Targeted gram-positive bacterium near-infrared fluorescent probe synthesized based on bacterial wall peptidoglycan as well as preparation method and application of targeted gram-positive bacterium near-infrared fluorescent probe

Abstract

The invention belongs to the technical field of fine chemical engineering, and discloses a Gram-positive bacterium targeting near-infrared fluorescent probe synthesized based on bacterial wall peptidoglycan as well as a preparation method and application of the Gram-positive bacterium targeting near-infrared fluorescent probe. The invention provides a near-infrared fluorescent probe for targeting gram-positive bacteria, which is synthesized on the basis of bacterial wall peptidoglycan. According to the near-infrared fluorescent probe targeting gram-positive bacteria, quinoline nitrile is used as a parent unit, so that bacterium wall confinement environment assembly lightening is realized; a D-lysine structural unit is used as a bacterial wall peptidoglycan binding group; the fluorescence emission of the dye is prolonged by introducing groups such as thiophene; an amphiphilic charge group containing sulfo betaine and the like is introduced to improve the dispersity of the probe, so that the background fluorescence interference of the probe is reduced, the marking efficiency is improved, and efficient washing-free imaging is realized; based on the molecular rational design, the probe has the advantages of long fluorescence emission wavelength (near infrared fluorescence emission), good selectivity, high bacterial marking efficiency and the like.

Description

Technical Field

[0001] The invention relates to the technical field of fine chemical industry, and in particular to a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, and a preparation method and application thereof. Background Art

[0002] The human intestinal ecosystem is composed of trillions of bacteria. These metabolites produced by the intestinal flora send signals to distal organs in the body, enabling intestinal bacteria to connect with the immune system, hormone system, brain (i.e., gut-brain axis), and metabolic and other functions of the host. This microbial-host communication is essential for maintaining important functions of a healthy host. Clinical studies have shown that intestinal flora are associated with a variety of diseases, including obesity, inflammatory diseases, and behavioral and physiological abnormalities associated with neurodevelopmental disorders. However, as "intestinal dark matter", intestinal bacteria are extremely difficult to culture alone in vitro and to be genetically modified, making it difficult to study them in real time in vitro or in vivo, especially in the specific labeling and in vivo imaging of the flora. There are great technical difficulties.

[0003] Since bacteria often have high turgor pressure, the bacterial wall is the core structure that maintains the bacterial shape and mechanical strength. Therefore, cell wall metabolism and synthesis are key control factors for bacterial growth and proliferation, and directly reflect bacterial activity. Near-infrared fluorescence imaging technology has the characteristics of high sensitivity and low cost, so it can track cell wall dynamics in bacteria with diverse phylogeny and morphology. However, due to the lack of suitable dye matrix and probe dispersion control strategy, most of the existing bacterial probes have problems such as short wavelength, insufficient bacterial wall labeling efficiency, and poor selectivity. Summary of the invention

[0004] The purpose of the present invention is to provide a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis and its preparation method and application, so as to solve the problems of most existing bacterial probes such as short wavelength, insufficient bacterial wall labeling efficiency, poor selectivity, etc.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, and the structural formula of the near-infrared fluorescent probe targeting Gram-positive bacteria is as follows:

[0007]

[0008] Wherein, R1 includes -CN, -COOH, R2 includes

[0009] In R2, n is an integer between 0 and 2, and m is an integer ≥ 1; R3 includes

[0010] The present invention also provides a method for preparing the near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, comprising the following steps:

[0011] (1) mixing compound A, an amine substance and a first solvent and performing a first reaction to obtain an intermediate compound B;

[0012] (2) mixing the intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinolin-1(2H)-yl)propanoic acid and a second solvent and performing a second reaction to obtain an intermediate compound C;

[0013] (3) mixing the intermediate compound C, N-(tert-butyloxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and a third solvent and performing a third reaction to obtain an intermediate compound D;

[0014] (4) mixing the intermediate compound D and the mixed solvent and performing a fourth reaction to obtain a near-infrared fluorescent probe targeting Gram-positive bacteria;

[0015] The structural formula of the 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 the compound A, intermediate compound B, intermediate compound C and intermediate compound 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 reaction, the second reaction, the third reaction and the fourth reaction are independently carried out under a protective atmosphere, and the protective atmosphere independently comprises helium and / or argon.

[0017] Preferably, the first solvent includes o-dichlorobenzene; the amine substance includes N,N-dimethylpropylamine sulfonate or trimethylamine; the dosage ratio of compound A, amine substance 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 time of the first reaction is 5-7 h.

[0018] Preferably, the second solvent comprises acetonitrile; the usage ratio of the intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propanoic 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 includes N,N-dimethylformamide; the dosage ratio of the intermediate compound C, N-(tert-butyloxycarbonyl)-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 time of the third reaction is 2-4 h.

[0020] Preferably, the mixed solvent comprises trifluoroacetic acid and dichloromethane, and the volume ratio of trifluoroacetic acid to dichloromethane is 1-2:1; the dosage ratio of the intermediate compound D to the mixed solvent is 0.01-0.4 mmol:4-9 mL; the temperature of the fourth reaction is 20-30° C., and the time of the fourth reaction is 2-4 h.

[0021] The present invention also provides the use of the targeted Gram-positive bacteria near-infrared fluorescent probe based on bacterial wall peptidoglycan synthesis in the preparation of a reagent for distinguishing and marking Gram-positive bacteria and Gram-negative bacteria.

[0022] The present invention also provides the use of the targeted Gram-positive bacteria near-infrared fluorescent probe based on bacterial wall peptidoglycan synthesis in the preparation of reagents used for dynamically marking the metabolic synthesis of Gram-positive bacterial cell walls.

[0023] The present invention also provides the use of the targeted Gram-positive bacteria near-infrared fluorescent probe based on bacterial wall peptidoglycan synthesis in the preparation of reagents used for non-invasive labeling of positive bacterial flora in vivo imaging in animals.

[0024] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention provides a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis. The near-infrared fluorescent probe targeting Gram-positive bacteria uses quinoline nitrile as the parent unit to realize the assembly and lighting of the bacterial wall confined environment; the D-lysine structural unit is used as the bacterial wall peptidoglycan binding group; the dye fluorescence emission is extended by introducing groups such as thiophene; the dispersion of the probe is improved by introducing amphiphilic charged groups such as sulfonate betaine, thereby reducing the background fluorescence interference of the probe, improving the labeling efficiency, and realizing efficient wash-free imaging; based on the above molecular rational 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 targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis provided by the present invention does not show fluorescence emission in an aqueous physiological environment. It is synthesized through the bacterial peptidoglycan network. After labeling the bacterial wall, the probe shows a significant (90 times, high signal-to-noise ratio) near-infrared fluorescence signal enhancement effect; the probe can specifically distinguish Gram-positive bacteria from Gram-negative bacteria, and has the characteristics of activation lighting, rapidity, and no need for washing during the Gram-positive bacteria labeling process; 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 the bacterial wall of Gram-positive bacteria, thereby obtaining high-fidelity visualization information such as real-time synthesis and morphology of the bacterial wall of Gram-positive bacteria; 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 with increasing dimethyl sulfoxide concentration in a dimethyl sulfoxide aqueous solution;

[0029] Figure 2 This is an enlarged effect diagram of the fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 with increasing dimethyl sulfoxide concentration in a dimethyl sulfoxide aqueous solution;

[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 graphs are different fluorescence labeling results of the Gram-positive bacteria targeted near-infrared fluorescent probe obtained in Example 1 on Gram-positive bacteria and Gram-negative bacteria, wherein a is a graph of fluorescence labeling results of different Gram-positive bacteria, and b is a graph of fluorescence labeling results of different Gram-negative bacteria;

[0032] Figure 5 The real-time dynamic fluorescence imaging diagram of the growth of the bacterial wall of Gram-positive bacteria by the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, wherein a is a real-time dynamic fluorescence imaging diagram of the growth of the bacterial wall of Bacillus subtilis, and b is a real-time dynamic fluorescence imaging diagram of the growth of the bacterial wall of Staphylococcus aureus;

[0033] Figure 6 This is a live imaging image of the positive bacterial flora in C57BL / 6J male mice using the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, wherein the concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria corresponding to a is 0.5 mM, and the concentration of the near-infrared fluorescent probe targeting Gram-positive bacteria corresponding to b is 1 mM;

[0034] Figure 7 This is an in vivo imaging image of the positive bacterial flora in the intestine of C57BL / 6J male mice using the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1. DETAILED DESCRIPTION

[0035] The present invention provides a near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, and the structural formula of the near-infrared fluorescent probe targeting Gram-positive bacteria is as follows:

[0036]

[0037] Wherein, R1 includes -CN, -COOH, R2 includes

[0038] In R2, n is an integer between 0 and 2, and m is an integer ≥ 1; R3 includes

[0039]

[0040] The present invention also provides a method for preparing the near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis, comprising the following steps:

[0041] (1) mixing compound A, an amine substance and a first solvent and performing a first reaction to obtain an intermediate compound B;

[0042] (2) mixing the intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinolin-1(2H)-yl)propanoic acid and a second solvent and performing a second reaction to obtain an intermediate compound C;

[0043] (3) mixing the intermediate compound C, N-(tert-butyloxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and a third solvent and performing a third reaction to obtain an intermediate compound D;

[0044] (4) mixing the intermediate compound D and the mixed solvent and performing a fourth reaction to obtain a near-infrared fluorescent probe targeting Gram-positive bacteria;

[0045] The structural formula of the 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 the compound A, intermediate compound B, intermediate compound C and intermediate compound 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 the present invention, the first reaction, the second reaction, the third reaction and the fourth reaction are independently carried out under a protective atmosphere, and the protective atmosphere independently includes helium and / or argon.

[0047] In the present invention, the first solvent includes o-dichlorobenzene; the amine substance includes N,N-dimethylpropylamine sulfonate or trimethylamine; the dosage ratio of the compound A, N,N-dimethylpropylamine sulfonate and the first solvent is preferably 1-3mmol:2-10mmol:40-60mL, more preferably 1.5-2.5mmol:4-8mmol:45-55mL, more preferably 2-2.2mmol:5-6mmol:50mL; the temperature of the first reaction is preferably 120-150°C, more preferably 125-145°C, more preferably 130-140°C; the time of the first reaction is preferably 5-7h, more preferably 5.5-6.5h, more preferably 6h.

[0048] In step (1) of the present invention, the product obtained by the first reaction is filtered, washed and dried in sequence to obtain an intermediate product B;

[0049] The reagent used for washing includes dichloromethane; the drying temperature is preferably 50-80°C, more preferably 60-70°C, and more preferably 65°C.

[0050] The reaction equation of step (1) of the present invention is as follows:

[0051]

[0052] In the present invention, the second solvent includes acetonitrile; the dosage ratio of the intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propanoic acid and the second solvent is preferably 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, more preferably 1.8-2 mmol: 2-2.2 mmol: 25-26 mL; the temperature of the second reaction is preferably 70-90°C, more preferably 75-85°C, more preferably 80-82°C; the time of the second reaction is preferably 5-7h, more preferably 5.5-6.5h, more preferably 6h.

[0053] In step (2) of the present invention, the product obtained by the second reaction is sequentially subjected to spin drying, column chromatography separation and drying to obtain an intermediate product C;

[0054] The purpose of spin drying 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, and 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 of step (2) of the present invention is as follows:

[0056]

[0057] In step (3) of the present invention, the specific steps of mixing the intermediate compound C, N-(tert-butyloxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent are: firstly, the intermediate compound C and N-(tert-butyloxycarbonyl)-D-lysine are mixed to obtain a mixture; then, the dicyclohexylcarbodiimide and the third solvent are mixed to obtain a mixed solution; finally, the mixed solution is added dropwise to the mixture; the dropping speed is preferably 0.1 to 0.2 mL / min, and more preferably 0.15 mL / min.

[0058] In the present invention, the third solvent includes N,N-dimethylformamide; the dosage ratio of the intermediate compound C, N-(tert-butyloxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent is preferably 0.02-0.6mmol:0.02-0.6mmol:0.2-0.6mmol:10-30mL, further preferably 0.3-0.5mmol:0.3-0.5mmol:0.3-0.5mmol:15-25mL, more preferably 0.35-0.4mmol:0.4-0.45mmol:0.4mmol:18-20mL; the temperature of the third reaction is preferably 20-30°C, further preferably 22-28°C, more preferably 25-27°C; the time of the third reaction is preferably 2-4h, further preferably 2.5-3.5h, more preferably 3h.

[0059] In step (3) of the present invention, the product obtained by the third reaction is successively spin-dried and dried to obtain an intermediate product D;

[0060] The purpose of spin drying is to remove the third solvent; the drying temperature is preferably 50-80°C, more preferably 60-70°C, and more preferably 65°C.

[0061] The reaction equation of step (3) of the present invention is as follows:

[0062]

[0063] In the present 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 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 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 more preferably 25-26°C; the time of the fourth reaction is preferably 2-4 h, more preferably 2.5-3.5 h, and more preferably 3 h.

[0064] In step (4) of the present invention, the product obtained by the fourth reaction is sequentially spin-dried and purified by column chromatography;

[0065] The purpose of spin drying 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, and more preferably 98:2.

[0066] The reaction equation of step (4) of the present invention is as follows:

[0067]

[0068] The present invention also provides the use of the targeted Gram-positive bacteria near-infrared fluorescent probe based on bacterial wall peptidoglycan synthesis in the preparation of a reagent for distinguishing and marking Gram-positive bacteria and Gram-negative bacteria.

[0069] The present invention also provides the use of the targeted Gram-positive bacteria near-infrared fluorescent probe based on bacterial wall peptidoglycan synthesis in the preparation of reagents used for dynamically marking the metabolic synthesis of Gram-positive bacterial cell walls.

[0070] The present invention also provides the use of the targeted Gram-positive bacteria near-infrared fluorescent probe based on bacterial wall peptidoglycan synthesis in the preparation of reagents used for non-invasive labeling of positive bacterial flora in vivo imaging in animals.

[0071] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0072] Example 1

[0073] The synthesis of near-infrared fluorescent probe targeting Gram-positive bacteria is as follows:

[0074] (1) Compound A (626 mg, 2 mmol) and N,N-dimethylpropylamine sulfonate (500 mg, 3 mmol) were placed in a 100 mL round-bottom flask, and 50 mL of ultra-dry o-dichlorobenzene was used as the solvent. The mixture was stirred at 140 °C for 6 h under argon protection. After the reaction was completed, the filter cake was filtered and washed with dichloromethane (DCM) three times, and then dried at 60 °C to obtain 428 mg of a yellowish solid intermediate compound B. The yield was 53.5%. The product was directly used in the next step without purification.

[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) The intermediate compound B1 (800 mg, 2 mmol) and 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinolin-1(2H)-yl)propionic acid (630 mg, 2 mmol) were placed in a 100 mL two-necked bottle, and 25 mL of ultra-dry CH3CN was used as the solvent. The mixture was refluxed at 80°C for 6 h under argon protection. After the reaction was completed, the solvent was dried by spin column chromatography using dichloromethane and methanol in a volume ratio of 99:1, and dried at 60°C to obtain 598 mg of a 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) The intermediate compound C1 (373 mg, 0.463 mmol) and N-(tert-butyloxycarbonyl)-D-lysine (107 mg, 0.463 mmol) were placed in a 50 mL two-necked flask, and a mixed solution containing 0.4 mmol of dicyclohexylcarbodiimide (DCC) and 20 mL of ultra-dry N,N-dimethylformamide (DMF) as a solvent was added dropwise at a rate of 0.2 mL / min. The mixture was reacted at room temperature for 6 h under argon protection. After the reaction, the solvent was dried by spin drying and dried at 60° C. to obtain 236 mg of a purple-black solid intermediate compound D1. The product was directly used in the next step without purification.

[0080] (4) The intermediate compound D1 (236 mg, 0.23 mmol) was placed in a 50 mL single-mouth bottle, and 6 mL of trifluoroacetic acid (TFA) and dichloromethane (DCM) were used as a mixed solvent (the volume ratio of trifluoroacetic acid (TFA) and dichloromethane (DCM) was 1:1) and reacted at room temperature for 3 h. After the reaction was completed, trifluoroacetic acid (TFA) was removed by rotary evaporation to obtain a 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 synthesis of near-infrared fluorescent probe targeting Gram-positive bacteria is as follows:

[0085] (1) Compound A (313 mg, 1 mmol) and trimethylamine (590 mg, 10 mmol) were placed in a 100 mL round-bottom flask, and 50 mL of ultra-dry o-dichlorobenzene was used as the solvent. The mixture was stirred at 140 °C for 6 h under argon protection. After the reaction was completed, the filter cake was washed with dichloromethane (DCM) three times and dried at 60 °C to obtain 148.41 mg of a yellowish solid intermediate compound B2. The yield was 51%. The product was directly used in the next step without purification.

[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-dihydroquinolin-1(2H)-yl)propanoic acid (150 mg, 0.48 mmol) were placed in a 100 mL two-necked bottle, and 25 mL of ultra-dry CH3CN was used as solvent. The mixture was refluxed at 80°C for 6 h under argon protection. After the reaction, the solvent was dried by spin column chromatography using dichloromethane and methanol in a volume ratio of 99:1, and 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) The intermediate compound C2 (34 mg, 0.057 mmol) and N-(tert-butyloxycarbonyl)-D-lysine (12 mg, 0.057 mmol) were placed in a 50 mL two-necked flask, and a mixed solution containing 0.4 mmol of dicyclohexylcarbodiimide (DCC) and 20 mL of ultra-dry N,N-dimethylformamide (DMF) as a solvent was added dropwise at a rate of 0.2 mL / min. The mixture was reacted at room temperature for 6 h under argon protection. After the reaction, the solvent was dried by spin drying and dried at 60° C. to obtain 42 mg of a purple-black solid intermediate compound D2. The product was directly used in the next step without purification.

[0090] (4) The intermediate compound D2 (42 mg, 0.051 mmol) was placed in a 50 mL single-mouth bottle, and 6 mL of trifluoroacetic acid (TFA) and dichloromethane (DCM) were used as a mixed solvent (the volume ratio of trifluoroacetic acid (TFA) and dichloromethane (DCM) was 1:1) and reacted at room temperature for 3 hours. After the reaction was completed, trifluoroacetic acid (TFA) was removed by rotary evaporation to obtain a 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 an aggregated state:

[0094] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was dissolved in analytically pure dimethyl sulfoxide to prepare 1.0×10 -3 Then prepare 2 mL of dimethyl sulfoxide aqueous solutions with dimethyl sulfoxide (DMSO) contents of 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 0% respectively. Take 11 portions of the above stock solutions with a volume of 20 μL each and add them to the prepared dimethyl sulfoxide aqueous solution. After mixing evenly, transfer to an optical quartz cuvette (10×10 mm) to test its fluorescence spectrum. The results are as follows Figure 1 and Figure 2 shown.

[0095] Figure 1 This is a fluorescence emission spectrum of the Gram-positive bacteria targeted near-infrared fluorescent probe obtained in Example 1 with increasing dimethyl sulfoxide concentration in a dimethyl sulfoxide aqueous solution. Figure 2This is an enlarged effect diagram of the fluorescence emission spectrum of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 with increasing dimethyl sulfoxide concentration in a dimethyl sulfoxide aqueous solution;

[0096] Depend on Figure 1 It can be seen that, with 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, which is located in the near-infrared region, and the Stokes shift reaches 300nm; and the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 aggregates in a dimethyl sulfoxide aqueous solution, resulting in enhanced fluorescence, and has typical aggregation fluorescence enhancement characteristics. Figure 2 It can be seen that the near-infrared fluorescent probe 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 standard MTT method was used to test the cytotoxicity of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1. The specific experimental steps are as follows: First, HeLa cells were plated in a 96-well plate at a cell density of 1×10 4 cells / well and cultured overnight in a cell culture incubator. Different concentrations of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 (0μM, 5μM, 10μM, 20μM, 40μM, 100μM, 100μL / well) were added to different wells, and dimethyl sulfoxide (DMSO) diluted in culture medium (volume concentration of 0.5%, 100μL / well) was used as a negative control group. The cells were cultured in a cell culture incubator for 24h, and then 10μL MTT solution (5mg / mL) was added to each well. After incubation for 4h, the liquid in the 96-well plate was removed, and dimethyl sulfoxide (DMSO) (100μL / well) was added, and finally the assay was performed using an enzyme reader. The results are as shown in the figure. Figure 3 shown.

[0100] Figure 3 This is a graph showing 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 was increased to 100 μM, the cell survival rate reached 91%, indicating that the cytotoxicity of the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was very low.

[0102] Application Example 3

[0103] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can distinguish between Gram-positive bacteria and Gram-negative bacteria:

[0104] Four different Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermis) and four different Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae) were selected for labeling, and then the labeling effect was observed using a fluorescence confocal microscope. The specific experimental steps are as follows: The targeted Gram-positive bacteria near-infrared fluorescent probe obtained in Example 1 was dissolved in sterile water to make 1.0×10 -2 M stock solution. The stock solution was directly added to the exponential bacterial culture to a final concentration of 0.5 mM of the near-infrared fluorescent probe targeting Gram-positive bacteria, and then incubated at 37°C for 1 min with shaking. No washing or fixation was required, and the bacteria were imaged immediately. 1 μL of bacterial solution was added to the slide at room temperature, and finally imaged using a confocal microscope. The imaging results are shown in Figure 4 shown.

[0105] Figure 4 These are graphs showing different fluorescence labeling results of the Gram-positive bacteria targeted near-infrared fluorescent probe obtained in Example 1 for Gram-positive bacteria and Gram-negative bacteria, wherein a is a graph showing the fluorescence labeling results of different Gram-positive bacteria, and b is a graph showing the fluorescence 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, but does not label Gram-negative bacteria. This proves that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can accurately distinguish Gram-positive bacteria from Gram-negative bacteria.

[0107] Application Example 4

[0108] Example 1 The obtained Gram-positive bacteria targeted near-infrared fluorescent probe dynamically labels the cell wall metabolism and synthesis of Gram-positive bacteria:

[0109] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was studied to determine whether it could monitor the synthesis of Gram-positive peptidoglycan in real time. The specific experimental steps were as follows: the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 was dissolved in sterile water to prepare 1.0×10 -2 M stock solution. A mixture of LB medium (Lysogeny broth), agarose, and preheated liquid of the stock solution was added to the cavity slide to a final concentration of 1 mM of the near-infrared fluorescent probe targeting Gram-positive bacteria. After the mixture solidified, Bacillus subtilis and Staphylococcus aureus were loaded on top of the agarose pad, respectively, and then covered with a coverslip. As Gram-positive bacteria grew in the slide, real-time labeling was observed using a time-lapse fluorescence microscope. The results are shown in Figure 5 shown.

[0110] Figure 5 The real-time dynamic fluorescence imaging diagram of the growth of the bacterial wall of Gram-positive bacteria by the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, wherein a is a real-time dynamic fluorescence imaging diagram of the growth of the bacterial wall of Bacillus subtilis, and b is a real-time dynamic fluorescence imaging diagram of the growth of the bacterial wall of Staphylococcus aureus; Figure 5 In a, the left picture at 0 min is the observation result under non-fluorescence, and the right picture is the observation result under fluorescence; the left picture at 35 min is the observation result under fluorescence, and the right picture is the observation result under non-fluorescence. Figure 5 In b, at 6 min, the left picture is the observation result under non-fluorescence, and the right picture is the observation result under fluorescence; at 11 min, the left picture is the observation result under fluorescence, and the right picture is the observation result under non-fluorescence.

[0111] Depend on Figure 5 From a, we can see that Bacillus subtilis is reproducing by binary fission. As time goes by, the fission ring gradually divides one bacterium into two. Figure 5 b It can be seen that Staphylococcus aureus is multiplying by binary fission, and as time goes by, one bacterium gradually splits into two identical ones. This proves that the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 can monitor the synthesis process of Gram-positive bacterial peptidoglycan in real time.

[0112] Application Example 5

[0113] The near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1 non-invasively labeled the positive bacterial flora in the living intestines of IBD enteritis 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; the stock solution was used for gavage of two groups of C57BL / 6J male mice (one group of IBD enteritis model mice, and one group of wt normal mice), and the concentration of the stock solution was 1 mM. After gavage, MARS was used to perform overall fluorescence imaging of the two groups of C57BL / 6J male mice, and the fluorescence changes over time were monitored simultaneously. One C57BL / 6J male mouse from each group was dissected, and fluorescence imaging of the intestine 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 shown.

[0115] Figure 6 These are the in vivo imaging images of the positive bacterial flora in C57BL / 6J male mice using the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, wherein a corresponds to the imaging image of three IBD enteritis model mice using the near-infrared fluorescent probe targeting Gram-positive bacteria over time, and b corresponds to the imaging image of three wt normal mice using the near-infrared fluorescent probe targeting Gram-positive bacteria over time. Figure 7 This is an in vivo imaging image of the positive bacterial flora in the intestine of C57BL / 6J male mice using the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1.

[0116] Depend on Figure 6 It can be seen that after gavage with the near-infrared fluorescent probe targeting Gram-positive bacteria obtained in Example 1, the fluorescent signal first appeared in the stomach of the mouse, and then the signal moved to the colon area as time went by. In addition, the fluorescence intensity of the three IBD enteritis mice was always higher than that of the three wt normal mice, indicating that the number of positive bacteria in the intestines of IBD enteritis mice was higher than that of wt normal mice. Figure 7 It can be seen that the fluorescence intensity of the Gram-positive bacteria targeted near-infrared fluorescent probe obtained in Example 1 is higher in IBD enteritis mice, and weaker in wt normal mice. This shows that the Gram-positive bacteria targeted near-infrared fluorescent probe obtained in Example 1 has good biological penetration and can be further applied to biological in vivo testing.

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe 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 includes -CN, -COOH, R2 includes In R2, n is an integer between 0 and 2, and m is an integer ≥ 1; R3 includes 2. The method for preparing the near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 1, characterized in that: The steps include: (1) mixing compound A, an amine substance and a first solvent and performing a first reaction to obtain an intermediate compound B; (2) mixing the intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinolin-1(2H)-yl)propanoic acid and a second solvent and performing a second reaction to obtain an intermediate compound C; (3) mixing the intermediate compound C, N-(tert-butyloxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and a third solvent and performing a third reaction to obtain an intermediate compound D; (4) mixing the intermediate compound D and the mixed solvent and performing a fourth reaction to obtain a near-infrared fluorescent probe targeting Gram-positive bacteria; The structural formula of the 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 the compound A, intermediate compound B, intermediate compound C and intermediate compound D, R1 includes -CN, -COOH, R2 includes In R2, n is an integer from 0 to 2, and m is an integer ≥1.

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 reaction, the second reaction, the third reaction and the fourth reaction are independently carried out under a protective atmosphere, and the protective atmosphere independently comprises 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 substance includes N,N-dimethylpropylamine sulfonate or trimethylamine; the dosage ratio of compound A, amine substance and first solvent is 1-3mmol:2-10mmol:40-60mL; the temperature of the first reaction is 120-150°C, and the time of the first reaction is 5-7h.

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 usage ratio of the intermediate compound B, 3-(6-(5-formylthiophen-2-yl)-3,4-dihydroquinoline-1(2H)-yl)propanoic 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 dosage ratio of the intermediate compound C, N-(tert-butyloxycarbonyl)-D-lysine, dicyclohexylcarbodiimide and the third solvent is 0.02-0.6mmol:0.02-0.6mmol:0.2-0.6mmol:10-30mL; the temperature of the third reaction is 20-30°C, and the time of the third reaction is 2-4h.

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 includes trifluoroacetic acid and dichloromethane, and the volume ratio of trifluoroacetic acid to dichloromethane is 1-2:1; the usage ratio of the intermediate compound D to the mixed solvent is 0.01-0.4 mmol:4-9 mL; the temperature of the fourth reaction is 20-30° C., and the time of the fourth reaction is 2-4 h.

8. Use of the near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 1 in the preparation of a reagent for differentially labeling Gram-positive bacteria and Gram-negative bacteria.

9. Use of the near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis according to claim 1 in the preparation of reagents for dynamically labeling Gram-positive bacterial cell wall metabolic synthesis.

10. Use of the near-infrared fluorescent probe targeting Gram-positive bacteria based on bacterial wall peptidoglycan synthesis as claimed in claim 1 in the preparation of reagents for non-invasive labeling of positive bacterial flora in vivo imaging in animals.

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