Differential modification method and use for polypeptide

Abstract

The present invention provides a differential modification method and use for a polypeptide. The differential modification method for a polypeptide comprises: step S1. providing an aromatic aldehyde and a polypeptide, and mixing the aromatic aldehyde and the polypeptide in a reaction solvent, so that an aldehyde group of the aromatic aldehyde reacts with an N-terminus of the polypeptide to generate a dihydroimidazolone intermediate reaction solution; and step S2. adding an alkaline substance and fluorosulfonyl azide to the dihydroimidazolone intermediate reaction solution for an azide reaction to obtain a modified polypeptide reaction solution. By means of the reactivity difference of polypeptide terminal groups, the present application achieves differential modifications of the N-terminus and C-terminus of the polypeptide separately by means of a one-pot two-step method in a similar pH environment, especially the modification of amino groups at two termini of a polypeptide having lysine at the C-terminus with different groups separately. The specificity is improved, the reaction conditions are simple and mild, the route is short, the operation is convenient, large-scale production is easily implemented, and the cost is saved.

Description

Differential modification methods and applications of peptides Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically, to a method for differential modification of peptides and its application. Background Technology

[0002] Polypeptides are compounds formed by multiple amino acids linked together in a specific sequence via peptide bonds, and are intermediate products of protein hydrolysis. Polypeptides play a crucial role in various aspects of human growth, development, immune regulation, and metabolism, possessing vital biological functions. For example, some polypeptides act as hormones, participating in physiological regulatory processes; insulin, for instance, is a polypeptide hormone that regulates blood sugar levels. In the medical field, polypeptides are widely used in drug development. Some polypeptide drugs exhibit high specificity and low toxicity, enabling them to precisely target specific sites and treat various diseases. Furthermore, polypeptides are also major active ingredients in some medicinal plants. Therefore, the analysis and research of polypeptides are of great significance for drug development and the exploration of life sciences.

[0003] Modification of peptides can significantly improve their pharmacokinetic properties, enhance their stability, improve their targeting, and reduce their toxicity, which is of great significance in drug development, biochemical research and other fields.

[0004] Since 2000, the availability of natural peptides has been continuously enriched, particularly driven by venom-derived peptidomics and novel chemical modification methods, which have facilitated the discovery of new peptide drugs. The emergence of new technologies such as multifunctional peptides, bound peptides, coupled peptides, oral peptides, long-acting peptides, and delivery systems has greatly propelled the development of the peptide drug field. For example, introducing Group 7 halogen elements (such as F and Cl) into peptide molecules has significantly altered their biological properties; modification methods such as PEGylation have been developed. Furthermore, the use of gene editing and synthetic biology techniques for peptide design and modification at the gene level, as well as the use of artificial intelligence algorithms to predict and optimize peptide modification schemes, have also contributed to the development of peptide modification technology.

[0005] Differential modification of peptide ends refers to the application of different chemical modifications to the two ends (usually the N-terminus and C-terminus) of a peptide to alter its properties, functions, or activities. There are various methods to achieve differential modification of peptide ends. For example, by differentially protecting and removing the α-amino group of the N-terminal amino acid, and then condensing this α-amino group with the C-terminal carboxyl group of the peptide, high-quality cyclic peptides can be achieved. This modification can improve the stability, bioavailability, or binding ability of the peptide to specific targets. Alternatively, specific chemical reactions or reagents with special functional groups can be used to selectively modify the N-terminus and C-terminus of the peptide, introducing different chemical groups, molecules, or other structures. Differential modification of peptide ends is of great significance in drug development, biochemistry, and other fields. It can: 1. Improve the pharmacokinetic properties of peptides, such as prolonging their half-life in vivo; 2. Enhance the specific binding of peptides to targets; 3. Reduce immunogenicity; 4. Confer new functions or activities to peptides; 5. Improve the physicochemical properties of peptides, such as solubility. Different peptides may require the selection of appropriate biterminal differential modification methods and modifying groups based on their specific applications and purposes to achieve the desired effects. When modifying peptides, factors such as modification efficiency, selectivity, impact on peptide structure and function, and the mildness of reaction conditions need to be considered. Simultaneously, thorough characterization and biological activity evaluation of the modified peptides are necessary to ensure that the modification achieves the intended goals. The development trend of biterminal differential modification technology for peptides will help promote the wider application and innovation of peptide modification technology in fields such as drug development, biomaterials, and diagnostics.

[0006] With the continuous advancement of chemistry and biotechnology, developing more precise and efficient modification methods can reduce adverse effects on peptide structure and function. Introducing more diverse functional groups at both ends of peptides can integrate multiple functions such as therapy, diagnosis, and imaging to meet complex medical needs. Developing bi-terminal differentially modified peptides that can respond to specific biological signals or environmental stimuli in vivo enables intelligent drug release and precise treatment. Combining with advanced nanomaterials or nanocarriers can improve peptide delivery efficiency and targeting. Customizing specific bi-terminal differentially modified peptides based on individual patients' disease characteristics and physiological conditions can achieve more precise personalized treatment. This involves deep interdisciplinary integration of chemistry, biology, medicine, and materials science, driving technological innovation and development. More bi-terminal differentially modified peptide drugs will enter clinical trials and be applied in more disease areas, such as neurological diseases and cardiovascular diseases. Greater emphasis will be placed on environmental protection and sustainability during the modification process, reducing the use of harmful substances and the generation of waste.

[0007] Currently, existing technologies for peptide modification encompass a variety of strategies and methods, each with unique technical solutions and application advantages. These mainly include chemical modification techniques, biological modification techniques, and combinatorial modification techniques. Among them, (I) chemical modification techniques include: 1. Acylation and alkylation: By introducing acyl or alkyl groups onto amino acid residues of peptides, the hydrophobicity, charge distribution, and stability of the peptides are altered. This technique typically requires mild reaction conditions to avoid damaging the active structure of the peptide. Selecting appropriate acylation or alkylation reagents, as well as optimizing the solvent, temperature, and reaction time, are crucial to ensuring the modification effect and product purity; 2. Phosphorylation: Adding phosphate groups to specific amino acid residues (such as serine, threonine, and tyrosine) to regulate the bioactivity and signal transduction function of the peptide. Phosphorylation modification techniques include methods using chemical phosphorylation reagents or enzyme catalysis. Enzyme catalysis generally has higher selectivity and site specificity, but chemical methods may be more advantageous in large-scale production. Precise control of the phosphorylation site and extent is crucial for achieving the desired biological effects; 3. PEGylation: Linking polyethylene glycol (PEG) molecules to peptides increases their water solubility, prolongs their in vivo half-life, and reduces immunogenicity. PEGylation techniques involve selecting PEG reagents with appropriate molecular weight and structure, and optimizing reaction conditions to achieve efficient and specific linkage. Commonly used methods include N-terminal PEGylation, C-terminal PEGylation, and side-chain PEGylation, each requiring selection based on the peptide's structural and functional requirements; 4. Cycling modification: Forming cyclic structures in peptides by forming peptide bonds or introducing other chemical linkers can increase conformational stability and resistance to protease degradation. Cycling reaction conditions need optimization to avoid affecting peptide activity; 5. Disulfide bond modification: Introducing or adjusting the position of disulfide bonds in peptides can enhance their structural stability and folding accuracy, requiring precise control of disulfide bond formation conditions. (II) Biomodification technologies include: 1. Glycosylation: Linking glycans to peptides via enzymatic reactions, thereby altering their immunogenicity, targeting, and bioactivity. Glycosylation modification requires the selection of suitable glycosyltransferases and reaction substrates, while optimizing reaction conditions to ensure proper glycan linkage and structural integrity. Furthermore, strict quality control and characterization of the glycosylated products are necessary to ensure they meet pharmaceutical standards. 2. Protein fusion: Fusing peptides with other proteins or peptide fragments to endow them with new functions or improve their properties. This technique typically involves genetic engineering to construct expression vectors for fusion proteins and express and purify them in appropriate host cells. The fusion sites and sequence need careful design to avoid affecting the structure and function of the peptide and its fusion partner. 3. Site-directed mutagenesis: Altering specific bases in the peptide-encoding gene through genetic engineering techniques to achieve amino acid substitution.Targeted modification of peptide properties and functions requires effective gene cloning and expression systems. (III) Combination modification techniques: To achieve more complex and comprehensive improvement of peptide performance, a combination of multiple modification techniques is often employed. For example, combining phosphorylation with polyethylene glycol (PEG) can both regulate the bioactivity of peptides and improve their pharmacokinetic properties. When designing combinatorial modification techniques, it is necessary to fully consider the interactions and synergies between different modifications, while optimizing the reaction sequence and conditions to ensure the final product possesses the expected properties and functions. Linking specific antibody fragments to the N-terminus of a peptide enables precise targeting of tumor cells. Simultaneously, modifying the C-terminus with a cell-penetrating peptide sequence significantly improves the efficiency of drug entry into tumor cells, providing a new strategy for cancer treatment. Introducing a photosensitive group at the N-terminus of a peptide allows for photocontrolled drug release; while linking a small molecule with anti-inflammatory activity to the C-terminus enables precise treatment and controlled release at inflammatory sites. Modifying the N-terminus of a peptide with a polyethylene glycol (PEG) chain increases its circulating half-life. Binding a receptor that specifically recognizes pathogens to the C-terminus can be used for the diagnosis and treatment of infectious diseases. Adding a group capable of binding to metal ions to the N-terminus of a peptide enables metal ion detection. Linking a chemical structure that enhances peptide stability to the C-terminus can improve the accuracy and reliability of detection. Many research findings demonstrate the wide application and enormous potential of peptide dual-terminal differential modification technology in the biomedical field.

[0008] Although there are many existing peptide modification technologies and many methods are relatively mature, differential modification of peptide ends still has obvious limitations: 1. Differential modification of ends requires stepwise execution: The conditions for two-end modification are incompatible, so it needs to be carried out separately, and each modification requires purification before the other end can be modified; 2. Peptide two-end modification lacks specificity: The current mainstream method is to couple the N-terminal amino group, but if there are multiple lysine residues in the peptide molecule, the amino residues on the lysine residues will also react, making differential modification impossible; 3. Multi-step operation, cumbersome process, time-consuming, labor-intensive, and costly.

[0009] In view of this, the present invention is hereby proposed. Summary of the Invention

[0010] The main objective of this invention is to provide a method and application for differential modification of peptides, in order to solve the problems in the prior art where differential modification of peptides at both ends requires stepwise purification, especially when lysine is present in the peptide molecule, differential modification cannot be achieved, and the process is cumbersome and costly.

[0011] To achieve the above objectives, according to one aspect of the present invention, a method for differential modification of peptides is provided, the method comprising: step S1, providing an aromatic aldehyde and a peptide, wherein the aromatic aldehyde and the peptide are mixed in a reaction solvent such that the aldehyde group of the aromatic aldehyde reacts with the N-terminus of the peptide to generate a dihydroimidazolone intermediate reaction solution; step S2, adding an alkaline substance and fluorosulfonyl azide (FSO2N3) to the dihydroimidazolone intermediate reaction solution to perform an azide reaction to obtain a double-terminated peptide reaction solution; wherein the structure of the aromatic aldehyde is shown in formula (I), the N-terminus of the peptide compound has a terminal group shown in formula (II), and the C-terminus of the peptide compound has at least one amino group (NH2).

[0012] *NH-CO-C(R2)-NH(R3) Formula (II)

[0013] R1 is independently selected from at least one of hydrogen, halogen, amino, hydroxyl, mercapto, cyano, nitro, substituted or unsubstituted C1-C20 alkylazido, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamine, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl.

[0014] When n≥2, multiple R1s can be the same or different; and when n=2 and Ar is a trivalent pyrazolium group, two R1s can connect with each other and together with the atoms on the pyrazolium ring to form substituted or unsubstituted 5-7 aryl, substituted or unsubstituted 5-7 heteroaryl, substituted or unsubstituted 5-7 cycloalkyl, or substituted or unsubstituted 5-7 heteroalkyl.

[0015] R2 is independently selected from at least one of hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamine, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl.

[0016] R3 is independently selected from at least one of hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamino, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl; and R2 and R3 may be connected to each other and together with the C atom and NH to form a substituted or unsubstituted 5-7 membered heterocyclic group.

[0017] Furthermore, Ar is selected from (n+1) valent phenyl, naphthyl, phenidyl, quinolinyl, pyridyl, pyrimidinyl, pyridazinyl, triazinyl, pyrazolyl, imidazoleyl, thiazolyl, oxazolyl, and selenozolyl.

[0018] Furthermore, Ar is selected from (n+1) valent phenyl, pyridyl, naphthyl, imidazolyl, and pyrazolyl.

[0019] Furthermore, R1 is selected from C1-C6 alkylazido or C1-C6 alkyl, and the C atom in the alkyl group may be selectively replaced by an S atom or an O atom.

[0020] Furthermore, R2 is selected from C1-C6 alkyl groups.

[0021] Furthermore, R3 is selected from hydrogen.

[0022] Furthermore, R4 is selected from C1-C6 alkylene groups.

[0023] Furthermore, the aromatic aldehyde is selected from at least one of the following structural compounds:

[0024] Furthermore, the N-terminal terminal group of the polypeptide compound is selected from at least one of the following structures:

[0025] Furthermore, the C-terminus of the polypeptide compound has at least one of the following terminal groups:

[0026] Further, in step S1, the molar ratio of the polypeptide to the aromatic aldehyde compound is 100:1-1:1000, preferably 1:2-1:200.

[0027] Furthermore, the reaction solvent is selected from at least one of PBS solution, water, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, and ethanol, preferably a mixed solution of PBS solution and dimethyl sulfoxide, and more preferably the volume ratio of PBS solution to dimethyl sulfoxide is 1-2:1.

[0028] Furthermore, the reaction temperature between the aldehyde group of the aromatic aldehyde and the N-terminus of the polypeptide is 0-100℃, 35-40℃, and the reaction time is 2-16h, preferably 8-10h.

[0029] Further, in step S2, the alkaline substance includes at least one of cesium carbonate, cesium bicarbonate, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, lithium carbonate, lithium phosphate, dipotassium hydrogen phosphate, sodium phosphate, disodium hydrogen phosphate, DBU, and imidazole, preferably potassium bicarbonate or sodium carbonate.

[0030] Furthermore, the molar ratio of the alkaline substance to the polypeptide is 0.1-10000, preferably 2-10.

[0031] Furthermore, the molar ratio of FSO2N3 to the polypeptide is 0.1-10000, preferably 2-10.

[0032] Furthermore, the temperature of the azide reaction is 0-100℃, preferably 10-40℃, and the reaction time is 5-60 min, preferably 20-40 min.

[0033] Furthermore, the above method also includes step S3, which involves purifying the modified peptide reaction solution to obtain the modified peptide.

[0034] Furthermore, the purification method is liquid chromatography separation.

[0035] According to a second aspect of the present invention, a modified polypeptide obtained by the differential modification method of the polypeptide provided in the first aspect is provided.

[0036] According to a third aspect of the present invention, the application of the above-described differential modification method of peptides in the fields of drug molecule synthesis, development of biological probes and biomarkers, or sequencing equipment is provided.

[0037] By applying the technical solution of this application, the differential modification method for peptides provided by this application utilizes the reactivity differences of the two-terminal groups of peptides to achieve differential modification of the N-terminus and C-terminus of peptides in a one-pot two-step process under similar pH conditions. In particular, it modifies different groups at both ends of the amino groups of peptides with lysine at the C-terminus, thereby improving specificity. The reaction conditions are simple, the route is short, the operation is convenient, and it is easy to realize large-scale production and save costs. Moreover, the reaction conditions are mild, which is particularly suitable for unprotected natural peptides. This is beneficial for the direct detection, modification and transformation of natural peptide compounds, and thus has great potential application value in the fields of drug molecule synthesis, development of biological probes and biolabeling reagents or sequencing equipment. Detailed Implementation

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0039] As analyzed in the background section of this application, many existing peptide modification techniques exist, but all have significant limitations. For example, the acid-base differences between the N-terminal and C-terminal modification conditions of peptide compounds make pH adjustment difficult, requiring stepwise modifications. Furthermore, N-terminal modification reagents can modify the C-terminus or the side chains of peptide intermediates. Especially when lysine residues are present in the peptide molecule, the C-terminal modification is affected by the previous N-terminal modification, leading to a decrease in conversion rate. Additionally, the process is cumbersome, time-consuming, labor-intensive, and costly. To address these issues, this application provides a method for differentiated peptide modification at both ends and its application.

[0040] In a first typical embodiment of this application, a method for differential modification of a peptide is provided. The method includes: step S1, providing an aromatic aldehyde and a peptide; mixing the aromatic aldehyde and the peptide in a first organic solvent such that the aldehyde group of the aromatic aldehyde reacts with the N-terminus of the peptide to generate a dihydroimidazolium intermediate reaction solution; step S2, adding an alkaline substance and FSO2N3 (fluorosulfonyl azide) to the dihydroimidazolium intermediate reaction solution to perform an azide reaction to obtain a modified peptide reaction solution; wherein, the structure of the aromatic aldehyde is shown in formula (I); the N-terminus has a terminal group shown in formula (II); and the C-terminus has at least one amino group (NH2).

[0041] *NH-CO-C(R2)-NH(R3) Formula (II)

[0042] Wherein, n is an integer between 1 and 5, such as 1, 2, 3, 4, or 5; Ar is a (n+1) valence C6-C10 aryl or C3-C9 heteroaryl; R1 is independently selected from hydrogen, halogen, amino, hydroxyl, mercapto, cyano, nitro, substituted or unsubstituted C1-C20 alkylazido, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted At least one of the following: C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamine, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl; when n≥2, the plurality of R1s can be the same or different; and when n=2, and Ar is a trivalent pyrazolyl group, two R1s can be connected to each other and together with atoms on the pyrazolyl ring to form substituted or unsubstituted 5-7-membered aryl, substituted or unsubstituted 5-7-membered heteroaryl, substituted or unsubstituted 5-7-membered cycloalkyl, and substituted or unsubstituted 5-7-membered heterocycloalkyl; R2 is independently selected. At least one of the following: hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamine, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl. An example in which R3 is independently selected from at least one of hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamino, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl; and R2 and R3 may be connected to each other and together with the C atom and NH to form a substituted or unsubstituted 5-7 membered heterocyclic group.

[0043] In this application, "substituted or unsubstituted" means substituted by one or more substituents selected from the following: deuterium; halogen group; nitrile group; nitro group; hydroxyl group; carbonyl group; ester group; imide group; amino group; phosphine oxide group; alkoxy group; aryloxy group; alkyl thio group; aryl thio group; alkyl sulfonyl group; silyl group; boron group; alkyl group; cycloalkyl group; alkenyl group; aryl group; aralkyl group; arylenyl group; alkylaryl group; alkylamine group; aralkylamine group; heteroarylamine group; arylamine group; arylphosphine group; or heterocyclic group containing at least one of N, O, Se, P, B, Si, As and S, or without substituents, or substituted by substituents linked by two or more substituents of the exemplified substituents, or without substituents. Specifically, when R1 and R2 are independently substituted or unsubstituted C1-C10 amide groups, the O atom can be selectively substituted by the N or S atom, and the C atom can be selectively substituted by the N, S, or O atom; when R1 and R2 are independently substituted or unsubstituted C1-C10 alkyl groups, the C atom can be substituted by the N, S, or O atom.

[0044] In this application, "C1-C10 alkyl azide" refers to "C1-C10 alkyl-N3".

[0045] In this application, "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon derived from aromatic hydrocarbons, such as phenyl or naphthyl. "Heteroaryl" refers to an aryl monocyclic or fused polycyclic aromatic hydrocarbon containing at least one heteroatom selected from the group consisting of N, O, Se, P, B, Si, As, and S, such as furanyl, pyrroleyl, pyrazolyl, thiazolyl, and oxazolyl.

[0046] In this application, "heterocyclic alkyl" refers to a monocyclic or fused polycyclic compound containing a ring backbone atom selected from the group consisting of N, O, Se, P, B, Si, As, and S, such as tetrahydropyrrole, piperidine, tetrahydrofuran, tetrahydropyran, dihydrofuran, biotin, or imidazolidinone.

[0047] Furthermore, in this application, the range of carbon atom numbers can be extended from the lower limit to the upper limit, such as C1-C10, which means that the number of carbon atoms can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0048] In this application, "C1-C10 alkyl" can be either straight-chain alkyl or branched-chain alkyl, and other groups can also be straight-chain or branched, which will not be elaborated here.

[0049] In this application, when R2 and R3 can be connected to each other and together with C atoms and NH to form substituted or unsubstituted 5-7 membered heterocyclic groups, the ring backbone contains 1-5 atoms independently selected from oxygen (O), sulfur (S), selenium (Se), nitrogen (N), phosphorus (P), arsenic (As), boron (B), and silicon (Si). Similarly, when Ar is a trivalent pyrazolium group, the two R1s can be connected to each other and together with atoms on the pyrazolium ring to form substituted or unsubstituted 5-7 membered cycloalkyl groups, or substituted or unsubstituted 5-7 membered heterocyclic cycloalkyl groups, the ring backbone contains 1-5 atoms independently selected from oxygen (O), sulfur (S), selenium (Se), nitrogen (N), phosphorus (P), arsenic (As), boron (B), and silicon (Si).

[0050] By applying the technical solution of this application, the differential modification method for peptides provided in this application utilizes the reactivity differences of the two-terminal groups of peptides to achieve differential modification of the N-terminus and C-terminus of peptides in a one-pot two-step process under similar pH conditions. In particular, it modifies different groups at both ends of the amino groups of peptides with lysine at the C-terminus, thereby improving specificity. The reaction conditions are simple, the route is short, the operation is convenient, and it is easy to realize large-scale production and save costs. Moreover, the reaction conditions are mild, making it particularly suitable for unprotected natural peptides. This is beneficial for the direct detection, modification, and transformation of natural peptide compounds, and thus has great potential application value in the fields of drug molecule synthesis, biological probes, and biolabeling reagent development.

[0051] In some embodiments, the Ar in the aromatic aldehyde is selected from (n+1) valent phenyl, naphthyl, phenidyl, quinolinyl, pyridyl, pyrimidinyl, pyridazinyl, triazinyl, pyrazolyl, imidazoleyl, thiazolyl, oxazolyl, and selenozolyl. In particular, when Ar is selected from (n+1) valent phenyl, pyridyl, naphthyl, imidazoleyl, or pyrazolyl, it is more conducive to reacting with the N-terminus of the polypeptide compound.

[0052] In some embodiments, R1 is a C1-C6 alkyl azide group, such as *CH3-N3, *CH3CH2-N3, *CH3CH2CH2-N3, *CH3CH(CH3)-N3, etc.; or, R1 is a C1-C6 alkyl group, and the C atom in the alkyl group can be selectively replaced by an S atom or an O atom, such as R1 being methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, *-CH2-SS-CH2-CH3, etc., to improve the reaction efficiency of aromatic aldehydes.

[0053] In some embodiments, R2 is a C1-C6 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, or isopentyl; R3 is H; or when R2 and R3 are connected to each other and together with C atoms and NH to form substituted or unsubstituted 5-7 membered heterocyclic groups, it is more conducive to the N-terminus of the polypeptide compound reacting with aromatic aldehydes.

[0054] In some embodiments, R4 is a C1-C6 alkylene group, such as methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, etc., to facilitate the introduction of an azide group at the C-terminus of the polypeptide compound and improve reaction efficiency.

[0055] In some specific embodiments, the aromatic aldehyde is selected from at least one of the following structural compounds:

[0056] In some specific embodiments, the N-terminal terminal group of the polypeptide compound is selected from at least one of the following structures:

[0057] In some specific embodiments, the C-terminus of the polypeptide compound has at least one of the following terminal groups:

[0058] In step S1 above, in order to further improve the modification efficiency of the N-terminus of the peptide, the preferred molar ratio of peptide to aromatic aldehyde is 100:1-1:1000, especially when the molar ratio of peptide compound to aromatic aldehyde is 1:2-1:100, it is more conducive to improving the reaction solution of dihydroimidazolidinone intermediates.

[0059] Typical, but not limiting, molar ratios of polypeptides to aromatic aldehydes are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:80, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, or any range of two such values.

[0060] The reaction solvents used to provide the reaction environment for the reaction between aromatic aldehydes and peptides are not specifically limited, and include, but are not limited to, any one or a mixture of two or more of the following: PBS solution, water, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, and ethanol. In particular, when the reaction solvent is a mixture of PBS solution and dimethyl sulfoxide, especially when the volume ratio of PBS solution to dimethyl sulfoxide is 1-2:1, it is more conducive to improving the efficiency of the reaction between the aldehyde group of the aromatic aldehyde and the N-terminus of the peptide.

[0061] To further improve the efficiency of the reaction between the aldehyde group of an aromatic aldehyde and the N-terminus of a polypeptide, the preferred reaction temperature is 0-100℃, more preferably 35-40℃, and the preferred reaction time is 2-16h, more preferably 8-10h. Specifically, the reaction temperature can be any range of 0℃, 5℃, 10℃, 20℃, 30℃, 32℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 50℃, 60℃, 80℃, 100℃, or any combination of two of these values, and the reaction time can be any range of 2h, 3h, 4h, 4.5h, 5h, 5.5h, 6h, 8h, 10h, 12h, 16h, or any combination of two of these values.

[0062] In step S2 above, to further improve the efficiency of the azide reaction, an alkaline substance is added to the reaction solution of dihydroimidazolone intermediates. The alkaline substance includes, but is not limited to, any one or a mixture of two or more of cesium carbonate, cesium bicarbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, lithium carbonate, lithium phosphate, dipotassium hydrogen phosphate, sodium phosphate, disodium hydrogen phosphate, DBU (1,8-diazabicyclo[5,4,0]undec-7-ene), and imidazole. In particular, when the alkaline substance is sodium bicarbonate, dipotassium bicarbonate, or cesium bicarbonate, it is more conducive to improving the efficiency of the azide reaction.

[0063] In some embodiments, the molar ratio of the alkaline substance to the polypeptide compound is 0.1-10000, such as 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1000, 2000, 5000, 8000, 10000, or any range of two values; especially when the molar ratio of the alkaline substance to the polypeptide compound is 2-10, it is more conducive to improving the efficiency of the azide reaction.

[0064] In step S2 above, in order to further improve the efficiency of C-terminal modification of the polypeptide compound, the molar ratio of FSO2N3 to the polypeptide compound is preferably 0.1-10000, such as 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 800, 1000, 2000, 5000, 8000, 10000 or any range of two values; especially when the molar ratio of FSO2N3 to the polypeptide compound is 2-10, it is more conducive to improving the C-terminal modification efficiency of the polypeptide compound.

[0065] In some embodiments, the temperature for the azide reaction is 0-100°C, preferably 10-40°C, and the reaction time is 5-60 min, preferably 20-40 min, to further promote the azide reaction and improve the conversion rate of the double-terminated modified peptides. Specifically, the temperature for the azide reaction is such as 0°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 40°C, 50°C, 60°C, 80°C, 100°C, or any range of two such values; the reaction time is such as 5 min, 10 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, 45 min, 50 min, 55 min, 60 min, or any range of two such values.

[0066] In some embodiments, the above-described differential modification method for peptides further includes step S3, purifying the modified peptide reaction solution to obtain the modified peptide. Specifically, the purification method is not limited, but choosing to purify the modified peptide reaction solution using liquid chromatography is particularly beneficial for further improving the purity and separation efficiency of the modified peptide product, further simplifying the operation steps, and reducing economic costs.

[0067] For example, taking a peptide compound with a C-terminus lysine residue as an example, its dual-terminal differential modification method includes the following steps: Step 1: N-terminal specific modification of the peptide: Using a lysine C-terminal restriction endonuclease to selectively digest the protein to obtain a peptide with a C-terminus lysine residue as a template peptide, peptide P2 undergoes a nucleophilic reaction with P1 (a heteroaromatic aldehyde) to generate an imine. Subsequently, under alkaline conditions, the N-pair imine of the ortho-amide undergoes a nucleophilic reaction to convert to dihydroimidazolone P3, completing the N-terminal amino-specific modification of the peptide. The reaction process can be represented as follows:

[0068] The specific operating method is as follows: Add a solvent (preferably a mixture of one or more of the following: PBS solution, water, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, ethanol, etc., with a volume ratio of PBS and dimethyl sulfoxide of 1:1) to a reaction tube. Then add peptide P2 and reagent P1 (the ratio of peptide P2 to reagent P1 can be any ratio from 100:1 to 1:1000, preferably any ratio from 1:2 to 1:200). React at 37°C for 5 hours to obtain intermediate C. No post-treatment is required; it can be directly used for the next conversion step.

[0069] The second step is specific modification of the C-terminal amino group of the peptide: After obtaining the key intermediate P3, the C-terminal amino group of intermediate P3 is azidiated to obtain the modified peptide P4. The reaction process can be represented as follows:

[0070] The specific operating method is as follows: Add a base (selected from cesium carbonate, cesium bicarbonate, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, lithium carbonate, potassium phosphate, dipotassium hydrogen phosphate, sodium phosphate, disodium hydrogen phosphate, DBU, imidazole, preferably potassium bicarbonate or sodium carbonate; the amount of base added is any ratio from 0.1 to 10,000 equivalents, preferably any ratio from 2 to 10 equivalents) and FSO2N3 (the amount of FSO2N3 added is any ratio from 0.1 to 10,000 equivalents, preferably any ratio from 2 to 10 equivalents) directly to the dihydroimidazolidinone P3 reaction solution obtained in the first step reaction, and react at room temperature for 5-30 minutes to obtain the target modified polypeptide P4 reaction solution.

[0071] The third step is to separate and purify the modified peptide P4 reaction solution by liquid chromatography to obtain pure P4.

[0072] In a second typical embodiment of this application, the application of the above-mentioned method for differential modification of peptide ends is also provided in the fields of drug molecule synthesis, biological probe and biomarker reagent development.

[0073] By applying the technical solution of this application, the method for differential modification of peptide ends provided by this application utilizes the reactivity differences of peptide end groups to achieve differential modification of the N-terminus and C-terminus of peptides in a one-pot, two-step process under similar pH conditions. In particular, it modifies different groups at both ends of the amino groups of peptides with lysine at the C-terminus, thereby improving specificity. The method is not only simple in reaction conditions, short in route, convenient in operation, and easy to achieve large-scale production and save costs, but also has mild reaction conditions, making it particularly suitable for unprotected natural peptides. This is beneficial for the direct detection, modification, and transformation of natural peptide compounds, and thus has great potential application value in the fields of drug molecule synthesis, biological probes, and biolabeling reagent development.

[0074] The beneficial effects of this application will be further illustrated below with reference to embodiments and comparative examples.

[0075] Example 1

[0076] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0077] The "equiv." mentioned above refers to "equivalence".

[0078] The specific reaction conditions were as follows: 1.0 mL of PBS (pH = 7.5) solution was added to a sterile centrifuge tube, followed by pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC1 (50 μL, 100 mM, 5.0 equiv.). The mixture was incubated at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC1-1. LC-MS: m / z [M+H] + Theoretical value: 711.36, found[M+H] + (Measured value): 711.47; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC1-1-N3 was 92% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 737.35, found[M+H] + (Measured value): 737.31.

[0079] Example 2

[0080] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0081] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and modification reagent PC2 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC2-1. LC-MS: m / z [M+H] + Theoretical calcd: 766.38, found[M+H] + (Measured value): 766.41; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC2-1-N3 was 90% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 792.37, found[M+H] + (Measured value): 792.42.

[0082] Example 3

[0083] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0084] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and modification reagent PC3 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC3-1. LC-MS: m / z [M+H] + Theoretical calcd: 817.35, found[M+H] + (Measured value): 817.45; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC3-1-N3 was 88% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 843.34, found[M+H] + (Measured value): 843.32.

[0085] Example 4

[0086] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0087] 1.0 mL of PBS (pH = 7.5) solution was added to a sterile centrifuge tube, followed by pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC4 (50 μL, 100 mM, 5.0 equiv.). The mixture was incubated at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC4-1. LC-MS: m / z [M+H] + Theoretical value: 711.36, found[M+H] + (Measured value): 711.47; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC4-1-N3 was 85% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 737.35, found[M+H] + (Measured value): 737.31.

[0088] Example 5

[0089] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0090] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC5 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC5-1. LC-MS: m / z [M+H] + Theoretical value: 711.36, found[M+H] + (Measured value): 711.47; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC5-1-N3 was 76% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 737.35, found[M+H] + (Measured value): 737.31.

[0091] Example 6

[0092] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0093] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and modification reagent PC6 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC6-1. LC-MS: m / z [M+H] + Theoretical value: 712.36, found[M+H] + (Measured value): 712.37; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC6-1-N3 was 91% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 738.35, found[M+H]+ (Measured value): 738.33.

[0094] Example 7

[0095] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0096] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and modification reagent PC7 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC7-1. LC-MS: m / z [M+H] + Theoretical value: 712.36, found[M+H] + (Measured value): 712.41; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC7-1-N3 was 86% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 738.35, found[M+H] + (Measured value): 738.36.

[0097] Example 8

[0098] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0099] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and modification reagent PC8 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC8-1. LC-MS: m / z [M+H] + Theoretical calcd: 713.35, found[M+H] + (Measured value): 713.40; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC8-1-N3 was 53% (purity >99%). LC-MS: m / z [M+H]+ Theoretical calcd: 739.34, found[M+H] + (Measured value): 739.36.

[0100] Example 9

[0101] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0102] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC9 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC9-1. LC-MS: m / z [M+H] + Theoretical calcd: 735.36, found[M+H] + (Measured value): 735.37; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC9-1-N3 was 75% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 761.35, found[M+H] + (Measured value): 761.39.

[0103] Example 10

[0104] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0105] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC10 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC10-1. LC-MS: m / z [M+H] + Theoretical calcd: 735.36, found[M+H] +(Measured value): 735.41; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC10-1-N3 was 68% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 761.35, found[M+H] + (Measured value): 761.34.

[0106] Example 11

[0107] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0108] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC11 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC11-1. LC-MS: m / z [M+H] + Theoretical calcd: 755.35, found[M+H] + (Measured value): 755.37; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC11-1-N3 was 56% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 781.34, found[M+H] + (Measured value): 781.37.

[0109] Example 12

[0110] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0111] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC12 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC12-1. LC-MS: m / z [M+H] +Theoretical calcd: 778.35, found[M+H] + (Measured value): 778.39; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC12-1-N3 was 69% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 804.34, found[M+H] + (Measured value): 804.35.

[0112] Example 13

[0113] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0114] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC13 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC13-1. LC-MS: m / z [M+H] + Theoretical calcd: 846.34, found[M+H] + (Measured value): 846.41; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC13-1-N3 was 67% (purity >99%). LC-MS: m / z [M+H] + Theoretical value: 872.33, found[M+H] + (Measured value): 872.37.

[0115] Example 14

[0116] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0117] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC14 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC14-1. LC-MS: m / z [M+H] + Theoretical calcd: 800.32, found[M+H] + (Measured value): 800.37; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC14-1-N3 was 81% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 826.31, found[M+H] + (Measured value): 826.30.

[0118] Example 15

[0119] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0120] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC15 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC15-1. LC-MS: m / z [M+H] + Theoretical calcd: 777.38, found[M+H] + (Measured value): 777.39; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC15-1-N3 was 88% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 803.37, found[M+H] + (Measured value): 803.41.

[0121] Example 16

[0122] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0123] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC16 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC16-1. LC-MS: m / z [M+H] + Theoretical calcd: 791.40, found[M+H] + (Measured value): 791.44; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC16-1-N3 was 72% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 817.39, found[M+H] + (Measured value): 817.43.

[0124] Example 17

[0125] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0126] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 1 (AAWFK, 50 μL, 20 mM) and the modifying reagent PC17 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC17-1. LC-MS: m / z [M+H] + Theoretical calcd: 778.38, found[M+H] + (Measured value): 778.41; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC17-1-N3 was 51% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 804.37, found[M+H] + (Measured value): 804.40.

[0127] Example 18

[0128] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0129] 1.0 mL of PBS (pH = 7.5) solution was added to a sterile centrifuge tube, followed by decapeptide 2 (PQGGACAGLK, 50 μL, 20 mM) and the modifying reagent PC2 (50 μL, 100 mM, 5.0 equiv.). The mixture was incubated at 37 °C with shaking for 10 h to obtain the target polypeptide intermediate PC2-2. LC-MS: m / z [2M+2H] 2+ calcd (theoretical value): 1044.49, 1044.99, found[2M+2H] 2+ (Measured value): 1044.81; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC2-2-N3 was 91% (purity >99%). LC-MS: m / z [2M+2H] 2+ calcd (theoretical value): 1070.48, 1070.98, found[2M+2H] 2+ (Measured value): 1070.94.

[0130] Example 19

[0131] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0132] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add decapeptide 2 (PQGGACAGLK, 50 μL, 20 mM) and modification reagent PC1 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC1-2. LC-MS: m / z [2M+2H] 2+ Theoretical value: 989.98.48, found[2M+2H] 2+(Measured value): 990.18; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC1-2-N3 was 91% (purity >99%). LC-MS: m / z [2M+2H] 2+ calcd (theoretical value): 1015.47, found[2M+2H] 2+ (Measured value): 1015.76; Subsequently, dibenzocyclooctylcarboxylic acid (40 μL, 50 mM) was added for a click reaction, and the reaction was carried out with shaking at room temperature for 2 hours. HPLC analysis showed that the conversion rate of PC1-2-DBCO was 89%, and LC-MS showed m / z [2M+2H]. 2+ Theoretical value: 1321.07, found[2M+2H] 2+ (Measured value): 1320.74.

[0133] Example 20

[0134] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0135] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add pentapeptide 3 (YYEDK, 50 μL, 20 mM) and the modifying reagent PC2 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC2-3. LC-MS: m / z [M+H] + Theoretical calcd: 861.35, found[M+H] + (Measured value): 861.37; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equivalent) and FSO2N3 (30 μL, 100 mM, 3.0 equivalent) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC2-3-N3 was 93% (purity >99%). LC-MS: m / z [M+H]+calcd (theoretical value): 887.34, found [M+H] + (Measured value): 887.38.

[0136] Example 21

[0137] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0138] Add 1.0 mL of PBS (pH = 7.5) solution to a sterile centrifuge tube, then add tetradecapeptide 4 (RSVLDNRARFRGRK, 50 μL, 20 mM) and the modifying reagent PC2 (50 μL, 100 mM, 5.0 equiv.). Incubate at 37 °C with shaking for 10 h to obtain the target peptide intermediate PC2-4. LC-MS: m / z [M+3H] 3+ calcd:625.69,found:625.96; LC-MS:m / z[M+4H] 4+ Theoretical calcd: 469.51, found[M+4H] 4+ (Measured value): 469.79; No separation was required. KHCO3 (50 μL, 100 mM, 5.0 equiv.) and FSO2N3 (30 μL, 100 mM, 3.0 equiv.) were added directly, and the reaction was continued with shaking at room temperature for 30 min. HPLC analysis showed that the conversion rate of the target peptide PC2-4-N3 was 90% (purity >99%). LC-MS: m / z [M+2H] 2+ Theoretical calcd: 951.02, found[M+2H] 2+ (Measured value): 951.01; LC-MS: m / z [M+3H] 3+ Theoretical calcd: 634.35, found [M+3H] 3+ (Measured value): 634.64.

[0139] Example 22

[0140] This embodiment provides a method for differential modification of peptides, the reaction process of which is as follows:

[0141] The nucleic acid sequence O1 (GCTTCTCGTGXXTTTTTTTTCTCTC: the sequence shown indicates that the 3' end of the nucleic acid sequence O1 has a dibenzocyclooctynyl modification, and X is a deoxynucleoside without a base) was dissolved in 250 mM phosphate solution (pH = 5.5) to prepare a concentration of 200 μM. An equal volume of the modification reagent PC2 (500 μM, 2.5 equiv.) was added. After reacting at room temperature for 2 hours, the mixture was desalted and purified using a nucleic acid purification column (NEB T1030L). After lyophilization, the intermediate O1-PC2 was obtained with a conversion rate of 83% (purity > 95%). LC-MS: m / z [M+H] + calcd (theoretical value): 8047.0, found[M+H] +(Measured value): 8047.0; 100 μL of PBS (pH = 7.5) solution was added to a sterile centrifuge tube, followed by tetrapeptide 5 (WMFK, 10 μL, 20 μM) and intermediate O1-PC2 (10 μL, 100 μM, 5.0 equiv.). The mixture was incubated at 37 °C with shaking for 10 h to obtain the target polypeptide intermediate O1-PC2-5, with a conversion rate of 75%. LC-MS: m / z [M+H] + Theoretical calcd: 8639.9, found[M+H] + (Measured value): 8638.7. The reaction solution did not require separation and purification; KHCO3 (10 μL, 100 μM, 5.0 equiv.) and FSO2N3 (20 μL, 30 μM, 3.0 equiv.) were directly added. After shaking for 30 min at room temperature, the reaction solution was desalted and purified using a nucleic acid purification column (NEB T1030L). Lyophilization yielded the target peptide O1-PC2-5-N3 with a conversion rate of 97% (purity >99%). LC-MS: m / z [M+H] + Theoretical calcd: 8665.9, found[M+H] + (Measured value): 8665.7.

[0142] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The differential modification method for peptides provided in this application utilizes the reaction differences of the two-terminal groups of peptides to achieve differential modification of the N-terminus and C-terminus of peptides in a one-pot two-step method under similar pH conditions. In particular, it modifies different groups at both ends of the amino groups of peptides with lysine at the C-terminus, thereby improving specificity. The reaction conditions are simple, the route is short, the operation is convenient, and it is easy to achieve large-scale production and save costs. Moreover, the reaction conditions are mild, which is particularly suitable for unprotected natural peptides. This is beneficial for the direct detection, modification and transformation of natural peptide compounds, and thus has great potential application value in the fields of drug molecule synthesis, biological probes and biolabeling reagent development.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for differential modification of peptides, characterized in that, The method includes: Step S1: Provide an aromatic aldehyde and a polypeptide, wherein the aromatic aldehyde and the polypeptide are mixed in a reaction solvent such that the aldehyde group of the aromatic aldehyde reacts with the N-terminus of the polypeptide to generate a dihydroimidazolone intermediate reaction solution. Step S2: Add an alkaline substance and fluorosulfonyl azide to the dihydroimidazolide intermediate reaction solution to carry out an azide reaction to obtain the modified peptide reaction solution. The structure of the aromatic aldehyde is shown in formula (I): The N-terminus of the polypeptide has a terminal group as shown in formula (II): *NH-CO-C(R2)-NH(R3) Formula (II) The C-terminus of the polypeptide has at least one amino group; Where n is an integer between 1 and 5; Ar is a (n+1) valence C6-C10 aryl or C3-C9 heteroaryl; R1 is independently selected from at least one of hydrogen, halogen, amino, hydroxyl, mercapto, cyano, nitro, substituted or unsubstituted C1-C20 alkylazido, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamine, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. When n≥2, multiple R1s can be the same or different; and when n=2 and Ar is a trivalent pyrazolium group, two R1s can connect with each other and together with the atoms on the pyrazolium ring to form substituted or unsubstituted 5-7 aryl, substituted or unsubstituted 5-7 heteroaryl, substituted or unsubstituted 5-7 cycloalkyl, or substituted or unsubstituted 5-7 heteroalkyl. R2 is independently selected from hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 amide, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamine, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. At least one of the following: R3 is independently selected from at least one of hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 alkylthio, substituted or unsubstituted C1-C10 alkylamino, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 aryloxy, substituted or unsubstituted C6-C10 arylthio, substituted or unsubstituted C6-C10 arylamino, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl; and R2 and R3 may be connected to each other and together with the C atom and NH to form a substituted or unsubstituted 5-7 membered heterocyclic group.

2. The method for differential modification of peptides according to claim 1, characterized in that, The Ar is selected from (n+1) valent phenyl, naphthyl, phenidyl, quinolinyl, pyridyl, pyrimidinyl, pyridazinyl, triazinyl, pyrazolyl, imidazoleyl, thiazolyl, oxazolyl, and selenozolyl; Preferably, Ar is selected from (n+1) valent phenyl, pyridyl, naphthyl, imidazole, and pyrazolyl.

3. The method for differential modification of peptides according to claim 1, characterized in that, R1 is selected from C1-C6 alkylazido or C1-C6 alkyl, and the C atom in the alkyl group may be selectively replaced by S or O atoms; And / or, R2 is selected from C1-C6 alkyl groups; And / or, R3 is selected from hydrogen.

4. The method for differential modification of peptides according to claim 1, characterized in that, The aromatic aldehyde is selected from at least one of the following structural compounds:

5. The method for differential modification of peptides according to claim 1, characterized in that, The N-terminal terminal group of the polypeptide compound is selected from at least one of the following structures: And / or, the C-terminus of the polypeptide compound has at least one of the following terminal groups:

6. The method for differential modification of peptides according to claim 1, characterized in that, In step S1, the molar ratio of the polypeptide to the aromatic aldehyde is 100:1-1:1000, preferably 1:2-1:

200. And / or, the reaction solvent is selected from at least one of PBS solution, water, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, methanol, and ethanol, preferably a mixed solution of PBS solution and dimethyl sulfoxide, and more preferably the volume ratio of PBS solution to dimethyl sulfoxide is 1-2:1; And / or, the temperature at which the aldehyde group of the aromatic aldehyde reacts with the N-terminus of the polypeptide is 0-100°C, preferably 35-40°C; the reaction time is 2-16 h, preferably 8-10 h.

7. The method for differential modification of peptides according to claim 1, characterized in that, In step S2, the alkaline substance includes at least one of cesium carbonate, cesium bicarbonate, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, lithium carbonate, lithium phosphate, dipotassium hydrogen phosphate, sodium phosphate, disodium hydrogen phosphate, DBU, and imidazole, preferably potassium bicarbonate or sodium carbonate. And / or, the molar ratio of the alkaline substance to the polypeptide is 0.1-10000, preferably 2-10.

8. The method for differential modification of peptides according to claim 1, characterized in that, The molar ratio of fluorosulfonyl azide to the polypeptide compound is 0.1-10000, preferably 2-10; And / or, the temperature of the azide reaction is 0-100℃, preferably 10-40℃; the time of the azide reaction is 5-60 min, preferably 20-40 min.

9. The method for differential modification of peptides according to any one of claims 1 to 8, characterized in that, The method further includes step S3, which involves purifying the modified polypeptide reaction solution to obtain the modified polypeptide; Preferably, the purification method is liquid chromatography separation.

10. A modified polypeptide obtained by a differential modification method of any one of claims 1 to 9.

11. A method for differentially modifying a polypeptide according to any one of claims 1 to 9, or the application of the modified polypeptide according to claim 10 in the fields of drug molecule synthesis, development of biological probes and biomarkers, or sequencing equipment.

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