Bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed by D-lysine derivatives and its application method

Abstract

The present invention relates to a bioluminescent detection system and application method of a bacterial cell wall nascent peptidoglycan constructed by a D-lysine derivative, and the system includes a D-fluorescein derivative and a D-lysine derivative. The fluorescein derivative and the amino acid derivative involved in the present invention have a simple synthesis method, and can realize the detection of nascent peptidoglycan in bacterial cell walls using bioluminescent technology, and bioluminescent detection of bacteria in mice. The synthesis method is simple, and luciferase can be used to detect nascent peptidoglycan in cell walls in bacteria and mice. Recognition will not be disturbed by coexisting ions in the environment, and has very high selectivity. Specific click reaction can occur with fluorescein derivatives, and bioluminescent detection is performed using luciferase, with high sensitivity, specificity and tissue penetration.

Description

Technical Field

[0001] The invention relates to a bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed by a D-lysine derivative and an application method. Background Art

[0002] The bacterial cell wall consists of a membrane layer and a rigid yet flexible scaffold called peptidoglycan. Peptidoglycan provides mechanical strength, allowing bacteria to resist environmental damage and dissolution due to internal expansion. Peptidoglycan also plays a key role in maintaining bacterial cell morphology. The role of peptidoglycan in bacterial reproduction and its value as an antibiotic target have aroused new interest in the study of peptidoglycan biosynthesis. However, before a clear understanding of the synthesis and dynamics of peptidoglycan is achieved, there are still major knowledge gaps that need to be addressed. The emergence of fluorescent molecular probes and imaging technology has greatly advanced the study of peptidoglycan synthesis and modification. However, since fluorescent molecular probe detection requires excitation from exogenous excitation light, the imaging background signal is high, and the fluorescence imaging photobleaching is severe and the tissue penetration is poor, it is difficult to effectively detect living bodies.

[0003] Bioluminescence detection technology is simple, has a high signal-to-noise ratio, strong tissue penetration, and high sensitivity. It has become one of the effective means to develop rapid detection of various species. At present, there have been many reports on fluorescent probe compounds for the detection of peptidoglycan, but there have been no reports on the use of bioluminescent probes to detect bacterial and living peptidoglycan. Therefore, it is obviously of great value to design a bioluminescent probe to detect bacterial cell wall peptidoglycan. Summary of the invention

[0004] Technical issues to be solved

[0005] In order to avoid the shortcomings of the prior art, the present invention proposes a bioluminescent detection system for bacterial cell wall nascent peptidoglycan constructed with a D-lysine derivative, provides a preparation method of the bioluminescent detection system for bacterial cell wall nascent peptidoglycan, and is used for detecting peptidoglycan in bacteria and living organisms.

[0006] Technical Solution

[0007] A bioluminescent detection system for bacterial cell wall nascent peptidoglycan constructed by D-lysine derivatives, characterized in that the system comprises a mixture of D-luciferin derivatives and D-lysine derivatives.

[0008] The ratio of the D-fluorescein derivative to the D-lysine derivative is in the range of 1:1-20.

[0009] A D-lysine derivative in the system is characterized by the structural formula:

[0010]

[0011] A method for preparing the D-lysine derivative, characterized by the following steps:

[0012] Step 1: Place 4-azido-2,3,5,6-tetrafluorobenzoic acid in dichloromethane, add oxalyl chloride dropwise at room temperature, add a catalytic amount of DMF, stir at room temperature for 1 to 12 hours, and evaporate to dryness to obtain a crude product of 4-azido-2,3,5,6-tetrafluorobenzoyl;

[0013] The molar ratio of 4-azido-2,3,5,6-tetrafluorobenzoic acid to oxalyl chloride is 1:2;

[0014] Step 2: Dissolve the crude 4-azido-2,3,5,6-tetrafluorobenzoyl chloride in dry tetrahydrofuran, and add it dropwise to N-(tert-butyloxycarbonyl)-D-lysine and potassium bicarbonate aqueous solution; stir at room temperature overnight, and concentrate to remove tetrahydrofuran;

[0015] The molar ratio of 4-azido-2,3,5,6-tetrafluorobenzoic acid to N-(tert-butyloxycarbonyl)-D-lysine is 1:2, and the molar ratio of 4-azido-2,3,5,6-tetrafluorobenzoic acid to potassium bicarbonate is 1:2-5;

[0016] Step 3: Separate the intermediate obtained in step 2 by column chromatography, dissolve it in a small amount of ethyl acetate, add saturated hydrogen chloride ethyl acetate solution to remove Boc, then a white solid precipitates, collect the white solid by filtration to obtain the D-lysine derivative DAz.

[0017] A D-fluorescein derivative in the system, characterized in that: the structural formula of the D-fluorescein derivative is:

[0018]

[0019] A method for preparing the D-luciferin derivative, characterized by the following steps:

[0020] Step 1): dissolving a mixture of 2-diphenylphosphinobenzoic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in dry dichloromethane and stirring at room temperature for 30 minutes; adding the compound 2-cyano-6-hydroxybenzothiazole to the reaction solution and stirring at room temperature for 0 to 12 hours; monitoring the reaction by thin layer chromatography and separating by column chromatography to obtain an intermediate;

[0021] Step 2): Dissolve the intermediate compound in a dichloromethane / methanol solution, add a mixture of D-cysteine ​​hydrochloride and potassium carbonate dissolved in ultrapure water / methanol, and react at room temperature for 0 to 1 hour; add aqueous hydrochloric acid to a pH value of 2-3, filter out the precipitate and rinse with ice water, and separate by column chromatography to obtain a white D-fluorescein derivative TL-PDC.

[0022] An application method of the bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed by the D-lysine derivative is characterized by the following steps: detecting nascent peptidoglycan in bacterial cell walls, and performing bioluminescent detection on bacteria in mice.

[0023] Beneficial Effects

[0024] The present invention proposes a bioluminescent detection system for newly formed peptidoglycan in bacterial cell walls constructed by a D-lysine derivative and an application method thereof, wherein the system comprises a D-luciferin derivative and a D-lysine derivative. The luciferin derivative and the amino acid derivative involved in the present invention have a simple synthesis method, and can realize the detection of newly formed peptidoglycan in bacterial cell walls by bioluminescent technology, as well as bioluminescent detection of bacteria in mice.

[0025] 1. The D-luciferin derivatives and D-lysine derivatives of the present invention have a simple synthesis method and can be used to detect the newly formed peptidoglycan in the cell wall of bacteria and mice using luciferase.

[0026] 2. The recognition of the fluorescein derivatives and non-natural amino acid derivatives of the present invention will not be interfered by coexisting ions in the environment and has very high selectivity.

[0027] 3. The non-natural amino acid derivatives of the present invention, as essential components for the synthesis of peptidoglycan, can undergo specific click reactions with luciferin derivatives, and use luciferase for bioluminescence detection, which has high sensitivity, specificity and tissue penetration. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention provides a synthetic route for the D-fluorescein derivative TL-PDC and the D-lysine derivative DAz.

[0029] Figure 2 This is the selective luminescence response detection of TL-PDC and DAz in the bioluminescence detection system of the present invention.

[0030] Figure 3 In the bioluminescence detection system of the present invention, TL-PDC selectively responds to interferences for detection and quantification.

[0031] Figure 4The luminescent response of newly synthesized peptidoglycan detected by TL-PDC in the bioluminescent detection system of the present invention is shown in (a) the luminescent intensity and quantitative analysis of Escherichia coli and (b) the luminescent intensity and quantitative analysis of Staphylococcus aureus.

[0032] Figure 5 The effect of the retention time of Escherichia coli on the luminescence intensity and quantitative analysis in the bioluminescence detection system of the present invention DETAILED DESCRIPTION

[0033] The present invention will now be further described with reference to the embodiments and the accompanying drawings:

[0034] Embodiment 1:

[0035] In the bioluminescent detection system of bacterial cell wall nascent peptidoglycan of the present invention, the D-luciferin derivative as shown in formula I has a synthetic route as follows: Figure 1 The specific synthesis method steps include:

[0036] A mixture of 2-(diphenylphosphino)benzoic acid (462.0 mg, 1.5 mmol), DMAP (31.0 mg, 0.25 mmol) and EDCI (288.0 mg, 1.5 mmol) was dissolved in dry DCM (15 mL) and stirred at room temperature for 30 minutes. Then 6-hydroxybenzothiazole-2-carbonitrile (264.0 mg, 1.5 mmol) was added and stirred at room temperature overnight. Concentrate under reduced pressure and purify by column chromatography to obtain the intermediate PCN shown in formula III with a yield of 71%; then the intermediate PCN (232.0 mg, 0.5 mmol) was dissolved in 4 mL DCM: MeOH (1: 1, v: v), a mixture of D-cysteine ​​hydrochloride (D-Cys, 165.5 mg, 0.6 mmol) and potassium carbonate (138.0 mg, 0.6 mmol) was dissolved in 5 mL H2O: MeOH (1: 1, v: v) and added to the mixture, and stirred at room temperature for 15 minutes. The organic solution was removed under reduced pressure, and then 0.1 M aqueous hydrochloric acid was added until pH = 3. The precipitate was filtered off and washed with water. The crude product was dried in vacuum and purified by column chromatography to obtain TL-PDC shown in formula I with a yield of 38%.

[0037]

[0038] The D-lysine derivative shown in formula II has a synthetic route as follows Figure 1 The specific synthesis method steps include:

[0039] Weigh 4-azido-2,3,5,6-tetrafluorobenzoic acid (470.0 mg, 2.0 mmol) and dissolve it in dry DCM (15 mL), and add oxalyl chloride (344.0 mL, 4.0 mmol) and DMF (2 μL) dropwise. The reaction mixture was stirred at room temperature for 30 minutes and evaporated under reduced pressure. The mixture was dissolved in dry THF (10 mL), and N-(tert-butyloxycarbonyl)-D-lysine (738.0 mg, 3.0 mmol) and KHCO3 (350.0 mg, 3.5 mmol) were added to the solution under stirring at 0°C. The reaction mixture was stirred at room temperature overnight and then dried in vacuo. Column chromatography purification gave a yellow oily intermediate, which was then dissolved in 1 mL of ethyl acetate, 1 mL of saturated hydrogen chloride-ethyl acetate solution was added, stirred at room temperature overnight, and the white precipitate was collected by filtration to obtain the D-lysine derivative shown in Formula II, with a total yield of 26%.

[0040]

[0041] 1 H NMR(500MHz,DMSO-d6)δ8.29(s,1H),8.13(d,J=9.0Hz,1H),7.86(s,1H),7.65(s,2H),7.41 (s,6H),7.25(s,1H),7.24(s,4H),6.96(s,1H),5.76(s,1H),3.55(dd,J=22.4,12.7Hz,2H).

[0042] 13 C NMR (126MHz, CDCl3) δ164.67,160.64,150.54,149.01,141.86,141.64,140.95, 137.59,137.51,136.11,134.35,134.12,133.96,133.04,132.90,132.53,131.41,128.80, 128.78,128.60,128.54,128.32,124.61,121.33,114.65,31.45.

[0043] The bioluminescent detection system of bacterial cell wall nascent peptidoglycan of the present invention can be used in the presence of luciferase, ATP and Mg 2+ In the presence of , a collectable bioluminescent signal is generated. The present invention mainly solves the defect that it is difficult to realize the in vivo imaging of the newly formed peptidoglycan of the bacterial cell wall in previous reports. Figure 2 For the bioluminescent bacterial cell wall peptidoglycan detection system of the present invention, the selective luminescence response of TL-PDC to DAZ was measured in PBS buffer solution. Figure 2As shown in the figure, at 37°C, when TL-PDC and DAz coexist, Staudinger ligation reaction occurs to release D-luciferin, and a strong light signal is generated under the catalysis of luciferase. The results show that TL-PDC can recognize DAz and successfully release D-luciferin, generating a light signal under the action of luciferase.

[0044] Figure 3 For the bioluminescence detection system of the present invention, the selectivity of related substances existing in organisms was studied. Several common substances in organisms were selected: control group, H2S donor (NaHS, Na2S), glutathione, FeCl2, glycine, H2O2, L-tyrosine, NaClO, L-ascorbic acid. At 37°C, the selectivity of TL-PDC to interfering substances and DAz was tested. Figure 3 As shown, the results showed that although the concentration of the interfering substance was large, D-luciferin could not be produced and the intensity of the light signal produced was negligible.

[0045] comprehensive Figure 2 and Figure 3 The results show that in the bioluminescence detection system of the present invention, TL-PDC has a high specific selectivity for DAz.

[0046] Embodiment 2:

[0047] Figure 4 The luminescent response of TL-PDC to newly synthesized peptidoglycan in the bioluminescent detection system of bacterial cell wall nascent peptidoglycan of the present invention. Escherichia coli and Staphylococcus aureus were incubated with different concentrations of DAz until OD 600 = 2.5, wash several times to remove free DAz, add TL-PDC (20 μM) and incubate for 20 minutes, the luminescence signal gradually increases with the DAz concentration, and quantitative normalization analysis is performed, such as Figure 4 As shown, the study showed that there was a significant correlation between the luminescence intensity and the DAz concentration.

[0048] Embodiment 3:

[0049] Figure 5 For the imaging application of the bioluminescent detection system of bacterial cell wall nascent peptidoglycan in living mice, DAz was co-incubated with Escherichia coli until OD 600 =2.5, 2 mL was taken for multiple washing to remove free DAz, and E. coli was injected into mice by gavage. In order to study the effect of E. coli retention time in mice on the luminescence intensity, TL-PDC (20 μM) was injected into the stomach after one hour and three hours, respectively. Figure 5As shown, different luminescence intensities were obtained. Studies have shown that if bacteria stay in the mouse body for too long, it will lead to bacterial loss, thus affecting the luminescence intensity.

[0050] From the above, it can be seen that the bioluminescent detection system of bacterial cell wall nascent peptidoglycan described in the present invention.

[0051] In the system, TL-PDC cannot be recognized by luciferase for oxidative luminescence. When coexisting with DAz, TL-PDC in the system reacts with DAz by Staudinger ligation, exposing the phenolic hydroxyl group to release free D-luciferin, which generates bioluminescent signals under the catalysis of luciferase. In vitro PBS buffer solution studies show that TL-PDC has good selectivity and high sensitivity for DAz. The experimental results show that DAz can be effectively metabolized and fused into the peptidoglycan of the cell wall by the bacterial cell's own synthesis machinery, and further obtain a good luminescent signal at the bacterial cell level, which can effectively detect the nascent peptidoglycan labeled with aromatic azide. In addition, this system can be used for in vivo imaging of exogenously labeled intestinal bacteria in mice and quantification of luminescence intensity. The experiment shows that this method has certain potential value in biological experiments related to bacterial detection.

[0052] Other embodiments

[0053] Embodiment 4:

[0054]

[0055] Embodiment 5:

[0056]

[0057] Embodiment 6:

[0058]

[0059] Embodiment 7:

[0060]

[0061] Embodiment 8:

[0062]

[0063] Embodiment 9:

[0064]

[0065] Embodiment 10:

[0066]

[0067] Embodiment 11:

[0068]

[0069] Embodiment 12:

[0070]

[0071] Embodiment 13:

[0072]

[0073] Embodiment 14:

[0074]

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bioluminescent detection system for bacterial cell wall nascent peptidoglycan constructed with D-lysine derivatives, characterized by: The system includes a mixture of a D-fluorescein derivative and a D-lysine derivative; the structural formula of the D-lysine derivative is: The structural formula of the D-luciferin derivative is:

2. The bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed by D-lysine derivatives according to claim 1, characterized in that: The ratio of the D-fluorescein derivative to the D-lysine derivative is in the range of 1:1-20.

3. The bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed by D-lysine derivatives according to claim 1, characterized in that: The preparation steps of the D-lysine derivative are as follows: Step 1: Place 4-azido-2,3,5,6-tetrafluorobenzoic acid in dichloromethane, add oxalyl chloride dropwise at room temperature, add a catalytic amount of DMF, stir at room temperature for 1 to 12 hours, and evaporate to dryness to obtain a crude product of 4-azido-2,3,5,6-tetrafluorobenzoyl; The molar ratio of 4-azido-2,3,5,6-tetrafluorobenzoic acid to oxalyl chloride is 1:2; Step 2: Dissolve the crude 4-azido-2,3,5,6-tetrafluorobenzoyl chloride in dry tetrahydrofuran, and add it dropwise to N-(tert-butyloxycarbonyl)-D-lysine and potassium bicarbonate aqueous solution; stir at room temperature overnight, and concentrate to remove tetrahydrofuran; The molar ratio of 4-azido-2,3,5,6-tetrafluorobenzoic acid to N-(tert-butyloxycarbonyl)-D-lysine is 1:2, and the molar ratio of 4-azido-2,3,5,6-tetrafluorobenzoic acid to potassium bicarbonate is 1:2-5; Step 3: Separate the intermediate obtained in step 2 by column chromatography, dissolve it in a small amount of ethyl acetate, add saturated hydrogen chloride ethyl acetate solution to remove Boc, then a white solid precipitates, collect the white solid by filtration to obtain the D-lysine derivative DAz.

4. The bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed according to the D-lysine derivative of claim 1, characterized in that: The preparation steps of the D-luciferin derivative are as follows: Step 1): dissolving a mixture of 2-diphenylphosphinobenzoic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in dry dichloromethane, and stirring at room temperature for 30 minutes; adding the compound 2-cyano-6-hydroxybenzothiazole to the reaction solution, and stirring at room temperature for 0 to 12 hours; monitoring the reaction by thin layer chromatography, and separating by column chromatography to obtain an intermediate; Step 2): dissolve the intermediate compound in a dichloromethane / methanol solution, add a mixture solution of D-cysteine ​​hydrochloride and potassium carbonate dissolved in ultrapure water / methanol, and react at room temperature for 0 to 1 hour; add an aqueous solution of hydrochloric acid to a pH value of 2-3, filter out the precipitate and rinse with ice water, and separate by column chromatography to obtain a white D-fluorescein derivative TL-PDC.

5. A method for using the bioluminescent detection system of bacterial cell wall nascent peptidoglycan constructed by the D-lysine derivative according to claim 1, characterized in that The steps are as follows: for the detection of nascent peptidoglycan in bacterial cell walls and for bioluminescent detection of bacteria in mice.

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