A method for selectively modifying carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides and its application

Abstract

The present invention discloses a method for selective modification of carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides and their applications. The present invention reacts carbon-terminal protected glycine or nitrogen-terminal glycine polypeptides with specific aromatic ketone compounds, uses the nitrogen end of glycine as the modification site, and prepares nitrogen-containing aromatic heterocycles by heating reaction to complete the selective modification of glycine. The method provided by the present invention significantly improves the modification yield of carbon-terminal protected glycine. The method is simple, has a short reaction time, a low substrate dosage, high selectivity for glycine, can avoid interference from amino acids such as lysine and histidine, and solves the problems of strong interference of adjacent amino acids on modification, poor chemical selectivity, and low conversion rate. It provides more methods for improving the bioavailability of polypeptide drugs, developing new targeted drugs, and polypeptide labeling.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound synthesis and polypeptide labeling, and more specifically relates to a method for selectively modifying carbon-terminally protected glycine and nitrogen-terminally protected glycine polypeptides and their applications. Background Art

[0002] Glycine, the simplest amino acid, has extensive applications and significant research value in chemical synthesis and peptide pharmaceuticals. Its simple structure, high reactivity, and excellent biocompatibility make it a key component in drug synthesis, peptide modification, and nanocarrier construction. Glycine is a key intermediate in the synthesis of many drugs, including antacids and antidotes, metal ion chelators, buffers, and stabilizers. Modification of glycine not only enhances the functional activity of modified peptides but also allows for cell-penetrating peptide tracking and protein purification / detection. For example, the use of modified glycyllysine nanoparticles can enhance the targeting and cell penetration of genes or drugs. Furthermore, glycine-modified peptides (such as Fmoc-Gly-Gly) have demonstrated antimicrobial and tumor cell apoptosis-inducing abilities.

[0003] The N-terminal α-amino group of a peptide is a common site for chemical modification of peptides and has good nucleophilicity. For example, N-terminal glycine labeling has been used as a method for identifying inhibitors of active substances and has also been identified as a target for proteasome degradation. Currently developed chemical modification strategies for the N-terminus of peptides include acylation, alkylation, arylation, azidation, etc. However, existing glycine modification methods generally have the disadvantages of low yield and harsh conditions. For example, in an existing study, glycine-N-methylamide hydrochloride was reacted with dibenzoylmethane in a microwave reactor at 130°C for 5 hours, and the final yield of the fluorescent pyrrole ring was only 21% (Liu H, Deol H, Raeisbahrami A, et al. A Method for Rigorously Selective Capture and Simultaneous Fluorescent Labeling of N-Terminal Glycine Peptides [J]. Journal of the American Chemical Society, 2024(20):146.).

[0004] Furthermore, because the amino group of N-terminal glycine shares similar chemical reactivity to the side-chain amino or amide groups of other amino acids (such as lysine and glutamine), its modification is susceptible to interference from other reactive groups, leading to nonspecific side reactions. For example, existing research has reported a method for achieving specific double modification of the N-termini of peptides via a three-component [3+2] cycloaddition reaction of aldehydes and maleimides under copper-catalyzed conditions. While this method overcomes interference from the ε-amino group of lysine, achieving this selectivity (based on steric hindrance) requires the use of a specific triethylamine base and catalyst, requiring increased base concentration and catalyst dosage. This harsh reaction conditions can also result in the labeling of other N-termini. Currently, there is a lack of glycine modification technologies that avoid interference from adjacent amino acids, offer excellent selectivity, and achieve high conversion rates. Therefore, developing new, efficient, and highly selective strategies for N-terminal glycine peptide modification is crucial for the development of new drugs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of strong interference of adjacent amino acids on modification, poor chemical selectivity and low conversion rate in existing carbon-terminal protected glycine modification technology, and provide a method for selective modification of carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides and their application.

[0006] The purpose of the present invention is to provide a method for selectively modifying carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides.

[0007] Another object of the present invention is to provide a compound modified by the above method.

[0008] Another object of the present invention is to provide an application of the compound as a fluorescent probe.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a method for selectively modifying carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides, comprising dissolving the carbon-terminal protected glycine or nitrogen-terminal glycine polypeptide and an aromatic ketone compound in an organic solvent, adding an additive, and reacting at 100-110°C for 2-4 hours.

[0011] The carbon-terminal protected glycine or nitrogen-terminal glycine polypeptide is selected from:

[0012] or ; Wherein R1 is selected from one of the following structural formulas:

[0013]

[0014] The structural formula of the aromatic ketone compound is as follows:

[0015] ; wherein R2 is selected from hydrogen, halogen, methyl, methoxy, cyano, and R3 is selected from pyridine, benzopyridine, pyrazine, benzothiazole and derivatives thereof;

[0016] The additive is selected from one of trifluoroacetate copper hydrate, trifluoroacetic acid, and p-toluenesulfonic acid;

[0017] The organic solvent is selected from one of anisole, N,N-dimethylformamide, dimethyl sulfoxide, o-dichlorobenzene and xylene.

[0018] The present invention uses the nitrogen end of glycine as the modification site, mixes and dissolves with an aromatic ketone compound, adds an additive, and prepares a nitrogen-containing aromatic heterocycle by heating reaction to complete the selective modification of glycine; at the same time, an iron catalyst is further added during the reaction, and the modification is carried out under specific reaction conditions, which can further improve the chemical selectivity and reaction efficiency of the glycine modification strategy, shorten the reaction time, and increase the carbon-end protected glycine modification yield, which is as high as 99%; and the modification method provided by the present invention is simple, has a short reaction time, a low substrate dosage, high selectivity for glycine, can avoid interference from amino acids such as lysine and histidine, and solves the problems of strong interference of adjacent amino acids on modification, poor chemical selectivity, and low conversion rate; and the product modified by the method of the present invention does not need to be modified, has fluorescent properties, and can be used for glycine detection and cell imaging after labeling glycine polypeptides. The present invention provides more methods for improving the bioavailability of polypeptide drugs, developing new targeted drugs, and polypeptide labeling.

[0019] The chemical reaction formula of the reaction between the aromatic ketone compound and glycine is as follows ( Figure 1 ):

[0020]

[0021] The modification principle of the method of the present invention is that aromatic ketone compounds and glycine, under the action of additives, form nitrogen-containing aromatic heterocycles with fluorescent properties by heating, thereby completing the selective modification of glycine.

[0022] Preferably, the structural formula of the carbon-terminal protected glycine or nitrogen-terminal glycine polypeptide is as follows:

[0023]

[0024]

[0025] Preferably, in the method, the iron catalyst is added together with the additive for reaction.

[0026] Preferably, the iron catalyst is selected from one of ferroferric oxide, nano iron powder, nano nickel ferrite, nano cobalt ferrite, nano zinc ferrite, and ferric oxide.

[0027] More preferably, the iron catalyst is ferrosoferric oxide.

[0028] Preferably, the additive and the iron catalyst are reacted together as follows: the additive is copper trifluoroacetate hydrate or trifluoroacetic acid; the iron catalyst is ferrosoferric oxide; and the solvent is anisole.

[0029] Preferably, the organic solvent is selected from one of anisole, o-dichlorobenzene and xylene.

[0030] More preferably, the organic solvent is anisole.

[0031] Preferably, the aromatic ketone compound is selected from any one of the compounds of the following structural formulas:

[0032]

[0033] wherein R is selected from hydrogen, halogen, methyl, methoxy, and cyano.

[0034] More preferably, the aromatic ketone compound is selected from any one of the compounds of the following structural formulas:

[0035]

[0036] wherein R is selected from hydrogen, halogen, methyl, methoxy, and cyano.

[0037] Preferably, the amount of the carbon-terminally protected glycine or nitrogen-terminally protected glycine polypeptide added is 1.2-2.0 equivalents of the aromatic ketone compound.

[0038] Preferably, the amount of the additive added is 0.8-2.0 equivalents of the aromatic ketone compound.

[0039] Preferably, the iron catalyst is added in an amount of 0.1-0.25 equivalents of the aromatic ketone compound.

[0040] Preferably, the amount of the organic solvent is 3-6 ml / mmol of aromatic ketone marker.

[0041] Further preferably, in the modification of a carbon-terminal protected glycine or nitrogen-terminal glycine polypeptide, the aromatic ketone compound is used as 1.0 equivalent, the feeding amount of glycine or nitrogen-terminal glycine polypeptide is 1.5 equivalents; the feeding amount of the iron catalyst is 0.2 equivalents, and the feeding amount of the additive is 1.1 equivalents; the amount of the solvent used is 5.0 ml / mmol of the aromatic ketone compound; the iron catalyst and additive are preferably ferrosoferric oxide and copper trifluoroacetate hydrate, and the reaction conditions are heating at 105 ° C for 4 h.

[0042] In addition, the present invention also provides a compound, the structural formula of the compound is shown below:

[0043]

[0044] wherein R4 is selected from hydrogen, halogen, ether, and cyano; and R5 is selected from halogen and methyl.

[0045] Preferably, the structural formula of the compound is as follows:

[0046]

[0047] The present invention also protects the application of the compound as a fluorescent probe.

[0048] The present invention has the following beneficial effects:

[0049] The present invention discloses a method for selective modification of carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides and their applications. The present invention reacts carbon-terminal protected glycine or nitrogen-terminal glycine polypeptides with specific aromatic ketone compounds, uses the nitrogen end of glycine as the modification site, and prepares nitrogen-containing aromatic heterocycles by heating reaction to complete the selective modification of glycine. The method provided by the present invention significantly improves the modification yield of carbon-terminal protected glycine. The method is simple, has a short reaction time, a low substrate dosage, high selectivity for glycine, can avoid interference from amino acids such as lysine and histidine, and solves the problems of strong interference of adjacent amino acids on modification, poor chemical selectivity, and low conversion rate. It provides more methods for improving the bioavailability of polypeptide drugs, developing new targeted drugs, and polypeptide labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the chemical reaction formula of the reaction between aromatic ketone compounds and glycine and polypeptides with glycine at the nitrogen end.

[0051] Figure 2 This is a graph showing the chemical reaction process of different aromatic ketone compounds with glycylglycine methyl ester hydrochloride and the yield results of the products.

[0052] Figure 3 This is a graph showing the reaction process of 2-benzoylpyridine with glycylglycine having different carbon-terminal protecting groups and the yield of the product.

[0053] Figure 4 This is a graph showing the reaction process of 2-benzoylpyridine for peptides with different carbon end protections and the product yield results.

[0054] Figure 5 These are the ultraviolet absorption spectra and fluorescence emission spectra of some modified products.

[0055] Figure 6 These are the ultraviolet absorption spectra and fluorescence emission spectra of some modified products.

[0056] Figure 7 These are the ultraviolet absorption spectra and fluorescence emission spectra of some modified products.

[0057] Figure 8 These are the ultraviolet absorption spectra and fluorescence emission spectra of some modified products.

[0058] Figure 9 These are the ultraviolet absorption spectra and fluorescence emission spectra of some modified products. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0061] Example 1 Preparation of aromatic ketone compounds

[0062] The aromatic ketone compound prepared in this example is synthesized according to the method reported in the existing literature (Song S, Li Z, Wang L, et al. Photoredox and NHC Enabled Deoxygenative Alcohol Homologation via Formal 1,2-Addition [J]. Organic Letters, 2024, 26 (1): 5.). The subsequent preparation of 2-benzoylpyridine, 3- (4-bromobenzyl) pyridine and other different aromatic ketone compounds used in Example 4 is also prepared according to this embodiment. The specific method is as follows: 1.18 g of 2-cyano-5-methylpyridine and 25 mL of tetrahydrofuran are added to a 100 mL round-bottom flask, nitrogen protection is provided, and the reaction system is placed in an ice-water bath and stirred for 5 minutes. Subsequently, 4.0 mL of a tetrahydrofuran solution (3M) of phenylmagnesium bromide is added dropwise under ice-bath conditions. After the addition is complete, the temperature is raised to room temperature and the reaction is allowed to react for 6 hours. After the reaction was complete, 25 mL of 6 M hydrochloric acid was added to quench the reaction, and the pH was adjusted to 8 with 2N sodium hydroxide. The organic phase was separated, and the aqueous phase was extracted three times with 20 mL of ethyl acetate. The combined organic phases were washed with 50 mL of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using an eluent of ethyl acetate / petroleum ether (1:9). Purification afforded the final product as a yellow solid in a 55% yield.

[0063] Synthesis route:

[0064]

[0065] NMR results: 1 H NMR (500 MHz, Chloroform-d) δ 8.57 - 8.51 (m, 1H), 8.08 -8.03 (m, 2H), 7.97 (d, J = 8.0 Hz, 1H), 7.70 (ddd, J = 8.0, 1.5, 0.7 Hz, 1H),7.61 - 7.56 (m, 1H), 7.48 (t, J = 7.8 Hz, 2H), 2.45 (s, 3H).

[0066] Example 2 2-Benzoylpyridine Modified Glycine Methyl Ester Hydrochloride

[0067] To a 15 mL glass test tube, 0.2 mmol of 2-benzoylpyridine, 0.3 mmol of glycine methyl ester hydrochloride, and 1 mL of anisole were added as solvent. Subsequently, 0.04 mmol of ferrosoferric oxide and 0.22 mmol of copper trifluoroacetate hydrate were added. After all reactants were added, the test tube was placed in an oil bath and heated at 105°C with magnetic stirring for 4 h. After completion of the reaction, the reaction system was cooled to room temperature and transferred to a separatory funnel with 20 mL of ethyl acetate and 20 mL of water. The organic phase was separated, and the aqueous phase was extracted three times with 20 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The dried organic phases were concentrated by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using a 1 / 2 ratio of ethyl acetate to petroleum ether as the eluent. The final product was obtained as a white solid in an 84% yield.

[0068] Synthesis route:

[0069]

[0070] NMR results: 1 H NMR (500 MHz, Chloroform-d) δ 9.40 (d, J = 7.1 Hz, 1H),7.97 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H),7.36 (t, J = 7.3 Hz, 1H), 7.13 (t, J = 7.9 Hz, 1H), 6.96 (t, J = 6.9 Hz, 1H), 4.05 (s, 3H).

[0071] 13 C NMR (126 MHz, Chloroform-d) δ 160.05, 134.14, 133.59, 130.79,128.72, 127.56, 127.44, 126.32, 125.80, 123.32, 118.62, 115.75, 52.19.

[0072] Example 3 2-Benzoylpyridine Modified Glycylglycine Methyl Ester Hydrochloride

[0073] To a 15 mL glass test tube, 0.2 mmol of 2-benzoylpyridine, 0.3 mmol of glycylglycine methyl ester hydrochloride, and 1 mL of anisole were added as solvent. Subsequently, 0.04 mmol of ferrosoferric oxide and 0.22 mmol of copper trifluoroacetate hydrate were added. After all reactants were added, the test tube was placed in an oil bath and heated at 105°C with magnetic stirring for 4 h. After completion of the reaction, the reaction system was cooled to room temperature and transferred to a separatory funnel with 20 mL of ethyl acetate and 20 mL of water. The organic phase was separated, and the aqueous phase was extracted three times with 20 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The dried organic phases were concentrated by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using a 1 / 4 ratio of ethyl acetate to petroleum ether as the eluent. The final product was obtained as a white solid in an 83% yield.

[0074] Synthesis route:

[0075]

[0076] NMR results: 1 H NMR (500 MHz, Chloroform-d) δ 9.47 (d, J = 7.2 Hz, 1H),7.88 – 7.92 (m, 4H), 7.48 (t, J = 7.8 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H),7.04 (dd, J = 9.2, 6.5 Hz, 1H), 6.85 (t, J = 6.9 Hz, 1H), 4.28 (d, J = 5.9Hz, 2H), 3.80 (s, 3H).

[0077] 13C NMR (126 MHz, Chloroform-d) δ 170.19, 159.80, 134.01, 132.14,130.08, 128.81, 128.38, 127.27, 126.97, 125.81, 122.50, 118.35, 115.01,52.40, 40.79.

[0078] Example 4 3-(4-bromobenzyl)pyridine modified glycylglycine methyl ester hydrochloride

[0079] To a 15 mL glass test tube, 0.2 mmol of 3-(4-bromobenzyl)pyridine, 0.3 mmol of glycylglycine methyl ester hydrochloride, and 1 mL of anisole were added as solvent. Subsequently, 0.04 mmol of ferrosoferric oxide and 0.22 mmol of copper trifluoroacetate hydrate were added. After all reactants were added, the test tube was placed in an oil bath and heated at 105°C with magnetic stirring for 4 h. After completion of the reaction, the reaction system was cooled to room temperature and transferred to a separatory funnel with 20 mL of ethyl acetate and 20 mL of water. The organic phase was separated, and the aqueous phase was extracted three times with 20 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The dried organic phases were concentrated by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using a 1 / 4 ratio of ethyl acetate to petroleum ether as the eluent. The final product was obtained as a white solid with a yield of 88%.

[0080] Synthesis route:

[0081]

[0082] NMR results: 1 H NMR (500 MHz, Chloroform-d) δ 9.47 (d, J = 7.2 Hz, 1H), 7.92 – 7.82 (m, 2H), 7.75 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.06(dd, J = 9.2, 6.5 Hz, 1H), 6.86 (t, J = 6.9 Hz, 1H), 4.28 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H).

[0083] 13C NMR (126 MHz, Chloroform-d) δ 170.17, 159.66, 132.99, 131.89,130.79, 130.07, 128.52, 128.31, 125.95, 122.92, 121.06, 118.06, 115.07,52.43, 40.78.

[0084] Example 5 2-Benzoylpyridine Modified Glycyl-L-Valine Methyl Ester Hydrochloride

[0085] To a 15 mL glass test tube, 0.2 mmol of 2-benzoylpyridine, 0.3 mmol of glycyl-L-valine methyl ester hydrochloride, and 1 mL of anisole were added as solvent. Subsequently, 0.04 mmol of ferrosoferric oxide and 0.22 mmol of copper trifluoroacetate hydrate were added. After all reactants were added, the test tube was placed in an oil bath and heated at 105°C with magnetic stirring for 4 h. After completion of the reaction, the reaction system was cooled to room temperature and transferred to a separatory funnel with 20 mL of ethyl acetate and 20 mL of water. The organic phase was separated, and the aqueous phase was extracted three times with 20 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The dried organic phases were concentrated by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using a 1:8 ratio of ethyl acetate to petroleum ether as the eluent. The final product was obtained as a white solid with a yield of 88%.

[0086] Synthesis route:

[0087]

[0088] NMR results: 1 H NMR (500 MHz, Chloroform-d) δ 9.49 (d, J = 7.2 Hz, 1H), 7.87 – 7.92 (m, 4H), 7.50 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H), 7.03(dd, J = 9.2, 6.5 Hz, 1H), 6.84 (t, J = 6.8 Hz, 1H), 4.74 (dd, J = 9.2, 5.5Hz, 1H), 3.79 (s, 3H), 2.33 (dq, J = 13.7, 6.9 Hz, 1H), 1.06 (dd, J = 6.9,3.0 Hz, 6H).

[0089] 13C NMR (126 MHz, Chloroform-d) δ 172.21, 159.54, 134.00, 131.99,130.01, 128.74, 128.47, 127.19, 126.99, 125.80, 122.34, 118.25, 114.83,56.96, 52.13, 31.35, 19.13, 18.02.

[0090] Example 6 Optimization of reaction conditions and yield results for C-terminally protected peptides

[0091] Based on the reaction conditions of Example 3, the reaction conditions for the carbon-terminus-protected polypeptide were optimized using 2-benzoylpyridine and glycylglycine methyl ester hydrochloride. The reaction conditions were adjusted by single-factor changes: the type and amount of catalyst, solvent, and additive, as well as the reaction temperature, time, etc., and the yield of the reaction product was statistically analyzed after the reaction.

[0092] The yield calculation formula is x% = (product weight / mg) / (0.2 mmol × product molecular weight) × 100%;

[0093] The reaction formula is:

[0094]

[0095] A single factor variable test was conducted based on the reaction method conditions of Example 3. The specific experimental conditions and statistical yield results are shown in Table 1.

[0096] Table 1 Optimization results of reaction conditions for C-terminus protected peptides

[0097]

[0098] Note: In the table, “Trace” means trace; “None” means no reaction; “ / ” means no content.

[0099] Further, a single factor experiment was carried out under the conditions of reaction temperature of 105 ° C and reaction time of 4 h. The adjusted variable conditions are shown in Table 2. Combined with the results in Table 1, it shows that the catalysts using ferroferric oxide, nano-iron powder, nano-nickel ferrite, nano-cobalt ferrite, nano-zinc ferrite, and ferric oxide, the solvent using anisole, and the additives using copper trifluoroacetate hydrate, trifluoroacetic acid, and p-toluenesulfonic acid have higher yields and are more conducive to the reaction. The optimal reaction conditions are: temperature 100-110 ° C, time 2-4 h.

[0100] Table 2 Optimization results of reaction conditions for C-terminus protected peptides

[0101]

[0102] Note: In the table, “Trace” means trace; “None” means no reaction; “ / ” means no content.

[0103] Example 7 Yields of Iron-Catalyzed Reactions of Different Aromatic Ketones

[0104] The preparation methods of different aromatic ketone compounds were referred to Example 1. After synthesis and NMR characterization according to the methods reported in existing literature, the raw materials used were shown in Table 3 below. Subsequently, according to the reaction method of 2-benzoylpyridine modified glycylglycine methyl ester hydrochloride provided in Example 3, the aromatic ketone raw materials were replaced and the reactions were carried out respectively. The synthetic routes are as follows: Figure 2 As shown, the products prepared were verified by nuclear magnetic resonance and then the yields of the reaction products were measured and statistically analyzed.

[0105] The yield calculation formula is x% = (product weight / mg) / (0.2 mmol × product molecular weight) × 100%;

[0106] The results are shown in Table 3 and Figure 2 As shown, it was shown that aromatic ketone compounds and the nitrogen end of glycine formed nitrogen-containing heterocycles under iron-catalyzed conditions, and most aromatic ketone compounds had high yields, proving that they can be used to modify nitrogen-terminal glycine peptides.

[0107] Table 3 Yields of iron-catalyzed reactions of different aromatic ketone compounds

[0108]

[0109] Example 8 Yields of Iron-Catalyzed Reaction of Glycylglycine with Different Carbon-Terminal Protecting Groups

[0110] In this example, glycylglycine with different carbon terminal protecting groups was used for the reaction, as shown in Table 4. 2-benzoylpyridine was used as the aromatic ketone compound, and different glycylglycine methyl ester hydrochlorides were replaced. The reaction was carried out according to the method of Example 3 and the products shown in Table 4 were prepared after nuclear magnetic resonance characterization. The synthetic route is as follows: Figure 3 As shown, the yields of the reaction products were statistically analyzed with reference to the calculation method of Example 7.

[0111] The results are shown in Table 4 and Figure 3 As shown, it is shown that glycine dipeptides with different carbon-terminal protecting groups can be modified, and most of them have high yields, which further verifies the feasibility of the present invention.

[0112] Table 4 Iron-catalyzed yields of glycylglycine with different carbon-terminal protecting groups

[0113]

[0114] Example 9 Yields of Iron-Catalyzed Reaction of Peptides with Different C-Terminus Protection

[0115] In this example, peptides with different carbon end protections were used for the reaction, as shown in Table 5. 2-benzoylpyridine was used as the aromatic ketone compound, and the peptides with different carbon end protections were replaced. The reaction was carried out according to the method of Example 2 and the NMR characterization was performed to prepare the products shown in Table 5. The synthetic route is as follows: Figure 4 As shown, the yields of the reaction products were statistically analyzed with reference to the calculation method of Example 7.

[0116] The results are shown in Table 5 and Figure 4 As shown, it is shown that the N-terminal glycine polypeptides protected by carbon-terminal methyl ester can be modified, and most of them have a high yield, which further verifies the application effect of the present invention in N-terminal glycine polypeptides.

[0117] Table 5 Yields of iron-catalyzed reactions of peptides with different carbon-terminal protections

[0118]

[0119] Example 10 Detection of fluorescence properties of some products

[0120] In this example, the fluorescence properties of some of the modified products were evaluated by fluorescence spectroscopy, revealing the regulation of photophysical properties by the electronic effects of substituents. The results are shown in Table 6, indicating that the substituents on the benzene ring and pyridine ring have little effect on the maximum absorption wavelength (λmax = 340-349 nm), while the Stokes shift (SS = 49-121 nm) can be significantly regulated by changing the substituents. The UV absorption and fluorescence emission spectra of some of the modified products are shown in Figure 6. Figures 5 to 9 As shown, the modified product has fluorescent properties and can be used as a fluorescent probe. This result lays a theoretical and technical foundation for the design of multifunctional N-terminal glycine fluorescent probes and proves the practical application of such molecules in living cell imaging or drug targeting tracking.

[0121] Table 6 Fluorescence property test results of some products

[0122]

[0123] Example 11 Reaction selectivity experimental results

[0124] The interference effects of different amino acids on the reaction were systematically investigated by adding amino acid methyl esters of different structures to a standard reaction system. The specific procedure was to add 1.5 equivalents of amino acid methyl ester hydrochloride (excluding glycine) (lysine methyl ester hydrochloride, arginine methyl ester hydrochloride, histidine methyl ester hydrochloride, threonine methyl ester hydrochloride, and valine methyl ester hydrochloride) to the reaction system based on the reaction in Example 3. The reaction was carried out under the same conditions and post-treatment, and the yield was calculated using the method in Example 7.

[0125] Synthetic route for the reaction:

[0126]

[0127] The experimental results are shown in Table 7. When lysine methyl ester containing ε-amino group was introduced, the target product still maintained an isolated yield of 79%, indicating that the lysine side chain amino group and the N-terminal α-amino group did not participate in the competitive reaction. After adding arginine methyl ester containing guanidine group, the yield of the target product dropped to only 66%, confirming that the strong basic side chain group of arginine did not induce side reactions. Even if Fe 3+ Strongly coordinating histidine methyl ester (imidazolyl) and threonine methyl ester (hydroxyl) groups still yielded 53% of the target product, eliminating the significant influence of these two coordinating groups on catalyst activity. When valine methyl ester, with an alkyl side chain, was used, the product yield reached 81%, essentially in line with the model, confirming the inertness of N-terminal non-glycine peptides. No byproducts were detected in all parallel experiments, fully demonstrating that the system can accurately recognize N-terminal glycine peptides while exhibiting excellent selectivity against other N-terminal amino acid peptides and reactive side chain groups. This property provides an important experimental basis for site-specific modification in complex peptide mixtures.

[0128] Table 7 Selectivity results of modification reaction

[0129]

[0130] In summary, the present invention reacts a carbon-terminally protected glycine or nitrogen-terminally protected glycine polypeptide with a specific aromatic ketone compound, uses the nitrogen terminus of glycine as the modification site, and prepares a nitrogen-containing aromatic heterocycle by heating reaction to complete the selective modification of glycine. The method provided by the present invention significantly improves the modification yield of carbon-terminally protected glycine. The method is simple, has a short reaction time, a low substrate dosage, high selectivity for glycine, can avoid interference from amino acids such as lysine and histidine, and solves the problems of strong interference of adjacent amino acids on modification, poor chemical selectivity, and low conversion rate. It provides more methods for improving the bioavailability of polypeptide drugs, developing new targeted drugs, and polypeptide labeling.

[0131] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for selectively modifying carbon-terminal protected glycine and nitrogen-terminal glycine polypeptides, characterized in that: Dissolve the carbon-terminal protected glycine or nitrogen-terminal glycine peptide and aromatic ketone compound in an organic solvent, add additives and iron catalyst, and react at 100-110°C for 2-4 hours. The carbon-terminal protected glycine or nitrogen-terminal glycine polypeptide is selected from: or ; wherein R1 is selected from one of the following structural formulas: The aromatic ketone compound is selected from any one of the compounds of the following structural formulas: wherein R is selected from hydrogen, halogen, methyl, methoxy, and cyano; The additive is copper trifluoroacetate hydrate; The organic solvent is anisole; The iron catalyst is ferrosoferric oxide.

2. The method according to claim 1, characterized in that The feeding amount of the carbon-terminal protected glycine or nitrogen-terminal glycine polypeptide is 1.2-2.0 equivalents of the aromatic ketone compound.

3. The method according to claim 1, characterized in that The amount of the additive added is 0.8-2.0 equivalents of the aromatic ketone compound.

4. The method according to claim 1, characterized in that The feeding amount of the iron catalyst is 0.1-0.25 equivalents of the aromatic ketone compound.

5. A compound, characterized in that The structural formula of the compound is shown below: wherein R4 is selected from hydrogen, halogen, ether, and cyano; and R5 is selected from halogen and methyl.

6. Use of the compound according to claim 5 as a fluorescent probe.

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