JP5649081B2 - Complex of multiple metal nanoparticles and apoprotein derived from hemoprotein dimer - Google Patents

Description

  The present invention relates to a complex of a plurality of metal nanoparticles and an apoprotein derived from a hemoprotein dimer.

  Metal nanoparticles are attracting attention as wiring materials, dyes, and sensors. This complex of metal nanoparticles and protein can be widely applied to sensing, imaging, protein activity control, etc., and is attracting attention as a new biomaterial. It has been reported that a complex of a metal nanoparticle and a protein is produced from the metal nanoparticle and a protein by electrostatic interaction (for example, Non-Patent Document 1).

  However, a complex in which the distance between metal nanoparticles is kept specific via a hemoprotein has not been known.

Author: Takashi Hayashi et al., Title: "Creation of gold nanoparticle-heme protein polymer complex", Journal name: 24th Symposium on Biological Functions and 12th Symposium on Biotechnology, 2009, p. 202

  Therefore, an object of the present invention is to provide a complex of a metal nanoparticle and a protein in which the distance between the metal nanoparticles is kept specific via a heme protein.

The present invention is a complex of a plurality of metal nanoparticles and an apoprotein derived from a hemoprotein dimer,
The metal nanoparticles are modified at least one place by a group represented by formula (I) and / or formula (II),
By binding the heme contained in the group represented by the formula (I) and / or the formula (II) modified with the metal nanoparticles to the apoprotein derived from the heme protein dimer from which heme has been removed. A complex in which a plurality of the metal nanoparticles and the protein are bound.

In the formula (I) and the formula (II),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 means a group bonded to -CO- in formula (I) and formula (II). And
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 _{- (CH 2) n1 -,} - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2 _{) n1 -O- (CH 2) n2} -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 -, Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
The heme protein dimer is a mutant in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of a natural heme protein.

  The complex of the present invention is a complex of a metal nanoparticle and a protein in which the distance between the metal nanoparticles is kept specific via a heme protein.

FIG. 1 is a UV-vis spectrum of gold nanoparticles modified with citric acid prepared in Example 1 (2). FIG. 2 is a transmission electron micrograph of gold nanoparticles modified with citric acid produced in Example 1 (2). FIG. 3 is a UV-vis spectrum of gold nanoparticles modified with Compound 1 produced in Example 1 (2). FIG. 4 is a transmission electron micrograph of gold nanoparticles modified with Compound 1 produced in Example 1 (2). FIG. 5 is a UV-vis spectrum for quantifying Compound 1 contained in the modified gold nanoparticles produced in Example 1 (2). FIG. 6 is a size exclusion chromatography chart of a dimer of cytochrome b ₅₆₂ mutant produced in Example 1 (3). FIG. 7 is a UV-vis spectrum of an apoprotein obtained by removing heme from a dimer of the cytochrome b ₅₆₂ mutant produced in Example 1 (3). FIG. 8 shows the agarose gel electricity of the composite obtained in Example 1 (4), the composite obtained in Comparative Example 1, and the gold nanoparticles modified with Compound 1 produced in Example 1 (2). Migration. FIG. 9A is a TEM photograph of the composite obtained in Example 1 (4). FIG. 9B is a TEM photograph of the composite obtained in Example 1 (4). FIG. 9 (c) is a TEM photograph of the composite obtained in Example 1 (4). FIG.9 (d) is the TEM photograph of the composite_body | complex obtained in Example 1 (4). 10A is a TEM photograph of the composite obtained in Comparative Example 1. FIG. FIG. 10B is a TEM photograph of the composite obtained in Comparative Example 1. FIG. 10C is a TEM photograph of the composite obtained in Comparative Example 1. FIG. 11 is an SEM photograph of the composite obtained in Example 1 (4). FIG. 12 is an SEM photograph of the composite obtained in Comparative Example 1. FIG. 13 shows the complex obtained in Example 2 (3), the complex obtained in Comparative Example 1, the gold nanoparticles modified with Compound 1 produced in Example 1 (2), and lipoic acid. Agarose gel electrophoresis of modified gold nanoparticles. FIG. 14 is a TEM photograph of the composite obtained in Example 2 (3). FIG. 15A shows the measurement results of dynamic light scattering (DLS) of gold nanoparticles modified with lipoic acid. FIG. 15B shows the measurement result of dynamic light scattering (DLS) of the composite obtained in Comparative Example 1. FIG. 15C shows the measurement result of dynamic light scattering (DLS) of the composite obtained in Example 2 (3). FIG. 16 is a UV-vis spectrum of cytochrome b ₅₆₂ polymer (III-3). FIG. 17 is a UV-vis spectrum of silver nanoparticles modified with citric acid prepared in Example 3 (1). FIG. 18 is a transmission electron micrograph of silver nanoparticles modified with citric acid produced in Example 3 (1). FIG. 19 is a transmission electron micrograph of silver nanoparticles modified with Compound 1 produced in Example 3 (1). FIG. 20 is a UV-vis spectrum for confirming the presence of Compound 1 contained in the modified silver nanoparticles produced in Example 3 (1). FIG. 21 (a) is a TEM photograph of the composite obtained in Example 3 (2). FIG. 21 (b) is a TEM photograph of the composite obtained in Example 3 (2). FIG. 22 is a TEM photograph of the composite obtained in Comparative Example 2.

  The present invention, as described above, is a complex of a plurality of metal nanoparticles and an apoprotein derived from a heme protein dimer, wherein the metal nanoparticles are represented by formula (I) and / or formula (II): Heme contained in the group represented by formula (I) and / or formula (II) modified with one or more sites by the group represented and modified with the metal nanoparticles, and the amount of heme protein from which heme has been removed It is a complex in which a plurality of the metal nanoparticles and the protein are bound by binding to apoprotein derived from the body.

  In the present invention, the hemoprotein dimer is preferably a dimer of cytochrome, hemoglobin, myoglobin, or peroxidase.

  In the present invention, the metal nanoparticles include gold nanoparticles, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, cadmium sulfide (CdS) nanoparticles, cadmium selenide (CdSe) nanoparticles, cadmium telluride (CdTe). ) Nanoparticles, zinc sulfide (ZnS) nanoparticles, zinc selenide (ZnSe) nanoparticles, or zinc telluride (ZnTe) nanoparticles or cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe) ), Zinc sulfide (ZnS), zinc selenide (ZnSe), and zinc telluride (ZnTe), core / shell type nanoparticles composed of two types selected from the group consisting of zinc telluride (ZnTe) are preferable.

The present invention also provides a method for producing the composite of the present invention,
A group represented by formula (I) and / or formula (II) is obtained by reacting a plurality of metal nanoparticles with a disulfide represented by formula (I ′) and / or formula (II ′). To modify one or more places
A plurality of the modified metal nanoparticles are reacted with apoprotein derived from a heme protein dimer from which heme has been removed, and the metal nanoparticles are modified by the formula (I) and / or the formula (II). When the heme contained in the group to be bonded to the apoprotein derived from the heme protein dimer from which heme has been removed, a complex in which a plurality of the metal nanoparticles and the protein are bound is obtained.

  The group represented by the formula (I) and / or the formula (II) may modify the metal nanoparticles at one or more positions. When modifying metal nanoparticles at two or more locations with the group, all types are groups represented by formula (I), all types are groups represented by formula (II), or formula (I) It may be a mixture of the group represented by the group represented by the formula (II).

  In the present invention, “lower” means 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, unless otherwise specified.

  As the “lower alkyl group”, the residue of a linear or branched alkane having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, neopentyl, hexyl, etc. Means group. Preferable examples of the lower alkyl group include alkyl having 1 to 5 carbon atoms. Preferable alkyl having 1 to 5 carbon atoms includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, neopentyl and the like.

  As the “lower alkenyl group”, vinyl, allyl, isopropenyl, 1-, 2- or 3-butenyl, 1-, 2-, 3- or 4-pentenyl, 1-, 2-, 3-, 4- or Examples thereof include straight-chain or branched alkenyl groups having 2 to 6 carbon atoms such as 5-hexenyl. Preferred lower alkenyl groups include vinyl.

  As the complex, in the formulas (I) and (II), when Y is represented by -Z1-Y1-Z2-Y2-, Y is, for example, No. represented by the following table. 1-No. There are 27 formulas.

  When Y is represented by -Z1-Y1-Z2-Y2-, no. Groups represented by the formulas 1, 2, 3, 10, 11, 12, 19, 20, 21 are preferred. Groups represented by the formulas 1, 2, and 3 are more preferable.

  As the complex, in the formulas (I) and (II), when Y is represented by -Z1-Y1-, Y is, for example, No. represented by the following table. 30-No. There are 35 formulas.

  When Y is represented by -Z1-Y1-, groups represented by the formulas of Nos. 30 and 31 in Table 2 are preferred. The group represented by the formula of 30 is more preferable.

Moreover, as said composite_body | complex, in said Formula (I) and Formula (II), R < ¹ >, R < ² >, R < ³ > and R < ⁴ > respectively independently mean a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 means a group bonded to -CO- in formula (I) and formula (II). And
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (CH 2) n1 -O- (CH 2) n2 -O- (CH 2) n3 - or _{- (CH} 2) n1 -O- ( _{CH 2) n2 -O- (CH 2} ) n3 -O- (CH 2) n4 - a group represented by where n1, n2, n3, and n4 are integers from 1 to 20 independently Means
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
The hemoprotein dimer is preferably a mutant in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of a natural hemoprotein.

  The complex in the present invention may take the form of a solvate, which is also included in the scope of the present invention. Preferred solvates include hydrates, ethanol solvates, dimethyl sulfide solvates, dimethylformamide solvates and pyridine solvates.

The “apoprotein derived from a heme protein dimer” is a mutant in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of a natural heme protein. The natural heme protein is not particularly limited as long as it is a protein having one or more heme, and examples thereof include cytochrome, hemoglobin, myoglobin, and peroxidase. Examples of the cytochrome include cytochrome b ₅₆₂ , cytochrome b ₅ and cytochrome P450 _CAM , and among them, cytochrome b ₅₆₂ is preferable. Examples of the hemoglobin include hemoglobin (human), hemoglobin (bovine), hemoglobin (equine), and hemoglobin (rat). Among them, hemoglobin (human) is preferable. Examples of the myoglobin include myoglobin (sus scrofa), myoglobin (equine), myoglobin (human), and myoglobin (sperm whale). Among them, myoglobin (sperm whale) and myoglobin (sus scrofa) are preferable. Examples of the peroxidase include horseradish peroxidase, chloroperoxidase, and catalase. Among them, horseradish peroxidase is preferable.

In the present invention, a mutant in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of the natural heme protein is, for example, cytochrome (a) one of the amino acid sequences represented by SEQ ID NO: 1 A protein comprising an amino acid sequence in which an amino acid is substituted with cysteine; (b) one or two amino acids in the amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, A protein consisting of an added amino acid sequence and functioning as cytochrome. The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of natural cytochrome b ₅₆₂ . The amino acid sequence of natural cytochrome b ₅₆₂ is not limited to the amino acid sequence represented by SEQ ID NO: 1, and an appropriate one may be selected in the Protein Data Bank. Specific examples include ID number 1QPU (June 2, 1999), and the amino acid sequence represented by SEQ ID NO: 1 corresponds to that of ID number 1QPU. The one amino acid residue that is replaced by cysteine is preferably an amino acid residue that is located outside the structure when the cytochrome takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of the heme contained in the cytochrome. In order to perform such substitution, for example, in the amino acid sequence of SEQ ID NO: 1, the first to 106th amino acid residues, preferably 60, 62, 63, 66, 67, 70, 73, 74, 76, 78, The 89th, 90th, 96th, 99th and 100th amino acid residues, more preferably the 60th, 63rd, 66th, 67th and 100th amino acid residues can be replaced with cysteine residues.

  In addition, for example, in the case of hemoglobin, such a hemoprotein variant is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO: 10 is substituted with cysteine, ( b) an amino acid sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 9 or 10 is substituted with cysteine, and hemoglobin A protein that functions as (human). The amino acid sequence represented by SEQ ID NO: 9 is the amino acid sequence of the natural hemoglobin α subunit, and the amino acid sequence represented by SEQ ID NO: 10 is the amino acid sequence of the β subunit of natural hemoglobin. The amino acid sequence of natural hemoglobin is not limited to the amino acid sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10, and an appropriate one may be selected in the Protein Data Bank. Specific examples include those with ID number 1GZX (July 8, 2002), and the amino acid sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10 correspond to those with ID number 1GZX. The one amino acid residue that can be replaced with cysteine is preferably an amino acid residue that is located outside the structure when the hemoglobin takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of the heme contained in the hemoglobin.

  For example, in the case of myoglobin, such a variant of heme protein is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 2 is substituted with cysteine, and (b) SEQ ID NO: It means an amino acid sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence in which one amino acid of the amino acid sequence represented by 2 is substituted with cysteine, and which functions as myoglobin. The amino acid sequence represented by SEQ ID NO: 2 is an amino acid sequence of wild-type myoglobin (sperm whale). The amino acid sequence represented by SEQ ID NO: 2 further includes Met as an amino acid residue before the first amino acid residue Val of the natural amino acid sequence (the amino acid sequence of the protein actually extracted from whale). . Furthermore, aspartic acid, which is the 122nd amino acid residue in the natural amino acid sequence, corresponds to the 123rd amino acid sequence represented by SEQ ID NO: 2, but the amino acid residue is replaced by asparagine. Yes. The amino acid sequence of wild-type myoglobin is not limited to the amino acid sequence represented by SEQ ID NO: 2, and an appropriate one may be selected in the Protein Data Bank. Specific examples include those with ID number 2 MBW (June 20, 1996), and the amino acid sequence represented by SEQ ID NO: 2 corresponds to that with ID number 2 MBW. The one amino acid residue that can be replaced by cysteine is preferably an amino acid residue that is located outside the structure when the myoglobin takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of the heme contained in the myoglobin. In order to perform such substitution, for example, in the amino acid sequence of SEQ ID NO: 2, the first to 154th amino acid residues, preferably the ninth, 13, 17, 54, 103, 107, 114, 118, 122, 126, The 127th, 134th, 141st and 148th amino acid residues, more preferably the 126th and 134th amino acid residues can be replaced with cysteine residues.

  In addition, for example, in the case of horseradish peroxidase, such a variant of heme protein is (a) a protein comprising an amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 3 is substituted with cysteine, (b) A protein comprising an amino acid sequence in which one or two amino acids are deleted, substituted or added in the amino acid sequence in which one amino acid of the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, and having horseradish peroxidase activity Means. The amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of natural horseradish peroxidase. The amino acid sequence of natural horseradish peroxidase is not limited to that represented by SEQ ID NO: 3, and an appropriate one may be selected in Protein Data Bank. Specific examples include those with ID number 1ATJ (February 4, 1998), and the amino acid sequence represented by SEQ ID NO: 3 corresponds to that with ID number 1ATJ. The one amino acid residue to be replaced with cysteine is preferably an amino acid residue that is located outside the structure when the horseradish peroxidase takes a three-dimensional structure. Moreover, it is necessary that the replacement with a cysteine residue does not affect the structure of heme contained in the horseradish peroxidase.

  As the complex, as described above, the hemoprotein dimer is preferably a cytochrome, hemoglobin, myoglobin, or peroxidase dimer, and is a cytochrome having the amino acid sequence represented by SEQ ID NO: 1. More preferably, the one amino acid residue is the 63rd histidine or myoglobin having the amino acid sequence represented by SEQ ID NO: 2, and the one amino acid residue is the 126th alanine. .

  As said metal nanoparticle, a commercially available thing may be used, and it may manufacture and obtain with reference to literature. Examples of such documents include G. Frens, Nature (London) Phys. Sci. 1973, 241, 20.

  In the composite, the metal nanoparticles include gold nanoparticles, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, cadmium sulfide (CdS) nanoparticles, cadmium selenide (CdSe) nanoparticles, cadmium telluride (CdTe). ) Nanoparticles, zinc sulfide (ZnS) nanoparticles, zinc selenide (ZnSe) nanoparticles, or zinc telluride (ZnTe) nanoparticles or cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe) ), Zinc sulfide (ZnS), zinc selenide (ZnSe), and zinc telluride (ZnTe), core / shell type nanoparticles composed of two types selected from the group consisting of zinc telluride (ZnTe) are preferable.

  The particle size of the metal nanoparticles is, for example, 1 nm to 2500 nm, preferably 1 nm to 100 nm, more preferably 1 nm to 20 nm.

In the complex (R), R ¹ , R ² , R ³ and R ⁴ in the formula (I) and / or formula (II) each independently represents a lower alkyl group or a lower alkenyl group. , Y is a group represented by the formula —NH— (CH ₂ ) _n1 —NH—CO— (CH ₂ ) _n2 —, wherein n1 and n2 each independently represents an integer of 1 to 20 , -NH- means a group that binds to -CO- in formula (I) and / or formula (II), the hemoprotein dimer is a dimer of cytochrome or myoglobin, and the metal nano More preferably, the particles are gold nanoparticles or silver nanoparticles.

In the complex (R), R ¹ , R ² , R ³ and R ⁴ in the formula (I) and / or formula (II) each independently represents a lower alkyl group or a lower alkenyl group. , Y is a group represented by the formula —NH— (CH ₂ ) _n1 —NH—CO— (CH ₂ ) _n2 —, wherein n1 and n2 each independently represents an integer of 1 to 20 , -NH- means a group that binds to -CO- in formula (I) and / or formula (II), and the heme protein dimer is cytochrome having the amino acid sequence represented by SEQ ID NO: 1. A dimer in which the one amino acid residue is the 63rd histidine or myoglobin having the amino acid sequence represented by SEQ ID NO: 2 and the one amino acid residue is the 126th alanine so Ri, wherein the metal nanoparticles, and even more preferably gold nanoparticles or silver nanoparticles.

  Next, an example of the manufacturing method of the composite_body | complex of this invention is demonstrated.

  As described above, the composite of the present invention is obtained by reacting (a) a plurality of metal nanoparticles with a disulfide represented by the formula (I ′) and / or the formula (II ′) to form metal nanoparticles. (A) apoprotein derived from a hemoprotein dimer that has been modified with one or more groups represented by (I) and / or formula (II), and (b) a plurality of the modified metal nanoparticles and heme removed And the heme contained in the group represented by the formula (I) and / or the formula (II) modified with the metal nanoparticles, and the apoprotein derived from the heme protein dimer from which heme has been removed, By binding, a complex in which a plurality of the metal nanoparticles and the protein are bound can be obtained.

  (A) reacting a plurality of metal nanoparticles with a disulfide compound represented by formula (I ′) and / or formula (II ′) to form metal nanoparticles according to formula (I) and / or formula (II) The process of modifying one or more places with the group represented can be performed as follows, for example.

  First, a solution of a plurality of metal nanoparticles, for example, a metal nanoparticle protected with citric acid, is made alkaline, and an excessive amount of a disulfide compound represented by formula (I ′) and / or formula (II ′) is added thereto. Mix and incubate with excess amount of lipoic acid. Thereafter, the unreacted disulfide compound represented by the formula (I ′) and / or the formula (II ′) and the unreacted lipoic acid are removed, and the metal nanoparticles are converted into the formula (I) and / or the formula ( It can be modified at one or more positions by the group represented by II). It is known that when metal nanoparticles and disulfide compounds (disulfide and lipoic acid) are mixed, the disulfide bond is cleaved, and the metal nanoparticles and lipoic acid are bonded via the “-S-” bond. Based on being.

  In the step (a), the reaction between the metal nanoparticles and the disulfide represented by the formula (I ′) and / or the formula (II ′) is carried out in a solvent under an inert gas (eg, argon, nitrogen, etc.) atmosphere. Can be done.

  In the step (a), the reaction solvent is not limited. For example, polar solvents such as alcohols (eg, methanol, ethanol, etc.), water, dimethyl sulfoxide, dimethylformamide, etc., buffer solutions (eg, Tris-HCl buffer solution, phosphorus Acid buffer, borate buffer, carbonate buffer) and the like.

  In the step (a), the reaction temperature is not limited, and is, for example, 0 ° C to 100 ° C, preferably 4 ° C to 40 ° C, more preferably 4 ° C to 10 ° C. In the step (a), the reaction time is not limited, but is, for example, 1 minute to 60 hours, preferably 1 hour to 12 hours, more preferably 6 hours to 10 hours.

  In the step (a), when an excessive amount of the disulfide compound represented by the formula (I ′) and / or the formula (II ′) is mixed with an excessive amount of lipoic acid, the metal nanoparticle is used instead of the lipoic acid. Other protective agents that can modify the particles may be used. Examples of such a protective agent include alkanethiol and mercaptopropionic acid.

  The disulfide compound represented by the formula (I ′) and / or the formula (II ′) used in the step (a) can be produced, for example, as follows. For simplification, only an example of the disulfide compound represented by the formula (I ′) is shown, but the disulfide compound represented by the formula (II ′) which is a positional isomer can be produced in the same manner.

In the above formula,
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 means a group bonded to -CO- in formula (I) and formula (II). And
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 _{- (CH 2) n1 -,} - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2 _{) n1 -O- (CH 2) n2} -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 -, Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
-O (P) is a protecting group for a carboxyl group.

  The heme body represented by the formula (III) and the disulfide body (IV) are coupled to obtain a compound represented by the formula (V). From this compound of formula (V), O (P) can be deprotected, and then M can be introduced into heme to obtain the disulfide represented by formula (I ').

  The mutant gene in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of a natural heme protein is based on the base sequence of the gene of the natural heme protein, using guinea pig total RNA or the like. Cloning may be performed, or DNA may be chemically synthesized using the phosphoramidite method. The cloning method is not particularly limited, and can be performed using, for example, a commercially available cloning kit. The mutant gene may be introduced into a host cell.

  Examples of the host cell include animal cells, plant cells, insect cells, yeasts, bacteria, and the like. Examples of the gene introduction method include a lithium acetate method, a calcium phosphate method, a method using a liposome, electroporation, a method using a viral vector, a micropipette injection method, and the like. Alternatively, it may be introduced using an integration type into a host chromosome or an artificial chromosome or plasmid type capable of autonomous replication / distribution.

  The gene to be introduced into the host cell is preferably operably linked to necessary regulatory sequences so that it is constitutively or arbitrarily expressed in the host cell. The regulatory sequence is a base sequence necessary for expression of the gene operably linked in a host cell. For example, examples of regulatory sequences suitable for eukaryotic cells include promoters, polyadenylation signals, enhancers. Etc. Said operatively connected means that the components are juxtaposed so that they can perform their functions.

  When the complex of the present invention was observed with a transmission electron microscope (TEM), it was spread two-dimensionally (see FIG. 9D). From observation by this TEM, it can be estimated that the composite of the present invention has, for example, the following network structure.

Since the composite of the present invention has a two-dimensionally expanded structure as described above, for example, when it is placed on a substrate, it may be thinly and two-dimensionally expanded. When protein is removed by baking, plasma irradiation, washing with acidic aqueous solution, etc. on the substrate, gold nanoparticles, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, cadmium sulfide (CdS) nanoparticles, selenization Cadmium (CdSe) nanoparticles, cadmium telluride (CdTe) nanoparticles, zinc sulfide (ZnS) nanoparticles, zinc selenide (ZnSe) nanoparticles, or zinc telluride (ZnTe) nanoparticles or cadmium sulfide (CdS), Core / shell type composed of two types selected from the group consisting of cadmium selenide (CdSe), cadmium telluride (CdTe), zinc sulfide (ZnS), zinc selenide (ZnSe) and zinc telluride (ZnTe) Only metal nanoparticles, such as the nanoparticles, remain on the substrate. If it does so, it is possible to obtain the board | substrate with which the metal nanoparticle arrange | positioned at intervals is arrange | positioned. Such a substrate has a large metal surface area and can save the amount of metal when used as a catalyst.
In addition, the composite on the substrate is expected to have conductivity, and application to molecular diodes, molecular circuits and the like is also expected.

  Furthermore, since the composite on the substrate has metal nanoparticles arranged at intervals (preferably at equal intervals), cadmium sulfide (CdS) nanoparticles, zinc sulfide (ZnS) nanoparticles, etc. are used as metal nanoparticles. When is used, application to semiconductor ultrafine particles (quantum dots) is also expected.

  The present invention may also be a complex of metal nanoparticles and heme protein-derived apoprotein. Specifically, in the complex of the metal nanoparticle and the heme protein-derived apoprotein, the metal nanoparticle is modified at least one place by a group represented by formula (I) and / or formula (II), The metal nanoparticle is formed by binding the heme contained in the group represented by the formula (I) and / or the formula (II) modified with the metal nanoparticle and the apoprotein derived from the heme protein from which heme has been removed. And a complex of heme protein-derived apoprotein.

In the formula (I) and the formula (II),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 means a group bonded to -CO- in formula (I) and formula (II). And
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 _{- (CH 2) n1 -,} - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2 _{) n1 -O- (CH 2) n2} -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 -, Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ .

In the complex of the metal nanoparticles and heme protein-derived apoprotein, R ¹ , R ² , R ³ , R ⁴ , Y and the metal nanoparticles in formula (I) and / or formula (II) This is the same as R ¹ , R ² , R ³ , R ⁴ , Y and the metal nanoparticles in the complex of the metal nanoparticles and the apoprotein derived from the hemoprotein dimer. The “hemoprotein-derived apoprotein” is the same as the “hemoprotein dimer-derived apoprotein”.

As the complex of the metal nanoparticles and the apoprotein derived from heme protein, in the formula (I) and / or formula (II), R ¹ , R ² , R ³ and R ⁴ are each independently Y represents a lower alkyl group or a lower alkenyl group, and Y is a group represented by the formula —NH— (CH ₂ ) _n1 —NH—CO— (CH ₂ ) _n2 —, wherein n1 and n2 are each independently In the formula (I) and / or formula (II), -NH- means a group that binds to -CO-, and the heme protein is cytochrome or myoglobin, More preferably, the metal nanoparticles are gold nanoparticles or silver nanoparticles.

In addition, as the complex of the metal nanoparticles and the apoprotein derived from heme protein, R ¹ , R ² , R ^3, and R ⁴ in the formula (I) and / or formula (II) are each independently A lower alkyl group or a lower alkenyl group, and Y is a group represented by the formula —NH— (CH ₂ ) _n1 —NH—CO— (CH ₂ ) _n2 —, wherein n1 and n2 are Each independently represents an integer of 1 to 20, and —NH— represents a group bonded to —CO— in formula (I) and / or formula (II), and the hemoprotein is represented by SEQ ID NO: 1. Cytochrome having the amino acid sequence represented by the above, wherein the one amino acid residue is the 63rd histidine, or myoglobin having the amino acid sequence represented by SEQ ID NO: 2, Acid residue is a 126th alanine, the metal nanoparticles, and even more preferably gold nanoparticles or silver nanoparticles.

  Next, an example of a method for producing a complex of the metal nanoparticles and apoprotein derived from heme protein will be described.

The complex of the metal nanoparticles and the apoprotein derived from heme protein is
(A) A plurality of metal nanoparticles are reacted with a disulfide compound represented by the formula (I ′) and / or the formula (II ′) to express the metal nanoparticles by the formula (I) and / or the formula (II). (B) a plurality of the modified metal nanoparticles and a heme protein-derived apoprotein from which heme has been removed reacted to modify the metal nanoparticles (I) ) And / or the heme contained in the group represented by the formula (II) and the apoprotein derived from the heme protein from which heme has been removed, thereby binding the metal nanoparticles to the protein. You can get a body.

  Apoproteins derived from heme proteins from which heme has been removed can be obtained by methods known in the literature, for example, Teale, FWJ Biochim. Biophys. Acta. 1959, 35, 543. and E. Itagaki, and LP Hager, J. Biol. Chem. According to the method described in 1966, 241, 3687.

  Further, the complex of the metal nanoparticle and the heme protein polymer having an apoprotein derived from a heme protein at the end is at least one site depending on the group represented by the formula (I) and / or the formula (II). Heme protein which is modified and is modified with the metal nanoparticles modified by the group represented by the formula (I) and / or the formula (II), and the heme protein from which the heme has been removed and the apoprotein derived from the heme protein It is a complex in which a metal nanoparticle and a heme protein polymer having an apoprotein derived from a heme protein are bonded to each other by binding to the polymer.

In the formula (I) and the formula (II),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 means a group bonded to -CO- in formula (I) and formula (II). And
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 _{- (CH 2) n1 -,} - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2 _{) n1 -O- (CH 2) n2} -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 -, Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ .

In the complex of the metal nanoparticle and a hemoprotein polymer having an apoprotein derived from hemoprotein at its end, R ¹ , R ² , R ³ , R ⁴ , Y and in formula (I) and / or formula (II) The metal nanoparticles are the same as R ¹ , R ² , R ³ , R ⁴ , Y and metal nanoparticles in a complex of a plurality of metal nanoparticles and an apoprotein derived from a hemoprotein dimer. In addition, examples of the “hemoprotein polymer having an apoprotein derived from heme protein at the end” include a protein polymer having the following formula.

In the above formulas (IA) and (IB),
R ¹¹ , R ¹² , R ¹³ and R ¹⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Z is a group represented by -Y11-Y12-, wherein Y11 means a group bonded to -CO- in formula (IA) and formula (IB);
Y11 is selected from the group consisting of O, NH and S;
Y12 has the formula _{- (CH 2) n1 -,} - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 - (CH 2 _{) n1 -, - (CH 2} ) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2) n1 -O _{- (CH 2) n2 -O- (} CH 2) n3 - or _- represented by _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
M ¹ is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
m and n are each an integer, 1 ≦ m + n ≦ 1000,
X ¹ means a protein portion in a hemoprotein mutant, and the hemoprotein mutant is represented by the formula (VII);

The mutant is obtained by replacing one amino acid residue with a cysteine residue in the amino acid sequence of a natural heme protein. Wherein in the variant, -SH means a side chain thiol group of the cysteine residue, wherein Heme means a natural heme formula HS-X ¹ refers to apo of the mutants.

  In the present invention, the protein polymer represented by the general formulas (IA) and (IB) includes both a random copolymer and a block copolymer, and is not limited to either one.

Examples of the complex of the metal nanoparticle and the hemoprotein polymer having an apoprotein derived from hemoprotein at the end include R ¹ , R ² , R ³ and R ^{4 in the} formula (I) and / or formula (II). Each independently represents a lower alkyl group or a lower alkenyl group, and Y is a group represented by the formula —NH— (CH ₂ ) _n1 —NH—CO— (CH ₂ ) _n2 —, n1 and n2 each independently represents an integer of 1 to 20, -NH- represents a group bonded to -CO- in formula (I) and / or formula (II),
A heme protein polymer having a terminal apoprotein derived from heme protein is a polymer represented by the formula (IA),
In the formula (IA), R ¹¹ , R ¹² , R ¹³ and R ¹⁴ each independently represents a lower alkyl group or a lower alkenyl group, and Y 11 is selected from the group consisting of O, NH and S ,
Y12 has the formula _{- (CH 2) n1 -,} - (CH 2) n1 -O- (CH 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 —O— (CH ₂ ) _n3 —O— (CH ₂ ) _n4 —, wherein n1, n2, n3 and n4 each independently represents an integer of 1 to 20,
More preferably, the heme protein is cytochrome or myoglobin, and the metal nanoparticles are gold nanoparticles or silver nanoparticles.

In addition, as a complex of the metal nanoparticle and a heme protein polymer having an apoprotein derived from heme protein at the end, in the formula (I) and / or formula (II), R ¹ , R ² , R ³ and R ⁴ each independently represents a lower alkyl group or a lower alkenyl group, Y is a group represented by the formula —NH— (CH ₂ ) _n1 —NH—CO— (CH ₂ ) _n2 —, Here, n1 and n2 each independently represent an integer of 1 to 20, -NH- represents a group bonded to -CO- in formula (I) and / or formula (II),
A heme protein polymer having a terminal apoprotein derived from heme protein is a polymer represented by the formula (IA),
In the formula (IA), R ¹¹ , R ¹² , R ¹³ and R ¹⁴ each independently represents a lower alkyl group or a lower alkenyl group, Y 11 represents O or NH, and Y 12 represents a formula _{(CH 2) n1 -, -} (CH 2) n1 -O- (CH 2) n2 -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH _{2) n3 -O- (CH 2)} n4 - a group represented by where n1, n2, n3 and n4 denotes an integer of 1 to 20 independently,
The hemoprotein is cytochrome having the amino acid sequence represented by SEQ ID NO: 1, and the one amino acid residue is the 63rd histidine, or myoglobin having the amino acid sequence represented by SEQ ID NO: 2. More preferably, the one amino acid residue is the 126th alanine, and the metal nanoparticles are gold nanoparticles or silver nanoparticles.

  Next, an example of a method for producing a complex of the metal nanoparticles and a heme protein polymer having an apoprotein derived from heme protein at the end will be described.

A complex of the metal nanoparticles and a heme protein polymer having an apoprotein derived from heme protein at its end,
(A) A plurality of metal nanoparticles are reacted with a disulfide compound represented by the formula (I ′) and / or the formula (II ′) to express the metal nanoparticles by the formula (I) and / or the formula (II). (B) reacting a plurality of the modified metal nanoparticles with a heme protein polymer having a terminal apoprotein derived from heme protein from which heme has been removed, The heme contained in the group represented by the formula (I) and / or the formula (II) modified with the particle is bonded to the heme protein polymer having the apoprotein derived from the heme protein from which the heme has been removed terminated. Thus, a complex in which the metal nanoparticles and the heme protein polymer having the apoprotein at the end are bound can be obtained.

  The heme protein polymer having an apoprotein derived from a heme protein from which heme has been removed terminated in accordance with a method known in the literature, for example, the method described in Angew. Chem., Int. Ed., 48, 1271-1274 (2009). Can be manufactured.

  The complex of the metal nanoparticles and the heme protein-derived apoprotein has a structure in which the cofactor heme serving as the active site of the hemoprotein is close to the metal nanoparticles. Therefore, this complex can efficiently transfer electrons to the metal nanoparticles by utilizing the electron transfer function of the hemoprotein. This complex is a bioelectrode that combines the functions of heme proteins and their variants (binding to gas molecules such as oxygen molecules, electron transfer, catalytic reactions including oxidation reactions) and the characteristics of metal nanoparticles. Application in the field of materials is conceivable.

  A complex of the metal nanoparticle and a hemoprotein polymer having an apoprotein derived from hemoprotein at its end has a loosely assembled structure. In other words, the composite has an arrangement in which the distance between the metal nanoparticles is relatively long. For example, when this composite is placed on a substrate, the metal nanoparticles may spread two-dimensionally at an appropriate distance. When protein is removed by baking, plasma irradiation, washing with acidic aqueous solution, etc. on the substrate, gold nanoparticles, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, cadmium sulfide (CdS) nanoparticles, selenization Cadmium (CdSe) nanoparticles, cadmium telluride (CdTe) nanoparticles, zinc sulfide (ZnS) nanoparticles, zinc selenide (ZnSe) nanoparticles, or zinc telluride (ZnTe) nanoparticles or cadmium sulfide (CdS), Core / shell type composed of two types selected from the group consisting of cadmium selenide (CdSe), cadmium telluride (CdTe), zinc sulfide (ZnS), zinc selenide (ZnSe) and zinc telluride (ZnTe) Only metal nanoparticles, such as the nanoparticles, remain on the substrate. If it does so, it is possible to obtain the board | substrate with which the metal nanoparticle was arrange | positioned at a wide space | interval of about several dozen nm to 100 nm. Such a substrate can save the amount of metal when the metal nanoparticles are arranged in a wide area and used as a catalyst.

The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by the following examples.
Various spectra were measured using the following instrument.
The nuclear magnetic resonance (NMR) spectrum was measured using a Bruker DPX-400 nuclear magnetic resonance apparatus (400 MHz), and the residual signal of the measurement solvent was used as an internal standard. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) was measured using an Applied Biosystems Mariner API-TOF workstation (Workstation). The ultraviolet-visible absorption spectrum was measured using a self-recording spectrophotometer UV-2550 or UV-3150 manufactured by Shimadzu Corporation. The pH of the aqueous solution was measured using a pH meter F-52 manufactured by Horiba. For size exclusion chromatography (SEC), an AKTA _FPLC system (exclusion limit: 1.0 × 10 ⁵ Da) manufactured by GE Healthcare was connected, and detection was performed using a UPC-900 detector. For electrophoresis measurement, measurement was performed using a fully automated electrophoresis system Phassystem manufactured by GE Healthcare. The transmission electron microscope (TEM) was measured using Hitachi H-7650 ZeroA manufactured by Hitachi.

The starting material was produced with reference to the following documents.
Protoporphyrin IX mono t-butyl ester 2: T. Matsuo, T. Hayashi, Y. Hisaeda, J. Am. Chem. Soc. 124, 11234 (2002).
As other general reagents, commercially available products were used as they were.

(1) Production of Compound Represented by Formula 1 and its Regioisomer The compound represented by Formula 1 and its regioisomer were produced according to Scheme 2 below.

  In scheme 2, each “positional isomer” is as follows. The positional isomer of Compound 6 is Compound 6 ′, the positional isomer of Compound 7 is Compound 7 ′ and Compound 7 ″, the positional isomer of Compound 8 is Compound 8 ′ and Compound 8 ″, and the positional isomer of Compound 1 is Compound 1 ′ and Compound 1 ″.

(I) Synthesis of Compound 2 and its positional isomer
R. Schneider, F. Schmitt, C. Frochot, Y. Fort, N. Lourette, F. Guillemin, J.-F. Mueller, M. Barberi-Heyob, Bioorg. Med. Chem. 2005, 13, 2799-2808 According to the procedure, compound 2 was synthesized.

(Ii) Synthesis of compound 3 and its positional isomer
According to B. Danieli, A. Giardini, G. Lesma, D. Passarella, B. Peretto, A. Sacchetti, A. Silvani, G. Pratesi, F. Zunino, J. Org. Chem. 2006, 71, 2848-2853 Compound 3 was synthesized.

(Iii) Compound 4 and a nitrogen atmosphere of its regioisomer, Compound 2 and its regioisomer (152mg, 9.51x10 ^-4 mol), Compound 3 and its regioisomer (100mg, 4.76x10 ^-4 mol ) And N, N-diisopropylethylamine (DIPEA) (170 μL, 9.76 × 10 ⁻⁴ mol) were dissolved in methylene chloride (20 mL). The solution was cooled by an ice bath, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (182 mg, 9.51 × 10 ⁻⁴ mol) in methylene chloride (20 mL) was added thereto. . The solution was stirred at room temperature for 18 hours. This solution was washed 5 times with 10% aqueous citric acid solution (50 mL), 5 times with aqueous sodium bicarbonate solution (50 mL), and 5 times with water (50 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain Compound 4 and its regioisomer (140 mg, 60%, colorless, solid).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.91 (s, 2H), 6.75 (s, 2H), 3.03 (m, 4H), 2.95 (m, 4H), 2.86 (t, 4H, J = 5.94 Hz), 2.43 (t, 4H, J = 5.94 Hz).

(Iv) Synthesis of Compound 5 and its Regioisomer Compound 4 and its regioisomer (100 mg, 2.02 × 10 ⁻⁴ mol) were dissolved in methylene chloride (35 mL) under a nitrogen atmosphere, and trifluoroacetic acid (6 mL) was dissolved. added. The solution was stirred at room temperature for 30 minutes. The solvent was distilled off from this solution under reduced pressure to obtain Compound 5 and its positional isomer.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.12 (s, 2H), 7.74 (s, 6H), 3.26 (m, 4H), 2.88 (m, 4H), 2.85 (t, 4H, J = 7.39 Hz), 2.45 (t, 4H, J = 7.39 Hz).

(V) Synthesis of Compound 6 and its positional isomer
According to T. Matsuo, T. Hayashi, Y. Hisaeda, J. Am. Chem. Soc. 2002, 124, 11234-11235, compound 6 and its positional isomer were synthesized.

(Vi) Synthesis of Compound 7 and its Regioisomer Under a nitrogen atmosphere, Compound 6 (42 mg, 8.10 × 10 ⁻⁵ mol), O- (benzotriazol-1-yl) -N, N, N ′, N′— Tetramethyluronium hexafluorophosphate (HBTU) (123 mg, 3.24 × 10 ⁻⁴ mol), DIPEA (120 μL, 6.89 × 10 ⁻⁴ mol) and 1-hydroxybenzotriazole (HOBt) (43 mg, 3.24 × 10 ⁻⁴ mol) ) Was dissolved in DMF (15 mL). The solution was cooled with an ice bath, and a DMF solution (10 mL) of compound 5 and its regioisomer (42 mg, 8.10 × 10 ⁻⁵ mol) was added dropwise thereto. The solution was stirred at room temperature for 18 hours. The solution was diluted with methylene chloride and washed 3 times with 5% aqueous citric acid (50 mL), 3 times with aqueous sodium bicarbonate (50 mL), and 3 times with water (50 mL). The organic layer was separated and dehydrated with 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) to obtain Compound 7 and its regioisomer (40 mg, 33%, purple, solid).

¹ H NMR (400 MHz, pyridine-d ₅ ) δ10.2-10.7 (m, 8H), 8.40-8.21 (m, 4H), 6.40-6.21 (m, 4H), 6.18-6.10 (m, 4H), 4.40-4.31 (m, 8H), 3.65-3.25 (m, 40H), 2.78-2.70 (m, 4H), 2.20-2.10 (m, 4H), 1.24 (s, 18H), −3.85 (br, 4H) .

(Vii) Synthesis of Compound 8 and its positional isomer
Compound 7 (40 mg, 2.67 × 10 ⁻⁵ mol) was dissolved in methylene chloride (20 mL), and trifluoroacetic acid (4 mL) was added thereto. The solution was stirred at room temperature for 7 hours. The solvent was distilled off from this solution under reduced pressure, and the resulting residue was washed with ether to obtain Compound 8 and its regioisomer (29 mg, 80%, purple, solid).

¹ H NMR (400 MHz, pyridine-d ₅ ) δ10.0-9.82 (m, 8H), 8.33-8.28 (m, 4H), 6.36-6.28 (m, 4H), 6.11-6.08 (m, 4H), 4.37-4.33 (m, 8H), 3.60-3.28 (m, 40H), 2.81-2.80 (m, 4H), 2.20-2.15 (m, 4H), -3.85 (br, 4H);
UV-vis (MeOH) λmax / nm (absorbance) 628 (0.0077), 577 (0.015), 540 (0.020), 508 (0.024), 404 (0.30).

(Viii) Synthesis of Compound 1 and its positional isomer Compound 8 and its positional isomer (29 mg, 2.10 × 10 ⁻⁴ mol), iron (II) chloride monohydrate (150 mg, 7.54 × 10 ⁻⁴ mol) and Sodium hydrogen carbonate (10 mg) was dissolved in a mixed solvent of chloroform and methanol (CHCl ₃ / MeOH = 2/1, 40 mL) saturated with nitrogen, and heated to reflux. After 3 hours, the reaction mixture was cooled to room temperature and the solvent was distilled off under reduced pressure. The residue was washed with 0.1 M hydrochloric acid and then with ether to give compound 1 and its regioisomer (25 mg, 80%, purple, solid).

ESI-TOF MS (positive mode) m / z 1490.33 (M + H) ⁺ , C ₇₈ H ₈₂ Fe ₂ N ₁₂ O ₈ S ₂ is calculated as 1490.45;
UV-vis (pyridine) λmax / nm (absorbance) 555 (0.013), 522 (0.014), 407 (0.12).

(2) Synthesis of gold nanoparticles modified with Compound 1 and its regioisomer (i) Synthesis of gold nanoparticles modified with citric acid All glassware used was aqua regia (concentrated hydrochloric acid: concentrated nitric acid = 3 1). A 1 mM chloroauric acid aqueous solution (100 mL) was heated to reflux. A 38.8 mM sodium citrate aqueous solution (10 mL) was quickly added to this aqueous solution, and reflux was further performed for 30 minutes. The reaction mixture was allowed to cool at room temperature to obtain citric acid-protected gold nanoparticles. Formation of gold nanoparticles was confirmed by obtaining a UV-vis spectrum having an absorption maximum in the vicinity of 520 nm, which is characteristic of surface plasmons (FIG. 1). Moreover, the production | generation of the gold nanoparticle was also confirmed from the transmission electron micrograph (FIG. 2).

(Ii) Synthesis of gold nanoparticles modified with compound 1 and its positional isomers To a solution of gold nanoparticles protected with citric acid, 1M aqueous sodium hydroxide solution was added to adjust the pH to 11. A solution of Compound 1 and its regioisomer (940 μg, 6.29 × 10 ⁻⁷ mol) and lipoic acid (2340 μg, 1.03 × 10 ⁻⁵ mol) dissolved in DMSO (1 mL) and gold nanoparticles (about 13 nM, 40 mL) And incubated overnight in the dark at room temperature. This solution was centrifuged at 14,000 rpm for 15 minutes to remove excess compound 1 and its positional isomer and lipoic acid. A sodium hydroxide aqueous solution of pH 11 was added to the residue to redisperse the heme-modified gold nanoparticles. This washing operation was repeated a total of 5 times to obtain gold nanoparticles modified with Compound 1 and its positional isomer. FIG. 3 shows a UV-vis spectrum of the obtained gold nanoparticle modified with compound 1 and its positional isomer, and FIG. 4 shows a transmission electron micrograph.

Quantification of Compound 1 and its regioisomer on the modified gold nanoparticles was performed by UV-vis measurement. When potassium cyanide is added to the modified gold nanoparticle solution, the gold nanoparticles are decomposed into K [Au (CN) ₄ ], and strong absorption derived from surface plasmons disappears. 1M aqueous potassium cyanide solution (30 μL) was added to the modified gold nanoparticle solution (30 μL) and incubated for 2 minutes to complete the reaction. The absorption at around 520 nm derived from the gold nanoparticles disappeared from the UV-vis spectrum of the reaction solution, and the absorption of heme biscyanide complex of Compound 1 and its positional isomer was confirmed (FIG. 5). Since the extinction coefficient at 421 nm of the heme biscyanide complex was 6.8 × 10 ⁴ M ⁻¹ cm ⁻¹ , 37 compounds 1 and their positional isomers that modified the gold nanoparticle surface were quantified.

(3) Generation of the prepared site-directed mutant of the synthesis of cytochrome _{b 562} apoprotein heme from dimer mutants have been removed (i) Cytochrome _{b 562} mutant (H63C) is Takara (TaKaRa) Co. The PCR was performed by polymerase chain reaction (PCR) using an LA PCR in vitro mutagenesis kit according to the attached protocol. Escherichia coli TG1 having an expression plasmid (pUC118) of wild-type cytochrome b ₅₆₂ (hereinafter abbreviated as b ₅₆₂ ) was mass-cultured to prepare a plasmid, which was used as a template for preparing an H63C mutant. Mutation site introduction primer (SEQ ID NO: 5)

(Underlined portion is mismatched base pair) and M13 primer M4 (SEQ ID NO: 6) (5′-GTTTTCCCAGTCACGAC-3 ′), or M13 primer RV SEQ ID NO: 7 (5′-CAGGAAACAGCTATGAC-3 ′) and MUT4 primer SEQ ID NO: 8 ( 5'-GGCCAGTGCCTAGCTTACAT-3 '), and the first stage DNA amplification by PCR was performed in each system. A hetero double-stranded DNA was prepared from these two first-stage PCR products, and the second-stage amplification by PCR of the hetero double-stranded DNA was performed using M13 primer RV and M13 primer M4. A DNA fragment having the base sequence of the _b562 mutant was excised with restriction enzymes EcoRI and HindIII and specifically ligated to the EcoRI / HindIII sites of the pUC118 vector. Thereafter, Escherichia coli strain TG1 was transformed with the obtained expression plasmid. The base sequence of the H63C mutant was confirmed by DNA sequencing (SEQ ID NO: 4). At that time, a mutation was also observed at the position of Ala37, but this mutation was unchanged (GCC was mutated to GCG). Variant protein using the E. coli strain TG1, literature (Y. Kawamata, S. Machida, T. Ogawa, K. Horie, T. Nagamune, J. Lumin. 98, 141 (2002)) according to the expression of the wild-type b ₅₆₂ And expressed in large quantities by the same operation. The expressed protein is purified by cation exchange column (CM-52, 2.7 cm × 18 cm) and gel filtration column (Sephadex G-50, 1.5 cm × 100 cm), Rz = A ₄₁₈ / A ₂₈₀ ≧ 6 0.0 fraction (H63C stock solution) was collected and used in the following experiments.

(Ii) Synthesis of apoprotein in which heme is removed from dimer of cytochrome b ₅₆₂ mutant At the purification stage of H63C, most of the protein forms disulfide by cysteine residue and exists as a dimer. Dimers of cytochrome b ₅₆₂ mutant were fractionated by size exclusion chromatography using a Superdex 7510 / 300GL column (FIG. 6). A 100 mM histidine solution is added to the dimer solution of the obtained cytochrome b ₅₆₂ mutant, 0.1 M hydrochloric acid is added until pH 1.8, and hemin contained in the dimer of the cytochrome b ₅₆₂ mutant is added. Liberated. Hemin was extracted with 2-butanone and dialyzed against 50 mM Tris-HCl buffer to obtain apoprotein from which heme was removed from the dimer of cytochrome b ₅₆₂ mutant (FIG. 7).

(4) from a dimer of the gold nanoparticles and cytochrome _{b 562} variants of synthetic 50mM complex with apoprotein heme is removed Tris-HCl buffer of cytochrome _{b 562} mutant dissolved in (pH 7.3) A solution of gold nanoparticles modified with Compound 1 and its positional isomer (gold nanoparticles about 3 μM, 2 μL) is added to apoprotein (about 100 μM, 8 μL) from which heme has been removed from the dimer, and gold nanoparticles and A complex with the apoprotein from which heme was removed from the dimer of the cytochrome b ₅₆₂ mutant was obtained.

[Comparative Example 1]
Gold nanoparticles and cytochrome _b Tris-HCl buffer synthetic 50mM complex with apoprotein heme has been removed from the ₅₆₂ (pH 7.3) heme cytochrome _{b 562} dissolved in the removed the apoprotein (approximately 10 [mu] M, 8 μL) is added a gold nanoparticle solution modified with compound 1 and its positional isomer (gold nanoparticle approximately 3 μM, 2 μL), and a complex of the gold nanoparticle and the apoprotein in which heme is removed from cytochrome b ₅₆₂ is obtained. Obtained.

[Physical properties of the composite obtained in Example 1 (4) and the composite obtained in Comparative Example 1]
(1) Agarose gel electrophoresis The complex obtained in Example 1 (4) was confirmed by agarose gel electrophoresis. Glycerol was added 10% to the solution containing the complex and added to 1.5% agarose in TBE buffer. Electrophoresis was performed at 100 V and 80 mA for 30 minutes. The obtained electrophoresis gel is shown in FIG. In FIG. 8, lane 1 is a gold nanoparticle modified with compound 1 and its positional isomer (Example 1 (2)), lane 2 is a composite obtained in Comparative Example 1, and lane 3 is Example 1 ( It is an electrophoresis of the complex obtained in 4). From FIG. 8, it was confirmed that the complex of the present invention was a complex of gold nanoparticles and apoprotein obtained by removing heme from the dimer of cytochrome b ₅₆₂ mutant.

(2) Measurement by Transmission Electron Microscope (TEM) The solution of the composite obtained in Example 1 (4) was dropped on a copper grid carrying a collodion support film and dried sufficiently. Ultrapure water was dropped on the grid and absorbed with filter paper. This operation was repeated twice to remove the salt. This grid was set in a TEM measuring device and measured at an acceleration voltage of 100 kV. The obtained TEM photograph is shown to Fig.9 (a)-(d). FIG. 10 is a TEM photograph of the composite obtained in Comparative Example 1. From FIG. 9, since the gold nanoparticles were associated by the apo body of the hemoprotein dimer, and a gap of 2 to 5 nm was observed between the gold nanoparticles, it was mediated through the hemoprotein dimer. It was confirmed to be a gold nanoparticle aggregate.

(3) Measurement by Scanning Electron Microscope (SEM) The solution of the composite obtained in Example 1 (4) was dropped onto a copper grid carrying an elastic carbon support film and sufficiently dried. Ultrapure water was dropped on the grid and absorbed with filter paper. This operation was repeated twice to remove the salt. This grid was set in an SEM measuring apparatus (JEOL JSM-6701F) and measured at an acceleration voltage of 5.0 kV. The obtained SEM photograph is shown in FIG. FIG. 12 is an SEM photograph of the composite obtained in Comparative Example 1. From FIG. 11, it was confirmed that in the case where the apoprotein of the hemoprotein dimer is present, the gold nanoparticles are aggregated, whereas in the case of the non-dimer, the aggregation is hardly observed.

  (1) The compound represented by the formula (III-3) was produced according to the following scheme 3 with reference to Example 3 of an international patent application (application number PCT / JP2009 / 0554569, international publication number WO2009 / 113551). .

The mutant (VII-2) is a cytochrome b ₅₆₂ mutant (H63C, produced in Example 1 (3) (i)). In the mutant, -SH means a side chain thiol group of the cysteine residue, Heme means natural heme, and formula HS-X '' means an apo form of the mutant.

  In scheme 3, each “positional isomer” is as follows. The positional isomer of Compound 1 (8) is Compound 1 (8) ′, the positional isomer of Compound (I-4) is Compound (I-4) ′, and the positional isomer of Compound (II-4) is Compound (II). -4) '.

The polymer (III-3) is represented by the following formula.

  In the above formula, m and n are integers, and m + n is 10.

(2) Synthesis of cytochrome b ₅₆₂ polymer 10 mM dithiothreitol (3 mL) was added to H63C by adding 200 mM to a dimer (200 mL) of cytochrome b ₅₆₂ (H63C) dissolved in 50 mM Tris-HCl buffer (pH 7.3). The disulfide bond by the cysteine residue contained was reduced to obtain a monomer (VII-2) of H63C. Ultrafiltration was performed twice with 50 mM Tris-HCl buffer (pH 7.3) containing 50 mM of 10 mM dithiothreitol and once with 50 mM Tris-HCl buffer (pH 7.3), and excess dithiothreitol was gel filtered. Removed with column. Next, maleimide hem (1 (8) in Scheme 3 and its positional isomer, 1.6 × 10 ⁻⁶ mol) dissolved in DMSO was added to a solution of reduced form of H63C (3.4 mL), and the mixture was stirred at room temperature for 2 hours. did. A hydrochloric acid aqueous solution is added to the reaction solution containing the cytochrome b ₅₆₂ monomer (I-4) and its positional isomer until the pH becomes 1.8 to liberate natural heme, and the cytochrome b ₅₆₂ monomer (II-4) and its position are released. An isomer was obtained. Heme liberated with 2-butanone was extracted. The extraction was repeated 5 times. The cytochrome b ₅₆₂ polymer (III-3) surface-modified with maleimide heme was obtained by dialysis against 50 mM Tris-HCl buffer (pH 7.3). From the UV-vis spectrum shown in FIG. 16, it was confirmed that a cytochrome b ₅₆₂ polymer was formed.

(3) Synthesis of complex of gold nanoparticle and cytochrome b ₅₆₂ polymer Gold nanoparticle modified with compound 1 in 8 μL of cytochrome b ₅₆₂ polymer (about 200 μM) dissolved in 50 mM Tris-HCl buffer (pH 7.3) By adding a solution (gold nanoparticles about 3 μM, 2 μL), a composite in which cytochrome b ₅₆₂ polymer was laminated on the surface of the gold nanoparticles was obtained.

[Physical properties of the composite obtained in Example 2 (3) and the composite obtained in Comparative Example 1]
(1) Agarose gel electrophoresis A complex in which cytochrome b ₅₆₂ polymer was laminated on the gold nanoparticle surface was confirmed by agarose gel electrophoresis. Glycerol was added 10% to the solution containing the complex and added to 1.5% agarose in TBE buffer. Electrophoresis was performed at 100 V and 80 mA for 30 minutes. The obtained electrophoresis gel is shown in FIG. In FIG. 13, lane 1 is a gold nanoparticle modified only with lipoic acid, lane 2 is a gold nanoparticle modified with compound 1 and its positional isomer (Example 1 (2)), and lane 3 is a comparative example. The complex obtained in 1 and lane 4 are electrophoresis of the complex obtained in Example 2 (3). From FIG. 13, it was confirmed that the composite of the present invention was a composite of gold nanoparticles and cytochrome b ₅₆₂ polymer.

(2) Measurement by transmission electron microscope (TEM) The solution of the composite obtained in Example 2 (3) was dropped on a copper grid on which a collodion support film was supported and sufficiently dried. Ultrapure water was dropped on the grid and absorbed with filter paper. This operation was repeated twice to remove the salt. This grid was set in a TEM measuring device and measured at an acceleration voltage of 100 kV. The obtained TEM photograph is shown in FIG. From FIG. 14, it was confirmed that the cytochrome b ₅₆₂ polymer was bonded to the gold nanoparticles and maintained an interval of several tens of nm or more without association between the gold nanoparticles.

(3) Measurement by dynamic light scattering (DLS) Gold nanoparticles modified with lipoic acid, the composite obtained in Comparative Example 1, the DLS measurement results of the composite obtained in Example 2 (3), They are shown in FIGS. 15 (a), 15 (b), and 15 (c), respectively. The average radii of the respective particles were 27 nm, 53 nwm, and 75 nm, and the composite obtained in Example 2 (3) confirmed that the cytochrome b ₅₆₂ polymer was bound to the gold nanoparticles.

(1) Synthesis of silver nanoparticles modified with compound 1 (i) Synthesis of silver nanoparticles modified with citric acid Wash all glassware used with aqua regia (concentrated hydrochloric acid: concentrated nitric acid = 3: 1). did. A 0.25 mM aqueous silver nitrate solution (100 mL) and a 0.31 mM aqueous sodium citrate solution (100 mL) were cooled to 4 ° C., mixed and stirred. A 0.25 mM aqueous sodium borohydride solution (6 mL) was added to the aqueous solution and stirred at 4 ° C. for 10 minutes. After being allowed to cool at room temperature, the reaction mixture was refluxed for 90 minutes. The reaction mixture was allowed to cool at room temperature to obtain citric acid-protected silver nanoparticles. Formation of silver nanoparticles was confirmed by obtaining a UV-vis spectrum having an absorption maximum in the vicinity of 396 nm, which is characteristic of surface plasmons (FIG. 17). Moreover, the production | generation of the silver nanoparticle was also confirmed from the transmission electron micrograph (FIG. 18).

(Ii) Synthesis of Silver Nanoparticles Modified with Compound 1 and Regioisomers Excess sodium citrate and sodium borohydride were removed by centrifuging a solution of silver nanoparticles protected with citric acid. An aqueous sodium hydroxide solution was added to the residue obtained after centrifugation, and the pH of the resulting solution was adjusted to 11. Compound 1 dissolved in DMSO (1 mL) and its regioisomer (1 mg, 6.69 × 10 ⁻⁷ mol, prepared in Example 1 (1)) and lipoic acid (2340 μg, 1.76 × 10 ⁻⁵ mol) Added to particle solution (8 nM, 80 mL) and incubated overnight at 4 ° C. in the dark. The solution was centrifuged at 4400 rpm to concentrate the silver nanoparticle solution. The resulting concentrated solution was subjected to gel filtration using LH20 to remove excess compound 1, its positional isomer and lipoic acid, and silver nanoparticles modified with compound 1 were obtained. FIG. 19 shows a transmission electron micrograph of the obtained silver nanoparticles modified with Compound 1 and its positional isomer.

The presence of Compound 1 and its positional isomer on the modified silver nanoparticles was confirmed by UV-vis measurement. When potassium cyanide is added to the modified silver nanoparticle solution, the silver nanoparticles are decomposed into K [Ag (CN) ₂ ], whereby strong absorption derived from surface plasmons disappears. 1M potassium cyanide aqueous solution (75 μL) was added to the modified silver nanoparticle solution (25 μL) and incubated for 5 minutes to complete the reaction. Absorption near 400 nm derived from silver nanoparticles disappeared from the UV-vis spectrum of the reaction solution, confirming the absorption of the heme biscyanide complex of Compound 1 (FIG. 20). Therefore, the presence of Compound 1 on the modified silver nanoparticles could be confirmed from FIG.

(2) from a dimer of the silver nanoparticles and cytochrome _{b 562} variants of synthetic 50mM complex with apoprotein heme is removed Tris-HCl buffer of cytochrome _{b 562} mutant dissolved in (pH 7.3) A solution of silver nanoparticles modified with compound 1 and its positional isomer (silver nanoparticles) on apoprotein (about 2 μM, 24 μL, prepared in Example 1 (3) (ii)) from which heme has been removed from the dimer About 900 nM, 5 μL) was added to obtain a complex of silver nanoparticles and apoprotein from which heme was removed from the dimer of cytochrome b ₅₆₂ mutant.

[Comparative Example 2]
A solution (5 μL) of silver nanoparticles modified with Compound 1 and its positional isomer to apoprotein (about 2 μM, 24 μL) from which heme has been removed from cytochrome b ₅₆₂ dissolved in 50 mM Tris-HCl buffer (pH 7.3). ) Was added to obtain a complex of silver nanoparticles and apoprotein from which heme was removed from cytochrome b ₅₆₂ .

(3) Measurement by transmission electron microscope (TEM) The solution of the composite obtained in Example 3 (2) was dropped on a copper grid carrying a collodion support film and dried sufficiently. Ultrapure water was dropped on the grid and absorbed with filter paper. This operation was repeated twice to remove the salt. This grid was set in a TEM measuring device and measured at an acceleration voltage of 100 kV. The obtained TEM photographs are shown in FIGS. 21 (a) and 21 (b). FIG. 22 is a TEM photograph of a complex of heme protein obtained in Comparative Example 2 with an aposome. In FIG. 22, formation of silver nanoparticle aggregates could not be confirmed. On the other hand, in FIG. 21 (a) and FIG. 21 (b), since the silver nanoparticles are associated by a heme protein dimer, and a gap of 2-5 nm was observed between the silver nanoparticles, It was confirmed to be an aggregate of silver nanoparticles via heme protein.

  The composite of the present invention can be disposed on a substrate and used as a catalyst.

SEQ ID NO: 1 cytochrome b ₅₆₂
SEQ ID NO: 2 Sperm whale wild-type myoglobin
SEQ ID NO: 3 Horseradish peroxidase SEQ ID NO: 4 H63C cytochrome b ₅₆₂
SEQ ID NO: 5 mutation site introduction primer SEQ ID NO: 6 M13 primer M4
SEQ ID NO: 7 M13 primer RV
SEQ ID NO: 8 MUT4 primer SEQ ID NO: 9 hemoglobin α subunit (human)
SEQ ID NO: 10 hemoglobin β subunit (human)

Claims (4)

A complex of a plurality of metal nanoparticles and an apoprotein derived from a hemoprotein dimer,
The metal nanoparticles are modified at least one place by a group represented by formula (I) and / or formula (II),
By binding the heme contained in the group represented by the formula (I) and / or the formula (II) modified with the metal nanoparticles to the apoprotein derived from the heme protein dimer from which heme has been removed. A complex in which a plurality of the metal nanoparticles and the protein are bound.

In the formula (I) and the formula (II),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 means a group bonded to -CO- in formula (I) and formula (II). And
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 _{- (CH 2) n1 -,} - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2 _{) n1 -O- (CH 2) n2} -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 -, Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ ,
The heme protein dimer is a mutant in which one amino acid residue is replaced with a cysteine residue in the amino acid sequence of a natural heme protein.   The complex according to claim 1, wherein the hemoprotein dimer is a dimer of cytochrome, hemoglobin, myoglobin, or peroxidase. The metal nanoparticles are gold nanoparticles, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, cadmium sulfide (CdS) nanoparticles, cadmium selenide (CdSe) nanoparticles, cadmium telluride (CdTe) nanoparticles, zinc sulfide. (ZnS) nanoparticles, zinc selenide (ZnSe) nanoparticles, or zinc telluride (ZnTe) nanoparticles, or cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe), zinc sulfide (ZnS) ), Zinc selenide (ZnSe), and zinc telluride (ZnTe), which are core / shell type nanoparticles composed of two types selected from the group consisting of zinc telluride (ZnTe). It is a manufacturing method of the composite_body | complex of Claim 1, Comprising:
A group represented by formula (I) and / or formula (II) is obtained by reacting a plurality of metal nanoparticles with a disulfide represented by formula (I ′) and / or formula (II ′). To modify one or more places
A plurality of the modified metal nanoparticles are reacted with apoprotein derived from a heme protein dimer from which heme has been removed, and the metal nanoparticles are modified by the formula (I) and / or the formula (II). A method for producing a complex in which a plurality of the metal nanoparticles and the protein are bound by binding the heme contained in the group to be bound to the apoprotein derived from the heme protein dimer from which heme has been removed .

In the formula (I ′) and formula (II ′),
R ¹ , R ² , R ³ and R ⁴ each independently represents a hydrogen atom, a lower alkyl group or a lower alkenyl group,
Y is a group represented by -Z1-Y1-Z2-Y2- or -Z1-Y1-, wherein Z1 is a group bonded to -CO- in formula (I ') and formula (II'). Means
Y1 and Y2 are _{independently, - (CH 2) n1 -} , - (C 6 H 4) n5 -, - (CH 2) n1 - (C 6 H 4) n5 -, - (C 6 H 4) n5 _{- (CH 2) n1 -,} - (CH 2) n1 - (C 6 H 4) n5 - (CH 2) n2 -, - (CH 2) n1 -O- (CH 2) n2 -, - (CH 2 _{) n1 -O- (CH 2) n2} -O- (CH 2) n3 - or _{- (CH 2) n1 -O- (} CH 2) n2 -O- (CH 2) n3 -O- (CH 2) n4 -, Wherein n1, n2, n3, n4 and n5 each independently represents an integer of 1 to 20,
Z1 is selected from the group consisting of O, NH and S;
Z2 is selected from the group consisting of NHCO, CONH and NH;
M is selected from the group consisting of Fe ²⁺ , Fe ³⁺ , Zn ²⁺ , Co ²⁺ , Co ³⁺ , Mn ²⁺ and Mn ³⁺ .