Esterase activated chemiluminescent probe for microbiological detection as well as preparation method and application of esterase activated chemiluminescent probe

Abstract

The invention belongs to the technical field of micromolecular chemiluminescent materials, and particularly relates to an esterase activated chemiluminescent probe for microbiological detection as well as preparation and application of the esterase activated chemiluminescent probe. The chemiluminescent probe disclosed by the invention is a compound prepared from an adamantane-dioxetane skeleton (AD-CL), and the chemiluminescent probe comprises two compounds which are named as ACCL or BUCL; the compound with the structure can be activated by esterase to generate chemiluminescence. The esterase for microbial detection comprises enzymes which are contained in escherichia coli, staphylococcus aureus and salmonella and can hydrolyze acetyl ester groups, butyryl ester groups, hexanoyl ester groups or capryloyl ester groups. The chemiluminescent probe can be used for responding to various esterases in vitro, and can also be used for microbiological detection and clinical disease diagnosis.

Description

Technical Field

[0001] The invention belongs to the technical field of small molecule chemiluminescent materials, and specifically relates to an esterase-activated chemiluminescent probe for microbial detection and a preparation method and application thereof. Background Art

[0002] Microbial infection is an important issue in food safety. Traditional microbial detection methods in the current national food safety standards include bacterial culture, antibody assay, and PCR-based detection. However, these methods often produce some false negative results, which affect clinical diagnosis [Kong MM, Yang B, Gong CJ, Wang H, Li X, Zhao KS, Li JJ, Wu F, Liu X, Hu Z. J Appl Microbiol. 2017, 123(1): 287-294]. Studies have shown that for the purpose of morphological maintenance or normal physiological function, all bacteria can express different esterases, either structural esterases or inducible esterases. For example, Bacillus microsus can express acetyl xylan esterase, and recombinant Escherichia coli can express pyrethroid esterase, etc. [Patil Sadichha, Kulkarni Sayali. Int. J. Curr. Microbiol. App. Sc. 2013, 2(7): 135-146]; some bacteria also express esterases that are related to their drug resistance, such as Escherichia coli expressing an erythromycin esterase, which mediates its resistance to macrolide drugs [Zieliński M, Park J, Sleno B, BerghuisAM. Nat Commun. 2021, 12(1): 1732.]. Therefore, the detection of microbial-related esterases is of great significance for food safety issues caused by microbial infection. For example, the research team of Li et al. developed a fluorescent probe for detecting the esterase activity expressed by Moraxella catarrhalis [Lee U, Kim YH, Yoon KS, Kim Y. Anal Chem. 2020, 92(24): 16051-16057]. Therefore, the development of a simple and rapid chemiluminescence method based on esterase activity will provide effective support for microbial detection and clinical disease diagnosis.

[0003] Chemiluminescence is a phenomenon in which a chemical reaction produces energy that excites a molecule to an excited state and then luminesces when it returns to the ground state. Compared with fluorescence and colorimetry, chemiluminescence avoids the use of external light sources for excitation, so it has low background, amplifiable signals, and is not easily affected by light scattering and autofluorescence, thus achieving high sensitivity. However, most chemiluminescence is suitable for in vitro and alkaline conditions. Molecules with an adamantane-dioxetane structure can produce chemiluminescence in a phosphate buffer at around pH 7, and the energy can be stored in a closed protective group connected to a phenolic group. When a specific molecule is present, the protective group falls off and the energy is released, so that the luminescence measurement of a specific molecule can be performed under physiological conditions. After the research of Professor Schaap in 1987 [Schaap, AP; Handley, RS; Giri, BP Tetrahedron Lett. 1987, 28, 935; Schaap, AP; Chen, T.-S.; Handley, RS; DeSilva, R.; Giri, BP Tetrahedron Lett. 1987, 28, 1155.; Schaap, AP; Sandison, MD; Handley, RS Tetrahedron Lett. 1987, 28, 1159] and Professor Shabat's research group in 2016 [Green O, Hananya EN, Gutkin S, Bauer CR, Shabat D. ACS Central Sci., 2017, 4, 349-58.], it has become possible to use this chemiluminescent skeleton for in vivo imaging of biological molecules [Blau, R., Shelef, O., Shabat, D. et al.Nat.Rev.Bioeng.,2023,1,648–664;FuA,WangH,Huo T,et al.Anal Chem.2021;93(16):6501-6507]. It has the characteristics of no need for external light source, simple method, imaging analysis and low cost.

[0004] In view of the characteristics of the chemiluminescent analysis method described above and the development of the adamantane-dioxetane chemiluminescent system, and in view of the fact that microorganisms have esterase activity, the present invention designed and synthesized two chemiluminescent probes ACCL and BUCL that can be activated by esterase for microbial detection and clinical sample analysis. No complicated reagents, instruments and pretreatment operations are required, and the detection results can be obtained within 1 hour, which is simple and fast. Summary of the invention

[0005] The present invention aims to provide an esterase-activated chemiluminescent probe with sensitive detection, simplicity and rapidity, a preparation method thereof and application thereof in microbial detection and clinical sample analysis.

[0006] The esterase-activated chemiluminescent probe provided by the present invention is a compound containing an adamantane-dioxetane skeleton (AD-CL) and has the following general structural formula:

[0007]

[0008] Where R is CH 3 - or CH 3 CH 2 CH 2 -; correspondingly, named ACCL or BUCL;

[0009] The compound of this structure can be activated by esterase to produce chemiluminescence.

[0010] The present invention also provides a method for preparing the above-mentioned esterase-activated chemiluminescent probe, which specifically comprises the following steps:

[0011] (1) dissolving the compound skeleton AD-CL in an organic solvent, adding an acid-binding agent, lowering the temperature of the reaction solution to -5 to 5°C (preferably -1 to 2°C), then adding an acylating agent acetyl chloride or n-butyryl chloride, and monitoring the reaction process by thin layer chromatography; after the reaction is completed, adding a small amount of organic solvent to dilute the reaction solution, and washing it with a saturated sodium chloride solution for multiple times, combining the organic phases, drying over anhydrous sodium sulfate, and removing the solvent by vacuum rotary evaporation to obtain an intermediate compound; the acylating agent is acetyl chloride, and the corresponding intermediate compound is recorded as p-ACCL, and the acylating agent is n-butyryl chloride, and the corresponding intermediate compound is recorded as p-BUCL;

[0012] (2) dissolving the compound p-ACCL or the compound p-BUCL in an organic solvent, adding an appropriate amount of methylene blue, continuously introducing oxygen, and irradiating with yellow light to react, and monitoring the reaction by high performance liquid chromatography;

[0013] (3) After the reaction is completed, the solvent is removed by vacuum rotary evaporation and purified by thin layer preparative chromatography to obtain compound ACCL or BUCL, both of which are light yellow oils;

[0014] Wherein, the structural formula of the compound AD-CL is as follows:

[0015]

[0016] The structural formulas of the compound p-ACCL and the compound p-BUCL are as follows:

[0017]

[0018] In the present invention, the organic solvent is selected from N,N-dimethylformamide (DMF), dichloromethane (DCM) and dimethyl sulfoxide (DMSO), preferably DCM;

[0019] Step (1), the acid binding agent is potassium carbonate or triethylamine, preferably triethylamine;

[0020] In step (1), the equivalents of the acid-binding agent and the acylating agent are both 2.0 (based on the equivalent of compound AD-CL, p-ACCL or p-BUCL being 1.0);

[0021] In step (2), oxygen is directly introduced into the reaction solution, and the reaction temperature ranges from 18 to 35°C.

[0022] In step (2), the conditions of high performance liquid chromatography are as follows: the chromatographic column is a C18 liquid chromatography column; the mobile phase is acetonitrile in phase A and ammonium formate aqueous solution (pH=3) containing glacial acetic acid in phase B; the flow rate is 1.0 mL / min;

[0023] In steps (1), (2) and (3), the thin layer chromatography reaction conditions are n-hexane or petroleum ether: ethyl acetate = (2-5): 1;

[0024] The present invention also provides the use of the chemiluminescent probe in microbial detection. The detection concentration of the microorganism is 10 3 -10 7 cfu / mL, and the concentration of the probe was 20 μM-200 μM.

[0025] The esterases for microbial detection include enzymes contained in Escherichia coli, Staphylococcus aureus and Salmonella that can hydrolyze acetyl ester groups, butyryl ester groups, hexanoyl ester groups or octanoyl ester groups. This type of chemiluminescent probe can be used to respond to various esterases in vitro, and can also be used for microbial detection and clinical disease diagnosis.

[0026] The microorganisms are Escherichia coli, Staphylococcus aureus and Salmonella.

[0027] After activation, the chemiluminescent probe has fluorescent properties and can be used for confocal imaging of microorganisms at a concentration of 20μM-200μM.

[0028] The chemiluminescent probe ACCL of the present invention can be used for detecting leukocyte esterase in clinical vaginitis samples, and the probe concentration is 20 μM-200 μM.

[0029] The esterase-activated chemiluminescent probe designed and synthesized by the present invention for microbial detection can be turned on and release light signals only when the probe encounters esterase (such as acetylesterase, butyrylesterase or octanoylesterase), and can be used for the analysis of esterase activity in vitro, and can also be used for esterase detection in microorganisms and leukocyte esterase analysis in clinical vaginal inflammation samples. The use of adamantane-dioxetane chemiluminescent probe in the field of microbial detection and clinical sample analysis is innovative and groundbreaking, and further expanding its scope of application will be an important development direction in the field of food safety and clinical disease diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the synthetic route of the chemiluminescent probe ACCL.

[0031] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of the chemiluminescent probe ACCL.

[0032] Figure 3 This is the synthetic route of the chemiluminescent probe BUCL.

[0033] Figure 4 This is the H NMR spectrum of the chemiluminescent probe BUCL.

[0034] Figure 5 The chemiluminescence kinetic curves of probes ACCL and BUCL are shown in Figure 2.

[0035] Figure 6 Esterase selectivity of probes ACCL and BUCL.

[0036] Figure 7 The ACCL probe is used for microbial detection and the activated probe is used for imaging analysis of microorganisms.

[0037] Figure 8 The probe ACCL is used for the detection of leukocyte esterase in clinical vaginal inflammation samples. DETAILED DESCRIPTION

[0038] The present invention will be further described below by way of embodiments in conjunction with the accompanying drawings.

[0039] The abbreviations represent the names of the compounds: adamantane-dioxetane skeleton (AD-CL), dichloromethane (DCM), petroleum ether (PE), ethyl acetate (EA), and dimethyl sulfoxide (DMSO).

[0040] Example 1: Synthesis of esterase-activated adamantane-dioxetane chemiluminescent probes ACCL and BUCL;

[0041] (I) Synthesis of compound ACCL. The synthetic route is shown in the attached Figure 1 The specific steps are:

[0042] Synthesis of compound p-ACCL:

[0043] Compound AD-CL (100 mg, 1.0 eq) was dissolved in 6 mL DCM, triethylamine (82 μL, 2.0 eq) was added, the mixture was cooled to 0 °C in an ice bath, and then acetyl chloride (59 μL, 2.0 eq) was added dropwise. After the addition was complete, there was no significant change in the reaction solution, and the reaction was monitored by thin layer chromatography (PE: EA = 3: 1). After the reaction was completed, an appropriate amount of DCM was added to dilute it, and it was washed with saturated sodium chloride solution (10 mL, 3 times). The organic phases were combined, dried over anhydrous sodium sulfate solid, and the solvent was removed by vacuum rotary evaporation to obtain a crude compound p-ACCL (100 mg, colorless oil). MS (ESI-): m / z C 24 H 27 C1O 5 : 430.15 (theoretical value), 429.15 [MH]-.

[0044] Synthesis of compound ACCL:

[0045] Compound p-ACCL (100 mg) was dissolved in 8 mL DCM, a small amount of methylene blue was added, oxygen was continuously introduced, and yellow light was irradiated. The reaction was monitored by high performance liquid chromatography. After the reaction was completed, the compound was purified by thin layer preparative chromatography to obtain compound ACCL (43 mg, light yellow oil). MS (ESI-): m / z C 24 H 27 C1O 7 :462.14 (theoretical value), visible 461.22 [MH] - . 1 H NMR (600 MHz, CDCl 3 )δ8.04(d,J=8.4Hz,1H),7.68(d,J=16.1Hz,1H),7.66(d,J=8.5Hz,1H),6.53(d,J= 16.1Hz,1H),3.82(s,3H),3.21(s,3H),2.44(d,J=4.4Hz,3H),2.25(d,J=12.9Hz,1H ),1.99(s,1H),1.84(dd,J=10.2,7.7Hz,2H),1.74–1.71(m,2H),1.63(dd,J=12.8,10.3Hz,3H),1.47(dd,J=12.9,2.4Hz,1H),1.37–1.33(m,1H),1.29–1.25(m,3H), as attached Figure 2 shown.

[0046] (II) Synthesis of compound BUCL. The synthetic route is shown in the attached Figure 3 As shown, the specific steps are:

[0047] Synthesis of compound p-BUCL:

[0048] Compound AD-CL (40 mg, 1.0 eq) was dissolved in 3 mL of anhydrous DCM, triethylamine (29 μL, 2.0 eq) was added, the mixture was cooled to 0 °C in an ice bath, and then n-butyryl chloride (21 μL, 2.0 eq) was added dropwise. After the addition was complete, there was no significant change in the reaction solution, and the reaction was monitored by thin layer chromatography (PE: EA = 3: 1). After the reaction was completed, an appropriate amount of DCM was added to dilute it, and it was washed with saturated sodium chloride solution (6 mL, 3 times). The organic phases were combined, dried over anhydrous sodium sulfate solid, and the solvent was removed by vacuum rotary evaporation to obtain a crude compound p-BUCL (86 mg, colorless oil).

[0049] Synthesis of compound BUCL:

[0050] The compound p-BUCL (38 mg) was dissolved in 8 mL of dichloromethane, a small amount of methylene blue was added, oxygen was continuously introduced, and yellow light was irradiated. The reaction was monitored by high performance liquid chromatography. After the reaction was completed, the compound was purified by thin layer preparative chromatography to obtain the compound BUCL (21 mg, light yellow oil). MS (ESI-): m / z C 26 H 31 C1O 7 =478.18 (theoretical value), found 478.50. 1H NMR (600MHz, CDCl3) δ8.02 (d, J = 8.4Hz, 1H), 7.66 (dd, J = 31.5, 12.2Hz, 1H), 6.52 (d, J = 16.1Hz, 1H), 3.82 (d, J = 1.4Hz, 1H), 3.21 (s, 1H), 2.62 (d, J = 56.3Hz, 1H), 2.26 (dd, J = 13. 5,3.1Hz,1H),2.00–1.95(m,1H),1.83(d,J=15.0Hz,1H),1.68–1.58(m,7H),1.48–1.43(m,1H),1.41–1.37(m,1H),1.35–1.32(m,1H),1.16–1.10(m,1H),0.90–0.79(m,2H), as attached Figure 4 shown.

[0051] Example 2: Determination of chemiluminescence kinetic curves of probes ACCL and BUCL

[0052] Prepare 4mM DMSO solution of probes ACCL and BUCL as stock solution, dilute the 4mM probe solution (DMSO) with phosphate buffer containing 1% DMSO to obtain a 40μM probe solution. Dilute the mother solution of carboxylesterase 2 (CES2) with phosphate buffer to obtain 20μg / mL CES2. Then add 100μL of 0 and 20μg / mL CES2 solution to the wells of a black 96-well plate, add 100μL of probe solution (40μM), and immediately use a fluorescent chemiluminescence instrument to measure, and record the chemiluminescence kinetic curves of probes ACCL and BUCL, as shown in the attached figure. Figure 5 As shown, the probes ACCL and BUCL can be used for the determination of carboxylesterase 2.

[0053] Example 3: Selectivity study of probes ACCL and BUCL

[0054] In order to study the selectivity of the two probes ACCL and BUCL for different esterases and other proteases, two highly homologous carboxylesterases 1 and 2 (CES1 and CES2) and a dipeptide kinase IV (DPPⅣ), which is also a serine hydrolase, were used as model enzymes to study the esterase detection selectivity of the probes and evaluate their application range. First, DPPⅣ (2μg / mL), CES1 (10μg / mL), and CES2 (10μg / mL) solutions were prepared by diluting with phosphate buffer, and 100μL of the above prepared solutions or 100μL PBS (blank experimental group) were taken and added to a black 96-well plate. Finally, 100μL of the probe ACCL and BUCL solutions (40μM, 2% DMSO, PH 7.5) were added to each well. The signal was immediately measured using a fluorescent chemiluminescence instrument. The results are shown in the figure. Figure 6 As shown in the figure, the probe ACCL can respond to two esterases, CES1 and CES2, but has a low response to DPPIV. Therefore, compared with non-esterase proteases, it has a higher response ability to esterases and has a certain selectivity; the probe BUCL not only has no obvious response to DPPIV, but also has selectivity in the determination of CES1 and CES2 among esterases. The response signal intensity of the probe BUCL to CES2 is 10 times that of CES1. Therefore, ACCL, which has a better ability to determine esterase spectra, was selected for the detection of esterases in microorganisms in the future; and BUCL is suitable for the detection of CES2-related diseases in vivo.

[0055] Example 4: Application of probe ACCL in microbial detection

[0056] In order to study the application of chemiluminescent probe ACCL in microbial detection, Escherichia coli was cultured in liquid culture medium, and the bacterial concentration after expansion was calculated by colony counting method and ultraviolet spectrophotometry; the supernatant was removed by centrifugation, and the Escherichia coli was collected and washed 2-3 times with physiological saline or phosphate buffer, and the concentration was prepared to 10 7 cfu / mL of bacterial solution, and then diluted with normal saline to 5×10 6 , 2.5×10 6 , 1.25×10 6 , 6.25×10 5 and 3.12×10 5 cfu / mL of bacterial solution, add the probe ACCL solution (100μM) and immediately measure the chemiluminescent signal. Then, take another 100cfu / mL of E. coli and place it in a confocal observation dish, add the probe ACCL, and perform laser confocal imaging analysis after 1-2 hours of reaction. The activated probe has a fluorescent signal, which can observe the growth status of E. coli for a long time, and has a stronger response ability to E. coli in the active growth period. The results are as follows Figure 7 As shown in A: The probe ACCL can be used for esterase detection in microorganisms. 5 -10 7 cfu / mL concentration range was linearly correlated (y = 12.29x-10.078, R 2 =0.9964), with good reproducibility (RSD≤15%). Figure 7 As shown in B: It shows that the probe ACCL can still remain in the microorganism after activation for fluorescence observation. It is worth noting that the fluorescence signal displayed in the microorganism in the division stage is significantly stronger than that in the microorganism with low activity, suggesting that esterase can characterize the activity state of the microorganism to a certain extent.

[0057] Example 5: Application of the probe ACCL in the determination of leukocyte esterase in clinical vaginitis samples

[0058] To study the application of chemiluminescent probe ACCL in clinical cases of microbial infection. The probe was used to detect 17 clinical vaginitis samples. The number of leukocytes was observed by culture in clinical examination, and leukocyte esterase was determined by colorimetry. The clinical determination results were grouped according to the number of leukocytes and leukocyte esterase results: A was negative / weakly positive group (- / +), the number of leukocytes was ≤30, and the leukocyte esterase was 0 / 1 level; B was medium-strong or strong positive group, the number of leukocytes was >30, and the leukocyte esterase was 2 / 3 level. Subsequently, the probe ACCL was used to detect leukocyte esterase. The 4mM probe ACCL solution was diluted to 40μM with phosphate buffer containing 1% DMSO. Then, the mother solution of leukocyte esterase (LE) was diluted with phosphate buffer to obtain 1μg / mL LE as a positive control, and the clinical samples were directly diluted 10 times with phosphate buffer before determination. 100 μL of each sample was added to a black-bottom 96-well plate, and then 100 μL of 40 μM probe was added to immediately measure the chemiluminescent signal. The results of the chemiluminescent probe method were compared with the clinical data, such as Figure 8 As shown, the two groups of data are correlated, and the results based on the chemical probe assay have significant differences between the negative group and the positive group (**p<0.001).

Claims

1. An esterase-activated chemiluminescent probe for microbial detection, characterized in that: It is a compound containing an adamantane-dioxetane skeleton (AD-CL) and has the following general structural formula: Wherein, R is CH3- or CH3CH2CH2-; correspondingly, it is named ACCL or BUCL; this structural compound can be activated by esterase to produce chemiluminescence.

2. A method for preparing the esterase-activated chemiluminescent probe according to claim 1, characterized in that: The specific steps are: (1) The compound skeleton AD-CL is dissolved in an organic solvent, an acid-binding agent is added, the temperature of the reaction solution is lowered to -10 to 5°C, and then an acylating agent acetyl chloride or n-butyryl chloride is added, and the reaction process is monitored by thin layer chromatography; after the reaction, a small amount of organic solvent is added to dilute the reaction solution, and the solution is washed with a saturated sodium chloride solution for multiple times, the organic phases are combined, dried over anhydrous sodium sulfate, and the solvent is removed by vacuum rotary evaporation to obtain an intermediate compound; the acylating agent is acetyl chloride, and the corresponding intermediate compound is recorded as p-ACCL, and the acylating agent is n-butyryl chloride, and the corresponding intermediate compound is recorded as p-BUCL; (2) dissolving the compound p-ACCL or the compound p-BUCL in an organic solvent, adding methylene blue, continuously introducing oxygen, and irradiating with yellow light to react, and monitoring the reaction process by high performance liquid chromatography; (3) After the reaction is completed, the solvent is removed by vacuum rotary evaporation and purified by thin layer preparative chromatography to obtain compound ACCL or BUCL, both of which are light yellow oils; Wherein, the structural formula of the compound AD-CL is as follows: The structural formulas of the compound p-ACCL and the compound p-BUCL are as follows:

3. The preparation method according to claim 2, characterized in that: The organic solvent is selected from N,N-dimethylformamide, dichloromethane and dimethyl sulfoxide.

4. The preparation method according to claim 2, characterized in that: In step (1), the acid-binding agent is potassium carbonate or triethylamine; the equivalents of the acid-binding agent and the acylating agent are both 2.0, with the equivalent of compound AD-CL, p-ACCL or p-BUCL being 1.0 as the standard.

5. The preparation method according to claim 2, characterized in that: In step (2), the oxygen is directly introduced into the reaction solution, and the reaction temperature ranges from 18 to 35°C.

6. The preparation method according to claim 2, characterized in that: In step (2), the conditions of high performance liquid chromatography are: the chromatographic column is a C18 liquid chromatography column; the mobile phase: phase A is acetonitrile, and phase B is an aqueous solution of ammonium formate containing glacial acetic acid; the flow rate is 1.0 mL / min.

7. The preparation method according to claim 2, characterized in that: The conditions of the thin layer chromatography detection reaction involved are n-hexane or petroleum ether: ethyl acetate = (2-5):

1.

8. The use of the esterase-activated chemiluminescent probe in microbial detection as claimed in claim 1, wherein the detection concentration of the microorganism is 10 3 -10 7 cfu / mL, and the concentration of the probe was 20 μM-200 μM.

9. The use according to claim 8, wherein the microorganism is Escherichia coli, Staphylococcus aureus or Salmonella.

10. The use according to claim 8, further used for confocal imaging analysis, wherein the concentration of the probe is 20 μM-200 μM.

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