Near-infrared cyanine probe for detecting penicillin G acylase as well as preparation method and application of near-infrared cyanine probe

Abstract

The invention belongs to the technical field of medicines, and discloses a near-infrared cyanine probe for detecting penicillin G acylase as well as a preparation method and application of the near-infrared cyanine probe. According to the research, a near-infrared fluorescent probe Cy-NEO-PA responding to penicillin G acylase (PGA) is developed, so that the influence of environmental factors on the formation of an acinetobacter baumannii biological membrane is observed. Research results show that glucose inhibits the generation of PGA to enhance the formation of a biological membrane, while phenylacetic acid (PAA) stimulates the generation of PGA and inhibits the formation of the biological membrane. The observation results highlight the excellent capability of Cy-NEO-PA in accurately measuring PGA kinetics, and reveal the key effect of PGA in biological membrane development. In addition, the Cy-NEO-PA has good biocompatibility, strong active oxygen generation, efficient photo-thermal conversion and bacterial targeting ability, so that the Cy-NEO-PA becomes a promising drug for resisting bacterial infection and promoting wound healing through photo-thermal / photodynamic therapy.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to the detection of penicillin G acylase and the biomedical technology of combating bacterial infection. Specifically, it relates to the design of a near-infrared cyanine fluorescent probe, as well as its preparation method and its application in exploring biofilm regulation and combating bacterial infection. Background Art

[0002] Acinetobacter baumannii (A. baumannii) is a key Gram-negative strain among ESKAPE pathogens and poses a major threat to global public health due to its robust biofilm-forming ability, which can enhance drug resistance and virulence. 1-3 The extracellular polymeric matrix within the biofilm acts as a protective barrier, hindering the penetration of antimicrobial drugs, thereby leading to enhanced bacterial resistance. 4-6 . Although the use of antibiotics is the main treatment method, the emergence of multidrug-resistant strains, especially "ESKAPE" resistance, has become an important cause of morbidity and mortality in patients, and there is an urgent need to explore new alternative antimicrobial treatments. Therefore, understanding the regulatory mechanism of biofilm formation of Acinetobacter baumannii and developing new antimicrobial treatment strategies are crucial to achieve successful treatment of drug-resistant bacteria. Antimicrobial phototherapy (PT), especially photodynamic therapy (PDT) and photothermal therapy (PTT), has become a promising method for antimicrobial infection due to its precise spatiotemporal selectivity, non-invasiveness, low drug resistance and few side effects. 7-15 However, the development of phototherapeutic agents (PTAs) with bacterial targeting specificity remains a huge scientific challenge. Ideal PTAs should be able to selectively generate reactive oxygen species (ROS) and photothermal conversion, show low activity in normal tissues, but show strong activity at sites of bacterial infection, thereby achieving selective and efficient phototherapy. Highly selective and efficient phototherapy technologies can combat bacterial infections, effectively target drug-resistant bacteria and disrupt biofilm formation.

[0003] Penicillin G acylase (PGA) has been widely used in pharmaceutical industry for more than 50 years. It is used in the synthesis of key intermediates for the industrial production of β-lactam antibiotics and is also widely used to remove residual penicillin in the environment. 16-19 In recent years, PGA has also been studied for site-specific modification of insulin. 20 Although the structure, physicochemical properties, and industrial applications of PGA enzymes are well understood, the physiological roles of PGA in bacterial signaling and pathogenesis are often overlooked. 21Recent studies have shown that acylases from Gram-negative bacteria have potent hydrolytic activity against N-acyl-homoserine lactones (AHLs), quorum sensing signaling molecules, thereby reducing virulence during biofilm formation. 22,23 In Acinetobacter baumannii, AHL signaling molecules play a crucial role in intercellular communication and significantly affect its biofilm formation. 24-27 Therefore, it is envisioned that PGA may play an important role in this process. However, the specific role of PGA in A. baumannii biofilm formation is still unclear, and further research in this area will have important implications for antibacterial and anti-biofilm therapy.

[0004] Several small molecule fluorescent probes have been reported for detecting PGA activity and screening natural PGA inhibitors, but they are rarely applicable to bacterial imaging and antibacterial therapy. 28-34 . In this study, we developed a new bacteria-targeted near-infrared (NIR) fluorescent probe, Cy-NEO-PA, for real-time detection of PGA activity in Acinetobacter baumannii. This probe can be used to visualize how environmental factors affect PGA activity and biofilm formation in Acinetobacter baumannii. The study found that glucose can inhibit PGA activity and promote biofilm formation, while phenylacetic acid (PAA) can enhance PGA activity and inhibit biofilm formation. These findings help to elucidate the complex relationship between PGA activity, environmental factors and biofilm formation.

[0005] Furthermore, Cy-NEO-PA exhibits targeted activation at the site of bacterial infection, efficiently generating ROS and producing a photothermal conversion effect, making it a promising phototherapeutic agent for combating bacterial infection and promoting wound healing through synergistic photothermal / photodynamic therapy. PGA-responsive probes, as a very promising photosensitizer, have significant potential in treating bacterial infections and combating biofilm formation. Summary of the Invention

[0006] The purpose of the present invention is to develop new tools to explore the formation and regulation of biofilms and innovative antibacterial strategies. The present invention has developed a penicillin G acylase (PGA) responsive near-infrared (NIR) fluorescent probe Cy-NEO-PA for investigating the effects of environmental factors on PGA activity and biofilm formation in Acinetobacter baumannii (A. baumannii). In addition, Cy-NEO-PA has good biocompatibility, potent reactive oxygen species (ROS) generation, efficient photothermal conversion efficiency, and precise bacterial targeting capabilities, making it a promising phototherapy agent for combating bacterial infections and promoting wound healing through combined photothermal (PTT) / photodynamic (PDT) therapy. To achieve the above objectives, in a first aspect, the present invention provides a PGA-responsive fluorescent probe Cy-NEO-PA having a structure as shown in formula (1); the product probe Cy-NEO-NH2 has a structure as shown in formula (3);

[0007]

[0008]

[0009] In a second aspect, the present invention also provides a method for preparing the above-mentioned probe, comprising the following steps:

[0010]

[0011] (a) DIPEA, dry DMF, -40℃, 3h; (b) 1) CH3COONa, C2H5OH, 30min; 2) 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium bromide, 80℃, 3h.

[0012] (1) Compound C1 was dissolved in 20 mL of ultra-dry DMF, and then DIPEA was added. Compound C2 was slowly added at -40°C under argon protection. The mixture was stirred at -40°C for 3 hours. After the reaction was completed, the reaction solution was poured into 1000 mL of water for recrystallization. The resulting solid was filtered and dried to obtain Compound C3;

[0013] (2) Compound C5 was dissolved in anhydrous ethanol and CH3COONa was added. Compound C4 was slowly added and stirred at room temperature for 30 min under argon protection. Then 1-(5-carboxypentyl)-2,3,3-trimethyl-3-hydrogen-indole-1-bromide was added, the reaction temperature was raised to 80°C, and stirred under argon protection for 3 h. After the reaction was completed, the solution was cooled to room temperature, filtered, and distilled under reduced pressure. It was then diluted with DCM, extracted with water 5 times, dried over anhydrous Na2SO4, and concentrated. Compound 1 was obtained by purification by silica gel column chromatography;

[0014]

[0015] (a) NaH, dry DMF, 40℃, 3h; (b) NaH, dry DMF, 80℃, 3h; (c) i. EDCI, HOBT, DIPEA, DCM, 25℃, 3h; ii. TFA, 0℃, 15min.

[0016] (3) Compound C3 was dissolved in ultra-dry DMF and 60% NaH was added. The reaction solution was stirred at room temperature under argon protection for 10 minutes. Compound 1 was added, the temperature was raised to 40°C, and the reaction was continued for 3 hours. After the reaction was completed, the reaction solution was diluted with DCM and extracted with water 5 times. The organic layer was dried over anhydrous sodium sulfate and concentrated. Product 2 was purified by silica gel column chromatography;

[0017] (4) Compound 1,1-dimethylethyl (4-hydroxyphenyl) carbamate was dissolved in DMF and 60% NaH was added. The mixture was stirred at room temperature under argon for 10 min, compound 1 was added, the temperature was raised to 80°C, and the mixture was stirred for 3 h. After the reaction, the solution was diluted with 15 mL of DCM and extracted with water five times. The organic layer was dried over anhydrous sodium sulfate and concentrated. The product 3 was purified by silica gel column chromatography.

[0018] (5) Compound 2 was dissolved in DCM, and then EDCI and HOBT were added. DIPEA was activated under argon at room temperature for 30 min. Subsequently, compound C6 was added and stirred for 3 h. After the reaction was completed, the reaction solution was diluted with DCM and extracted 5 times with saturated NaCl solution. The organic layer was then dried over anhydrous Na2SO4 and concentrated by vacuum distillation. The residue was diluted with TFA in an argon ice bath, and the reaction mixture was stirred in an ice bath for 15 min and then concentrated. The product Cy-NEO-PA was purified by high performance liquid chromatography;

[0019] (6) Compound Cy-NH2 was dissolved in DCM, and then EDCI and HOBT were added. DIPEA was stirred at room temperature under argon for 30 min. Subsequently, compound C6 was added and stirred for 3 h. After the reaction was completed, the reaction was diluted with DCM and extracted five times with saturated NaCl solution. The organic layer was dried over anhydrous Na2SO4 and concentrated by vacuum distillation. TFA was added to the residue, and the reaction mixture was stirred in an ice bath for 15 min and then dried by spin drying. The product Cy-NEO-NH2 was obtained by purification by high performance liquid chromatography.

[0020] In a third aspect, the present invention provides the photophysical properties of the Cy-NEO-PA probe and its responsiveness to PGA.

[0021] In a fourth aspect, the present invention provides real-time detection and imaging of Acinetobacter baumannii PGA activity.

[0022] In a fifth aspect, the present invention provides the effects of environmental factors on PGA activity and biofilm formation.

[0023] In a sixth aspect, the present invention provides the PTT / PDT properties of Cy-NEO-PA and its phototherapeutic antibacterial activity against Acinetobacter baumannii and its biofilms.

[0024] In a seventh aspect, the present invention provides Cy-NEO-PA in vivo photodynamic and photothermal synergistic treatment of bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The mechanism of Cy-NEO-PA in measuring PGA activity and its use as an antibacterial phototherapeutic agent is presented.

[0026] Figure 2 Shows the spectrum of Cy-NEO-PA and its response to PGA.

[0027] Figure 3 Confocal fluorescence imaging of Acinetobacter baumannii was achieved using Cy-NEO-PA.

[0028] Figure 4 Represents the effects of different environmental factors on PGA activity and biofilm formation.

[0029] Figure 5 Showing the photothermal and photodynamic properties of Cy-NEO-PA and Cy-NEO-NH2.

[0030] Figure 6 It represents the study of the antibacterial activity of Cy-NEO-PA under laser irradiation.

[0031] Figure 7 It represents the study of the anti-biofilm activity of Cy-NEO-PA under laser irradiation.

[0032] Figure 8 Represents near-infrared laser-mediated photodynamic and photothermal synergistic therapy in a mouse skin infection model. DETAILED DESCRIPTION

[0033] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0034] In the first aspect, the present invention provides a PGA-responsive fluorescent probe Cy-NEO-PA having a structure as shown in formula (1); the product probe Cy-NEO-NH2 having a structure as shown in formula (3);

[0035]

[0036]

[0037] Example 1 Preparation steps of compounds Cy-NEO-PA and Cy-NEO-NH2:

[0038]

[0039] Preparation Example 1, Synthesis of Compound C3

[0040] Compound C1 (218.26 mg, 2 mmol) was dissolved in 20 mL of ultra-dry DMF, followed by the addition of DIPEA (310.18 mg, 2.4 mmol). Compound C2 (309.18 mg, 2 mmol) was slowly added at -40°C under argon protection, and the mixture was stirred at -40°C for 3 hours. After completion of the reaction, the reaction solution was poured into 1000 mL of water for recrystallization. The resulting solid was filtered and dried to afford compound C3 (400 mg) as a white solid in an 89.9% yield. 1 H NMR (400MHz, DMSO-D6) δ9.90(s,1H),9.16(s,1H),7.37(d,J=8.8Hz,2H),7.33-7.28(m,4H),7.26-7.20(m,1H),6.68(d,J=8.8Hz,2H),3.57(s,2H). 13 C NMR(150MHz,DMSO)δ168.32,153.27,136.29,130.88,129.03,128.25,126.42,120.86,115.02,43.20.HRMS(m / z)[M+H] + :Calcd.for C 14 H 14 O2N 228.1019; found228.1020.

[0041] Preparation Example 2, Synthesis of Compound 1

[0042] Compound C5 (10 g, 57.93 mmol) was dissolved in anhydrous ethanol (579.3 mL), and CH3COONa (9.6 g, 115.9 mmol) was added. Compound C4 (18.2 g, 57.93 mmol) was slowly added, and the mixture was stirred at room temperature for 30 min under argon. Then, 1-(5-carboxypentyl)-2,3,3-trimethyl-3-hydrogen-indole-1-bromide (20.6 g, 57.93 mmol) was added. The reaction temperature was raised to 80°C, and the mixture was stirred under argon for 3 h. After the reaction, the solution was cooled to room temperature, filtered, and evaporated under reduced pressure. The residue was diluted with DCM, extracted five times with water, dried over anhydrous Na2SO4, and concentrated. Compound 1 was purified by silica gel column chromatography (4% MeOH / DCM) with a yield of 28% (11 g). 1 H NMR (400MHz, CDCl3) δ8.40-8.29(m,2H),7.43-7.38(m,2H),7.38-7.35(m,2H),7.27-7.24(m,1H), 7.23-7.20(m,1H),7.18(d,J=7.9Hz,1H),7.14(d,J=7.8Hz,1H),6.22(d,J=14.1Hz,1H),6.11(d,J= 14.0Hz,1H),4.21-4.07(m,4H),2.76-2.66(m,4H),2.63(t,J=7.1Hz,2H),1.99(p,J=5.7Hz,2H),1. 86(p,J=6.6Hz,2H),1.82-1.75(m,2H),1.70(s,12H),1.55(p,J=7.7Hz,2H),1.43(p,J=6.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ175.75,173.03,171.44,150.99,145.27,144.19,142.07, 141.91,141.20,141.12,129.10,128.93,127.92,127.50,125.71,125.20,122. 39,122.34,111.25,110.50,101.81,100.35,49.60,49.30,44.77,39.61,34.86 ,28.25,28.21,26.99,26.68,26.62,26.36,24.68,20.79,12.40.HRMS(m / z)[M] + :Calcd.for C 38 H 46 O2N2 + ,597.3242;found 597.3246.

[0043] Preparation Example 3, Synthesis of Compound 2

[0044] Compound C3 (100.54 mg, 0.442 mmol) was dissolved in ultra-dry DMF (4.42 mL) and 60% NaH (29.5 mg, 0.737 mmol) was added. The reaction mixture was stirred at room temperature under argon for 10 minutes, and compound 1 (200 mg, 0.295 mmol) was added. The temperature was raised to 40°C and the reaction was allowed to proceed for 3 hours. After completion of the reaction, the reaction mixture was diluted with DCM and extracted five times with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The resulting product 2 was purified by silica gel column chromatography (5% MeOH / DCM) with a yield of 78% (200 mg). 1 H NMR (400MHz, CDCl3) δ10.87(s,1H),7.97(d,J=9.1Hz,2H),7.91(d,J=14.5Hz,2H),7.44(d,J=7.3Hz,2H),7.33-7.27 (m,2H),7.24-7.19(m,3H),7.18-7.14(m,2H),7.13-7.08(m,2H),7.04(d,J=8.2Hz,1H),6.98(d,J=8.0Hz,1H),6.88( d,J=9.1Hz,2H),5.92(d,J=13.6Hz,1H),5.83(d,J=14.2Hz,1H),3.98-3.88(m,4H),3.87(s,2H),2.67-2.57(m,4H), 2.34(t,J=7.4Hz,2H),2.05-1.95(m,2H),1.76-1.62(m,4H),1.44-1.37(m,2H),1.34(t,J=7.2Hz,3H),1.29(s,12H). 13 C NMR (150MHz, CDCl3) δ172.11,171.80,170.42,165.02,156.17,142.62,142.44,142.02,14 1.57,141.19,140.97,135.35,133.75,129.55,128.65,128.57,126.86,125.15,122.40,12 2.34,122.26,122.22,114.53,110.36,110.12,99.37,99.12,49.16,44.16,43.96,39.22,3 3.97,33.90,29.72,27.87,27.80,26.86,26.27,24.39,24.25,21.02,12.09.HRMS(m / z)[M]+ :Calcd.for C 52 H 58 O4N3 + 788.4422; found788.4387.

[0045] Preparation Example 4, Synthesis of Compound 3

[0046] Compound 1,1-dimethylethyl (4-hydroxyphenyl) carbamate (46.3 mg, 0.221 mmol) was dissolved in DMF (2.21 mL), and 60% NaH (8.83 mg, 0.221 mmol) was added. The mixture was stirred at room temperature under argon for 10 minutes, and compound 1 (100 mg, 0.147 mmol) was added. The temperature was raised to 80°C and stirred for 3 hours. After the reaction, the solution was diluted with 15 mL of DCM and extracted with water five times. The organic layer was dried over anhydrous sodium sulfate and concentrated. The product 3 was purified by silica gel column chromatography (6% MeOH / DCM) in an 88.7% yield (110.9 mg). 1 H NMR (600MHz, CDCl3) δ7.94(d,J=14.21Hz,1H),7.89(d,J=14.03Hz,1H),7.47-7.42(m,2H),7.37-7.31(m,2H),7.26-7.24(m,2 H),7.20(d,J=7.43Hz,1H),7.16(dd,1H),7.09(d,J=7.79Hz,1H),7.02(d,J=7.89Hz,1H),7.00-6.97(m,2H),6.07(d,J=14.40 Hz,1H),5.92(d,J=14.12Hz,1H),4.05-3.97(m,4H),2.72(t,J=5.87Hz,2H),2.67(t,J=5.96Hz,2H),2.54(t,J=7.20Hz,2H),2 .08-2.02(m,2H),1.83-1.78(m,2H),1.77-1.71(m,2H),1.48(s,12H),1.38(t,J=7.01Hz,3H),1.35(s,9H),1.26-1.22(m,2H). 13C NMR (150MHz, CDCl3) δ175.74,172.77,170.68,164.95,155.66,153.21,143.19,141.98,141.83,1 41.60,141.10,141.02,133.58,128.83,128.61,125.38,124.70,123.03,122.63,122.28,122.21, 120.64,114.87,110.87,109.85,100.52,98.70,80.35,49.30,48.85,44.54,39.13,34.55,29.73, 28.39,27.94,27.91,26.84,26.19,24.51,24.44,24.38,21.09,12.14.HRMS(m / z)[M]+:Calcd.for C 49 H 60 O5N3 + ,770.4527;found 770.4485.

[0047] Preparation Example 5, Synthesis of Compound Cy-NEO-PA

[0048] Compound 2 (20.43 mg, 0.0235 mmol) was dissolved in DCM (2.35 mL), followed by the addition of EDCI (9.01 mg, 0.047 mmol), HOBT (5.42 mg, 0.047 mmol), and DIPEA (6.075 mg, 0.047 mmol) and activation under argon at room temperature for 30 min. Subsequently, compound C6 (31.4 mg, 0.0259 mmol) was added, and the mixture was stirred for 3 h. After completion of the reaction, the reaction solution was diluted with DCM and extracted five times with saturated NaCl solution. The organic layer was then dried over anhydrous NaSO and concentrated by vacuum distillation. The residue was diluted with TFA (2.35 mL) in an argon ice bath. The reaction mixture was stirred in an ice bath for 15 min and then concentrated. Cy-NEO-PA was purified by HPLC (68% acetonitrile / water) to yield 16 mg, with a yield of 50.25%. 1H NMR(600MHz,CD3OD)δ8.02(d,J=14.2Hz,1H),7.97(d,J=14.0Hz,1H),7.59(d,J=9.2Hz,2H),7.39(t,J=7.7Hz,2H),7.37-7.34(m,2H),7.34-7.32(m,2H),7.31(t,J=2.7Hz,1H),7.30-7.26(m,2H),7.24(t,J=1.8Hz,1H),7.23(t,J=2.4Hz,1H),7.22(t,J=1.7Hz,1H),7.20(t,J=7.8Hz,1H),7.10-7.06(m,2H),6.17(d,J=14.3Hz,1H),6.10(d,J=14.1Hz,1H),5.83(d,J=3.9Hz,1H),5.40(d,J=5.1Hz,1H),5.30(d,J=1.7Hz,1H),4.40(dd,J=5.3,3.8Hz,1H),4.30-4.27(m,1H),4.25-4.19(m,2H),4.15(t,J=6.7Hz,2H),4.14(t,J=3.1Hz,1H),4.08(d,J=5.5Hz,1H),4.06(t,J=5.2Hz,2H),4.04-4.00(m,1H),4.00-3.97(m,1H),3.88(t,J=9.0Hz,1H),3.69-3.67(m,1H),3.66(d,J=2.9Hz,2H),3.64(s,4H),3.59-3.54(m,1H),3.51(d,J=3.9Hz,1H),3.49(d,J=3.8Hz,1H),3.44-3.41(m,2H),3.41-3.39(m,1H),3.38-3.35(m,1H),3.35-3.33(m,1H),3.25(d,J=3.6Hz,1H),3.23(d,J=3.5Hz,1H),3.17(dd,J=13.5,8.0Hz,1H),2.73(s,4H),2.48-2.42(m,1H),2.31-2.23(m,2H),2.08(t,J=12.8Hz,1H),2.05-2.00(m,2H),1.80(p,J=7.5Hz,2H),1.71-1.64(m,2H),1.50-1.42(m,2H),1.39(d,J=19.1Hz,1H),1.36(s,6H),1.35(s,9H),1.29(d,J=6.0Hz,4H). 13C NMR (150MHz, CD3OD) δ175.29,172.40,171.71,170.73,163.96,156.46,142.5 1,142.20,141.56,141.41,141.27,140.94,135.32,133.30,129.40,128.68,1 28.39,128.26,128.16,126.54,125.00,124.53,122.02,121.67,121.59,114.47,110.46,110.25,107.99,99.54,99.01,95.73,95.47,85.18,81.95,76.50, 75.19,74.22,72.58,72.06,71.60,70.69,70.35,68.02,67.78,60.80,53.58,51.43,49.60,49.01,48.82,48.69,43.39,43.10,40.42,40.22,38.74,35.46, 31.61,29.40,29.37,29.29,29.17,29.14,29.01,28.87,28.07,26.79,26.59, 26.34,25.47,25.26,23.77,23.70,22.28,20.98,12.99,10.94.HRMS(m / z)[M] + :Calcd.for C 75 H 103 O 15 N 10 + 1383.7599; found 1383.7662.

[0049] Preparation Example 6, Synthesis of Compound Cy-NEO-NH2

[0050] Compound Cy-NH2 (20 mg, 0.0235 mmol) was dissolved in DCM (2.35 mL), followed by the addition of EDCI (9.01 mg, 0.047 mmol), HOBT (5.42 mg, 0.047 mmol), and DIPEA (6.075 mg, 0.047 mmol). The mixture was stirred at room temperature under argon for 30 minutes. Subsequently, compound C6 (31.4 mg, 0.0259 mmol) was added and stirred for 3 hours. After completion of the reaction, the reaction mixture was diluted with DCM and extracted five times with saturated NaCl solution. The organic layer was dried over anhydrous Na2SO4 and concentrated by distillation under reduced pressure. TFA (2.35 mL) was added to the residue, and the reaction mixture was stirred in an ice bath for 15 minutes before being spin-dried to dryness. The product, Cy-NEO-NH2, was purified by HPLC (acetonitrile / water ratio 44%) to yield 17 mg, with a yield of 57.12%. 1H NMR(600MHz,CD3OD)δ8.00(d,J=14.2Hz,1H),7.95(d,J=14.0Hz,1H),7.40(t,J=7.4Hz,2H),7.37(t,J=7.7Hz,2H),7.29(d,J=7.9Hz,1H),7.27-7.23(m,2H),7.23(s,1H),7.21(t,J=7.7Hz,2H),7.19-7.15(m,2H),6.19(d,J=14.3Hz,1H),6.12(d,J=14.1Hz,1H),5.82(d,J=3.9Hz,1H),5.40(d,J=5.1Hz,1H),5.30(d,J=1.8Hz,1H),4.39(q,J=5.4,3.7Hz,1H),4.30-4.27(m,1H),4.22(t,1H),4.20(t,J=6.4Hz,1H),4.17(d,J=7.2Hz,2H),4.14(t,J=3.1Hz,1H),4.08(t,J=7.3Hz,2H),4.06(t,J=5.3Hz,1H),4.03-4.00(m,1H),4.00-3.98(m,1H),3.88(t,1H),3.69-3.67(m,2H),3.66-3.62(m,2H),3.57-3.55(m,1H),3.51(d,J=3.9Hz,1H),3.49(d,J=3.9Hz,1H),3.45-3.43(m,1H),3.43-3.42(m,1H),3.41-3.40(m,1H),3.38(d,1H),3.37-3.35(m,1H),3.35-3.32(m,1H),3.28-3.26(m,1H),3.24(t,J=3.6Hz,1H),3.21-3.16(m,1H),2.74(p,J=6.3Hz,4H),2.48-2.43(m,1H),2.30-2.25(m,2H),2.11-2.06(m,1H),2.06-2.01(m,2H),1.81(p,J=7.8Hz,2H),1.68(q,J=6.5Hz,2H),1.47(q,J=8.0Hz,2H),1.41-1.38(m,2H),1.37(d,J=4.4Hz,12H),1.35(s,3H),1.29(d,J=6.0Hz,2H). 13C NMR (150MHz, CD3OD) δ176.74,173.80,173.16,164.98,158.99,143.70,143.63,143.02,142.67,142.36,130.85,129.91,129.7 8,126.52,126.07,123.69,123.42,123.37,123.03,122.96,117.05,111.99,111.79,109.50,101.12,100.62,97.22,96.98,86 .66,83.45,77.97,76.67,75.70,74.07,73.53,73.02,72.14,71.84,69.46,69.26,69.23,62.26,55.02,52.89,51.03,50.44,5 0.27,50.13,44.89,41.86,41.67,40.23,36.92,29.47,28.25,28.06,27.80,26.70,25.21,25.15,22.38,12.40.HRMS(m / z)[M] + :Calcd.for C 67 H 97 O 14 N 10 + 1265.7180; found1265.7242.

[0051] Pharmacological experiments

[0052] Experimental Example 1: Spectral properties of Cy-NEO-PA and its response to PGA enzyme.

[0053] Prepare 5 mM stock solutions of Cy-NEO-PA and Cy-NEO-NH2 in DMSO. Dilute the probe stock solutions to 10 μM in DMSO or TRIS buffer containing 1.5% DMSO. Measure the absorption and fluorescence spectra. Figure 2 .

[0054] Experimental Example 2: Application of the probe Cy-NEO-PA to achieve confocal fluorescence imaging of Acinetobacter baumannii.

[0055] Acinetobacter baumannii ATCC 19606 and Bio-53272 were cultured in NB medium and LB medium at 37°C for 12 h, respectively. The two strains cultured overnight were collected and washed twice with Tris buffer. Cy-NEO-PA (10 μM) was incubated with ATCC 19606 and Bio-53272 (OD 0.000) in the presence or absence of the PGA inhibitor penicillin (100 mM).600 =0.5) and incubated. After 1 h of incubation, the supernatant was centrifuged and washed three times with Tris-buffered saline. 8 μL of the suspension was added to an 8-well plate covered with agarose gel to obtain bacterial samples for confocal imaging. Fluorescence images were obtained using a confocal microscope (Leica TCS SP8X) with an HC PL Apo6 3× oil objective and a λ ex =670nm,λ em =750-800nm, see Figure 3 .

[0056] Experimental Example 3: Effects of different environmental factors on PGA activity and biofilm formation.

[0057] To evaluate the effects of different environmental factors on PGA activity in Acinetobacter baumannii (Bio-53272), LB medium was supplemented with different substances: 50 mM KCl, 50 mM MgCl2, 10 mM CaCl2, 1% glucose (Glu), 1% galactose (Gal), 1% glycine (Gly), 1% glycerol (Gl), or 0.05% PAA. Cy-NEO-PA (10 μM) was added to Bio-53272 (OD 600 =0.1) were co-incubated under different culture medium conditions. After 1 hour, the fluorescence intensity (λ ex =740nm,λ em =785nm). The blank group was treated with Cy-NEO-PA dissolved in Tris-buffered saline containing 1.5% DMSO and 0.1% Kolliphor RH40 at a final concentration of 10μM. Relative PGA activity (%) of each group = (Fl blank -Fl experimental ) / (Fl blank -Fl control ).

[0058] Acinetobacter baumannii (Bio-53272) was cultured overnight in LB medium supplemented with different concentrations of glucose and phenylacetic acid. The control group consisted of bacteria cultured in LB medium without glucose and phenylacetic acid, and the other groups were experimental groups. The bacteria were resuspended in appropriate culture medium to a concentration of 1×10 8CFU / mL concentration, added to 96-well plates (100 μL per well), and incubated at 37°C for 48 hours. After incubation, the supernatant was removed, and the biofilm was washed with 100 μL of sterile PBS buffer and dried. The biofilm was fixed with 100 μL of anhydrous methanol for 20 minutes, and then stained with 100 μL of 0.4% crystal violet staining solution at room temperature for 30 minutes. After that, it was washed with 100 μL of sterile PBS buffer to remove the unbound crystal violet staining solution, and the biofilm was dried. The staining of the biofilms in each group was observed under an optical microscope, and 200 μL of 33% acetic acid was added to release the bound crystal violet dye. abs = 595nm to measure the absorbance of each well. Biofilm survival rate (%) = OD 595 (Experimental group) / OD 595 (Control group). Results are shown in Figure 4 .

[0059] Experimental Example 4: Photothermal and photodynamic properties of Cy-NEO-PA and Cy-NEO-NH2.

[0060] In order to evaluate the ability of Cy-NEO-PA and Cy-NEO-NH2 to generate reactive oxygen species (ROS) in solution, we used the ROS fluorescent probe DCFH-DA (2,7-dichlorofluorescein diacetate) for detection. First, after adding 10mM NaOH under light-proof conditions, DCFH-DA (1mM) was hydrolyzed to generate DCFH at room temperature. Then Tris buffer (pH 7.4) was added to terminate the deacetylation process. The DCFH solution was further diluted to 40μM with Tris buffered saline and stored frozen. Subsequently, Cy-NEO-PA or Cy-NEO-NH2 was diluted to 10μM with the above DCFH solution in a 96-well plate. Then, laser (λ ex =785nm,1.2W / cm 2 ) for 30 s, and the fluorescence intensity (λ ex =480nm,λ em =525nm). The experiment was repeated three times independently. The results are shown in Figure 5 .

[0061] Experimental Example 5-6: Study on the antibacterial and anti-biofilm activities of Cy-NEO-PA under laser irradiation.

[0062] Scanning electron microscopy was used to observe the effects of Cy-NEO-PA on Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii at the bacterial and biofilm levels.

[0063] Bacterial cultures were collected, washed with Tris-buffered saline, and resuspended in Tris buffer containing 1.5% DMSO and 0.1% Kolliphor RH40. Experimental and control groups were set up. The experimental group of bacteria was co-incubated with 10 μM Cy-NEO-PA, while the control group was incubated alone without the probe. Laser (785 nm, 1.2 W / cm 2 After 1 min of irradiation, the samples were fixed with 2.5% glutaraldehyde solution, dehydrated with gradient ethanol, dried with CO2 critical point, and subjected to SEM imaging after gold spraying.

[0064] In the biofilm level experiment, the strains were cultured in the optimal medium at 37 °C for 12 h and then diluted to 10 8 CFU / mL and inoculated into 96-well plates. After incubation for 24 h, Cy-NEO-PA was added at a final concentration of 5 μM or 10 μM. After incubation for 1 h, laser (785 nm, 1.2 W / cm 2 ) for 1 minute and incubate at 37°C for 24 hours. Discard the supernatant, fix the biofilm with anhydrous methanol, and stain with crystal violet. Finally, add 33% acetic acid to release the bound crystal violet dye. Measure the absorbance at 595 nm using a microplate reader to calculate the biofilm biomass (%) = OD 595 (sample) / OD 595 (control).

[0065] Calcein-AM / PI staining experiments were used to evaluate the effects of Cy-NEO-PA on ATCC 19606 and Bio-53272 at the bacterial and biofilm levels. The bacterial treatment methods for the control and experimental groups were the same as those for the scanning electron microscopy experiments. After laser irradiation, 200 μL of Calcein-AM / PI detection working solution was given to each bacterial group. Confocal fluorescence imaging was performed after incubation for 30 minutes. The biofilm pretreatment method was the same as the scanning electron microscopy imaging experimental method. 200 μL of Calcein-AM / PI detection working solution was added to each well, and confocal fluorescence imaging was performed after incubation for 30 minutes (Calcein-AM: λ ex =494nm,λ em =500-525nm; PI-DNA: λ ex =535nm,λ em =600-700nm). The results are shown in Figure 6-7 .

[0066] Experimental Example 7: Bacteria-targeted probe-mediated photodynamic and photothermal synergistic therapy for skin infection in mice.

[0067] Twenty-four 6-8 week old BALB / c mice were randomly divided into four groups: PBS, PBS+Laser (785 nm, 1.2 W / cm2 ,5min), Cy-NEO-PA, Cy-NEO-PA+Laser (785nm, 1.2W / cm 2 After the mice were anesthetized, they were depilated with depilatory cream, and a full-thickness wound of approximately 0.8 cm was created on the back skin. 2 , and then subcutaneously injected with bacterial suspension (10 8 CFU / mL). 50 μL (20 μM) of the probe was injected in situ for 4 consecutive days, and then the wound was irradiated with laser for 5 minutes. The wound area was monitored using a digital camera every other day to evaluate the healing rate. The results are shown in Figure 8 .

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Claims

1. A near-infrared cyanine fluorescent probe, characterized in that: The hemicyanine fluorescent probe has a structure shown in formula (1):

2. Use of the near-infrared cyanine fluorescent probe according to claim 1 in detecting the activity of penicillin acylase (PGA), EC 3.5.1.

11.

3. The method for preparing the near-infrared cyanine fluorescent probe according to claim 1, characterized in that: The method comprises conjugating a compound represented by formula (2) with a modified neomycin in an organic solvent under nucleophilic reaction conditions; 4. Use of the near-infrared cyanine fluorescent probe according to claim 1 in the preparation of a phototherapy antibacterial agent, characterized in that: The pathogenic bacteria or the target pathogenic microorganisms of the photodynamic and photothermal antibacterial treatments are Gram-positive bacteria, Gram-negative bacteria or fungi.

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

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