CN113621018B

CN113621018B - Biological macromolecule conjugate and preparation method and application thereof

Info

Publication number: CN113621018B
Application number: CN202110941534.4A
Authority: CN
Inventors: 贾凌云; 彭强; 臧柏林
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2023-06-09
Anticipated expiration: 2041-08-17
Also published as: CN113621018A

Abstract

The invention relates to a preparation method of a biological macromolecule conjugate, which comprises the following steps: (a) Providing a biomacromolecule comprising an aldehyde group, the biomacromolecule comprising a protein and a polypeptide; (b) providing an amide derivative of a milbelic acid; and (c) coupling the biomacromolecule containing aldehyde group with amide derivative of Mi's acid to obtain biomacromolecule conjugate. The invention also relates to amide derivatives of Mitiglinide having general formula I and to biomacromolecule conjugates obtained by the above preparation method. The method has mild reaction conditions, high reaction rate and stable reaction products, can realize the fixed-point coupling of small molecular compounds (medicines, fluorophores and polymers) and the directional immobilization of proteins, and has no adverse effect on the structure and the function of the proteins.

Description

Biological macromolecule conjugate and preparation method and application thereof

Technical Field

The invention relates to the field of biochemistry, in particular to a biomacromolecule conjugate, a preparation method and application thereof.

Background

Site-directed conjugation of proteins is a key technology for the preparation of protein drugs, diagnostic imaging reagents and biofunctionalized materials. Due to its good bio-orthogonality, aldehyde groups are widely used as functional groups for site-directed coupling of proteins.

Most of the existing aldehyde group modification technologies adopt nucleophilic reagents based on amino groups, hydrazine groups or oxyamino groups as coupling reagents of aldehyde groups, but the existing aldehyde group modification technologies have the defects of severe reaction conditions, low reaction rate, low product stability and the like. Since nucleophiles based on amino, hydrazino or oxyamino groups have a smaller nucleophilicity (greater pKa value), the reaction can only be carried out slower under acidic conditions; formed is an unstable carbon-nitrogen double bond, which is susceptible to hydrolysis under acidic conditions and thus unstable. In particular, patent US5633351a and EP0913691A1 disclose a protein aldehyde coupling method based on schiff bases, but the reaction is reversible, the reaction efficiency is slow and the product is easily hydrolytically unstable. Other protein aldehyde coupling reactions typically require the presence of organic solvents that remain extremely detrimental to the structure and function of the protein (see non-patent literature: J.Am. Chem. Soc.,2010,132,9546-9548; chem. Commun.; 2011,47,9066-9068; chem. Commun.; 2012,48,11079-11081; org. Lett.; 2015,17,1361-1364).

In addition, non-patent literature (org. Lett.2020april 03;22 (7): 2626-2629) reports methods for protein lysine modification based on Michael addition receptors as Michael addition receptors. In addition, patent US5243053a and EP0206673A2 disclose the preparation of derivatives of milbezier acid and a series of derivatives. In addition, patent EP0578849A1 and non-patent literature (Tetrahedron Letters,1978,19 (20): 1759-1762) disclose the use of Mirabilitic acid and aldehyde reactions in organic syntheses.

In particular, patent US5633351A, EP0913691A1 discloses a schiff base-based protein aldehyde coupling method, but the reaction is reversible, the reaction efficiency is slow and the product is easily hydrolyzed and unstable. Other protein aldehyde coupling reactions typically require the presence of organic solvents that remain extremely detrimental to the structure and function of the protein (see J.Am. Chem. Soc.,2010,132,9546-9548; chem. Commun.,2011,47,9066-9068; chem. Commun.,2012,48,11079-11081; org. Lett.,2015,17,1361-1364). Non-patent literature (Omar Boutureira et al chem. Rev.2015,115, 2174-2195) reviews the disadvantages of non-site-directed uncontrollable modification of existing proteins, resulting in non-uniformity of the product, large impact on protein structure and function, and poor reproducibility of the reaction.

Aiming at the technical problems of severe reaction conditions, low reaction efficiency, unstable products, uneven products, large influence on protein structure and function and poor reaction reproducibility of protein aldehyde group coupling strategies in the prior art, a biomacromolecule conjugate, a preparation method and application thereof need to be developed to solve the technical problems, so that the aldehyde group coupling reaction conditions are mild, the reaction rate is high, the reaction products are stable, and the controllable fixed point modification of the protein is realized so as not to have adverse influence on the structure and function of the protein.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, in one aspect, the present invention provides a method for preparing a biomacromolecule conjugate, comprising the steps of:

(a) Providing aldehyde group-containing biomacromolecules including proteins and polypeptides;

(b) Providing an amide derivative of Mitiglinide;

(c) The biological macromolecule containing aldehyde group and amide derivative of Mi's acid are coupled to obtain biological macromolecule conjugate.

According to a preferred embodiment of the present invention, wherein step (c) is carried out at a pH of 2.5 to 10.5 and a temperature of-20 ℃ to 70 ℃, the pH is preferably 3 to 10 and 4 to 9, more preferably 4 to 7, still more preferably 6 to 7 and most preferably 6.5, the temperature is preferably-10 ℃ to 60 ℃ and-0 ℃ to 50 ℃, more preferably 10 ℃ to 40 ℃ and most preferably 25 ℃ to 37 ℃.

According to a preferred embodiment of the present invention, the aldehyde group-containing biomacromolecule in step (a) comprises a protein having an aldehyde group.

According to a preferred embodiment of the present invention, the biomacromolecule conjugate in step (c) comprises a protein aldehyde conjugate

Wherein the R group is a functional group coupled with protein and comprises a fluorophore, a chemotherapeutic drug and a radiotherapy drug, wherein the fluorophore, the chemotherapeutic drug and the radiotherapy drug comprise an alkyl group with 1 to 50 carbon atoms, an alkenyl group with 2 to 50 carbon atoms and/or an aryl or heteroaryl group with 6 to 50 carbon atoms.

According to a preferred embodiment of the invention, wherein the amide derivative of Mitiglinide has the following general formula I:

wherein R is ₁ Is an alkyl group having 1 to 50 carbon atoms, a cycloalkyl group having 2 to 50 carbon atoms, an alkenyl group having 2 to 50 carbon atoms, an alkynyl group having 2 to 50 carbon atoms, an aryl or heteroaryl group having 6 to 50 carbon atoms, and a heteroatom is selected from N, O, S and P; r is R ₂ Is- (CH) ₂ ) _n CONHR ₃ And n is a positive integer from 1 to 60, preferably from 1 to 30, more preferably from 1 to 20 and most preferably from 1 to 10, R ₃ Is hydrogen, alkyl having 1 to 50 carbon atoms, cycloalkyl having 2 to 50 carbon atoms, alkenyl having 2 to 50 carbon atoms, alkynyl having 2 to 50 carbon atoms, aryl or heteroaryl having 6 to 50 carbon atoms, and a heteroatom selected from N, O, S and P; r is R ₁ 、R ₂ And R is ₃ Each can be R ₄ Substituted, R ₄ Is fluorine, chlorine, bromine, iodine, an alkyl group having 1 to 50 carbon atoms, a cycloalkyl group having 2 to 50 carbon atoms, an aryl or heteroaryl group having 6 to 50 carbon atoms, and a heteroatom is selected from N, O, S and P.

According to a preferred embodiment of the present invention, wherein the amide derivative of Mitiglinide comprises

According to a preferred embodiment of the invention, wherein R ₁ To R ₃ Preferably an alkyl group having 1 to 50 carbon atoms, more preferably an alkyl group having 1 to 30 carbon atoms, and most preferably an alkyl group having 1 to 15 carbon atoms; r is R ₄ Preferred are fluorine, chlorine, bromine, iodine, alkyl groups having 1 to 50 carbon atoms, more preferred are fluorine, chlorine, bromine, iodine, alkyl groups having 1 to 30 carbon atoms, and most preferred are alkyl groups having 1 to 10 carbon atoms.

According to a preferred embodiment of the present invention, wherein the amide derivative of Miq acid is prepared from Miq acid having the following formula II with an amide condensing agent, H ₂ N-R ₃ The reaction is carried out at normal temperature and normal pressure to obtain the product:

wherein R is ₅ Is- (CH) ₂ ) _n COOH, n is a positive integer from 1 to 60, preferably from 1 to 30, more preferably from 1 to 20 and most preferably from 1 to 10, and R ₅ Can be R ₄ Substitution; the amide condensing agent is selected from the group consisting of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, O-benzotriazol-N, N, -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate, 2-succinimidyl-1, 3-tetramethylurea tetrafluoroborate 2- (5-norbornene-2, 3-dicarboximide) -1, 3-tetramethylurea tetrafluoroboric acid quaternary ammonium salt or benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate is preferred.

According to a preferred embodiment of the invention, wherein the biomacromolecule is a protein, and preferably the protein has an amino acid sequence which is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and 100% identical to the amino acid sequence of SEQ ID No.1, preferably 95%, 96%, 97%, 98%, 99% and 100%, more preferably 98%, 99% and 100%, most preferably 100%.

According to a preferred embodiment of the present invention, wherein the aldehyde groups of the protein include aldehyde groups obtained by N-terminal oxidation reaction or amine transfer reaction of the protein, protein aldehyde group tags obtained by an enzymatic method, and aldehyde groups contained in unnatural amino acids in the protein.

According to a preferred embodiment of the invention, wherein the protein aldehyde group coupling comprises a fluorophore, a chemotherapeutic agent, a radiotherapeutic agent, a polypeptide, a protein, a nucleic acid, a coupling of an affinity tag, and immobilization of a protein.

According to a preferred embodiment of the present invention, wherein the biomacromolecule comprising aldehyde groups comprises a formylglycine containing protein, the formylglycine containing protein being obtained by interaction of a cysteine containing protein with a formylglycine generating enzyme.

In another aspect, the present invention relates to the use of the above-mentioned biomacromolecule conjugate for protein immobilization, wherein the above-mentioned migratory acid having the general formula II is used for immobilization of biomacromolecules containing amino groups, wherein the immobilization is performed by condensing carboxyl groups of the migratory acid having the general formula II with amino groups of biomacromolecules, preferably proteins and polypeptides, most preferably proteins, at normal temperature and pressure, and the immobilization carrier comprises agarose gel, ferroferric oxide magnetic microspheres, silica microspheres and metal surfaces.

In addition, the invention relates to an amide derivative of the Mitiglinide with the general formula I and a biomacromolecule conjugate obtained according to the preparation method.

Effects of the invention

According to the method, the reaction condition is mild, the reaction rate is high, the reaction product is stable, and the corresponding defects of the traditional protein aldehyde group modification method are overcome; by the method, the fixed-point coupling of small molecular compounds (including drugs, fluorophores and polymers) and the directional immobilization of proteins can be realized, and the structure and the functions of the proteins are not adversely affected.

Drawings

The invention is further described below with reference to the accompanying drawings, but the invention is not limited thereto. Wherein:

FIG. 1 shows a schematic representation of the coupling reaction of an aldehyde group-containing protein with a Mirabilide amide derivative according to one embodiment of the invention.

FIG. 2A shows an electrophoresis diagram of the expression and purification of a protein according to an embodiment of the present invention (lane M represents an electrophoresis Marker (reference standard), lane 1 is a cell after induction of expression, lane 2 is a cell disruption supernatant, lane 3 is a cell fragment, and lane 4 is a purified protein); FIG. 2B shows a graph of measured molecular weight versus theoretical molecular weight for the protein of FIG. 2A prior to introduction of aldehyde groups; FIG. 2B shows a graph of the measured molecular weight versus theoretical molecular weight obtained for the protein of FIG. 2A after the introduction of aldehyde groups.

Fig. 3 shows a schematic representation of a process for the preparation of a derivative of milbehenic acid according to one embodiment of the invention.

FIG. 4 shows a graph of reaction conversion versus pH for a protein bearing aldehyde groups reacted with a Mitiglinide derivative under different pH conditions according to one embodiment of the present invention.

FIG. 5A shows a graph of measured molecular weight versus theoretical molecular weight for a protein conjugate with an aldehyde group obtained at pH 7.0 according to one embodiment of the present invention; FIG. 2B shows a graph of measured molecular weight versus theoretical molecular weight for a protein conjugate with an aldehyde group obtained at pH 2.8 according to another embodiment of the present invention; FIG. 2C shows a graph of the measured molecular weight versus the theoretical molecular weight of a protein conjugate with aldehyde groups obtained at pH 10.0 according to yet another embodiment of the present invention.

FIG. 6 shows a graph of molar ellipticity versus wavelength of a protein bearing aldehyde groups before and after coupling according to one embodiment of the present invention.

FIG. 7 shows a reaction scheme for protein immobilization by Mild acid according to one embodiment of the invention.

FIG. 8 shows a graph of protein immobilization versus time according to one embodiment of the invention.

FIG. 9 shows a schematic representation of the coupling reaction of an aldehyde group-containing protein with a Mirabilide amide derivative according to another embodiment of the invention.

FIG. 10A shows an electrophoresis diagram of the expression and purification of a protein according to another embodiment of the present invention (lane M represents an electrophoresis Marker (reference standard), lane 1 is a cell after induction of expression, lane 2 is a cell disruption supernatant, lane 3 is a cell fragment, and lane 4 is a purified protein); FIG. 10B shows a graph of measured molecular weight versus theoretical molecular weight for the protein of FIG. 10A prior to introduction of aldehyde groups; FIG. 10C shows a graph of measured molecular weight versus theoretical molecular weight for the protein of FIG. 10A after the introduction of aldehyde groups.

FIG. 11 shows a graphical representation of the efficient, highly specific recognition of tumor cells by coupling fluorophores to aldehyde group containing proteins according to another embodiment of the present invention.

FIG. 12 shows a schematic representation of the coupling reaction of an aldehyde group-containing protein with a Mirabilide acid amide derivative according to yet another embodiment of the invention.

FIG. 13A shows an electrophoresis diagram of the expression and purification of a protein according to still another embodiment of the present invention (lane M represents an electrophoresis Marker (reference standard), lane 1 is a cell after induction of expression, lane 2 is a cell disruption supernatant, lane 3 is a cell fragment, and lane 4 is a purified protein); FIG. 13B shows a graph of measured molecular weight versus theoretical molecular weight for the protein of FIG. 13A prior to introduction of aldehyde groups; FIG. 13C shows a graph of the measured molecular weight versus theoretical molecular weight for the protein of FIG. 13A after the introduction of aldehyde groups.

FIG. 14 shows a graph of cell viability as a function of protein conjugate concentration, according to one embodiment of the invention.

FIG. 15A shows a mass spectrum of a protein based on green fluorescent protein after introduction of aldehyde groups after coupling of fluorophores according to one embodiment of the present invention; FIG. 15B shows a mass spectrum of a protein based on an anti-EGFR nanobody after introduction of an aldehyde group after coupling with a fluorophore according to an embodiment of the invention; fig. 15C shows a mass spectrum of a protein after coupling a fluorophore based on an anti- β2 microglobulin nanobody after introduction of an aldehyde group according to an embodiment of the invention.

FIG. 16A shows a mass spectrum of a protein based on green fluorescent protein after introduction of aldehyde groups after coupling a chemotherapeutic agent according to one embodiment of the present invention; FIG. 16B shows a mass spectrum of a protein based on anti-EGFR nanobody after introduction of aldehyde groups after coupling with a chemotherapeutic agent according to one embodiment of the invention; figure 16C shows a mass spectrum of a protein after coupling a chemotherapeutic drug based on an anti- β2 microglobulin nanobody after introduction of an aldehyde group in accordance with one embodiment of the invention.

FIGS. 17A, 17B and 17C are graphical representations showing the molecular weight changes at pH 7, pH 2.8 and pH 10.0, respectively, of the resulting coupled products after coupling fluorophores based on anti-EGFR nanobodies after introduction of aldehyde groups, according to one embodiment of the invention.

FIG. 18 shows a graph of molar ellipticity as a function of wavelength before and after coupling to fluorophores for anti-EGFR nanobodies according to an embodiment of the invention.

Detailed Description

In order that the present invention may be more clearly understood, the following detailed description will be made with reference to the accompanying drawings, which illustrate, but do not limit the invention in any way.

It should be noted that, the numerical ranges in the present invention include the end values and any point value between the end values. For example, the numerical range 1 to 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; the numerical range 0.1 to 0.9 includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9.

The term "biomacromolecule" as used herein refers to a macromolecule such as a protein, polypeptide, nucleic acid, polysaccharide, etc., that is present within a cell of an organism. Each biological macromolecule has thousands to hundreds of thousands atoms, and the molecular weight is from tens of thousands to millions. Biological macromolecules are mostly polymerized from simple constituent structures, the constituent units of proteins and polypeptides are amino acids, the constituent units of nucleic acids are nucleotides, and the constituent units of polysaccharides are monosaccharides. From the chemical structure, proteins and polypeptides are formed by dehydrating and condensing alpha-L-amino acids, nucleic acids are formed by dehydrating and condensing purine and pyrimidine bases, sugar D-ribose or 2-deoxy-D-ribose and phosphoric acid, and polysaccharides are formed by dehydrating and condensing monosaccharides.

The term "aldehyde group-containing biomacromolecule" as used herein refers to the biomacromolecule described above containing one or more aldehyde groups.

The term "protein" as used herein is a covalent polypeptide chain formed by the end-to-end condensation of amino acids. Proteins include natural proteins and synthetic proteins. Each of the natural proteins has its own unique spatial structure or three-dimensional structure, which is commonly referred to as the conformation of the protein, i.e., the structure of the protein. The molecular structure of proteins can be divided into four classes: (1) primary structure: linear amino acid sequences that make up the polypeptide chain of the protein; (2) secondary structure: stable structures, mainly alpha helices and beta sheets, formed by means of hydrogen bonds between c=o and N-H groups between different amino acids; (3) tertiary structure: a three-dimensional structure of a protein molecule formed by the arrangement of a plurality of secondary structural elements in a three-dimensional space; (4) four-stage structure: are used to describe the formation of functional protein complex molecules from interactions between different polypeptide chains (subunits). In addition to these structural layers, proteins can be transformed in a number of similar structures to perform their biological functions. For functional structural changes, these tertiary or quaternary structures are generally described by chemical conformations, and the corresponding structural transformations are referred to as conformational changes.

In the present invention, commonly used proteins include, but are not limited to: (1) Green Fluorescent Protein (GFP), a β -barrel protein 1, consisting of 238 amino acids, having the amino acid sequence of SEQ ID No.1, with a molecular weight of about 27kDa; GFP was isolated from jellyfish Aequorea victoria; GFP can convert blue fluorescence from aequorin by chemical action into green fluorescence 2 by energy transfer. (2) An anti-epidermal growth factor receptor nanobody having the amino acid sequence of SEQ ID No.2, having a molecular weight of about 18.00834kDa. (3) A nanobody against β2 microglobulin having the amino acid sequence of SEQ ID No.3 and molecular weight of 18.45833kDa. These proteins are available from the company Shanghai, inc., and their amino acid sequences are as follows:

amino acid sequence of SEQ ID No.1 of green fluorescent protein:

GSSHHHHHHSSGLVPRGSHMSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTLVTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSVLSKDPNEKRDHMVL LEFVTAAGIT HGMDELYK GGGGSLCTPSR

the amino acid sequence of SEQ ID NO.2 of the anti-EGF receptor nanobody:

AEFQVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSEPKTPKPQPQPQPQPQPNPTTEHHHHHHGGGGSLCTPSR

amino acid sequence of SEQ ID No.3 of nanobody against β2 microglobulin:

AQVQLQESGGGSVQAGGSLRLSCAASGYTDSRYCMAWFRQAPGKEREWVARINSGRDITYYADSVKGRFTFSQDNAKNTVYLQMDSLEPEDTATYYCATDIPLRCRDIVAKGGDGFRYWGQGTQVTVSSEPKTPKPQPQPQPQPQPNPTTEHHHHHHGGGGSLCTPSR

the term "polypeptide" as used herein refers to a peptide consisting of three or more amino acid molecules, the peptide being a compound formed by joining together α -amino acids in peptide bonds, and being a proteolytic intermediate product. The molecular weight of the polypeptide is lower than 10,000Da, and the polypeptide can penetrate through a semipermeable membrane and is not precipitated by trichloroacetic acid and ammonium sulfate. In addition, peptides composed of 2 to 10 amino acids are referred to as oligopeptides (also referred to as small molecule peptides); peptides consisting of 10 to 50 amino acids are called polypeptides; peptides consisting of more than 50 amino acids are called proteins. Any polypeptide can be used in the art as the polypeptide of the present invention, and one skilled in the art can select a polypeptide of an appropriate structure as required to impart a corresponding functional effect.

The term "Mielic acid" as described herein has the following formula II:

wherein R is ₁ Is an alkyl group having 1 to 50 carbon atoms, a cycloalkyl group having 2 to 50 carbon atoms, an alkenyl group having 2 to 50 carbon atoms, an alkynyl group having 2 to 50 carbon atoms, an aryl or heteroaryl group having 6 to 50 carbon atoms, and a heteroatom is selected from N, O, S and P; r is R ₅ Is- (CH) ₂ ) _n COOH, n is a positive integer from 1 to 60, preferably from 1 to 30, more preferably from 1 to 20 and most preferably from 1 to 10; and R is ₁ And R is ₅ Each can be R ₄ Substituted, R ₄ Is fluorine, chlorine, bromine, iodine, an alkane having 1 to 50 carbon atomsA group, cycloalkyl having 2 to 50 carbon atoms, aryl or heteroaryl having 6 to 50 carbon atoms, and a heteroatom selected from N, O, S and P.

The term "amide derivative of Mitiglinide" as described herein has the following general formula I:

wherein R is ₁ As described above; r is R ₂ Is- (CH) ₂ ) _n CONHR ₃ And n is a positive integer from 1 to 60, preferably from 1 to 30, more preferably from 1 to 20 and most preferably from 1 to 10, R ₃ Is hydrogen, alkyl having 1 to 50 carbon atoms, cycloalkyl having 2 to 50 carbon atoms, alkenyl having 2 to 50 carbon atoms, alkynyl having 2 to 50 carbon atoms, aryl or heteroaryl having 6 to 50 carbon atoms, and a heteroatom selected from N, O, S and P; r is R ₁ 、R ₂ And R is ₃ Each can be R as described above ₄ And (3) substitution.

In the present invention, common amide derivatives of Mirabilic acid include, but are not limited to, for example, mirabilic acid amide derivatives having the structure:

(wherein, R group can be any functional molecule such as fluorophores, drugs, etc.);

the term "coupling reaction" as used herein is the process of performing a chemical reaction from two organic (bio) chemical units to obtain one organic (bio) molecule. The chemical reaction herein includes a reaction of a grignard reagent with an electrophile, a reaction of a lithium reagent with an electrophile, an electrophilic and nucleophilic reaction on an aromatic ring, and the like. Coupling reactions herein generally refer to aldehyde coupling reactions of proteins or polypeptides.

According to the present invention, the aldehyde group of the protein or polypeptide includes an aldehyde group obtained by an N-terminal oxidation reaction or amine transfer reaction of the protein or polypeptide, an aldehyde group tag of the protein or polypeptide obtained by an enzymatic method, and an unnatural amino acid containing an aldehyde group in the protein or polypeptide. In addition, according to the present invention, aldehyde coupling of proteins or polypeptides includes coupling of fluorophores, chemotherapeutic drugs, radiotherapeutic drugs, polypeptides, proteins, nucleic acids, affinity tags, and immobilization of proteins. The immobilization carrier comprises agarose gel, ferroferric oxide magnetic microsphere, silicon dioxide microsphere and metal surface.

For other terms of the present invention, those skilled in the art will generally understand their ordinary meaning in the art, unless otherwise indicated.

In the art, the reaction of Mirabilic acid and aldehyde group belongs to common organic synthesis reaction, but does not give corresponding hint to the invention, which is the first unexpected discovery of the present inventors. The reasons are as follows:

first, the type of Mitsubishi acid derivatives mentioned in the present invention has not been reported in the literature, and patent EP0578849A1 discloses only one 1, 3-dioxane-4, 6-dione derivative in which the two groups attached to the ring carbon atom number 2 are each C _1-5 Alkyl or phenyl and hydrogen or C _1-4 An alkyl group.

Secondly, although there are a number of applications of the reaction of Mitsubishi acid and its derivatives with aldehyde groups in the field of organic synthesis, the reaction with protein aldehyde groups has not been reported yet. The structure of Mitsui acid is used as a raw material in organic synthesis, whereas in the present invention, the structure of Mitsui acid is a functional group that provides a reaction with an aldehyde group of a protein, thereby realizing functional derivatization of the protein, so that the functions of the structure of Mitsui acid in organic synthesis are significantly different from those of the present invention.

Moreover, due to the specific microenvironment and steric hindrance of proteins, the aldehyde group properties and small molecule aldehyde groups are greatly different, so that aldehyde group reactions in organic synthesis cannot be suggested for protein aldehyde group reactions. For example, aldehyde groups and amino groups react very rapidly in organic synthesis and a catalyst can be used to increase the reaction rate, whereas aldehyde groups and amino groups react very slowly in proteins and the catalyst is not effective (see non-patent literature: j.am. Chem. Soc.2011, 133, 16127-16135). Therefore, it is thought that the aldehyde coupling reaction of the biomacromolecule (including protein and polypeptide) of the present invention has an insurmountable technical obstacle according to the common organic synthesis reaction, and thus the advantages obtained by the present invention are difficult to be expected from the prior art.

In particular, protein conjugates play a key role in a variety of fields. For example, protein-coupled imaging molecules are used for targeted imaging and specific recognition in the field of biological imaging; in the field of tumor treatment, antibody molecules are coupled with chemotherapeutic drugs for targeted killing of tumors; in the field of biological detection, proteins are immobilized on a carrier to prepare a protein chip, so that high-flux and high-specificity target detection is realized. The main challenges of current protein coupling are mainly how to achieve protein coupling while ensuring that the structure and function of the protein is not destroyed, and ensuring the uniformity and reproducibility of the conjugate. Conventional protein coupling generally adopts side chain groups of amino acids (such as lysine and cysteine) endogenous to the protein as coupling groups, but because the amino acids widely exist in the protein, random coupling of the protein can cause problems of easily influenced structure and function of the protein, poor uniformity and reproducibility of conjugates and the like. While aldehyde groups are not common in proteins, site-directed coupling of proteins can be achieved by introducing aldehyde groups in specific positions of the protein and using them as coupling groups. The site-directed conjugation of proteins overcomes the disadvantages of conventional protein conjugation methods, thereby minimizing the impact on the structure and function of the protein and ensuring the uniformity and reproducibility of the conjugates. In sharp contrast to the present invention, most of the existing aldehyde group modification techniques use nucleophiles based on amino groups, hydrazine groups or oxamine groups as coupling reagents for aldehyde groups, but they have disadvantages of severe reaction conditions, slow reaction rate, low product stability, etc. In this regard, the present invention developed a novel protein aldehyde coupling strategy as described herein.

The unexpected findings of the inventor solve the defects existing in the prior art, such as the technical problems of severe reaction conditions, low reaction efficiency, unstable products and the like in the prior protein aldehyde group coupling strategy; realizes the controllable fixed point modification of the protein, and solves the technical problems of non-uniformity of products, great influence on the structure and the function of the protein, poor reaction reproducibility and the like in the prior art.

For the same reasons as above, the present inventors have also unexpectedly found a novel use of a carrier having a Michaelis acid structure for immobilizing a protein. The protein immobilization has the advantages that: the protein immobilization can improve the stability of the protein, is beneficial to the separation and recovery and reutilization of the protein, or endows the material with a certain biological function. Common examples are: immobilized enzymes, antibody chips, and the like.

In addition, for the same reasons as described above, the present inventors have also unexpectedly found that the protein aldehyde group coupling method can also be applied to protein modification compounds (such as fluorophores, chemotherapeutics, radiotherapeutic drugs, polypeptides, proteins, nucleic acids, and affinity tags).

Preferred embodiments of the present invention will be described in further detail below with reference to the attached drawings, but the scope of the present invention is not limited thereby.

Aldehyde group coupling reaction

According to a preferred embodiment of the present invention, as shown in FIG. 1, a protein having an aldehyde group is coupled with a Mirabilide derivative. In particular, proteins with aldehyde groups

With Mi's acid amide derivatives->

(wherein, R group can be any functional molecule, such as fluorophor, medicine, etc.), and the aldehyde coupling reaction is carried out at pH of 2.5-10.5 and temperature of minus 20-70 ℃. During the reaction, the peripheral electron of the No. 2 ring carbon atom of the 1, 3-dioxane-4, 6-dione of the Mi's acid amide derivative is attracted by carbonyl oxygen and ether bond oxygen on the ring and amide group on the side chain of the ring to carry positive charge, and then the No. 2 ring carbon atom with positive charge attacks aldehyde group on proteinCarbonyl group on the amino acid radical to finally obtain protein aldehyde conjugate +.>

In particular, for the milbeac amide derivatives, R is a common functional group coupled to proteins, including fluorophores, chemotherapeutics, radiotherapeutic drugs, and the like. For example, in these structures of fluorophores and chemotherapeutics, alkyl groups, benzene rings, double bonds, etc. are generally included. The number of alkyl groups directly bonded to the amide bond may be 1 or more, and is preferably a positive integer of 1 to 60 and 1 to 30, more preferably 1 to 20, and most preferably 1 to 10.

In fact, provided that it has a Michaelis acid structure parent nucleus

The aldehyde group coupling reaction can be carried out with biological macromolecules such as proteins or polypeptides with aldehyde groups.

Moreover, the reaction conditions of the aldehyde group coupling reaction are mild, and the reaction temperature is in the range of-20 ℃ to 70 ℃, preferably-10 ℃ to 60 ℃ and-0 ℃ to 50 ℃, more preferably 10 ℃ to 40 ℃ and most preferably 25 ℃ to 37 ℃ in consideration of the aldehyde group coupling conversion rate of the protein; the pH is in the range of 2.5 to 10.5, preferably 3 to 10 and 4 to 9, more preferably 4 to 7, still more preferably 6 to 7 and most preferably 6.5. Compared with other aldehyde group coupling methods, the reaction has the advantages of mild reaction conditions, high reaction rate and stable coupling product. In addition, from the viewpoint of the aldehyde group coupling conversion rate of the protein, the time is 2 to 24 hours, preferably 4 to 24 hours, more preferably 6 to 24 hours, and most preferably 12 to 24 hours.

Relation between aldehyde coupling conversion and pH

The aldehyde coupling conversion of proteins at different pH is shown in FIG. 4. As can be seen from fig. 4, the pH ranges corresponding to the aldehyde coupling conversion rate of the protein from high to low are in order: 6 to 7, 5 to 6, 4 to 5, 7 to 8 and 8 to 9.

Influence of aldehyde-based coupling reactions on the structure and function of proteins

In addition, it can be seen from FIG. 5 that no significant hydrolysis of the resulting protein aldehyde conjugate occurs, either at acidic, neutral or basic pH conditions, as can be demonstrated from the measured molecular weight of the resulting conjugate at different pH and the relationship of the measured molecular weight to the theoretical molecular weight at the same pH (see FIGS. 5A, 5B and 5C and FIGS. 17A, 17B and 17C).

In addition, the effect of the coupling of proteins bearing aldehyde groups to Mi's acid derivatives on protein structure and function can be determined by circular dichroism. In particular, the protein-coupled product was diluted to a final concentration of 0.03mg/ml in 10mM phosphate buffer and added to a 1mM cuvette for circular dichroism scanning. The instrument used in the circular dichroism measuring method is a MOS-500 circular dichroism spectrometer (Bio-Logic Science Instrument); the operating parameters are a spectral scan wavelength in the range 190nm to 250nm with a scan step size of 1nm. As shown in fig. 6 and 18, the obtained circular dichroism spectrum did not change significantly before and after the aldehyde group coupling, indicating that the coupling reaction of the protein with aldehyde group and the milbeacid derivative did not have a significant effect on the protein structure. Thus, the coupling reaction of proteins bearing aldehyde groups with the miglyotic acid derivatives does not adversely affect the structure and function of the protein.

Immobilization of proteins

In the present invention, common immobilization carriers include agarose gel, ferroferric oxide magnetic microspheres, silica microspheres, and metal surfaces. According to a preferred embodiment of the present invention, the present invention synthesizes an immobilized agarose gel carrier containing a Mitiglinide structure by a solid phase synthesis method (see FIG. 7, which is typically performed at normal temperature and pressure, and pH of 6.5).

Specifically, the synthesis precursor, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide was dissolved in dimethyl sulfoxide/phosphate buffer (pH 7.4) 1:1 in solution. The solution was added to the amino-activated gel and mixed by inversion at room temperature. After the reaction was completed, the gel was washed with water, 1M sodium chloride solution, and water in this order, and the gel was stored in 20% ethanol solution, and left to stand for use.

The immobilization carrier is washed with an immobilization buffer. The protein with aldehyde groups was dissolved in MES buffer (pH 6.5) and added to the immobilization carrier. The mixture was inverted and shaken at room temperature and sampled at regular time. The protein concentration difference in the supernatant before and after immobilization was measured by BCA method to determine the amount of immobilized protein. Protein immobilization was measured at various time points (see fig. 8).

Through protein immobilization, the stability of the protein can be improved, and the separation, recovery and reutilization of the protein are facilitated, or certain biological functions are endowed to the material.

Introduction of aldehyde groups into proteins

With respect to proteins having aldehyde groups, they are formed by introducing aldehyde groups into the protein. There are many ways of introducing aldehyde groups into proteins, including, but not limited to, aldehyde groups obtained by N-terminal oxidation or amine transfer of proteins, aldehyde labels of proteins obtained by enzymatic methods, and aldehyde groups contained in unnatural amino acids in proteins.

For example, aldehyde groups are produced by using formylglycine generating enzymes. Specifically, formylglycine generating enzymes convert cysteines in proteins to formylglycines, thereby generating aldehyde groups, as shown in the following figures:

it follows that the molecular weight is reduced by about 18 after successful introduction of aldehyde groups in the protein, i.e. after conversion of the protein without aldehyde groups into a protein with aldehyde groups. Thus, by determining whether the amount of decrease in molecular weight is about 18 (e.g., 17 to 19), it can be confirmed whether aldehyde groups have been successfully introduced into the protein.

As shown in fig. 2A to 2C, fig. 2A is an expression purification electrophoresis diagram of green fluorescent protein (described in the examples below) for determining successful preparation of green fluorescent protein. FIG. 2B is a graph showing that the green fluorescent protein prepared was further confirmed to be correct from the viewpoint of molecular weight, wherein the measured molecular weight (29765.79) was consistent with the theoretical molecular weight (29766.32). Moreover, successful introduction of aldehyde groups into proteins was confirmed by the resulting change in the molecular weight of fig. 2B compared with that of fig. 2C (29765.79-29747.61 =18.18 for the change in the measured molecular weight; 29766.32-29748.22 =18.1 for the change in the theoretical molecular weight).

Preparation of Mitiglinide derivatives

As shown in FIG. 3, according to a preferred embodiment of the present invention, mitsubishi acid is obtained in the presence of an amide condensing agent (e.g., 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) at normal temperature and pressure

And H is ₂ N-R is subjected to condensation reaction to obtain the Mitiglinide derivative +.>

Amide condensing agent

In general, amidation reaction is difficult to occur, and a condensing agent is required to promote the reaction. The reaction principle is that carboxyl is activated first and then reacted with amine to obtain amide.

As for the amide condensing agent, the following four types of amide condensing agents are commonly used: (1) active esters, such as Carbonyldiimidazole (CDI). (2) Carbodiimide condensing agents such as Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDCI). (3) Onium salt condensing agents are classified into two types, that is, carbonium salt and phosphonium salt, depending on the type of salt; for example, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), 6-chlorobenzotriazol-1, 3-tetramethyluronium Hexafluorophosphate (HCTU), 2-succinimidyl-1, 3-tetramethyluronium tetrafluoroborate (TSTU) 2- (5-norbornene-2, 3-dicarboximide) -1, 3-tetramethylurea tetrafluoroborate quaternary ammonium salt (TNTU), O- (7-azabenzotriazol-1-yl) -bis (tetrahydropyrrolyl) carbonium hexafluorophosphate (HAPyU), O- (benzotriazol-1-yl) -N, N, N ', N' -dipyrromethene urea Hexafluorophosphate (HBPYU), benzotriazol-1-oxy tris (dimethylamino) phosphonium hexafluorophosphate (BOP), hexamethylphosphoramide (HMPA), benzotriazol-1-yl-oxy tripyrrolidinylphosphine hexafluorophosphate (PyBOP), and the like. (4) Organophosphorus condensing agents such as diethyl cyanophosphate (DECP), bis (2-oxo-3-oxazolidinyl) hypophosphorous acid chloride (BOP-Cl), and the like. (5) Other condensing agents such as triphenylphosphine-polyhalogenated methane, triphenylphosphine-hexachloroacetone, triphenylphosphine-NBS, 3-acyl-2-thiothiazoline, tris (2, 6-dimethoxyphenyl) bismuth, and the like.

In the present invention, the commonly used amide condensing agent may be selected from the group consisting of 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, O-benzotriazol-N, N, -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate, 2-succinimidyl-1, 3-tetramethylurea tetrafluoroborate 2- (5-norbornene-2, 3-dicarboximide) -1, 3-tetramethylurea tetrafluoroboric acid quaternary ammonium salt or benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate is preferred.

Measurement method

Electrophoresis measurement:

1. 15% release gum was formulated as follows in table 1. After the gel was separated to completion, the water was poured out and the residual water was sucked dry with clean filter paper. 4% concentrated glue is prepared, and a clean comb is immediately inserted to avoid generating bubbles. After the concentrated glue is coagulated, the comb is pulled out. And (3) installing an electrophoresis tank, and respectively adding a proper amount of Tris-glycine electrophoresis buffer solution into the upper tank and the lower tank. Avoiding bubbles at the bottom of the glue.

TABLE 1 formula of separating gel and concentrated gel

Reagent(s)	Release adhesive (15%)	Concentrated glue (4%)
			Liquid A	7.5mL	1.34mL
Liquid B	3.75mL	0mL
			Liquid C	0ml	2ml
Ultrapure water	3.75mL	4.6mL
			10% ammonium persulfate	75μL	60μL
TEMED	5μL	10μL
			Total volume of	15mL	8mL

2. To 100. Mu.l of the electrophoresis sample was added 25. Mu.l of 5 Xloading buffer and the mixture was boiled in water for 10min. Mu.l of electrophoresis sample was added to each well of the electrophoresis gel.

3. The electrophoresis apparatus is opened, 92V is used for the voltage of the concentrated gel section, after the bromophenol blue indicator enters the separation gel, the voltage is increased to 120V, the electrophoresis is ended when the bromophenol blue indicator reaches the bottom of the separation gel, the power supply is turned off, and the gel is taken out.

4. Dyeing and decoloring: washing the gel surface after electrophoresis with double distilled water, pouring the gel surface into a dyeing liquid (the formula of the dyeing liquid and the decoloring liquid are shown in the table 2 below), and placing the gel surface in a water bath at 90 ℃ for 20-30 min; after this time, the mixture was rinsed with double distilled water and poured into a decolorization solution, decolorized on an equilibrium decolorization shaker, and after the decolorization was completed, photographic analysis was performed using a gel imaging system ChemiDoc xrs+ (BIORAD corporation, usa).

TABLE 2 dyeing and bleaching liquid formulations

Reagent(s)	Dyeing liquid	Decoloring liquid
			Coomassie brilliant blue R-250	1g	0g
Industrial ethanol	0ml	300ml
			Isopropyl alcohol	250mL	0mL
Industrial acetic acid	100ml	100ml
			Single distilled water	650mL	600mL

And (II) circular dichroism spectrum measurement:

the coupled product was centrifuged through an Amicon 15 ml ultrafiltration centrifuge tube 4500g to replace the background solution with 10mM phosphate buffer (pH 7.4) at a protein concentration of 0.3mg/ml. Mu.l of the protein solution was added to a 1mm optical path cuvette and placed in a multifunctional circular dichroism spectrometer MOS-500 (French BioLogic Science Instruments company) to scan a circular dichroism spectrum between 190 and 250nm, with a scanning step size of 1nm and a slit width of 5nm. The molar ellipticity was calculated using Biokine in combination with the dichoroprot software to analyze the measured circular dichroism spectrum ratio, calculate the molar ellipticity at different wavelengths and plot the curve (e.g., fig. 6). By comparing the changes in the curves before and after coupling, it is possible to obtain that the coupling has no effect on the conformation of the protein.

(III) definition of molar ellipticity and correlation between molar ellipticity and circular dichroism measurement:

raw CD spectrum data obtained when testing circular dichroism in experiments are typically expressed in milli-degrees mdeg (Y-axis). For proteins, the molar ellipticity is generally used in literature in ordinate and in units of deg.cm ² Dmol-1. The molar ellipticity relative to the original mdeg unit takes into account the optical path of the cuvette, the concentration of the protein and the number of amino acid residues of the protein, and its calculation formula is as follows:

where X is the raw data measured by circular dichroism, the unit is medg, l is the optical path, c is the concentration of the protein, and m is the total number of amino acid residues of the protein. The molar ellipticity is obtained by taking all experimental parameters into consideration, so that the data obtained under different experimental conditions are comparable. The conversion in the experiment was accomplished by Biokine in combination with the dichroprot software.

(IV) relationship between circular dichroism spectrum and protein structure:

the peptide bond of the protein has circular dichroism in the interval of 190-250 nm detection wavelength, and the circular dichroism spectrum of the protein can reflect the change of the secondary structure of the protein.

Examples

The present invention is described in detail below by way of examples. However, the present invention is not limited to these embodiments, and those skilled in the art can make various modifications, changes, and variations to these embodiments within the scope of the present invention.

In the following examples, unless otherwise indicated, the methods are conventional; unless otherwise indicated, both the materials and reagents are commercially available.

Example 1: introduction of aldehyde groups into green fluorescent proteins

Green fluorescent protein (protein sequence shown as SEQ ID NO.1, synthesized by Shanghai) with aldehyde group tag was synthesized and cloned into pET28a (+) expression vector, then transformed into E.coli T7 buffer (DE 3), and cultured and induced to express in Terrific Broth medium. The cells were collected by centrifugation at 8000 Xg for 5 min and resuspended in disruption buffer (20 mM phosphate, pH 7.4, 500mM sodium chloride, 20mM imidazole). Cell disruption was performed using a high pressure homogenization disruptor (AH-NANO, ATS Engineering limited) and cell disruption supernatant and cell debris were collected by centrifugation at 8000 Xg for 30 minutes. Purifying cell disruption supernatant by metal ion affinity chromatography column HisTrap HP 5ml (GE Healthcare), and using purified FGE

Ultra-15.10K ultrafiltration tube (Millipore) was concentrated to 10mg/ml by ultrafiltration and the solution was changed to phosphate buffered saline (pH 7.4) and frozen at-80℃until use. As shown in FIG. 2A, successful expression of the target protein was determined by SDS-PAGE analysis of the cells, cell disruption supernatant, cell debris, and purified protein after induction of expression. Determination by high resolution liquid chromatography-mass spectrometry The protein of interest was expressed correctly (found molecular weight: 29765.79, theoretical molecular weight: 29766.32, table 7 below).

0.1mM aldehyde-tagged green fluorescent protein was added to triethanolamine buffer (50 mM triethanolamine, 50mM sodium chloride, 2mM dithiothreitol, 0.01mM copper sulfate, pH 9.0), and 0.01mM formylglycine generating enzyme was added and incubated at room temperature for 24h. After completion of the reaction, use is made of

The green fluorescent protein stock solution was concentrated to phosphate buffered saline (pH 7.4) using Ultra-15 10K ultrafiltration tubes (Millipore) and frozen at-80℃until use. As shown in FIGS. 2B and 2C, table 7, successful introduction of aldehyde groups was determined by high resolution LC-MS (measured molecular weight: 29747.61, theoretical molecular weight: 29748.22).

The amino acid sequence of SEQ ID NO. 1:

example 2: coupling reaction of Mitsubishi acid derivative and green fluorescent protein with aldehyde group

(1) Preparation of Mitiglinide derivatives

The synthesis route of the Mitiglinide derivative is shown in FIG. 3. The synthetic precursors (99 mg) and 2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate (190.12 mg) were dissolved in 2 ml dimethylformamide, 66.07 μl of 2,4, 6-trimethylpyridine were added and stirred at room temperature for 15 minutes. In addition, the compound H with amino group ₂ N-R (wherein R is a fluorophore) was dissolved in 1 ml of dimethylformamide, 132.15. Mu.l of 2,4, 6-trimethylpyridine was added thereto, and the solution was slowly added to the reaction solution and reacted with stirring for 2 hours. Purifying by silica gel column chromatography and preparative liquid chromatography to obtain the product.

(2) Coupling reaction of Mitsubishi acid derivative and protein having aldehyde group

At room temperature, 0.1mM of green fluorescent protein having aldehyde groups was dissolved in 50mM MES buffer, 1mM of Mild acid derivative was added and the reaction was carried out at different pH for 3 hours. And analyzing the product by adopting high-resolution liquid chromatography-mass spectrometry. As shown in fig. 4, milbezier acid has the highest coupling efficiency (expressed by coupling conversion) under near neutral conditions (pH 6.5). The aldehyde group coupling conversion of the green fluorescent protein as a function of pH is shown in Table 3.

TABLE 3 summary of experimental data concerning aldehyde coupling conversion and pH for green fluorescent protein

pH range	Aldehyde group coupling conversion of proteins
		pH 4-5	68-72％
pH 5-6	75-79％
		pH 6-7	76-83％
pH 7-8	28-34％
		pH 8-9	3-10％

From Table 3 above, it is understood that the pH is preferably 4 to 9, more preferably 4 to 7 and most preferably 6 to 7, particularly 6.5, from the viewpoint of the aldehyde group coupling conversion rate of the protein.

The resulting coupling product of the Mitiglinide derivative and the protein aldehyde group was exchanged by ultrafiltration to acidic (pH 2.8), neutral (pH 7.0) and basic (pH 10.0) solutions, incubated at room temperature for 48h, and the presence or absence of hydrolysis of the coupling product was determined by high resolution liquid chromatography-mass spectrometry. As shown in fig. 5A, 5B and 5C, the aldehyde-based coupling product did not undergo significant hydrolysis under both acidic, neutral and basic conditions.

In addition, the effect of the coupling of aldehyde groups and Mitsubishi acid derivatives on the protein structure was determined by circular dichroism. The coupled protein product was diluted to a final concentration of 0.03mg/ml in 10mM phosphate buffer and added to a 1mM cuvette for circular dichroism scanning. As shown in FIG. 6, the obtained circular dichroism spectrum did not change significantly before and after the reaction, indicating that the coupling of aldehyde groups and Mitiglinide derivatives did not have a significant effect on the protein structure.

In addition, 0.1mM of green fluorescent protein having aldehyde groups was dissolved in 50mM MES (pH 6.5) buffer, 1mM of Mild acid derivative was added and the reaction was carried out under different temperature conditions for 3 hours. The products were analyzed using a linear ion trap-high resolution LC-MS LTQ Orbitrap XL (available from Thermo Scientific technologies Co.). The relationship between the aldehyde group coupling conversion rate of the green fluorescent protein and the temperature change is shown in Table 4.

TABLE 4 summary of experimental data concerning the aldehyde coupling conversion of green fluorescent protein versus temperature

As is clear from Table 4 above, the temperature is preferably-10℃to 60℃and-0℃to 50℃more preferably 10℃to 40℃and most preferably 25℃to 37℃from the viewpoint of the aldehyde group coupling conversion rate of the protein.

In addition, 0.1mM of green fluorescent protein having aldehyde groups was dissolved in 50mM MES buffer (pH 6.5), 1mM of Mild acid derivative was added and the reaction was carried out at room temperature for various times. The products were analyzed using a linear ion trap-high resolution LC-MS LTQ Orbitrap XL (available from Thermo Scientific technologies Co.). The relationship between the aldehyde group coupling conversion rate of the green fluorescent protein and the time is shown in Table 5.

TABLE 5 summary of experimental data on aldehyde coupling conversion of green fluorescent protein versus time

Time (hours)	Yield (%)
		2	55
4	76
		6	89
12	100
		24	100

From the above Table 5, the time is preferably 2 to 24 hours, more preferably 4 to 24 hours and 6 to 24 hours and most preferably 12 to 24 hours from the viewpoint of the aldehyde group coupling conversion rate of the protein.

Example 3: immobilization of proteins Using vectors with Mitsubishi acid Structure

As shown in FIG. 7, the solid phase synthesis method was used to synthesize an immobilized agarose gel carrier containing a Mirabilitic acid structure. 25 mg of the synthesis precursor, 0.77 mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 1.15 mg of N-hydroxysuccinimide were dissolved in 2 ml of dimethyl sulfoxide/phosphate buffer (pH 7.4) 1:1 solution. This solution was added to 1g of the amino-activated gel and mixed for 2.5 hours at room temperature with inversion. After the reaction was completed, the gel was washed sequentially with 10 volumes of water, 1M sodium chloride solution, and water, and the gel was stored in 20% ethanol solution, and stored at 4 ℃ for use.

0.1g of the immobilization support was washed with 10 volumes of immobilization buffer (50 mM MES, pH 6.5). Green fluorescent protein (0.2 ml,7 mg/ml) with aldehyde groups was dissolved in MES buffer (pH 6.5) and added to the immobilization carrier. The mixture was inverted and shaken at room temperature and sampled at regular time. The protein concentration difference in the supernatant before and after immobilization was measured by BCA method to determine the amount of immobilized protein. Protein immobilization was measured at various time points as shown in fig. 8 and table 6 below.

TABLE 6 summary of experimental data for protein immobilization at various time points

Time (hours)	Protein immobilization amount (mg/g carrier)	Immobilization efficiency (%)
			1	1.7	13
3	3.1	24
			6	3.6	29
12	4.4	33
			24	5.4	40
36	6.3	50

From the above tables 6 and 8, it is understood that protein immobilization can improve the stability of protein, facilitate separation and recovery of protein for reuse, or impart a certain biological function to the material. Common proteins are immobilized, for example, with immobilized enzymes, antibody chips, and the like.

Example 4: introduction of aldehyde group into anti-EGFR nanobody

The corresponding procedure was the same as in example 1 except that the green fluorescent protein of example 1 was replaced with an anti-EGFR nanobody (protein sequence shown in SEQ ID NO.2, manufactured by Biotechnology (Shanghai) Co., ltd.) and a Mielic acid derivative was prepared as shown in FIG. 9. Successful expression of the protein of interest was determined by SDS-PAGE analysis of the induced expression cells, cell disruption supernatant, cell debris and purified protein. The correct expression of the target protein was determined by high resolution LC-MS analysis (found molecular weight: 18008.34, theoretical molecular weight: 18008.58, as shown in Table 7 and FIG. 10 below). In addition, successful introduction of aldehyde groups was determined by high resolution LC-MS (found molecular weight: 17990.99, theoretical molecular weight: 17990.58, as shown in Table 7 and FIG. 10 below).

The amino acid sequence of SEQ ID NO. 2:

example 5: coupling of Mitiglinide derivative and anti-EGFR nanobody with aldehyde group and application of antibody conjugate in cell imaging

1. Coupling:

the anti-EGFR nanobody can target the EGFR high-expression tumor cells, and can realize the efficient and high-specificity recognition of the tumor cells by coupling fluorophores to the protein. In this experiment, a Michaelis acid derivative with a fluorescent group is added into an anti-EGF receptor nanobody solution with an aldehyde group for reaction, as shown in FIG. 9. The reaction conditions are as follows: the reaction time was 12 hours at room temperature at pH 6.5. After the reaction was completed, the reaction solution was added to an Amicon 15 ml ultrafiltration centrifuge tube, and 4500g was centrifuged to replace the coupled product with phosphate buffer at pH 7.4. The molecular weight of the protein before and after coupling was measured by a linear ion trap-high resolution LC-MS (available from Thermo Scientific technology Co., ltd.) and matched with the theoretical molecular weight (theoretical molecular weight before coupling: 18538.22, measured molecular weight: 18537.74; theoretical molecular weight after coupling: 18458.33, measured molecular weight after coupling: 18458.99).

In the same manner as in example 2, the stability of the above anti-epidermal growth factor receptor nanobody-coupled product at various pH was verified (as shown in FIG. 17, pH of FIG. 17A is 7, pH of FIG. 17B is 2.8, pH of FIG. 17C is 10.0; also shown in Table 10 below). From the above, it was found that the molecular weight of the conjugated product of anti-EGF receptor nanobody was 18458.99 at pH 7.4, and 18537.68, 18537.49 and 18537.99 at pH 7, 2.8 and pH 10.0, respectively. Therefore, the actually measured molecular weight change of the anti-EGF receptor nanobody coupled product at different pH values is not obviously changed; similarly, the theoretical molecular weight of the coupled product at different pH values also did not change significantly. This suggests that the anti-EGFR nanobody coupled product is structurally stable at different pH values and its function is not adversely affected.

In the same manner as in example 2, it was confirmed by circular dichroism that the above anti-epidermal growth factor receptor nanobody had no effect on the antibody structure before and after coupling (as shown in fig. 18).

2. Cell imaging:

about 5X 10 in number ⁴ Tumor cells a431 cells highly expressing the epidermal growth factor receptor of (1% penicillin and streptomycin, and 10% fetal bovine serum) were inoculated in DMEM medium and cultured overnight in a cell culture incubator. The coupling product (1. Mu.M concentration) was added to the medium and incubated for 1 hour at 37 ℃. The cells were then washed twice with phosphate buffer and imaged using a laser scanning confocal microscope (Olympus FV 1000) at an excitation wavelength of 405 nm. As shown in fig. 11, the coupled product can realize the targeted imaging of tumor cells with high expression of the epidermal growth factor receptor.

Example 6: introduction of aldehyde groups in nanobodies against beta 2 microglobulin

The procedure was the same as in example 1 except that the green fluorescent protein of example 1 was replaced with a nanobody against β2 microglobulin (the protein sequence is shown in SEQ ID NO.3, which was synthesized by Shanghai Co., ltd.). Successful expression of the protein of interest was determined by SDS-PAGE analysis of the induced expression cells, cell disruption supernatant, cell debris and purified protein. The correct expression of the target protein was determined by high resolution LC-MS analysis (found molecular weight: 18458.99, theoretical molecular weight: 18458.33, as shown in Table 7 and FIG. 13 below). In addition, successful introduction of aldehyde groups was determined by high resolution LC-MS (found molecular weight: 18439.73, theoretical molecular weight: 18440.33, as shown in Table 7 and FIG. 13 below).

The amino acid sequence of SEQ ID NO. 3:

example 7: coupling of Mi's acid derivatives and nanobodies against beta 2 microglobulin bearing aldehyde groups, and use of the antibody conjugates in chemotherapeutic agents

1. Coupling:

the anti-EGFR nanobody can target the tumor cells with EGFR high expression, and the tumor cells can be targeted and killed by coupling the chemotherapeutic drugs. The coupling reaction is shown in FIG. 12, and the reaction process is the same as that of example 5. The molecular weight of the protein before and after coupling was measured by a linear ion trap-high resolution LC-MS (available from Thermo Scientific technology Co., ltd.) and matched to the theoretical molecular weight (theoretical molecular weight before coupling: 18538.22, measured molecular weight: 18537.74; theoretical molecular weight after coupling: 18700.25, measured molecular weight after coupling: 18700.52; as shown in FIG. 13).

2. Tumor cell killing:

about 5X 10 in number ⁴ Tumor cells a431 cells highly expressing the epidermal growth factor receptor of (1% penicillin and streptomycin, and 10% fetal bovine serum) were inoculated in DMEM medium and added to 96-well plates. After overnight incubation in the incubator, different concentrations (0.02-2.5. Mu.M) of conjugate were added to each well and incubation continued overnight in the incubator. MTT (thiazole blue) was dissolved in phosphate buffer at a concentration of 5mg/ml and 200. Mu.l per well was added in 96-well plates. The cells were further placed in an incubator for 4 hours, after which the medium was decanted and 200. Mu.l of dimethyl sulfoxide (DMSO) was added, and the absorbance at 570nm was measured per well using a microplate reader (Bio-Rad microplate reader). Cell viability was calculated from the following formula:

the cell survival graph is shown in fig. 14, and the conjugate can efficiently realize the targeted killing of the tumor cells with high expression of the EGF receptor.

The principle of detection of cell viability by MTT assay is summarized as follows: succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water insoluble blue-violet crystalline Formazan (Formazan) and deposits in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value of the formazan can be measured at 570nm wavelength by an enzyme-linked immunosorbent assay, so that the number of living cells can be indirectly reflected. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers.

In addition, fig. 15 shows mass spectra of proteins based on green fluorescent protein, anti-epidermal growth factor receptor nanobody, and anti- β2 microglobulin nanobody after coupling fluorophores, respectively, after introducing aldehyde groups; table 8 below shows a summary of molecular weight data for proteins based on green fluorescent protein after aldehyde group introduction, anti-epidermal growth factor receptor nanobody and anti- β2 microglobulin nanobody before and after coupling of fluorophores, respectively; table 9 below shows a summary of molecular weight data for proteins based on green fluorescent protein after aldehyde group introduction, anti-epidermal growth factor receptor nanobody and anti- β2 microglobulin nanobody before and after coupling of chemotherapeutic drugs, respectively.

TABLE 7 summary of molecular weight data for proteins before and after the introduction of aldehyde groups for Green fluorescent protein, anti-EGFR nanobody and anti-beta 2 microglobulin nanobody, respectively

TABLE 8 summary of molecular weight data for proteins based on green fluorescent protein after aldehyde group introduction, anti-EGFR nanobody and anti-beta 2 microglobulin nanobody before and after coupling fluorophore, respectively

TABLE 9 summary of molecular weight data for proteins based on green fluorescent protein after aldehyde group introduction, anti-EGFR nanobody and anti-beta 2 microglobulin nanobody before and after coupling chemotherapeutic drug, respectively

TABLE 10 stability data for anti-EGFR nanobody conjugated fluorophore products at different pH conditions

pH	Theoretical molecular weight	Measured molecular weight
			7.0	18538.22	18537.68
2.8	18538.22	18537.49
			10.0	18538.22	18537.99

The foregoing detailed description and description of the preferred embodiments of the invention have been presented by way of specific examples. It will be appreciated by persons skilled in the art that the invention is not limited to the preferred embodiments and specific examples described above. Modifications, substitutions or changes can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, which also overcomes the technical problems objectively existing in the present invention, and achieves the objects and advantageous technical effects of the present invention.

Sequence listing

<110> university of Dalian theory of engineering

<120> a biomacromolecule conjugate, and preparation method and use thereof

<130> 2021-08-16

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 268

<212> PRT

<213> Artificial Sequence

<400> 1

Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg

1 5 10 15

Gly Ser His Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro

20 25 30

Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val

35 40 45

Arg Gly Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys

50 55 60

Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val

65 70 75 80

Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His

85 90 95

Met Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val

100 105 110

Gln Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg

115 120 125

Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu

130 135 140

Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu

145 150 155 160

Glu Tyr Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln

165 170 175

Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp

180 185 190

Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly

195 200 205

Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser

210 215 220

Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu

225 230 235 240

Glu Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr

245 250 255

Lys Gly Gly Gly Gly Ser Leu Cys Thr Pro Ser Arg

260 265

<210> 2

<211> 166

<212> PRT

<213> Artificial Sequence

<400> 2

Ala Glu Phe Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln

1 5 10 15

Thr Gly Gly Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser

20 25 30

Arg Ser Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

35 40 45

Glu Phe Val Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala

50 55 60

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

65 70 75 80

Thr Val Asp Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile

85 90 95

Tyr Tyr Cys Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr

100 105 110

Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu

115 120 125

Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro Gln Pro

130 135 140

Asn Pro Thr Thr Glu His His His His His His Gly Gly Gly Gly Ser

145 150 155 160

Leu Cys Thr Pro Ser Arg

165

<210> 3

<211> 168

<212> PRT

<213> Artificial Sequence

<400> 3

Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Asp Ser Arg

20 25 30

Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp

35 40 45

Val Ala Arg Ile Asn Ser Gly Arg Asp Ile Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Phe Ser Gln Asp Asn Ala Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asp Ser Leu Glu Pro Glu Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Thr Asp Ile Pro Leu Arg Cys Arg Asp Ile Val Ala Lys Gly

100 105 110

Gly Asp Gly Phe Arg Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro

130 135 140

Gln Pro Asn Pro Thr Thr Glu His His His His His His Gly Gly Gly

145 150 155 160

Gly Ser Leu Cys Thr Pro Ser Arg

165

Claims

1. A method of preparing a biomacromolecule conjugate comprising the steps of:

(a) Providing a biological macromolecule containing aldehyde groups, wherein the biological macromolecule containing aldehyde groups comprises proteins, and the amino acid sequence of the proteins is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;

(b) Providing an amide derivative of Mitiglinide;

(c) Coupling the aldehyde group-containing biomacromolecule with the amide derivative of the Mi's acid to obtain the biomacromolecule conjugate,

Wherein the step (c) is carried out at a pH of 2.5-10.5 and a temperature of-20-70 ℃;

the biomacromolecule conjugate in step (c) comprises a protein aldehyde conjugate

Wherein the R group is a functional group coupled with a protein and comprises a fluorophore, a chemotherapeutic drug and a radiotherapy drug, wherein the fluorophore, the chemotherapeutic drug and the radiotherapy drug comprise an alkyl group with 1 to 50 carbon atoms, an alkenyl group with 2 to 50 carbon atoms and/or an aryl or heteroaryl group with 6 to 50 carbon atoms.

2. The preparation method according to claim 1, wherein the pH is 3 to 10 and the temperature is-10 to 60 DEG C

3. The preparation method according to claim 1, wherein the pH is 4-9 and the temperature is 0-50 ℃.

4. The preparation method according to claim 1, wherein the pH is 4-7 and the temperature is 10-40 ℃.

5. The preparation method according to claim 1, wherein the pH is 6-7 and the temperature is 25-37 ℃.

6. The process according to claim 1, wherein the pH is 6.5.

7. The preparation method according to claim 1, wherein the biological macromolecule containing an aldehyde group comprises a formylglycine-containing protein obtained by interacting a cysteine-containing protein with a formylglycine generating enzyme.

8. Use of a biomacromolecule conjugate according to any one of claims 1 to 7 for the preparation of a protein chip, wherein a conjugate having the formula

Is immobilized on an amino group-containing biomacromolecule by the amino group-containing acid having the formula +.>

The condensation is carried out on the carboxyl of the Mielic acid and the amino of the biomacromolecule at normal temperature and normal pressure, the biomacromolecule is selected from protein, the amino acid sequence of the protein is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and the immobilized carrier comprises agarose gel, ferroferric oxide magnetic microsphere, silicon dioxide microsphere and metal surface.

9. A biomacromolecule conjugate obtainable by the preparation method according to any one of claims 1 to 7.