] Dual donor molecules and NO molecules, wherein the gas donor molecules can self-assemble with photocatalyst molecules (photosensitizers) to prepare nano-assemblies with a particle size of 40-200 nm;

[0053] The invention discloses a method for preparing a double-donor single-component and NO donor molecule, including a method for preparing an NO donor molecule and a CO donor molecule or a caged CO donor molecule.

[0054] Nanoassembly preparation methods include dissolution and mixing of organic molecules and amphiphilic block copolymers, magnetic stirring, thin film hydration / dialysis, etc.

[0055] The nano-assemblies loaded with photocatalyst molecules (photosensitizers) can simultaneously release CO and NOCO / NO donor molecules under light conditions, and have the structures of formula VI, VII and VIII:

[0056]

[0057]

[0058] The CO / NO donor molecule of the present invention has an N-nitrosoamine group in the structure as the NO releasing part and an oxygen / thioflavonol as the CO releasing part.

[0059] The present invention provides a NO donor molecule having a structure of Formula IX:

[0060]

[0061] In the NO donor molecule of the present invention, the N-nitrosoamine group in the structure is the NO releasing part, and the caged flavonol part is the non-functionalized CO releasing part.

[0062] The nanoassembly obtained by co-assembling the gas donor molecule and the photocatalyst molecule (photosensitizer molecule) is a platform for triggering CO / NO release by oxygen-resistant red light photoredox catalysis. The photocatalyst molecule (photosensitizer molecule) has the following chemical structural characteristics:

[0063]

[0064] Example 1: Synthesis of NO Donor Carboxylate Molecules

[0065]

[0066] Preparation: First, in N 2 Under protection, NBNO (7.5 g, 35.51 mmol) and 4-NPC (10.71 g, 53.27 mmol) were dissolved in dry DCM (100 mL), and then TEA (7.19 g, 71.03 mmol) was added with a syringe. After 6 h of reaction, 60 mL of water was added, and DCM (3X30 mL) was used for extraction 3 times. The organic phases were combined, rotary evaporated, and the crude product was separated by column chromatography. Yield: 7.8 g (58%), white solid A;

[0067] The second step is to 2 Under protection, A (7.8 g, 20.72 mmol) was dissolved in dry THF, and then diethanolamine (3.27 g, 31.09 mmol) was added to the solution of A using a syringe. After 8 h of reaction, the crude product was separated by column chromatography. Yield: 6.4 g (90%), yellow crystalline solid B;

[0068] The third step is to 2 Under protection, B (6.4 g, 18.70 mmol) and 4-NPC (9.4 g, 46.76 mmol) were dissolved in dry DCM (70 mL), and then DIPEA (7.3, 56.12 mmol) was added. The crude product was separated by column chromatography. Yield: 6.2 g (40%), light brown crystalline solid C.

[0069] The structures and purities of A, B, and C were verified by NMR hydrogen and carbon spectra. The results are as follows: Figure 1 , 2 , as shown in Figure 3.

[0070] Example 2: Synthesis of MCNO donor molecules

[0071]

[0072] Preparation method: In N 2 Under the protection of OFlaV-NH 2(200 mg, 0.433 mmol) was added to dry DMF (10 mL), and then TEA (43.8 mg, 0.433 mmol) was added dropwise, and stirred at room temperature for 30 min. Compound A (168 mg, 0.433 mmol) was then dissolved in dry DMF (3 mL) and added dropwise to the above reaction system. After 12 h, the solution was washed with brine and the crude product was purified by column chromatography. Yield: 110 mg (43%), yellow colloidal solid. Its structure and purity were verified by NMR hydrogen spectrum and carbon spectrum, and the results were as follows: Figure 4 shown.

[0073] Example 3: Synthesis of MHC NO1-1 donor molecule

[0074]

[0075] Preparation method: In N 2 Under protection, SFlav-NH 2 (300 mg, 0.6283 mmol) was added to dry DMF (10 mL), and then TEA (63 mg, 0.6283 mmol) was added dropwise, and stirred at room temperature for 30 min. Compound A (236 mg, 0.6283 mmol) was then dissolved in dry DMF (3 mL) and added dropwise to the above reaction system. After 12 h, the product was purified by column chromatography, yield: 184 mg (48%), dark red solid.

[0076] Its structure and purity were verified by NMR hydrogen spectrum and carbon spectrum. The results were as follows: Figure 5 shown.

[0077] Example 4: Synthesis of MHCNO1-2 donor molecules

[0078]

[0079] Preparation method: In N 2 Under protection, SFlav-NH 2 (323 mg, 0.892 mmol) was added to dry DCM (10 mL), and then TEA (90 mg, 0.892 mmol) was added dropwise and stirred at room temperature for 30 min. Then C (300 mg, 0.446 mmol) solution was added dropwise to the above reaction system. After 12 h, the product was purified by column chromatography. Yield: 187 mg (37%), dark brown solid.

[0080] Its structure and purity were verified by NMR hydrogen spectrum and carbon spectrum. The results were as follows: Figure 6 shown.

[0081] Example 5: Synthesis of CMCNO donor molecules

[0082]

[0083] Preparation: In the first step, OFlav-Boc (1.0 g, 2.23 mmol) was mixed with anhydrous K 2 CO 3 (0.34 g, 2.46 mmol) was added to DMF (10 mL), followed by benzyl bromide (0.42 g, 2.46 mmol), and the mixture was stirred at room temperature for 10 h. The reaction mixture was filtered to remove K 2 CO 3 , washed with brine, extracted with DCM. The crude product was purified by column chromatography, yield: 0.73 mg (60.8%), caged OFlav-Boc bright yellow powder;

[0084] In the second step, caged OFlav-Boc (0.5 g, 0.93 mmol) was dissolved in chloroform (30 mL). Then trifluoroacetic acid (1.6 g, 13.96 mmol) was added dropwise, stirred at room temperature for 2 h, and purified. Yield: 310 mg (76.1%), caged OFlav-NH 2 Brown solid;

[0085] The third step is to 2 Under protection, caged OFlav-NH 2 (70 mg, 0.16 mmol), then TEA (16 mg, 0.16 mmol) was added dropwise, and stirred at room temperature for 30 min. Compound A (60 mg, 0.16 mmol) was then dissolved in dry DMF (3 mL) and added dropwise to the reaction system. After 12 h, the product was purified. Yield: 53 mg (49.0%), caged MCNO (CMCNO) was a white solid.

[0086] Its structure and purity were verified by NMR hydrogen spectrum and carbon spectrum. The results were as follows: Figure 7 shown.

[0087] Example 6: Nanoassembly

[0088] The donor and photocatalyst molecules (photosensitizer molecules) are dissolved in an organic solvent as a co-solvent (tetrahydrofuran and dimethyl sulfoxide), added to water (such as ultrapure water, Wahaha water) at a certain temperature and stirring speed, and the organic solvent is removed by dialysis to obtain nanoassemblies loaded with gas donors and photocatalyst molecules (photosensitizer molecules), hereinafter referred to as MHCNO1-2 / PC, MHCNO1-1 / PC and MCNO / PC micelles, while the control group assembly (single gas donor) is also prepared by the same method. Abbreviated CMCNO / PC and SFlav-Boc / PC micelles.

[0089] The details are as follows:

[0090] The gas donor (2 mM), photocatalyst (0.2 mM) and diblock copolymer (20 mg) were dissolved in 1 mL of tetrahydrofuran-dimethyl sulfoxide co-solvent (1:1), injected into pure water with high-speed stirring at room temperature, and the flash-precipitated assembly was placed in a 14000 Da dialysis bag and dialyzed in water at room temperature for 8 hours to obtain the nanoassembly.

[0091] Figure 8 and Fig. 9 Hydrated particle size and transmission electron microscopy (TEM) characterization of each of the gas donor components are shown.

[0092] Example 7: Photoredox catalysis triggers CO and NO release

[0093] CO release test:

[0094] The CO release level was monitored using a commercially available CO detector (DragerPac6500).

[0095] NO release test:

[0096] NO release was determined by Griess test.

[0097] Fig.10 Shown is the release of (a) CO and (b) NO from the nanocarriers under oxygen-tolerant photoredox catalysis.

[0098] Example 8: Photoredox catalytically triggered CO / NO release for antibacterial applications

[0099] Antibacterial experiment: For each bacterial strain, 1-2 individual colonies were inoculated into fresh tryptone soy broth (TSB) medium and incubated at 37°C for 15 hours. Then, 50 μL of bacterial suspension was added to 5 mL of fresh TSB and incubated at 37°C to the logarithmic growth phase (OD600 = 0.5-0.7), the bacteria were collected by centrifugation, washed twice with sterile PBS and centrifuged (10,000 rpm, 5 minutes; 4°C), and the bacterial concentration was adjusted to 1.5 × 106 CFU / mL with sterile PBS. Different assemblies (100 μL) were added to a 96-well plate, 50 μL of bacterial suspension was added to each well, the 96-well plate was incubated at 37°C for 10 min, and then exposed to light or not, the 96-well plate was further incubated at 37°C for 30 min and diluted 100 times with sterile PBS, the bacterial suspension (20 μL) was placed on a TSB agar plate and incubated at 37°C for 15 h, the number of bacterial colonies was recorded, and each experiment was repeated 3 times independently.

[0100] Fig.11Photoredox-catalyzed CO and NO release is shown to kill (a) S. aureus, (b) E. coli, and (c) MRSA.

[0101] The experimental results show that the release of CO / NO by photoredox catalysis can effectively kill Gram-positive bacteria, Gram-negative bacteria and drug-resistant Gram-negative bacteria.

[0102] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0103] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for releasing CO / NO by oxygen-resistant light redox catalysis, characterized in that: The invention comprises three CO / NO donor molecules and one NO donor molecule, wherein the gas donor molecules are prepared by covalent bonding and loaded into the nanoassembly together with the photocatalyst (photosensitizer molecule) by self-assembly; the release of carbon monoxide (CO) and nitric oxide (NO) from a single component is triggered under the action of the photoredox catalyst; the CO release motif and the NO release motif are directly coupled to prepare a single component molecule containing the CO and NO release motifs; The molecular structures of NO donor carboxylates include Formula I and Formula II; The structures of CO donor molecules include Formula III and Formula IV; The caged CO donor molecular structure includes Formula V.

2. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 1, characterized in that: The single-component molecule obtained by direct conjugation of the NO donor molecule and the CO donor molecule is a dual-donor molecule, and the single-component molecule obtained by direct coupling of the NO donor molecule and the caged CO donor molecule is a single NO donor molecule.

3. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 2, characterized in that: The dual donor single component molecule has the structure of Formula VI-VIII.

4. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 3, characterized in that: The molecular weights of the dual donor molecules are: VI has a molecular weight of 584.15; VII has a molecular weight of 600.13; and VII has a molecular weight of 1120.

26.

5. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 4, characterized in that: The NO donor molecular structure comprises formula IX.

6. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 5, characterized in that: The molecular weight of the NO donor molecule is: IX molecular weight 674.

20.

7. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 6, characterized in that: The dual donor molecules and NO molecules, wherein the gas donor molecules can be self-assembled with photocatalyst molecules (photosensitizers) to prepare nano-assemblies with a particle size of 40-200 nm.

8. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 7, characterized in that: Dissolution and mixing of the organic molecules and the amphiphilic block copolymers, magnetic stirring, thin film hydration / dialysis, etc.

9. The method for releasing CO / NO by oxygen-resistant light redox catalysis according to claim 8, characterized in that: The nano-assembly loaded with photocatalyst molecules (photosensitizer) can release CO and NO simultaneously under light conditions.

10. Use of CO / NO released by the method for releasing CO / NO by oxygen-resistant photo-redox catalysis according to any one of claims 1 to 9 in killing Gram-positive and Gram-negative bacterial pathogens.

Images