Near-infrared hemicyanine probe for detecting bacterial leucine aminopeptidase as well as preparation method and application of near-infrared hemicyanine probe

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a preparation method and application of a near-infrared hemicyanine fluorescent probe Hcy-NEO-Leu for detecting bacterial leucine aminopeptidase. The research aims to develop a near-infrared fluorescent probe Hcy-NEO-Leu capable of responding to leucine aminopeptidase (LAP), a series of lycorine derivatives are screened, the influence of the lycorine derivatives on the activity of the leucine aminopeptidase and the influence of the lycorine derivatives on biological membrane formation are researched, and finally a compound LY-58 with the inhibition effect superior to that of an inhibitor ubenimex is successfully screened. The near-infrared fluorescent probe Hcy-NEO-Leu not only has high sensitivity, good water solubility and excellent biocompatibility, but also has a bacterium targeting capability. Due to the characteristics, the probe Hcy-NEO-Leu can achieve implant infection imaging in vitro and in vivo, and the probe Hcy-NEO-Leu has important significance on later surgical debridement and treatment. In addition, the probe has wide application prospects in the fields of inhibitor screening, in-vivo and in-vitro imaging and the like, and a new breakthrough is brought to research and clinical practice in the field of medicine.

Description

Technical Field

[0001] This invention relates to the field of bacterial leucine aminopeptidase detection, specifically to a near-infrared semicyanine fluorescent probe, a method for preparing the near-infrared semicyanine fluorescent probe, its application in detecting leucine aminopeptidase activity, and its application in biomembrane imaging and inhibitor screening. Background Technology

[0002] Antibiotic resistance caused by overuse has gradually become a major challenge threatening human health. Besides traditionally known mechanisms of antibiotic resistance, such as efflux pumps, modifying enzymes, and target mutations... [1] The formation of bacterial biofilms also constitutes an important line of defense against attacks from antibiotics and the immune system. [2,3] Biofilms are formed by microbial communities embedded in their extracellular polymeric matrix (EPS). These microbial communities irreversibly attach to living or non-living surfaces, exhibiting complex spatial organization and metabolic interactions. Biofilm formation is a continuous and multi-stage phenomenon, beginning with the initial attachment of free microorganisms, followed by the formation of microcolonies and cell growth, maturation, or development, ultimately enabling the biofilm to diffuse and colonize new surfaces. Compared to planktonic bacteria, bacteria within biofilms are significantly more resistant to adverse external environments due to the protection of the EPS. The threat posed by these persistent bacterial biofilms continues to challenge public health. [4] .

[0003] Statistics show that up to 80% of human bacterial infections are closely related to biofilms. [5] These biofilm-related diseases not only increase morbidity and mortality rates but also impose a significant economic burden on patients. Typical biofilm-related diseases include respiratory diseases, autologous valvular endovascular inflammation, chronic otitis media, eye infections, chronic wounds, diabetic foot ulcers, urinary tract infections, and gingivitis. [6-9] In addition, biofilms can also form on non-biological surfaces, such as orthopedic prostheses, artificial heart valves, coronary artery stents, intravascular and urinary catheters, neurosurgical devices, cochlear implants and breast implants, dentures, and medical devices such as ventricular assist devices and eye devices.

[10] These infections associated with bacterial biofilms are often chronic and difficult to cure completely.

[0004] Currently, several technologies are available for detecting bacterial biofilms, including mass spectrometry imaging. [11-14] Raman

[15] and infrared spectrum

[16] Scanning electrochemical microscopy technique [17,18] and fluorescence imaging technology [19,20]Among these, fluorescence imaging technology, with its unique advantages such as high sensitivity, high resolution, and real-time imaging capabilities, shows great potential in the field of biofilm detection. Therefore, in this study, we focused on developing an activated near-infrared fluorescent probe and selected a near-infrared fluorescent nucleus, which not only increased tissue penetration but also enabled real-time imaging of bacterial biofilms. This innovative method provides strong technical support for the early diagnosis and effective treatment of biofilm-related diseases.

[0005] Pseudomonas aeruginosa (PAO1) is the leading cause of chronic infections, particularly cystic fibrosis and other hospital-acquired infections. These chronic infections often manifest as biofilm infections, which are persistent and difficult to cure. Leucine aminopeptidase (LAP) is one of the most abundant proteins in the biofilm matrix. LAP expression not only promotes the production of Psl polysaccharides in biofilms but is also closely related to biofilm development and participates in important biological processes such as bacterial virulence and nutrient acquisition. Therefore, selecting P. aeruginosa leucine aminopeptidase as a research subject to develop a visualization tool for rapid diagnosis and real-time monitoring of bacterial biofilms has clinical and scientific significance for subsequent surgical debridement and treatment. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of limited tissue penetration in traditional imaging methods, and to provide a leucine aminopeptidase-activated near-infrared fluorescent probe with high specificity, high sensitivity and simple synthesis route, as well as its preparation method and application.

[0007] In a first aspect, the present invention provides the structure of the LAP-responsive fluorescent probe Hcy-NEO-Leu as shown in formula (1); and the structure of the product probe Hcy-NEO-OH as shown in formula (3);

[0008]

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

[0010]

[0011] (a)1) K2CO3, DMF, 50℃, 30min; 2) 50℃, 3.5h; (b) EEDQ, DCM, 25℃, 2h; (c) PBr3, DCM, 0℃, 15min.

[0012] (1) Compound C2 was dissolved in 5 mL of DMF, and then potassium carbonate was added. After stirring at room temperature for 30 minutes, compound C1 was added, and the reaction mixture was stirred at 50 °C for 3.5 hours. After the reaction was completed, the reaction solution was poured into 50 mL of DCM for dilution, washed with 0.1 N hydrochloric acid and saturated saline, dried the organic phase, filtered and concentrated under reduced pressure, and purified by column chromatography to obtain compound C3;

[0013] (2) Compound C4 was dissolved in DCM, and C5 and EEDQ were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was poured into 1000 mL of DCM and washed with deionized water and saturated saline. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound C6, which was directly used in the next step.

[0014] (3) Compound C6 was dissolved in DCM, and then phosphorus tribromide was added. The reaction mixture was stirred at 0°C for 15 minutes. An equal volume of NaHCO3 was added to terminate the reaction. The combined organic phases were dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and purified by chromatographic column to obtain product C7.

[0015]

[0016] (a) Et3N, DCM, 25℃, 3h; (b) Et3N, DMF, 25℃, 12h.

[0017] (4) Compound C3 was dissolved in DCM and added to a reaction flask, followed by the addition of compound C8 and triethylamine. The reaction mixture was then stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was poured into 12 mL of DCM and washed with saturated saline and deionized water. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound C9, which was directly used in the next step.

[0018] (5) Compound C9 was dissolved in DMF, followed by the addition of compound C10 and triethylamine. The reaction system was stirred at room temperature for 2 hours. After the reaction was completed, the reaction solution was poured into 12 mL of DCM and washed with saturated saline and deionized water. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound C11.

[0019]

[0020] (a) TFA, 0℃, 15min; (b) DIPEA, DMF, 60℃, 12h.

[0021] (6) Trifluoroacetic acid was added dropwise to the dry round-bottom flask containing compound C11. The reaction system was continued to react at 0°C for 15 minutes. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was directly purified by column chromatography to obtain compound Hcy-NEO-OH;

[0022] (7) Compound C7 was dissolved in DMF and added to a reaction flask, followed by compound C11 and DIPEA. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was poured into 14.2 mL of DCM and washed with deionized water and saturated saline. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was added to TFA, and the reaction mixture was stirred in an ice bath for 15 min and then evaporated to dryness. The product Hcy-NEO-Leu was purified by high performance liquid chromatography.

[0023] Thirdly, this invention provides the photophysical properties of the Hcy-NEO-Leu probe and its responsiveness to LAP.

[0024] Fourthly, this invention provides a method for screening LAP-active enzyme inhibitors, and discovers highly active LAP inhibitors. The structure of the lycorine derivative LY-58 is shown in formula (4); the spectral data of the lycorine derivative LY-58 are as follows: 1 H NMR (400MHz, Acetone-d6) δ10.04(s,1H,NH),7.82(s,1H,Ar-H),7.37-7.11(m,4H,Ar-H) ,6.90(s,1H,H-11),6.63(s,1H,H-8),6.04(s,1H,H-2),5.94(s,2H,H-12),5.62-5.59(m ,1H,H-3),4.80-4.72(m,1H,H-1),4.38(s,1H,OH),4.11(d,J=14.1Hz,H-7),3.43(d,J=1 4.1Hz,H-7),3.31-3.27(m,1H),2.87-2.81(m,1H),2.67-2.53(m,3H),2.35-2.28(m,1H). 13 C NMR (100MHz, Acetone-d6) δ188.22,147.17,146.92,131.01,129.66,129.39,125.89,123.54,1 22.74,107.91,106.01,101.76,68.92,68.81,61.76,57.82,54.36,43.07,29.34.HRMS(ESI)m / z calcd for C 23 H 23 N₂O₄S[M+H]+ 423.1373, found 423.1358.

[0025]

[0026] Fifthly, the present invention provides real-time detection and imaging of LAP activity in Pseudomonas aeruginosa.

[0027] In a sixth aspect, the present invention provides real-time imaging of bacterial biofilm infection in vivo by Hcy-NEO-Leu. Attached Figure Description

[0028] Figure 1 This demonstrates the mechanism by which Hcy-NEO-Leu measures LAP activity and its application in in vivo and in vitro imaging.

[0029] Figure 2 This indicates the optical properties of Hcy-NEO-Leu and its response to LAP.

[0030] Figure 3 This indicates that the Hcy-NEO-Leu probe was used to screen for inhibitors of LAP active enzymes.

[0031] Figure 4 This demonstrates the use of Hcy-NEO-Leu to achieve confocal fluorescence imaging of Pseudomonas aeruginosa.

[0032] Figure 5 This indicates that the probe Hcy-NEO-Leu enables confocal 3D fluorescence imaging of Pseudomonas aeruginosa biofilms.

[0033] Figure 6 This indicates in vitro imaging of common implant infections by the probe.

[0034] Figure 7 This indicates that the probe is used to image skin implants in mice in real time. Detailed Implementation

[0035] 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.

[0036] In a first aspect, the present invention provides the structure of the LAP-activated fluorescent probe Hcy-NEO-Leu as shown in formula (1); and the structure of the product probe Hcy-NEO-OH as shown in formula (3);

[0037]

[0038] Example 1: Preparation steps of compounds Hcy-NEO-Leu and Hcy-NEO-OH:

[0039]

[0040]

[0041] Preparation Example 1: Synthesis of Compound C3

[0042] Compound C2 (121.96 mg, 1.108 mmol) was dissolved in DMF and added to a reaction flask. Potassium carbonate (306.15 mg, 2.215 mmol) was then added to the flask, and the mixture was stirred at room temperature for 30 minutes. Compound C1 (379 mg, 0.554 mmol) was then added, and the reaction mixture was stirred at 50 °C for 3.5 hours. After the reaction was complete, the reaction solution was poured into dichloromethane and washed with 0.1 N hydrochloric acid and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give compound C3, 93 mg, in a yield of 29.7%. 1 H NMR(400MHz, Methanol-d4)δ8.73(d,J=14.7Hz,1H),7.64(d,J=7.4Hz,1H),7.52(s,1H),7.51 (s,1H),7.43(q,1H),7.42(d,J=2.6Hz,1H),7.40(d,J=4.7Hz,1H),6.86(t,1H),6.84(t,1H),6 .45(d,J=14.8Hz,1H),4.33(t,J=7.5Hz,2H),3.35(s,1H),2.78(t,2H),2.71(t,J=6.1Hz,2H) ,2.32(t,J=7.2Hz,2H),1.98-1.87(m,4H),1.81(s,6H),1.75-1.67(m,2H),1.57-1.48(m,2H). 13 C NMR (151MHz, CD3OD) δ178.40,177.56,163.72,156.12,146.52,143.25,143.03,136.26,130.48,130.22,128.10,127.49,123.81,116.3 2,115.97,115.67,113.65,104.02,102.98,51.77,45.91,34.91,29.96,28.55,28.38,27.34,25.75,25.08,21.70.HRMS(ESI)m / zcalcd for C 31 H 34 N1O4 + 484.2482 [M] +;found:484.2474.

[0043] Preparation Example 2, Synthesis of Compound C7

[0044] Compound C4 (2312.9 mg, 10 mmol) was dissolved in DCM and added to a reaction flask, followed by Boc-L-leucine (1231.6 mg, 10 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline EEDQ (4945.8 mg, 20 mmol). The reaction mixture was then stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was poured into dichloromethane and washed with deionized water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound C6, which was directly used in the next step.

[0045] Compound C6 (1682.2 mg, 5 mmol) was dissolved in DCM, followed by the addition of phosphorus tribromide (1353.5 mg, 5 mmol). The reaction mixture was stirred at 0 °C for 15 minutes. The reaction was terminated by adding an equal volume of NaHCO3. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give product C7. The two-step yield was 56.5%. 1 H NMR(400MHz,Chloroform-d)δ8.65(s,1H),7.47(d,2H),7.28(d,J=8.0Hz,2H),5.04(d,J=7.9Hz,1H),4.45(s,2H ),4.27(s,1H),1.79-1.70(m,2H),1.62-1.52(m,1H),1.45(s,9H),0.97(d,J=6.2Hz,3H),0.95(d,J=6.0Hz,3H); 13 C NMR(151MHz, CDCl3)δ171.21,156.59,138.19,133.49,129.87,120.01,80.83,53.98,40.64,33.62,28.47,24.92,23.11,22.01.HRMS(ESI)m / zcalcd for C 18 H 28 BrN2O3 + 399.1278 [M] + ;found:399.1267.

[0046] Preparation Example 3: Synthesis of Compound C11

[0047] Compound C3 (20 mg, 0.0356 mmol) was dissolved in DCM and added to a reaction flask, followed by the addition of N,N'-disuccinimidyl carbonate (9.12 mg, 0.0356 mmol) and triethylamine (6.46 μL, 0.0427 mmol). The reaction mixture was then stirred at room temperature for 2 hours. After the reaction was complete, the reaction mixture was poured into dichloromethane and washed with saturated brine and deionized water. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound C9, which was directly used in the next step.

[0048] Compound C9 (15 mg, 0.0227 mmol) was dissolved in N,N-dimethylformamide, followed by the addition of compound C10 (27.6 mg, 0.0227 mmol) and dropwise addition of triethylamine (10.3 μL, 0.0682 mmol). The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was poured into dichloromethane (12 mL) and washed with saturated brine and deionized water. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give compound C11. The two-step yield was 39.8%. 1H NMR(600MHz,Methanol-d4)δ8.76(d,J=14.8Hz,1H),7.64(d,J=7.4Hz,1H),7.56-7.50(m,2H),7.45-7.43(m,2H),7.43-7.41(m,1H),6.87(s,1H),6.85(d,J=2.3Hz,1H),6.46(d,J=14.8Hz,1H),5.43(s,1H),5.07(s,1H),4.89-4.86(m,1H),4.33(t,J=7.4Hz,2H),4.30-4.26(m,1H),4.07-4.01(m,1H),3.98(d,J=13.6Hz,1H),3.95-3.91(m,1H),3.91-3.84(m,2H),3.75(s,1H),3.74-3.69(m,1H),3.59-3.55(m,1H),3.55-3.51(m,2H),3.51-3.48(m,2H),3.47(s,1H),3.46-3.42(m,1H),3.40-3.36(m,1H),3.36-3.32(m,2H),3.29-3.26(m,2H),3.25-3.21(m,1H),3.15(t,J=9.2Hz,1H),3.06(t,J=10.4Hz,1H),2.80(t,2H),2.73(t,2H),2.42-2.31(m,2H),1.99-1.89(m,5H),1.78-1.72(m,2H),1.63-1.57(m,1H),1.57-1.51(m,3H),1.44(s,12H),1.43(s,6H),1.43(s,9H),1.42(s,18H),1.40(s,9H),1.32-1.27(m,6H),0.94-0.86(m,3H); 13C NMR (151MHz, CD3OD) δ178.49,176.08,163.67,163.58,158.86,158.46,158.2 3,157.87,156.19,146.73,143.25,143.03,136.28,130.83,130.45,130.30,1 28.13,127.66,123.76,116.39,115.82,115.66,113.71,112.15,104.03,102.98,100.61,98.98,88.46,80.98,80.88,80.71,80.66,80.33,80.30,79.80,7 6.24, 75.59, 74.46, 73.47, 73.38, 72.67, 72.61, 71.66, 71.57, 68.99, 56.94, 53.60, 52.53, 51.79, 51.41, 45.94, 44.02, 42.75, 41.96, 36.54, 35.68, 34.94, 33.05,30.82,30.73,30.61,30.59,30.45,30.32,30.00,29.02,28.90,28.84, 28.80,28.75,28.48,28.42,28.10,27.50,26.75,25.13,21.76.HRMS(ESI)m / z calcd for C 84 H 127 N8O 27 + :1679.8805[M] + Found: 1679.8807.

[0049] Preparation Example 4: Synthesis of compound Hcy-NEO-OH

[0050] Trifluoroacetic acid was added dropwise to compound C11. The reaction system was continued to react at 0 °C for 15 minutes. After the reaction was completed, the mixture was directly concentrated under reduced pressure, and the residue was directly purified by column chromatography to give compound Hcy-NEO-OH, 12 mg, with a yield of 48.6%. ¹H NMR (600 MHz, Methanol-d⁴) δ 8.77 (d, J = 14.7 Hz, 1H), 7.64 (d, 1H), 7.52 (t, J = 7.0 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H), 7.47–7.41 (m, 3H), 6.89–6.88 (m, 1H), 6.86 (t, J = 2.1 Hz, 1H), 6.45 (d, J = 14.7 Hz, 1H), 5.82 (d, J = 2.4 Hz, 1H), 5 .40(d,J=5.1Hz,1H),5.29(s,1H),4.39(t,J=5.2Hz,1H),4.32(t,J=7.5Hz,2H),4.27(dd,J=6.9,2.9Hz,1H),4 .24-4.19(m,2H),4.13(q,J=2.4,1.9Hz,1H),4.05(t,J=4.6Hz,1H),4.02-3.97(m,2H),3.88(t,J=9.0Hz,1H), 3.67-3.65(m,2H),3.65-3.62(m,1H),3.52-3.47(m,2H),3.43(d,J=4.8Hz,1H),3.42(q,J=5.8,4.8Hz,2H),3. 39(d,J=4.6Hz,1H),3.38-3.35(m,1H),3.33(d,J=8.8Hz,1H),3.28-3.22(m,2H),3.17(dd,J=13.4,7.9Hz,1H) ,2.79(t,J=6.1Hz,2H),2.71(t,J=6.2Hz,2H),2.48-2.42(m,1H),2.28(dd,J=9.2,6.4Hz,2H),2.07(q,J=12.6 ,11.7Hz,1H),1.97-1.93(m,2H),1.93-1.87(m,2H),1.82(s,6H),1.73-1.66(m,2H),1.51(p,J=7.6Hz,2H); 13C NMR (151MHz, CD3OD) δ178.45,176.66,163.86,163.71,156.25,146.78,143.25,143.02,136.57,136 .54,130.50,130.18,128.07,127.58,123.79,116.41,115.91,115.63,113.51,109.41,103.78,102.95,97.20,96.94,86.60,83.42,77.93,76.57,75.70,74.03,73.04,72.13,71.82,69.44,69.24,55.02,52.87,51.74,5 1.02,50.25,45.88,41.87,41.83,41.65,36.86,29.94,29.41,28.48,28.45,27.74,26.65,25.05,21.71.HRMS(ESI)m / z calcd for C54H79N8O15+:1679.5659[M]+; found:1679.5663.

[0051] Preparation Example 5: Synthesis of compound Hcy-NEO-Leu

[0052] Compound C7 (11.4 mg, 0.0284 mmol) was dissolved in DMF and transferred to a reaction flask. Compound C11 (25 mg, 0.0142 mmol) and N,N-diisopropylethylamine (DIPEA, 12.4 μL, 0.0710 mmol) were then added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was poured into dichloromethane and washed with deionized water and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography before being used directly in the next step.

[0053] Trifluoroacetic acid was added dropwise to the product from the previous step. The reaction system was continued to react at 0°C for 15 minutes. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was directly purified by column chromatography to give compound Hcy-NEO-Leu, 15.09 mg, with a yield of 31.9%. 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.

[0054] Pharmacological experiments

[0055] Experimental Example 1: Spectral properties of probe Hcy-NEO-Leu and its response to LAP

[0056] 5 mM stock solutions of Hcy-NEO-Leu and Hcy-NEO-OH were prepared in DMSO. The stock solutions were diluted to 10 μM with DMSO or Tris buffer containing 1.5% DMSO. Absorption and fluorescence spectra were measured, and the enzyme kinetics and specificity of the probes for LAP were further investigated. See [link to relevant documentation]. Figure 2 .

[0057] Experimental Example 2: Screening of LAP enzyme inhibitors using the probe Hcy-NEO-Leu

[0058] The probe was used to evaluate LAP enzyme inhibitors in *Pseudomonas aeruginosa*. First, the strain was cultured overnight, and Hcy-NEO-Leu (10 μM) was mixed with *Pseudomonas aeruginosa* supernatant (OD200). 600 =0.5) Co-incubated under different concentrations of lycorine derivatives. After 3 h, the fluorescence intensity (λ) of 1) probe; 2) positive probe (Hcy-NEO-OH); 3) probe + bacterial supernatant; and 4) probe + bacterial supernatant + inhibitor group was measured using an ELISA reader. ex =660nm, λ em =705-850nm). This experiment discovered LY-58, a highly active LAP inhibitor.

[0059] Pseudomonas aeruginosa and its leucine aminopeptidase knockout strain (ΔLAP) were cultured overnight in medium supplemented with ubenimex (1000 μM, a known enzyme inhibitor) and LY-58 (50 μM). The control group consisted of bacteria cultured in LB medium without the inhibitor, and the remaining groups were experimental groups. The bacteria were diluted in LB medium to OD0.05. 600 =0.01, added to 200 μL per well of a 96-well plate, and incubated at 37°C for 48 h. After incubation, the supernatant was removed, and the biofilm was washed with 200 μL of sterile Tris buffer and dried. The biofilm was fixed with 100 μL of anhydrous methanol for 20 min, and then stained with 100 μL of 0.4% crystal violet staining solution at room temperature for 30 min. Afterwards, it was washed with sterile Tris buffer to remove unbound crystal violet staining solution, and the biofilm was dried. The staining of each group of biofilms was observed under an optical microscope. 200 μL of 33% acetic acid was added to release the bound crystal violet dye, and the λ was measured using a microplate reader. abs =Absorbance at 595 nm in each well. Calculate biofilm viability (%) = OD 595 (Experimental group) / OD 595 (Control group). Results are shown below. Figure 3 .

[0060] Experiment Example 3: Confocal fluorescence imaging of Pseudomonas aeruginosa using the probe Hcy-NEO-Leu.

[0061] Pseudomonas aeruginosa (PAO1) was cultured in LB medium at 37°C for 12 h. The strain cultured overnight was collected, and its OD value was adjusted. 600 Up to 0.5. Subsequently, with or without the addition of the LAP inhibitor ubenimex (1 mM) / LY-58 (50 μM), Hcy-NEO-Leu (10 μM) was reacted with Pseudomonas aeruginosa (OD) 600=0.5) Co-incubate. After incubation for 3 hours, collect the bacterial strain by centrifugation, wash 3 times with Tris buffer, and add 8 μL of the suspension to an 8-well plate covered with agarose gel to obtain bacterial samples for confocal imaging.

[0062] The frozen Raw 264.7 cells were rapidly thawed, and 1640 medium was added to dilute the cryopreservation solution. The cell suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The cells were then dispersed in 5 mL of 1640 medium and cultured in a cell culture incubator until optimal cell culture was achieved. Next, the cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 4 Raw 264.7 cells were added to 8-well plates at a density of cells / well and cultured in a cell culture incubator for 24 hours. Bacterial PAO1 (OD100) cells were added under inhibitor-free / in-presence conditions. 600 =0.5) Incubate for 30 min. Then replace the original 1640 cell culture medium with bacterial suspension and add Hcy-NEO-Leu to a final concentration of 10 μM. Co-culture Raw 264.7 cells with bacteria for 1 h. After culture, replace the bacterial suspension with phenol red-free 1640 medium and wash the cells twice. Confocal fluorescence imaging (λ) was performed using a confocal laser scanning microscope (Leica TCS SP8X, HC PL Apo 63× oil immersion). ex =660nm, λ em =700-800nm). Results are shown in Figure 4 .

[0063] Experiment Example 4: Confocal fluorescence imaging of Pseudomonas aeruginosa biofilm using the probe Hcy-NEO-Leu.

[0064] Pseudomonas aeruginosa (PAO1) and its leucine aminopeptidase knockout strain (ΔLAP) were cultured in LB medium at 37°C for 12 h to reach the logarithmic growth phase. Subsequently, the bacteria were diluted in LB medium to OD0.05. 600 =0.01. Next, the diluted bacterial suspension was added to 8-well plates (200 μL per well), and four experimental conditions were set up: 1) PAO1 group; 2) ΔLAP group; 3) PAO1 + ubenimex group; 4) PAO1 + LY-58 group. After each group was cultured under the same conditions for 48 h, the culture medium was discarded, and the cells were washed and resuspended with sterile Tris buffer for 3D imaging of the biofilm. Confocal fluorescence imaging (λ) was performed using a confocal laser scanning microscope (Leica TCS SP8X, HC PL Apo 63× oil immersion). ex =660nm, λ em =700-800nm). Results are shown in Figure 5 .

[0065] Experiment Example 5: In vitro imaging of common implants using probes

[0066] Common implants (including catheters, silicone, staples, or surgical sutures) approximately 2 mm in length were placed in a PAO1 bacterial solution with an initial OD value of 0.01 and cultured for 48 hours to obtain implants with attached biofilms. Subsequently, the biofilm-bearing implant material was transferred to a Tris buffer solution (containing 1.5% DMSO) with a final probe concentration of 10 μM and incubated for 3 hours. After rinsing the implants three times with sterile Tris buffer, they were imaged using a low-power microscope or a confocal laser scanning microscope (CLSM imager). Results are shown below. Figure 6 .

[0067] Experiment Example 6: Real-time probe imaging of implant infection in mice

[0068] The ability of the probe Hcy-NEO-Leu to image biofilm infection in vivo was evaluated by constructing a mouse implant biofilm infection model. First, a catheter approximately 5 mm long was placed in a PAO1 bacterial solution with an initial OD value of 0.01 and cultured for 48 hours to induce PAO1 biofilm formation on the catheter surface. Subsequently, the catheter containing the mature PAO1 biofilm was subcutaneously implanted into the left side of 6-8 week old BALB / c mice, while an implant uninfected with PAO1 was implanted into the right side, successfully establishing a mouse implant biofilm infection model. After 5 hours of incubation, the probe (25 μM, 50 μL) was injected orally into both sides of the mouse. Using a small animal three-dimensional optical in vivo imaging and X-ray computed tomography scanner (PerkinElmer), the mice were imaged at different time intervals (0.5 hours, 1 hour, 3 hours, and 5 hours). Fluorescence imaging data were analyzed using the Living Image 4.3.1 software package (PerkinElmer). Elliptical regions indicate the total radiometric efficiency (ROI) of the region of interest (p / sec / cm²). 2 / sr) / (μM / cm 2 See results. Figure 7 .

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Claims

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

2. The application of the near-infrared semi-fluorochrome in detecting the activity of bacterial leucine aminopeptidase (LAP, EC 3.4.11.1) according to claim 1.

3. The method for preparing the near-infrared hemicyanine fluorescent probe according to claim 1, characterized in that, The method comprises conjugating a compound shown in formula (2) with the modified neomycin under nucleophilic reaction conditions in an organic solvent; 4. The application of the near-infrared semi-fluorochrome in detecting and imaging the activity of leucine aminopeptidase according to claim 1, which involves contacting a sample to be detected with the near-infrared semi-fluorochrome according to claim 1, the leucine part of the fluorochrome being capable of specifically recognizing the leucine aminopeptidase in the sample to be detected, and the activity level of the leucine aminopeptidase in the sample to be detected being accurately indicated by detecting the fluorescence intensity of the semi-fluorochrome under specific excitation light after the contacting.

5. The detection method according to claim 4, the near-infrared semi-fluorochrome according to claim 1 being capable of being used for bacterial imaging, bacterial biofilm imaging and screening of aminopeptidase inhibitors after being hydrolyzed by leucine aminopeptidase.

Images