A near-infrared fluorescent probe for targeting gram-positive bacteria based on bacterial cell wall peptidoglycan synthesis and a preparation method and application thereof
By designing a near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, the problems of short wavelength and insufficient labeling efficiency of existing probes have been solved, achieving efficient and selective labeling and real-time imaging of Gram-positive bacteria.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA UNIV OF SCI & TECH
- Filing Date
- 2024-08-09
- Publication Date
- 2026-06-02
AI Technical Summary
Existing bacterial probes have short wavelengths, insufficient efficiency in labeling bacterial walls, and poor selectivity, making it difficult to conduct real-time studies of gut bacteria in vitro or in vivo.
A near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis was designed. Quinolinonitrile was used as the parent unit, D-lysine structural unit was used as the binding group, thiophene and other groups were introduced to prolong fluorescence emission, and sulfonic acid betaine was added to improve dispersibility, thus creating a molecularly rational probe.
It achieves long near-infrared fluorescence emission wavelength, good selectivity, and high labeling efficiency, and can specifically distinguish between Gram-positive and Gram-negative bacteria, providing a stable fluorescence signal, suitable for long-term real-time imaging of bacterial walls and in vivo imaging.
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Figure CN119330951B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fine chemical technology, and in particular to a near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, its preparation method, and its application. Background Technology
[0002] The human gut ecosystem comprises trillions of bacteria. These gut microbiota produce metabolites that signal to distant organs, enabling them to connect with the immune system, hormonal system, brain (the gut-brain axis), and the host's metabolism and other functions. This microbe-host communication is crucial for maintaining vital functions in a healthy host. Clinical studies have shown that gut microbiota is associated with a variety of diseases, including obesity, inflammatory diseases, and behavioral and physiological abnormalities related to neurodevelopmental disorders. However, as the "dark matter" of the gut, gut bacteria present significant challenges in in vitro culture and transgenic manipulation, making real-time in vitro or in vivo studies difficult, particularly in terms of specific labeling and in vivo imaging.
[0003] Because bacteria often possess high turgor pressure, and the bacterial wall is a core structure maintaining bacterial shape and mechanical strength, cell wall metabolism and synthesis are key control factors for bacterial growth and proliferation, directly reflecting bacterial activity. Near-infrared fluorescence imaging technology is characterized by high sensitivity and low cost, enabling the tracking of cell wall dynamics in phylogenetic and morphologically diverse bacteria. However, due to the lack of suitable dye parent and probe dispersion control strategies, most existing bacterial probes suffer from problems such as short wavelengths, insufficient bacterial wall labeling efficiency, and poor selectivity. Summary of the Invention
[0004] The purpose of this invention is to provide a near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, its preparation method and application, in order to solve the problems of short wavelength, insufficient bacterial wall labeling efficiency and poor selectivity of most existing bacterial probes.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] This invention provides a near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis. The structural formula of the near-infrared fluorescent probe for targeting Gram-positive bacteria is as follows:
[0007]
[0008] Among them, R1 includes -CN, -COOH, R2 includes
[0009] In R2, n is an integer from 0 to 2, and m is an integer ≥ 1; R3 includes
[0010] This invention also provides a method for preparing the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, comprising the following steps:
[0011] (1) Compound A, amines and first solvent are mixed and subjected to the first reaction to obtain intermediate compound B;
[0012] (2) Intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid and a second solvent were mixed and subjected to a second reaction to obtain intermediate compound C;
[0013] (3) The intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent were mixed and the third reaction was carried out to obtain the intermediate compound D;
[0014] (4) After mixing intermediate compound D and mixed solvent, a fourth reaction is carried out to obtain a near-infrared fluorescent probe targeting Gram-positive bacteria.
[0015] The structural formula of compound A is: The structural formula of the intermediate compound B is: The structural formula of the intermediate compound C is: The structural formula of the intermediate compound D is: In compounds A, B, C, and D, R1 includes -CN, -COOH, R2 includes In R2, n is an integer from 0 to 2, and m is an integer ≥ 1.
[0016] Preferably, the first, second, third, and fourth reactions are carried out independently under a protective atmosphere, which may include helium and / or argon.
[0017] Preferably, the first solvent includes o-dichlorobenzene; the amine includes N,N-dimethylpropylamine sulfonate or trimethylamine; the ratio of compound A, amine and first solvent is 1-3 mmol: 2-10 mmol: 40-60 mL; the temperature of the first reaction is 120-150 °C, and the reaction time is 5-7 h.
[0018] Preferably, the second solvent comprises acetonitrile; the ratio of the amount of the intermediate compound B, 3-(6-(5-formylthiophene-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid, and the second solvent is 0.1–3 mmol: 0.1–3 mmol: 20–30 mL; the temperature of the second reaction is 70–90 °C, and the time of the second reaction is 5–7 h.
[0019] Preferably, the third solvent comprises N,N-dimethylformamide; the ratio of the intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide, and the third solvent is 0.02–0.6 mmol: 0.02–0.6 mmol: 0.2–0.6 mmol: 10–30 mL; the temperature of the third reaction is 20–30 °C, and the reaction time is 2–4 h.
[0020] Preferably, the mixed solvent comprises trifluoroacetic acid and dichloromethane, with a volume ratio of 1 to 2:1; the ratio of intermediate compound D to the mixed solvent is 0.01 to 0.4 mmol to 4 to 9 mL; the temperature of the fourth reaction is 20 to 30 °C, and the reaction time is 2 to 4 h.
[0021] The present invention also provides the application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis in the preparation of reagents for distinguishing between Gram-positive and Gram-negative bacteria.
[0022] This invention also provides the application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis in the preparation of reagents for dynamically labeling the metabolic synthesis of Gram-positive bacterial cell walls.
[0023] This invention also provides the application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis in the preparation of reagents for in vivo imaging of positive bacterial flora in non-invasively labeled animals.
[0024] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] (1) This invention provides a near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis. This near-infrared fluorescent probe for targeting Gram-positive bacteria uses quinolinonitrile as the parent unit to achieve assembly and illumination of the bacterial wall confined environment; D-lysine structural units serve as the bacterial wall peptidoglycan binding groups; the fluorescence emission of the dye is prolonged by introducing groups such as thiophene; and the dispersibility of the probe is improved by introducing amphipathic groups such as sulfonic acid betaine, thereby reducing background fluorescence interference, improving labeling efficiency, and achieving efficient wash-free imaging. Based on the above rational molecular design, the probe has many advantages such as long fluorescence emission wavelength (near-infrared fluorescence emission), good selectivity, and high bacterial labeling efficiency.
[0026] (2) The near-infrared fluorescent probe for Gram-positive bacteria based on bacterial wall peptidoglycan synthesis provided by this invention does not exhibit fluorescence emission in an aqueous physiological environment. Synthesized through a bacterial peptidoglycan network, the probe shows a significant (90-fold, high signal-to-noise ratio) near-infrared fluorescence signal enhancement effect after labeling the bacterial wall. The probe can specifically distinguish between Gram-positive and Gram-negative bacteria, and has the characteristics of activation, rapid illumination, and no washing required during the labeling process of Gram-positive bacteria. The probe can provide a stable fluorescence emission signal in fluorescence confocal imaging and can be used for long-term real-time imaging of bacterial walls. The probe has many advantages such as long fluorescence emission wavelength, good photostability, high signal-to-noise ratio, good water solubility and biocompatibility, and can achieve specific and precise positioning of Gram-positive bacterial walls, thereby obtaining high-fidelity visualization information on the real-time synthesis and morphology of Gram-positive bacterial walls. The probe has deeper biological penetration and can achieve in vivo imaging and long-term tracking of mouse intestinal flora through near-infrared fluorescence imaging. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0028] Figure 1 The fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 as the concentration of dimethyl sulfoxide in dimethyl sulfoxide aqueous solution is continuously increased;
[0029] Figure 2 This is a magnified view of the fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 as the concentration of dimethyl sulfoxide in the aqueous solution of dimethyl sulfoxide increases.
[0030] Figure 3This is a diagram showing the cytotoxicity characterization results of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1;
[0031] Figure 4 The images show the results of different fluorescent labeling of Gram-positive and Gram-negative bacteria by the near-infrared fluorescent probe for targeting Gram-positive bacteria obtained in Example 1. In the images, a represents the fluorescent labeling results of different Gram-positive bacteria, and b represents the fluorescent labeling results of different Gram-negative bacteria.
[0032] Figure 5 The images show real-time dynamic fluorescence imaging of the growth of Gram-positive bacterial walls by the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1. Among them, a is a real-time dynamic fluorescence imaging of the growth of the bacterial wall of Bacillus subtilis, and b is a real-time dynamic fluorescence imaging of the growth of the bacterial wall of Staphylococcus aureus.
[0033] Figure 6 The image shows the in vivo imaging of Gram-positive bacteria in male C57BL / 6J mice by the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1. In the image, the concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria in a is 0.5 mM, and the concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria in b is 1 mM.
[0034] Figure 7 This is a live imaging image of Gram-positive bacteria in the intestine of a male C57BL / 6J rat obtained from Example 1 using a near-infrared fluorescent probe targeting Gram-positive bacteria. Detailed Implementation
[0035] This invention provides a near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis. The structural formula of the near-infrared fluorescent probe for targeting Gram-positive bacteria is as follows:
[0036]
[0037] Among them, R1 includes -CN, -COOH, R2 includes
[0038] In R2, n is an integer from 0 to 2, and m is an integer ≥ 1; R3 includes
[0039]
[0040] This invention also provides a method for preparing the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, comprising the following steps:
[0041] (1) Compound A, amines and first solvent are mixed and subjected to the first reaction to obtain intermediate compound B;
[0042] (2) Intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid and a second solvent were mixed and subjected to a second reaction to obtain intermediate compound C;
[0043] (3) The intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent were mixed and the third reaction was carried out to obtain the intermediate compound D;
[0044] (4) After mixing intermediate compound D and mixed solvent, a fourth reaction is carried out to obtain a near-infrared fluorescent probe targeting Gram-positive bacteria.
[0045] The structural formula of compound A is: The structural formula of the intermediate compound B is: The structural formula of the intermediate compound C is: The structural formula of the intermediate compound D is: In compounds A, B, C, and D, R1 includes -CN, -COOH, R2 includes In R2, n is an integer from 0 to 2, and m is an integer ≥ 1.
[0046] In this invention, the first reaction, the second reaction, the third reaction and the fourth reaction are carried out independently under a protective atmosphere, which includes helium and / or argon.
[0047] In this invention, the first solvent comprises o-dichlorobenzene; the amine comprises N,N-dimethylpropylamine sulfonate or trimethylamine; the preferred ratio of compound A, N,N-dimethylpropylamine sulfonate and the first solvent is 1-3 mmol: 2-10 mmol: 40-60 mL, more preferably 1.5-2.5 mmol: 4-8 mmol: 45-55 mL, and even more preferably 2-2.2 mmol: 5-6 mmol: 50 mL; the preferred temperature of the first reaction is 120-150°C, more preferably 125-145°C, and even more preferably 130-140°C; the preferred reaction time is 5-7 h, more preferably 5.5-6.5 h, and even more preferably 6 h.
[0048] In step (1) of the present invention, the product obtained from the first reaction is sequentially filtered, washed and dried to obtain intermediate product B;
[0049] The washing reagent includes dichloromethane; the drying temperature is preferably 50-80°C, more preferably 60-70°C, and even more preferably 65°C.
[0050] The reaction equation for step (1) of this invention is as follows:
[0051]
[0052] In this invention, the second solvent comprises acetonitrile; the preferred ratio of the intermediate compound B, 3-(6-(5-formylthiophene-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid, and the second solvent is 0.1–3 mmol: 0.1–3 mmol: 20–30 mL, more preferably 1.5–2.5 mmol: 1.5–2.5 mmol: 22–28 mL, and even more preferably 1.8–2 mmol: 2–2.2 mmol: 25–26 mL; the preferred temperature of the second reaction is 70–90 °C, more preferably 75–85 °C, and even more preferably 80–82 °C; the preferred reaction time is 5–7 h, more preferably 5.5–6.5 h, and even more preferably 6 h.
[0053] In step (2) of the present invention, the product obtained from the second reaction is sequentially subjected to rotary evaporation, column chromatography separation and drying to obtain intermediate product C;
[0054] The purpose of rotary evaporation is to remove the second solvent; the reagents used for column chromatography separation include dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 97-99:1-3, more preferably 98:2; the drying temperature is preferably 50-80°C, more preferably 60-70°C, and more preferably 65°C.
[0055] The reaction equation for step (2) of this invention is as follows:
[0056]
[0057] In step (3) of the present invention, the specific steps for mixing intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent are as follows: first, intermediate compound C and N-(tert-butoxycarbonyl)-D-lysine are mixed to obtain a mixture; then, dicyclohexylcarbodiimide and the third solvent are mixed to obtain a liquid mixture; finally, the liquid mixture is added dropwise to the mixture; the dropwise addition rate is preferably 0.1-0.2 mL / min, more preferably 0.15 mL / min.
[0058] In this invention, the third solvent comprises N,N-dimethylformamide; the preferred molar ratio of the intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide, and the third solvent is 0.02–0.6 mmol: 0.02–0.6 mmol: 0.2–0.6 mmol: 10–30 mL, more preferably 0.3–0.5 mmol: 0.3–0.5 mmol: 0.3–0.5 mmol: 15–25 mL, and even more preferably 0.35–0.4 mmol: 0.4–0.45 mmol: 0.4 mmol: 18–20 mL; the preferred temperature of the third reaction is 20–30 °C, more preferably 22–28 °C, and even more preferably 25–27 °C; the preferred reaction time is 2–4 h, more preferably 2.5–3.5 h, and even more preferably 3 h.
[0059] In step (3) of the present invention, the product obtained from the third reaction is sequentially subjected to rotary evaporation and drying to obtain intermediate product D;
[0060] The purpose of rotary drying is to remove the third solvent; the drying temperature is preferably 50-80°C, more preferably 60-70°C, and even more preferably 65°C.
[0061] The reaction equation for step (3) of this invention is as follows:
[0062]
[0063] In this invention, the mixed solvent comprises trifluoroacetic acid and dichloromethane, and the volume ratio of trifluoroacetic acid to dichloromethane is preferably 1-2:1, more preferably 1.2-1.8:1, and even more preferably 1.5-1.6:1; the amount ratio of the intermediate compound D to the mixed solvent is preferably 0.01-0.4 mmol:4-9 mL, more preferably 0.2-0.3 mmol:5-8 mL, and even more preferably 0.25-0.28 mmol:6-7 mL; the temperature of the fourth reaction is preferably 20-30°C, more preferably 22-28°C, and even more preferably 25-26°C; the time of the fourth reaction is preferably 2-4 h, more preferably 2.5-3.5 h, and even more preferably 3 h.
[0064] In step (4) of the present invention, the product obtained from the fourth reaction is purified sequentially by rotary evaporation and column chromatography;
[0065] The purpose of rotary evaporation is to remove the mixed solvent; the reagents used for column chromatography separation include dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 97-99:1-3, more preferably 98:2.
[0066] The reaction equation for step (4) of this invention is as follows:
[0067]
[0068] The present invention also provides the application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis in the preparation of reagents for distinguishing between Gram-positive and Gram-negative bacteria.
[0069] This invention also provides the application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis in the preparation of reagents for dynamically labeling the metabolic synthesis of Gram-positive bacterial cell walls.
[0070] This invention also provides the application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis in the preparation of reagents for in vivo imaging of positive bacterial flora in non-invasively labeled animals.
[0071] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0072] Example 1
[0073] The specific steps for synthesizing a near-infrared fluorescent probe targeting Gram-positive bacteria are as follows:
[0074] (1) Compound A (626 mg, 2 mmol) was prepared. N,N-dimethylpropylamine sulfonate (500 mg, 3 mmol) was placed in a 100 mL round-bottom flask, and 50 mL of ultra-dry o-dichlorobenzene was used as solvent. The mixture was stirred at 140 °C for 6 h under argon protection. After the reaction was completed, the mixture was filtered, and the filter cake was washed three times with dichloromethane (DCM). The cake was then dried at 60 °C to obtain 428 mg of a light yellow solid intermediate compound B. The yield was 53.5%. The product required no purification and was directly used in the next step.
[0075] Mass spectrometry(ESI-MS,m / z):[M+Na] + calcd.for[C 20 H 24 N4O3S+
[0076] Na] + 423.1461; found, 423.1465.
[0077] (2) Intermediate compound B1 (800 mg, 2 mmol) and 3-(6-(5-formylthiophene-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid (630 mg, 2 mmol) were placed in a 100 mL two-necked flask, and refluxed at 80 °C for 6 h under argon protection with 25 mL of ultra-dry CH3CN as solvent. After the reaction was completed, the solvent was evaporated, and the mixture was separated by column chromatography with dichloromethane and methanol in a volume ratio of 99:1. The solution was dried at 60 °C to obtain 598 mg of dark red solid intermediate compound C1. The yield was 43.2%.
[0078] 1 H-NMR (400MHz, CDCl3, ppm): δ = 9.16 (d, J = 8.40Hz, 1H, Ph-H), 7.75 (t, J = 8.00Hz, 1H, Ph-H), 7.50 (d, J = 15.60Hz, 1H, Alkene-H), 7.46 (t, J = 8.8 0Hz,1H,Ph-H),7.36(dd,J=8.40Hz,1H,Ph-H),7.25(d,J=8.00Hz,1H,Ph-H),7.20(d,J=8.00Hz,1H,Ph-H),7.11(s,1H,Ph-H),6.74(d,J=15.60 Hz,1H,Alkene-H),6.62(d,J=8.80Hz,1H,Ph-H),5.30(s,1H,Ph-H),4.40(q,J=6.80Hz,2H,-CH2-),3.66(t,J=5.20Hz,2H,-CH2-),3.35(t,J=5 Mass spectrometry(ESI-MS,m / z):[M+Na] + calcd.for[C 37 H 39 N5O5S2+H] + 698.2393; found, 698.2380.
[0079] (3) Intermediate compound C1 (373 mg, 0.463 mmol) and N-(tert-butoxycarbonyl)-D-lysine (107 mg, 0.463 mmol) were placed in a 50 mL two-necked flask. 20 mL of a mixture containing 0.4 mmol of dicyclohexylcarbodiimide (DCC) was added dropwise at a rate of 0.2 mL / min using ultradry N,N-dimethylformamide (DMF) as solvent. The mixture was reacted at room temperature for 6 h under argon protection. After the reaction was complete, the solvent was evaporated and dried at 60 °C to obtain 236 mg of a purplish-black solid intermediate compound D1. The product does not require purification and can be directly used in the next step.
[0080] (4) Intermediate compound D1 (236 mg, 0.23 mmol) was placed in a 50 mL single-necked flask and reacted at room temperature for 3 h with 6 mL of a mixture of trifluoroacetic acid (TFA) and dichloromethane (DCM) as the solvent (the volume ratio of TFA to DCM was 1:1). After the reaction was completed, TFA was removed by rotary evaporation to obtain the crude product. The crude product was separated by column chromatography using dichloromethane and methanol in a volume ratio of 99:1 to obtain 64 mg of a purple-black solid near-infrared fluorescent probe targeting Gram-positive bacteria. The yield was 64%.
[0081] 1 H-NMR (400MHz, DMSO-d6, ppm): δ = 8.94 (d, J = 8.40Hz, 1H, Ph-H), 8.10 (d, J = 8.80Hz, 1H, Ph-H), 7.93 (d, J = 7.20Hz, 1H, Ph-H), 7.68 (d, J = 15.60Hz, 1H, Alk ene-H),7.62(d,J=8.00Hz,1H,Ph-H),7.54(d,J=3.80Hz,1H,thiophene-H),7.39(dd,J=8.60Hz,1H,Ph-H),7.33(d,J=3.80Hz,1H,thiophene-H),7.28( s,1H,Ph-H),7.07(d,J=15.60Hz,1H,Alkene-H),7.04(s,1H,Ph-H),6.66(d ,J=8.84Hz,1H,Ph-H),4.56(q,J=6.80Hz,2H,-CH2-),3.66(t,J=5.2Hz,2H,- CH2-),3.35(t,J=5.6Hz,2H,-CH2-),2.80(t,J=6.4Hz,2H,-CH2-),2.62(t, J=7.2Hz,2H,-CH2-),2.00-1.96(m,2H,-CH2-),1.28(t,J=5.6Hz,3H,-CH3).
[0082] Mass spectrometry(ESI-MS,m / z):[M+H] + calcd.for[C 44 H 51 N9O7S+H] + 827.3376; found, 827.3351.
[0083] Example 2
[0084] The specific steps for synthesizing a near-infrared fluorescent probe targeting Gram-positive bacteria are as follows:
[0085] (1) Compound A (313 mg, 1 mmol) was prepared. The mixture was placed in a 100 mL round-bottom flask with trimethylamine (590 mg, 10 mmol) and 50 mL of ultra-dry o-dichlorobenzene as solvent. The mixture was stirred at 140 °C for 6 h under argon protection. After the reaction was complete, the mixture was filtered, and the filter cake was washed three times with dichloromethane (DCM). The cake was then dried at 60 °C to obtain 148.41 mg of a light yellow solid intermediate, compound B2. The yield was 51%. The product required no purification and was directly used in the next step.
[0086] Mass spectrometry(ESI-MS,m / z):[M] + calcd.for[C 18 H 21 N4] + 293.1761; found, 293.1756.
[0087] (2) Compound B2 (225 mg, 0.77 mmol) and 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid (150 mg, 0.48 mmol) were placed in a 100 mL two-necked flask, and refluxed at 80 °C for 6 h under argon protection with 25 mL of ultra-dry CH3CN as solvent. After the reaction was completed, the solvent was evaporated, and the mixture was separated by column chromatography with dichloromethane and methanol in a volume ratio of 99:1. The solution was dried at 60 °C to obtain 34 mg of dark red solid intermediate compound C2. The yield was 13.31%.
[0088] 1H-NMR (400MHz, CDCl3, ppm): δ = 9.16 (d, J = 8.40Hz, 1H, Ph-H), 7.75 (t, J = 8.00Hz, 1H, Ph-H), 7.50 (d, J = 15.60Hz, 1H, Alkene-H), 7.46 (t, J = 8. 80Hz,1H,Ph-H),7.36(dd,J=8.40Hz,1H,Ph-H),7.25(d,J=8.00Hz,1H,Ph-H),7.20(d,J=8.00Hz,1H,Ph-H),7.11(s,1H,Ph-H),6.74(d,J=15. 60Hz,1H,Alkene-H),6.62(d,J=8.80Hz,1H,Ph-H),5.30(s,1H,Ph-H),4.40(q,J=6.80Hz,2H,-CH2-),3.66(t,J=5.20Hz,2H,-CH2-),3.35(t, J=5.60Hz,2H,-CH2-),2.80(t,J=6.40Hz,2H,-CH2-),2.62(t,J=7.20Hz,2H,-CH2-),2.00-1.96(m,2H,-CH2-),1.28(t,J=5.60Hz,3H,-CH3).
[0089] (3) Intermediate compound C2 (34 mg, 0.057 mmol) and N-(tert-butoxycarbonyl)-D-lysine (12 mg, 0.057 mmol) were placed in a 50 mL two-necked flask. 20 mL of a mixture containing 0.4 mmol of dicyclohexylcarbodiimide (DCC) was added dropwise at a rate of 0.2 mL / min using ultradry N,N-dimethylformamide (DMF) as solvent. The mixture was reacted at room temperature for 6 h under argon protection. After the reaction was complete, the solvent was evaporated, and the mixture was dried at 60 °C to obtain 42 mg of a purplish-black solid intermediate compound D2. The product does not require purification and can be directly used in the next step.
[0090] (4) The intermediate compound D2 (42 mg, 0.051 mmol) was placed in a 50 mL single-necked flask and reacted at room temperature for 3 h with 6 mL of a mixture of trifluoroacetic acid (TFA) and dichloromethane (DCM) as the solvent (the volume ratio of TFA to DCM was 1:1). After the reaction was completed, the trifluoroacetic acid (TFA) was removed by rotary evaporation to obtain the crude product. The crude product was separated by column chromatography using dichloromethane and methanol in a volume ratio of 99:1 to obtain 22 mg of a purple-black solid near-infrared fluorescent probe targeting Gram-positive bacteria. The yield was 60%.
[0091] 1H-NMR (400MHz, DMSO-d6, ppm): δ = 8.94 (d, J = 8.40Hz, 1H, Ph-H), 8.10 (d, J = 8.80Hz, 1H, Ph-H), 7.93 (d, J = 7.20Hz, 1H, Ph-H), 7.68 (d, J = 15.60Hz, 1H, Alk ene-H),7.62(d,J=8.00Hz,1H,Ph-H),7.54(d,J=3.80Hz,1H,thiophene-H),7.39(dd,J=8.60Hz,1H,Ph-H),7.33(d,J=3.80Hz,1H,thiophene-H),7.28( s,1H,Ph-H),7.07(d,J=15.60Hz,1H,Alkene-H),7.04(s,1H,Ph-H),6.66(d ,J=8.84Hz,1H,Ph-H),4.56(q,J=6.80Hz,2H,-CH2-),3.66(t,J=5.2Hz,2H,- CH2-),3.35(t,J=5.6Hz,2H,-CH2-),2.80(t,J=6.4Hz,2H,-CH2-),2.62(t, J=7.2Hz,2H,-CH2-),2.00-1.96(m,2H,-CH2-),1.28(t,J=5.6Hz,3H,-CH3).
[0092] Application Example 1
[0093] Fluorescence spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 in the aggregated state:
[0094] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ ppm solution. -3 A stock solution of M was prepared. Then, 2 mL of each of the following dimethyl sulfoxide (DMSO) aqueous solutions were prepared: 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0%. Eleven 20 μL portions of the above stock solution were added to the prepared DMSO aqueous solutions, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to measure their fluorescence spectra. The results are as follows: Figure 1 and Figure 2 As shown.
[0095] Figure 1 The fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 as the concentration of dimethyl sulfoxide in the aqueous solution is continuously increased. Figure 2This is a magnified view of the fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 as the concentration of dimethyl sulfoxide in the aqueous solution of dimethyl sulfoxide increases.
[0096] Depend on Figure 1 It can be seen that, using 500nm as the excitation wavelength, the maximum emission peak of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 is located at 800nm, in the near-infrared region, with a Stokes shift of 300nm; furthermore, the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 aggregates in dimethyl sulfoxide aqueous solution, resulting in enhanced fluorescence, exhibiting typical aggregation fluorescence enhancement characteristics. Figure 2 It can be seen that the near-infrared fluorescent probe for targeting Gram-positive bacteria obtained in Example 1 has almost no background fluorescence in aqueous solution and has little imaging interference.
[0097] Application Example 2
[0098] Cytotoxicity of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1:
[0099] The cytotoxicity of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was tested using the standard MTT assay. The specific experimental steps are as follows: First, HeLa cells were seeded into 96-well plates at a cell density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured overnight in a cell culture incubator. Different concentrations of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 (0 μM, 5 μM, 10 μM, 20 μM, 40 μM, 100 μM, 100 μL / well) were added to different wells. Dimethyl sulfoxide (DMSO) diluted in culture medium (0.5% v / well, 100 μL / well) served as a negative control. Cells were cultured in a cell culture incubator for 24 h, and then 10 μL of LTT solution (5 mg / mL) was added to each well. After incubation for 4 h, the liquid in the 96-well plate was removed, and DMSO (100 μL / well) was added. Finally, the results were measured using a microplate reader. Results are as follows: Figure 3 As shown.
[0100] Figure 3 The figure shows the cytotoxicity characterization results of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1.
[0101] Depend on Figure 3 It can be seen that when the concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 is increased to 100 μM, the cell survival rate reaches 91%, indicating that the cytotoxicity of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 is very low.
[0102] Application Example 3
[0103] The near-infrared fluorescent probe for targeting Gram-positive bacteria obtained in Example 1 can differentiate between Gram-positive and Gram-negative bacteria:
[0104] Four different Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis) and four different Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae)) were selected for labeling, and the labeling effect was observed using a fluorescence confocal microscope. The specific experimental steps are as follows: The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was dissolved in sterile water to prepare a 1.0 × 10⁻⁶ ppm solution. -2 M stock solution. The stock solution was added directly to the exponential phase bacterial culture until the final concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria reached 0.5 mM. The culture was then incubated with shaking at 37°C for 1 min without washing or fixation, and the bacteria were immediately imaged. 1 μL of bacterial culture was added to a glass slide at room temperature, and finally, confocal microscopy was used for imaging. The imaging results are shown below. Figure 4 As shown.
[0105] Figure 4 The images show the results of different fluorescent labeling of Gram-positive and Gram-negative bacteria by the near-infrared fluorescent probe for targeting Gram-positive bacteria obtained in Example 1. In the images, a represents the fluorescent labeling results of different Gram-positive bacteria, and b represents the fluorescent labeling results of different Gram-negative bacteria.
[0106] Depend on Figure 4 It can be seen that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can clearly fluorescently label the bacterial wall of Gram-positive bacteria, while showing no labeling for Gram-negative bacteria. This demonstrates that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can accurately distinguish between Gram-positive and Gram-negative bacteria.
[0107] Application Example 4
[0108] The near-infrared fluorescent probe obtained in Example 1 dynamically labels the cell wall metabolism and synthesis of Gram-positive bacteria:
[0109] To investigate whether the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can monitor the peptidoglycan synthesis process of Gram-positive bacteria in real time, the specific experimental steps are as follows: The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was dissolved in sterile water to prepare a 1.0 × 10⁻⁶ ppm solution. -2 M stock solution. A mixture of LB medium (Lysogeny broth), agarose, and preheated stock solution was added to a cavity slide until the final concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria was 1 mM. After the mixture solidified, Bacillus subtilis and Staphylococcus aureus were loaded onto the top of the agarose pad, respectively, and then covered with a coverslip. Real-time labeling was observed using a time-lapse fluorescence microscope as the Gram-positive bacteria grew in the slide. Results are shown below. Figure 5 As shown.
[0110] Figure 5 The images show real-time dynamic fluorescence imaging of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, where a is a real-time dynamic fluorescence imaging of the bacterial wall growth of Bacillus subtilis, and b is a real-time dynamic fluorescence imaging of the bacterial wall growth of Staphylococcus aureus. Figure 5 In Figure a, at 0 min, the left image shows the observation results under non-fluorescence conditions, and the right image shows the observation results under fluorescence conditions; at 35 min, the left image shows the observation results under fluorescence conditions, and the right image shows the observation results under non-fluorescence conditions. Figure 5 In Figure b, at 6 min, the left image shows the observation results under non-fluorescence conditions, and the right image shows the observation results under fluorescence conditions; at 11 min, the left image shows the observation results under fluorescence conditions, and the right image shows the observation results under non-fluorescence conditions.
[0111] Depend on Figure 5 As shown in section a, Bacillus subtilis reproduces through binary fission; over time, the dividing ring gradually separates one bacterium into two. Figure 5 As shown in b, Staphylococcus aureus reproduces through binary fission, with one bacterium gradually dividing into two identical individuals over time. This demonstrates that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can monitor the peptidoglycan synthesis process of Gram-positive bacteria in real time.
[0112] Application Example 5
[0113] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was used to non-invasively label the intestinal positive flora of IBD colitis mice and wt normal mice:
[0114] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was dissolved in sterile water to prepare a stock solution. Two groups of C57BL / 6J male mice (one group with IBD enteritis model mice and one group of weight-controlled normal mice) were administered the stock solution by gavage. The concentration of the stock solution was 1 mM. After gavage, whole-body fluorescence imaging of the two groups of C57BL / 6J male mice was performed using MARS, and the fluorescence change over time was monitored. One C57BL / 6J male mouse from each group was dissected, and fluorescence imaging of the intestines was performed. Before the imaging experiment, the mice were anesthetized with 2.5% isoflurane gas. The results are as follows: Figure 6 and Figure 7 As shown.
[0115] Figure 6 The images shown are in vivo images of Gram-positive bacteria in C57BL / 6J male mice obtained in Example 1 using the near-infrared fluorescent probe targeting Gram-positive bacteria. In the images, a corresponds to the imaging of three IBD enteritis model mice over time using the near-infrared fluorescent probe targeting Gram-positive bacteria, and b corresponds to the imaging of three wt normal mice over time using the near-infrared fluorescent probe targeting Gram-positive bacteria. Figure 7 This is a live imaging image of Gram-positive bacteria in the intestine of a male C57BL / 6J rat obtained from Example 1 using a near-infrared fluorescent probe targeting Gram-positive bacteria.
[0116] Depend on Figure 6 It can be seen that after gavage administration of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, the fluorescence signal first appeared in the stomach of the mice, and then shifted to the colon region over time. Furthermore, the fluorescence intensity in the three IBD enteritis mice was consistently higher than that in the three wt normal mice, indicating that the number of Gram-positive bacteria in the intestines of the IBD enteritis mice was higher than that in the wt normal mice. Figure 7 It was found that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 exhibited higher fluorescence intensity in IBD enteritis mice and weaker fluorescence in wt% normal mice. This indicates that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 has excellent bio-penetration and can be further applied to in vivo biological testing.
[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, characterized in that, The structural formula of the near-infrared fluorescent probe targeting Gram-positive bacteria is as follows: ； Wherein, R1 is selected from -CN or R2 is selected from , , or In R2, n is an integer from 0 to 2, and m is 3; R3 is selected from... , , or .
2. The method for preparing a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis as described in claim 1, characterized in that, Includes the following steps: (1) Compound A, amines and the first solvent are mixed and subjected to the first reaction to obtain intermediate compound B; (2) The intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid and the second solvent were mixed and subjected to a second reaction to obtain the intermediate compound C; (3) The intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent are mixed and the third reaction is carried out to obtain the intermediate compound D; (4) After mixing intermediate compound D and mixed solvent, a fourth reaction is carried out to obtain a near-infrared fluorescent probe targeting Gram-positive bacteria; The structural formula of compound A is: The structural formula of the intermediate compound B is: The structural formula of the intermediate compound C is: The structural formula of the intermediate compound D is: In compounds A, B, C, and D, R1 is selected from -CN, or... R2 is selected from , , or In R2, n is an integer between 0 and 2, and m is 3.
3. The method for preparing a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 2, characterized in that, The first, second, third, and fourth reactions are carried out independently under a protective atmosphere, which includes helium and / or argon.
4. The method for preparing a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 2 or 3, characterized in that, The first solvent includes o-dichlorobenzene; the amine includes N,N-dimethylpropylamine sulfonate or trimethylamine; the ratio of compound A, amine and first solvent is 1~3 mmol: 2~10 mmol: 40~60 mL; the temperature of the first reaction is 120~150℃, and the time of the first reaction is 5~7 h.
5. The method for preparing a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 4, characterized in that, The second solvent includes acetonitrile; the ratio of the amount of the intermediate compound B, 3-(6-(5-formylthiophene-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propionic acid, and the second solvent is 0.1~3 mmol: 0.1~3 mmol: 20~30 mL; the temperature of the second reaction is 70~90 °C, and the time of the second reaction is 5~7 h.
6. The method for preparing a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 2, 3, or 5, characterized in that, The third solvent includes N,N-dimethylformamide; the ratio of the intermediate compound C, N-(tert-butoxycarbonyl)-D-lysine, dicyclohexylcarbodiimide, and the third solvent is 0.02~0.6 mmol: 0.02~0.6 mmol: 0.2~0.6 mmol: 10~30 mL; the temperature of the third reaction is 20~30℃, and the reaction time is 2~4 h.
7. The method for preparing a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 6, characterized in that, The mixed solvent comprises trifluoroacetic acid and dichloromethane, with a volume ratio of 1 to 2:1; the ratio of intermediate compound D to the mixed solvent is 0.01 to 0.4 mmol to 4 to 9 mL; the temperature of the fourth reaction is 20 to 30 °C, and the reaction time is 2 to 4 h.
8. The application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis as described in claim 1 in the preparation of reagents for distinguishing between Gram-positive and Gram-negative bacteria.
9. The application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis as described in claim 1 in the preparation of reagents for dynamically labeling the metabolic synthesis of Gram-positive bacterial cell walls.
10. The application of the near-infrared fluorescent probe for targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis as described in claim 1 in the preparation of reagents for in vivo imaging of positive bacterial flora in non-invasively labeled animals.