Neurotensin cyclic peptide, its preparation method and application
By constructing a neurostatin cyclic peptide and using disulfide bonds to form a cyclic peptide structure, the problem of easy degradation of the peptide was solved, the stability and activity were improved, and the research potential in the treatment of obesity and eating disorders was enhanced.
Patent Information
- Application Number
- CN202411355241.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2044-09-27
AI Technical Summary
Existing peptide drugs are easily degraded by proteases, resulting in poor stability and difficulty in effectively playing physiological functions such as treating obesity and eating disorders.
By constructing a neurostatin cyclic peptide and using disulfide bonds to form a cyclic peptide structure, the amino and carboxyl groups at both ends of the polypeptide are prevented from being degraded, thereby improving stability and activity.
The stability and activity of neurostatin cyclic peptides were improved, enhancing their research potential in treating obesity and eating disorders.
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Figure CN119192295B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of bioengineering, in particular to a neuronostatin cyclic peptide and a preparation method and application thereof. BACKGROUND
[0002] Neuronostatin (NST) is an endogenous neuropeptide with certain physiological functions, which is determined by Samson et al. through a new bioinformatics method, searching for cleavage sites in the precursor of the known polypeptide Somatostatin (SST), and then screening the evolutionary conserved sequences of more than 200 different potential peptides, and finally verified by experiments.
[0003] It is proved that NST and SST are co-expressed in various types of tissue cells, including hypothalamic neurons, pancreatic δ cells, and oxyntic mucosal wall cells. However, although NST and SST are co-expressed in various types of tissue cells and are encoded by the same gene, their contents in each tissue show significant differences, with the highest content of NST in the spleen, followed by the pancreas, the brain, and the hypothalamus, and SST is most abundant in the hypothalamus. Therefore, it can be inferred that there are different processing processes in different tissues. Given their co-expression in multiple different tissues and significant expression differences, it is suggested that NST may also play an important and different function from SST, like SST.
[0004] However, NST cannot act on the known five SST receptors, so NST may play some other important functions in brain neural functions, and is different from SST. Preliminary studies have shown this: in cultured rat hypothalamic neurons, SST can reduce calcium influx, while NST can increase intracellular calcium concentration. And NST induces the expression of early response genes c-Jun and c-Fos in many different tissues, and is proved to be involved in the discharge of hypothalamic neurons in vitro, regulate neural migration, and increase mean arterial pressure and inhibit light-induced feeding and drinking in rats after intracerebroventricular administration. And feeding and drinking are related to the expression of satiety molecules Nefatin-1 and NST.
[0005] The pancreas plays a very important role in the regulation of blood glucose. In the pancreas, NST can be produced in delta cells, which in turn stimulates the production and release of glucagon, especially under low glucose conditions. In addition, intraperitoneal injection of NST in male mice can cause accumulation of c-Jun in islets, which is consistent with the activation of alpha cells. In cultured pancreatic alpha cells, impaired expression of GPR107 leads to the loss of the ability of NST to increase proglucagon mRNA levels, and PKA phosphorylation is also blocked. Therefore, in isolated rat islets, NST must promote the release of glucagon and the expression of glucagon mRNA through GPR107, and endocrine interaction between pancreatic alpha and beta cells to regulate glucose homeostasis. Consistent with this, in the alpha TC1-9 cell line of isolated rat pancreas, high-dose neurotensin (4000 nmol / kg) can promote low glucose-induced glucagon release and significantly increase glucagon mRNA levels. Further, NST was shown to significantly increase proglucagon mRNA levels in cultured alpha cells, and in a low glucose environment, it can promote glucagon release in a concentration-dependent manner. In addition, NST administration can delay glucose clearance in rat models and significantly inhibit insulin response in vivo. Therefore, in blood glucose regulation, NST at least one role is to limit insulin secretion, promote glucagon secretion and maintain blood glucose levels.
[0006] Commonly used anxiety assessment tests include forced swimming test and elevated plus maze test, etc. In the mouse forced swimming test, intracerebroventricular injection of NST (4-40 nmol / kg) leads to an increase in the resting time of mice, and central melanocortin and GABA type A receptor (GABAA) produce a depressive effect. In rats, intracerebroventricular injection of NST (24 nmol / kg) produces anxiolytic effects.
[0007] In addition to the physiological functions described above, NST plays an important role in appetite, pain, neuroendocrine, etc. It is even a promising new therapeutic target for prostate cancer.
[0008] As a new drug carrier and therapeutic molecule with its unique advantages, polypeptides have been widely used in tumor imaging, immunotherapy and cancer treatment in recent years. However, polypeptides are easily degraded by proteases, which can recognize the amino and carboxyl groups at both ends of the polypeptide backbone, so polypeptides can easily lose their function. However, compared with linear peptides, cyclic peptides have higher stability. Because the formation of cyclic peptides through disulfide bonds can eliminate the amino or carboxyl groups at both ends of the polypeptide, it can avoid degradation and thus prolong the half-life of the polypeptide. SUMMARY
[0009] In order to solve the above technical problems, the present application provides a neurotensin ring peptide and a preparation method and application thereof, the method constructs a more stable and more active NST ring peptide, which not only improves the convenience of subsequent NST related research, but also can be used as a research basis for developing new potential drugs for treating obesity and eating disorders (such as binge eating).
[0010] To this end, the present application provides the following technical solutions,
[0011] In a first aspect, the present application provides, in optional embodiments, a preparation method of a neurotensin ring peptide, comprising the following steps:
[0012] S1: adding a dichloromethane solution containing Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine into the resin to perform a condensation reaction, removing the Fmoc protecting group after the reaction is completed, and obtaining a peptide chain;
[0013] S2: after washing the peptide chain, adding a mixed solution of trifluoroethanol and dichloromethane to shake and filter, and obtaining a linear peptide crude product;
[0014] S3: adding a dichloromethane solution containing hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and diisopropylethylamine into the linear peptide crude product to perform a cyclization reaction, vacuum concentration and purification, and obtaining a cyclization peptide crude product;
[0015] S4: after C-terminal amidation and side chain deprotection of the cyclization peptide crude product, separating and purifying to obtain the neurotensin ring peptide.
[0016] Further, in step S4, the method of C-terminal amidation is that the cyclization peptide crude product is dissolved in N,N-dimethylformamide, hydroxybenzotriazole and diisopropylcarbodiimide, incubated at room temperature for 30 min, then the cyclization peptide crude product is activated, and then an N,N-dimethylformamide solution containing aminobutyric acid is added to react for 4 hours.
[0017] Further, in step S4, the method of side chain deprotection is that the crude product after C-terminal amidation is added to a moderate solution of trifluoroacetic acid and dichloromethane to react for 4 hours.
[0018] Preferably, the molar ratio of the trifluoroacetic acid and dichloromethane is 1:3.
[0019] Further, in step S4, the method of separation and purification is that the crude product after side chain deprotection is dissolved in a 40% acetonitrile aqueous solution, and then HPLC purification is performed.
[0020] Preferably, the HPLC purified mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution.
[0021] Preferably, in step S1, the Fmoc-amino acid comprises one or more of Fmoc-S-trityl-L-cysteine, Fmoc-L-alanine, Fmoc-L-leucine, Fmoc-O-t-butyl-L-serine, Fmoc-N-trityl-L-glutamine, Fmoc-L-phenylalanine, N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine and Fmoc-L-proline; and / or, the molar ratio of the Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine is 1:1:2.
[0022] Preferably, in step S2, the molar ratio of the trifluoroethanol and dichloromethane is 1:4; and / or, in step S3, the molar ratio of the hydroxybenzotriazole, benzotriazole-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphate and diisopropylethylamine is 1:1:2.
[0023] In a second aspect, the present application provides, in optional embodiments, a neurostatin cyclic peptide prepared by the above preparation method.
[0024] In a third aspect, the present application provides, in optional embodiments, an application of the neurostatin cyclic peptide prepared by the above preparation method or the above neurostatin cyclic peptide in preparing a drug for treating obesity and eating disorders (such as binge eating disorder).
[0025] Compared with the prior art, the present application has one or more of the following beneficial effects:
[0026] The present method constructs a more stable and more active NST cyclic peptide, which not only improves the convenience of subsequent NST related research, but also can be used as a research basis for developing a new potential drug for treating obesity and eating disorders (such as binge eating disorder). BRIEF DESCRIPTION OF DRAWINGS
[0027] Figure 1 The synthesis route flow chart of Example 1 of the present application is shown in the following figure:
[0028] Figure 2 The HPLC chromatogram in Example 1 of the present application is shown in the following figure:
[0029] Figure 3The structural formula of the neurotensin cyclic peptide in the embodiment 1 of the present application, wherein A indicates the site of adding cysteine at both ends of the linear NST, and the cyclic peptide is still kept the C-terminal amidation because the C-terminal amidation is very important for the function of the linear NST peptide, and the box indicated by B marks the still kept C-terminal amidation.
[0030] Figure 4 The HPLC chromatogram of the neurotensin cyclic peptide in the embodiment 2 of the present application.
[0031] Figure 5 The effects of the neurotensin and the neurotensin cyclic peptide on the blood glucose of zebrafish in the embodiment 2 of the present application, wherein * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.
[0032] Figure 6 The effects of the neurotensin and the neurotensin cyclic peptide on the blood glucose of zebrafish in the embodiment 2 of the present application, wherein * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.
[0033] Figure 7 The effects of the neurotensin and the neurotensin cyclic peptide on the open field behavior of zebrafish in the embodiment 2 of the present application, wherein * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.
[0034] Figure 8 The thermal imaging diagram of the effects of the neurotensin and the neurotensin cyclic peptide on the open field behavior of zebrafish in the embodiment 2 of the present application.
[0035] Figure 9 The effects of the neurotensin and the neurotensin cyclic peptide on the behavior of zebrafish in the light-dark box in the embodiment 2 of the present application, wherein * represents P<0.05, ** represents P<0.01, *** represents P<0.001, # represents P<0.05, ## represents P<0.01, and ### represents P<0.001. DETAILED DESCRIPTION
[0036] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.
[0037] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0038] The materials and reagents used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0039] In the following examples, the Fmoc-amino acid is Fmoc-S-trityl-L-cysteine (Fmoc-Cys(Trt)-OH), Fmoc-L-alanine (Fmoc-Ala-OH), Fmoc-L-leucine (Fmoc-Leu-OH), Fmoc-O-tert-butyl-L-serine (Fmoc-Ser(tBu)-OH), Fmoc-N-trityl-L-glutamine (Fmoc-Gln(Trt)-OH), Fmoc-L-phenylalanine (Fmoc-Phe-OH), N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH), and Fmoc-L-proline (Fmoc-Pro-OH).
[0040] Example 1
[0041] This example provides a preparation method of a neurotensin cyclic peptide, and the specific flow is shown in Figure 1 , which comprises the following steps:
[0042] S1: (1) A certain amount of resin is weighed and placed in a synthesis reaction tube, DCM is used to soak the resin for 30 minutes with slow stirring, and finally the resin is dried.
[0043] (2) An appropriate amount of Fmoc-Cys(Trt)-OH and DIPEA in DCM solution is added, and the reaction is oscillated at room temperature overnight.
[0044] (3) Each of DCM (10 ml) and DMF (10 ml) is washed for 3 times, 3 minutes each time.
[0045] (4) An appropriate amount of methanol is added to react for 20 minutes to block the unreacted resin.
[0046] (5) Each of DCM (10 ml) and DMF (10 ml) is washed for 3 times, 3 minutes each time.
[0047] (6) Deprotection: 20% piperidine in DMF solution (10 ml) is added, oscillated at room temperature for 10 minutes, and repeated twice to remove the Fmoc protecting group on the amino group of the amino acid.
[0048] (7) Each of DCM (10 ml) and DMF (10 ml) is washed for 3 times, 3 minutes each time.
[0049] (8) Condensation: according to the polypeptide sequence, Fmoc-amino acid, HCTU and DIPEA are sequentially dissolved in an appropriate amount of NMP in a molar ratio of 1:1:2, and added to the reaction tube, and oscillated at 35°C for 1 hour.
[0050] (9) Take a small amount of resin, add ninhydrin reagent and heat at 110°C for 5 min. If the resin is colorless, the condensation reaction is complete. The next step can be continued, otherwise continue the condensation. This step is to ensure the formation of the correct peptide chain.
[0051] (10) Repeat steps (6)-(9) until all amino acids are connected. Repeat step (6) to remove the Fmoc protecting group.
[0052] S2: (1) After the synthesis of the peptide chain, wash thoroughly with anhydrous ether and dry, add an appropriate amount of TFE-DCM (molar ratio of 1:4) solution, shake at room temperature overnight.
[0053] (2) Filter, collect the filtrate and concentrate under reduced pressure to obtain the linear peptide crude product. HPLC analysis is performed.
[0054] S3: (1) Dissolve HOBt (5 molar amounts), PyBOP (5 molar amounts) and DIPEA (10 molar amounts) in an appropriate amount of DCM, slowly drop into the DCM solution of the linear peptide.
[0055] (2) After the dropwise addition is completed, warm to room temperature under natural conditions, and slowly stir the reaction for 1 h.
[0056] (3) Concentrate it under reduced pressure, then purify by gel column and concentrate under reduced pressure.
[0057] S4: (1) After the last Cys is added and deprotected, perform acylation reaction. First, add an appropriate amount of DMF, then add an appropriate amount of HOBt and DIC, stir to dissolve completely. Incubate at room temperature for 30 min to fully activate the polypeptide.
[0058] (2) Add an appropriate amount of amino butyric acid DMF solution to the reaction tube and stir uniformly at room temperature for 4 h of stirring reaction or overnight at 4°C. Concentrate under reduced pressure.
[0059] (3) Add the crude cyclic peptide to a TFA-DCM (1:3) solution, shake at room temperature for 4 h to remove the side chain protecting group.
[0060] (4) Dissolve the obtained crude C-terminal amide cyclized peptide in 40% acetonitrile aqueous solution and purify by preparative RP HPLC. The analytical column type is SHIMADZU Inertsil ODS-SP (4.6*250mm*5um), the mobile phase A is 0.1% TFA aqueous solution, B is 0.1% TFA acetonitrile solution, the detection wavelength is 220 nm, and the flow rate is 1 ml / min. Elution mode: gradient elution, the initial concentration of B phase is 15%. The gradient elution program is shown in Table 1, and the HPLC chromatogram is shown in Figure 2 . The separation and elution results of NST cyclic peptides are shown in Figure 2The target peak of NST cyclic peptide appeared at 11.632 min and the peak area reached 98.30%, while the non-NST cyclic peptide only accounted for 1.70%. The final freeze-drying obtained a white powder, which was the pure product, i.e. the neurestin cyclic peptide (NST cyclic peptide).
[0061] The structural formula of the neurestin cyclic peptide is shown in Figure 3 , and the position of A indicates the addition of cysteine at both ends of the linear NST. Since the C-terminal amidation is very important for the function of the linear NST peptide, the constructed cyclic peptide still retains the C-terminal amidation. The box marked by B indicates the still retained C-terminal amidation.
[0062] Table 1 Gradient elution program
[0063] Time Module Action Value 0 Pumps B.Conc 15 25 Pumps B.Conc 75 25.01 Pumps B.Conc 100 30 Pumps B.Conc 100 30.01 Pumps Stop
[0064] Table 2 Gradient elution results
[0065] RT Area %Area Height 1 11.5 7307 0.10 6378 2 11.632 7037157 98.30 819266 3 12.036 114725 1.6 11161
[0066] Example 2
[0067] Zebrafish intraperitoneal injection
[0068] Preparation of anesthetic platform: a thin and soft sponge block with a size of about 5 cm in length and width is slotted in the center, which can accommodate an adult zebrafish, and is soaked in an anesthetic box for standby. Hypothermia anesthesia: place the zebrafish to be injected into the anesthetic box with water temperature of 26-28℃, put in 1 adult zebrafish to be injected, and add a small amount of crushed ice to gradually reduce the water temperature. At this time, the zebrafish shows reduced movement, accompanied by continuous temperature drop, the zebrafish gill cover breathing gradually weakens, the swimming weakens, and when the water temperature drops to about 12℃, the movement completely stops and the gill cover movement slows down until it stops, and finally the zebrafish loses balance and floats out of the water.
[0069] The prepared slotted sponge is placed in the anesthetic box in advance, and after the zebrafish floats out of the water and no longer swims, the zebrafish is placed in the sponge slot with the back down, then the microsyringe is quickly and stably inserted into the midline between the pelvic fins of the zebrafish, and the depth of insertion is ensured to inject the polypeptide into the abdominal cavity of the zebrafish. After injection, the fish is quickly taken out of the sponge slot and placed back into the independent warm water (26-28℃), and the movement ability is restored within a few seconds. The fish in the same batch is ensured to be quickly injected within a short time to avoid affecting the normal activity of the fish body due to long-term hypothermia anesthesia. When the second batch of intraperitoneal injection is performed, the water in the anesthetic box is replaced with warm water, and the cooling is re-performed.
[0070] Zebrafish blood glucose test
[0071] The zebrafish under the same feeding conditions were weighed, and the zebrafish with similar body weight were selected and placed alone and fasted for 72 h, so that the blood glucose of all zebrafish was reduced to the baseline level, and the initial blood glucose of the zebrafish was as consistent as possible.
[0072] The experiment was divided into 3 groups, namely the control group injected with PBS, the NST injection group, and the NST cyclic peptide injection group, 10 fish in each group.
[0073] During the experiment, after low-temperature anesthesia, PBS, NST (10 mM, 4 uL) or NST cyclic peptide (10 mM, 4 uL) was injected intraperitoneally, respectively, and the tail artery was cut off at 15 min, 30 min, 1 h, 2 h, 3 h and 4 h after injection, so that the blood flowed out, and then the SANNUO three-nuo blood glucose meter and Sinocare three-nuo blood glucose test strip were used to determine the blood glucose.
[0074] Results: Zebrafish intraperitoneally injected with different concentrations of NST cyclic peptide (0.1 mM, 1 mM, 10 mM, 100 mM / 4 uL) showed a dose-dependent blood glucose increasing effect in the blood glucose determination experiment, and there was a significant difference compared with the control group (PBS injection group). Intraperitoneal injection of 10 mM, 4 uL of NST cyclic peptide showed the maximum blood glucose increasing effect at 15 min after injection, while lower and higher doses could not show better blood glucose increasing effect (see Figure 4 ).
[0075] Intraperitoneal injection of 10 mM, 4 uL of NST cyclic peptide had a longer sustained effect compared with injection of NST group, and there was a significant difference at 2 h and 3 h after injection (see Figure 5 ).
[0076] Zebrafish feeding experiment
[0077] The experimental setup includes six round fish tanks, each loaded with 500 ml of distilled water to establish a uniform experimental baseline. Zebrafish are evenly grouped, with 15 fish per group, forming two main groups: control group, NST injection group, and NST cyclic peptide injection group, with three repeated experiments within each group, each with 5 fish. An equal amount of freshly incubated Daphnia magna is introduced into each fish tank. The initial number of Daphnia magna is determined as follows: First, thoroughly mix the water in the fish tank to evenly distribute the Daphnia magna, then at five randomly selected locations, use a pipette to draw 1 ml of water sample, for a total of 5 ml. By counting the number of Daphnia magna in these 5 ml of water sample, the initial total number of Daphnia magna in the entire fish tank is calculated, and this step is plotted as the initial total amount graph. During the experiment, at 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours, the above water sampling and counting process is repeated to determine the remaining number of Daphnia magna. By subtracting the remaining amount at each time point from the initial total amount, the actual feeding amount of zebrafish within each time interval can be accurately determined.
[0078] Results: After intraperitoneal injection of 10 mM, 4 μL of NST cyclic peptide in zebrafish, the experimental results show that NST cyclic peptide has the same inhibitory feeding effect as NST, with significant differences from the control group at 1 h and 2 h after injection, and significant differences from the NST injection group at 1 h after injection (see results in Figure 6 ).
[0079] Behavioral measurement of zebrafish to observe the effect of NST cyclic peptide on cognitive behavior
[0080] (1) Open field
[0081] S1: Grouping: Zebrafish are grouped, with 15 fish per group, divided into experimental group 1 (injected with NST), experimental group 2 (injected with NST cyclic peptide), and control group (injected with PBS).
[0082] S2: Drug administration: Zebrafish are adapted to standard conditions for a period of time to ensure they are in a stable state. Zebrafish in experimental group 1 are injected intraperitoneally with 4 μL of NST solution, zebrafish in experimental group 2 are injected intraperitoneally with 4 μL of NST cyclic peptide solution, and the control group is injected with an equal amount of PBS (phosphate buffered solution) as an experimental control.
[0083] S3: Open field experiment: Ensure that the open field experiment device is located in a quiet, evenly lit environment. After adjusting the equipment, the treated zebrafish were individually placed in the center of the open field experiment device from the breeding container, given a short adaptation period, and then the behavior was recorded for 10 minutes. During the experiment observation, the zebrafish treated with PBS injection in the control group were observed first, and then the zebrafish in the experimental group were observed. A high-definition camera was used to record the swimming trajectory of the zebrafish in the tank from the top throughout the experiment. The entire process was recorded to ensure that all behavioral details could be traced back. Then, the behavior was analyzed by the dedicated video analysis software EthoVision.
[0084] Results: By studying the effects of Neuronostatin on the spontaneous behavior, exploratory behavior and tension of zebrafish in a novel environment, the open field experiment results showed that NST and NST cyclic peptides had inhibitory effects on the behavior of zebrafish. The results are shown in Figure 7 and Figure 8 , which shows the relationship between the injection of NST and NST cyclic peptides and the swimming speed and distance of zebrafish.
[0085] (2) Light-dark box
[0086] S1: Grouping: The zebrafish were divided into groups, with 15 zebrafish per group, and divided into experimental group 1 (injected with NST), experimental group 2 (injected with NST cyclic peptide), and control group (injected with PBS).
[0087] S2: Drug treatment: The zebrafish were adapted to standard conditions for a period of time to ensure that they were in a stable state. The zebrafish in experimental group 1 were injected with 4 μL of NST solution, the zebrafish in experimental group 2 were injected with 4 μL of NST cyclic peptide solution, and the control group was injected with an equal amount of PBS (phosphate buffered solution) as an experimental control.
[0088] S3: Light-dark box experiment: The experiment was conducted in a quiet, well-lit environment to ensure that the light outside the light-dark box was stable and would not cause additional stimulation to the zebrafish. The zebrafish were gently removed from the breeding container and placed in the dark box using a fishing net. The experiment was conducted in a quiet, independent environment by quickly closing the door curtain to reduce external interference. The zebrafish were individually placed in the light-dark box device, given a short adaptation period, and then the video recording equipment was started to record the behavior for 10 minutes, including the time spent in the light and dark areas and the number of crossings. During the experiment observation, the zebrafish treated with PBS injection in the control group were observed first, and then the zebrafish in the experimental group were observed. A high-definition camera was used to record the swimming trajectory of the zebrafish in the light-dark box from the top throughout the experiment. The behavior of each zebrafish in the light-dark box for 10 minutes was recorded to ensure that all behavioral details could be traced back. Then, the behavior was analyzed by the dedicated video analysis software EthoVision.
[0089] Results: By studying the effects of Neuronostatin and Neuronostatin cyclic peptide on zebrafish in the light-dark box experiment, the anxiety level and photophobic behavior of zebrafish can be analyzed, and the results of the light-dark box experiment are shown in Figure 9 , Figure 9 It is shown that there is no significant difference between the cyclic peptide injection and the PBS injection, and the injection of nst will cause anxiety.
[0090] Although the principles of the present application have been described in detail with reference to the preferred embodiments thereof, it is to be understood that the above-described embodiments are merely illustrative of the present application and are not intended to limit the scope of the present application. The details in the embodiments do not constitute a limitation on the scope of the present application. Any equivalent changes, simple replacements, etc. based on the technical solutions of the present application, without departing from the spirit and scope of the present application, fall within the protection scope of the present application.
Claims
1. A process for the preparation of a neurotensin cyclic peptide, characterized in that, Comprising the following steps: S1: adding a dichloromethane solution containing Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine into the resin, performing condensation reaction, removing the Fmoc protecting group after the reaction is completed, and obtaining a peptide chain; S2: after washing the peptide chain, adding a mixed solution of trifluoroethanol and dichloromethane, shaking and filtering to obtain a linear peptide crude product; S3: adding a dichloromethane solution containing hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and diisopropylethylamine into the linear peptide crude product, performing cyclization reaction, vacuum concentration and purification to obtain a cyclized peptide crude product; S4: after C-terminal amidation and side chain deprotection of the cyclized peptide crude product, separating and purifying to obtain the neprilysin cyclic peptide; The method for C-terminal amidation is: adding N,N-dimethylformamide, hydroxybenzotriazole and diisopropylcarbodiimide into the cyclized peptide crude product, incubating at room temperature for 30 min, activating the cyclized peptide crude product, then adding an N,N-dimethylformamide solution containing aminobutyric acid, and reacting for 4 hours; The Fmoc-amino acid is Fmoc-S-trityl-L-cysteine, Fmoc-L-alanine, Fmoc-L-leucine, Fmoc-O-tert-butyl-L-serine, Fmoc-N-trityl-L-glutamine, Fmoc-L-phenylalanine, N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine and Fmoc-L-proline; The structural formula of the neprilysin cyclic peptide is shown in Formula 1: Formula 1.
2. The process for the preparation of a neurotensin cyclic peptide according to claim 1, characterized in that, In step S4, the method for side chain deprotection is: adding the crude product after C-terminal amidation into a moderate solution of trifluoroacetic acid and dichloromethane, and reacting for 4 hours.
3. The process for the preparation of a neurotensin cyclic peptide according to claim 2, characterized in that, The molar ratio of the trifluoroacetic acid and dichloromethane is 1:
3.
4. The process for the preparation of a neurotensin cyclic peptide according to claim 1, characterized in that, In step S4, the method for separation and purification is: dissolving the crude product after side chain deprotection in a 40% acetonitrile aqueous solution, and then performing HPLC purification.
5. The process for the preparation of a neurotensin cyclic peptide according to claim 4, characterized in that, The mobile phase A for HPLC purification is a 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution.
6. The process for the preparation of a neurotensin cyclic peptide according to claim 1, characterized in that, In step S1, the molar ratio of the Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine is 1:1:
2.
7. The process for the preparation of a neurotensin cyclic peptide according to claim 1, characterized in that, In step S2, the molar ratio of the trifluoroethanol and dichloromethane is 1:4; and / or, In step S3, the molar ratio of the hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and diisopropylethylamine is 1:1:
2.
8. A neurotensin cyclic peptide, characterized in that, Prepared by the preparation method of any one of claims 1-7.
9. Use of the neprilysin cyclic peptide prepared by the preparation method of any one of claims 1-7 or the neprilysin cyclic peptide of claim 8 in the preparation of a medicament for treating obesity.