WO2005077984A1 - Process for producing organic compound and antibody for use therein - Google Patents

Abstract

A process for producing an organic compound with high yield, which is applicable to a compound of complex structure; and an antibody suitable thereto. It has been found to use an antibody capable of recognizing a target compound of reaction as an abzyme. Thus, as an abzyme can be obtained without the need to use a transition state compound or transition state homologue whose synthesis is difficult, application thereof can be made to a compound of complex structure. Further, by the use of high discriminating power of antibody, effective application can be made to a reaction in which it is desired to selectively obtain specified one among multiple products. For example, especially preferred application can be made to an asymmetric synthesis, an addition reaction to unsaturated bond, etc. in which it is desired to selectively obtain specified one among optical isomers (enantiomer), diastereomers, geometrical isomers, etc.

Description

 Specification

 Method for producing organic compound and antibody used therefor

 Technical field

 The present invention relates to a method for producing an organic compound and an antibody used for the method.

 Background art

 [0002] In the field of organic chemistry, particularly in the field of pharmacy, it is an industrially important task to supply a target compound at a high yield and at low cost. In particular, a compound that does not exist in nature or a compound that exists only in a trace amount is required to be synthesized at a high yield by an artificial synthesis method. However, in the case of a compound having a complex structure, it is often impossible to synthesize a compound at an extremely low yield because of difficulties in controlling side reactions. For example, for vinblastine, which is naturally contained as a trace component (vinblastine, sometimes referred to as VLB), various synthetic routes have already been published, but problems such as yield and cost can be sufficiently solved. There is no synthetic method yet (see Non-patent Documents 15 and 8).

 [0003] On the other hand, as an example of the use of antibodies for organic synthesis, V ヽ ゎ yuru abzyme or abzyme (see, eg, Lerner et al. (See non-patent document 6) and Schultz et al. (See non-patent document 7). Synthetic methods using catalytic antibodies) are widely known. In this synthesis method, the antibody is used as a catalyst for accelerating the reaction by reducing the free energy in the transition state of the reaction using an antibody that recognizes the transition state compound of the target reaction. This is the synthesis method used. By taking advantage of the high discriminating ability of antibodies, it is expected that complex compounds that are difficult to synthesize by general synthesis methods can be obtained in high yields.

 [0004] However, although a number of abzymes of reactions have been produced so far, their application range has been limited to the synthesis of compounds having relatively simple structures.

For example, in Non-Patent Document 7, Schultz et al. Performed a hydrolysis reaction of a carbonate ester (a) using Abzym as shown in the following Scheme 1. At this time, Schultz et al. First synthesized a transition state analog (c) of the reaction of scheme 1, and further prepared a monoclonal antibody using the complex of the transition state analog and the protein as an antigen, and then prepared the monoclonal antibody. Using the antibody as an abzyme, the hydrolysis reaction of Scheme 1 was performed as described above. [Formula 11]

[0006] Various organic reactions using Abzym have been developed since the publication of Non-Patent Document 7 by Schultz et al., But first of all, a transition state compound or a transition state analog of the target reaction (transition state analog) was developed. ) Needed to be obtained. However, when the target compound or its precursor has a complicated structure, it is often very difficult to synthesize a transition state compound or a transition state analog of the target reaction. For this reason, the synthesis of organic compounds by Abzaim was expected as a new synthesis method, but its application was limited to compounds with relatively simple structures.

 [0007] Thus, up to now, there have been promising methods for synthesizing organic compounds having a complicated structure in high yield, but there are still problems to be solved.

 [0008] Non-patent literature l: Atta-ur-Rahman; Basha, A .; Ghazala, M. Tetrahedron Lett., 1976, 27, 2351-2354.

 Non-Patent Document 2: Kuehne, M.E .; Matson, P.A .; Bornmann, W.G.J.Org.Chem.,

1991, 56, 513-528.

Non-Patent Document 3: Bornmann, WG; Kuehne, MEJ Org.Chem., 1992, 57, 1752-1760. Non-Patent Document 4: Magnus, P .; Mendoza, JS; Stamford, A .; Ladlow, M .; Willis, PJ Am. Chem. Soc, 1992, 114, 10232-10245.

 ^^ Patent Document 5: Yokoshima, S .; Ueda, T .; Kobayashi, S .; Sato, A .; Kuboyama,

T .; Tokuyama, H .; Fukuyama, T. J. Am. Chem. Soc, 2002, 124, 2137-2139.

 Non-Patent Document 6: Tramontano, A .; Janda, K.D .; Lerner, R.A.Science, 1986, 234, 1566-1570.

 Non-Patent Document 7: Pollack, S.J .; Jacobs, J.W; Schultz, P.G.Science, 1986, 234, 1570-1573.

 Non-Patent Document 8: Langlois, N .; Potier, P.J.C.S.Chem.Commun "1979, 582-583.

 Disclosure of the invention

 [0009] Therefore, an object of the present invention is to provide a method for producing an organic compound at a high yield, which can be applied to a compound having a complicated structure, and an antibody suitable for the method. In order to solve the above-mentioned problems, a production method of the present invention is a method for producing an organic compound, wherein the precursor of the organic compound is produced in the presence of an antibody capable of recognizing the organic compound to be produced. This is a production method for converting into an organic compound.

 [0010] With the above configuration, the method for producing an organic compound of the present invention can be applied to a compound having a complicated structure, and the organic compound to be produced (hereinafter referred to as "the target compound") Or simply referred to as “target compound”) can be obtained in high yield. Brief Description of Drawings

 [FIG. 1] FIG. 1 is a graph showing the results of ELISA measurement of deacetylVLB-TG complex-sensitized mice.

 FIG. 2 is a graph showing column chromatography separation of antibody MAb-10-A9.

 FIG. 3 is a graph showing HPLC sampling of a reaction using the antibody of the present invention.

 FIG. 4 is a graph showing HPLC sampling of a reaction using the antibody of the present invention.

FIG. 5 is a graph showing HPLC sampling of a reaction using the antibody of the present invention. FIG. 6 is a graph showing HPLC sampling of a reaction using the antibody of the present invention.

 FIG. 7 is a graph showing sampling by ESI-MS of a reaction using the antibody of the present invention.

 BEST MODE FOR CARRYING OUT THE INVENTION

Next, an embodiment of the present invention will be described.

 [0013] As described above, the conventional Abzyme uses an antibody that recognizes a transition state of a target reaction. However, the inventors of the present invention used an antibody that recognizes the target compound instead of the transition state of the reaction as an abzyme! /, And obtained an idea, and synthesized based on this idea. The present inventors have found that the target compound can be produced under low pressure and mild conditions, and have reached the present invention. As a result, an abzyme can be obtained without using a transition state compound or a transition state analog which is difficult to synthesize, so that the production method of the present invention can be applied to a compound having a complicated structure. Further, by utilizing the high discriminating ability of the antibody, the target compound can be obtained in high yield as described above.

 [0014] The production method of the present invention may consist only of a conversion reaction from the precursor to the organic compound, but may further include other reaction steps as appropriate. Further, the organic compound to be produced may be used to induce another compound by another reaction step.

 [0015] The conversion reaction from the precursor to the organic compound is not particularly limited. For example, at least a group force including an addition reaction, an elimination reaction, a transfer reaction, an oxidation-reduction reaction, a condensation reaction, and a decomposition reaction is also selected. Preferably, it involves one reaction. Also, by utilizing the high discriminating ability of the antibody, it can be effectively used for a reaction to selectively obtain a specific product among a plurality of products. For example, an optical isomer (enantiomer), It is particularly preferable to use the compound for asymmetric synthesis, addition reaction to an unsaturated bond, or the like in which a specific one of diastereomers and geometric isomers is desired to be selectively obtained. Conventional Abzym has the effect of reducing the free energy in the transition state of the reaction by recognizing the transition state compound of the reaction.However, in the present invention, the target compound is used instead of the transition state compound of the reaction. Recognition can increase the selectivity of the reaction.

[0016] In the production method of the present invention, the conversion reaction from the precursor to the organic compound may be a seed. It is applicable to various organic compounds, and the organic compound to be produced is not particularly limited.

V is preferably, for example, a compound represented by the following formula (I).

[Formula 12]

In the formula (I),

R ¹ is a hydrogen atom, a linear or branched alkyl group having 16 carbon atoms, or a linear or branched acyl group having 16 carbon atoms,

R ² is a hydrogen atom, a linear or branched alkoxy group having 16 carbon atoms, or an amino group;

R ³ is a hydrogen atom, a linear or branched alkyl group having 16 carbon atoms, or a linear or branched acyl group having 116 carbon atoms;

R ⁴ is a hydrogen atom, a C 16 linear or branched alkyl group, or a C 16 linear or branched acyl group,

R ⁵ is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms,

R ⁶ is a hydrogen atom, a linear or branched alkoxy group having 16 carbon atoms, or an amino group;

R ⁷ is a hydrogen atom or a linear or branched alkyl group having 16 carbon atoms.

In the present invention, the “straight or branched alkyl group having 1 to 6 carbon atoms” is not particularly limited. However, examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group and a tert-butyl group. Examples of the `` or branched alkoxy group '' include, but are not particularly limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. can give. Examples of the “C6-C16 straight-chain or branched acyl group” include, but are not particularly limited to, for example, formyl group, acetyl group, propionyl group, isoptyryl group, valeryl group, and isovaleryl group. The acyl group of 1 means a formyl group.

 Further, among the compounds represented by the formula (I), compounds represented by the following formula (III) are more preferable.

 [Formula 13]

In equation (ΠΙ),

R ¹ is a methyl group or a formyl group,

R ² is a methoxy group or an amino group,

R ³ is a hydrogen atom or an acetyl group.

Among the target compounds represented by the formula (III), vinblastine, vincristine and And vindesine are particularly preferred. These are all compounds that are frequently used for applications such as antitumor drugs.

In the production method of the present invention, when the target compound of the formula (I) is synthesized in the presence of an antibody, the production method of the antibody is not particularly limited, and is, for example, as follows. That is, first, a compound represented by the following formula (IX) is prepared, and it is further converted to a compound represented by the following formula (VII). In the formulas (VII) and (IX),

And R ⁴ —R ⁷ are the same as those in the above formula (I).

[Formula 14]

[Formula 15]

[0020] The method for producing the compounds represented by the formulas (VII) and (IX) is not particularly limited! However, for example, synthesis of the compound (I) itself is preferred because it is simple and convenient. In this case, first, the same compound as the target compound (I) must be prepared.However, when the compound (I) is a natural compound, it may be obtained through an extraction / sales route from natural sources or the like. However, in the case of a non-naturally occurring compound, it may be synthesized by any method. If the compound (I) can be obtained in a small amount, the compound (I) can be produced again by the production method of the present invention. The method for obtaining the compounds (IX) and (VII) from the compound (I) is not particularly limited.For example, the compound (IX) is produced by reacting the compound (I) with hydrazine, and further producing the compound (IX). More preferably, the hydrochloride is reacted with sodium nitrite to produce compound (VII).

Then, a complex is obtained by a condensation reaction between the compound of the formula (VII) and the protein, and the target antibody is produced using the complex as an antigen. The protein is not particularly limited, but is preferably, for example, serum albumin, thyroglobulin, or ovalbumin. This antibody can be used for the production of the target compound (I) as many times as possible without losing the activity. [0022] The precursor of the compound represented by the formula (I) is not particularly limited, but may be synthesized. For example, a compound represented by the following formula (Π) or (π ′) is preferable from the viewpoint of the sharpness and the like.

[Formula 16]

[Formula 17]

In the formulas (II) and (ΙΓ), R ¹ —R ⁷ are the same as in the formula (I). Similarly, the precursor of the target compound represented by the formula (III) is preferably a compound represented by the following formula (IV) or (IV ′).

[Formula 18]

In the formulas (IV) and (IV), R ¹ —R ³ are the same as in the formula (III). When the organic compound to be produced is vinblastine, vincristine, or vindesine, the precursor is preferably the corresponding compound of the formula (IV) or (IV), respectively. These precursors The reaction conditions for the conversion reaction into the organic compound are not particularly limited. For example, the reaction is preferably performed in the presence of the antibody, an oxidizing agent and a reducing agent. The transition metal element preferably contains at least one of oxygen, air, and a transition metal element, and the transition metal element is preferably Fe (II), Rh (III), or Mn (III). ), Co (III), Ce (IV),

 It is particularly preferred that the group forces consisting of Mo (VI) and Cu (II) also include at least one selected. The oxygen may be a simple substance or an element contained in an oxygen compound. Further, the transition metal element may be, for example, an element contained in an appropriate transition metal compound. Examples of the reducing agent include a hydrogen compound, mercaptoethanol, dithiothreitol (dithiothreito or dithiothreitol), glutathione, and NADH.

 2, NADPH, Nico

 It is more preferable that the group strength consisting of 2 tinamide, FAD and FMN also include at least one selected from the group. NAD represents nicotinamide adenine dinucleotide, NADP represents -cotinamide adenine dinucleotide phosphate, FAD represents flavin adenine dinucleotide, and FMN represents flavin mononucleotide. The hydrogen compound is NaBH CN,

 Three

 NaBH, then iBH, Zn (BH), Me NBH (OAc), then iBH (sec-Bu), KBH (sec-Bu), then iBHEt

 4 4 4 2 2 3 3 3 3

, And LiAlH (tert-BuO) force or group force

 Three

 preferable.

 Hereinafter, vinblastine and its synthesis will be described as an example of the target compound and its synthesis by the production method of the present invention.

 [0024] Vinblastine (VLB) has a structure represented by the following chemical formula (1), and is a dimer contained in the leaves of Catharanthus roseus (L.) Don. As a trace component. Indole alkaloid. Its antitumor activity has been used in clinical settings as an important cancer drug for strong bladder cancer, testicular tumor, malignant lymphoma, cystic sarcoma, neuroblastoma and breast cancer. However, since it is a component that exists only in trace amounts in nature, its supply is limited and its cost is high. Therefore, many organic synthesizers have tried to synthesize VLB. As described above, various synthetic routes have already been found, and many have been published in international academic journals (for example, see Non-Patent Documents 115).

[Formula 20]

However, due to the complexity of the structure of VLB, a synthesis method that can sufficiently solve the problems such as yield and cost has been used until now. Many researchers have adopted the method of synthesizing VLB (l) via a monomeric polymerization reaction, which is present in a relatively high content in the plant-Catharanthus roseus (L.) Dmk. did. However, each of these syntheses requires a number of steps and requires strict control of chemical reaction conditions to suppress side reactions and the like. For example, according to Non-Patent Document 8, it has been known that VLB (l) can be obtained by subjecting anhydro VLB (2) to air oxidation as shown in Scheme 2 below. However, the yield of leurosidine (5) and leurosine (6) is higher than that of VLB (l), and the yield of VLB (l) is extremely high even under optimal conditions. Trace (less than 1%). In Non-Patent Document 8, it was presumed that VLB (l) in this reaction was generated via a conjugated iminium intermediate (3) and enamine (4).

[Formula 21]

 Scheme 2

On the other hand, applying Abzym to the synthesis of a natural organic compound having a complex structure such as VLB (l), as described above, makes it difficult to synthesize a transition state analog of the desired reaction. For some reason, it seemed almost impossible. However, the present inventors have found that VLB can be synthesized in high yield by using an antibody that recognizes not the transition state analog but the VLB itself.

 Hereinafter, the power of briefly explaining the synthesis of VLB according to the present invention This is merely an example, and the present invention is not limited to this. That is, first, a complex is synthesized using deacetylVLB as a hapten, and a hapten and a protein, and the complex is used as an antigen to immunize a mouse to produce a monoclonal antibody that recognizes VLB. Meanwhile, anhydrovinblastine or anhydroVLB (2) described in the above scheme 2 is prepared. This compound is readily obtained from the monomeric indole alkaloids vindoline and catharanthine. Then, this anhydrovinblastine is reacted in the presence of the monoclonal antibody, oxygen and NBHCN as shown in Scheme 3 below to obtain VLB (l).

 Three

[Formula 22]

Anhydrovinblastine (2) is a compound that can be easily obtained. As described above, it has been conventionally difficult to obtain VLB (l) in high yield. However, according to the method of the present invention, it is possible to convert anhydrovinblastine (2) to VLB (l) stereoselectively and regioselectively in one step. Also, for example, VLB (l) can be produced from a conjugated iminium intermediate (3) instead of anhydrovinblastine (2). As described above, according to the present invention, an industrial technology for producing a useful natural conjugate in a short time, which has a complicated structure and conventionally required many steps and strict reaction conditions for its synthesis, has been established. can do. In addition, the present invention is not limited to natural conjugates, and can be used as a method for synthesizing a compound that does not exist in nature.

 Next, in the production method of the present invention, the organic compound to be produced may be, for example, a compound represented by the following formula (V), and among them, taxol (paclitaxel) is particularly preferred. .

[Formula 23]

In the formula (V), R ^{8 to} R ¹³ are each independently a hydrogen atom or a linear or branched alkyl group having 16 carbon atoms.

The method for producing the antibody for producing the compound (V) is not particularly limited, and is, for example, as follows. That is, first, the same compound as the target compound (V) is prepared, and it is converted into a compound represented by the following formula (VIII). In the formula (VIII), R ⁸ — R ¹³ are the same as in the formula (V). The method for first obtaining the same compound as the target compound (V) is, for example, as described for the compound (I). The method for obtaining the compound (VIII) from the compound (V) is not particularly limited. For example, a method of reacting the compound (V) with succinic anhydride is preferable.

[Formula 24]

Then, a complex is obtained by a condensation reaction between the compound (VIII) and the protein, and the target antibody is produced using the complex as an antigen. The protein is not particularly limited, but is preferably, for example, serum albumin, thyroglobulin, or ovalbumin. This antibody can be used for the production of compound (V) any number of times as long as the activity is not lost.

 [0032] The precursor of the compound (V) is not particularly limited. However, for example, when the target compound, which is preferably a compound represented by the following formulas (Via) and (VIb), is taxol, The precursors are more preferably the corresponding compounds of the formulas (Via) and (VIb) below.

 [Formula 25]

(VIb) In the formula (Via), R ″ is an ether group or a thioether group having a carbon number of 16 and may be saturated, unsaturated, linear or branched, and in the formula (VIb), R ⁸ — R ¹³ is the same as in the formula (V) R ¹⁴ is a methylsulfur group (-SCH) or a buroxy group (_OCH = CH)

 32 is particularly preferred.

 Next, the antibody of the present invention, that is, the antibody used in the production method of the present invention and a general production method thereof will be described more specifically.

 [0034] The method for producing the antibody of the present invention is not particularly limited, and a known antibody production method or the like may be appropriately applied. Since the conventional Abzyme is an antibody that recognizes the transition state of the target reaction, it was necessary to first synthesize the transition state compound of the reaction or an analog thereof as a hapten as described above. In the case of the antibody of the present invention, there is basically no need for such a method, but except for this point, the antibody is produced by referring to a conventional method for producing Abzym.

 [0035] A specific example of the method for producing the antibody of the present invention is as follows. First, the antigen is not particularly limited. However, since the antibody is an antibody that recognizes a target compound in a reaction using the same, it is convenient to use, for example, the target compound or a derivative thereof as a pentane. . That is, the antibody is preferably an antibody produced using a complex obtained by binding an organic compound (target compound) or a derivative thereof to be produced by the production method of the present invention and a protein as an antigen. In this case, first, the same compound as the target compound may be obtained, and the composite may be produced using the same as a raw material. In some cases, a compound having a structure similar to, but not identical to, the target compound may be used for the production of the antibody. Once the target antibody is obtained, it can be used for the production of the target compound any number of times as long as its activity is not lost.

[0036] The method for producing an antibody of the present invention is characterized in that a complex obtained by binding an organic compound or a derivative thereof to be produced by the production method of the present invention and a protein is administered to an animal, Preferably, the method further comprises a step of extracting the antibody-producing cells from the animal, and fusing the antibody-producing cells with myeloma cells to produce a hybridoma, and culturing the hybridoma in vitro. Is more preferable. Although the antibody-producing cells can be used as they are, it is better to use hybridomas when culturing cells. And has advantages such as easy maintenance. More preferably, the method further comprises the step of administering the hybridoma to another animal to obtain ascites containing the target antibody from the animal. The animal to which the hybridoma is administered is not particularly limited, but a mouse is particularly preferable from the viewpoint of easy availability and easy handling. Similarly, the animal to which the complex is administered is more preferably a mouse. The protein in the complex is more preferably albumin, globulin or hemocyanin from the viewpoint of water solubility, antigenicity, and the like, and particularly preferably is serum albumin, thyroglobulin, or ovalbumin.

 [0037] In general, when an anti-nopten antibody is produced using a complex of a hapten and a protein, the properties of the antibody are affected by the binding site between the hapten and the protein in the complex. It is believed that. Specifically, the discriminating ability of the anti-hapten antibody is considered to be strong near the binding site between the hapten and the protein and weaker at a portion farther away. Therefore, also in the case of the complex used for producing the antibody of the present invention, it is preferable to appropriately select the binding site between the hapten and the protein in consideration of the reaction site in the reaction using the antibody. In the present invention, the type of the hapten may be appropriately selected according to the type of the target compound in the reaction using the antibody.For example, as described above, the condensation reaction between the compound (VII) or the compound (VIII) and the protein may be performed. The composite obtained by the above method can be used. In particular, a complex obtained by binding any one of vinblastine, vincristine, vindesine, taxol, and their derivatives to a protein is suitable for the synthesis of the corresponding hapten or an analog thereof.

 [0038] Further, as the antibody of the present invention, a monoclonal antibody is also preferable in terms of the ability to use a polyclonal antibody, and the viewpoint of the target reaction yield and selectivity.

[0039] The production method of the present invention using the antibody of the present invention can be applied to various organic compounds as described above. In the conversion reaction to the organic compound to be produced, in addition to the precursor of the organic compound and the antibody, any reactant can be appropriately used depending on the type of the reaction. The reaction temperature and the reaction time of the conversion reaction to the organic compound are not particularly limited, and may be appropriately set.However, due to its properties, the reaction can be performed under mild conditions as compared with a general organic reaction. One of the advantages. Example 1

 Next, examples of the present invention will be described. In this example, an example of preparing a monoclonal antibody using vinblastine and an example of a method for synthesizing vinblastine using the same will be described. In the following data, the nuclear magnetic resonance (NMR) spectrum was measured using EX-400 (trade name, manufactured by Nippon Electronics Co., Ltd. (JEOL), 400 MHz). Chemical shifts are expressed in parts per million (ppm). Tetramethylsilane (TMS) was used as the internal standard Oppm. Coupling constants (J) are given in Hertz and the abbreviations s, d, t, q, m and br are singlet, doublet, doublet, triplet and quadruple, respectively. Represents a line (quartet), a multiple line (multiplet) and a wide line (broad). The infrared (IR) spectrum was measured using FTIR-8400 (P / N206-71000) (trade name) manufactured by Shimadzu Corporation. The UV absorbance measurement (or UV-visible absorption spectrum measurement) was performed using a U-2000 Spectrophotometer (trade name) manufactured by Hitachi, Ltd. Mass spectrometry (MS) was measured in ESI-MS, positive mode using a mass spectrometer (product name API-3000) manufactured by Applied Biosystems Japan Co., Ltd. (Applied Biosystems).

 [0041] [1] Synthesis of deacetylVLB-protein complex

 In synthesizing vinblastine (VLB), first, a complex of protein and deacetylVLB, a VLB derivative, was produced according to the following procedure (0— (V). Selection of protein used for complex of hapten and protein) As described above, water solubility and antigenicity are important factors, but in this example, thyroglobulin is used in consideration of high antigenicity.

 (thyroglobulin or TG) was used as a protein for antibody production (immunization). In addition, for detection of the prepared antibodies, a complex of deoxy VLB and deacetylVLB was used in consideration of the high water solubility. In addition, several methods have been tried for the synthesis of a complex of a VLB derivative and a protein. In this example, the method that yielded the best results is shown.

 [0042] (0 Induction reaction of vinblastine sulfate (VLB sulfate) to free base

 Commercially available VLB sulfate (purchased by Wako Pure Chemical Industries, Ltd.) 10 mg (l 1 μmol) is dissolved in 1 mL of distilled water, and 6N (6 mol / L) NHOH is added to the aqueous solution to adjust the pH to 10, and the aqueous solution is dissolved. inside

 Four

The released VLB was extracted three times with 3 mL each of CH C1. After washing the extract three times with each lmL of distilled water, Dried over Na SO. After drying, the extract is filtered by suction and concentrated under reduced pressure to give white powdery crystals.

twenty four

 8.69 mg (yield: 97.2%) of VLB (l) were obtained. This reaction is described in "Barnett, C.J .; Cullinan, u.J .; Gerzon, K .; Hoying, R. Shi; Jones, W.E .; Newlon, W.M .; Poore, u.A .;

Robinson, R 丄.; Sweeney, MJ; Todd, GC; J. Med. Chem., 1978, 21, 88-96. ". The chemical structural formula of VLB (l) was Also shown below are the instrumental analysis data for this compound: In the NMR data, the numbers attached to the carbon atom (C) and the nitrogen atom (N) indicate the carbon atom and the nitrogen atom attached to the chemical formula below, respectively. Corresponding to the number!

[Formula 26]

 VLB (1)

IR (neat) cm: 3469,2946,1741,1614,1503,1460,1227,748;

 1H-NMR (400MHz, CDCl): δ 0.82 (3H, J = 7.2Hz, 21-CH), 0.89 (3H, J = 7.6Hz, 21'-CH)

 3 3

 ), 2.11 (3H, s, C -OCOCH), 2.71 (3H, s, N -CH), 3.61 (3H, s, C — COOCH),

 4 3 1 3 18 '3

3.79 (3H, s, C -OCH), 5.30 (lH, d, J = 10.4Hz, C-H), 5.47 (lH, s, C -H),

 16 3 6 4

 5.84 (lH, m, C-H), 6.11 (lH, s, C-H), 6.64 (lH, s, C-H), 7.12 (3H, m, C-H, C

 7 17 14 11 '12'

— HandC — H), 7.52 (lH, d, J = 7.6Hz, C — H), 8.04 (lH, brs, indoleNH).

 13 '14'

 (ii) Synthesis of deacetylvinblastine (deacetylVLB) monohydrazide

8.69 mg (11.3 μmol) of VLB (1) (free base) obtained as described above was added to anhydrous hydrazine. It was dissolved in a mixed solution of 0.22 mL and anhydrous methanol l.OmL, and stirred at 60 ° C for 24 hours under an Ar gas flow. After stirring, the reaction solution was cooled, and 3.0 mL of distilled water was added. Release the released product to each CH C1

 twenty two

The mixture was extracted three times with 3.0 mL, and the extract was washed with 1 OmL of distilled water, then with 1 OmL of satd. NaCl, and then dried over NaSO. The extract was concentrated under reduced pressure to give deacetylVLB as pale yellow powder.

 twenty four

6.98 mg (yield: 82.0%) of monohydrazide was obtained. This reaction is described in "Barnett, CJ; Shi ullinan, uJ; Gerzon, K .; Hoying, R. Shi; Jones, WE; Newlon, WM; Poore, GA; Robinson, RL; Sweeney, MJ; Todd, GC; J. Med. Chem., 1978, 21, 88-96. " deacetylVLB

The chemical structural formula of monohydrazide is described below, and the instrumental analysis data of this compound is shown. In the NMR data, the numbers attached to the carbon atom (C) and the nitrogen atom (N) correspond to the numbers of the carbon atom and the nitrogen atom, respectively, in the following chemical formula.

[Formula 27]

IR (neat) cm: 3743,3467,2934,1723,1658,1614,1503,1460,1229,748;

1H-NMR (270MHz, CDCl): δ 0.87- 0.94 (6H, m, 21- CH and21,-CH), 2.74 (3H, s, N

 3 3 3

 -CH), 3.60 (3H, s, C — COOCH), 3.78 (3H, s, C — OCH), 4.15 (lH, s, C— H),

 3 18 '3 16 3 4

 5.81 (2H, m, C—HandC—H), 6.09 (lH, s, C—H), 6.60 (lH, s, C—H), 7.13 (3H, m, C

 6 7 17 14 1:

-H, C -HandC — H), 7.52 (lH, d, J = 5.1Hz, C — H), 8.03 (lH, brs, indoleNH), 8.23 (lH, brs, — CONHNH).

 One two

 [0046] (iii) Synthesis of deacetylVLB-acid azide

 Dissolve 6.98 mg (9.08 μmol) of deacetylVLB monohydrazide in a mixture of 0.35 mL of methanol and 1.20 mL of lN (lmol / L) HCl, cool to 4 ° C, and calorie 7.95 mg of NaNO.

 2

 Stirred for lOmin. Add 5% NaHCO to the reaction solution that turned reddish brown until it became pH-powered.

 3 The released product was quickly extracted three times with 3.0 mL each of CH C1 and then dried over Na 2 SO 4.

 2 2 2 4

 This extract was concentrated under reduced pressure to about lOmL to obtain a concentrated solution of deacetylVLB-acid azide. In addition, this reaction, ί3 ~, Barnett, shi. J .; shi ullinan, uJ; Gerzon, K .; Hoying, RC; Jones, WE; Newlon, WM; Poore, GA; Robinson, RL; Sweeney, MJ; The method was performed with reference to the method described in Todd, GC; J. Med. Chem., 1978, 21, 88-96. The IR ^ vector peak value of deacetylVLB-acid azide is shown below.

[0047] IR (neat) cm " ¹ : 3645,3358,2920,2135,1714,1651,1504,1455,747.

 Next, using the above deacetylVLB-acid azide, deacetylVLB-Bovine Serum

 Albumin (BSA) complex and deacetylVLB-thyroglobulin (TG) complex were synthesized. The 0.1 M phosphate buffer (pH 6.2) used in the following steps (iv) and (V) was 13.28 g of sodium dihydrogen phosphate (NaHPO), 5.33 g of disodium hydrogen phosphate (Na HPO) and EDTA

 2 4 2 4

 • Prepared by dissolving 1.87 g of 2Na in 1.00 L of Milli-Q water (EDTA stands for ethylenediaminetetraacetic acid). The number of VLB bonds in each of the above complexes was calculated by ultraviolet absorbance measurement, and this calculation was performed by preparing a calibration curve of concentration versus absorbance at observation wavelengths of 280 nm and 310 nm for each compound of VLB, BSA and TG. These were compared with the measured values of the ultraviolet absorbance of each complex. BSA did not show absorption at the observed wavelength of 310.

 [0049] (iv) Synthesis of deacetylVLB-Bovine Serum Albumin (BSA) complex

 BSA (purchased from SIGMA-ALDRICH) 17.9 mg to 2.0 mL of 0.1 N (0.1 mol / L) Na HPO

 The solution was dissolved, and a few drops of 0.1N (0.1 mol / L) NaOH were added to adjust the pH to 9. On the other hand, 200 μL of anhydrous dioxane was added to the CHCI solution (l.OmL) of deacetylvinblastine-acid azide obtained in step (iii).

 twenty two

 In addition, the mixture was concentrated under reduced pressure to distill off CH C1. Azide dioxane solution obtained in this way

 twenty two

200 L was added dropwise to the BSA solution, and the mixture was stirred at room temperature for 3 hr while maintaining the pH at 9. Anti The reaction solution was purified using a 2 × 35 cm Sephadex G-25M (Pharmacia Biotech) column with 0.1 M phosphate buffer (pH 6.2) to obtain a deacetylVLB-BSA complex (10 mL, 1.39 mg / mL). . As a result of ultraviolet absorption measurement, 3.2 mol of deacetylVLB was bound to 1 mol of BSA. This reaction was performed by referring to the method described in "Conrad, RA; Cullinan, GJ; Gerzon, K .; Poore, GAJ Med. Chem., 1979, 22, 391-400."

(V) Synthesis of deacetylVLB-thyroglobulin (TG) complex

 First, azide dioxane solution was prepared in the same manner as in step (iv) (synthesis of deacetylVLB-Bovine Serum Albumin (BSA) complex). Meanwhile, 17.9 mg of TG (purchased from SIGMA-ALDRICH) was dissolved in 2.0 mL of 0.1 N (0.1 mol / L) Na HPO,

 twenty four

 Several drops of 0.1N (0.1 mol / L) NaOH were added to adjust the pH to 9. 200 L of the azide dioxane solution was added dropwise to the TG solution, and the resulting mixture was stirred at room temperature for 3 hours while maintaining the pH at 9. The reaction solution was purified using a 2 × 35 cm Sephadex G-25M (Pharmacia Biotech) column with 0.1 M phosphate buffer (pH 6.2) to obtain a deacetylVLB-TG complex (16 mL, 0.60 mg / mL). . As a result of ultraviolet absorbance measurement, 8.21 mol of deacetylVLB was bound to 1 mol of TG in this product.

[2] Preparation of anti-vinblastine monoclonal antibody

 Using the deacetylVLB-TG complex synthesized as described above, an anti-vinblastine monoclonal antibody was prepared. The antibody titer was measured using the above deacetylVLB-BSA complex.

 [0052] (0) Sensitization of mice with 0 deacetylVLB-TG complex and collection of serum

For each of a total of 11 Balb / c mice (7 weeks old, female), 100 µg of TG-VLB (deacetylVLB-TG complex) was emulsified with Freund's complete adjuvant (CFA) in 0.1 M sodium phosphate buffer 100 Immunization (sensitization) was performed by intradermal administration together with L. Thereafter, a booster immunization was performed in a similar manner three times at 2-week intervals. The administered TG-VLB differs depending on the mouse. For mouse Nos. 9-11, TG-VLB to which 8.21 mol of deacetylVLB was bound by the above-mentioned synthesis method was administered. On the other hand, blood was collected at 0, 14, 28 and 42 days after the first sensitization, and the antibody titer of the obtained serum (antiserum) was measured by ELISA. as a result Of the 11 Balb / c mice, No. 9-11 showed a particularly remarkable increase in antibody titer. Figure 1 graphically shows the measurement results for mouse Nos. 9-11. Using the cells collected from this mouse No. 9-11, a hybridoma was prepared and cultured. The specific operation of the measurement by the ELISA method is described below.

(Ii) Measurement of antibody titer by ELISA method

 In this measurement, the following buffer “buffer P” was prepared by adding 1.00 L of Milli-Q water to NaHPO

 2 4 2

It was prepared by dissolving O (0.55 g), Na HPO-12H0 (2.29 g) and NaCl (5.84 g). buffer

 2 4 2

 The sodium phosphate concentration of P is 10 mM, the NaCl concentration is 0.1 M, and the pH is 7.0. `` Buffer A '' is prepared by adding 1.00 L of Milli-Q water to NaHPO2ΗO (0.55 g), NaHPO-12H0 (2.29 g), NaCl (5.84 g),

 2 4 2 2 4 2

 It was prepared by dissolving BSA (1.00 g), NaN (1.00 g), and MgCl (0.095 g). buffer A

 3 2

 BSA concentration is 0.1% (W / W), NaN concentration is 0.1% (W / W), MgCl concentration is lmM, pH is 7.0

 3 2

 . The “dilution buffer” was prepared by adding 1.00 g (0.1 wt%) of BSA to 1.00 L of buffer P.

 [0054] The measurement was performed as follows. That is, first, it was manufactured as described above.

 DeacetylVLB—BSA complex in 0.1M sodium phosphate buffer (pH7.5)

 The solution was diluted to 10 g / mL with a solution of 0.1% (W / W) NaN. Next, this deacetylVLB- BSA

 Three

The complex solution was placed in a 96-well plate at 150 L / well, and allowed to stand at 4 ° C. After standing, discard the supernatant, wash the remaining solid-phased protein with buffer P (10 mM phosphate buffer), add buffer A (0.1 wt% BSA protein solution) at L per well, and incubate at 4 ° C for 2 hours. Blocked. Discarded again supernatant after washing the residual solid phase protein in buffer P, 10 ³ times with serum (buffer A was also collected the sensitized mice force thereto, it was diluted 10 ⁴ fold or 10 ⁵ fold solution ) Was added and the mixture was incubated at 37 ° C for 2 hours. After washing it further with buffer P, secondary antibody labeled with horseradish oxidase (POD) (goat (anti-mouse IgG) Fab'-POD, sunao ¾ anti-mouse IgG (H + L-chain) ( goat Ig / rabno conj. peroxidase Lot. 310) diluted to 20.8 ng / mL with a dilution buffer was added to 150 μL, and incubated at 37 ° C for 1.5 hours, followed by washing with buffer P and 100 μL of an aqueous rangenamine (OPD) solution (7.5 mM OPD + 50 mM AcONa) was added, and the mixture was incubated at 37 ° C for 45 minutes, and 50 μL of an aqueous 1.2 MHSO (containing 2.4 g of NaSO in 1 L) solution was added thereto. The absorbance at 492nm It was measured. In addition, this ELISA measurement is based on "Kohno, T .; Tanaka, H .; Watabe, K .; Yamashita, S .; Sezaki, H .; Nadai, T .; Sugie, Y .; Ogouchi, T. Microbiol. Immunol , 1999, 43, 253-258. ".

 (Iii) Production of mouse hybridoma

 A mouse hybridoma was prepared as follows.

 (Iii-1) Culture of mouse myeloma cells

 First, mouse myeloma cells were cultured. That is, first, non-secretory mouse myeloma cells SP2 / 0-Agl4 were transferred to a GIT medium (Nihon

The cells were cultured in 75 mL of Pharmaceutical Co. Ltd. Tokyo, Japan (ie, manufactured by Nippon Pharmaceutical Co., Ltd.) to obtain 3 million cells (cell density: 4.0 × 10 ⁴ cells / mL). This was the first day of culture. On the second day of culture, the number of cells is 6 million (cell density: 8.0 × 10 ⁴ cells / mL), and on the third day of culture, the number of cells is 18.75 million (cell density: 2.5 × 10 ⁵ cells / mL). On the fourth day of culture, 33 million mouse myeloma cells (cell density: 2.1 × 10 ⁵ cells / mL) were obtained. The GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.) is described in more detail in "Murakami, H .; Masui, H .; Sato, GH;

 Natl. Acad. Sci. USA, 1982, 79, 1158-1162. "Sueoka, N .; Chow, TP; Sitoka, TK Proc. Natl. Acad. Was used.

 (Iii-2) Collection of macrophages

While the mouse myeloma cells were cultured, macrophages (peritoneal cells) were collected from each of the sensitized Balb / c mice Nos. 9 to 11 in which the antibody titer was significantly increased. That is, first, 10 mL of GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.) was placed in an injection cylinder (manufactured by Terumo Corporation, trade name: Terumosyringe), and 70% ethanol was sprayed on each of the mice No. 9-11. Then, the mouse's abdominal hair was clipped (approximately 1 cm in diameter), and a 26G injection needle was attached to the syringe, and 10 mL of GIT medium was intraperitoneally administered. The abdomen of this mouse was rubbed for 5 minutes, and the GIT medium was collected in an intraperitoneal Petri dish and centrifuged at 1200 rpm for 5 minutes. The macrophages obtained by discarding the supernatant were added with 10 mL of PBS, and centrifuged again under the same conditions. After performing this centrifugation again, macrophages were mixed with regional GIT medium + penicillin streptomycin (diluted 100-fold). The cells were cultured at 37 ° C in a cuvette. This method referred to "Lane, RD; Crissman, RS; Ginn, S. Method EnzymoL, 1986, 121, 183-192."

(Iii-3) Cell fusion

 Next, cell fusion was performed. That is, first, 70% ethanol was sprayed on each of the above-mentioned Balb / c mice Nos. 9 to 11, and they were killed by dislocation of the cervical spine in a clean bench. Next, the mouse was laparotomized to remove the spleen, and the spleen was placed in an ice-cooled RPMI1640 medium and loosened. It was filtered through a metal mesh, transferred to a 15 mL centrifuge tube, and centrifuged at 1200 rpm for 5 minutes. After discarding the supernatant, 5 mL of ridging reagent warmed to 37 ° C was added, and incubated at 37 ° C for 2 minutes.Furthermore, 5 mL of RPMI was added and centrifuged at 1200 rpm for 5 minutes. . After discarding the supernatant again, add 5 mL of the ridging reagent warmed to 37 ° C again, incubate at 37 ° C for 2 minutes, remove 5 mL of RPMI, centrifuge at 1200 rpm for 5 minutes to increase the spleen cell power. Red blood cells were removed. Further, the supernatant was discarded, 10 mL of RPMI was added, and after pipetting, centrifugation at 1200 rpm for 5 minutes was repeated twice to remove the ridging reagent.

 On the other hand, SP2 / 0-Agl4 mouse myeloma cells cultured as described above were transferred to a 50 mL centrifuge tube, and centrifuged at 1200 rpm for 5 minutes. Further, the supernatant was discarded, 10 mL of RPMI was washed, pipetting, and centrifugation at 1200 rpm for 5 minutes were repeated twice to remove the medium used for culturing the SP2 / 0_Agl4 mouse myeloma cells.

 [0060] Each of the spleen cells thus obtained and SP2 / 0-Agl4 mouse myeloma cells was

5 mL of RPMI was added and the number of cells was counted using a hemocytometer for cell fusion. Cell fusion was performed according to the method of Roche Diagnostics (Mannheim, Germany). That is, first, the spleen cells and SP2 / 0-Agl4 mouse myeloma cells were placed in one 50 mL centrifuge tube at a cell ratio of 5: 1, centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Polyethylene Glycol 1500 in 75mM HEPES (Roche Co., Ltd., trade name) warmed to 37 ° C and kept in a water bath placed in a clean bench, and warmed to 37 ° C The mixture was stirred with a pipette while adding 1 mL of (PEG-1500) over 1 minute. After finishing the PEG-1500, the contents were further agitated with a pipette for 2 minutes while rotating the centrifuge tube in a water bath, and RPMI was further continued while rotating the centrifuge tube and stirring the contents. 4 mL for 4 minutes I got a lot of calories. Then, 10 mL of RPMI preliminarily heated to 37 ° C. was added thereto, gently suspended, and centrifuged at 900 rpm for 5 minutes. When the centrifuge was stopped, the rotation speed was gradually reduced over about 5 minutes and stopped in order to prevent damage to the cells due to the brake. Cell fusion was performed in this manner to obtain the desired hybridoma.

[0061] The obtained hybridomas were added to 10% FCS and HAT

(50-fold dilution) was added to the culture medium, and the spleen cells were suspended at a concentration of 12 × 10 ⁶ / mL, and the macrophages collected in advance as described above were added to a 96-well plate that had been prepared. Was. The GIT + 10% FCS + HAT medium was further cultivated, the spleen cell concentration was adjusted to 1 × 10 V mL and the volume was L / well, and the cells were cultured at 37 ° C. in a CO incubator.

 2

 (Iii-4) Claw Jung of mouse hybridoma producing anti-vinblastine monoclonal antibody

 After the colony formation of the above-mentioned (i) and (ii), the antibody titer of the culture supernatant was measured by ELISA. From the colonies in which anti-vinblastine monoclonal antibody production was confirmed, Sanko Pure Chemical (Tokyo,

In accordance with the guidelines of (Japan), antibody-producing hybridomas (positive hybridomas) were cloned using S-Clone medium. The hybridoma supernatant was periodically assayed for the presence of anti-vinblastine antibodies. As a result, the desired antibody-producing wells were cloned from the wells of mouse No. 9 to 5 well and mouse No. l (½ ^ 27 well, mouse No. l ^ 20we ^) by the limiting dilution method. Positive clones were confirmed by binding to deacetylVLB-BSA by ELISA, and two anti-vinblastine monoclonal antibodies MAb-9-E7 and MAb-9-D were obtained from three wells of mouse No. 9. In addition, two anti-vinblastine monoclonal antibodies MAb-10-A9 and MAb-9-10F10 were obtained from the nine wells of mouse No. 10. On the other hand, the desired antibody was obtained from llwell of mouse No. ll. The ELISA measurement was performed in the same manner as in the above (ii) except that culture supernatant 20 / zL and buffer A 130 L were used instead of serum 150 L. The results are shown in Table 1 below. A summary is shown in Section 13.

[table 1] Preparation of hybridoma

[Table 2] Limit dilution cloning of the above hybridomas

[Table 3]

Antibody titer by ELISA method

 Anti-VLB monoclonal antibody antibody titer (observed wavelength 490 nm)

MAb— 9 -E 7 0.453

 MAb- 9 -D 1 2 0.657

 MAb-1 0 -A 9 2-1 7

MAb-1 0 -F 1 0 1.72 [0063] [3] Mass production of anti-vinblastine monoclonal antibody MAb-10-A9 using mouse ascites

Of the anti-vinblastine monoclonal antibodies MAb-9-E7, MAb-9-D, MAb-10-A9 and MAb-9-10F10, the MAb with the highest affinity for deacetylVLB-BSA by ELISA measurement Nine Balb / c nude mice (5 weeks old, female) were intraperitoneally administered with 9 hybridomas (2 × 10 ⁸ ) producing the 10-A9 antibody, and 8 days later, 8 mL of ascites was obtained. On the other hand, Protein A Sepharose 4 Fast Flow (trade name of Pharmacia Biotech) ImL is suspended in 1.5 mL of 1.5 M glycine-NaOH buffer (pH 8.9) containing 3 M NaCl, and after filling the column, 1.5 M containing 3 M NaCl Equilibrated with glycine-NaOH buffer (pH 8.9). Then, ImL of the mouse ascites was diluted with ImL of 1.5M glycine-NaOH buffer (pH 8.9) containing 3M NaCl, and the mixture was added to the equilibrated column. Each fraction was eluted with 1.5 M glycine-NaOH buffer (pH 8.9) containing 3 M NaCl while collecting ImL, and unbound protein was washed away. Next, elution was performed with lOOmM citrate-NaOH buffer (pH6.0), lOOmM citrate-NaOH buffer (pH5.0), and lOOmM citrate-NaOH buffer (pH4.0). The absorbance of each fraction was monitored by ultraviolet absorbance measurement (observation wavelength: 280 nm). Figure 2 shows the results graphically. As shown in the figure, the absorption of unbound protein in the fraction at pH 8.9 and the absorption of the target antibody in the fraction at pH 6.0 are shown. Further, this column chromatography separation was performed 5 times in total using the mouse ascites by ImL to obtain an anti-VLB monoclonal antibody IgGl (6.75 mg in 20.6 mL of 100 mM citrate-NaOH buffer, pH 6.0). .

 [4] Synthesis of VLB (l) using monoclonal antibody as reaction field

 VLB (l) was synthesized using the monoclonal antibody (anti-VLB monoclonal antibody IgGKMAb-10-A9) obtained as described above. The chemical structural formulas of the following compounds (1)-(6) are all shown in Scheme 2 above.

(0 Synthesis of anhydroVLB (2)

First, anhydroVLB (2), a precursor of VLB (l), was synthesized. That is, first, 5.0 mL of distilled water was poured into a 20-mL eggplant-shaped flask, and cooled with ice while publishing argon gas. There, catalanthin sulfate (catharanthine '1/2 H SO) 4.9π ^ (12.7 mol) is distilled Dissolve 0.54 mL of water, 5.8 mg (12.7 μmol) of vindoline in 0.54 mL of distilled water and 10.8 / zL of 2N HCl, and distill 0.223 g (0.825 mmoL) of FeCl6ΗO

 3 2

 A solution dissolved in 1.4 mL of water was added, and the mixture was stirred under an argon stream and ice-cooling for 3 hours. After stirring, a solution prepared by dissolving 0.33 g (4.03 mmol) of NaOAc in 1.35 mL of distilled water and 6.05 mg (0.16 mmol) of NaBH in a solid state were added, and the mixture was stirred under an argon stream and ice-cooling for 30 minutes. this

Four

 The reaction solution was extracted four times with 6.0 mL of EtOAc, dried over anhydrous Na 2 SO and concentrated under reduced pressure. Obtained

 twenty four

 After dissolving 2.0 mU of CHC1 in the residue, the CHC1 layer was washed with 1.5 mL of satd.

 3 3 3

 , And dried over anhydrous Na SO and concentrated under reduced pressure. Cold methanol is added to the obtained residue.

 twenty four

 2.0 mL was collected and mpl78.3-184.1 ° C, to obtain anhydroVLB (2) 9.8π ^ (12.4 mol, yield: 97.6%) as white powdery crystals. Thus, anhydroVLB (2) could be obtained in a single step with high yield, regardless of catharanthin and bindlinka. This synthesis was performed with reference to Japanese Patent Application Laid-Open No. 2-048582. The following shows the instrumental analysis data of this compound. In the NMR data, the numbers attached to the carbon atom (C) and the nitrogen atom (N) conform to the numbers attached to the VLB (l) chemical formula.

[0066] IR (neat) cm " ¹ : 3393,2926,1734,1616,1231,748;

¹ H-NMR (270MHz, CDCl) δ: 0.80 (3H, t, J = 7.3Hz, 21- CH),

 3 3

 1.00 (3H, t, J = 7.8Hz, 21'-CH), 2.10 (3H, s, C- OCOCH), 2.71 (3H, s, N -CH),

 3 4 3 1 3

 3.61 (3H, s, C -COOCH), 3.79 (3H, s, C -COOCH), 3.81 (3H, s, C — OCH),

 18 '3 3 3 16 3

 5.27 (lH, d, J = 10.2Hz, C -H), 5.45 (lH, s, C-H), 5.48 (lH, m, C -H), 5.82 (lH, m, C

 6 4 3 '7

-H), 6.11 (lH, s, C -H), 6.59 (lH, s, C-H), 7.13 (3H, m, C-H, C-HandC-H),

 17 14 11 '12' 13 '

7.49 (lH, m, C-H), 8.03 (lH, brs, indoleNH).

 14'

APCI-MS m / z 793.5 (M + H ⁺ ).

(Ii) Synthesis of VLB using monoclonal antibody as reaction field

 Next, the monoclonal antibody (anti- VLB monoclonal antibody)

 VLB (l) was synthesized using IgGl (MAb-10-A9)) and anhydroVLB (2).

[0068] Prior to the synthesis, VLB (1), anhydroVLB (2), leurosidine (5) and leurosine (6) were injected with HPLC as a standard sample and confirmed Rt (retention time). At the same time, a calibration curve of concentration versus peak area was created. The Rt confirmed for each compound is shown as VLB (l) was Rt = 21.1min, anhydroVLB (2) was Rt = 22.6min, leurosidine (5) was Rt = 20.9min, and leurosine (6) was Rt = 20.4min. The HPLC conditions are shown below.

[0069] HPLC conditions:

 The column used was Xterra (trade name) MSC5 ^ m Column (2.1 X 150 mm) manufactured by Waters.

 8 The mobile phase was prepared by diluting 85% HPO (5 mL) and 5 M NaOH (12 mL) to H0 to make 100 mL.

 3 4 2

 Using a gradient system of a mixture of (Solution A, pH 2.5) and MeOH, the composition was changed over time as follows. 0- lOmin, 20-40% MeOH; 10-14 min, 40-50% MeOH; 14-19 min,

50% MeOH; 19-23min, 50-95% MeOH; 23-23.lmin, 95-100% MeOH;

 23.1-30min, 100% MeOH.

 The flow rate was 0.4 mL / min, the column temperature was 40 ° C, the solvent temperatures were 40 ° C, and the wavelength (detection wavelength) was 265 nm.

Hereinafter, a synthesis example of VLB (l) will be described.

 (Ii-1) in an lOOmM citrate-NaOH buffer, anhydroVLB (2) (l equivalent), ant

 Reaction using monoclonal antibody (1 equivalent), NaBHCN (4 equivalents) and oxygen

 Three

 anhydroVLB (2) (1.13nmoL), anti-VLB monoclonal antibody

 IgGl (MAb-10-A9) (1.13 nmol) and NaBHCN (4.50 nmol) were added to citrate-NaOH buffer (67

 Three

 μL, lOOmM, pH 6.0, containing 10% acetonitrile) into a small test tube (18 mm ID X 40 mm, with an oxygen balloon attached to the top) and vigorously stir at 25 ° C. (A double-sided cross-shaped Teflon (registered trademark) coated rotor, 10 mm wide X 8 mm high was used as a stirrer). The progress of the reaction was monitored by HPLC. The same HPLC conditions were used as described above. To monitor the progress of the reaction, more specifically, at 1, 2, 3 and 4 hours after the start of the reaction, take 6 μL of the reaction solution, mix 3.0 μL of acetonitrile and 3.0 μL of Α solution, The mixture was obtained by sonication for 3 minutes, and 10 L of the mixture was injected into an HPLC instrument. The composition of the solution A is as shown in the HPLC conditions. The production of VLB (l) was confirmed by co-injection of the reaction solution and a VLB (l) standard sample (Rt, 21.2 min) by HPLC.

[0072] The graph in FIG. 3 shows the results of HPLC sampling 4 hours after the start of the reaction. The vertical axis is the relative intensity of the peak, the horizontal axis is the retention time, and reference numeral 1 in the figure is the VLB (l) peak, Symbol 2 is the peak of anhydroVLB (2), and symbol 6 is the peak of leurosine (6). The numbers near the peaks indicate the respective retention times. The yield of each substance was calculated from the area of the obtained peak and the previously prepared calibration curve. The yield of VLB (l) was 16.0%. Since the recovery of the starting material, anhydroVLB (2), was 80.6%, it was concluded that the conversion to VLB (l) was 82.2%. In addition, it was found that leurosidine (Rt, 20.9 min), a C-4 'isomer of VLB (l), was not produced or, if produced, was negligible. Was. Further, the yield of leurosine (Rt, 20.4 min) was 0.4%. According to the conventional method (Non-Patent Document 8), leurosidine (5) and leurosine (6) have higher yields, and the yield of VLB (l) is less than 1%, which is very small. It can be said that the yield and reaction selectivity of VLB (l) are very excellent. As described above, according to the production method of the present invention, it was possible to selectively synthesize VLB (l) from anhydroVLB (2) in a high yield. The above reaction is simple because the above reaction proceeds smoothly only by mixing the respective reactants and stirring at room temperature.

(Ii-2) lOOmM citrate- NaOH buffer in anhydroVLB (2) (l equivalent), ant

 Reaction using monoclonal antibody (l / 3 equivalent), NaBHCN (4/3 equivalent) and oxygen

 Three

 Next, the same reaction was carried out by changing the amount ratio of each reactant. That is, anhydroVLB (2) (1.13 nmol containing anti-VLB monoclonal antibody

 IgGl (MAb-10-A9) (0.38 nmol) and NaBHCN (1.50 nmol) in citrate-NaOH buffer (67

 Three

 μL, lOOmM, pH6.0, containing 10% acetonitrile) into a small test tube (18mm ID X 40mm, oxygen balloon mounted on top) and at 25 degrees And reacted. FIG. 4 shows an HPLC diagram 4 hours after the reaction. The meanings of the symbols and numerical values in the figure are the same as in FIG. When the yield of each substance was calculated in the same manner as (iH), the yield of VLB (l) was 15.5%, and the recovery of anhydroVLB (2) was 61.7%. That is, also in the present synthesis example, good results according to (ii-1) were obtained.

(Ii-3) anhydroVLB (2) (l equivalent), NaBH CN (4 equivalent) and 100 mM citrate-NaOH buffer

 Three

 Reaction using oxygen

For comparison with the production method of the present invention, an antibody-free synthesis was also performed. That is, anhydroVLB (2) (1.13 nmol) and NaBHCN (4.50 nmol) were added to citrate-NaOH buffer (67 μL , 100 mM, pH 6.0, containing 10% acetonitrile), placed in a small test tube (18 mm ID X 40 mm, with an oxygen balloon attached to the top), and reacted at 25 ° C as described above. I let it. FIG. 5 shows an HPLC diagram 4 hours after the reaction. The meanings of the symbols and numerical values in the figure are the same as in FIG. When the yield of each substance was calculated in the same manner as in GH), the yield of VLB (l) was 3.9%, which was significantly lower than that in the case where the antibody was used.

(Ii-4) lOOmM NH OAc— anhydroVLB (2) (1 equivalent) in AcOH buffer, ant to VLB

 Four

 Reaction using monoclonal antibody (1 equivalent), NaBHCN (4 equivalents) and oxygen

 Three

 The reaction was performed in the same manner as in (ii-1) except that the solvent was changed. That is, first, 2 mL of lOOmM citrate-NaOH buffer (pH 6.0) containing anti-VLB monoclonal antibody IgGl (MAb-10- 109) (1.86 M) was mixed with Vivascience VIVASPIN 2 mL Concentrator (trade name) (Membrane: 50,000 PES) and concentrated 20-fold. LOOmM NH in the concentrated buffer

 Four

Add 1.8 mL of OAc-AcOH buffer (pH 6.0), concentrate again 10-fold with VIVASPIN 2 mL Concentrator (Membrane: 50,000 PES), and add lOOmM NH OAc- containing anti-VLB monoclonal antibody IgGl (MAb-10-A9). AcOH buffer (pH 6.0) (22.5 μM) was prepared.

 Four

 . Next, this anti-VLB monoclonal antibody IgGl (MAb-10-A9) (1.13 nmol), anhydroVLB (2) (1.13 nmol) and NaBHCN (4.50 nmol) were added to NH OAc-Ac OH buffer (67 μm).

 3 4

 L, lOOmM, pH 6.0, containing 10% acetonitrile), placed in a small test tube (18 mm ID X 40 mm, with an oxygen balloon attached to the top) and stirred vigorously at 25 degrees ( A double-sided cross-shaped Teflon (registered trademark) coated rotor, 10 mm wide X 8 mm high was used as a stirrer.

After 1, 2, 3, and 4 hours from the start of the above reaction, a 3 μL sample of the reaction solution was sampled, diluted with 85 L of acetonitrile-milli-Q water mixed solvent (1: 1), and then subjected to mass spectrometry ( API-3000, ES micro MS, positive mode). Fig. 7 shows the spectrum diagram. Fig. 7 (a) is a spectrum diagram 1 hour after the start of the reaction, Fig. 7 (b) is a spectrum diagram 2 hours after the start of the reaction, and Fig. 7 (c) is a spectrum diagram 4 hours after the start of the reaction. is there. Symbols 1, 2 and 3 in the figure indicate the peaks of the conjugated products (1) and (2) and the peaks considered to be the conjugated compound (3), respectively. M / z value. As shown in the figure, one hour after the reaction, the peak (m / z) which seems to be a conjugated iminium intermediate (3) together with the starting material anhydroVLB (2) (m / z 793.5) 791.3) was confirmed. Two hours later, the peak of the conjugated iminium intermediate (3) (m / z 791.5) was superior to the peak of anhydroVLB (2) (m / z 793.0). Also, a VLB (l) peak (m / z 811.5) appeared. After 4 hours, a strong peak derived from VLB (l) (m / z 811.5) was observed.

 After 4 hours of the reaction, 6 μL of the reaction solution was taken, 3.0 μL of acetonitrile and 3.0 μL of the solution were mixed, and the mixture was subjected to ultrasonication for 3 minutes to obtain a mixture. Of these, 10 / z L was collected, injected into HPLC and analyzed. The measurement conditions are as shown below.

 [0078] HPLC conditions:

 The column used was a YMC YMC-Pack C-AP (trade name) 5 μm Column (4.6 X 150 mm).

 8

 It was.

 The mobile phase was prepared by diluting 85% H PO (5 mL) and 5 M NaOH (12 mL) to H0 to make 100 mL.

 3 4 2

 Using a gradient system of a mixture of (Solution A, pH 2.5) and MeOH, the composition was changed over time as follows. 0- lOmin, 20-40% MeOH; 10-14 min, 40-50% MeOH; 14-19 min,

50% MeOH; 19-23min, 50-95% MeOH

 The flow rate was 2.0 mL / min, the column temperature was 40 ° C, and the solvent temperatures were 40 ° C. Wavelength (detection wavelength) was 265 nm.

 FIG. 6 shows the HPLC diagram. Symbol 1 in the figure is the peak of VLB (l), and symbol 2 is

 This is the peak of anhydroVLB (2). For VLB (l) and anhydro VLB (2), a calibration curve was prepared by measuring a standard sample under the above conditions in advance, and Rt was Rt = 14.4 for VLB (l). It was confirmed that Rt = 15.4min for min and anhydro VLB (2). When the yield of VLB (l) (Rt, 14.4 min) was calculated from the calibration curve and the peak area of the HPLC chart in FIG. 6, a good yield of about 22.5% was obtained.

 [0080] As described above, in this example, by reacting anhydroVLB (2) with NaBHCN and oxygen in the presence of anti-vinblastine monoclonal ant¾ody, VLB (l) was converted at a conversion rate of about 82%.

 Three

 Could be obtained. Furthermore, in this reaction, leurosine (6), a leurosidine (5) -epoxy form, which is a C-4'epimer, was hardly produced, and it was proved that the reaction progressed to the reactivity ^ hemoselective. In addition, since the ion peak m / z 791.3 was detected by mass spectrometry, it is considered that the synergistic iminium intermediate (3) is an intermediate in the production process of (1).

[0081] (iii) Synthesis of conjugated iminium intermediate (3) Furthermore, VLB (l) was synthesized using a conjugated iminium intermediate (3) instead of anhydroVLB (2). First, Duangteraprecha, S .; Hirata, K .; Morihara, E .; Nakae, M .; Katayama, H .; Honda, M .; Miyamoto, KJ Ferment. Bioeng., 83 (3), 227-232 (1997) Hirata, K .; Duangteraprecha, S .; Morihara, E .; Honda, M .; Akagi, T .; Nakae, M .; Katayama, H .; Miyamoto, K. Biotechnol. Lett., 19 (1 ), 53-57 (1997). A conjugated iminium intermediate (3) was synthesized using near-ultraviolet light irradiation as described below.

That is, first, catalanthin (0.75 μmol) and vindoline hydrochloride (0.75 mol) were dissolved in methanol in a 20-ml eggplant-shaped flask, and the mixture was dried by blowing argon gas. After DMSCXlOO / zl) was completely dissolved in the mixture, 3 ml of 0.1 M Tris-HCl buffer (pH 7.0) was added thereto, followed by thorough stirring. Then add FMN aqueous solution (250 M) and MnCl aqueous solution (50 mM) respectively.

 2

 1 ml was added (final concentration 0.15 mM). The reaction solution was irradiated with near-ultraviolet light having a maximum absorption at 370 nm at a distance of 10 cm from the vessel and reacted for 10 minutes to obtain an iminium intermediate (3).

[0083] Since it is difficult to directly detect the iminium intermediate (3) obtained in the above reaction, the production was confirmed as follows, and the yield was calculated. That is, first, a reaction liquid 5001 is collected, 100 μl of NaBH (72 mM) is added, and 0.5N (0.5 mol / L) NaOH is added to pHIO to obtain a pHIO.

Four

 The iminium intermediate (3) was converted to AnhydroVLB (2). Next, this was extracted with ethyl acetate, and the ethyl acetate extract was further concentrated under argon gas. The obtained residue was dissolved in methanol (100 μl), and 5 μl of the residue was injected into an HPLC device and analyzed. As a result, it was found that AnhydroVLB (2) was obtained at a yield of 65% based on the starting materials catalantin and vindoline hydrochloride. That is, the iminium intermediate (3) was obtained in a yield of about 65%. The HPLC conditions are shown below.

[0084] HPLC conditions:

 The column used was Xterra (trade name) MSC5 ^ m Column (2.1 X 150 mm) manufactured by Waters.

 8 The mobile phase was prepared by diluting 85% H PO (5 mL) and 5 M NaOH (12 mL) into H

 3 4 2

The composition was changed over time as follows using a graient system of a mixture of the solution (solution A, pH 2.5) and MeOH. 0-10min, 20-40% MeOH; 10-14min, 40-50% MeOH; 14-19min, 50% MeOH; 19-23min, 50-95% MeOH; 23-23.lmin, 95-100% MeOH; 23.1-30min, 100% MeOH.

 Flow rate 0.4mL / min, column temperature 40 ° C, solvent temperatures 40 ° C

The Wavelength (detection wavelength) was 265 nm.

[0085] Thus, the conjugated iminium intermediate (3) could be easily synthesized from vindoline hydrochloride and catharanthine by a known method.

[0086] (iv) Conversion reaction of iminium intermediate (3) (1 equivalent) to VLB (l) using anti-VLB monoclonal antibody (1 equivalent) and NADH (80 equivalents)

 Next, VLB (l) was synthesized using the conjugated iminium intermediate (3) obtained as described above. That is, first, the reaction solution 101 obtained by the above-described near-ultraviolet light irradiation reaction was collected in a 20-ml eggplant-shaped flask, and Milli-Q water (H 0 (\ 1 (3) was added to make 4 ml. Mess up, anti- VLB

 2

 Monoclonal antibody IgGl (MAb-10-A9) (1.5ηπιο1) 500; ζ 1 and then add NADH (240 μ Μ) 100 μl every 1 hour for 5 hours for a total of 500 μl Reacted for 5 hours. After the completion of the reaction, 500 1 of the reaction solution was collected. To this, 0.5N (0.5 mol / L) NaOH was added to make pHIO, and ethyl acetate 5001 was added, followed by sonication for 3 minutes. The mixture was further vigorously stirred using a magnetic rotator, and then the organic layer was separated. Further, ethyl acetate 5001 was added to the separated aqueous layer again, and the same extraction operation as described above was repeated. Then, the first and second ethyl acetate extraction solutions were combined, and argon gas was blown to evaporate the solvent. After adding and dissolving 20 μl of methanol to the obtained residue, 20 μl was poured into an HPLC device for analysis. The yield of VLB (l) for which the calibration curve force was also calculated was 46.7%. The HPLC conditions are shown below.

[0087] HPLC conditions:

 The column used was Xterra (trade name) MSC5 ^ m Column (2.1 X 150 mm) manufactured by Waters.

 8 The mobile phase was prepared by diluting 85% H PO (5 mL) and 5 M NaOH (12 mL) into H

 3 4 2

 The composition was changed over time as follows using a graient system of a mixture of the solution (solution A, pH 2.5) and MeOH. 0-10min, 20-40% MeOH; 10-14min, 40-50% MeOH;

14-19min, 50% MeOH; 19-23min, 50-95% MeOH; 23-23.lmin, 95-100% MeOH; 23.1-30 min, 100% MeOH.

 The flow rate was 0.4 mL / min, the column temperature was 40 ° C, the solvent temperatures were 40 ° C, and the wavelength (detection wavelength) was 265 nm.

[0088] As described above, the conjugated iminium intermediate (3) was used as a starting material for anti-VLB monoclonal

 The reaction was performed in the presence of antibody IgGl Mab-10-A9 to obtain the desired vinblastine (VLB) (1) in high yield. The conjugated iminium intermediate (3) of the starting material could be easily synthesized from vindoline hydrochloride and catharanthine in a known manner Example 2

 [0089] In this example, an example of preparing a monoclonal antibody using taxol as a hapten and an example of synthesizing taquinol using the same will be described. The nuclear magnetic resonance (NMR) spectrum was measured using EX-500 (trade name, manufactured by Nihon Electronics Co., Ltd. (trade name), 500 MHz). Other instrumental analysis conditions and the like are the same as in Example 1 unless otherwise indicated.

 [0090] [1] Synthesis of 2'-succinyltaxo protein complex

 2'-sucdnyltaxol was synthesized as described below, and a complex thereof with a protein was synthesized. In this example, a thyroglobulin (thyroglobulin or TG) complex and an ovalbumin (ovalbumin or OVA) complex were synthesized and used for antibody production (immunization). For the detection of the prepared antibody, a complex with a serum albumin (Bovine Serum Albumin or BSA) was used.

 [0091] (0 2 Synthesis of succinyltaxol

 20 mg (23.4 μmol) of taxol (purchased from Wako Pure Chemical Industries, Ltd.) and 36 mg (359.7 μmol) of succinic anhydride were dissolved in 480 μL of pyridine, and the mixture was stirred at room temperature under a stream of Ar at room temperature. The reaction solution was concentrated under reduced pressure, and extracted three times with CHC1. The extract is concentrated under reduced pressure and

 Three

 (Developing solvent: CHC1: MeOH = 10: 1) to give 2'-succinyltaxol as white powdery crystals

 Three

20.1πι _§ (21.1 / ζ πιο1, yield: 89%) was obtained. This reaction is described in "(a) Jyh-Gang, L .;

 Bi—Xing, C; Peter, B.S .; Bernard, F.E. and ancer Res., 1993, 53,

1388-1391. "And" (b) Neal, FM; David, GIKJ Nat. Prod., 1988, 51, 298-306. ". 2'-succinyltaxol Chemistry The structural formula is shown, and ¹ H-NMR data of this compound is shown. In the data, the number attached to the carbon atom (C) corresponds to the number of the carbon atom attached to the following chemical formula, respectively.

 [Formula 28]

H-NMR (500 MHz, CDCl): δ 1.13 (3H, s, C -H), 1.22 (3H, s, C -H), 1.67 (3H, s, C

 3 17 3 16 3 li

-H), 1.91 (3H, s, C -H), 2.17 (2H, m, C-H), 2.21 (3H, s, — OCOCH),

 3 18 3 14 2 3

 2.34 (2H, m, C-H), 2.44 (3H, s,-OCOCH), 2.63 (4H, m, C-OCOCH CH),

 6 2 3 2 '2 2

 3.80 (lH, d, J = 7.05Hz, C-H), 4.20 (0.5H, d, J = 8.25Hz, C-H),

 3 20 2

 4.30 (0.5H, d, J = 8.55Hz, C — H), 4.43 (lH, dd, J = 6.75andll.0Hz, C -H),

 20 2 7

 4.97 (lH, brd, J = 8.55Hz, C-H), 5.53 (lH, d, J = 3.35Hz, C-H), 5.68 (lH, d, J = 7.00Hz, C

 5 2 ':

-H), 5.98 (lH, dd, J = 3.05and9.15Hz, C-H), 6.23 (lH, brt, J = 8.55Hz, C-H),

 3 '13

 6.29 (lH, s, C-H), 7.08 (lH, d, J = 9.15Hz, C-NH), 7.37 (8H, m, C-NBz'C-Ph),

 10 3 '3' 3 '

 7.50 (3H, m, C-OBz), 7.76 (2H, d, J = 7.35Hz, C-NBz), 8.13 (2H, d, J = 7.30Hz, C

 2 3 '2

-OBz).

(Ii) Synthesis of 2'-succinyltaxol-Bovine Serum Albumin (BSA) complex First, 30.0 mg (0.44 μmol) of BSA (purchased from Nakarai Tester Co., Ltd.) was dissolved in 1.2 mL of 50 mM sodium phosphate buffer (pH 7.0) to obtain a BSA solution. On the other hand,

2 succinyltaxol 2.1 mg (2.2 μmol) ¾ rdimethylformamide dissolved in 0.1 mL; ^ then 丄 -ethyl-3- (3-dimethylaminopropyl) carbodumide hydrochloride 0.46 mg (2.4 μmol) and N-hydroxy-sulfosuccinimide 2.1 mg (9.6 μmol) was stirred at 25 ° C. for 1 hour under an Ar gas flow. The reaction solution thus obtained was added dropwise to the above BSA solution, and the mixture was stirred at room temperature. After the reaction, the BSA solution was centrifuged to remove the insoluble fraction. The soluble fraction in a fractionation molecular weight of 10,000 concentrated by ultrafiltration, by adding a 50mM sodium phosphate buffer + 0.15M sodium chl O ride (pH7.0), the solvent was replaced. Ultrafiltration and concentration were performed again with a molecular weight cut off of 10,000 to obtain 2'-succinyltaxo BSA complex (4.0 mL, 5.62 mg / mL). This reaction is described in "Paul, GG; TGJ, R; Gary, SB; Carolyn, B 丄.; Jilanne, BBJ

 Immunol. Methods., 1993, 158, 5-15 ".

[0094] (iii) Synthesis of succinyltaxoH: hyroglobulin (TG) complex

 30.0 mg (0.44 μmol) of TG (purchased from SIGMA-ALDRICH) was dissolved in 1.2 mL of 50 mM sodium phosphate buffer (pH 7.0) to prepare a TG solution. On the other hand,

Dissolve 2.1 mg (2.2 μmol) of 2'-succinyltaxol (l) in 0.6 mL of dimethylformamide, then 0.46 mg (2.4 μmol) of 丄 -ethyl-3- (3-dimethylaminopropyl) carbodumide hydrochloride and 2.1 mg of N-hydroxy-sulfosuccinimide (9.6 μmol) was stirred at 25 ° C. for 1 hour under an Ar gas flow. The reaction solution thus obtained was dropped into the above-mentioned TG solution, and stirred at room temperature overnight. After the reaction, the TG solution was centrifuged to remove the insoluble fraction. The soluble fraction in a fractionation molecular weight of 10,000 concentrated by ultrafiltration, by adding a 50mM sodium phosphate buffer + 0.15M sodium chl O ride (pH7.0), the solvent was replaced. Ultrafiltration and concentration were performed again at a molecular weight cut off of 10,000 to obtain 2'-succinyltaxo BSA complex (2.6 mL, 9.00 mg / mL).

[0095] (iv) Synthesis of succinyltaxo ovalbumin (OVA) complex

 17.0 mg (0.38 μmol) of OVA (purchased from Nakarai Tester Co., Ltd.) was dissolved in 5.2 mL of 50 mM sodium phosphate buffer (pH 7.0) to prepare an OVA solution. On the other hand,

Dissolve 9.0 mg (9.4 μmol) of 2'-succinyltaxol (l) in 2.6 mL of dimethylformamide, and add 2.0 mg (10.4 μmol) of ethyl-ethyl— 3- (3-dimethylaminopropyl) carbodumide hydrochloride 9.0 mg (41.4 μmol) of N-hydroxy-sulfosuccinimide was dried and stirred at 25 ° C. for 1 hour under an Ar gas flow. The reaction solution thus obtained was added dropwise to the above-mentioned OVA solution, and the mixture was stirred at room temperature. After the reaction, the OVA solution was centrifuged to remove the insoluble fraction. The soluble fraction was concentrated by ultrafiltration with a molecular weight cut-off of 10,000, and 50 mM sodium phosphate buffer + 0.15M sodium chloride (pH 7.0) was added to replace the solvent. Ultrafiltration was performed again with a molecular weight cut-off of 10,000 to obtain 2'-succinyltaxo OVA complex (3.1 mL, 0.853 mg / mL).

[0096] [2] Preparation of anti-taxol monoclonal antibody

 (0) Sensitization of mice with the 2-succinyltaxo OVA complex and the 2-succinyltaxo KTG complex and collection of serum

 2'-succinyltaxo OVA complex was added to 4 Balb / c mice (five weeks old, female, these were Balb / c No. 4 mice), and 2'-succinyltaxoKTG complex was added to 4 Balb / c mice. / c mice (5 weeks old, female, these are Balb / c No. 5-8 mice) and 3 DDY mice (5 weeks old, female, these are DDY No.l-3 mice) And Freund's complete adjuvant were administered intradermally at 50 g / 100 μL each. Thereafter, the same administration was performed at a two-week interval for a total of four times. On the other hand, blood was collected at 0, 14, 28 and 42 days after the initial sensitization, and the anti-taxol antibody titer of the obtained serum was measured by ELISA. The conditions and operation for measurement by the ELISA method are the same as those in Example 1, but will be described below.

(Ii) Measurement of antibody titer by ELISA

 2'-succinyltaxo BSA complex solution diluted to 10 g / mL with 0.1 M sodium phosphate buffer + 0.1% sodium azide (pH 7.5) is placed in a 96-well plate at 150 / z L / well at 4 ° C / well. It was left still and solidified. Discard the solution, wash each well with buffer P [10 mM sodium phosphate buffer + 0.1 M sodium chloride (pH 7.0)], and then buffer A [buffer P + 0.1% (w / w) BSA + 0.1% sodium azide + lmM magnesium dichloride (pH 7.0)] for 2 hours. Next, each well was washed with buffer P, and 150 μL of a 1000-fold diluted buffer A of serum obtained from the sensitized mouse was also added thereto, followed by incubation at 37 ° C. for 2 hours. After washing each well with buffer P, a peroxidase-labeled secondary antibody [anti-mouse IgG] diluted to 20.8 ng / mL with dilution buffer [buffer P + 0.1% (w / w) BSA (pH 7.0)]

(H + L—chainXgoat IgG / Fab ') conj. Peroxidase Lot. Incubated at ° C for 1.5 hr. After washing with buffer P, OPD solution [7.5mM

 100 μL of o-phenylenediamine + 0.25 mg / mL BSA + 0.025% hydrogen peroxide] was added, and the mixture was incubated at 37 ° C. for 45 minutes. To this was added 100 μL of 1.2 M sulluric acid + 2.4 g / L sodium sulfite, and the absorbance at 492 nm was measured. This ELISA assay is described in "Kohno, T .; Tanaka, H .; Watabe, K .; Yamashita, S .; beza i, H .; Nadai, T .; Sugie, Y .; Ogouchi, T. Microbiol. Immunol. , 1999, 43, 253-258. "

 [0098] (iii) Production of mouse hybridoma

 (iii-1) Culture of mouse myeloma cells

Mouse myeloma cells (Sp2 / 0-Agl4) were cultured in GIT medium (Nihon Pharmaceutical Co. Ltd. Tokyo, Japan, manufactured by Nippon Pharmaceutical Co., Ltd.) + 1% penicillin-streptomysin, and the cell density was adjusted to 1.0 × 10 ⁴ —1.0 X. It was maintained at 10 ⁵ / mL. Mouse myeloma cells having a cell count of 30 million or more and a survival rate of 90% or more were prepared for the cell fusion operation for one mouse. The GIT medium is described in detail in "Murakami, H .; Masui, H .; Sato, GH; Sueoka, N .; Chow, TP; Sueoka, TK Proc. Natl. Acad. Sci. USA, 1982, 79, 1158-1162. " In addition, the same GIT medium described below was used in all cases.

 [0099] (iii-2) Collection of macrophages

 First, 10 mL of GIT medium was placed in a syringe (Termo Syringe, manufactured by Terumo Corporation), and 70% ethanol was sprayed on mice with an increased antibody titer. The GIT medium (10 mL) was intraperitoneally administered using a 26G injection needle (diameter: about 1 cm). The mouse's abdomen was rubbed for 5 minutes, and the GIT medium was recovered from the intraperitoneal cavity in a petri dish. This was centrifuged at 1200 rpm for 5 minutes. GIT medium +10 mL was added to the macrophages obtained by discarding the supernatant, followed by centrifugation again under the same conditions. This centrifugation was performed again, GIT medium + 1% penicillin-streptomysin was added to the macrophage, 100 μL each was seeded in a 96-well plate, and cultured at 37 ° C. in a CO incubator. This method is described in "Lane, R.D .;

 2

 Crissman, R.S .; Ginn, S. Method EnzymoL, 1986, 121, 183-192. "

[0100] (iii-3) Cell fusion Next, cell fusion was performed. That is, first, the mouse was sprayed with 70% ethanol, and the cervical vertebra was dislocated in a clean bench. The laparotomy was performed, and the removed spleen was placed in an ice-cooled GIT medium, and the spleen was loosened. After filtration through a metal mesh and transfer to a 15 mL centrifuge tube, the mixture was centrifuged at 1200 rpm for 5 minutes. Using spleen cells and Sp2 / 0-Agl4 mouse myeloma cells at a ratio of 5: 1, cell fusion was performed using PEG-1500 according to the method of Roche Diagnostics (Mannheim, Germany). The obtained hybridomas were cultured in a 96-well plate containing the macrophages collected as described above, using GIT medium + 10% fetal calf serum + 2% HAT medium + 1% penicillin-streptomysin. ° C, in CO incubator

 2

 And cultured. This method referred to "Lane, R.D .; Crissman, R.S .; Ginn, S. Method EnzymoL, 1986, 121, 183-192." The conditions and operations other than those described above for cell fusion are the same as in Example 1.

 [0101] (iii-4) Cloning of mouse hybridoma

 After the colony formation, the antibody titer of the culture supernatant was measured by ELISA.From the colony in which anti-taxol monoclonal antibody production was confirmed, GIT medium + 10% fetal was added according to the guidelines of Sanko Junyaku (Tokyo, Japan). Cloning of antibody-producing hybridomas was performed using calf serum + 1% penicillin-streptomysin. As a result, the antibody production wells were lwell from DDY No.1 mouse, 2well from DDY No.2 mouse, 4well from DDY No.3 mouse, lwell from Balb / c No.2 mouse, and lwell, Balb / c No. 3 wells were obtained from 5 mice, 3 wells from Balb / c No. 6 mice, 2 wells from Balb / c No. 7 mice, and 4 wells from Balb / c No. 8 mice. From these wells, the desired hybridoma was further clawed by the limiting dilution method. One anti-taxol monoclonal antibody MAT-01-G12 was obtained from one well of DDY 1 mouse. The ELISA measurement was performed in the same manner as (ii) except that 20 μL of culture supernatant and 130 L of buffer A were used instead of 150 μL of serum.

 [0102] [3] Mass production of anti-taxol monoclonal antibody MAT-01-G12 using mouse ascites

2 × 10 ⁷ hybridomas producing the cloned MAT-01-G12 antibody were intraperitoneally administered to 5 Balb / c nude mice (five weeks old, female) to obtain 8 mL of ascites. On the other hand, 2 mL of Protein A Sepharose 4 Fast Flow (trade name of Pharmacia Biotech) was The suspension was suspended in 1 mL of 1.5 M glycine-NaOH buffer (pH 8.9) containing NaCl, packed in a column, and equilibrated with 1.5 M sodium glycinate + 3 M sodium chloride (pH 8.9). Then, the collected ascites was diluted 2-fold with 1.5 M sodium glycinate + 3 M sodium chloride (pH 8.9) and added to the equilibrated column. While collecting 2 mL of each fraction, the fraction was eluted with 1.5 M sodium glycinate buffer + 3 M sodium chloride (pH 8.9), and unbound protein was washed away. Next, elution was performed with lOOmM sodium citrate buffer (pH 6.0), lOOmM sodium citrate buffer (pH 5.0), and lOOmM sodium citrate buffer (pH 4.0). This operation was performed twice using 4 mL of ascites fluid, and the anti-taxol monoclonal antibody

 IgGl (13.22 mg in 4 mL of 100 mM sodium citrate buffer, pH 6.0) was obtained.

[0103] When this monoclonal antibody anti-taxol monoclonal antibody IgGl was used for taxol synthesis, good results were obtained.

 Industrial potential

As described above, according to the present invention, it is possible to provide a method for producing an organic compound at a high yield, which can be applied to a compound having a complicated structure, and an antibody suitable for the method. According to the production method of the present invention, for example, rare natural organic compounds such as vinblastine and taxol, which are used in clinical settings as important anticancer agents, can be synthesized in a short step. It is applicable to various compounds as well as products. The production method of the present invention can provide the target compound at low cost because it uses a reaction with excellent yield and selectivity, and can be applied to all fields of pharmacy and organic chemistry. It is useful in pharmaceutical technology fields such as drug discovery science and fine chemicals.

Claims

Claims [1] A method for producing an organic compound, the method comprising converting a precursor of the organic compound into the organic compound in the presence of an antibody capable of recognizing the organic compound to be produced [2] 2. The method according to claim 1, wherein the conversion reaction from the precursor to the organic compound includes at least one reaction selected from the group consisting of an addition reaction, an elimination reaction, a transfer reaction, an oxidation-reduction reaction, a condensation reaction, and a decomposition reaction. The method according to 1. [3] The production method according to claim 1, wherein the organic compound to be produced is a compound represented by the following formula (I).

 [Chemical 1]

In the formula (I),

R ¹ is a hydrogen atom, a linear or branched alkyl group having 16 carbon atoms, or a linear or branched acyl group having 116 carbon atoms;

R ² is a hydrogen atom, a linear or branched alkoxy group having 16 carbon atoms, or an amino group;

R ³ is a hydrogen atom, a linear or branched alkyl group having 16 carbon atoms, or a linear or branched acyl group having 116 carbon atoms; R ⁴ is a hydrogen atom, a linear or branched alkyl group having 16 carbon atoms, or a linear or branched acyl group having 116 carbon atoms;

R ⁵ is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms,

R ⁶ is a hydrogen atom, a linear or branched alkoxy group having 16 carbon atoms, or an amino group;

R ⁷ is a hydrogen atom or a linear or branched alkyl group having 16 carbon atoms.

The production method according to claim 3, wherein the precursor of the compound represented by the formula (I) is a compound represented by the following formula (II).

[Formula 2]

In formula (II),-is the same as in formula (I).

4. The production method according to range 3, wherein the precursor of the compound represented by the formula (I) is a compound represented by the following formula (II).

[Formula 3]

In the formula (Π,), R ¹ — R ⁷ are the same as in the formula (I).

4. The production method according to claim 3, wherein the organic compound to be produced is a compound represented by the following formula (III) among the compounds represented by the formula (I).

[Formula 4]

(III) In equation (m),

R ¹ is a methyl group or a formyl group,

R ² is a methoxy group or an amino group,

R ³ is a hydrogen atom or an acetyl group.

7. The production method according to range 6, wherein the precursor of the compound represented by the formula (III) is a compound represented by the following formula (IV).

[Formula 5]

In the formula (IV), R ¹ —R ³ are the same as in the formula (III).

7. The method according to claim 6, wherein the precursor of the compound represented by the formula (III) is a compound represented by the following formula (IV ′).

[Formula 6]

In the formula (IV,), R ¹ —R ³ are the same as in the formula (III).

[9] The production method according to claim 6, wherein the organic compound to be produced is vinblastine, vincristine, or vindesine.

[10] The claim according to claim 7, wherein the organic compound to be produced is vinblastine, vincristine or vindesine, and a precursor thereof is a compound of the formula (IV) corresponding to the organic compound. Production method.

[11] The organic compound according to claim 8, wherein the organic compound to be produced is vinblastine, vincristine or vindesine, and a precursor thereof is a compound of the formula (IV ′) corresponding to the organic compound. Production method.

12. The production method according to claim 4, wherein the precursor is converted to the organic compound in the presence of the antibody, an oxidizing agent, and a reducing agent.

13. The production method according to claim 12, wherein the oxidizing agent includes at least one selected from the group consisting of oxygen, air, and a transition metal element.

[14] A group force in which the transition metal element is composed of Fe (II), Rh (III), Mn (III), Co (III), Ce (IV), Mo (VI) and Cu (II) is also selected. 14. The production method according to claim 13, comprising at least one of:

[15] The reducing agent may be a hydrogen compound, mercaptoethanol, dithiothreitol (dithiothreitol or dithiothreitol), glutathione, NADH, NADPH, nicotinamide, FAD or the like. 13. The production method according to claim 12, including at least one in which a group force comprising FMN and FMN is also selected.

 [16] The hydrogen compound is selected from the group consisting of NaBHCN, NaBH, LiBH, Zn (BH), MeNBH (OAc),

 3 4 4 4 2 2 3

 Selected from the group consisting of LiBH (sec-Bu), KBH (sec-Bu), LiBHEt, and LiAlH (tert-BuO).

 3 3 3 3

 16. The production method according to claim 15, comprising at least one selected from the group consisting of:

[17] The production method according to claim 1, wherein the organic compound to be produced is a compound represented by the following formula (V).

 [Formula 7]

(V) In the formula (V), R ^{8 to} R ¹³ are each independently a hydrogen atom or a linear or branched alkyl group having 16 carbon atoms.

 18. The production method according to claim 17, wherein the precursor of the compound represented by the formula (V) is a compound represented by the following formulas (Via) and (VIb).

[Formula 8]

In the formula (Via), R ¹⁴ is an ether group or a thioether group having 16 carbon atoms. In the formula (VIb), R ⁸ — R may be saturated, unsaturated, linear, or branched. ¹³ is the same as the formula (V) described in claim 17.

[19] The production method according to claim 17, wherein the organic compound to be produced is taxol (paclitaxel).

 [20] The production method according to claim 18, wherein the organic compound to be produced is taxol, and the precursor thereof is the corresponding compound of the above formulas (Via) and (VIb).

 [21] An antibody used in the production method according to claim 1, which is capable of recognizing the organic compound to be produced.

 [22] The antibody according to claim 21, which is a monoclonal antibody.

 23. The antibody according to claim 21, wherein the antibody is produced using a complex obtained by binding the organic compound or its derivative to be produced and a protein as an antigen.

 24. The antibody according to claim 23, wherein the complex is a complex obtained by a condensation reaction between a compound represented by the following formula (VII) and a protein.

[Formula 9]

(VI I) In the formula (VII), R and R ⁴ —R ⁷ are the same as those in the formula (I) described in claim 3.

24. The antibody according to claim 23, wherein the complex is a complex obtained by a condensation reaction between a compound represented by the following formula (VIII) and a protein.

[Formula 10]

Wherein (VIII), IT one R ¹³ is the same as the formula (V) according to claim 17 of the claims.

24. The antibody according to claim 23, wherein the protein is pepsin serum albumin, thyroglobulin, or ovalbumin.