Preparation method and antibacterial application of leucine aminopeptidase response type near-infrared cyanine probe

Abstract

The invention belongs to the technical field of medicines, and discloses a near-infrared fluorescent probe Cy-NEO-Leu for leucine aminopeptidase responsiveness as well as a preparation method and application of the near-infrared fluorescent probe Cy-NEO-Leu. The parent nucleus of the probe is of a heptamethyl cyanine structure, and the probe has high molar absorption coefficient, high fluorescence quantum yield and excellent phototherapy characteristics. The introduction of a neomycin group can increase the water solubility of the probe, and a neomycin multi-ammoniation structure exists in a polycation form under physiological conditions and can target negatively charged cell walls of gram-negative bacteria, so that the probe Cy-NEO-Leu is enriched around the bacteria, can be specifically recognized by leucine aminopeptidase and can be hydrolyzed and activated; and photo-thermal and photodynamic sterilization effects are achieved under laser irradiation. Due to the good biocompatibility, the remarkable active oxygen generation capacity, the excellent photo-thermal conversion efficiency and the unique property of accurately targeting bacteria, the Cy-NEO-Leu is expected to play an important role in a photo-thermal / photodynamic combined treatment scheme, and is used for resisting diseases caused by bacteria and biological membranes and accelerating the healing process of wounds.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the specific response of bacterial leucine aminopeptidase and the biomedical field of combating bacterial infection. More specifically, it relates to the design of a near-infrared cyanin fluorescent probe, its preparation method, and its application in combating bacteria and their biofilm infections. Background Technology

[0002] Pseudomonas aeruginosa is a very important group of Gram-negative opportunistic pathogens. Its rich metabolic pathways and regulatory genes enable it to exhibit great adaptability in various environments. 1-3 Its heterotrophic nature, diverse toxic factors, and high antibiotic resistance make it difficult to completely eliminate the bacteria from the patient's body, leading to various serious acute and chronic infections with high morbidity and mortality. Therefore, there is an urgent need to research novel antibacterial treatment strategies. 4-5 The combined use of photodynamic therapy (PDT) and photothermal therapy (PTT) is considered a promising approach to combating microbial infections due to its precise time-space selection, non-invasiveness, low drug resistance, and relatively few side effects. However, developing phototherapy agents with bacterial targeting specificity remains a formidable scientific challenge. 6-9 In this study, we constructed a novel bacterial-targeting near-infrared fluorescent probe, Cy-NEO-Leu, based on leucine aminopeptidase highly expressed in *Pseudomonas aeruginosa*. This probe specifically reacts with LAP, enabling visualization of LAP activity. Furthermore, Cy-NEO-Leu exhibits highly efficient reactive oxygen species (ROS) generation and photothermal conversion after interaction with LAP, showing promise as a novel phototherapy agent for the synergistic treatment of bacterial biofilm infections and accelerated wound healing through photothermal / photodynamic therapy. Summary of the Invention

[0003] The purpose of this invention is to develop new strategies for combating bacteria and their biofilm infections. This invention has successfully developed a near-infrared (NIR) fluorescent probe, Cy-NEO-Leu, with leucine aminopeptidase (LAP) responsiveness. Given its excellent biocompatibility, strong ROS generation capacity, superior photothermal conversion efficiency, and precise bacterial targeting characteristics, Cy-NEO-Leu is a highly promising phototherapy agent for combating bacteria and their biofilm infections and promoting wound healing in combined photothermal (PTT) / photodynamic (PDT) therapy. To achieve the above objectives, in a first aspect, this invention provides the structure of the LAP-responsive fluorescent probe Cy-NEO-Leu as shown in formula (1):

[0004]

[0005] Secondly, the present invention also provides a method for preparing the above-mentioned probe, comprising the following steps:

[0006]

[0007] (a) i.EDCI, HOBT, DIPEA, DCM, 25℃, 30min; ii.Pd / C, 12N HCl, H2, MeOH, 25℃, 30min.

[0008] (1) Compound C2 was dissolved in 75 mL of DCM, followed by the addition of EDCI, HOBT, and DIPEA. After stirring at room temperature for 20 minutes, compound C1 was added. The reaction was terminated after stirring for another 10 minutes. The reaction mixture was diluted with DCM and the organic phase was extracted five times with saturated NaCl aqueous solution. The organic phase was dried over anhydrous NaSO4 and then filtered under vacuum. The residue was dissolved in MeOH, followed by the addition of Pd / C and 12N HCl, while replacing all the air in the reaction system with H2. The reaction was then stirred at room temperature for 30 minutes until the reaction was complete. The reaction solution was filtered, vacuum filtered, and purified by silica gel column chromatography to obtain a white solid compound C3.

[0009]

[0010] (a) NaH, dry DMF, 25℃, 3h; (b) i. EDCI, HOBT, DIPEA, DCM, 25℃, 3h; ii. TFA, 0℃, 15min.

[0011] (2) Compound C3 was dissolved in DMF, and then NaH was added. The mixture was stirred thoroughly for 10 minutes. Then, compound 1 was added, and the mixture was stirred at room temperature for 3 hours until the reaction was complete. The reaction product was diluted with DCM, and the organic phase was extracted five times with saturated NaCl aqueous solution. The product was dried with anhydrous NaSO4 and then evaporated to dryness. Purification by silica gel column chromatography yielded a green solid compound 2.

[0012] (3) Compound 2 was dissolved in DCM, and then EDCI, HOBT, and DIPEA were added sequentially, with stirring continued at room temperature for 30 minutes. After adding compound C4, stirring was continued at room temperature for 3.5 hours to complete the reaction. The reaction solution was diluted with DCM, and the organic phase was extracted five times with saturated NaCl. Then, it was dried over anhydrous NaSO4, filtered, and evaporated to dryness. TFA was added to the residue, and the mixture was stirred in an ice bath for 20 minutes. The reaction was then quenched with DCM and evaporated to dryness. The green solid product Cy-NEO-Leu was obtained by high performance liquid chromatography purification.

[0013] Thirdly, this invention provides the photophysical properties of the Cy-NEO-Leu probe and its responsiveness to LAP.

[0014] Fourthly, this invention provides real-time detection and imaging of LAP activity in Pseudomonas aeruginosa.

[0015] Fifthly, the present invention provides the PTT / PDT properties of Cy-NEO-Leu and its phototherapy antibacterial activity against Pseudomonas aeruginosa and its biofilm.

[0016] In a sixth aspect, the present invention provides an evaluation of the level of immune factors produced by bacterial-stimulated cells after treatment.

[0017] In a seventh aspect, the present invention provides Cy-NEO-Leu in vivo photodynamic and photothermal synergistic therapy for bacterial biofilm infection. Attached Figure Description

[0018] Figure 1 This indicates the mechanism of Cy-NEO-Leu in antibacterial phototherapy.

[0019] Figure 2 The spectrum of Cy-NEO-Leu and its response to LAP are shown.

[0020] Figure 3 This demonstrates the use of Cy-NEO-Leu to achieve confocal fluorescence imaging of Pseudomonas aeruginosa.

[0021] Figure 4 This indicates the photothermal / photodynamic properties of Cy-NEO-Leu and its products, as well as their antibacterial / antibiofilm effects.

[0022] Figure 5 This indicates an in vitro evaluation of the immune cell response to immune factors after different treatments.

[0023] Figure 6 This indicates that the probe molecules mediated a photodynamic and photothermal synergistic treatment of a mouse skin bacterial biofilm infection model under near-infrared laser irradiation. Detailed Implementation

[0024] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0025] In a first aspect, the present invention provides the structure of the LAP-responsive fluorescent probe Cy-NEO-Leu as shown in formula (1);

[0026]

[0027] Example 1: Preparation steps of compound Cy-NEO-Leu:

[0028]

[0029] Preparation Example 1: Synthesis of Compound C3

[0030] Compound C2 (1741 mg, 7.5 mmol) was dissolved in DCM (75 mL), followed by the addition of EDCI (1924 mg, 10.03 mmol), HOBT (1355 mg, 10.03 mmol), and DIPEA (1.746 mL, 10.03 mmol). The mixture was stirred at room temperature for 20 min. Then, C1 (1000 mg, 5.0 mmol) was added. The mixture was stirred at room temperature for 10 min until the reaction was complete. The reaction solution was diluted with DCM, and the organic phase was extracted five times with a saturated NaCl aqueous solution. The organic phase was then dried over anhydrous NaSO4 and evaporated to dryness. The residue (1615 mg, 3.91 mmol) was dissolved in MeOH, followed by the addition of Pd / C (161.5 mg) and 12N HCl (241.2 μL, 7.82 mmol), while simultaneously replacing all air in the reaction system with H2. The mixture was stirred at room temperature for 30 min until the reaction was complete. The reaction solution was filtered and evaporated to dryness. After purification by silica gel column chromatography (2% MeOH / DCM), a white solid C3 (417 mg) was obtained, with a yield of 27.8%. 1 H NMR(400MHz,CD3OD)δ7.32(d,J=8.9Hz,2H),6.73(d,J=8.9Hz,2H),4.24-4.16(m,1H) ,1.78-1.69(m,1H),1.58(dd,J=9.0,5.6Hz,2H),1.44(s,9H),0.97(t,J=5.9Hz,6H). 13 C NMR(101MHz,CD3OD)δ173.92,157.92,155.55,131.27,123.62,116.24,80.63,55.08,42.52,28.74,26.01,23.52,22.06.HRMS(m / z)[M+H] + :Calcd.for C 17 H 27 N2O4 + 323.1965; found 323.1969.

[0031] Preparation Example 2, Synthesis of Compound 2

[0032] Compound C3 (232 mg, 0.719 mmol) was dissolved in DMF (7.2 mL), followed by the addition of NaH (47.9 mg, 1.197 mmol). After stirring for 10 min, compound 1 (286 mg, 0.478 mmol) was added. The reaction was carried out at room temperature with stirring for 3 h until completion. The reaction solution was diluted with DCM, and the organic phase was extracted five times with saturated NaCl aqueous solution. The organic phase was dried over anhydrous NaSO4 and evaporated to dryness. Purification by silica gel column chromatography (6% MeOH / DCM) yielded green solid 2 (55 mg), with a yield of 40.1%. 1 H NMR (400MHz, CDCl3) δ9.56 (s, 1H), 7.94 (d, J = 5.7Hz, 1H), 7.90 (d, J = 6.1Hz, 1H), 7.75 (d, J = 8.5Hz, 2H), 7.32 (t, J = 7.7Hz, 2H), 7.27(s,1H),7.25(s,1H),7.17(t,J=7.3Hz,2H),7.05(d,J=8.1Hz,1H),7.01(d,J=8.0Hz,1H),6.96(d,J=8.7Hz,2H),5.96(d,J =14.1Hz,1H),5.89(d,J=14.1Hz,1H),4.29(s,1H),4.02-3.90(m,4H),2.69-2.60(m,4H),2.38(t,J=7.1Hz,2H),2.02(d,J=9.0 Hz,2H),1.80-1.69(m,4H),1.69-1.58(m,4H),1.49(s,1H),1.37(s,9H),1.33(s,12H),1.25(s,1H),0.91(s,3H),0.90(s,3H). 13C NMR (151MHz, CDCl3) δ176.33,172.64,172.18,171.34,165.25,161.89,161.66,156.31,156.20,143.00,14 2.26,142.15,141.78,141.22,141.04,133.98,128.84,128.68,125.41,125.03,122.70,122.49,122.40,12 2.34,114.62,110.74,109.97,99.91,98.79,54.45,49.44,49.13,44.55,41.39,39.18,34.29,29.82,29.48 ,29.44,28.37,27.96,27.94,26.97,26.30,24.92,24.52,24.39,23.24,21.82,21.13,12.17.HRMS(m / z)[M] + :Calcd.for C 55 H 71 N4O6 + 883.5368; found 883.5364.

[0033] Preparation Example 3: Synthesis of compound Cy-NEO-Leu

[0034] Compound 2 (50 mg, 0.056 mmol) was dissolved in DCM, followed by the addition of EDCI (21.70 mg, 0.113 mmol), HOBT (13.04 mg, 0.113 mmol), and DIPEA (19.73 μL, 0.113 mmol). The mixture was stirred at room temperature for 30 min. Then, compound C4 (75.62 mg, 0.062 mmol) was added. The mixture was stirred at room temperature for 3.5 h until the reaction was complete. The reaction solution was diluted with DCM, and the organic phase was extracted five times with saturated NaCl. The solution was dried over anhydrous NaSO4, filtered, and evaporated to dryness. TFA was added to the residue, and the mixture was stirred in an ice bath for 20 min until the reaction was complete. The reaction was quenched with DCM, and the solution was evaporated to dryness. The solution was purified by high-performance liquid chromatography (68% acetonitrile / water) to give a green solid Cy-NEO-Leu (30 mg), with a yield of 27.6%. 1H NMR(600MHz,CD3OD)δ8.03-8.00(m,1H),8.00-7.96(m,1H),7.66(s,1H),7.64(s,1H),7.41-7.38(m,2H),7.38-7.35(m,2H),7.28(d,J=8.2Hz,1H),7.24(t,1H),7.21(q,2H),7.13(s,1H),7.12(s,1H),6.18(d,J=14.2Hz,1H),6.12(d,J=14.1Hz,1H),5.84(d,J=3.7Hz,1H),5.41(d,J=4.9Hz,1H),5.30(s,1H),4.41(t,J=4.5Hz,1H),4.32-4.27(m,1H),4.27-4.20(m,2H),4.19-4.15(m,2H),4.14(t,1H),4.11-4.04(m,3H),4.01(t,2H),3.99-3.97(m,1H),3.89(t,J=8.8Hz,1H),3.67(d,J=3.2Hz,2H),3.64(d,J=2.8Hz,1H),3.50(d,J=3.6Hz,1H),3.49(d,J=3.7Hz,1H),3.45-3.42(m,2H),3.41(t,J=3.3Hz,1H),3.38(dd,J=13.5,7.8Hz,1H),3.35-3.32(m,1H),3.27(d,J=3.6Hz,1H),3.25(d,1H),3.20-3.15(m,1H),2.78-2.69(m,4H),2.66(s,1H),2.43(d,J=10.9Hz,1H),2.28(dd,J=9.1,6.3Hz,2H),2.11-2.00(m,3H),1.84-1.79(m,2H),1.78(dd,1H),1.76(dd,J=4.8,3.2Hz,1H),1.75-1.72(m,1H),1.72-1.70(m,1H),1.69-1.66(m,1H),1.47(q,J=8.7Hz,2H),1.42(d,J=12.5Hz,1H),1.38-1.35(m,15H),1.33-1.30(m,1H),1.30-1.27(m,2H),1.25-1.20(m,1H),1.00(t,6H),0.92-0.83(m,1H). 13C NMR (151MHz, CD3OD) δ176.74,173.72,173.19,168.83,165.25,158.25,143.81,143.63,143.04,142.86,142.68,142.38,133.90,1 29.89,129.78,126.43,126.04,123.52,123.39,123.34,123.08,116.15,111.94,111.80,109.39,101.01,100.61,97.17,96.84,8 6.58,83.31,78.01,76.66,75.63,73.99,73.12,72.15,71.76,69.51,69.24,55.08,53.68,52.89,51.12,50.41,50.30,50.13,44. 89,41.91,41.72,41.67,40.19,36.93,28.25,28.06,27.80,26.70,25.51,25.23,25.17,23.17,22.42,22.05,12.39.HRMS(m / z)[M] + :Calcd.for C 73 H 108 N 11 O 15 + 1378.8021; found 1378.8020.

[0035] Pharmacological experiments

[0036] Experimental Example 1: Spectral properties of Cy-NEO-Leu and its response to LAP enzyme.

[0037] Cy-NEO-Leu and Cy-NEO-NH2 were dissolved in DMSO to prepare a 5 mM stock solution. The probe stock solution was diluted to 10 μM with Tris buffer and contained 1.5% DMSO. The absorption and fluorescence emission spectra were measured, see [link to relevant documentation]. Figure 2 .

[0038] Experimental Example 2: Confocal fluorescence imaging of Pseudomonas aeruginosa using the probe Cy-NEO-Leu.

[0039] Pseudomonas aeruginosa (PAO1) was cultured overnight in LB medium for 12 h. The strain was collected by centrifugation and washed twice with Tris buffer. In the absence / within the presence of the inhibitor Bestatin (1 mM), 10 μM Cy-NEO-Leu was mixed with PAO1 (OD200). 600=0.5) co-incubate. After 1 h, rinse the incubated strain twice, take 8 μL and add it to an eight-well plate and cover with an agarose gel. Confocal fluorescence imaging is performed using a confocal microscope (Leica TCS SP8X, HC PL Apo 63× oil immersion). λ ex =670nm, and λ em =690-800nm, see Figure 3 .

[0040] Experimental Example 3: Photothermal / photodynamic properties of Cy-NEO-Leu and its products and their antibacterial / antibiofilm effects.

[0041] The detection of ROS generated after irradiation of Cy-NEO-Leu and Cy-NEO-NH2 was performed using DCFH-DA dye. DCFH-DA (1 mM) was hydrolyzed with NaOH (10 mM) at room temperature for 30 min to form DCFH. DCFH (1 mM) was diluted to 40 μM with Tris buffer and stored at low temperature. Cy-NEO-Leu and Cy-NEO-NH2 were diluted to 10 μM with the above DCFH Tris solution and added to 96-well plates. The ROS generated after irradiation of Cy-NEO-Leu and Cy-NEO-NH2 was detected using a laser (λ). ex =785nm, 1.2W / cm 2 Irradiate for 5 seconds, then measure the fluorescence intensity (λ). ex =480nm,λ em =525nm). The process was repeated 6 times, with a total illumination time of 30s.

[0042] PAO1 was cultured overnight in LB medium for 12 hours. Group 1 consisted of PAO1 (OD10 ... 600 =0.5); Group 2 is PAO1 (OD 600 =0.5)+Laser(λ ex =785nm, 1.2W / cm 2 Group 3 consisted of PAO1 (OD1, 1 min); Group 3 consisted of PAO1 (OD1, 1 min). 600 =0.5)+Cy-NEO-Leu(10μM); Group 4 is PAO1(OD 600 =0.5)+Cy-NEO-Leu(10μM)+Laser(λ ex =785nm, 1.2W / cm 2 After treatment, each group was incubated with PI staining reagent at a final concentration of 5 μg / mL for 30 min. Fluorescence intensity (λ) was measured using a microplate reader. ex =535nm,λ em =615nm). At the same time, 100μL of the bacterial suspension after each of the above treatments was spread on LB solid medium, incubated for 12h, and photographed.

[0043] The cultured bacterial solution was resuspended in LB solid medium to obtain OD. 600 =0.01. Add 200 μL of the above bacterial culture to a 96-well plate and incubate for 24 h. Perform the following operations: Group 1 is a PAO1 biofilm; Group 2 is a PAO1 biofilm + Laser(λ) ex =785nm, 1.2W / cm 2 Group 3 consisted of PAO1 biofilm + Cy-NEO-Leu (10 μM); Group 4 consisted of PAO1 biofilm + Cy-NEO-Leu (10 μM) + Laser (λ). ex =785nm, 1.2W / cm 2 (1 min). After the above treatment, incubation continued for 24 h. Then, the biofilm supernatant was discarded, and bacteria not bound to the biofilm were rinsed with PBS. The biofilm was fixed with anhydrous methanol and then stained with crystal violet. Finally, the crystal violet bound to the biofilm was dissolved in 33% glacial acetic acid-water solution, and the biofilm biomass was determined by absorbance at 595 nm. Biofilm quantification was expressed as biofilm viability, calculated using the following formula: Biofilm viability (%) = OD 595 (sample) / OD 595 (Control group), results are shown in Figure 4 .

[0044] Experimental Example 4: Detection of the release of immune factors by bacterial-stimulated immune cells.

[0045] RAW 264.7 cells were revived and passaged into 24-well plates, with approximately 20,000 cells per well. Simultaneously, the bacteria in each group underwent corresponding pretreatments: Group 1 was a negative control group with RAW 264.7 cells alone; Group 2 was a positive control group with RAW 264.7 cells supplemented with LPS (1 mg / mL); Group 3 involved bacterial incubation for 2 hours; Group 4 involved co-incubation of bacteria with Cy-NEO-Leu (10 μM) for 2 hours; and Group 5 involved 2 hours of bacterial incubation followed by laser irradiation (λ). ex =785nm, 1.2W / cm 2 Group 6: Bacteria were co-incubated with Cy-NEO-Leu (10 μM) for 2 h and then subjected to laser irradiation (λ). ex =785nm, 1.2W / cm 2 (1 min). Then, the bacterial cultures of each group were added to the cell culture medium at an MOI of 10. After stimulation for 24 h, the cell supernatant from each group was collected, centrifuged at 10000 rpm at 4°C for 10 min, and the expression levels of the corresponding immune factors were detected using an ELISA kit. The results are shown in the figure. Figure 5 .

[0046] Experimental Example 5: Synergistic treatment of skin infection in mice mediated by aminopeptidase-responsive probes and photodynamic / photothermal and immune responses.

[0047] BALB / c mice (6-8 weeks old, male, 20g) were purchased from Huafukang Biotechnology Co., Ltd. (Beijing). Surgery was performed on the back of the mice to create a fossa with an area of ​​approximately 0.8 cm². 2 The wound was open. Subsequently, 50 μL of Pseudomonas aeruginosa biofilm (OD) was injected into the wound. 600 =6). After 24 hours, each group was treated: Group 1 received an injection of 50 μL PBS; Group 2 received an injection of 50 μL PBS + Laser (λ). ex =785nm, 1.2W / cm 2 Group 3 was injected with 50 μL Cy-NEO-Leu (20 μM); Group 4 was injected with 50 μL Cy-NEO-Leu (20 μM) + Laser (λ). ex =785nm, 1.2W / cm 2 (5 min). Each group was treated once a day for three consecutive days. Wound photographs and wound area were recorded daily using a handheld camera. On day 12, blood samples were collected from each group for routine blood tests. Immediately afterwards, skin tissue from the mouse wounds was collected for hematoxylin-eosin (H&E) staining, Masson staining, and IL-6 and TNF-alpha immunofluorescence. Skin tissue from each group was weighed consistently and then homogenized in the same volume of PBS buffer. The suspension was spread onto LB solid medium, and the bacterial growth was observed after 24 hours. Results are shown below. Figure 6 .

[0048] References

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Claims

1. A near-infrared cyanin-based fluorescent probe, characterized in that, The cyanin-based fluorescent probe has the structure shown in formula (1):

2. The near-infrared cyanin fluorescent probe of claim 1 specifically responds to leucine aminopeptidase, LAP, EC 3.4.11.1, thereby generating photothermal and photodynamic properties under near-infrared laser irradiation, and is used in combination to combat bacterial and biofilm infections.

3. The method for preparing the near-infrared cyanin-based fluorescent probe according to claim 1, characterized in that, The method involves conjugating the compound of formula (2) with the modified neomycin in an organic solvent under nucleophilic reaction conditions; 4. The application of the near-infrared cyanin fluorescent probe according to claim 1 in the preparation of phototherapy antibacterial agents, characterized in that, The pathogenic bacteria or the target pathogenic microorganism of the photodynamic and photothermal antibacterial treatment are Gram-positive bacteria, Gram-negative bacteria, or fungi.

5. The application according to claim 4, characterized in that, The pathogenic microorganisms are ESKAPE pathogens, including Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Klebsiella pneumoniae, and are used as phototherapy antibacterial agents.

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