JP5190915B2 - Protein-encapsulating spherical transition metal complex and method for producing the same - Google Patents

Description

  The present invention has a hollow shell formed from a transition metal atom and a bidentate organic ligand, and one of the bidentate organic ligands is formed by binding a protein via a linking group. The present invention relates to a protein-encapsulated spherical transition metal complex formed so that the protein is oriented inside a hollow shell, and a method for producing the same.

  The present inventors are examining self-organization utilizing a coordinate bond between an organic ligand and a transition metal ion. Since the coordinate bond has an appropriate binding force and the directionality is clearly defined, it is possible to spontaneously and quantitatively construct a molecular assembly whose structure is precisely controlled. In addition, since the coordination number and the bond angle can be controlled in accordance with the type of metal and the oxidation number, various coordination-bonding structures can be obtained (Non-Patent Documents 3 to 5).

  For example, when planar four-coordinate Pd (II) ions are used, the direction of coordination bond can be defined as 90 degrees, and by using the panel-like organic ligand (L), the ligand It is possible to obtain spherical palladium complexes having hollow structures of various sizes according to (Non-Patent Documents 6 to 11).

  If it becomes possible to encapsulate a protein in such a precisely controlled hollow shell, it will not only be used for protein stabilization but also for the development of new information acquisition methods for protein structure. I can expect.

R. M.M. Kramer, C.I. Li, D.D. C. Carter, M.M. O. Stone, R.M. R. Naik, J .; Am. Chem. Soc. , 2004, 126, 13283 T.A. Douglas, E .; Strable, D.M. Willits, A.D. Aitouchen, M.A. Libera, M .; Young, Adv. Mater. , 2002, 14, 415 P. J. et al. Stang, B.M. Olynuk, Acc. Chem. Res. 1997, 30, 507 M.M. Fujita, Chem. Soc. Rev. 1998, 27, 417 B. Olynuk, A .; Fechtencotter, P.M. J. et al. Stang, J. et al. Chem. Soc. Dalton Trans. 1998, 1707 M.M. Fujita, K .; Umemoto, M.M. Yoshizawa, N .; Fujita, T .; Kusukawa, K. et al. Biradha, Chem. Commun. , 2001, 509 M.M. Fujita, D .; Oguro, M .; Miyazawa, H .; Oka, K .; Yamaguchi, K .; Ogura, Nature, 1995, 378, 469 N. Takeda, K .; Umemoto, K.M. Yamaguchi, M .; Fujita, Nature, 1999, 398, 794 K. Umemoto, H.M. Tsukui, T .; Kusukawa, K. et al. Biradha, M .; Fujita, Angew. Chem. Int. Ed. , 2001, 40, 2620 M.M. Aoyagi, S .; Tashiro, M .; Tominaga, K .; Biradha, M .; Fujita, Chem. Commun. , 2002, 2036 T.A. Yamaguchi, S .; Tashiro, M .; Tominaga, M .; Kawano, T .; Ozeki, M .; Fujita, J .; Am. Chem. Soc. , 2004, 126, 10818

  The present invention has been made as part of such research and development by the present inventors, and has a hollow shell formed of a transition metal atom and a bidentate organic ligand, One of the ligands is formed by binding a protein via a linking group, and the protein-encapsulating spherical transition metal complex formed so that the protein is oriented inside the hollow shell, the bidentate organic coordination It is an object to provide a child and a manufacturing method thereof.

The present inventors tried to encapsulate ubiquitin, which is a kind of protein, using the internal space (hollow shell) of the M ₁₂ L ₂₄ spherical complex having a hollow shell of precisely controlled size. As a result, 1- (2- {2,6-bis [4- (pyridyl) phenylethynyl] phenoxy} ethyl) pyrrole-2,5-dione linked to ubiquitin, trimethyl {4-methyl- When 3,5-bis [4- (4-pyridyl) phenylethynyl] benzyl} ammonium nitrate and Pd (II) ions were mixed at a molar ratio of 1:23:12, ubiquitin was found inside the hollow shell of the complex. The present inventors have found that a spherical transition metal complex arranged in can be efficiently obtained, and generalizing this finding has led to the completion of the present invention.

Thus, according to the first aspect of the present invention, the protein-encapsulated globular transition metal complex according to any one of the following (1) to (6) is provided.
(1) A spherical transition metal complex having a hollow shell, wherein the hollow shell has n transition metal atoms (n represents an integer of 6 to 60) and 2n bidentate organic arrangement. Formed from a ligand, and one of the bidentate organic ligands is formed by binding a protein through a linking group, and the protein is oriented in a hollow shell. A protein-encapsulated globular transition metal complex characterized in that
(2) The protein-encapsulated globular transition metal complex according to (1), wherein n is 6, 12, 24, 30 or 60.

(3) From the transition metal compound (M), the bidentate organic ligand (L1) formed by binding the protein via a linking group, and the bidentate organic ligand (L2), the protein has a hollow shell. Formula: M _n L2 _(2n-1) L1 (n represents the same meaning as described above, and a plurality of Ms and L2s may be the same as each other. The protein-encapsulated globular transition metal complex according to (1) or (2), which may be different from each other.
(4) The transition metal atom constituting the transition metal complex is a kind selected from the group consisting of Ti, Fe, Co, Ni, Cu, Zn, Ru, Rh, Pd, Cd, Os, Ir, and Pt. The protein-encapsulated globular transition metal complex according to any one of (1) to (3).
(5) The bidentate organic ligand (L1) is represented by the formula (I)

{Wherein X represents an ethynylene group or a p-phenylene group;
t represents an integer of 2 to 6, and a plurality of Xs may be the same or different.
R ¹ and R ² each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m1 and m2 each independently represents an integer of 0 to 4. When m1 and m2 are 2 or more, a plurality of R ^{1 s} and R ^{2 s} may be the same or different.
A ₁ represents the following formulas (a-1) to (a-4):

[In the formula, R ³ represents a formula: —YD-Pro (wherein Y represents a single bond or a divalent linking group, and D represents O, S, NH, or O— (C═O D-Pro represents a portion of the protein represented by Pro-DH excluding H.
R ⁴ may have a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, or a substituent. An amino group, an aminoalkyl group which may have a substituent, or a quaternized aminoalkyl group is represented.
m3 represents an integer of 0 to 3, and m4 represents an integer of 0 to 2. When m3 is 2 or more, or when m4 is 2, even a plurality of R ⁴ with each other are the same, may be different from each other.
Q is, -Nr1- (r1 represents a hydrogen atom, an alkyl group, an aryl group or an acyl group.), - O -, - C (= O) -, - S-, or -SO ₂ - represents a. ] The kind of group represented by this is shown. }, Wherein the bidentate organic ligand (L2) is represented by the formula (II)

{Wherein X and n represent the same meaning as described above.
R ⁵ and R ⁶ each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m5 and m6 each independently represents an integer of 0 to 4. m5, when m6 is 2 or more, plural R ⁵ together, even R ⁶ together are each identical or may be different phases.
A ₂ represents the following formulas (a-5) to (a-8)

(In the formula, Q represents the same meaning as described above.
R ⁷ has a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, a carboxyalkyl group, and a substituent. An amino group that may be substituted, an aminoalkyl group that may have a substituent, or a quaternized aminoalkyl group.
R ⁸ represents a hydrogen atom, a halogen atom, an alkyl group, or a haloalkyl group.
m7 represents an integer of 0 to 3, and m8 represents an integer of 0 to 2. When m7 is 2 or more, or when m8 is 2, a plurality of R ⁷ may be the same or different. ). } The protein-encapsulated globular transition metal complex according to any one of (1) to (4), which is a compound represented by
(6) Y is the following formula

(Wherein m9 represents an integer of 1 to 10), and the protein-encapsulated globular transition metal complex according to (5),

According to the second aspect of the present invention, there is provided the following method (7) for producing a protein-encapsulating globular transition metal complex.
(7) A transition metal compound (M), a bidentate organic ligand (L1) represented by the formula (I) and a bidentate organic ligand (L2) represented by the formula (II), 1 mol of bidentate organic ligand (L1) and bidentate organic ligand (L2) (2n-1) per n mol of transition metal compound (M) (n represents an integer of 6 to 60) The method for producing a protein-encapsulated globular transition metal complex according to any one of (1) to (6), wherein the reaction is performed in a molar ratio.

According to the third aspect of the present invention, the following compounds (8) and (9) are provided.
(8) Formula (I)

{Wherein X represents an ethynylene group or a p-phenylene group;
n1 represents an integer of 1 to 6, and when n1 is 2 or more, a plurality of Xs may be the same or different.
R ¹ and R ² each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m1 and m2 each independently represents an integer of 0 to 4. When m1 and m2 are 2 or more, a plurality of R ^{1 s} and R ^{2 s} may be the same or different.
A ₁ represents the following formulas (a-1) to (a-4):

[In the formula, R ³ represents a formula: —YD-Pro (wherein Y represents a single bond or a divalent linking group, and D represents O, S, NH, or O— (C═O D-Pro represents a portion of the protein represented by Pro-DH excluding H.
R ⁴ may have a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, or a substituent. An amino group, an aminoalkyl group which may have a substituent, or a quaternized aminoalkyl group is represented.
m3 represents an integer of 0 to 3, and m4 represents an integer of 0 to 2. When m3 is 2 or more, or when m4 is 2, even a plurality of R ⁴ with each other are the same, may be different from each other.
Q is, -Nr1- (r1 represents a hydrogen atom, an alkyl group, an aryl group or an acyl group.), - O -, - C (= O) -, - S-, or -SO ₂ - represents a. ] The kind of group represented by this is shown. } The compound shown.

(9) Y is the following formula

（式中、ｍ９は１〜１０の整数を表す。）で表される基であることを特徴とする（８）に記載の化合物。

(Wherein, m9 represents an integer of 1 to 10), and the compound according to (8),

According to the fourth aspect of the present invention, the following compound (10) is provided.
(10) Formula (II)

{Wherein X represents an ethynylene group or a p-phenylene group;
n1 represents an integer of 1 to 6, and when n1 is 2 or more, a plurality of Xs may be the same or different.
R ⁵ and R ⁶ each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m5 and m6 each independently represents an integer of 0 to 4. m5, when m6 is 2 or more, plural R ⁵ together, even R ⁶ together are each identical or may be different phases.
A ₂ represents the following formulas (a-5) to (a-8)

(In the formula, Q represents the same meaning as described above.
R ⁷ has a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, a carboxyalkyl group, and a substituent. An amino group that may be substituted, an aminoalkyl group that may have a substituent, or a quaternized aminoalkyl group.
R ⁸ represents a hydrogen atom, a halogen atom, an alkyl group, or a haloalkyl group.
m10 represents an integer of 1 to 3, and m11 represents an integer of 1 to 2. When m10 is 2 or more, or when m11 is 2, a plurality of R ⁷ may be the same or different. ). } The compound shown.

According to the first aspect of the present invention, a protein has a hollow shell with a precisely controlled size, and a protein binds to one of the bidentate organic ligands forming the shell via a linking group, A protein-encapsulated globular transition metal complex having a special structure in which a protein is oriented inside the hollow shell is provided.
According to the present invention, since a protein can be encapsulated in a precisely controlled hollow, it can be used not only for protein stabilization but also for development of a new information acquisition method for protein structure. .

According to the second aspect of the present invention, a spherical transition metal capable of efficiently producing a nanometer-scale spherical transition metal complex containing the protein inside the spherical structure (the spherical transition metal complex of the present invention) without requiring a complicated step. A method for producing the complex is provided.
According to the third and fourth aspects of the present invention, a novel bidentate organic ligand is provided. Some of these can be used as a raw material for producing the spherical transition metal complex of the present invention.

  Hereinafter, the present invention will be described in detail by dividing into 1) a protein-encapsulated spherical transition metal complex, 2) a method for producing a protein-encapsulated spherical transition metal complex, and 3) a bidentate organic ligand.

1) Protein-encapsulated spherical transition metal complex The protein-encapsulated spherical transition metal complex of the present invention (hereinafter sometimes simply referred to as “spherical transition metal complex”) is a spherical transition metal complex having a hollow shell, The shell is formed of n transition metal atoms and 2n bidentate organic ligands, and one of the bidentate organic ligands is formed by binding a protein via a linking group. And the protein is formed so as to be oriented inside the hollow shell.

Here, n is an integer of 6 to 60.
In the spherical transition metal complex of the present invention, since the self-assembly easily proceeds, the n is preferably 6, 12, 24, 30 or 60, more preferably 6 or 12. 12 is particularly preferred.

  The spherical transition metal complex of the present invention is formed by self-organization utilizing a coordinate bond between a transition metal ion and a bidentate organic ligand. Since the coordinate bond has an appropriate binding force and the directionality is clearly defined, it is possible to spontaneously and quantitatively construct a molecular assembly whose structure is precisely controlled. In addition, since the coordination number and the bond angle can be controlled in accordance with the type of metal and the oxidation number, various coordination bond structures can be obtained.

As the spherical transition metal complex of the present invention, there are 2n transition metal compounds (M), 1 bidentate organic ligand (L1) formed by binding a protein via a linking group, and bidentate organic coordination. A compound represented by the formula: M _n L2 _(2n−1) L1 formed by self-organization so that the protein is oriented inside the hollow shell by 2n−1 children (L2) is preferable. Here, n represents the same meaning as described above. The plurality of Ms and L2s may be the same or different from each other, but are preferably the same.

  The size of the hollow shell of the spherical transition metal complex of the present invention is preferably 5 to 15 nm in diameter because the protein is encapsulated.

(1) Transition metal atom Although it does not restrict | limit especially as a transition metal atom which comprises the spherical transition metal complex of this invention, Ti, Fe, Co, Ni, Cu, Zn, Ru, Rh, Pd, Cd, Os, Ir And a platinum group atom such as Ru, Rh, Pd, Os, Ir, and Pt is preferable because Ru can be easily formed as a four-coordinated complex. , Pd, and Pt are more preferable, and Pd is particularly preferable.
The valence of the transition metal atom is usually 0 to 4, preferably 2. The coordination number is usually 4 to 6, preferably 4.

(2) Bidentate organic ligands The bidentate organic ligands constituting the spherical transition metal complex of the present invention are a bidentate organic ligand (L1) and a bidentate organic ligand (L2).
That is, when a bidentate organic ligand is reacted with a transition metal atom in a self-organizing manner to form a spherical transition metal complex, the protein is encapsulated in one of the bidentate organic ligands used. A bidentate organic ligand (bidentate organic ligand (L1)) bonded through a linking group is used.

(I) Bidentate organic ligand (L1)
The bidentate organic ligand (L1) is a bidentate organic ligand formed by binding a protein through a linking group. In the formed spherical transition metal complex, the protein is oriented inside the hollow shell. The compound represented by the following formula (I) is preferable as long as it can be used.

The compound represented by the formula (I) has a bridge portion next to the pyridyl group, forms a wide space between the pyridyl groups at both ends while maintaining planarity, and is bonded to this space via a linking group. The structure in which the formed protein is located.
In formula (I), X represents an ethynylene group or a p-phenylene group.
t represents an integer of 2 to 6, and a plurality of Xs may be the same or different.
Specific examples of the group represented by the formula:-(X) t- are shown below.

R ¹ and R ² each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.

Examples of the halogen atom for R ¹ and R ² include a fluorine atom, a chlorine atom, and a bromine atom.
As the alkyl group of the alkyl group which may be substituted, methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, t-butyl group, n-pentyl group, n-hexyl group, n-octyl group , N-nonyl group, n-decyl group and the like, and an alkyl group having 1 to 10 carbon atoms. Examples of the substituent of the alkyl group which may be substituted include a halogen atom, an alkoxyl group, and a phenyl group which may have a substituent.

  The alkoxyl group of the optionally substituted alkoxyl group has 1 to 10 carbon atoms such as methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, t-butoxy group, pentyloxy group, hexyloxy group and the like. An alkoxyl group is mentioned. Examples of the substituent of the alkoxyl group which may be substituted include a halogen atom and a phenyl group which may have a substituent.

m1 and m2 each independently represents an integer of 0 to 4. When m1 and m2 are 2 or more, a plurality of R ^{1 s} and R ^{2 s} may be the same or different.

A ₁ represents one type of groups represented by the following formulas (a-1) to (a-4).

In formulas (a-1) to (a-4), R ³ represents a group represented by the formula: —YD-Pro.
In the formula, Y represents a single bond or a divalent linking group. The divalent linking group is not particularly limited as long as it can bind to D. For _{example, -O- (CH 2) a- (} a is the same in the following represents an integer of _{1~8.), - O- (CH} 2) a-C (= O) -, - C (= O) _{-, - O- (CH 2)} a-NH -, - NH-, and wherein (y)

The group etc. which are represented by these are mentioned. In formula (y), m9 represents an integer of 1 to 10, preferably 1 to 3.
Among these, the group represented by the formula (y) is preferable because the protein can be easily bound and detached, and hardly denatured or decomposed when bound to the protein.

  D represents O, S, NH, or O— (C═O), and D-Pro represents a portion excluding H of the protein represented by Pro-DH.

Specific examples of the Pro-DH _{is, Pro-OH, Pro-NH} 2, Pro-COOH, Pro-SH , and the like.
For example, Pro-SH is a protein obtained by mutating a protein having C-terminal methionine or a protein having C-terminal glycine into cysteine. Note that Pro-SH may form a dimer by forming an S—S bond. In that case, it reduces and uses it as a monomer.

  Proteins generally have a molecular weight of 4,000 or more among those polycondensated (polypeptide) by dehydration between the carboxyl group and amino group of the L-α-amino acid. The protein used in the present invention has a size that fits in the hollow shell formed by the spherical transition metal complex and has 50 to 500 amino acid residues, and it is desired to obtain new information on its structure. Is preferred.

Specific examples of such proteins include ubiquitin, for example.
Ubiquitin consists of 76 amino acids and is a small protein with an outer diameter of slightly over 3 nm. It is known to be stable against organic solvents, enzymes, heat, etc., and is considered to be relatively easy to handle. . Ubiquitin is used for the modification of other proteins in vivo and is an important protein that controls many life phenomena such as proteolysis, cell cycle, signal transduction, DNA repair, transcriptional regulation, and metabolic control. In 2004, the Nobel Prize in Chemistry was awarded for “Discovery of Proteolysis via Ubiquitin”, and the structure has attracted attention.

R ⁴ has a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, a carboxyalkyl group, and a substituent. An amino group that may be substituted, an aminoalkyl group that may have a substituent, or a quaternized aminoalkyl group.

Of R ^4, halogen atom, optionally substituted alkyl groups, as good alkoxyl group which may be substituted include the same ones as exemplified for R ^1.
Examples of the alkyl group of the hydroxyalkyl group, carboxylalkyl group, optionally substituted aminoalkyl group, and quaternized aminoalkyl group include alkyl groups having 1 to 6 carbon atoms such as a methyl group, an ethyl group, and a propyl group. Groups.

That is, examples of the hydroxyalkyl group include a hydroxymethyl group, a 2-hydroxyethyl group, and a 3-hydroxy-n-propyl group.
Examples of the carboxyalkyl group include a carboxymethyl group, a 2-carboxylethyl group, and a 3-carboxy-n-propyl group.

  Examples of the aminoalkyl group which may have a substituent include an aminomethyl group, a methylaminomethyl group, and a dimethylaminomethyl group.

The quaternized aminoalkyl ^{_{group, -CH 2 -N + Me 3,}} -CH 2 -N + Et 3, -CH 2 CH 2 -N + Me 3, like _-CH 2 CH 2 ^-N + Et ₃ is (Me represents a methyl group, Et represents an ethyl group).

  Examples of the amino group which may have a substituent include an amino group, a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, an acetylamino group, a benzoylamino group, a mesylamino group, and a tosylamino group. It is done.

m3 represents an integer of 0 to 3, and m4 represents an integer of 0 to 2. When m3 is 2 or more, or when m4 is 2, even a plurality of R ⁴ with each other are the same, may be different from each other.

Q is, -Nr1 -, - O -, - C (= O) -, - S-, or -SO ₂ - represents a.
In the group represented by the formula: -Nr1-, r1 represents a hydrogen atom, an alkyl group, an aryl group or an acyl group.
Examples of the alkyl group for r1 include a methyl group, an ethyl group, a propyl group, and an isopropyl group. Examples of the aryl group include a phenyl group and a p-methylphenyl group. Examples of the acyl group include an acetyl group, a propionyl group, and a benzoyl group.

  As the bidentate organic ligand (L1) used in the present invention, since a spherical transition metal complex having a size preferable for encapsulating a protein is obtained, and handling is easy, the following formula (I-1) ) And (I-2) are preferred, and compounds represented by formulas (I-1a) and (I-2a) are particularly preferred.

In the formula, D-Pro and R ³ represent the same meaning as described above.
The bidentate organic ligand (L1) can be produced by applying a known synthesis method. For example, among the compounds represented by the formula (I), the compound represented by the following formula (I-3) is converted into a compound represented by the formula: Pro as shown in the following formula: It can be produced by reacting a protein represented by -DH.

In the formula, R ¹ , m 1, X, t, Pro-D, and Y represent the same meaning as described above.
A ₁ ′ is a group that satisfies A ₁ ′ −R ₃ = A ₁ .
Y ′ represents a precursor of the linking group Y, which reacts with Pro-DH to become Pro-DY.
_{Specifically, -OH, -O- (CH 2)} a-OH (a is hereinafter the same represents an integer of _{1~8.), - O- (CH} 2) a-COOH, -COOH, - _{O- (CH 2) a-NH} 2, -NH 2, and the formula (y ')

(Wherein, m9 represents the same meaning as described above).
Examples of the reaction for obtaining the compound represented by the formula (I-3) by reacting the compound represented by the formula (III) with Pro-DH include known esterification reaction, dehydration reaction, Michael addition reaction and the like. Can be mentioned.

For example,
(A) When the protein to be used is represented by Pro-OH, it is reacted with Y ′ having a COOH group at the terminal portion to form Pro-O—C (═O) — A compound represented by the formula (I-3) can be obtained.
(B) When the protein to be used is represented by Pro-NH ₂ , it is reacted with Y ′ having a COOH group at the end to form Pro-NH—C (═O) — A compound represented by formula (I-3) can be obtained.
(C) When the protein to be used is represented by Pro-COOH, it is reacted with Y ′ having an OH group at the terminal portion to form Pro-C (═O) —O— A compound represented by the formula (I-3) can be obtained.
(D) When the protein to be used is represented by Pro-SH, it is reacted with Y ′ having —CH═CH— at the terminal portion (Michael addition reaction),

The compound represented by the formula (I-3) can be obtained by forming the partial structure represented by
Among these, in the present invention, the method of Michael addition reaction using the protein represented by Pro-SH as the protein in the above (d) is preferable from the viewpoint of simplicity and the like.

  The compound represented by the formula (III) can be prepared by a known method (K. Sonogashira, Y. Tohda, N. Hagihara, Tetrahedron Lett., 1975, 4467; J. F. Nguefack, V Bolitt, D. Sinou, Tetrahedron Lett., 1996, 31, 5527).

In the formula, R ¹ , m 1, X, t, A ₁ ′ and Y ′ represent the same meaning as described above. Y ″ represents Y ′ or a precursor of Y ′ (including a group in which Y ′ is protected with a protecting group). Z represents a halogen atom such as a chlorine atom, a bromine atom, or an iodine atom.

  That is, first, the bidentate organic ligand represented by the formula (VI) is obtained by the Sonogashira cross-coupling reaction between the compound represented by the formula (IV) and the compound represented by the formula (V). In the case where Y ″ is a group obtained by protecting Y ′ with a protecting group, the protecting group can be appropriately deprotected to synthesize a compound represented by the formula (III).

The compound represented by the formula (VI) is prepared by using a base, a palladium catalyst such as Pd (PhCN) ₂ Cl ₂ / P (t-Bu) ₃ , Pd (PPh ₃ ) ₄ , and the first iodide in a suitable solvent. Obtained by reacting 4- (4-pyridyl) phenylacetylenes represented by formula (III) (or a salt thereof) with a compound represented by formula (IV) in the presence of a copper salt such as copper. be able to.

  The above reaction is an example in which two compounds represented by the formula (IV) are reacted at once to produce a compound having the same bridge portion. A compound having two different bridge portions substituted with different substituents can be obtained by reacting the corresponding compound represented by the formula (IV) stepwise under similar reaction conditions.

  Examples of the base used here include amines such as dimethylamine, diethylamine, diisopropylamine, triethylamine, and diisopropylethylamine.

  Examples of the solvent to be used include ethers such as 1,4-dioxane, diisopropyl ether, tetrahydrofuran and 1,2-dimethoxyethane; amides such as dimethylformamide; sulfoxides such as dimethyl sulfoxide; nitriles such as acetonitrile; It is done.

  The reaction temperature is usually in the temperature range from 0 ° C. to the boiling point of the solvent used, preferably 10 ° C. to 70 ° C., and the reaction time is usually from several minutes to several tens of hours depending on the reaction scale and the like.

  When Y ″ is a group obtained by protecting Y ′ with a protecting group, the protecting group is appropriately deprotected by a known method, and when Y ″ is a precursor of Y ′, a substitution reaction or the like. By performing the desired deformation, the compound represented by the formula (III) can be obtained.

  The compound represented by the formula (IV) and the compound represented by the formula (V) can be produced by a known method. Commercial products can also be used.

(Ii) bidentate organic ligand (L2)
The bidentate organic ligand (L2) is capable of forming a spherical transition metal complex in a self-organizing manner with the transition metal atom and is not particularly limited as long as no protein is bound thereto. ) And a compound represented by the following formula (II) in which the size of the ligand is substantially the same.

  The compound represented by the formula (II) has a plurality of ethynylene groups and / or p-phenylene groups next to the pyridyl group, and maintains planarity between the pyridyl groups at both ends. It has a structure with a wide space. That is, the compound represented by the formula (I) is a compound not bound with a protein.

In formula (II), X and t represent the same meaning as described above.
R ⁵ and R ⁶ each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, or a nitro group, similar to R ¹ and R ² .
m5 and m6 each independently represents an integer of 0 to 4. m5, when m6 is 2 or more, plural R ⁵ together, even R ⁶ together are each identical or may be different phases.

A ₂ represents one type of group represented by the following formulas (a-5) to (a-8).

In formulas (a-5) to (a-8), Q represents the same meaning as described above.
R ⁷ is the same as the above R ⁴ , halogen atom, optionally substituted alkyl group, optionally substituted alkoxyl group, cyano group, nitro group, hydroxyl group, hydroxyalkyl group, carboxyl group, carboxylalkyl A group, an amino group which may have a substituent, an aminoalkyl group which may have a substituent, or a quaternized aminoalkyl group;

As will be described later, in the reaction for forming the spherical transition metal complex, it is preferable to use water or a mixed solvent of water and an organic solvent as a reaction solvent so that the protein is not denatured. Therefore, in order to improve the solubility of the bidentate organic ligand (L2) in these solvents, at least one of R ⁷ has a hydroxyl group, a hydroxyalkyl group, a carboxyl group, a carboxylalkyl group, and a substituent. It is preferably a hydrophilic group such as an optionally substituted amino group, an optionally substituted aminoalkyl group, or a quaternized aminoalkyl group, and is preferably a quaternized aminoalkyl group. More preferred.
The counter ion of the quaternized aminoalkyl group is preferably the same as the anion of the transition metal group compound that is a raw material for producing the spherical transition metal complex, as will be described later.

m7 represents an integer of 0 to 3, and m8 represents an integer of 0 to 2. When m7 is 2 or more, or when m8 is 2, a plurality of R ⁷ may be the same or different.

R ⁸ represents a hydrogen atom, a halogen atom, an alkyl group, or a haloalkyl group.
Examples of the halogen atom for R ⁸ include a fluorine atom, a chlorine atom, and a bromine atom.
As the alkyl group, methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, t-butyl group, n-pentyl group, n-hexyl group, n-octyl group, n-nonyl group, n-decyl C1-C20 alkyl groups, such as group, etc. are mentioned.
Examples of the haloalkyl group include a chloromethyl group, a dichloromethyl group, a bromomethyl group, and a trifluoromethyl group.

  Among these, the bidentate organic ligand (L2) is preferably a compound represented by the following formulas (II-1) and (II-2).

  In the formula, r represents an alkyl group having 1 to 6 carbon atoms such as a methyl group, an ethyl group or a propyl group; a phenyl group which may have a substituent such as a phenyl group or a 4-methylphenyl group. r is preferably an alkyl group having 1 to 6 carbon atoms.

G ⁻ represents a counter ion such as Br ⁻ , Cl ⁻ , NO ₃ ⁻ , ClO ₄ ⁻ , BF ₄ ⁻ , SbF ₄ ⁻ , PF ₆ ⁻ , AsF ₆ ⁻ , SiF ₆ ²⁻ , and CH ₃ CO ₂ ^−. .

The bidentate organic ligand (L2) can be produced by the same method as the compound represented by the formula (III). For example, the compound represented by the formula (II-3) in which R ⁷ is a group having a hydrophilic trialkylammonium cation can be produced, for example, according to the reaction formula shown below.

(Wherein r, X, t and R ⁸ have the same meaning as described above.)
That is, the compound represented by the formula (VII) is converted into a benzyl bromide derivative using triphenylphosphine and carbon tetrabromide, and then an aqueous trialkylamine solution (Nr ₃ aq.) Is added without isolation. A compound represented by the formula (II-3) having a trialkylammonium cation group can be synthesized.

  In addition, although the counter ion of the compound represented by the formula (II-3) is a bromine ion, the counter ion may be exchanged by a known method if necessary.

2) Method for Producing Spherical Transition Metal Complex The method for producing the spherical transition metal complex of the present invention comprises a transition metal compound (M), a bidentate organic ligand (L1) represented by the formula (I) and a formula (I II) the bidentate organic ligand (L2) and the transition metal compound (M) n mol (n represents an integer of 6 to 60), and the bidentate organic ligand (L1). 1 mol of bidentate organic ligand (L2) is reacted at a ratio of (2n-1) mol.

  The transition metal compound (M) used in the present invention is not particularly limited as long as it can form a spherical transition metal complex in a self-organizing manner with the bidentate organic ligands (L1) and (L2). Metal compounds are preferred.

  As a transition metal atom which comprises a transition metal compound (M), the thing similar to what was illustrated as a transition metal atom of the spherical transition metal complex of the said this invention is mentioned.

  Specific examples of the transition metal compound (M) include transition metal halides, nitrates, hydrochlorides, sulfates, acetates, methanesulfonates, trifluoromethanesulfonates, p-toluenesulfonates, and the like. It is done. Among these, nitrates and trifluoromethanesulfonates of transition metals are preferred because the desired hollow transition metal complex can be obtained efficiently.

The ratio of the transition metal compound (M) to the bidentate organic ligands (L1) and (L2) can be appropriately set according to the composition of the target spherical transition metal complex. For example, when it is desired to obtain a protein-encapsulating spherical transition metal complex having the composition of the formula: M ₁₂ (L1) (L2) ₂₃ , bidentate organic ligand (L1) with respect to 12 mol of the transition metal compound (M) 1 mol and bidentate organic ligand (L2) may be reacted at a ratio of 23 mol.

  The reaction of the transition metal compound (M) with the bidentate organic ligands (L1) and (L2) can be carried out in a suitable solvent. As the solvent, those which do not denature the protein are preferable. Specifically, water or a mixed solvent of water and a polar organic solvent is preferable.

  Examples of polar organic solvents to be used include nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide (DMSO); amides such as N, N-dimethylformamide; tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, and the like. Ethers; alcohols such as methanol, ethanol and isopropyl alcohol; ketones such as acetone and methyl ethyl ketone; cellosolves such as ethyl cellosolve; These solvents can be used alone or in combination of two or more.

The reaction between the transition metal compound (M) and the bidentate organic ligands (L1) and (L2) proceeds smoothly in a temperature range from 0 ° C. to the boiling point of the solvent used.
The reaction time is several minutes to several days.

  After completion of the reaction, normal post-treatment operations in synthetic organic chemistry are performed, and if desired, purification by known separation and purification means such as column purification by ion exchange resin, distillation, recrystallization, etc., the desired spherical transition Metal complexes can be isolated.

The counter ion of the obtained spherical transition metal complex is usually an anion of the transition metal compound (M) to be used, but the counter ion is used for the purpose of improving crystallinity or improving the stability of the spherical transition metal complex. May be replaced. Examples of such counter ions include PF ₆ ⁻ , ClO ₄ ⁻ , SbF ₄ ⁻ , AsF ₆ ⁻ , BF ₄ ⁻ , SiF ₆ ^{2− and the} like.

The structure of the obtained spherical transition metal complex is as follows: ¹ H-NMR, DOSY NMR, ¹³ C-NMR, IR spectrum, mass spectrum, visible light absorption spectrum, UV absorption spectrum, reflection spectrum, X-ray crystal structure analysis, elemental analysis It can confirm by well-known analysis means, such as.

DOSY NMR is a technique for separating signals by a diffusion coefficient, and signals can be separated by the size of the structure. That is, it is considered that it can be confirmed that the protein is encapsulated in the spherical transition metal complex by confirming whether the formed spherical transition metal complex and the encapsulated protein are moving with the same diffusion coefficient.
As described above, the spherical transition metal complex of the present invention can be efficiently produced by an extremely simple operation. Therefore, mass synthesis on a gram scale is also possible.

  The spherical transition metal complex of the present invention has a certain size on the nanometer scale, and the protein bound to the bidentate organic ligand (L1) is oriented precisely inside the complex spherical structure. Has a special structure.

  As described above, since the spherical transition metal complex of the present invention is a protein in which a hollow shell having a precisely controlled size is encapsulated, not only protein stabilization but also new information on protein structure is provided. Expected to contribute to the development of acquisition methods.

3) Bidentate organic ligand The bidentate organic ligand of the present invention is a compound represented by the above formula (I ′) (hereinafter referred to as “compound (I ′)”) and the above (II ′). Compound (hereinafter referred to as “compound (II ′)”).
Compound (I ′) is a compound represented by formula (I) wherein t is n1 (n1 represents an integer of 1 to 6). Compound (II ′) is a compound in which t is n1 (n1 represents an integer of 1 to 6) in the compound represented by the formula (II).
The bidentate organic ligand of the present invention is a novel compound and can be produced in the same manner as the bidentate organic ligands (L1) and (L2).

  Next, the present invention will be described in more detail by way of examples. However, the present invention is not limited to the examples.

(Equipment)
(1) Measurement of ¹ H-NMR spectrum
¹ H-NMR spectrum was measured with a Bruker DRX 500 (500 MHz) NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard).

(2) Measurement of ¹³ C-NMR spectrum
^{The 13} C-NMR spectrum was measured using a Bruker DRX 500 (125 MHz) NMR spectrometer.

(3) Measurement of mass spectrum The CSI-MS spectrum (cold spray ionization mass spectrometry) was measured using JEOL, JMS-700C (4-sector (BE / BE) -tandem mass spectrometer).

(4) MALDI-TOF MS
MALDI-TOF MS was measured using a mass spectrometer (Applied Biosystem Voyager DE-STR).

(5) Melting | fusing point Melting | fusing point was measured using melting | fusing point measuring device: Yanaco MP-500V.
(4) Elemental analysis Elemental analysis of carbon, hydrogen, and nitrogen was performed using Yanaco MT-6.

(5) IR spectrum The IR spectrum was measured using DIGILAB Scientific FTS-2000 (KBr).

(Reagents)
Unless otherwise specified, solvents and reagents include Tokyo Chemical Industry Co., Ltd., Wako Pure Chemical Industries, Ltd., or Sigma-Aldrich Co. The commercial product of was used.

Example 1 Synthesis of Ligand 4a (1) Synthesis of N- (2-hydroxyethyl) maleimide (i) Synthesis of N-methoxycarbonylmaleimide

  Methyl chloroformate (3.40 mL, 44. mmol) in a solution of maleimide (3.88 g, 40.0 mmol) and N-methylmorpholine (4.82 mL, 44.0 mmol) in ethyl acetate (240 mL) at 0 ° C. 0 mmol) was added and stirred for 1 hour. The precipitate was filtered off and the filtrate was concentrated under reduced pressure. The obtained residue was recrystallized from a mixed solvent of chloroform and n-hexane to obtain the title compound as a white solid (6.10 g, 39.3 mmol). Yield 98%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 6.84 (s, 2H), 3.99 (s, 3H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 165.6 (C), 148.1 (C), 135.3 (CH), 54.3 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3093, 2972, 1764, 1713, 1442, 1337, 1265, 1139, 1036, 921, 842, 769, 696, 641, 595

<References>
Helvetica Chimica Acta, 58, 536, 1975

(Ii) Synthesis of N- (2-hydroxyethyl) maleimide N-methoxycarbonylmaleimide (3.10 g, 20.0 mmol) and 2-aminoethanol (1.25 g, 20.5 mmol) were added to sodium bicarbonate (2.52 g). , 30.0 mmol) in water (150 mL) and the whole volume was stirred at room temperature for 30 minutes. The reaction solution was extracted with chloroform (200 mL × 6). The residue obtained by concentrating the organic layer was recrystallized from a mixed solvent of chloroform and n-hexane to obtain the title compound as a white solid (1.45 g, 10.3 mmol). Yield 52%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 6.70 (s, 2H), 3.82-3.77 (m, 2H), 3.76-3.72 (m, 2H), 2.04 (t, J = 5.8Hz, 1H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 171.1 (C), 134.3 (CH), 61.0 (CH ₂ ), 40.7 (CH ₂ )

IR (KBr, cm ⁻¹ ) 3260, 3114, 2961, 1712, 1444, 1407, 1363, 1322, 1160, 1068, 973, 853, 833, 695.

(2) Synthesis of compound 4a

(I) Synthesis of acetic acid 2,6-bis [4- (4-pyridyl) phenylethynyl] phenyl ester Tri-t-butylphosphine (0.462 mL, 0.184 mmol; 10 wt% n-hexane solution) and diisopropylamine (2.0 mL, 14 mmol), 1-acetoxy-2,6-dibromobenzene (441 mg, 1.50 mmol), 4- (4-pyridyl) phenylacetylene (716 mg, 4.00 mmol), Pd (PhCN) ₂ Cl ₂ (34.5 mg, 0.090 mmol) and a solution of copper (I) iodide (11.5 mg, 0.0060 mmol) in 1,4-dioxane (25 mL) and the whole volume was added at 45 ° C. under an argon atmosphere. Stir for 12 hours. Ethyl acetate (40 mL) was added to the reaction mixture, and the insoluble material was filtered off. Water (100 mL) and ethylenediamine (2 mL) were added to the filtrate, and the ethyl acetate layer was separated. After drying the organic layer with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure.
The obtained residue was purified by silica gel column chromatography (CHCl ₃ : methanol = 50: 1 (volume ratio)) to obtain the title compound as a white solid (650 mg, 1.33 mmol). Yield 88%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.69 (d, J = 5.3 Hz, 4H), 7.67-7.61 (m, 8H), 7.59 (d, J = 7.7 Hz, 2H), 7.51 (d, J = 5.9 Hz, 4H), 7.27 (t, J = 7.9 Hz, 1H), 2.47 (s, 3H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 168.1 (C), 152.4 (C), 150.4 (CH), 147.3 (C), 138.4 (C), 133.0 (CH), 132.4 (CH), 127.1 (CH), 126.1 (CH), 123.6 (C), 121.5 (CH), 118.2 (C), 94 0.0 (C), 85.3 (C), 20.7 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3032, 2215, 1171, 1594, 1516, 1488, 1438, 1402, 1369, 1181, 1151, 1072, 902, 811, 682, 530

(Ii) Synthesis of 1- (2- {2,6-bis [4- (pyridyl) phenylethynyl] phenoxy} ethyl) pyrrole-2,5-dione (4a) Diisopropyl azodicarboxylate (0.089 mL, 0 .45 mmol) is added to a solution of 2,6-bis [4- (4-pyridyl) phenylethynyl] phenol (168 mg, 0.375 mmol) and triphenylphosphine (110 mg, 0.42 mmol) in THF (20 mL), The mixture was stirred at room temperature for 30 minutes under an argon atmosphere. Then, N- (2-hydroxyethyl) maleimide (59.2 mg, 0.42 mmol) was added to this solution, and the whole volume was stirred at room temperature for 17 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 50: 1 (volume ratio)) to obtain the title compound as a white solid (42.9 mg, 0.0750 mmol). Yield 20%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.69 (d, J = 6.1 Hz, 4H), 7.737.67 (m, 8H), 7.54 (d, J = 6 .2 Hz, 4H), 7.50 (d, J = 7.7 Hz, 2H), 7.11 (t, J = 7.7 Hz, 1H), 6.53 (s, 2H), 4.54 (d , J = 6.2 Hz, 2H), 4.07 (d, J = 6.2 Hz, 2H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 170.5 (C), 160.0 (C), 150.5 (CH), 147.4 (C), 138.1 (C), 134.0 (CH), 133.9 (CH), 132.5 (CH), 127.0 (CH), 124.0 (CH), 123.9 (C), 121.4 (CH), 117 .5 (C), 93.6 (C), 86.5 (C), 69.8 (CH ₂ ), 37.9 (CH ₂ )

IR (KBr, cm ⁻¹ ) 3029, 2211, 1711, 1596, 1489, 1439, 1408, 1325, 1227, 1169, 1102, 1070, 1020, 812, 696

(3) Synthesis of ligand 2a

  First, ubiquitin (Ubiquitin-SH) used as a protein to be encapsulated in a spherical transition metal complex was prepared. Ubiquitin is a protein consisting of 76 residues, but this time a mutated C-terminal glycine to cysteine was used.

  First, cDNA that encodes the protein of interest is introduced into the host E. coli using an expression vector that has been incorporated into the multicloning site of a vector from which GST has been removed (pGEX-6P-1: GE Healthcare Bioscience). did. Escherichia coli was cultured using LB medium containing ampicillin, and protein was expressed by adding IPTG. After culturing, Escherichia coli was pulverized and produced in the order of an anion exchange column, a cation exchange column, and dialysis, thereby obtaining a target ubiquitin having a cysteine residue at the C-terminus.

The amino acid sequence of the obtained mutant ubiquitin is as follows.
MQIFVKTLGKTITLEVEPSTDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTSDYNIQKESTLHLVLLRRGC

  Since the cultured ubiquitin formed an S—S bond between ubiquitins, a monomer was first prepared by reducing the S—S bond using 5 mM tris (2-carboxyethyl) phosphine (TCEP). That is, tris (2-carboxyethyl) phosphine (TCEP) was added to a 20 mM Tris buffer solution (15 mL) of mutated ubiquitin (22.4 mg, 2.60 μmol) at 37 ° C. for 2 hours. Stir slowly. It was confirmed by MALDI-TOF MS that the solution contained monomerized ubiquitin. After adjusting the pH of the solution to 8.0 with 1 M aqueous sodium hydroxide solution, a solution of compound 4a (22.8 mg, 39.9 μmol) in THF (10 mL) was added, and the mixture was added at 37 ° C. for 2 hours and at room temperature for 12 hours. Stir.

  The reaction mixture was purified by reversed phase HPLC (on an Inertsil Peptide C18 semi-preparative column (20 mm × 250 mm), eluent: 0.1% trifluoroacetic acid (TFA) aqueous solution and acetonitrile), lyophilized, The title compound was obtained as a white powder (15 mg, 1.6 μmol). Yield 63%.

MALDI-TOF-MS: calcd for [M + Na] ⁺ 9205.4, found 9203.7

Example 2 Synthesis of Ligand 1a

(1) Synthesis of 3,5-dibromo-4-methylphenyl) methanol A THF-like (20 mL) -like solution of 3,5-dibromo-4-methylbenzoic acid (2.35 g, 8.00 mmol) was added to 1.0 M ( BH ₃ -THF) THF solution (18.0 mmol, was added dropwise over 30 minutes 18.0 mL). The reaction solution was slowly poured into a saturated aqueous sodium hydrogen carbonate solution. The reaction mixture was extracted with ethyl acetate (200 mL × 3). The organic layer was collected and the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from a mixed solvent of chloroform and n-hexane to obtain the title compound as a white solid (1.95 g, 6.97 mmol). Yield 87%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 7.52 (s, 2H), 4.62 (d, J = 5.8 Hz, 2H), 2.56 (s, 3H), 1. 70 (t, J = 6.0 Hz, 1H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 141.2 (C), 136.5 (C), 130.1 (CH), 125.3 (C), 63.6 (CH ₂ ) , 23.4 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3314, 3202, 2928, 1745, 1594, 1541, 1431, 1391, 126, 1216, 1195, 1064, 1031, 992, 866, 730, 687, 652

(2) Synthesis of {4-methyl-3,5-bis [4- (4-pyridyl) phenylethynyl] phenyl} methanol Tri-t-butylphosphine (0.462 mL, 0.186 mmol; 10 wt% n-hexane solution ), And diisopropylamine (1.5 mL, 10.7 mmol) to (3,5-dibromo-4methylphenyl) methanol (420 mg, 1.50 mmol), 4- (4-ethynylphenyl) pyridine (716 mg, 4. 00 mmol), Pd (PhCN) ₂ Cl ₂ (34.5 mg, 0.090 mmol), and copper iodide (I) (11.5 mg, 0.060 mmol) in 1,4-dioxane (25 mL) solution, The whole volume was stirred at 45 ° C. for 16 hours under an argon atmosphere. Ethyl acetate (30 mL) was added to the reaction solution, and the insoluble material was filtered off. Water (80 mL) and ethylenediamine (1 mL) were added to the filtrate, and the ethyl acetate layer was separated. The organic layer was dried over anhydrous sodium sulfate and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 40: 1 (volume ratio)) to obtain the title compound as a white solid (634 mg, 1.33 mmol). Yield 89%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.68 (d, J = 6.2 Hz, 4H), 7.677.65 (m, 8H), 7.55 (s, 2H), 7.53 (d, J = 6.2 Hz, 4H), 4.71 (d, J = 5.5 Hz, 2H), 2.74 (s, 3H), 1.94 (d, J = 5.9 Hz) , 1H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 150.4 (CH), 147.4 (C), 141.4 (C), 138.5 (C), 138.0 (C), 132.3 (CH), 130.7 (CH), 127.0 (CH), 124.1 (C), 123.7 (C), 121.4 (CH), 93.1 (C), 89 .5 (C), 64.3 (CH ₂ ), 19.1 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3273, 3039, 2203, 1597, 1566, 1489, 1403, 1329, 1290, 1226, 1112, 1064, 997, 81, 686, 566

(3) Synthesis of trimethyl {4-methyl-3,5-bis [4- (4-pyridyl) phenylethynyl] benzyl} ammonium bromide Triphenylphosphine (367 mg, 1.40 mmol) and carbon tetrabromide (663 mg, 3.00 mmol) in dry THF (150 mL) of {4-methyl-3,5-bis [4- (4-pyridyl) phenylethynyl] phenyl} methanol (238 mg, 0.500 mmol) at room temperature under an argon atmosphere. Sequentially added to the solution. By stirring the whole volume for 3 hours, it was confirmed by MALDI-TOF MS that 4-methyl-3,5-bis [4- (4-pyridyl) phenylethynyl] benzyl bromide was quantitatively formed ( Not isolated).

  To this solution, trimethylamine (7.0 mL, 30 mmol; 4.3 M aqueous solution) was added, and the whole volume was stirred at room temperature for 20 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was washed with ethyl acetate and water, and then dried in vacuo to give the title compound as a white solid (207 mg, 0.345 mmol). Yield 69%

¹ H-NMR (500 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 8.75 (d, J = 5.3 Hz, 4H), 7.98 (d, J = 8.3 Hz, 2H), 7.91 (D, J = 4.8 Hz, 4H), 7.83 (s, 2H), 7.78 (d, J = 8.2 Hz, 2H), 4.56 (s, 2H), 3.09 (s , 9H), 2.77 (s, 3H)

¹³ C-NMR (125 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 148.8 (CH), 147.3 (C), 143.5 (C), 137.0 (C), 136.1 (CH ), 132.2 (CH), 127.4 (CH), 126.8 (C), 123.3 (C), 123.0 (C), 121.6 (CH), 93.9 (C) , 88.5 (C), 66.5 (CH ₂ ), 52.0 (CH ₃ ), 18.9 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3429, 2361, 2206, 1632, 1599, 1490, 1401, 1289, 1214, 1069, 1004, 878, 812, 685

(4) Synthesis of trimethyl {4-methyl-3,5-bis [4- (4-pyridyl) phenylethynyl] benzyl} ammonium nitrate (1a) Trimethyl {4-methyl-3,5-bis [4- To a solution of (4-pyridyl) phenylethynyl] benzyl} ammonium bromide (77.5 mg, 0.130 mmol) and silver nitrite (22.8 mg, 0.133 mmol) in acetonitrile (10 mL) and water (40 mL), The whole volume was stirred at 40 ° C. for 3 hours under an argon atmosphere.
The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound as a white solid (47 mg, 0.081 mmol). Yield 62%.

¹ H-NMR (500 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 8.69 (d, J = 4.7 Hz, 4H), 7.94 (d, J = 8.3 Hz, 2H), 7.82 (S, 2H), 7.79 (d, J = 4.6 Hz, 4H), 7.76 (d, J = 8.4 Hz, 2H), 4.53 (s, 2H), 3.09 (s , 9H), 2.77 (s, 3H)

¹³ C-NMR (125 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 150.2 (CH), 145.8 (C), 143.5 (C), 137.5 (C), 136.0 (CH ), 132.1 (CH), 127.2 (CH), 126.8 (C), 123.3 (C), 122.5 (C), 121.1 (CH), 94.0 (C) , 88.3 (C), 66.6 (CH ₂ ), 52.0 (CH ₃ ), 18.9 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3032, 2929, 2208, 1633, 1597, 1539, 1490, 1343, 1215, 1113, 993, 880, 812, 723, 684

Example 3 Synthesis of Ligand 2b

(1) Synthesis of 2,6-bis [4- (4-pyridylethynyl) phenylethynyl] phenylacetate Tri-t-butylphosphine (0.062 mL, 0.025 mmol; 10% n-hexane solution) and diisopropylamine ( 0.20 mL, 1.4 mmol), 1-acetoxy-2,6-dibromobenzene (58.8 mg, 0.200 mmol), 1-ethynyl-4- (pyridylethynyl) benzene (101.6 mg, 0.500 mmol) , Pd (PhCN) ₂ Cl ₂ (4.60 mg, 0.012 mmol), and copper (I) iodide (1.52 mg, 0.0080 mmol) in 1,4-dioxane (1.5 mL) Was stirred at 45 ° C. for 12 hours under an argon atmosphere.

Ethyl acetate (10 mL) was added to the reaction mixture, the insoluble material was filtered off, water (30 mL) was added to the filtrate, washed with ethylenediamine (1 mL), and extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (CHCl ₃ : methanol = 50: 1 (volume ratio)) to obtain the title compound as a white solid (86.1 mg, 0.160 mmol). Yield 80%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.62 (d, J = 5.3 Hz, 4H), 7.57 (d, J = 7.9 Hz, 2H), 7.56-7 .49 (m, 8H), 7.39 (d, J = 5.7 Hz, 4H), 7.26 (t, J = 7.7 Hz, 1H), 2.45 (s, 3H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 168.1 (C), 152.4 (C), 149.9 (CH), 133.1 (CH), 131.9 (CH), 131.7 (CH), 131.1 (C), 126.1 (CH), 125.5 (CH), 123.5 (C), 122.4 (C), 118.1 (C), 94 0.0 (C), 93.3 (C), 88.6 (C), 86.0 (C), 20.7 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3038, 2217, 1768, 1590, 1509, 1430, 1406, 1368, 1187, 1077, 1011, 904, 837, 820, 789, 547

(2) Synthesis of 1- (2- {2,6-bis [4- (4-pyridylethynyl) phenylethynyl] phenoxy} ethyl) pyrrole-2,5-dione (4b) diisopropyl azodicarboxylate (0. 032 mL, 0.16 mmol), 2,6-bis [4- (4-pyridylethynyl) phenylethynyl] phenol (69 mg, 0.14 mmol), triphenylphosphine (36.7 mg, 0.14 mmol) in THF (10 mL) ) Was added to the solution and the whole volume was stirred at room temperature for 30 minutes under an argon atmosphere.

Next, N- (2-hydroxyethyl) maleimide (19.8 mg, 0.14 mmol) was added to the solution, and the mixture was stirred at room temperature for 10 hours under an argon atmosphere.
The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 50: 1 (volume ratio)) to give the title compound as a white solid (46.5 mg, 0.075 mmol). Got as. Yield 54%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.63 (d, J = 5.5 Hz, 4H), 7.61-7.55 (m, 8H), 7.48 (d, J = 7.7 Hz, 2H), 7.39 (d, J = 5.6 Hz, 4H), 7.10 (t, J = 7.7 Hz, 1H), 6.51 (s, 2H), 4.51 (D, J = 6.0 Hz, 2H), 4.04 (d, J = 6.1 Hz, 2H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 170.5 (C), 160.0 (C), 149.9 (CH), 134.0 (CH), 133.9 (CH), 131.9 (CH), 131.8 (CH), 131.2 (C), 125.6 (CH), 124.0 (CH), 123.9 (C), 122.2 (C), 117 .4 (C), 93.7 (C), 93.5 (C), 88.5 (C), 87.3 (C), 69.9 (CH ₂ ), 37.9 (CH ₂ )

IR (KBr, cm ⁻¹ ) 3038, 2217, 1711, 1589, 1509, 1439, 1406, 1230, 1168, 1102, 1017, 969, 823, 696, 546
(3) Synthesis of ligand 2b

  Tris (2-carboxyethyl) phosphine was added to a 20 mM Tris buffer solution (15 mL) of mutated ubiquitin (22.4 mg, 2.60 μmol). By slowly stirring the whole volume at 37 ° C. for 2 hours, the formation of monomerized ubiquitin in the solution was confirmed by MALDI-TOF MS.

The solution was adjusted to pH 8.0 with 0.1 M aqueous sodium hydroxide solution, and a solution of compound 4b (25.0 mg, 39.9 μmol) in THF (10 mL) was added thereto, and the mixture was stirred at 37 ° C. for 2 hours at room temperature. Stir for 12 hours.
The reaction mixture was purified by reverse phase HPLC (on an Inertsil Peptide C18 semi-preparative column (20 mm × 250 mm), eluent: 0.1 wt% trifluoroacetic acid (TFA) aqueous solution and acetonitrile), and then lyophilized. The title compound was obtained as a white powder (14 mg, 1.5 μmol). Yield 57%.

MALDI-TOF-MS calcd for [M + Na] ⁺ 9253.4, found 9248.9.

Example 5 Synthesis of Ligand 1b

(1) Synthesis of {4-, methyl-3,5-bis [4- (4-pyridylethynyl) phenylethynyl] phenyl} methanol Tri-t-butylphosphine (0.462 mL, 0.0186 mmol; 10 wt% n- Hexane solution), and diisopropylamine (1.5 mL, 11 mmol) to (3,5-dibromo-4-methylphenyl) methanol (420 mg, 1.50 mmol), 4- (4-ethynylphenylethynyl) pyridine (762 mg, 3.75 mmol), Pd (PhCN) ₂ Cl ₂ (35 mg, 0.090 mmol), and copper iodide (I) (11 mg, 0.060 mmol) in 1,4-dioxane (15 ml) solution. The mixture was stirred at 50 ° C. for 14 hours under an argon atmosphere. Ethyl acetate (30 mL) was added to the reaction mixture, and the insoluble material was filtered off. Water (50 mL) was added to the filtrate, washed with ethylenediamine (1 mL), and extracted with ethyl acetate. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 40: 1 (volume ratio)) to obtain the title compound as a white solid (703 mg, 1.34 mmol). Yield 89%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.61 (d, J = 5.6 Hz, 4H), 7.55 (s, 8H), 7.53 (s, 2H), 7. 39 (d, J = 5.6 Hz, 4H), 4.69 (s, 2H), 2.71 (s, 3H), 1.94 (s, 1H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 149.8 (CH), 141.4 (C), 138.6 (C), 131.9 (CH), 131.6 (CH), 131.2 (C), 130.7 (CH), 125.5 (CH), 124.1 (C), 123.6 (C), 122.1 (C), 93.5 (C), 93 .2 (C), 90.2 (C), 88.5 (C), 64.3 (CH ₂ ), 19.0 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3289, 3041, 2873, 2218, 1590, 1540, 1510, 1407, 1213, 1102, 1049, 997, 835, 750, 636, 540

Elemental _{analysis: Calcd for C 38 H 24 N} 2 O · 1.5H 2 O: C, 82.74; H, 4.93; N, 5.08.
Found: C, 82.54; H, 4.57; N, 4.80

(2) Synthesis of trimethyl {4-methyl-3,5-bis [4- (4-pyridylethynyl) phenylethynyl] benzyl} ammonium bromide Triphenylphosphine (367 mg, 1.40 mmol) and carbon tetrabromide (597 mg, 1.80 mmol) in dry THF (150 mL) of {4-methyl-3,5-bis [4- (4-pyridylethynyl) phenylethynyl] phenyl} methanol (314 mg, 0.600 mmol) at room temperature under an argon atmosphere. ) Sequentially added to the solution.

  It was confirmed by MALDI-TOF MS that 4-methyl-3,5-bis [4- (4-pyridylethynyl) phenylethynyl] benzyl bromide was quantitatively formed by stirring the whole volume for 3 hours. (Not isolated.)

  To this solution, trimethylamine (7.0 mL, 30 mmol; 4.3 M aqueous solution) was added, and the whole volume was stirred at room temperature for 15 hours. After the solvent was distilled off under reduced pressure, the resulting residue was washed with ethyl acetate and water and dried in vacuo to give the title compound as a white solid (295 mg, 0.456 mmol). Yield 76%.

¹ H-NMR (500 MHz, CD ₃ CN, 27 ° C.) δ ppm: 8.61 (d, J = 4.8 Hz, 4H), 7.71 (s, 2H), 7.64 (s, 8H), 7 .46 (d, J = 4.6 Hz, 4H), 4.49 (s, 2H), 3.09 (s, 9H), 2.78 (s, 3H)

¹³ C-NMR (125 MHz, CD ₃ CN, 27 ° C.) δ ppm: 151.1 (CH), 145.7 (C), 137.2 (CH), 133.1 (CH), 132.8 (CH) , 131.5 (C), 127.1 (C), 126.4 (CH), 125.3 (C), 124.4 (C), 123.5 (C), 95.1 (C), 93.6 (C), 89.9 (C), 89.5 (C), 68.9 (CH ₂ ), 53.5 (CH ₃ ), 19.7 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3431, 3010, 2218, 1590, 1510, 1487, 1407, 1215, 988, 878, 820, 637, 538

Elemental _{analysis: Calcd for C 41 H 32 N} 3 Br · 1.0H 2 O · 1.0EtOAc: C, 71.80; H, 5.62; N, 5.58
Found: C, 71.94; H, 5.57; N, 5.66

(3) Synthesis of trimethyl {4-methyl-3,5-bis [4- (4-pyridylethynyl) phenylethynyl] benzyl} ammonium nitrate trimethyl {4-methyl-3,5-bis [4- (4- Pyridylethynyl) phenylethynyl] benzyl} ammonium bromide (120 mg, 0.186 mmol), and a mixed solution of silver nitrate (32.6 mg, 0.192 mmol) in acetonitrile (10 mL) and water (60 mL) were added to the whole volume in an argon atmosphere. Under stirring at 40 ° C. for 12 hours. The reaction mixture was filtered and dried in vacuo to give the title compound as a white solid (111 mg, 0.176 mmol). Yield 95%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.60 (d, J = 5.9 Hz, 4H), 7.62 (s, 2H), 7.55-7.51 (m, 8H) ), 7.37 (d, J = 5.6 Hz, 4H), 4.81 (s, 2H), 3.28 (s, 9H), 2.72 (s, 3H)

¹³ C-NMR (125 MHz, CD ₃ CN, 27 ° C.) δ ppm: 149.8 (CH), 145.0 (C), 135.7 (CH), 131.9 (CH), 131.7 (CH) , 131.0 (C), 125.5 (CH), 125.1 (C), 124.9 (C), 123.3 (C), 122.5 (C), 94.9 (C), 93.3 (C), 88.73 (C), 88.68 (C), 68.3 (CH ₂ ), 53.0 (CH ₃ ), 19.3 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3400, 3034, 2217, 1591, 1540, 1509, 1489, 1407, 1360, 1336, 1215, 1103, 988, 880, 819, 636, 539

Elemental _{analysis: Calcd for C 41 H 32 N} 4 O 3 · 1.8H 2 O: C, 74.48; H, 5.43; N, 8.47
Found: C, 74.67; H, 5.29; N, 8.16

Example 6 Synthesis of Ligand 2c (1) Synthesis of 1- (2- {2,6-bis [(4-pyridyl) ethynyl] phenoxy} ethynyl) pyrrole-2,5-dione (4c)

Diisopropyl azodicarboxylate (0.21 mL, 1.2 mmol) was added to 2,6-bis (4-pyridylethynyl) phenol (294 mg, 1.00 mmol) and triphenylphosphine (262 mg, 1.00 mmol) in THF ( 10 mL) solution and the whole volume was stirred for 20 minutes at room temperature under argon atmosphere. N- (2-hydroxyethyl) maleimide (141 mg, 1.00 mmol) was added thereto, and the whole was stirred at room temperature for 12 hours under an argon atmosphere. The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 50: 1 (volume ratio)) to give the title compound as a white solid (310 mg, 0.740 mmol). . Yield 74%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.65 (d, J = 5.6 Hz, 4H), 7.53 (d, J = 7.5 Hz, 2H), 7.46 (d , J = 5.6 Hz, 4H), 7.13 (t, J = 7.6 Hz, 1H), 6.55 (s, 2H), 4.50 (t, J = 6.0 Hz, 2H), 4 .04 (d, J = 6.0 Hz, 2H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 170.4 (C), 160.5 (C), 149.9 (CH), 134.7 (CH), 134.1 (CH), 131.0 (C), 125.6 (CH), 124.1 (CH), 116.8 (C), 91.5 (C), 89.1 (C), 70.0 (CH ₂ ), 37.9 (CH ₂ )

IR (KBr, cm ⁻¹ ) 3462, 3049, 2946, 2215, 1708, 1593, 1539, 1446, 1412, 1324, 1238, 1171, 1019, 990, 969, 817, 695

Elemental _{analysis: Calcd for C 26 H 17 N} 3 O 3: C, 74.45; H, 4.09; N, 10.02
Found: C, 74.20; H, 4.09; N, 9.81

(2) Synthesis of ligand 2c

  Tris (2-carboxyethyl) phosphine was added to a 20 mM Tris buffer solution (20 mL) of mutated ubiquitin (28.4 mg, 3.30 μmol) and stirred at 37 ° C. for 2 hours. Production of monomerized ubiquitin was confirmed by MALDI-TOF MS. The pH was adjusted to 8.0 with 1 M aqueous sodium hydroxide solution, and THF (10 mL) of compound 4c (68.2 mg, 110 μmol) was added. The whole volume was stirred at 37 ° C. for 2 hours and at room temperature for 12 hours.

  The reaction mixture was purified by reverse phase HPLC (on an Inertsil Peptide C18 semi-preparative column (20 mm × 250 mm), eluent: 0.1 wt% trifluoroacetic acid (TFA) aqueous solution and acetonitrile), and then lyophilized. The title compound was obtained as a white powder (24 mg, 2.6 μmol). Yield 80%.

MALDI-TOF-MS calcd for [M + Na] ⁺ 9053.3, found 9054.8

Example 9 Synthesis of Ligand 1c

(1) Synthesis of {4-methyl-3,5-bis [(4-pyridyl) ethynyl] phenyl} methanol Tri-t-butylphosphine (1.84 mL, 0.745 mmol; 10 wt% n-hexane solution, and diisopropyl Amine (9.0 mL, 64 mmol) was added to (3,5-dibromo-4-methylphenyl) methanol (1.34 g, 4.79 mmol), 4-ethynylpyridine hydrochloride (2.18 g, 15.6 mmol), Pd. (PhCN) ₂ Cl ₂ (138 mg, 0.360 mmol) and a solution of copper (I) iodide (45.7 mg, 0.2400 mmol) in 1,4-dioxane (30 mL) were added under an argon atmosphere. The mixture was stirred for 24 hours at 40 ° C. Ethyl acetate (40 mL) was added to the reaction mixture, the insoluble material was filtered off, and water (100 mL) was added to the filtrate. . Additionally, washed with ethylenediamine (3 mL), and extracted with ethyl acetate The organic layer was collected, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure the resulting residue was purified by silica gel column chromatography (CHCl _3:. Methanol = 50: 1 (volume ratio)) to give the title compound as a white solid (1.15 g, 3.54 mmol), yield 74%.

¹ H-NMR (500 MHz, CDCl ₃ , 27 ° C.) δ ppm: 8.63 (d, J = 5.9 Hz, 4H), 7.57 (s, 2H), 7.40 (d, J = 6.0 Hz) , 4H), 4.71 (d, J = 4.9 Hz, 2H), 2.69 (s, 3H), 1.86 (t, J = 5.6 Hz, 1H)

¹³ C-NMR (125 MHz, CDCl ₃ , 27 ° C.) δ ppm: 149.9 (CH), 142.0 (C), 138.8 (C), 131.4 (CH), 131.2 (C), 125.5 (CH), 122.9 (C), 92.1 (C), 91.0 (C), 64.1 (CH ₂ ), 19.0 (CH ₃ )

IR (KBr, cm ⁻¹ ) 3290, 2858, 2208, 1594, 1538, 1490, 1417, 1215, 1113, 1064, 998, 869, 819, 742, 554

(2) Synthesis of trimethyl (4-methyl-3,5-bis [(4-pyridyl) ethynyl] benzyl) ammonium bromide Triphenylphosphine (1.73 g, 6.60 mmol) and carbon tetrabromide (2.98 g) , 9.00 mmol) in a dry THF (200 mL) -like solution of {4-methyl-3,5-bis [(4-pyridyl) ethynyl] phenyl} methanol (930 mg, 3.00 mmol) under argon atmosphere at room temperature. Were added sequentially. By stirring the whole volume for 3 hours, it was confirmed by MALDI-TOF MS that 4-methyl-3,5-bis [(4-pyridyl) ethynyl] benzyl bromide was quantitatively formed (not isolated). ).

  To this solution, trimethylamine (14.0 mL, 60 mmol; 4.3 M aqueous solution) was added and stirred at room temperature for 18 hours. The solvent was removed from the reaction solution under reduced pressure, and the resulting residue was washed with ethyl acetate and water, and then dried in vacuo to give the title compound as a white solid (1.21 g, 2.79 mmol). Yield 97%.

¹ H-NMR (500 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 8.69 (d, J = 6.1 Hz, 4H), 7.89 (s, 2H), 7.59 (d, J = 6 .1 Hz, 4H), 4.58 (s, 2H), 3.09 (s, 9H), 2.74 (s, 3H)

¹³ C-NMR (125 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 150.0 (CH), 144.2 (C), 136.9 (CH), 129.5 (C), 127.0 (C ), 125.2 (CH), 122.6 (C), 91.7 (C), 90.6 (C), 66.3 (CH ₂ ), 51.9 (CH ₃ ), 18.9 ( CH ₃₎

IR (KBr, cm ⁻¹ ) 3412, 3005, 2214, 1593, 1535, 1488, 1405, 1211, 1063, 988, 881, 821, 742, 549

(2) Synthesis of trimethyl (4-methyl-3,5-bis [(4-pyridyl) ethynyl] benzyl) ammonium trifluoromethanesulfonate (1c)

  Trimethyl (4-methyl-3,5-bis [(4-pyridyl) ethynyl] benzyl) ammonium bromide (259 mg, 0.600 mmol), and silver salt of trifluoromethanesulfonic acid (AgOTf) (162 mg, 0.630 mmol) Acetonitrile (60 mL) was stirred at 40 ° C. for 5 hours under an argon atmosphere. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound as a white solid (295 mg, 0.589 mmol). Yield 98%.

¹ H-NMR (500 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 8.69 (d, J = 5.7 Hz, 4H), 7.88 (s, 2H), 7.59 (d, J = 6 .0Hz, 4H), 4.53 (s, 2H), 3.07 (s, 9H), 2.74 (s, 3H)

¹³ C-NMR (125 MHz, DMSO-d ₆ , 27 ° C.) δ ppm: 150.6 (CH), 144.8 (C), 137.5 (CH), 130.1 (C), 127.5 (C ), 125.8 (CH), 123.2 (C), 92.2 (C), 91.2 (C), 67.0 (CH ₂ ), 52.5 (CH ₃ ), 19.4 ( CH ₃₎

IR (KBr, cm ⁻¹ ) 3041, 2155, 1594, 1489, 1408, 1257, 1224, 1160, 1030, 988, 880, 820, 639, 573, 547
(Reference 1)
The obtained bidentate organic ligands 2a, 2b, and 2c were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE is a technique for separating proteins according to molecular weight according to the size of the eyes using a molecular sieve obtained by polymerizing a mixed solution of acrylamide and N, N′-methylenebisacrylamide. The sample was boiled to unfold the higher-order structure of the protein, and SDS (sodium dodecyl sulfate) was added to give the protein a negative charge corresponding to the molecular weight (= amino acid chain length). By applying a voltage to this, the protein moves in the anode direction and can be fractionated by the molecular weight. Finally, the protein was colored blue with Coomassie Brilliant Blue.

  FIG. 1 shows the result of SDS-PAGE performed on the ligand 2c. In FIG. 1, a is a molecular weight marker, b is ubiquitin before reduction, c and d are ubiquitin after reduction by TCEP, and ei is ligand 2c. Ubiquitin before reduction has a large spot near the 14400 marker. Considering that the molecular weight of ubiquitin is about 8600, this is considered to be a dimer of ubiquitin in which thiol groups form an S—S bond. As a result of reducing this with TCEP, the spot of the dimer disappeared and only a spot having a molecular weight of about 6000 was found, so that it was confirmed that all SS bonds were reduced and became monomers. . Further, only one spot was confirmed for 2c bonded to the ligand, and it was confirmed that a ubiquitin-linked ligand was obtained without the presence of decomposition products.

(Reference 2)
Subsequently, in order to determine the solvent used for the complex formation reaction, the influence of the solvent on the folding structure of ubiquitin was examined. Information on the protein folding structure can be read from the width of the chemical shift value in which a signal exists by measurement of ¹ H-NMR. When the folding structure is maintained, the chemical shift value of the amide proton is observed in a wide range because the environment in which each proton exists is different. On the other hand, when the protein is denatured, it is known that the width of the chemical shift observed for the amide protons becomes narrow due to the random coil shape.

The ¹ H-NMR spectrum of ubiquitin is shown in FIG. Ubiquitin is known to take a stable folding structure in _{H 2} O, amide protons in _{H 2} O was observed in the region of 9.5 ppm-7.0 ppm (FIG. 2 (a)). On the other hand, in the spectrum in DMSO, it was observed as a broad peak at 8.5 ppm to 7.0 ppm, and it can be said that the region was narrower than in the case of ubiquitin in water (FIG. 2B). When ubiquitin was dissolved in a mixed solvent of water and acetonitrile (H ₂ O / CD ₃ CN = 1: 1), the amide proton was observed at 9.5 ppm to 7.0 ppm, and was observed in a region of almost the same width as water. (FIG. 2 (c)).

  From the above results, it was confirmed that ubiquitin denatured in DMSO, but almost maintained a folding structure in a mixed solvent of water and acetonitrile. Therefore, it was found that it is preferable to use a mixed solvent of water-acetonitrile for the formation of the spherical complex in order to encapsulate ubiquitin while maintaining the folding structure.

(Example 10) Production of spherical transition metal complex (3a)

Ligand (1a), ubiquitin-linked ligand (2a), and Pd (NO ₃ ) ₂ are mixed at a ratio of 23: 1: 12, and D ₂ O: CD ₃ CN = 1: 1 (volume ratio) The spherical complex (3a) was produced by gently stirring in a mixed solvent at 45 ° C. for 3 hours.

The DOSY NMR for these reaction solutions was measured. FIG. 3 shows the DOSY spectrum of the ubiquitin-linked ligand (2a) and the spectrum after complex formation.
From the spectrum after complex formation, the diffusion coefficient of the ligand skeleton is observed at log D = -10.12, and is substantially equal to the diffusion coefficient of the spherical complex formed only from (1a) (log D = -10.10). The formation of a spherical complex with a diameter of 6.3 nm was confirmed.

  On the other hand, the diffusion coefficient of the ubiquitin-linked ligand (2a) before complex formation was observed at log D = -10.08. From the spectrum after complex formation, it was found that the diffusion coefficient of ubiquitin was logD = -10.12, and the ubiquitin motion was slightly slower than that before complex formation. It should be noted that the diffusion coefficient of ubiquitin is almost equal to the diffusion coefficient of the spherical complex, and it can be seen that it moves at the same speed as the spherical complex.

(Example 11) Production of spherical transition metal complex (3b)

  In the same manner as in Example 10, a spherical transition metal complex (3b) was produced, and DOSY NMR of the reaction solution was measured. FIG. 4 shows the DOSY spectrum of the ubiquitin-linked ligand (2b) and the spectrum after complex formation.

  After the complex formation, the diffusion coefficient of the signal derived from the coordination skeleton was observed at -10.26, confirming the formation of a spherical complex having a diameter of 7.3 nm. For the ubiquitin-linked ligand (2b), the diffusion coefficient before complex formation was observed at log D = -10.08.

  On the other hand, by forming a spherical complex, it can be seen that the diffusion coefficient of ubiquitin is greatly changed to log D = -10.26, and the movement of ubiquitin is slow. Also here, the diffusion coefficient of ubiquitin after the formation of the spherical complex is observed at almost the same value as the diffusion coefficient of the spherical complex, and it can be seen that it moves at the same speed as the spherical complex.

  From the above results, it was strongly suggested that ubiquitin moves in the same manner as a spherical complex having diameters of 6.3 nm and 7.3 nm, respectively. In addition, the diffusion coefficient of the formed spherical complex is almost the same as the diffusion coefficient of the spherical complex into which ubiquitin is not introduced. Therefore, ubiquitin is not present outside the spherical complex, but is included in the spherical complex. It is thought that there is.

  A molecular model of a spherical complex encapsulating ubiquitin is shown in FIG. 5 (in the figure, the cation portion on the outer surface and the methyl group on the inner surface are omitted). Ubiquitin is encapsulated in spherical complexes of 6.3 nm and 7.3 nm, respectively, and it can be seen that both provide a sufficiently wide space for the size of ubiquitin.

Also in the following experiment by DOSY NMR, the result of supporting the inclusion of ubiquitin in a spherical complex was obtained.
First, a spherical complex having a diameter of 7.3 nm was formed from the ligand (1a). To this complex, ubiquitin not linked with a ligand was added and stirred at room temperature, and then DOSY NMR was measured. As a result, the diffusion coefficient of the added ubiquitin was observed in a larger region than that of the spherical complex, and it was found that ubiquitin was smaller in size and faster in diffusing motion than the spherical complex. That is, when ubiquitin and a spherical complex coexist in a solution, it was shown that these diffusion coefficients are observed separately. This supports that the fact that the spherical complex and ubiquitin were observed with the same diffusion coefficient in the previous experiment can be interpreted as ubiquitin being encapsulated in the spherical complex.
(Reference Example 3)

Ligand (1c), ligand (2c) and Pd (OTf) ₂ were mixed at a ratio of 23: 1: 12, and in a solvent of D ₂ O: CD ₃ CN = 2: 1 (volume ratio). The mixture was stirred at 45 ° C. for 3 hours to form a spherical complex having a diameter of 4.5 nm.

The obtained reaction solution was subjected to ¹ H-NMR and DOSY NMR measurements. ^{In 1} H-NMR, a signal converged with a diffusion coefficient of log D = -10.15 was observed in the region of 8-10 ppm, whereby formation of an M ₁₂ L ₂₄ spherical complex could be confirmed. On the other hand, since the diffusion coefficient of ubiquitin was observed at log D = -10.20, it can be seen that the movement of ubiquitin is slower than that of the spherical complex. That is, it was suggested that ubiquitin moves separately from the spherical complex and is not encapsulated in the spherical complex. The reason why the inclusion in the spherical complex did not occur is considered to be because there was no wide space for enclosing ubiquitin in the spherical complex having a diameter of 4.5 nm.

  From these results, it is not possible to encapsulate ubiquitin in a complex of 4.5 nm whose internal space is not sufficient, and it is possible to encapsulate ubiquitin in spherical complexes of 7.3 nm and 6.3 nm in diameter. I knew it was possible.

It is a figure which shows the result of having performed SDS-PAGE about the ligand 2c. ^{1 is} a ¹ H-NMR spectrum of ubiquitin. (A) is a figure measured in H ₂ O, (b) is a figure measured in DMSO, (c) is a mixed solvent of water and acetonitrile (H ₂ O / CD ₃ CN = 1: It is the figure measured in 1). It is a figure which shows the DOSY spectrum of a ubiquitin connection ligand (2a), and the spectrum after complex formation. It is a figure which shows the DOSY spectrum of a ubiquitin connection ligand (2b), and the spectrum after complex formation. It is a figure which shows the molecular model of the spherical complex which included ubiquitin. (A) is a case where ubiquitin is encapsulated in a spherical complex having a hollow size of 6.3 nm, and (b) is a case where ubiquitin is encapsulated in a spherical complex having a hollow size of 7.3 nm. Each is shown.

Claims (6)

Have a hollow shell, the transition metal compound (M), the bidentate organic ligand proteins through a linking group formed by bonding (L1), and from a bidentate organic ligand (L2), said protein Is formed in a self-organized manner so that the inside of the hollow shell is oriented: M _n L2 _(2n−1) L1 (n represents an integer of 6 to 60, and a plurality of Ms, L2s are be the same, respectively, a spherical transition metal complex is Ru represented by may be different from each other.)
The bidentate organic ligand (L1) has the formula (I)

{Wherein X represents an ethynylene group or a p-phenylene group,
t represents an integer of 2 to 6, and a plurality of Xs may be the same or different.
R ¹ and R ² each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m1 and m2 each independently represents an integer of 0 to 4. When m1 and m2 are 2 or more, a plurality of R ^{1 s} and R ^{2 s} may be the same or different.
A ₁ represents the following formulas (a-1) to (a-4):

[Wherein R ³ represents a formula: —YD-Pro (wherein Y represents a single bond or —O— (CH ₂ ) _a — (a represents an integer of 1 to 8. _same), Te _{- O- (CH 2) a -C} (= O) -, - C (= O) -, - O- (CH 2) a -NH -, - NH-, and the following formula

(Wherein, m9 represents an integer of 1 to 10) represents a divalent linking group selected from the group consisting of groups represented by: D represents O, S, NH, or O— (C═O D-Pro represents a portion excluding H of the protein represented by Pro-DH. ) Represents a group represented by
R ⁴ may have a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, or a substituent. An amino group, an aminoalkyl group which may have a substituent, or a quaternized aminoalkyl group is represented.
m3 represents an integer of 0 to 3, and m4 represents an integer of 0 to 2. When m3 is 2 or more, or when m4 is 2, even a plurality of R ⁴ with each other are the same, may be different from each other.
Q is, -Nr1- (r1 represents a hydrogen atom, an alkyl group, an aryl group or an acyl group.), - O -, - C (= O) -, - S-, or -SO ₂ - represents a. ] The kind of group represented by this is shown. } Is a compound represented by
The bidentate organic ligand (L2) has the formula (II)

{Wherein X and t represent the same meaning as described above.
R ⁵ and R ⁶ each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m5 and m6 each independently represents an integer of 0 to 4. m5, when m6 is 2 or more, plural R ⁵ together, even R ⁶ together are each identical or may be different phases.
A ₂ represents the following formulas (a-5) to (a-8)

(In the formula, Q represents the same meaning as described above.
R ⁷ has a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, a carboxyalkyl group, and a substituent. An amino group that may be substituted, an aminoalkyl group that may have a substituent, or a quaternized aminoalkyl group.
R ⁸ represents a hydrogen atom, a halogen atom, an alkyl group, or a haloalkyl group.
m7 represents an integer of 0 to 3, and m8 represents an integer of 0 to 2. When m7 is 2 or more, or when m8 is 2, a plurality of R ⁷ may be the same or different. ). } The protein inclusion globular transition metal complex characterized by the above-mentioned.   The protein-encapsulated globular transition metal complex according to claim 1, wherein n is 6, 12, 24, 30 or 60. The transition metal atom constituting the transition metal complex is a kind selected from the group consisting of Ti, Fe, Co, Ni, Cu, Zn, Ru, Rh, Pd, Cd, Os, Ir, and Pt. The protein-encapsulated globular transition metal complex according to claim 1 or 2 . Transition metal compound (M), bidentate organic ligand (L1) represented by formula (I) and bidentate organic ligand (L2) represented by formula (II) (M) A ratio of 1 mol of bidentate organic ligand (L1) and (2n-1) mol of bidentate organic ligand (L2) with respect to n mol (n represents an integer of 6 to 60). The method for producing a protein-encapsulated globular transition metal complex according to any one of claims 1 to 3 , wherein Formula (I)

{Wherein X represents an ethynylene group or a p-phenylene group,
n1 represents an integer of 1 to 6, and when n1 is 2 or more, a plurality of Xs may be the same or different.
R ¹ and R ² each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m1 and m2 each independently represents an integer of 0 to 4. When m1 and m2 are 2 or more, a plurality of R ^{1 s} and R ^{2 s} may be the same or different.
A ₁ represents the following formulas (a-1) to (a-4):

[Wherein R ³ represents a formula: —YD-Pro (wherein Y represents a single bond or —O— (CH ₂ ) _a — (a represents an integer of 1 to 8. _same), Te _{- O- (CH 2) a -C} (= O) -, - C (= O) -, - O- (CH 2) a -NH -, - NH-, and the following formula

(Wherein, m9 represents an integer of 1 to 10) represents a divalent linking group selected from the group consisting of groups represented by: D is O, S, NH, or O— (C═O D-Pro represents a portion excluding H of the protein represented by Pro-DH. ) Represents a group represented by
R ⁴ may have a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group, a nitro group, a hydroxyl group, a hydroxyalkyl group, a carboxyl group, or a substituent. An amino group, an aminoalkyl group which may have a substituent, or a quaternized aminoalkyl group is represented.
m3 represents an integer of 0 to 3, and m4 represents an integer of 0 to 2. When m3 is 2 or more, or when m4 is 2, a plurality of R4s may be the same or different.
Q is, -Nr1- (r1 represents a hydrogen atom, an alkyl group, an aryl group or an acyl group.), - O -, - C (= O) -, - S-, or -SO ₂ - represents a. ] The kind of group represented by this is shown. } The compound shown. Formula (II ' )

{Wherein X represents an ethynylene group or a p-phenylene group,
n1 represents an integer of 1 to 6, and when n1 is 2 or more, a plurality of Xs may be the same or different.
R ⁵ and R ⁶ each independently represents a halogen atom, an optionally substituted alkyl group, an optionally substituted alkoxyl group, a cyano group or a nitro group.
m5 and m6 each independently represents an integer of 0 to 4. m5, when m6 is 2 or more, plural R ⁵ together, even R ⁶ together are each identical or may be different phases.
A ₂ represents the following formulas (a-5) to (a-8)

(In the formula, Q represents the same meaning as described above.
R ⁷ represents a quaternized aminoalkyl group.
R ⁸ represents a hydrogen atom, a halogen atom, an alkyl group, or a haloalkyl group.
m10 represents an integer of 1 to 3, and m11 represents an integer of 1 to 2. When m10 is 2 or more, or when m11 is 2, a plurality of R ⁷ may be the same or different. ). } The compound shown.

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