A method and system for identifying the structure of a polypeptide drug containing modified side chains

Abstract

The application discloses a kind of methods and systems for identifying the overall structure of polypeptide drug containing modified side chain, belong to polypeptide drug analysis technical field.The present application is to solve the technical problem that there is no identification scheme for the overall spatial structure of polypeptide drug containing modified side chain in the prior art, by collecting polypeptide drug spectrum data and carrying out main body analysis;Based on the main body attribution result, identify and attribute side chain atoms, determine their covalent structure;Further analyze the spatial correlation signal and the connection region structure signal, respectively determine the side chain spatial structure, the covalent connection relationship between side chain and main body and the relative spatial structure relationship;Finally, the above information is integrated to realize the identification of the overall three-dimensional structure of the drug.The present application can realize the accurate characterization of the complete spatial structure of polypeptide drug containing modified side chain, and provide core technical support for drug quality control, consistency evaluation and pharmacological mechanism research.

Description

Technical Field

[0001] This invention belongs to the field of peptide drug analysis technology, and particularly relates to a method and system for identifying modified side chains of peptide drugs based on high-field nuclear magnetic resonance technology. Background Technology

[0002] Currently, over 7,000 natural peptides have been identified as playing crucial roles in physiological functions such as human hormone activity, neurotransmission, growth factors, ion channel ligands, and anti-infection. Natural peptides offer advantages as drugs, including high safety, high tolerability, high efficacy, and lower product complexity. However, many natural peptides, when used alone, often exhibit drawbacks such as easy hydrolysis, easy aggregation, and poor duration of action. Therefore, side-chain modification is necessary to optimize their pharmaceutical properties. For example, blockbuster peptide biologics such as smegglutide and liraglutide are GLP-1 receptor agonists modified from human glucagon-like peptide-1 (GLP-1). Targeted modification can effectively enhance the drug's resistance to degradation and significantly prolong its duration of action.

[0003] The molecular structure of peptide biopharmaceuticals is the material basis for their efficacy, and accurate identification of molecular structure is a mandatory review item in the legal process of drug application. Therefore, establishing a systematic structural identification method for peptide biopharmaceuticals is of great practical necessity and significant application value. In the identification of peptide structures containing modified side chains, in addition to completing the structural analysis of the peptide body, a comprehensive identification of its side chain modification groups is also required. The structural identification of the peptide body can be carried out using existing mature technologies. Compared with the structural identification of conventional compounds, the identification of the side chain modification structure of peptide biopharmaceuticals is more complex, specifically in the following three aspects: First, it is necessary to follow the identification paradigm of conventional compounds to achieve accurate characterization of the covalent structure of the side chain modification groups; second, if the modification groups have characteristics such as large molecular weight and long carbon chain length, it is also necessary to further clarify their spatial conformation characteristics; third, it is necessary to focus on analyzing the connection mode between the side chain modification groups and the peptide body, as well as the relative spatial arrangement relationship between them. Only by integrating all the above identification information can the complete molecular structure of peptide drugs containing modified side chains be obtained. To date, there is still a lack of systematic identification methods for the side chain modification structures of such peptide biopharmaceuticals.

[0004] In the structural identification of peptide biopharmaceuticals, these substances generally exhibit the inherent characteristic of being difficult to crystallize, making structural analysis challenging using X-ray crystallography. Furthermore, their relatively low molecular weight also exceeds the effective detection range of cryo-electron microscopy. Based on these limitations, liquid-phase nuclear magnetic resonance (LPN) technology undoubtedly becomes the optimal technique for the structural identification of peptide biopharmaceuticals.

[0005] Furthermore, liquid-phase nuclear magnetic resonance (NMR) technology uses a liquid system as the detection environment, which can simulate the actual state of peptide biopharmaceuticals during production, transportation, and storage to the greatest extent possible, thereby significantly improving the accuracy and reliability of the detection results. By integrating one-dimensional and two-dimensional homonuclear and heteronuclear covalent correlation and spatial correlation experimental techniques of NMR, and combining them with existing mature NMR techniques in the field of peptide drug structure identification, a complete technical solution can be constructed that combines the identification of the main peptide structure with the identification of modified side chain structures, including the covalent structure, spatial conformation, and spatial arrangement of the modifying groups relative to the main peptide structure. This enables comprehensive structural identification of peptide drugs containing modified side chains. Summary of the Invention

[0006] The purpose of this invention is to provide a method and system for identifying the structure of peptide drugs with modified side chains based on high-field nuclear magnetic resonance technology, which can achieve the overall structural identification of the peptide body and the modified side chain portion.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A method for identifying the overall structure of a polypeptide drug containing modified side chains, comprising the following steps: One-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples were collected to obtain raw spectral data of all-atom nuclear magnetic resonance. Based on the original spectral data, the chemical shifts of the polypeptide host were assigned, and the three-dimensional structure of the polypeptide host was obtained through analysis. Based on the chemical shift assignment results of the polypeptide body, the chemical shifts of related atoms in the modified side chain are identified and assigned from the original spectral data, and the covalent structure of the modified side chain is determined. Based on the chemical shift assignment results of related atoms in the modified side chain, the spatial correlation spectrum in the original spectral data was analyzed to determine the spatial structure of the modified side chain; Based on the chemical shift assignments of related atoms in the polypeptide host and modified side chains, the structural signals of the linking regions are analyzed to determine the covalent linkage relationship between the modified side chains and the polypeptide host. Based on the chemical shift assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, the spatial distance constraint signal is analyzed to determine the relative spatial structure relationship between the modified side chain and the polypeptide host. Based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent linkage relationship, and the relative spatial structure relationship, the overall three-dimensional structure of the polypeptide drug is determined.

[0009] Furthermore, one-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectra of the peptide drug samples were acquired to obtain raw all-atom NMR spectral data, including: 1D of collecting peptide drug samples1 H spectrum, 2D 1 H- 13 C HSQC spectrum, 2D 1 H- 1 H TOCSY spectrum, 2D 1 H- 1 HNOESY spectra, as well as targeted heteronuclear spectra collected based on the types of elements contained in the side chains, are used to obtain raw spectral data of whole-atom nuclear magnetic resonance.

[0010] Furthermore, a high-field nuclear magnetic resonance spectrometer was used to acquire raw spectral data, with a proton resonance frequency of not less than 700 MHz, and equipped with an ultra-low temperature probe.

[0011] Furthermore, the targeted heteronuclear spectrum includes: 2D spectra collected when the side chain contains nitrogen. 1 H- 15 N HSQC spectrum.

[0012] Furthermore, the 2D 1 H- 13 C HSQC spectra include acquisition parameters set separately for aliphatic and aromatic regions; the 2D 1 H- 1 The mixing time for the H TOCSY spectrum was set to 80 ms; the 2D 1 H- 1 The mixing time of the H NOESY spectrum is dynamically adjusted within the range of 200~400 ms according to the molecular weight of the peptide drug.

[0013] Furthermore, based on the original spectral data, the chemical shifts of the polypeptide matrix were assigned, and the three-dimensional structure of the polypeptide matrix was obtained through analysis, including: Analyze the original spectral data to extract the chemical shift assignments and spatial distance constraints of the peptides, and extract dihedral constraints based on the chemical shift assignments; when assigning chemical shifts, use... 1 H- 1 H TOCSY, 1 H- 1 H COSY and 1 H- 13 CHSQC identifies amino acid residue types and chemical shifts, from 1 H- 1 In the H NOESY spectrum, all amino acid residues were identified, including the amino group H in the main chain and the α-positions of the preceding and following amino acids. 1 H's NOE information, combined with 1 H- 1 H NOESY experiments determine the sequence linkage information between amino acid residues; analyze 1 H-1 The distance constraint information between hydrogen atoms extracted from the H NOESY spectrum was used to calculate the initial structure of the peptide sample in vacuum, and the spatial structure of the peptide sample in the presence of solvent was obtained based on the initial structure. Based on the usage of the spatial structure's statistical distance constraints, the violation of the angle and distance constraints of the statistical structure is determined, and the root mean square deviation, energy data, and Laplace plot data of the structure are calculated to determine the three-dimensional structure of the polypeptide host.

[0014] Furthermore, based on the chemical shift assignment results of the polypeptide body, the chemical shifts of related atoms in the modified side chain are identified and assigned from the original spectral data, and the covalent structure of the modified side chain is determined, including: Based on the chemical shift assignment of the polypeptide host, using 1D 1 H spectrum, 2D 1 H- 13 C HSQC spectrum and 2D 1 H- 1 HTOCSY spectrum is used for signal cross-verification, and in 2D 1 H- 1 H NOESY spectroscopy was used for auxiliary verification. The main peptide signal was identified and subtracted from the original spectral data, and the chemical shifts of relevant atoms in the modified side chain were assigned to determine the covalent structure of the modified side chain.

[0015] Furthermore, based on the chemical shift assignments of related atoms in the modified side chains, the spatial correlation spectra in the original spectral data were analyzed to determine the spatial structure of the modified side chains, including: Based on the chemical shift assignments of atoms related to the modified side chains, the 2D... 1 H- 1 H NOESY spectra, including analysis of covalently topologically distant but three-dimensionally adjacent atomic pairs, to determine the spatial structure of modified side chains.

[0016] Furthermore, based on the chemical shift assignments of related atoms in the polypeptide host and modified side chains, the structural signals of the linker regions are analyzed to determine the covalent linkage between the modified side chains and the polypeptide host, including: Based on the chemical shift assignments of related atoms in the polypeptide host and the modified side chain, covalent bond connection signals or NOE spatial correlation signals between the terminal atoms of the polypeptide host side chain and the head atoms of the modified side chain are identified and extracted. The covalent connection relationship between the modified side chain and the polypeptide host is determined based on the extracted signals.

[0017] Furthermore, based on the chemical shift assignments of related atoms in the three-dimensional structure of the polypeptide host and the modified side chains, spatial distance constraint signals are analyzed to determine the relative spatial structural relationship between the modified side chains and the polypeptide host, including: Based on the 3D structure of the polypeptide host and the chemical shift assignments of related atoms in the modified side chains, the 2D structure was analyzed. 1 H- 1 H NOESY spectra were used to obtain information on the relative spatial constraints between the modified side chain and the polypeptide body, and to determine the relative spatial structural relationship between the modified side chain and the polypeptide body.

[0018] Furthermore, the overall three-dimensional structure of the peptide drug was determined, including: Based on the distance constraint data between all atoms, the optimal conformation is obtained, and a three-dimensional structural diagram of all atoms is drawn based on the optimal conformation.

[0019] A system for identifying the overall structure of a polypeptide drug containing modified side chains, comprising: High-field nuclear magnetic resonance spectrometer is used to acquire one-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples and obtain raw spectral data of all-atom nuclear magnetic resonance. The polypeptide matrix analysis module is used to assign chemical shifts of the polypeptide matrix based on the original spectral data and to analyze and obtain the three-dimensional structure of the polypeptide matrix. The side chain covalent identification module is used to identify and assign the chemical shifts of related atoms in the modified side chain from the original spectral data based on the chemical shift assignment results of the polypeptide body, and to determine the covalent structure of the modified side chain. The side chain conformation analysis module is used to analyze the spatial correlation spectrum in the original spectral data based on the chemical shift assignment results of the atoms related to the modified side chain, and to determine the spatial structure of the modified side chain. The covalent linkage identification module is used to analyze the structural signals of the linkage region based on the chemical shift assignment results of related atoms of the polypeptide host and the modified side chain, and to determine the covalent linkage relationship between the modified side chain and the polypeptide host. The relative spatial structure identification module is used to analyze the spatial distance constraint signal based on the chemical displacement assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, and to determine the relative spatial structure relationship between the modified side chain and the polypeptide host. The overall structure identification module is used to determine the overall three-dimensional structure of the polypeptide drug based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent connection relationship, and the relative spatial structure relationship.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: In existing technologies, only structural identification methods exist for peptide drugs without modified side chains; no structural identification technology has been established applicable to peptide drugs containing modified side chains. This invention proposes a novel method for structural identification of peptide drugs containing modified side chains based on high-field nuclear magnetic resonance (NMR) technology. Building upon mature techniques for three-dimensional structural analysis of the peptide matrix and characterization of the covalent structure of the modified side chains, this method further adds identification steps for the spatial conformation of the modified side chains, the connection mode between the modified side chains and the peptide matrix, and their relative spatial arrangement. Ultimately, this achieves accurate identification of the complete three-dimensional structure of peptide drugs containing modified side chains. Attached Figure Description

[0021] Figure 1 This is a flowchart of a method for identifying the structure of a modified side-chain polypeptide drug proposed in this invention; Figure 2 This is a system architecture diagram for identifying the structure of a modified side-chain polypeptide drug proposed in this invention; Figure 3 This is a schematic diagram of the three-dimensional structure of smegglutinin in an embodiment of the present invention. Detailed Implementation

[0022] The present invention will be further described below with reference to exemplary embodiments, which are not intended to limit the invention. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

[0023] One specific embodiment of the present invention provides a method for identifying the overall structure of a polypeptide drug containing modified side chains, such as... Figure 1 As shown, it includes the following steps: One-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples were collected to obtain raw spectral data of all-atom nuclear magnetic resonance. Based on the original spectral data, the chemical shifts of the polypeptide host were assigned, and the three-dimensional structure of the polypeptide host was obtained through analysis. Based on the chemical shift assignment results of the polypeptide body, the chemical shifts of related atoms in the modified side chain are identified and assigned from the original spectral data, and the covalent structure of the modified side chain is determined. Based on the chemical shift assignment results of related atoms in the modified side chain, the spatial correlation spectrum in the original spectral data was analyzed to determine the spatial structure of the modified side chain; Based on the chemical shift assignments of related atoms in the polypeptide host and modified side chains, the structural signals of the linking regions are analyzed to determine the covalent linkage relationship between the modified side chains and the polypeptide host. Based on the chemical shift assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, the spatial distance constraint signal is analyzed to determine the relative spatial structure relationship between the modified side chain and the polypeptide host. Based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent linkage relationship, and the relative spatial structure relationship, the overall three-dimensional structure of the polypeptide drug is determined.

[0024] In an optional embodiment of the present invention, one-dimensional and two-dimensional nuclear magnetic resonance spectra of a polypeptide drug sample are acquired to obtain raw all-atom nuclear magnetic resonance spectral data, including: 1D of collecting peptide drug samples 1 H spectrum, 2D 1 H- 13 C HSQC spectrum, 2D 1 H- 1 H TOCSY spectrum, 2D 1 H- 1 HNOESY spectra, as well as targeted heteronuclear spectra collected based on the types of elements contained in the side chains, are used to obtain raw spectral data of whole-atom nuclear magnetic resonance.

[0025] In one optional embodiment of the present invention, a high-field nuclear magnetic resonance spectrometer is used to acquire raw spectral data, with a proton resonance frequency of not less than 700 MHz, and is equipped with an ultra-low temperature probe.

[0026] In an optional embodiment of the present invention, the targeted heteronuclear spectrum includes: 2D spectra collected when the side chain contains nitrogen. 1 H- 15 N HSQC spectrum.

[0027] In an optional embodiment of the present invention, the 2D 1 H- 13 C HSQC spectra include acquisition parameters set separately for aliphatic and aromatic regions; the 2D 1 H- 1 The mixing time for the H TOCSY spectrum was set to 80 ms; the 2D 1 H- 1 The mixing time of the H NOESY spectrum is dynamically adjusted within the range of 200~400 ms according to the molecular weight of the peptide drug.

[0028] In an optional embodiment of the present invention, the chemical shift assignment of the polypeptide host is completed based on the original spectral data, and the three-dimensional structure of the polypeptide host is obtained by analysis, including: Analyze the original spectral data to extract the chemical shift assignments and spatial distance constraints of the peptides, and extract dihedral constraints based on the chemical shift assignments; when assigning chemical shifts, use... 1 H-1 H TOCSY, 1 H- 1 H COSY and 1 H- 13 CHSQC identifies amino acid residue types and chemical shifts, from 1 H- 1 In the H NOESY spectrum, all amino acid residues were identified, including the amino group H in the main chain and the α-positions of the preceding and following amino acids. 1 H's NOE information, combined with 1 H- 1 H NOESY experiments determine the sequence linkage information between amino acid residues; analyze 1 H- 1 The distance constraint information between hydrogen atoms extracted from the H NOESY spectrum was used to calculate the initial structure of the peptide sample in vacuum, and the spatial structure of the peptide sample in the presence of solvent was obtained based on the initial structure. Based on the usage of the spatial structure's statistical distance constraints, the violation of the angle and distance constraints of the statistical structure is determined, and the root mean square deviation, energy data, and Laplace plot data of the structure are calculated to determine the three-dimensional structure of the polypeptide host.

[0029] In an optional embodiment of the present invention, based on the chemical shift assignment results of the polypeptide body, the chemical shifts of related atoms in the modified side chain are identified and assigned from the original spectral data to determine the covalent structure of the modified side chain, including: Based on the chemical shift assignment of the polypeptide host, using 1D 1 H spectrum, 2D 1 H- 13 C HSQC spectrum and 2D 1 H- 1 HTOCSY spectrum is used for signal cross-verification, and in 2D 1 H- 1 H NOESY spectroscopy was used for auxiliary verification. The main peptide signal was identified and subtracted from the original spectral data, and the chemical shifts of relevant atoms in the modified side chain were assigned to determine the covalent structure of the modified side chain.

[0030] In an optional embodiment of the present invention, based on the chemical shift assignment results of related atoms in the modified side chain, the spatial correlation spectrum in the original spectral data is analyzed to determine the spatial structure of the modified side chain, including: Based on the chemical shift assignments of atoms related to the modified side chains, the 2D... 1 H- 1 H NOESY spectra, including analysis of covalently topologically distant but three-dimensionally adjacent atomic pairs, to determine the spatial structure of modified side chains.

[0031] In an optional embodiment of the present invention, based on the chemical shift assignment results of related atoms of the polypeptide host and the modified side chain, the structural signal of the linking region is analyzed to determine the covalent linking relationship between the modified side chain and the polypeptide host, including: Based on the chemical shift assignments of related atoms in the polypeptide host and the modified side chain, chemical bond connection signals or NOE spatial correlation signals between the terminal atoms of the polypeptide host side chain and the head atoms of the modified side chain are identified and extracted. The covalent connection relationship between the modified side chain and the polypeptide host is determined based on the extracted signals.

[0032] In an optional embodiment of the present invention, based on the chemical shift assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, the spatial distance constraint signal is analyzed to determine the relative spatial structural relationship between the modified side chain and the polypeptide host, including: Based on the 3D structure of the polypeptide host and the chemical shift assignments of related atoms in the modified side chains, the 2D structure was analyzed. 1 H- 1 H NOESY spectra were used to obtain information on the relative spatial constraints between the modified side chain and the polypeptide body, and to determine the relative spatial structural relationship between the modified side chain and the polypeptide body.

[0033] In an optional embodiment of the present invention, determining the overall three-dimensional structure of the polypeptide drug includes: Based on the distance constraint data between all atoms, the optimal conformation is obtained, and a three-dimensional structural diagram of all atoms is drawn based on the optimal conformation.

[0034] Another specific embodiment of the present invention provides a system for identifying the overall structure of a polypeptide drug containing modified side chains, such as... Figure 2 As shown, it includes: High-field nuclear magnetic resonance spectrometer is used to acquire one-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples and obtain raw spectral data of all-atom nuclear magnetic resonance. The polypeptide matrix analysis module is used to assign chemical shifts of the polypeptide matrix based on the original spectral data and to analyze and obtain the three-dimensional structure of the polypeptide matrix. The side chain covalent identification module is used to identify and assign the chemical shifts of related atoms in the modified side chain from the original spectral data based on the chemical shift assignment results of the polypeptide body, and to determine the covalent structure of the modified side chain. The side chain conformation analysis module is used to analyze the spatial correlation spectrum in the original spectral data based on the chemical shift assignment results of the atoms related to the modified side chain, and to determine the spatial structure of the modified side chain. The covalent linkage identification module is used to analyze the structural signals of the linkage region based on the chemical shift assignment results of related atoms of the polypeptide host and the modified side chain, and to determine the covalent linkage relationship between the modified side chain and the polypeptide host. The relative spatial structure identification module is used to analyze the spatial distance constraint signal based on the chemical displacement assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, and to determine the relative spatial structure relationship between the modified side chain and the polypeptide host. The overall structure identification module is used to determine the overall three-dimensional structure of the polypeptide drug based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent connection relationship, and the relative spatial structure relationship.

[0035] The following is a specific example.

[0036] This embodiment specifically provides a method for identifying the overall structure of a polypeptide drug containing modified side chains, such as... Figure 3 As shown, the specific steps include: 1. Collect one-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples to obtain raw all-atom nuclear magnetic resonance spectral data.

[0037] In this embodiment, 6.43 mg of semaglutide sample was dissolved in 500 μL of a phosphate buffer system (pH 7.4) containing 10% heavy water and internal standard DSS. Then, 1D samples were acquired using a 950 MHz NMR instrument. 1 H-spectrum, 2D 1 H- 1 H TOCSY, aliphatic and aromatic 1 H- 13 C HSQC and 1 H- 1 H NOESY spectra were obtained at an experimental temperature of 298 K. The mixing times for the TOCSY and NOESY experiments were 80 ms and 200 ms, respectively. 2D data were acquired. 1 H- 15 N HSQC spectroscopy is used to modify side chains. 15 Assignment of the chemical shift of N.

[0038] 2. Assign chemical shifts to the polypeptide matrix based on the original spectral data, and analyze to obtain the three-dimensional structure of the polypeptide matrix.

[0039] In this embodiment, to facilitate result analysis during the analysis process, smegglutide is named according to the following rules: The fatty acid acylated side chain of smegglutide includes a fatty acid side chain, a linker, and a spacer, which are respectively one 1,18-octadecane fatty acid diacid (represented by 17-carboxyheptadecanoyl4'), one γ-glutamic acid group (represented by Glu3'), and two 8-amino-3,6-dioxanoic acid groups (represented by AEEAc1' for those near the polypeptide side chain and by AEEAc2' for those near the linker). When using NMR for structural analysis, the atoms in the polypeptide are named according to a common naming convention, starting with the atom connected to the nitrogen atom in the peptide bond, and sequentially numbered α, β, γ, δ, ε, ζ, η. For convenience, the letters A, B, G, D, E, Z, and H are used in place of the Greek letters in the software. Therefore, the carbon and hydrogen atoms at each position of the polypeptide residue are represented as CA, HA, CB, HB, CG, HG, etc., while the nitrogen atom in the polypeptide backbone is represented by N, and the hydrogen atom on the nitrogen atom is represented by HN. Although the fatty acid acylated side chain is not composed of amino acids, it is also connected by peptide bonds, and the atoms are named according to the amino acid naming convention. All letter notations are case-insensitive. For ease of structural calculation, the amino acid sequence of the polypeptide is numbered 1-31, corresponding to GLP-1 numbers 7-37.

[0040] In this embodiment, the chemical shifts and structural analysis of the main peptide region were performed. The results show that, except for the N-terminus... 1 H, 15 N and carbonyl groups 13 Outside of C, all 1 H atoms and connected to 1 H atom 13 C atom and 15 The chemical shifts of the nitrogen atoms were precisely assigned, with 99% of the atoms assigned chemical shifts. The main peptide structure tends to form an α-helix-like structure, but is divided into three parts by residues Thr7 and Gly16. The His1-Thr7 part is mainly composed of random loops, the Ser8-Gly16 part contains one α-helix (Ser8-Ser12) and random loops, and the Gln17-Gly31 part contains two short α-helices (Gln11-Lys20, Ala24-Arg30) and other random loops. The two structural turns of smegglutinin allow most of its hydrophobic side chains to point in the same direction, forming a hydrophobic core, which is an important reason why it can exist in aqueous solution, resulting in a reasonable solution structure.

[0041] 3. Based on the chemical shift assignment results of the polypeptide host, the chemical shifts of the relevant atoms of the modified side chain are identified and assigned from the original spectral data to determine the covalent structure of the modified side chain.

[0042] In this embodiment, 1D 1H spectrum and 2D spectrum are used. 1 H- 1 H TOCSY spectrum, aliphatic 1 H- 13 C HSQC and 2D 1 H- 15 NHSQC spectroscopy was used to assign the chemical shifts of modified side chain atoms, while also employing 2D... 1 H- 1 ¹H NOESY spectroscopy was used to assist in verification. The results showed that, except for the fatty acid acylated side chain 17-carboxyheptadecanoyl4', which had significant overlap due to similar chemical shifts, resulting in only chemical shift assignments near the two ends, the other fatty acid acylated side chains... 1 H atoms and connected to 1 H atom 13 C atom and 15 The chemical shifts of the N atoms were assigned (Table 1), and all chemical shift values ​​conformed to the theoretical side chain structure. Based on the chemical shift assignments, 2D... 1 H- 1 H TOCSY spectroscopy yielded correlation signals within 58 side chains (Table 2), indicating that the structure of each of the four parts of the side chain conforms to the theory.

[0043] 4. Based on the chemical shift assignments of related atoms in the modified side chain, analyze the spatial correlation spectra in the original spectral data to determine the spatial structure of the modified side chain.

[0044] In this embodiment, 2D analysis 1 H- 1 1H NOESY spectroscopy yielded 52 correlation signals within the side chains (Table 3), most of which were between AEEAc1' and AEEAc2', between AEEAc2' and Glu3', and between Glu3' and 17-carboxyheptadecanoyl4', indicating that the connection order between the four parts of the side chains is consistent with the theory. Furthermore, the ends of AEEAc1' and 17-carboxyheptadecanoyl4' are spatially constrained, with 17-carboxyheptadecanoyl4' ultimately oriented towards the polypeptide matrix. Therefore, the covalent structure of the fatty acid acylated side chains is consistent with expectations. Simultaneously, NMR analysis did not reveal typical positional isomers caused by incorrect side chain connection positions.

[0045] 5. Based on the chemical shift assignments of related atoms in the polypeptide host and modified side chains, the structural signals of the linkage regions are analyzed to determine the covalent linkage relationship between the modified side chains and the polypeptide host.

[0046] In this embodiment, based on the chemical shift assignments of the smegglutinin polypeptide moiety and the fatty acid acylated side chains, 2D analysis was performed. 1 H- 1 2D TOCSY spectroscopy can obtain the cross signals of the hydrogen at the ε position of the main peptide residue Lys20 and the hydrogen at the ζ position of the modified side chain AEEAc1' (Table 4), 2D 1 H- 1 The H TOCSY spectrum provides information on covalent bonding. The hydrogen at the ε-position of AEEAc1' is theoretically the hydrogen most closely covalently bonded to the Lys20 side chain, making the presence of a crossover signal reasonable. Furthermore, analysis of 2D... 1 H- 1 1H NOESY spectroscopy revealed multiple pairs of clear, non-overlapping distance constraint signals between the hydrogen atoms of the side chain AEEAc1' and the ε and ζ positions of Lys20 (Table 5), indicating that these residues are spatially close. Combined with the correct connection of each part of the side chain in steps 5-6, this shows that the fatty acid acylated side chain has the same modification connection relationship as expected.

[0047] 6. Based on the chemical shift assignments of related atoms in the three-dimensional structure of the polypeptide host and the modified side chains, the spatial distance constraint signal is analyzed to determine the relative spatial structural relationship between the modified side chains and the polypeptide host.

[0048] In this embodiment, by analyzing 2D 1 H- 1102 distance constraints were obtained between the four side chain moieties and the main peptide mass from the 1H NOESY spectroscopy (Table 6). Spatial constraints exist between side chains AEEAc1' and AEEAc2' and Leu14, Ala18, Ile23, Leu26, and Val27 of the peptide. Leu14, Ala18, and Ile23 are spatially close to Lys20, while Ile23, Leu26, and Val27 are located on the same side of the Lys20 side chain. Spatial constraints also exist between side chain Glu3' and peptides Tyr13, Leu14, Phe22, Ile23, Trp25, Leu26, and Val27. Spatial constraints also exist between side chain 17-carboxyheptadecanoyl4' and peptides Phe6, Tyr13, Leu14, Phe22, Ile23, Trp25, Leu26, and Val27. This demonstrates that the side chains primarily exhibit spatial constraints with the hydrophobic residues of the polypeptide moiety, and the closer to the end of the side chain, the more hydrophobic residues are spatially constrained by the polypeptide moiety. From the perspective of the polypeptide's three-dimensional structure, these hydrophobic side chains all point in the same direction, forming a hydrophobic core. The side chain orientation initially follows the direction of the third α-helix of the polypeptide, then changes direction at the Glu3' position, stabilizing the hydrophobic portion of 17-carboxyheptadecanoyl4' within the hydrophobic core.

[0049] 7. Based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent linkage relationship, and the relative spatial structure relationship, determine the overall three-dimensional structure of the polypeptide drug.

[0050] In this embodiment, based on the information obtained from the above steps, the overall structure of the polypeptide drug containing the modified side chain can be obtained, as shown in the schematic diagram below. Figure 3 As shown.

[0051] Table 1. Assignment of chemical shifts in the side chains modified with smegglutinin Table 2. Information related to covalent modification of side chains Table 3. Information related to modified sidechain space Table 4. Covalent information between modified side chains and the Lys20 polypeptide host. Table 5. Spatial information related to modified side chains and the Lys20 host peptide. Table 6. Relative spatial structure information of modified side chains and polypeptide body Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Appropriate modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the protection scope of the present invention, which is defined by the claims.

Claims

1. A method for identifying the overall structure of a polypeptide drug containing modified side chains, characterized in that, Includes the following steps: One-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples were collected to obtain raw spectral data of all-atom nuclear magnetic resonance. Based on the original spectral data, the chemical shifts of the polypeptide host were assigned, and the three-dimensional structure of the polypeptide host was obtained through analysis. Based on the chemical shift assignment results of the polypeptide body, the chemical shifts of related atoms in the modified side chain are identified and assigned from the original spectral data, and the covalent structure of the modified side chain is determined. Based on the chemical shift assignment results of related atoms in the modified side chain, the spatial correlation spectrum in the original spectral data was analyzed to determine the spatial structure of the modified side chain; Based on the chemical shift assignments of related atoms in the polypeptide host and modified side chains, the structural signals of the linking regions are analyzed to determine the covalent linkage relationship between the modified side chains and the polypeptide host. Based on the chemical shift assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, the spatial distance constraint signal is analyzed to determine the relative spatial structure relationship between the modified side chain and the polypeptide host. Based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent linkage relationship, and the relative spatial structure relationship, the overall three-dimensional structure of the polypeptide drug is determined.

2. The method as described in claim 1, characterized in that, One-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectra of peptide drug samples were collected to obtain raw all-atom NMR spectral data, including: 1D of collecting peptide drug samples 1 H spectrum, 2D 1 H- 13 C HSQC spectrum, 2D 1 H- 1 H TOCSY spectrum, 2D 1 H- 1 The raw spectral data of whole-atom nuclear magnetic resonance are obtained by using H NOESY spectra and targeted heteronuclear spectra collected based on the types of elements contained in the side chains.

3. The method as described in claim 2, characterized in that, The 2D 1 H- 13 C HSQC spectra include acquisition parameters set separately for aliphatic and aromatic regions; the 2D 1 H- 1 The mixing time for the H TOCSY spectrum was set to 80 ms; the 2D 1 H- 1 The mixing time of the H NOESY spectrum is dynamically adjusted within the range of 200~400 ms according to the molecular weight of the peptide drug.

4. The method as described in claim 2, characterized in that, Based on the original spectral data, the chemical shifts of the polypeptide matrix were assigned, and the three-dimensional structure of the polypeptide matrix was obtained through analysis, including: Analyze the original spectral data to extract the chemical shift assignments and spatial distance constraints of the peptides, and extract dihedral constraints based on the chemical shift assignments; when assigning chemical shifts, use... 1 H- 1 H TOCSY, 1 H- 1 H COSY and 1 H- 1 H HSQC identifies amino acid residue types and chemical shifts, from 1 H- 1 In the H NOESY spectrum, all amino acid residues were identified, including the amino group H in the main chain and the α-positions of the preceding and following amino acids. 1 H's NOE information, combined with 1 H- 1 H NOESY experiments determine the sequence linkage information between amino acid residues; analyze 1 H- 1 The distance constraint information between hydrogen atoms extracted from the H NOESY spectrum was used to calculate the initial structure of the peptide sample in vacuum, and the spatial structure of the peptide sample in the presence of solvent was obtained based on the initial structure. Based on the usage of the spatial structure's statistical distance constraints, the violation of the angle and distance constraints of the statistical structure is determined, and the root mean square deviation, energy data, and Laplace plot data of the structure are calculated to determine the three-dimensional structure of the polypeptide host.

5. The method as described in claim 2, characterized in that, Based on the chemical shift assignments of the polypeptide matrix, the chemical shifts of relevant atoms in the modified side chains are identified and assigned from the original spectral data, determining the covalent structure of the modified side chains, including: Based on the chemical shift assignment of the polypeptide host, using 1D 1 H spectrum, 2D 1 H- 13 C HSQC spectrum and 2D 1 H- 1 H TOCSY spectrum was used for signal cross-verification, and 2D was used for signal cross-verification. 1 H- 1 H NOESY spectroscopy was used for auxiliary verification. The main peptide signal was identified and subtracted from the original spectral data, and the chemical shifts of relevant atoms in the modified side chain were assigned to determine the covalent structure of the modified side chain.

6. The method as described in claim 2, characterized in that, Based on the chemical shift assignments of relevant atoms in the modified side chains, the spatial correlation spectra in the original spectral data were analyzed to determine the spatial structure of the modified side chains, including: Based on the chemical shift assignments of atoms related to the modified side chains, the 2D... 1 H- 1 H NOESY spectra, including analysis of covalently topologically distant but three-dimensionally adjacent atomic pairs, to determine the spatial structure of modified side chains.

7. The method as described in claim 2, characterized in that, Based on the chemical shift assignments of relevant atoms in the polypeptide host and modified side chains, the structural signals of the linker regions are analyzed to determine the covalent linkage between the modified side chains and the polypeptide host, including: Based on the chemical shift assignments of related atoms in the polypeptide host and the modified side chain, covalent bond connection signals or NOE spatial correlation signals between the terminal atoms of the polypeptide host side chain and the head atoms of the modified side chain are identified and extracted. The covalent connection relationship between the modified side chain and the polypeptide host is determined based on the extracted signals.

8. The method as described in claim 2, characterized in that, Based on the chemical shift assignments of related atoms in the three-dimensional structure of the polypeptide host and the modified side chains, spatial distance constraint signals are analyzed to determine the relative spatial structural relationship between the modified side chains and the polypeptide host, including: Based on the 3D structure of the polypeptide host and the chemical shift assignments of related atoms in the modified side chains, the 2D structure was analyzed. 1 H- 1 HNOESY spectra were used to obtain information on the relative spatial constraints between the modified side chain and the polypeptide body, and to determine the relative spatial structural relationship between the modified side chain and the polypeptide body.

9. The method as described in claim 1 or 2, characterized in that, Determining the overall three-dimensional structure of a peptide drug, including: Based on the distance constraint data between all atoms, the optimal conformation is obtained, and a three-dimensional structural diagram of all atoms is drawn based on the optimal conformation.

10. A system for identifying the overall structure of a polypeptide drug containing modified side chains, characterized in that, include: High-field nuclear magnetic resonance spectrometer is used to acquire one-dimensional and two-dimensional nuclear magnetic resonance spectra of peptide drug samples and obtain raw spectral data of all-atom nuclear magnetic resonance. The polypeptide matrix analysis module is used to assign chemical shifts of the polypeptide matrix based on the original spectral data and to analyze and obtain the three-dimensional structure of the polypeptide matrix. The side chain covalent identification module is used to identify and assign the chemical shifts of related atoms in the modified side chain from the original spectral data based on the chemical shift assignment results of the polypeptide body, and to determine the covalent structure of the modified side chain. The side chain conformation analysis module is used to analyze the spatial correlation spectrum in the original spectral data based on the chemical shift assignment results of the atoms related to the modified side chain, and to determine the spatial structure of the modified side chain. The covalent linkage identification module is used to analyze the structural signals of the linkage region based on the chemical shift assignment results of related atoms of the polypeptide host and the modified side chain, and to determine the covalent linkage relationship between the modified side chain and the polypeptide host. The relative spatial structure identification module is used to analyze the spatial distance constraint signal based on the chemical displacement assignment results of the three-dimensional structure of the polypeptide host and the related atoms of the modified side chain, and to determine the relative spatial structure relationship between the modified side chain and the polypeptide host. The overall structure identification module is used to determine the overall three-dimensional structure of the polypeptide drug based on the above-mentioned three-dimensional structure of the polypeptide body, the covalent structure of the modified side chain, the spatial structure of the modified side chain, the covalent connection relationship, and the relative spatial structure relationship.

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