Targeted polypeptide probe based on arginine group selective fluorine-18 labeling as well as preparation method and application of targeted polypeptide probe

Abstract

The invention discloses a targeted polypeptide probe based on arginine group selective fluorine-18 labeling and a preparation method and application thereof, and belongs to the field of nuclear medicine imaging and tumor diagnosis. According to the invention, a new arginine group is introduced into a targeting polypeptide, and guanidyl exposed on the basis of arginine in a peptide chain is combined with a small-molecule radioactive fluorine-18 marker, so that the fluorine-18-labeled targeting polypeptide probe as well as the preparation method and the application of the fluorine-18-labeled targeting polypeptide probe are prepared, and the preparation of a nuclide polypeptide conjugate is realized. The nuclide polypeptide conjugate has the advantages of mild preparation method, accurate quantification and strong visualization, retains the physicochemical properties and in-vivo metabolic behavior of the targeting polypeptide to a greater extent, overcomes the problems of high temperature, high metabolic rate and the like in the existing marking method, provides accurate reference information for early diagnosis and treatment monitoring of various tumors, and has a good application prospect. The clinical transformation and popularization are easy.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine imaging, and in particular relates to a targeted polypeptide probe based on selective fluorine-18 labeling of an arginine group, and a preparation method and application thereof. Background Art

[0002] Positron emission tomography (PET) technology, as an important means of modern medical imaging, plays a key role in disease diagnosis and therapeutic efficacy evaluation due to its advantages of non-invasiveness and molecular-level imaging. By tracking the distribution of radionuclide probes in the body, this technology can reflect physiological functions and pathological changes in real time. However, the technical bottleneck of probe development has restricted the further development of PET technology, making functional peptide molecules with high specificity, good pharmacokinetic properties and biosafety a research hotspot. Peptide-conjugated radionuclide drugs provide new possibilities for precise imaging and treatment of tumors by specifically recognizing tumor cell surface receptors.

[0003] In the development of PET-peptide conjugates, targeted linear peptides are linear peptide molecules that are obtained through rational design or screening and are capable of specifically binding to specific targets (such as proteins, nucleic acids, cell surface receptors, etc.). These peptides are usually composed of natural or unnatural amino acids and have clear targeting and functionality. They are widely used in drug development, molecular probes, diagnostic tools, and other fields, especially in the field of radionuclide applications. Their small molecular weight, simple structure, and ease of chemical modification give them excellent tissue penetration and rapid in vivo clearance. By specifically recognizing disease-related receptors or biomarkers, they can achieve precise targeted delivery and significantly improve the enrichment efficiency of radionuclides in target tissues. Their good biocompatibility and low immunogenicity make them highly safe for clinical application. At the same time, the mature and cost-effective synthesis process of linear peptides facilitates large-scale preparation, providing an ideal molecular carrier for integrated radionuclide diagnosis and treatment. These characteristics give targeted linear peptides broad application prospects in tumor diagnosis (such as PET imaging) and targeted radiotherapy.

[0004] 18 F is the most widely used radionuclide for peptide labeling in PET imaging. It has ideal physicochemical properties: a high positron yield of 97%, a short positron range of only 0.5 mm in water, and a moderate half-life (t 1 / 2 =110min). It is built with carbon atoms as the skeleton. 18The F labeling process is widely used due to its engineering advantages such as single-step operation, rapid reaction (usually completed within half an hour), simple process and easy standardized mass production. However, this process has extremely stringent requirements on the reaction environment. Slight changes in pH, temperature and solvent system may affect the labeling effect, which makes it difficult to be compatible with polypeptide molecules containing unstable groups or complex secondary structures. In contrast, fluoride ions can form strong coordination bonds with many metal cations, and their interaction with Al (670kJ / mol) is stronger than that of most metals. Under aqueous conditions, metal fluorides can coordinate with chelating groups, so it is expected to be used [ 18 F]AlF 2+ Chelating with peptides modified with chelating groups to achieve one-step peptide 18 However, this labeling method requires high temperature labeling, which may lead to the possibility of peptide fragmentation, reducing its radiochemical purity and causing the peptide to circulate in the body. 18 The risk of F isotope dissociation, and the strong water solubility of chelating groups such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), can make peptides more hydrophilic, leading to disadvantages such as rapid metabolism and poor retention in the body, which in turn reduces the targeting of the peptide itself. Therefore, this labeling method has certain limitations for the preparation of new fluorine-18 targeted probes. Therefore, the development and optimization of stable fluorine-18 labeling of targeted peptides under mild labeling conditions and the adjustment of the physical properties of the peptide chain are urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a targeted polypeptide probe based on selective fluorine-18 labeling of arginine groups, as well as a preparation method and application thereof.

[0006] The present invention provides an additional option for the preparation of fluorine-18 labeled targeted polypeptide probes. The present invention is based on the combination of the exposed guanidine group of arginine in the peptide chain with a small molecule radioactive fluorine-18 label to prepare a fluorine-18 labeled targeted polypeptide probe and its preparation method and application. The preparation conditions of this radionuclide polypeptide conjugate are extremely mild, and the entire process is close to physiological conditions, which avoids the damage to the structure and function of the protein or polypeptide caused by the harsh environment to the greatest extent, perfectly retains the natural physiological activity of the straight-chain polypeptide, and lays a solid foundation for subsequent biological experiments and medical applications. In addition, the present invention is universally applicable to the modification of various types of polypeptides based on the fact that arginine is common in polypeptides and is easy to combine with known specific targeting peptide chains. Subsequent [ 18The F]FPG small molecule radioactive label undergoes a mild biological click reaction with arginine, thereby cleverly introducing an imidazole group. The introduction of this key structural unit may change the molecule's physical and chemical properties such as lipophilicity, thereby affecting its ability to penetrate biological membranes and the efficiency of its cell uptake. For example, the introduction of groups such as imidazole and imidazole cations into naphthalimide derivatives can increase the lipophilicity of the compound, which is beneficial for improving the cell's absorption of these compounds, thereby indirectly increasing the cell binding rate and making its metabolic pathway more prone to liver and intestinal metabolism, thereby improving its retention in the body. In addition, the five-membered aromatic ring structure of imidazole contains two nitrogen atoms (1 and 3), of which the 3-position nitrogen has a lone pair of electrons and can react with metal ions (such as heme iron Fe 3+ ) coordination, and tumor cells grow rapidly and have active metabolism, which may require more Fe 3+ To support its rapid proliferation, the successful introduction of imidazole groups is beneficial to the retention of polypeptide probes in tumor tissues, and combined with the targeting property of the polypeptide itself, the retention and accumulation of targeted polypeptide probes prepared by this method in tumor sites in the body are greatly improved.

[0007] The optimized polypeptide molecules of the present invention can effectively recognize and bind to receptors or antigens specific to the surface of tumor cells, can highlight and clearly outline the contours of the tumor, and strictly distinguish tumor tissue from surrounding normal tissue. The retention time of the targeted polypeptide probe provided by the present invention at the tumor site is significantly prolonged. In addition, the anchoring effect of the targeted polypeptide probe is very good. It can be firmly bound to tumor cells and will not easily fall off or be metabolized and decomposed even in a complex in vivo environment. This good anchoring performance ensures that the probe can function stably at the tumor site, and is expected to show a higher signal-to-noise ratio and better imaging quality in molecular imaging, providing a more powerful tool for early diagnosis and treatment monitoring of the disease.

[0008] The technical solution adopted by the present invention comprises the following steps:

[0009] The present invention first provides a method for preparing a targeting polypeptide probe based on selective fluorine-18 labeling of an arginine group, which comprises the following steps:

[0010] 1) Introducing an arginine group into the targeted linear peptide to obtain a targeted linear peptide containing arginine;

[0011] 2) 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate dissolved in DMSO solvent was mixed with [ 18 F]KF / Kryptofix[2,2,2] for reaction;

[0012] 3) Adding DMSO containing iodine to the product obtained in step 2) and heating the mixture for reaction, the reaction product was subjected to solid phase extraction and purification to obtain [18 F]FPG;

[0013] 4) The targeted linear peptide with arginine from step 1) was dissolved and the mixture containing [ 18 The DMSO solution of F]FPG was mixed with nitrogen, and the temperature was raised to 35-40°C for reaction. The reaction product was purified to obtain the targeting polypeptide probe.

[0014] According to a preferred embodiment of the present invention, the targeted linear peptide containing arginine in step 1) can be obtained by Fmoc solid-phase synthesis, and the targeted linear peptide containing arginine is one or more of KTLLPTPR (shown in SEQ ID No. 1), LTVSPWYR (shown in SEQ ID No. 2), and CPKSNNGVCR (shown in SEQ ID No. 3), and R in the polypeptide sequence is the arginine group introduced by the present invention.

[0015] According to a preferred embodiment of the present invention, in step 2), the 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate and [ 18 The mass ratio of KF / Kryptofix[2,2,2] is 2-3:1, 18 The radioactivity of [F]KF / Kryptofix[2,2,2] is 1-2ci; the reaction conditions are 90-120℃ for 10-30min.

[0016] According to a preferred embodiment of the present invention, the mass ratio of iodine to 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate is 1-5:1; and the heating reaction condition in step 2) is 130° C. for 10 min.

[0017] According to a preferred embodiment of the present invention, the reaction product in step 3) is subjected to solid phase extraction, comprising: diluting the reaction product to 10-30 ml with pure water and then subjecting it to Oasis TM Solid phase extraction was performed using a PLUS Short HLB cartridge. The HLB cartridge was eluted with 1-2 ml of DMSO to obtain the crude product, which was then diluted with pure water and purified using a preparative column.

[0018] According to a preferred embodiment of the present invention, the purification in step 3) is performed by high performance liquid chromatography separation and purification, specifically: acetonitrile is used as mobile phase A, water containing 0.1% H3PO4 is used as mobile phase B, and gradient elution is performed, wherein the volume ratio of mobile phase A is linearly increased from 30% to 100%, and the mobile phase ratio is 30-100% of phase A; the product is collected and solid phase extraction is performed with HLB cartridge, and eluted with DMSO to obtain [ 18 F]FPG.

[0019] According to a preferred embodiment of the present invention, in step 4), a linear peptide containing arginine is dissolved in HEPES buffer and mixed with [ 18 After mixing the DMSO solution of F]FPG, the pH is controlled at 8-9; the closed reaction temperature range is 35-40°C, and the reaction time is 10-30 minutes; the mass ratio of the targeting polypeptide containing arginine to 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate is 1-3:1.

[0020] According to a preferred embodiment of the present invention, the purification in step 4) comprises collecting the reaction product and isolating and purifying it using high-performance liquid chromatography to obtain a targeted polypeptide probe. Preferably, the purification comprises using acetonitrile as mobile phase A and water containing 0.1% H₃PO₄ as mobile phase B, using gradient elution, wherein the volume proportion of mobile phase A increases linearly from 30% to 100%, with the mobile phase proportion being a gradient of 30-100%. The collected product is subjected to solid-phase extraction using an HLB cartridge and eluted with DMSO to obtain the targeted polypeptide probe.

[0021] The present invention also provides a targeting polypeptide probe prepared by the method.

[0022] The present invention also provides the use of the targeting polypeptide probe in preparing tumor nuclear medicine imaging reagents.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] existing 18 F-labeled tumor nuclear medicine imaging probes need to be labeled at high temperatures and exist when circulating in the body. 18 There is the risk of F isotope dissociation, and the strong water solubility of chelating groups such as NOTA can affect the physicochemical properties of the modified peptide. Furthermore, the relatively simple and linear structure of linear peptides makes them more susceptible to protease recognition and cleavage in vivo, resulting in rapid metabolism. Due to this rapid metabolism, targeted linear peptides have a short retention time in the target tissue. In molecular imaging, ideal targeting molecules must remain in the target tissue for a sufficient period of time for effective imaging. However, the rapid metabolism of targeted linear peptides makes it difficult for them to remain in the target tissue for a long time, thus affecting the quality and accuracy of imaging. The poor anchoring effect and limited receptor selectivity of targeted linear peptides can reduce diagnostic accuracy. In the diagnosis of diseases such as tumors, targeted molecules must precisely bind to receptors in diseased tissues. However, these shortcomings of targeted linear peptides make precise targeting difficult, increasing the risk of misdiagnosis.

[0025] To overcome the above technical bottlenecks, the present invention creatively proposes to introduce a new arginine group into the targeting polypeptide, and to combine the exposed guanidine group of arginine in the peptide chain with a small molecule radioactive fluorine-18 marker to prepare a fluorine-18 labeled targeting polypeptide probe and its preparation method and application, thereby realizing the preparation of nuclide polypeptide conjugates. The present invention optimizes the reaction system parameters to achieve 18 The efficient covalent binding of the F isotope to the targeting peptide increases the diversity of peptide probe labeling methods and enhances its in vitro stability. Furthermore, the optimized peptide molecules significantly prolong their retention time at the tumor site, demonstrating the exceptional anchoring effectiveness of this targeted peptide probe. It firmly binds to the tumor cell surface and resists easy detachment or metabolic degradation, even in the complex in vivo environment. This excellent anchoring performance ensures the probe's stable function at the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below with reference to the accompanying drawings and examples.

[0027] Figure 1 [ 18 [F] Radiolabeling protocol of F-KTLLPTPR.

[0028] Figure 2 The high performance liquid chromatography analysis of Example 1 of the present invention [ 18 F]F-KTLLPTPR.

[0029] Figure 3 [ 18 PET imaging of F]F-KTLLPTPR in KPC pancreatic cancer subcutaneous tumor model 1 h after administration (A), under conventional aluminum fluoride labeling scheme [ 18 F] PET imaging of F-KTLLPTPR in the KPC pancreatic cancer subcutaneous tumor model 1 hour after administration (B).

[0030] Figure 4 [ 18 [F] Radiolabeling protocol for F-LTVSPWYR.

[0031] Figure 5 For Example 2 of the present invention [ 18 Radiolabeled purity of [F]F-LTVSPWYR.

[0032] Figure 6 For Example 2 of the present invention [ 18 [F]F-LTVSPWYR PET imaging in the U-87MG subcutaneous human glioma model.

[0033] Figure 7 [18 [F] Radiolabeling protocol for F-CPKSNNGVCR.

[0034] Figure 8 For Example 3 of the present invention [ 18 Radiolabeled purity of [F]-CPKSNNGVCR.

[0035] Figure 9 For Example 3 of the present invention [ 18 [F]F-CPKSNNGVCR PET imaging in a subcutaneous colorectal cancer model. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are described below, but the embodiments of the present invention are not limited thereto.

[0037] The mouse KrasLSL-G12D p53LSL-R172H Pdx1-Cre (KPC) cell line was kindly provided by the laboratory of the First Affiliated Hospital of Zhejiang University School of Medicine. It was derived from pancreatic cancer in genetically engineered KPC mice and cultured in an incubator at 37°C and 5% CO2. The KPC tumor model was constructed by inoculating KPC cells (5×10 6 100 μL of PBS were suspended and injected subcutaneously into the right upper limb of shaved C57BL / 6 mice (18-20 g). U-87MG cell line (human glioma cell line) and HCT-116 cell line (human colorectal cancer cell line) were purchased from the Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (located in Beijing, China) and cultured in an incubator at 37°C and 5% CO2. U-87MG and HCT-116 tumor models were created by inoculating U-87MG (5 × 10 6 ), HCT-116 cells (5×10 6 ) were suspended in 100 μL of PBS and injected subcutaneously into the right upper limb of BALB / C nude mice (18-20 g). 4-Acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate was prepared by the laboratory of the First Affiliated Hospital of Jiangnan University School of Medicine. 1-(4-(Dimethylamino)phenyl)ethanone (163 mg, 1 mmol) was dissolved in dichloromethane (10 mL) in a 50 mL round-bottom flask equipped with a magnetic stirrer and cooled to 0°C in an ice bath. Methyl trifluoromethanesulfonate (0.23 mL, 2.0 mmol) was added dropwise to the stirred solution, and the ice bath was removed. Ethyl acetate was added dropwise to precipitate, which was separated using a Buchner funnel. The solid was then rinsed several times with ethyl acetate to obtain a white solid (328 mg, 66.4% yield).

[0038] The reagents described in the present invention: DMSO, / Kryptofix[2,2,2], dichloromethane, ethyl acetate, methyl trifluoromethanesulfonate, 1-(4-(dimethylamino)phenyl)ethanone, etc. were all purchased from the Titan Technology Exploration Platform.

[0039] Example 1 18 Radiolabeling of F]F-KTLLPTPR

[0040] [ 18 The radiolabeling protocol of [F]F-KTLLPTPR is shown in the attached figure. Figure 1 As shown, 15 mg of 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate was dissolved in 0.5 ml of DMSO and 6.25 mg of [ 18 F]KF / Kryptofix[2,2,2] was heated to 110°C for 10 minutes, then cooled to room temperature. 16mg of iodine in 1ml of DMSO was added, and the mixture was heated to 130°C for 20 minutes. The bottle was cooled with nitrogen, and 0.1ml of saturated Na2S2O3 solution was added. The mixture was diluted to 20ml with pure water and then subjected to solid-phase extraction using an OasisTM PLUS Short HLB cartridge. The HLB cartridge was eluted with 1ml of DMSO to obtain the crude product, which was then diluted to 10ml with pure water and purified using a preparative column. High-performance liquid chromatography (HPLC) was used for separation and purification using acetonitrile as mobile phase A and H2O containing 0.1% H3PO4 as mobile phase B, using a gradient elution system. The volume ratio of mobile phase A increased linearly from 30% to 100% (the volume ratio of mobile phase B decreased linearly from 70% to 0). The product was purified using a liquid chromatography column XBridge BEH C18 OBD Prep Column (5 μm, 10 mm × 250 mm) with a flow rate of 3 ml / min. TM After solid phase extraction with PLUS Short HLB cartridge and secondary purification with HPLC, [ 18 F]FPG.

[0041] A new arginine group was introduced into the targeted linear peptide KTLLPTP, i.e., the KTLLPTPR sequence obtained by Fmoc solid phase synthesis. 5 mg of KTLLPTPR was dissolved in 9 mL of HE PES Buffer (pH 8.5), and 1 mL of [ 18 F]FPG DMSO solution was mixed with nitrogen, heated to 37°C, and reacted for 20 min. After cooling to room temperature, the product was purified and collected using a preparative column. The separation conditions were the same as those for [ 18The product was collected and subjected to solid phase extraction using a C18 cartridge, and then washed with ethanol to obtain the purified product. 18 F]F-KTLLPTPR was subjected to the next step of analysis. Its radiochemical purity was verified by HPLC, and the peak time was 11.36min ( Figure 2 ).

[0042] Will[ 18 PET scan was performed after F]F-KTLLPTPR was injected into the KPC pancreatic cancer subcutaneous tumor model. PET imaging is shown in the figure below. Figure 3 As shown in A, the tumor site is indicated by a red circle. Compared with the fluoroaluminum labeling scheme of NOTA (Gao H, Ma Z, Zhu Z, et al. Comparative study of [ (18) F]AlF-NOTA-FAPI-RGD and[ (18) F]FDG / [ (18) F]AlF-NOTA-FAPI-04PET / CT in renal cell carcinoma[J]. Theranostics, 2025, 15(12):5790-5800.) obtained [ 18 F]F-NOTA-KTLLPTP comparison revealed that the KTLLPTP polypeptide sequence was modified with the ARG group. 18 F markers, injections [ 18 One hour after F]F-KTLLPTPR, the tumor uptake value reached 2.45±0.15%ID / g, and the tumor-muscle ratio (T / M) was 8.49±0.35, while [ 18 F]F-NOTA-PTP was 1.38±0.19%ID / g, and the tumor-muscle ratio (T / M) was 4.13±0.48. 18 F]F-KTLLPTPR can image tumors with high specificity and maintain prolonged retention at the tumor site. This high signal-to-noise ratio imaging can be used to accurately diagnose pancreatic cancer.

[0043] Example 2 18 Radiolabeling of [F]F-LTVSPWYR

[0044] [ 18 The radiolabeling protocol for [F]F-LTVSPWYR is shown in the attached figure. Figure 4 As shown, 15 mg of 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate was dissolved in 0.5 ml of DMSO and 6.25 mg of [ 18F]KF / Kryptofix[2,2,2] was heated to 110°C for 10 minutes. 16 mg of iodine dissolved in 1 ml of DMSO was added, and the mixture was heated to 130°C for 20 minutes. The bottle was cooled with nitrogen, and 0.1 ml of a saturated Na₂S₂O₃ solution was added. The mixture was diluted to 20 ml with pure water and then subjected to solid-phase extraction using an Oasis™ PLUSShort HLB cartridge. The HLB cartridge was eluted with 1 ml of DMSO to obtain the crude product, which was then diluted to 10 ml with pure water and purified using a preparative column. High-performance liquid chromatography (HPLC) was used for separation and purification using acetonitrile as mobile phase A and H₂O containing 0.1% H₃PO₄ as mobile phase B. Gradient elution was employed, with the volume proportion of mobile phase A increasing linearly from 30% to 100% (and the volume proportion of mobile phase B decreasing linearly from 70% to 0). The product was purified using a liquid chromatography column XBridge BEH C18OBD Prep Column (5 μm, 10 mm × 250 mm) with a flow rate of 3 ml / min. TM After solid phase extraction with PLUS Short HLB cartridge and secondary purification with HPLC, [ 18 F]FPG.

[0045] A new arginine group was introduced into the targeted linear peptide LTVSPWY, i.e., the LTVSPWYR sequence was obtained by Fmoc solid phase synthesis. 5 mg of LTVSPWYR was dissolved in 9 mL of HEPES Buffer (pH 8.5), and 1 mL of [ 18 F]FPG DMSO solution was mixed with nitrogen, heated to 37°C, and reacted for 20 min. After cooling to room temperature, the product was purified and collected using a preparative column. The separation conditions were the same as those for [ 18 The product was collected and subjected to solid phase extraction using a C18 cartridge, and then washed with ethanol to obtain the purified product. 18 F]F-LTVSPWYR was subjected to the next step of analysis. Its radiochemical purity was verified by HPLC, and the peak time was 8.62 min ( Figure 5 ).

[0046] Will[ 18 PET scans were performed after F]F-LTVSPWYR was injected into the U-87MG human glioma subcutaneous tumor model. PET images are shown in the figure below. Figure 6 As shown, the tumor site is indicated by a red circle and the injection [ 18One hour after F]F-LTVSPWYR, the tumor uptake value reached 1.96±0.21%ID / g, and the tumor-muscle ratio (T / M) was 3.92±0.28. 18 The F-labeling allows for highly specific tumor imaging and prolonged retention at the tumor site. This high signal-to-noise ratio imaging can be used to accurately diagnose pancreatic cancer.

[0047] Example 3 18 Radiolabeling of [F]F-CPKSNNGVCR

[0048] [ 18 The radiolabeling protocol of [F]F-CPKSNNGVCR is shown in the attached figure. Figure 7 As shown, 15 mg of 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate was dissolved in 0.5 ml of DMSO and 6.25 mg of [ 18 F]KF / Kryptofix[2,2,2] was heated to 110°C for 10 minutes, then cooled to room temperature. 16mg of iodine dissolved in 1ml of DMSO was added, and the mixture was heated to 130°C for 20 minutes. The bottle was cooled with nitrogen, and 0.1ml of a saturated Na2S2O3 solution was added. The mixture was diluted to 20ml with pure water and then subjected to solid-phase extraction using an OasisTM PLUS Short HLB cartridge. The HLB cartridge was eluted with 1ml of DMSO to obtain the crude product. The crude product was diluted to 10ml with pure water and analyzed by radioactive high-performance liquid chromatography to separate the products. xBndgepBEH C18 OBD TM The product was purified and collected using a prem Column preparative column with acetonitrile as mobile phase A and H2O containing 0.1% H3PO4 as mobile phase B. Gradient elution was employed, with the volume ratio of mobile phase A increasing linearly from 30% to 100% (the volume ratio of mobile phase B decreasing linearly from 70% to 0). The flow rate was maintained at 3 ml / min. The product was collected and passed through an Oasis TM After solid phase extraction with PLUS Short HLB cartridge and secondary purification with HPLC, [ 18 F]FPG.

[0049] A new arginine group was introduced into the targeted linear peptide CPKSNNGVC, i.e., the CPKSNNGVCR sequence was obtained by Fmoc solid phase synthesis. 5 mg of CPKSNNGVCR was dissolved in 9 mL of HEPES Buffer (pH 8.5), and 1 mL of [ 18F]FPG DMSO solution was mixed with nitrogen, heated to 37°C, and reacted for 20 min. After cooling to room temperature, the product was purified and collected using a preparative column. The separation conditions were the same as those for [ 18 The product was collected and subjected to solid phase extraction using a C18 cartridge, and then washed with ethanol to obtain the purified product. 18 F]F-CPKSNNGVCR was used for the next analysis. Its radiochemical purity was verified by HPLC, and the peak time was 13.37min ( Figure 8 ).

[0050] Will[ 18 PET scan was performed after F]F-CPKSNNGVCR was injected into the HCT-116 colorectal cancer subcutaneous tumor model. PET imaging is shown in the figure below. Figure 9 As shown, the tumor site is indicated by a red circle and the injection [ 18 At 1 hour after F]F-CPKSNNGVCR, the tumor uptake value reached 2.72±0.28%ID / g, and the tumor-muscle ratio (T / M) was 8.36±0.32. 18 The labeling of F allows for highly specific tumor imaging and prolonged retention at the tumor site. This high signal-to-noise ratio imaging can be used to accurately diagnose colorectal cancer.

[0051] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing a targeting polypeptide probe based on selective fluorine-18 labeling of arginine groups, characterized in that: The steps include: 1) Introducing an arginine group into the targeted linear peptide to obtain a targeted linear peptide containing arginine; 2) reacting 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate dissolved in DMSO with [18F]KF / Kryptofix[2,2,2]; 3) adding DMSO containing elemental iodine to the product obtained in step 2), heating the mixture for reaction, and subjecting the reaction product to solid phase extraction and purification to obtain [18F]FPG; 4) The targeted linear peptide with arginine from step 1) was dissolved, and the DMSO solution containing [18F]FPG obtained from step 3) was added, mixed with nitrogen, and heated to 35-40°C for reaction. The reaction product was purified to obtain the targeted polypeptide probe.

2. The preparation method according to claim 1, characterized in that In step 1), the targeted linear peptide containing arginine is one or more of KTLLPTPR, LTVSPWYR, and CPKSNNGVCR, and R in the sequence is an introduced arginine group.

3. The preparation method according to claim 1, characterized in that In step 2), the mass ratio of 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate to [18F]KF / Kryptofix[2,2,2] is 2-3:1, and the radioactivity of [18F]KF / Kryptofix[2,2,2] is 1-2ci; the reaction conditions are 90-120°C for 10-30min.

4. The preparation method according to claim 1, characterized in that The mass ratio of iodine to 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate is 1-5; and the heating reaction condition in step 3) is 130° C. for 10 min.

5. The preparation method according to claim 1, characterized in that The reaction product in step 3) is subjected to solid phase extraction, including: diluting the reaction product to 10-30 ml with pure water and then subjecting it to solid phase extraction using an Oasis™ PLUS Short HLB cartridge. The HLB cartridge is then eluted with 1-2 ml of DMSO to obtain a crude product, which is then diluted with pure water and purified using a preparative column.

6. The preparation method according to claim 1, characterized in that The purification in step 3) was performed by high performance liquid chromatography (HPLC), specifically using acetonitrile as mobile phase A and water containing 0.1% H₃PO₄ as mobile phase B, with gradient elution, wherein the volume proportion of mobile phase A was linearly increased from 30% to 100%, with a gradient of 30-100% mobile phase A. The collected product was subjected to solid phase extraction using an HLB cartridge and eluted with DMSO to obtain [₁₈]FPG.

7. The preparation method according to claim 1, characterized in that In step 4), the linear peptide containing arginine is dissolved in HEPES buffer, mixed with a DMSO solution of [18F]FPG, and the pH is controlled at 8 to 9; the closed reaction temperature range is 35-40°C, and the reaction time is 10 to 30 minutes; the mass ratio of the targeted linear peptide containing arginine to 4-acetyl-N,N,N-trimethylanilinium trifluoromethanesulfonate is 1-3:

1.

8. The preparation method according to claim 1, characterized in that The purification in step 4) is as follows: collecting the reaction products, and separating and purifying them by high performance liquid chromatography to obtain the targeted polypeptide probe.

9. A targeting polypeptide probe prepared by the method according to any one of claims 1 to 8.

10. Use of the targeted polypeptide probe according to claim 9 in the preparation of tumor nuclear medicine imaging reagents.

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