Method for fixed-point synthesis of protein-active molecule conjugate

Abstract

The invention provides a biological coupling technology, particularly provides a protein covalent modification technology, namely provides a protein part-linker conjugate which can be used as an intermediate for synthesizing a protein-active molecule conjugate. The invention also provides a preparation method of the protein part-linker conjugate, and a method for synthesizing a protein-active molecule conjugate by using the protein part-linker conjugate as an intermediate. The method has the advantages of higher conversion rate and fewer byproducts.

Description

A method for site-specific synthesis of protein-active molecule conjugates

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. CN202210880480.X filed on July 25, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to bioconjugation technology, and more specifically, to protein covalent modification technology. Specifically, the present invention provides a protein moiety-linker conjugate that can be used as an intermediate in the synthesis of protein-active molecule conjugates. The present invention also relates to a method for preparing the protein moiety-linker conjugate, and a method for synthesizing protein-active molecule conjugates using the protein moiety-linker conjugate as an intermediate. The method of the present invention has a higher conversion rate and fewer byproducts. Background Art

[0004] Chemical modification of proteins has a wide range of biological applications. In recent years, small molecule modification has been applied to protein analysis (Nature Protocols 2007, 2(6), 1414-1425) and cell imaging (Nature Methods 2006, 3(8), 591-596). Biopolymer modification contributes to the rational design of protein drugs (Chemical Society Reviews 2018, 47(24), 8998-9014). In addition, inspired by the successful application of antibody-drug conjugates (ADCs), antibody-oligonucleotide conjugates (AOCs) also have application prospects in the field of targeted delivery of oligonucleotides (J Control Release 2016, 237, 1-13).

[0005] Covalent modification of proteins while maintaining their function has always been a goal of chemical biology research. Typically, lysine and cysteine ​​are selected for protein modification due to their reactive side chains. However, the overall frequency of lysine in proteins is approximately 5.9%. If the labeling amount and site are not accurately controlled, this method will usually produce a mixture of modified sites and even change the original biological function of the protein (Nature Biotechnology 2008, 26(8), 925-932). On the other hand, cysteine ​​usually maintains the active protein structure by forming disulfide bonds. Modification of cysteine ​​sites may be detrimental to maintaining the correct folding of proteins (Trends in biochemical sciences 2003, 28(4), 210-4). To avoid these problems, site-specific modification methods such as amber codon suppression (ACS chemical biology 2019, 14(4), 603-612), protein end labeling (Nature Protocols 2013, 8(9), 1800-1807), enzyme labeling (The Journal of biological chemistry 2017, 292(38), 15622-15635) and affinity tag-mediated labeling (Angewandte Chemie International Edition 2019, 58(17), 5592-5597) have been reported. However, these methods often require the intervention of complex protein translation processes to insert reaction sites or enzyme mediation.

[0006] Existing macromolecular modification of proteins using 2-pyridinecarboxaldehyde derivatives often has a conversion rate of only about 50%, and multiple adduct byproducts are generated (Bioconjugate Chemistry 2019, 30(9), 2427-2434). Moreover, the stability of the modified products is also poor. After incubation at 37 degrees for 12 hours, about 30% of the modified products of 2-pyridinecarboxaldehyde were degraded (Nature Chemical Biology 2015, 11(5), 326-331).

[0007] Therefore, there is an urgent need in the art for a new method for synthesizing protein-nucleic acid conjugates, which has advantages such as higher conversion rate and fewer by-products compared with existing methods.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a protein part-linker conjugate, which can be used as an intermediate for synthesizing a protein-active molecule conjugate, a method for preparing the protein part-linker conjugate, and a method for synthesizing a protein-nucleic acid conjugate using the protein part-linker conjugate as an intermediate. The method of the present invention is based on the property of a 2-pyridine formaldehyde derivative to specifically label the nitrogen terminus (N terminus) of a protein, and generates a protein part-linker conjugate as an intermediate under the mediation of a polyhistidine tag (His tag) located at the N terminus and a maleimide derivative, thereby further obtaining a protein-active molecule conjugate. Compared with the previously reported method (mAbs 2018, 10 (5), 712-719), the method of the present invention has a higher conversion rate and fewer by-products. In the present invention, while maintaining site-specific conjugation, the conversion rate and conjugate stability are significantly improved, wherein the conversion rate is increased to more than 80%, and the conjugate degradation rate is less than 5%.

[0010] In one aspect, the present invention provides a protein moiety-linker conjugate having a structure as shown in Formula (I):

[0011] wherein R1 is selected from hydrogen, a hydrocarbon group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; the hydrocarbon group optionally has one or more substituents selected from a hydrocarbon group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; the hydrocarbon group is preferably an alkyl group, an aryl group, or a combination thereof; wherein the alkyl group optionally has one or more substituents selected from an aryl group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; the aryl group optionally has one or more substituents selected from an alkyl group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; or R1 is selected from a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, and a nucleic acid-modified group;

[0012] R2 is selected from the side chain portion of any natural amino acid;

[0013] R3 is selected from hydrogen, alkyl, alkoxy, hydroxy, amino, and amine;

[0014] R4 is selected from alkylene, arylene, PEG;

[0015] R5 is selected from hydrogen and alkyl;

[0016] R6 is selected from the side chain portion of any natural amino acid.

[0017] In some embodiments, the protein moiety-linker conjugate of the present invention has the following structure:

[0018] In some embodiments, in the protein moiety-linker conjugates of the invention, the protein moiety is an antibody or a fragment thereof.

[0019] In some embodiments, in the protein moiety-linker conjugates of the present invention, the protein moiety has a molecular weight ranging from 8 kDa to 160 kDa.

[0020] In one aspect, the present invention provides a method for preparing a protein moiety-linker conjugate, comprising the steps of:

[0021] (1) Modifying the N-terminus of a protein with a short peptide containing a His tag to obtain a modified protein;

[0022] (2) reacting the modified protein with an azide-containing 2-pyridinecarboxaldehyde derivative or a maleimide derivative to obtain the protein portion-linker conjugate.

[0023] In some embodiments, in the method of preparing a protein moiety-linker conjugate of the present invention, the short peptide containing a His tag has a sequence of Xaa-His-Xaa-His, where Xaa is any natural amino acid residue.

[0024] In some embodiments, in the method of preparing the protein moiety-linker conjugate of the present invention, the azide-containing 2-pyridinecarboxaldehyde derivative is a 6-(azidomethyl)-2-pyridinecarboxaldehyde derivative, preferably 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde.

[0025] In some embodiments, in the method of preparing a protein moiety-linker conjugate of the present invention, the maleimide derivative has the following structure:

[0026] wherein R1 is selected from hydrogen, hydrocarbon, amine, amino, hydroxyl, and carboxyl, the hydrocarbon optionally having one or more substituents selected from hydrocarbon, amine, amino, hydroxyl, and carboxyl, the hydrocarbon is preferably an alkyl, an aryl, or a combination thereof, wherein the alkyl optionally has one or more substituents selected from aryl, amine, amino, hydroxyl, and carboxyl, the aryl optionally has one or more substituents selected from alkyl, amine, amino, hydroxyl, and carboxyl, or R1 is a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, or a nucleic acid-modified group;

[0027] R5 is selected from hydrogen and alkyl.

[0028] In some embodiments, in the method of preparing a protein moiety-linker conjugate of the present invention, the maleimide derivative is selected from

[0029] In some embodiments, in the method of preparing a protein moiety-linker conjugate of the present invention, the modified protein, the azide-containing 2-pyridinecarboxaldehyde derivative, and the maleimide derivative are reacted in a substance amount ratio of 1:5-60:5-40.

[0030] In some embodiments, in the method for preparing a protein moiety-linker conjugate of the present invention, the molar ratio of the azide-containing 2-pyridinecarboxaldehyde derivative to the maleimide derivative is in the range of 30:1 to 1:1.

[0031] In some embodiments, in the method of preparing a protein moiety-linker conjugate of the present invention, the reaction of the modified protein with the azide-containing 2-pyridinecarboxaldehyde derivative or the maleimide derivative is carried out at pH 6.0-9.0.

[0032] In one aspect, the present invention provides use of a protein moiety-linker conjugate in preparing a protein-active molecule conjugate.

[0033] In some embodiments, when the protein moiety-linker conjugate of the present invention is used to prepare a protein-active molecule conjugate, the active molecule is selected from a nucleic acid molecule, a fluorescent molecule, a PEG molecule, a biotin molecule, a polypeptide molecule, and a small drug molecule.

[0034] In one aspect, the present invention provides a method for synthesizing a protein-active molecule conjugate, comprising the following steps:

[0035] The protein part-linker conjugate of the present invention is reacted with the active molecule modified with an alkyne group to obtain a protein-active molecule conjugate.

[0036] In some embodiments, in the method for synthesizing a protein-active molecule conjugate of the present invention, the active molecule is selected from a nucleic acid molecule, a fluorescent molecule, a PEG molecule, a biotin molecule, a polypeptide molecule, and a small drug molecule.

[0037] In some embodiments, in the method for synthesizing a protein-active molecule conjugate of the present invention, the active molecule is a nucleic acid molecule.

[0038] In some embodiments, in the method for synthesizing a protein-active molecule conjugate of the present invention, the alkyne-modified active molecule is a nucleic acid molecule modified with an alkyne-containing group at the 3' end, the 5' end, or any position between the two ends.

[0039] In some embodiments, in the method for synthesizing a protein-active molecule conjugate of the present invention, the alkyne-modified active molecule is a nucleic acid molecule modified with dibenzocyclooctyne at the 3' end or the 5' end or at any position between the two ends.

[0040] In some embodiments, in the method for synthesizing a protein-active molecule conjugate of the present invention, the nucleic acid molecule is selected from natural DNA; natural RNA; nucleic acids with base modifications such as 2'-F, 2'-OMe, 2'-MOE, etc.; nucleic acids with biotin, fluorescent groups, polypeptides, PEG, and phosphorylation modifications; and the nucleic acid molecule includes single-stranded nucleic acid or double-stranded nucleic acid.

[0041] In some embodiments, in the method for synthesizing a protein-active molecule conjugate of the present invention, the nucleic acid molecule has a length of 10 to 100 nt.

[0042] In one aspect, the present invention provides a protein-active molecule conjugate obtained by the method for synthesizing a protein-active molecule conjugate according to the present invention.

[0043] In some embodiments, in the protein-active molecule conjugate obtained by the method for synthesizing a protein-active molecule conjugate according to the present invention, the active molecule is selected from nucleic acid molecules, fluorescent molecules, PEG molecules, biotin molecules, polypeptide molecules, and small drug molecules.

[0044] In some embodiments, in the protein-active molecule conjugate obtained by the method for synthesizing a protein-active molecule conjugate according to the present invention, the active molecule is a nucleic acid molecule.

[0045] In one aspect, the present invention provides a protein-nucleic acid conjugate having the following structure:

[0046] wherein R1 is selected from hydrogen, a hydrocarbon group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; the hydrocarbon group optionally has one or more substituents selected from a hydrocarbon group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; the hydrocarbon group is preferably an alkyl group, an aryl group, or a combination thereof; wherein the alkyl group optionally has one or more substituents selected from an aryl group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; the aryl group optionally has one or more substituents selected from an alkyl group, an amine group, an amino group, a hydroxyl group, and a carboxyl group; or R1 is selected from a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, and a nucleic acid-modified group;

[0047] R2 is selected from the side chain portion of any natural amino acid;

[0048] R3 is selected from hydrogen, alkyl, alkoxy, hydroxy, amino, and amine;

[0049] R4 is selected from alkylene, arylene, PEG;

[0050] R5 is selected from hydrogen and alkyl;

[0051] R6 is selected from the side chain portion of any natural amino acid;

[0052] n is an integer selected from 1-8.

[0053] In some embodiments, the protein-nucleic acid conjugate of the present invention has the following structure:

[0054] In some embodiments, in the protein-nucleic acid conjugates of the present invention, the protein portion is an antibody or a fragment thereof.

[0055] In some embodiments, in the protein-nucleic acid conjugate of the present invention, the molecular weight of the protein portion ranges from 8 kDa to 160 kDa.

[0056] In some embodiments, in the protein-nucleic acid conjugates of the present invention, the nucleic acid molecule is selected from natural DNA; natural RNA; nucleic acids with base modifications such as 2'-F, 2'-OMe, 2'-MOE, etc.; nucleic acids with biotin, fluorescent groups, polypeptides, PEG, and phosphorylation modifications; and the nucleic acid molecule includes single-stranded nucleic acid or double-stranded nucleic acid.

[0057] In some embodiments, in the protein-nucleic acid conjugate of the present invention, the nucleic acid molecule has a length of 10 to 100 nt. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG1 shows a synthetic route for preparing protein-nucleic acid conjugates according to the method of the present invention.

[0059] FIG2 shows a preparation route of azide-containing 2-pyridinecarboxaldehyde derivatives as N-terminal labeling probes (NLPs).

[0060] FIG3 shows a synthetic route for oligonucleotides with DBCO modification at the 3′ end.

[0061] FIG4 shows the HPLC analysis spectrum of the purified 3′-end DBCO-modified oligonucleotide.

[0062] FIG5 shows the mass spectrometry detection spectrum of an oligonucleotide modified with an amino group at the 3' end and a biotin at the 5' end.

[0063] FIG6 shows the mass spectrometry detection spectrum of an oligonucleotide modified with DBCO at the 3' end and biotin at the 5' end.

[0064] FIG. 7 shows a synthetic route for preparing protein moiety-linker conjugates according to the method of the present invention.

[0065] Figure 8 shows the mass spectrometry analysis of maltose binding protein and its various modifications before, during, and after the reaction, wherein: a is the mass spectrometry analysis of maltose binding protein; b is the mass spectrometry analysis of maltose binding protein modified with azide small molecules; and c is the mass spectrometry analysis of maltose binding protein-nucleic acid coupling.

[0066] FIG9 shows the LC-MS analysis of the sample after protein-nucleic acid conjugate digestion.

[0067] FIG10 shows the coupling efficiency of maltose binding protein with nucleic acid after being treated and modified with different maleimide derivative small molecules.

[0068] Figure 11 shows the protein-nucleic acid coupling efficiency after small molecule modification in different pH solutions.

[0069] FIG12 shows the protein-nucleic acid coupling efficiency after modification with different equivalents of small molecules.

[0070] FIG13 shows the protein-nucleic acid coupling efficiency after small molecule modification in different buffer salt systems.

[0071] FIG14 shows the protein-nucleic acid coupling efficiency after small molecule modification by adding different additives.

[0072] FIG15 shows the coupling efficiency of maltose binding protein after being treated and modified with different maleimide derivative small molecules with nucleic acid and the ratio of the conjugate after heating.

[0073] FIG16 shows the coupling efficiency of green fluorescent protein, ubiquitin protein, and small ubiquitin-like modifier protein with nucleic acid after being modified by maleimide derivative small molecules.

[0074] FIG17 shows the HPLC analysis spectrum of the purified 65 nt long-chain oligonucleotide modified with DBCO at the 3′ end.

[0075] FIG18 shows the mass spectrometry detection spectrum of a long-chain oligonucleotide modified with an amino group at the 3' end and a biotin at the 5' end.

[0076] FIG19 shows the mass spectrometry detection spectrum of a long-chain oligonucleotide modified with DBCO at the 3′ end and biotin at the 5′ end.

[0077] FIG20 shows the protein-nucleic acid coupling efficiency of maltose binding protein and nucleic acids of different lengths.

[0078] FIG21 shows the modification ratio of the heavy chain of the antibody-nucleic acid conjugate.

[0079] FIG22 shows the comparison of IC50 values ​​of the antibodies and the obtained antibody-nucleic acid conjugates subjected to Elisa assay. DETAILED DESCRIPTION

[0080] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. It will be understood by those skilled in the art that the methods and materials described below are exemplary only and should not be construed as limiting the scope of the present invention.

[0081] The inventors have discovered that, through the combined mediation of a His tag and a maleimide derivative modified at the N-terminus of a protein, a 6-(azidomethyl)-2-pyridinecarboxaldehyde derivative can be linked to the N-terminus of a protein to form a protein moiety-linker conjugate. On the one hand, the protein moiety-linker conjugate can be used as an intermediate to further react with an active molecule, such as a biomolecule including a nucleic acid or a small drug molecule, thereby forming a protein-active molecule conjugate. Compared to the prior art, this method improves the conversion rate and reduces the generation of by-products. On the other hand, by containing a maleimide derivative containing an active molecule moiety, the protein moiety-linker conjugate itself can already include an active molecule moiety, which will be described in detail below.

[0082] The protein that can be used in the method of the present invention is not particularly limited. When used in this article, the term "protein" or "protein" covers any type of protein, refers to any polymer of amino acids of any length, and therefore also covers peptides, for example, peptides containing only 2 to 100 amino acids, whereby the term "protein" or "protein" can be used interchangeably with the term "polypeptide". When used in this article, the term "protein" is generally used to describe macromolecules comprising an amino acid chain, and also describes macromolecules comprising multiple amino acid chains. In some embodiments, the present invention includes full-length proteins, fragments or domains of proteins. In addition, when used in this article, the term "protein" refers to a linear amino acid chain, or refers to an amino acid chain that has been processed and folded into a functional protein. In some embodiments, proteins can be obtained by separating and purifying from cells that naturally produce proteins, cutting with enzymes (such as proteolytic enzymes), and / or by recombinant expression of nucleic acids encoding proteins or fragments. In some embodiments, proteins of the present invention and / or their fragments can be obtained by chemical synthesis or other known schemes for producing proteins and their fragments. In some embodiments, the protein is preferably an antibody and its fragment. In some embodiments, the protein preferably has a molecular weight range of 8 kDa to 160 kDa and an isoelectric point of 5.0 to 9.5.

[0083] When used herein, the term "antibody" has a broad meaning, specifically including monoclonal antibodies (including full-length antibodies with an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Antibodies may be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG21, IgG22, IgG33, IgG34, IgG35, IgG36, IgG37, IgG38, IgG39, IgG40, IgG41, IgG42, IgG43, IgG44, IgG45, IgG46, IgG47, IgG48, IgG49, IgG51, IgG5 2a , IgG3, IgG4, IgA1, IgA2, etc.).

[0084] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[0085] As used herein, the term "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; nanobodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0086] In the present invention, the N-terminus of a protein is modified with a short peptide containing a His tag.

[0087] In some embodiments, the His-tag-containing peptide has a sequence of Xaa-His-Xaa-His, where Xaa is any naturally occurring amino acid residue. There are no particular limitations on Xaa, which can be any naturally occurring amino acid residue. Thus, in the protein moiety-linker conjugates of the present invention, R2 and R6 can be side chain moieties of any naturally occurring amino acid.

[0088] As used herein, the term "natural amino acid" refers to any of the common, naturally occurring amino acids found in naturally occurring proteins. Examples of natural amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0089] In some embodiments, the His-tag portion of a short peptide containing a His-tag is attached to the N-terminus of the protein.

[0090] In some embodiments, residue Xaa in the short peptide containing the His tag has a free -NH2 group when attached to the N-terminus of the protein.

[0091] The short peptide containing the His tag can be linked to the N-terminus of the protein by methods known in the art, and the present application has no particular limitation thereto.

[0092] After obtaining the protein modified with the short peptide containing the His tag, it is reacted with an azide-containing 2-pyridinecarboxaldehyde derivative or a maleimide derivative to obtain the protein moiety-linker conjugate of the present invention.

[0093] As used herein, the term "hydrocarbyl" refers to a group containing only hydrogen and carbon atoms. The hydrocarbyl may contain one or more double and / or triple carbon-carbon bonds and may contain cyclic groups in combination with straight or branched groups. The hydrocarbyl may contain 1 to 20 carbon atoms. In some embodiments, the hydrocarbyl contains 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. As used herein, the hydrocarbyl includes alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkynyl, aryl, and combinations thereof.

[0094] When used in this article, the term "alkyl" refers to a saturated hydrocarbon group obtained by losing a hydrogen atom from a saturated alkane. The alkyl group can be linear, branched, cyclic or a combination thereof. When the alkyl group is cyclic, it may also be referred to as a "cycloalkyl group". The alkyl group typically has 1 to 20 carbon atoms. In some embodiments, the alkyl group comprises 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6 or 1 to 4 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including n-propyl, isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, tert-butyl), n-pentyl, isopentyl, n-hexyl, cyclohexyl, n-heptyl, n-octyl and ethylhexyl etc. In some embodiments, the alkyl group includes methyl, ethyl, propyl and butyl etc.

[0095] As used herein, the term "alkenyl" refers to an unsaturated hydrocarbon group obtained by losing a hydrogen atom from an unsaturated olefin, having one or more carbon-carbon double bonds. An alkenyl group typically has 1 to 20 carbon atoms. In some embodiments, an alkenyl group comprises 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Non-limiting examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl.

[0096] As used herein, the term "alkynyl" refers to an unsaturated hydrocarbon group obtained by losing a hydrogen atom from an unsaturated alkyne, which has one or more carbon-carbon triple bonds. Alkynyl groups typically have 1 to 20 carbon atoms. In some embodiments, alkynyl groups comprise 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0097] As used herein, the term "alkylene" refers to a saturated hydrocarbon group derived from a saturated alkane by losing two hydrogen atoms. An alkylene group can be linear, branched, cyclic, or a combination thereof. When an alkylene group is cyclic, it may also be referred to as a "cycloalkylene group." An alkylene group often has 1 to 20 carbon atoms. In some embodiments, an alkylene group comprises 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. An alkylene group can be derived from a corresponding saturated alkane by losing two hydrogen atoms on the same carbon atom or by losing one hydrogen atom on different carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methylene (i.e., -CH2-), ethylene (i.e., -CH2CH2- or -CH(CH3)-), propylene (i.e., -CH2CH2CH2-, -CH(CH2CH3)-, or -CH(CH3)CH2-), and butylene (i.e., -CH2CH2CH2CH2-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, or -CH(CH2CH3)CH2-).

[0098] When used in this article, term " alkoxy " refers to the group with formula -OR, wherein R is alkyl, and it is directly connected with oxy group.Alkyl is as defined above.Alkoxy usually has 1 to 20 carbon atoms.In some embodiments, alkoxy comprises 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6 or 1 to 4 carbon atoms.The non-limiting example of alkoxy comprises methoxy, ethoxy, propoxy (comprising n-propoxy and isopropoxy), butoxy (comprising n-butoxy, isobutoxy, sec-butoxy and tert-butoxy), pentyloxy, hexyloxy etc.In some embodiments, alkoxy comprises methoxy, ethoxy, propoxy and butoxy etc.

[0099] As used herein, the term "aryl" refers to an aromatic group comprising one ring or two or three fused rings. The aryl group may have one to five rings attached to or fused to the aromatic ring. Other ring structures may be aromatic, non-aromatic, or a combination thereof. The aryl group may have 6 to 20 carbon atoms. In some embodiments, the aryl group comprises 6 to 18, 6 to 16, 6 to 14, 6 to 12, or 6 to 10 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, biphenyl, terphenyl, anthracenyl, naphthyl, acenaphthenyl, anthraquinone, indenyl, isoindenyl, indanyl, phenanthrenyl, anthracenyl, pyrenyl, and fluorenyl. The aryl group may optionally be substituted with halogen, C 1-6 Alkyl, C 3-10 In some embodiments, aryl groups include phenyl, naphthyl, anthracenyl, and the like.

[0100] As used herein, the term "arylene" refers to a divalent group formed by removing a hydrogen atom from the aromatic ring portion of an aryl group as described above. An arylene group may have 6-20 carbon atoms. In some embodiments, an arylene group comprises 6 to 18, 6 to 16, 6 to 14, 6 to 12, or 6 to 10 carbon atoms. In some embodiments, aryl groups include phenylene, naphthylene, and the like.

[0101] As used herein, the term "amino" refers to a group in which the hydrogen of an amino group is replaced by one or two hydrocarbon groups. Amine groups include secondary amino groups (-NHR a ) or tertiary amino group (-NR a R b , where R a and R b The amino groups may be the same or different. Non-limiting examples of amino groups include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, 2-propylamino, 1-propylamino, di(n-propyl)amino, di(isopropyl)amino, methyl-n-propylamino, tert-butylamino, and the like. As used herein, the term "amino" may be used interchangeably with "primary amino."

[0102] As used herein, the term "halogen" or "halo" refers to chlorine, bromine, fluorine, or iodine. The term "acyl" refers to an alkylcarbonyl or arylcarbonyl substituent. The term "acylamino" refers to an amide group attached at the nitrogen atom (i.e., R-CO-NH-). The term "carbamoyl" refers to an amide group attached at the carbonyl carbon atom (i.e., NH2-CO-). The nitrogen atom on the acylamino or carbamoyl substituent is optionally substituted. The term "sulfonamido" refers to a sulfonamide substituent attached through a sulfur or nitrogen atom. The term "amino" refers to an -NH2 group. The term "hydroxy" refers to an -OH group. The term "carboxyl" refers to a -CO2H group.

[0103] The number of carbon atoms in a hydrocarbyl group such as alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkynyl, and aryl is indicated herein by the prefix "C x -C y ” or “C x-y " represents, where x is the minimum number of carbon atoms in the group and y is the maximum number of carbon atoms in the group. For example, "C1-C4 alkyl" or "C 1-4 "Alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0104] In the present invention, the azide-containing 2-pyridinecarboxaldehyde derivative is used as a N-terminal labeling probe (NLP), which will be bound to the nitrogen terminus of the protein to mediate the subsequent coupling reaction with the nucleic acid molecule.

[0105] The azide-containing 2-pyridinecarboxaldehyde derivative has the following structural formula:

[0106] wherein R3 is selected from hydrogen, alkyl, alkoxy, hydroxyl, amino, and amine; R4 is selected from alkylene, arylene, PEG (polyethylene glycol repeating unit, i.e., -[OCH2CH2] m -, m can be an integer of 1-20, 1-16, 1-12, 1-8, 1-6, 1-4); R7 is selected from hydrogen, alkyl, alkoxy.

[0107] R7 is optionally substituted at positions 3 and 5 of the pyridine ring. Optionally, the azide-containing 2-pyridinecarboxaldehyde derivative may have one or two identical or different R7 groups.

[0108] In some embodiments, R3 is selected from alkyl, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, butyl. Alkoxy is selected from C1-C8 alkoxy, C1-C6 alkoxy, C1-C4 alkoxy, preferably methoxy, ethoxy, propoxy, butoxy.

[0109] In some embodiments, R4 is selected from alkylene, for example, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, preferably methylene, ethylene, propylene, butylene. Arylene is for example C6-C 14 Arylene, C6-C 12 Arylene, C6-C 10 The arylene group is preferably a phenylene group.

[0110] In some embodiments, R7 is selected from alkyl, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, butyl. Alkoxy is selected from alkoxy, for example, C1-C8 alkoxy, C1-C6 alkoxy, C1-C4 alkoxy, preferably methoxy, ethoxy, propoxy, butoxy.

[0111] In some embodiments, R3, R4, and R7 are each optionally further substituted.

[0112] In some embodiments, the azide-containing 2-pyridinecarboxaldehyde derivative is a 6-(azidomethyl)-2-pyridinecarboxaldehyde derivative.

[0113] In this article, 6-(azidomethyl)-2-pyridinecarboxaldehyde derivatives include 6-(azidomethyl)-2-pyridinecarboxaldehyde, and derivatives having one to multiple substituents on the pyridine ring. In some embodiments, the substituent is preferably an alkoxy group, such as a C1-C8 alkoxy group, a C1-C6 alkoxy group, a C1-C4 alkoxy group, specifically a methoxy group, an ethoxy group, etc.

[0114] In some embodiments, the 6-(azidomethyl)-2-pyridinecarboxaldehyde derivative is selected from

[0115] Synthesis of 6-(azidomethyl)-2-pyridinecarboxaldehyde derivatives can be performed by those skilled in the art using well-known methods. For example, a bromine-substituted 2-pyridinecarboxaldehyde compound can be reacted with an azide to produce an azide-containing 2-pyridinecarboxaldehyde derivative. Specifically, in some embodiments, 6-(bromomethyl)-4-methoxypyridinecarboxaldehyde can be reacted with sodium azide in a solvent such as DMF for several hours under heating to produce 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde for use as an NLP.

[0116] In the present invention, the maleimide derivative has the following structure:

[0117] Wherein, R1 is selected from hydrogen, hydrocarbon, amine, amino, hydroxyl, and carboxyl, and the hydrocarbon group optionally has one or more substituents selected from hydrocarbon, amine, amino, hydroxyl, and carboxyl. The hydrocarbon group is preferably an alkyl, aryl, or a combination thereof, wherein the alkyl group optionally has one or more substituents selected from aryl, amine, amino, hydroxyl, and carboxyl, and the aryl group optionally has one or more substituents selected from alkyl, amine, amino, hydroxyl, and carboxyl, or R1 is a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, or a nucleic acid-modified group; and R5 is selected from hydrogen and alkyl.

[0118] In some embodiments, the hydrocarbon group selected from R1 is, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, butyl. The alkyl group may further have one or more identical or different C6-C 14 Substituents of aryl, amine, amino, hydroxyl and carboxyl groups.

[0119] In some embodiments, R1 is selected from hydrocarbon groups such as C6-C 14 Aryl, C6-C 12 Aryl, C6-C 10 The aryl group is preferably a phenyl group or a naphthyl group. The aryl group may further have one or more identical or different substituents selected from C1-C8 alkyl groups, amine groups, amino groups, hydroxyl groups, and carboxyl groups.

[0120] In some embodiments, the alkyl group selected from R5 is, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, or butyl.

[0121] In some embodiments, R1 and R5 are each optionally further substituted.

[0122] In some embodiments, examples of maleimide derivatives include the following compounds, but the present invention is not limited thereto.

[0123] The modified protein, the azide-containing 2-pyridinecarboxaldehyde derivative, and the maleimide derivative are reacted at a molar ratio of 1:5-60:5-40, and the reaction is continued overnight to obtain the protein moiety-linker conjugate of the present invention. In some embodiments, the molar ratio of the reactants is preferably 1:10:5.

[0124] In some embodiments, the amount ratio of the azide-containing 2-pyridinecarboxaldehyde derivative and the maleimide derivative is in the range of 30:1 to 1:1, for example, 30:1, 20:1, 15:1, 12:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, etc., preferably 4:1 to 1:1, and more preferably 2:1.

[0125] In some embodiments, the reaction of the modified protein with the azide-containing 2-pyridinecarboxaldehyde derivative or maleimide derivative is carried out in a buffer system. Buffer systems suitable for use in the present invention include, but are not limited to, BES buffer, Bis-Tris buffer, HEPES buffer, MOPS buffer, Phos buffer, TAPS buffer, Tris buffer, and the like. In some embodiments, the buffer system is preferably a phosphate buffer system (Phos buffer).

[0126] In some embodiments, the reaction of the modified protein with the azide-containing 2-pyridinecarboxaldehyde derivative or maleimide derivative is carried out at pH 6.0-9.0, for example, pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0; pH 6.5-8.5, pH 7.0-8.0; pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0; preferably pH 7.2-7.8, more preferably pH 7.4, 7.5, 7.6.

[0127] Thus, the present inventors have successfully obtained a novel protein moiety-linker conjugate having a structure as shown in formula (I):

[0128] wherein R1 is selected from hydrogen, a hydrocarbon group, an amine group, an amino group, a hydroxyl group, and a carboxyl group, wherein the hydrocarbon group optionally has one or more substituents selected from a hydrocarbon group, an amine group, an amino group, a hydroxyl group, and a carboxyl group. The hydrocarbon group is preferably an alkyl group, an aryl group, or a combination thereof, wherein the alkyl group optionally has one or more substituents selected from an aryl group, an amine group, an amino group, a hydroxyl group, and a carboxyl group, and the aryl group optionally has one or more substituents selected from an alkyl group, an amine group, an amino group, a hydroxyl group, and a carboxyl group, or R1 is selected from a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, and a nucleic acid-modified group;

[0129] R2 is selected from the side chain portion of any natural amino acid;

[0130] R3 is selected from hydrogen, alkyl, alkoxy, hydroxy, amino, and amine;

[0131] R4 is selected from alkylene, arylene, PEG;

[0132] R5 is selected from hydrogen and alkyl;

[0133] R6 is selected from the side chain portion of any natural amino acid.

[0134] In some embodiments, the hydrocarbon group selected from R1 is, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, butyl. The alkyl group may further have one or more identical or different C6-C 14 Substituents of aryl, amine, amino, hydroxyl and carboxyl groups.

[0135] In some embodiments, R1 is selected from hydrocarbon groups such as C6-C 14 Aryl, C6-C 12 Aryl, C6-C 10The aryl group is preferably a phenyl group or a naphthyl group. The aryl group may further have one or more identical or different substituents selected from C1-C8 alkyl groups, amine groups, amino groups, hydroxyl groups, and carboxyl groups.

[0136] In a preferred embodiment, R1 is selected from propyl, ethylamino, propionic acid, valeric acid, anilino, benzoic acid, tolyl, and hydroxyl.

[0137] In a preferred embodiment, R2 is selected from the methionine side chain moiety.

[0138] In some embodiments, R3 is selected from alkyl, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, butyl. Alkoxy is selected from C1-C8 alkoxy, C1-C6 alkoxy, C1-C4 alkoxy, preferably methoxy, ethoxy, propoxy, butoxy.

[0139] In a preferred embodiment, R3 is selected from methoxy.

[0140] In some embodiments, R4 is selected from alkylene, for example, C1-C8 alkylene, C1-C6 alkylene, C1-C4 alkylene, preferably methylene, ethylene, propylene, butylene. Arylene is for example C6-C 14 Arylene, C6-C 12 Arylene, C6-C 10 The arylene group is preferably a phenylene group.

[0141] In a preferred embodiment, R4 is selected from methylene.

[0142] In some embodiments, the alkyl group selected from R5 is, for example, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, preferably methyl, ethyl, propyl, or butyl.

[0143] In a preferred embodiment, R5 is selected from methyl.

[0144] In a preferred embodiment, R6 is selected from a histidine side chain moiety.

[0145] In some embodiments, R1, R3, R4, and R5 are each optionally further substituted.

[0146] In a preferred embodiment, n is an integer selected from 1-8, for example 1, 2, 3, 4, 5, 6, 7, 8.

[0147] In a preferred embodiment, the protein moiety-linker conjugate has the structure shown below:

[0148] In some embodiments, R1 of the protein moiety-linker conjugates of the present invention is a bioactive molecule selected from a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, or a nucleic acid-modified group, thereby being suitable for various biological, chemical, medical, and pharmaceutical applications. For example, fluorescent groups include, but are not limited to, common fluorescent groups such as FAM, ROX, and CY5; PEG-modified groups include, but are not limited to, PEG4000 and PEG5000; polypeptide-modified groups include, but are not limited to, GRKKRRQRRRPQ; and small drug molecules include, for example, MMAE (Monomethyl auristatin E) and DM1 (Myotonic dystrophy type 1).

[0149] In one aspect, the protein moiety-linker conjugate of the present invention is further linked to an active molecule via an azide group to form a protein-active molecule conjugate. In the present invention, the active molecule includes but is not limited to a nucleic acid molecule, a fluorescent molecule, a PEG molecule, a biotin molecule, a polypeptide molecule, a small drug molecule, and the like. Thus, the protein-active molecule conjugate obtained after being linked to the protein moiety-linker conjugate may include the following groups, for example, fluorescent groups including but not limited to common fluorescent groups such as FAM, ROX, and CY5; PEG modification groups including but not limited to PEG4000, PEG5000, and the like; polypeptide modification groups such as GRKKRRQRRRPQ, and the like; and small drug molecules such as MMAE (Monomethyl auristatin E) and DM1 (Myotonic dystrophy type 1).

[0150] In the present invention, the connection between the protein moiety-linker conjugate and the active molecule is carried out via a reaction between an azide group and an alkynyl group. Therefore, the active molecule needs to be modified before coupling with the protein moiety-linker conjugate. In some embodiments, the active molecule is modified with a compound containing an alkynyl group. As used herein, the term "alkynyl-containing compound" refers to a compound containing one or more carbon-carbon triple bonds. Alkyne-containing compounds include, but are not limited to, straight-chain alkyne compounds, substituted straight-chain alkyne compounds, cycloalkyne compounds, substituted cycloalkyne compounds, and the like. The active molecule is modified with an alkynyl-containing compound to have an alkynyl substituent containing one or more carbon-carbon triple bonds.

[0151] In a preferred embodiment, the compound containing an alkynyl group is preferably dibenzocyclooctyne carboxylic acid.

[0152] In some embodiments, the active molecule is a nucleic acid molecule.

[0153] In a preferred embodiment, the nucleic acid is modified to have a substituent selected from diphenylcyclooctynyl, bicyclo[6.1.0]nonynyl, and the like.

[0154] In a preferred embodiment, dibenzocyclooctyne carboxylic acid is used to obtain dibenzocyclooctyne-modified nucleic acids.

[0155] The nucleic acid that can be used in the method for the present invention is not particularly limited. When used in this article, the term "nucleic acid" encompasses any type of nucleic acid, refers to any polymer of nucleotides (e.g., deoxyribonucleotides, ribonucleotides) of any length, wherein containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The term "non-natural nucleotides" or "modified nucleotides" refers to nucleotides comprising modified nitrogenous bases, sugar or phosphate groups, or nucleotides incorporating non-natural parts into their structure. Examples of non-natural nucleotides include dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated and fluorescently labeled nucleotides. The term "nucleic acid" includes DNA and RNA, such as but not limited to genomic or chromosomal DNA, plasmid DNA, amplified DNA, cDNA, total RNA, mRNA, small RNA, etc. The term "nucleic acid" is intended to include natural DNA and / or RNA molecules, or synthetic DNA and / or RNA molecules. In the present invention, the backbone of the nucleic acid can contain sugars and phosphate groups commonly found in RNA or DNA, or modified or substituted sugars or phosphate groups. The nucleic acid can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, such as base modifications such as 2'-F, 2'-OMe, and 2'-MOE. Nucleic acids can also have other modifications, including but not limited to biotin, fluorescent groups, polypeptides, PEG, phosphorylation, and the like.

[0156] In some embodiments, nucleic acid can be obtained by separating and purifying from the cell comprising nucleic acid by natural origin.In some embodiments, nucleic acid can be obtained by chemical synthesis or other known schemes for the production of nucleic acid.In some embodiments, nucleic acid has a length of 10 to 100nt, for example, 10 to 90nt, 10 to 80nt, 10 to 70nt, 10 to 60nt, 10 to 50nt, 10 to 40nt, 10 to 30nt, a length of 10 to 20nt, for example, 10nt, 20nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, the degree of 100nt, for example, the length of any integer nt between 10 to 100nt.

[0157] The nucleic acid modification reaction can be carried out by those skilled in the art according to a well-known method, wherein the amino-modified nucleic acid is reacted with a compound containing an alkynyl group.

[0158] In the present invention, the amino modification position can be the 3' end, 5' end, or any position between the two ends of the nucleic acid. Amino-modified nucleic acids can be synthesized by those skilled in the art according to well-known methods, or commercially available amino-modified nucleic acid products can be purchased.

[0159] In some embodiments, an amino-modified nucleic acid is reacted with an alkynyl-containing carboxylic acid compound of the following structural formula in the presence of an amine to produce an alkynyl-containing modified nucleic acid:

[0160] wherein n=1-8, such as 1-6, 1-4, such as 1, 2, 3 and 4. The choice of amine can be determined by those skilled in the art based on common knowledge, as long as it can catalyze the connection reaction between the carboxyl group and the amino group.

[0161] In a preferred embodiment, the 3'-terminal amino-modified nucleic acid is reacted with dibenzocyclooctyne carboxylic acid of the following structural formula in the presence of an amine:

[0162] Dibenzocyclooctyne carboxylic acid.

[0163] In the above reaction, the nucleic acid and the alkyne-containing carboxylic acid compound are reacted at a molar ratio of 200:1-20:1, for example, 200:1, 150:1, 100:1, 80:1, 60:1, 40:1, or 20:1.

[0164] In the present invention, when the amino group is modified at the 3' or 5' end of the nucleic acid, the corresponding other end can be unmodified or modified with a modifying group commonly used in the art, such as biotin, FLAG TAG, etc., to obtain corresponding biological properties. When the amino group is modified at any position between the two ends of the nucleic acid, each end can independently be unmodified or modified with a modifying group commonly used in the art, such as biotin, FLAG TAG, etc., to obtain corresponding biological properties.

[0165] After the above reaction, a nucleic acid modified with an alkyne-containing group at the 3' end, the 5' end, or any position between the two ends is obtained.

[0166] After obtaining the active molecule modified with an alkynyl group, it is reacted with the protein moiety-linker conjugate of the present invention, for example, at room temperature overnight, to obtain a protein-active molecule conjugate.

[0167] In some embodiments, the reaction of the protein moiety-linker conjugate with the active molecule modified with an alkynyl group is carried out in a buffer system. Buffer systems suitable for use in the present invention include, but are not limited to, BES buffer, Bis-Tris buffer, HEPES buffer, MOPS buffer, Phos buffer, TAPS buffer, Tris buffer, and the like. In some embodiments, the buffer system is preferably a phosphate buffer system (Phos buffer).

[0168] In some embodiments, the reaction of the protein moiety-linker conjugate with the active molecule modified with an alkynyl group is carried out at pH 6.0-9.0, e.g., pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0; pH 6.5-8.5, pH 7.0-8.0; pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0; preferably pH 7.2-7.8, more preferably pH 7.4, 7.5, 7.6.

[0169] In some embodiments, the active molecule is a nucleic acid molecule, and the structure of the nucleic acid molecule modified with an alkynyl group is shown in the following formula:

[0170] in Represents the nucleic acid portion.

[0171] Thus, in some embodiments, the protein moiety-linker conjugate is linked to a nucleic acid molecule modified with an alkynyl group to produce a protein-nucleic acid conjugate having the following structural formula:

[0172] Wherein, R1, R2, R3, R4, R5, R6, and n are as defined above.

[0173] In a preferred embodiment, the protein-nucleic acid conjugate has the following structure:

[0174] Example

[0175] The technical solutions of the present invention are further described in detail below through examples and in conjunction with the accompanying drawings. Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade; the experimental methods and experimental conditions used are all conventional experimental methods and experimental conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer's instructions.

[0176] instrument

[0177] The PCR instrument model was A37834, purchased from Thermo Fisher Scientific.

[0178] HPLC used LC-20AD, purchased from Shimadzu.

[0179] LC-MS was performed using a 1260 Infinity II HPLC and a 6230 TOF, both purchased from Agilent.

[0180] Example 1. Preparation of 2-pyridinecarboxaldehyde derivatives containing an azide group (NLP)

[0181] The reaction is shown in Figure 2.

[0182] Experimental procedure: Dissolve brominated compound 1 (11.5 mg, 50 μmol) in 80 μL DMF solution, add NaN3 (6.5 mg, 100 μmol), and heat at 60°C for 16 hours. Add 500 μL of water to the reaction solution, and extract with ethyl acetate three times. The organic layers are combined and concentrated under reduced pressure to obtain the crude product of compound 2, 6-(azidomethyl)-4-methoxypicolinaldehyde, as a dark yellow liquid. The crude product can be used without further purification.

[0183] Example 2. Preparation of diphenylcyclooctyne-modified nucleic acids

[0184] The reaction is shown in Figure 3.

[0185] Experimental Procedure: 6.1 mg of dibenzocyclooctyne carboxylic acid (DBCO acid) was dissolved in 160 μL of DMSO. 4 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 4 mg of s-NHS (sulfosuccinimide) were added and activated at room temperature for 30 minutes. After activation, 200 nmol of a 3'-amino- and 5'-biotin-modified nucleic acid (dissolved in 500 μL of pH 7.54 phosphate buffer) and 340 μL of DMSO were added. The reaction was shaken overnight at room temperature in a shaker at 1200 rpm. After settling, a crude sample of DBCO-modified nucleic acid was obtained. This crude sample was purified using a 20-minute 10-80% acetonitrile gradient HPLC. The purified sample was lyophilized to obtain pure DBCO-modified nucleic acid. 0.5 nmol of the purified nucleic acid was analyzed by HPLC and LC-MS, respectively. The HPLC gradient was 5-50% acetonitrile over 6 minutes, and the LC-MS gradient was 5-80% acetonitrile over 1.5 minutes. The HPLC analysis spectrum is shown in Figure 4. The mass spectrometry spectra are shown in Figures 5 and 6.

[0186] The sequence of the nucleic acid with biotin at the 5' end and amino acid at the 3' end:

[0187]

[0188] in: <pcbt>5'-end photocleavage biotin modification, <3am> 3'-end amino modification (Genscript)

[0189] Table 1. Molecular weight results of the samples tested by mass spectrometry

[0190] Conclusion: Using the conditions provided, dibenzocyclooctyne (DBCO)-modified nucleic acids can be obtained.

[0191] Example 3. Preparation of protein moiety-linker conjugates

[0192] In this example, the protein moiety-linker conjugates of the present invention were prepared using the preparation method of the present invention from different proteins. The proteins used in this example were maltose binding protein, green fluorescent protein, ubiquitin protein, and small ubiquitin modifier protein. The amino acid sequence of the protein modified with an N-terminal His tag is shown below:

[0193] H6-MBP: (SEQ ID NO: 2)

[0194] H6-GFP: (SEQ ID NO: 3)

[0195] H6-Ubiquitin: (SEQ ID NO: 4)

[0196] H6-SUMO: (SEQ ID NO: 5)

[0197] The reaction is shown in Figure 7.

[0198] Experimental steps:

[0199] Step 1: 3 nmol of protein was added to 30 μL of 50 mM phosphate buffer solution at pH 7.4, 30 nmol of compound 2 (NLP, dissolved in 1 μL of DMSO) obtained in Example 1 and 15 nmol of 3-maleimidopropionic acid (dissolved in 1 μL of DMSO) were added, and the mixture was reacted at 37°C overnight in a PCR instrument;

[0200] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. The washing process was repeated three times to remove excess small molecule compounds to obtain a protein-linker conjugate.

[0201] Table 2 shows the molecular weight results of reactants and products detected by mass spectrometry when maltose binding protein was used.

[0202] Table 2: Mass spectrometry molecular weight results of the samples to be tested

[0203] Conclusion: Using the provided conditions, NLP and 3-maleimidopropionic acid were simultaneously added to the protein, and a protein moiety-linker conjugate could be obtained.

[0204] Example 4. Synthesis of protein-nucleic acid conjugates

[0205] In this example, a protein-nucleic acid conjugate was prepared by reacting the protein moiety-linker conjugate of the present invention with a modified nucleic acid.

[0206] Experimental steps:

[0207] Step 3: To the protein-linker conjugate obtained in Example 3, dissolved in 30 μL of 50 mM phosphate buffer (pH 7.4), was added 4.5 nmol of a nucleic acid modified with DBCO at the 3' end and biotin at the 5' end. The reaction was allowed to proceed overnight at room temperature to obtain a protein-nucleic acid conjugate. When maltose binding protein was used, mass spectrometry analysis revealed a molecular weight of 50,124, which matched the theoretical value.

[0208] Conclusion: Using the provided conditions, the protein moiety-linker conjugate of the present invention reacts with the modified nucleic acid to prepare a protein-nucleic acid conjugate.

[0209] The mass spectrometry analysis of maltose binding protein and its various modifications before, during and after the reaction is shown in FIG8 .

[0210] Example 5. Peptide analysis test

[0211] In this example, the position of the modification site of the linker moiety on the protein was detected.

[0212] Experimental steps: Use trypsin to digest the protein-nucleic acid conjugate sample, and use liquid chromatography-mass spectrometry to detect and analyze the site, type, and ratio of post-translational modifications in the sample.

[0213] Figure 9 shows that when maltose binding protein is modified by 6-(azidomethyl)-4-methoxypicolinaldehyde and 3-maleimidopropionic acid, the modification sites are both located at the two last amino acids from the N-terminus. This also confirms that according to the method of the present invention, the azide-containing 2-pyridinecarboxaldehyde derivative and the maleimide derivative jointly form an imidazolidinone ring structure with the last two amino acid residues of the short peptide containing the His tag attached to the N-terminus of the protein.

[0214] Example 6. Screening of maleimide derivative small molecules

[0215] In this example, the conversion rate of the synthesis reaction of the present invention under the mediation of different maleimide derivative small molecules was tested. The maleimide derivative small molecules used in this example include

[0216] The reaction is shown in Figure 10, and the reaction steps are the same as those described in Examples 3 and 4.

[0217] The protein used in this example is maltose binding protein, the short peptide containing a His tag is Met(His)6, the 2-pyridinecarboxaldehyde derivative containing an azide group is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0218] Experimental steps:

[0219] Step 1: 3 nmol of protein was added to 30 μL of 50 mM phosphate buffer solution at pH 7.4, followed by 30 nmol of compound 2 (NLP, dissolved in 1 μL of DMSO) obtained in Example 1 and 15 nmol of a maleimide derivative small molecule (dissolved in 1 μL of DMSO), and the reaction was carried out in a PCR instrument at 37°C overnight.

[0220] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein-linker conjugate.

[0221] Step 3: Add 4.5 nmol of nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of pH 7.4 50 mM phosphate buffer solution, and react at room temperature overnight to obtain a protein-nucleic acid conjugate.

[0222] The ratio of protein-nucleic acid conjugates was determined by SDS-PAGE gel, and the results are shown in FIG10 .

[0223] in conclusion:

[0224] The reaction in step 1 of the synthesis method of the present invention has a good conversion rate under the mediation of various maleimide derivative small molecules, among which 3-maleimidopropionic acid has the highest conversion rate.

[0225] Example 7. pH Optimization of Reaction Solution

[0226] In this example, the conversion rate of the synthesis reaction of the present invention at different pH values ​​was tested.

[0227] The reaction steps of this example are the same as those of Example 6, wherein the protein used is maltose binding protein, the short peptide containing a His tag is Met(His)6, the azide-containing 2-pyridinecarboxaldehyde derivative is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, the maleimide derivative is 3-maleimidopropionic acid, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0228] Experimental steps:

[0229] Step 1: 3 nmol of protein was added to 30 μL of 50 mM phosphate buffer solution at pH 6-9, 30 nmol of compound 2 (NLP) obtained in Example 1 (dissolved in 1 μL of DMSO) and 15 nmol of 3-maleimidopropionic acid (dissolved in 1 μL of DMSO) were added, and the reaction was carried out in a PCR instrument at 37°C overnight;

[0230] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein-linker conjugate.

[0231] Step 3: Add 4.5 nmol of nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of pH 7.4 50 mM phosphate buffer solution, and react at room temperature overnight to obtain a protein-nucleic acid conjugate.

[0232] The ratio of protein-nucleic acid conjugates was determined by SDS-PAGE gel, and the results are shown in FIG11 .

[0233] in conclusion:

[0234] The reaction in step 1 of the synthesis method of the present invention can be well performed in a buffer solution with a pH range of 6-9.

[0235] Example 8. Equivalent Optimization of Small Molecules

[0236] In this example, the conversion rate of the synthesis reaction of the present invention with the participation of different equivalents of azide-containing 2-pyridinecarboxaldehyde derivative small molecules and maleimide derivative small molecules was tested.

[0237] The reaction steps of this example are the same as those of Example 6, wherein the protein used is maltose binding protein, the short peptide containing a His tag is Met(His)6, the azide-containing 2-pyridinecarboxaldehyde derivative is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, the maleimide derivative is 3-maleimidopropionic acid, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0238] Experimental steps:

[0239] Step 1: 3 nmol of protein was added to 30 μL of 50 mM phosphate buffer solution at pH 7.4, 30 nmol of compound 2 (NLP, dissolved in 1 μL of DMSO) obtained in Example 1 and 15 nmol of 3-maleimidopropionic acid (dissolved in 1 μL of DMSO) were added, and the reaction was carried out in a PCR instrument at 37°C overnight.

[0240] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein moiety-linker conjugate.

[0241] Step 3: Add 4.5 nmol of nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of 50 mM phosphate buffer solution at pH 7.4, and react at room temperature overnight to obtain a protein-nucleic acid conjugate.

[0242] The above reaction steps were repeated using different equivalents of NLP and 3-MPA (3-maleimidopropionic acid) as shown in Table 3. The ratio of protein-nucleic acid conjugates was determined by SDS-PAGE gel, and the results are shown in Figure 12.

[0243] Table 3: Different equivalents of NLP (N-terminal labeling probe) and 3-MPA (3-maleimidopropionic acid)

[0244] As shown in Figure 12:

[0245] The reaction in step 1 of the synthesis method of the present invention has a good conversion rate when different equivalents of small molecules are involved.

[0246] Example 9. Compatibility of the reaction with different buffer salts

[0247] In this example, the compatibility of the synthesis reaction of the present invention in different buffer salts was tested. The buffer salt systems used in this example include BES, Bis-Tris, HEPES, MOPS, Phos, TAPS, and Tris.

[0248] The reaction steps of this example are the same as those of Example 6, wherein the protein used is maltose binding protein, the short peptide containing a His tag is Met(His)6, the azide-containing 2-pyridinecarboxaldehyde derivative is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, the maleimide derivative is 3-maleimidopropionic acid, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0249] Experimental steps:

[0250] Step 1: 3 nmol of protein was added to 30 μL of 50 mM buffer solutions of different pH 7.4, 30 nmol of compound 2 (NLP, dissolved in 1 μL DMSO) obtained in Example 1 and 15 nmol of 3-maleimidopropionic acid (dissolved in 1 μL DMSO) were added, and the reaction was carried out in a PCR instrument at 37°C overnight.

[0251] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein moiety-linker conjugate.

[0252] Step 3: Add 4.5 nmol of a nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of 50 mM phosphate buffer (pH 7.4). The reaction was allowed to proceed overnight at room temperature to obtain a protein-nucleic acid conjugate. The protein-nucleic acid conjugate ratio was determined by SDS-PAGE, as shown in Figure 13.

[0253] in conclusion:

[0254] The reaction in step 1 of the synthesis method of the present invention is compatible with various buffer salt systems.

[0255] Example 10. Compatibility of reaction with different additives

[0256] This example tests the compatibility of various additives in the synthesis reaction of the present invention. The additives used in this example include urea, guanidine hydrochloride, imidazole, 3-methylindole, phenol, and SDS, which mimic the side chain structures of common amino acids. The results of this example demonstrate that the synthesis method of the present invention is highly specific, with only short peptides containing a His tag modified at the N-terminus of the protein. The amino acid side chains present in the protein do not affect the efficiency of the coupling reaction of the present invention.

[0257] The reaction steps of this example are the same as those of Example 6, wherein the protein used is maltose binding protein, the short peptide containing a His tag is Met(His)6, the azide-containing 2-pyridinecarboxaldehyde derivative is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, the maleimide derivative is 3-maleimidopropionic acid, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0258] Experimental steps:

[0259] Step 1: 3 nmol of protein was added to 30 μL of 50 mM phosphate buffer solution at pH 7.4, and 5 mM of a small molecule additive was additionally added. 30 nmol of compound 2 (NLP, dissolved in 1 μL DMSO) obtained in Example 1 and 15 nmol of 3-maleimidopropionic acid (dissolved in 1 μL DMSO) were added, and the reaction was carried out in a PCR instrument at 37°C overnight.

[0260] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein moiety-linker conjugate.

[0261] Step 3: Add 4.5 nmol of a nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of 50 mM phosphate buffer (pH 7.4). The reaction was allowed to proceed overnight at room temperature to obtain a protein-nucleic acid conjugate. The protein-nucleic acid conjugate ratio was determined by SDS-PAGE, as shown in Figure 14.

[0262] in conclusion:

[0263] The reaction in step 1 of the synthesis method of the present invention is compatible with a variety of commonly used small molecule additives in biology, and these small molecule additives will not affect the efficiency of the coupling reaction of the present invention, which means that the amino acid side chains included in the protein will not affect the efficiency of the coupling reaction of the present invention.

[0264] Example 11. Thermal stability test of protein-nucleic acid conjugates

[0265] In this example, the effect of the addition of maleimide derivatives on the thermal stability of protein-nucleic acid conjugates was tested.

[0266] Experimental steps:

[0267] Following steps similar to those of Example 6, without adding the maleimide derivative, a protein-nucleic acid conjugate was obtained, wherein the intermediate had the following structure:

[0268] The protein-nucleic acid conjugates obtained without the addition of maleimide derivatives and the protein-nucleic acid conjugates obtained according to Example 6 using different maleimide derivative small molecules were heated at 95° C. for 10 minutes in a PCR instrument. The ratio of the protein-nucleic acid conjugates before and after heating was determined by SDS-PAGE gel. The results are shown in FIG15 .

[0269] in conclusion:

[0270] The conjugate obtained without using maleimide derivative small molecules has poor thermal stability, with a degradation rate of 50% after heating at 95°C for 10 minutes. However, the thermal stability of the protein-nucleic acid conjugate obtained by adding maleimide derivative small molecules using the method of the present invention is significantly improved.

[0271] Example 12. Protein substrate test

[0272] In this example, the synthetic reaction of the present invention was tested for other proteins with polyhistidine tags at the N-terminus. The proteins used in this example included green fluorescent protein, ubiquitin, and small ubiquitin modifier proteins.

[0273] The reaction steps of this example are the same as those of Example 6, wherein the short peptide containing a His tag used is Met(His)6, the 2-pyridinecarboxaldehyde derivative containing an azide group is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, the maleimide derivative is 3-maleimidopropionic acid, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0274] Experimental steps:

[0275] Step 1: Add 3 nmol of protein to 30 μL of 50 mM phosphate buffer solution at pH 7.4, add 30 nmol of compound 2 obtained in Example 1 (NLP, dissolved in 1 μL DMSO, 60 nmol is required for small ubiquitin modifier protein) and 3-maleimidopropionic acid (dissolved in 1 μL DMSO, 120 nmol is required for green fluorescent protein, 15 nmol is required for ubiquitin protein, and 30 nmol is required for small ubiquitin modifier protein), and react in a PCR instrument at 37°C overnight.

[0276] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein moiety-linker conjugate.

[0277] Step 3: Add 4.5 nmol of a nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of 50 mM phosphate buffer (pH 7.4). The reaction was allowed to proceed overnight at room temperature to obtain a protein-nucleic acid conjugate. The protein-nucleic acid conjugate ratio was determined by SDS-PAGE, as shown in Figure 16.

[0278] in conclusion:

[0279] Different proteins with polyhistidine tags at their N-termini can be coupled with nucleic acids according to the synthesis method of the present invention to obtain protein-nucleic acid conjugates with a high yield.

[0280] Example 13. Long-chain nucleic acid coupling compatibility test

[0281] In this example, the synthesis reaction of the present invention was tested for a 65-base long-chain nucleic acid modified with DBCO at the 3' end and biotin at the 5' end.

[0282] The synthesis method of a long-chain oligonucleotide modified with DBCO at the 3' end and biotin at the 5' end was the same as in Example 2, and the reaction is shown in Figure 3. The HPLC analysis spectrum is shown in Figure 17. The mass spectrometry detection spectra are shown in Figures 18 and 19.

[0283] The sequence of the long chain nucleic acid with biotin at the 5' end and amino acid at the 3' end:

[0284] in: <pcbt>5'-end photocleavage biotin modification, <3am> 3'-end amino modification) (Genscript)

[0285] Table 4. Molecular weight results of the samples tested by mass spectrometry

[0286] Experimental steps:

[0287] Step 1: Add 3 nmol of protein to 30 μL of 50 mM phosphate buffer solution at pH 7.4, add 30 nmol of compound 2 obtained in Example 1 (NLP, dissolved in 1 μL DMSO, 60 nmol is required for small ubiquitin modifier protein) and 3-maleimidopropionic acid (dissolved in 1 μL DMSO, 120 nmol is required for green fluorescent protein, 15 nmol is required for ubiquitin protein, and 30 nmol is required for small ubiquitin modifier protein), and react in a PCR instrument at 37°C overnight.

[0288] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the protein moiety-linker conjugate.

[0289] Step 3: Add 4.5 nmol of a long-chain nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the protein-linker conjugate dissolved in 30 μL of 50 mM phosphate buffer, pH 7.4. The reaction was allowed to proceed overnight at room temperature to obtain a protein-nucleic acid conjugate. The ratio of the protein-nucleic acid conjugate product was determined by SDS-PAGE gel, as shown in Figure 20.

[0290] in conclusion:

[0291] Nucleic acid molecules with a longer length, such as 65 nt nucleic acid molecules, can be coupled with a protein moiety-linker conjugate according to the synthesis method of the present invention to obtain a protein-nucleic acid conjugate with a higher yield.

[0292] Example 14. Antibody substrate testing

[0293] In this example, the synthetic reaction of the present invention was tested for antibodies having a polyhistidine tag at the N-terminus of the heavy chain. The antibodies used in this example included anti-EGFR and anti-HER2 antibodies.

[0294] The reaction steps of this example are the same as those of Example 6, wherein the short peptide containing a His tag used is Met(His)6, the 2-pyridinecarboxaldehyde derivative containing an azide group is 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde, the maleimide derivative is 3-maleimidopropionic acid, and the modified nucleic acid is the diphenylcyclooctyne-modified nucleic acid prepared in Example 2.

[0295] Experimental steps:

[0296] Step 1: 3 nmol of antibody was added to 100 μL of 50 mM phosphate buffer solution at pH 7.4, and 120 nmol of compound 2 (NLP, dissolved in 4 μL of DMSO) obtained in Example 1 and 60 nmol of 3-maleimidopropionic acid (dissolved in 1 μL of DMSO) were added, and the mixture was reacted at 37°C in a PCR instrument overnight.

[0297] Step 2: The reaction solution was transferred to a 0.5 mL ultrafiltration tube and diluted to 500 μL with phosphate buffer. The sample was concentrated to 30 μL by centrifugation at 12,000 rpm for 10 minutes. This process was repeated three times to remove excess small molecule compounds to obtain the antibody-linker conjugate.

[0298] Step 3: Add 24 nmol of a nucleic acid modified with DBCO at the 3' end and biotin at the 5' end to the antibody-linker conjugate dissolved in 30 μL of 50 mM phosphate buffer (pH 7.4). Incubate overnight at room temperature to obtain a protein-nucleic acid conjugate. The modification ratio of the heavy chain of the antibody-nucleic acid conjugate was determined by SDS-PAGE, as shown in Figure 21.

[0299] Step 4: The antibody and the obtained antibody-nucleic acid conjugate were subjected to ELISA assay and the IC50 values ​​were compared. The results are shown in FIG22 .

[0300] Antibody sequence:

[0301] Anti-EGFR antibody (Genscript)

[0302] Heavy chain: (SEQ ID NO: 7)

[0303] Light chain: (SEQ ID NO: 8)

[0304] Anti-HER2 antibody (Genscript)

[0305] Heavy chain: (SEQ ID NO: 9)

[0306] Light chain: (SEQ ID NO: 10)

[0307] in conclusion:

[0308] An antibody with a polyhistidine tag at the N-terminus of the heavy chain can be coupled with a nucleic acid according to the synthesis method of the present invention to obtain an antibody-nucleic acid conjugate, which has a high yield and does not affect the antigen binding ability of the antibody itself.

[0309] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.< / pcbt> < / pcbt>

Claims

1. A protein moiety-linker conjugate having a structure as shown in formula (I): in, R1 is selected from hydrogen, hydrocarbon, amine, amino, hydroxyl, carboxyl, the hydrocarbon optionally having one or more substituents selected from hydrocarbon, amine, amino, hydroxyl, carboxyl, the hydrocarbon is preferably an alkyl, aryl, or a combination thereof, wherein the alkyl optionally has one or more substituents selected from aryl, amine, amino, hydroxyl, carboxyl, and the aryl optionally has one or more substituents selected from alkyl, amine, amino, hydroxyl, carboxyl, or R1 is selected from a fluorescent group, a PEG modification group, a biotin modification group, a polypeptide modification group, or a nucleic acid modification group; R2 is selected from the side chain portion of any natural amino acid; R3 is selected from hydrogen, alkyl, alkoxy, hydroxy, amino, and amine; R4 is selected from alkylene, arylene, PEG; R5 is selected from hydrogen and alkyl; R6 is selected from the side chain portion of any natural amino acid.

2. The protein moiety-linker conjugate according to claim 1, having the following structure:

3. The protein moiety-linker conjugate according to claim 1 or 2, wherein The protein portion is an antibody or a fragment thereof.

4. The protein moiety-linker conjugate according to any one of claims 1 to 3, wherein The molecular weight of the protein portion ranges from 8 kDa to 160 kDa.

5. A method for preparing the protein moiety-linker conjugate according to any one of claims 1 to 4, comprising the following steps: (1) Modifying the N-terminus of a protein with a short peptide containing a His tag to obtain a modified protein; (2) reacting the modified protein with an azide-containing 2-pyridinecarboxaldehyde derivative or a maleimide derivative to obtain the protein portion-linker conjugate.

6. The preparation method according to claim 5, wherein The short peptide containing the His tag has a sequence of Xaa-His-Xaa-His, where Xaa is any natural amino acid residue.

7. The preparation method according to claim 5 or 6, wherein The azide-containing 2-pyridinecarboxaldehyde derivative is a 6-(azidomethyl)-2-pyridinecarboxaldehyde derivative, preferably 6-(azidomethyl)-4-methoxypyridinecarboxaldehyde.

8. The method according to any one of claims 5 to 7, wherein The maleimide derivative has the following structure: wherein R1 is selected from hydrogen, hydrocarbon, amine, amino, hydroxyl, and carboxyl, the hydrocarbon optionally having one or more substituents selected from hydrocarbon, amine, amino, hydroxyl, and carboxyl, the hydrocarbon is preferably an alkyl, an aryl, or a combination thereof, wherein the alkyl optionally has one or more substituents selected from aryl, amine, amino, hydroxyl, and carboxyl, the aryl optionally has one or more substituents selected from alkyl, amine, amino, hydroxyl, and carboxyl, or R1 is a fluorescent group, a PEG-modified group, a biotin-modified group, a polypeptide-modified group, or a nucleic acid-modified group; R5 is selected from hydrogen and alkyl.

9. The method according to any one of claims 5 to 8, wherein The maleimide derivative is selected from 10. The method according to any one of claims 5 to 9, wherein: The modified protein, the azide-containing 2-pyridinecarboxaldehyde derivative and the maleimide derivative are reacted at a substance amount ratio of 1:5-60:5-40.

11. The method according to any one of claims 5 to 10, wherein: The molar ratio of the azide-containing 2-pyridinecarboxaldehyde derivative to the maleimide derivative is in the range of 30:1 to 1:

1.

12. The method according to any one of claims 5 to 11, wherein: The reaction of the modified protein with the azide-containing 2-pyridinecarboxaldehyde derivative and the maleimide derivative is carried out at pH 6.0-9.

0.

13. Use of the protein moiety-linker conjugate according to any one of claims 1 to 4 in the preparation of a protein-active molecule conjugate.

14. The use according to claim 13, wherein The active molecules are selected from nucleic acid molecules, fluorescent molecules, PEG molecules, biotin molecules, polypeptide molecules, and small drug molecules.

15. A method for synthesizing a protein-active molecule conjugate, comprising the following steps: The protein moiety-linker conjugate according to any one of claims 1 to 4 is reacted with an alkyne-modified active molecule to obtain a protein-active molecule conjugate.

16. The method for synthesizing a protein-active molecule conjugate according to claim 15, wherein: The active molecules are selected from nucleic acid molecules, fluorescent molecules, PEG molecules, biotin molecules, polypeptide molecules, and small drug molecules.

17. The method for synthesizing a protein-active molecule conjugate according to claim 15 or 16, wherein: The active molecule is a nucleic acid molecule.

18. The method for synthesizing a protein-active molecule conjugate according to claim 15 or 16, wherein: The alkyne-modified active molecule is a nucleic acid molecule modified with an alkyne-containing group at the 3' end, the 5' end, or any position between the two ends.

19. The method for synthesizing a protein-active molecule conjugate according to claim 15 or 16, wherein: The alkynyl-modified active molecule is a nucleic acid molecule modified with diphenylcyclooctyne at the 3' end or the 5' end or at any position between the two ends.

20. The method for synthesizing a protein-active molecule conjugate according to any one of claims 17 to 19, wherein: The nucleic acid molecule is selected from natural DNA; natural RNA; nucleic acids with base modifications such as 2'-F, 2'-OMe, 2'-MOE, etc.; nucleic acids with biotin, fluorescent groups, polypeptides, PEG, and phosphorylation modifications; and the nucleic acid molecule includes single-stranded nucleic acid or double-stranded nucleic acid.

21. The method for synthesizing a protein-active molecule conjugate according to any one of claims 17 to 20, wherein: The nucleic acid molecule has a length of 10 to 100 nt.

22. A protein-active molecule conjugate obtained according to the method for synthesizing a protein-active molecule conjugate according to any one of claims 15 to 20.

23. The protein-active molecule conjugate according to claim 22, wherein The active molecules are selected from nucleic acid molecules, fluorescent molecules, PEG molecules, biotin molecules, polypeptide molecules, and small drug molecules.

24. The protein-active molecule conjugate according to claim 23, wherein The active molecule is a nucleic acid molecule.

25. A protein-nucleic acid conjugate having the following structure: in, R1 is selected from hydrogen, hydrocarbon, amine, amino, hydroxyl, carboxyl, the hydrocarbon optionally having one or more substituents selected from hydrocarbon, amine, amino, hydroxyl, carboxyl, the hydrocarbon is preferably an alkyl, aryl, or a combination thereof, wherein the alkyl optionally has one or more substituents selected from aryl, amine, amino, hydroxyl, carboxyl, and the aryl optionally has one or more substituents selected from alkyl, amine, amino, hydroxyl, carboxyl, or R1 is selected from a fluorescent group, a PEG modification group, a biotin modification group, a polypeptide modification group, or a nucleic acid modification group; R2 is selected from the side chain portion of any natural amino acid; R3 is selected from hydrogen, alkyl, alkoxy, hydroxy, amino, and amine; R4 is selected from alkylene, arylene, PEG; R5 is selected from hydrogen and alkyl; R6 is selected from the side chain portion of any natural amino acid; n is an integer selected from 1-8.

26. The protein-nucleic acid conjugate according to claim 25, having the following structure:

27. The protein-nucleic acid conjugate according to claim 25 or 26, wherein The protein portion is an antibody or a fragment thereof.

28. The protein-nucleic acid conjugate according to any one of claims 25 to 27, wherein The molecular weight of the protein portion ranges from 8 kDa to 160 kDa.

29. The protein-nucleic acid conjugate according to any one of claims 25 to 28, wherein The nucleic acid molecule is selected from natural DNA; natural RNA; nucleic acids with base modifications such as 2'-F, 2'-OMe, 2'-MOE, etc.; nucleic acids with biotin, fluorescent groups, polypeptides, PEG, and phosphorylation modifications; and the nucleic acid molecule includes single-stranded nucleic acid or double-stranded nucleic acid.

30. The protein-nucleic acid conjugate according to any one of claims 25 to 29, wherein The nucleic acid molecule has a length of 10 to 100 nt.

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