Biotin-labeled agents, biotin-labeled proteins, and immunoassay reagents containing biotin-labeled agents

Abstract

To provide a labeling agent which labels proteins at distinct sites and is free from deactivation.SOLUTION: A biotin labeling agent is provided, having a biotin structure represented by a formula (1) below at one end, and a dibromopyridazinedione group represented by a formula (2) below at the other end.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a biotin labeling agent for labeling various proteins such as antibodies, and more particularly to a biotin labeling agent suitable for proteins having disulfide bonds. [Background technology]

[0002] Biotin is known to bind specifically and strongly to certain proteins such as streptavidin, and is used in immunohistochemistry and protein extraction and purification. For example, by attaching streptavidin conjugated with a dye or pigment-producing enzyme to a biotin-labeled antibody, target antigen molecules can be detected and quantified. Furthermore, by reacting a biotin-labeled protein with streptavidin conjugated with a dye or pigment-producing enzyme, the biotin and streptavidin bind, enabling the visual detection of the target protein. Additionally, by utilizing the four biotin-binding pockets of streptavidin, as shown in Figure 3, it is possible to immobilize a biotin-labeled antibody on the unbiotin-bound portion of streptavidin by binding it to a metal substrate such as Au with a biotinylation reagent. Various other proteins besides streptavidin are known to bind specifically to biotin and are used in a similar manner.

[0003] Conventionally, biotin-labeling reagents for proteins such as antibodies have included reagents that target lysine and cysteine ​​residues of proteins and peptides. Because lysine residues are abundant on the protein surface, biotin labeling of the amino group on the side chain of lysine residues is random, making quantitative determination difficult. On the other hand, while the labeling position of cysteine ​​residues is more limited than that of lysine residues due to their lower abundance, biotin binds to the sulfhydryl group formed by cleaving the disulfide bond, which destabilizes the protein structure after labeling, leading to decreased activity or, in some cases, inactivation.

[0004] Note that a compound that labels various compounds such as fluorescent dyes to disulfide bonds instead of biotin labeling is proposed in Non-Patent Document 1. This compound has a structure in which a dibromo-pyridazinedione group is bonded to the other end of a chain compound having an alkyne group at a terminal, and the dibromo-pyridazinedione group binds to the sulfhydryl terminal generated by cleaving the disulfide bond of a protein. As a result, the two sulfur atoms derived from the original disulfide bond are linked (re-crosslinked) via the pyridazinedione group. On the other hand, various compounds can be bound by the highly reactive alkyne group, whereby different types of labeling can be performed on proteins.

[0005] However, the compound disclosed in Non-Patent Document 1 has a poor production yield and, in the first place, cannot be directly used for biotin labeling. That is, for biotin labeling, a reaction for adding a biotin structure to the terminal alkyne is required, but Non-Patent Document 1 does not describe or suggest this.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a biotin labeling agent that has a clear labeling position on a protein and does not cause reduction or loss of the inherent activity of the protein due to labeling.

Means for Solving the Problems

[0008] The biotin labeling agent of the present invention that solves the above problems is a biotin labeling agent having a biotin structure (formula (1)) at one end and a dibromopyridazinedione group (formula (2)) at the other end (hereinafter referred to as a monobiotin labeling agent), or a biotin labeling agent having a branched structure with three terminals, where two of the three terminals each have a biotin structure (formula (1)) and the other terminal has a dibromopyridazinedione group (formula (2)) (hereinafter referred to as a double biotin labeling agent).

[0009] Formula (1) [ka] Formula (2) [ka]

[0010] The monobiotin labeling agent of the present invention is a linear compound having a dibromopyridazinedione group at one end and a biotin structure at the other end, while the double biotin labeling agent is a branched compound having dibromopyridazinedione groups at two ends and a biotin structure at the other end; both are novel compounds. Both are used as biotin labeling agents with the intention of utilizing the biotin structure which has a specific affinity for biotin-binding proteins such as streptavidin. In the following description, when monobiotin labeling agents and double biotin labeling agents are not distinguished, they will simply be referred to as biotin labeling agents.

[0011] Furthermore, the present invention provides proteins labeled with the biotin labeling agent described above, and immunoassay reagents containing the biotin labeling agent. [Effects of the Invention]

[0012] The present invention provides a novel compound that binds to disulfide groups (SS bonds) contained in proteins and biotin-labels the proteins. This compound has high affinity for the biotin-binding proteins mentioned above and can be used in immunohistochemistry, immunoassay reagents, protein extraction and isolation, etc. Furthermore, since the biotin labeling agent of the present invention binds to the disulfide groups of proteins, the labeling site is clearly defined, and quantitative accuracy can be obtained, for example, when used as an immunoassay reagent. Moreover, since the biotin labeling agent of the present invention binds to both ends of the cleaved disulfide group, the disulfide group can be re-crosslinked, and the structural stability of the protein to be labeled can be maintained.

[0013] Furthermore, the biotin labeling agent of the present invention can be obtained stably in higher yield compared to compounds that bind to disulfide groups disclosed in Non-Patent Document 1, etc., and since it has a biotin structure at its terminus, it does not require reaction with another compound to bind to biotin-binding proteins and can be used directly as a biotin labeling agent. [Brief explanation of the drawing]

[0014] [Figure 1] A diagram illustrating the binding of a protein disulfide group to a biotin labeling agent. [Figure 2] This figure illustrates an example of the application of antibodies labeled with the biotin-labeling agent of the present invention. [Figure 3] A diagram showing an example of an antibody immobilization reagent. [Figure 4] Figure showing the results of SDS-PAGE in Experimental Example 1. [Figure 5] The figure shows the results of Western blotting in Experimental Example 1 (left: streptavidin binding activity, right: antigen binding activity). [Figure 6] Figure showing the results of SDS-PAGE in Experimental Example 2. [Figure 7] The figure shows the results of Western blotting in Experimental Example 2, with streptavidin binding activity on the left and antigen binding activity on the right. [Figure 8] Diagram illustrating the structure of the biotin-labeled VHH antibody. [Figure 9]This figure shows the results of SDS-PAGE (left) and Western blotting (right) for Experimental Example 3. [Figure 10] Figure showing the immobilization of monobiotin-labeled VHH antibody in the SPR interaction analysis of Experimental Example 3. [Figure 11] Figure showing the antigen-binding ability of monobiotin-labeled VHH antibody in SPR interaction analysis of Experimental Example 3. [Figure 12] This figure shows the evaluation of the antigen-binding activity of raw material Fab by the SPR method in Experimental Example 4. [Figure 13] This figure shows the evaluation of the antigen-binding activity of monobiotin-labeled Fab using the SPR method in Experimental Example 4. [Figure 14] This figure shows the results of the evaluation of the antigen-binding activity of double biotin-labeled Fab using the SPR method in Experimental Example 4. [Figure 15] This figure shows the results of the evaluation of the streptavidin-binding activity of monobiotin-labeled Fab using the SPR method in Experimental Example 4. [Figure 16] Figure showing the evaluation of streptavidin binding activity of double biotin-labeled Fab by the SPR method in Experimental Example 4. [Modes for carrying out the invention]

[0015] The following describes embodiments of the biotin-labeled agent and the protein labeled therewith according to the present invention.

[0016] As described above, the biotin labeling agent of the present invention includes a monobiotin compound having one biotin structure (formula (1)) and a doublebiotin compound having two biotin structures, both of which have a dibromopyridazinedione group represented by formula (2) at their ends. As shown in Figure 1, this dibromopyridazinedione group reattaches the SS group of the protein to be labeled via the pyridazinedione group through a substitution reaction between two Br and the sulfhydryl terminus after the cleavage of the disulfide group. This makes it possible to maintain the structural stability of the protein even when biotin labeling is performed after the cleavage of the SS group.

[0017] [ka] [ka]

[0018] The biotin structure at the other end, similar to the biotin shown in formula (3) below, exhibits high affinity for biotin-binding proteins such as streptavidin, and can be used in various immune reactions, antibody and antigen detection, and separation while bound to biotin-binding proteins such as streptavidin. [ka]

[0019] In the biotin labeling agent of the present invention, the biotin structure (one of two if there are two) and the dibromopyridazinedione group are linked, for example, by a linking chain A. Here, the linking chain A has as an element C 1~5 Alkylene, polyethylene glycol with 1 to 20 repeating units ((-CH2CH2O-) 1~20 ), or a combination thereof, with the number of elements being one to six.

[0020] Here, when the number of elements is between 2 and 6, each element may be directly bonded, or it may be linked via an amide bond, a triazole group, or a secondary amine. Furthermore, the bonds between all elements may be of the same type or different types.

[0021] Furthermore, the double biotin labeling agent has a structure in which a secondary amine is contained in one of the linking sites connecting elements of linking chain A, and the hydrogen atom bonded to the secondary amine is substituted with that at one end of linking chain B, and one of the two biotin structures, i.e., the biotin structure not linked to linking chain A (formula (2)), is linked to the other end of linking chain B. Linking chain B has C as an element. 1~5 Alkylene, polyethylene glycol with 1 to 20 repeating units ((-CH2CH2O-) 1~20), or a combination thereof, and the number of elements is any one of 1 to 3.

[0022] Also for linking chain B, each element may be directly bonded, or linked via any one of an amide bond, a triazole group, and a secondary amine. Further, the bonds between all elements may be of the same bonding mode or may be different.

[0023] When the number of elements in linking chain A and linking chain B is at the maximum (the number of elements in A = 6, the number of elements in B = 3) is represented by a general formula, the monobiotin labeling agent and the double biotin labeling agent are represented by the following formulae (I) and (II).

[0024]

Chemical Formula

Chemical Formula

[0025] In equation (II), the connecting chain B is connected to the central connecting portion X of the connecting chain A. 3 The case where linking chain B is attached is shown, but if the linking site of linking chain B is a secondary amine, then as shown by the arrow in the formula, X 1 ~X 5 It can be connected to any of the following.

[0026] Typical examples of the monobiotin labeling agent and double biotin labeling agent of the present invention include the biotin labeling agents represented by the following chemical formulas (IA) and (IIA).

[0027] [ka] This monobiotin labeling agent IA is a compound in which j elements (j=1 to 5) from among the multiple elements of A and one element (alkylene) are linked by an amide bond.

[0028] [ka] In the formula, k = one of 1 to 3, and m is 1 or 2. This double biotin labeling agent IIA has a binding site X in formula II. 3 It is a secondary amine, and the linking site X 2 and Y 1 Both have a characteristic structure called a triazole group.

[0029] The biotin labeling agent of the present invention labels proteins by binding a dibromopyridazinedione group to the disulfide group of the protein to be labeled. Unlike conventional labeling agents that bind to lysine residues, which have the problem of not being able to determine which position on the protein is biotin-labeled, the number of biotin molecules bound is more clearly defined, making it useful for quantitative analysis. Furthermore, conventional biotin labeling methods that bind biotin to cleaved disulfide groups can destabilize the protein structure, leading to decreased activity and potentially inactivation. However, with the biotin labeling agent of the present invention, the disulfide bond after cleavage is rejoined (bypassed) via the pyridazinedione group, thus stabilizing the protein structure and preventing decreased activity and inactivation.

[0030] <Biotin-labeled proteins and reagents> The biotin-labeled protein of the present invention is a protein labeled with the monobiotin labeling agent or double biotin labeling agent described above, that is, a protein to which the biotin labeling agent is bound in such a way that the disulfide bonds of the protein are rejoined. As shown in Figure 1, the two Br groups of the dibromopyridazinedione group of the biotin labeling agent bind to the protein by substitution with the sulfhydryl terminus after the disulfide group of the protein to be labeled has been cleaved, thereby rejoining the disulfide group. This results in a biotin-labeled protein with a stable structure.

[0031] The proteins to be labeled are not particularly limited as long as they have disulfide bonds within the molecule, and include various antibodies (or antigens), such as IgA, IgE, IgG, IgM, ScFv-Fc, (Fab')2, Minibody, Fab, Diabody, scFv, and VHH.

[0032] The biotin-labeled proteins of the present invention can be applied to immunostaining, protein extraction, and purification by attaching fluorescent dyes, enzymes, or beads via biotin-binding proteins.

[0033] Here, "biotin-binding proteins" refer to proteins that specifically bind to biotin, including, for example, avidin, streptavidin, neutraavidin, AVR protein (Biochem.J.,2002,363, 609-617), bradavidin (J.Biol.Chem.,2005,280, 13250-13255), rizavidin (Biochem.J., 2007,405, 397-405), tamavidin (WO02 / 072817), and their variants, modifications, monomers, and multimers (conjugates), as well as other proteins with high affinity for biotin.

[0034] The biotin-binding protein according to this embodiment may have one or more (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) amino acid substitutions, insertions, deletions, and / or additions in its amino acid sequence, as long as it has specific binding ability to biotin, and any of the amino acids may be modified with sugar chains such as glycosyl groups or other substituents.

[0035] Taking streptavidin as the biotin-binding protein as an example, as shown in Figure 2, for instance, by biotin-labeling an antibody with the biotin-labeling agent of the present invention and then reacting it with streptavidin conjugated with a fluorescent dye, the antibody can be stained with a fluorescent dye, allowing for visualization of the immunoassay. Conversely, the biotin-labeling agent may be stained with a fluorescent dye via streptavidin before being reacted with the antibody. Similarly, enzymes can be bound to biotin-labeled antibodies via streptavidin, and can be applied to immunostaining (dye amplification), ELISA, and Western blotting. Furthermore, by attaching beads to the biotin-labeled protein, the insolubility of the beads, or, if magnetism is imparted, the magnetism, can be used to extract and purify a specific protein.

[0036] The immunoassay reagents of the present invention are reagents used in tests including the immunostaining (dye amplification), ELISA, and Western blotting described above, and include the biotin labeling agent, biotin-labeled protein, or a biotin-binding protein bound to the biotin structure of the biotin labeling agent. Figure 2 shows the case where the biotin-binding protein is streptavidin, but similar reagents can be made for biotin-binding proteins other than streptavidin as described above, and such reagents are also included in the present invention.

[0037] Furthermore, the biotin-labeled protein of the present invention can also be used as a reagent with antibodies immobilized on a substrate. An example of an antibody-immobilized reagent is shown in Figure 3. In this example, an Au substrate is prepared, and a biotin-labeled alkylthiol is immobilized on the substrate, for example, by binding a thiol group to Au. In addition, the biotin structure of the biotin-labeling agent, which is at a free end, is bound to streptavidin, thereby immobilizing the streptavidin. On the other hand, an antibody labeled with the labeling agent of the present invention is prepared, and this is bound to the streptavidin in the tetrameric streptavidin that does not have a bound biotin structure. As a result, the antibody is immobilized on the substrate. Such an antibody-immobilized substrate can be used in immunoassays.

[0038] In this example as well, even with biotin-binding proteins other than streptavidin, it is possible to immobilize antibodies labeled with the labeling agent of the present invention onto a substrate by immobilizing the other biotin-binding protein onto the substrate.

[0039] <Method for producing biotin-labeled agents> The following describes an example of a method for producing the biotin-labeled agent of the present invention.

[0040] The biotin labeling agent of the present invention uses biotin as a starting material to synthesize compound (IV) or compound (V), which will be the main structure of the biotin labeling agent. Compound (IV) (n=3) can be synthesized from biotin in a yield of 90% or more (Tantama et al., J. Am. Chem. Soc., 2008, 130, 15733-15787, Zong et al., J. Am. Chem. Soc. 2019, 141, 8450-8461), and compound (V) (n=3) can be synthesized from compound (IV) in a yield of 95% or more (Wooyoung et al., Chem. & Biol., 2010, 17, 537-547, Peng-Yu et al., Chem. Asian. J., 2011, 6, 2762-2775).

[0041] [ka] [ka]

[0042] In equations (IV) and (V), Z is -(CH2CH2O) as an element. 1~20 - or contains an alkylene group having 1 to 5 carbon atoms, and each element is either directly bonded or bonded by an amide bond, a triazole group, or a secondary amine. That is, when the number of elements n is 6, Z is represented as Z=-Z1-X1-Z2-X2-Z3-X3-Z4-X4-Z5-X5-Z6-, where the elements are Z1 to Z6 and the bonding sites are X1 to X5. When n is the starting material for the production of monobiotin labeling agents, n is one of n=1 to 6. When producing double biotin labeling agents, compounds with n=1 to 6 and compounds with n=1 to 3 are prepared as starting materials.

[0043] The monobiotin labeling agent (compound (I)) can be produced by a condensation reaction between compound (V) and an activated compound (VI) having a dibromopyridazinedione group.

[0044] [ka]

[0045] The double biotin labeling agent (Formula (II)) can be produced by using a compound (Formula (VII)) obtained by introducing an alkyne group to the amino group of 3-amino-1-propanol, and using a hysgen cycloaddition reaction to produce a dimer in which a starting compound (Formula (IV)) containing a biotin structure is linearly linked around an amine. Then, the terminal hydroxyl group linked to the central amine nitrogen is converted to an amino group, and a compound (Formula (VI)) containing a dibromopyridazinedione group is bonded to the amino group by a condensation reaction.

[0046] [ka]

[0047] The above manufacturing method is just one example. For instance, the above describes the case where a single starting compound (IV) is used as a double biotin labeling agent to produce a homodimer, but the linking chain (Z) n The heterodimer can also be formed using two different starting compounds (IV) with different compositions, and in this case, it is possible to link the two compounds in a linear chain by a similar reaction. Furthermore, after the production of monobiotin, it is also possible to introduce an alkyne group into the secondary amine contained in the linking chain A and attach compound (IV), which contains the biotin structure, via a hysgen cycloaddition reaction. [Examples]

[0048] The following describes examples of the biotin-labeling agent of the present invention and proteins labeled thereby.

[0049] <Experimental Example 1> Double biotin labeling of Fab 1. Synthesis of double biotin labeling agent The double biotin labeling agent (II) was synthesized using the following reaction equation. [ka] The synthesis procedure is described in detail below.

[0050] <Synthesis of compound (VIII)> Compound (IV) (24.4 g, 54.9 mmol) obtained according to the method described in the literature [Bioconjugate Chem. 2006, 17, 52-57] was dissolved in DMF (220 mL). Compound (VII) (4.15 g, 27.5 mmol) obtained according to the method described in the literature [Angew. Chem. Int. Ed. 2014, 53, 5872-5876.], aqueous solution of sodium L-ascorbate (5.99 g, 30.2 mmol, 20 mL), and aqueous solution of copper(II) sulfate pentahydrate (0.75 g, 3.02 mmol, 5 mL) were added, and the mixture was stirred at room temperature for 42 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: ethyl acetate:methanol = 1:0 → 1:2) to obtain compound (VIII) (16.5 g, 15.9 mmol) in 58% yield. R f 0.30 [100%(v) methanol]; 1 H NMR (400 MHz, methanol-d4) δ value (ppm): 7.99 (s, 2H), 4.61-4.56(m, 4H), 4.49 (ddd, J = 7.8, 5.0, 0.9 Hz, 2H), 4.30 (dd, J = 7.9, 4.5 Hz, 2H), 3.94-3.88 (m, 4H), 3.61 (q, J = 1.3 Hz, 9H), 3.58(d, J = 4.6 Hz, 10H), 3.52 (t, J = 5.5 Hz, 4H), 3.34 (d, J = 1.7 Hz, 7H), 3.31 (s, 2H), 3.20 (ddd, J = 8.8, 5.8, 4.4 Hz, 2H), 2.92 (dd, J = 12.8, 5.0 Hz, 2H), 2.70 (d, J = 12.7 Hz, 2H), 2.57 (t, J = 7.1 Hz, 2H), 2.20 (t, J = 7.4 Hz, 4H), 1.82-1.53 (m, 11H), 1.43 (p, J = 7.4 Hz, 4H);13 ¹³C NMR (100 MHz, methanol-d4) δ values ​​(ppm): 176.1, 166.1, 145.2, 126.1, 71.6, 71.5, 71.5, 71.3, 70.6, 70.4, 63.4, 61.6, 61.6, 57.0, 51.4, 41.1, 40.3, 36.7, 30.8, 29.8, 29.5, 26.8; MALDI-TOFMS (m / z) [M+H] + Calculated value 1040.54, Detected value 1040.47, [M+Na] + Calculated value: 1062.52, Detected value: 1062.45.

[0051] <Synthesis of compound (IX)> Compound (VIII) (16.2 g, 15.6 mmol), triethylamine (1.74 g, 17.2 mmol), and methanesulfonyl chloride (1.97 g, 17.2 mmol) were dissolved in DMF (234 mL) and stirred at room temperature for 15 hours under light-shielding conditions. Then, sodium azide (5.17 g, 79.5 mmol) was added and the mixture was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent:ethyl acetate:methanol = 1:2) to obtain compound (IX) (7.87 g, 7.39 mmol) in 47% yield. f 0.20 [1:2 (v / v) ethyl acetate-methanol]; 1H NMR (400 MHz, methanol-d4) δ value (ppm): 8.00 (s, 2H), 4.59 (t, J = 5.1 Hz, 4H), 4.51-4.46 (m, 2H), 4.30 (dd, J = 7.9, 4.5 Hz, 2H), 3.91 (t, J = 5.1, 4H), 3.79 (s, 4H), 3.64-3.55 (m, 18H), 3.52 (t, J = 5.5 Hz, 4H), 3.34 (d, J = 1.7 Hz, 9H), 3.20 (ddd, J = 8.9, 5.9, 4.4 Hz, 2H), 2.92 (dd, J = 12.8, 5.0, 2H), 2.70 (d, J = 12.7 Hz, 2H), 2.59 (t, J = 7.1, 2H), 2.20 (t, J = 7.4, 4H), 1.84-1.52 (m, 10H), 1.43 (p, J = 7.2 Hz, 4H); 13 ¹³C NMR (100 MHz, methanol-d4) δ values ​​(ppm): 176.1, 166.1, 144.9, 126.2, 71.5, 71.5, 71.5, 71.3, 70.6, 70.4, 63.4, 61.6, 61.6, 57.0, 51.4, 41.1, 40.3, 36.7, 30.7, 29.8, 29.5, 26.8; MALDI-TOFMS (m / z) [M+Na] + Calculated value 1087.53, detected value 1087.41, [M-N2+Na] + Calculated value: 1059.52, Detected value: 1059.34.

[0052] <Synthesis of double biotin-labeled agent (II)> Compound (IX) (400 mg, 0.375 mmol) was dissolved in methanol (4 mL), and 20% palladium hydroxide-activated carbon (500 mg) was added. The mixture was stirred at room temperature under a hydrogen atmosphere for 20 hours. After Celite filtration, the filtrate was concentrated under reduced pressure, and the residue (X) (291 mg, R) was obtained. f0.10 [100% (v) methanol]) was obtained. Compound (VI) (127 mg, 0.28 mmol) and residue (X) (291 mg, 0.28 mmol) obtained according to the method described in the literature [Org. Biomol. Chem. 2018, 16, 1359-1366.] were dissolved in DMF (4 mL) and stirred at room temperature for 21 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: ethyl acetate: methanol = 1:0 → 0:1) to obtain double biotin labeling agent (II) (134 mg, 0.097 mmol) in 35% yield. f 0.30 [100%(v) methanol]; 1 H NMR (400 MHz, methanol-d4) δ value (ppm): 7.98 (s, 2H), 4.59 (t, J = 5.1 Hz, 4H), 4.54-4.39 (m, 4H), 4.30 (dd, J = 7.9, 4.5 Hz, 2H) 3.91 (t, J = 5.4 Hz, 5H), 3.74 (s, 3H), 3.67 (s, 2H), 3.65-3.54 (m, 18H), 3.52 (t, J = 5.4 Hz, 5H), 3.31 (s, 1H)m 3.19 (ddt, J = 8.9, 6.7, 4.5 Hz, 2H), 2.92 (dd, J = 12.8, 5.0 Hz, 2H), 2.79 (d, J = 2.1 Hz, 1H), 2.70 (d, J = 12.7 Hz, 2H), 2.66-2.58 (m, 2H), 2.52-2.48 (m, 2H), 2.21 (t, J = .4 Hz, 4H), 1.65 (dddt, J = 38.7, 24.3, 13.9, 6.8 Hz, 8H), 1.43 (p, J = 7.3 Hz, 4H); 13¹³C NMR (100 MHz, methanol-d4) δ values ​​(ppm): 176.1, 172.1, 166.1, 154.8, 154.5, 145.3, 136.7, 136.5, 126.1, 71.6, 71.5, 71.5, 71.3, 70.6, 70.4, 63.4, 61.6, 57.0, 51.9, 51.4, 49.9, 45.5, 41.1, 40.4, 38.9, 36.8, 35.7, 35.0, 29.8, 29.5, 27.3, 26.9, 26.3; MALDI-TOFMS (m / z) [M+H] + Calculated value 1377.43, Detected value 1377.43, [M+Na] + Calculated value 1399.41, Detected value 1399.41, [M+K] + Calculated value: 1415.38, Detected value: 1415.39.

[0053] 2. Preparation of double biotin-labeled Fab Fab was double-biotin labeled using the double-biotin labeling agent (II) obtained by the above synthesis. Fab was identified using anti-mouse polyclonal IgG (H+L), goat, and affinity-purified Fab fragments (Jackson Immuno Research Laboratories, Inc.).

[0054] First, a BBS solution of Fab fragments (26.0 μM, 1.3 mg / mL, 28.3 μL) was mixed with a BBS solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (26.1 mM, 21.8 μL) and incubated at 37°C for 1.5 hours. After ultrafiltration, a DMF solution of the double biotin labeling agent (IIA) (6 mM, 4.90 μL) and BBS buffer (65.1 μL) were added and incubated at 21°C for 21 hours. After ultrafiltration, double biotin-labeled Fab was obtained.

[0055] The buffers used were BBS buffer (25 mM boric acid, 25 mM sodium chloride, 0.5 mM ethylenediaminetetraacetic acid, pH 8.0), PBS buffer (TaKaRa's "Phosphate Buffer Saline (PBS) Tablets, pH 7.4" dissolved in 1000 mL of ultrapure water), or TBS-T buffer (TaKaRa's "Tris Buffered Saline with Tween 20 (TBS-T) Tablets, pH 7.6" dissolved in 500 mL of ultrapure water). Ultrafiltration was performed using a Merck Amicon Ultra 0.5 mL centrifugal filter (3000 MWCO) according to the product protocol.

[0056] Polyacrylamide electrophoresis (SDS-PAGE) was performed to confirm the formation of double biotin-labeled Fab cells. PAGE was performed using 12% separation gels and 4% concentration gels, and after electrophoresis, the gels were stained with Barrett CBB Stain One (Nacalai Tesque). Precision Plus Dual Extra Standard (Bio-Rad Laboratories) was used as the molecular weight marker in SDS-PAGE. Printgraph CMOS I (ATTO) was used for gel imaging. The results are shown in Figure 4.

[0057] In Figure 4, each lane, from left to right, shows the molecular weight marker, the starting material Fab, the Fab after the TCEP reaction (reduction), and the Fab after double biotin labeling. As shown in the figure, the molecular weight of the starting material Fab (approximately 50 kDa) is halved (approximately 25 kDa) after the reduction reaction, indicating that the SS bond was cleaved and separated into light and heavy chains. Furthermore, after the reaction with the double biotin labeling agent (far right lane), many bands appeared with the same molecular weight as the starting material Fab (approximately 50 kDa), confirming that the light and heavy chains were re-crosslinked by the double biotin labeling agent. The re-crosslinking rate was calculated from the intensity ratio of the two bands detected in the far right lane (band with a molecular weight similar to that of the starting material Fab / band with a molecular weight similar to that of the reduced Fab), resulting in a result of approximately 79%. This confirms that double biotin labeling is performed with high efficiency by the above reaction.

[0058] 3. Activity test Western blotting was performed to confirm the activity of double biotin-labeled Fab. For gel transfer in Western blotting, a TransBlot Turbo Transfer Pack 0.2 μm PVDF was used and the gel was transferred using the TransBlot Turbo system. After transfer, the PVDF membrane was immersed in Bullet Blocking One (for Western blotting) (Nacalai Tesque) (50 mL / gel) and blocked by permeation at room temperature for 30 minutes. To detect streptavidin binding activity and antigen binding activity by fluorescence, reagents pre-labeled with Cy3 dye, streptavidin and antigen (mouse IgG antibody), respectively, were used.

[0059] For evaluating antigen-binding activity, Cy is used as a fluorescently labeled antigen. TM 3-conjugeted ChromPure Mouse IgG, whole molecule (Jackson Immunoresearch Laboratories, Inc.) was used to evaluate streptavidin binding activity. TM3-conjugeted Streptavidin (Jackson Immunoresearch Laboratories, Inc.) was used and diluted with TBS-T buffer (10 μL / gel). A PVDF membrane was immersed in this solution and shaken at room temperature for 1 hour. The reaction mixture was discarded, and 50 mL of TBS-T buffer was added and shaken for 5 minutes. This washing procedure was repeated three times. The resulting gels were subjected to fluorescence gel imaging (excitation wavelength 532 nm, fluorescence wavelength 670 nm) using a Typhoon 9400 (Amersham Bioscience). The results of Western blotting are shown in Figure 5.

[0060] The fluorescent gel image on the left side of Figure 5 shows the results of the streptavidin binding activity test. Since a fluorescent band was detected only in the far right lane, it indicates that the double biotin labeling agent bound to the reduced Fab, resulting in the expression of the intended streptavidin binding activity. Of the detected fluorescent bands, the upper band (around 50 kDa) represents the streptavidin binding activity of the double biotin-labeled Fab, while the lower band (around 25 kDa) represents the streptavidin binding activity of the double biotin-labeled Fab light chain or the double biotin-labeled Fab heavy chain. These results demonstrate that Fab labeled with the double biotin labeling agent of the present invention possesses sufficient binding activity to streptavidin.

[0061] The fluorescent gel image on the right side of Figure 5 shows the results of the antigen-binding activity test. Two fluorescent bands were detected around 50 kDa; one was from the raw material Fab, and the other was from the double-biotin-labeled Fab. This result confirms that the antigen-binding ability of Fab is maintained without being impaired by double-biotin labeling.

[0062] <Experimental Example 2> Monobiotin labeling of Fab 1. Synthesis of monobiotin labeling agents

[0063] The monobiotin labeling agent (I) was synthesized using the following reaction equation. [ka]

[0064] The following describes the details of the synthesis procedure. Compound (IV) (1.02 g, 2.30 mmol), obtained according to the method described in the literature [Bioconjugate Chem. 2006, 17, 52-57], was dissolved in methanol (10 mL), and 20% palladium hydroxide-activated carbon (1.25 g) was added. The mixture was stirred at room temperature under a hydrogen atmosphere for 6 hours. After Celite filtration, the filtrate was concentrated under reduced pressure, and the residue (V) (836 mg, R) was obtained. f Compound (I) (292 mg, 0.39 mmol) was obtained in 24% yield by silica gel chromatography (eluent:chloroform:methanol = 6:1). The residue (V) (778 mg) and compound (VI) (719 mg, 1.59 mmol) were dissolved in DMF (10 mL) and stirred at room temperature for 14 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent:chloroform:methanol = 6:1). f 0.30 [4:1 (v / v) chloroform-methanol]; 1 H NMR (400 MHz, methanol-d4) δ value (ppm): 4.57-4.41 (m, 3H), 4.31 (dd, J = 7.9, 4.4 Hz, 1H), 3.68 (s, 3H), 3.66-3.62 (m, 4H), 3.62-3.46 (m, 8H), 3.41-3.30 (m, 4H), 3.21 (ddd, J = 8.9, 5.9, 4.4 Hz, 1H), 2.93 (dd, J = 12.7, 5.0, 1H), 2.69 (dd, J = 12.7, 5.0 Hz, 1H), 2.60 (t, J = 6.8 Hz, 2H), 2.22 (t, J = 7.4 Hz, 2H), 1.81-1.53 ​​(m, 4H), 1.51-1.38 (m, 2H); 13¹³C NMR (100 MHz, methanol-d4) δ values ​​(ppm): 176.1, 166.1, 154.8, 154.5, 136.8, 136.5, 71.6, 71.2, 70.6, 70.4, 63.4, 61.6, 57.0, 45.5, 41.1, 40.5, 40.3, 36.8, 35.6, 34.7, 29.8, 29.5, 26.8, 26.3, 18.4; MALDI-TOFMS (m / z) [M+H] + Calculated value 757.11, Detected value 757.11, [M+Na] + Calculated value 779.11, Detected value 779.09, [M+K] + Calculated value: 795.06, Detected value: 765.09.

[0065] 2. Preparation of monobiotin-labeled Fab Monobiotin-labeled Fab was prepared using the same Fab antibody as that used for double biotin labeling, and the same method as in Experimental Example 1. Unless otherwise specified, the reagents, solvents, equipment, and instruments other than the monobiotin labeling agent were the same as those used for the double biotin-labeled Fab described above.

[0066] In the same manner as in Experimental Example 1, a BBS solution of reduced Fab (26.0 μM, 1.3 mg / mL, 28.3 μL) was mixed with TCEP (26.1 mM, 21.8 μL) and incubated at 37°C for 1.5 hours. After ultrafiltration, a DMF solution of the above monobiotin labeling agent (I) (15 mM, 3.43 μL) and BBS buffer (66.6 μL) were added and incubated at 21°C for 18 hours. After ultrafiltration, monobiotin-labeled Fab was obtained.

[0067] To confirm the generation of monobiotin-labeled Fab and its antigen-binding activity, SDS-PAGE was performed under the same conditions as for double-biotin-labeled Fab. The results are shown in Figure 6.

[0068] In Figure 6, the four lanes, from left to right, show the molecular weight marker, the starting material Fab, the Fab after the TCEP reaction (reduction), and the Fab after monobiotin labeling. In the far right lane, a band was observed at the same position as the starting material Fab (approximately 50 kDa), indicating that monobiotin-labeled Fab was produced. The re-crosslinking rate was calculated to be approximately 78%. In other words, it was confirmed that monobiotin labeling was performed with high efficiency by the above reaction, similar to double biotin labeling.

[0069] 3. Activity test To confirm the activity of monobiotin-labeled Fab, Western blotting was performed under the same conditions as for double-biotin-labeled Fab. The results are shown in Figure 7.

[0070] The fluorescent gel image on the left of Figure 7 shows the results of the streptavidin binding activity test. Since a fluorescent band was detected only in the far right lane, it indicates that the monobiotin labeling agent bound to the reduced Fab, resulting in the expression of the intended streptavidin binding activity. Similar to the double biotin labeling case, the upper band (around 50 kDa) of the detected fluorescent bands represents the streptavidin binding activity of monobiotin-labeled Fab, while the lower band (around 25 kDa) represents the streptavidin binding activity of the monobiotin-labeled Fab light chain or the monobiotin-labeled Fab heavy chain. From these results, it was confirmed that Fab labeled with the monobiotin labeling agent of the present invention possesses sufficient binding activity to streptavidin.

[0071] The fluorescent gel image on the right side of Figure 7 shows the results of the antigen-binding activity test. Two fluorescent bands were detected around 50 kDa; one was from the raw material Fab, and the other was from monobiotin-labeled Fab. From these results, it was confirmed that, similar to double biotin labeling, monobiotin labeling preserved the antigen-binding ability of Fab without impairing it.

[0072] <Experimental Example 3> Monobiotin Labeling of VHH Antibody Using the monobiotin labeling agent (I) synthesized in Experimental Example 2, we performed monobiotin labeling of the VHH antibody and confirmed and evaluated its binding activity.

[0073] 1. Preparation of monobiotin-labeled VHH and confirmation of streptavidin binding activity. VHH antibodies are derived from the antigen-binding domain of heavy chain antibodies from camelid animals. They are expected to be a viable alternative to conventional antibodies due to their advantages, including a small molecular weight (15 kDa), high thermal stability, the ability to be recombinantly produced using E. coli or yeast, and good handling in protein engineering. As shown in Figure 8, VHH antibodies have a disulfide bond within their molecule, and in this experiment, this disulfide bond was targeted for monobiotin labeling.

[0074] The VHH antibody used was produced by Protein Express based on the information described in the literature [Behar et al., Protein Eng. Des. Sel., 2008, 21, 1-10] (anti-FcγRIII VHH antibody, C21). Unless otherwise specified, the reagents, solvents, equipment, and instruments other than the VHH antibody were the same as those used in the double biotin-labeled Fab described above.

[0075] A PBS solution of VHH antibody (147 μM, 2.2 mg / mL, 8.4 μL) was mixed with a BBS solution of TCEP (15.0 mM, 8.2 μL) and BBS buffer (33.5 μL), and incubated at 37°C for 1.5 hours. After ultrafiltration, a DMF solution of monobiotin labeling agent (I) (10 mM, 5.0 μL) and BBS buffer (65.0 μL) were added, and incubated at 21°C for 21 hours. After ultrafiltration, monobiotin-labeled VHH antibody was obtained.

[0076] The reduction reaction of the VHH antibody and the monobiotin labeling process were analyzed using SDS-PAGE (15% separation gel, 4% concentration gel). Western blotting was also performed, and bands containing biotin structures were detected by fluorescence using Cy3-labeled streptavidin. The results of SDS-PAGE and Western blotting are shown in Figure 9.

[0077] The left side of Figure 9 shows the results of SDS-PAGE, and the right side shows the results of Western blotting. In both figures, the four lanes, from left to right, show the molecular weight marker, the starting material VHH, the reduced VHH, and the monobiotin-labeled VHH. In the SDS-PAGE results, the molecular weight differences between the VHHs were small, and no significant differences were observed in the band positions. However, the Western blotting results shown on the right side of Figure 9 show a fluorescent band detected in the far right lane, indicating binding activity with streptavidin, thus confirming the formation of monobiotin-labeled VHH.

[0078] 2. Evaluation of antigen-binding activity of monobiotin-labeled VHH To quantitatively evaluate the antigen-binding activity of the monobiotin-labeled VHH described above, interaction analysis experiments were performed using the SPR method. The measurement device used was a Biacore X100 (GE Healthcare Life Sciences). First, the monobiotin-labeled VHH solution was passed through a Sensor Chip SA (Cytiva) pre-immobilized with streptavidin, and the monobiotin-labeled VHH antibody was immobilized on the substrate. The results are shown in Figure 10.

[0079] In the graph shown in Figure 10, the vertical axis represents the amount immobilized on the chip (RU), and the horizontal axis represents time (elapsed time from the start of the flow experiment). The black circles indicate the points in time when VHH mixtures of different concentrations were added. VHH mixtures of 9.2 ng / ml, 92 ng / ml, and 0.92 μg / ml were added at time intervals. The concentrations of the VHH mixtures were calculated from the UV absorbance at 280 nm. The amount immobilized increased sharply (by approximately 700 RU) immediately after the 0.92 μg / ml VHH mixture was introduced, indicating that monobiotin-labeled VHH was successfully immobilized on streptavidin on the substrate.

[0080] After binding monobiotin-labeled VHH to the SA chip, the mixture was flowed while gradually increasing the antigen (CD16) concentration, and the binding affinity between the antigen and VHH was measured by surface plasmon resonance. The results are shown in Figure 11.

[0081] The vertical axis of the sensorgram shown in Figure 11 represents response (RU), and the horizontal axis represents time (s). The sensorgram confirmed that the amount of antigen bound increases in a concentration-dependent manner. Furthermore, the dissociation constant (K) was identified from the sensorgram. D When the value was calculated, K D The value is 1.1 × 10 -7 The result was M. This confirmed that the antigen-binding activity is maintained even when the disulfide site of VHH is labeled with biotin.

[0082] <Experimental Example 4> Comparison of binding activity of monobiotin-labeled and double-biotin-labeled agents

[0083] The binding affinity to the antigen and to streptavidin was evaluated using the SPR method.

[0084] First, the antigen of the Fab antibody is immobilized as a ligand and then the sensor chip CM5 (Biacore) is used. TH Using a 3D filter, raw material Fab, monobiotin-labeled Fab, and double biotin-labeled Fab were passed through the filter as analytes, and the dissociation constant was measured by surface plasmon analysis. The results are shown in Figures 12 (raw material Fab), 13 (monobiotin-labeled Fab), and 14 (double biotin-labeled Fab). In Figures 12 to 14, a Fab concentration-dependent change in the sensorgram was observed. From the sensorgram, K D When the value was calculated, the K of the raw material Fab was D The value is 7.0 × 10 -9 In contrast to M, monobiotin-labeled Fab is K D The value is 9.9 × 10 -9 M, K of double biotin-labeled Fab D The value is 1.1 × 10 -8 At level M, it was confirmed that the activity was almost completely preserved.

[0085] Furthermore, tetrameric streptavidin was passed through monobiotin-labeled Fab and double-biotin-labeled Fab bound to immobilized antigens on the sensor chip, and the binding activity to streptavidin was measured. The results are shown in Figure 15 (monobiotin-labeled Fab) and Figure 16 (double-biotin-labeled Fab). In Figures 15 and 16, streptavidin concentration-dependent changes in the sensorgram were observed. From the sensorgram, K D When the value was calculated, the K of monobiotin-labeled Fab D The value is 4.4 × 10 -13 M, K of double biotin-labeled Fab D The value is 4.4 × 10 -14 The result was M. In other words, it was confirmed that double biotin-labeled Fab binds to streptavidin 10 times more strongly than monobiotin-labeled Fab.

[0086] Experimental examples 1 to 4 confirmed that the biotin labeling agent of the present invention can biotin-label the disulfide moiety of proteins, that the thiol is re-crosslinked, stabilizing the protein structure and maintaining its activity, and that incorporating two biotin structures into the molecule increases the binding affinity to streptadipine tenfold.

Claims

1. A biotin labeling agent having a biotin structure (formula (1)) at one end and a dibromopyridazinedione group (formula (2)) at the other end, wherein the biotin structure and the dibromopyridazinedione group are linked by a linking chain A, and the linking chain A includes, as elements, C1 to C5 alkylene, polyethylene glycol ((-CH2CH2O-)1 to 20) having 1 to 20 repeating units, or a combination thereof, and the number of elements is 1 to 6. Formula (1) 【Chemistry 1】 Formula (2) 【Chemistry 2】

2. A biotin labeling agent having a branched structure with three terminals, wherein two of the three terminals have a biotin structure (formula (1)) and the other terminal has a dibromopyridazinedione group (formula (2)), wherein the biotin structure at one terminal and the dibromopyridazinedione group at the other terminal are linked by a connecting chain A, and the connecting chain A includes, as elements, C1 to C5 alkylene, polyethylene glycol ((-CH2CH2O-)1 to 20) with 1 to 20 repeating units, or a combination thereof, and the number of elements is 1 to 6. Formula (1) 【Chemistry 1】 Formula (2) 【Chemistry 2】

3. A biotin labeling agent according to claim 1 or 2, The biotin labeling agent is characterized in that, when the number of elements in the linked chain A is 2 to 6, each element is either directly linked or linked via an amide bond, a triazole group, or a secondary amine.

4. A biotin labeling agent according to claim 2, The aforementioned linking chain A contains a secondary amine, and a hydrogen atom bonded to the secondary amine is substituted with one end of linking chain B, and the other end of linking chain B is connected to a biotin structure at one end that is not connected to linking chain A. Linked chain B has C as an element. １～５ A biotin labeling agent characterized by comprising an alkylene, polyethylene glycol having 1 to 20 repeating units, or a combination thereof, wherein the number of elements is one to three.

5. A biotin labeling agent according to claim 4, The biotin labeling agent is characterized in that, when the number of elements in the linking chain B is two or three, each element is either directly linked or linked via an amide bond, a triazole group, or a secondary amine.

6. A biotin-labeled protein using the biotin-labeling agent described in claim 1 or claim 2.

7. A biotin-labeled protein according to claim 6, Proteins include IgA, IgE, IgG, IgM, ScFv-Fc, (Fab') 2 A biotin-labeled protein characterized by being one of the antibodies or antibody fragments selected from small molecule antibodies (Minibody), Fab, bispecific antibodies (Diabody), single-chain variable region fragments (scFv), and VHH.

8. An immunoassay reagent comprising the biotin labeling agent according to claim 1 or claim 2.

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