WO2020220218A1 - 一种化合物、含有所述化合物的产品及其在γ-谷氨酰转肽酶检测中的用途 - Google Patents

一种化合物、含有所述化合物的产品及其在γ-谷氨酰转肽酶检测中的用途 Download PDF

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WO2020220218A1
WO2020220218A1 PCT/CN2019/085051 CN2019085051W WO2020220218A1 WO 2020220218 A1 WO2020220218 A1 WO 2020220218A1 CN 2019085051 W CN2019085051 W CN 2019085051W WO 2020220218 A1 WO2020220218 A1 WO 2020220218A1
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compound
glutamyl transpeptidase
detection
fluorescence
ratio
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French (fr)
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李富友
周晓波
吴勇
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Shanghai Taywell Biotechnology Co Ltd
Fudan University
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Shanghai Taywell Biotechnology Co Ltd
Fudan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/572—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
    • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention belongs to the field of fluorescence detection. Specifically, the present invention relates to a compound, a product containing the compound, a preparation method thereof, and use in the detection of ⁇ -glutamyl transpeptidase.
  • ⁇ -glutamyl transpeptidase is a protease distributed on the cell membrane, which plays an important role in the metabolism of glutathione in the organism.
  • ⁇ -glutamyl transpeptidase is an important indicator for the diagnosis of liver and gallbladder diseases, and is widely used in the diagnosis and prognosis of liver diseases and other organ diseases.
  • Abnormally elevated expression of ⁇ -glutamyl transpeptidase in the local microenvironment of tissues is often associated with some malignant tumors.
  • ⁇ -glutamyl transpeptidase has been detected in tumor tissues such as liver cancer, ovarian cancer, lung cancer, and squamous cell carcinoma. High expression of peptidase.
  • Such fluorescent molecules are relatively complex The biological samples are susceptible to interference from background fluorescence signals and tissue scattering, and it is difficult to achieve accurate detection of GGT content in biological samples.
  • Luo et al. [c1] reported a variety of GGT detection molecules with different structures. Such molecules are usually “Turn on” fluorescent probes, and their response to GGT presents the characteristics of increased fluorescence intensity in a single band. This leads to the fact that when such molecules are used to detect the GGT content in tissues, the detection signal is easily affected by the concentration of the probe, which reduces the accuracy of detection.
  • kits in the prior art use colorimetry and fluorescence enhancement methods to detect GGT.
  • these two methods are applied to biological samples (especially tissues), it is difficult to eliminate probe concentration, excitation light power, etc.
  • the influence of external environmental factors on the detection signal makes it difficult to accurately detect the GGT content in the biological sample (especially tissue).
  • the ratio fluorescence detection method usually uses the optical signal of one waveband as the internal reference in the fluorescence detection, and the optical signal of the other waveband is used as the detection signal.
  • the ratio of the fluorescence intensity is used as the output signal, which can eliminate the interference of the change of the external environment on the value of the output signal to a certain extent.
  • the ratio detection method is used to detect the analyte with higher accuracy, and the dual-wavelength fluorescence ratio change as the detection signal will facilitate the construction of quantification of GGT in the complex biological system Method of detection.
  • fluorescent probes used for ratio detection of GGT there are few reports on fluorescent probes used for ratio detection of GGT, and these fluorescent probes have slow response speed and low fluorescence ratio change. Therefore, these probes are difficult to be used for the detection of content in tissue samples.
  • the purpose of the present invention is to provide a new compound as a fluorescent probe, a product containing the compound, a preparation method thereof, and use in GGT detection in biological samples (especially tissues).
  • the fluorescent probe of the present invention ie, the compound of formula (I) of the present invention, the same below
  • reacts quickly and has high sensitivity and the product containing the fluorescent probe of the present invention detects the content of GGT in the tissue by the fluorescence ratio detection method It can not only eliminate the interference of the external environment on the detection signal to a certain extent, but also greatly improve the detection accuracy, and its ratio signal change multiple can reach 170 times, which makes the sensitivity of the fluorescent probe significantly improved .
  • the fluorescent probe of the present invention has a very fast response speed to GGT, which can completely respond within 10 minutes, which makes the product of the present invention applicable to point-of-care testing tissues. GGT content.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same Or differently selected from:
  • n and n can be the same or different integers from 1-18; or
  • X 1 , X 2 , X 3 , X 4 and X 5 may be the same or differently selected from: NH, NCH 3 , O, S, Se, C(CH 3 ) 2 and Si(CH 3 ) 2 .
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same or differently selected from:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same or differently selected from:
  • the compound is:
  • a product which contains the compound defined in the first aspect of the present invention.
  • the product also contains additives and instructions for use.
  • the additive is selected from one or more of the following: sodium azide, glycerin, thimerosal and Tween.
  • the product is a kit.
  • the compound and each additive are each in the form of a separate formulation, or the compound and each additive are in the form of a composition.
  • a use which includes:
  • the compound defined in the first aspect of the present invention or the product defined in the second aspect of the present invention is provided, and the compound or product is used for detecting ⁇ -glutamyl transglutamin in biological samples. Peptidase content or fluorescence imaging of ⁇ -glutamyl transpeptidase in biological samples.
  • the fifth aspect of the present invention there is provided a method for detecting the content of ⁇ -glutamyl transpeptidase in a biological sample, wherein the compound defined in the first aspect of the present invention or the compound defined in the second aspect of the present invention is used.
  • the product includes the following steps:
  • concentration range of the compound or the compound in the product is 0.01-10 mM.
  • the concentration of the ⁇ -glutamyl transpeptidase is 1-500 mU/mL; the series of ⁇ -glutamyl transpeptidase with different concentrations -The volume of the glutamyl transpeptidase standard reaction solution is 0.1-10 mL; in the series of ⁇ -glutamyl transpeptidase standard reaction solutions of different concentrations, the molar concentration of the compound stock solution is 0.01- 10mM, the volume is 5-200 ⁇ L, and sodium azide is optionally used during the incubation, the molar concentration of the sodium azide is 0.01-10mM, the volume is 5-200 ⁇ L; the ⁇ -glutamyl The standard reaction solution of the transpeptidase is prepared by dissolving the ⁇ -glutamyl transpeptidase standard stock solution in water, and the concentration of the ⁇ -glutamyl transpeptidase standard stock solution is 1000-20000
  • the method can be used for clinical biological sample analysis.
  • the clinical samples are tissues and tissue sections, and the concentration of the compound is 10 nM-50 ⁇ M.
  • the detection range of the method for the detection of ⁇ -glutamyl transpeptidase is 0.01-1000 mU/mL.
  • the present invention has high sensitivity as a fluorescent probe (ie, the compound of formula (I) of the present invention, the same below), the detection limit in solution can reach 10mU/L, and the product containing the fluorescent probe of the present invention has dual channels
  • the collection of ratio fluorescence signal can reduce the interference of the imaging signal caused by problems such as probe concentration and tissue scattering to a certain extent.
  • the method of fluorescence ratio detection is used to detect ⁇ -glutamyl transpeptide in biological samples (especially tissues). Enzyme content detection can not only eliminate the interference of the external environment on the detection signal to a certain extent, but also greatly improve the detection accuracy. Its ratio signal change multiple can reach 170 times, which makes the sensitivity of the fluorescent probe Significantly improved.
  • the fluorescent probe of the present invention has a very fast response speed to ⁇ -glutamyl transpeptidase, which can completely respond within 10 minutes, which makes the product of the present invention can be used to detect ⁇ -glutamyl in the side bed. Transpeptidase content.
  • the fluorescent probe of the present invention is a purple solid, which is dark purple in the solution, and after reacting with ⁇ -glutamyl transpeptidase, the solution can be observed to be obviously light yellow with naked eyes.
  • the fluorescent probe of the present invention has high fluorescence quantum efficiency.
  • the color reaction only occurs in the presence of ⁇ -glutamyl transpeptidase during application, and other common inorganic salts, amino acids, hydrolases, etc. are not Interference occurs, and at the same time, the fluorescent probe of the present invention has good biocompatibility, has a linear relationship with the response of ⁇ -glutamyl transpeptidase, and can be used for the quantitative determination of the enzyme.
  • the fluorescent probe of the present invention has good solubility, and its mother liquid product prepared with an organic solvent can be directly used for the detection of ⁇ -glutamyl transpeptidase in a buffer system.
  • Figure 1 shows the products of fluorescent molecules in the currently commercialized fluorescence detection kits.
  • Figure 2 shows the UV-Vis absorption spectrum of the kit of the present invention containing compound C3 in response to ⁇ -glutamyl transpeptidase over time.
  • Figure 3 shows the emission spectrum of the kit of the present invention containing compound C3 in response to ⁇ -glutamyl transpeptidase over time.
  • Figure 4 is a graph showing the fluorescence response of the kit of the present invention containing compound C3 to different concentrations of ⁇ -glutamyl transpeptidase.
  • concentration of ⁇ -glutamyl transpeptidase in the figure is 0, 1, and 5 from top to bottom. , 10, 20, 25, 30, 40, 50, 75 and 100U/L.
  • Figure 5 is a working curve of the ratio value of the kit of the present invention containing compound C3 against the concentration of ⁇ -glutamyl transpeptidase.
  • Fig. 6 is the anti-interference test of the ratio value of the kit of the present invention containing compound C3 to the detection of ⁇ -glutamyl transpeptidase (emission spectrum under 5 ⁇ W excitation light power).
  • Figure 7 is the anti-interference test of the ratio value of the kit of the present invention containing compound C3 to the detection of ⁇ -glutamyl transpeptidase (emission spectrum under 20 ⁇ W excitation light power).
  • Fig. 8 is the anti-interference test of the ratio value of the kit of the present invention containing compound C3 to the detection of ⁇ -glutamyl transpeptidase (emission spectrum under 40 ⁇ W excitation light power).
  • Figure 9 shows the ratio of the ratio value of the kit of the present invention containing compound C3 to the anti-interference test of ⁇ -glutamyl transpeptidase detection, and the ratio value under different excitation light powers.
  • Figure 10 The fluorescence response graph of the kit of the present invention containing compound C3 with a probe concentration of 10 ⁇ M to different concentrations of ⁇ -glutamyl transpeptidase, the concentration of ⁇ -glutamyl transpeptidase in the figure is from top to bottom
  • the lower order is 0, 1, 5, 10, 20, 25, 30, 40, 50, 75 and 100 U/L.
  • Fig. 11 is a working curve of the fluorescence ratio value measured with a probe concentration of 10 ⁇ M in the kit of the invention containing compound C3 versus the concentration of ⁇ -glutamyl transpeptidase.
  • Figure 12 is a graph showing the fluorescence response of a solution containing compound C3 with a probe concentration of 2 ⁇ M to different concentrations of ⁇ -glutamyl transpeptidase.
  • concentration of ⁇ -glutamyl transpeptidase in the figure is from the top From the bottom to the bottom are 0, 1, 5, 10, 20, 25, 30, 40, 50, 80 U/L.
  • Fig. 13 is a working curve of the fluorescence ratio value of the solution containing the compound C3 of the present invention prepared with a probe concentration of 2 ⁇ M versus the concentration of ⁇ -glutamyl transpeptidase.
  • Fig. 14 is a comparison of GGT content in clinical samples detected by the kit of the present invention containing compound C3 and the commercial GGT detection kit.
  • Fig. 15 is a graph showing the time-dependent changes in fluorescence intensity values of the kit of the present invention and the commercial GGT detection kit containing compound C3 in response to GGT.
  • Figure 16 is a comparison of the parameters of the effect of the kit of the present invention and the commercial GGT detection kit containing compound C3 with GGT.
  • Figure 17 is a working curve of the fluorescence intensity of a commercial GGT detection kit as a function of GGT.
  • Figure 18 is a confocal imaging image of the kit of the present invention containing compound C3 used in HOSEpiC and SKOV3 cells, respectively.
  • Figure 19 shows the fluorescence imaging image of the detection system of the present invention on the tissue removed during the resection operation of a clinical ovarian cancer patient.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Figure 20 shows the fluorescence imaging diagram of the detection system of the present invention on the tissue removed during the resection operation of clinical ovarian cancer patients.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Figure 21 shows the fluorescence imaging image of the detection system of the present invention on the tissue removed during the resection of clinical ovarian cancer patients.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Figure 22 shows the fluorescence imaging image of the detection system of the present invention on the tissue removed during the resection of clinical ovarian cancer patients.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Figure 23 shows the fluorescence imaging image of the detection system of the present invention on the tissues removed during the resection of clinical ovarian cancer patients.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Fig. 24 shows the fluorescence imaging diagram of the removed tissue in the clinical uterine cyst resection by the detection system of the present invention.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Figure 25 shows the fluorescence imaging diagram of the removed tissue in the clinical uterine cystectomy by the detection system of the present invention.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Fig. 26 shows the fluorescence imaging diagram of the removed tissue in the clinical uterine cyst resection by the detection system of the present invention.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • Figure 27 shows the fluorescence imaging diagram of the detection system of the present invention on the tissue removed during the resection of a clinical uterine cyst.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging chart (ratio imaging chart derived from the ratio of the signal intensity of the green light channel to the signal intensity of the red light channel)
  • Fig. 28 shows the fluorescence imaging image of the detection system of the present invention on the tissue removed during the resection of clinical ovarian cancer patients.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging chart (ratio imaging chart derived from the ratio of the signal intensity of the green light channel to the signal intensity of the red light channel)
  • Fig. 29 shows the fluorescence imaging image of the detection system of the present invention on the tissue removed during the resection of clinical ovarian cancer patients.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging chart (ratio imaging chart derived from the ratio of the signal intensity of the green light channel to the signal intensity of the red light channel)
  • Figure 30 shows the fluorescence imaging of the tissue removed in the clinical ovarian cancer resection by the detection system of the present invention.
  • the four pictures in the figure from left to right are the tissue brightfield imaging map, the green light channel imaging map (the collected light signal band is 510-560nm), the red light channel imaging map (the collected light signal band is 620-690nm) and the ratio Imaging map (ratio imaging map derived from the ratio of the intensity of the green channel light signal to the signal intensity of the red channel).
  • biological sample refers to various sample types obtained from patients (for example, patients with cancer) or normal individuals, including any cells or extracellular fluids such as blood, serum, plasma, urine Or other fluid samples, such as saliva, peritoneal or pleural fluid, cerebrospinal fluid, gastric or colorectal fluid, lymph, synovial fluid, interstitial fluid, amniotic fluid, physiological secretions, tears, mucus, sweat, milk, semen, Vaginal secretions and fluids that ooze from ulcers and other surfaces.
  • cells or extracellular fluids such as blood, serum, plasma, urine Or other fluid samples, such as saliva, peritoneal or pleural fluid, cerebrospinal fluid, gastric or colorectal fluid, lymph, synovial fluid, interstitial fluid, amniotic fluid, physiological secretions, tears, mucus, sweat, milk, semen, Vaginal secretions and fluids that ooze from ulcers and other surfaces.
  • biological sample also includes tissues, including but not limited to tumors (including benign tumors (for example, cysts, including but not limited to: ovarian cysts and uterine cysts) or malignant tumors (ie, cancer)) tissues, or normal Organ tissue or tumor organ tissue (for example, heart, liver, spleen, lung, kidney, brain, etc.), or a tissue section of the tissue.
  • tumors including benign tumors (for example, cysts, including but not limited to: ovarian cysts and uterine cysts) or malignant tumors (ie, cancer)
  • normal Organ tissue or tumor organ tissue for example, heart, liver, spleen, lung, kidney, brain, etc.
  • tissue section of the tissue for example, heart, liver, spleen, lung, kidney, brain, etc.
  • cancer refers to a new organism or tumor caused by abnormal uncontrolled cell growth.
  • cancer includes diseases involving both premalignant cancer cells and malignant cancer cells.
  • Non-limiting examples include: brain cancer, lung cancer, liver cancer, spleen cancer, kidney cancer (such as renal cell carcinoma and renal pelvic cancer), lymph node cancer, small bowel cancer, pancreatic cancer, blood cell cancer, bone cancer, colon cancer/colorectal cancer Cancer, stomach cancer, breast cancer, endometrial cancer, cervical cancer, prostate cancer, testicular cancer, ovarian cancer, central nervous system cancer, skin cancer, head and neck cancer, esophageal cancer, bone marrow cancer or squamous cell cancer, or blood cancer , Such as leukemia (eg, acute and chronic leukemia or acute promyelocytic leukemia) lymphoma, multiple myeloma, myelodysplasia, myeloproliferative disease or refractory anemia.
  • leukemia eg, acute and chronic leukemia or
  • point-of-care testing refers to clinical testing or bedside testing performed next to a patient, also known as point-of-care testing.
  • halogen refers to fluorine, chlorine, bromine and iodine, preferably fluorine or chlorine.
  • Tween refers to polysorbate (polyoxyethylene sorbitan fatty acid ester), including, but not limited to: Tween 20 (TWEEN-20), Tween 21 (TWEEN-21 ), Tween 40 (TWEEN-40), Tween 60 (TWEEN-60), Tween 61 (TWEEN-61), Tween 80 (TWEEN-80), Tween 81 (TWEEN-81), Tween 85 (TWEEN-85).
  • Tween 20 TWEEN-20
  • Tween 21 TWEEN-21
  • Tween 40 TWEEN-40
  • Tween 60 TWEEN-60
  • Tween 61 TWEEN-61
  • Tween 80 TWEEN-80
  • Tween 81 TWEEN-81
  • TWEEN-85 Tween 85
  • patient or “individual” as used herein refers to humans, companion animals (such as dogs, cats, and horses), edible animals (such as cows, pigs, sheep), zoo animals, marine animals, poultry and other similar Animal species.
  • ratio value refers to the ratio of signal intensities in fluorescence imaging images of biological samples (for example, tissues) measured at different fluorescence wavelength bands (for example, at (510-560 nm and 620-690 nm)).
  • biological samples for example, tissues
  • fluorescence wavelength bands for example, at (510-560 nm and 620-690 nm
  • room temperature refers to 25°C ⁇ 2°C. At the same time, if the experiment temperature is not specified, it is room temperature.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same Or differently selected from:
  • m and n can be the same or different integers from 1 to 18, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 Or 18 or an integer in any range between them, ie 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 2-3, 2-4, 2-5, 2-6, 2-7, 2- 8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3- 18, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 3-14, 4-15, 4-16, 4-17, 4-18, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5
  • X 1 , X 2 , X 3 , X 4 and X 5 may be the same or differently selected from: NH, NCH 3 , O, S, Se, C(CH 3 ) 2 and Si(CH 3 ) 2 .
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same or differently selected from:
  • X 1 , X 2 and X 5 may be the same or differently selected from: NH, NCH 3 , O, S, Se, C(CH 3 ) 2 and Si(CH 3 ) 2 .
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same or differently selected from:
  • halogen is fluorine or chlorine.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same or differently selected from:
  • halogen is fluorine or chlorine.
  • R 9 is halogen, preferably fluorine or chlorine
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 12 , R 13 , R 14 , R 15 and R 16 may be the same or differently selected from:
  • X 1 , X 2 and X 5 may be the same or different selected from: NH, NCH 3 , O, and S.
  • R 9 is fluorine
  • X 1 , X 2 and X 5 may be selected from O and S the same or differently.
  • R 9 is fluorine
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 12 , R 13 and R 14 are
  • X 1 , X 2 and X 5 may be the same or different selected from: O and S, preferably O.
  • the compound is:
  • a product which contains the compound defined in the first aspect of the present invention.
  • the product also contains additives and instructions for use.
  • the additive is selected from one or more of the following: sodium azide, glycerin, thimerosal and Tween.
  • the product is a kit.
  • the compound and each additive are each in the form of a separate formulation, or the compound and each additive are in the form of a composition.
  • the usage guide relates to the following specific steps described in the fifth aspect of the present invention.
  • a use which includes:
  • the biological sample is normal cells, tumor cells, normal tissues or tumor tissues.
  • the biological sample is cancer tissue, such as those cancerous tissues described in the above definition of cancer, such as tissues selected from ovarian cancer, liver cancer, lung cancer, squamous cell carcinoma, endometrial cancer, and cervical cancer .
  • the biological sample is normal cells, tumor cells, normal tissues or tumor tissues.
  • the biological sample is cancer tissue, such as those cancerous tissues described in the above definition of cancer, such as tissues selected from ovarian cancer, liver cancer, lung cancer, squamous cell carcinoma, endometrial cancer, and cervical cancer .
  • the tumor detection includes the detection of benign tumors and malignant tumors as defined in the above definition section (ie, cancer, the cancer is as defined in the above definition section). Further, the tumor detection refers to side-bed detection of ovarian cancer, liver cancer, lung cancer, squamous cell carcinoma, endometrial cancer, cervical cancer, etc.
  • the compound defined in the first aspect of the present invention or the product defined in the second aspect of the present invention is provided, and the compound or product is used for detecting ⁇ -glutamyl transglutamin in biological samples.
  • the content of peptidase is used for fluorescence imaging of ⁇ -glutamyl transpeptidase in biological samples or for tumor detection.
  • the tumor detection includes the detection of benign tumors and malignant tumors as defined in the above definition section (ie, cancer, the cancer is as defined in the above definition section). Further, the tumor detection refers to side-bed detection of ovarian cancer, liver cancer, lung cancer, squamous cell carcinoma, endometrial cancer, cervical cancer, etc.
  • the fifth aspect of the present invention there is provided a method for detecting the content of ⁇ -glutamyl transpeptidase in a biological sample, wherein the compound defined in the first aspect of the present invention or the compound defined in the second aspect of the present invention is used.
  • the product includes the following steps:
  • concentration range of the compound or the compound in the product is 0.01-10 mM.
  • the concentration of the ⁇ -glutamyl transpeptidase is 1-500 mU/mL (for example, 5, 10, 15, 20, 25, 30, 40, 50, 80, 100, 200, 300, 400mU/mL and any range between them, including but not limited to: 5-400mU/mL, 5-300mU/mL, 5-200mU/ mL, 5-100mU/mL); the volume of a series of standard reaction solutions of ⁇ -glutamyl transpeptidase with different concentrations is 0.1-10mL (for example, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL , 8mL, 9mL or 10mL); in the series of standard reaction solutions of ⁇ -glutamyl transpeptidase at different concentrations, the molar concentration of the compound stock solution is 0.01-10 mM, and the volume is 5-200 ⁇ L (
  • the method can be used for clinical biological sample analysis.
  • the clinical samples are tissues and tissue sections, and the concentration of the compound is 10 nM-50 ⁇ M.
  • the detection range of the method for the detection of ⁇ -glutamyl transpeptidase is 0.01-1000 mU/mL.
  • Example 7 The kit described in Example 6 is used to detect ⁇ -glutamyl transpeptidase in solution
  • Figures 2 and 3 show the UV-visible absorption and fluorescence emission spectrum response diagrams of the kit of the present invention to ⁇ -glutamyl transpeptidase, respectively.
  • Figure 2 shows the changes in the absorption spectrum after the probe solution reacted with ⁇ -glutamyl transpeptidase (50mU/mL)
  • Figure 3 shows the probe solution reacted with ⁇ -glutamyl transpeptidase (50mU/mL).
  • the ⁇ -glutamyl transpeptidase standard stock solution in the above step (1) as the sample to be tested. Under different excitation light power, test the emission spectrum of the sample. Detect the fluorescence intensity ratio values of the different emission bands (510-560nm and 620-690nm) of the sample to be tested according to the method described in step (2) above. As shown in Figures 6 to 8, the intensity of the fluorescence signal in the 510-560nm band increases with the increase of the excitation light power, and the fluorescence intensity in the 510-560nm band also increases with the increase of the GGT content.
  • ⁇ -glutamyl transpeptidase standard stock solution in the above step (1) as the sample to be tested.
  • Configure probe solutions of different concentrations to test the fluorescence spectra of samples in response to different concentrations of GGT. Detect the ratio of fluorescence intensity at different emission wavelengths (510-560nm and 620-690nm) of the sample to be tested according to the method described in step (2) above. Compare Figure 4 (the concentration of probe used in the test is 5 ⁇ M) and Figure 10. As shown in Figure 12, the 510-560nm band fluorescence signal intensity changes, it is not difficult to find that under the same GGT concentration, as the concentration of the probe in the solution used increases, the fluorescence intensity of this band also increases.
  • ⁇ -glutamyl transpeptidase standard stock solution in the above step (1) As the sample to be tested. Configure probe solutions of different concentrations, and test the response time of samples to different concentrations of GGT by testing the fluorescence spectra of the probe solutions. According to the method described in the above step (2), the trend of the fluorescence intensity ratio of the different emission bands (510-560nm and 620-690nm) of the sample to be tested is detected over time. Under the same conditions, test the response time of a commercial kit (purchased from Sigma-Aldrich, product number: MAK090) to different concentrations of GGT, and probe the commercial kit sample at 460nm (excitation wavelength 405nm) according to the method described in step (2) above.
  • a commercial kit purchased from Sigma-Aldrich, product number: MAK090
  • the higher response speed of the kit of the present invention to GGT is due to the higher maximum reaction rate (V max ) and higher catalytic efficiency (K cat ) of the probe used in the kit of the present invention under enzyme catalysis.
  • V max maximum reaction rate
  • K cat catalytic efficiency
  • Example 8 Use of the ⁇ -glutamyl transpeptidase detection kit 1 described in Example 6 in tumor cell detection
  • DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin to culture cells (cultured
  • the cells are human ovarian epithelial cells HOSEpiC and human ovarian cancer cells SKOV3). Wash the cells with DMEM before use.
  • the kit of the present invention is used for human ovarian epithelial cells HOSEpiC (low expression of ⁇ -glutamyl transpeptidase), human ovarian cancer cell SKOV3 (high expression of ⁇ -glutamyl transpeptidase) and GGT Confocal imaging of human ovarian cancer cell SKOV3 treated with inhibitor (GGs Top, purchased from Shanghai Yuanxi Biotechnology Co., Ltd.). The above cells were purchased from Tongpai (Shanghai) Biotechnology Co., Ltd.
  • the average ratio values measured in the imaging images of cells in different groups were 3.12, 1.48, 0.33 and 0.87, respectively.
  • the above results indicate that the difference in the average ratio value of the cells in different grouped imaging images is caused by the difference in GGT activity in the different grouped cells.
  • the kit of the present invention can well identify tumor cells by measuring the content of ⁇ -glutamyl transpeptidase in the cells.
  • Example 9 The use of the ⁇ -glutamyl transpeptidase detection kit described in Example 6 in the detection of ovarian cancer by bedside
  • the detection of ovarian cancer in the tissue by the kit of the present invention specifically follows the following steps:
  • the tumor tissue removed from the clinical tumor resection operation the obtained tissue is evenly divided, washed several times, and the solution containing fluorescent probe (0-50 ⁇ M) diluted with the mother solution of the kit is incubated for 1-10min .
  • the imaging device is the self-built imaging system of the research group, see the patent number CN2013394004Y, the entire content of the patent is incorporated herein by reference
  • the ratio signal is the ratio of the signal intensity of different fluorescent bands.
  • the kit of the present invention (the first kit described in Example 6) is used for the detection of GGT content in surgically removed tissues of patients with ovarian cancer and uterine cysts.
  • Figures 19-23 are imaging images of surgically removed tissues from patients with ovarian cancer (the ratio values of Figures 19-23 are 3.25, 2.73, 2.94, 3.12, 3.19), and the tissues in Figures 19-23 have passed clinical trials Pathological analysis was determined to be a tumor tissue;
  • Figure 24-27 is an imaging map of surgically removed tissue from a patient with a uterine cyst (the ratio values of Figure 24-27 are 0.37, 0.28, 0.33, 0.39), and the Figure 24-27 The tissue in has been identified as non-tumor tissue through clinicopathological rate analysis.
  • the ratio values in the tissue ratio imaging map are 2.3, 1.9, and 0.35, respectively.
  • the tissues in Figure 28-30 are tumor tissue, tumor tissue and non-tumor tissue in order, and the results are consistent with the results obtained by clinical pathological analysis.
  • the above results indicate that the kit of the present invention has the characteristics of rapid response, high sensitivity and strong anti-interference ability, and can be used for side-bed detection for accurately distinguishing tumor tissue and non-tumor tissue.

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Abstract

涉及具有下述式(1)结构的化合物、含有所述化合物的产品及它们的制备方法和在γ-谷氨酰转肽酶检测中的用途。

Description

一种化合物、含有所述化合物的产品及其在γ-谷氨酰转肽酶检测中的用途 技术领域
本发明属于荧光检测领域。具体地，本发明涉及一种化合物、含有所述化合物的产品及它们的制备方法和在γ-谷氨酰转肽酶检测中的用途。
背景技术
在临床肿瘤切除手术过程中，对肿瘤部位的准确定位并切除有利于病人术后生活质量的保证及相对良好的预后。在临床医学诊断和基础生物医学研究中，组织切片分析被广泛的用于确定切缘位置以及术后的病理分析。冷冻切片准确性较高，是目前临床中用途最为广泛的切片分析方法，然而这种方法比较耗费时间和人力，难以应用于术中肿瘤定位。荧光成像技术成像速度快、灵敏度高等，这些优点使荧光成像技术能够对组织中与肿瘤相关的生物分子实现快速、准确的定量，进而达到对肿瘤组织快速诊断的目的。
γ-谷氨酰转肽酶(GGT)是一种分布在细胞膜上的蛋白酶，其在生物体内的谷胱甘肽的代谢过程中具有重要的作用。在临床上，γ-谷氨酰转肽酶是诊断肝胆疾病的重要指标，被广泛用途于肝病及其它脏器疾病的诊断和预后检测。组织局部微环境的γ-谷氨酰转肽酶表达异常升高常常与一些恶性肿瘤相关，在肝癌、卵巢癌、肺癌、鳞状细胞癌等肿瘤组织都都有检测到γ-谷氨酰转肽酶的高表达。因此，对生物样品中γ-谷氨酰转肽酶进行定量检测具有重要的意义。免疫化学的方法是目前准确度和灵敏度最高的GGT检测方法，但是其操作繁琐，成本高，耗时较长；比色法是目前IFCC推荐使用的方法，其商业化用途最为广泛，其问题在于无法用于对相对复杂的生物环境如活体组织中或者细胞GGT含量的测定。目前商品化的荧光检测试剂盒主要是基于香豆素、萘酰亚胺等荧光分子的产品，请参见图1(Luo Z等人ChemBioChem,2019,20,474-487)，这类荧光分子在相对复杂的生物样品中容易受到背景荧光信号和组织散射的干扰，难以实现对生物样品中GGT含量的准确检测。Luo等人[c1]报道了多种不同结构的GGT检测分子，这类分子通常是“Turn on”型荧光探针，其对GGT的响应呈现单个波段 荧光强度升高的特点。这导致这类分子用于组织中GGT含量的检测时，其检测信号容易受到探针浓度的影响，使检测的准确性降低。
因此，现有技术中的试剂盒都是采用比色法和荧光增强法对GGT进行检测，这两种方法在生物样品(特别是组织)中应用时，难以消除探针浓度、激发光功率等外界环境因素对检测信号的影响，难以对所述生物样品(特别是组织)中的GGT含量进行准确的检测。相比于以单波长强度变化对待测物进行检测的方法，比率荧光检测方法在荧光检测中通常是以一个波段的光信号作为内参，另一个波段的光信号作为检测信号，以两个波段的荧光强度的比值作为输出信号，从而可以在一定程度上消除外界环境的变化对输出信号数值的干扰。因此，在相对复杂的生物体系中，利用比率检测方法对待测物进行检测，具有更高的准确性，且以双波长荧光比率变化作为检测信号将有利于构建对复杂生物体系中的GGT进行定量检测的方法。目前，用于比率检测GGT的荧光探针鲜有报道，并且这些荧光探针响应速度慢、荧光比率变化倍数低，因此这些探针难以用于对组织样本中含量的检测。现有技术中存在对GGT检测具有反应快速和高灵敏度的作为荧光探针的化合物以及含有所述化合物的新型的高灵敏度、抗干扰能力强的检测生物样品(特别是组织)中GGT的产品的迫切需求。
发明内容
为解决上述技术问题，本发明的目的在于提供一种新的作为荧光探针的化合物、含有所述化合物的产品及其制备方法和在生物样品中(特别是组织)GGT检测中的用途。本发明荧光探针(即，本发明的式(I)化合物，下同)反应快速且具有高灵敏度，且含有本发明荧光探针的产品通过荧光比率检测的方法对组织中GGT的含量进行检测，它不仅可以在一定程度上消除外界环境对检测信号的干扰，在检测准确度上有很大的提升，而且其比率信号变化倍数能达到170倍，这使得荧光探针的灵敏度得到显著地提高。另外，更重要的是，本发明荧光探针对GGT的响应速度非常快，在10min内即可完全响应，这使得本发明产品可以应用于旁床检测(point-of-care testing)组织中的GGT含量。
在本发明的第一方面，提供一种化合物，所述化合物具有下述式(1)结构；
Figure PCTCN2019085051-appb-000001
其中，
Figure PCTCN2019085051-appb-000002
选自：
Figure PCTCN2019085051-appb-000003
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
H、卤素、
Figure PCTCN2019085051-appb-000004
Figure PCTCN2019085051-appb-000005
m和n可以相同地或不同地为1-18的整数；或
Figure PCTCN2019085051-appb-000006
也可以相同地或不同地选自：
Figure PCTCN2019085051-appb-000007
X 1、X 2、X 3、X 4和X 5可以相同地或不同地选自：NH、NCH 3、O、S、Se、C(CH 3) 2和Si(CH 3) 2。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000008
选自：
Figure PCTCN2019085051-appb-000009
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
H、卤素、
Figure PCTCN2019085051-appb-000010
Figure PCTCN2019085051-appb-000011
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000012
选自：
Figure PCTCN2019085051-appb-000013
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相 同地或不同地选自：
卤素、
Figure PCTCN2019085051-appb-000014
其中当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16为
Figure PCTCN2019085051-appb-000015
时，n＝1-5；当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16相同地或不同地选自
Figure PCTCN2019085051-appb-000016
时，n＝12-18。
进一步地，在所述式(1)化合物中，所述化合物为：
Figure PCTCN2019085051-appb-000017
在本发明的第二方面，提供一种产品，所述产品包含上述本发明第一方面所定义的化合物。
进一步地，所述产品还包含添加剂和使用指南。
进一步地，所述添加剂选自下述的一种或多种：叠氮化钠、甘油、硫柳汞和吐温。
进一步地，所述产品为试剂盒。
进一步地，所述化合物和所述每一种添加剂各自呈单独制剂形式存在，或者所述化合物和所述每一种添加剂呈组合物的形式存在。
在本发明的第三方面，提供一种用途，其包括：
上述本发明第一方面所定义的化合物在制备用于检测生物样品中的γ-谷氨酰转肽酶含量的产品中的用途。
上述本发明第一方面所定义的化合物在制备用于生物样品中的γ-谷氨酰转 肽酶荧光成像的产品中的用途。
在本发明的第四方面，提供上述本发明的第一方面所定义的化合物或上述本发明第二方面所定义的产品，所述化合物或产品用于检测生物样品中的γ-谷氨酰转肽酶含量或用于生物样品中的γ-谷氨酰转肽酶荧光成像。
在本发明的第五方面，提供一种生物样品中的γ-谷氨酰转肽酶含量的检测方法，其中使用上述本发明第一方面所定义的化合物或上述本发明第二方面所定义的产品，包括如下步骤：
(1)制作标准曲线：在激发光激发下，测定一系列不同浓度γ-谷氨酰转肽酶标准反应液在不同发射波长处的荧光强度，记为F 1和F 2，取两者之间的比率值，记为R，以γ-谷氨酰转肽酶的浓度C为横坐标，比率值R为纵坐标，绘制标准曲线；和
(2)测定生物样品中γ-谷氨酰转肽酶的含量：将待测生物样本与所述化合物的储备液或在所述产品中的所述化合物的储备液孵育，经过一段时间(优选地5-30分钟)，取出待测生物样品，洗涤后(例如，用PBS洗涤)，用荧光成像仪分别记录不同通道(例如，荧光通道F1通道和F2通道)的荧光成像图，对两个荧光通道的荧光成像图进行比率处理，读取得到的比率图像中不同位置处的比率值，即根据标准曲线算出生物样品不同位置处γ-谷氨酰转肽酶的含量。
进一步地，其中所述化合物或所述产品中的所述化合物的浓度范围为0.01-10mM。
进一步地，所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液中，所述γ-谷氨酰转肽酶的浓度为1-500mU/mL；所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液的体积均为0.1-10mL；所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液中，所述化合物的储备液的摩尔浓度为0.01-10mM，体积均5-200μL，且在孵育过程中还任选地使用叠氮化钠，所述叠氮化钠的摩尔浓度为0.01-10mM，体积为5-200μL；所述γ-谷氨酰转肽酶的标准反应液由γ-谷氨酰转肽酶标准储备液溶于水中配成，所述γ-谷氨酰转肽酶标准储备液的浓度为1000-20000mU/mL。
进一步地，所述方法可用于临床生物样品分析。
进一步地，所述临床样品为组织和组织切片，所述化合物的浓度为10nM-50μM。
进一步地，所述方法用于γ-谷氨酰转肽酶检测的检测范围为0.01-1000mU/mL。
有益效果：
(1)本发明作为荧光探针(即，本发明的式(I)化合物，下同)具有高灵敏度，在溶液中检测限可达到10mU/L，且含有本发明荧光探针的产品双通道比率荧光信号的收集，可以在一定程度上降低由于探针浓度、组织散射等问题对成像信号造成的干扰，通过荧光比率检测的方法对生物样品(特别是组织)中γ-谷氨酰转肽酶的含量进行检测，它不仅可以在一定程度上消除外界环境对检测信号的干扰，在检测准确度上有很大的提升，其比率信号变化倍数能达到170倍，这使得荧光探针的灵敏度得到显著提高。
(2)本发明荧光探针对γ-谷氨酰转肽酶的响应速度非常快，在10min内即可完全响应，这使得本发明产品可以应用于旁床检测组织中的γ-谷氨酰转肽酶含量。
(3)本发明荧光探针是紫色的固体，在溶液中呈深紫色，与γ-谷氨酰转肽酶反应后，肉眼即可观察到溶液变为明显的浅黄色。
(4)本发明荧光探针具有较高的荧光量子效率，应用时显色反应仅在γ-谷氨酰转肽酶存在的条件下发生，其它常见的无机盐、氨基酸、水解酶等均不产生干扰，同时本发明荧光探针具备好的生物相容性，对γ-谷氨酰转肽酶的响应呈线性关系，可以用于该酶的定量测定。
(5)本发明荧光探针溶解性好，其用有机溶剂配成的母液产品可直接用于缓冲体系中γ-谷氨酰转肽酶的检测。
附图说明
图1显示目前商品化的荧光检测试剂盒中的荧光分子的产品。
图2为含有化合物C3的本发明试剂盒对γ-谷氨酰转肽酶响应随时间变化的紫外-可见吸收光谱。
图3为含有化合物C3的本发明试剂盒对γ-谷氨酰转肽酶响应随时间变化的发射光谱。
图4为含有化合物C3的本发明试剂盒对不同浓度γ-谷氨酰转肽酶的荧光响 应图，图中γ-谷氨酰转肽酶的浓度从上到下依次为0、1、5、10、20、25、30、40、50、75和100U/L。
图5为含有化合物C3的本发明试剂盒的比率值对γ-谷氨酰转肽酶浓度的工作曲线。
图6为含有化合物C3的本发明试剂盒的比率值对γ-谷氨酰转肽酶检测的抗干扰性测试(在5μW激发光功率下的发射光谱)。
图7为含有化合物C3的本发明试剂盒的比率值对γ-谷氨酰转肽酶检测的抗干扰性测试(在20μW激发光功率下的发射光谱)。
图8为含有化合物C3的本发明试剂盒的比率值对γ-谷氨酰转肽酶检测的抗干扰性测试(在40μW激发光功率下的发射光谱)。
图9为含有化合物C3的本发明试剂盒的比率值对γ-谷氨酰转肽酶检测的抗干扰性测试，不同激发光功率下的比率值。
图10含有化合物C3的本发明试剂盒配成探针浓度为10μM的溶液对不同浓度γ-谷氨酰转肽酶的荧光响应图，图中γ-谷氨酰转肽酶的浓度从上到下依次为0、1、5、10、20、25、30、40、50、75和100U/L。
图11为含有化合物C3的本发明试剂盒配成探针浓度为10μM的溶液测得的荧光比率值对γ-谷氨酰转肽酶浓度的工作曲线。
图12为含有化合物C3的本发明试剂盒配成探针浓度为2μM的溶液对不同浓度γ-谷氨酰转肽酶的荧光响应图，图中γ-谷氨酰转肽酶的浓度从上到下依次为0、1、5、10、20、25、30、40、50、80U/L。
图13为含有化合物C3的本发明试剂盒配成探针浓度为2μM的溶液测得的荧光比率值对γ-谷氨酰转肽酶浓度的工作曲线。
图14为含有化合物C3的本发明试剂盒与商业化GGT检测试剂盒检测临床样本中GGT含量对比。
图15为含化合物C3的本发明试剂盒和商业化GGT检测试剂盒对GGT响应的荧光强度值随时间变化的曲线。
图16为含化合物C3的本发明试剂盒和商业化GGT检测试剂盒与GGT作用的参数对比。
图17为商业化GGT检测试剂盒的荧光强度随GGT变化的工作曲线。
图18为含有化合物C3的本发明试剂盒分别用于HOSEpiC和SKOV3细胞的共聚焦成像图。
图19显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图20显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图21显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图22显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图23显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图24显示本发明检测系统对临床子宫囊肿患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集 光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图25显示本发明检测系统对临床子宫囊肿患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图26显示本发明检测系统对临床子宫囊肿患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
图27显示本发明检测系统对临床子宫囊肿患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)
图28显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)
图29显示本发明检测系统对临床卵巢癌患者切除手术中切除组织的荧光成像图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)
图30显示本发明检测系统对临床卵巢癌切除手术中切除组织的荧光成像 图。图中四张图片从左至右依次为组织明场成像图、绿光通道成像图(收集光信号波段为510-560nm)、红光通道成像图(收集光信号波段为620-690nm)和比率成像图(根据绿光通道光信号强度与红光通道信号强度的比值导出的比率成像图)。
发明详述
定义
除非另外定义，否则在本文中使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。
在本文中使用的术语“生物样品”是指得自患者(例如，患有癌症的患者)或正常个体的各种样品类型，包括任意的细胞或细胞外液，例如血液、血清、血浆、尿或其它液体样品，例如唾液、腹膜液或胸膜液、脑脊髓液、胃液或结肠直肠流体、淋巴液、滑液、间隙液、羊膜液、生理分泌物、泪液、粘液、汗液、乳、精液、阴道分泌物和来自溃疡和其它表面渗出的流体。术语“生物样品”还包括组织，所述组织包括但不限于肿瘤(包括良性肿瘤(例如，囊肿，包括但不限于：卵巢囊肿和子宫囊肿)或恶性肿瘤(即，癌症))组织，或者正常器官组织或肿瘤器官组织(例如，心、肝、脾、肺、肾、脑等)，或者所述组织的组织切片。术语“生物样品”还包括构成生物体内胞外隔室的胞外基质和细胞外液。它不仅包括临床样品(例如，所述组织和组织切片)，而且包括细胞培养物和组织培养物和来源于它们的细胞及其子代。
在本文中使用的术语“癌症”是指由异常的不受控制的细胞生长引起的新生物或肿瘤。术语“癌症”包括同时涉及恶化前癌细胞和恶性癌细胞的疾病。非限制性的例子包括：脑癌、肺癌、肝癌、脾癌、肾癌(例如肾细胞癌和肾骨盆癌)、淋巴结癌、小肠癌、胰腺癌、血细胞癌、骨癌、结肠癌/结肠直肠癌、胃癌、乳腺癌、子宫内膜癌、宫颈癌、前列腺癌、睾丸癌、卵巢癌、中枢神经系统癌、皮肤癌、头颈癌、食管癌、骨髓癌或鳞状细胞癌，或者血液性癌症，例如白血病(例如，急性和慢性白血病或急性早幼粒细胞性白血病)淋巴瘤、多发性骨髓瘤、脊髓发育不良、骨髓组织增生性疾病或顽固性贫血。
在本文中使用的术语“旁床检测(point-of-care testing)”是指在患者旁边 进行的临床检测或床边检测，也称作床旁检测或即时检测。
在本文使用的术语“卤素”是指氟、氯、溴和碘，优选地为氟或氯。
在本文中使用的术语“吐温”是指聚山梨酯(聚氧乙烯失水山梨醇脂肪酸酯)，包括、但不限于：吐温20(TWEEN-20)、吐温21(TWEEN-21)、吐温40(TWEEN-40)、吐温60(TWEEN-60)、吐温61(TWEEN-61)、吐温80(TWEEN-80)、吐温81(TWEEN-81)、吐温85(TWEEN-85)。
在本文中使用的术语“患者”或“个体”是指人类、伴侣动物(如狗、猫及马)、可食用动物(如奶牛、猪、羊)、动物园动物、海洋动物、禽类及其它类似动物种类。
在本文使用的术语“比率值”是指不同荧光波段(例如，在(510-560nm和620-690nm))测得的生物样品(例如，组织)的荧光成像图中的信号强度的比值。当所述比值处于0.2-0.8之间，则所述生物样品被认为是正常组织(阴性)，当所述比值处于1.2以上将其判断为肿瘤组织(阳性)。如果所述比值处于0.8-1.2之间，则为无法判断的组织。
在本文中使用的术语“室温”是指25℃±2℃。同时，若没有具体指明实验温度，均为室温。
在本文中使用的术语“约”是指该术语所修饰的数值的±10％，更优选为±5％，最优选为±2％，因此本领域的普通技术人员能够清楚地根据所修饰的数值确定术语“约”的范围。
在本发明的第一方面，提供一种化合物，所述化合物具有下述式(1)结构；
Figure PCTCN2019085051-appb-000018
其中，
Figure PCTCN2019085051-appb-000019
选自：
Figure PCTCN2019085051-appb-000020
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
H、卤素、
Figure PCTCN2019085051-appb-000021
Figure PCTCN2019085051-appb-000022
m和n可以相同地或不同地为1-18的整数，包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18或者它们之间的任何范围内的整数，即1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、1-11、1-12、1-13、1-14、1-15、1-16、1-17、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、2-11、2-12、2-13、2-14、2-15、2-16、2-17、2-18、3-4、3-5、3-6、3-7、3-8、3-9、3-10、3-11、3-12、3-13、3-14、3-15、3-16、3-17、3-18、4-5、4-6、4-7、4-8、4-9、4-10、4-11、4-12、4-13、3-14、4-15、4-16、4-17、4-18、5-6、5-7、5-8、5-9、5-10、5-11、5-12、5-13、5-14、5-15、5-16、5-17、5-18、6-7、6-8、6-9、6-10、6-11、6-12、6-13、6-14、6-15、6-16、6-17、6-18、7-8、7-9、7-10、7-11、7-12、7-13、7-14、7-15、7-16、7-17、7-18、8-9、8-10、8-11、8-12、8-13、8-14、8-15、8-16、8-17、8-18、9-10、9-11、9-12、9-13、9-14、9-15、9-16、9-17、9-18、10-11、10-12、 10-13、10-14、10-15、10-16、10-17、10-18、11-12、11-13、11-14、11-15、11-16、11-17、11-18、12-13、12-14、12-15、12-16、12-17、12-18、13-14、13-15、13-16、13-17、13-18、14-15、14-16、14-17、14-18、15-16、15-17、15-18、16-17、16-18或17-18；
Figure PCTCN2019085051-appb-000023
也可以相同地或不同地选自：
Figure PCTCN2019085051-appb-000024
X 1、X 2、X 3、X 4和X 5可以相同地或不同地选自：NH、NCH 3、O、S、Se、C(CH 3) 2和Si(CH 3) 2。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000025
选自：
Figure PCTCN2019085051-appb-000026
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
H、卤素、
Figure PCTCN2019085051-appb-000027
Figure PCTCN2019085051-appb-000028
m和n的定义如上所述；
X 1、X 2和X 5可以相同地或不同地选自：NH、NCH 3、O、S、Se、C(CH 3) 2和Si(CH 3) 2。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000029
选自：
Figure PCTCN2019085051-appb-000030
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
H、卤素、
Figure PCTCN2019085051-appb-000031
其中当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16为
Figure PCTCN2019085051-appb-000032
时，n＝1-5，而当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16相同地或不同地选自
Figure PCTCN2019085051-appb-000033
时，n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
进一步地，所述卤素为氟或氯。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000034
选自：
Figure PCTCN2019085051-appb-000035
Figure PCTCN2019085051-appb-000036
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
卤素、
Figure PCTCN2019085051-appb-000037
其中当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16为
Figure PCTCN2019085051-appb-000038
时，n＝1-5，而当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16相同地或不同地选自
Figure PCTCN2019085051-appb-000039
时，n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
进一步地，所述卤素为氟或氯。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000040
选自：
Figure PCTCN2019085051-appb-000041
R 9为卤素，优选为氟或氯；
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
Figure PCTCN2019085051-appb-000042
其中当R 1、R 2、R 3、R 4、R 5、 R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16为
Figure PCTCN2019085051-appb-000043
时，n＝1-5，而当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 12、R 13、R 14、R 15和R 16相同地或不同地选自
Figure PCTCN2019085051-appb-000044
Figure PCTCN2019085051-appb-000045
时，n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
X 1、X 2和X 5可以相同地或不同地选自：NH、NCH 3、O和S。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000046
选自：
Figure PCTCN2019085051-appb-000047
R 9为氟；
R 1为
Figure PCTCN2019085051-appb-000048
其中当R 1为
Figure PCTCN2019085051-appb-000049
时，n＝1-5，且当R 1为
Figure PCTCN2019085051-appb-000050
时，n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 12、R 13和R 14可以相同地或不同地选自：
Figure PCTCN2019085051-appb-000051
Figure PCTCN2019085051-appb-000052
其中n＝1-5；
R 15和R 16均为
Figure PCTCN2019085051-appb-000053
n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
X 1、X 2和X 5可以相同地或不同地选自：O和S。
进一步地，在所述式(1)化合物中，
Figure PCTCN2019085051-appb-000054
选自：
Figure PCTCN2019085051-appb-000055
R 9为氟；
R 1为
Figure PCTCN2019085051-appb-000056
其中当R 1为
Figure PCTCN2019085051-appb-000057
时，n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 12、R 13和R 14为
Figure PCTCN2019085051-appb-000058
R 15和R 16均为
Figure PCTCN2019085051-appb-000059
n＝12-18，优选n＝13-18、14-18、15-18、16-18、17-18或18；
X 1、X 2和X 5可以相同地或不同地选自：O和S，优选为O。
进一步地，在所述式(1)化合物中，所述化合物为：
Figure PCTCN2019085051-appb-000060
在本发明的第二方面，提供一种产品，所述产品包含上述本发明第一方面所定义的化合物。
进一步地，所述产品还包含添加剂和使用指南。
进一步地，所述添加剂选自下述的一种或多种：叠氮化钠、甘油、硫柳汞和吐温。
进一步地，所述产品为试剂盒。
进一步地，所述化合物和所述每一种添加剂各自呈单独制剂形式存在，或者所述化合物和所述每一种添加剂呈组合物的形式存在。
进一步地，所述使用指南涉及下述本发明第五方面所述的具体步骤。
在本发明的第三方面，提供一种用途，其包括：
上述本发明第一方面所定义的化合物在制备用于检测生物样品中的γ-谷氨酰转肽酶含量的产品中的用途。进一步地，所述生物样品是正常细胞、肿瘤细胞、正常组织或肿瘤组织。进一步地，所述生物样品是癌症组织，如在上述关于癌症定义中所述的那些癌症的组织，例如选自卵巢癌、肝癌、肺癌、鳞状细胞癌、子宫内膜癌、宫颈癌的组织。
上述本发明第一方面所定义的化合物在制备用于生物样品中的γ-谷氨酰转肽酶荧光成像的产品中的用途。进一步地，所述生物样品是正常细胞、肿瘤细胞、正常组织或肿瘤组织。进一步地，所述生物样品是癌症组织，如在上述关于癌症定义中所述的那些癌症的组织，例如选自卵巢癌、肝癌、肺癌、鳞状细胞癌、子宫内膜癌、宫颈癌的组织。
上述本发明第一方面所定义的化合物在制备用于肿瘤检测的产品中的用途。进一步地，所述肿瘤检测包括对在上述定义部分所定义的良性肿瘤和恶性肿瘤(即，癌症，所述癌症如在上述定义部分所定义)的检测。进一步地，所述肿瘤检测是指卵巢癌、肝癌、肺癌、鳞状细胞癌、子宫内膜癌、宫颈癌等的旁床检测。
在本发明的第四方面，提供上述本发明的第一方面所定义的化合物或上述本发明第二方面所定义的产品，所述化合物或产品用于检测生物样品中的γ-谷氨酰转肽酶含量或用于生物样品中的γ-谷氨酰转肽酶荧光成像或用于肿瘤检测。进一步地，所述肿瘤检测包括对在上述定义部分所定义的良性肿瘤和恶性肿瘤(即，癌症，所述癌症如在上述定义部分所定义)的检测。进一步地，所述肿瘤检测是指卵巢癌、肝癌、肺癌、鳞状细胞癌、子宫内膜癌、宫颈癌等的旁床检测。
在本发明的第五方面，提供一种生物样品中的γ-谷氨酰转肽酶含量的检测方 法，其中使用上述本发明第一方面所定义的化合物或上述本发明第二方面所定义的产品，包括如下步骤：
(1)制作标准曲线：在激发光激发下，测定一系列不同浓度γ-谷氨酰转肽酶标准反应液在不同发射波长处(例如，510-560nm和620-690nm)的荧光强度，记为F 1和F 2，取两者之间的比率值，记为R，以γ-谷氨酰转肽酶的浓度C为横坐标，比率值R为纵坐标，绘制标准曲线；和
(2)测定生物样品中γ-谷氨酰转肽酶的含量：将待测生物样本与所述化合物的储备液或在所述产品中的所述化合物的储备液孵育，经过一段时间(优选地5-30分钟)，取出待测生物样品，洗涤后(例如，用PBS洗涤)，用荧光成像仪分别记录不同通道(例如，荧光通道F1通道和F2通道)的荧光成像图，对两个荧光通道的荧光成像图进行比率处理，读取得到的比率图像中不同位置处的比率值，即根据标准曲线算出生物样品不同位置处γ-谷氨酰转肽酶的含量。
进一步地，其中所述化合物或所述产品中的所述化合物的浓度范围为0.01-10mM。
进一步地，所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液中，所述γ-谷氨酰转肽酶的浓度为1-500mU/mL(例如，5、10、15、20、25、30、40、50、80、100、200、300、400mU/mL以及它们之间的任意范围，包括但不限于：5-400mU/mL、5-300mU/mL、5-200mU/mL、5-100mU/mL)；所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液的体积均为0.1-10mL(例如，1mL、2mL、3mL、4mL、5mL、6mL、7mL、8mL、9mL或10mL)；所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液中，所述化合物的储备液的摩尔浓度为0.01-10mM，体积均5-200μL(例如，50μL、100μL或200μL)且在孵育过程中还任选地使用叠氮化钠，所述叠氮化钠的摩尔浓度为0.01-10mM，体积为5-200μL(例如，50μL、100μL或200μL)；所述γ-谷氨酰转肽酶的标准反应液由所述γ-谷氨酰转肽酶标准储备液溶于水中配成，所述γ-谷氨酰转肽酶标准储备液的浓度为1000-20000mU/mL(例如，10000mU/mL)。
进一步地，所述方法可用于临床生物样品分析。
进一步地，所述临床样品为组织和组织切片，所述化合物的浓度为10nM-50μM。
进一步地，所述方法用于γ-谷氨酰转肽酶检测的检测范围为0.01-1000mU/mL。
具体实施方式
为了更好的理解本发明专利的内容，下面通过具体的实施例和附图来进一步说明本发明的技术方案，具体包括合成、性质测定、光谱实验以及成像实验。但这些实施实例并不限于本发明。
实施例1、γ-谷氨酰转肽酶响应型荧光探针C3的制备
Figure PCTCN2019085051-appb-000061
化合物A3(0.5克)溶于20毫升甲醇溶液中，室温下加入B1(0.35克)的水溶液(10毫升)于其中，室温下继续反应12小时。反应结束后，旋蒸除去溶剂，所测粗产物通过高效液相色谱(乙腈/水＝2/1)分离提纯得到化合物C1(0.21克，产率28％)。结构确证如下： 1H NMR(400MHz,D 2O,δ)7.77(d,J＝9.6Hz,2H),6.87(dd,J＝9.6Hz,J＝2Hz,2H),6.46(d,J＝2Hz,2H),4.31(dd,J＝8Hz,J＝4.8Hz,1H),3.6-3.2(m,12H),2.3-2.05(m,3H),1.9-1.8(m,12H)。
实施例2、γ-谷氨酰转肽酶响应型荧光探针C5的制备
Figure PCTCN2019085051-appb-000062
化合物A5(0.46克)溶于20毫升甲醇溶液中，室温下加入B1(0.35克)的水溶液(12毫升)于其中，室温下继续反应12小时。反应结束后，旋蒸除去溶剂，所测粗产物通过高效液相色谱(乙腈/水＝3/2)分离提纯得到化合物C5(0.1克，产率29％)。结构确证如下： 1H NMR(400MHz,D 2O,δ)8.37(d,J＝9.0Hz,1H),8.29(d,J＝15Hz,1H),8.26(d,J＝9.0Hz,1H),8.06(d,J＝15Hz,1H),7.84(t,J ＝8.3Hz,1H),7.76(m,2H),6.95(dd,J1＝2.4Hz,J2＝9.6Hz,1H),6.69(d,J＝2.2Hz,1H),4.22(s,3H),4.03(m,1H),3.6-3.2(m,9H),2.06(t,2H),1.9-1.7(m,2H),1.32(d,J＝7.2Hz,3H)。
实施例3、γ-谷氨酰转肽酶响应型荧光探针C7的制备
Figure PCTCN2019085051-appb-000063
化合物A7(0.4克)溶于20毫升甲醇溶液中，室温下加入B1(0.45克)的水溶液(8毫升)于其中，室温下继续反应24小时。反应结束后，旋蒸除去溶剂，所测粗产物通过高效液相色谱(乙腈/水＝4/1)分离提纯得到化合物C7(0.1克，产率23％)。结构确证如下： 1H NMR(400MHz,D 2O,δ)7.28(d,J＝9.6Hz,1H),6.77(d,J＝9.6Hz,1H),4.04(m,1H),3.6-3.2(m,3H),2.05(t,2H),1.9-1.8(m,2H),1.32(d,J＝7.2Hz,3H).
实施例4、γ-谷氨酰转肽酶响应型荧光探针C13的制备
Figure PCTCN2019085051-appb-000064
化合物A13(0.5克)溶于20毫升甲醇溶液中，室温下加入B1(0.55克)的水溶液(8.2毫升)于其中，室温下继续反应18小时。反应结束后，旋蒸除去溶剂，所测粗产物通过高效液相色谱(乙腈/水＝3/1)分离提纯得到化合物C13(0.1克，产率23％)。结构确证如下： 1H NMR(400MHz,D 2O,δ)8.92(d,J＝8.4Hz,2H),8.02(d,J＝8.4Hz,2H),7.48(m,4H),6.97(d,J＝7.6Hz,J＝2Hz,2H),6.27(dd,J＝2Hz,2H),4.03(m,1H),3.6-3.2(m,3H),2.27(s,6H)2.06(t,2H),1.9-1.7(m,2H),1.32(d,J＝7.2Hz,3H),1.02(s,12H).
实施例5、γ-谷氨酰转肽酶响应型荧光探针C14的制备
Figure PCTCN2019085051-appb-000065
化合物A14(0.8克)溶于20毫升甲醇溶液中，室温下加入B2(0.45克)的水溶液(20毫升)于其中，室温下继续反应36小时。反应结束后，旋蒸除去溶剂，所测粗产物通过高效液相色谱(乙腈/水＝1/1)分离提纯得到化合物C14(0.27克，产率27％)。结构确证如下： 1H NMR(400MHz,D 2O,δ)8.92(d,J＝8.4Hz,2H),8.02(d,J＝8.4Hz,2H),7.48(m,4H),6.97(d,J＝7.6Hz,J＝2Hz,2H),6.27(dd,J＝2Hz,2H),4.04(m,1H),3.8-3.2(m,3H),2.59-2.22(m,12H),2.05(t,2H),2.0-1.6(m,8H),1.32(d,J＝7.2Hz,3H).1.25-0.89(m,104H)
实施例6、γ-谷氨酰转肽酶检测试剂盒的制备
称取化合物C3(4.8mg，0.01mmol)、叠氮化钠(0.01mg)、甘油(1mg)、硫柳汞(0.05mg)、吐温-20(0.01mg)，溶于40mL超纯水中，配成荧光探针储备液(又称母液，探针浓度：250μM)，即得到γ-谷氨酰转肽酶检测试剂盒一。
称取化合物C13(4.9mg，0.01mmol)和叠氮化钠(0.01mg)，溶于200mL超纯水中，配成荧光探针储备液(探针浓度：50μM)，即得到γ-谷氨酰转肽酶检测试剂盒二。
实施例7.实施例6所述试剂盒一用于检测溶液中γ-谷氨酰转肽酶
本发明试剂盒检测γ-谷氨酰转肽酶具体按照以下步骤进行：
(1)荧光探针光谱响应性质研究：将20μL的上述试剂盒母液滴加到2mL的50mU/mLγ-谷氨酰转肽酶溶液和0.01M磷酸盐缓冲溶液中，37℃下反应，测量紫外-可见吸收光谱和荧光发射光谱随反应时间的变化。荧光发射光谱测定时以488nm为激发光；激发和发射狭缝宽度均为10nm；电压400V。吸收光谱由Perkin-Elmer公司Lamda750型紫外可见吸收光谱仪测得；荧光光谱由PTI公司QM40荧光光谱仪测得。如图2和图3所示分别为本发明试剂盒对γ-谷氨酰转肽酶的紫外可见吸收及荧光发射光谱响应图。图2为探针溶液与γ-谷氨酰转肽酶(50mU/mL)反应后的吸收光谱变化，图3为探针溶液与γ-谷氨酰转肽酶(50mU/mL)反应后的发射光谱变化。
(2)制作标准曲线：
在激发光激发下，测定浓度分别为0、5、10、15、20、25、30、40、50、80和100mU/mL的γ-谷氨酰转肽酶标准反应液的发射光谱(如图4)，在不同发光波段(510-560nm和620-690nm)的荧光强度的比值，记为R(510-560nm波段的荧光强度比620-690nm波段的荧光强度)。以γ-谷氨酰转肽酶的浓度C为横坐标，比率值R为纵坐标，绘制标准曲线(如图5)。
(3)检测待测样品中γ-谷氨酰转肽酶的含量：
将上述步骤(1)中γ-谷氨酰转肽酶标准储备溶液替换为待测样品，按照上述步骤(2)所述方法检测所述待测样品不同发射波段(510-560nm和620-690nm)的荧光强度比率值，记为R'，将所述R'带入所述步骤(2)所得标准曲线，即可得到待测样品中γ-谷氨酰转肽酶的含量。
(4)γ-谷氨酰转肽酶检测试剂盒检测GGT含量的可靠性研究
将上述步骤(1)中γ-谷氨酰转肽酶标准储备溶液作为待测样品。在不同激发光功率下，测试样品的发射光谱。按照上述步骤(2)所述方法检测所述待测样品不同发射波段(510-560nm和620-690nm)的荧光强度比率值。如图6至图8所示，510-560nm波段荧光信号的强度随激发光功率的增加而增加，同时510-560nm波段的荧光强度也随GGT含量的增加而增加。这表明根据单个波段荧光强度的变化检测GGT含量的方法容易受到激发光功率的影响，而导致检测结果出现偏差。而本试剂盒所采用的比率检测方法证明(图9)该方法所得出的检测结果相比于单波段荧光变化检测GGT的方法，受到激发光功率的影响小得多。因此，相比于单波长信号变化的荧光探针，本试剂盒中使用的比率荧光探针的输出信号不受激发光功率变化的影响，具有更高的稳定性。
将上述步骤(1)中γ-谷氨酰转肽酶标准储备溶液作为待测样品。配置不同浓度的探针溶液，测试样品对不同浓度GGT响应后的荧光光谱。按照上述步骤(2)所述方法检测所述待测样品不同发射波长处((510-560nm和620-690nm)的荧光强度比率值。比较图4(测试所用探针浓度为5μM)、图10和图12中510-560nm波段荧光信号的强度变化，不难发现，在相同GGT浓度的条件下，随着使用的溶液中探针浓度的增加，该波段荧光强度也相应增加。而根据图5(测试所用探针浓度为5μM)、图11和图13中所示的荧光比率值对GGT浓度变化 的工作曲线可以得出，探针浓度的变化对工作曲线的斜率以及得出的检测结果都没有明显的影响。这说明本试剂盒所述的比率检测GGT的方法，可以消除探针浓度变化对检测结果准确性的影响。
将上述步骤(1)中γ-谷氨酰转肽酶标准储备溶液替换为临床血清样本。按照上述步骤(2)所述方法检测所述待测样品不同发射波段(510-560nm和620-690nm)的荧光强度比率值。将本试剂盒测出的GGT含量与用目前商业化的单波长荧光检测GGT的试剂盒(购自Sigma-Aldrich公司，货号：MAK090)及ELISA检测GGT试剂盒(购自上海酶研生物科技有限公司，货号：EK-H10297)检出的GGT含量进行比较(图14)。该结果表明，比率探针对实际样本中GGT含量的检测结果与ELISA方法检出的结果更为接近，也具有更低的标准偏差。
(5)γ-谷氨酰转肽酶检测试剂盒与商用试剂盒检测速度和灵敏度比较
将上述步骤(1)中γ-谷氨酰转肽酶标准储备溶液作为待测样品。配置不同浓度的探针溶液，通过测试探针溶液的荧光光谱，测试样品对不同浓度GGT的响应时间。按照上述步骤(2)所述方法检测所述待测样品不同发射波段(510-560nm和620-690nm)的荧光强度比率值随时间变化的趋势。在同样条件下，测试商用试剂盒(购自Sigma-Aldrich公司，货号：MAK090)对不同浓度GGT的响应时间，按照上述步骤(2)所述方法，探测商用试剂盒样品在460nm(激发波长405nm)处的荧光强度数值随时间变化的趋势。如图15所示，在GGT过量的情况下，本发明所述试剂盒在10分钟内即可反应完全，并且比率变化倍数能够达到170倍，而商用试剂盒响应完全所需时间为25分钟，荧光强度的变化倍数为30倍。该结果表明，本发明试剂盒所述荧光探针对GGT的响应速度和荧光变化的灵敏度都要显著优于该商用试剂盒。另外，如图16所示，通过上述响应速度测试得到本发明试剂盒所用荧光探针和商用试剂盒所用荧光探针与GGT相互作用的各项参数。本发明试剂盒对GGT具有更高的响应速度是由于本发明试剂盒所用探针在酶催化下具有更高的最大反应速率(V max)和更高的酶催化效率(K cat)。根据图5和图17可知，本发明试剂盒所述探针对GGT检测限为10mU/L，而测得商用试剂盒的检测限为53mU/L。上述结果表明，本发明所述试剂盒比商用荧光法GGT检测试剂盒具有显著更高的灵敏度和检测速度。
实施例8、实施例6所述γ-谷氨酰转肽酶检测试剂盒一在肿瘤细胞检测中的 用途
本发明试剂盒检测细胞中γ-谷氨酰转肽酶具体按照以下步骤进行：
i、在37℃和5％CO 2条件下，用含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100μg/mL的链霉素的DMEM培养基培养细胞(培养的细胞为人卵巢上皮细胞HOSEpiC和人卵巢癌细胞SKOV3)。细胞使用之前用DMEM清洗。
ii、在细胞中加入1mL DMEM，再加入实施例6所述γ-谷氨酰转肽酶检测试剂盒一的母液(10μL)孵育0.5h后进行共聚焦荧光成像(激光共聚焦扫描显微镜厂家为Olympus，型号为Fluo View FV1000)，在激发光激发下，收集不同荧光波段(510-560nm和620-690nm)的荧光强度。比率信号为不同波段荧光信号强度的比值。根据上述实施例7中所述的荧光比率值对γ-谷氨酰转肽酶浓度的工作曲线，我们可以测得细胞中的γ-谷氨酰转肽酶含量。
如图18所示为本发明试剂盒分别用于人卵巢上皮细胞HOSEpiC(γ-谷氨酰转肽酶低表达)、人卵巢癌细胞SKOV3(γ-谷氨酰转肽酶高表达)和GGT抑制剂(GGs Top，购自上海源溪生物科技有限公司)处理后的人卵巢癌细胞SKOV3的共聚焦成像图，以上细胞均购自通派(上海)生物科技有限公司。不同分组(SKOV3、20μM抑制剂培养的SKOV3、100μM抑制剂培养的SKOV3以及正常细胞HOSEpiC)中的细胞成像图所测得的平均比率值分别为3.12，1.48，0.33和0.87。上述结果说明，不同分组成像图中细胞的平均比率值的不同是由于不同分组细胞中GGT活性的不同造成的。基于肿瘤细胞和正常细胞中GGT含量的不同，上述结果表明本发明试剂盒可以通过对细胞中的γ-谷氨酰转肽酶含量的测定对肿瘤细胞进行良好地识别。
实施例9、实施例6中所述的γ-谷氨酰转肽酶检测试剂盒一在旁床检测卵巢癌中的用途
本发明试剂盒检测组织中卵巢癌具体按照以下步骤进行：
1.从临床肿瘤切除手术中取下的肿瘤组织，将所得组织均匀切分，清洗几遍后，用试剂盒母液稀释后配成的含荧光探针(0-50μM)的溶液孵育1-10min。
2.用活体成像仪(成像仪为课题组自行搭建成像系统，参见专利的专利号为CN2013394004Y，该专利全部内容通过引用并入本文)对组织进行荧光成像，收集不同荧光波段((510-560nm和620-690nm))的荧光信号。比率信号为不 同荧光波段信号强度的比值。
3.对已通过病理分析确认为肿瘤组织和正常组织的临床样本，按照步骤(1)和(2)中所述方法对组织进行荧光成像，统计不同组织的成像图中比率信号的平均比率值(图19-27)。将所得数据统计后可得，肿瘤组织成像图中比率值为3.1±0.5，正常组织成像图中的比率值为0.5±0.3。
4.将上述步骤3中所述已通过病理确认过的组织替换为待测临床样本。按照步骤(1)和(2)中所述方法对组织进行荧光成像(图28-30)。将测得的组织成像图的比率值与步骤(3)中所得的已知病理的组织的比率值进行对比，如果其数值大小处于在0.2-0.8之间，则该组织被认为是正常组织(阴性)，如果其数值大小处于1.2以上将其判断为肿瘤组织(阳性)。如果数值落在0.8-1.2之间，则为无法判断的组织。
如图19-30所示为本发明试剂盒(实施例6所述试剂盒一)用于卵巢癌和子宫囊肿患者手术切除组织GGT含量的检测。其中图19-23为卵巢癌患者手术切除的组织的成像图(图19-23的比率值依次为3.25、2.73、2.94、3.12、3.19)，并且所述图19-23中的组织已经通过临床病理分析被确定织为肿瘤组织；图24-27为子宫囊肿患者手术切除的组织的成像图(图24-27的比率值依次为0.37、0.28、0.33、0.39)，并且所述图24-27中的组织已经通过临床病理率分析被确定为非肿瘤组织。在图28-30中，组织比率成像图中的比率值分别为2.3、1.9和0.35。按照本试剂盒所述方法进行判断，图28-30中的组织依次为肿瘤组织、肿瘤组织和非肿瘤组织，该结果与临床病理分析得出的结果一致。上述结果表明利用本发明所述的试剂盒具有反应迅速、高灵敏度和抗干扰能力强等特点，可以用于对肿瘤组织和非肿瘤组织精确辨别的旁床检测。

Claims (17)

  1. 化合物，所述化合物具有下述式(1)结构；
    Figure PCTCN2019085051-appb-100001
    其中，
    Figure PCTCN2019085051-appb-100002
    选自：
    Figure PCTCN2019085051-appb-100003
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 10、R 11、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
    H、卤素、
    Figure PCTCN2019085051-appb-100004
    Figure PCTCN2019085051-appb-100005
    m和n可以相同地或不同地为1-18的整数；或
    Figure PCTCN2019085051-appb-100006
    也可以相同地或不同地选自：
    Figure PCTCN2019085051-appb-100007
    X 1、X 2、X 3、X 4和X 5可以相同地或不同地选自：NH、NCH 3、O、S、Se、 C(CH 3) 2和Si(CH 3) 2。
  2. 根据权利要求1所述的化合物，其中
    Figure PCTCN2019085051-appb-100008
    选自：
    Figure PCTCN2019085051-appb-100009
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
    H、卤素、
    Figure PCTCN2019085051-appb-100010
    Figure PCTCN2019085051-appb-100011
  3. 根据权利要求2所述的化合物，其中R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16可以相同地或不同地选自：
    卤素、
    Figure PCTCN2019085051-appb-100012
    其中当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16为
    Figure PCTCN2019085051-appb-100013
    时，n＝1-5；当R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9、R 12、R 13、R 14、R 15和R 16相同地或不同地选自
    Figure PCTCN2019085051-appb-100014
    时，n＝12-18。
  4. 根据权利要求1-3中任一项所述的化合物，所述化合物为：
    Figure PCTCN2019085051-appb-100015
  5. 产品，所述产品包含权利要求1-4中任一项的化合物。
  6. 根据权利要求5所述的产品，所述产品还包含添加剂和使用指南。
  7. 根据权利要求6所述的产品，所述添加剂选自下述的一种或多种：叠氮化钠、甘油、硫柳汞和吐温。
  8. 根据权利要求5-7中任一项的产品，所述产品为试剂盒。
  9. 根据权利要求8所述的产品，其中所述化合物和所述每一种添加剂各自呈单独制剂形式存在，或者所述化合物和所述每一种添加剂呈组合物的形式存在。
  10. 根据权利要求1-4中任一项的化合物在制备用于检测生物样品中的γ-谷氨酰转肽酶含量的产品中的用途。
  11. 根据权利要求1-4中任一项的化合物在制备用于生物样品中的γ-谷氨酰转肽酶荧光成像的产品中的用途。
  12. 生物样品中的γ-谷氨酰转肽酶含量的检测方法，其中使用权利要求1-4中任一项所述的化合物或权利要求5-9中任一项所述的产品，包括如下步骤：
    (1)制作标准曲线：在激发光激发下，测定一系列不同浓度γ-谷氨酰转肽酶标准反应液在不同发射波长处的荧光强度，记为F 1和F 2，取两者之间的比率值，记为R，以γ-谷氨酰转肽酶的浓度C为横坐标，比率值R为纵坐标，绘制标准曲线；和
    (2)测定生物样品中γ-谷氨酰转肽酶的含量：将待测生物样本与所述化合物的储备液或在所述产品中的所述化合物的储备液孵育，经过一段时间，取出待 测生物样品，洗涤后，用荧光成像仪分别记录不同通道的荧光成像图，对两个荧光通道的荧光成像图进行比率处理，读取得到的比率图像中不同位置处的比率值，即根据标准曲线算出生物样品不同位置处γ-谷氨酰转肽酶的含量。
  13. 根据权利要求12所述的检测方法，其中所述化合物或所述产品中的所述化合物的浓度范围为0.01-10mM。
  14. 根据权利要求13所述的检测方法，其中所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液中，所述γ-谷氨酰转肽酶的浓度为1-500mU/mL；
    所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液的体积均为0.1-10mL；
    所述一系列不同浓度的γ-谷氨酰转肽酶标准反应液中，所述化合物的储备液的摩尔浓度为0.01-10mM，体积5-200μL，且在孵育过程中还任选地使用叠氮化钠，所述叠氮化钠的摩尔浓度为0.01-10mM，体积为5-200μL。
  15. 根据权利要求12-14中的任一项所述的检测方法，所述方法可用于临床生物样品分析。
  16. 根据权利要求15所述的检测方法，所述临床样品为组织和组织切片，所述化合物的浓度为10nM-50μM。
  17. 根据权利要求12-14和16中的任一项所述的检测方法，所述方法用于γ-谷氨酰转肽酶检测的检测范围为0.01-1000mU/mL。
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