CN112409322A

CN112409322A - GGT activated chemiluminescent probe and synthesis method and application thereof

Info

Publication number: CN112409322A
Application number: CN201910783411.5A
Authority: CN
Inventors: 叶德举; 安瑞冰; 魏世轩
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2021-02-26
Anticipated expiration: 2039-08-23
Also published as: CN112409322B

Abstract

The invention discloses a chemiluminescent probe, which comprises an amino acid substrate gamma-Glu (adamantane) -1, 2-dioxetane chemiluminescent group capable of being specifically hydrolyzed by GGT and a self-cleavable connecting group. The invention also discloses a synthetic method and application of the chemiluminescent probe. The chemiluminescence probe provided by the invention has extremely high signal-to-noise ratio in imaging, and can be used for realizing high sensitivity and high signal-to-noise ratio of detection on serum, cell and living body levels.

Description

GGT activated chemiluminescent probe and synthesis method and application thereof

Technical Field

The invention belongs to the field of biological probes, and relates to a chemiluminescent probe and a synthetic method and application thereof.

Background

Gamma-glutamyl transpeptidase (GGT) is a protease present on the surface of cell membranes that hydrolyzes Glutathione (GSH) from outside the cell to produce cysteine (cysteine) for the re-synthesis of GSH inside the cell. GGT plays an important role in maintaining the homeostatic balance of GSH and cysteine in cells, and thus plays an important biological role in many physiological and pathological processes, and abnormal changes in GGT activity are often associated with many diseases, such as liver function impairment, asthma, diabetes, and cancer. Also, GGT is commonly used in the diagnosis of liver and obstructive biliary diseases as an index for clinical routine examination of blood. In addition, GGT is overexpressed in many types of malignancies, such as liver, neck, and ovarian cancers, due to its ability to promote tumor cell proliferation, metastasis, and resistance. Therefore, the ability to accurately detect the activity of GGT and to visualize the location of its distribution will aid in the diagnosis of disease and the guided evaluation of cancer treatment.

At present, many methods for detecting GGT activity have been developed, such as high-performance liquid chromatography, electrochemical method, colorimetric method, and fluorescence method. The fluorescence detection method has the advantages of high sensitivity, simplicity in operation, non-invasiveness and the like, and can be used for detecting in vitro samples such as blood samples and pathological tissue samples, and can also be used for performing visual imaging detection on the expression activity of GGT on a living body level. Based on this, in recent years, researchers have developed many fluorescent probes capable of detecting GGT activity with high sensitivity. For example, Urano, Inc. of Japan developed a GGT fluorescent probe that can be specifically activated by GGT and emits strong green fluorescence under excitation of external excitation light. By means of direct spraying, the method is successfully applied to imaging detection of mouse abdominal cavity ovarian cancer. Recently, two GGT near-infrared fluorescent probes were also developed for imaging detection of GGT activity in mouse subcutaneous tumor-bearing cells. Although the existing fluorescent probe can carry out imaging detection on GGT activity at the living body level, the existing fluorescent probe has the defects of low tissue penetrability, easy photobleaching, easy generation of tissue background fluorescence interference under external light excitation and the like.

Disclosure of Invention

In order to overcome the defects of the fluorescent probe, the invention provides a chemiluminescent probe which comprises an amino acid substrate gamma-Glu (gamma-glutamic acid) capable of being specifically hydrolyzed by GGT, an (adamantane) -1, 2-dioxetane chemiluminescent group and a self-cleavable connecting group.

In one embodiment, the (adamantane) -1, 2-dioxetane-based chemiluminescent group is substituted with a methyl acrylate group.

In one embodiment, the (adamantane) -1, 2-dioxetane-based chemiluminescent group is an (adamantane) -1, 2-dioxetane-based chemiluminescent group is 3- (2' -spiroadamantane) -4-methoxy-4- (2 "-chloro-3" -hydroxy-4 "-methyl acrylate) -phenyl-1, 2-dioxetane.

In one embodiment, the self-cleavable linking group is para-aminobenzyl alcohol.

The invention also provides a method for synthesizing the chemiluminescent probe, which is realized by the following process S1,

directly reacting the compound 6((E) -3- (4- (((1R,3R,5R,7S) -adamantan-2-ene) (methoxy) methyl) -3-chloro-2-hydroxyphenyl) acrylic acid methyl ester (methyl (E) -3- (4- (((1R,3R,5R,7S) -adaman-2-ylene) (methoxy) -3-chloro-2-hydroxyphenyl) acrylate)) and the compound 7(N5- (4- (bromomethyl) phenyl) -N2- (tert-butoxycarbonyl) -D-glutamic acid diphenylmethyl ester (benzyl N5- (4- (bromomethyl) phenyl) -N2- (tert-butoxycarbyl) -D-glutaminate)) to obtain the compound 8(N5- (4- ((3- (((1R), 3R,5R,7S) -adamantan-2-ene) (methoxy) methyl) -2-chloro-6- ((E) -3-methoxy-3-oxoprop-1-en-1-yl) phenoxy) methyl) phenyl) -N2- (tert-butoxycarbonyl) -D-glutamic acid diphenylmethyl ester (benzyl N5- (4- ((3- (((1R,3R,5R,7S) -adaman-2-ylene) (methoxy) methyl) -2-chloro-6- ((E) -3-methoxy-3-oxop-1-en-1-yl) phenoxy) methyl) -N2- (tert-butoxy-carbonyl) -D-glutamine)), then, the singlet oxygen is oxidized to obtain a compound 9(N2- (tert-butoxycarbonyl) -N5- (4- ((2-chloro-6- ((E) -3-methoxy-3-oxoprop-1-en-1-yl) -3- ((1R,3S,5R,7R) -4' -methoxyhelix [ adamantane-2,3' - [1,2] dioxane ] -4' -yl) phenoxy) methyl) phenyl) -D-glutamic acid diphenylmethyl ester (benzyl N2- (tert-butoxycarbnyl) -N5- (4- ((2-chloro-6- ((E) -3-methoxy-3-oxoprop-1-en-1-yl) -3- ((1R,3S,5R,7R) -4' -methoxylpiro [ adamantane-2,3' - [1,2] dioxan ] -4' -yl) phenoxy) methyl) phenyl) -D-glutaminate ], compound 9 is deprotected to obtain probe compound 1(N5- (4- ((2-chloro-6- ((E) -3-methoxy-3-oxoprop-1-en-1-yl) -3- ((1R,3S,4' S,5S,7S) -4' -methoxyhelix [ adamantane-2,3' - [1,2] dioxane ] -4' -yl) phenyl) methyl) phenoxy) -L-glutamic acid diphenylmethyl ester (benzylphenyl N5- (4- ((2-chloro-6- ((E) -3-methoxy-3-oxoprop)), and the product is obtained -1-en-1-yl) -3- ((1R,3S,4'S,5S,7S) -4' -methoxyspro [ adamantane-2,3'- [1,2] dioxetan ] -4' -yl) phenoxy) methyl) phenyl) -L-glutamine)).

In one embodiment, the synthetic scheme comprises the steps of:

(1) mixing compound 6, compound 7 and K₂CO₃And KI is dissolved in anhydrous N, N-dimethylformamide, stirred at room temperature and reacted for 5 hours, after the reaction is completed, ethyl acetate and saturated saline are added into a reaction system for extraction, an organic phase is collected and dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure rotary evaporation, and the obtained solid is purified by column chromatography to obtain a white compound 8 solid;

(2) dissolving the compound 8 in dichloromethane, adding methylene blue, filling oxygen into a reaction system in a bubbling mode under ice bath, irradiating by using a white light LED lamp, and after complete reaction, decompressing and removing the solvent to obtain a crude product of a compound 9;

(3) and dissolving the crude product of the compound 9 in a mixed solvent of dichloromethane/trifluoroacetic acid/TIPSH, carrying out deprotection reaction at 0 ℃, adding saturated sodium carbonate to adjust the pH value to be neutral after the reaction is finished, and separating the crude product by a prepared liquid phase to obtain a white solid probe 1 compound.

The chemiluminescence probe can be used for measuring the content of GGT in serum.

The chemiluminescent probes of the present invention may be used for the chemiluminescent use of GGT activity in living cells.

The chemiluminescent probe of the present invention may be used in the chemiluminescent determination of the number of tumor cells.

The chemiluminescent probe of the present invention may be used in the chemiluminescence detection of GGT activity at the living body level.

Unlike conventional fluorescence, chemiluminescence is a phenomenon in which photons are generated by chemical excitation, and it does not require real-time excitation by external light to achieve emission of photons. Due to the fact that interference of sample background fluorescence is avoided, the signal to noise ratio of chemiluminescence imaging is extremely high, and the method can be used for achieving high sensitivity and high signal to noise ratio of detection on a living body level.

The chemiluminescent probe can be used for carrying out chemiluminescence detection on GGT activity at serum, cell and living body levels.

Brief description of the drawings

Figure 1. chemical structure of GGT activated chemiluminescent probe 1 and its possible chemical transformation processes after its response to GGT.

FIG. 2 (a) UV-VIS absorption and fluorescence spectra of Probe 1(10 μ M) in enzyme assay buffer (PBS with 5% DMSO, pH7.4) before (solid line) and after 6 hours incubation (dashed line) with GGT (250U/L). (b) HPLC analysis of Probe 1 (10. mu.M) before (solid line) and after (dashed line) incubation at 37T for 6h with GGT (250U/L). (c) Chemiluminescence spectra and chemiluminescence photographs (inset) of probe 1(10 μ M) after incubation for 30min at 37 ℃ before (lower curve) and after addition of GGT (100U/L) (upper curve). (d) Chemiluminescence kinetics curves for probe 1(10 μ M) at 250min incubation before (lower curve) and after (upper curve) addition of GGT (100U/L) at 37 ℃.

FIG. 3 stability analysis of Probe 1. (a) PBS buffer with Probe 1 (10. mu.M) or PBS buffer without Probe 1 was incubated at 37 ℃ for a time course of 6 hours change in background chemiluminescent emission intensity. Values are expressed as mean ± standard deviation (n ═ 3). (b) Probe 1 (10. mu.M) was incubated in PBS buffer, DMEM with 10% Fetal Bovine Serum (FBS) in the absence of GGT at 37 ℃ for 0 and 6 hours of chemiluminescence enhancement. Values are expressed as mean ± standard deviation (n ═ 3). (c) Results of HPLC analysis of Probe 1 (10. mu.M) after incubation for 6 hours at 37 ℃ in DMEM, DMEM containing 10% Fetal Bovine Serum (FBS), PBS buffer and PBS buffer containing 100U/L GGT.

FIG. 4 (a) images of chemiluminescence after incubation of Probe 1 (10. mu.M) in GGT digestion buffer with various concentrations of GGT (0,2.5,5,10,25,50,100 and 250U/L) for 10 minutes at 37 ℃. (b) The average chemiluminescence intensity of each group in panel (a) is plotted as a linear fit to the GGT concentration (0-250U/L). (c) The chemiluminescence enhancement factor after incubation of probe 1 (10. mu.M) with different types of enzymes at 37 ℃ for 25min in digestion buffer. 1. The probe 1 itself; 2. probe 1+ trypsin (2 μ g/mL); 3. probe 1+ ALP (100U/L); 4. probe 1+ MMP-2(10 nM); 5. probe 1+ caspase-3 (1. mu.g/mL); 6. probe 1+ cathepsin B (2. mu.g/mL); 7. probe 1+ GGT (100U/L); 8. probe 1+ GGT (100U/L) + GGsTop (200. mu.M). (d) Probe 1 shows chemiluminescence intensity after incubation of healthy mouse serum diluted 10-fold with GGT digestion buffer solution and mouse serum treated with LPS at 37 ℃ for 10 minutes. (e) GGT activity in mouse serum was calculated from chemiluminescence intensity in panel d. P < 0.05.

FIG. 5 (a) cell survival after incubation of OVCAR5, U87MG and HUVEC cells for 24 hours at different concentrations of probe 1(0,10, 20. mu.M). (b) Indicated viable cells (approximately 2X 10 per well)⁴One) real-time measurement of chemiluminescence intensity after incubation with probe 1(10 μ M). I: OVCAR5, II: U87MG, III: HUVEC, IV: U87MG + GGsTop (200. mu.M), V: OVCAR5+ GGsTop (200. mu.M). Values are expressed as mean ± standard deviation (n ═ 3). (c) Chemiluminescent imaging of the indicated cells in the culture plates referred to in panel (a). The chemiluminescent imaging was obtained at 1 hour.

Figure 6 OVCAR5 tumor cells were detected using probe 1 at in vitro and in vivo levels, respectively. (a) Different numbers of OVCAR5 cells (0, 2X 10 cells per well)²、5×10²、1×10³、2×10³、5×10³、1×10⁴、2×10⁴And 4X 10⁴Individual cells) were incubated with probe 1(10 μ M) at 37 ℃ for 30 minutes before chemiluminescence imaging. (b) The linear fit curve of mean chemiluminescence intensity versus number of cells in panel (a). Values are expressed as mean ± standard deviation (n ═ 3). (c) Mice were injected subcutaneously with different numbers of OVCAR5 cells (0, 5X 10) mixed with Probe 1 (10. mu.M)²、1×10³、2×10³、5×10³、1×10⁴、2×10⁴Individual cells) followed by chemiluminescence imaging. The arrow indicates the injection site. (d) The logarithm of the average chemiluminescence intensity quantified in graph (c) is a linear fit to the logarithm of the number of cells.

FIG. 7 investigation of tissue penetration depth. (a) Chemiluminescence and fluorescence imaging images of probe 1(10 μ M) after incubation with GGT (250U/L) for 20 minutes at 37 ℃ followed by coverage with chicken tissue of different thicknesses (0,5,10,15,20mm) wherein the chemiluminescence imaging acquisition time was 0.75s and the fluorescence imaging was taken with 420nm excitation and with a 570nm emission filter (b) signal to noise ratio (SBR) analysis of the luminescence signal in panel (a): the dashed circle indicates the selected background area.

FIG. 8 in vivo GGT chemiluminescence imaging. (a) U87MG tumor mice were subjected to whole body real-time chemiluminescence imaging of mice injected with the probe via tail vein (100. mu.M, 100. mu.L) or pre-intratumorally injected GGsTop (10mM, 50. mu.L GGsTop) followed by intravenous injection of the probe. The dashed circle represents the selected background area and the arrow represents the location of the tumor. (b) Graph (a) mean chemiluminescence intensity of each group was varied with time. (c) Panel (a) TBR of the mouse imaging signals shown in each group. Values are expressed as mean ± standard deviation (n ═ 3). P <0.05, P < 0.001.

Detailed Description

The present invention is illustrated in detail by the following examples.

Reagent and apparatus

All chemicals and solvents were purchased from Bailingwei technologies, Shanghai, into Industrial development, Inc., and Sigma-Aldrich. The analytical solvent and the reagent are chromatographically pure, and the conventional reagent is analytically pure and is not further purified. High sugar Dulbeccoo's Modified Eagle Medium (DMEM), high sugar Roswell Park Memori (RPMI)1640Medium, Fetal Bovine Serum (FBS), Trypsin (Trypsin) and 3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kits were purchased from KeyGen Biotech (south Beijing, China). U87MG, OVCAR5 and HUVEC cells were purchased from shanghai life science research institute cell banks, chinese academy of sciences.

Nuclear magnetic spectrum (¹H NMR and¹³c NMR) determined by a 400MHz Bruker Avance III 400spectrometer, the solvent being DMSO-d₆Or CD₃OD, chemical shift (. delta.) in ppm, singlet, doublet, triplet, quartet, dd (doublelet of do)ublets) peaks, multiplets and broad peaks are denoted by s, d, t, q, dd, m and br, respectively; coupling constants (J) are in Hz; the number of hydrogens was determined by integration of the map and marked nH. High Performance Liquid Chromatography (HPLC) analysis was performed by Thermo Scientific Dionex Ultimate 3000 with mobile phase of acetonitrile and water containing 1 ‰ trifluoroacetic acid; UV-visible absorption spectra were measured using Ocean Optics Maya 2000 Pro; fluorescence spectra were measured using a Hitachi F-7000Fluorescence Spectrophotometer; the cell fluorescence pictures were taken with a Leica TCS SP8 laser confocal fluorescence microscope (Leica TCS SP8confocal laser scanning microscope);

design, Synthesis and characterization of probes

Fig. 1 shows the structural design of the probe. The probe mainly comprises an amino acid substrate gamma-Glu which can be specifically hydrolyzed by GGT, a 3- (2 '-spiral adamantane) -4-methoxy-4- (2' -chlorine-3 '-hydroxyl-4' -methyl acrylate) -phenyl-1, 2-dioxetane chemiluminescence group (3) with strong emission and a self-cleavable connecting group p-aminobenzyl alcohol. The enzyme digestion substrate gamma-Glu is widely used for a recognition group of a GGT fluorescent probe, and the 3- (2 '-spiral adamantane) -4-methoxy-4- (2' -chlorine-3 '-hydroxyl-4' -methyl acrylate) -phenyl-1, 2-dioxetane chemiluminescence group (3) can emit yellow green fluorescence under physiological conditions and has strong chemiluminescence efficiency, and can be used for living body imaging. In addition, the self-breaking connecting group on the aminobenzyl alcohol helps to reduce the influence of the steric hindrance of the probe and helps the gamma-Glu part to extend into the deep and narrow active site of GGT enzyme. When the gamma-Glu substrate is hydrolyzed by GGT, the probe is converted to intermediate 2, which in turn triggers a 1, 6-elimination reaction of the aminobenzyl alcohol linker to form the caged dioxetane phenol chemiluminescent group (3). Under physiological conditions at pH7.4, the chemiluminescent group (3) will deprotonate allowing the cleavage of the peroxy bond to give the fluorescent product 5 and the adamantanone moiety, with a chemical excitation process accompanied by emission of light with a maximum emission wavelength of 540 nm. Therefore, GGT enzyme can selectively activate and enhance chemiluminescence of probe 1, and can realize detection of GGT activity in cells and living bodies.

Reaction conditions are as follows:

i Compound 6, Compound 7, K₂CO₃,KI,DMF,91％；

II methylene blue, LED white light;

III 10％TFA,2.5％TIPSH,87.5％DCM(v/v),0℃,22％。

first, the synthesis of the probe was performed according to the synthetic route in route S1. First, compounds 6 and 7, which are chloro-substituted at the C2 position and strongly electron-withdrawing methacrylate-substituted at the C6 position, were synthesized according to the literature methods reported previously. Compound 8 is obtained by direct reaction of

compounds

6 and 7, followed by oxidation with singlet oxygen to give compound 9. And deprotecting the compound 9 to obtain the final probe compound 1.

In the synthesis, the method for simultaneously removing the tert-butyloxycarbonyl group and the benzhydryl group is generally to carry out deprotection by using a trifluoroacetic acid solution with a certain concentration at room temperature, and under the condition, the molecular structure of the probe is extremely easy to hydrolyze and damage, so that the yield is extremely low. The synthesis method adopts the relatively mild reaction condition of trifluoroacetic acid with concentration of 10% at 0 ℃ to carry out deprotection reaction, adopts sodium carbonate solution to neutralize trifluoroacetic acid in a reaction system at low temperature after the reaction is completed, then utilizes reduced pressure rotary evaporation to remove dichloromethane solvent in the reaction system, adds DMF or methanol to the final aqueous solution to dissolve products, utilizes a preparation liquid phase to carry out separation, uses an acetonitrile/water system without trifluoroacetic acid as a mobile phase, and then carries out vacuum freeze drying, thus obtaining the probe molecule with high yield and high purity.

All compounds have chemical structures represented by¹H NMR，¹³C NMR, matrix assisted laser desorption ionization time-of-flight mass spectrometry and high resolution mass spectrometry were characterized.

Synthesis of Compound 6:

the compound 6 can be obtained by reaction according to the synthetic method reported in the documents ACS cent.Sci.2017,3, 349-358.

Synthesis of compound 7:

the compound 7 can be obtained by reaction according to the synthetic method reported in the literature anal. chem.2018,90, 2875-2883.

Synthesis of compound 8:

the reaction mixture was purified by mixing Compound 6(50mg,0.11mmol), Compound 7(128mg, 1.2mmol), K₂CO₃(22mg,0.22mmol) and KI (183mg,1.1mmol) were dissolved in 10mL of anhydrous N, N-dimethylformamide, and the reaction was stirred at room temperature for 5 hours. The progress of the reaction was monitored by thin layer chromatography (developer system, petroleum ether: ethyl acetate: 3: 1). After completion of the reaction, 30mL of ethyl acetate and saturated brine (200 mL. times.3) were added to the reaction system to conduct extraction, the organic phase was collected and dried over anhydrous sodium sulfate, the solvent was removed by rotary evaporation under reduced pressure, and the obtained solid was purified by column chromatography to obtain Compound 8 as a white solid (89mg, yield 91%).¹H NMR(400MHz,CDCl₃)δ8.71(s,1H),7.92(d,J＝16.20Hz,1H),7.59(d,J＝7.76Hz,2H),7.45-7.42(m,3H),7.38-7.27(m,11H),7.07(d,J＝7.96Hz,1H),6.91(s,1H),6.43(d,J＝16.16Hz,1H),5.43(d,J＝7.44Hz,1H),4.97(d,J＝4.48Hz,1H),4.47(t,J＝4.47Hz,1H),3.80(s,3H),3.33(s,3H),2.43-2.36(m,2H),2.07-1.61(m,16H),1.46(s,9H).¹³C NMR(100MHz,CDCl₃)δ171.14,170.99,167.20,156.52,153.67,139.35,139.30,139.10,138.93,138.21,138.12,132.52,131.94,129.71,128.68,128.61,128.26,127.77,127.19,127.07,125.11,119.87,80.91,78.60,77.22,75.73,57.27,52.86,51.84,39.19,39.04,38.61,37.05,33.75,32.95,30.09,29.72,28.28.MS:calcd.For C₅₂H₅₇ClN₂O₉Na⁺[(M+Na)⁺]:911.3650；found MALDI-MS:m/z 911.3464.HRMS found:m/z 911.3652

Synthesis of Probe 1

Compound 8(0.43mmol,230mg) was dissolved in 20mL of dichloromethane, methylene blue (0.006mmol,2mg) was added, and the reaction system was sparged with oxygen and cooled with a white LED (50 mW/cm) using a white LED (0.006mmol,2mg) on an ice bath²) And (5) irradiating by using a lamp. The progress of the reaction was monitored by high performance liquid chromatography. After completion of the reaction, the solvent was removed by evaporation under reduced pressure to give a crude product of Compound 9 (241 mg). Dissolve crude Compound 9 in 10mL dichloromethane/trifluoroacetic acidThe deprotection reaction is carried out in a mixed solvent of TIPSH (volume ratio 87.5/10/2.5) at 0 ℃, and the reaction is monitored by a high performance liquid phase, so that the reaction product is single under the condition. After completion of the reaction, saturated sodium carbonate (2M) was added to adjust pH to neutral, the organic solvent was removed, and the crude product was isolated via preparative liquid phase and lyophilized to give probe 1 compound as a white solid (59mg, 21% in two-step total yield).¹H NMR(400MHz,CDCl₃)δ9.53(s,1H),8.35(s,2H),7.81(s,1H),7.75(d,J＝15.4Hz,1H),7.53-7.42(m,3H),7.23-7.15(m,2H),6.38(d,J＝15.0Hz,1H),4.70(s,1H),3.93(s,1H),3.69(s,1H),3.12(s,3H),2.95(s,1H),2.69-2.55(m,2H),2.34-2.16(m,2H),1.92-1.39(m,14H).¹³C NMR(100MHz,CDCl₃)δ171.96,167.15,154.16,138.57,138.40,135.29,131.43,131.34,129.61,128.92,127.61,125.14,120.79,120.16,111.59,96.20,77.22,75.92,52.00,49.61,36.50,33.85,33.54,33.13,33.03,32.55,32.13,31.46,29.71,26.09,25.78.MS:calcd.For C₃₄H₄₁ClN₂O₉ ⁺[(M+H)⁺]:655.2422found MALDI-MS:m/z 655.1790.HRMS found:m/z 655.2420。

Comparative example 1

Compound 8(0.11mmol,60mg) was dissolved in 20mL of dichloromethane, methylene blue (0.0015mmol,0.5mg) was added, and oxygen was bubbled through the reaction system under ice bath by white light LED (50 mW/cm)²) And (5) irradiating by using a lamp. The progress of the reaction was monitored by high performance liquid chromatography. After completion of the reaction, the solvent was removed by evaporation under reduced pressure to give a crude product of Compound 9 (62 mg). The crude compound 9 was dissolved in 5mL of a mixed solvent of dichloromethane/trifluoroacetic acid/TIPSH (volume ratio: 87.5/10/2.5), and the deprotection reaction was carried out at room temperature, and the reaction was monitored by high performance liquid chromatography to find the production of a hydrolysis product. After completion of the reaction, the solvent was spun off and the crude product was analyzed by HPLC to show almost complete hydrolysis of probe 1.

Comparative example 2

Compound 8(0.11mmol,58mg) was dissolved in 20mL of dichloromethane, methylene blue (0.0015mmol,0.5mg) was added, and the reaction system was sparged with oxygen and white LED (50 mW/cm) with white light LED (0.0015mmol,0.5mg) under ice bath²) And (5) irradiating by using a lamp. The progress of the reaction was monitored by high performance liquid chromatography. Inverse directionAfter completion of the reaction, the solvent was removed by evaporation under reduced pressure to give the crude compound 9 (61 mg). Dissolving the crude compound 9 in 5mL of a mixed solvent of dichloromethane/trifluoroacetic acid/TIPSH (volume ratio of 47.5/50/2.5), carrying out deprotection reaction at 0 ℃, and monitoring the reaction by using a high performance liquid phase to find that hydrolysis products are generated in the reaction process. After complete conversion of compound 9, the solvent was spun off and the crude product was analyzed by HPLC to show almost complete hydrolysis of probe 1.

Comparative example 3

Compound 8(0.11mmol,58mg) was dissolved in 20mL of dichloromethane, methylene blue (0.0015mmol,0.5mg) was added, and the reaction system was sparged with oxygen and white LED (50 mW/cm) with white light LED (0.0015mmol,0.5mg) under ice bath²) And (5) irradiating by using a lamp. The progress of the reaction was monitored by high performance liquid chromatography. After completion of the reaction, the solvent was removed by evaporation under reduced pressure to give a crude product of Compound 9 (63 mg). Dissolving the crude compound 9 in 5mL of a mixed solvent of dichloromethane/trifluoroacetic acid/TIPSH (volume ratio of 47.5/50/2.5), carrying out deprotection reaction at 0 ℃, and monitoring the reaction by using a high performance liquid phase to find that hydrolysis products are generated in the reaction process. After the compound 9 was reacted completely, saturated sodium carbonate (2M) was added to adjust the pH to neutral, and then the organic solvent was spin-dried to obtain a crude aqueous solution, which was separated and lyophilized via a preparative liquid phase to obtain a white solid compound of probe 1 (2.8mg, 4% in two-step total yield).

Probe performance testing

General operation method for detecting GGT by probe

The probes were dissolved in DMSO to prepare 20mM stock solutions and stored at-20 ℃ until use. For use, the mixture was diluted with GGT digestion buffer (10mM PBS containing 5% DMSO, pH7.4) to the desired concentration. Typically, probes (20. mu.M) were dissolved in 100. mu.L of GGT digest buffer, 100. mu.L of GGT digest buffer containing different GGT concentrations was added, the probe final working concentration was 10. mu.M, and incubated at 37 ℃ for the indicated time. Chemiluminescent imaging of the probe solution was acquired by the IVIS luminea XR III system. The reactivity of the probe and the GGT is characterized by high performance liquid chromatography, ultraviolet-visible absorption spectrum, fluorescence spectrum and chemiluminescence spectrum. Wherein the collection range of the ultraviolet-visible absorption spectrum is 200-1000nm, the fluorescence spectrum is collected by a Hitachi fluorescence spectrometer, the excitation wavelength is 350nm, and the emission wavelength is 450-700 nm. The chemiluminescence spectra were collected by a Hitachi fluorescence spectrometer with a collection range of 450-700 nm.

Chemiluminescence responsiveness assay of probe to GGT

The response of probes (10. mu.M) to GGT was first studied in GGT digestion buffer (5% DMSO in PBS, pH 7.4).

As shown in FIG. 2a, the probe itself has weak UV absorption and fluorescence emission in the range above 350nm, and after 6h incubation with GGT, a strong UV absorption band appears at 400nm, accompanied by a significant increase in fluorescence at 540 nm. Meanwhile, HPLC analysis of a reaction solution of a probe (10 μ M) and GGT (100U/L) showed that the probe (retention time TR ═ 10.4min) was almost completely converted to compound 5 (retention time TR ═ 7min) after the reaction with GGT (fig. 2 b). Next, the reaction change of the probe before and after the addition of GGT was investigated. As shown in FIG. 2c, the chemiluminescence of the probe is in a quenching state, and after the GGT enzyme is added for reaction for 30min, the chemiluminescence intensity is remarkably enhanced, and the maximum emission wavelength is 540 nm. The chemiluminescence intensity at the maximum emission wavelength is enhanced by a factor of up to 876, so that the reaction solution exhibits a strong yellowish green emission in the absence of excitation light (FIG. 2c inset). Next, time-dependent changes in chemiluminescence were examined before and after the reaction of the probe (10. mu.M) with GGT (100U/L). FIG. 2d shows the kinetics of a typical probe 1 reaction with GGT, with the chemiluminescence intensity of the probe reaching a maximum around 30min and then beginning to decay gradually. Whereas in the absence of GGT, the probe produced little chemiluminescence throughout the measurement. The above results indicate that GGT can efficiently activate probes, resulting in a significant increase in chemiluminescent intensity.

Stability testing of probes

To investigate its stability, probe 1(10 μ M) was dissolved in 200 μ L of PBS buffer (1 × 7.4, containing 5% DMSO), high-glucose DMEM, and DMEM medium of 10% Fetal Bovine Serum (FBS), respectively, and then added to 96-well plates, each set of three duplicate wells. The chemiluminescence of each well before and after the probe was incubated in the above buffer system for 6h was measured with a microplate reader at 37 ℃ and the change of each well solution after 6h was further analyzed with high performance liquid chromatography. In addition, the chemiluminescence time-dependent change of PBS buffer alone or PBS buffer containing probe 1 (10. mu.M) was recorded with a microplate reader every 5 minutes for 6 hours in triplicate wells.

Probe 1 was incubated in several physiological environments to test the stability of the probe. As can be seen from FIG. 3, when the probe was incubated in PBS buffer, DMEM medium and DMEM medium containing 10% Fetal Bovine Serum (FBS) for more than 6 hours, no significant change in chemiluminescence of the probe was observed, and no decomposition product of the probe was observed when the solution was subjected to HPLC analysis, indicating that the probe maintained high stability in a complex physiological environment.

Detection sensitivity determination of probes for GGT

Adding a probe 1(20 mu M,100 mu L) solution into a 96-well plate, then adding 100 mu L PBS enzyme digestion buffer solution containing different GGT concentrations, wherein the final working concentration of the probe is 10 mu M, the final concentration of the GGT enzyme is 0,2.5,5,10,25,50,100 and 250U/L respectively, and each concentration group has three multiple wells. The 96-well plate was incubated at 37 ℃ for 10min, and fluorescence imaging and chemiluminescence imaging were performed by the IVIS Lumia XR III system, respectively. Fluorescence imaging was performed with 420nm excitation and 570nm collection using a filter. The chemiluminescence imaging adopts a full-acceptance optical filter mode for acquisition, and the acquisition time is 0.75 second. Fluorescence intensity and chemiluminescence intensity were obtained by ROI measurement with IVIS Lumia XR III system software and fitted linearly to GGT concentration. After fitting, the slope k of each fitting straight line is obtained, and the detection limit LOD is 3 δ/k. Where δ is the standard deviation of the intensity values measured for the 11 blank probe well solutions.

Probe 1 was first incubated with various concentrations of GGT (0,5,10,25,50,100 and 250U/L) at 37 ℃ for 30min, followed by chemiluminescent imaging of the above solution using the IVIS Lumia XR III imaging system. As shown in FIG. 4a, the chemiluminescence intensity of the probe increased with increasing GGT concentration. Such asFIG. 4b shows that the intensity of the quantified chemiluminescent signal is linear with GGT concentration (0-250U/L) and the linear equation is y-3.652 x (U/L) +0.1803, R²0.9999. The LOD of the detection limit of the probe to GGT is calculated to be 16mU/L, which indicates that the probe can be used for high-sensitivity detection of GGT.

Specificity of probes for GGT detection

Adding a probe 1(20 mu M,100 mu L) solution into a 96-well plate, then adding 100 mu L PBS enzyme digestion buffer solution containing different enzymes, wherein the final working concentration of the probe is 10 mu M, and the different enzymes are cathepsin B (2.0 mu g/mL), trypsin (2 mu g/mL), MMP-2(10nM), ALP (100U/L), caspase-3(1 mu g/mL), GGT (100U/L), GGT (100U/L) and an inhibitor thereof GGsTop (200 mu M), and each group has three multiple wells. After incubating the 96-well plate at 37 ℃ for 25 minutes, the chemiluminescence intensity of 360-700nm was collected immediately by a microplate reader.

The chemiluminescent changes of the comparative probes were observed after incubation of the probes with GGT, GGT and its inhibitor GGsTop, and other representative biological enzymes such as cathepsin B, trypsin (trypsin), metalloproteinase (MMP-2), alkaline phosphatase (ALP), and cysteine protease (caspase-3), respectively, at 37 ℃. As shown in fig. 4c, only the addition of GGT activated the probe and caused a significant increase in chemiluminescent intensity, while in the presence of GGT inhibitor (200 μ M), the activation of the probe by GGT was significantly inhibited. The addition of other enzymes did not cause significant enhancement of the chemiluminescence intensity of the probe, indicating that the probe has strong specificity to GGT.

Application of the Probe

Determination of GGT content in serum

GGT is an important biomarker, is an index of clinical blood detection, and is commonly used for diagnosing liver dysfunction and malignant tumor diseases. In addition, studies have reported that endotoxin (LPS) in the gram-negative bacterial wall causes acute hepatitis in mice. Therefore, the probe is applied to the detection of GGT activity in the serum of healthy mice and mice with LPS-induced liver inflammation.

6 immunodeficient female BALB/c nude mice aged 6-8 weeks were selected for serum GGT assay studies. 6 mice were randomly divided into two groups: LPS treated group and healthy group. For healthy control group, three healthy mice were collected via orbital sinus, and blood was stored in 1.5mL Eppendorf blood collection tube containing EDTA pre-cooled in ice bath and centrifuged at 3000rpm at 4 ℃ to obtain serum of healthy mice. For the LPS-treated group, three mice were each injected with LPS (10mg/kg) intraperitoneally, and 24 hours later, treated by the above procedure to obtain mouse sera of the LPS-treated group. During determination, 20 mu L of mouse serum is added into a 96-well plate, then 180 mu L of enzyme digestion buffer solution containing the probe 1 is added, the final working concentration of the probe is 10 mu M, and each group comprises three multiple wells. After incubation at 37 ℃ for 10min, the chemiluminescence intensity at this time was collected with a microplate reader. The standard curve was obtained by an additional standard method, i.e., 20. mu.L of control serum was diluted in 180. mu.L of GGT digestion buffer, and GGsTop (500. mu.M) and 2.5, 5.0 and 10.0U/L GGT were added, respectively. These solutions were incubated with probe 1(10 μ M) for 10 minutes at 37 ℃ and the chemiluminescence intensity of each well was recorded. GGT activity is calculated according to a fitted linear equation of chemiluminescence intensity and GGT concentration of each group.

As shown in FIGS. 4d and 4e, the average chemiluminescence intensity of 599. + -. 125(RLU) generated after the probe was incubated with 10-fold diluted serum from healthy mice was calculated to be 0.16. + -. 0.038U/L in GGT according to the standard curve obtained by the standard method, and thus the activity of GGT in serum from healthy mice was 1.6U/L. When LPS (10mg/kg) was intraperitoneally injected into the mice, the GGT content in the serum was increased to 3.8U/L.

Cell culture

U87MG brain glioma cells and HUVEC human umbilical vein vascular endothelial cells were cultured using high glucose DMEM medium (Gibco) containing 10% fetal bovine serum and 1% double antibody. OVCAR5 ovarian cancer cells were cultured using high-sugar RPMI 1640medium containing 10% fetal bovine serum and 1% double antibody. All cells were in the presence of 5% CO₂Culturing in a constant temperature and humidity incubator at 37 ℃.

Cytotoxicity test

U87MG, OVCAR5 and HUVEC cells (-5000 per well) were seeded in 96-well plates, respectively. After incubation 24, the medium was removed and 100. mu.L of DMEM or 1640 complete medium containing different concentrations of probe 1(10,20, 30. mu.M) was added again.After an additional 24 hours of incubation, 50. mu.L of MTT (1 in PBS) reagent was added to each well and incubation was continued for 4 hours at 37 ℃. Subsequently, the culture containing the MTT reagent was removed, 150. mu.L of DMSO was added to each well to dissolve the formed crystalline formazan solid, and the absorbance (OD) at 490nm was detected by a microplate reader. Absorbance (OD) of blank wells without any treatment_control) The percentage of viability activity of the cells per well can be determined from OD/OD, set as control wells_controlX 100% was calculated and each experiment was repeated three times.

Chemiluminescent detection of GGT activity in living cells

In order to verify whether the probe can detect the GGT activity in living cells, three cell lines, namely a human ovarian cancer cell OVCAR5, a human brain glioma cell U87MG and a normal human umbilical vein epithelial cell HUVEC, are selected firstly, and the cytotoxicity of the probe on the cells is examined through an MTT cell proliferation experiment.

As shown in FIG. 5a, after adding probe 1 at various concentrations (0,10, 20. mu.M) and incubating for 24 hours, probe 1 showed no strong biotoxicity to all cell lines, indicating that the probe did not affect the viability of the cells and could be used to detect GGT activity in living cells.

To verify the ability of probe 1 to detect GGT activity in cells, U87MG, OVCAR5 and HUVEC cells (-5000 per well) were seeded separately in 96-well plates in triplicate wells per set. After 24 hours of incubation, the medium was removed and 200. mu.L of GGT digestion buffer containing 10. mu.M probe was added to each well. To inhibit GGT activity in cells, U87MG and OVCAR5 cells were incubated with GGsTop (200 μ M) for 30min, after which the medium containing GGsTop was removed and 200 μ L of GGT digestion buffer containing 10 μ M probe was added to each well. Immediately after addition of the probe, Spark was used^TMAnd (3) acquiring the chemiluminescence intensity of each hole at 37 ℃ in real time by using a 10M microplate reader, wherein the time interval is 1min, and the duration is about 1 h. In addition, after the cells were incubated with the probe at 37 ℃ for 1 hour, chemiluminescence images of the cell solution were collected by the IVIS Lumina XR III system for 1 min.

Since probe 1 has very low background signal and high chemiluminescence activation times after reaction with GGT, the probe can detect GGT activity on cells in real time without washing after addition. As shown in fig. 5b, both GGT-overexpressed OVCAR5 cells and U87MG cells showed a gradual increase in chemiluminescence intensity over time after incubation of probe 1(10 μ M), with the chemiluminescence intensity reaching a maximum at about 15 minutes and then continuing to emit for more than 45 minutes. In contrast, the chemiluminescence intensity of these two cells was greatly reduced after the addition of GGsTop (200. mu.M) to previously inhibit GGT activity in the cells. Similarly, HUVEC-negative GGT cells produced weaker chemiluminescence after incubation of probe 1. After 60 min incubation with probe 1, the cells were subjected to chemiluminescent imaging and the results are shown in figure 5c, with GGT over-expressed OVCAR5 cells and U87MG cells both producing brighter imaging signals than GGT negative HUVEC cells.

Chemiluminescence assay for probe number of tumor cells

For in vitro tumor cell numbers, different numbers of OVCAR5 cells (0,200,500,1000,2000,5000,10000,20000 and 40000 cells/well) were first seeded into 96-well plates, respectively, and incubated overnight to allow adherent growth of the cells. The medium was then removed from each well and 200. mu.L of GGT cleavage buffer containing 10. mu.M probe was added to each well. After incubating the cell solution at 37 ℃ for 30min, collecting chemiluminescence images of the cell solution by an IVIS Lumina XR III system for 1 min. Chemiluminescence intensities were measured by ROI measurement using IVIS Lumia XR III system software and plotted against cell number.

For tumor cell number detection at the living level, OVCAR5 cells of different cell numbers (0,500,1000,2000,5000,10000 and 20000) were first mixed with 50. mu.L of the digestion buffer containing 10. mu.M probe 1, and the above mixed solution was immediately injected subcutaneously into nude mice. After 30min, the mice were gas anesthetized with isoflurane, followed by collection of a whole body chemiluminescence image of the mice using the IVIS Lumia XR III system. The chemiluminescence intensity at the injection site was measured by ROI measurement using IVIS Lumia XR III system software and was analyzed by plotting the correlation of chemiluminescence intensity with cell number using a log coordinate system.

As shown in fig. 6a and 6b, the chemiluminescence intensity of the well solution gradually increased with the increase of the number of cells, and the chemiluminescence intensity was in a good linear relationship with the number of OVCAR5 cells (200-. In addition, the results of the OVCAR5 cell imaging assay at the in vivo level showed (fig. 6c and 6d) that the chemiluminescence intensity at the injection site gradually increased with the increase in the number of cells, and that the chemiluminescence intensity was significantly stronger in the presence of 500 OVCAR5 cells in combination with the probe than that of the probe alone, indicating that the probe is also suitable for highly sensitive chemiluminescence imaging assay of OVCAR5 cells at the in vivo level.

Tissue penetration

Adding the enzyme digestion buffer solution of the probe (20 mu M,100 mu L) into a 96-well plate, then adding 100 mu L of the enzyme digestion buffer solution containing 500U/L GGT, wherein the final working concentration of the probe is 10 mu M, and the final concentration of the enzyme is 250U/L. The solution wells were then covered with muscle tissue of different thickness (0,5,10,15 and 20mm), each tissue covering three replicates. After incubation at 37 ℃ for 20min, chemiluminescent and fluorescence images were collected using the IVIS Lumia XR III system, respectively. The chemiluminescence image acquisition mode is open filter collection, and the acquisition time is 0.75 s. Fluorescence imaging was performed with 420nm excitation and 570nm collection using a filter.

As shown in FIG. 7, the chemiluminescent intensity and the fluorescence intensity of the probe solution both decreased gradually with increasing thickness of the coated chicken tissue due to the absorption and diffraction effects of the tissue on the light. However, when 20mm thick chicken tissue was overlaid on top of the sample solution, the chemiluminescent signal was still visible and the signal to noise ratio was 43 times higher. Whereas the fluorescence signal almost disappeared when only 5mm of chicken tissue was covered above the sample, at which the signal-to-noise ratio was below 1.2. The results show that the chemiluminescence of the probe has deeper tissue penetration depth than the fluorescence of the probe, so the probe is more suitable for in vivo imaging detection.

Chemiluminescence detection of GGT activity at in vivo level

Immunodeficient 6-8 week-old female BALB/C nude mice were injected subcutaneously 3 on the right posterior thigh outer side×10⁶U87MG brain glioma cells, establishing xenograft U87MG tumors. When the average tumor volume reaches about 150mm³When the mice were randomly divided into two groups (n-3). Probe 1 (100. mu.M, 100. mu.l physiological saline) was injected via the tail vein. To inhibit GGT activity in tumors, GGsTop (10mm, 50 μ L) was injected directly into tumors 1 hour before intravenous injection of probe. The mice whole body chemiluminescence images were acquired at 0,30, and 60 minutes after tail vein injection of probe 1 using the IVIS-lumiaxrliii system (fully open filter mode) for 60 seconds.

As shown in FIG. 8, 30 minutes after tail vein injection of the probe, a strong chemiluminescent signal appeared at the tumor site in the mice, with a signal to noise ratio of about 2.9 times, and the intensity of the chemiluminescent signal decreased to near background signal intensity after the next 30 minutes. Whereas the prior intratumoral injection of GGT inhibitors significantly suppressed the intratumoral chemiluminescence intensity, resulting in an approximately 3-fold decrease in chemiluminescence intensity, with a signal-to-noise ratio of approximately 1.1-fold. The results show that the probe 1 can be activated by endogenously expressed tumor GGT after tail vein injection into mice, and is used for real-time and non-invasive chemiluminescence imaging detection of in-vivo level GGT positive tumors.

Claims

1. A chemiluminescent probe comprises an amino acid substrate gamma-Glu capable of being specifically hydrolyzed by GGT, an (adamantane) -1, 2-dioxetane chemiluminescent group and a self-cleavable connecting group.

2. The chemiluminescent probe of claim 1 wherein the (adamantane) -1, 2-dioxetane based chemiluminescent group is substituted with a methyl acrylate group.

3. The chemiluminescent probe of claim 1 wherein the chemiluminescent group of (adamantane) -1, 2-dioxetane is 3- (2' -spiroadamantane) -4-methoxy-4- (2 "-chloro-3" -hydroxy-4 "-methyl acrylate) -phenyl-1, 2-dioxetane.

4. The chemiluminescent probe of claim 1 wherein the self-cleavable linking group is para-aminobenzyl alcohol.

5. The method for synthesizing a chemiluminescent probe according to claim 1 is achieved by the following scheme S1,

the compound 6 and the compound 7 directly react to obtain a compound 8, then the compound 8 is oxidized by singlet oxygen to obtain a compound 9, and the compound 9 is deprotected to obtain a probe compound 1.

6. The method for synthesizing a chemiluminescent probe of claim 3 comprising the steps of:

7. Use of a chemiluminescent probe according to claim 1 for the determination of GGT levels in serum.

8. Use of the chemiluminescent probe of claim 1 for the chemiluminescence of GGT activity in living cells.

9. Use of the chemiluminescent probe of claim 1 for the chemiluminescent determination of tumor cell number.

10. Use of the chemiluminescent probe of claim 1 for the chemiluminescent detection of GGT activity at the living body level.