A method for iron-catalyzed modification of unprotected glycine or N-terminal glycine peptides

Abstract

This invention discloses an iron-catalyzed method for modifying unprotected glycine or N-terminal glycine peptides. Using the N-terminus of glycine as the modification site, unprotected glycine and the peptide are modified by heating with 2-benzoylpyridine in the presence of an iron catalyst and additives to form a fluorescent nitrogen-containing aromatic heterocycle, thus completing the modification of unprotected glycine or unprotected N-terminal glycine peptides. This invention overcomes the deficiency of requiring carboxyl-terminal protection when labeling unprotected glycine or glycine-containing peptides. The provided modification method eliminates the need for protection followed by deprotection, is simple, has a short reaction time, and high yield. As a novel N-terminal glycine peptide modification and labeling technology, this invention can be effectively used for the modification of unprotected N-terminal glycine peptides, establishing a theoretical foundation for improving peptide drug bioavailability, developing new targeted drugs, and peptide labeling and tracking.

Description

Technical Field

[0001] This invention belongs to the field of compound synthesis and peptide labeling technology. More specifically, it relates to an iron-catalyzed method for modifying unprotected glycine or N-terminal glycine peptides. Background Technology

[0002] Glycine is the simplest amino acid in structure and one of the non-essential amino acids for the human body. It is widely distributed in nature, especially abundant in proteins. Glycine is not only a key component of protein synthesis but also plays an important role in various biological processes, such as participating in the synthesis of purines, glutathione, and heme, and acting as a neurotransmitter to regulate nerve activity in the central nervous system. Glycine's unique structure gives it high flexibility, making it crucial in the secondary structure of proteins. Furthermore, glycine is widely used in the food, pharmaceutical, and cosmetic industries. In the food industry, it is used as a sweetener and preservative; in the pharmaceutical field, glycine is used to treat certain metabolic diseases and improve sleep quality.

[0003] Glycine modification methods are widely used in organic synthesis, peptide chemistry, and drug design. Modifying glycine can enhance the stability and pharmacokinetic properties of peptides, such as regulating the hydrophilic-hydrophobic balance of peptides to adapt to different routes of administration (such as transmembrane or sustained release). Modification can also regulate the structure and function of peptides. Fluorescent labels (such as FITC), biotin, or radioisotopes can be attached to the nitrogen-terminal glycine for tracking (such as cell imaging) or purification (such as affinity chromatography). The common modification methods currently include: 1. Modification of amino groups (N-modification): By introducing acyl groups, alkyl groups, sulfonyl groups, etc., taking N-acylation as an example, the specific steps are: (1) Protecting the carboxyl group: Esterifying the carboxyl group of glycine (such as methyl ester, ethyl ester) or converting it into an amide (to avoid the interference between amino and carboxyl groups); (2) Amino modification: Under alkaline conditions (such as NaHCO3), treat with acyl chloride or acid anhydride to generate N-acylated glycine ester (such as N-acetylglycine methyl ester); (3) Deprotection: Hydrolyze the ester group (such as NaOH / H2O) to obtain N-acylated glycine. II. Modification of carboxyl groups (C-modification): forming esters, amides, reducing to alcohols, etc. Taking esterification as an example: (1) Protecting the amino group: using Boc (tert-butyloxycarbonyl) or Fmoc (fluorenylmethoxycarbonyl); (2) Activating the carboxyl group: using DCC (dicyclohexylcarbodiimide) or EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to activate the carboxyl group and condense it with alcohol to form an ester; (3) Deprotection: removing Boc under acidic conditions (such as TFA) to obtain glycine ester. III. Simultaneous modification of both ends also requires first protecting the amino group (such as Boc), then modifying the carboxyl group (such as esterification), and then deprotecting the amino group and modifying it (such as alkylation).

[0004] Direct modification with unprotected glycine presents several drawbacks: (1) self-condensation reaction: amino and carboxyl groups readily form dipeptides (e.g., glycylglycine), reducing the yield of the target product; (2) poor reaction selectivity: if the modifier reacts simultaneously with both amino and carboxyl groups (e.g., bifunctional crosslinking agents), the product becomes complex and difficult to separate; (3) increased side reactions: for example, during esterification, the free amino group may catalyze ester hydrolysis or react with carboxyl activators (e.g., DCC). Therefore, existing glycine modification methods require N- or C-modification protection followed by deprotection to improve selectivity and yield, strengthen reaction conditions, and avoid side reactions.

[0005] Glycine modification typically requires protection strategies to improve selectivity and yield. However, existing methods for glycine modification are cumbersome and demanding, requiring protection followed by deprotection, and even after protection, low yields persist. Therefore, it is necessary to develop more methods for modifying unprotected glycine and unprotected N-terminal glycine peptides to address the aforementioned problems associated with unprotected glycine or its peptides. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the problems of cumbersome steps, harsh conditions and low yield of existing methods for modifying unprotected glycine or its peptides, as well as the shortcomings of unprotected glycine modification methods, and to provide an iron-catalyzed method for modifying unprotected glycine and nitrogen-terminated glycine peptides.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention provides an iron-catalyzed method for modifying unprotected glycine or N-terminal glycine peptides. The unprotected glycine or N-terminal glycine peptide to be modified is dissolved with 2-benzoylpyridine in an organic solvent, and then an iron catalyst and additives are added. The reaction is carried out at 95~105℃ for 3~9h to complete the modification of unprotected glycine or N-terminal glycine peptides.

[0009] The structural formula of the unprotected glycine or unprotected N-terminal glycine polypeptide is as follows:

[0010] In the formula, R1 is selected from the following structural formulas:

[0011] One of them;

[0012] The chemical reaction formula for the reaction of 2-benzoylpyridine with unprotected glycine is as follows:

[0013] ;

[0014] The additive is selected from one of copper trifluoroacetate hydrate, trifluoroacetic acid, and p-toluenesulfonic acid;

[0015] The iron catalyst is selected from one of ferric chloride, ferric fluoride, and ferric bromide.

[0016] This invention provides a method for modifying unprotected glycine and N-terminal glycine peptides. The method involves dissolving unprotected glycine and N-terminal glycine peptides in a mixture with 2-benzoylpyridine, adding an iron catalyst and additives, and then performing specific modification under specific reaction conditions. The iron catalyst used improves the chemoselectivity and reaction efficiency of the unprotected glycine modification strategy, overcoming the deficiency of requiring carboxyl-terminal protection when labeling glycine-containing peptides. It eliminates the need for protection followed by deprotection, is simple in method, requires only a trace amount of iron catalyst, has a short reaction time, and achieves high yield. This method, as a novel N-terminal glycine peptide modification and labeling technology, can be effectively used for the modification of unprotected N-terminal glycine peptides, establishing a theoretical basis for improving peptide drug bioavailability, developing new targeted drugs, and peptide labeling and tracking.

[0017] Furthermore, the structural formula of the 2-benzoylpyridine is as follows:

[0018] .

[0019] The principle of the modification method provided by this invention is as follows: 2-benzoylpyridine reacts with unprotected glycine or unprotected N-terminal glycine peptides under the action of an iron catalyst and additives, and is heated to form a nitrogen-containing aromatic heterocycle with fluorescent properties, thereby completing the specific modification of glycine or the N-terminal glycine of the unprotected peptide.

[0020] Furthermore, the N-terminal glycine peptides used in this invention are all amidation products, with each amino acid residue having an amino group as the substitution site, forming an amide bond. This is a stabilization modification, and the resulting amidation-modified final product does not require further modification. The modification is required for the final function, which can improve the stability of the peptide or regulate its activity, and can ultimately be used for targeted detection of markers. For example, by targeting amino acids or N-terminal glycine peptides, the modified glycine peptides have fluorescent properties and can be used for cell imaging. The fluorescent group needs to be present throughout the process and does not require further modification.

[0021] Preferably, the organic solvent is anisole, N,N-2-methylformamide, dimethyl sulfoxide, xylene, or o-dichlorobenzene.

[0022] Preferably, the unprotected glycine or unprotected N-terminal glycine polypeptide is selected from compounds with the following structural formulas:

[0023]

[0024] More preferably, the unprotected glycine or unprotected N-terminal glycine polypeptide is selected from compounds with the following structural formulas:

[0025]

[0026] Preferably, the iron catalyst is iron bromide.

[0027] Preferably, the additive is trifluoroacetic acid.

[0028] Preferably, the organic solvent is anisole.

[0029] Preferably, the amount of unprotected glycine or unprotected N-terminal glycine polypeptide fed is 1.2-2.0 equivalents of 2-benzoylpyridine.

[0030] Preferably, the amount of iron catalyst fed is 0.05-0.25 equivalents of 2-benzoylpyridine.

[0031] Preferably, the amount of the additive is 1.0-2.0 equivalents of 2-benzoylpyridine.

[0032] Preferably, the amount of the organic solvent used is 3-6 ml / mmol of 2-benzoylpyridine.

[0033] More preferably, in the modification of unprotected glycine or its polypeptide, 2-benzoylpyridine is used as 1.0 equivalent, the amount of nitrogen-terminated glycine polypeptide is 1.4 equivalent, the amount of iron catalyst is 0.2 equivalent, and the amount of additive is 2.0 equivalent; the amount of solvent used is 5.0 ml / mmol of 2-benzoylpyridine; the iron catalyst and additive are preferably ferric bromide and trifluoroacetic acid, and the reaction conditions are heating at 95°C for 4 h.

[0034] This invention provides the application of the above modification method in the modification of glycine at the nitrogen terminus of unprotected peptides.

[0035] In addition, the present invention provides a compound obtained by the above method, the structural formula of which is shown below:

[0036] In the formula, R2 is selected from the following structures:

[0037] One of them.

[0038] This invention also protects the use of the above-mentioned compounds as fluorescent probes.

[0039] The present invention has the following beneficial effects:

[0040] This invention utilizes the aromatic ketone compound 2-benzoylpyridine to modify unprotected glycine and unprotected N-terminal glycine peptides under specific reaction conditions. Using the N-terminus of glycine as the modification site, under the action of 2-benzoylpyridine, an iron catalyst, and additives, a fluorescent nitrogen-containing aromatic heterocycle is formed upon heating, achieving specific modification of unprotected glycine or its peptides. This invention overcomes the deficiency of requiring carboxyl-terminal protection during the modification and labeling of unprotected glycine and unprotected N-terminal glycine peptides, eliminating the need for protection followed by deprotection. The method is simple, requires only a trace amount of iron catalyst, has a short reaction time, and high yield. The modification method provided by this invention, as a novel unprotected glycine modification technology, can directly modify unprotected glycine and unprotected N-terminal glycine peptides in a one-step reaction without the need for protection and deprotection, establishing a theoretical basis for improving the bioavailability of peptide drugs, developing new targeted drugs, and peptide labeling and tracking. Attached Figure Description

[0041] Figure 1 It is the chemical reaction formula for the reaction of 2-benzoylpyridine with unprotected glycine and nitrogen-terminated glycine polypeptide.

[0042] Figure 2 This is a diagram showing the iron-catalyzed reaction process and product yield results of 2-benzoylpyridine for different nitrogen-terminated unprotected glycine peptides.

[0043] Figure 3 These are the UV absorption and fluorescence emission spectra of the modified product. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] Example 1: 2-Benzoylpyridine-modified glycyl-L-leucine

[0047] In a 15 mL glass test tube, 0.2 mmol of 2-benzoylpyridine, 0.28 mmol of glycyl-L-leucine, and 0.6 mL of anisole were added as solvents, followed by 0.04 mmol of ferric bromide and 0.4 mmol of trifluoroacetic acid. After all reactants were added, the test tube was placed in an oil bath and heated magnetically at 95 °C for 4 h with stirring. After the reaction was complete, the reaction system was cooled to room temperature, and the system was transferred to a separatory funnel using 20 mL of ethyl acetate and 20 mL of water to separate the organic phase. The aqueous phase was extracted three times with 20 mL of ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate. The dried organic phase was concentrated by vacuum distillation to obtain a crude product. The crude product was purified by recrystallization from 20 mL of petroleum ether and 20 mL of ethyl acetate to obtain a pure gray solid with a yield of 81%.

[0048] Synthesis route:

[0049] MRI results: 1 H NMR (500 MHz, DMSO-d6) δ 9.43 (d, J = 7.2 Hz, 1H), 8.56 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 9.2 Hz, 1H), 8.03 – 7.96 (m, 2H), 7.50 (t,J = 7.8 1.91 (ddd, J= 12.9, 10.4, 4.1 Hz, 1H), 1.76 – 1.63 (m, 2H), 0.92 (t, J = 5.9 Hz, 6H).

[0050] 13 C NMR (126 MHz, DMSO-d6) δ 173.96, 159.14, 133.78, 130.51, 129.32,128.83, 128.37, 127.07, 126.57, 125.30, 123.28, 118.47, 115.48, 50.05, 24.54,23.02, 21.30.

[0051] Example 2: Optimization of reaction conditions and yield results for unprotected peptides

[0052] Based on the reaction conditions of Example 1, the optimization study of the reaction conditions of unprotected peptides with 2-benzoylpyridine and glycyl-L-leucine was carried out. The modification reaction was carried out by adjusting the reaction conditions: catalyst type and amount, solvent amount, reactant amount, additive amount, reaction temperature, time, etc. The specific condition parameters are shown in Table 1 below, and the conversion rate of the reaction product was calculated by the following formula.

[0053] The yield calculation formula is x% = (product weight / mg) / (0.2 mmol × product molecular weight) × 100%;

[0054] The reaction formula is:

[0055]

[0056] A single-factor variable experiment was conducted based on the reaction method conditions of Example 1. The specific experimental conditions and statistical yield results are shown in Table 1.

[0057] Table 1 Results of Optimization of Reaction Conditions for Unprotected Peptides

[0058]

[0059] Note: In the table, "Trace" indicates trace amount; "None" indicates no response; " / " indicates no content.

[0060] Further, single-factor experiments were conducted at a reaction temperature of 105℃ and a reaction time of 4 h. The adjusted variable conditions are shown in Table 2. Combined with the results in Table 1, it is shown that using ferric chloride, ferric fluoride, and ferric bromide as catalysts, anisole as solvent, and trifluoroacetic acid as additive resulted in higher yields and were more conducive to the reaction. The optimal reaction conditions were: temperature 90~105℃ and time 3~4 h.

[0061] Table 2 Results of Optimization of Reaction Conditions for Unprotected Peptides

[0062]

[0063] Note: In the table, "Trace" indicates trace amounts; "None" indicates no reaction.

[0064] Example 3: Yields of iron-catalyzed reactions of peptides with different nitrogen-terminated unprotected ends

[0065] In this embodiment, glycine or its polypeptides with different nitrogen-terminal unprotected forms were used for the reaction. The reaction was carried out according to the method of Example 1. The starting material was the aromatic ketone compound 2-benzoylpyridine, which replaced glycine or its polypeptides with different nitrogen-terminal unprotected forms. After the reaction, the products were prepared according to the NMR characterization, as shown in Table 3 below. The synthetic route is as follows. Figure 2As shown, the yield calculation formula is x% = (product weight / mg) / (0.2 mmol × product molecular weight) × 100%.

[0066] The results are shown in Table 3 and Figure 2 As shown, the modification method based on Example 1 is illustrated, demonstrating that different unprotected glycine or its N-terminal glycine polypeptides used for characterization can be modified, with most exhibiting high yields.

[0067] Table 3. Iron-catalyzed conversion efficiency of peptides with different nitrogen-terminal unprotected ends.

[0068]

[0069] Example 4: Detection of the fluorescence properties of the product

[0070] In this embodiment, the fluorescence properties of the product obtained after modification by the method in Example 1 were evaluated using a fluorescence spectrometer. The statistical results are shown in Table 4, which shows that the maximum absorption wavelength λmax of the product obtained by modifying glycyl-L-leucine with 2-benzoylpyridine is 346 nm, and the Stokes shift SS is 99 nm. Its ultraviolet absorption spectrum and fluorescence emission spectrum are shown in Table 4. Figure 3 As shown, the modified product exhibits fluorescent properties and can be used as a fluorescent probe for live-cell imaging or drug targeting.

[0071] Table 4 Results of fluorescence property detection of products

[0072]

[0073] In summary, this invention provides an iron-catalyzed method for modifying unprotected glycine or N-terminal glycine peptides. Using 2-benzoylpyridine, and under specific reaction conditions, an iron catalyst and additives are added to specifically modify unprotected glycine or unprotected N-terminal glycine peptides. The chemical reaction formula for the modification reaction is as follows: Figure 1 As shown, 2-benzoylpyridine and unprotected glycine or its polypeptide form a nitrogen-containing heterocycle under iron catalysis. The product exhibits fluorescent properties and can be used as a fluorescent probe. This invention overcomes the deficiency of requiring carboxyl-terminal protection when labeling glycine or glycine-containing polypeptides. The provided modification method can complete the modification in a single step, without the need for protection followed by deprotection. The method is simple, requires only a trace amount of iron catalyst, has a short reaction time, good selectivity, and high yield. The method provided by this invention, as a novel glycine modification and labeling technology, achieves high yields for modifying nitrogen-terminated glycine polypeptides, establishing a theoretical basis for improving the bioavailability of polypeptide drugs, developing new targeted drugs, and polypeptide labeling and tracking.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for modifying unprotected glycine or N-terminal glycine polypeptides by iron catalysis, characterized in that, The unprotected glycine or unprotected N-terminal glycine peptide to be modified is dissolved in an organic solvent with 2-benzoylpyridine, and then an iron catalyst and additives are added. The reaction is carried out at 90~105℃ for 3~9h to complete the modification of unprotected glycine or unprotected N-terminal glycine peptide. The structural formula of the unprotected glycine or unprotected N-terminal glycine polypeptide is as follows: In the formula, R1 is selected from the following structural formulas: One of them; The chemical reaction formula for the reaction of 2-benzoylpyridine with unprotected glycine is as follows: ； The additive is trifluoroacetic acid; The iron catalyst is selected from one of ferric chloride, ferric fluoride, and ferric bromide; The organic solvent is selected from one of anisole, N,N-2-methylformamide, dimethyl sulfoxide, xylene, and o-dichlorobenzene.

2. The modification method according to claim 1, characterized in that, The iron catalyst is iron bromide.

3. The modification method according to claim 1, characterized in that, The organic solvent is anisole.

4. The modification method according to claim 1, characterized in that, The amount of unprotected glycine or unprotected N-terminal glycine polypeptide fed is 1.2-2.0 equivalents of 2-benzoylpyridine.

5. The modification method according to claim 1, characterized in that, The amount of the additive is 1.0-2.0 equivalents of 2-benzoylpyridine.

6. The modification method according to claim 1, characterized in that, The amount of iron catalyst fed is 0.05-0.25 equivalents of 2-benzoylpyridine.

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