Pentapeptide compound fluorescent probe and application thereof in identifying pre-birth injury and post-fatality injury

Abstract

The invention provides a pentapeptide compound fluorescent probe and application thereof to identification of injury before life and injury after death, and belongs to the technical field of forensic pathology, the pentapeptide compound fluorescent probe comprises a targeted pentapeptide CREKA and a dicyanomethylene-4H-pyran dicyanomethylene-4H-pyran fluorescence labeling compound; the targeted pentapeptide CREKA and the dicyanomethylene-4H-pyran dicyanomethylene-4H-pyran fluorescence labeling compound are in covalent linkage with each other; the pentapeptide compound fluorescent probe provided by the invention has the advantages of high specificity aiming at the fibrin-fibronectin compound, rapidness and convenience in detection, wide application range and the like, and can be effectively applied to rapid identification of pre-birth injury and post-fatality injury. The pentapeptide compound fluorescent probe can solve the problem that a traditional method is insufficient in accuracy under the conditions of atypical damage form change, incomplete field conditions and the like, and provides a new technical support for forensic pathological damage identification.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic pathology, and in particular relates to a pentapeptide complex fluorescent probe and its application in differentiating antemortem injuries from postmortem injuries. Background Art

[0002] The accurate identification of antemortem injuries from postmortem injuries is a key content in forensic identification work, and is of great significance for case detection and forensic expertise. In actual case inspections, especially when death occurs shortly after the injury, or when the wound is contaminated and interfered by other environmental factors, the vital reactions observed by traditional naked-eye and histological methods are often not obvious, making it difficult to draw conclusions in a timely and accurate manner. When the conditions at the grass-roots level are limited, relying on naked-eye observation for identification is still the norm, but its reliability is insufficient. Existing identification methods include using Martius scarlet blue staining to show that fibrin is red, but the detection rate is only about 70%, and there are certain limitations in convenience, specificity and accuracy. Histological or immunological detection of indicators such as blood coagulation and inflammatory cell infiltration in damaged tissues requires high experimental conditions and is not suitable for large-scale use by grass-roots forensic doctors.

[0003] Current research on the targeting pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) mostly focuses on the cancer field, for tumor imaging or drug delivery. There has been no report on its application in differentiating antemortem injuries from postmortem injuries in the forensic field. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a pentapeptide complex fluorescent probe and its application in differentiating antemortem injuries from postmortem injuries. The pentapeptide complex fluorescent probe provided by the present invention has the advantages of high specificity for fibrin-fibronectin complexes, rapid and convenient detection, wide application range, etc., and can be effectively applied to the rapid differentiation of antemortem injuries from postmortem injuries. This pentapeptide complex fluorescent probe can solve the problem of insufficient accuracy of traditional methods in cases where the injury morphology changes atypically and the on-site conditions are imperfect, providing new technical support for forensic pathology injury identification.

[0005] The present invention provides a pentapeptide complex fluorescent probe, which includes a targeting pentapeptide CREKA and a dicyanomethylene-4H-pyran fluorescent labeling compound; the targeting pentapeptide CREKA and the dicyanomethylene-4H-pyran fluorescent labeling compound are covalently linked.

[0006] Preferably, the structural formula is as shown in Formula I:

[0007]

[0008] The present invention provides a diagnostic reagent for differentiating antemortem injuries from postmortem injuries, which includes the above-mentioned pentapeptide complex fluorescent probe.

[0009] Preferably, the diagnostic reagent is a liquid preparation, and the use concentration of the diagnostic reagent is 0.8 to 1.2 mg / ml.

[0010] The present invention provides the application of the pentapeptide complex fluorescent probe and the diagnostic reagent in differentiating antemortem injuries from postmortem injuries.

[0011] Preferably, it includes the following steps:

[0012] 1) Apply the diagnostic reagent on the surface of the wound of the corpse.

[0013] 2) Perform fluorescence detection on the wound of the corpse by near-infrared imaging. If fluorescence is present, it is determined as an antemortem injury; if no fluorescence is present, it is determined as a postmortem injury.

[0014] Preferably, the application method in step 1) is spraying or coating.

[0015] Preferably, the excitation wavelength of the fluorescence detection in step 2) is 560 nm, and the emission wavelength is 704 nm.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a pentapeptide complex fluorescent probe, which includes a targeting pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) and a dicyanomethylene-4H-pyran dicyanomethylene-4H-pyran-based fluorescent labeling compound; the present invention utilizes the property that the targeting pentapeptide CREKA can specifically bind to the fibrin-fibronectin complex, and can specifically detect the fibrin-fibronectin complex; and the bleeding and blood coagulation processes during life will form an obvious fibrin-fibronectin network structure at the tissue injury site, which can provide a specific target for differentiating antemortem injuries, thereby realizing accurate differentiation of antemortem injuries. In addition, the present invention uses fluorescence detection, which is more rapid and convenient; the fluorescence color development is clear, without cumbersome specimen preparation and color development processes, and the preliminary identification can be quickly completed on-site or in the autopsy room. Further, the near-infrared I region fluorescence signal utilized by the present invention has strong penetration and low autofluorescence background, and relatively clear results can still be obtained in complex environments or under mild contamination conditions.

[0017] The diagnostic reagent for differentiating antemortem injuries from postmortem injuries provided by the present invention has strong practicability, is suitable for grass-roots forensic doctors, and can quickly make a judgment in cooperation with a portable reaction device, solving the problem of "difficult diagnosis of antemortem and postmortem injuries in a short time and complex environment". Description of the Drawings

[0018] Figure 1 is the synthesis route of the pentapeptide complex fluorescent probe;

[0019] Figure 2 is the high-resolution mass spectrum of the pentapeptide complex fluorescent probe;

[0020] Figure 3 Schematic diagram of experimental animal model of antemortem injury / postmortem injury;

[0021] Figure 4 It is a schematic diagram of fluorescence imaging. After the diagnostic reagent of the present invention is sprayed, the fluorescence difference contrast between antemortem and postmortem injury sites is photographed by a near-infrared imager. Detailed implementation manners

[0022] The present invention provides a pentapeptide complex fluorescent probe, which comprises a targeting pentapeptide CREKA and a dicyanomethylene-4H-pyran fluorescent labeling compound; the targeting pentapeptide CREKA and the dicyanomethylene-4H-pyran fluorescent labeling compound are covalently linked.

[0023] In the present invention, the targeting pentapeptide CREKA is cysteine-arginine-glutamic acid-lysine-alanine, which can specifically bind to the fibrin-fibronectin complex. Fibrin is transformed from plasma fibrinogen under the action of thrombin and can form a complex with fibronectin (FN) at the wound; the bleeding and blood coagulation processes before death will form an obvious fibrin-fibronectin network structure at the tissue injury site, which can provide a specific target for identifying antemortem injury; the present invention precisely utilizes the targeting pentapeptide and the fibrin-fibronectin complex generated during the formation of antemortem injury to achieve the purpose of identification.

[0024] In the present invention, the structural formula of the pentapeptide complex fluorescent probe is shown in Formula I:

[0025]

[0026] The present invention also provides a diagnostic reagent for differentiating antemortem injury from postmortem injury, which comprises the pentapeptide complex fluorescent probe described above.

[0027] In the present invention, the diagnostic reagent is preferably a liquid preparation, and the use concentration of the diagnostic reagent is preferably 0.8 - 1.2 mg / ml, more preferably 0.9 - 1.1 mg / ml, and even more preferably 1.0 mg / ml. In the present invention, the diagnostic reagent is preferably obtained by dissolving the pentapeptide complex fluorescent probe in water.

[0028] The present invention provides the application of the pentapeptide complex fluorescent probe and the diagnostic reagent described above in differentiating antemortem injury from postmortem injury.

[0029] In the present invention, the application preferably comprises the following steps: 1) applying the diagnostic reagent on the surface of the corpse wound; 2) performing fluorescence detection on the corpse wound with a near-infrared imager.

[0030] In the present invention, first, the diagnostic reagent is applied to the surface of the corpse wound, and the application method is preferably spraying or coating; the application amount of the diagnostic reagent is preferably 0.5-1.5 ml, and more preferably 0.8-1.2 ml.

[0031] After applying the diagnostic reagent in the present invention, near-infrared imaging is used to perform fluorescence detection on the corpse wound. If fluorescence is detected, it is an ante-mortem injury; if no fluorescence is detected, it is a post-mortem injury. In the present invention, the excitation wavelength of the fluorescence detection is 560 nm, and the emission wavelength is 704 nm; the present invention does not have special limitations on the instrument and equipment used for the near-infrared imaging, as long as it is equipped with a near-infrared imaging system. In the specific implementation process of the present invention, a small animal in vivo imager is used.

[0032] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0033] Example 1

[0034] The preparation method of the pentapeptide complex fluorescent probe, and the synthesis route is as Figure 1 shown.

[0035] 2-Methyl-4H-chromenone (2)

[0036] Suspend 1-(2-hydroxyphenyl)ethanone (10.0 g) in ethyl acetate (200 mL), replace the air with nitrogen, and continuously stir under a nitrogen atmosphere. Add sodium hydride (8.0 g) and stir at room temperature for 4 hours. The gray-green solid formed by the reaction is filtered and then resuspended in 200 mL of deionized water, and the pH of the solution is adjusted to neutral. The aqueous phase is extracted with ethyl acetate (200 mL), and the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a brown solid crude product (7.2 g, yield 55%), and this crude product is directly used for the next reaction.

[0037] Suspend compound 1 (7.2 g) in 10 times its volume of acetic acid, and slowly add sulfuric acid (5 mL). After the reaction mixture is refluxed at 120 °C for 30 minutes, it is poured into 800 mL of ice water, and the pH is adjusted to neutral with sodium carbonate solution. Extract twice with dichloromethane (200 mL), combine the organic phases, dry over sodium sulfate, filter, and concentrate to obtain product 2 as a gray solid (4.8 g, yield 78.8%). The hydrogen spectrum is as follows: 1 H NMR (300 MHz, CDCl3) δ 8.18 (dd, J = 7.9, 1.7 Hz, 1H), 7.64 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.45–7.34 (m, 2H), 6.18 (d, J = 0.9 Hz, 1H), 2.39 (d, J = 0.8 Hz, 3H).

[0038] 2-Methyl-4H-chromene-4-ylidene malononitrile (3)

[0039] Compound 2 (4.8 g) and malononitrile (2.40 g, 36.2 mmol) were co-suspended in 25 mL of acetic anhydride and refluxed at 140 °C for 14 hours. After the solvent was removed by distillation under reduced pressure, the residue was re-suspended in 80 mL of water and refluxed for an additional 0.5 hour. The mixture was extracted three times with dichloromethane (100 mL each time), and the combined organic phases were dried and concentrated. The residue was purified by flash silica gel column chromatography (eluent: 30% ethyl acetate / hexane) to afford compound 3 (2.3 g, yield 35.93%) as an orange solid. The 1H NMR spectrum was as follows: 1 1H NMR (300 MHz, CDCl3) δ 8.92 (dt, J = 8.3, 1.4 Hz, 1H), 7.74 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.53–7.40 (m, 2H), 6.77–6.68 (m, 1H), 2.46 (s, 3H).

[0040] (E)-2-(2-(4-Hydroxystyryl)-4H-chromene-4-ylidene) malononitrile (4)

[0041] Compound 3 (400 mg) and 4-hydroxybenzaldehyde (300 mg) were dissolved in ethanol (34 mL), and piperidine (0.6 mL) was added. The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. Then the mixture was heated to reflux for 17 hours, and then cooled to room temperature and concentrated. The crude product was purified by silica gel column chromatography (eluent: 40% ethyl acetate / hexane) to afford compound 4 (252 mg, yield 42%) as a red solid. The 1H NMR spectrum was as follows: 1 1H NMR (300 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.72 (dd, J = 8.3, 1.4 Hz, 1H), 7.91 (ddd, J = 8.5, 7.1, 1.5 Hz, 1H), 7.78 (dd, J = 8.5, 1.4 Hz, 1H), 7.73–7.56 (m, 4H), 7.26 (d, J = 16.0 Hz, 1H), 6.94 (s, 1H), 6.91–6.81 (m, 2H).

[0042] (E)-4-[2-(4-(Dicyanomethylene)-4H-benzopyran-2-yl)vinyl]phenyl acrylate (5)

[0043] Compound 4 (100 mg) was dissolved in dichloromethane (DCM, 5 mL), followed by the addition of triethylamine (TEA, 90 mg), and then acryloyl chloride (117 mg) was slowly added dropwise. The mixture was stirred at room temperature for 10 minutes. The probe TP-NIR was purified by silica gel column chromatography (eluent: dichloromethane:n-hexane = 7:3) to obtain yellow solid compound 5 (107 mg, yield 92%). The 1H NMR spectrum is as follows: 1 1H NMR (300 MHz, CDCl3) δ 8.93 (dd, J = 8.4, 1.3 Hz, 1H), 7.77 (ddd, J = 8.5, 7.2, 1.4 Hz, 1H), 7.68–7.56 (m, 4H), 7.48 (ddd, J = 8.4, 7.2, 1.3 Hz, 1H), 7.28–7.22 (m, 2H), 6.89 (s, 1H), 6.81 (d, J = 16.0 Hz, 1H), 6.67 (dd, J = 17.3, 1.2 Hz, 1H), 6.36 (dd, J = 17.3, 10.4 Hz, 1H), 6.09 (dd, J = 10.4, 1.2 Hz, 1H).

[0044] (4S,7R)-4-[(S)-2-[R-2-Amino-3-mercaptopropionamido]-5-guanidinopentanamido]-7-[(S)-1-carboxyethylcarbamoyl]-11-{[2-(4-[(E)-2-(4-dicyanomethylene-4H-benzopyran-2-yl)vinyl]phenoxy)-2-oxoethyl]amino}-5-oxoundecanoic acid (I)

[0045] Compound 5 (50 mg) was dissolved in DMF (2 mL), then tris(2-carboxyethyl)phosphine (25 mg) and CREAK (130 mg) were added, and the mixture was stirred at room temperature for 1 hour under nitrogen protection. The mixture was poured into ethyl acetate, and a large amount of solid precipitated. The solid was filtered by suction, and the filter cake was eluted with ethyl acetate to obtain yellow solid compound I (117 mg, yield 85%). HRMS calcd for C 46 H 57 N 10 O 11 S + ([M+H] + ): 957.3919, found: 957.3927.

[0046] The synthesized and purified compound 1 (i.e., the pentapeptide complex fluorescent probe) powder was dissolved in water to prepare a 1 mg / ml mixture, which is the diagnostic reagent for differentiating antemortem injuries from postmortem injuries.

[0047] Example 2

[0048] Construction of the experimental animal model of antemortem injury / postmortem injury

[0049] By preparing models of antemortem injuries and postmortem injuries on Sprague-Dawley rats (6w - 8w, 250 ± 10g, Vital River Laboratory Animal Technology Co., Ltd., Beijing), the antemortem injury model was established by inflicting injuries at different time intervals (such as 12h, 6h, 2h, 0.5h before death, etc.) and then sacrificing the animals; the postmortem injury model was established by inflicting injuries on dead rats.

[0050] After anesthetizing the rats with sodium pentobarbital (40mg / kg, intraperitoneal injection), shaving the hair on their backs, and then using a scalpel to make a wound 1.5 - 2.5 cm in size, 1.0 ml (1.0 mg / ml) of the diagnostic reagent prepared in the example was sprayed or applied at the wound site. Then, a small animal in vivo imaging system (IVIS Spectrum CT Imaging System, PerkinElmer.Inc., USA) or a corresponding near-infrared imaging system was used to observe the fluorescence signal; the fluorescence detection results of different time periods and different treatment groups were recorded and analyzed. The results are as Figure 3 shown. In the antemortem group, fluorescence could be shown in the fluorescence system, and there was a certain linear relationship between the fluorescence intensity and the extension of the injury time. In the postmortem group, no fluorescence appeared at the injured wound, regardless of the injury time.

[0051] It is proved that the diagnostic reagent provided by the present invention has a significant discrimination degree for differentiating antemortem injuries from postmortem injuries.

[0052] As can be seen from the above examples, the pentapeptide complex fluorescent probe provided by the present invention has the advantages of high specificity for fibrin-fibronectin complex, rapid and convenient detection, wide application range, etc., and can be effectively applied to the rapid differentiation of antemortem injuries from postmortem injuries.

[0053] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A pentapeptide complex fluorescent probe, characterized in that: The invention comprises a targeting pentapeptide CREKA and a dicyanomethylene-4H-pyran fluorescent labeling compound; the targeting pentapeptide CREKA and the dicyanomethylene-4H-pyran fluorescent labeling compound are covalently connected.

2. The pentapeptide complex fluorescent probe according to claim 1, characterized in that: The structural formula is shown in Formula I 3. A diagnostic reagent for distinguishing antemortem injuries from postmortem injuries, characterized in that: It includes the pentapeptide complex fluorescent probe according to claim 1 or 2.

4. The diagnostic reagent according to claim 3, characterized in that The diagnostic reagent is a liquid preparation, and the usage concentration of the diagnostic reagent is 0.8-1.2 mg / ml.

5. Use of the pentapeptide complex fluorescent probe according to claim 1 or 2, or the diagnostic reagent according to claim 3 or 4 in distinguishing antemortem injuries from postmortem injuries.

6. The use according to claim 5, characterized in that: The following steps are involved: 1) applying the diagnostic reagent of claim 5 to the wound surface of a cadaver; 2) Use near-infrared imaging to perform fluorescence detection on the wounds on the corpse. If there is fluorescence, it is determined to be an injury before death; if there is no fluorescence, it is determined to be an injury after death.

7. The use according to claim 6, characterized in that: The application method of step 1) is spraying or coating.

8. The use according to claim 6 or 7, characterized in that: Step 2) The excitation wavelength of the fluorescence detection is 520-560 nm, and the emission wavelength is 690-704 nm.

Images